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1.  Medical students’ experiences learning intimate physical examination skills: a qualitative study 
BMC Medical Education  2014;14:39.
Background
Intimate physical examination skills are essential skills for any medical graduate to have mastered to an appropriate level for the safety of his or her future patients. Medical schools are entrusted with the complex task of teaching and assessing these skills for their students. The objectives of this study were to explore a range of medical students’ experiences of learning intimate physical examination skills and to explore their perceptions of factors which impede or promote the learning of these skills.
Methods
Individual semi-structured interviews (N = 16) were conducted with medical students in years two to five from the University of Newcastle, as part of a larger research project investigating how medical students develop their attitudes to gender and health. This was a self-selected sample of the entire cohort who were all invited to participate. A thematic analysis of the transcribed data was performed.
Results
Students reported differing levels of discomfort with their learning experiences in the area of intimate physical examination and differing beliefs about the helpfulness of these experiences. The factors associated with levels of discomfort and the helpfulness of the experience for learning were: satisfaction with teaching techniques, dealing with an uncomfortable situation and perceived individual characteristics in both the patients and the students. The examination causing the greatest reported discomfort was the female pelvic examination by male students.
Conclusions
Student discomfort with the experience of learning intimate physical examination skills may be common and has ongoing repercussions for students and patients. Recommendations are made of ways to modify teaching technique to more closely match students’ perceived needs.
doi:10.1186/1472-6920-14-39
PMCID: PMC3943277  PMID: 24575827
Medical student; Intimate physical examination; Learning experiences; Teaching techniques
2.  Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in C. elegans 
PLoS ONE  2013;8(12):e83450.
An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.
doi:10.1371/journal.pone.0083450
PMCID: PMC3861484  PMID: 24349511
3.  Integration of β-catenin, Sirtuin and FOXO Signaling Protects from Mutant Huntingtin Toxicity 
One of the current challenges of neurodegenerative disease research is to determine whether signaling pathways that are essential to cellular homeostasis might contribute to neuronal survival and modulate the pathogenic process in human disease. In C. elegans, sir-2.1/SIRT1 overexpression protects neurons from the early phases of expanded polyglutamine (polyQ) toxicity, and this protection requires the lonfity-promoting factor daf-16/FOXO. Here, we show this neuroprotective effect also requires the DAF-16/FOXO partner bar-1/β-catenin and putative DAF-16 regulated gene, ucp-4, the sole mitochondrial uncoupling protein (UCP) in nematodes. These results fit with a previously-proposed mechanism in which the β-catenin, FOXO and SIRT1 proteins may together regulate gene expression and cell survival. Knockdown of β-catenin enhanced the vulnerability to cell death of mutant-huntingtin striatal cells derived from the HdhQ111 knock-in mice. In addition, this effect was compensated by SIRT1 overexpression and accompanied by the modulation of neuronal UCP expression levels, further highlighting a cross-talk between β-catenin and SIRT1 in the modulation of mutant polyQ cytoxicity. Taken together, these results suggest that integration of β-catenin, sirtuin and FOXO signaling protects from the early phases of mutant huntingtin toxicity.
doi:10.1523/JNEUROSCI.0277-12.2012
PMCID: PMC3780431  PMID: 22956852
4.  Single-Neuron Sequencing Analysis of L1 Retrotransposition and Somatic Mutation in the Human Brain 
Cell  2012;151(3):483-496.
Summary
A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Since recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of 3 normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.
doi:10.1016/j.cell.2012.09.035
PMCID: PMC3567441  PMID: 23101622
5.  A Transcriptional Regulatory Role of the THAP11–HCF-1 Complex in Colon Cancer Cell Function 
Molecular and Cellular Biology  2012;32(9):1654-1670.
The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes, including transcriptional regulation, but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown. In this work, we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohistochemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases. Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation, and profiling of gene expression in these cells identified a novel gene set composed of 80 differentially expressed genes, 70% of which were derepressed by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor 1) and recruit HCF-1 to target promoters. Importantly, THAP11-mediated gene regulation and its chromatin association require HCF-1, while HCF-1 recruitment at these genes requires THAP11. Collectively, these data provide the first characterization of THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11–HCF-1 complex may be an important transcriptional and cell growth regulator in human colon cancer.
doi:10.1128/MCB.06033-11
PMCID: PMC3347244  PMID: 22371484
6.  A pilot study to evaluate assisted freehand ultrasound elasticity imaging in the sizing of early breast cancer: a comparison of B-mode and AFUSON elasticity ultrasound with histopathology measurements 
The British Journal of Radiology  2011;84(1007):1011-1019.
Objective
This pilot study investigates the role of assisted-freehand ultrasound (AFUSON) elasticity imaging of the breast in assessing the contour, size and area of 23 early breast cancers by making comparison of AFUSON with the equivalent B-mode ultrasound images and gold standard histopathology slides.
Methods
The B-mode, AFUSON and digitised histopathology slides of three early breast cancers were compared for contour, size and area with histopathology scans. AFUSON features that corresponded to areas of known malignant change on the histopathology slides were regarded as diagnostic. These diagnostic criteria were then applied to the B-mode and AFUSON elasticity images of all 23 breast cancers in the pilot study without having the availability of the histopathology scans for reference. Corresponding diameters were measured and the results were compared with the equivalent measurements on the scans of the histology slides. The results were tabulated in histogram form. Diagnostic confidence levels were evaluated.
Results
Size dimension accuracy increased from 66% using B-mode alone to 82% using combined B-mode and AFUSON elasticity images. Tumour area accuracy was also increased. A small number of cases had a striking visual similarity of shape on AFUSON elasticity scans and histopathology slides.
Conclusion
In spite of the shortfalls in this study, AFUSON elasticity imaging was capable of acquiring some high-quality images that showed strong correlation between AFUSON elasticity and scans of histology slides. Further studies will be carried out to refine the technique and determine if it has a role in the diagnosis and management of breast cancer.
doi:10.1259/bjr/13216246
PMCID: PMC3473697  PMID: 21632651
7.  Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence 
The inactivation of ERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.
doi:10.1128/AAC.05720-11
PMCID: PMC3318373  PMID: 22252807
8.  Equine Stomachs Harbor an Abundant and Diverse Mucosal Microbiota 
Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 were Firmicutes (>83% reads/sample), mainly Streptococcus spp., Lactobacillus spp. and, Sarcina spp. Cluster 2 was more diverse, with predominantly Proteobacteria, Bacteroidetes, and Firmicutes, consisting of Actinobacillus spp. Moraxella spp., Prevotella spp., and Porphyromonas spp. Helicobacter sp. sequences were not identified in any of 53,920 reads. FISH (n = 9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual.
doi:10.1128/AEM.06252-11
PMCID: PMC3318809  PMID: 22307294
9.  Methylene Blue Protects against TDP-43 and FUS Neuronal Toxicity in C. elegans and D. rerio 
PLoS ONE  2012;7(7):e42117.
The DNA/RNA-binding proteins TDP-43 and FUS are found in protein aggregates in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and related dementia, but little is known about the neurotoxic mechanisms. We have generated Caenorhabditis elegans and zebrafish animal models expressing mutant human TDP-43 (A315T or G348C) or FUS (S57Δ or R521H) that reflect certain aspects of ALS including motor neuron degeneration, axonal deficits, and progressive paralysis. To explore the potential of our humanized transgenic C. elegans and zebrafish in identifying chemical suppressors of mutant TDP-43 and FUS neuronal toxicity, we tested three compounds with potential neuroprotective properties: lithium chloride, methylene blue and riluzole. We identified methylene blue as a potent suppressor of TDP-43 and FUS toxicity in both our models. Our results indicate that methylene blue can rescue toxic phenotypes associated with mutant TDP-43 and FUS including neuronal dysfunction and oxidative stress.
doi:10.1371/journal.pone.0042117
PMCID: PMC3407135  PMID: 22848727
10.  TDP-1/TDP-43 Regulates Stress Signaling and Age-Dependent Proteotoxicity in Caenorhabditis elegans 
PLoS Genetics  2012;8(7):e1002806.
TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS–associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16–dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
doi:10.1371/journal.pgen.1002806
PMCID: PMC3390363  PMID: 22792076
11.  PAM50 assay and the three-gene model for identifying the major and clinically relevant molecular subtypes of breast cancer 
It has recently been proposed that a three-gene model (SCMGENE) that measures ESR1, ERBB2, and AURKA identifies the major breast cancer intrinsic subtypes and provides robust discrimination for clinical use in a manner very similar to a 50-gene subtype predictor (PAM50). However, the clinical relevance of both predictors was not fully explored, which is needed given that a ~30 % discordance rate between these two predictors was observed. Using the same datasets and subtype calls provided by Haibe-Kains and colleagues, we compared the SCMGENE assignments and the research-based PAM50 assignments in terms of their ability to (1) predict patient outcome, (2) predict pathological complete response (pCR) after anthracycline/taxane-based chemotherapy, and (3) capture the main biological diversity displayed by all genes from a microarray. In terms of survival predictions, both assays provided independent prognostic information from each other and beyond the data provided by standard clinical–pathological variables; however, the amount of prognostic information was found to be significantly greater with the PAM50 assay than the SCMGENE assay. In terms of chemotherapy response, the PAM50 assay was the only assay to provide independent predictive information of pCR in multivariate models. Finally, compared to the SCMGENE predictor, the PAM50 assay explained a significantly greater amount of gene expression diversity as captured by the two main principal components of the breast cancer microarray data. Our results show that classification of the major and clinically relevant molecular subtypes of breast cancer are best captured using larger gene panels.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-012-2143-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s10549-012-2143-0
PMCID: PMC3413822  PMID: 22752290
Breast cancer; Microarrays; PAM50; Prognosis; Gene expression
12.  Study protocol: The registrar clinical encounters in training (ReCEnT) study 
BMC Family Practice  2012;13:50.
Background
Patient encounters are the core learning activity of Australian general practice (family practice) training. Exposure to patient demographics and presentations may vary from one general practice registrar (vocational trainee) to another. This can affect comprehensiveness of training. Currently, there is no mechanism to systematically capture the content of GP registrar consultations. The aim of the Registrar Clinical Encounters in Training (ReCEnT) study is to document longitudinally the nature and associations of consultation-based clinical and educational experiences of general practice registrars.
Methods/design
This is an ongoing prospective multi-site cohort study of general practice registrars’ consultations, entailing paper-based recording of consultation data. The study setting is general practices affiliated with three geographically-based Australian general practice regional training providers. Registrars record details of 60 consecutive consultations. Data collected includes registrar demographics, details of the consultation, patient demographics, reasons for encounter and problems managed. Problems managed are coded with the International Classification of Primary Care (second edition) classification system. Additionally, registrars record educational factors related to the encounter. The study will follow the clinical exposure of each registrar six-monthly over the 18 months to two years (full-time equivalent) of their general practice training program.
Conclusions
The study will provide data on a range of factors (patient, registrar and consultation factors). This data will be used to inform a range of educational decisions as well as being used to answer educational research questions. We plan to use ReCEnT as a formative assessment tool for registrars and help identify and address educational needs. The study will facilitate program evaluation by the participating training providers and thus improve articulation of educational programs with practice experience. From the research point of view it will address an evidence gap – the in-practice clinical and educational experience of general practice trainees, determinants of these experiences, and the determinants of registrars’ patterns of practice (for example, prescribing practice) over the course of their training.
doi:10.1186/1471-2296-13-50
PMCID: PMC3507666  PMID: 22672139
13.  Concordance among gene expression-based predictors for ER-positive breast cancer treated with adjuvant tamoxifen 
Annals of Oncology  2012;23(11):2866-2873.
Background
ER-positive (ER+ ) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only.
Methods
Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan–Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance.
Results
All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%–100%).
Conclusions
Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.
doi:10.1093/annonc/mds080
PMCID: PMC3477878  PMID: 22532584
breast cancer; genomics; luminal; mammaprint; oncotype; PAM50
14.  Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons 
BMC Genomics  2012;13:91.
Background
A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD.
Results
Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified.
Conclusions
Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD.
doi:10.1186/1471-2164-13-91
PMCID: PMC3331833  PMID: 22413862
15.  National Athletic Trainers' Association Position Statement: Prevention of Pediatric Overuse Injuries 
Journal of Athletic Training  2011;46(2):206-220.
Abstract
Objective:
To provide certified athletic trainers, physicians, and other health care professionals with recommendations on best practices for the prevention of overuse sports injuries in pediatric athletes (aged 6–18 years).
Background:
Participation in sports by the pediatric population has grown tremendously over the years. Although the health benefits of participation in competitive and recreational athletic events are numerous, one adverse consequence is sport-related injury. Overuse or repetitive trauma injuries represent approximately 50% of all pediatric sport-related injuries. It is speculated that more than half of these injuries may be preventable with simple approaches.
Recommendations:
Recommendations are provided based on current evidence regarding pediatric injury surveillance, identification of risk factors for injury, preparticipation physical examinations, proper supervision and education (coaching and medical), sport alterations, training and conditioning programs, and delayed specialization.
doi:10.4085/1062-6050-46.2.206
PMCID: PMC3070508  PMID: 21391806
adolescents; children; chronic injuries; microtrauma; growth; development
16.  Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans 
PLoS ONE  2012;7(2):e31321.
Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.
doi:10.1371/journal.pone.0031321
PMCID: PMC3283630  PMID: 22363618
17.  Molecular Epidemiology and Phylogeny Reveal Complex Spatial Dynamics in Areas Where Canine Parvovirus Is Endemic ▿† 
Journal of Virology  2011;85(15):7892-7899.
Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described.
doi:10.1128/JVI.01576-10
PMCID: PMC3147911  PMID: 21593180
18.  Functional emergence of the hippocampus in context fear learning in infant rats 
Hippocampus  2010;20(9):1037-1046.
The hippocampus is a part of the limbic system and is important for the formation of associative memories, such as acquiring information about the context (e.g. the place where an experience occurred) during emotional learning (e.g. fear conditioning). Here, we assess whether the hippocampus is responsible for pups’ newly emerging context learning. In all experiments, postnatal day (PN) 21 and PN24 rat pups received 10 pairings of odor-0.5mA shock or control unpaired odor-shock, odor only and shock only. Some pups were used for context, cue or odor avoidance tests, while the remaining pups were used for c-Fos immunohistochemistry to assess hippocampal activity during acquisition. Our results show that cue and odor avoidance learning were similar at both ages, while contextual fear learning and learning-associated hippocampal (CA1, CA3 and dentate gyrus) activity (c-Fos) only occurred in PN24 paired pups. To assess a causal relationship between the hippocampus and context conditioning, we infused muscimol into the hippocampus, which blocked acquisition of context fear learning in the PN24 pups. Muscimol or vehicle infusions did not affect cue learning or aversion to the odor at PN21 or PN24. The results suggest that the newly emerging contextual learning exhibited by PN24 pups is supported by the hippocampus.
doi:10.1002/hipo.20702
PMCID: PMC2891848  PMID: 19739248
fear conditioning; odor; cue; shock; odor aversion; development
19.  Redefining Lumpectomy Using a Modification of the “Sick Lobe” Hypothesis and Ductal Anatomy 
Objectives. The “Sick Lobe” hypothesis states that breast cancers evolve from entire lobes or portions of lobes of the breast where initiation events have occurred early in development. The implication is that some cancers are isolated events and others are truly multi-focal but limited to single lobar-ductal units. Methods. This is a single surgeon retrospective review of early stage breast cancer lumpectomy patients treated from 1/2000 to 2/2005. Ductal endoscopy was used direct lumpectomy surgical margins by defining ductal anatomy and mapping proliferative changes within the sick lobe for complete excision. Results. Breast conservation surgery for stage 0–2 breast cancer with an attempt to perform endoscopy in association with therapeutic lumpectomy was performed in 554 patients (successful endoscopy in 465 cases). With an average followup of >5 years for the entire group, annual hazard rate for local failure in traditional lumpectomy without ductal mapping was 0.97%/yr. and for lumpectomy with ductal mapping and excision of entire sick lobe was 0.18%/yr. With endoscopy, 42% of patients were found to have extensive disease within their “sick lobe.” Conclusions. Targeting breast cancer lumpectomy using endoscopy and excision of regional associated proliferation seems associated with lower recurrence in this non-randomized series.
doi:10.4061/2011/726384
PMCID: PMC3262561  PMID: 22295232
20.  Transitions in Sensitive Period Attachment Learning in Infancy: The Role of Corticosterone 
Survival of altricial infants, including humans and rats, depends on attachment to the caregiver – a process that requires infants to recognize, learn, and remember their attachment figure. The demands of a dynamic environment combined with a maturing organism requires frequent neurobehavioral reorganization. This restructuring of behavior and its supporting neural circuitry can be viewed through the unique lens of attachment learning in rats in which preference learning is enhanced and aversion learning is attenuated. Behavioral restructuring is well adapted to securing the crucial infant-caregiver relationship regardless of the quality of care. With maturation and the end of the infant-caregiver attachment learning period, the complex interplay of neural structures, hormones, and social behavior coordinates the developing rat’ s eventual transition to life outside of the nest. Nevertheless, early-life environmental and physiological stressors can alter the resilient nature of this system, particularly in respect to the amygdala, and these changes may provide important clues to understanding the lasting effects of early stress.
doi:10.1016/j.neubiorev.2009.11.010
PMCID: PMC2848912  PMID: 19931556
21.  Nuclear factor-kappa B pathway and response in a phase II trial of bortezomib and docetaxel in patients with recurrent and/or metastatic head and neck squamous cell carcinoma 
Annals of Oncology  2009;21(4):864-870.
Background: Our previous study has shown that nuclear factor-kappa B (NF-κB)-signaling pathway was associated with a higher rate of recurrence in head and neck squamous cell carcinoma (HNSCC). The combination of bortezomib, an NF-κB inhibitor by inhibition of proteasomes, plus docetaxel was assessed for efficacy and toxicity.
Materials and methods: Patients with recurrent and/or metastatic HNSCC were enrolled on a phase II bortezomib/docetaxel trial (bortezomib 1.6 mg/m2 and docetaxel 40 mg/m2 on days 1 and 8 of a 21-day cycle). Response was assessed using RECIST. Tissue specimens were evaluated for the presence of human papillomavirus (HPV) and expression of NF-κB-associated genes.
Results: Twenty-one of 25 enrolled patients were assessable for response; one partial response (PR, 5%), 10 stable disease (SD, 48%) and 10 progressive disease (PD, 48%). Patients with PR/SD had significantly longer survival compared with patients with PD and the regimen was well tolerated. Only one of 20 tumors was positive for HPV. Patients with PD had higher expression of NF-κB and epidermal growth factor receptor-associated genes in their tumors by gene expression analysis.
Conclusion: Further understanding of treatment resistance and interactions between bortezomib and docetaxel may provide novel approaches in managing HNSCC.
doi:10.1093/annonc/mdp390
PMCID: PMC2844946  PMID: 19850643
bortezomib; docetaxel; epidermal growth factor receptor; head and neck squamous cell carcinoma; nuclear factor-kappa B
22.  Pre-Risk HIV-Prevention Paradigm Shift: The Feasibility and Acceptability of the Parents Matter! Program in HIV Risk Communities 
Public Health Reports  2010;125(Suppl 1):38-46.
SYNOPSIS
Objectives
Many youth begin human immunodeficiency virus (HIV) sexual risk behaviors in preadolescence, yet risk-reduction programs are typically implemented in middle or late adolescence, missing an important window for prevention. Parent-based programming may play an important role in reaching youth early with prevention messages. One such program is the Parents Matter! Program (PMP), a five-session theory- and evidence-based intervention for parents of children aged 9 to 12 years. A randomized controlled trial showed PMP to be efficacious in promoting effective parent-child communication about sexuality and sexual risk reduction. We assessed the feasibility and acceptability of PMP when implemented under typical programmatic circumstances in communities at high risk for HIV infection.
Methods
We selected 15 sites (including health departments, local education agencies, community-based organizations, and faith-based organizations) throughout the U.S. and Puerto Rico to participate in delivering PMP. Sites were provided training, program materials, and ongoing technical assistance. We collected multilevel data to assess the feasibility of program implementation and delivery, program relevance, and satisfaction with PMP activities and materials.
Results
PMP was successfully implemented and evaluated in 13 of 15 sites; 76% of parents attended at least four of five sessions. Organization-, facilitator-, and parent-level data indicated the feasibility and acceptability of PMP, and overall high satisfaction with PMP activities and materials.
Conclusion
The results of this project demonstrate that HIV pre-risk prevention programs for parents can be implemented and embraced by a variety of community organizations in HIV at-risk communities. The time to embrace parents as partners in public health HIV-prevention efforts has come.
PMCID: PMC2788407  PMID: 20408386
23.  Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function▿  
The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.
doi:10.1128/AEM.02158-09
PMCID: PMC2863451  PMID: 20305029
24.  Anomalous Origin and Retropulmonary Course of an Atherosclerotic Stenosed Left Circumflex Coronary Artery 
We here present the case of a rarely seen anomalous origin and retropulmonary course of the left circumflex artery from the proximal right coronary artery. The patient suffered from coronary ischemia due to stenotic lesions both in the aberrant circumflex coronary artery and in the first and second diagonal branches. Coronary bypass operation was performed.
doi:10.1155/2010/490858
PMCID: PMC2989650  PMID: 21151519
25.  Chromatin Binding of SRp20 and ASF/SF2 and Dissociation from Mitotic Chromosomes Is Modulated by Histone H3 Serine 10 Phosphorylation 
Molecular cell  2009;33(4):450-461.
SUMMARY
Histone H3 serine 10 phosphorylation is a hallmark of mitotic chromosomes but its full function remains to be elucidated. We report here that two SR protein splicing factors, SRp20 and ASF/SF2, associate with interphase chromatin, are released from hyperphosphorylated mitotic chromosomes but reassociate with chromatin late in M-phase. Inhibition of Aurora B kinase diminished histone H3 serine 10 phosphorylation and increased SRp20 and ASF/SF2 retention on mitotic chromosomes. Unexpectedly, we also found that HP1 proteins interact with ASF/SF2 in mitotic cells. Strikingly, siRNA-mediated knockdown of ASF/SF2 caused retention of HP1 proteins on mitotic chromatin. Finally, ASF/SF2-depleted cells released from a mitotic block displayed delayed G0/G1 entry, suggesting a functional consequence of these interactions. These findings underscore the evolving role of histone H3 phosphorylation and demonstrate a direct, functional and histone modification-regulated association of SRp20 and ASF/SF2 with chromatin.
doi:10.1016/j.molcel.2009.02.003
PMCID: PMC2667802  PMID: 19250906

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