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1.  Genomic Confirmation of Hybridisation and Recent Inbreeding in a Vector-Isolated Leishmania Population 
PLoS Genetics  2014;10(1):e1004092.
Although asexual reproduction via clonal propagation has been proposed as the principal reproductive mechanism across parasitic protozoa of the Leishmania genus, sexual recombination has long been suspected, based on hybrid marker profiles detected in field isolates from different geographical locations. The recent experimental demonstration of a sexual cycle in Leishmania within sand flies has confirmed the occurrence of hybridisation, but knowledge of the parasite life cycle in the wild still remains limited. Here, we use whole genome sequencing to investigate the frequency of sexual reproduction in Leishmania, by sequencing the genomes of 11 Leishmania infantum isolates from sand flies and 1 patient isolate in a focus of cutaneous leishmaniasis in the Çukurova province of southeast Turkey. This is the first genome-wide examination of a vector-isolated population of Leishmania parasites. A genome-wide pattern of patchy heterozygosity and SNP density was observed both within individual strains and across the whole group. Comparisons with other Leishmania donovani complex genome sequences suggest that these isolates are derived from a single cross of two diverse strains with subsequent recombination within the population. This interpretation is supported by a statistical model of the genomic variability for each strain compared to the L. infantum reference genome strain as well as genome-wide scans for recombination within the population. Further analysis of these heterozygous blocks indicates that the two parents were phylogenetically distinct. Patterns of linkage disequilibrium indicate that this population reproduced primarily clonally following the original hybridisation event, but that some recombination also occurred. This observation allowed us to estimate the relative rates of sexual and asexual reproduction within this population, to our knowledge the first quantitative estimate of these events during the Leishmania life cycle.
Author Summary
Sexual reproduction is predicted to be a rare event in Leishmania parasites, as evidenced by detection of rare parasite hybrids in natural populations using molecular methods. Recently, a sexual cycle has been detected experimentally in parasites within the sand fly vector (that transmits this pathogenic microorganism to mammalian species including man, causing human leishmaniasis). In this study, we have used whole genome sequencing to investigate genetic variation at the highest level of resolution in Leishmania parasites isolated from sand flies in a defined focus of leishmaniasis in southeast Turkey. Using a range of analytical tools, we show that variation in these parasites arose following a single cross between two diverse strains and subsequent recombination between the progeny, despite mainly clonal reproduction in the parasite population. We have thus been able to derive quantitative estimates of the relative rates of sexual and asexual reproduction during the Leishmania life cycle for the first time, information that will be critical to our understanding of the epidemiology and evolution of this genus.
doi:10.1371/journal.pgen.1004092
PMCID: PMC3894156  PMID: 24453988
2.  Extracellular matrix synthesis in vascular disease: hypertension, and atherosclerosis 
Journal of Biomedical Research  2013;28(1):25-39.
Extracellular matrix (ECM) within the vascular network provides both a structural and regulatory role. The ECM is a dynamic composite of multiple proteins that form structures connecting cells within the network. Blood vessels are distended by blood pressure and, therefore, require ECM components with elasticity yet with enough tensile strength to resist rupture. The ECM is involved in conducting mechanical signals to cells. Most importantly, ECM regulates cellular function through chemical signaling by controlling activation and bioavailability of the growth factors. Cells respond to ECM by remodeling their microenvironment which becomes dysregulated in vascular diseases such hypertension, restenosis and atherosclerosis. This review examines the cellular and ECM components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
doi:10.7555/JBR.27.20130064
PMCID: PMC3904172  PMID: 24474961
extracellular matrix; vascular disease
3.  An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea 
BMC Biotechnology  2013;13:112.
Background
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.
Results
To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.
Conclusion
GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
doi:10.1186/1472-6750-13-112
PMCID: PMC3878110  PMID: 24359319
Vaccine; Halophiles; Archaea; Luciferase
4.  External Beam Accelerated Partial-Breast Irradiation Using 32 Gy in 8 Twice-Daily Fractions: Five-Year Results of a Prospective Study 
PURPOSE
External-beam accelerated partial breast irradiation (APBI) is an increasingly popular technique following treatment of patients for early-stage breast cancer with conventional breast-conserving therapy. Here we present 5-year results of a prospective trial.
METHODS AND MATERIALS
From 10/2003 through 11/2005, 98 evaluable patients with Stage I breast cancer were enrolled on the first dose-step (32 Gy delivered in 8 twice-daily fractions) of a prospective, multi-institutional, dose-escalation clinical trial of three-dimensional conformal external-beam APBI (3D-APBI).
The median age was 61 years; the median tumor size was 0.8 cm; 89% of tumors were estrogen receptor positive; 10% had a triple-negative phenotype; and 1% had a HER-2-positive subtype. Median follow-up was 71 months (range, 2–88 months; interquartile range 64–75 months).
RESULTS
Five patients developed IBTR, for a 5-year actuarial IBTR rate of 5% (95% confidence interval, 1–10%). Three of these occurred in patients with triple-negative disease and 2 in non-triple-negative patients, for 5-year actuarial IBTR rates of 33% (0–57%) and 2% (0–6%; p<0.0001), respectively. On multivariate analysis, triple-negative phenotype was the only predictor of IBTR, with borderline statistical significance after adjusting for tumor grade (p=0.0537).
CONCLUSIONS
Overall outcomes were excellent, particularly for patients with estrogen receptor positive disease. Patients in this study with triple-negative breast cancer had a significantly higher IBTR rate than patients with other receptor phenotypes when treated with 3D-APBI. Larger, prospective 3D-APBI clinical trials should continue evaluating the effect of hormone receptor phenotype on IBTR rates.
doi:10.1016/j.ijrobp.2012.04.019
PMCID: PMC3455124  PMID: 22652104
1. Breast cancer; 2. Triple-negative; 3. 3D conformal; 4. Prospective trial; 5. PBI
5.  Accelerated Partial Breast Irradiation With Low-Dose-Rate Interstitial Implant Brachytherapy After Wide Local Excision: 12-Year Outcomes From a Prospective Trial 
Purpose
To evaluate the long-term toxicity, cosmesis, and local control of accelerated partial breast irradiation with implant brachytherapy after wide local excision for Stage T1N0 breast cancer (BCa).
Materials and Methods
Between 1997 and 2001, 50 patients with Stage T1N0M0 BCa were treated in a Phase I–II protocol using low-dose-rate accelerated partial breast irradiation with implant brachytherapy after wide local excision and lymph node surgery. The total dose was escalated in three groups: 50 Gy (n = 20), 55 Gy (n = 17), and 60 Gy (n = 13). Patient- and physician-assessed breast cosmesis, patient satisfaction, toxicity, mammographic abnormalities, repeat biopsies, and disease status were prospectively evaluated at each visit. Kendall’s tau (τβ) and logistic regression analyses were used to correlate outcomes with dose, implant volume, patient age, and systemic therapy.
Results
The median follow-up period was 11.2 years (range, 4–14). The patient satisfaction rate was 67%, 67% reported good-excellent cosmesis, and 54% had moderate-severe fibrosis. Higher dose was correlated with worse cosmetic outcome (τβ 0.6, p < .0001), lower patient satisfaction (τβ 0.5, p < .001), and worse fibrosis (τβ 0.4, p = .0024). Of the 50 patients, 35% had fat necrosis and 34% developed telangiectasias ≥1 cm2. Grade 3–4 late skin and subcutaneous toxicities were seen in 4 patients (9%) and 6 patients (13%), respectively, and both correlated with higher dose (τβ 0.3–0.5, p ≤ .01). One patient had Grade 4 skin ulceration and fat necrosis requiring surgery. Mammographic abnormalities were seen in 32% of the patients, and 30% underwent repeat biopsy, of which 73% were benign. Six patients had ipsilateral breast recurrence: five elsewhere in the breast, and one at the implant site. One patient died of metastatic BCa after recurrence. The 12-year actuarial local control, recurrence-free survival, and overall survival rate was 85% (95% confidence interval, 70–97%), 72% (95% confidence interval, 54–86%), and 87% (95% confidence interval, 73–99%), respectively.
Conclusion
Low-dose-rate accelerated partial breast irradiation with implant brachytherapy provides acceptable local control in select early-stage BCa patients. However, treatment-related toxicity and cosmetic complications were significant with longer follow-up and at higher doses.
doi:10.1016/j.ijrobp.2011.09.003
PMCID: PMC3786258  PMID: 22099046
Accelerated partial breast irradiation; Implant brachytherapy; Early-stage breast cancer; Low-dose-rate; Long-term cosmesis
6.  PATTERNS AND RISK FACTORS OF LOCOREGIONAL RECURRENCE IN T1-T2 NODE NEGATIVE BREAST CANCER PATIENTS TREATED WITH MASTECTOMY: IMPLICATIONS FOR POSTMASTECTOMY RADIOTHERAPY 
Purpose
Postmastectomy radiation therapy (PMRT) can reduce locoregional recurrences (LRR) in high-risk patients, but its role in the treatment of lymph node negative (LN−) breast cancer remains unclear. The aim of this study was to identify a subgroup of T1-T2 breast cancer patients with LN− who might benefit from PMRT.
Methods and Materials
We retrospectively reviewed 1,136 node-negative T1-T2 breast cancer cases treated with mastectomy without PMRT at the Massachusetts General Hospital between 1980 and 2004. We estimated cumulative incidence rates for LRR overall and in specific subgroups, and used Cox proportional hazards models to identify potential risk factors.
Results
Median follow-up was 9 years. The 10-year cumulative incidence of LRR was 5.2% (95% CI: 3.9–6.7%). Chest wall was the most common (73%) site of LRR. Tumor size, margin, patient age, systemic therapy, and lymphovascular invasion (LVI) were significantly associated with LRR on multivariate analysis. These five variables were subsequently used as risk factors for stratified analysis. The 10-year cumulative incidence of LRR for patients with no risk factors was 2.0% (95% CI: 0.5–5.2%), whereas the incidence for patients with three or more risk factors was 19.7% (95% CI: 12.2–28.6%).
Conclusion
It has been suggested that patients with T1-T2N0 breast cancer who undergo mastectomy represent a favorable group for which PMRT renders little benefit. However, this study suggests that select patients with multiple risk factors including LVI, tumor size ≥2 cm, close or positive margin, age ≤50, and no systemic therapy are at higher risk of LRR and may benefit from PMRT.
doi:10.1016/j.ijrobp.2011.01.015
PMCID: PMC3722592  PMID: 21420245
Breast cancer; Mastectomy; Postmastectomy radiation; Risk factors; Locoregional recurrence
7.  Repeatability of aerobic capacity measurements in Parkinson Disease 
Purpose
Maximal or peak aerobic capacity (VO2peak) during a maximal effort graded exercise test (GXT) is considered by many to be the “gold standard” outcome for assessing the impact of exercise training on cardiorespiratory fitness. The reliability of this measure in Parkinson Disease (PD) has not been established, where the degree of motor impairment can vary greatly and is influenced by medications. This study examined the reliability of VO2peak during maximal effort GXT in subjects with PD.
Methods
Seventy healthy middle-aged and older subjects with PD, Hoehn and Yahr stage 1.5 to 3 underwent a screening/acclimatization maximal effort treadmill test followed by 2 additional maximal effort treadmill tests with repeated measurements of VO2peak. A third VO2peak test was performed in a subset of 21 subjects.
Results
The mean VO2peak measurement was 2.4% higher in the second test compared to the first test (21.42 ± 4.3 ml/kg/min versus 21.93 ± 4.50 ml/kg/min, mean ± SD, p=0.03). The intraclass correlation coefficients (ICC) for VO2peak expressed either as ml/kg/min or L/min was highly reliable, with ICC of 0.90 and 0.94 respectively. The maximum heart rate (ICC of 0.91), and final speed achieved during the tests (ICC of 0.94) were also highly reliable, with the respiratory quotient (RQ) being the least reliable of the parameters measured (ICC of 0.65).
Conclusion
Our results demonstrate that measurement of VO2peak is reliable and repeatable in subjects with mild to moderate PD, thereby validating use of this parameter for assessing the effects of exercise interventions on cardiorespiratory fitness.
doi:10.1249/MSS.0b013e31822432d4
PMCID: PMC3701959  PMID: 21606869
graded exercise test; reliability; cardiorespiratory fitness; maximal oxygen uptake; Parkinson Disease
8.  Correction: Chronic Intrinsic Transient Tracheal Occlusion Elicits Diaphragmatic Muscle Fiber Remodeling in Conscious Rodents 
PLoS ONE  2013;8(5):10.1371/annotation/4d84ef28-da1b-4dba-9df6-e8f2ec51cced.
doi:10.1371/annotation/4d84ef28-da1b-4dba-9df6-e8f2ec51cced
PMCID: PMC3660615
9.  Inspiratory Muscle Training in a Child with Nemaline Myopathy and Organ Transplantation 
Objective
To report the use of inspiratory muscle strength training (IMST) to treat repeated ventilatory insufficiency in a child with nemaline myopathy (NM) who underwent cardiac and renal transplantation.
Design
Case report.
Setting
Pediatric intensive care unit of a tertiary care university teaching hospital.
Intervention
IMST was provided five days weekly for two weeks, accompanied by progressive weaning from non-invasive ventilation.
Measurements and Main Results
Maximal inspiratory pressure (MIP) increased from −36.7 cm H2O to −77.8 cm H2O, accompanied by improved inspiratory flow, volume, pressure activation and power. During the training period, the patient weaned from continuous non-invasive ventilatory assist to her pre-operative level of ventilatory function.
Conclusions
Inspiratory muscle training may be a beneficial component of care for children with NM who experience acute ventilatory insufficiency.
doi:10.1097/PCC.0b013e3181dde680
PMCID: PMC3633211  PMID: 20407395
Myopathy, nemaline; Respiratory insufficiency; Muscle weakness; Respiratory muscles; Strength training; Organ transplantation
10.  Basal Subtype of Invasive Breast Cancer is Associated with a Higher Risk of True Recurrence after Conventional Breast-Conserving Therapy 
Purpose
To determine whether breast cancer subtype is associated with patterns of ipsilateral breast tumor recurrence (IBTR), either true recurrence (TR) or elsewhere local recurrence (ELR), among women with pT1-T2 invasive breast cancer (IBC) who receive breast-conserving therapy (BCT)
Methods and Materials
From 1/1998 to 12/2003, 1223 women with pT1-T2N0-3 IBC were treated with BCT (lumpectomy + whole breast radiation). Ninety percent of patients received adjuvant systemic therapy, but none received trastuzumab. Biologic subtype was approximated using estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2): luminal A (ER+ or PR+ and HER-2-), luminal B (ER+ or PR+ and HER-2+), HER-2 (ER- and PR- and HER-2+), and basal (ER- and PR- and HER-2-). Imaging, pathology and operative reports were reviewed by two physicians independently, including an attending breast radiologist. Readers were blinded to subtype and outcome. TR was defined as IBTR within the same quadrant and within three centimeters of the primary tumor. All others were ELR.
Results
At median follow-up of 70 months, 24 patients developed IBTR (5-year cumulative incidence 1.6%), including 15 TR and 9 ELR. At 5 years, basal (4.4%) and HER-2 (9%) subtypes had a significantly higher incidence of TR compared with luminal B (1.2%) and luminal A (0.2%) subtypes (p<0.0001). On multivariate analysis, basal subtype (HR 4.8, p=0.01), younger age at diagnosis (HR 0.97, p=0.05), and increasing tumor size (HR 2.1. p=0.04) were independent predictors of TR. Only younger age (HR 0.95, p=0.01) significantly predicted for ELR.
Conclusions
Basal and HER2 subtypes are significantly associated with higher rates of TR among women with pT1-T2 IBC after BCT. Younger age predicts for both TR and ELR. Strategies to reduce TR in basal breast cancers, such as increased boost doses, concomitant radiation and chemotherapy, or targeted therapy agents, should be explored.
doi:10.1016/j.ijrobp.2011.02.061
PMCID: PMC3161144  PMID: 21601377
breast cancer; biologic subtype; basal; triple negative; local recurrence; true recurrence; elsewhere recurrence
11.  Sentinel lymph node biopsy at the time of mastectomy does not increase the risk of lymphedema: implications for prophylactic surgery 
Women diagnosed with or at high risk for breast cancer increasingly choose prophylactic mastectomy. It is unknown if adding sentinel lymph node biopsy (SLNB) to prophylactic mastectomy increases the risk of lymphedema. We sought to determine the risk of lymphedema after mastectomy with and without nodal evaluation. 117 patients who underwent bilateral mastectomy were prospectively screened for lymphedema. Perometer arm measurements were used to calculate weight-adjusted arm volume change at each follow-up. Of 234 mastectomies performed, 15.8 % (37/234) had no axillary surgery, 63.7 % (149/234) had SLNB, and 20.5 % (48/234) had axillary lymph node dissection (ALND). 88.0 % (103/117) of patients completed the LEFT-BC questionnaire evaluating symptoms associated with lymphedema. Multivariate analysis was used to assess clinical characteristics associated with increased weight-adjusted arm volume and patient-reported lymphedema symptoms. SLNB at the time of mastectomy did not result in an increased mean weight-adjusted arm volume compared to mastectomy without axillary surgery (p = 0.76). Mastectomy with ALND was associated with a significantly greater mean weight-adjusted arm volume change compared to mastectomy with SLNB (p < 0.0001) and without axillary surgery (p = 0.0028). Patients who underwent mastectomy with ALND more commonly reported symptoms associated with lymphedema compared to those with SLNB or no axillary surgery (p < 0.0001). Patients who underwent mastectomy with SLNB or no axillary surgery reported similar lymphedema symptoms. Addition of SLNB to mastectomy is not associated with a significant increase in measured or self-reported lymphedema rates. Therefore, SLNB may be performed at the time of prophylactic mastectomy without an increased risk of lymphedema.
doi:10.1007/s10549-012-2231-1
PMCID: PMC3563357  PMID: 22941538
Prophylactic mastectomy; Breast cancer-related lymphedema; Sentinel lymph node biopsy; Bilateral mastectomy; Arm swelling
12.  Functional Analysis of Leishmania Cyclopropane Fatty Acid Synthetase 
PLoS ONE  2012;7(12):e51300.
The single gene encoding cyclopropane fatty acid synthetase (CFAS) is present in Leishmania infantum, L. mexicana and L. braziliensis but absent from L. major, a causative agent of cutaneous leishmaniasis. In L. infantum, usually causative agent of visceral leishmaniasis, the CFAS gene is transcribed in both insect (extracellular) and host (intracellular) stages of the parasite life cycle. Tagged CFAS protein is stably detected in intracellular L. infantum but only during the early log phase of extracellular growth, when it shows partial localisation to the endoplasmic reticulum. Lipid analyses of L. infantum wild type, CFAS null and complemented parasites detect a low abundance CFAS-dependent C19Δ fatty acid, characteristic of a cyclopropanated species, in wild type and add-back cells. Sub-cellular fractionation studies locate the C19Δ fatty acid to both ER and plasma membrane-enriched fractions. This fatty acid is not detectable in wild type L. major, although expression of the L. infantum CFAS gene in L. major generates cyclopropanated fatty acids, indicating that the substrate for this modification is present in L. major, despite the absence of the modifying enzyme. Loss of the L. infantum CFAS gene does not affect extracellular parasite growth, phagocytosis or early survival in macrophages. However, while endocytosis is also unaffected in the extracellular CFAS nulls, membrane transporter activity is defective and the null parasites are more resistant to oxidative stress. Following infection in vivo, L. infantum CFAS nulls exhibit lower parasite burdens in both the liver and spleen of susceptible hosts but it has not been possible to complement this phenotype, suggesting that loss of C19Δ fatty acid may lead to irreversible changes in cell physiology that cannot be rescued by re-expression. Aberrant cyclopropanation in L. major decreases parasite virulence but does not influence parasite tissue tropism.
doi:10.1371/journal.pone.0051300
PMCID: PMC3519623  PMID: 23251490
13.  Genome-Wide Screen Identifies Drug-Induced Regulation of the Gene Giant Axonal Neuropathy (Gan) in a Mouse Model of Antiretroviral-Induced Painful Peripheral Neuropathy 
Painful peripheral neuropathy is a debilitating complication of the treatment of HIV with nucleoside reverse transcriptase inhibitors (NRTIs). Patients are living longer with these drugs; however many develop excruciating, unremitting, and often treatment-limiting neuropathy that is resistant to conventional pain management therapies. Improving patient comfort and quality of life is paramount and depends on a clearer understanding of this devastating side effect. The mechanisms underlying the development of NRTI-induced neuropathy, however, remain unclear. Using a mouse model of NRTI-induced neuropathy, the authors conducted an unbiased whole-genome microarray screen to identify molecular targets in the spinal dorsal horn, which is the location where integration of ascending sensory transmission and descending modulatory effects occur. Analysis of the microarray data identified a change in the gene giant axonal neuropathy 1 (Gan1). Mutation of this gene has been linked to the development of giant axonal neuropathy (GAN), a rare autosomal recessive condition characterized by a progressive sensorimotor neuropathy. Gan1 has not been previously linked to nerve pathologies in other populations. In this study, downregulation of the Gan1 gene and the gene protein product, gigaxonin, was validated via quantitative polymerase chain reaction ([qPCR] gene expression) and Western blot analyses (protein level). Our report is the first to suggest that Gan1 might be a novel molecular target in the development of NRTI-induced peripheral neuropathy with implications for new therapeutic approaches to preventing or reducing a significant side effect of HIV treatment.
doi:10.1177/1099800409332726
PMCID: PMC3513273  PMID: 19398414
microarray; painful peripheral neuropathy; chronic pain; gigaxonin; giant axonal neuropathy; HIV/AIDS; HAART; NRTI
14.  Chronic Intrinsic Transient Tracheal Occlusion Elicits Diaphragmatic Muscle Fiber Remodeling in Conscious Rodents 
PLoS ONE  2012;7(11):e49264.
Background
Although the prevalence of inspiratory muscle strength training has increased in clinical medicine, its effect on diaphragm fiber remodeling is not well-understood and no relevant animal respiratory muscle strength training-rehabilitation experimental models exist. We tested the postulate that intrinsic transient tracheal occlusion (ITTO) conditioning in conscious animals would provide a novel experimental model of respiratory muscle strength training, and used significant increases in diaphragmatic fiber cross-sectional area (CSA) as the primary outcome measure. We hypothesized that ITTO would increase costal diaphragm fiber CSA and further hypothesized a greater duration and magnitude of occlusions would amplify remodeling.
Methodology/Principal Findings
Sprague-Dawley rats underwent surgical placement of a tracheal cuff and were randomly assigned to receive daily either 10-minute sessions of ITTO, extended-duration, 20-minute ITTO (ITTO-20), partial obstruction with 50% of cuff inflation pressure (ITTO-PAR) or observation (SHAM) over two weeks. After the interventions, fiber morphology, myosin heavy chain composition and CSA were examined in the crural and ventral, medial, and dorsal costal regions. In the medial costal diaphragm, with ITTO, type IIx/b fibers were 26% larger in the medial costal diaphragm (p<0.01) and 24% larger in the crural diaphragm (p<0.05). No significant changes in fiber composition or morphology were detected. ITTO-20 sessions also yielded significant increases in medial costal fiber cross-sectional area, but the effects were not greater than those elicited by 10-minute sessions. On the other hand, ITTO-PAR resulted in partial airway obstruction and did not generate fiber hypertrophy.
Conclusions/Significance
The results suggest that the magnitude of the load was more influential in altering fiber cross-sectional area than extended-duration conditioning sessions. The results also indicated that ITTO was associated with type II fiber hypertrophy in the medial costal region of the diaphragm and may be an advantageous experimental model of clinical respiratory muscle strength training.
doi:10.1371/journal.pone.0049264
PMCID: PMC3486807  PMID: 23133678
15.  Development of a Data Collection Instrument for Violent Patient Encounters against Healthcare Workers 
Introduction:
Healthcare and social workers have the highest incidence of workplace violence of any industry. Assaults toward healthcare workers account for nearly half of all nonfatal injuries from occupational violence. Our goal was to develop and evaluate an instrument for prospective collection of data relevant to emergency department (ED) violence against healthcare workers.
Methods:
Participants at a high-volume tertiary care center were shown 11 vignettes portraying verbal and physical assaults and responded to a survey developed by the research team and piloted by ED personnel addressing the type and severity of violence portrayed. Demographic and employment groups were compared using the independent-samples Mann-Whitney U Test.
Results:
There were 193 participants (91 male). We found few statistical differences when comparing occupational and gender groups. Males assigned higher severity scores to acts of verbal violence versus females (mean M,F=3.08, 2.70; p<0.001). While not achieving statistical significance, subgroup analysis revealed that attending physicians rated acts of verbal violence higher than resident physicians, and nurses assigned higher severity scores to acts of sexual, verbal, and physical violence versus their physician counterparts.
Conclusion:
This survey instrument is the first tool shown to be accurate and reliable in characterizing acts of violence in the ED across all demographic and employment groups using filmed vignettes of violent acts. Gender and occupation of ED workers does not appear to play a significant role in perception of severity workplace violence.
doi:10.5811/westjem.2011.12.6795
PMCID: PMC3556953  PMID: 23358263
16.  Age, Breast Cancer Subtype Approximation, and Local Recurrence After Breast-Conserving Therapy 
Journal of Clinical Oncology  2011;29(29):3885-3891.
Purpose
Prior results of breast-conserving therapy (BCT) have shown substantial rates of local recurrence (LR) in young patients with breast cancer (BC).
Patients and Methods
We studied 1,434 consecutive patients with invasive BC who received BCT from December 1997 to July 2006. Ninety-one percent received adjuvant systemic therapy; no patients received trastuzumab. Five BC subtypes were approximated: estrogen receptor (ER) or progesterone receptor (PR) positive, HER2 negative, and grades 1 to 2 (ie, luminal A); ER positive or PR positive, HER2 negative, and grade 3 (ie, luminal B); ER or PR positive, and HER2 positive (ie, luminal HER2); ER negative, PR negative, and HER2 positive (ie, HER2); and ER negative, PR negative, and HER2 negative (ie, triple negative). Actuarial rates of LR were calculated by using the Kaplan-Meier method.
Results
Median follow-up was 85 months. Overall 5-year cumulative incidence of LR was 2.1% (95% CI, 1.4% to 3.0%). The 5-year cumulative incidence of LR was 5.0% (95% CI, 3.0% to 8.3%) for age quartile 23 to 46 years; 2.2% (95% CI, 1.0% to 4.6%) for ages 47 to 54 years; 0.9% (95% CI, 0.3% to 2.6%) for ages 55 to 63 years; and 0.6% (95% CI, 0.1% to 2.2%) for ages 64 to 88 years. The 5-year cumulative incidence of LR was 0.8% (95% CI, 0.4% to 1.8%) for luminal A; 2.3% (95% CI, 0.8% to 5.9%) for luminal B; 1.1% (95% CI, 0.2% 7.4%) for luminal HER2; 10.8% (95% CI, 4.6% to 24.4%) for HER2; and 6.7% (95% CI, 3.6% to 12.2%) for triple negative. On multivariable analysis, increasing age was associated with decreased risk of LR (adjusted hazard ratio, 0.97; 95% CI, 0.94 to 0.99; P = .009).
Conclusion
In the era of systemic therapy and BC subtyping, age remains an independent prognostic factor after BCT. However, the risk of LR for young women appears acceptably low.
doi:10.1200/JCO.2011.36.1105
PMCID: PMC3189090  PMID: 21900114
17.  COMPARISON OF DIFFERENT TRUNK ENDURANCE TESTING METHODS IN COLLEGE‐AGED INDIVIDUALS 
Objective:
Determine the reliability of two different modified (MOD1 and MOD2) testing methods compared to a standard method (ST) for testing trunk flexion and extension endurance.
Participants:
Twenty‐eight healthy individuals (age 26.4 ± 3.2 years, height 1.75 ± m, weight 71.8 ± 10.3 kg, body mass index 23.6 ± 3.4 m/kg2).
Method:
Trunk endurance time was measured in seconds for flexion and extension under the three different stabilization conditions. The MOD1 testing procedure utilized a female clinician (70.3 kg) and MOD2 utilized a male clinician (90.7 kg) to provide stabilization as opposed to the ST method of belt stabilization.
Results:
No significant differences occurred between flexion and extension times. Intraclass correlations (ICCs3,1) for the different testing conditions ranged from .79 to .95 (p <.000) and are found in Table 3. Concurrent validity using the ST flexion times as the gold standard coefficients were .95 for MOD1 and .90 for MOD2. For ST extension, coefficients were .91 and .80, for MOD1 and MOD2 respectively (p <.01).
Conclusions:
These methods proved to be a reliable substitute for previously accepted ST testing methods in normal college‐aged individuals. These modified testing procedures can be implemented in athletic training rooms and weight rooms lacking appropriate tables for the ST testing.
Level of Evidence:
3
PMCID: PMC3474305  PMID: 23091786
Core; stabilization; trunk endurance
18.  A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei 
Biochimica et Biophysica Acta  2012;1823(7):1178-1191.
The small GTPase Arl6 is implicated in the ciliopathic human genetic disorder Bardet–Biedl syndrome, acting at primary cilia in recruitment of the octomeric BBSome complex, which is required for specific trafficking events to and from the cilium in eukaryotes. Here we describe functional characterisation of Arl6 in the flagellated model eukaryote Trypanosoma brucei, which requires motility for viability. Unlike human Arl6 which has a ciliary localisation, TbARL6 is associated with electron-dense vesicles throughout the cell body following co-translational modification by N-myristoylation. Similar to the related protein ARL-3A in T. brucei, modulation of expression of ARL6 by RNA interference does not prevent motility but causes a significant reduction in flagellum length. Tubulin is identified as an ARL6 interacting partner, suggesting that ARL6 may act as an anchor between vesicles and cytoplasmic microtubules. We provide evidence that the interaction between ARL6 and the BBSome is conserved in unicellular eukaryotes. Overexpression of BBS1 leads to translocation of endogenous ARL6 to the site of exogenous BBS1 at the flagellar pocket. Furthermore, a combination of BBS1 overexpression and ARL6 RNAi has a synergistic inhibitory effect on cell growth. Our findings indicate that ARL6 in trypanosomes contributes to flagellum biogenesis, most likely through an interaction with the BBSome.
Graphical abstract
Highlights
► The BBSome-associated protein ARL6 localises to vesicles in Trypanosoma brucei. ► T. brucei ARL6 is N-myristoylated. ► RNAi knockdown causes a decrease in flagellum length but does not affect motility. ► TbARL6 binds to tubulin and has a relatively low affinity for guanine nucleotides. ► The BBSome subunit BBS1 and ARL6 are functionally linked in trypanosomes.
doi:10.1016/j.bbamcr.2012.05.007
PMCID: PMC3793860  PMID: 22609302
Arf, ADP-ribosylation factor; Arl, ADP-ribosylation factor-like; Arl6ip, Arl6 interacting protein; BBS, Bardet–Biedl syndrome; BBS1, Bardet–Biedl syndrome 1 protein; BSF, bloodstream form; ConA, Concanavalin A; GEF, guanine nucleotide exchange factor; GPCR, G-protein coupled receptor; HRG4, human retinal gene 4; IFT, intraflagellar transport; ITC, isothermal titration calorimetry; MANT, N-methylanthraniloyl; MAP2, microtubule associated protein 2; NES, nuclear export signal; NLS, nuclear localisation signal; NMT, myristoyl-CoA:protein N-myristoyltransferase; PCF, procyclic form; PCM1, pericentriolar material 1; PFR, paraflagellar rod; PM, plasma membrane; RNAi, RNA interference; RP2, retinitis pigmentosa protein 2; TAP, tandem affinity purification; TiEM, transmission immuno-electron microscopy; Trypanosoma brucei; Arl6; BBSome; BBS1; Flagellum; Tubulin
19.  The Effect of Class II Transactivator Mutations on Bleomycin-Induced Lung Inflammation and Fibrosis 
IFN-γ expression increases during the inflammatory response after bleomycin injury in mice. IFN-γ deficiency attenuates lung inflammation and fibrosis. Because IFN-γ stimulates class II transactivator (CIITA) expression, which activates major histocompatibility class (MHC) II and represses collagen expression, it was hypothesized that CIITA mediates IFN-γ action after bleomycin injury. To test this hypothesis, two CIITA mouse lines, one carrying a mutation of the leucine-rich region of CIITA (CIITA C−/−) and one with a deletion extending into the GTP-binding domain (CIITA G−/−), were used. IFN-γ treatment of lung cells isolated from both strains of mice induced mutant CIITA expression, which did not activate MHC II transcription. Collagen expression was similar in both mutant mouse strains and comparable to C57BL/6 (wild-type) mice. When mice were exposed to intratracheal bleomycin, both strains of CIITA mutant mice retained body weight and altered inflammation at 14 days after bleomycin injury compared with bleomycin-treated wild-type mice. However, there was no difference in fibrosis as judged by histology, mRNA, and protein expression of lungs. Bronchoalveolar lavage cells from CIITA C−/− and C57BL/6 lungs were examined at 3, 7, and 14 days after bleomycin injury. CD4 mRNA expression in bronchoalveolar lavage cells was down-regulated, whereas IL-4 and IL-10 expression was up-regulated, in CIITA C−/− mice, indicating a diminished, skewed Th2 response. The expression of IFN-γ was the same in all mice tested. Combined, our data suggest that CIITA mutations altered the immune response without affecting fibrosis.
doi:10.1165/rcmb.2009-0416OC
PMCID: PMC3135849  PMID: 20705943
collagen; CIITA; IFN-γ; fibrosis; bleomycin
20.  Pompe disease gene therapy 
Human Molecular Genetics  2011;20(R1):R61-R68.
Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.
doi:10.1093/hmg/ddr174
PMCID: PMC3095055  PMID: 21518733
21.  A Standardized Method for Quantification of Developing Lymphedema in Patients Treated for Breast Cancer 
Purpose
The lack of standard method to quantify developing breast cancer related lymphedema (BCRL) impedes the progress in research and clinical practice. We therefore developed a simple and practical formula for quantifying both the asymmetry of upper extremities' volumes and their temporal changes.
Methods & Materials
We present the analysis of bilateral perometer measurements of the upper extremity in a series of 677 women who prospectively underwent lymphedema screening during their treatment for unilateral breast cancer at Massachusetts General Hospital between August 2005 and November 2008. Four sources of variation are analyzed: between repeated measurements on the same arm at the same session, between both arms at the baseline (pre-operative) visit, in follow-up measurements, and between patients. We analyze the effects of hand dominance, time since diagnosis and surgery, age, weight, and body mass index (BMI).
Results
The statistical distribution of variation of measurements suggests that the ratio of volume ratios is most appropriate for quantification of both asymmetry and temporal changes. Therefore, we present the formula for Relative Volume Change (RVC): RVC=(A2U1)/(U2A1)-1, where A1, A2 are arm volumes on the side of the treated breast at two different time points, and U1, U2 are volumes on the contralateral side. RVC is not significantly associated with hand dominance, age, or time since diagnosis. Baseline weight correlates (P=0.0016) with higher RVC; however, baseline BMI or weight changes over time do not.
Conclusions
We propose the use of RVC formula to assess the presence and the course of BCRL in clinical practice and research.
doi:10.1016/j.ijrobp.2010.01.001
PMCID: PMC2952286  PMID: 20605339
lymphedema; quantification; standardized method; perometer; breast cancer
22.  The Orthologue of Sjögren's Syndrome Nuclear Autoantigen 1 (SSNA1) in Trypanosoma brucei Is an Immunogenic Self-Assembling Molecule 
PLoS ONE  2012;7(2):e31842.
Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.
doi:10.1371/journal.pone.0031842
PMCID: PMC3282761  PMID: 22363749
23.  Therapeutic Vaccination With Recombinant Adenovirus Reduces Splenic Parasite Burden in Experimental Visceral Leishmaniasis 
The Journal of Infectious Diseases  2012;205(5):853-863.
Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani–infected BALB/c mice, HASPB- and KMP11-specific CD8+ T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ+CD8+ T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.
doi:10.1093/infdis/jir842
PMCID: PMC3274377  PMID: 22301630
24.  The feasibility of using natural language processing to extract clinical information from breast pathology reports 
Objective:
The opportunity to integrate clinical decision support systems into clinical practice is limited due to the lack of structured, machine readable data in the current format of the electronic health record. Natural language processing has been designed to convert free text into machine readable data. The aim of the current study was to ascertain the feasibility of using natural language processing to extract clinical information from >76,000 breast pathology reports.
Approach and Procedure:
Breast pathology reports from three institutions were analyzed using natural language processing software (Clearforest, Waltham, MA) to extract information on a variety of pathologic diagnoses of interest. Data tables were created from the extracted information according to date of surgery, side of surgery, and medical record number. The variety of ways in which each diagnosis could be represented was recorded, as a means of demonstrating the complexity of machine interpretation of free text.
Results:
There was widespread variation in how pathologists reported common pathologic diagnoses. We report, for example, 124 ways of saying invasive ductal carcinoma and 95 ways of saying invasive lobular carcinoma. There were >4000 ways of saying invasive ductal carcinoma was not present. Natural language processor sensitivity and specificity were 99.1% and 96.5% when compared to expert human coders.
Conclusion:
We have demonstrated how a large body of free text medical information such as seen in breast pathology reports, can be converted to a machine readable format using natural language processing, and described the inherent complexities of the task.
doi:10.4103/2153-3539.97788
PMCID: PMC3424662  PMID: 22934236
Breast pathology reports; clinical decision support; natural language processing
25.  Impaired Economy of Gait and Decreased Six-Minute Walk Distance in Parkinson's Disease 
Parkinson's Disease  2011;2012:241754.
Changes in the biomechanics of gait may alter the energy requirements of walking in Parkinson's Disease (PD). This study investigated economy of gait during submaximal treadmill walking in 79 subjects with mild to moderate PD and the relationship between gait economy and 6-minute walk distance (6 MW). Oxygen consumption (VO2) at the self-selected treadmill walking speed averaged 64% of peak oxygen consumption (VO2 peak). Submaximal VO2 levels exceeded 70% of VO2 peak in 30% of the subjects. Overall the mean submaximal VO2 was 51% higher than VO2 levels expected for the speed and grade consistent with severe impairment in economy of gait. There was an inverse relationship between economy of gait and 6MW (r = −0.31, P < 0.01) and with the self-selected walking speed (r = −0.35, P < 0.01). Thus, the impairment in economy of gait and decreased physiologic reserve result in routine walking being performed at a high percentage of VO2 peak.
doi:10.1155/2012/241754
PMCID: PMC3171762  PMID: 21922051

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