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1.  Breast cancer subtypes: response to radiotherapy and potential radiosensitisation 
The British Journal of Radiology  2013;86(1023):20120601.
Radiotherapy (RT) is of critical importance in the locoregional management of early breast cancer. Over 50% of patients receive RT at some time during the treatment of their disease, equating to over 500 000 patients worldwide receiving RT each year. Unfortunately, not all patients derive therapeutic benefit and some breast cancers are resistant to treatment, as evidenced by distant metastatic spread and local recurrence. Prediction of individual responses to RT may allow a stratified approach to this treatment permitting those patients with radioresistant tumours to receive higher doses of RT (total and/or tumour cavity boost doses) and/or radiosensitising agents to optimise treatment. Also, for those patients unlikely to respond at all, it would prevent harmful side effects occurring for no therapeutic gain. More selective targeting would better direct National Health Service resources, ease the burden on heavily used treatment RT machines and reduce the economic cost of cancer treatment. Unfortunately, there are no robust and validated biomarkers for predicting RT outcome. We review the available literature to determine whether classification of breast cancers according to their molecular profile may be used to predict successful response to, or increased morbidity from, RT. Class-specific biomarkers for targeting by radiosensitising agents are also discussed.
doi:10.1259/bjr.20120601
PMCID: PMC3608055  PMID: 23392193
2.  Review of Brucellosis Cases from Laboratory Exposures in the United States in 2008 to 2011 and Improved Strategies for Disease Prevention 
Journal of Clinical Microbiology  2013;51(9):3132-3136.
Five laboratory-acquired brucellosis (LAB) cases that occurred in the United States between 2008 and 2011 are presented. The Centers for Disease Control and Prevention (CDC) reviewed the recommendations published in 2008 and the published literature to identify strategies to further prevent LAB. The improved prevention strategies are described.
doi:10.1128/JCM.00813-13
PMCID: PMC3754678  PMID: 23824776
3.  A mechanism of benefit of soy genistein in asthma: inhibition of eosinophil p38-dependent leukotriene synthesis 
Summary
Background
Dietary intake of the soy isoflavone genistein is associated with reduced severity of asthma, but the mechanisms responsible for this effect are unknown.
Objective
To determine whether genistein blocks eosinophil leukotriene C4 (LTC4) synthesis and to evaluate the mechanism of this effect, and to assess the impact of a 4-week period of soy isoflavone dietary supplementation on indices of eosinophilic inflammation in asthma patients.
Methods
Human peripheral blood eosinophils were stimulated in the absence and presence of genistein, and LTC4 synthesis was measured. 5-lipoxygenase (5-LO) nuclear membrane translocation was assessed by confocal immunofluorescence microscopy. Mitogen-activated protein (MAP) kinase activation was determined by immunoblot. Human subjects with mild-to-moderate persistent asthma and minimal or no soy intake were given a soy isoflavone supplement (100 mg/day) for 4 weeks. The fraction of exhaled nitric oxide (FENO) and ex vivo eosinophil LTC4 production were assessed before and after the soy isoflavone treatment period.
Results
Genistein inhibited eosinophil LTC4 synthesis (IC50 80 nm), blocked phosphorylation of p38 MAP kinase and its downstream target MAPKAP-2, and reduced translocation of 5-LO to the nuclear membrane. In patients with asthma, following 4 weeks of dietary soy isoflavone supplementation, ex vivo eosinophil LTC4 synthesis decreased by 33% (N = 11, P = 0.02) and FENO decreased by 18% (N = 13, P = 0.03).
Conclusion
At physiologically relevant concentrations, genistein inhibits eosinophil LTC4 synthesis in vitro, probably by blocking p38- and MAPKAP-2-dependent activation of 5-LO. In asthma patients, dietary soy isoflavone supplementation reduces eosinophil LTC4 synthesis and eosinophilic airway inflammation. These results support a potential role for soy isoflavones in the treatment of asthma.
doi:10.1111/j.1365-2222.2007.02862.x
PMCID: PMC3873088  PMID: 17979994
asthma; eosinophils; genistein; 5-lipoxygenase; soy isoflavones
4.  Physiological effects of housing density on C57BL/6J mice over a 9-month period1 
Journal of animal science  2012;90(13):5182-5192.
The National Research Council has consistently recommended housing densities for animals used in science and agriculture. For mice, the recommended density is 77.4 cm2 (12 in2) for a 15–25 gm mouse. The Council noted that its recommendations were based on “best professional judgment” and encouraged alternatives that were data driven. As part of a continual effort of The Jackson Laboratory to ensure the health and well-being of production and research mice while promoting cost-effective, state-of-the-art research, several density-driven studies have been conducted by lab researchers. The objectives of this study were to determine the effect of housing density on parameters related to mouse physiology and air quality in the cages and to assess the value of specific measured parameters in such studies. The study discussed in this report monitored C57BL/6J mice in individually ventilated cages from weaning until 9 months of age. Housing densities were equivalent to 66.4 and 36.8 cm2/mouse (10.3 and 5.7 in2), representing increases in density of 17% and 110%, respectively, over the National Research Council recommendation. Clinical physiological parameters representing general health and well-being were measured. Hematological traits, plasma lipids and glucose, growth, bone mineral density and percent body fat did not differ between densities. In the more densely housed mice, however, adrenal glands were significantly smaller, heart rates were significantly lower, and food consumption was less. Cage air microenvironment was evaluated for ammonia, carbon dioxide, temperature and humidity in cages changed weekly or every 2 weeks. The cage microenvironment remained within acceptable limits at the higher density of mice at both cage-changing frequencies. The results suggest that mice housed in individually ventilated cages for up to 9 months at up to twice the density currently recommended by the National Research Council show no measurable adverse effects. Continued re-evaluation of the recommendation by measuring additional relevant parameters of health and general well-being and studying additional strains is warranted.
doi:10.2527/jas.2012-5417
PMCID: PMC3561486  PMID: 22952366
adrenal weight; animal husbandry; cage air microenvironment; heart rate; mouse housing density; animal well-being
5.  Effects of Valproic Acid on Organic Acid Metabolism in Children: A Metabolic Profiling Study 
Clinical pharmacology and therapeutics  2011;89(6):10.1038/clpt.2011.47.
Young children are at increased risk for valproic acid (VPA) hepatotoxicity. Urinary organic acid profiles, as a measure of mitochondrial function, were obtained in children 3.5 to 17.3 years old treated for seizure disorders with valproic acid (VPA; n=52). Age-matched patients treated with carbamazepine (CBZ; n=50) and untreated healthy children (n=22) served as controls. Age-related changes in organic acid profiles were observed in all three groups. Although untreated and CBZ control subjects were not distinguished by the principal component analysis (PCA) scores plot, a distinct boundary was apparent between the VPA and control/CBZ groups. Inter-individual variability in VPA-induced alterations in endogenous pathways reflecting branched chain amino acid metabolism and oxidative stress was observed. The data suggest that more detailed metabolomic analysis may provide novel insights into biological mechanisms and predictive biomarkers for children at highest risk for serious toxicity.
doi:10.1038/clpt.2011.47
PMCID: PMC3822904  PMID: 21544075
6.  Genomic Insights into the Immune System of the Sea Urchin 
Science (New York, N.Y.)  2006;314(5801):952-956.
Comparative analysis of the sea urchin genome has broad implications for the primitive state of deuterostome host defense and the genetic underpinnings of immunity in vertebrates. The sea urchin has an unprecedented complexity of innate immune recognition receptors relative to other animal species yet characterized. These receptor genes include a vast repertoire of 222 Toll-like receptors, a superfamily of more than 200 NACHT domain–leucine-rich repeat proteins (similar to nucleotide-binding and oligomerization domain (NOD) and NALP proteins of vertebrates), and a large family of scavenger receptor cysteine-rich proteins. More typical numbers of genes encode other immune recognition factors. Homologs of important immune and hematopoietic regulators, many of which have previously been identified only from chordates, as well as genes that are critical in adaptive immunity of jawed vertebrates, also are present. The findings serve to underscore the dynamic utilization of receptors and the complexity of immune recognition that may be basal for deuterostomes and predicts features of the ancestral bilaterian form.
doi:10.1126/science.1134301
PMCID: PMC3707132  PMID: 17095692
7.  A Review of the Elements of Human Well-Being with an Emphasis on the Contribution of Ecosystem Services 
Ambio  2012;41(4):327-340.
Natural ecosystems perform fundamental life-support services upon which human civilization depends. However, many people believe that nature provides these services for free and therefore, they are of little or no value. While we do not pay for them, we pay significantly for their loss in terms of wastewater treatment facilities, moratoriums on greenhouse gases, increased illnesses, reduced soil fertility and losses in those images of nature that contribute to our basic happiness. Little is understood about the well-being benefits of the natural environment and its ecosystem services. The interwoven relationship of ecosystems and human well-being is insufficiently acknowledged in the wider philosophical, social, and economic well-being literature. In this article, we discuss an approach to examine human well-being and the interactions of its four primary elements—basic human needs, economic needs, environmental needs, and subjective well-being—and ecosystem services.
doi:10.1007/s13280-012-0256-7
PMCID: PMC3393065  PMID: 22581385
Ecosystem services; Human well-being; Sustainability; Ecological economics; Subjective happiness
8.  Aggregation of Sea Urchin Phagocytes Is Augmented In Vitro by Lipopolysaccharide 
PLoS ONE  2013;8(4):e61419.
Development of protocols and media for culturing immune cells from marine invertebrates has not kept pace with advancements in mammalian immune cell culture, the latter having been driven by the need to understand the causes of and develop therapies for human and animal diseases. However, expansion of the aquaculture industry and the diseases that threaten these systems creates the need to develop cell and tissue culture methods for marine invertebrates. Such methods will enable us to better understand the causes of disease outbreaks and to develop means to avoid and remedy epidemics. We report a method for the short-term culture of phagocytes from the purple sea urchin, Strongylocentrotus purpuratus, by modifying an approach previously used to culture cells from another sea urchin species. The viability of cultured phagocytes from the purple sea urchin decreases from 91.6% to 57% over six days and phagocyte morphology changes from single cells to aggregates leading to the formation of syncytia-like structures. This process is accelerated in the presence of lipopolysaccharide suggesting that phagocytes are capable of detecting this molecular pattern in culture conditions. Sea urchin immune response proteins, called Sp185/333, are expressed on the surface of a subset of phagocytes and have been associated with syncytia-like structures. We evaluated their expression in cultured phagocytes to determine their possible role in cell aggregation and in the formation of syncytia-like structures. Between 0 and 3 hr, syncytia-like structures were observed in cultures when only ∼10% of the cells were positive for Sp185/333 proteins. At 24 hr, ∼90% of the nuclei were Sp185/333-positive when all of the phagocytes had aggregated into syncytia-like structures. Consequently, we conclude that the Sp185/333 proteins do not have a major role in initiating the aggregation of cultured phagocytes, however the Sp185/333 proteins are associated with the clustered nuclei within the syncytia-like structures.
doi:10.1371/journal.pone.0061419
PMCID: PMC3629189  PMID: 23613847
9.  Cenozoic climate change and diversification on the continental shelf and slope: evolution of gastropod diversity in the family Solariellidae (Trochoidea) 
Ecology and Evolution  2013;3(4):887-917.
Recent expeditions have revealed high levels of biodiversity in the tropical deep-sea, yet little is known about the age or origin of this biodiversity, and large-scale molecular studies are still few in number. In this study, we had access to the largest number of solariellid gastropods ever collected for molecular studies, including many rare and unusual taxa. We used a Bayesian chronogram of these deep-sea gastropods (1) to test the hypothesis that deep-water communities arose onshore, (2) to determine whether Antarctica acted as a source of diversity for deep-water communities elsewhere and (3) to determine how factors like global climate change have affected evolution on the continental slope. We show that although fossil data suggest that solariellid gastropods likely arose in a shallow, tropical environment, interpretation of the molecular data is equivocal with respect to the origin of the group. On the other hand, the molecular data clearly show that Antarctic species sampled represent a recent invasion, rather than a relictual ancestral lineage. We also show that an abrupt period of global warming during the Palaeocene Eocene Thermal Maximum (PETM) leaves no molecular record of change in diversification rate in solariellids and that the group radiated before the PETM. Conversely, there is a substantial, although not significant increase in the rate of diversification of a major clade approximately 33.7 Mya, coinciding with a period of global cooling at the Eocene–Oligocene transition. Increased nutrients made available by contemporaneous changes to erosion, ocean circulation, tectonic events and upwelling may explain increased diversification, suggesting that food availability may have been a factor limiting exploitation of deep-sea habitats. Tectonic events that shaped diversification in reef-associated taxa and deep-water squat lobsters in central Indo-West Pacific were also probably important in the evolution of solariellids during the Oligo-Miocene.
doi:10.1002/ece3.513
PMCID: PMC3631403  PMID: 23610633
Biogeography; deep sea; Eocene–Oligocene transition; phylogeny
10.  Decreased dopamine activity predicts relapse in methamphetamine abusers 
Molecular psychiatry  2011;17(9):918-925.
Studies in methamphetamine (METH) abusers showed that the decreases in brain dopamine (DA) function might recover with protracted detoxification. However, the extent to which striatal DA function in METH predicts recovery has not been evaluated. Here we assessed whether striatal DA activity in METH abusers is associated with clinical outcomes. Brain DA D2 receptor (D2R) availability was measured with PET and [11C]raclopride in sixteen METH abusers both after placebo and after challenge with 60 mg oral methylphenidate (to measure DA release) to assess if it predicted clinical outcomes. For this purpose METH abusers were tested within 6 months of last METH use and then followed up for 9 months of abstinence. In parallel, 15 healthy controls were tested. METH abusers had lower D2R availability in caudate than controls. Both METH abusers and controls showed decreased striatal D2R availability after MPH and these decreases were smaller in METH than in controls in left putamen. The 6 METH abusers who relapsed during the follow up period had lower D2R availability in dorsal striatum than controls and had no D2R changes after MPH challenge. The 10 METH abusers who completed detoxification did not differ from controls neither in striatal D2R availability nor in MPH-induced striatal dopamine changes. These results provide preliminary evidence that low striatal DA function in METH abusers is associated with a greater likelihood of relapse during treatment. Detection of the extent of DA dysfunction may be helpful in predicting therapeutic outcomes.
doi:10.1038/mp.2011.86
PMCID: PMC3261322  PMID: 21747399
methamphetamine; dopamine release; positron emission tomography; relapse; early withdrawal
12.  Primary cervical cancer screening with HPV testing compared with liquid-based cytology: results of round 1 of a randomised controlled trial – the HPV FOCAL Study 
British Journal of Cancer  2012;107(12):1917-1924.
Background:
Round 1 data of human papillomavirus (HPV) FOCAL, a three-arm, randomised trial, which aims to establish the efficacy of HPV DNA testing as a primary screen for cervical cancer, are presented.
Methods:
The three arms are: Control arm – liquid based cytology with atypical squamous cells of unknown significance (ASC-US) triage with hrHPV testing; Intervention Arm – hrHPV at entry with liquid-based cytology (LBC) triage of hrHPV positives, with exit screen at 4 years; Safety check arm – hrHPV at entry with LBC triage of hrHPV positives with exit screen at 2 years.
Results:
A total of 6154 women were randomised to the control arm and 12 494 to the HPV arms (intervention and safety check). In the HPV arm, the baseline cervical intraepithelial neoplasia (CIN)2+ and CIN3+ rate was 9.2/1000 (95%CI; 7.4, 10.9) and 4.8/1000 (95%CI; 3.6, 6.1), which increased to 16.1/1000 (95%CI 13.2, 18.9) for CIN2+ and to 8.0/1000 (95%CI; 5.9, 10.0) for CIN3+ after subsequent screening of HPV-DNA-positive/cytology-negative women. Detection rate in the control arm remained unchanged after subsequent screening of ASC-US-positive/hrHPV DNA-negative women at 11.0/1000 for CIN2+ and 5.0/1000 for CIN3+.
Conclusion:
After subsequent screening of women who were either hrHPV positive/cytology negative or ASC-US positive/HPV negative, women randomised to the HPV arms had increased CIN2+ detection compared with women randomised to the cytology arm.
doi:10.1038/bjc.2012.489
PMCID: PMC3516684  PMID: 23169286
HPV; cervical cancer; screening; randomised trial; North America
13.  Clinical severity and quality of life in children and adolescents with Rett syndrome 
Neurology  2011;77(20):1812-1818.
Objective:
The clinical features and genetics of Rett syndrome (RTT) have been well studied, but examination of quality of life (QOL) is limited. This study describes the impact of clinical severity on QOL among female children and adolescents with classic RTT.
Methods:
Cross-sectional and longitudinal analyses were conducted on data collected from an NIH-sponsored RTT natural history study. More than 200 participants from 5 to 18 years of age with classic RTT finished their 2-year follow-up at the time of analysis. Regression models after adjustment for their MECP2 mutation type and age at enrollment were used to examine the association between clinical status and QOL.
Results:
Severe clinical impairment was highly associated with poor physical QOL, but worse motor function and earlier age at onset of RTT stereotypies were associated with better psychosocial QOL; conversely, better motor function was associated with poorer psychosocial QOL.
Conclusions:
Standard psychosocial QOL assessment for children and adolescents with RTT differs significantly with regard to their motor function severity. As clinical trials in RTT emerge, the Child Health Questionnaire 50 may represent one of the important outcome measures.
doi:10.1212/WNL.0b013e3182377dd2
PMCID: PMC3233210  PMID: 22013176
14.  Association between Intimate Partner Violence and Health Behaviors of Female Emergency Department Patients 
Introduction
We assessed the correlation between intimate partner violence (IPV) and health behaviors, including seat belt use, smoke alarm in home, handgun access, body mass index, diet, and exercise. We hypothesized that IPV victims would be less likely to have healthy behaviors as compared to women with similar demographics.
Methods
All adult female patients who presented to 3 Atlanta-area emergency department waiting rooms on weekdays from 11AM to 7PM were asked to participate in a computer-based survey by trained research assistants. The Universal Violence Prevention Screen was used for IPV identification. The survey also assessed seatbelt use, smoke alarm presence, handgun access, height, weight, exercise, and diet. We used chi-square tests of association, odds ratios, and independent t-tests to measure associations between variables.
Results
Participants ranged from 18 to 68 years, with a mean of 38 years. Out of 1,452 respondents, 155 patients self-identified as white (10.7%), and 1,218 as black (83.9%); 153 out of 832 women who were in a relationship in the prior year (18.4%) screened positive for IPV. We found significant relationships between IPV and not wearing a seatbelt (p<0.01), handgun access (p<0.01), and eating unhealthy foods (p<0.01).
Conclusion
Women experiencing IPV are more likely to exhibit risky health behaviors than women who are not IPV victims.
doi:10.5811/westjem.2012.3.11747
PMCID: PMC3415833  PMID: 22900126
15.  Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice 
Nutrition & Diabetes  2012;2(7):e36-.
Objective:
Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM).
Design:
Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks–26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet.
Results:
The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs.
Conclusion:
This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.
doi:10.1038/nutd.2012.10
PMCID: PMC3408641  PMID: 23448719
diabetic retinopathy; omega-3 PUFAs; electroretinography
18.  Morphological predictors of BRCA1 germline mutations in young women with breast cancer 
British Journal of Cancer  2011;104(6):903-909.
Background:
Knowing a young woman with newly diagnosed breast cancer has a germline BRCA1 mutation informs her clinical management and that of her relatives. We sought an optimal strategy for identifying carriers using family history, breast cancer morphology and hormone receptor status data.
Methods:
We studied a population-based sample of 452 Australian women with invasive breast cancer diagnosed before age 40 years for whom we conducted extensive germline mutation testing (29 carried a BRCA1 mutation) and a systematic pathology review, and collected three-generational family history and tumour ER and PR status. Predictors of mutation status were identified using multiple logistic regression. Areas under receiver operator characteristic (ROC) curves were estimated using five-fold stratified cross-validation.
Results:
The probability of being a BRCA1 mutation carrier increased with number of selected histology features even after adjusting for family history and ER and PR status (P<0.0001). From the most parsimonious multivariate model, the odds ratio for being a carrier were: 9.7 (95% confidence interval: 2.6–47.0) for trabecular growth pattern (P=0.001); 7.8 (2.7–25.7) for mitotic index over 50 mitoses per 10 high-powered field (P=0.0003); and 2.7 (1.3–5.9) for each first-degree relative with breast cancer diagnosed before age 60 years (P=0.01).The area under the ROC curve was 0.87 (0.83–0.90).
Conclusion:
Pathology review, with attention to a few specific morphological features of invasive breast cancers, can identify almost all BRCA1 germline mutation carriers among women with early-onset breast cancer without taking into account family history.
doi:10.1038/bjc.2011.41
PMCID: PMC3065278  PMID: 21343941
BRCA1; early-onset breast cancer; tumour morphology
19.  Activated KrasG12D is associated with invasion and metastasis of pancreatic cancer cells through inhibition of E-cadherin 
British Journal of Cancer  2011;104(6):1038-1048.
Background:
Pancreatic cancer (PC) harbours an activated point mutation (KrasG12D) in the Kras proto-oncogene that has been demonstrated to promote the development of PC.
Methods:
This study was designed to investigate the effect of the oncogenic KrasG12D allele on aggressiveness and metastatic potential of PC cells. We silenced the oncogenic KrasG12D allele expression in CD18/HPAF and ASPC1 cell lines by stable expression of shRNA specific to the KrasG12Dallele.
Results:
The KrasG12D knockdown cells exhibited a significant decrease in motility (P<0.0001), invasion (P<0.0001), anchorage-dependent (P<0.0001) and anchorage-independent growth (P<0.0001), proliferation (P<0.005) and an increase in cell doubling time (P<0.005) in vitro and a decrease in the incidence of metastases upon orthotopic implantation into nude mice. The knockdown of the KrasG12D allele led to a significant increase in the expression of E-cadherin (mRNA and protein) both in vitro and in vivo. This was associated with a decrease in the expression of phoshpo-ERK-1/2, NF-κB and MMP-9, and transcription factors such as δEF1, Snail and ETV4. Furthermore, the expression of several proteins involved in cell survival, invasion and metastasis was decreased in the KrasG12D knockdown cells.
Conclusions:
The results of this study suggest that the KrasG12D allele promotes metastasis in PC cells partly through the downregulation of E-cadherin.
doi:10.1038/bjc.2011.31
PMCID: PMC3065271  PMID: 21364589
activated Kras; invasion; metastasis; motility; pancreatic cancer
20.  Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin 
Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A–G) which differ from each other by 34–64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.
doi:10.1093/protein/gzq111
PMCID: PMC3038462  PMID: 21149386
botulism; botulinum neurotoxin; molecular evolution; single-chain Fv; yeast display
21.  Chromosomes with delayed replication timing lead to checkpoint activation, delayed recruitment of Aurora B and chromosome instability 
Oncogene  2006;26(13):1852-1861.
Certain chromosome rearrangements display a significant delay in chromosome replication timing (DRT) that is associated with a subsequent delay in mitotic chromosome condensation (DMC). DRT/DMC chromosomes are common in tumor cells in vitro and in vivo and occur frequently in cells exposed to ionizing radiation. A hallmark for these chromosomes is the delayed phosphorylation of serine 10 of histone H3 during mitosis. The chromosome passenger complex, consisting of multiple proteins including Aurora B kinase and INCENP is thought to be responsible for H3 phosphorylation, chromosome condensation and the subsequent segregation of chromosomes. In this report, we show that chromosomes with DRT/DMC contain phosphorylated Chk1, consistent with activation of the S–M phase checkpoint. Furthermore, we show that INCENP is recruited to the DRT/DMC chromosomes during all phases of mitosis. In contrast, Aurora B kinase is absent on DRT/DMC chromosomes when these chromosomes lack serine 10 phosphorylation of H3. We also show that mitotic arrest deficient 2 (Mad2), a member of the spindle assembly checkpoint, is present on DRT/DMC chromosomes at a time when the normally condensed chromosomes show no Mad2 staining, indicating that DRT/DMC activates the spindle assembly checkpoint. Finally, cells with DRT/DMC chromosomes have centrosome amplification, abnormal spindle assembly, endoreduplication and significant chromosome instability.
doi:10.1038/sj.onc.1209995
PMCID: PMC3285441  PMID: 17001311
replication checkpoint; chromosome passenger complex; genomic instability; spindle assembly checkpoint; endoreduplication; chromosome instability
23.  Innate Immune Complexity in the Purple Sea Urchin: Diversity of the Sp185/333 System 
The California purple sea urchin, Strongylocentrotus purpuratus, is a long-lived echinoderm with a complex and sophisticated innate immune system. There are several large gene families that function in immunity in this species including the Sp185/333 gene family that has ∼50 (±10) members. The family shows intriguing sequence diversity and encodes a broad array of diverse yet similar proteins. The genes have two exons of which the second encodes the mature protein and has repeats and blocks of sequence called elements. Mosaics of element patterns plus single nucleotide polymorphisms-based variants of the elements result in significant sequence diversity among the genes yet maintains similar structure among the members of the family. Sequence of a bacterial artificial chromosome insert shows a cluster of six, tightly linked Sp185/333 genes that are flanked by GA microsatellites. The sequences between the GA microsatellites in which the Sp185/333 genes and flanking regions are located, are much more similar to each other than are the sequences outside the microsatellites suggesting processes such as gene conversion, recombination, or duplication. However, close linkage does not correspond with greater sequence similarity compared to randomly cloned and sequenced genes that are unlikely to be linked. There are three segmental duplications that are bounded by GAT microsatellites and include three almost identical genes plus flanking regions. RNA editing is detectible throughout the mRNAs based on comparisons to the genes, which, in combination with putative post-translational modifications to the proteins, results in broad arrays of Sp185/333 proteins that differ among individuals. The mature proteins have an N-terminal glycine-rich region, a central RGD motif, and a C-terminal histidine-rich region. The Sp185/333 proteins are localized to the cell surface and are found within vesicles in subsets of polygonal and small phagocytes. The coelomocyte proteome shows full-length and truncated proteins, including some with missense sequence. Current results suggest that both native Sp185/333 proteins and a recombinant protein bind bacteria and are likely important in sea urchin innate immunity.
doi:10.3389/fimmu.2012.00070
PMCID: PMC3342357  PMID: 22566951
echinoid; invertebrate; evolution; innate immunity; coelomocyte; microsatellites; RNA editing; gene family
24.  Effect of a Targeted Women's Health Intervention in an Inner-City Emergency Department 
Objective. To evaluate the effect of an Emergency Department (ED) based, educational intervention for at-risk health behaviors. Methods. A randomized trial over a one-year period. African American women, aged 21–55, presenting to the ED waiting room were eligible. Each participant took a computer-based survey on health risk behaviors. Participants who screened positive on any of four validated scales (for IPV, nicotine, alcohol, or drug dependence) were randomized to standard information about community resources (control) or to targeted educational handouts based upon their screening results (intervention). Participants were surveyed at 3 months regarding contacts with community resources and harm-reduction actions. Results. 610 women were initially surveyed; 326 screened positive (13.7% for IPV, 40.1% for nicotine addiction, 26.6% for alcohol abuse, and 14.4% for drug abuse). 157 women were randomized to intervention and 169 to control. Among women who completed follow-up (n = 71), women in the Intervention Group were significantly more likely to have contacted local resources (37% versus 9%, P = 0.04) and were more likely to have taken risk-reducing action (97% versus 79%, P = 0.04). Conclusion. Targeted, brief educational interventions may be an effective method for targeting risk behaviors among vulnerable ED populations.
doi:10.1155/2011/543493
PMCID: PMC3235772  PMID: 22203904
25.  The role of SMAD4 in early-onset colorectal cancer 
Objective
Chromosomal loss within the region of 18q and loss of SMAD4 expression have been reported to be frequent somatic events during colorectal cancer tumour progression; however, their associations with age at onset have not been widely studied.
Method
We analysed 109 tumours from a population-based case-family study based on colorectal cancers diagnosed before the age of 45 years. These patients with early-onset colorectal cancer had been previously screened for germ-line mismatch repair gene mutations, microsatellite instability (that included the mononucleotide repeat in TGFβRII) and somatic k-ras mutations. We measured SMAD4 protein expression using immunohistochemistry and SMAD4 copy number using quantitative real-time PCR.
Results
Loss of SMAD4 protein expression was observed in 27/109 (25%) of cancers tested and was more commonly observed in rectal tumours (15/41, 36%) when compared with tumours arising in the colon (11/66, 17%) (P = 0.04). There was no association between SMAD4 protein expression and TGFβR11 mutation status, SMAD4 copy number, family history, MSI status, tumour stage or grade.
Conclusion
Loss of SMAD4 expression is a common feature of early-onset colorectal tumours as it is in colorectal cancers diagnosed in other age-groups. Taken together, the molecular pathways (genetic and epigenetic) now known to be involved in early-onset colorectal cancer only explain a small proportion of the disease and require further exploration.
doi:10.1111/j.1463-1318.2009.01779.x
PMCID: PMC3228835  PMID: 19183329
Early-onset colorectal cancer; SMAD4; 18q; copy number

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