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1.  The crystallization of apo-form UMP kinase from Xanthomonas campestris is significantly improved in a strong magnetic field 
A bacterial UMP kinase from the plant pathogen X. campestris pathovar campestris has been overexpressed in E. coli, purified and crystallized in a strong magnetic field. The crystals diffracted to 2.35 Å.
Bacterial UMP kinases (UMPKs) are crucial enzymes that are responsible for microbial UTP biosynthesis. Interestingly, eukaryotic and prokaryotic cells use different enzymes for UMP-phosphorylation reactions. Prokaryotic UMPKs are thus believed to be potential targets for antimicrobial drug development. Here, the cloning, expression and crystallization of SeMet-substituted XC1936, a bacterial UMPK from Xanthomonas campestris pathovar campestris, are reported. The crystallization of the apo-form UMPK was found to be significantly improved in a strong magnetic field; the crystals diffracted to a resolution of 2.35 Å, a dramatic improvement over the original value of 3.6 Å. Preliminary structural analyses of apo-form XC1936 using crystals grown in a strong magnetic field clearly reveal well defined loop regions involved in substrate-analogue binding that were previously not visible. Crystallization in a strong magnetic field thus was found to be indispensable in determining the flexible region of the XC1936 UMPK structure.
doi:10.1107/S1744309107018787
PMCID: PMC2335002  PMID: 17565191
Xanthomonas campestris; UMPK; optimum solubility screening; crystallization in a magnetic field
2.  Brilliant Sm, Eu, Tb, and Dy Chiral Lanthanide Complexes with Strong Circularly Polarized Luminescence 
The synthesis, characterization, and luminescent behavior of trivalent Sm, Eu, Dy, and Tb complexes of two enantiomeric, octadentate, chiral, 2-hydroxyisophthalamide ligands are reported. These complexes are highly luminescent in solution. Functionalization of the achiral parent ligand with a chiral 1-phenylethylamine substituent on the open face of the complex in close proximity to the metal center yields complexes with strong circularly polarized luminescence (CPL) activity. This appears to be the first example of a system utilizing the same ligand architecture to sensitize four different lanthanide cations and display CPL activity. The luminescence dissymmetry factor, glum, recorded for the Eu(III) complex is one of the highest values reported, and this is the first time the CPL effect has been demonstrated for a Sm(III) complex with a chiral ligand. The combination of high luminescence intensity with CPL activity should enable new bioanalytical applications of macromolecules in chiral environments.
doi:10.1021/ja064902x
PMCID: PMC2647558  PMID: 17199285
3.  Effects of 3'-deoxynucleoside 5'-triphosphate concentrations on chain termination by nucleoside analogs during human immunodeficiency virus type 1 reverse transcription of minus-strand strong-stop DNA. 
Journal of Virology  1996;70(2):712-720.
We have compared the effects of nucleoside analogs in quiescent and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) exposed to human immunodeficiency virus type 1 (HIV-1) with those of their triphosphorylated derivatives in cell-free HIV-1 reverse transcription assays. We observed a substantial decrease in synthesis of early minus-strand proviral DNA products in HIV-1-infected, quiescent PBMC exposed to each of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and 2',3'-dideoxy-3'-thiacytidine (3TC), in comparison with nontreated, infected controls. In contrast, no such diminution was observed when PHA-stimulated, HIV-1-infected PBMC were treated with the same drugs. This result was attributed to previously reported findings that PHA-stimulated PBMC possessed larger deoxynucleoside triphosphate (dNTP) pools than quiescent cells did. To further investigate this subject, a cell-free HIV-1 reverse transcription reaction involving HIV-1 RNA genomic template, recombinant purified HIV-1 reverse transcriptase, all four dNTPs and either tRNA3Lys or a deoxyoligonucleotide as primer was used to monitor chain termi-nation mediated by 2',3'-dideoxynucleoside triphosphates (ddNTPs) during synthesis of minus-strand strong-stop DNA. Augmented chain termination was observed with decreasing concentrations of both ddNTP and dNTP when the ratio of dNTP to ddNTP was fixed. We also found that both the number and strength of reverse transcription pause sites were increased at low concentrations of dNTPs and when a deoxyoligonucleotide primer was used in place of the cognate primer, tRNA3Lys. Preferential incorporation of ddATP was observed dur-ing reverse transcription opposite a distinct pause site in a short synthetic RNA template. These results con-firm the notion that the antiviral activities of ddNTP are dependent on both cellular dNTP pools and the state of cellular activation. Pausing of HIV-1 reverse transcriptase during reverse transcription, altered by dNTP concentrations, may be a mechanism that controls the position and extent of incorporation of nucleoside analogs.
PMCID: PMC189871  PMID: 8551607
4.  Complete Genome Sequence of the Strong Mutator Salmonella enterica subsp. enterica Serotype Heidelberg Strain B182 
Journal of Bacteriology  2012;194(13):3537-3538.
In bacteria, normal mutation frequencies are mostly around 10−10 per base pair. However, there exists natural isolates, called “mutators,” that exhibit permanent mutation occurrences up to 1,000-fold greater than usual. As mutations play essential roles, particularly in the evolution of antibiotic resistance, bacteria showing elevated mutation rates could have an important responsibility in the emergence of antibiotic resistance, especially in the clinical background. In this announcement, we report the first complete genome sequence of the Salmonella enterica subsp. enterica serotype Heidelberg B182 mutator strain, isolated from bovine feces (France), which consists of a 4,750,465-bp circular chromosome (cB182_4750; GC, 52.2%) and one circular plasmid of 37,581 bp (pB182_37; GC, 42.8%).
doi:10.1128/JB.00498-12
PMCID: PMC3434739  PMID: 22689230
5.  A common prostate cancer risk variant 5’ of MSMB (microseminoprotein-beta) is a strong predictor of circulating MSP (microseminoprotein) in multiple populations 
Background
Beta-microseminoprotein (MSP) is one of the three most abundantly secreted proteins of the prostate, and has been suggested as a biomarker for prostate cancer risk. A common variant, rs10993994, in the 5’ region of the gene which encodes MSP (MSMB), has recently been identified as a risk factor for prostate cancer.
Methods
We examined the association between rs10993994 genotype and MSP levels in a sample of 500 prostate cancer-free men from four racial/ethnic populations in the Multiethnic Cohort (European Americans, African Americans, Latinos, and Japanese Americans). Generalized linear models were used to estimate the association between rs10993994 genotype and MSP levels.
Results
We observed robust associations between rs10994994 genotype and MSP levels in each racial/ethnic population (all P<10−8) with carriers of the C allele having lower geometric mean MSP levels (ng/mL) (CC/CT/TT genotypes: European Americans, 28.8/20.9/10.0; African Americans, 29.0/21.9/10.9; Latinos, 29.2/17.1/8.3; and Japanese Americans 25.8/16.4/6.7). We estimated the variant accounts for 30–50% of the variation in MSP levels in each population. We also observed significant differences in MSP levels between populations (P=3.5×10−6), with MSP levels observed to be highest in African Americans and lowest in Japanese Americans.
Conclusions
Rs10993994 genotype is strongly associated with plasma MSP levels in multiple racial/ethnic populations.
Impact
This supports the hypothesis that rs10993994 may be the biologically functional allele.
doi:10.1158/1055-9965.EPI-10-0427
PMCID: PMC3041671  PMID: 20736317
MSMB; beta-microseminoprotein; prostate; genetic; multiethnic
6.  Long term voluntary wheel running is rewarding and produces plasticity in the mesolimbic reward pathway 
Behavioural brain research  2010;217(2):354-362.
The mesolimbic reward pathway is implicated in stress-related psychiatric disorders and is a potential target of plasticity underlying the stress resistance produced by repeated voluntary exercise. It is unknown, however, whether rats find long-term access to running wheels rewarding, or if repeated voluntary exercise reward produces plastic changes in mesolimbic reward neurocircuitry. In the current studies, young adult, male Fischer 344 rats allowed voluntary access to running wheels for 6 weeks, but not 2 weeks, found wheel running rewarding, as measured by conditioned place preference (CPP). Consistent with prior reports and the behavioral data, 6 weeks of wheel running increased ΔFosB/FosB immunoreactivity in the nucleus accumbens (Acb). In addition, semi quantitative in situ hybridization revealed that 6 weeks of wheel running, compared to sedentary housing, increased tyrosine hydroxylase (TH) mRNA levels in the ventral tegmental area (VTA), increased delta opioid receptor (DOR) mRNA levels in the Acb shell, and reduced levels of dopamine receptor (DR)-D2 mRNA in the Acb core. Results indicate that repeated voluntary exercise is rewarding and alters gene transcription in mesolimbic reward neurocircuitry. The duration-dependent effects of wheel running on CPP suggest that as the weeks of wheel running progress, the rewarding effects of a night of voluntary wheel running might linger longer into the inactive cycle thus providing stronger support for CPP. The observed plasticity could contribute to the mechanisms by which exercise reduces the incidence and severity of substance abuse disorders, changes the rewarding properties of drugs of abuse, and facilitates successful coping with stress.
doi:10.1016/j.bbr.2010.11.005
PMCID: PMC3021978  PMID: 21070820
Exercise; physical activity; conditioned place preference; ventral tegmental area; nucleus accumbens; FosB
7.  Delta-1 Activation of Notch-1 Signaling Results in HES-1 Transactivation 
Molecular and Cellular Biology  1998;18(12):7423-7431.
The Notch receptor is involved in many cell fate determination events in vertebrates and invertebrates. It has been shown in Drosophila melanogaster that Delta-dependent Notch signaling activates the transcription factor Suppressor of Hairless, leading to an increased expression of the Enhancer of Split genes. Genetic evidence has also implicated the kuzbanian gene, which encodes a disintegrin metalloprotease, in the Notch signaling pathway. By using a two-cell coculture assay, we show here that vertebrate Dl-1 activates the Notch-1 cascade. Consistent with previous data obtained with active forms of Notch-1 a HES-1-derived promoter construct is transactivated in cells expressing Notch-1 in response to Dl-1 stimulation. Impairing the proteolytic maturation of the full-length receptor leads to a decrease in HES-1 transactivation, further supporting the hypothesis that only mature processed Notch is expressed at the cell surface and activated by its ligand. Furthermore, we observed that Dl-1-induced HES-1 transactivation was dependent both on Kuzbanian and RBP-J activities, consistent with the involvement of these two proteins in Notch signaling in Drosophila. We also observed that exposure of Notch-1-expressing cells to Dl-1 results in an increased level of endogenous HES-1 mRNA. Finally, coculture of Dl-1-expressing cells with myogenic C2 cells suppresses differentiation of C2 cells into myotubes, as previously demonstrated for Jagged-1 and Jagged-2, and also leads to an increased level of endogenous HES-1 mRNA. Thus, Dl-1 behaves as a functional ligand for Notch-1 and has the same ability to suppress cell differentiation as the Jagged proteins do.
PMCID: PMC109323  PMID: 9819428
8.  Phase I trial of recombinant adenovirus gene transfer in lung cancer. Longitudinal study of the immune responses to transgene and viral products. 
Journal of Clinical Investigation  1997;100(9):2218-2226.
Animal studies indicate that the use of replication-deficient adenovirus for human gene therapy is limited by host antivector immune responses that result in transient recombinant protein expression and blocking of gene transfer when rechallenged. Therefore, we have examined immune responses to an adenoviral vector and to the beta-galactosidase protein in four patients with lung cancer given a single intratumor injection of 10(9) plaque-forming units of recombinant adenovirus. The beta-galactosidase protein was expressed in day-8 tumor biopsies from all patients at variable levels. Recombinant virus DNA was detected by PCR in day-30 and day-60 tumor biopsies from all patients except patient 1. A high level of neutralizing antiadenovirus antibodies was detected in patient 1 before Ad-beta-gal injection whereas it was low (patient 3) or undetectable in the other two patients. All patients developed potent CD4 type 1 helper T cell (Th1) responses to adenoviral particles which increased gradually over time after injection. Antiadenovirus cytotoxic T lymphocyte responses were consistently boosted in the two patients examined (patients 3 and 4). Sustained production of anti-beta-galactosidase IgG was observed in all patients except patient 1. Consistent with anti-beta-gal antibody production, all patients except patient 1 developed intense, dose-dependent Th1 responses to soluble beta-galactosidase which increased over time. Strong beta-galactosidase-specific cytotoxic T lymphocyte responses were detected in patients 2, 3, and 4. Our results clearly show that despite the intensity of antiadenovirus responses, transgene protein expression was sufficient to induce strong and prolonged immunity in three patients. Recombinant adenovirus injected directly into the tumor is a highly efficient vector for immunizing patients against the transgene protein.
PMCID: PMC508417  PMID: 9410899
9.  Detection of Lipid-Rich Prostate Circulating Tumour Cells with Coherent Anti-Stokes Raman Scattering Microscopy 
BMC Cancer  2012;12:540.
Background
Circulating tumour cells (CTC) are an important indicator of metastasis and associated with a poor prognosis. Detection sensitivity and specificity of CTC in the peripheral blood of metastatic cancer patient remain a technical challenge.
Methods
Coherent anti-Stokes Raman scattering (CARS) microscopy was employed to examine the lipid content of CTC isolated from the peripheral blood of metastatic prostate cancer patients. CARS microscopy was also employed to evaluate lipid uptake and mobilization kinetics of a metastatic human prostate cancer cell line.
Results
One hundred CTC from eight metastatic prostate cancer patients exhibited strong CARS signal which arose from intracellular lipid. In contrast, leukocytes exhibited weak CARS signal which arose mostly from cellular membrane. On average, CARS signal intensity of prostate CTC was 7-fold higher than that of leukocytes (P<0.0000001). When incubated with human plasma, C4-2 metastatic human prostate cancer cells exhibited rapid lipid uptake kinetics and slow lipid mobilization kinetics. Higher expression of lipid transport proteins in C4-2 cells compared to non-transformed RWPE-1 and non-malignant BPH-1 prostate epithelial cells further indicated strong affinity for lipid of metastatic prostate cancer cells.
Conclusions
Intracellular lipid could serve as a biomarker for prostate CTC which could be sensitively detected with CARS microscopy in a label-free manner. Strong affinity for lipid by metastatic prostate cancer cells could be used to improve detection sensitivity and therapeutic targeting of prostate CTC.
doi:10.1186/1471-2407-12-540
PMCID: PMC3519750  PMID: 23171028
10.  The Relationship Between Human Papillomavirus Status and Other Molecular Prognostic Markers in Head and Neck Squamous Cell Carcinomas 
Purpose
To evaluate the relationship between human papillomavirus (HPV) status and known prognostic makers for head and neck cancers including tumor hypoxia, epidermal growth factor receptor (EGFR) expression and intratumoral T-cell levels and to determine the prognostic impact of these markers by HPV status.
Methods and Materials
HPV status in 82 evaluable head and neck squamous cell carcinomas patients was determined by pyrosequencing and related to p16INK4a staining and treatment outcomes. It was correlated with tumor hypoxia (tumor pO2 and carbonic anhydrase [CAIX] staining), EGFR status, and intratumoral lymphocyte expression (CD3 staining).
Results
Forty-four percent of evaluable tumors had strong HPV signal by pyrosequencing. There was a significant relationship between strong HPV signal and p16INK4a staining as well as oropharynx location. The strong HPV signal group fared significantly better than others, both in time to progression (TTP, p = 0.008) and overall survival (OS, p = 0.004) for all patients and for the oropharyngeal subset. Positive p16INK4a staining was associated with better TTP (p = 0.014) and OS (p = 0.00002). There was no relationship between HPV status and tumor pO2 or CAIX staining. However, HPV status correlated inversely with EGFR reactivity (p = 0.0006) and directly with CD3(+) T-lymphocyte level (p = 0.03). Whereas CAIX and EGFR overexpression were negative prognostic factors regardless of HPV status, CD3(+) T-cell levels was prognostic only in HPV(−) tumors.
Conclusion
HPV status was a prognostic factor for progression and survival. It correlated inversely with EGFR expression and directly with T-cell infiltration. The prognostic effect of CAIX and EGFR expression was not influenced by HPV status, whereas intratumoral T-cell levels was significant only for HPV(−) tumors.
doi:10.1016/j.ijrobp.2009.02.015
PMCID: PMC2768774  PMID: 19427557
HPV; Head and neck cancer; p16INK4a; EGFR; Hypoxia; CD3
11.  Variation in genetic admixture and population structure among Latinos: the Los Angeles Latino eye study (LALES) 
BMC Genetics  2009;10:71.
Background
Population structure and admixture have strong confounding effects on genetic association studies. Discordant frequencies for age-related macular degeneration (AMD) risk alleles and for AMD incidence and prevalence rates are reported across different ethnic groups. We examined the genomic ancestry characterizing 538 Latinos drawn from the Los Angeles Latino Eye Study [LALES] as part of an ongoing AMD-association study. To help assess the degree of Native American ancestry inherited by Latino populations we sampled 25 Mayans and 5 Mexican Indians collected through Coriell's Institute. Levels of European, Asian, and African descent in Latinos were inferred through the USC Multiethnic Panel (USC MEP), formed from a sample from the Multiethnic Cohort (MEC) study, the Yoruba African samples from HapMap II, the Singapore Chinese Health Study, and a prospective cohort from Shanghai, China. A total of 233 ancestry informative markers were genotyped for 538 LALES Latinos, 30 Native Americans, and 355 USC MEP individuals (African Americans, Japanese, Chinese, European Americans, Latinos, and Native Hawaiians). Sensitivity of ancestry estimates to relative sample size was considered.
Results
We detected strong evidence for recent population admixture in LALES Latinos. Gradients of increasing Native American background and of correspondingly decreasing European ancestry were observed as a function of birth origin from North to South. The strongest excess of homozygosity, a reflection of recent population admixture, was observed in non-US born Latinos that recently populated the US. A set of 42 SNPs especially informative for distinguishing between Native Americans and Europeans were identified.
Conclusion
These findings reflect the historic migration patterns of Native Americans and suggest that while the 'Latino' label is used to categorize the entire population, there exists a strong degree of heterogeneity within that population, and that it will be important to assess this heterogeneity within future association studies on Latino populations. Our study raises awareness of the diversity within "Latinos" and the necessity to assess appropriate risk and treatment management.
doi:10.1186/1471-2156-10-71
PMCID: PMC3087512  PMID: 19903357
12.  Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes 
Nucleic Acids Research  2004;32(15):4687-4695.
In order to determine the contribution of modified bases on the efficiency with which tRNALys,3 is used in vitro as the HIV-1 replication primer, the properties of synthetic derivatives prepared by three independent methods were compared to the natural, i.e. fully modified, tRNA. When prepared directly by in vitro run-off transcription, we show here that the predominant tRNA species is 77 nt, representing a non-templated addition of a single nucleotide. As a consequence, this aberrant tRNA inefficiently primes (−) strand strong stop DNA synthesis from the primer binding site (PBS) on the HIV-1 viral RNA genome to which it must hybridize. In contrast, correctly sized tRNALys,3 can be prepared by (i) total chemical synthesis and ligation of ‘half’ tRNAs, (ii) transcription of a cassette whose DNA template contained strategically placed 2′-O-Methyl-containing ribonucleotides and (iii) processing from a larger precursor by means of targeted cleavage with Escherichia coli RNase H. When each of these 76 nt tRNAs was supplemented into a (−) strand strong stop DNA synthesis reaction utilizing the HXB2 strain of HIV-1, the amount of product obtained was comparable to that from the fully modified counterpart. Parallel assays monitoring early events in (−) strand strong stop DNA synthesis using either the HXB2 or Mal strain of HIV-1 RNA as the template indicated little difference in the pattern or total product amount when primed with either natural or synthetic tRNALys,3. In addition, nuclease mapping of PBS-bound tRNA suggests inter-molecular base pairing between bases of the tRNA anticodon domain and the U-rich U5-IR loop of the viral 5′ leader region is less stable on the HIV-1HXB2 genome than the HIV-1Mal isolate.
doi:10.1093/nar/gkh813
PMCID: PMC516074  PMID: 15342789
13.  Evolution of dominance mechanisms at a butterfly mimicry supergene 
Nature Communications  2014;5:5644.
Genetic dominance in polymorphic loci may respond to selection; however, the evolution of dominance in complex traits remains a puzzle. We analyse dominance at a wing-patterning supergene controlling local mimicry polymorphism in the butterfly Heliconius numata. Supergene alleles are associated with chromosomal inversion polymorphism, defining ancestral versus derived alleles. Using controlled crosses and the new procedure, Colour Pattern Modelling, allowing whole-wing pattern comparisons, we estimate dominance coefficients between alleles. Here we show strict dominance in sympatry favouring mimicry and inconsistent dominance throughout the wing between alleles from distant populations. Furthermore, dominance among derived alleles is uncoordinated across wing-pattern elements, producing mosaic heterozygous patterns determined by a hierarchy in colour expression. By contrast, heterozygotes with an ancestral allele show complete, coordinated dominance of the derived allele, independently of colours. Therefore, distinct dominance mechanisms have evolved in association with supergene inversions, in response to strong selection on mimicry polymorphism.
The evolution of genetic dominance in polymorphic traits remains poorly understood. Here, the authors show that distinct dominance mechanisms have evolved in association with supergene inversions controlling wing pattern in Heliconius butterflies, in response to strong selection favouring mimicry.
doi:10.1038/ncomms6644
PMCID: PMC4263167  PMID: 25429605
14.  Is puberty a risk factor for back pain in the young? a systematic critical literature review 
Background
Back pain is a common condition that starts early in life and seems to increase markedly during puberty. A systematic review was performed in order to investigate the link between puberty and back pain, using some Bradford Hill criteria for causality.
Objectives
We sought to obtain answers to the following questions: 1) Is there an association between puberty and back pain? If so, how strong is this association? And do the results remain unchanged also when controlling for age and sex? 2) Are the results of the studies consistent? 3) Is there a dose–response, showing a link between the increasing stages of puberty and the subsequent prevalence of back pain? 4) Is there a temporal link between puberty and back pain?
Design
A systematic critical literature review.
Methods
Systematic searches were made in March 2014 in PubMed, Embase, CINAHL and PsycINFO including longitudinal or cross-sectional studies on back pain for subjects <19 years, written in French or English. The review process followed the AMSTAR recommendations. Interpretation was made using some of the Bradford-Hill criteria for causality.
Results
Four articles reporting five studies were included, two of which were longitudinal. 1) Some studies show a weak and others a strong positive association between puberty and back pain, which remains after controlling for age and sex; 2) Results were consistent across the studies; 3) There was a linear increase of back pain according to the stage of puberty 4) Temporality has not been sufficiently studied.
Conclusion
All our criteria for causality were fulfilled or somewhat fulfilled indicating the possibility of a causal link between puberty and back pain. Future research should focus on specific hypotheses, for example investigating if there could be a hormonal or a biomechanical aspect to the development of back pain at this time of life.
Electronic supplementary material
The online version of this article (doi:10.1186/s12998-014-0027-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12998-014-0027-6
PMCID: PMC4200222  PMID: 25328668
Back pain; Puberty; Adolescent; Cause; Aetiology; Systematic review
15.  Optimizing Tyrosine Kinase Inhibitor Therapy in Gastrointestinal Stromal Tumors: Exploring the Benefits of Continuous Kinase Suppression 
The Oncologist  2013;18(11):1192-1199.
This review discusses data supporting continuous kinase suppression with imatinib for the treatment of gastrointestinal stromal tumors and key issues, including response to imatinib reintroduction, effect of treatment interruption on secondary resistance to imatinib, and prognostic factors associated with sustained response to imatinib. Results from recent studies provide a strong rationale for continuous imatinib treatment for 3 years following surgical resection and long-term continuous administration in advanced or metastatic gastrointestinal stromal tumors.
Background.
The oral tyrosine kinase inhibitor (TKI) imatinib has revolutionized the treatment of gastrointestinal stromal tumors (GISTs), most of which harbor oncogenic mutation in genes that encode the receptor tyrosine kinases KIT or PDGFA. Imatinib is the standard of care for patients with advanced GIST and for patients with primary GIST at significant risk of recurrence after surgery.
Design.
This review discusses data supporting continuous kinase suppression with imatinib and key issues, including response to imatinib reintroduction, effect of treatment interruption on secondary resistance to imatinib, and prognostic factors associated with sustained response to imatinib.
Results.
Long-term follow-up results of the B2222 study and updated results of the BFR14 trial demonstrate that continuous imatinib treatment in patients with advanced GIST is associated with reduced risk of progression. For patients progressing on or intolerant of imatinib, continuing therapy with TKIs sunitinib followed by regorafenib is recommended. In the adjuvant setting, final results of the trial by the Scandinavian Sarcoma Group and the Sarcoma Group of the Arbeitsgemeinschaft Internistische Onkologie demonstrate that 3 years of adjuvant imatinib, compared with 1 year, significantly reduces the risk of recurrence and improves overall survival of patients with KIT-positive GIST at high risk of recurrence.
Conclusions.
Maintenance of therapy with TKIs is the key to successful treatment of GIST. Results from recent studies provide a strong rationale for continuous imatinib treatment for 3 years following surgical resection and long-term continuous administration in advanced or metastatic GIST.
doi:10.1634/theoncologist.2012-0361
PMCID: PMC3825304  PMID: 24136010
Gastrointestinal stromal tumor; Imatinib; Tyrosine kinase inhibitor; KIT; Metastatic; Adjuvant
16.  Novel YAP1-TFE3 Fusion Defines a Distinct Subset of Epithelioid Hemangioendothelioma 
Genes, chromosomes & cancer  2013;52(8):775-784.
Conventional epithelioid hemangioendotheliomas (EHE) have a distinctive morphologic appearance and are characterized by a recurrent t(1;3) translocation, resulting in a WWTR1-CAMTA1 fusion gene. We have recently encountered a fusion-negative subset characterized by a somewhat different morphology, including focally well-formed vasoformative features, which was further investigated for recurrent genetic abnormalities. Based on a case showing strong TFE3 immunoreactivity, FISH analysis for TFE3 gene rearrangement was applied to the index case as well as to 9 additional cases, selected through negative WWTR1-CAMTA1 screening. A control group, including 18 epithelioid hemangiomas, 9 pseudomyogenic HE and 3 epithelioid angiosarcomas, was also tested. TFE3 gene rearrangement was identified in 10 patients, with equal gender distribution and a mean age of 30 years old. The lesions were located in somatic soft tissue in 6 cases, lung in 3 and one in bone. One case with available frozen tissue was tested by RNA sequencing and FusionSeq data analysis to detect novel fusions. A YAP1-TFE3 fusion was thus detected, which was further validated by FISH and RT-PCR. YAP1 gene rearrangements were then confirmed in 7 of the remaining 9 TFE3-rearranged EHEs by FISH. No TFE3 structural abnormalities were detected in any of the controls. The TFE3-rearranged EHEs showed similar morphologic features with at least focally, well-formed vascular channels, in addition to a variably solid architecture. All tumors expressed endothelial markers, as well as strong nuclear TFE3. In summary we are reporting a novel subset of EHE occurring in young adults, showing a distinct phenotype and YAP1-TFE3 fusions.
doi:10.1002/gcc.22073
PMCID: PMC4089994  PMID: 23737213
TFE3; YAP1; epithelioid hemangioendothelioma; WWTR1
17.  DNA-encoded nucleosome occupancy is associated with transcription levels in the human malaria parasite Plasmodium falciparum 
BMC Genomics  2014;15(1):347.
Background
In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite.
Results
Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite’s erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes.
Conclusions
The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-347) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-347
PMCID: PMC4035074  PMID: 24885191
Malaria; Cell cycle; Nucleosome; Transcription; Sequence
18.  Haemoproteus iwa in Great Frigatebirds (Fregata minor) in the Islands of the Western Indian Ocean 
PLoS ONE  2014;9(5):e97185.
Blood parasites of the sub-genus Haemoproteus have been reported in seabirds, in particular in species in the Suliformes order. These parasites are transmitted by hippoboscid flies of the genus Olfersia; strong specificity has been suggested between the vector and its vertebrate host. We investigated the prevalence of Haemoproteus infection in Suliformes and hippoboscid flies in two oceanic islands of the Western Indian Ocean: Europa and Tromelin. In total, 209 blood samples were collected from great frigatebirds (Fregata minor), masked boobies (Sula dactylatra) and red-footed boobies (Sula sula). Forty-one hippoboscid flies were also collected from birds. Seventeen frigatebirds and one fly collected on Europa tested positive for the presence of Haemoproteus parasites by polymerase chain reaction. Phylogenetic analyses based on partial sequences of the Cytochrome b gene showed that parasites were closely related to Haemoproteus iwa reported from frigatebirds in the Pacific Ocean and in the Caribbean. Plasmodium was also detected in a frigatebird on Europa; however, its placement on the phylogenetic tree could not be resolved. We provide strong support for transmission of blood parasites in seabirds in the Western Indian Ocean and suggest that migrations between the Pacific and the Indian oceans could favor the large-scale distribution of Haemoproteus iwa in frigatebird populations.
doi:10.1371/journal.pone.0097185
PMCID: PMC4014603  PMID: 24810172
19.  Protective Efficacy of Passive Immunization with Monoclonal Antibodies in Animal Models of H5N1 Highly Pathogenic Avian Influenza Virus Infection 
PLoS Pathogens  2014;10(6):e1004192.
Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype often cause severe pneumonia and multiple organ failure in humans, with reported case fatality rates of more than 60%. To develop a clinical antibody therapy, we generated a human-mouse chimeric monoclonal antibody (MAb) ch61 that showed strong neutralizing activity against H5N1 HPAI viruses isolated from humans and evaluated its protective potential in mouse and nonhuman primate models of H5N1 HPAI virus infections. Passive immunization with MAb ch61 one day before or after challenge with a lethal dose of the virus completely protected mice, and partial protection was achieved when mice were treated 3 days after the challenge. In a cynomolgus macaque model, reduced viral loads and partial protection against lethal infection were observed in macaques treated with MAb ch61 intravenously one and three days after challenge. Protective effects were also noted in macaques under immunosuppression. Though mutant viruses escaping from neutralization by MAb ch61 were recovered from macaques treated with this MAb alone, combined treatment with MAb ch61 and peramivir reduced the emergence of escape mutants. Our results indicate that antibody therapy might be beneficial in reducing viral loads and delaying disease progression during H5N1 HPAI virus infection in clinical cases and combined treatment with other antiviral compounds should improve the protective effects of antibody therapy against H5N1 HPAI virus infection.
Author Summary
The H5N1 highly pathogenic avian influenza virus has been circulating in poultry in Asia, the Middle East, and Africa since its first appearance in southern China in 1996. This virus occasionally infects humans with a high case mortality rate and poses a significant pandemic threat. Since neutralizing antibodies generally play a major role in protective immunity against influenza viruses, antibody therapy is a potential option for preventing highly lethal infection with the H5N1 virus in humans. Here we evaluated the protective potential of a human-mouse chimeric monoclonal antibody with strong neutralizing activity against H5N1 viruses in mouse and nonhuman primate models of lethal H5N1 virus infection. The therapeutic use of the neutralizing antibody resulted in reduced viral loads and improved survival in animals infected with highly pathogenic H5N1 viruses. It was noted that the protective effects were more prominent in immunosuppressed macaques, which might provide a model of protection against severe clinical disease in immunocompromised patients. In addition, combination therapy together with an antiviral drug reduced the selection of escape mutants. Collectively, this study suggests that antibody therapy may have beneficial effects in clinical cases of H5N1 HPAI virus infection in humans.
doi:10.1371/journal.ppat.1004192
PMCID: PMC4055766  PMID: 24945244
20.  Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques 
PLoS Pathogens  2013;9(12):e1003810.
The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4+ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4+ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.
Author Summary
Human Immunodeficiency Virus infection is predominantly transmitted by mucosal exposure, after sexual intercourse. Although substantial progresses have been recently achieved in our understanding of the mechanisms of HIV mucosal transmission, many questions remain. Semen is one of the major sources for HIV which contains both cell-free viral particles and viral infected cells. However, today, the role of cell-associated virus has been largely understudied. We provide here a detailed characterization of the semen leukocyte populations in the highly relevant experiment model of SIV infection of macaques. We demonstrate that the major target cells for the virus, CD4+ T cells and macrophages, are present in macaques semen at all stages of infection. Both cell types are productively infected in vivo and are endowed with adhesion and migration markers that may facilitate virus transmission during sexual exposure. The acute phase of infection is associated with a strong seminal inflammation that may increase semen leukocytes infectivity. This work supports for a role of cell-associated virus in HIV transmission which needs to be considered for the design of prevention strategies.
doi:10.1371/journal.ppat.1003810
PMCID: PMC3861532  PMID: 24348253
21.  Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications 
Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.
doi:10.1007/s10295-013-1323-1
PMCID: PMC3824566  PMID: 24061566
Invertase; Yarrowia lipolytica; Sucrose; Citric acid; Secretion
22.  Masculinization of the X Chromosome in the Pea Aphid 
PLoS Genetics  2013;9(8):e1003690.
Evolutionary theory predicts that sexually antagonistic mutations accumulate differentially on the X chromosome and autosomes in species with an XY sex-determination system, with effects (masculinization or feminization of the X) depending on the dominance of mutations. Organisms with alternative modes of inheritance of sex chromosomes offer interesting opportunities for studying sexual conflicts and their resolution, because expectations for the preferred genomic location of sexually antagonistic alleles may differ from standard systems. Aphids display an XX/X0 system and combine an unusual inheritance of the X chromosome with the alternation of sexual and asexual reproduction. In this study, we first investigated theoretically the accumulation of sexually antagonistic mutations on the aphid X chromosome. Our results show that i) the X is always more favourable to the spread of male-beneficial alleles than autosomes, and should thus be enriched in sexually antagonistic alleles beneficial for males, ii) sexually antagonistic mutations beneficial for asexual females accumulate preferentially on autosomes, iii) in contrast to predictions for standard systems, these qualitative results are not affected by the dominance of mutations. Under the assumption that sex-biased gene expression evolves to solve conflicts raised by the spread of sexually antagonistic alleles, one expects that male-biased genes should be enriched on the X while asexual female-biased genes should be enriched on autosomes. Using gene expression data (RNA-Seq) in males, sexual females and asexual females of the pea aphid, we confirm these theoretical predictions. Although other mechanisms than the resolution of sexual antagonism may lead to sex-biased gene expression, we argue that they could hardly explain the observed difference between X and autosomes. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms displaying an alternative mode of sex chromosome inheritance to understanding the forces shaping chromosome evolution.
Author Summary
Males and females differ in their optimal values for most phenotypic traits, which makes intra-locus genetic conflicts among sexes common. Sex chromosomes have a sex-biased transmission, a pattern which might create favourable conditions for the spread of sexually antagonistic alleles (i.e. alleles beneficial for one sex but deleterious for the other). Yet, expectations for genetic systems with unusual inheritance of sex chromosomes may differ from those derived from standard systems (e.g. XY). Here we demonstrate theoretically that in organisms such as aphids, which alternate sexual and asexual reproduction and display an unusual inheritance of the X chromosome, male-beneficial sexually antagonistic alleles accumulate preferentially on that chromosome, while asexual female-beneficial alleles accumulate on autosomes. Theoretical models suggest that the evolution of sex-biased gene expression may solve such sexual conflicts, by restricting the product of a sexually antagonistic allele to the sex it benefits. We show that in the pea aphid, the genomic location (X versus autosomes) of genes with a sex-biased expression fits predictions derived from this hypothesis. On top of reporting a strong masculinization of the aphid X chromosome, our study highlights the relevance of organisms with an alternative mode of sex chromosome inheritance to understanding the evolutionary forces shaping chromosome evolution.
doi:10.1371/journal.pgen.1003690
PMCID: PMC3738461  PMID: 23950732
23.  The genetic structure of a Venturia inaequalis population in a heterogeneous host population composed of different Malus species 
Background
Adaptation, which induces differentiation between populations in relation to environmental conditions, can initiate divergence. The balance between gene flow and selection determines the maintenance of such a structure in sympatry. Studying these two antagonistic forces in plant pathogens is made possible because of the high ability of pathogens to disperse and of the strong selective pressures exerted by their hosts. In this article, we analysed the genetic structure of the population of the apple scab fungus, Venturia inaequalis, in a heterogeneous environment composed of various Malus species. Inferences were drawn from microsatellite and AFLP data obtained from 114 strains sampled in a single orchard on nine different Malus species to determine the forces that shape the genetic structure of the pathogen.
Results
Using clustering methods, we first identified two specialist subpopulations: (i) a virulent subpopulation sampled on Malus trees carrying the Rvi6 resistance gene; and (ii) a subpopulation infecting only Malus trees that did not carry this resistance gene. A genome scan of loci on these two subpopulations did not detect any locus under selection. Additionally, we did not detect any other particular substructure linked to different hosts. However, an isolation-by-distance (IBD) pattern at the orchard scale revealed free gene flow within each subpopulation.
Conclusions
Our work shows a rare example of a very strong effect of a resistance gene on pathogen populations. Despite the high diversity of Malus hosts, the presence of Rvi6 seems sufficient to explain the observed genetic structure. Moreover, detection of an IBD pattern at the orchard scale revealed a very low average dispersal distance that is particularly significant for epidemiologists and landscape managers for the design of scab control strategies
doi:10.1186/1471-2148-13-64
PMCID: PMC3626921  PMID: 23497223
Gene flow; Isolation-by-distance (IBD); Apple scab; Adaptation; Spatial genetic structure
24.  Regulatory T Lymphocytes Are Associated with Less Aggressive Histologic Features in Microsatellite-Unstable Colorectal Cancers 
PLoS ONE  2013;8(4):e61001.
Background
Colorectal cancers (CRCs) with microsatellite instability (MSI) are associated with a good prognosis and a high density of tumor-infiltrating lymphocytes (TILs). We have undertaken to determine the link between TIL densities and MSI CRC histologic features.
Patients and Methods
Using tissue microarrays, T-cell sub-population infiltration, including T cells (CD3), cytotoxic T cells (CD8) and regulatory T cells (FoxP3) were studied in 86 MSI CRCs. We separately analyzed TILs of the stromal and epithelial compartments in the tumor center, the tumoral invasion margin and associated normal tissue.
Results
For FoxP3+ TIL density in the tumor center stromal compartment, we found a strong negative correlation with T4 stage (p = 0.01), node invasion (p<0.001) and VELIPI (vascular emboli, lymphatic invasion and perinervous invasion) criteria (p = 0.002).
Conclusion
The strong correlation between regulatory T cell density and the absence of VELIPI criteria suggests that this sub-group of T cells is preferentially associated with less invasive tumors.
doi:10.1371/journal.pone.0061001
PMCID: PMC3626697  PMID: 23613769
25.  Tibia shaft fractures: costly burden of nonunions 
Background
Tibia shaft fractures (TSF) are common for men and women and cause substantial morbidity, healthcare use, and costs. The impact of nonunions on healthcare use and costs is poorly described. Our goal was to investigate patient characteristics and healthcare use and costs associated with TSF in patients with and without nonunion.
Methods
We retrospectively analyzed medical claims in large U.S. managed care claims databases (Thomson Reuters MarketScan®, 16 million lives). We studied patients ≥ 18 years old with a TSF diagnosis (ICD-9 codes: 823.20, 823.22, 823.30, 823.32) in 2006 with continuous pharmaceutical and medical benefit enrollment 1 year prior and 2 years post-fracture. Nonunion was defined by ICD-9 code 733.82 (after the TSF date).
Results
Among the 853 patients with TSF, 99 (12%) had nonunion. Patients with nonunion had more comorbidities (30 vs. 21, pre-fracture) and were more likely to have their TSF open (87% vs. 70%) than those without nonunion. Patients with nonunion were more likely to have additional fractures during the 2-year follow-up (of lower limb [88.9% vs. 69.5%, P < 0.001], spine or trunk [16.2% vs. 7.2%, P = 0.002], and skull [5.1% vs. 1.3%, P = 0.008]) than those without nonunion. Nonunion patients were more likely to use various types of surgical care, inpatient care (tibia and non-tibia related: 65% vs. 40%, P < 0.001) and outpatient physical therapy (tibia-related: 60% vs. 42%, P < 0.001) than those without nonunion. All categories of care (except emergency room costs) were more expensive in nonunion patients than in those without nonunion: median total care cost $25,556 vs. $11,686, P < 0.001. Nonunion patients were much more likely to be prescribed pain medications (99% vs. 92%, P = 0.009), especially strong opioids (90% vs. 76.4%, P = 0.002) and had longer length of opioid therapy (5.4 months vs. 2.8 months, P < 0.001) than patients without nonunion. Tibia fracture patterns in men differed from those in women.
Conclusions
Nonunions in TSF’s are associated with substantial healthcare resource use, common use of strong opioids, and high per-patient costs. Open fractures are associated with higher likelihood of nonunion than closed ones. Effective screening of nonunion risk may decrease this morbidity and subsequent healthcare resource use and costs.
doi:10.1186/1471-2474-14-42
PMCID: PMC3573940  PMID: 23351958
Tibia shaft fractures; Nonunions; Healthcare resource utilization; Costs; Opioids

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