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1.  The association between expressions of Ras and CD68 in the angiogenesis of breast cancers 
Cancer Cell International  2015;15(1):17.
Objective
Angiogenesis is a critical step of breast cancer metastasis. Oncogenic Ras promotes the remodeling of cancer microenviroment. Tumor-associated macrophages (TAMs) are a prominent inflammatory cell population emerging in the microenviroment and facilitating the angiogenesis and metastasis. In the present study, we tried to investigate the relationship between the expression of Ras and infiltration of TAM, both of which could further promote angiogenesis.
Methods
Expressions of Ras, CD68 and CD34 were assessed by immunohistochemistry. The infiltration of macrophages was evaluated by counting the number of CD68+ cells. Vessel endothelial cells were defined as CD34+ cells. Angiogenesis vascularity was defined by microvessel density (MVD) assay through counting the number of vessels per field counted in the area of highest vascular density. The Kaplan–Meier survival analysis was used to estimate the overall survival (OS). Macrophages were derived from monocytes in the presence of macrophage colony-stimulating-factor (MCSF). Breast cancer cells were treated with macrophage-conditioned medium (MCM) and tested the expressions of K-, H- and N-Ras by using realtime-PCR.
Results
Ras positive status was correlated with ER, PR and Her-2 positivity, larger tumour size and lymph node metastasis, as well as higher TNM stages. A higher number of CD68+ cells was correlated with larger tumour size, higher TNM stages and Her-2 positivity. Both Ras positivity and infiltration of CD68+ macrophages correlated with poor OS. The number of CD68+ cells was positively correlated with the expression of Ras. Treatment with MCM did not up-regulate but repressed the expression of Ras. Both up-regulation of Ras and infiltration of TAMs correlated with increased MVD.
Conclusion
Expression of Ras and infiltration of TAM were positively correlated, and both participated in angiogenesis. Elevated Ras could be responsible for the infiltration of TAM.
doi:10.1186/s12935-015-0169-1
PMCID: PMC4326448
Breast cancer; Ras; TAM; CD34; Angiogenesis
2.  Analyses of Alternatively Processed Genes in Ciliates Provide Insights into the Origins of Scrambled Genomes and May Provide a Mechanism for Speciation 
mBio  2015;6(1):e01998-14.
ABSTRACT 
Chromosome rearrangements occur in a variety of eukaryotic life cycles, including during the development of the somatic macronuclear genome in ciliates. Previous work on the phyllopharyngean ciliate Chilodonella uncinata revealed that macronuclear β-tubulin and protein kinase gene families share alternatively processed germ line segments nested within divergent regions. To study genome evolution in this ciliate further, we characterized two additional alternatively processed gene families from two cryptic species of the ciliate morphospecies C. uncinata: those encoding histidine acid phosphatase protein (Hap) and leishmanolysin family protein (Lei). Analyses of the macronuclear Hap and Lei sequences reveal that each gene family consists of three members in the macronucleus that are marked by identical regions nested among highly divergent regions. Investigation of the micronuclear Hap sequences revealed a complex pattern in which the three macronuclear sequences are derived either from a single micronuclear region or from a combination of this shared region recombined with additional duplicate micronuclear copies of Hap. We propose a model whereby gene scrambling evolves by gene duplication followed by partial and reciprocal degradation of the duplicate sequences. In this model, alternative processing represents an intermediate step in the evolution of scrambled genes. Finally, we speculate on the possible role of genome architecture in speciation in ciliates by describing what might happen if changes in alternatively processed loci occur in subdivided populations.
IMPORTANCE  Genome rearrangements occur in a variety of eukaryotic cells and serve as an important mechanism for generating genomic diversity. The unusual genome architecture of ciliates with separate germline and somatic nuclei in each cell, provides an ideal system to study further principles of genome evolution. Previous analyses revealed complex forms of chromosome rearrangements, including gene scrambling and alternative processing of germ line chromosomes. Here we describe more complex rearrangements between germ line and somatic chromosomes than previously seen in alternatively processed gene families. Drawing on the present and previous findings, we propose a model in which alternative processing of duplicated micronuclear regions represents an intermediate stage in the evolution of scrambled genes. Under this model, alternative processing may provide insights into a mechanism for speciation in ciliates. Our data on gene scrambling and alternative processing also enhance views on the dynamic nature of genomes across the eukaryotic tree of life.
IMPORTANCE 
Genome rearrangements occur in a variety of eukaryotic cells and serve as an important mechanism for generating genomic diversity. The unusual genome architecture of ciliates with separate germline and somatic nuclei in each cell, provides an ideal system to study further principles of genome evolution. Previous analyses revealed complex forms of chromosome rearrangements, including gene scrambling and alternative processing of germ line chromosomes. Here we describe more complex rearrangements between germ line and somatic chromosomes than previously seen in alternatively processed gene families. Drawing on the present and previous findings, we propose a model in which alternative processing of duplicated micronuclear regions represents an intermediate stage in the evolution of scrambled genes. Under this model, alternative processing may provide insights into a mechanism for speciation in ciliates. Our data on gene scrambling and alternative processing also enhance views on the dynamic nature of genomes across the eukaryotic tree of life.
doi:10.1128/mBio.01998-14
PMCID: PMC4324306  PMID: 25650397
3.  Effect of Different Tumbling Marination Treatments on the Quality Characteristics of Prepared Pork Chops 
The effect of different tumbling marination treatments (control group, CG; conventional static marination, SM; vacuum continuous tumbling marination, CT; vacuum intermittent tumbling marination, IT) on the quality characteristics of prepared pork chops was investigated under simulated commercial conditions. The CT treatment increased (p<0.05) the pH value, b* value, product yield, tenderness, overall flavor, sensory juiciness and overall acceptability in comparison to other treatments for prepared boneless pork chops. The CT treatment decreased (p<0.05) cooking loss, shear force value, hardness, gumminess and chewiness compared with other treatments. In addition, CT treatment effectively improved springiness and sensory color more than other treatments. However, IT treatment achieved the numerically highest (p<0.05) L* and a* values. These results suggested that CT treatment obtained the best quality characteristics of prepared pork chops and should be adopted as the optimal commercial processing method for this prepared boneless pork chops.
doi:10.5713/ajas.14.0511
PMCID: PMC4283172  PMID: 25557823
Tumbling; Continuous; Intermittent; Quality Characteristic; Prepared Pork Chop
4.  The Value of Perioperative Imaging in Patients with Uterine Sarcomas 
Gynecologic oncology  2009;115(1):37-40.
Objective
To explore the yield and impact of perioperative imaging on management among patients undergoing surgical resection and treatment of uterine sarcomas.
Methods
A retrospective chart review was done for women with histologically confirmed uterine sarcomas treated at Barnes Jewish Hospital/Washington University from 2001–2007. Descriptive statistics, Cox multivariate models, and Kaplan-Meier plots were used to evaluate associations and survival.
Results
A total of 92 patients were identified and 55(60%) were diagnosed with stage III–IV disease. Perioperative imaging was obtained in 84 (91%) cases, including chest x-ray in 66 (72%), computerized tomography (CT) of the abdomen and pelvis in 59 (64%), chest CT in 33 (36%), positron emission tomography (PET) in 8 (9%), and CT of the head, pelvic magnetic resonance imaging (MRI), or bone scan in a total of 2 (2.2%). Imaging identified abnormalities concerning for metastases in 30 (32%) studies. Thirty-four recurrences have been documented, and 21 (62%) of these treatment failures were extrapelvic. Multivariate analysis of this series noted that tomographic evidence of extrauterine disease predicted recurrence (p=0.028) and incomplete surgical resection (p=0.003, HR 6.0 95% CI 1.9–19.9) predicted disease free survival. Imaging contributed to change in surgical and postsurgical treatment decisions in 8 (9%) patients.
Conclusion
Pretreatment imaging studies change management in a minority of patients with newly diagnosed uterine sarcomas.
doi:10.1016/j.ygyno.2009.06.013
PMCID: PMC4306556  PMID: 19577795
5.  Expression, purification, crystallization and preliminary X-ray diffraction analysis of Arabidopsis thaliana Deg8 
Crystals of Deg8, an ATP-independent serine endopeptidase from A. thaliana, were monoclinic, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, β = 132.4°, and diffracted to 2.0 Å resolution.
Arabidopsis thaliana Deg8, an ATP-independent serine endopeptidase, is involved in the repair of photosystem II (PSII), specifically the degradation of the photo-damaged PSII reaction centre D1 protein. To understand the molecular mechanism underlying the participation of Deg8 in the degradation of the photo-damaged D1 protein, the structure of Deg8 is needed. Until recently, however, no structure of Deg8 had been solved. In this study, Deg8 from A. thaliana was cloned, overexpressed and purified in Escherichia coli. Crystallization was performed at 277 K using tribasic sodium citrate as the precipitant and the crystals diffracted to 2.0 Å resolution, belonging to space group C2 with unit-cell parameters a = 129.5, b = 124.2, c = 93.3 Å, α = γ = 90, β = 132.4°. Assuming one trimer in the asymmetric unit, the Matthews coefficient and the solvent content were calculated to be 2.35 Å3 Da−1 and 47.6%, respectively.
doi:10.1107/S1744309112048774
PMCID: PMC3539708  PMID: 23295491
Deg8; photosystem II; Arabidopsis thaliana
6.  Optimizing parameters in clinical trials with a randomized start or withdrawal design 
Disease-modifying (DM) trials on chronic diseases such as Alzheimer’s disease (AD) require a randomized start or withdrawal design. The analysis and optimization of such trials remain poorly understood, even for the simplest scenario in which only three repeated efficacy assessments are planned for each subject: one at the baseline, one at the end of the trial, and the other at the time when the treatments are switched. Under the assumption that the repeated measures across subjects follow a trivariate distribution whose mean and covariance matrix exist, the DM efficacy hypothesis is formulated by comparing the change of efficacy outcome between treatment arms with and without a treatment switch. Using a minimax criterion, a methodology is developed to optimally determine the sample size allocations to individual treatment arms as well as the optimum time when treatments are switched. The sensitivity of the optimum designs with respect to various model parameters is further assessed. An intersection-union test (IUT) is proposed to test the DM hypothesis, and determine the asymptotic size and the power of the IUT. Finally, the proposed methodology is demonstrated by using reported statistics on the placebo arms from several recently published symptomatic trials on AD to estimate necessary parameters and then deriving the optimum sample sizes and the time of treatment switch for future DM trials on AD.
doi:10.1016/j.csda.2013.07.013
PMCID: PMC3804275  PMID: 24159249
Alzheimer’s disease; Disease-modifying trials; Intersection-union test; Minimax criterion; Random intercept and slope models; Randomized start design
7.  Phylogenomic analyses support the bifurcation of ciliates into two major clades that differ in properties of nuclear division 
Ciliates are a diverse assemblage of eukaryotes that have been the source of many discoveries including self-splicing RNAs, telomeres and trans-splicing. While analyses of ciliate morphology have given rise to robust hypotheses on relatively shallow level relationships, the deeper evolutionary history of ciliates is largely unknown. This is in part because studies to date have focused on only a single locus, small subunit ribosomal DNA (SSU-rDNA). In the present study, we use a taxon-rich strategy based on multiple loci from GenBank and recently completed transcriptomes to assess deep phylogenetic relationships among ciliates. Our phylogenomic data set includes up to 537 taxa, all of which have been sampled for SSU-rDNA and a subset of which have LSU-rDNA and up to 7 protein-coding sequences. Analyses of these data support the bifurcation of ciliates as suggested by SSU-rDNA, with one major clade defined by having somatic macronuclei that divide with intranuclear microtubules (Intramacronucleata) and the other clade containing lineages that either divide their macronuclei with microtubules external to the macronucleus or are unable to divide their macronuclei (Postciliodesmatophora). These multigene phylogenies provide a robust framework for interpreting the evolution of innovations across the ciliate tree of life.
doi:10.1016/j.ympev.2013.10.001
PMCID: PMC3950933  PMID: 24121125
Phylogenomic analysis; macronucleus; Ciliophora; Postciliodesmatophora; Intramacronucleata
8.  Effectiveness and safety of Humalog Mix 50/50 versus Humalog Mix 75/25 in Chinese patients with type 2 diabetes 
Humalog Mix 75/25 insulin analog is widely used in the People’s Republic of China to treat type 2 diabetes mellitus, but the Humalog Mix 50/50 analog is not as yet widely used or assessed. The purpose of this 12-week, parallel-group, randomized, treat-to-target study was to evaluate the difference in clinical efficacy, safety, and outcome of treatment between Humalog Mix 50/50 and Humalog Mix 75/25 analogs in Chinese patients with type 2 diabetes mellitus. In total, 146 insulin-naïve patients with type 2 diabetes mellitus and aged 18–75 years were randomized and treated twice a day with either Humalog Mix 50/50 (group A) or Humalog Mix 75/25 (group B). We monitored levels of fasting blood glucose, 2-hour postprandial blood glucose, and glycosylated hemoglobin (HbA1c) in patients in both groups prior to and 3 months post treatment, the average time to achieve target blood glucose level, and frequency of hypoglycemic episodes during treatment. We found that group A showed better glycemic control as per fasting blood glucose and 2-hour postprandial blood glucose than group B. Moreover, HbA1c levels in group A (5.5%±1.4%) were lower by 1.0%±0.1% (P<0.05) compared with those in group B (6.5%±1.5%). The time to achieve glucose control was shorter (P<0.05) in group A (12.6±3.6 days) than in group B (22.3±4.7 days). Regarding safety, no significant adverse events or severe hypoglycemia on treatment was observed in either group. Additionally, the 1:1 ratio of Humalog Mix 50/50 showed a trend towards fewer episodes of nocturnal hypoglycemia. Thus, compared with Humalog Mix 75/25, the high-proportion premix insulin analog, Humalog Mix 50/50 showed better glycemic control, achieved target blood glucose levels more rapidly and without an increase in hypoglycemic episodes in Chinese type 2 diabetic individuals and is recommended for use in clinical practice.
doi:10.2147/TCRM.S75602
PMCID: PMC4278736  PMID: 25565854
type 2 diabetes mellitus; Chinese type 2 diabetics; Humalog Mix 50/50; Humalog Mix 75/25; premixed insulin lispro 50/50; premixed insulin lispro 75/25
9.  Identification of a novel gene fusion (BMX-ARHGAP) in gastric cardia adenocarcinoma 
Diagnostic Pathology  2014;9(1):218.
Background
Gastric cardia adenocarcinoma (GCA) is one of the major causes of cancer related mortality worldwide. We aim to provide new understanding in the pathogenesis of GCA through investigations on gene expression alterations.
Methods
We preformed RNA-Seq for one pair of GCA and matched non-tumor tissues. Differentially expressed genes (DEGs) and fusion genes were acquired. PCR and gel analysis in additional 14 pairs of samples were performed to validate the chimeric transcripts.
Results
1590 up-regulated and 709 down-regulated genes were detected. Functional analysis revealed that these DEGs were significantly overrepresented in gene ontology items of cell cycle, tumor invasion and proliferation. Moreover, we firstly discovered 3 fusion genes in GCA, including BMX-ARHGAP, LRP5- LITAF and CBX3-C15orf57. The chimeric transcript BMX-ARHGAP was validated and recurrently occurred in 4/15 independent tumor tissues.
Conclusions
Our results may provide new understanding of GCA and biomarkers for further therapeutic studies.
Virtual Slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_218
doi:10.1186/s13000-014-0218-4
PMCID: PMC4282731  PMID: 25499959
Gastric cardia adenocarcinoma (GCA); RNA-Seq; Gene fusion
10.  Fine scale Spatial-temporal cluster analysis for the infection risk of Schistosomiasis japonica using space-time scan statistics 
Parasites & Vectors  2014;7(1):578.
Background
Marching towards the elimination of schistosomiasis in China, both the incidence and prevalence have witnessed profound decline over the past decades, with the strategy shifting from morbidity control to transmission control. The current challenge is to find out hotspots of transmission risk for precise targeted control in low-prevalence areas. This study assessed the risk at the village level, using the spatial and temporal characteristics of Schistosomiasis japonica in Anhui province from 2006 to 2012.
Method
The comprehensive database was generated from annual surveillance data at village level in Anhui province between 2006 and 2012, comprising schistosomiasis prevalence among humans and cattle, occurrence rate of infected environments and incidence rate of acute schistosomiasis. The database parameters were matched with geographic data of the study area and fine scale spatial-temporal cluster analysis based on retrospective space-time scan statistics was used to assess the clustering pattern of schistosomiasis. The analysis was conducted by using SaTScan 9.1.1 and ArcGIS 10.0. A spatial statistical modelling was carried out to determine the spatial dependency of prevalence of human infection by using Geoda 1.4.3.
Result
A pronounced decline was found in the prevalence of human infection, cattle infection, occurrence rate of environment with infected vector snails and incidence rate of acute schistosomiasis from 2006 to 2012 by 48.6%, 71.5%, 91.9% and 96.4%, respectively. Meanwhile, all 4 indicators showed a statistically significant clustering pattern both in time and space, with a total of 16, 6, 8 and 4 corresponding clustering foci found respectively. However, the number of clustering foci declined with time, and none was found after year 2010. All clustering foci were mainly distributed along the Yangtze River and its connecting branches. The result shows that there is a direct spatial relationship between prevalence of human infection and the other indicators.
Conclusion
A decreasing trend in space-time clustering of schistosomiasis endemic status was observed between 2006 and 2012 in Anhui province. Nevertheless, giving the complexity in schistosomiasis control, areas within the upper-stream of Yangtze River in Anhui section and its connecting branches should be targeted for effective implementation of control strategies in the future.
doi:10.1186/s13071-014-0578-3
PMCID: PMC4273478  PMID: 25491192
Fine scale spatial-temporal scan statistics; Schistosomiasis japonica; Infection risk analysis; Anhui province
11.  Accounting for eXentricities: Analysis of the X Chromosome in GWAS Reveals X-Linked Genes Implicated in Autoimmune Diseases 
PLoS ONE  2014;9(12):e113684.
Many complex human diseases are highly sexually dimorphic, suggesting a potential contribution of the X chromosome to disease risk. However, the X chromosome has been neglected or incorrectly analyzed in most genome-wide association studies (GWAS). We present tailored analytical methods and software that facilitate X-wide association studies (XWAS), which we further applied to reanalyze data from 16 GWAS of different autoimmune and related diseases (AID). We associated several X-linked genes with disease risk, among which (1) ARHGEF6 is associated with Crohn's disease and replicated in a study of ulcerative colitis, another inflammatory bowel disease (IBD). Indeed, ARHGEF6 interacts with a gastric bacterium that has been implicated in IBD. (2) CENPI is associated with three different AID, which is compelling in light of known associations with AID of autosomal genes encoding centromere proteins, as well as established autosomal evidence of pleiotropy between autoimmune diseases. (3) We replicated a previous association of FOXP3, a transcription factor that regulates T-cell development and function, with vitiligo; and (4) we discovered that C1GALT1C1 exhibits sex-specific effect on disease risk in both IBDs. These and other X-linked genes that we associated with AID tend to be highly expressed in tissues related to immune response, participate in major immune pathways, and display differential gene expression between males and females. Combined, the results demonstrate the importance of the X chromosome in autoimmunity, reveal the potential of extensive XWAS, even based on existing data, and provide the tools and incentive to properly include the X chromosome in future studies.
doi:10.1371/journal.pone.0113684
PMCID: PMC4257614  PMID: 25479423
12.  Natural antioxidants for health promotion and disease prevention 
doi:10.3389/fphar.2014.00266
PMCID: PMC4251313
natural antioxidants; reactive oxygen species; herbs; spices; chronic disease
13.  Molecular cloning, expression, purification and crystallographic analysis of zebrafish THEM2 
The THEM2 protein from zebrafish has successfully been expressed, purified and crystallized. Diffraction data were collected to a resolution of 1.8 Å.
Thioesterase superfamily member 2 (THEM2) is essential for cell proliferation of mammalian cells. It belongs to the hotdog-fold thioesterase superfamily and catalyzes the hydrolysis of the thioester bonds of acyl-CoA in vitro. In this study, THEM2 protein from zebrafish (fTHEM2) was expressed in Escherichia coli and purified by Ni-affinity and gel-filtration chromatography. fTHEM2 crystals were obtained using the sitting-drop vapour-diffusion method with PEG 10 000 as precipitant. X-ray diffraction data were collected to 1.80 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 77.1, b = 74.4, c = 96.6 Å, β = 93.7°.
doi:10.1107/S1744309112043813
PMCID: PMC3509980  PMID: 23192039
THEM2; thioesterase superfamily member 2; Danio rerio
14.  Phase II Study of Bevacizumab and Pemetrexed for Recurrent or Persistent Epithelial Ovarian, Fallopian Tube or Primary Peritoneal Cancer 
Gynecologic oncology  2013;131(3):535-540.
Objective
To evaluate the efficacy and safety of combination bevacizumab/pemetrexed for the treatment of recurrent epithelial ovarian cancer (EOC).
Methods
Platinum-sensitive or -resistant patients with recurrent or persistent EOC were eligible if they had received up to 2 prior chemotherapy regimens, including a platinum/taxane regimen without prior bevacizumab. Pemetrexed 500 mg/m2 IV and bevacizumab 15 mg/kg IV were administered every 3 weeks. The primary endpoint was 6-month progression-free survival (PFS); other endpoints included toxicities, PFS and overall survival (OS).
Results
Thirty-four patients received a median of 7 treatment cycles (range, 2–26). Median follow-up was 25.7 months (range, 3.0–47.2). Six month progression-free survival (PFS) was 56% (95%CI: 38–71). The following response rates were documented (%; 95%CI): 0 complete response, 14 partial responses (41%; 25–59), 18 stable disease (53%; 35–70) and 2 progressive disease (6%; 1–20). Median PFS was 7.9 months (95%CI, 4.6–10.9), with a median OS of 25.7 months (95% CI, 15.4–29.8). Twenty-two patients (64.7%) had a platinum-free interval (PFI) of >6 months prior to enrollment. Grade 3–4 hematologic toxicities included neutropenia (50%), leukopenia (26%), thrombocytopenia (12%) and anemia (9%). Non-hematologic grade 3–4 toxicities included metabolic (29%), constitutional (18%), pain (18%) and gastrointestinal (15%). Two patients developed hematologic malignancies within one year of treatment.
Conclusions
Combination bevacizumab/pemetrexed is an active option for both platinum-sensitive and -resistant recurrent EOC. Further investigation of cost and novel toxicities associated with this regimen may be warranted.
doi:10.1016/j.ygyno.2013.09.032
PMCID: PMC3939037  PMID: 24096113
15.  A non-invasive screening technique for type 1 autoimmune pancreatitis 
Introduction
Type 1 autoimmune pancreatitis (AIP) is the pancreatic manifestation of a systemic fibroinflammatory IgG4-related disease. Accurate diagnosis of AIP can avoid major hepatobiliary and pancreatic surgery as it respond dramatically to corticosteroid therapy.
Aim
This research investigated the feasibility of using peripheral blood cell immunohistochemistry, serum IgG4, T-cell receptor (TCR) and serum isoelectric focusing electrophoresis in the screening of type 1 autoimmune pancreatitis (AIP).
Material and methods
The peripheral blood from 3 type 1 AIP patients, 10 pancreatic cancer patients and 40 normal controls was collected. Sediment smears were jointly incubated with anti-IgG4 and anti-IgG. The percentage of IgG4/IgG positive cells was counted and serum TCR and IgG4 were detected through the whole process. After serum isoelectric focusing electrophoresis, anti-IgG4 and anti-IgG were used to confirm the components of serum.
Results
In the serum isoelectric focusing electrophoresis, IgG4 and IgG strips showed mirrored distribution in type 1 AIP patients, while there were no strips in the normal controls and pancreatic cancer. Compared with pancreatic tumor patients and healthy controls, serum TCR was significant increased in AIP. The percentage of IgG4/IgG positive cells of peripheral blood cell immunohistochemistry was related to serum IgG4 and hormone therapy reactions.
Conclusions
Peripheral blood cell immunohistochemistry, serum IgG4, TCR and serum isoelectric focusing electrophoresis is suitable for the screening of type 1 AIP and monitoring its response assessment.
doi:10.5114/wiitm.2014.44290
PMCID: PMC4280412  PMID: 25561988
type 1 autoimmune pancreatitis; peripheral blood cell immunohistochemistry; serum IgG4; TCR; serum isoelectric focusing electrophoresis
16.  Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation 
Retrovirology  2014;11(1):101.
Background
Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses.
Results
The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations.
Conclusions
Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.
doi:10.1186/s12977-014-0101-0
PMCID: PMC4264250  PMID: 25407514
HIV-1; Fitness; Immune escape mutation; Compensatory mutation; Cytotoxic T lymphocytes; Transmitted/founder virus; Reversion
17.  DCE-MRI assessment of the effect of Epstein-Barr virus-encoded latent membrane protein-1 targeted DNAzyme on tumor vasculature in patients with nasopharyngeal carcinomas 
BMC Cancer  2014;14(1):835.
Background
EBV-encoded latent membrane protein 1 (EBV-LMP1) is an important oncogenic protein for nasopharyngeal carcinoma (NPC) and has been shown to engage a plethora of signaling pathways. Correspondingly, an LMP1-targeted DNAzyme was found to inhibit the growth of NPC cells both in vivo and in vitro by suppressing cell proliferation and inducing apoptosis. However, it remains unknown whether an LMP1-targeted DNAzyme would affect the vasculature of NPC. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has been applied in the clinical trials of anti-angiogenic drugs for more than ten years, and Ktrans has been recommended as a primary endpoint. Therefore, the objective of the current study was to use DCE-MRI to longitudinally study the effect of an EBV-LMP1-targeted DNAzyme on the vasculature of patients with NPC.
Methods
Twenty-four patients were randomly divided into two groups: a combined treatment group (radiotherapy + LMP1-targeted DNAzyme) and a radiotherapy alone group (radiotherapy + normal saline). DCE-MRI scans were conducted 1 ~ 2 days before radiotherapy (Pre-RT), during radiotherapy (RT 50 Gy), upon completion of radiotherapy (RT 70 Gy), and three months after radiotherapy (3 months post-RT). Parameters of vascular permeability and intra- and extravascular volumes were subsequently obtained (e.g., Ktrans, kep, ve) using nordicICE software.
Results
Both Ktrans and kep values for NPC tumor tissues decreased for both groups after treatment. Moreover, a statistically significant difference in Ktrans values at the pre-therapy and post-therapy timepoints emerged earlier for the combined treatment group (RT 50 Gy, P =0.045) compared to the radiotherapy alone group (3 months post-RT, P = 0.032). For the kep values, the downward trend observed for both the combined treatment group and the radiotherapy alone group were similar. In contrast, ve values for all of the tumor tissues increased following therapy.
Conclusions
The EBV-LMP1-targeted DNAzyme that was tested was found to accelerate the decline of Ktrans values for patients with NPC. Correspondingly, the LMP1-targeted DNAzyme treatments were found to affect the angiogenesis and microvascular permeability of NPC.
Trial registration
ClinicalTrials.gov: NCT01449942. Registered 6 October 2011.
doi:10.1186/1471-2407-14-835
PMCID: PMC4246516  PMID: 25407966
Nasopharyngeal neoplasm; Epstein-Barr virus encoded latent membrane protein-1; Magnetic resonance imaging; Dynamic contrast-enhanced magnetic resonance imaging; Molecular therapy; DNAzyme
18.  Expression of P-gp in acute myeloid leukemia and the reversal function of As2O3 on drug resistance 
Oncology Letters  2014;9(1):177-182.
To study the expression of P-glycoprotein (P-gp) and the reversal function of As2O3, the active ingredient of arsenic, on drug resistance in acute myeloid leukemia (AML) patients, P-gp and cluster of differentiation 34 (CD34) were examined in primary mononuclear and resistant cells, with or without As2O3. In addition, multidrug resistance gene 1 (MDR1) mRNA expression was investigated in K562/D cells and AML patients. In total, 28.6% of newly-treated (NT) patients and 59.1% of relapsed/refractory (RR) patients were P-gpfunction+, and 31.7% of NT patients and 59.1% of RR patients were CD34+. The positivity rate of P-gpfunction and CD34+ expression in the RR group were significantly higher compared with that in the NT group (P<0.05). In addition, higher CD34+, P-gpexpression+ and P-gpfunction+ values were observed in older patients compared with younger patients. MDR1 expression was downregulated in certain patients following treatment with AS2O3. In the present study, the overexpression of P-gp was the primary cause of drug resistance in the AML patients, and MDR1 expression was downregulated by As2O3 in primary leukemia and drug-resistant cells.
doi:10.3892/ol.2014.2692
PMCID: PMC4247107  PMID: 25435954
acute myeloid leukemia; P-gp; As2O3; MDR1
19.  L-Glutamate Supplementation Improves Small Intestinal Architecture and Enhances the Expressions of Jejunal Mucosa Amino Acid Receptors and Transporters in Weaning Piglets 
PLoS ONE  2014;9(11):e111950.
L-Glutamate is a major oxidative fuel for the small intestine. However, few studies have demonstrated the effect of L-glutamate on the intestinal architecture and signaling of amino acids in the small intestine. The aim of this study was to investigate the effects of dietary L-glutamate supplementation on the intestinal architecture and expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets. A total of 120 weaning piglets aged 35±1 days with an average body weight at 8.91±0.45 kg were randomly allocated to two treatments with six replicates of ten piglets each, fed with diets containing 1.21% alanine, or 2% L-glutamate. L-Glutamate supplementation increased the activity of glutamate oxaloacetate transaminase (GOT) in the jejunal mucosa. Also, the mRNA expression level of jejunal mucosa glutamine synthetase (GS) was increased by L-glutamate supplementation. The height of villi in duodenal and jejunal segments, and the relative mRNA expression of occludin and zonula occludens protein-1 (ZO-1) in jejunal mucosa were increased by dietary L-glutamate supplementation. L-Glutamate supplementation increased plasma concentrations of glutamate, arginine, histidine, isoleucine, leucine, methionine, phenylalanine and threonine. L-Glutamate supplementation also increased the relative mRNA expression of the jejunal mucosa Ca2+-sensing receptor (CaR), metabotropic glutamate receptor 1 (mGluR1) and metabotropic glutamate receptor 4 (mGluR4), and neutral amino acid transporter B0-like (SLC1A5) in the jejunal mucosa. These findings suggest that dietary addition of 2% L-glutamate improves the intestinal integrity and influences the expression of amino acid receptors and transporters in the jejunum of weaning, which is beneficial for the improvement of jejunal nutrients for digestion and absorption.
doi:10.1371/journal.pone.0111950
PMCID: PMC4219819  PMID: 25368996
20.  Enalapril Mitigates Radiation-Induced Pneumonitis and Pulmonary Fibrosis if Started 35 Days after Whole-Thorax Irradiation 
Radiation research  2013;180(5):546-552.
Victims of a radiological attack or nuclear accident may receive high-dose, heterogeneous exposures from radiation to the chest that lead to lung damage. Our goal is to develop countermeasures to mitigate such injuries. We used WAG/RijCmcr rats receiving 13 Gy to the whole thorax to induce pulmonary fibrosis within 210 days. The angiotensin converting enzyme (ACE) inhibitor enalapril was evaluated as a mitigator of these injuries at two doses (18 and 36 mg/m2/day) and 8 schedules: starting at 7, 35, 70, 105 and 140 days and continuing to 210 days or starting at 7 days and stopping at 30, 60 or 90 days after whole-thorax irradiation. The earliest start date at 7 days after irradiation would provide an adequate window of time for triage and dosimetry. Survival after 35 days, as permitted by our Institutional Animal Care and Use Committee (IACUC) was also recorded as a primary end point of pneumonitis. Pulmonary fibrosis was evaluated using the Sircol biochemical assay to measure lung collagen. Our results indicated that a short course of either dose of enalapril from 7–90 days improved survival. However, pulmonary fibrosis was only mitigated by the higher dose of enalapril (36 mg/m2/day). The latest effective start date for the drug was 35 days after irradiation. These results indicate that ACE inhibitors can be started at least a month after irradiation for mitigation of pneumonitis and/or pulmonary fibrosis.
doi:10.1667/RR13350.1
PMCID: PMC3895940  PMID: 24131041
21.  Prevalence of Sleep Disturbances in Children with Neurofibromatosis Type 1 
Journal of child neurology  2013;28(11):1400-1405.
Children with neurodevelopmental disorders are at increased risk for sleep issues, which affect quality of life, cognitive function, and behavior. To determine the prevalence of sleep problems in children with the common neurodevelopmental disorder, neurofibromatosis type 1, a cross-sectional study was performed on 129 affected subjects and 89 unaffected siblings, age 2-17 years, using the Sleep Disturbance Scale for Children questionnaire. Children with neurofibromatosis type 1 were significantly more likely to have disturbances in initiating and maintaining sleep, arousal, sleep-wake transition, and hyperhidrosis, but not problems with abnormal sleep breathing, or excessive somnolence. While the overall sleep scores were higher in children with neurofibromatosis type 1, this was not related to a co-existing attention deficit disorder, cognitive impairment, or stimulant medication use. Collectively, these results demonstrate that children with neurofibromatosis type 1 are more likely to have sleep disturbances, and support the use of appropriate interventions for this at-risk population.
doi:10.1177/0883073813500849
PMCID: PMC3805763  PMID: 24065580
NF1; sleep disturbances; neurocutaneous disorders; brain tumor
22.  Role of PON2 in innate immune response in an acute infection model 
Molecular genetics and metabolism  2013;110(3):362-370.
N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC12-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC12-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC12-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC12-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC12-HSL. PON2 expression (message and protein) is not altered in response to 3OC12-HSL in macrophages. 3OC12-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6×107CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.
doi:10.1016/j.ymgme.2013.07.003
PMCID: PMC3800229  PMID: 23911207
Paraoxonase 2; Pseudomonas aeruginosa; Quorum sensing; Mitochondrial oxidative stress; Endoplasmic reticulum stress
23.  Identification of plant-derived natural products as potential inhibitors of the Mycobacterium tuberculosis proteasome 
Background
The Mycobacterium tuberculosis (Mtb) proteasome has been established as a viable target for the development of anti-tuberculosis agents. In this study, the inhibitory activities of 100 plant-derived natural products on the Mtb proteasome were analyzed to identify novel potential inhibitors.
Methods
The fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC can be hydrolyzed by the proteasome to release free AMC, the fluorescence of which is proportional to the proteasome activity. The inhibitory activities of 100 natural products (each at a final concentration of 200 μM) were detected by this method using MG132 as a positive control.
Results
Twelve of these natural products (10 of which were flavonoids) inhibited the activity of the Mtb proteasome by more than 65%. Comparison of the structural differences between the flavonoids with good inhibitory activity and those without inhibitory activity revealed that the hydroxyl at the flavonoid C ring C-3 or the hydroxyl/methoxyl at the flavonoid A ring C-6 were critical for the inhibition of proteasomal activity.
Conclusions
These data indicate that flavonoids represent a basis for rational structural design in the process of novel anti-tuberculosis drug discovery.
doi:10.1186/1472-6882-14-400
PMCID: PMC4203866  PMID: 25315519
Mycobacterium tuberculosis; Proteasome inhibitor; Plant-derived natural products; Flavonoids
24.  Synergistic actions of olomoucine and bone morphogenetic protein-4 in axonal repair after acute spinal cord contusion 
Neural Regeneration Research  2014;9(20):1830-1838.
To determine whether olomoucine acts synergistically with bone morphogenetic protein-4 in the treatment of spinal cord injury, we established a rat model of acute spinal cord contusion by impacting the spinal cord at the T8 vertebra. We injected a suspension of astrocytes derived from glial-restricted precursor cells exposed to bone morphogenetic protein-4 (GDAsBMP) into the spinal cord around the site of the injury, and/or olomoucine intraperitoneally. Olomoucine effectively inhibited astrocyte proliferation and the formation of scar tissue at the injury site, but did not prevent proliferation of GDAsBMP or inhibit their effects in reducing the spinal cord lesion cavity. Furthermore, while GDAsBMP and olomoucine independently resulted in small improvements in locomotor function in injured rats, combined administration of both treatments had a significantly greater effect on the restoration of motor function. These data indicate that the combined use of olomoucine and GDAsBMP creates a better environment for nerve regeneration than the use of either treatment alone, and contributes to spinal cord repair after injury.
doi:10.4103/1673-5374.143431
PMCID: PMC4239774  PMID: 25422646
nerve regeneration; spinal cord injury; olomoucine; glial-restricted precursor-derived astrocytes; glial scar; cavity; axonal regeneration; neural regeneration
25.  Adoptive Transfer of Heme Oxygenase-1 (HO-1)–Modified Macrophages Rescues the Nuclear Factor Erythroid 2–Related Factor (Nrf2) Antiinflammatory Phenotype in Liver Ischemia/Reperfusion Injury 
Molecular Medicine  2014;20(1):448-455.
Macrophages are instrumental in the pathophysiology of liver ischemia/reperfusion injury (IRI). Although Nrf2 regulates macrophage-specific heme oxygenase-1 (HO-1) antioxidant defense, it remains unknown whether HO-1 induction might rescue macrophage Nrf2-dependent antiinflammatory functions. This study explores the mechanisms by which the Nrf2–HO-1 axis regulates sterile hepatic inflammation responses after adoptive transfer of ex vivo modified HO-1 overexpressing bone marrow–derived macrophages (BMMs). Livers in Nrf2-deficient mice preconditioned with Ad-HO-1 BMMs, but not Ad-β-Gal-BMMs, ameliorated liver IRI (at 6 h of reperfusion after 90 min of warm ischemia), evidenced by improved hepatocellular function (serum alanine aminotransferase [sALT] levels) and preserved hepatic architecture (Suzuki histological score). Treatment with Ad-HO-1 BMMs decreased neutrophil accumulation, proinflammatory mediators and hepatocellular necrosis/apoptosis in ischemic livers. Moreover, Ad-HO-1 transfection of Nrf2-deficient BMMs suppressed M1 (Nos2+) while promoting the M2 (Mrc-1/Arg-1+) phenotype. Unlike in controls, Ad-HO-1 BMMs increased the expression of Notch1, Hes1, phosphorylation of Stat3 and Akt in IR-stressed Nrf2-deficient livers as well as in lipopolysaccharide (LPS)-stimulated BMMs. Thus, adoptive transfer of ex vivo generated Ad-HO-1 BMMs rescued Nrf2-dependent antiinflammatory phenotype by promoting Notch1/Hes1/Stat3 signaling and reprogramming macrophages toward the M2 phenotype. These findings provide the rationale for a novel clinically attractive strategy to manage IR liver inflammation/damage.
doi:10.2119/molmed.2014.00103
PMCID: PMC4212016  PMID: 25014792

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