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1.  Item Response Theory Analysis of the Patient Satisfaction with Cancer-Related Care Measure: A Psychometric Investigation in A Multicultural Sample of 1,296 Participants 
We developed and validated a Patient Satisfaction with Cancer-Related Care (PSCC) measure using classical test theory methods. The present study applied item response theory (IRT) analysis to determine item-level psychometric properties, facilitate development of short forms, and inform future applications for the PSCC.
We applied unidimensional IRT models to PSCC data from 1,296 participants (73% female; 18 to 86 years). An unconstrained graded response model (GRM) and a Rasch Model were fitted to estimate indices for model comparison using likelihood ratio (LR) test and information criteria. We computed item and latent trait parameter estimates, category and operating characteristic curves, and tested information curves for the better fitting model.
The GRM fitted the data better than the Rasch Model (LR=828, df=17, p<0.001). The log-Likelihood (−17390.38 vs. −17804.26) was larger, and the AIC and BIC were smaller for the GRM compared to the Rash Model (AIC=34960.77 vs. 35754.73; BIC=35425.80 vs. 36131.92). Item parameter estimates (IPEs) showed substantial variation in items’ discriminating power (0.94 to 2.18). Standard errors of the IPEs were small (threshold parameters mostly around 0.1; discrimination parameters: 0.1 to 0.2), confirming the precision of the IPEs.
The GRM provides precise IPEs that will enable comparable scores from different subsets of items, and facilitate optimal selections of items to estimate patients’ latent satisfaction level. Given the large calibration sample, the IPEs can be used in settings with limited resources (e.g., smaller samples) to estimate patients’ satisfaction.
PMCID: PMC4256077  PMID: 24664356
Psychometrics; Measure development; Psychometric validation; Item Response Theory; Cancer Disparities; Race-ethnicity
2.  rpoS-Regulated Core Genes Involved in the Competitive Fitness of Salmonella enterica Serovar Kentucky in the Intestines of Chickens 
Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry.
PMCID: PMC4277580  PMID: 25362062
3.  Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells 
Cellular and Molecular Immunology  2014;12(4):424-434.
Gouty arthritis is an inflammatory disease that is caused by an accumulation of monosodium urate (MSU) crystals in the joints. MSU is capable of activating the nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome, leading to interleukin-1β (IL-1β) secretion. Reactive oxygen species (ROS) are major mediators of the NLRP3/IL-1β interaction. Although nuclear factor E2-related factor 2 (Nrf2) is recognized as a transcription factor that is involved in the response to oxidative stress, the effect of MSU on Nrf2 and on Nrf2-mediated antioxidant enzymes remains unclear. The treatment of THP-1 monocytes using phorbol 12-myristate 13-acetate (PMA) was shown to initiate inflammatory responses. Here, we showed that THP-1 cells, following treatment with MSU crystals, significantly increased IL-1β release, NLRP3 inflammasome activation and ROS production. MSU also promoted the nuclear translocation of Nrf2 and activated lysosomal destabilization. Moreover, the levels of heme oxygenase-1 (HO-1) in gene and protein expressions were upregulated by MSU. MSU-induced IL-1β secretion and NLRP3 inflammasome activation were inhibited by the knockdown of Nrf2 and via the HO-1 inhibitor zinc (II) protoporphyrin IX (ZnPP). In addition, HO-1 inhibition increased the level of superoxide anion production and the consumption of glutathione. These findings suggest that Nrf2 and HO-1 mediate redox homeostasis and interact with pro-inflammatory factors in MSU-challenged THP-1 cells, thereby providing new insight into how MSU-induced gouty inflammation is mediated by specific mechanisms that are involved in the Nrf2/Ho-1 antioxidant signaling pathway.
PMCID: PMC4496538  PMID: 25109682
HO-1; monosodium urate; NLRP3 inflammasome; Nrf2; THP-1 cells
4.  Diversity and Persistence of Salmonella enterica Strains in Rural Landscapes in the Southeastern United States 
PLoS ONE  2015;10(7):e0128937.
Salmonellosis cases in the in the United States show distinct geographical trends, with the southeast reporting among the highest rates of illness. In the state of Georgia, USA, non-outbreak associated salmonellosis is especially high in the southern low-lying coastal plain. Here we examined the distribution of Salmonella enterica in environmental waters and associated wildlife in two distinct watersheds, one in the Atlantic Coastal Plain (a high case rate rural area) physiographic province and one in the Piedmont (a lower case rate rural area). Salmonella were isolated from the two regions and compared for serovar and strain diversity, as well as distribution, between the two study areas, using both a retrospective and prospective design. Thirty-seven unique serovars and 204 unique strain types were identified by pulsed-field gel electrophoresis (PFGE). Salmonella serovars Braenderup, Give, Hartford, and Muenchen were dominant in both watersheds. Two serovars, specifically S. Muenchen and S. Rubislaw, were consistently isolated from both systems, including water and small mammals. Conversely, 24 serovars tended to be site-specific (64.8%, n = 37). Compared to the other Salmonella serovars isolated from these sites, S. Muenchen and S. Rubislaw exhibited significant genetic diversity. Among a subset of PFGE patterns, approximately half of the environmental strain types matched entries in the USA PulseNet database of human cases. Ninety percent of S. Muenchen strains from the Little River basin (the high case rate area) matched PFGE entries in PulseNet compared to 33.33% of S. Muenchen strains from the North Oconee River region (the lower case rate area). Underlying the diversity and turnover of Salmonella strains observed for these two watersheds is the persistence of specific Salmonella serovars and strain types that may be adapted to these watersheds and landscapes.
PMCID: PMC4489491  PMID: 26131552
5.  Huaier Aqueous Extract Induces Hepatocellular Carcinoma Cells Arrest in S Phase via JNK Signaling Pathway 
Huaier aqueous extract, the main active constituent of Huaier proteoglycan, has antihepatocarcinoma activity in experimental and clinical settings. However, the potential and associated antihepatoma mechanisms of Huaier extract are not yet fully understood. Therefore, in this study, we aimed to elucidate the inhibitory proliferation effect of Huaier extract on apoptosis and cycle of HepG2 and Bel-7402 cells. Our data demonstrated that incubation with Huaier extract resulted in a marked decrease in cell viability dose-dependently. Flow cytometric analysis showed that a 48 h treatment of Huaier extract caused cell apoptosis. Typical apoptotic nucleus alterations were observed with fluorescence microscope after Hoechst staining. Immunoblot analysis further demonstrated that Huaier extract activated caspase 3 and PARP. Additionally, Huaier extract inhibited the activity of p-ERK, p-p38, and p-JNK in terms of MAPK. Furthermore, Huaier extract induced HCC cells arrest in S phase and decreased the cycle related protein expression of β-catenin and cyclin D1. Studies with JNK specific inhibitor, SP600125, showed that Huaier extract induced S phase arrest and decreased β-catenin and cyclin D1 expression via JNK signaling pathway. In conclusion, we verify that Huaier extract causes cell apoptosis and induces hepatocellular carcinoma cells arrest in S phase via JNK pathway, which advances our understanding on the molecular mechanisms of Huaier extract in hepatocarcinoma management.
PMCID: PMC4504122
7.  Axitinib versus sorafenib as a second-line therapy in Asian patients with metastatic renal cell carcinoma: results from a randomized registrational study 
OncoTargets and therapy  2015;8:1363-1373.
This registrational trial evaluated the efficacy, safety, and patient-reported outcomes of axitinib versus sorafenib as a second-line treatment in Asian patients with clear-cell metastatic renal cell carcinoma (mRCC).
In this open-label, multicenter study, previously treated Asian patients with clear-cell mRCC were stratified by Eastern Cooperative Oncology Group performance status and prior therapy and randomized in a 2:1 ratio to receive axitinib (5 mg twice daily) or sorafenib (400 mg twice daily). The primary end point was progression-free survival (PFS) assessed by a masked independent review committee.
A total of 204 Asian patients received axitinib (n=135) or sorafenib (n=69). Median PFS (95% confidence interval [CI]) was 6.5 (4.7–9.1) months with axitinib versus 4.8 (3.0–6.5) months with sorafenib (hazard ratio, 0.731; 95% CI, 0.506–1.058; one-sided P=0.0531). The objective response rate (95% CI) was 23.7% (16.8%–31.8%) with axitinib versus 10.1% (4.2%–19.8%) with sorafenib. Common, grade ≥3, all-causality adverse events were hypertension (19.3%), weight decrease (5.2%), and proteinuria (5.2%) with axitinib and hypertension (8.7%) and palmar-plantar erythrodysesthesia (7.2%) with sorafenib. In a time-to-deterioration composite end point of death, progression, and worsening of Functional Assessment of Cancer Therapy Kidney Symptom Index score, patients treated with axitinib demonstrated a 17%–24% risk reduction compared with sorafenib-treated patients.
Axitinib is clinically active and well tolerated in previously treated Asian patients with mRCC, consistent with the results from the global Phase III trial. These results establish axitinib as a second-line treatment option for Asian patients with mRCC.
PMCID: PMC4467642  PMID: 26089686
axitinib; renal cell carcinoma; sorafenib; vascular endothelial growth factor receptor inhibitor
8.  A standardized autopsy procurement allows for the comprehensive study of DIPG biology 
Oncotarget  2015;6(14):12740-12747.
Diffuse intrinsic pontine glioma (DIPG) is one of the least understood and most deadly childhood cancers. Historically, there has been a paucity of DIPG specimens for molecular analysis. However, due to the generous participation of DIPG families in programs for postmortem specimen donation, there has been a recent surge in molecular analysis of newly available tumor specimens. Collaborative efforts to share data and tumor specimens have resulted in rapid discoveries in other pediatric brain tumors, such as medulloblastoma, and therefore have the potential to shed light on the biology of DIPG. Given the generous gift of postmortem tissue donation from DIPG patients, there is a need for standardized postmortem specimen accrual to facilitate rapid and effective multi-institutional molecular studies.
We developed and implemented an autopsy protocol for rapid procurement, documenting and storing these specimens. Sixteen autopsies were performed throughout the United States and Canada and processed using a standard protocol and inventory method, including specimen imaging, fixation, snap freezing, orthotopic injection, or preservation. This allowed for comparative clinical and biological studies of rare postmortem DIPG tissue specimens, generation of in vivo and in vitro models of DIPG, and detailed records to facilitate collaborative analysis.
PMCID: PMC4494970  PMID: 25749048
Diffuse Intrinsic Pontine Glioma (DIPG); Brainstem Glioma; Autopsy; Histone 3; Orthotopic Injection
9.  Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum 
The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated.
In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption.
These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.
PMCID: PMC4453273  PMID: 25981633
Shikimic acid pathway; Corynebacterium glutamicum; Shikimate production; Synthetic biology; Genetic modules; Ribosome binding site (RBS)
10.  Detection of K-ras Mutations in Predicting Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase (EGFR-TK) Inhibitor in Patients with Metastatic Colorectal Cancer 
PLoS ONE  2015;10(5):e0101019.
Epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors are useful in treating different advanced human cancers; however, their clinical efficacy varies. This study detected K-ras mutations to predict the efficacy of EGFR-TK inhibitor cetuximab treatment on Chinese patients with metastatic colorectal cancer (mCRC). A total of 87 patients with metastatic colorectal cancer were treated with cetuximab for 2-16 months, in combination with chemotherapy between August 2008 and July 2012, and tissue samples were used to detect K-ras mutations. The data showed that K-ras mutation occurred in 27/87 (31%). The objective response rates and disease control rate in K-ras wild type and mutant patients were 42% (25/60) versus 11% (3/27) (p<0.05) and 60% (36/60) versus 26% (7/27) (p<0.05), respectively. Patients with the wild-type K-ras had significantly higher median survival times and progression-free survival, than patients with mutated K-ras (21 months versus 17 months, p=0.017; 10 months versus 6 months, p=0.6). These findings suggest that a high frequency of K-ras mutations occurs in Chinese mCRC patients and that K-ras mutation is required to select patients for eligibility for cetuximab therapy. Further prospective studies using a large sample size are needed to confirm these preliminary findings.
PMCID: PMC4423938  PMID: 25950441
11.  Radiation-driven lipid accumulation and dendritic cell dysfunction in cancer 
Scientific Reports  2015;5:9613.
Dendritic cells (DCs) play important roles in the initiation and maintenance of the immune response. The dysfunction of DCs contributes to tumor evasion and growth. Here we report our findings on the dysfunction of DCs in radiation-induced thymic lymphomas, and the up-regulation of the expression of the lipoprotein lipase (LPL) and the fatty acid binding protein (FABP4), and the level of triacylglycerol (TAG) in serum after total body irradiation, which contribute to DCs lipid accumulation. DCs with high lipid content showed low expression of co-stimulatory molecules and DCs-related cytokines, and were not able to effectively stimulate allogeneic T cells. Normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the function of DCs. A high-fat diet promoted radiation-induced thymic lymphoma growth. In all, our study shows that dysfunction of DCs in radiation-induced thymic lymphomas was due to lipid accumulation and may represent a new mechanism in radiation-induced carcinogenesis.
PMCID: PMC4413852  PMID: 25923834
12.  Acupuncture Therapy for Sudden Sensorineural Hearing Loss: A Systematic Review and Meta-Analysis of Randomized Controlled Trials 
PLoS ONE  2015;10(4):e0125240.
Acupuncture has commonly been used in China, either alone or in combination with Western medicine, to treat sudden sensorineural hearing loss (SSHL). The purpose of this systematic review is to assess the efficacy and safety of acupuncture therapy for patients with SSHL.
We searched PubMed, the Cochrane Library, Embase, China National Knowledge Internet (CNKI), Database for Chinese Technical Periodicals (VIP), and Chinese Biomedical literature service system (SinoMed) to collect randomized controlled trials of acupuncture for SSHL published before July 2014. A meta-analysis was conducted according to the Cochrane systematic review method using RevMan 5.2 software. The evidence level for each outcome was assessed using the GRADE methodology.
Twelve trials involving 863 patients were included. A meta-analysis showed that the effect of manual acupuncture combined with Western medicine comprehensive treatment (WMCT) was better than WMCT alone (RR 1.33, 95%CI 1.19–1.49) and the same as the effect of electroacupuncture combined with WMCT (RR 1.33, 95%CI 1.19–1.50). One study showed a better effect of electroacupuncture than of WMCT (RR 1.34, 95%CI 1.24–1.45). For mean changes in hearing over all frequencies, the meta-analysis showed a better effect with the combination of acupuncture and WMCT than with WMCT alone (MD 10.85, 95%CI 6.84–14.86). However, the evidence levels for these interventions were low or very low due to a high risk of bias and small sample sizes in the included studies.
There was not sufficient evidence showing that acupuncture therapy alone was beneficial for treating SSHL. However, interventions combining acupuncture with WMCT had more efficacious results in the treatment of SSHL than WMCT alone. Electroacupuncture alone might be a viable alternative treatment besides WMCT for SSHL. However, given that there were fewer eligible RCTs and limitations in the included trials, such as methodological drawbacks and small sample sizes, large-scale RCTs are required to confirm the current findings regarding acupuncture therapy for SSHL.
PMCID: PMC4412536  PMID: 25919000
13.  Arginine Starvation Impairs Mitochondrial Respiratory Function in ASS1-Deficient Breast Cancer Cells 
Science signaling  2014;7(319):ra31.
Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer bio-samples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.
PMCID: PMC4229039  PMID: 24692592
14.  Hepatic Expression of Detoxification Enzymes Is Decreased in Human Obstructive Cholestasis Due to Gallstone Biliary Obstruction 
PLoS ONE  2015;10(3):e0120055.
Background & Aims
Levels of bile acid metabolic enzymes and membrane transporters have been reported to change in cholestasis. These alterations (e.g. CYP7A1 repression and MRP4 induction) are thought to be adaptive responses that attenuate cholestatic liver injury. However, the molecular mechanisms of these adaptive responses in human obstructive cholestasis due to gallstone biliary obstruction remain unclear.
We collected liver samples from cholestatic patients with biliary obstruction due to gallstones and from control patients without liver disease (n = 22 per group). The expression levels of bile acid synthetic and detoxification enzymes, membrane transporters, and the related nuclear receptors and transcriptional factors were measured.
The levels of bile acid synthetic enzymes, CYP7B1 and CYP8B1, and the detoxification enzyme CYP2B6 were increased in cholestatic livers by 2.4-fold, 2.8-fold, and 1.9-fold, respectively (p<0.05). Conversely, the expression levels of liver detoxification enzymes, UGT2B4/7, SULT2A1, GSTA1-4, and GSTM1-4, were reduced by approximately 50% (p<0.05) in human obstructive cholestasis. The levels of membrane transporters, OSTβ and OCT1, were increased 10.4-fold and 1.8-fold, respectively, (p<0.05), whereas those of OSTα, ABCG2 and ABCG8 were all decreased by approximately 40%, (p<0.05) in human cholestatic livers. Hepatic nuclear receptors, VDR, HNF4α, RXRα and RARα, were induced (approximately 2.0-fold, (p<0.05) whereas FXR levels were markedly reduced to 44% of control, (p<0.05) in human obstructive cholestasis. There was a significantly positive correlation between the reduction in FXR mRNA and UGT2B4/7, SULT2A1, GSTA1, ABCG2/8 mRNA levels in livers of obstructive cholestatic patients (p<0.05).
The levels of hepatic detoxification enzymes were significantly decreased in human obstructive cholestasis, and these decreases were positively associated with a marked reduction of FXR levels. These findings are consistent with impaired detoxification ability in human obstructive cholestasis.
PMCID: PMC4370735  PMID: 25798860
15.  Primary hepatic neuroendocrine tumor with multiple liver metastases: A case report with review of the literature 
We herein present a case involving a 41-year-old woman in whom ultrasound examination revealed multiple liver hemangiomas more than 3 years ago. Follow-up ultrasound examination revealed that the masses had significantly increased; the largest was located in the right lobe (about 8.2 cm × 7.4 cm × 6.0 cm). Abdominal multidetector computed tomography revealed multiple well-circumscribed, heterogeneous, hypodense masses (largest, 6.4 cm × 6.3 cm × 5.0 cm) with significant contrast enhancement during the arterial and portal phases and with contrast wash-out and peripheral enhancement during the delayed phases. Magnetic resonance images demonstrated multiple well-circumscribed, heterogeneous, hypointense hepatic masses with significant contrast enhancement (largest, 6.4 cm × 6.5 cm × 5.1 cm); multiple enlarged porta lymph nodes; and multiple slightly enlarged retroperitoneal lymph nodes. Histological and immunohistochemical examination of the right mass biopsy specimen suggested a malignant neoplasm that had originated from a neuroendocrine cell type (grade 2 well-differentiated neuroendocrine carcinoma). After performing a systemic examination to exclude metastasis from an extrahepatic primary site, we considered that the masses had arisen from a primary hepatic neuroendocrine tumor with multiple liver metastases. The patient underwent transcatheter arterial chemoembolization using a combination of oxaliplatin (150 mg) mixed with one bottle of gelatin sponge particles (560-710 μm) and lipiodol (6 mL). Primary neuroendocrine tumors of the liver are extremely rare. This case is interesting because of the rarity of this neoplasm and previous misdiagnosis as multiple liver hemangiomas. Previously reported cases in the literature are also reviewed.
PMCID: PMC4356938  PMID: 25780316
Liver; Neoplasms - primary; Neuroendocrine tumor; Metastatic; Immunohistochemical; Imaging; Transcatheter arterial chemoembolization
16.  Sequence Variation in tcdA and tcdB of Clostridium difficile: ST37 with Truncated tcdA Is a Potential Epidemic Strain in China 
Journal of Clinical Microbiology  2014;52(9):3264-3270.
Clostridium difficile is a well-known nosocomial infectious pathogen. Research on C. difficile infection has primarily focused on strains such as the hypervirulent PCR ribotype 027 (sequence type 1 [ST1]) emerging in Europe and North America. However, other new emerging ribotypes in some countries have attracted attention, such as PCR ribotype 17 (ST37) in Asia and Latin America. We collected 70 strains and sequenced their toxin genes, tcdA and tcdB. Multilocus sequence typing (MLST) was used to study their population structure. In addition, tcdA and/or tcdB sequences of 25 other isolates were obtained from GenBank. Single nucleotide polymorphisms (SNPs) were identified and analyzed. Phylogenetic analyses were performed to study toxin gene evolution. All tcdA and tcdB sequences were divided into 1 of 16 types (denoted A01 to -16 and B01 to -16, respectively). Hypervirulent strain RT027 is A13B12, and RT078 is A14B10, whereas the newly epidemic strain RT017 is A15B13. SNP analysis suggests the possibility of recombination in tcdB, perhaps through horizontal gene transfer. SNPs were also found in the sequences corresponding to the PCR primers widely used for toxin detection. Our study shows that ST037 shares a few genotypic features in its tcdA and tcdB genes with some known hypervirulent strains, indicating that they fall into a unique clade. Our findings can be used to map the relationships among C. difficile strains more finely than can be done with less sensitive methods, such as toxinotyping or even MLST, to reveal their inherent epidemiological characteristics.
PMCID: PMC4313148  PMID: 24958798
17.  Genetic Profiling by Single-Nucleotide Polymorphism-Based Array Analysis Defines Three Distinct Subtypes of Orbital Meningioma 
Orbital meningiomas can be classified as primary optic nerve sheath (ON) meningiomas, primary intraorbital ectopic (Ob) meningiomas and spheno-orbital (Sph-Ob) meningiomas based on anatomic site. Single-nucleotide polymorphism (SNP)-based array analysis with the Illumina 300K platform was performed on formalin-fixed, paraffin-embedded tissue from 19 orbital meningiomas (5 ON, 4 Ob and 10 Sph-Ob meningiomas). Tumors were World Health Organization (WHO) grade I except for two grade II meningiomas, and one was NF2-associated. We found genomic alterations in 68% (13 of 19) of orbital meningiomas. Sph-Ob tumors frequently exhibited monosomy 22/22q loss (70%; 7/10) and deletion of chromosome 1p, 6q and 19p (50% each; 5/10). Among genetic alterations, loss of chromosome 1p and 6q were more frequent in clinically progressive tumors. Chromosome 22q loss also was detected in the majority of Ob meningiomas (75%; 3/4) but was infrequent in ON meningiomas (20%; 1/5). In general, Ob tumors had fewer chromosome alterations than Sph-Ob and ON tumors. Unlike Sph-Ob meningiomas, most of the Ob and ON meningiomas did not progress even after incomplete excision, although follow-up was limited in some cases. Our study suggests that ON, Ob and Sph-Ob meningiomas are three molecularly distinct entities. Our results also suggest that molecular subclassification may have prognostic implications.
PMCID: PMC4324373  PMID: 24773246
chromosome 22; cytogenetics; NF2; orbital meningioma; optic nerve sheath; SNP array
18.  Deleterious Effects of Minocycline after in vivo Target Deprivation of Thalamocortical Neurons in the Immature, Metallothionein-Deficient Mouse Brain 
Journal of neuroscience research  2009;87(6):1356-1368.
Compared to adults, immature metallothionein I & II knockout (MT−/−) mice incur greater neuronal loss and a more rapid rate of microglia accumulation following target deprivation-induced injury. Since minocycline has been proposed to inhibit microglial activation and associated production of neuroinflammatory factors, we investigated its ability to promote neuronal survival in the immature, metallothionein-deficient brain. Following ablation of the visual cortex, 10-day-old MT−/− mice were treated with minocycline or saline and sacrificed 24 or 48 hours after injury. Using stereological methods, the number of microglia and neurons were estimated in the ipsilateral dorsal lateral geniculate nucleus (dLGN) by an investigator blinded to the treatment. No effect on neuronal survival was observed at 24 hours, but 48 hours after injury an unanticipated but significant minocycline-mediated increase in neuronal loss was detected. Further, while failing to inhibit microglial accumulation, minocycline treatment increased the proportion of amoeboid microglia in the ipsilateral dLGN. To understand the molecular mechanisms underlying this neurotoxic response, we identified minocycline-mediated changes in the expression of three potentially pro-apoptotic/ inflammatory genes: growth arrest- and DNA damage-inducible gene 45γ (GADD45γ); interferon-inducible protein 1 (IFI1) and cytokine induced growth factor (CTGF). We also observed increased mitogen-activated protein kinase (MAPK) p38 phosphorylation with minocycline treatment. Although minocycline inhibited calpain activity at 12 hours post-injury, this effect was not sustained at 24 hours. Together, these results help to explain how minocycline has a deleterious effect on neuronal survival in this injury model.
PMCID: PMC4333151  PMID: 19115404
microglia; minocycline; metallothionein; traumatic brain injury
19.  Oral administration of oleanolic acid, isolated from Swertia mussotii Franch, attenuates liver injury, inflammation, and cholestasis in bile duct-ligated rats 
Background & aims: Oleanolic acid is abundantly distributed in Swertia mussotii Franch, a Chinese traditional herb for the treatment of jaundice. However, the hepatoprotective role of oleanolic acid in obstructive cholestasis and its underlying molecular mechanism are unclear. Methods: Normal rats and bile duct-ligated (BDL) rats were given oleanolic acid and serum biochemistry, bile salts, and pro-inflammatory factors were measured, as well as the expression levels of liver bile acid synthesis and detoxification enzymes, membrane transporters, nuclear receptors, and transcriptional factors. Results: Oral administration of oleanolic acid at 100 mg/kg did not cause rat liver injury. However, it significantly reduced the serum levels of alanine aminotransferase (ALT) on days 7 and 14, aspartate aminotransferase (AST) and TNF-α on day 14, and alkaline phosphatase (ALP) and IL-1β on days 3, 7, and 14 in the BDL rats. Furthermore, the serum levels of total bile acid (TBA) and bile acids, including CDCA, CA, DCA, and Tα/βMCA were significantly reduced by oleanolic acid on day 3 in the BDL rats. In addition, the expression levels of detoxification enzymes Cyp3a, Ugt2b, Sult2a1, Gsta1-2, and Gstm1-3, membrane transporters Mrp3, Mrp4, Ostβ, Mdr1, Mdr2, and Bsep, nuclear receptors Pxr, Vdr, Hnf4α, Rxrα, Rarα, Lxr, and Lrh-1, and transcriptional factors Nrf2, Hnf3β, and Ahr were significantly increased in oleanolic acid-treated rats. Conclusion: We demonstrated that the oral administration of oleanolic acid attenuates liver injury, inflammation, and cholestasis in BDL rats. The anti-cholestatic effect may be associated with the induction of hepatic detoxification enzymes and efflux transporters mediated by nuclear receptors and transcriptional factors.
PMCID: PMC4402745  PMID: 25932098
Oleanolic acid; obstructive cholestasis; synthetic enzyme; detoxification enzyme; hepatic efflux transporter; nuclear receptor; transcriptional factor
20.  Deregulated microRNAs in triple-negative breast cancer revealed by deep sequencing 
Molecular Cancer  2015;14:36.
MicroRNAs (miRNAs) are short, non-coding RNA molecules that play critical roles in human malignancy. However, the regulatory characteristics of miRNAs in triple-negative breast cancer, a phenotype of breast cancer that does not express the genes for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, are still poorly understood.
In this study, miRNA expression profiles of 24 triple-negative breast cancers and 14 adjacent normal tissues were analyzed using deep sequencing technology. Expression levels of miRNA reads were normalized with the quantile-quantile scaling method. Deregulated miRNAs in triple-negative breast cancer were identified from the sequencing data using the Student’s t-test. Quantitative reverse transcription PCR validations were carried out to examine miRNA expression levels. Potential target candidates of a miRNA were predicted using published target prediction algorithms. Luciferase reporter assay experiments were performed to verify a putative miRNA-target relationship. Validated molecular targets of the deregulated miRNAs were retrieved from curated databases and their associations with cancer progression were discussed.
A novel 25-miRNA expression signature was found to effectively distinguish triple-negative breast cancers from surrounding normal tissues in a hierarchical clustering analysis. We documented the evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNAs in triple-negative breast cancer. Two of these miRNA clusters (miR-143-145 at 5q32 and miR-497-195 at 17p13.1) were markedly down-regulated in triple-negative breast cancer, while the other five miRNA clusters (miR-17-92 at 13q31.3, miR-183-182 at 7q32.2, miR-200-429 at 1p36.33, miR-301b-130b at 22q11.21, and miR-532-502 at Xp11.23) were up-regulated in triple-negative breast cancer. Moreover, miR-130b-5p from the miR-301b-130b cluster was shown to directly repress the cyclin G2 (CCNG2) gene, a crucial cell cycle regulator, in triple-negative breast cancer cells. Luciferase reporter assays showed that miR-130b-5p-mediated repression of CCNG2 was dependent on the sequence of the 3′-untranslated region. The findings described in this study implicate a miR-130b-5p-CCNG2 axis that may be involved in the malignant progression of triple-negative breast cancer.
Our work delivers a clear picture of the global miRNA regulatory characteristics in triple-negative breast cancer and extends the current knowledge of microRNA regulatory network.
Electronic supplementary material
The online version of this article (doi:10.1186/s12943-015-0301-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4351690  PMID: 25888956
Triple-negative breast cancer; Deep sequencing; MicroRNA expression; MicroRNA cluster; miR-130b-5p; CCNG2
21.  Cloning of a caffeoyl-coenzyme A O-methyltransferase from Camellia sinensis and analysis of its catalytic activity*  
Epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) present in leaves of Camellia sinensis has many beneficial biological activities for human health. However, EGCG3"Me occurs naturally in tea leaves in extremely limited quantities. Finding an enzyme from C. sinensis to catalyze the synthesis of EGCG3"Me is an alternative method to make up for the scarcity of EGCG3"Me in natural situations. In the present study, a complementary DNA (cDNA) encoding region and genomic DNA of the caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) gene were isolated from C. sinensis (designated CsCCoAOMT). Nucleotide sequence analysis of CsCCoAOMT revealed an open reading frame of 738 bp that encodes a polypeptide with a predicted molecular weight of 28 kDa, which correlated well with the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The full-length DNA sequence (2678 bp) contained five exons and four introns. The deduced amino acid sequence of CsCCoAOMT shared 92% identity with CCoAOMTs from Codonopsis lanceolata and Betula luminifera. The catalytic activity of CsCCoAOMT was analyzed. Three monomethylated epigallocatechin-3-O-gallate (EGCG) compounds (EGCG4"Me, EGCG3"Me, and EGCG3'Me) were produced by CsCCoAOMT with K m in the micromolar range. Real-time polymerase chain reaction (RT-PCR) experiments indicated that the CsCCoAOMT transcript was present at low levels during the early stages of leaf maturity (the first leaf and bud on a shoot) but the relative expression was augmented at advanced stages of leaf maturity (the third or fourth leaf on a shoot), which accorded well with changes in EGCG3"Me content in fresh leaves. Hence, we concluded that CsCCoAOMT catalyzes the syntheses of methylated EGCGs.
PMCID: PMC4322421  PMID: 25644465
Tea (Camellia sinensis); O-methyltransferase; CsCCoAOMT; Prokaryotic expression; Catalytic activity; Methylated epigallocatechin-3-O-gallate (EGCG)
22.  Unraveling the kinetic diversity of microbial 3-dehydroquinate dehydratases of shikimate pathway 
AMB Express  2015;5:7.
3-Dehydroquinate dehydratase (DHQase) catalyzes the conversion of 3-dehydroquinic acid to 3-dehydroshikimic acid of the shikimate pathway. In this study, 3180 prokaryotic genomes were examined and 459 DHQase sequences were retrieved. Based on sequence analysis and their original hosts, 38 DHQase genes were selected for chemical synthesis. The selected DHQases were translated into new DNA sequences according to the genetic codon usage bias by both Escherichia coli and Corynebacterium glutamicum. The new DNA sequences were customized for synthetic biological applications by adding Biobrick adapters at both ends and by removal of any related restriction endonuclease sites. The customized DHQase genes were successfully expressed in E. coli, and functional DHQases were obtained. Kinetic parameters of Km, kcat, and Vmax of DHQases were determined with a newly established high-throughput method for DHQase activity assay. Results showed that DHQases possessed broad strength of substrate affinities and catalytic capacities. In addition to the DHQase kinetic diversities, this study generated a DHQase library with known catalytic constants that could be applied to design artificial modules of shikimate pathway for metabolic engineering and synthetic biology.
PMCID: PMC4314829  PMID: 25852984
3-Dehydroquinate dehydratase (DHQase); Kinetic constants; Shikimate pathway; Biobrick; Synthetic biology
23.  Receptor interacting protein 140 attenuates endoplasmic reticulum stress in neurons and protects against cell death 
Nature communications  2014;5:4487.
Inositol 1, 4, 5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) triggers many physiological responses in neurons and when uncontrolled can cause ER stress that contributes to neurological disease. Here we show that the unfolded protein response (UPR) in neurons induces rapid translocation of nuclear receptor-interacting protein 140 (RIP140) to the cytoplasm. In the cytoplasm, RIP140 localizes to the ER by binding to the IP3R. The carboxyl-terminal RD4 domain of RIP140 interacts with the carboxyl-terminal gate-keeping domain of the IP3R. This molecular interaction disrupts the IP3R's “head-tail” interaction, thereby suppressing channel opening and attenuating IP3R-mediated Ca2+ release. This contributes to a rapid suppression of the ER stress response and provides protection from apoptosis in both hippocampal neurons in vitro and in an animal model of ER stress. Thus, RIP140 translocation to the cytoplasm is an early response to ER stress and provides protection against neuronal death.
PMCID: PMC4200015  PMID: 25066731
IP3R; RIP140; ER stress; hippocampal neuron; calcium homeostasis
24.  Beta-Amyloid, Phospho-Tau and Alpha-Synuclein Deposits Similar to Those in the Brain Are Not Identified in the Eyes of Alzheimer’s and Parkinson’s Disease Patients 
Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders, and are characterized by deposition of specific proteins in the brain. If similar abnormal protein deposits are present in the eye, it would facilitate noninvasive diagnosis and monitoring of disease progression. We therefore evaluated expression of proteins associated with AD and PD pathology in postmortem eyes and brains in a case-control study. Eyes from 11 cases of AD, 6 cases of PD or PD with dementia, and 6 age-matched controls were retrieved from the autopsy archives of The Johns Hopkins Hospital. Immunostains for β-amyloid, phospho-tau and α-synuclein and Congo red stains were performed in the same laboratory in both brains and eyes. No amyloid deposits or abnormal tau accumulations were detected in the lens, retina or other structures in the eyes of AD patients. Eyes also lacked definite Lewy bodies or Lewy neurites in either PD or AD cases. Patchy cytoplasmic α-synuclein positivity was seen in the retina of AD, PD and control cases, but did not correlate with the presence or extent of Lewy body pathology in the brain. Abnormal protein aggregations characteristic of AD and PD are thus not commonly present in the retinas or lens of affected patients when assayed using the same protocols as in the brain. This suggests that β-amyloid, phospho-tau nd α-synuclein either do not deposit in the eye in a manner analogous to brain, or are present at lower levels or in different forms.
PMCID: PMC3976129  PMID: 23714377
amyloid; eye; lens; synuclein; tau
25.  Benzoate Metabolism Intermediate Benzoyl Coenzyme A Affects Gentisate Pathway Regulation in Comamonas testosteroni 
Applied and Environmental Microbiology  2014;80(13):4051-4062.
A previous study showed that benzoate was catabolized via a coenzyme A (CoA)-dependent epoxide pathway in Azoarcus evansii (R. Niemetz, U. Altenschmidt, S. Brucker, and G. Fuchs, Eur. J. Biochem. 227:161-168, 1995), but gentisate 1,2-dioxygenase was induced. Similarly, we found that the Comamonas testosteroni strain CNB-1 degraded benzoate via a CoA-dependent epoxide pathway and that gentisate 1,2-dioxygenase (GenA) was also induced when benzoate or 3-hydroxybenzoate served as a carbon source for growth. Genes encoding the CoA-dependent epoxide (box genes) and gentisate (gen genes) pathways were identified. Genetic disruption revealed that the gen genes were not involved in benzoate and 3-hydroxybenzoate degradation. Hence, we investigated gen gene regulation in the CNB-1 strain. The PgenA promoter, a MarR-type regulator (GenR), and the GenR binding site were identified. We found that GenR took gentisate, 3-hydroxybenzoate, and benzoyl-CoA as effectors and that binding of GenR to its target DNA sequence was prohibited when these effectors were present. In vivo studies showed that the CNB-1 mutant that lost benzoyl-CoA synthesis was not able to activate PgenA promoter, while transcription of genA was upregulated in another CNB-1 mutant that lost the ability to degrade benzoyl-CoA. The finding that benzoyl-CoA (a metabolic intermediate of benzoate degradation) and 3-hydroxybenzoate function as GenR effectors explains why GenA was induced when CNB-1 grew on benzoate or 3-hydroxybenzoate. Regulation of gentisate pathways by MarR-, LysR-, and IclR-type regulators in diverse bacterial groups is discussed in detail.
PMCID: PMC4054210  PMID: 24771026

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