Objective: To investigate the inhibitory effect of plasmid-based survivin-specific short hairpin RNA and GRIM-19 on the growth of Hep-2 laryngeal cancer cells. Methods: The plasmid expressing survivin-specific short hairpin RNA (shRNA) and GRIM-19 (p-siRNA survivin/GRIM-19) was prepared and transfected into Hep-2 cells with Lipofectamine 2000. The mRNA and protein expression of surviving and GRIM-19 were measured with RT-PCR and western blot assay, respectively. MTT assay was employed to detect the proliferation of Hep-2 cells, and flow cytometry and AO/EB assay were done to determine the apoptosis of Hep-2 cells. Results: In the p-siRNA survivin/GRIM-19, the mRNA and protein expression of survivin was markedly reduced by 54.4% and 42.2%, and the reduction in protein expression of surviving was more obvious than that in the p-siRNA survivin group (37%) (P<0.05). The protein expression of GRIM-19 was markedly enhanced when compared with the control group (P<0.01). MTT assay revealed the proliferation of Hep-2 cells undergoing transfection with p-siRNA survivin/GRIM-19 was markedly inhibited, and the inhibition rate was as high as 79%, which was higher than that in the psi-survivin group (45%) and p-GRIM-19 group (35%). AO/EB assay and flow cytometry indicated that the apoptotic cells in the p-siRNA survivin/GRIM-19 group were dramatically increased as compared to the psi-survivin group and p-GRIM-19 group. Conclusion: The p-siRNA survivin/GRIM-19 has marked decrease in survivin expression and dramatic increase in GRIM-19 expression. Moreover, silencing of survivin and over-expression of GRIM-19 can significantly inhibit the growth and induce the apoptosis of Hep-2 in vitro and in vivo.
Co-expression plasmid; gene silencing; survivin; laryngeal cancer
The rapid discovery of novel viruses using next generation sequencing (NGS) technologies including DNA-Seq and RNA-Seq, has greatly expanded our understanding of viral diversity in recent years. The timely identification of novel viruses using NGS technologies is also important for us to control emerging infectious diseases caused by novel viruses. In this study, we identified a novel duck coronavirus (CoV), distinct with chicken infectious bronchitis virus (IBV), using RNA-Seq. The novel duck-specific CoV was a potential novel species within the genus Gammacoronavirus, as indicated by sequences of three regions in the viral 1b gene. We also performed a survey of CoVs in domestic fowls in China using reverse-transcription polymerase chain reaction (RT-PCR), targeting the viral nucleocapsid (N) gene. A total of 102 CoV positives were identified through the survey. Phylogenetic analysis of the viral N sequences suggested that CoVs in domestic fowls have diverged into several region-specific or host-specific clades or subclades in the world, and IBVs can infect ducks, geese and pigeons, although they mainly circulate in chickens. Moreover, this study provided novel data supporting the notion that some host-specific CoVs other than IBVs circulate in ducks, geese and pigeons, and indicated that the novel duck-specific CoV identified through RNA-Seq in this study is genetically closer to some CoVs circulating in wild water fowls. Taken together, this study shed new insight into the diversity, distribution, evolution and control of avian CoVs.
ZBRK1, a zinc finger protein that interacts with breast cancer 1 (BRCA1) and KRAB-ZFP-associated protein 1 (KAP1), has been suggested to serve as a tumor suppressor via repression of tumor metastasis/invasion. To date, the detailed molecular mechanisms for how BRCA1 and KAP1 participate in ZBRK1-mediated transcriptional repression, metastasis and invasion as well as the associated clinical relevance remain unclear. In this study, we demonstrated that both the N- and C-terminal domains of ZBRK1 are important for inhibiting cell proliferation and anchorage-independent growth in cervical cancer. Specifically, the N-terminal KRAB domain of ZBRK1 displayed a more crucial role in inhibiting metastasis and invasion through modulation of KAP1 function in a transcriptionally dependent manner. The loss of ZBRK1 results in an increase of KAP1 expression, which enhanced migration and invasion of cervical cancer cells both the in vitro and in vivo. Moreover, an inverse correlation of expression levels was observed between ZBRK1 and KAP1 following tumor progression from in situ carcinoma to invasive/metastatic cervical cancer specimens. Taken together, the current results indicate that a loss of ZBRK1 contributes to the increased expression of KAP1, potentiating its role to enhance metastasis and invasion.
Angiotensinogen is the precursor of angiotensin II, which is associated with ischemia-reperfusion injury. Angiotensin II reduces liver regeneration after hepatectomy and causes dysfunction and failure of reduced-size liver transplants. However, the regulation of angiotensinogen during liver regeneration is still unclear.
To investigate the regulation of angiotensinogen during liver regeneration for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.
A mouse in vitro partial hepatectomy animal model was used to evaluate the expression of interleukin-6 (IL-6) and angiotensinogen during liver regeneration. Serum IL-6 and angiotensinogen were detected by enzyme immunoassay (EIA). Angiotensinogen mRNA was detected by RT-PCR. Tissue levels of angiotensinogen protein were detected by Western blot analysis. Primary cultures of mouse hepatocytes were used to investigate IL-6-induced angiotensinogen. Chemical inhibitors were used to perturb signal transduction pathways. Synthetic double-stranded oligodeoxynucleotides (ODNs) were used as ‘decoy’ cis-elements to investigate transcription. Ki 67 staining and quantification were used to verify liver regeneration.
In the in vivo model, the levels of serum IL-6 and angiotensinogen correlated. In the in vitro model, IL-6 transcriptionally regulated angiotensinogen expression. Additionally, IL-6 mediated angiotensinogen expression through the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and JAK/p38 signaling. Decoy ODN analyses revealed that STAT3 and nuclear factor-kB (NF-kB) also played critical roles in the transcriptional regulation of angiotensinogen by IL-6. IL-6-mediated signaling, JAK2, STAT3 and p38 inhibitors reduced angiotensinogen expression in the partially hepatectomized mice.
During liver regeneration, IL-6-enhanced angiotensinogen expression is dependent on the JAK/STAT3 and JAK/p38/NF-kB signaling pathways. Interruption of the molecular mechanisms of angiotensinogen regulation may be applied as the basis of therapeutic strategies for preventing angiotensin II-related ischemia-reperfusion injury during liver regeneration.
Using a large, nationally representative longitudinal sample of Chinese aged 65 and older, this study examines the effects of childhood, adult, and community socioeconomic conditions on mortality and several major health outcomes. The role of social mobility is also tested. We find that childhood socioeconomic conditions exert long-term effects on functional limitations, cognitive impairment, self-rated health, and mortality independent of adult and community socioeconomic conditions. Achieved conditions matter for most outcomes as well, considering that adult and community socioeconomic conditions have additional impacts on health among Chinese elders. The majority of the effects of childhood conditions are not mediated by adult and community conditions. The results also show that social mobility and health in later life are linked in complex ways and that psychosocial factors have marginal explanatory power for the effects of socioeconomic conditions. Overall, this study provides new longitudinal evidence from China to support the notion that health and mortality at older ages are influenced by long-term and dynamic processes structured by the social stratification system. We discuss our findings in the context of the life course and ecological perspective, emphasizing that human development is influenced by a nexus of social experiences that impact individuals throughout life.
Socioeconomic status; Social mobility; Aging; Mortality; Self-rated health; Disability; China
To assess the prevalence of health care utilization in Mexico by Texas border residents and to identify the main contributing factors to their cross-border utilization of health care services.
Data and Methods
This study used primary data from a population-based telephone survey that was conducted in the whole Texas border area in 2008. The survey included responses from 1,405 adults. Multivariate logistic regression models were estimated to determine predictors of utilizing a wide range of health care services in Mexico.
Forty-nine percent of the sample reported having ever purchased medications in Mexico, followed by 41 percent for dentist visits, 37.3 percent for doctor visits, and 6.7 percent for inpatient care. The most significant predictors of health care utilization in Mexico were lack of U.S. health insurance coverage, dissatisfaction with the quality of U.S. health care, and poor self-rated health status.
The high prevalence of use of health care services in Mexico by Texas border residents is suggestive of unmet needs in health care on the U.S. side of the border. Addressing these unmet needs calls for a binational approach to improve the affordability, accessibility, and quality of health care in the U.S.–Mexico border region.
Health care utilization; uninsurance; U.S.–Mexico border; cross-border utilization
Patients with chronic kidney disease have abnormal energy expenditure and metabolism. The mechanisms underlying altered energy expenditure in uremia are unknown and remain to be elucidated. Irisin is a peroxisome proliferator-activated receptor γ coactivator 1-α–dependent myokine, and it increases energy expenditure in the absence of changes in food intake or activity. We hypothesize that chronic kidney disease patients have altered irisin levels. We measured resting irisin levels in 38 patients with stage 5 chronic kidney disease and in 19 age- and sex-matched normal subjects. Plasma irisin levels were significantly decreased in chronic kidney disease patients (58.59%; 95% CI 47.9%–69.2%, p<0.0001). The decrease in irisin levels was inversely correlated with the levels of blood urea nitrogen and creatinine. Further association analysis revealed that irisin level is independently associated with high-density lipoprotein cholesterol level. Our results suggest that chronic kidney disease patients have lower than normal irisin levels at rest. Furthermore, irisin may play a major role in affecting high-density lipoprotein cholesterol levels and abnormal energy expenditure in chronic kidney disease patients.
Objectives. To demonstrate the use of acupuncture in the lower limbs to treat myofascial pain of the upper trapezius muscles via a remote effect. Methods. Five adults with latent myofascial trigger points (MTrPs) of bilateral upper trapezius muscles received acupuncture at Weizhong (UB40) and Yanglingquan (GB34) points in the lower limbs. Modified acupuncture was applied at these points on a randomly selected ipsilateral lower limb (experimental side) versus sham needling on the contralateral lower limb (control side) in each subject. Each subject received two treatments within a one-week interval. To evaluate the remote effect of acupuncture, the range of motion (ROM) upon bending the contralateral side of the cervical spine was assessed before and after each treatment. Results. There was significant improvement in cervical ROM after the second treatment (P = 0.03) in the experimental group, and the increased ROM on the modified acupuncture side was greater compared to the sham needling side (P = 0.036). Conclusions. A remote effect of acupuncture was demonstrated in this pilot study. Using modified acupuncture needling at remote acupuncture points in the ipsilateral lower limb, our treatments released tightness due to latent MTrPs of the upper trapezius muscle.
AIM: To investigate the significance of Twist2 for colorectal cancer (CRC).
METHODS: In this study, 93 CRC patients were included who received curative surgery in Eastern Hepatobiliary Surgery Hospital from January 1999 to December 2010. Records of patients’ clinicopathological characteristics and follow up data were reviewed. Formalin-fixed, paraffin-embedded tissue blocks were used to observe the protein expression of Twist2 and E-cadherin by immunohistochemistry. Two independent pathologists who were blinded to the clinical information performed semiquantitative scoring of immunostaining. A total score of 3-6 (sum of extent + intensity) was considered as Twist2-positive expression. The expression of E-cadherin was divided into two levels (preserved and reduced). An exploratory statistical analysis was conducted to determine the association between Twist2 expression and clinicopathological parameters, as well as E-cadherin expression. Furthermore, the variables associated with prognosis were analyzed by Cox’s proportional hazards model. Kaplan-Meier analysis was used to plot survival curves according to different expression levels of Twist2.
RESULTS: Twist2-positive expression was observed in 66 (71.0%) samples and mainly located in the cytoplasm. Forty-three (46.2%) samples showed reduced expression of E-cadherin. There were no significant correlations between Twist2 expression and any of the clinicopathological parameters. However, Twist2-positive expression was significantly associated with reduced expression of E-cadherin (P = 0.040). Multivariate analysis revealed that bad M-stage [hazard ratio (HR) = 7.694, 95%CI: 2.927-20.224, P < 0.001] and Twist2-positive (HR = 5.744, 95%CI: 1.347-24.298, P = 0.018) were the independent risk factors for poor overall survival (OS), while Twist2-positive (HR = 3.264, 95%CI: 1.455-7.375, P = 0.004), bad N-stage (HR = 2.149, 95%CI: 1.226-3.767, P = 0.008) and bad M-stage (HR = 10.907, 95%CI: 4.937-24.096, P < 0.001) were independently associated with poor disease-free survival (DFS). Survival curves showed a definite trend for Twist2-negative patients to have longer OS and DFS than Twist2-negative patients, not only overall, but also for patients in different stages, especially for DFS of patients in stage III (P = 0.033) and IV (P = 0.026).
CONCLUSION: Our data suggests, for the first time, that Twist2 is a valuable prognostic biomarker for CRC, particularly for patients in stage III and IV.
Colorectal cancer; Prognostic biomarker; Twist2; Epithelial-mesenchymal transition; Immunohistochemstry
A central challenge in the fMRI based study of functional connectivity is distinguishing neuronally related signal fluctuations from the effects of motion, physiology, and other nuisance sources. Conventional techniques for removing nuisance effects include modeling of noise time courses based on external measurements followed by temporal filtering. These techniques have limited effectiveness. Previous studies have shown using multi-echo fMRI that neuronally related fluctuations are Blood Oxygen Level Dependent (BOLD) signals that can be characterized in terms of changes in R2* and initial signal intensity (S0) based on the analysis of echo-time (TE) dependence. We hypothesized that if TE-dependence could be used to differentiate BOLD and non-BOLD signals, non-BOLD signal could be removed to denoise data without conventional noise modeling. To test this hypothesis, whole brain multi-echo data were acquired at 3 TEs and decomposed with Independent Components Analysis (ICA) after spatially concatenating data across space and TE. Components were analyzed for the degree to which their signal changes fit models for R2* and S0 change, and summary scores were developed to characterize each component as BOLD-like or not BOLD-like. These scores clearly differentiated BOLD-like “functional network” components from non BOLD-like components related to motion, pulsatility, and other nuisance effects. Using non BOLD-like component time courses as noise regressors dramatically improved seed-based correlation mapping by reducing the effects of high and low frequency non-BOLD fluctuations. A comparison with seed-based correlation mapping using conventional noise regressors demonstrated the superiority of the proposed technique for both individual and group level seed-based connectivity analysis, especially in mapping subcortical-cortical connectivity. The differentiation of BOLD and non-BOLD components based on TE-dependence was highly robust, which allowed for the identification of BOLD-like components and the removal of non BOLD-like components to be implemented as a fully automated procedure.
fMRI; Resting state; Multi-echo; BOLD; TE dependence; ICA; Denoising; Subcortical; Connectivity
Differential diagnosis of high-grade dysplastic nodules (HGDN) and well-differentiated hepatocellular carcinoma (WDHCC) represents a challenge to experienced hepatic clinicians, radiologists and hepatopathologists.
The expression profiles of aminoacylase-1 (ACY1), sequestosome-1 (SQSTM1) and glypican-3 (GPC3) in low-grade dysplastic nodules (LGDN), HGDN and WDHCC were assessed by immunohistochemistry. The differential diagnostic performances of these three markers alone and in combination for HGDN and WDHCC were investigated by logistic regression models (HGDN = 21; WDHCC = 32) and validated in an independent test set (HGDN, n = 21; WDHCC n = 24). Postoperative overall survival and time to recurrence were evaluated by univariate and multivariate analyses in an independent set of 500 patients.
ACY1, SQSTM1 and GPC3 were differentially expressed in each group. For the differential diagnosis of WDHCC from HGDN, the sensitivity and specificity of the combination of ACY1 + SQSTM1 + GPC3 for detecting WDHCC were 93.8% and 95.2% respectively in the training set, which were higher than any of the three two-marker combinations. The validities of the four diagnostic models were further confirmed in an independent test set, and corresponding good sensitivity and specificity were observed. Interestingly, GPC3 expression in HCC tissues combined with serum α-fetoprotein (AFP) was found to be an independent predictor for overall survival and time to recurrence.
ACY1 + SQSTM1 + GPC3 combination represents a potentially valuable biomarker for distinguishing between WDHCC and HGDN using immunohistochemistry. Meanwhile, low GPC3 staining combined with positive serum AFP may play a practical role in predicting poor postoperative outcome and high tumor recurrence risk.
High grade dysplastic nodules; Well-differentiated hepatocellular carcinoma; Aminoacylase-1; Sequestosome-1; Glypican-3
CpG-ODN stimulates dendritic cells (DCs) to produce cytokines, which are important for pathogenesis of autoimmune disorders and vaccine strategy for cancer. CpG-ODN activates the TLR9/MyD88/TRAF6 cascade leading to activation of IKK-NF-κB and JNK, which are critical for production of pro-inflammatory cytokines. However, whether other molecules are involved in activation of CpG-ODN signaling is still not clear. Here we report that the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is involved in this activation process. DNA-PKcs-deficient DCs exhibited a defect in the IL-6 and IL-12 response to CpG-ODN in a dose- and time- dependent manner. Loss of DNA-PKcs impaired phosphorylation of IKK, IκBα, NF-κB and JNK in response to CpG-ODN. Interestingly, CpG-ODN was able to bind DNA-PKcs and induce its association and co-localization with TRAF6 in the absence of TLR9. Our data suggest that DNA-PKcs is a player in CpG-ODN signaling and may explain why DNA-PKcs is implicated in the pathogenic process of autoimmune disease.
This work aims to validate the clinical significance of coronary artery calcium score (CACS) in predicting coronary artery disease
(CAD) and cardiac events in 100 symptomatic patients (aged 37–87 years, mean 62.5, 81 males) that were followed up for a mean of 5 years.
Our results showed that patients with CAD and cardiac events had significantly higher CACS than those without CAD and cardiac events, respectively.
The corresponding data were 1450.42 ± 3471.24 versus 130 ± 188.29 (P < 0.001) for CAD, and 1558.67 ± 513.29 versus 400.46 ± 104.47 (P = 0.031)
for cardiac events. Of 72 patients with CAD, cardiac events were found in 56 (77.7%) patients.
The prevalence of cardiac events in our cohort was 13.3% for calcium score 0,
50% for score 11–100, 56% for score 101–400, 68.7% for score 401–1,000, and 75.0% for score >1000.
Increased CACS (>100) was also associated with an increased frequency of multi-vessel disease.
Nonetheless, 3 (20%) out of 15 patients with zero CACS had single-vessel disease.
Significant correlation (P < 0.001) was observed between CACS and CAD on a vessel-based analysis for coronary arteries.
It is concluded that CACS is significantly correlated with CAD and cardiac events.
We report here the complete genome sequence of a nonpathogenic and hemagglutination-negative avian paramyxovirus type 4 isolated from a duck in southern China. Phylogenetic analysis of the genome sequence indicated that the waterfowl virus possibly has evolved into the Eastern and Western Hemisphere lineages.
Describe self-reported and measured height, weight, and body mass index (BMI) stratified by sex and ethnicity in the United States, explore ethnic variations in the likelihood of under-reporting BMI, and investigate pathways linking race/ethnicity to the underassessment of BMI.
An observational study.
The entire United States.
Patients or Participants
Data were from the 2007–2008 National Health and Nutrition Examination Survey, a nationally representative sample of non-institutionalized civilian Americans.
Objectively measured and subjectively reported BMI.
Independent variables include race/ethnicity (non-Hispanic Whites, non-Hispanic Blacks, Hispanics, and others), sex, age groups (age 20–29, 30–49, 50–69, and ≥70), marital status (currently married vs other marital categories), education (less than high school, high school graduate or equivalent, some college, college graduate or above), and poverty income ratio (PIR).
This study confirmed that the use of reported BMI led to underestimates of the population prevalence of overweight and obesity due to the general tendency towards over-reporting height and under-reporting weight. Women were more likely than men to under-report BMI. And Whites were more likely than Blacks and Hispanics to under-report BMI. Other factors positively associated with higher likelihood of under-reporting of BMI included overweight and obese weight status, aged ≥60 years, and college education. Among women, family income was an additional positive covariate.
The results from this study underscore the need for frequently monitoring ethnic differences in validity of reported BMI and highlight the care which needs to be taken in making comparisons across sociodemographic groups based on reported BMI.
BMI; Validity; Self-reported; Measurement; Sex; Ethnicity
Adequate migration of Schwann cells (Sc) is crucial for axon-guidance in the regenerative process after peripheral nerve injury (PNI). Considering neuregulin-erbB-FAK signaling is an essential pathway participating in the regulation of Sc migration during development, the present study is aimed to examine whether neuregulin would exert its beneficial effects on adult following PNI and further determine the potential changes of downstream pathway engaged in neuro-regeneration by both in vitro and in vivo approaches.
Methodology and Principal Findings
Cultured RSC96 cells treated with neuregulin were processed for erbB2/3 immunofluorescence and FAK immunoblotings. The potential effects of neuregulin on Sc were assessed by cell adherence, spreading, and migration assays. In order to evaluate the functional significance of neuregulin on neuro-regeneration, the in vivo model of PNI was performed by chronic end-to-side neurorrhaphy (ESN). In vitro studies indicated that after neuregulin incubation, erbB2/3 were not only expressed in cell membranes, but also distributed throughout the cytoplasm and nucleus of RSC96 cells. Activation of erbB2/3 was positively correlated with FAK phosphorylation. Neuregulin also increases Sc adherence, spreading, and migration by 127.2±5.0%, 336.8±3.0%, and 80.0±5.7%, respectively. As for in vivo study, neuregulin significantly accelerates the speed of Sc migration and increases Sc expression in the distal stump of injured nerves. Retrograde labeling and compound muscle action potential recordings (CMAP) also showed that neuregulin successfully facilitates nerve regeneration by eliciting noticeably larger CMAP and promoting quick re-innervation of target muscles.
As neuregulin successfully improves axo-glial interaction by speeding Sc migration via the erbB2/3-FAK pathway, therapeutic use of neuregulin may thus serve as a promising strategy to facilitate the progress of nerve regeneration after PNI.
Porcine cytomegalovirus (PCMV) induces silent infection in adult pigs but more frequently causes fatal, generalized infection in newborn piglets. This study aimed to develop a new loop-mediated isothermal amplification (LAMP) method for the sensitive, rapid, and inexpensive detection of PCMV under field conditions.
Tissue obtained from nine-week-old PCMV-free Landrace pigs or pig samples from postmortem examinations were analyzed. The samples were found to have clinical signs and lesions consistent with inclusion body rhinitis. Six specific primers were designed by targeting the PCMV DNA polymerase (DPOL) DNA. The LAMP reaction was optimized in a water bath. The sensitivity and specificity of LAMP and polymerase chain reaction (PCR) were compared.
PCMV DNA was amplified at 65°C, and the result could be detected as early as 30 min into the reaction. Positive reactions could be visualized by the naked eye as a color change brought on by the addition of SYBR Green. The sensitivity and specificity of LAMP were found to be similar to those of the PCR.
LAMP is a high-throughput technique for the detection of PCMV and has a high specificity, sensitivity and simplicity; these factors make it suitable for detection of PCMV under field conditions.
Porcine cytomegalovirus; Loop-mediated isothermal amplification; Landrace pigs; Field detection
The prevalence rate of obesity in the United States has been persistently high in recent decades, and disparities in obesity risks are routinely observed. Both individual and contextual factors should be considered when addressing health disparities. This study examines how Latino-white spatial segregation is associated with the risk of obesity for Latinos and whites, whether neighborhood socioeconomic resources, the built environment, and subcultural orientation serve as the underlying mechanisms, and whether neighborhood context helps explain obesity disparities across ethnic and immigrant groups. The study was based on an extensive database containing self-reported BMI measures obtained from driver license records in Utah merged with census data and several GIS-based data. Multilevel analyses were performed to examine the research questions. For both men and women, Latino residential isolation is significantly and positively linked to the risk of obesity; after controlling for immigrant concentration, this effect gets amplified. Moreover, for men and women, the segregation effect is partly attributable to neighborhood SES and the built environment; and only for women is it partly attributable to obesity prevalence in the neighborhood. Place matters for individual risk of obesity for both men and women and there are multifarious pathways linking residence to obesity. Among the demographic, socioeconomic, physical, and cultural aspects of neighborhood context examined in this study, perhaps the most modifiable environment features that could prevent weight gain and its associated problems would be the built environmental factors such as greenness, park access, and mixed land use.
Obesity; Residential segregation; Immigrant enclave; Built environment
Single-shot EPI is the most common acquisition technique for whole-brain diffusion tensor imaging (DTI) studies in vivo. Higher field MRI systems are readily available and advantageous for acquiring DTI due to increased signal. One of the practical issues for DTI with single-shot EPI at high field is incomplete fat suppression resulting in a chemically-shifted fat artifact within the brain image. Unsuppressed fat is especially detrimental in DTI because the diffusion coefficient of fat is two orders of magnitude lower than that of parenchyma, producing brighter appearing fat artifacts with greater diffusion weighting. In this work, several fat suppression techniques were tested alone and in combination with the goal of finding a method that provides robust fat suppression and can be utilized in high-resolution single-shot EPI DTI studies. Combination of chemical shift saturation with slice-select gradient reversal within a dual-spin-echo diffusion preparation period was found to provide robust fat suppression at 3T.
The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaCCsp) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaCH16β, which was PhaCH16 bearing 30 point mutations derived from the middle region of PhaCCsp, accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaCH16β and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaCH16β (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaCH16 (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaCH16β (11.2 h) was 127-fold longer than that of PhaCH16 (5.3 min). Furthermore, the chimera PhaCH16β accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaCH16 (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaCH16 activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.
To investigate the effects of specific immunotherapy on the NKT cells in peripheral blood and the ability of NKT cells to proliferate in response to alpha-galactosylceramide (alpha-GalCer) in house-dust-mite- (HDM-) sensitized asthma children, peripheral blood mononuclear cells were isolated from 42 asthmatic children, of whom 24 were on specific immunotherapy (SIT) for more than a year and 20 were healthy. Compared with control group, the ratio of peripheral blood NKT and CD4+NKT cells was significantly decreased (P < 0.01) and was elevated in SIT asthma group (P < 0.05), respectively, but it was still less than the normal control group (P < 0.01). The level of IL-4 in serum secreted by NKT cells in asthma group was significantly higher than that of control group (P < 0.01), particularly apparent after 72 hours. The level of IL-4 in SIT group decreased significantly (P < 0.01). The level of IL-10 in serum secreted by NKT cells in asthma group was decreased significantly than that of the control group (P < 0.01) especially in 48 hours, while that of SIT group was increased significantly (P < 0.01). These results suggest that the pathogenesis of asthma may be related to the ratio and dysfunction of NKT and CD4+NKT cells.
Seedlessness is an important agronomic trait for citrus, and male sterility (MS) is one main cause of seedless citrus fruit. However, the molecular mechanism of citrus seedlessness remained not well explored.
An integrative strategy combining suppression subtractive hybridization (SSH) library with cDNA microarray was employed to study the underlying mechanism of seedlessness of a Ponkan mandarin seedless mutant (Citrus reticulata Blanco). Screening with custom microarray, a total of 279 differentially expressed clones were identified, and 133 unigenes (43 contigs and 90 singletons) were obtained after sequencing. Gene Ontology (GO) distribution based on biological process suggested that the majority of differential genes are involved in metabolic process and respond to stimulus and regulation of biology process; based on molecular function they function as DNA/RNA binding or have catalytic activity and oxidoreductase activity. A gene encoding male sterility-like protein was highly up-regulated in the seedless mutant compared with the wild type, while several transcription factors (TFs) such as AP2/EREBP, MYB, WRKY, NAC and C2C2-GATA zinc-finger domain TFs were down-regulated.
Our research highlighted some candidate pathways that participated in the citrus male gametophyte development and could be beneficial for seedless citrus breeding in the future.
Citrus; cDNA microarray; Differential transcript; Male sterility-like protein; Seedlessness
Hyperuricemia is an independent risk factor of nephropathy, but its role in renal transplant recipients (RTRs) is controversial.
Based on the methods of Cochrane systematic reviews, we searched MEDLINE (1948–2011.6), EMBASE (1956–2011.6), CBM (Chinese Biomedicine Database) (1978–2011.6) to identify cohort studies assessing the association between uric acid level and kidney allograft. Two authors independently screened the studies, assessed the risk of bias of included studies and extracted data. Unadjusted odds ratio(OR), mean difference (MD), adjusted hazard ratio (aHR) and their corresponding 95%CI were pooled to assess the effects of hyperuricemia on kidney allograft.
Twelve cohort studies were included and the quality was moderate to high based on the NEWCASTLE-OTTAWA quality assessment scale. RTRs with hyperuricemia had lower eGFR (P<0.0001, 95%CI−16.34∼6.14) and higher SCr (P<0.00001, 95%CI 0.17∼0.31) than those with normal uric acid level. Meta-analysis showed that hyperuricemia was a risk factor of chronic allograft nephropathy (Unadjusted OR = 2.85, 95%CI 1.84∼4.38, adjusted HR = 1.65, 95%CI 1.02∼2.65) and graft loss (Unadjusted OR = 2.29, 95%CI 1.55∼3.39; adjusted HR = 2.01, 95%CI 1.39∼2.94).
Current evidence suggests that hyperuricemia may be an independent risk factor of allograft dysfunction. Hyperuricemia may modestly increase the risk of poor outcomes of RTRs. Future research is needed to verify whether lowering uric acid level could improve the kidney function and prognosis of RTRs with hyperuricemia.
MicroRNAs (miRNAs) are small (~19-24nt) non-coding RNAs that play important roles in various biological processes. To date, the next-generation sequencing (NGS) technology has been widely used to discover miRNAs in plants and animals. Although evolutionary analysis is important to reveal the functional dynamics of miRNAs, few computational tools have been developed to analyze the evolution of miRNA sequence and expression across species, especially the newly emerged ones,
We developed miREvo, an integrated software platform with a graphical user interface (GUI), to process deep-sequencing data of small RNAs and to analyze miRNA sequence and expression evolution based on the multiple-species whole genome alignments (WGAs). Three major features are provided by miREvo: (i) to identify novel miRNAs in both plants and animals, based on a modified miRDeep algorithm, (ii) to detect miRNA homologs and measure their pairwise evolutionary distances among multiple species based on a WGA, and (iii) to profile miRNA expression abundances and analyze expression divergence across multiple species (small RNA libraries). Moreover, we demonstrated the utility of miREvo with Illumina data sets from Drosophila melanogaster and Arabidopsis, respectively.
This work presents an integrated pipline, miREvo, for exploring the expressional and evolutionary dynamics of miRNAs across multiple species. MiREvo is standalone, modular, and freely available at http://evolution.sysu.edu.cn/software/mirevo.htm under the GNU/GPL license.
Clopidogrel is a prodrug that undergoes in vivo bioactivation to show its antiplatelet effects. Recent studies have shown that cytochrome P450 (CYP), ATP-binding cassette transporters (ABCB1), and paraoxonase-1 (PON1) play crucial roles in clopidogrel bioactivation. Here, we aim to determine the effects of genetic polymorphisms of CYP (CYP 2C19*2, CYP 2C19*3, and CYP 2C19*17), ABCB1 (ABCB1 3435C>T, ABCB1 129T>C, and ABCB1 2677G>T/A), and PON1 (PON1 Q192R, PON1 L55M, and PON1 108C>T) on the development of stent thrombosis (ST) in patients receiving clopidogrel after percutaneous coronary intervention (PCI).
Methods and Results
We evaluated the incidence of ST (0.64%) in 4964 patients who were recruited in the CAPTAIN registry (Cardiovascular Atherosclerosis and Percutaneous TrAnsluminal INterventions). The presence of genetic polymorphisms was assessed in 20 subjects who developed ST after aspirin and clopidogrel therapy and in 40 age- and sex-matched control subjects who did not develop ST, which was documented after 9 months of angiographic follow-up. ST was acute in 5 subjects, subacute in 7, late in 7, and very late in 1. The presence of CYP 2C19*2 allele was significantly associated with ST (adjusted odds ratio [ORadj]: 4.20, 95% confidence interval [CI], 1.263–9.544; P = 0.031). However, genetic variations in PON1 and ABCB1 showed no significant association with ST.
We conclude that in a Taiwanese population, PON1 Q192R genotype is not associated with ST development after PCI. However, the presence of CYP 2C19*2 allele is a risk factor for ST development after PCI.