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1.  Fatty Acid Oxidation Disorders in a Chinese Population in Taiwan 
JIMD Reports  2013;11:165-172.
Background: Fatty acid oxidation (FAO) disorders are a heterogeneous group of inborn errors in the transportation and oxidation of fatty acids. FAO disorders were thought to be very rare in the Chinese population. Newborn screening for FAO disorders beginning in 2002 in Taiwan may have increased the diagnosis of this group of diseases.
Materials and Methods: Till 2012, the National Taiwan University Hospital Newborn Screening Center screened more than 800,000 newborns for FAO disorders. Both patients diagnosed through screening and patients detected after clinical manifestations were included in this study.
Results: A total of 48 patients with FAO disorders were identified during the study period. The disorders included carnitine palmitoyltransferase I deficiency, carnitine acylcarnitine translocase deficiency, carnitine palmitoyltransferase II deficiency, very long-chain acyl-CoA dehydrogenase deficiency, medium-chain acyl-CoA dehydrogenase deficiency, multiple acyl-CoA dehydrogenase deficiency, short-chain defects, and carnitine uptake defect. Thirty-nine patients were diagnosed through newborn screening. Five false-negative newborn screening cases were noted during this period, and four patients who were not screened were diagnosed based on clinical manifestations. The ages of all patients ranged from 6 months to 22.9 years (mean age 6.6 years). Except for one case of postmortem diagnosis, there were no other mortalities.
Conclusions: The combined incidence of FAO disorders estimated by newborn screening in the Chinese population in Taiwan is 1 in 20,271 live births. Newborn screening also increases the awareness of FAO disorders and triggers clinical diagnoses of these diseases.
PMCID: PMC3755561  PMID: 23700290
2.  Liquiritigenin alleviates mechanical and cold hyperalgesia in a rat neuropathic pain model 
Scientific Reports  2014;4:5676.
This study assessed the potential antinociceptive effects of liquiritigenin, a plant-derived compound with transient receptor potential melastatin 3 blocking activity in a rat model of persistent neuropathic pain. Chronic constriction injury (CCI) to the sciatic nerve was induced in male Sprague-Dawley rats to model human peripheral neuropathic pain. Liquiritigenin (1, 3, or 9 mg/kg) was administered intraperitoneally to examine the effects on mechanical, thermal, and cold hyperalgesia using the von Frey test, plantar test, and cold plate test, respectively. A rotarod test was also conducted to examine motor function. Liquiritigenin dose dependently alleviated mechanical, thermal and cold hyperalgesia. In addition, daily repeated treatment with liquiritigenin did not demonstrate significant antinociceptive tolerance in the measures of hyperalgesia. Within the doses studied, liquiritigenin did not significantly affect motor performance. These results suggest that liquiritigenin may be potentially useful novel treatments for neuropathic pain.
PMCID: PMC4097342  PMID: 25022218
3.  Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry 
Dynamic and cellular histomorphometry of trabeculae is the most biologically relevant way of assessing steady state bone health. Traditional measurement involves manual visual feature identification by a trained and qualified professional. Inherent with this methodology is the time and cost expenditure, as well as the subjectivity that naturally arises under human visual inspection. In this work, we propose a rapidly deployable, automated, and objective method for dynamic histomorphometry. We demonstrate that our method is highly effective in assessing cellular activities in distal femur and vertebra of mice which are injected with calcein and alizarin complexone 7 and 2 days prior to sacrifice. The mineralized bone tissues of mice are cryosectioned using a tape transfer protocol. A sequential workflow is implemented in which endogenous fluorescent signals (bone mineral, green and red mineralization lines), tartrate resistant acid phosphatase identified by ELF-97 and alkaline phosphatase identified by Fast Red are captured as individual tiled images of the section for each fluorescent color. All the images are then submitted to an image analysis pipeline that automates identification of the mineralized regions of bone and selection of a region of interest. The TRAP and AP stained images are aligned to the mineralized image using strategically placed fluorescent registration beads. Fluorescent signals are identified and are related to the trabecular surface within the ROI. Subsequently, the pipelined method computes static measurements, dynamic measurements, and cellular activities of osteoclast and osteoblast related to the trabecular surface. Our method has been applied to the distal femurs and vertebrae of 8 and 16 week old male and female C57Bl/6J mice. The histomorphometric results reveal a significantly greater bone turnover rate in female in contrast to male irrespective of age, validating similar outcomes reported by other studies.
PMCID: PMC4090931  PMID: 25019033
Histomorphometry; Enzymatic stains; Cryohistology; Image processing; Image analysis
4.  The Genomic Landscape of the Ewing Sarcoma Family of Tumors Reveals Recurrent STAG2 Mutation 
PLoS Genetics  2014;10(7):e1004475.
The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.
Author Summary
The Ewing sarcoma family of tumors is a group of aggressive cancers that primarily affects the pediatric and young adult population. Increasingly, genomics are being used to better define the disease biology and to identify targets for therapy in many cancer types. Here, we report one of the first and largest genomic studies to date in the Ewing sarcoma family of tumors. Using a combination of modern sequencing techniques in >100 samples, we discover that Ewing sarcomas have a genome that is less complex compared to most cancer types previously surveyed. We find that this cancer is frequently affected by mutations in STAG2, a gene that has recently gained attention due to its importance in the biology of several cancer types. We show that Ewing sarcoma patients whose tumors are affected by STAG2 loss may have a worse prognosis. Additionally, we identify a subset of tumors that were diagnosed as Ewing sarcoma that appear to be distinct from the majority based on genetic and molecular characteristics. Our findings help to define the genetic landscape of Ewing sarcoma and provide a starting point for improving individualization of diagnosis, prognosis and treatment in this cancer.
PMCID: PMC4091782  PMID: 25010205
5.  Tumor characteristics of breast cancer in predicting axillary lymph node metastasis 
Tumor characteristics was sought to be related to axillary lymph node metastasis (ALNM), the paramount prognostic factor in patients with invasive breast cancer. This study was aimed to identify the ALNM-associated tumor characteristics and to determine the predictive clinical pathway.
Data from 1325 patients diagnosed with invasive breast cancer between January 2004 and January 2010 were retrospectively reviewed. The structure equation model (SEM) was used to build the predictive clinical pathway.
Among the factors found in the final model, the status of human epidermal growth factor receptor 2 is the primary influence on ALNM through histology grade (β=0.18), followed by tumor size (β=0.16). Tumor size was highly relevant to lymphovascular invasion (LVI) and influenced ALNM through LVI (β=0.26), the strongest predictor of ALNM in the final model (β=0.46) and the highest risk of ALNM (odds ratio=9.282; 95% confidence interval: 7.218–11.936).
The structure equation model presented the relation of these important predictors, and might help physicians to assess axillary nodal condition and appropriate surgical procedures.
PMCID: PMC4099209  PMID: 24998473
Breast Neoplasms; Lymph Nodes; Lymphatic Metastasis - diagnosis; Neoplasm Grading; Neoplasm Invasiveness; Receptor; erbB-2
6.  SUMO-2 Promotes mRNA Translation by Enhancing Interaction between eIF4E and eIF4G 
PLoS ONE  2014;9(6):e100457.
Small ubiquitin-like modifier (SUMO) proteins regulate many important eukaryotic cellular processes through reversible covalent conjugation to target proteins. In addition to its many well-known biological consequences, like subcellular translocation of protein, subnuclear structure formation, and modulation of transcriptional activity, we show here that SUMO-2 also plays a role in mRNA translation. SUMO-2 promoted formation of the active eukaryotic initiation factor 4F (eIF4F) complex by enhancing interaction between Eukaryotic Initiation Factor 4E (eIF4E) and Eukaryotic Initiation Factor 4G (eIF4G), and induced translation of a subset of proteins, such as cyclinD1 and c-myc, which essential for cell proliferation and apoptosis. As expected, overexpression of SUMO-2 can partially cancel out the disrupting effect of 4EGI-1, a small molecule inhibitor of eIF4E/eIF4G interaction, on formation of the eIF4F complex, translation of the cap-dependent protein, cell proliferation and apoptosis. On the other hand, SUMO-2 knockdown via shRNA partially impaired cap-dependent translation and cell proliferation and promoted apoptosis. These results collectively suggest that SUMO-2 conjugation plays a crucial regulatory role in protein synthesis. Thus, this report might contribute to the basic understanding of mammalian protein translation and sheds some new light on the role of SUMO in this process.
PMCID: PMC4074059  PMID: 24971752
7.  Analysis of 30 Patients with Acupuncture-Induced Primary Inoculation Tuberculosis 
PLoS ONE  2014;9(6):e100377.
Primary inoculation tuberculosis is a skin condition that develops at the site of inoculation of Mycobacterium tuberculosis in tuberculosis-free individuals. This report describes the diagnosis, treatment and >1 year follow-up of 30 patients presenting with acupuncture-induced primary inoculation tuberculosis. Our data provide a deeper insight into this rare route of infection of tuberculosis. We also review effective treatment options.
PMCID: PMC4069069  PMID: 24959854
8.  Genomic insights into the serine protease gene family and expression profile analysis in the planthopper, Nilaparvata lugens 
BMC Genomics  2014;15(1):507.
The brown planthopper (Nilaparvata lugens) is one of the most destructive rice plant pests in Asia. N. lugens causes extensive damage to rice by sucking rice phloem sap, which results in hopper burn (complete death of the rice plants). Despite its importance, little is known about the digestion, development and defense mechanisms of this hemimetabolous insect pest. In this study, we aim to identify the serine protease (SP) and serine protease homolog (SPH) genes, which form a large family in eukaryotes, due to the potential for multiple physiological roles. Having a fully sequenced genome for N. lugens allows us to perform in-depth analysis of the gene structures, reveal the evolutionary relationships and predict the physiological functions of SP genes.
The genome- and transcriptome-wide analysis identified 90 putative SP (65) and SPH (25) genes in N. lugens. Detailed gene information regarding the exon-intron organization, size, distribution and transcription orientation in the genome revealed that many SP/SPH loci are closely situated on the same scaffold, indicating the frequent occurrence of gene duplications in this large gene family. The gene expression profiles revealed new findings with regard to how SPs/SPHs respond to bacterial infections as well as their tissue-, development- and sex-specific expressions.
Our findings provide comprehensive gene sequence resources and expression profiles of the N. lugens SP and SPH genes, which give insights into clarifying the potentially functional roles of these genes in the biological processes including development, digestion, reproduction and immunity.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-507) contains supplementary material, which is available to authorized users.
PMCID: PMC4085338  PMID: 24952583
Nilaparvate lugens; Serine protease; Serine protease homolog; Genome; Transcriptome; Expression profile
9.  Favorable effects of alemtuzumab on allospecific regulatory T-cell generation 
Human immunology  2011;73(2):141-149.
We studied the effects of alemtuzumab on T-regulatory cells (Tregs) during alloactivation, first by differences in depletion of various naive versus alloactivated cell subsets in peripheral blood of healthy volunteers, then by adding serial concentrations to human leukocyte antigen (HLA)–DR–matched and –mismatched responding and stimulating cells in mixed lymphocyte reaction (MLR). Lymphoproliferation inhibition and the development of proliferating carboxyfluorescein succinimidyl ester (CFSE)–diluted CD4+CD25highCD127−FOXP3+ (phenotypic) Tregs by flow cytometry were measured. Also, the ability of alemtuzumab-treated versus nontreated MLR generated CD4+CD127− cells to allospecifically inhibit MLRs and recruit additional responding Tregs was tested. We found a more pronounced refractoriness of alloactivated versus naive CD4+CD25high cells to alemtuzumab induced lymphodepletion. Alemtuzumab dose dependently inhibited lymphoproliferation while amplifying percentages of MLR-generated Tregs. This was somewhat augmented by human complement addition. CD127−CD4+ cells immunoselected after 7 days from alemtuzumab-treated MLRs allospecifically inhibited lymphoproliferation and recruited additional Tregs in fresh MLR-responding cells, similar to modulators derived from MLRs without drug addition (media). Addition of tacrolimus and sirolimus to alemtuzumab further inhibited MLR proliferation. However, Treg percentages were markedly higher with sirolimus. These results support the notion that alemtuzumab induces immunoregulation in naïve T cells undergoing alloactivation absent presensitization, especially used in conjunction with maintenance SRL.
PMCID: PMC4064370  PMID: 22154724
Mixed lymphocyte reaction; Regulatory T cells; FOXP3; Alemtuzumab; mTOR inhibition
10.  The relationship of anatomical and functional connectivity to resting state connectivity in primate somatosensory cortex 
Neuron  2013;78(6):1116-1126.
Studies of resting state activity in the brain have provoked critical questions about the brain’s functional organization but, its biological basis is not clear. Specifically, the relationships between interregional correlations in resting state measures of activity, neuronal functional connectivity and anatomical connectivity are much debated. To investigate these relationships, we have examined both anatomical and steady state functional connectivity within the hand representation of primary somatosensory cortex (areas 3b and 1) in anesthetized squirrel monkeys. The comparison of three data sets (fMRI, electrophysiological, anatomical) indicate two primary axes of information flow within SI: prominent interdigit interactions within area 3b and predominantly homotopic interactions between area 3b and area 1. These data support a strikingly close relationship between baseline functional connectivity and anatomical connections. This study is also the first to extend findings derived from large-scale cortical networks to the realm of local mm-scale networks.
PMCID: PMC3723346  PMID: 23791200
11.  ID2 predicts poor prognosis in breast cancer, especially in triple-negative breast cancer, and inhibits E-cadherin expression 
OncoTargets and therapy  2014;7:1083-1094.
Inhibitors of DNA-binding (ID) proteins are known as important modulators in the regulation of cell proliferation and differentiation. This study sought to investigate the prognostic value of ID proteins in breast cancer.
The prognostic role of ID proteins in human breast cancer was investigated in 250 breast cancers, via tissue microarrays. The messenger (m)RNA and protein levels of E-cadherin were examined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting, in cells overexpressing IDs. Dual-luciferase report assay was used to investigate the potential mechanism, and a migration assay was performed to investigate the influence of IDs on cell migratory activity.
The survival analysis with Kaplan–Meier and Cox regression showed that ID2 expression level, which correlated with estrogen receptor status and E-cadherin abundance, served as an independent prognostic factor for disease-free survival (DFS) (P=0.013). The prognostic value of ID2 for DFS was most significant in triple-negative breast cancer patients (P=0.009). We also found that ID2 was negatively correlated with E-cadherin expression by correlation analysis (P=0.020, Pearson’s R=−0.155). Subsequently, we explored the biological rationale and uncovered that the enforced expression of ID proteins could suppress E-cadherin expression significantly, thus increasing the migration ability of mammary epithelial cells. Then using a combination of ID2 and E-cadherin expression, the patients were classified into four subgroups with different DFS (P=0.023).
The overexpression of ID2 can be used as a prognostic marker in breast cancer patients, especially in triple-negative breast cancer patients. ID proteins were still, unexpectedly, revealed to inhibit E-cadherin abundance.
PMCID: PMC4069128  PMID: 24971018
breast cancer; prognosis; biomarker
13.  Gene expression signatures differentiate uterine endometrial stromal sarcoma from leiomyosarcoma 
Gynecologic oncology  2012;128(2):349-355.
Endometrial stromal sarcoma (ESS) and leiomyosarcoma (LMS) are the two most common uterine sarcomas, but both are rare tumors. The aim of the present study was to compare the global gene expression patterns of ESS and LMS.
Gene expression profiles of 7 ESS and 13 LMS were analyzed using the HumanRef-8 BeadChip from Illumina. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry.
Unsupervised hierarchical clustering using all 54,675 genes in the array separated ESS from LMS samples. We identified 549 unique probes that were significantly differentially expressed in the two malignancies by greater than 2-fold with 1% FDR cutoff using one-way ANOVA with Benjamini–Hochberg correction, of which 336 and 213 were overexpressed in ESS and LMS, respectively. Genes overexpressed in ESS included SLC7A10, EFNB3, CCND2, ECEL1, ITM2A, NPW, PLAG1 and GCGR. Genes overexpressed in LMS included CDKN2A, FABP3, TAGLN, JPH2, GEM, NAV2 and RAB23. The top 100 genes overexpressed in LMS included those coding for myosin light chain and caldesmon, but not the genes coding for desmin or actin. CD10 was not overexpressed in ESS. Results for selected genes were validated by quantitative real-time PCR and immunohistochemistry.
We present the first study in which gene expression profiling was shown to distinguish between ESS and LMS. The molecular signatures unique to each of these malignancies may aid in expanding the diagnostic battery for their differentiation, and may provide a molecular basis for prognostic studies and therapeutic target discovery.
PMCID: PMC4051229  PMID: 23178314
Diagnosis; Gene expression array; Uterine sarcoma
14.  Epstein-Barr Virus (EBV) Infection in Chinese Children: A Retrospective Study of Age-Specific Prevalence 
PLoS ONE  2014;9(6):e99857.
Epstein-Barr Virus (EBV) is a globally prevalent herpesvirus associated with infectious mononucleosis and many malignancies. The survey on EBV prevalence appears to be important to study EBV-related diseases and determine when to administer prophylactic vaccine. The purpose of this retrospective study was to collect baseline information about the prevalence of EBV infection in Chinese children.
Methodology/Principal Finding
We collected 1778 serum samples from healthy children aged 0 to 10, who were enrolled in conventional health and nutrition examinations without any EBV-related symptom in 2012 and 2013 in North China (n = 973) and South China (n = 805). We detected four EBV-specific antibodies, i.e., anti-VCA-IgG and IgM, anti-EBNA-IgG and anti-EA-IgG, by ELISA, representing all of the phases of EBV infection. The overall EBV seroprevalence in samples from North and South China were 80.78% and 79.38% respectively. The EBV seropositivity rates dropped slightly at age 2, and then increased gradually with age. The seroprevalence became stabilized at over 90% after age 8. In this study, the seroprevalence trends between North and South China showed no difference (P>0.05), and the trends of average antibody concentrations were similar as well (P>0.05).
EBV seroprevalence became more than 50% before age 3 in Chinese children, and exceed 90% after age 8. This study can be helpful to study the relationship between EBV and EBV-associated diseases, and supportive to EBV vaccine development and implementation.
PMCID: PMC4051769  PMID: 24914816
15.  Biomechanical comparison of different combinations of hook and screw in one spine motion unit - an experiment in porcine model 
The biomechanical performance of the hooks and screws in spinal posterior instrumentation is not well-characterized. Screw-bone interface failure at the uppermost and lowermost vertebrae is not uncommon. Some have advocated for the use of supplement hooks to prevent screw loosening. However, studies describing methods for combined hook and screw systems that fully address the benefits of these systems are lacking. Thus, the choice of which implant to use in a given case is often based solely on a surgeon’s experience instead of on the biomechanical features and advantages of each device.
We conducted a biomechanical comparison of devices instrumented with different combinations of hooks and screws. Thirty-six fresh low thoracic porcine spines were assigned to three groups (12 per group) according to the configuration used for of fixation: (1) pedicle screw; (2) lamina hook and (3) combination of pedicle screw and lamina hook. Axial pullout tests backward on transverse plane in the direction normal to the rods were performed using a material testing machine and a specially designed grip with self-aligned function.
The pullout force for the pedicle screws group was significantly greater than for the hooks and the combination (p < 0.05). However, no significant difference was found between the hooks and the combination (p > 0.05).
Pedicle screws achieve the maximal pullout strength for spinal posterior instrumentation.
PMCID: PMC4062888  PMID: 24913189
Pedicle screw; Lamina hook; Biomechanical study; Porcine model
16.  Isolation and Sequence Analysis of Highly Pathogenic Porcine reproductive and respiratory syndrome virus from Swine Herds in the Jilin Province of China 
Indian Journal of Virology  2013;24(1):90-92.
The aim of the present study was to determine the causative agent of infected swines in the Jilin province of China and assess its genetic characteristics. Virus was isolated from tissues suspected of being infected by porcine reproductive and respiratory syndrome virus (PRRSV) and inoculated onto MARC-145 cells. Virus detection was carried out by RT-PCR, immunofluorescence, electron microscopy and sequencing. The results showed that the isolate was the North American genotype PRRSV, termed the JL-04/12 strain, with a 15,320 bp genome. The homology of the amino acid sequences in two nonstructural proteins and GP2 to GP5, between strains JL-04/12 and HUN4, ranged from 97.2 to 99.3 %. However, JL-04/12 GP6 and N protein were identical in HP-PRRSV JXA1 and HUN4. JL-04/12 was characterized by two discontinuous deletions in Nsp2. We speculate that the isolate is a variant of highly pathogenic porcine reproductive and respiratory syndrome derived from strains in 2006–2008. Altogether, these results indicate that highly pathogenic porcine reproductive and respiratory syndrome virus still exists in the Jilin province of China.
PMCID: PMC3650200  PMID: 24426266
Porcine reproductive and respiratory syndrome virus; High pathogenicity; Isolation; Sequencing analysis; Nonstructural protein 2; China
17.  In vivo biochemistry: Applications for small molecule biosensors in plant biology 
Current opinion in plant biology  2013;16(3):389-395.
Revolutionary new technologies, namely in the areas of DNA sequencing and molecular imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes, receptors and transporters in vitro or in heterologous systems, and more recently been able to analyze their regulation at the transcriptional level, use GFP reporters to obtain insights into cellular and subcellular localization, and measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on location and dynamics of the substrates of enzymes, receptors and transporters, and on the regulation of these proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. Genetically encoded fluorescent protein-based sensors for ion and metabolite dynamics provide highly resolved spatial and temporal information, and are complemented by sensors for pH, redox, voltage, and tension. They serve as powerful tools for identifying missing processes (e.g. glucose transport across ER membranes), components (e.g. SWEET sugar transporters for cellular sugar efflux), and signaling networks (e.g. from systematic screening of mutants that affect sugar transport or cytosolic and vacuolar pH). Combined with the knowledge of properties of enzymes and transporters and their interactions with the regulatory machinery, biosensors promise to be key diagnostic tools for systems and synthetic biology.
PMCID: PMC3679211  PMID: 23587939
metabolic signaling; metabolic pathways; sugar signaling; imaging
18.  Y chromosome AZFc microdeletion may not affect the outcomes of ICSI for infertile males with fresh ejaculated sperm 
To explore whether the presence of a Y chromosome AZFc microdeletion confers any adverse effect on the outcomes of intracytoplasmic sperm injection (ICSI) with fresh ejaculated sperm.
A total of 143 oligozoospermia patients with Y chromosome AZFc microdeletion in ICSI cycles in a five-year period were studied. Infertile men with normal Y chromosome in ICSI at the same time-frame were used as controls matched to the study group for age of female, female’s body mass index, male’s age, infertility duration and number of oocytes retrieved. Retrospective case–control study was used.
There were no significant differences between groups in clinical outcomes of endometrial thickness, transferred embryos, good embryo rates, implantation rates, biochemical pregnancy rates, clinical pregnancy rates, ectopic pregnancy rates, miscarriage rates, preterm birth rates, the ratio of male and female babies, newborn body height, newborn weight, low birth weight and birth defects (P > 0.05). Patients with Y chromosome AZFc microdeletion had a lower fertilization rate (61.8 % vs. 67.8 %, P < 0.05) and higher cleaved embryo rate (94.0 % vs. 88.1 %, P < 0.05).
ICSI clinical outcomes for oligozoospermic patients with Y chromosome AZFc microdeletion are basically comparable to that of infertile patients with normal Y chromosomes. The results of ICSI were not affected by the AZFc deletion. Preimplantation genetic diagnosis (PGD) before ICSI for Y chromosome AZFc microdeletion may not be a justifiable regular procedure if the couples didn’t care the vertical transmission of Y chromosome deletion.
PMCID: PMC3696459  PMID: 23715876
Y chromosome microdeletion; AZF; ICSI; Male infertility; Clinical outcome; PGD
19.  Comparison of Next-Generation Sequencing and Clone-Based Sequencing in Analysis of Hepatitis B Virus Reverse Transcriptase Quasispecies Heterogeneity 
Journal of Clinical Microbiology  2013;51(12):4087-4094.
We previously reported that, based on clone-based sequencing (CBS), hepatitis B virus (HBV) heterogeneity within the reverse transcriptase (RT) region was a predictor of antiviral efficacy. Here, by comparing ultradeep pyrosequencing (UDPS), i.e., next-generation sequencing (NGS), with CBS in characterizing the genetic heterogeneity of HBV quasispecies within the RT region, we evaluated the performance of UDPS in the analysis of HBV viral populations. HBV genomic DNA was extracted from serum samples from 31 antiviral treatment-naive patients with chronic hepatitis B. The RT region quasispecies were analyzed in parallel using CBS and UDPS. Characterization of quasispecies heterogeneity was conducted using bioinformatics analysis. Quasispecies complexity values were calculated with the formula Sn = −Σi(pilnpi)/lnN. The number of qualified strains obtained by UDPS was much larger than that obtained by CBS (P < 0.001). Pearson analysis showed that there was a positive correlation of quasispecies complexity values at the nucleotide level for the two methods (P < 0.05), while the complexity value derived from UDPS data was higher than that derived from CBS data (P < 0.001). Study of the prevalences of variations within the RT region showed that CBS detected an average of 9.7 ± 1.1 amino acid substitutions/sample and UDPS detected an average of 16.2 ± 1.4 amino acid substitutions/sample. The phylogenetic analysis based on UDPS data showed more genetic entities than did that based on CBS data. Viral heterogeneity determination by the UDPS technique is more sensitive and efficient in terms of low-abundance variation detection and quasispecies simulation than that by the CBS method, although imperfect, and thus sheds light on the future clinical application of NGS in HBV quasispecies studies.
PMCID: PMC3838070  PMID: 24088859
20.  Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes 
Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol—excessive consumption of which is a major cause of pancreatitis in the West—normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC. We show that CCN2 production by activated PSC is associated with enhanced expression of microRNA-21 (miR-21) which was detected at high levels in activated PSC in a murine model of alcoholic chronic pancreatitis. A positive feedback loop between CCN2 and miR-21 was identified that resulted in enhancement of their respective expression as well as that of collagen α1(I). Both miR-21 and CCN2 mRNA were present in PSC-derived exosomes, which were characterized as 50–150 nm CD9-positive nano-vesicles. Exosomes from CCN2-GFP- or miR-21-GFP-transfected PSC were taken up by other PSC cultures, as shown by direct fluorescence or qRT-PCR for GFP. Collectively these studies establish miR-21 and CCN2 as participants in a positive feedback loop during PSC activation and as components of the molecular payload in PSC-derived exosomes that can be delivered to other PSC. Thus interactions between cellular or exosomal miR-21 and CCN2 represent novel aspects of fibrogenic regulation in PSC. Summary Chronic injury in the pancreas is associated with fibrotic pathology which is driven in large part by CCN2-dependent collagen production in pancreatic stellate cells. This study shows that CCN2 up-regulation in PSC is associated with increased expression of miR-21 which, in turn, is able to stimulate CCN2 expression further via a positive feedback loop. Additionally miR-21 and CCN2 were identified in PSC-derived exosomes which effected their delivery to other PSC. The cellular and exosomal miR-21-CCN2 axis is a novel component in PSC fibrogenic signaling.
PMCID: PMC4063995  PMID: 24464300
Alcohol; Connective tissue growth factor; Fibrosis; miR; Microvesicle; Pancreas
21.  Coactivator Recruitment of AhR/ARNT1 
A common feature of nuclear receptors (NRs) is the transformation of external cell signals into specific transcriptions of the signal molecule. Signal molecules function as ligands for NRs and, after their uptake, activated NRs form homo- or heterodimers at promoter recognition sequences of the specific genes in the nucleus. Another common feature of NRs is their dependence on coactivators, which bridge the basic transcriptional machinery and other cofactors to the target genes, in order to initiate transcription and to unwind histone-bound DNA for exposing additional promoter recognition sites via their histone acetyltransferase (HAT) function. In this review, we focus on our recent findings related to the recruitment of steroid receptor coactivator 1 (SRC1/NCoA1) by the estrogen receptor-α (ERα) and by the arylhydrocarbon receptor/arylhydrocarbon receptor nuclear translocator 1 (AhR/ARNT1) complex. We also describe the extension of our previously published findings regarding the binding between ARNT1.1 exon16 and SRC1e exon 21, via in silico analyses of androgen receptor (AR) NH2-carboxyl-terminal interactions, the results of which were verified by in vitro experiments. Based on these data, we suggest a newly derived tentative binding site of nuclear coactivator 2/glucocorticoid receptor interacting protein-1/transcriptional intermediary factor 2 (NCOA-2/ GRIP-1/TIF-2) for ARNT1.1 exon 16. Furthermore, results obtained by immunoprecipitation have revealed a second leucine-rich binding site for hARNT1.1 exon 16 in SRC1e exon 21 (LSSTDLL). Finally, we discuss the role of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an endocrine disruptor for estrogen related transcription.
PMCID: PMC4100201  PMID: 24950180
22.  OsRACK1 Is Involved in Abscisic Acid- and H2O2-Mediated Signaling to Regulate Seed Germination in Rice (Oryza sativa, L.) 
PLoS ONE  2014;9(5):e97120.
The receptor for activated C kinase 1 (RACK1) is one member of the most important WD repeat–containing family of proteins found in all eukaryotes and is involved in multiple signaling pathways. However, compared with the progress in the area of mammalian RACK1, our understanding of the functions and molecular mechanisms of RACK1 in the regulation of plant growth and development is still in its infancy. In the present study, we investigated the roles of rice RACK1A gene (OsRACK1A) in controlling seed germination and its molecular mechanisms by generating a series of transgenic rice lines, of which OsRACK1A was either over-expressed or under-expressed. Our results showed that OsRACK1A positively regulated seed germination and negatively regulated the responses of seed germination to both exogenous ABA and H2O2. Inhibition of ABA biosynthesis had no enhancing effect on germination, whereas inhibition of ABA catabolism significantly suppressed germination. ABA inhibition on seed germination was almost fully recovered by exogenous H2O2 treatment. Quantitative analyses showed that endogenous ABA levels were significantly higher and H2O2 levels significantly lower in OsRACK1A-down regulated transgenic lines as compared with those in wildtype or OsRACK1A-up regulated lines. Quantitative real-time PCR analyses showed that the transcript levels of OsRbohs and amylase genes, RAmy1A and RAmy3D, were significantly lower in OsRACK1A-down regulated transgenic lines. It is concluded that OsRACK1A positively regulates seed germination by controlling endogenous levels of ABA and H2O2 and their interaction.
PMCID: PMC4035261  PMID: 24865690
23.  Botch is a γ-glutamyl Cyclotransferase that Deglycinates and Antagonizes Notch 
Cell reports  2014;7(3):681-688.
Botch promotes embryonic neurogenesis through inhibition of Notch-1 signaling through inhibition of the initial S1 furin-like cleavage step of Notch maturation. The biochemical process by which Botch inhibits Notch maturation is not known. Here we show that Botch has γ-glutamyl cyclotransferase (GGCT) activity that deglycinates Notch, which prevents the S1 furin-like cleavage. Moreover, Notch is mono-glycinated on the γ-glutamyl carbon of glutamate 1669. The deglycinase activity of Botch is required for inhibition of Notch signaling, both in vitro and in vivo. When the γ-glutamyl-glycine at position 1669 of Notch is degylcinated it is replaced by 5-oxy-proline. These results reveal that Botch regulates Notch signaling through deglycination and identify a novel posttranslational modification of Notch that plays an important role in neurogenesis.
PMCID: PMC4031649  PMID: 24767995
24.  Iqcg Is Essential for Sperm Flagellum Formation in Mice 
PLoS ONE  2014;9(5):e98053.
Mammalian spermatogenesis comprises three successive phases: mitosis phase, meiosis phase, and spermiogenesis. During spermiogenesis, round spermatid undergoes dramatic morphogenesis to give rise to mature spermatozoon, including the condensation and elongation of nucleus, development of acrosome, formation of flagellum, and removal of excessive cytoplasm. Although these transformations are well defined at the morphological level, the mechanisms underlying these intricate processes are largely unknown. Here, we report that Iqcg, which was previously characterized to be involved in a chromosome translocation of human leukemia, is highly expressed in the spermatogenesis of mice and localized to the manchette in developing spermatids. Iqcg knockout causes male infertility, due to severe defects of spermiogenesis and resultant total immobility of spermatozoa. The axoneme in the Iqcg knockout sperm flagellum is disorganized and hardly any typical (“9+2”) pattern of microtubule arrangement could be found in Iqcg knockout spermatids. Iqcg interacts with calmodulin in a calcium dependent manner in the testis, suggesting that Iqcg may play a role through calcium signaling. Furthermore, cilia structures in the trachea and oviduct, as well as histological appearances of other major tissues, remain unchanged in the Iqcg knockout mice, suggesting that Iqcg is specifically required for spermiogenesis in mammals. These results might also provide new insights into the genetic causes of human infertility.
PMCID: PMC4029791  PMID: 24849454
25.  Patient-Controlled Epidural Levobupivacaine with or without Fentanyl for Post-Cesarean Section Pain Relief 
BioMed Research International  2014;2014:965152.
Purpose. The purpose of this study was to compare the analgesic properties of levobupivacaine with or without fentanyl for patient-controlled epidural analgesia after Cesarean section in a randomized, double-blinded study. Methods. We enrolled American Society of Anesthesiologists class I/II, full-term pregnant women at National Taiwan University Hospital who received patient-controlled epidural analgesia after Cesarean section between 2009 and 2010. Eighty women were randomly assigned into two groups. In group A, the 40 subjects received drug solutions made of 0.6 mg/ml levobupivacaine plus 2 mcg/ml fentanyl, and in group B the 40 subjects received 1 mg/ml levobupivacaine. Maintenance was self-administered boluses and a continuous background infusion. Results. There were no significant differences in the resting and dynamic pain scales and total volume of drug used between the two groups. Patient satisfaction was good in both groups. Conclusion. Our study showed that pure epidural levobupivacaine can provide comparative analgesic properties to the levobupivacaine-fentanyl combination after Cesarean section. Pure levobupivacaine may serve as an alternative pain control regimen to avoid opioid-related adverse events in parturients.
PMCID: PMC4055260  PMID: 24982917

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