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1.  Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery 
Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.
PMCID: PMC3831115  PMID: 24250247
chronic kidney disease; exosome; miRNA; urine; biomarker.
2.  Silencing the HaAK Gene by Transgenic Plant-Mediated RNAi Impairs Larval Growth of Helicoverpa armigera 
Insect pests have caused noticeable economic losses in agriculture, and the heavy use of insecticide to control pests not only brings the threats of insecticide resistance but also causes the great pollution to foods and the environment. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been is currently developed for protection against insect pests. In this study, we used this technology to silence the arginine kinase (AK) gene of Helicoverpa armigera (HaAK), encoding a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrate. Transgenic Arabidopsis plants producing HaAK dsRNA were generated by Agrobacterium-mediated transformation. The maximal mortality rate of 55% was reached when H. armigera first-instar larvae were fed with transgenic plant leaves for 3 days, which was dramatically higher than the 18% mortality recorded in the control group. Moreover, the ingestion of transgenic plants significantly retarded larval growth, and the transcript levels of HaAK were also knocked down by up to 52%. The feeding bioassays further indicated that the inhibition efficiency was correlated with the integrity and concentration of the produced HaAK dsRNA in transgenic plants. These results strongly show that the resistance to H. armigera was improved in transgenic Arabidopsis plants, suggesting that the RNAi targeting of AK has the potential for the control of insect pests.
PMCID: PMC4278256  PMID: 25552931
Arginine kinase; Helicoverpa armigera; RNAi; transgenic plants; double-stranded RNA; pest control.
3.  Adipose-Derived Stromal Cells Protect Intervertebral Disc Cells in Compression: Implications for Stem Cell Regenerative Disc Therapy 
Introduction: Abnormal biomechanics plays a role in intervertebral disc degeneration. Adipose-derived stromal cells (ADSCs) have been implicated in disc integrity; however, their role in the setting of mechanical stimuli upon the disc's nucleus pulposus (NP) remains unknown. As such, the present study aimed to evaluate the influence of ADSCs upon NP cells in compressive load culture.
Methods: Human NP cells were cultured in compressive load at 3.0MPa for 48 hours with or without ADSCs co-culture (the ratio was 50:50). We used flow cytometry, live/dead staining and scanning electron microscopy (SEM) to evaluate cell death, and determined the expression of specific apoptotic pathways by characterizing the expression of activated caspases-3, -8 and -9. We further used real-time (RT-) PCR and immunostaining to determine the expression of the extracellular matrix (ECM), mediators of matrix degradation (e.g. MMPs, TIMPs and ADAMTSs), pro-inflammatory factors and NP cell phenotype markers.
Results: ADSCs inhibited human NP cell apoptosis via suppression of activated caspase-9 and caspase-3. Furthermore, ADSCs protected NP cells from the degradative effects of compressive load by significantly up-regulating the expression of ECM genes (SOX9, COL2A1 and ACAN), tissue inhibitors of metalloproteinases (TIMPs) genes (TIMP-1 and TIMP-2) and cytokeratin 8 (CK8) protein expression. Alternatively, ADSCs showed protective effect by inhibiting compressive load mediated increase of matrix metalloproteinases (MMPs; MMP-3 and MMP-13), disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs; ADAMTS-1 and 5), and pro-inflammatory factors (IL-1beta, IL-6, TGF-beta1 and TNF-alpha).
Conclusions: Our study is the first in vitro study assessing the impact of ADSCs on NP cells in an un-physiological mechanical stimulation culture environment. Our study noted that ADSCs protect compressive load induced NP cell death and degradation by inhibition of activated caspase-9 and -3 activity; regulating ECM and modulator genes, suppressing pro-inflammatory factors and preserving CK8. Consequently, the protective impact of ADSCs found in this study provides an essential understanding and expands our knowledge as to the utility of ADSCs therapy for intervertebral disc regeneration.
PMCID: PMC4279089  PMID: 25561896
intervertebral disc; adipose-derived stromal cells; nucleus pulposus; compressive load.
4.  Peripheral Blood Mononuclear Cell Traffic Plays a Crucial Role in Mother-to-Infant Transmission of Hepatitis B Virus 
The role of peripheral blood mononuclear cells (PBMCs) in HBV intrauterine infection is not fully defined. Particularly the origin of PBMCs in HBV-infected neonates remains to be addressed. We carried out a population-based nested case-control study by enrolling 312 HBsAg-positive mothers and their babies. PBMC HBV DNA as well as serum HBsAg and HBV DNA was tested in cohort entry samples. Totally, 45.5% (142/312) of the newborns were found to be infected with HBV in perinatal transmission. 119 mother-infant pairs were identified to be different in the genetic profile of maternal and fetal PBMCs by AS-PCR and hemi-nested PCR. Among them, 57.1% (68/119) of the maternal PBMCs in index cases were positive for HBV DNA while 83.8% (57/68) of the HBV DNA positive maternal PBMCs passed the placental barrier and entered the fetus. Furthermore, maternal PBMC HBV infection was significantly associated with newborn infants HBV infection. PBMC traffic from mother to fetus resulted in a 9.5-fold increased risk of HBV infection in PBMC HBV DNA positive newborn infants. These data indicate that maternal PBMCs infected with HBV contribute to HBV intrauterine infection of newborn infants via PBMC traffic from mother to fetus.
PMCID: PMC4323366
Hepatitis B virus; mother-to-infant transmission; peripheral blood mononuclear cell; fetomaternal cellular traffic.
5.  Common Chromosomal Fragile Site Gene WWOX in Metabolic Disorders and Tumors 
WWOX, a gene that spans the second most common chromosomal fragile site (FRA16D), often exhibits homozygous deletions and translocation breakpoints under multiple cellular stresses induced by extrinsic or intrinsic factors, such as hypoxia, UV, and DNA damage regents. Loss of WWOX is closely related to genomic instability, tumorigenesis, cancer progression and therapy resistance. WWOX heterozygous knockout mice show an increased incidence of spontaneous or induced tumors. WWOX can interact via the WW domain with proteins that possess proline PPxY motifs and is involved in a variety of cellular processes. Accumulating evidence has shown that WWOX that contains a short-chain dehydrogenase/reductase (SDR) domain is involved in steroid metabolism and bone development. Reduced or lost expression of WWOX will lead to development of metabolic disease. In this review, we focus on the roles of WWOX in metabolic disorders and tumors.
PMCID: PMC3920169  PMID: 24520212
WW domain-containing oxidoreductase; metabolic disorders; tumorigenesis; endocrine and chemotherapy.
6.  Establishment of an Inflamed Animal Model of Diabetic Nephropathy 
Aims Inflammatory stress plays a crucial role in the progression of diabetic nephropathy (DN). This study aimed to establish a novel inflamed animal model of DN and to evaluate its significance in DN.
Methods Nondiabetic db/m mice and diabetic db/db mice were randomly divided into four groups: db/m, db/m+casein, db/db, and db/db+casein for eight weeks. Casein was subcutaneously injected to induce chronic inflammation. Body weight and albumin to creatinine ratio (ACR) in the urine were measured every week. The plasma levels of serum amyloid protein A (SAA) and tumour necrotic factor-α (TNF-α) were determined with the enzyme-linked immunosorbent assay. The morphological changes to the renal pathology and ultra-microstructures were checked by pathological staining and electron microscopy. Immunofluorescent staining and Western blotting were used to determine the protein expression of podocyte-specific molecules and inflammatory cytokines in kidneys.
Results ACR, plasma levels of SAA and TNF-α, protein expression of inflammatory cytokines, mesangial expansion, collagen accumulation, and foot process effacement in kidneys of casein-injected db/db mice were significantly increased compared with the db/db mice. Casein injection markedly decreased the protein expression of Wilms' tumor-1 and nephrin in kidneys of db/db mice, which are specific podocyte biomarkers, suggesting that chronic inflammation accelerates podocyte injuries in db/db mice. Interestingly, no obvious urinary protein, inflammatory cytokine expression, or histological changes in the kidneys of casein-injected db/m mice were found compared with the db/m mice.
Conclusion An inflamed animal model of DN was successfully established and may provide a useful tool for investigating the pathogenesis of DN under inflammatory stress.
PMCID: PMC3920170  PMID: 24520213
Diabetic nephropathy; inflammation; db/db mice; animal model
7.  DLG5 in Cell Polarity Maintenance and Cancer Development 
Failure in establishment and maintenance of epithelial cell polarity contributes to tumorigenesis. Loss of expression and function of cell polarity proteins is directly related to epithelial cell polarity maintenance. The polarity protein discs large homolog 5 (DLG5) belongs to a family of molecular scaffolding proteins called Membrane Associated Guanylate Kinases (MAGUKs). As the other family members, DLG5 contains the multi-PDZ, SH3 and GUK domains. DLG5 has evolved in the same manner as DLG1 and ZO1, two well-studied MAGUKs proteins. Just like DLG1 and ZO1, DLG5 plays a role in cell migration, cell adhesion, precursor cell division, cell proliferation, epithelial cell polarity maintenance, and transmission of extracellular signals to the membrane and cytoskeleton. Since the roles of DLG5 in inflammatory bowel disease (IBD) and Crohn's disease (CD) have been reviewed, here, our review focuses on the roles of DLG5 in epithelial cell polarity maintenance and cancer development.
PMCID: PMC4046881  PMID: 24910533
DLG5; epithelial cell polarity; EMT; cell proliferation; migration.
8.  Up-Regulated Lipocalin-2 in Pulmonary Hypertension Involving in Pulmonary Artery SMC Resistance to Apoptosis 
A key feature of pulmonary hypertension (PH) is the remodeling of small pulmonary arteries due to abnormal pulmonary artery smooth muscle cell (PASMC) proliferation and resistance to apoptosis. However, the cellular mechanisms underlying how PASMCs in the pathological condition of pulmonary hypertension become resistant to apoptosis remain unknown. It was recently reported that lipocalin 2 (Lcn2) is up-regulated in a wide array of malignant conditions, which facilitates tumorigenesis partly by inhibiting cell apoptosis. In this study, we observed that the expression levels of Lcn2 were significantly elevated in a rat PH model induced with monocrotaline and in patients with congenital heart disease-associated PH (CHD-PH) when compared with respective control. Therefore, we hypothesize that Lcn2 could regulate human PASMC (HPASMC) apoptosis through a mechanism. By the detection of DNA fragmentation using the TUNEL assay, the detection of Annexin V/PI-positive cells using flow cytometry, and the detection of cleaved caspase-3 and caspase-3 activity, we observed that Lcn2 significantly inhibited HPASMC apoptosis induced by serum withdrawal and H2O2 treatment. We also observed that Lcn2 down-regulated the proapoptotic protein Bax, decreased the levels of cellular ROS, and up-regulated the expression of superoxide dismutases (SOD1 and SOD2). In conclusion, Lcn2 significantly inhibits HPASMC apoptosis induced by oxidative stress via decreased intracellular ROS and elevated SODs. Up-regulation of Lcn2 in a rat PH model and CHD-PH patients may be involved in the pathological process of PH.
PMCID: PMC4115200  PMID: 25076856
apoptosis; oxidative stress; Lipocalin-2 (Lcn2); SOD; ROS; pulmonary hypertension (PH)
9.  microRNAs in Spinal Cord Injury: Potential Roles and Therapeutic Implications 
microRNAs (miRNAs) are a novel class of small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. miRNAs can modulate gene expression and thus play important roles in diverse neurobiological processes, such as cell differentiation, growth, proliferation and neural activity, as well as the pathogenic processes of spinal cord injury (SCI) like inflammation, oxidation, demyelination and apoptosis. Results from animal studies have revealed the temporal alterations in the expression of a large set of miRNAs following SCI in adult rats, and the expressional changes in miRNAs following SCI is bidirectional (increase or decrease). In addition, several miRNAs have distinct roles in prognosis of SCI (protective, detrimental and varied). Taken together, the existing evidence suggests that abnormal miRNA expression following SCI contributes to the pathogenesis of SCI, and miRNAs may become potential targets for the therapy of SCI.
PMCID: PMC4159691  PMID: 25210498
microRNA; spinal cord injury; therapy; diagnosis.
10.  Co-Expression Analysis of Fetal Weight-Related Genes in Ovine Skeletal Muscle during Mid and Late Fetal Development Stages 
Background: Muscle development and lipid metabolism play important roles during fetal development stages. The commercial Texel sheep are more muscular than the indigenous Ujumqin sheep.
Results: We performed serial transcriptomics assays and systems biology analyses to investigate the dynamics of gene expression changes associated with fetal longissimus muscles during different fetal stages in two sheep breeds. Totally, we identified 1472 differentially expressed genes during various fetal stages using time-series expression analysis. A systems biology approach, weighted gene co-expression network analysis (WGCNA), was used to detect modules of correlated genes among these 1472 genes. Dramatically different gene modules were identified in four merged datasets, corresponding to the mid fetal stage in Texel and Ujumqin sheep, the late fetal stage in Texel and Ujumqin sheep, respectively. We further detected gene modules significantly correlated with fetal weight, and constructed networks and pathways using genes with high significances. In these gene modules, we identified genes like TADA3, LMNB1, TGF-β3, EEF1A2, FGFR1, MYOZ1, and FBP2 correlated with fetal weight.
Conclusion: Our study revealed the complex network characteristics involved in muscle development and lipid metabolism during fetal development stages. Diverse patterns of the network connections observed between breeds and fetal stages could involve some hub genes, which play central roles in fetal development, correlating with fetal weight. Our findings could provide potential valuable biomarkers for selection of body weight-related traits in sheep and other livestock.
PMCID: PMC4183924  PMID: 25285036
Serial expression analysis; WGCNA; fetal development stages; fetal weight.
11.  The Steroid Receptor Coactivator-3 Is Required for Developing Neuroendocrine Tumor in the Mouse Prostate 
Neuroendocrine tumor cells (NETCs) are commonly observed in prostate cancer. Their presence is associated with castration resistance, metastasis and poor prognosis. Cellular and molecular mechanisms for NETC initiation and growth are unknown. TRAMP mice develop heterogeneous adenocarcinomas induced by expression of the SV40-T/t oncogene in prostate epithelial cells. Here, we demonstrate prostate tumors in TRAMP mice with a mixed genetic background are characterized mostly by atypical hyperplasia (AH) containing steroid receptor coactiator-3-positive, androgen receptor-positive and synaptophysin-negative (SRC-3+/AR+/Syp-) cells. Few SRC-3+/AR-/Syp+ NETCs are present in their prostates. We generated TRAMP mice in which SRC-3 was specifically ablated in AR+/Syp- prostatic epithelial cells (termed PE3KOT mice). In these animals, we observed a substantial reduction in SRC-3-/AR+/Syp- AH tumor growth. There was a corresponding increase in SRC-3-/AR+/Syp- phyllodes lesions, suggesting SRC-3 knockout can convert aggressive AH tumors with mostly epithelial tumor cells into less aggressive phyllodes lesions with mostly stromal tissue. Surprisingly, PE3KOT mice developed many more SRC-3+/AR-/Syp+ NETCs versus control TRAMP mice, indicating SRC-3 expression was retained in NETCs. In contrast, TRAMP mice with global SRC-3 knockout did not develop any NETC, indicating SRC-3 is required for developing NETC. Analysis of cell-differentiating markers revealed that these NETCs might not be derived from the mature AR-/Syp+ neuroendocrine cells or the AR+/Syp- luminal epithelial tumor cells. Instead, these NETCs might originate from the SV40-T/t-transformed intermediate/progenitor epithelial cells. In summary, SRC-3 is required for both AR+/Syp- AH tumor growth and AR-/Syp+ NETC development, suggesting SRC-3 is a target for inhibiting aggressive prostate cancer containing NETCs.
PMCID: PMC4202028  PMID: 25332686
coactivator; SRC-3/NCOA3; prostate epithelium; carcinogenesis; neuroendocrine cell; transgenic mouse model.
12.  Effects of Hypothyroidism on Expression of CRMP2B and ARPC5 during Development of the Rat Frontal Cortex 
Congenital hypothyroidism (CH) can lead to irreversible central nervous system (CNS) damage. However, the pathogenesis of the developmental brain disorders caused by CH has not been completely elucidated. ARPC5 and CRMP2 are closely associated with neurite outgrowth in brain development. Thus, the aim of the present study was to determine whether CRMP2B and ARPC5 expression is altered in the developing cerebral cortex of rats with CH. Control rats and rats with hypothyroidism were sacrificed at birth and at 15 days postpartum. We performed qRT-PCR to detect differences in the crmp2B and arpc5 mRNA expression in the right half of the frontal cortex of these rats. Western blotting was then used to detect differences in CRMP2B and ARPC5 protein expression. Furthermore, immunohistochemical analysis was performed on the left half of the frontal cortex to detect abnormal localization of CRMP2B and ARPC5. Results showed increased expression of the nuclear short isoform of CRMP2B and decreased expression of full-length CRMP2B and ARPC5 in cortical neurons of rats with hypothyroidism. These findings demonstrate that reduced levels of thyroid hormones can inhibit the expression of full-length CRMP2B and ARPC5 and promote nuclear transformation of the short isoform of CRMP2B. CRMP2B and ARPC5 may participate in CNS injury mediated by hypothyroidism by inducing neurite outgrowth inhibition and cytoskeletal protein disorganization.
PMCID: PMC3584917  PMID: 23459330
CRMP2B; ARPC5; congenital hypothyroidism; frontal cortex; rat.
13.  Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation 
DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.
PMCID: PMC3683939  PMID: 23781148
DOC-1R; CDK2; G1/S transition; cyclin E; cyclin A; CKI.
14.  BMP Receptor 1A Determines the Cell Fate of the Postnatal Growth Plate 
Bone morphogenic proteins (BMPs) are critical for both chondrogenesis and osteogenesis. Previous studies reported that embryos deficient in Bmp receptor (Bmpr)1a or Bmpr1b in cartilage display subtle skeletal defects; however, double mutant embryos develop severe skeletal defects, suggesting a functional redundancy that is essential for early chondrogenesis. In this study, we examined the postnatal role of Bmpr1a in cartilage. In the Bmpr1a conditional knockout (cKO, a cross between Bmpr1a flox and aggrecan-CreERT2 induced by a one-time-tamoxifen injection at birth and harvested at ages of 2, 4, 8 and 20 weeks), there was essentially no long bone growth with little expression of cartilage markers such as SOX9, IHH and glycoproteins. Unexpectedly, the null growth plate was replaced by bone-like tissues, supporting the notions that the progenitor cells in the growth plate, which normally form cartilage, can form other tissues such as bone and fibrous; and that BMPR1A determines the cell fate. A working hypothesis is proposed to explain the vital role of BMPR1A in postnatal chondrogenesis.
PMCID: PMC3807016  PMID: 24163588
BMPR1A; Growth plate; Cell fate; Chondrogenesis; Endochondral Bone.
15.  Enhanced Antitumor Efficacy of Gemcitabine by Evodiamine on Pancreatic Cancer via Regulating PI3K/Akt Pathway 
Evodiamine has therapeutic potential against cancers. This study was designed to investigate whether combination therapy with gemcitabine and evodiamine enhanced antitumor efficacy in pancreatic cancer. In vitro application of the combination therapy triggered significantly higher frequency of pancreatic cancer cells apoptosis, inhibited the activities of PI3K, Akt, PKA, mTOR and PTEN, and decreased the activation of NF-κB and expression of NF-κB-regulated products. In vivo application of the combination therapy induced significant enhancement of tumor cell apoptosis, reductions in tumor volume, and inhibited activation of mTOR and PTEN. In conclusion, evodiamine can augment the therapeutic effect of gemcitabine in pancreatic cancer through direct or indirect negative regulation of the PI3K/Akt pathway.
PMCID: PMC3226028  PMID: 22211100
evodiamine; gemcitabine; pancreatic cancer SW1990 cells; apoptosis; PTEN; mTOR
16.  Localization, Expression Change in PRRSV Infection and Association Analysis of the Porcine TAP1 Gene 
The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the lumen of the endoplasmic reticular and plays a critical role in the major histocompatibility complex (MHC) class I molecule-mediated antigenic presentation pathway. In this study, the porcine TAP1 gene was mapped to the pig chromosome 7 (SSC7) and was closely linked to the marker SSC2B02 (retention fraction=43%, LOD=15.18). Subcellular localization of TAP1 by transient transfection of PK15 cells indicated that the TAP1 protein might be located in the endoplasmic reticulum (ER) in pig kidney epithelial cells (PK-15). Gene expression analysis by semi-quantitative RT-PCR revealed that TAP1 was selectively expressed in some immune and immune-related tissues. Quantitative real-time PCR (qRT-PCR) analysis revealed that this gene was up-regulated after treatments that mimic viral and bacterial infection (polyriboinosinic-polyribocytidylic acid (poly(I:C)) and lipopolysaccharide (LPS), respectively). In addition, elevated TAP1 expression was detected after porcine reproductive and respiratory syndrome virus (PRRSV) infection in porcine white blood cells (WBCs). One single nucleotide polymorphism (SNP) in exon 3 of TAP1 was detected in a Landrace pig population by Bsp143I restriction enzyme digestion. Different genotypes of this SNP had significant associations (P<0.05) with the red blood cell distribution width (RDW) of 1-day-old (1 d) pigs (P=0.0168), the PRRSV antibody level (PRRSV Ab) (P=0.0445) and the absolute lymphocyte count (LYM#) (P=0.024) of 17 d pigs. Our results showed that the TAP1 gene might have important roles in swine immune responses, and these results provide useful information for further functional studies.
PMCID: PMC3226032  PMID: 22211104
Pig; TAP1; Localization; Expression; PRRSV; Association analyses
17.  Isolation and Identification of miRNAs in Jatropha curcas 
MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles by targeting mRNAs for silencing. To identify miRNAs in Jatropha curcas L, a bioenergy crop, cDNA clones from two small RNA libraries of leaves and seeds were sequenced and analyzed using bioinformatic tools. Fifty-two putative miRNAs were found from the two libraries, among them six were identical to known miRNAs and 46 were novel. Differential expression patterns of 15 miRNAs in root, stem, leave, fruit and seed were detected using quantitative real-time PCR. Ten miRNAs were highly expressed in fruit or seed, implying that they may be involved in seed development or fatty acids synthesis in seed. Moreover, 28 targets of the isolated miRNAs were predicted from a jatropha cDNA library database. The miRNA target genes were predicted to encode a broad range of proteins. Sixteen targets had clear BLASTX hits to the Uniprot database and were associated with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function. Four targets were identified for JcumiR004. By silencing JcumiR004 primary miRNA, expressions of the four target genes were up-regulated and oil composition were modulated significantly, indicating diverse functions of JcumiR004.
PMCID: PMC3303143  PMID: 22419887
Jatropha; Biofuel; miRNA; fatty acid synthesis.
18.  Cdc42-Interacting Protein-4 Promotes TGF-Β1-Induced Epithelial-Mesenchymal Transition and Extracellular Matrix Deposition in Renal Proximal Tubular Epithelial Cells 
Cdc42-interacting protein-4 (CIP4) is an F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family member that regulates membrane deformation and endocytosis, playing a key role in extracellular matrix (ECM) deposition and invasion of cancer cells. These processes are analogous to those observed during the initial epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. The role of CIP4 in renal tubular EMT and renal tubulointerstitial fibrosis was investigated over the course of the current study, demonstrating that the expression of CIP4 increased in the tubular epithelia of 5/6-nephrectomized rats and TGF-β1 treated HK-2 cells. Endogenous CIP4 evidenced punctate localization throughout the cytosol, with elevated levels observed in the perinuclear region of HK-2 cells. Subsequent to TGF-β1 treatment, CIP4 expression increased, forming clusters at the cell periphery that gradually redistributed into the cytoplasm. Simultaneously, EMT induction in cells was confirmed by the prevalence of morphological changes, loss of E-cadherin, increase in α-SMA expression, and secretion of fibronectin. Overexpression of CIP4 promoted characteristics similar to those commonly observed in EMT, and small interfering RNA (siRNA) molecules capable of CIP4 knockdown were used to demonstrate reversed EMT. Cumulatively, results of the current study suggest that CIP4 promotes TGF-β1-induced EMT in tubular epithelial cells. Through this mechanism, CIP4 is capable of inducing ECM deposition and exacerbating progressive fibrosis in chronic renal failure.
PMCID: PMC3385008  PMID: 22745576
Cdc42-interacting protein-4; epithelial-mesenchymal transition; renal proximal tubular epithelial cells; β-catenin; TGF-β1-induced
19.  Molecular and Biochemical Characterization of a Novel β-N-Acetyl-D-Hexosaminidase with Broad Substrate-Spectrum from the Aisan Corn Borer, Ostrinia Furnacalis 
Insect β-N-acetyl-D-hexosaminidases with broad substrate-spectrum (IBS-Hex) are the homologues of human β-N-acetyl-D-hexosaminidase A/B (HsHex A/ B). These enzymes are distributed in most insect species and vary in physiological roles. In this study, the gene encoding an IBS-Hex, OfHEX2, was cloned from the Asian corn borer, Ostrinia furnacalis. Recombinant OfHex2 was expressed in Pichia pastoris and purified to homogeneity. By structure-based sequence alignment, three sequence segments with high diversity among IBS-Hexs were firstly concluded. Furthermore, the residue pair N423-R424/ D452-L453 important for the specificity of human β-N-acetyl-D-hexosaminidase subunits α/β toward charged/ non-charged substrates was not conserved in OfHex2 and other IBS-Hexs. Unlike HsHex A, OfHex2 could not degrade charged substrates such as 4-methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide, ganglioside GM2 and peptidoglycan. OfHex2 showed a broad substrate-spectrum by hydrolyzing β1-2 linked N-acetyl-D-glucosamines from both α3 and α6 branches of biantennary N-glycan and β1-4 linked GlcNAc from chitooligosaccharides as well as β1-3 linked or β1-4 linked N-acetyl-D-galactosamine from oligosaccharides of glycolipids. Real-time PCR analysis demonstrated that the expression of OfHEX2 was up-regulated in the intermolt stages (both larva and pupa), and mainly occurred in the carcass rather than in the midgut during the feeding stage of fifth (final) instar larva. This study reported a novel IBS-Hex with specific biochemical properties, suggesting biodiversity of this class of enzymes.
PMCID: PMC3445047  PMID: 22991497
β-N-acetyl-D-hexosaminidase; insect; glycoside hydrolase; N-glycan; Ostrinia furnacalis.
20.  Sperm Nuclear Transfer and Transgenic Production in the Fish Medaka 
Sperm nuclear transfer or intracytoplasmic sperm injection (ICSI) is a powerful assisted reproductive technology (ART) for treating human male infertility. Controversial reports of increased birth defects have raised concerns about the ART's safety. The cause for birth defects, however, has remained elusive for analysis in human because of the sample size, male infertility genetics, physiological heterogeneity and associated procedures such as embryo manipulations. Animal models are required to evaluate factors leading to the increased birth defects. Here we report the establishment of medakafish model for ICSI and transgenic production. This small laboratory fish has high fecundity and easy embryology. We show that ICSI produced a 5% high percentage of fertile animals that exhibited both paternal and maternal contribution as evidenced by the pigmentation marker. Furthermore, when sperm were pre-incubated with a plasmid ubiquitously expressing RFP and subjected to ICSI, 50% of sperm nuclear transplants showed germline transmission. We conclude that medaka is an excellent model for ICSI to evaluate birth defects and that sperm nuclear transfer can mediate stable gene transfer at high efficiency. Although more demanding for experimentation, sperm-mediated transgenesis should be particularly applicable for aquaculture species with a lengthy generation time and/or a large adult body size.
PMCID: PMC3088289  PMID: 21547064
medaka; nuclear transfer; sperm; gene transfer; transgenesis
21.  Massive Production of All-female Diploids and Triploids in the Crucian Carp 
In many species of aquaculture importance, all-female and sterile populations possess superior productivity due to faster growth and a relatively homogenous size of individuals. However, the production of all-female and sterile fish in a large scale for aquaculture is a challenge in practice, because treatments necessary for gynogenesis induction usually cause massive embryonic and larval mortality, and the number of induced gynogens is too small for their direct use in aquaculture. Here we report the massive production of all-female triploid crucian carp by combining artificial gynogenesis, sex reversal and diploid-tetraploid hybridization. Previously, we have obtained an allotetraploid carp population (4n = 200) by hybridization between red crucian carp (Carassius auratus red var; ♀) and common carp (Cyprinus carpio; ♂). We induced all-female diploid gynogens of the Japanese crucian carp (Carassius cuvieri; 2n = 100). We also generated male diploid gynogens of the same species treated gynogenetic fry with 17-α-methyltestosterone, leading to the production of sex-revered gynogenetic males. Finally, these males were used to cross with the female diploid Japanese crucian carp gynogens and the allotetraploid females, resulting in the production of fertile all-female diploid Japanese crucian carp (2n=100) and sterile all-female triploid hybrids (3n = 150), respectively. Therefore, diploid crucian carp gynogenetic females and sex-reversed male together with an allotetraploid line provide an opportunity to produce all-female triploid populations in a large scale to meet demands in aquaculture industry.
PMCID: PMC3088291  PMID: 21547066
gynogenesis; sex reversal; all-female diploid; all-female triploid; Stem cell; fish breeding.
22.  Interleukin-8 Induces the Endothelial Cell Migration through the Activation of Phosphoinositide 3-Kinase-Rac1/RhoA Pathway 
Endothelial cell migration is essential for tumor angiogenesis, and interleukin-8 (IL-8) has been shown to play an important role in tumor growth, angiogenesis, and metastasis. This study aimed to investigate the molecular mechanism of IL-8 induced endothelial cell migration. Our results indicated that IL-8 induced a rapid rearrangement of the actin cytoskeleton in EA.Hy926 cells, generating extensions resembling membrane ruffling and stress fibers. These processes required parallel upregulation of the small GTPases Rac1 and RhoA. Moreover, we demonstrated that IL-8 activated PI3K following the same kinetics observed from IL-8 induction of cytoskeletal rearrangement, suggesting the participation of PI3K in these processes. Taken together, our study demonstrates that PI3K-Rac1/RhoA signaling pathway plays a vital role in IL-8 induced endothelial cell migration, and provides new insight into the molecular mechanisms by which IL-8 contributes to tumor angiogenesis and metastasis.
PMCID: PMC3133886  PMID: 21750647
Rac1; Interleukin-8; PI3-kinase; cell migration; tumor angiogenesis
23.  The Architecture and Complete Sequence of Mitochondrial Genome of an Assassin Bug Agriosphodrus Dohrni (Hemiptera: Reduviidae) 
The 16, 470 bp nucleotide sequence of the mitochondrial genome (mitogenome) of an assassin bug from the reduviid subfamily Harpactorinae, Agriosphodrus dohrni, has been revealed. The entire genome encodes for two ribosomal RNA genes (rrnL and rrnS), 22 transfer RNA (tRNA) genes, 13 protein-coding genes, and a control region. The nucleotide composition is biased toward adenine and thymine (A+T = 72.2%). Comparative analysis with two other reduviid species Triatoma dimidiata and Valentia hoffmanni, exhibited highly conserved genome architectures including genome contents, gene order, nucleotide composition, codon usage, amino acid composition, as well as genome asymmetry. All protein-coding genes use standard mitochondrial initiation codons (methionine and isoleucine), except that nad1 starts with GTG. All tRNAs have the classic clover-leaf structure, except that the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. Secondary structure comparisons of the two mitochondrial ribosomal subunits among sequenced assassin bugs show that the sequence and structure of rrnL is more conservative than that of rrnS. The presence of structural elements in the control region is also discussed, with emphasis on their implications in the regulation of replication and/or transcription of the reduviid mitogenome. The phylogenetic analyses indicated that within Reduviidae, Harpactorinae is a sister group to the Salyavatinae + Triatominae clade.
PMCID: PMC3133887  PMID: 21750648
Mitogenome; Agriosphodrus dohrni; Reduviidae; genome architecture; phylogenetic relationship
24.  MicroRNA-7 Inhibits the Growth of Human Non-Small Cell Lung Cancer A549 Cells through Targeting BCL-2 
MicroRNAs(miRNAs) are emerging as important regulators in tumorigenesis. Increasing evidences have indicated microRNA-7(miR-7) to be a potential tumor suppressor in several human cancers. However, only a limited number of target genes have been identified so far and its biological function in Non-Small Cell Lung Cancer (NSCLC) remains to be further elucidated. In the present study, we observed a reduction of miR-7 level in NSCLC cell lines. Overexpression of miR-7 not only suppressed NSCLC A549 cells proliferation, induced cell apoptosis and inhibited cell migration in vitro, but also reduced tumorigenicity in vivo. Bioinformatics predictions revealed a potential binding site of miR-7 on 3'UTR of BCL-2 and it was further confirmed by luciferase assay. Moreover, subsequent experiments showed that BCL-2 was downregulated by miR-7 at both transcriptional and translational levels. These results suggest that miR-7 regulates the expression of BCL-2 through direct 3'UTR interactions. Therefore, we postulate BCL-2 to be a novel target possibly involved in miR-7-mediated growth suppression and apoptosis of A549 cells. These findings may provide a basic rationale for the use of miR-7 in the treatment of NSCLC.
PMCID: PMC3133888  PMID: 21750649
miR-7; BCL-2; non-small cell lung cancer; A549 cells; apoptosis.
25.  Autosomal Interactions and Mechanisms of Pyrethroid Resistance in House Flies, Musca domestica 
Five BC1 lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.
PMCID: PMC3149284  PMID: 21814485
House fly lines; pyrethroid resistance; autosomes; Genetic crossing; Cytochrome P450s; Sodium Channels

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