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Ensemble methods such as bagging and random forests are ubiquitous in various fields, from finance to genomics. Despite their prevalence, the question of the efficient tuning of ensemble parameters has received relatively little attention. This article introduces a cross-validation method, Extrapolated Cross-Validation (ECV), for tuning the ensemble and subsample sizes in randomized ensembles. Our method builds on two primary ingredients: initial estimators for small ensemble sizes using out-of-bag errors and a novel risk extrapolation technique that leverages the structure of prediction risk decomposition. By establishing uniform consistency of our risk extrapolation technique over ensemble and subsample sizes, we show that ECV yields δ-optimal (with respect to the oracle-tuned risk) ensembles for squared prediction risk. Our theory accommodates general predictors, only requires mild moment assumptions, and allows for high-dimensional regimes where the feature dimension grows with the sample size. As a practical case study, we employ ECV to predict surface protein abundances from gene expressions in single-cell multiomics using random forests under a computational constraint on the maximum ensemble size. Compared to sample-split and K-fold cross-validation, ECV achieves higher accuracy by avoiding sample splitting. Meanwhile, its computational cost is considerably lower owing to the use of the risk extrapolation technique. for this article are available online. [ABSTRACT FROM AUTHOR]

Background: To investigate the role of interleukin (IL)‐17 in tissue and peripheral blood of perianal abscess and anal fistula. Methods: Patients with primary perianal abscess (n = 50) admitted to Jinhua Municipal Central Hospital between March 2003 and August 2004 were enrolled. Fifty patients with mixed haemorrhoids, who showed no perianal abscess or anal fistula, were also recruited as the control. After surgery, patients were followed up for 6 months. Protein and gene expression of IL‐17 was determined in surgically harvested anal tissues and peripheral blood, respectively. The relationship between IL‐17 and clinical pathological features were analysed. Results: As shown by immunohistochemistry of anorectal tissues, the positive rate of IL‐17 protein was higher in the perianal abscess group than in the control group. In patients with perianal abscess, the expression of IL‐17 significantly correlated with the diameter of the abscess (P = 0.013), the wound surface healing time (P = 0.010) and the progression into anal fistula (P = 0.003). For the gene expression of IL‐17 in peripheral blood cells, the level was significantly higher in patients with perianal abscess comparing to the control group (0.4350 ± 0.1190 versus 0.1785 ± 0.1230, P ≤ 0.001). Comparing to the recovery group, patients with their perianal abscess progressed to anal fistula showed higher levels of IL‐17 gene expression (P = 0.014). Conclusions: Expression of IL‐17 was increased in the anorectal tissues and peripheral blood of patients with perianal abscess and anal fistula. IL‐17 may play an important role in the pathogenesis of perianal abscess and anal fistula. [ABSTRACT FROM AUTHOR]

Oxidative stress is unavoidable for aerobic organisms. When abiotic and biotic stresses are encountered, oxidative damage could occur in cells. To avoid this damage, defense mechanisms must be timely and efficiently modulated. While the response to oxidative stress has been extensively studied in plants, little is known about how the activated response is switched off when oxidative stress is diminished. By studying Arabidopsis mutant paraquat tolerance3, we identified the genetic locus PARAQUAT TOLERANCE3 (PQT3) as a major negative regulator of oxidative stress tolerance. PQT3, encoding an E3 ubiquitin ligase, is rapidly down-regulated by oxidative stress. PQT3 has E3 ubiquitin ligase activity in ubiquitination assay. Subsequently, we identified PRMT4b as a PQT3-interacting protein. By histone methylation, PRMT4b upregulates the expression of APX1 and GPX1, encoding two key enzymes against oxidative stress. On the other hand, PRMT4b is recognized by PQT3 for targeted degradation via 26S proteasome. Therefore, we have identified PQT3 as an E3 ligase that acts as a negative regulator of activated response to oxidative stress and found that histone modification by PRMT4b at APX1 and GPX1 loci plays an important role in oxidative stress tolerance. [ABSTRACT FROM AUTHOR]

Ubiquitin ligase Nrdp1 (neuregulin receptor degradation protein 1) plays important roles in multiple physiological process because it can ubiquitinate various substrates such as ErbB3, BRUCE, MyD88, C/EBPβ, and Parkin, and so forth. In addition to the physiological function, it was also found to be involved in tumor progression. It has been shown that loss of Nrdp1 enhances breast cancer cell growth. Up to now, the role of Nrdp1 in glioma has not been elucidated. Here, we reported that Nrdp1 as well as cleaved caspase 3 was lower expressed in human glioma tissues comparing with the nontumorous. And then we found that the expression of Nrdp1 and cleaved caspase 3 was increased in the treatment of Temozolomide (TMZ), a drug for glioma chemotherapy. Further investigation indicated that transient transfection of Nrdp1 significantly promoted cell apoptosis by aggravating the degradation of BRUCE and activation of caspase 3. In addition, overexpression of Nrdp1 augmented TMZ induced apoptosis by evaluating the degradation of BRUCE and the activation of caspase 3, while silencing of Nrdp1 reduced the sensitivity to the TMZ by inhibiting the degradation of BRUCE and the activation of caspase 3 in human glioma cells. These observations show that Nrdp1 is a pro-apoptotic protein in human glioma and lower expression of Nrdp1 in human glioma may promote tumor progression by reducing apoptosis, suggesting that Nrdp1 may be an important regulator in the development of human glioma. © 2014 IUBMB Life, 66(10):704-710, 2014 [ABSTRACT FROM AUTHOR]

The expression levels of sorbose/sorbosone dehydrogenase genes (sdh and sndh) and the synthesis genes (pqqABCDEN) of the adjoint cofactor pyrroloquinoline quinone (PQQ) were genetically manipulated in Ketogulonigenium vulgare to increase the production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in the consortium of K. vulgare and Bacillus cereus. We found that overexpression of sdh–sndh alone in K. vulgare could not significantly enhance the production of 2-KLG, revealing the cofactor PQQ was required for the biosynthesis of 2-KLG. Various expression levels of PQQ were achieved by differential expression of pqqA, pqqABCDE and pqqABCDEN, respectively. The combinatorial expression of sdh/sndh and pqqABCDEN in K. vulgare enabled a 20% increase in the production of 2-KLG (79.1±0.6gl−1) than that of the parental K. vulgare (65.9±0.4gl−1) in shaking flasks. Our results demonstrated the balanced co-expression of both the key enzymes and the related cofactors was an efficient strategy to increase chemicals' biosynthesis. [ABSTRACT FROM AUTHOR]

Background: To investigate the effects of fluids resuscitation on pulmonary expression of aquaporin1 and aquaporin5 in a rat model of uncontrolled hemorrhagic shock and infection. Methods: Sixty Sprague-Dawley rats were randomly assigned to five groups, sham operation group (Group C) and four treated groups: no fluid resuscitation group (Group NF), groups resuscitated with Lactated Ringer's (LR),7.5% NaCl (HTS) and Hydroxyl ethyl starch (HES) respectively. Three-phased uncontrolled hemorrhagic shock and infection model was used. Phase I: Massive hemorrhage with a mean arterial pressure of 35–40 mmHg for 60 min, and followed by infection of lipopolysaccharide. Then some animals were resuscitated with solutions mentioned above, until 90 min. Phase II: At hemorrhagic shock 90 minutes, phase II of 60 minutes began with hemostasis and returning of all the initial shed blood. Phase III: Observation phase for 3.5 hours. After phase III, arterial blood gas analysis and the survival rates of the rats were recorded, Wet-to-dry lung weight ratio, BALF protein, pulmonary permeability index, and expressions of aquaporin1 and aquaporin5 were tested. Results: The expressions of aquaporin1 and aquaporin5 were decreased in treatment groups comparing with sham operation group. Group HES and Group HTS decreased pulmonary vascular permeability and Wet-to-dry lung weight ratio, improved arterial blood gas analysis and survival rates, and attenuated the decreased pulmonary expression of aquaporin1 and aquaporin5 after the “two-hit”, comparing with groups NF and LR,but these beneficial effects were blunted in group HTS. Conclusion: The expression of aquaporin1 and aquaporin5 may play important roles in formation of pulmonary edema. Resuscitation with HTS and HES, especially HES can reduce lung injury after hemorrhagic shock, partly by up-regulating the expressions of aquaporin1 and aquaporin5. [ABSTRACT FROM AUTHOR]

Plants have evolved complex processes to ward off attacks by insects. In parallel, insects have evolved mechanisms to thwart these plant defenses. To gain insight into mechanisms that mediate this arms race between plants and herbivorous insects, we investigated the interactions between gramine, a toxin synthesized by plants of the family Gramineae, and glutathione S transferase (GST), an enzyme found in insects that is known to detoxify xenobiotics. Here, we demonstrate that rice (Oryza sativa), a hydrophytic plant, also produces gramine and that rice resistance to brown planthoppers (Nilaparvata lugens, BPHs) is highly associated with in planta gramine content. We also show that gramine is a toxicant that causes BPH mortality in vivo and that knockdown of BPH GST gene nlgst1-1 results in increased sensitivity to diets containing gramine. These results suggest that the knockdown of key detoxification genes in sap-sucking insects may provide an avenue for increasing their sensitivity to natural plant-associated defense mechanisms. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stem cells (MSCs) therapies are emerging as a promising approach to therapeutic regeneration. Therapeutic persistence and reduced functional stem cells following cell delivery remain critical hurdles for clinical investigation due to the senescence of freshly isolated cells and extensive in-vitro passage. Methods: Cultured adipose-derived stem cells (ASCs) were derived from subcutaneous white adipose tissue isolated from mice fed a normal diet. We performed senescence-associated-β-galactosidase (SA-β-gal) staining, real-time PCR, and Westernblot to evaluate the levels related to cellular senescence markers. Results: The mRNA expression levels of senescence markers were significantly increased in the later passage of ASCs. We show that light activation reduced the expression of senescent genes, and SA-β-Gal in all cells at passages. Moreover, the light-activated ASCs-derived exosomes decrease the expression of senescence, and SA-β-Gal in the later passage cells. We further investigated the photoreceptive effect of Opsin3 (Opn3) in light-activated ASCs. Deletion of Opn3 abolished the differences of light activation in reduced expression of senescent genes, increased Ca 2+ influx, and cAMP levels. Conclusions: ASCs can undergo cellular senescence in-vitro passage. Photomodulation might be better preserved over senescence and Opn3-dependent activation in aged ASCs. Light-activated ASCs-derived exosomes could be served as e a new protective paradigm for cellular senescence in-vitro passage. AY8VxFmVMVxVLQ-spERSup Video Abstract [ABSTRACT FROM AUTHOR]

Abstract: Ceruloplasmin (CP) is essential for brain iron homeostasis. However, little is known about the effect of iron on CP expression in the brain. Also, the role of CP in brain iron transport has not been well determined. In this study, we investigated the effects of iron on CP expression and the role of CP in iron transport in the C6 rat glioma cells. Our data showed that treatment of the cells with iron (cell iron overload) or iron chelators (cell iron deficiency) did not induce a significant change in the expression of CP mRNA. However, western blotting analysis demonstrated that cell iron overload induced a significant decrease in CP protein content in the cells and that treatment with iron chelators led to a significant increase in CP protein level in the cells. These findings suggest a translational regulation of CP expression by iron in the cells. We also examined the effects of CP on iron transport in the cells. We found that glycosylphosphatidylinositol-anchored CP did not have any impact on iron uptake by normal iron or iron-deficient cells nor on iron release from normal iron or iron-sufficient cells. However, low concentrations of soluble CP (2–8μg/ml) increased iron uptake by iron-deficient C6 glioma cells, while the same concentrations of CP had no effect on iron uptake by normal iron cells and iron release from normal iron and iron-sufficient cells. The possible reason for the difference between our results in vitro and those obtained from in vivo studies was discussed. [Copyright &y& Elsevier]

Objective: Serum uric acid is associated with tumor progression and hepatocarcinogenesis. Here, we aimed to determine whether serum uric acid is related to the survival time of patients with hepatocellular carcinoma (HCC) and whether the inhibition of uric acid production affects the progression and survival of rats with HCC. Methods: The follow-up data of 288 patients with advanced HCC were analyzed. Ten purine metabolites in serum and liver samples of diethylnitrosamine (DEN)-induced HCC rats were quantitatively determined by an established UPLC-MS/MS method. On this basis, febuxostat, a specific inhibitor of xanthine oxidase (XOD), was used to interfere with HCC rats. Results: The serum uric acid level of HCC patients was significantly negatively correlated with survival days (r = -0.155). The median survival time was 133.5 days in the high uric acid group (>360 μmol/L, n = 80) and 176.0 days in the normal serum uric acid group (<360 μmol/L, n = 208, p = 0.0013). The levels of hypoxanthine, guanine, and uric acid; XOD activity; and xanthine dehydrogenase mRNA expression in the serum or liver samples of HCC rats were significantly upregulated compared with those in the control group. After febuxostat intervention in DEN-induced HCC rats, the number of atypical cells and inflammatory cells decreased significantly; the serum alpha fetoprotein level and Fisher's ratio tended to return to normal; the median survival time increased from 36 to 96 days (p = 0.08). In addition, serum malondialdehyde, superoxide dismutase, and glutathione activity nearly returned to the level of the healthy control group. Conclusion: The elevation of serum uric acid implies a risk of poor survival in advanced HCC patients and Febuxostat can reduce the generation of reactive oxygen species, thereby playing a role in delaying the progression of liver cancer. [ABSTRACT FROM AUTHOR]

Reovirus infection induces dramatic changes in host mRNA expression. We utilized oligonucleotide microarrays to measure cellular mRNA decay rates in mock- or reovirus-infected murine L929 cells to determine if changes in host mRNA expression are a consequence of reovirus-induced alterations in cellular mRNA stability. Our analysis detected a subset of cellular transcripts that were coordinately induced and stabilized following infection with the reovirus isolates c87 and c8, strains that led to an inhibition of cellular translation, but not following infection with Dearing, a reovirus isolate that did not negatively impact cellular translation. The induced and stabilized transcripts encode multiple regulators of TGF- β signaling, including components of the Smad signaling network and apoptosis/survival pathways. The coordinate induction, through mRNA stabilization, of multiple genes that encode components of TGF-β signaling pathways represents a novel mechanism by which the host cell responds to reovirus infection. [ABSTRACT FROM AUTHOR]

Growth arrest and DNA damage‑inducible‑β (Gadd45β) is a stress‑response protein involved in a number of processes, including cell cycle control, DNA repair, survival and death control, and stress signaling, depending on its interactions. Gadd45β expression is dysregulated in numerous types of cancer, functioning as either a tumor promoter or a tumor suppressor. However, the functions of Gadd45β in cholangiocarcinoma (CCA), particularly in metastasis, has not been studied. The immunohistochemical analysis of Gadd45β expression revealed that 75% of histological specimens from patients with CCA expressed high levels of Gadd45β, and that high Gadd45β expression was associated with metastasis. The role of Gadd45β in CCA was examined using siRNA‑mediated gene knockdown in HuCCA‑1, a human CCA cell line established from a Thai patient. The effects of Gadd45β downregulation upon cell viability and death, invasion, migration, matrix metalloproteinase (MMP) activity and epithelial‑mesenchymal transition (EMT) marker expression were investigated. Gadd45β knockdown impaired cell viability, which was associated with the induction of apoptosis. In addition, there was a marked reduction in invasion and migration, although MMP activity was unaffected. Impairment of these metastatic properties was accompanied by the decreased expression of EMT markers, including Slug, vimentin, claudin‑1 and zona occludens protein 1, whereas E‑cadherin expression was increased. The present study suggests that Gadd45β is involved in regulating the viability and the metastatic potential of CCA cells, which may be mediated by the modulation of the EMT pathway. [ABSTRACT FROM AUTHOR]

Current staging is inadequate for predicting clinical outcome of esophageal squamous cell carcinoma ( ESCC). Aberrant expression of LOXL2 and actin-related proteins plays important roles in ESCC. Here, we aimed to develop a novel molecular signature that exceeds the power of the current staging system in predicting ESCC prognosis. We found that LOXL2 colocalized with filamentous actin in ESCC cells, and gene set enrichment analysis ( GSEA) showed that LOXL2 is related to the actin cytoskeleton. An ESCC-specific protein-protein interaction ( PPI) network involving LOXL2 and actin-related proteins was generated based on genome-wide RNA-seq in 15 paired ESCC samples, and the prognostic significance of 14 core genes was analyzed. Using risk score calculation, a three-gene signature comprising LOXL2, CDH1, and FN1 was derived from transcriptome data of patients with ESCC. The high-risk three-gene signature strongly correlated with poor prognosis in a training cohort of 60 patients ( P = 0.003). In mRNA and protein levels, the prognostic values of this signature were further validated in 243 patients from a testing cohort ( P = 0.001) and two validation cohorts ( P = 0.021, P = 0.007). Furthermore, Cox regression analysis revealed that the signature was an independent prognostic factor. Compared with using the signature or TNM stage alone, the combined model significantly enhanced the accuracy in evaluating ESCC prognosis. In conclusion, our data reveal that the tumor-promoting role of LOXL2 in ESCC is mediated by perturbing the architecture of actin cytoskeleton through its PPIs. We generated a novel three-gene signature ( PPI interfaces) that robustly predicts poor clinical outcome in ESCC patients. [ABSTRACT FROM AUTHOR]

Background: Despite the identification of many signaling pathways involved in colorectal cancer (CRC) tumorigenesis, metastatic CRC still remains one of the major causes of cancer related death. NIK and IKKβ-binding protein (NIBP) is one of the key regulators of the NF-κB signaling pathway, which has been implicated in CRC metastasis. The aim of this study was to investigate the possible role of NIBP in CRC metastasis through its regulation of NF-κΒ and extracellular regulated kinase/c-Janus kinase (ERK/JNK) signaling pathways. Methods: In this study NIBP, phosphorylated (p)-p65, p-ERK1/2, and p-JNK1/2 expression was examined in 130 CRC, and 25 adenoma tissue samples were studied by immunohistochemistry. NIBP shRNA knockdown was performed in HCT116 cells, and NF-κB and ERK/JNK pathway activity was measured after TNF-α stimulation in vitro and in vivo. Results: We found that NIBP, p-p65, p-ERK1/2, and p-JNK1/2 expression was higher in late stages of CRC compared to early stages or adenomas. Expression of p-p65, p-IκBα, p-IκBβ, p-ERK1/2, and p-JNK1/2 was inhibited in TNF-α stimulated HCT116 cells following NIBP knockdown. Nevertheless, p-ERK1/2 expression in un-transfected and NIBP knockdown HCT116 cells remained the same in the absence of TNF-α stimulation. Furthermore, cell motility and invasion were reduced in HCT116 cells following NIBP knockdown even after TNF-α treatment. Finally, primary tumor weight and liver metastasis were reduced in nude mice with orthotopically transplanted NIBP knockdown of HCT116 cells. Conclusion: In conclusion, we demonstrated that NIBP knockdown reduces colorectal cancer metastasis through down-regulation of canonical NF-κΒ signaling and suppression of ERK and JNK signaling. [ABSTRACT FROM AUTHOR]

Conclusion: The large conductance Ca2+-activated K+ channels (BK) expression is decreased in the cochleae of age-related hearing loss (AHL) mice. BK channel may be associated with AHL. Objective: AHL is the most common among elderly persons. BK channels act as sensors for membrane voltage and intracellular Ca2+ and are essential for hearing. To investigate the distribution of BK channel in the cochleae of C57BL/6J mice, and the relationship between the expression of BK channel and the etiology of AHL. Methods: BK expression was studied in the cochleae of C57BL/6J mice at various ages (4, 12, 26, 52 weeks). The expressions of BK at the protein and mRNA levels were detected by immunofluorescence technique, western blot and quantitative real time PCR. Results: In comparison with 4-week-old mice, BK expressions in the cochleae at 12, 26 and 52 weeks of age were significantly and gradually decreased at both the protein and the mRNA levels. The immunofluorescence technique showed the BK channel was located in the hair cells and cells of the spiral ganglion, spiral ligament and stria vascularis and its expression also decreased with aging. [ABSTRACT FROM AUTHOR]

Fatty acid-binding protein (Fabp)-4 is a member of the FABP family. Mammalian fabp4 has been demonstrated to involve in inflammation and immunity, whereas the related data of fish fabp4 remain limited. Therefore, we further investigated the effects of fabp4 on immunity in Ctenopharyngodon idella. The fabp4 sequence spanned 405 bp was cloned first, sharing high identity to fabp4 from other fish and mammals. Fabp4 expression was the highest in the adipose tissue, followed by the heart, muscle, and liver. In vivo , lipopolysaccharide (LPS) triggered the expression of fabp4 , toll-like receptor (tlr)-22, interleukin (il)-1β, and tumor necrosis factor (tnf)-α in the kidney and spleen. In vitro , exposing C. idella CIK cells to LPS decreased their viability, and the expression of fabp4 was also increased by LPS. However, BMS309403, an inhibitor of FABP4, mitigated these effects. Furthermore, treating the cells with LPS or fabp4 overexpression plasmids resulted in reactive oxygen species (ROS) generation and upregulation of inflammatory genes expression, including tlr22 , type-I interferon (ifn-1), interferon regulatory factor (irf)-7, tnfα , il-1β , and interferon-β promoter stimulator 1. These effects were ameliorated by preincubation with BMS309403. Moreover, incubating the cells with glutathione reduced the production of ROS and the expression of inflammatory genes that were evoked by LPS and plasmid treatments. These results showed that fabp4 acts as a pro-inflammatory molecule via elevating ROS levels, providing a novel understanding of the molecular regulation of innate immunity in teleosts. [Display omitted] • Fabp4 gene was successfully cloned, sequenced and characterized from C. idella. • Fabp4 was widely distributed with variable abundance of expression among tissues. • Fabp4 regulated inflammation through ROS generation in C. idella. [ABSTRACT FROM AUTHOR]

Deregulation of the receptor tyrosine kinase Axl has been implicated in the progression of several human cancers. However, the role of Axl in prostate cancer remains poorly understood, and the therapeutic efficacy of Axl targeting remains untested. In this report we identified Axl as a new therapeutic target for prostate cancer. Axl is consistently overexpressed in prostate cancer cell lines and human prostate tumors. Interestingly, the blockage of Axl gene expression strongly inhibits proliferation, migration, invasion and tumor growth. Furthermore, inhibition of Axl expression by small interfering RNA regulates a transcriptional program of genes involved in cell survival, strikingly all connected to the nuclear factor-κB pathway. Additionally, blockage of Axl expression leads to inhibition of Akt, IKKα and IκBα phosphorylation, increasing IκBα expression and stability. Furthermore, induction of Akt phosphorylation by insulin-like growth factor 1 in Axl knockdown cells restores Akt activity and proliferation. Taken together, our results establish an unambiguous role for Axl in prostate cancer tumorigenesis with implications for prostate cancer treatment. [ABSTRACT FROM AUTHOR]

Gadd45 proteins modulate signaling in response to physiological and environmental stressors. Expression of gadd45 genes is rapidly induced by different stressors, including differentiation-inducing cytokines and genotoxic stress. Induction of gadd45 genes at the onset of myeloid differentiation suggested that Gadd45 protein(s) play a role in hematopoiesis, yet no apparent abnormalities were observed in either the bone marrow (BM) or peripheral blood compartments of mice deficient for either gadd45a or gadd45b. However, under conditions of hematological stress, including acute stimulation with cytokines, myelo-ablation and inflammation, both gadd45a-deficient and gadd45b-deficient mice exhibited deficiencies. This is discussed within the context of what is known about Gadd45 proteins in stress signaling, hematopoietic development and the innate immune response. Furthermore, myeloid enriched BM cells from gadd45a and gadd45b deficient mice were observed to be more sensitive to ultraviolet radiation (UVC), VP-16 and daunorubicin (DNR) induced apoptosis compared to wild-type (WT) cells, displaying defective G2/M arrest following exposure to UVC and VP-16, but not to DNR. Novel mechanisms that mediate the pro-survival functions of Gadd45 in hematopoietic cells following UV irradiation were demonstrated, involving activation of the Gadd45a-p38-NF-κB survival pathway and Gadd45b mediated inhibition of the stress response MKK4-JNK apoptotic pathway. The ramifications regarding the pathogenesis of different leukemias and the response of normal and malignant hematopoietic cells to chemo- and radiation-therapy, as well as other challenges to the hematopoietic compartment, are discussed. J. Cell. Physiol. 218: 26–31, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

SUMMARY: The identification of genes differentially regulated by ischemia will lead to an improved understanding of cell death pathways such as those involved in the neuronal loss observed following a stroke. Furthermore, the characterization of such pathways could facilitate the identification of novel targets for stroke therapy. We have used a novel approach to amplify differential gene expression patterns in a primary neuronal model of stroke by employing a lentiviral vector system to specifically bias the transcriptional activation of hypoxically regulated genes. Overexpression of the hypoxia-induced transcription factor subunits HIF-1α and HIF-2α elevated hypoxia-mediated transcription of many known HIF-regulated genes well above control levels. Furthermore, many potentially novel HIF-regulated genes were discovered that were not previously identified as hypoxically regulated. Most of the novel genes identified were activated by a combination of HIF-2α overexpression and hypoxic insult. These included several genes with particular importance in cell survival pathways and of potential therapeutic value. Hypoxic induction of HIF-2α may therefore be a critical factor in mediating protective responses against ischemic injury. Further investigation of the genes identified in this study may provide increased understanding of the neuronal response to hypoxia and may uncover novel therapeutic targets for the treatment of cerebral ischemia. [ABSTRACT FROM AUTHOR]

This study was designed to identify genes that regulate the transition from FSH- to LH-dependent development in the bovine dominant follicle (DF). Serial analysis of gene expression (SAGE) was used to compare the transcriptome of granulosa cells isolated from the most oestrogenic growing cohort follicle (COH), the newly selected DF and its largest subordinate follicle (SF) which is destined for atresia. Follicle diameter, follicular fluid oestradiol (E) and E:progesterone ratio confirmed follicle identity. Results show that there are 93 transcript species differentially expressed in DF granulosa cells, but only 8 of these encode proteins known to be involved in DF development. Most characterised transcripts upregulated in the DF are from tissue development genes that regulate cell differentiation, proliferation, apoptosis, signalling and tissue remodelling. Semiquantitative real-time PCR analysis confirmed seven genes with upregulated (P=0.05) mRNA expression in DF compared with both COH and SF granulosa cells. Thus, the new genes identified by SAGE and real-time PCR, which show enhanced mRNA expression in the DF, may regulate proliferation (cyclin D2; CCND2), prevention of apoptosis or DNA damage (growth arrest and DNA damage-inducible, Β; GADD45B), RNA synthesis (splicing factor, arginine/serine rich 9; SFRS9) and unknown processes associated with enhanced steroidogenesis (ovary-specific acidic protein; DQ004742) in granulosa cells of DF at the onset of LH-dependent development. Further studies are required to show whether the expression of identified genes is dysregulated when abnormalities occur during DF selection or subsequent development. [ABSTRACT FROM AUTHOR]