Highly Parallel Genome-Wide Expression Analysis of Single Mammalian Cells

We have developed a high-throughput amplification method for generating robust gene expression profiles using single cell or low RNA inputs.The method uses tagged priming and template-switching, resulting in the incorporation of universal PCR priming sites at both ends of the synthesized cDNA for global PCR amplification. Coupled with a whole-genome gene expression microarray platform, we routinely obtain expression correlation values of R(2)~0.76-0.80 between individual cells and R(2)~0.69 between 50 pg total RNA replicates. Expression profiles generated from single cells or 50 pg total RNA correlate well with that generated with higher input (1 ng total RNA) (R(2)~0.80). Also, the assay is sufficiently sensitive to detect, in a single cell, approximately 63% of the number of genes detected with 1 ng input, with approximately 97% of the genes detected in the single-cell input also detected in the higher input.In summary, our method facilitates whole-genome gene expression profiling in contexts where starting material is extremely limiting, particularly in areas such as the study of progenitor cells in early development and tumor stem cell biology

Effects of [mu]-opioids on neurons of the medical geniculate nucleus

Immunohistochemical and hybridization studies have revealed the presence of μ-, but not k- and δ-opioid receptors in the medial geniculate body. Using whole-cell patch clamp techniques, I examined the effects of opioid agonists on gerbil MGB neurons (P9-P16) in vitro. The opioid effects were concentration-dependent. Opioids produced different actions on the input conductance (G[sub i]) when applied in low, compared with high concentrations. The increase in G[sub i] might be due to increase in K⁺ conductance whereas the decrease in G[sub i] might be due to l[sub H] inactivation. When Gi was increased, the reversal potential was —65 mV; this implicates opioid actions on K+ , among other ion channels. In the case of decreased G[sub i], the opioid currents did not reverse from -100 mV to -50 mV, implying the involvement of cationic channels, other than K⁺ . DAMGO, a μ-selective opioid agonist, had a reversal potential that was similar to that observed when morphine increased G[sub i], implying that opioids activate u-opioid receptors in MGB neurons. Tetrodotoxin altered the concentration-dependent action of morphine. Here, the suggestion is morphine's actions involve neurons that presynaptic to the patch clamped neuron. Morphine application blocked spike-frequency adaptation and reduced firing rates in response to depolarizing current pulse injection. Morphine application may block Ca²⁺-mediated K⁺ channels that inhibited spike-frequency adaptation. Such blockade may be expected to increase the spike frequency. However, the increased conductance due to morphine would shunt the Na⁺ current, resulting in a lower spike frequency. The results of this study have revealed that opioids have both excitatory and inhibitory effects on MGB neurons.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat

Expression and function of HOXA genes in the normal ovary and ovarian carcinoma

Homeobox genes, which code for families of transcription factors, act at the top of genetic hierarchies. HOX genes specify positional identity during development, and in adult tissues, regulate differentiation and proliferation. Most ovarian carcinomas are derived from the ovarian surface epithelium (OSE). In contrast to other carcinomas, OSE differentiates further with neoplastic progression and acquires Mtillerian duct-derived epithelial properties. I tested the hypothesis that growth and differentiation in ovarian carcinogenesis are regulated by HOX genes. I studied three genes: HOXA4, A7 and A9. HOXA7 has been reported to play a role in the differentiation of ovarian cancers, while HOXA9 is expressed in normal oviductal epithelium which resembles ovarian serous adenocarcinomas. Furthermore, microarray studies showed that HOXA4 and HOXA7, but not HOXA9, were highly expressed in ovarian carcinomas. In this study, I produced a well-characterized polyclonal anti-HOXA7 antibody and investigated its distribution in ovarian carcinomas. HOXA7 expression was not related to grade. HOXA7 was expressed mainly in the cytoplasm, however, nuclear staining correlated with prognosis, suggesting that HOXA7 in different subcellular locations has different functions. HOXA4 expression in normal OSE was not consistently related to proliferation but increased at low cell density, low [Ca ] and with EGF stimulation, and HOXA4 siRNA enhanced motility. In ovarian cancer cultures, HOXA4 siRNA enhanced motility, increased proliferation in three-dimensional cultures and reduced CA125 secretion. These results imply that HOXA4 regulates differentiation and cell-cell interrelationships, and that increased HOXA4 expression may reflect a response to maintain homeostasis during neoplastic progression. The study was expanded to investigate HOXA7 distribution in human ovarian folliculogenesis. Granulosa cells were negative in primordial, but positive in primary follicles. As they matured, HOXA7 relocated to the cytoplasm. HOXA7 expression in granulosa cells increased with proliferation, and in culture it was regulated by GDF-9. These results indicate that, in folliculogenesis, HOXA7 expression undergoes cell typeand stage-specific changes, which are regulated, in part, by GDF-9. In summary, HOX genes contribute to the development of ovarian follicles and carcinomas. Controlling HOX genes may be one of the critical methods to overcome diseases associated with ovarian folliculogenesis and carcinogenesis.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

Serine Protease HtrA1 Associates with Microtubules and Inhibits Cell Migration▿

HtrA1 belongs to a family of serine proteases found in organisms ranging from bacteria to humans. Bacterial HtrA1 (DegP) is a heat shock-induced protein that behaves as a chaperone at low temperature and as a protease at high temperature to help remove unfolded proteins during heat shock. In contrast to bacterial HtrA1, little is known about the function of human HtrA1. Here, we report the first evidence that human HtrA1 is a microtubule-associated protein and modulates microtubule stability and cell motility. Intracellular HtrA1 is localized to microtubules in a PDZ (PSD95, Dlg, ZO1) domain-dependent, nocodazole-sensitive manner. During microtubule assembly, intracellular HtrA associates with centrosomes and newly polymerized microtubules. In vitro, purified HtrA1 promotes microtubule assembly. Moreover, HtrA1 cosediments and copurifies with microtubules. Purified HtrA1 associates with purified α- and β-tubulins, and immunoprecipitation of endogenous HtrA1 results in coprecipitation of α-, β-, and γ-tubulins. Finally, downregulation of HtrA1 promotes cell motility, whereas enhanced expression of HtrA1 attenuates cell motility. These results offer an original identification of HtrA1 as a microtubule-associated protein and provide initial mechanistic insights into the role of HtrA1 in theregulation of cell motility by modulating microtubule stability

A systematic review of observational studies of trifluridine/tipiracil (TAS-102) for metastatic colorectal cancer

Background: The treatment options for patients with therapy refractory metastatic colorectal cancer (mCRC) are sparse. TAS-102 (FTD/TPI) is a new oral anti-tumour agent composed of a nucleoside analogue, trifluridine, and a thymidine phosphorylase inhibitor, tipiracil, indicated for patients with mCRC who are refractory to standard therapies. This study summarizes published and unpublished experience with FTD/TPI in clinical practice settings. Patients and methods: The Medline/PubMed, Embase and Cochrane Library databases were searched to identify observational studies on FTD/TPI monotherapy for mCRC. Papers describing use of FTD/TPI monotherapy outside clinical trials in series of patients evaluable for effectiveness were eligible. The outcomes of interest were median progression free survival (mPFS), median overall survival (mOS) as well as mean PFS time restricted to six months (PFS6m) and mean OS time restricted to one year (OS1y). Results of the pooled analyses of observational studies were compared to the results of the Japanese phase II trial and the two phase III trials, RECOURSE and TERRA. Results: Seven published and two unpublished studies with 1008 patients from 64 centres were included for analysis. The pooled mPFS was 2.2 months (95% CI 2.1 to 2.3 months), and the pooled mOS was 6.6 months (95% CI 6.1 to 7.1 months). PFS6m was 2.9 months (95% CI 2.6 to 3.1 months) and OS1y was 6.8 (95% CI 6.0 to 7.5) months. While these results all reflect RECOURSE, the pooled mOS is lower than in the phase II trial and the OS1y is inferior to both the phase II trial and TERRA. Conclusion: This systematic review and a meta-analysis indicates that in real life settings, the survival benefit of FTD/TPI monotherapy in patients with therapy refractory mCRC reflects the outcomes in RECOURSE but is inferior to outcomes in the two Asian efficacy trials.What is already knownTAS 102 (Lonsurf) is an oral fixed dose combination of trifluridine (FTD) and tipiracil (TPI) indicated as salvage-line treatment in patients with therapy refractory metastatic colorectal cancer (mCRC). A Japanese phase II trial and two phase III trials, RECOURSE and TERRA, demonstrated that FTD/TPI prolonged overall survival.What this study addsThis systematic review and meta-analysis of real life data from 64 sites indicates that the effectiveness in daily clinical practice settings of FTD/TPI monotherapy in late stage mCRC reflects the outcomes in RECOURCE but is inferior to the outcomes in the Japanese phase II trial and TERRA. TAS 102 (Lonsurf) is an oral fixed dose combination of trifluridine (FTD) and tipiracil (TPI) indicated as salvage-line treatment in patients with therapy refractory metastatic colorectal cancer (mCRC). A Japanese phase II trial and two phase III trials, RECOURSE and TERRA, demonstrated that FTD/TPI prolonged overall survival. This systematic review and meta-analysis of real life data from 64 sites indicates that the effectiveness in daily clinical practice settings of FTD/TPI monotherapy in late stage mCRC reflects the outcomes in RECOURCE but is inferior to the outcomes in the Japanese phase II trial and TERRA.</p

HOX cofactors expression and regulation in the human ovary

Background: HOX cofactors enhance HOX binding affinities and specificities and increase HOX's unique functional activities. The expression and the regulation of HOX cofactors in human ovaries are unknown. Methods In this study, the expression of HOX cofactors, PBX1, PBX2, and MEIS1/2, were examined by using RT-PCR, immunofluorescence in cultured immortalized human granulosa (SVOG) cells. The distribution of these HOX cofactors in human ovaries was examined by immunohistochemistry. The effects of growth differentiation factor-9 (GDF-9) and follicle-stimulating hormone (FSH) on PBX2 in SVOG cells were investigated by western blot analysis. Binding activities of HOXA7 and PBX2 to the specific sequences in granulosa cells were determined by electrophoretic mobility shift assay (EMSA). Results and conclusion In SVOG cells, PBX1, PBX2 and MEIS1/2 were expressed during cell culture. In normal human ovaries, PBX1 and MEIS1/2 were expressed in granulosa cells at essentially all stages of follicular development. These cofactors were expressed in the nuclei of the granulosa cells from the primordial to the secondary follicles, whereas beyond multilayered follicles was observed in the cytoplasm. The co-expression of PBX1 and MEIS1/2 in granulosa cells in normal human ovaries suggested that MEIS1/2 might control PBX1 sublocalization, as seen in other systems. PBX2 was not expressed or weakly expressed in the primordial follicles. From the primary follicles to the preovulatory follicles, PBX2 expression was inconsistent and the expression was found in the granulosa cell nuclei. The PBX2 expression pattern is similar to HOXA7 expression in ovarian follicular development. Furthermore, FSH down-regulated, GDF-9 did not change PBX2 expression, but co-treatment of the granulosa cells with FSH and GDF-9 up-regulated PBX2 expression. These results implicated a role for PBX2 expression in the steroidogenic activities of granulosa cells in humans. Moreover, PBX2 and HOXA7 bound together to the Pbx sequence, but not to the EMX2 promoter sequence, in SVOG cells. Our findings indicate that HOX cofactors expression in normal human ovary is temporally and spatially specific and regulated by FSH and GDF-9 in granulosa cells. HOX proteins may use different HOX cofactors, depending on DNA sequences that are specific to the granulosa cells.Medicine, Faculty ofObstetrics and Gynaecology, Department ofNon UBCReviewedFacult

Top-15 signature genes.

<p>The table lists the genes with over-expression indicating higher hazard of death or recurrence, identified by Net-Cox and in the consensus ranking across the three datasets.</p