Polony analysis of gene expression in ES cells and blastocysts

Expression profiling of stem cells is challenging due to their small numbers and heterogeneity. The PCR colony (polony) approach has theoretical advantages as an assay for stem cells but has not been applied to small numbers of cells. An assay has been developed that is sensitive enough to detect mRNAs from small numbers of ES cells and from fractions of a single mouse blastocyst. Genes assayed include Oct3, Rex1, Nanog, Cdx2 and GLUT-1. The assay is highly sensitive so that multiple mRNAs from a single blastocyst were easily detected in the same assay. In its present version, the assay is an attractive alternative to conventional RT–PCR for profiling small populations of stem cells. The assay is also amenable to improvements that will increase its sensitivity and ability to analyze many cDNAs simultaneously

AMPK Regulation of Mouse Oocyte Meiotic Resumption in Vitro

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200–212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2′-Deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro

Metastatic disease to the breast: the Washington University experience

<p>Abstract</p> <p>Background</p> <p>Metastases to the breast occur rarely, but may be increasing in incidence as patients live longer with malignant diseases. The aim of this study is to characterize metastatic disease to the breast and to describe the management and prognosis of patients who present with this diagnosis.</p> <p>Methods</p> <p>A retrospective review of our institution's pathology and breast cancer databases was performed in order to identify patients with breast malignancies that were not of primary breast origin. Chart review provided additional information about the patients' primary malignancies and course of illness.</p> <p>Results</p> <p>Between 1991 and 2006, eighteen patients with metastatic disease to the breast of non-hematologic origin were identified and all had charts available for review. Among the 18 patients with disease metastatic to the breast, tissues of origin included 3 ovarian, 6 melanoma, 3 medullary thyroid, 3 pulmonary neuroendocrine, 1 pulmonary small cell, 1 oral squamous cell, and 1 renal cell. Overall mean survival after diagnosis of metastatic disease to the breast was 22.4 months. Treatment of metastases varied and included combinations of observation, surgery, radiation, and chemotherapy. Five patients (27.8%) required a change in management of their breast disease for local control.</p> <p>Conclusion</p> <p>Due to the variable course of patients with metastatic disease, a multi-disciplinary approach is necessary for each patient with disease metastatic to the breast to determine optimal treatment. Based on our review, many patients survive for long periods of time and local treatment of metastases to the breast may be beneficial in these patients to prevent local complications.</p

Myeloid-specific Asxl2 deletion limits diet-induced obesity by regulating energy expenditure

We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells\u27 inflammatory phenotype may impact obesity and its complications

Comparative analysis of diagnostic performance, feasibility and cost of different test-methods for thyroid nodules with indeterminate cytology

Since it is impossible to recognize malignancy at fine needle aspiration (FNA) cytology in indeterminate thyroid nodules, surgery is recommended for all of them. However, cancer rate at final histology is < 30%. Many different test-methods have been proposed to increase diagnostic accuracy in such lesions, including Galectin-3-ICC (GAL-3-ICC), BRAF mutation analysis (BRAF), Gene Expression Classifier (GEC) alone and GEC+BRAF, mutation/fusion (M/F) panel, alone, M/F panel+miRNA GEC, and M/F panel by next generation sequencing (NGS), FDG-PET/CT, MIBI-Scan and TSHR mRNA blood assay. We performed systematic reviews and meta-analyses to compare their features, feasibility, diagnostic performance and cost. GEC, GEC+BRAF, M/F panel+miRNA GEC and M/F panel by NGS were the best in ruling-out malignancy (sensitivity = 90%, 89%, 89% and 90% respectively). BRAF and M/F panel alone and by NGS were the best in ruling-in malignancy (specificity = 100%, 93% and 93%). The M/F by NGS showed the highest accuracy (92%) and BRAF the highest diagnostic odds ratio (DOR) (247). GAL-3-ICC performed well as rule-out (sensitivity = 83%) and rule-in test (specificity = 85%), with good accuracy (84%) and high DOR (27) and is one of the cheapest (113 USD) and easiest one to be performed in different clinical settings. In conclusion, the more accurate molecular-based test-methods are still expensive and restricted to few, highly specialized and centralized laboratories. GAL-3-ICC, although limited by some false negatives, represents the most suitable screening test-method to be applied on a large-scale basis in the diagnostic algorithm of indeterminate thyroid lesions