Impaired Akt phosphorylation in insulin-resistant human muscle is accompanied by selective and heterogeneous downstream defects.

AIMS/HYPOTHESIS: Muscle insulin resistance, one of the earliest defects associated with type 2 diabetes, involves changes in the phosphoinositide 3-kinase/Akt network. The relative contribution of obesity vs insulin resistance to perturbations in this pathway is poorly understood. METHODS: We used phosphospecific antibodies against targets in the Akt signalling network to study insulin action in muscle from lean, overweight/obese and type 2 diabetic individuals before and during a hyperinsulinaemic-euglycaemic clamp. RESULTS: Insulin-stimulated Akt phosphorylation at Thr309 and Ser474 was highly correlated with whole-body insulin sensitivity. In contrast, impaired phosphorylation of Akt substrate of 160 kDa (AS160; also known as TBC1D4) was associated with adiposity, but not insulin sensitivity. Neither insulin sensitivity nor obesity was associated with defective insulin-dependent phosphorylation of forkhead box O (FOXO) transcription factor. In view of the resultant basal hyperinsulinaemia, we predicted that this selective response within the Akt pathway might lead to hyperactivation of those processes that were spared. Indeed, the expression of genes targeted by FOXO was downregulated in insulin-resistant individuals. CONCLUSIONS/INTERPRETATION: These results highlight non-linearity in Akt signalling and suggest that: (1) the pathway from Akt to glucose transport is complex; and (2) pathways, particularly FOXO, that are not insulin-resistant, are likely to be hyperactivated in response to hyperinsulinaemia. This facet of Akt signalling may contribute to multiple features of the metabolic syndrome.

Conditional activation of Pik3ca(H1047R) in a knock-in mouse model promotes mammary tumorigenesis and emergence of mutations.

Oncogenic mutations in PIK3CA, which encodes the phosphoinositide-3-kinase (PI3K) catalytic subunit p110α, occur in ∼25% of human breast cancers. In this study, we report the development of a knock-in mouse model for breast cancer where the endogenous Pik3ca allele was modified to allow tissue-specific conditional expression of a frequently found Pik3ca(H1047R) (Pik3ca(e20H1047R)) mutant allele. We found that activation of the latent Pik3ca(H1047R) allele resulted in breast tumors with multiple histological types. Whole-exome analysis of the Pik3ca(H1047R)-driven mammary tumors identified multiple mutations, including Trp53 mutations that appeared spontaneously during the development of adenocarinoma and spindle cell tumors. Further, we used this model to test the efficacy of GDC-0941, a PI3K inhibitor, in clinical development, and showed that the tumors respond to PI3K inhibition.

AVAglio: Phase 3 trial of bevacizumab plus temozolomide and radiotherapy in newly diagnosed glioblastoma multiforme.

Despite treatment with the current standard-of-care therapies, patients with newly diagnosed glioblastoma multiforme (GBM) exhibit dismal prognoses. Bevacizumab has demonstrated activity in patients with recurrent GBM and phase 2 trials indicate that the combination of bevacizumab with standard-of-care therapy is feasible and active for patients with newly diagnosed GBM. Bevacizumab has been granted US approval for use as single-agent therapy for patients with progressive GBM following prior therapy, although it has not received approval for use in patients with GBM in Europe. Phase 3 studies have been initiated in patients with newly diagnosed GBM and are currently recruiting patients. We describe the protocol for the AVAglio phase 3 registration trial, which is designed to evaluate the efficacy and safety of combining bevacizumab with standard-of-care therapy in patients with newly diagnosed GBM.

Regional variation in gene expression in the healthy colon is dysregulated in ulcerative colitis.

OBJECTIVE: To investigate differential intestinal gene expression in patients with ulcerative colitis and in controls. DESIGN: Genome-wide expression study (41,058 expression sequence tags, 215 biopsies). SETTING: Western General Hospital, Edinburgh, UK, and Genentech, San Francisco, USA. PATIENTS: 67 patients with ulcerative colitis and 31 control subjects (23 normal subjects and 8 patients with inflamed non-inflammatory bowel disease biopsies). INTERVENTIONS: Paired endoscopic biopsies were taken from 5 specific anatomical locations for RNA extraction and histology. The Agilent microarray platform was used and confirmation of results was undertaken by real time polymerase chain reaction and immunohistochemistry. RESULTS: In healthy control biopsies, cluster analysis showed differences in gene expression between the right and left colon. (chi(2) = 25.1, p<0.0001). Developmental genes, homeobox protein A13 (HOXA13), (p = 2.3x10(-16)), HOXB13 (p<1x10(-45)), glioma-associated oncogene 1 (GLI1) (p = 4.0x10(-24)), and GLI3 (p = 2.1x10(-28)) primarily drove this separation. When all ulcerative colitis biopsies and control biopsies were compared, 143 sequences had a fold change of >1.5 in the ulcerative colitis biopsies (0.01>p>10(-45)) and 54 sequences had a fold change of <-1.5 (0.01>p>10(-20)). Differentially upregulated genes in ulcerative colitis included serum amyloid A1 (SAA1) (p<10(-45)) the alpha defensins 5 and 6 (DEFA5 and 6) (p = 0.00003 and p = 6.95x10(-7), respectively), matrix metalloproteinase 3 (MMP3) (p = 5.6x10(-10)) and MMP7 (p = 2.3x10(-7)). Increased DEFA5 and 6 expression was further characterised to Paneth cell metaplasia by immunohistochemistry and in situ hybridisation. Sub-analysis of the inflammatory bowel disease 2 (IBD2) and IBD5 loci, and the ATP-binding cassette (ABC) transporter genes revealed a number of differentially regulated genes in the ulcerative colitis biopsies. CONCLUSIONS: Key findings are the expression gradient in the healthy adult colon and the involvement of novel gene families, as well as established candidate genes in the pathogenesis of ulcerative colitis.

Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria.

Tumor cells with stem cell-like properties can be cultured from human glioblastomas by using conditions that select for the expansion of neural stem cells. We generated cell lines from glioblastoma specimens with the goal to obtain model systems for glioma stem cell biology. Unsupervised analysis of the expression profiles of nine cell lines established under neural stem cell conditions yielded two distinct clusters. Four cell lines were characterized by the expression of neurodevelopmental genes. They showed a multipotent differentiation profile along neuronal, astroglial and oligodendroglial lineages, grew spherically in vitro, expressed CD133 and formed highly invasive tumors in vivo. The other five cell lines shared expression signatures enriched for extracellular matrix-related genes, had a more restricted differentiation capacity, contained no or fewer CD133+ cells, grew semiadherent or adherent in vitro and displayed reduced tumorigenicity and invasion in vivo. Our findings show that stable, multipotent glioblastoma cell lines with a full stem-like phenotype express neurodevelopmental genes as a distinctive feature, which may offer therapeutic targeting opportunities. The generation of another distinct cluster of cell lines showing similarly homogeneous profiling but restricted stem cell properties suggests that different phenotypes exist, each of which may lead to the typical appearance of glioblastoma.

The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Optimized T7 amplification system for microarray analysis.

Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.

CPT-EIA assays for the detection of vancomycin resistant vanA and vanB genes in enterococci.

Cycling Probe Technology (CPT) was combined with a colorimetric enzyme-immuno assay (EIA) to develop two assays for the detection of vanA and vanB genes in vancomycin resistant enterococci (VRE). The CPT-EIA assay employs a gene-specific fluorescein labeled DNA-RNA-DNA probe that gets cleaved within the probe : target duplex. The cleaved DNA probe fragments dissociate from the target, making it available for further cycling. Following the separation of cleaved probe fragments, anti-fluorescein-horseradish peroxidase antibodies are used for the detection of uncleaved probes. The two CPT-EIA assays were used to screen a collection of 440 clinical isolates (Modrusan et al., 1999). All of the 154 VanA and 131 VanB isolates were correctly identified in the vanA and vanB CPT-EIA, respectively. The VanA and VanB isolates were differentiated from vancomycin sensitive enterococci (VSE) and also from the VanC isolates. In addition, an accurate VRE detection in the CPT-EIA assay was shown with cultures grown on eight different media.

Detection of vancomycin resistant genes vanA and vanB by cycling probe technology.

Cycling Probe Technology (CPT) has been used to develop gene-based assays for detection of vancomycin resistance genes vanA and vanB in enterococci (VRE). Cycling Probe Technology utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme RNase H when hybridized to its complementary DNA target. Conversion of full-length probe into the cleaved probe fragments is the basis for detection and quantification of the CPT reaction. Two gene-specific probes, each one unique to either the vanA or vanB gene, were utilized for development of vanA and vanB CPT assays, respectively. Both vanA and vanB CPT assays were used to determine the presence or absence of the corresponding gene in 440 clinical enterococcal isolates. The presence of vanA and vanB gene sequences was detected in 154 and 131 isolates, respectively. Phenotypic characterization of all isolates was determined through interpretation of conventional susceptibility data obtained with the disk diffusion method. Comparison between disk diffusion characterization and CPT assays revealed 11 discrepant isolates. The identity of these isolates was resolved by polymerase chain reaction (PCR) which confirmed the vanA and vanB CPT assay data. Therefore, compared to conventional phenotyping, both the vanA and vanB CPT assays appeared superior for accurate identification of VanA and VanB isolates.

Spermine-mediated improvement of cycling probe reaction.

Cycling probe technology (CPT) represents a simple method for detection of DNA target sequences. Cycling probe technology utilizes a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target. Probe cleavage in the presence or absence of target generates CPT product or background, respectively. Addition of non-homologous DNA into the CPT reaction affects the background and CPT product. Low amounts of human DNA (4-40 ng) result in high background while higher amounts (40-400 ng) inhibit the reaction. The simultaneous addition of spermine and EGTA into the CPT reaction containing human DNA resulted in a significant release of the inhibition and a reduction of background. The presence of spermine alone caused an increase of probe cleavage whereas addition of EGTA increased the specificity of the CPT. A possible mechanism by which spermine could lead to this improvement of CPT has been proposed. Using a membrane-binding assay, the authors demonstrated that human DNA competes with the probe for binding to RNase H. Furthermore, by using a DNA-agarose column, it has been shown that such RNase H-DNA binding can be disrupted by spermine. Within the CPT reaction, similar spermine-mediated displacement of RNase H from human DNA could lead to an improved CPT efficiency.

The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium.

Ovule development in Arabidopsis involves the formation of three morphologically defined proximal-distal pattern elements. Integuments arise from the central pattern element. Analysis of Bell 1 (Bel 1) mutant ovules indicated that BEL1 was required for integument development. Cloning of the BEL1 locus reveals that it encodes a homeodomain transcription factor. Prior to integument initiation, BEL1 RNA localizes to the central domain, providing molecular evidence for a central pattern element. Therefore, proximal-distal patterning of the ovule involves the regulated expression of the BEL1 gene that controls integument morphogenesis. A model for BEL1 function is evaluated with regard to new data showing the expression pattern of the floral homeotic gene AGAMOUS (AG) early in wild-type and BEL1 ovule development.

Homeotic Transformation of Ovules into Carpel-like Structures in Arabidopsis.

Ovules are specialized reproductive organs that develop within the carpels of higher plants. In Arabidopsis, mutations in two genes, BELL1 (BEL1) and APETALA2 (AP2), disrupt ovule development. In Bel1 ovules, the inner integument fails to form, the outer integument develops abnormally, and the embryo sac arrests at a late stage of megagametogenesis. During later stages of ovule development, cells of the outer integument of a Bel1 ovule sometimes develop into a carpel-like structure with stigmatic papillae and second-order ovules. The frequency of carpel-like structures was highest when plants were grown under conditions that normally induced flowering and was correlated with ectopic expression in the ovule of AGAMOUS (AG), an organ-identity gene required for carpel formation. Together, these results suggested that BEL1 negatively regulates AG late in ovule development. Likewise, mutants homozygous for the strong AP2 allele ap2-6 sometimes displayed structures with carpel-like features in place of ovules. However, such abnormal Ap2 ovules are much less ovulelike in morphology and form earlier than the Bel1 carpel-like structures. Because one role of the AP2 gene is to negatively regulate AG expression early in flower development, it is possible that AP2 works in a similar manner in the ovule. A novel ovule phenotype observed in Bel1/Ap2-6 double mutants suggested that BEL1 and AP2 genes function independently during ovule development.