Simon G. Talbot

The Transcription Factor STAT-1 Couples Macrophage Synthesis of 25-Hydroxycholesterol to the Interferon Antiviral Response

Summary Recent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.

Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid

We have produced a plasmid expression vector for the coat protein of RNA bacteriophage MS2. The vector has been modified to introduce a unique KpnI restriction site within the coat protein gene at a site corresponding to the most radially distant feature of the bacteriophage capsid, namely the top of the N-terminal beta-hairpin (between residues 15 and 16). Insertion of DNA oligonucleotides at this site allows the production of chimeric MS2 coat proteins having foreign peptide sequences expressed as the central part of the hairpin. We have produced chimeras with a number of different peptide sequences (up to 24 amino acids in length) chosen because of their known antigenic properties. The chimeric coat proteins self-assemble into largely RNA-free phage-like capsids in Escherichia coli and can be easily disassembled and reassembled in vitro. Such peptide-presenting particles may have a number of biotechnological applications, including use as a cost-effective, synthetic vaccine. We have tested the antigenicity of one such construct in vivo in mice and have shown that these particles are immunogenic and that antibody titres against the inserted peptide epitope can be obtained.

Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

BackgroundThe present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling.MethodsThe gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR.ResultsHierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4mRNA expression failed to show correlation with any of the relevant parameters.ConclusionThe characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.

Kaposi's Sarcoma-Associated Herpesvirus vCyclin Open Reading Frame Contains an Internal Ribosome Entry Site

ABSTRACT We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposis sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

Coagulation-factor deficiencies and abnormal bleeding in Noonan's syndrome

Noonans syndrome is characterised by a dysmorphic facies, congenital heart disease, and short stature, and is inherited as an autosomal dominant trait. Because abnormal bleeding has also been reported, we investigated a group of patients for coagulation-factor deficits. Of the 72 individuals studied (37 male, 35 female, mean age 11.4 years), 47 (65%) had a history of abnormal bruising or bleeding. 29 patients (40%) had a prolonged activated partial thromboplastin time. Specific abnormalities in the intrinsic pathway of coagulation (partial factor XI:C, XII:C, and VIII:C deficiencies) were found in 36 patients (50%). Multiple abnormalities among these 36 patients included combined factor XI:C and XII:C deficiencies (4 patients) and factor XI:C and VIII:C deficiencies (4), and 1 patient had combined factor VIII:C, XI:C, and XII:C deficiency. There was poor correlation between a history of abnormal bleeding and coagulation-factor deficit. In five families, similar coagulation-factor deficiencies were present in first-degree relatives with the syndrome. The pattern of inherited bleeding abnormalities seen in Noonans syndrome suggests autosomal regulation of the intrinsic coagulation pathway.

Casein Kinase II Alpha Subunit and C1-Inhibitor Are Independent Predictors of Outcome in Patients with Squamous Cell Carcinoma of the Lung

Purpose: Gene expression profiling has been shown to be a valuable tool for prognostication and identification of cancer-associated genes in human malignancies. We aimed to identify potential prognostic marker(s) in non-small cell lung cancers using global gene expression profiles. Experimental Design: Twenty-one previously untreated patients with non-small cell lung cancer were analyzed using the Affymetrix GeneChip high-density oligonucleotide array and comparative genomic hybridization. Identified candidate genes were validated in an independent cohort of 45 patients using quantitative real-time reverse transcription-PCR and Western blot analyses. Follow-up data for these patients was collected and used to assess outcome correlations. Results: Hierarchical clustering analysis yielded three distinct subgroups based on gene expression profiling. Cluster I consisted of 4 patients with adenocarcinoma and 1 with squamous cell carcinoma (squamous cell carcinoma); clusters II and III consisted of 6 and 10 patients with squamous cell carcinoma, respectively. Outcome analysis was performed on the cluster groups containing solely squamous cell carcinoma, revealing significant differences in disease-specific survival rates. Moreover, patients having a combination of advanced Tumor-Node-Metastasis stage and assigned to the poor prognosis cluster group (cluster II) had significantly poorer outcomes. Comparative genomic hybridization analysis showed recurrent chromosomal losses at 1p, 3p, 17, 19, and 22 and gains/amplifications at 3q, 5p, and 8q, which did not vary significantly between the cluster groups. We internally and externally validated a subset of 11 cluster II (poor prognosis)-specific genes having corresponding chromosomal aberrations identified by comparative genomic hybridization as prognostic markers in an independent cohort of patients with lung squamous cell carcinoma identifying CSNK2A1 and C1-Inh as independent predictors of outcome. Conclusion: CSNK2A1 and C1-Inh are independent predictors of survival in lung squamous cell carcinoma patients and may be useful as prognostic markers.

Investigation of the mechanism of enhanced effect of EGCG on huperzine A's inhibition of acetylcholinesterase activity in rats by a multispectroscopic method.

The mechanism of enhanced effect of (-)-epigallocatechin-3-gallate (EGCG) on huperzine As (HUP) inhibition of acetylcholinesterase (AChE) activity in rats was investigated. The inhibitory effects of HUP at 10 and 5 microg/kg on AChE activity were quite weak in the whole phase. In contrast, upon addition of EGCG (100 mg/kg) to the HUP 10 and 5 microg/kg groups, remarkably enhanced inhibitory effects with maximum inhibitory percentages of 90.94 and 88.13% were observed under the same conditions. EGCG also can greatly prolong the inhibitory time. The mechanism of the enhanced effects of EGCG on HUPs inhibition of AChE activity was investigated by steady fluorescence spectroscopy, infrared spectroscopy, and ultraviolet spectroscopy. HUP hardly interacted with the main transport protein, whereas there was a very strong binding interaction between EGCG and bovine serum albumin. The enhanced transport of HUP is a possible cause of the enhanced effect of EGCG on HUP bioactivity.

Probing sequence-specific RNA recognition by the bacteriophage MS2 coat protein.

We present the results of in vitro binding studies aimed at defining the key recognition elements on the MS2 RNA translational operator (TR) essential for complex formation with coat protein. We have used chemically synthesized operators carrying modified functional groups at defined nucleotide positions, which are essential for recognition by the phage coat protein. These experiments have been complemented with modification-binding interference assays. The results confirm that the complexes which form between TR and RNA-free phage capsids, the X-ray structure of which has recently been reported at 3.0 A, are identical to those which form in solution between TR and a single coat protein dimer. There are also effects on operator affinity which cannot be explained simply by the alteration of direct RNA-protein contacts and may reflect changes in the conformational equilibrium of the unliganded operator. The results also provide support for the approach of using modified oligoribonucleotides to investigate the details of RNA-ligand interactions.

Squamous Cell Carcinoma Related Oncogene/DCUN1D1 Is Highly Conserved and Activated by Amplification in Squamous Cell Carcinomas

Chromosomal amplification at 3q is common to multiple human cancers, but has a specific predilection for squamous cell carcinomas (SCC) of mucosal origin. We identified and characterized a novel oncogene, SCC-related oncogene (SCCRO), which is amplified along the 3q26.3 region in human SCC. Amplification and overexpression of SCCRO in these tumors correlate with poor clinical outcome. The importance of SCCRO amplification in malignant transformation is established by the apoptotic response to short hairpin RNA against SCCRO, exclusively in cancer cell lines carrying SCCRO amplification. The oncogenic potential of SCCRO is underscored by its ability to transform fibroblasts (NIH-3T3 cells) in vitro and in vivo. We show that SCCRO regulates Gli1--a key regulator of the hedgehog (HH) pathway. Collectively, these data suggest that SCCRO is a novel component of the HH signaling pathway involved in the malignant transformation of squamous cell lineage.

Lessons learned from simultaneous face and bilateral hand allotransplantation.

Summary: The performance of simultaneous vascularized composite allotransplantation procedures on patients requiring both the face and bilateral hands remains controversial. The authors present their separate institutional experiences with this challenging procedure in the interests of dispelling misconceptions regarding this intervention and forwarding their understanding of the issues related to concomitant vascularized composite allotransplantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.