InteBac: An integrated bacterial and baculovirus expression vector suite

The successful production of recombinant protein for biochemical, biophysical, and structural biological studies critically depends on the correct expression organism. Currently, the most commonly used expression organisms for structural studies are Escherichia coli (~70% of all PDB structures) and the baculovirus/ insect cell expression system (~5% of all PDB structures). While insect cell expression is frequently successful for large eukaryotic proteins, it is relatively expensive and time‐consuming compared to E. coli expression. Frequently the decision to carry out a baculovirus project means restarting cloning from scratch. Here we describe an integrated system that allows simultaneous cloning into E. coli and baculovirus expression vectors using the same PCR products. The system offers a flexible array of N‐ and C‐terminal affinity, solubilization and utility tags, and the speed allows expression screening to be completed in E. coli, before carrying out time and cost‐intensive experiments in baculovirus. Importantly, we describe a means of rapidly generating polycistronic bacterial constructs based on the hugely successful biGBac system, making InteBac of particular interest for researchers working on recombinant protein complexes.     

Genetic inactivation of the polycomb repressive complex 2 in T cell acute lymphoblastic leukemia

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In this study we report the presence of loss-of-function mutations and deletions of the EZH2 and SUZ12 genes, which encode crucial components of the Polycomb repressive complex 2 (PRC2), in 25% of T-ALLs. To further study the role of PRC2 in T-ALL, we used NOTCH1-dependent mouse models of the disease, as well as human T-ALL samples, and combined locus-specific and global analysis of NOTCH1-driven epigenetic changes. These studies demonstrated that activation of NOTCH1 specifically induces loss of the repressive mark Lys27 trimethylation of histone 3 (H3K27me3) by antagonizing the activity of PRC2. These studies suggest a tumor suppressor role for PRC2 in human leukemia and suggest a hitherto unrecognized dynamic interplay between oncogenic NOTCH1 and PRC2 function for the regulation of gene expression and cell transformation.     

Estradiol and tamoxifen stimulate LAM-associated angiomyolipoma cell growth and activate both genomic and nongenomic signaling pathways

Lymphangioleiomyomatosis (LAM) is a progressive lung disease affecting almost exclusively women. The reasons for this strong gender predisposition are poorly understood. Renal angiomyolipomas occur in 50-60% of sporadic LAM patients. The smooth muscle cells of pulmonary LAM and renal angiomyolipomas are nearly indistinguishable morphologically. Here, we report the first successful cell culture of a LAM-associated renal angiomyolipoma. The cells carried inactivating mutations in both alleles of the TSC2 gene and expressed estrogen receptor , estrogen receptor , and androgen receptor. To elucidate the cellular pathways through which steroid hormones influence LAM pathogenesis, we treated the cells with both estradiol and tamoxifen. Cell growth was stimulated by estradiol, associated with phosphorylation of p44/42 MAPK at 5 min and an increase in c-myc expression at 4 h. Tamoxifen citrate also stimulated cell growth, associated with increased phosphorylation of p44/42 MAPK and expression of c-myc, indicating that tamoxifen has agonist effects on angiomyolipoma cells. This response to tamoxifen in human angiomyolipoma cells differs from prior studies of Eker rat leiomyoma cells, possibly reflecting cell type or species differences in cells lacking tuberin. Our data provide the first evidence that estradiol stimulates the growth of angiomyolipoma cells, that tamoxifen has agonist effects in angiomyolipoma cells, and that estradiol and tamoxifen impact both genomic and nongenomic signaling pathways in angiomyolipoma cells. The responsiveness of angiomyolipoma cells to estradiol may be related to the underlying reasons that LAM affects primarily women.     

A sensitive, support-vector-machine method for the detection of horizontal gene transfers in viral, archaeal and bacterial genomes

In earlier work, we introduced and discussed a generalized computational framework for identifying horizontal transfers. This framework relied on a gene's nucleotide composition, obviated the need for knowledge of codon boundaries and database searches, and was shown to perform very well across a wide range of archaeal and bacterial genomes when compared with previously published approaches, such as Codon Adaptation Index and C + G content. Nonetheless, two considerations remained outstanding: we wanted to further increase the sensitivity of detecting horizontal transfers and also to be able to apply the method to increasingly smaller genomes. In the discussion that follows, we present such a method, Wn-SVM, and show that it exhibits a very significant improvement in sensitivity compared with earlier approaches. Wn-SVM uses a one-class support-vector machine and can learn using rather small training sets. This property makes Wn-SVM particularly suitable for studying small-size genomes, similar to those of viruses, as well as the typically larger archaeal and bacterial genomes. We show experimentally that the new method results in a superior performance across a wide range of organisms and that it improves even upon our own earlier method by an average of 10% across all examined genomes. As a small-genome case study, we analyze the genome of the human cytomegalovirus and demonstrate that Wn-SVM correctly identifies regions that are known to be conserved and prototypical of all beta-herpesvirinae, regions that are known to have been acquired horizontally from the human host and, finally, regions that had not up to now been suspected to be horizontally transferred. Atypical region predictions for many eukaryotic viruses, including the alpha-, beta- and gamma-herpesvirinae, and 123 archaeal and bacterial genomes, have been made available online at http://cbcsrv.watson.ibm.com/HGT_SVM/.     

Clinical Impact of Pancreatic Metastases from Renal Cell Carcinoma: A Multicenter Retrospective Analysis

Pancreatic metastases from renal cell carcinoma are uncommon and their prognostic significance is not well defined. In this analysis we evaluated the outcome of patients with pancreatic metastases treated with either targeted therapies or local treatment to the pancreas. Patients with pancreatic metastases from renal cell carcinoma treated between 1993 and 2014 were identified from 11 European centers. Clinical records were retrospectively reviewed. Kaplan-Meier method and log-rank test were used to evaluate progression-free survival and overall survival. Cox’s proportional hazard models were used for survival analysis. In total, 276 PM patients were evaluated, including 77 (28%) patients treated by either surgery or radiotherapy to the pancreas, and 256 (93%) who received systemic therapy. Median time from nephrectomy to diagnosis of pancreatic metastases was 91 months (IQR 54–142). Disease control rate after first-line TTs was 84%, with a median progression-free survival of 12 months (95% CI 10–14). Median overall survival was 73 months (95% CI 61–86) with a 5-year OS of 58%. Median OS of patients treated with local treatment was 106 months (95% CI 78–204) with a 5-year overall survival of 75%. On multivariable analysis, nephrectomy (HR 5.31; 95%CI 2.36–11.92; p<0.0001), Memorial Sloan Kettering/International Metastatic RCC Database Consortium prognostic score (HR 1.45, 95% CI 0.94–2.23 for intermediate vs good vs risk; HR 2.76 95%, CI 1.43–5.35 for poor vs good risk p = 0.0099) and pancreatic local treatment (HR 0.48; 95%CI 0.30–0.78 p = 0.0029) were associated with overall survival. Difference in median OS between patients with PM and that reported in a matched-control group of mRCC patients with extrapancreatic metastases was statistically significant (p < .0001). Pancreatic metastases from renal cell carcinoma usually occur years after nephrectomy, are associated with an indolent behavior and a prolonged survival. Targeted therapies and locoregional approaches are active and achieve high disease control rate.