We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.
Solid-state nanopores bear great potential to be used to probe single proteins; however, the passage of proteins through nanopores was found to be complex, and unexpected translocation behavior with respect to the passage direction, rate, and duration was observed. Here we study the translocation of a model protein (avidin) through silicon nitride nanopores focusing on the electrokinetic effects that facilitate protein transport across the pore. The nanopore zeta potential zeta(pore) and the protein zeta potential zeta(protein) are measured independently as a function of solution pH. Our results reveal that electroosmotic transport may enhance or dominate and reverse electrophoretic transport in nanopores. The translocation behavior is rationalized by accounting for the charging states of the protein and the pore, respectively; the resulting translocation direction can be predicted according to the difference in zeta potentials, zeta(protein) - zeta(pore). When electrophoresis and electroosmosis cancel each other out, diffusion becomes an effective (and bias-independent) mechanism which facilitates protein transport across the pore at a significant rate.
Mycobacterium tuberculosis (M. tuberculosis) infections cause 9.0 million new tuberculosis (TB) cases and 1.5 million deaths annually1. To search for sequence variants that confer risk of TB we tested 28.3 million variants identified through whole-genome sequencing of 2,636 Icelanders for association with TB (8,162 cases and 277,643 controls), pulmonary TB (PTB), and M. tuberculosis infection. We found association of three sequence variants in the HLA class II region: rs557011[T] (MAF=40.2%) with M. tuberculosis infection (OR =1.14, P=3.1×10-13) and PTB (OR=1.25, P=5.8×10-12) and rs9271378[G] (MAF=32.5%) with PTB (OR=0.78, P=2.5×10-12), both located between HLA-DQA1 and HLA-DRB1. Finally, a missense variant p.Ala210Thr in HLA-DQA1, (MAF=19.1%, rs9272785) shows association with M. tuberculosis infection (P=9.3×10-9, OR=1.14). The association of these variants with PTB was replicated in large samples of European ancestry from Russia and Croatia (P< 5.9×10-4). These findings demonstrate that the HLA class II region contributes to the complex genetic risk of tuberculosis, possibly through reduced presentation of protective M. tuberculosis antigens to T cells.
Measurements in stationary or mobile phases are fundamental principles in protein analysis. Although the immobilization of molecules on solid supports allows for the parallel analysis of interactions, properties like size or shape are usually inferred from the molecular mobility under the influence of external forces. However, as these principles are mutually exclusive, a comprehensive characterization of proteins usually involves a multi-step workflow. Here we show how these measurement modalities can be reconciled by tethering proteins to a surface via dynamically actuated nanolevers. Short DNA strands, which are switched by alternating electric fields, are employed as capture probes to bind target proteins. By swaying the proteins over nanometre amplitudes and comparing their motional dynamics to a theoretical model, the protein diameter can be quantified with Angström accuracy. Alterations in the tertiary protein structure (folding) and conformational changes are readily detected, and even post-translational modifications are revealed by time-resolved molecular dynamics measurements.
The objective of this article was to analyze 40 years of experience of colon interposition in the surgical treatment of caustic esophageal strictures from the standpoints of our long-term personal experience. Colon interposition has proved to be the most suitable type of reconstruction for esophageal corrosive strictures. The choice of colon graft is based on the pattern of blood supply, while the type of anastomosis is determined by the stricture level and the part of colon used for reconstruction. In the period between 1964 and 2004, colon interposition was performed in 336 patients with a corrosively scared esophagus, using the left colon in 76.78% of the patients. In 87.5% a colon interposition was performed, while in the remaining patients an additional esophagectomy with colon interposition had to be done. Hypopharyngeal strictures were present in 24.10% of the patients. Long-term follow-up results were obtained in the period between 1 to up to 30 years. Early postoperative complications occurred in 26.48% of patients, among which anastomosic leakage was the most common. The operative mortality rate was 4.16% and late postoperative complications were present in 13.99% of the patients. A long-term follow up obtained in 84.82% of the patients found excellent functional results in 75.89% of them. We conclude that a colon graft is an excellent esophageal substitute for patients with esophageal corrosive strictures, and when used by experienced surgical teams it provides a low rate of postoperative morbidity and mortality, and long-term good and functional quality of life.
A label-free method for the analysis of interactions of proteins with surface-tethered ligands is introduced. Short DNA levers are electrically actuated on microelectrodes by ac potentials, and their switching dynamics are measured in real-time by fluorescence energy transfer. Binding of proteins to ligands attached to the top of the DNA levers is detected by time-resolved measurements of the levers' dynamic motion. We demonstrate the quantitation of binding kinetics (k(on), k(off) rate constants), dissociation constants (K(D) in the pM regime), and the influence of competitive binders (EC(50) values). Moreover, the "switchSENSE" method reveals avidity effects and allows discriminating between analytes with one or more binding sites. In a comparative study, interactions of six hexa-histidine-tagged proteins with tris-nitrilotriacetic acid (NTA(3)) ligands are quantitated. Their binding kinetics and affinities are found to vary over up to 2 orders of magnitude, evidencing that the proteins' individual chemical environments significantly influence the His(6)-NTA(3) interaction.
Recent studies have indicated that the interleukin-12/interferon-g (IFN-g) axis is important in mycobacterial infection susceptibility. Using an intronic (CA) n polymorphic microsatellite marker within the IFN-g receptor-1 (IFNGR1) gene, we have compared the allelic frequencies of this marker in hospitalized tuberculosis patients (n ¼ 120) with that of controls (n ¼ 87) from Rijeka, Croatia. We identified 13 (CA) n alleles in the tuberculosis patients, whereas only 10 were found in the controls. A significant difference between one allelic marker and the control group was observed (P ¼ 0.02, 95% confidence interval 0.14-0.94), suggesting a possible protective association. In contrast, several other allelic markers showed a trend towards association with the disease. We also found a trend towards an increased frequency in homozygosity of one allelic marker in patients (11.7%) as compared with controls (4.6%). We conclude that there is no evidence for disease association of the IFNGR1 gene marker in Mendeliantype (single-allele) inheritance. However, our results also suggest that unidentified allelic variations in the IFNGR1 gene might elevate or decrease the risk in this ethnic population, as a part of the multigenic predisposition to tuberculosis.
Toll-like receptors (TLR) are overexpressed on many types of cancer cells, including colorectal cancer cells, but little is known about the functional relevance of these immune regulatory molecules in malignant settings. Here, we report frequent single-nucleotide polymorphisms (SNP) in the flagellin receptor TLR5 and the TLR downstream effector molecules MyD88 and TIRAP that are associated with altered survival in a large cohort of Caucasian patients with colorectal cancer (n ¼ 613). MYD88 rs4988453, a SNP that maps to a promoter region shared with the acetyl coenzyme-A acyl-transferase-1 (ACAA1), was associated with decreased survival of patients with colorectal cancer and altered transcriptional activity of the proximal genes. In the TLR5 gene, rs5744174/ F616L was associated with increased survival, whereas rs2072493/N592S was associated with decreased survival. Both rs2072493/N592S and rs5744174/F616L modulated TLR5 signaling in response to flagellin or to different commensal and pathogenic intestinal bacteria. Notably, we observed a reduction in flagellin-induced p38 phosphorylation, CD62L shedding, and elevated expression of interleukin (IL)-6 and IL-1b mRNA in human primary immune cells from TLR5 616LL homozygote carriers, as compared with 616FF carriers. This finding suggested that the well-documented effect of cytokines like IL-6 on colorectal cancer progression might be mediated by TLR5 genotype-dependent flagellin sensing. Our results establish an important link between TLR signaling and human colorectal cancer with relevance for biomarker and therapy development. Cancer Res; 73(24); 7232-42. Ó2013 AACR.
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