Approaching disorder-free transport in high-mobility conjugated polymers.

Conjugated polymers enable the production of flexible semiconductor devices that can be processed from solution at low temperatures. Over the past 25 years, device performance has improved greatly as a wide variety of molecular structures have been studied. However, one major limitation has not been overcome; transport properties in polymer films are still limited by pervasive conformational and energetic disorder. This not only limits the rational design of materials with higher performance, but also prevents the study of physical phenomena associated with an extended π-electron delocalization along the polymer backbone. Here we report a comparative transport study of several high-mobility conjugated polymers by field-effect-modulated Seebeck, transistor and sub-bandgap optical absorption measurements. We show that in several of these polymers, most notably in a recently reported, indacenodithiophene-based donor-acceptor copolymer with a near-amorphous microstructure, the charge transport properties approach intrinsic disorder-free limits at which all molecular sites are thermally accessible. Molecular dynamics simulations identify the origin of this long sought-after regime as a planar, torsion-free backbone conformation that is surprisingly resilient to side-chain disorder. Our results provide molecular-design guidelines for 'disorder-free' conjugated polymers.We gratefully acknowledge financial support from the Engineering and Physical Sciences Research Council (EPSRC) through a programme grant (EP/G060738/1) and the Technology Strategy Board (TSB) (PORSCHED project). D. Venkateshvaran acknowledges financial support from the Cambridge Commonwealth Trust through a Cambridge International Scholarship. K. Broch acknowledges post-doctoral fellowship support from the German Research Foundation (DFG). Mateusz Zelazny acknowledges funding from the NanoDTC in Cambridge. The work in Mons was supported by the European Commission / Région Wallonne (FEDER – Smartfilm RF project), the Interuniversity Attraction Pole program of the Belgian Federal Science Policy Office (PAI 7/05), Programme d’Excellence de la Région Wallonne (OPTI2MAT project) and FNRS-FRFC. D.B. and J.C. are FNRS Research Fellows.This is the accepted manuscript. The final version's available from Nature at http://www.nature.com/nature/journal/vaop/ncurrent/full/nature13854.html

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology

β-Lactam Resistance Response Triggered by Inactivation of a Nonessential Penicillin-Binding Protein

It has long been recognized that the modification of penicillin-binding proteins (PBPs) to reduce their affinity for β-lactams is an important mechanism (target modification) by which Gram-positive cocci acquire antibiotic resistance. Among Gram-negative rods (GNR), however, this mechanism has been considered unusual, and restricted to clinically irrelevant laboratory mutants for most species. Using as a model Pseudomonas aeruginosa, high up on the list of pathogens causing life-threatening infections in hospitalized patients worldwide, we show that PBPs may also play a major role in β-lactam resistance in GNR, but through a totally distinct mechanism. Through a detailed genetic investigation, including whole-genome analysis approaches, we demonstrate that high-level (clinical) β-lactam resistance in vitro, in vivo, and in the clinical setting is driven by the inactivation of the dacB-encoded nonessential PBP4, which behaves as a trap target for β-lactams. The inactivation of this PBP is shown to determine a highly efficient and complex β-lactam resistance response, triggering overproduction of the chromosomal β-lactamase AmpC and the specific activation of the CreBC (BlrAB) two-component regulator, which in turn plays a major role in resistance. These findings are a major step forward in our understanding of β-lactam resistance biology, and, more importantly, they open up new perspectives on potential antibiotic targets for the treatment of infectious diseases

Insights into the Molecular Basis of L-Form Formation and Survival in Escherichia coli

L-forms have been shown to occur among many species of bacteria and are suspected to be involved in persistent infections. Since their discovery in 1935, numerous studies characterizing L-form morphology, growth, and pathogenic potential have been conducted. However, the molecular mechanisms underlying the formation and survival of L-forms remain unknown. Using unstable L-form colonies of Escherichia coli as a model, we performed genome-wide transcriptome analysis and screened a deletion mutant library to study the molecular mechanisms involved in formation and survival of L-forms. Microarray analysis of L-form versus classical colonies revealed many up-regulated genes of unknown function as well as multiple over-expressed stress pathways shared in common with persister cells and biofilms. Mutant screens identified three groups of mutants which displayed varying degrees of defects in L-form colony formation. Group 1 mutants, which showed the strongest defect in L-form colony formation, belonged to pathways involved in cell envelope stress, DNA repair, iron homeostasis, outer membrane biogenesis, and drug efflux/ABC transporters. Four (Group 1) mutants, rcsB, a positive response regulator of colanic acid capsule synthesis, ruvA, a recombinational junction binding protein, fur, a ferric uptake regulator and smpA a small membrane lipoprotein were selected for complementation. Complementation of the mutants using a high-copy overexpression vector failed, while utilization of a low-copy inducible vector successfully restored L-form formation. This work represents the first systematic genetic evaluation of genes and pathways involved in the formation and survival of unstable L-form bacteria. Our findings provide new insights into the molecular mechanisms underlying L-form formation and survival and have implications for understanding the emergence of antibiotic resistance, bacterial persistence and latent infections and designing novel drugs and vaccines

Assessment of aortic stiffness by cardiovascular magnetic resonance following the treatment of severe aortic stenosis by TAVI and surgical AVR

Aortic stiffness is increasingly used as an independent predictor of adverse cardiovascular outcomes. We sought to compare the impact of transcatheter aortic valve implantation (TAVI) and surgical aortic valve replacement (SAVR) upon aortic vascular function using cardiovascular magnetic resonance (CMR) measurements of aortic distensibility and pulse wave velocity (PWV).A 1.5 T CMR scan was performed pre-operatively and at 6 m post-intervention in 72 patients (32 TAVI, 40 SAVR; age 76 ± 8 years) with high-risk symptomatic severe aortic stenosis. Distensibility of the ascending and descending thoracic aorta and aortic pulse wave velocity were determined at both time points. TAVI and SAVR patients were comparable for gender, blood pressure and left ventricular ejection fraction. The TAVI group were older (81 ± 6.3 vs. 72.8 ± 7.0 years, p < 0.05) with a higher EuroSCORE II (5.7 ± 5.6 vs. 1.5 ± 1.0 %, p < 0.05). At 6 m, SAVR was associated with a significant decrease in distensibility of the ascending aorta (1.95 ± 1.15 vs. 1.57 ± 0.68 × 10(-3)mmHg(-1), p = 0.044) and of the descending thoracic aorta (3.05 ± 1.12 vs. 2.66 ± 1.00 × 10(-3)mmHg(-1), p = 0.018), with a significant increase in PWV (6.38 ± 4.47 vs. 11.01 ± 5.75 ms(-1), p = 0.001). Following TAVI, there was no change in distensibility of the ascending aorta (1.96 ± 1.51 vs. 1.72 ± 0.78 × 10(-3)mmHg(-1), p = 0.380), descending thoracic aorta (2.69 ± 1.79 vs. 2.21 ± 0.79 × 10(-3)mmHg(-1), p = 0.181) nor in PWV (8.69 ± 6.76 vs. 10.23 ± 7.88 ms(-1), p = 0.301) at 6 m.Treatment of symptomatic severe aortic stenosis by SAVR but not TAVI was associated with an increase in aortic stiffness at 6 months. Future work should focus on the prognostic implication of these findings to determine whether improved patient selection and outcomes can be achieved

Exploiting protein flexibility to predict the location of allosteric sites

Background: Allostery is one of the most powerful and common ways of regulation of protein activity. However, for most allosteric proteins identified to date the mechanistic details of allosteric modulation are not yet well understood. Uncovering common mechanistic patterns underlying allostery would allow not only a better academic understanding of the phenomena, but it would also streamline the design of novel therapeutic solutions. This relatively unexplored therapeutic potential and the putative advantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drug research is receiving at present. A first step to harness the regulatory potential and versatility of allosteric sites, in the context of drug-discovery and design, would be to detect or predict their presence and location. In this article, we describe a simple computational approach, based on the effect allosteric ligands exert on protein flexibility upon binding, to predict the existence and position of allosteric sites on a given protein structure. Results: By querying the literature and a recently available database of allosteric sites, we gathered 213 allosteric proteins with structural information that we further filtered into a non-redundant set of 91 proteins. We performed normal-mode analysis and observed significant changes in protein flexibility upon allosteric-ligand binding in 70% of the cases. These results agree with the current view that allosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligand binding. Furthermore, we implemented an approach that achieves 65% positive predictive value in identifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set, 0.22 sensitivity), by combining the current analysis on dynamics with previous results on structural conservation of allosteric sites. We also analyzed four biological examples in detail, revealing that this simple coarse-grained methodology is able to capture the effects triggered by allosteric ligands already described in the literature. Conclusions: We introduce a simple computational approach to predict the presence and position of allosteric sites in a protein based on the analysis of changes in protein normal modes upon the binding of a coarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newly curated non-redundant set of 91 proteins with reported allosteric properties. The software developed in this work is available upon request from the authors

The role of the proteasome in the generation of MHC class I ligands and immune responses

The ubiquitin–proteasome system (UPS) degrades intracellular proteins into peptide fragments that can be presented by major histocompatibility complex (MHC) class I molecules. While the UPS is functional in all mammalian cells, its subunit composition differs depending on cell type and stimuli received. Thus, cells of the hematopoietic lineage and cells exposed to (pro)inflammatory cytokines express three proteasome immunosubunits, which form the catalytic centers of immunoproteasomes, and the proteasome activator PA28. Cortical thymic epithelial cells express a thymus-specific proteasome subunit that induces the assembly of thymoproteasomes. We here review new developments regarding the role of these different proteasome components in MHC class I antigen processing, T cell repertoire selection and CD8 T cell responses. We further discuss recently discovered functions of proteasomes in peptide splicing, lymphocyte survival and the regulation of cytokine production and inflammatory responses

Rad51 Inhibits Translocation Formation by Non-Conservative Homologous Recombination in Saccharomyces cerevisiae

Chromosomal translocations are a primary biological response to ionizing radiation (IR) exposure, and are likely to result from the inappropriate repair of the DNA double-strand breaks (DSBs) that are created. An abundance of repetitive sequences in eukaryotic genomes provides ample opportunity for such breaks to be repaired by homologous recombination (HR) between non-allelic repeats. Interestingly, in the budding yeast, Saccharomyces cerevisiae the central strand exchange protein, Rad51 that is required for DSB repair by gene conversion between unlinked repeats that conserves genomic structure also suppresses translocation formation by several HR mechanisms. In particular, Rad51 suppresses translocation formation by single-strand annealing (SSA), perhaps the most efficient mechanism for translocation formation by HR in both yeast and mammalian cells. Further, the enhanced translocation formation that emerges in the absence of Rad51 displays a distinct pattern of genetic control, suggesting that this occurs by a separate mechanism. Since hypomorphic mutations in RAD51 in mammalian cells also reduce DSB repair by conservative gene conversion and stimulate non-conservative repair by SSA, this mechanism may also operate in humans and, perhaps contribute to the genome instability that propels the development of cancer