Effect of protein thermostability on the cooperative function of split enzymes

Although the effects of mutational events on protein function cannot yet be predicted a priori, molecular evolution studies have shown that the tolerance of protein structure and function to random mutation is positively correlated with the thermostability of the protein mutated. To test whether thermostability also influences protein function upon random fission, I have characterized the function of split Bacillus subtilis and Thermotoga neapolitana adenylate kinases (AK Bs and AKTn, respectively), enzymes that are required to maintain adenine energy charge. Using libraries of split AKBs and AKTn variants, I show that mesophilic and thermophilic AK orthologs can be split at multiple sites into fragments that complement the growth of Escherichia coli with a temperature-sensitive AK at 40°C. However, I find that the fraction of split AKTn variants that function is ∼7-fold higher than that observed for split AKBs variants. I also find that AKTn can be split within the AMP-binding and LID domains to create functional variants, whereas AKBs can only be split within the AMP-binding domain. Biochemical and biophysical analysis of one pair of homologous split AK variants reveal that polypeptide fragments derived from the more thermostable AK exhibit greater secondary structure and enzymatic activity, suggesting that residual structure of these fragments could account for their retention in function. In addition, complementation studies show that the association and cooperative function of AKBs fragments with little residual structure can be increased by fusing these peptides to interacting proteins. Similarly, the interaction of a split AK Tn can be enhanced at a temperature (78°C) where the fragments are non-functional by fusion to proteins that interact. This split AKTn, which represents the first high-temperature protein fragment complementation assay (ht-PCA) for analyzing protein-protein interactions within a living thermophilic bacterium, is capable of detecting predicted interactions among Thermotoga maritima chemotaxis proteins. These findings show that split proteins with varying functions can be rapidly discovered by fragmenting orthologs with a range of thermostabilities. Moreover, the novel ht-PCA described herein will aid in creating genome-wide maps of thermophilic protein-protein interactions, studying the effects of temperature on biomolecular interactions, and engineering oligomeric thermostable nanomaterials

Using Joint Utilities of the Times to Response and Toxicity to Adaptively Optimize Schedule–Dose Regimes

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/101836/1/biom12065-sm-0001-SuppData.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/101836/2/biom12065.pd

Mechanical Reinforcement of Polymeric Fibers through Peptide Nanotube Incorporation

High aspect ratio nanotubular assemblies can be effective fillers in mechanically reinforced composite materials. However, most existing nanotubes used for structural purposes are limited in their range of mechanical, chemical, and biological properties. We demonstrate an alternative approach to mechanical reinforcement of polymeric systems by incorporating synthetic d,l-cyclic peptide nanotube bundles as a structural filler in electrospun poly d-, l-lactic acid fibers. The nanotube bundles self-assemble through dynamic hydrogen bonding from synthetic cyclic peptides to yield structures whose dimensions can be altered based on processing conditions, and can be up to hundreds of micrometers long and several hundred nanometers wide. With 8 wt % peptide loading, the composite fibers are >5-fold stiffer than fibers composed of the polymer alone, according to atomic force microscopy-based indentation experiments. This represents a new use for self-assembling cyclic peptides as a load-bearing component in biodegradable composite materials.Engineering and Applied Science

Co-infection of human parvovirus B19 with Plasmodium falciparum contributes to malaria disease severity in Gabonese patients

Background: High seroprevalence of parvovirus B19 (B19V) coinfection with Plasmodium falciparum has been previously reported. However, the impact of B19V-infection on the clinical course of malaria is still elusive. In this study, we investigated the prevalence and clinical significance of B19V co-infection in Gabonese children with malaria. Methods: B19V prevalence was analyzed in serum samples of 197 Gabonese children with P. falciparum malaria and 85 healthy controls using polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and direct DNA-sequencing. Results: B19V was detected in 29/282 (10.28%) of Gabonese children. B19V was observed more frequently in P. falciparum malaria patients (14.21%) in comparison to healthy individuals (1.17%) (

A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids

Synthetic biology approaches to bioremediation are a key sustainable strategy to leverage the self-replicating and programmable aspects of biology for environmental stewardship. The increasing spread of anthropogenic mercury pollution into our habitats and food chains is a pressing concern. Here, we explore the use of programmed bacterial biofilms to aid in the sequestration of mercury. We demonstrate that by integrating a mercury-responsive promoter and an operon encoding a mercury-absorbing self-assembling extracellular protein nanofiber, we can engineer bacteria that can detect and sequester toxic Hg2+ ions from the environment. This work paves the way for the development of on-demand biofilm living materials that can operate autonomously as heavy-metal absorbents.Engineering and Applied Science

Modulation of TGF-β/BMP-6 expression and increased levels of circulating smooth muscle progenitor cells in a type I diabetes mouse model

<p>Abstract</p> <p>Background</p> <p>Diabetic patients experience exaggerated intimal hyperplasia after endovascular procedures. Recently it has been shown that circulating smooth muscle progenitor cells (SPC) contribute to intimal hyperplasia. We hypothesized that SPC differentiation would be increased in diabetes and focused on modulation of TGF-β/BMP-6 signaling as potential underlying mechanism.</p> <p>Methods</p> <p>We isolated SPC from C57Bl/6 mice with streptozotocin-induced diabetes and controls. SPC differentiation was evaluated by immunofluorescent staining for αSMA and collagen Type I. SPC mRNA expression of TGF-β and BMP-6 was quantified using real-time PCR. Intima formation was assessed in cuffed femoral arteries. Homing of bone marrow derived cells to cuffed arterial segments was evaluated in animals transplanted with bone marrow from GFP-transgenic mice.</p> <p>Results</p> <p>We observed that SPC differentiation was accelerated and numeric outgrowth increased in diabetic animals (24.6 ± 8.8 vs 8.3 ± 1.9 per HPF after 10 days, p < 0.05). Quantitative real-time PCR showed increased expression of TGF-β and decreased expression of the BMP-6 in diabetic SPC. SPC were MAC-3 positive, indicative of monocytic lineage. Intima formation in cuffed arterial segments was increased in diabetic mice (intima/media ratio 0.68 ± 0.15 vs 0.29 ± 0.06, p < 0.05). In GFP-chimeric mice, bone marrow derived cells were observed in the neointima (4.4 ± 3.3 cells per section) and particularly in the adventitia (43.6 ± 9.3 cells per section). GFP-positive cells were in part MAC-3 positive, but rarely expressed α-SMA.</p> <p>Conclusions</p> <p>In conclusion, in a diabetic mouse model, SPC levels are increased and SPC TGF-β/BMP-6 expression is modulated. Altered TGF-β/BMP-6 expression is known to regulate smooth muscle cell differentiation and may facilitate SPC differentiation. This may contribute to exaggerated intimal hyperplasia in diabetes as bone marrow derived cells home to sites of neointima formation.</p

Las políticas de reequilibro territorial e innovación institucional en Madrid, 2015-2019

Lograr un mejor reparto de los recursos materiales es una tarea inseparable de una transformación institucional dirigida a un mejor reparto del poder y de la capacidad de decisión. Es preciso reconfigurar las instituciones para redefinir la relación entre estas y la ciudadanía. El espacio de cooperación entre las instituciones públicas y la sociedad civil organizada es un terreno privilegiado para lograr esa reconfiguración institucional. El Fondo de Reequilibrio Territorial se concibe como un primer paso en ese complejo camino y se relaciona con el proceso de descentralización y con el concepto de cooperación público-social. Debe entenderse en relación con otros desarrollos institucionales, como el Consejo Coordinador de los Distritos o los Foros Locales. Una tarea de estas características no puede realizarse desde un solo espacio institucional y en un solo mandato, pero en todo caso debe hacerse contando con la firme voluntad y dirección política del equipo de gobierno que quiera desempeñarla. El gobierno es una herramienta formidable para emprender un proceso de reequilibrio territorial, pero aun cuando no hay en el mismo voluntad política en este sentido existen posibilidades para estimular o condicionar la voluntad del equipo de gobierno: articular un amplio pacto entre los actores políticos, sociales, económicos, académicos, etc. que ponga la cuestión en el centro de la agenda política y eleve hasta lo impagable el coste político de seguir dando la espalda a una cuestión crucial para el futuro de la democracia.Lograr un mejor reparto de los recursos materiales es una tarea inseparable de una transformación institucional dirigida a un mejor reparto del poder y de la capacidad de decisión. Es preciso reconfigurar las instituciones para redefinir la relación entre estas y la ciudadanía. El espacio de cooperación entre las instituciones públicas y la sociedad civil organizada es un terreno privilegiado para lograr esa reconfiguración institucional.El Fondo de Reequilibrio Territorial se concibe como un primer paso en ese complejo camino y se relaciona con el proceso de descentralización y con el concepto de cooperación público-social. Debe entenderse en relación con otros desarrollos institucionales, como el Consejo Coordinador de los Distritos o los Foros Locales. Una tarea de estas características no puede realizarse desde un solo espacio institucional y en un solo mandato, pero en todo caso debe hacerse contando con la firme voluntad y dirección política del equipo de gobierno que quiera desempeñarla. El gobierno es una herramienta formidable para emprender un proceso de reequilibrio territorial, pero aun cuando no hay en el mismo voluntad política en este sentido existen posibilidades para estimular o condicionar la voluntad del equipo de gobierno: articular un amplio pacto entre los actores políticos, sociales, económicos, académicos, etc. que ponga la cuestión en el centro de la agenda política y eleve hasta lo impagable el coste político de seguir dando la espalda a una cuestión crucial para el futuro de la democracia

Targeted testing for acute HIV infection in North Carolina

Persons with acute HIV infection contribute disproportionately to HIV transmission. Identification of these persons is a critical public health challenge. We developed targeted approaches to detect HIV RNA in persons with negative serological tests

Особливості синтаксичної організації художньої прози Редьярда Кіплінга

Статья посвящена исследованию синтаксических конструкций, с помощью которых в художественной прозе Редьярда Киплинга отображаются особенности английской и индийской культур.Стаття присвячена дослідженню синтаксичних конструкцій, за допомогою яких у художній прозі Редьярда Кіплінга відображено особливості англійської та індійської культур.The article is dedicated to the investigation of the main syntactic constractions with the help of which in the prose of Rudyard Kipling there are depicted the peculiarities of English and Indian cultures