Purification and characterization of recombinant human renin for X-ray crystallization studies

<p>Abstract</p> <p>Background</p> <p>The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies.</p> <p>Results</p> <p>A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained.</p> <p>Conclusion</p> <p>This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.</p

Wear of human teeth: a tribological perspective

The four main types of wear in teeth are attrition (enamel-on-enamel contact), abrasion (wear due to abrasive particles in food or toothpaste), abfraction (cracking in enamel and subsequent material loss), and erosion (chemical decomposition of the tooth). They occur as a result of a number of mechanisms including thegosis (sliding of teeth into their lateral position), bruxism (tooth grinding), mastication (chewing), toothbrushing, tooth flexure, and chemical effects. In this paper the current understanding of wear of enamel and dentine in teeth is reviewed in terms of these mechanisms and the major influencing factors are examined. In vitro tooth wear simulation and in vivo wear measurement and ranking are also discussed

Host-linked soil viral ecology along a permafrost thaw gradient

Climate change threatens to release abundant carbon that is sequestered at high latitudes, but the constraints on microbial metabolisms that mediate the release of methane and carbon dioxide are poorly understood1,2,3,4,5,6,7. The role of viruses, which are known to affect microbial dynamics, metabolism and biogeochemistry in the oceans8,9,10, remains largely unexplored in soil. Here, we aimed to investigate how viruses influence microbial ecology and carbon metabolism in peatland soils along a permafrost thaw gradient in Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, 58% of which were detected in metatranscriptomes and presumed to be active. In silico predictions linked 35% of the viruses to microbial host populations, highlighting likely viral predators of key carbon-cycling microorganisms, including methanogens and methanotrophs. Lineage-specific virus/host ratios varied, suggesting that viral infection dynamics may differentially impact microbial responses to a changing climate. Virus-encoded glycoside hydrolases, including an endomannanase with confirmed functional activity, indicated that viruses influence complex carbon degradation and that viral abundances were significant predictors of methane dynamics. These findings suggest that viruses may impact ecosystem function in climate-critical, terrestrial habitats and identify multiple potential viral contributions to soil carbon cycling

Branch-and-lift algorithm for deterministic global optimization in nonlinear optimal control

This paper presents a branch-and-lift algorithm for solving optimal control problems with smooth nonlinear dynamics and potentially nonconvex objective and constraint functionals to guaranteed global optimality. This algorithm features a direct sequential method and builds upon a generic, spatial branch-and-bound algorithm. A new operation, called lifting, is introduced, which refines the control parameterization via a Gram-Schmidt orthogonalization process, while simultaneously eliminating control subregions that are either infeasible or that provably cannot contain any global optima. Conditions are given under which the image of the control parameterization error in the state space contracts exponentially as the parameterization order is increased, thereby making the lifting operation efficient. A computational technique based on ellipsoidal calculus is also developed that satisfies these conditions. The practical applicability of branch-and-lift is illustrated in a numerical example. © 2013 Springer Science+Business Media New York

Maximal planar networks with large clustering coefficient and power-law degree distribution

In this article, we propose a simple rule that generates scale-free networks with very large clustering coefficient and very small average distance. These networks are called {\bf Random Apollonian Networks}(RAN) as they can be considered as a variation of Apollonian networks. We obtain the analytic results of power-law exponent $\gamma =3$ and clustering coefficient $C={46/3}-36\texttt{ln}{3/2}\approx 0.74$, which agree very well with the simulation results. We prove that the increasing tendency of average distance of RAN is a little slower than the logarithm of the number of nodes in RAN. Since most real-life networks are both scale-free and small-world networks, RAN may perform well in mimicking the reality. The RAN possess hierarchical structure as $C(k)\sim k^{-1}$ that in accord with the observations of many real-life networks. In addition, we prove that RAN are maximal planar networks, which are of particular practicability for layout of printed circuits and so on. The percolation and epidemic spreading process are also studies and the comparison between RAN and Barab\'{a}si-Albert(BA) as well as Newman-Watts(NW) networks are shown. We find that, when the network order $N$(the total number of nodes) is relatively small(as $N\sim 10^4$), the performance of RAN under intentional attack is not sensitive to $N$, while that of BA networks is much affected by $N$. And the diseases spread slower in RAN than BA networks during the outbreaks, indicating that the large clustering coefficient may slower the spreading velocity especially in the outbreaks.Comment: 13 pages, 10 figure

Quantitative MRI evidence for altered structural remodelling of the temporal lobe in cryptogenic West syndrome

Objective: To explore the structure-function relation of the temporal lobe in newly diagnosed West syndrome of unknown cause (uWS). / Methods: Quantitative magnetic resonance imaging (3D structural MRI and diffusion tensor imaging, DTI) was analysed by voxel-based morphometry (VBM) and tractbased spatial statistics (TBSS) in 22 patients and healthy age-matched controls. The electrophysiological responsiveness of the temporal lobe was measured using the N100 auditory event-related potential (ERP) to a repeated 1000Hz tone. Neurocognitive function was assessed using the Bayley scales of infant development II (BSID-II). Tests followed first-line treatment with Vigabatrin (17) or high dose oral prednisolone (5). / Results: Total temporal lobe volume was similar in patients and controls. Patients had a smaller temporal stem (TS) [p < 0.0001] and planum temporale (PT) [p = 0.029] bilaterally. TS width asymmetry with a larger right-sided width in controls, was absent in patients [p = 0.033]. PT asymmetry was present in both groups, being larger on the right [p = 0.048]. VBM grey matter volume was increased at the left temporal lobe (superior and middle temporal gyri, the perirhinal cortex and medial temporal lobe) [p<0.005, family wise error-corrected]. VBM grey matter volume correlated with the duration of infantile spasms. [Pearson’s R = - 0.630, p = 0.009] DTI metrics did not differ between patients and controls on TBSS. Patients’ mean BSID-II scores were lower [p<0.001] and their auditory N100 ERP attenuated less than controls’ [p = 0.002]. / Significance: The functional networking and white matter development of the temporal lobe are impaired following infantile spasms. Treatment may promote structural plasticity within the temporal lobe following infantile spasms, manifest as increased grey matter volume on VBM. It remains to be investigated further whether this predicts patients’ longterm cognitive difficulties

Minor differences in body condition and immune status between avian influenza virus-infected and noninfected mallards: a sign of coevolution?

Wildlife pathogens can alter host fitness. Low pathogenic avian influenza virus (LPAIV) infection is thought to have negligible impacts on wild birds; however, effects of infection in free-living birds are largely unstudied. We investigated the extent to which LPAIV infection and shedding were associated with body condition and immune status in free-living mallards (Anas platyrhynchos), a partially migratory key LPAIV host species. We sampled mallards throughout the species\u27 annual autumn LPAIV infection peak, and we classified individuals according to age, sex, and migratory strategy (based on stable hydrogen isotope analysis) when analyzing data on body mass and five indices of immune status. Body mass was similar for LPAIV-infected and noninfected birds. The degree of virus shedding from the cloaca and oropharynx was not associated with body mass. LPAIV infection and shedding were not associated with natural antibody (NAbs) and complement titers (first lines of defense against infections), concentrations of the acute phase protein haptoglobin (Hp), ratios of heterophils to lymphocytes (H:L ratio), and avian influenza virus (AIV)-specific antibody concentrations. NAbs titers were higher in LPAIV-infected males and local (i.e., short distance) migrants than in infected females and distant (i.e., long distance) migrants. Hp concentrations were higher in LPAIV-infected juveniles and females compared to infected adults and males. NAbs, complement, and Hp levels were lower in LPAIV-infected mallards in early autumn. Our study demonstrates weak associations between infection with and shedding of LPAIV and the body condition and immune status of free-living mallards. These results may support the role of mallards as asymptomatic carriers of LPAIV and raise questions about possible coevolution between virus and host

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1

ABSTRACT The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays