Electrostatic Steering Accelerates C3d:CR2 Association.

Electrostatic effects are ubiquitous in protein interactions and are found to be pervasive in the complement system as well. The interaction between complement fragment C3d and complement receptor 2 (CR2) has evolved to become a link between innate and adaptive immunity. Electrostatic interactions have been suggested to be the driving factor for the association of the C3d:CR2 complex. In this study, we investigate the effects of ionic strength and mutagenesis on the association of C3d:CR2 through Brownian dynamics simulations. We demonstrate that the formation of the C3d:CR2 complex is ionic strength-dependent, suggesting the presence of long-range electrostatic steering that accelerates the complex formation. Electrostatic steering occurs through the interaction of an acidic surface patch in C3d and the positively charged CR2 and is supported by the effects of mutations within the acidic patch of C3d that slow or diminish association. Our data are in agreement with previous experimental mutagenesis and binding studies and computational studies. Although the C3d acidic patch may be locally destabilizing because of unfavorable Coulombic interactions of like charges, it contributes to the acceleration of association. Therefore, acceleration of function through electrostatic steering takes precedence to stability. The site of interaction between C3d and CR2 has been the target for delivery of CR2-bound nanoparticle, antibody, and small molecule biomarkers, as well as potential therapeutics. A detailed knowledge of the physicochemical basis of C3d:CR2 association may be necessary to accelerate biomarker and drug discovery efforts

Broad application of a simple and affordable protocol for isolating plant RNA

BACKGROUND: Standard molecular biological methods involve the analysis of gene expression in living organisms under diverse environmental and developmental conditions. One of the most direct approaches to quantify gene expression is the isolation of RNA. Most techniques used to quantify gene expression require the isolation of RNA, usually from a large number of samples. While most published protocols, including those for commercial reagents, are either labour intensive, use hazardous chemicals and/or are costly, a previously published protocol for RNA isolation in Arabidopsis thaliana yields high amounts of good quality RNA in a simple, safe and inexpensive manner. FINDINGS: We have tested this protocol in tomato and wheat leaves, as well as in Arabidopsis leaves, and compared the resulting RNA to that obtained using a commercial phenol-based reagent. Our results demonstrate that this protocol is applicable to other plant species, including monocots, and offers yield and purity at least comparable to those provided by commercial phenol-based reagents. CONCLUSIONS: Here, we show that this previously published RNA isolation protocol can be easily extended to other plant species without further modification. Due to its simplicity and the use of inexpensive reagents, this protocol is accessible and affordable and can be easily implemented to work on different plant species in laboratories worldwide

The Receptor-Like Kinase SERK3/BAK1 Is Required for Basal Resistance against the Late Blight Pathogen Phytophthora infestans in Nicotiana benthamiana

BACKGROUND The filamentous oomycete plant pathogen Phytophthora infestans causes late blight, an economically important disease, on members of the nightshade family (Solanaceae), such as the crop plants potato and tomato. The related plant Nicotiana benthamiana is a model system to study plant-pathogen interactions, and the susceptibility of N. benthamiana to Phytophthora species varies from susceptible to resistant. Little is known about the extent to which plant basal immunity, mediated by membrane receptors that recognise conserved pathogen-associated molecular patterns (PAMPs), contributes to P. infestans resistance. PRINCIPAL FINDINGS We found that different species of Phytophthora have varying degrees of virulence on N. benthamiana ranging from avirulence (incompatible interaction) to moderate virulence through to full aggressiveness. The leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1/SERK3 is a major modulator of PAMP-triggered immunity (PTI) in Arabidopsis thaliana and N. benthamiana. We cloned two NbSerk3 homologs, NbSerk3A and NbSerk3B, from N. benthamiana based on sequence similarity to the A. thaliana gene. N. benthamiana plants silenced for NbSerk3 showed markedly enhanced susceptibility to P. infestans infection but were not altered in resistance to Phytophthora mirabilis, a sister species of P. infestans that specializes on a different host plant. Furthermore, silencing of NbSerk3 reduced the cell death response triggered by the INF1, a secreted P. infestans protein with features of PAMPs. CONCLUSIONS/SIGNIFICANCE We demonstrated that N. benthamiana NbSERK3 significantly contributes to resistance to P. infestans and regulates the immune responses triggered by the P. infestans PAMP protein INF1. In the future, the identification of novel surface receptors that associate with NbSERK3A and/or NbSERK3B should lead to the identification of new receptors that mediate recognition of oomycete PAMPs, such as INF1.This work was supported by the Gatsby Charitable Foundation, BBSRC, Nuffield Foundation and the German Research Foundation (DFG). SS was supported by a personal research fellowship (SCHO1347/1-1). JPR is an Australian Research Council Future Fellow (FT0992129). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Anti-melanocortin-4 receptor autoantibodies in obesity

Background: The melanocortin-4 receptor (MC4R) is part of an important pathway regulating energy balance. Here we report the existence of autoantibodies (autoAbs) against the MC4R in sera of obese patients. Methods: The autoAbs were detected after screening of 216 patients' sera by using direct and inhibition ELISA with an N-terminal sequence of the MC4R. Binding to the native MC4R was evaluated by flow cytometry and pharmacological effects by measuring adenylyl cyclase activity. Results: Positive results in all tests were obtained in patients with overweight or obesity (prevalence: 3.6%) but not in normal weight patients. The selective binding properties of anti-MC4R autoAbs were confirmed by surface plasmon resonance and by immunoprecipitation with the native MC4R. Finally it was demonstrated that these autoAbs increased food intake in rats after passive transfer via intracerebroventricular injection. Conclusion: These observations suggest that inhibitory anti-MC4R autoAbs might contribute to the development of obesity in a small subpopulation of patients

Borrelia recurrentis employs a novel multifunctional surface protein with anti-complement, anti-opsonic and invasive potential to escape innate immunity

Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever

Risk of early versus later rebleeding from dural arteriovenous fistulas with cortical venous drainage

BACKGROUND: Cranial dural arteriovenous fistulas with cortical venous drainage are rare lesions that can present with hemorrhage. A high rate of rebleeding in the early period following hemorrhage has been reported, but published long-term rates are much lower. No study has examined how risk of rebleeding changes over time. Our objective was to quantify the relative incidence of rebleeding in the early and later periods following hemorrhage. METHODS: Patients with dural arteriovenous fistula and cortical venous drainage presenting with hemorrhage were identified from the multinational CONDOR (Consortium for Dural Fistula Outcomes Research) database. Natural history follow-up was defined as time from hemorrhage to first treatment, rebleed, or last follow-up. Rebleeding in the first 2 weeks and first year were compared using incidence rate ratio and difference. RESULTS: Of 1077 patients, 250 met the inclusion criteria and had 95 cumulative person-years natural history follow-up. The overall annualized rebleed rate was 7.3% (95% CI, 3.2-14.5). The incidence rate of rebleeding in the first 2 weeks was 0.0011 per person-day; an early rebleed risk of 1.6% in the first 14 days (95% CI, 0.3-5.1). For the remainder of the first year, the incidence rate was 0.00015 per person-day; a rebleed rate of 5.3% (CI, 1.7-12.4) over 1 year. The incidence rate ratio was 7.3 (95% CI, 1.4-37.7; CONCLUSIONS: The risk of rebleeding of a dural arteriovenous fistula with cortical venous drainage presenting with hemorrhage is increased in the first 2 weeks justifying early treatment. However, the magnitude of this increase may be considerably lower than previously thought. Treatment within 5 days was associated with a low rate of rebleeding and appears an appropriate timeframe

Towards a Mg lattice clock: Observation of the 1S0−^1S_{0}-3P0^3P_{0} transition and determination of the magic wavelength

We optically excite the electronic state $3s3p~^3P_{0}$ in $^{24}$Mg atoms, laser-cooled and trapped in a magic-wavelength lattice. An applied magnetic field enhances the coupling of the light to the otherwise strictly forbidden transition. We determine the magic wavelength, the quadratic magnetic Zeeman shift and the transition frequency to be 468.463(207)$\,$nm, -206.6(2.0)$\,$MHz/T$^2$ and 655 058 646 691(101)$\,$kHz, respectively. These are compared with theoretical predictions and results from complementary experiments. We also developed a high-precision relativistic structure model for magnesium, give an improved theoretical value for the blackbody radiation shift and discuss a clock based on bosonic magnesium.Comment: 5 pages, 3 figure

Borrelia valaisiana resist complement-mediated killing independently of the recruitment of immune regulators and inactivation of complement components

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae

Identification and functional characterisation of Complement Regulator Acquiring Surface Protein-1 of serum resistant Borrelia garinii OspA serotype 4

<p>Abstract</p> <p>Background</p> <p><it>B. burgdorferi </it>sensu lato (sl) is the etiological agent of Lyme borreliosis in humans. Spirochetes have adapted themselves to the human immune system in many distinct ways. One important immune escape mechanism for evading complement activation is the binding of complement regulators Factor H (CFH) or Factor H-like protein1 (FHL-1) to Complement Regulator-Acquiring Surface Proteins (CRASPs).</p> <p>Results</p> <p>We demonstrate that <it>B. garinii </it>OspA serotype 4 (ST4) PBi resist complement-mediated killing by binding of FHL-1. To identify the primary ligands of FHL-1 four CspA orthologs from <it>B. garinii </it>ST4 PBi were cloned and tested for binding to human CFH and FHL-1. Orthologs BGA66 and BGA71 were found to be able to bind both complement regulators but with different intensities. In addition, all CspA orthologs were tested for binding to mammalian and avian CFH. Distinct orthologs were able to bind to CFH of different animal origins.</p> <p>Conclusions</p> <p><it>B. garinii </it>ST4 PBi is able to evade complement killing and it can bind FHL-1 to membrane expressed proteins. Recombinant proteins BGA66 can bind FHL-1 and human CFH, while BGA71 can bind only FHL-1. All recombinant CspA orthologs from <it>B. garinii </it>ST4 PBi can bind CFH from different animal origins. This partly explains the wide variety of animals that can be infected by <it>B. garinii</it>.</p