Effect of rare coding variants in the CFI gene on Factor I expression levels.

Factor I (FI) is one of the main inhibitors of complement activity, and numerous rare coding variants have been reported in patients with age-related macular degeneration, atypical hemolytic uremic syndrome and C3 glomerulopathy. Since many of these variants are of unknown clinical significance, this study aimed to determine the effect of rare coding variants in the complement factor I (CFI) gene on FI expression. We measured FI levels in plasma samples of carriers of rare coding variants and in vitro in the supernatants of epithelial cells expressing recombinant FI. FI levels were measured in 177 plasma samples of 155 individuals, carrying 24 different rare coding variants in CFI. In carriers of the variants p.Gly119Arg, p.Leu131Arg, p.Gly188Ala and c.772G>A (r.685_773del), significantly reduced FI plasma levels were detected. Furthermore, recombinant FI expression levels were determined for 126 rare coding variants. Of these variants 68 (54%) resulted in significantly reduced FI expression in supernatant compared to wildtype (WT). The recombinant protein expression levels correlated significantly with the FI level in plasma of carriers of CFI variants. In this study, we performed the most comprehensive FI expression level analysis of rare coding variants in CFI to date. More than half of CFI variants lead to reduced FI expression, which might impair complement regulation in vivo. Our study will aid the interpretation of rare coding CFI variants identified in clinical practice, which is in particular important in light of patient inclusion in ongoing clinical trials for CFI gene supplementation in AMD.

Functional analyses of rare genetic variants in complement component C9 identified in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is a progressive disease of the central retina and the leading cause of irreversible vision loss in the western world. The involvement of abnormal complement activation in AMD has been suggested by association of variants in genes encoding complement proteins with disease development. A low-frequency variant (p.P167S) in the complement component C9 (C9) gene was recently shown to be highly associated with AMD; however, its functional outcome remains largely unexplored. In this study, we reveal five novel rare genetic variants (p.M45L, p.F62S, p.G126R, p.T170I and p.A529T) in C9 in AMD patients, and evaluate their functional effects in vitro together with the previously identified (p.R118W and p.P167S) C9 variants. Our results demonstrate that the concentration of C9 is significantly elevated in patients' sera carrying the p.M45L, p.F62S, p.P167S and p.A529T variants compared with non-carrier controls. However, no difference can be observed in soluble terminal complement complex levels between the carrier and non-carrier groups. Comparing the polymerization of the C9 variants we reveal that the p.P167S mutant spontaneously aggregates, while the other mutant proteins (except for C9 p.A529T) fail to polymerize in the presence of zinc. Altered polymerization of the p.F62S and p.P167S proteins associated with decreased lysis of sheep erythrocytes and adult retinal pigment epithelial-19 cells by carriers' sera. Our data suggest that the analyzed C9 variants affect only the secretion and polymerization of C9, without influencing its classical lytic activity. Future studies need to be performed to understand the implications of the altered polymerization of C9 in AMD pathology.

Relevance of low specific IgE levels to egg, milk and peanut in infancy.

BACKGROUND: IgE sensitization is usually associated with allergy-related diseases, but may also occur in asymptomatic individuals. The clinical importance of IgE antibody concentrations in the interval 0.1-0.34 kU/L in early life in relation to allergy development is poorly evaluated. OBJECTIVE: To assess the relevance of low specific IgE (s-IgE) to hen's egg, cow's milk and peanut at 6 months of age for development of sensitization and allergy-related disease during early childhood. METHODS: s-IgE concentrations to relevant allergens from blood samples taken at 6 months and 1, 2 and 5 years from children in the prospective ALADDIN cohort were divided into three categories: non-sensitized (<0.1 kU/L), low sensitized (0.1-0.34 kU/L) and sensitized (≥0.35 kU/L) and allergy-related disease assessed. RESULTS: A total of 372 children were included in this study. Compared with non-sensitized children at 6 months of age, children with low levels of allergen specific IgE (0.1-0.34 kU/L) to food allergens, especially to egg, at 6 months of age were associated with development of sensitization to aeroallergens at 5 years of age (10/14 [71%] vs 39/250 [15%]). In addition, children with low levels to egg or milk at 6 months were more often sensitized to the respective allergen at 1 year of age and, regarding low levels to egg, also to the development of eczema (6/18 [33%] vs 29/292 [10%]). CONCLUSION & CLINICAL RELEVANCE: IgE antibody concentrations in the interval 0.1-0.34 kU/L to food allergens in infancy seem to increase the probability of sensitization to aeroallergens and, regarding low levels to egg, also of eczema during early childhood. Thus, IgE levels during the first year of life, although below 0.35 kU/L, can provide additional allergy-related prognostic information.

Polyunsaturated fatty acids in plasma at 8 years and subsequent allergic disease.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) are hypothesized to modulate the risk of allergic disease. However, evidence from previous studies is inconclusive, and limited longitudinal data exist using circulating biomarkers of PUFA intake and metabolism. OBJECTIVE: We aimed to investigate associations between n-3 and n-6 PUFAs at age 8 years and asthma, rhinitis, and aeroallergen sensitization at age 16 years. METHODS: Proportions of n-3 PUFAs (very long-chain n-3 [VLC n-3; sum of eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid] and α-linolenic acid) and n-6 PUFAs (linoleic acid and arachidonic acid [AA]) in blood samples at age 8 years were measured for 940 children from the prospective Swedish birth cohort BAMSE (Children, Allergy, Milieu, Stockholm, Epidemiology). Allergic disease phenotypes were defined by using questionnaires and IgE measures at the ages of 8 and 16 years. Logistic regression was used to examine potential associations. RESULTS: A higher proportion of total VLC n-3 PUFAs in plasma at age 8 years was associated with a reduced risk of prevalent asthma, rhinitis, and aeroallergen sensitization at age 16 years and with incidence of asthma between 8 and 16 years (adjusted odds ratio, 0.67; 95% CI, 0.47-0.94). AA was associated with a reduced risk of asthma, aeroallergen sensitization, and allergic rhinitis. The findings were most evident for allergic phenotypes of asthma and rhinitis. Additionally, AA was associated with an increased probability of asthma and rhinitis remission between 8 and 16 years of age. CONCLUSION: Higher proportions of certain VLC n-3 and very long-chain n-6 PUFAs in plasma phospholipids at age 8 years were associated with a reduced risk of allergic disease at age 16 years.

Does asthma affect school performance in adolescents? Results from the Swedish population-based birth cohort BAMSE.

BACKGROUND: Asthma is common among schoolchildren and may influence quality of life and school attendance. However, it is unclear if asthma affects school performance. The aim of this study was to examine whether different phenotypes of asthma affect school performance during adolescence. METHODS: The study population consisted of 1715 adolescents from a population-based birth cohort, followed up to age 16 with questionnaires and clinical examinations. Asthma was defined as at least 4 wheeze episodes or at least 1 wheeze episode in combination with inhaled steroids in the last 12 months. School grades were obtained from Statistics Sweden, and logistic regression analysis was performed to investigate the association between the final overall grade from secondary school and asthma phenotypes. RESULTS: Among the adolescents, 20.8% have had ever asthma; 24.2% early transient, 47.2% school-age onset, and 24.2% persistent asthma. At 16 years, 7.8% had asthma; 71.7% multimorbidity and 73.9% allergic asthma. A statistically significant association for performing less well was seen for ever asthma (ORadj  = 1.43, 95% CI = 1.09-1.88). In analyses of asthma onset, an association was seen for school-age onset (ORadj  = 1.49, CI = 1.02-2.16) and a tendency for persistent asthma (ORadj  = 1.61, CI = 0.98-2.66), although with overlapping confidence intervals. Further, adolescents with uncontrolled asthma tended to perform less well (ORadj  = 2.60, CI = 0.87-7.80) compared to adolescents with partly controlled (ORadj  = 1.12, CI = 0.68-1.83) and fully controlled (ORadj  = 1.29, CI = 0.55-3.01) asthma. CONCLUSIONS: Our results indicate that asthma impairs school performance in adolescence. Moreover, some evidence suggests the adolescents with asthma during school age and with poorer asthma control to be more likely to perform less well.

C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Snapshot of cobalt, chromium and nickel exposure in dental technicians.

BACKGROUND: It is not fully understood where and how people are exposed to sensitizing metals. Much can be learnt from studying occupational settings where metals are handled. OBJECTIVES: To quantify cobalt (Co), chromium (Cr) and nickel (Ni) exposure on the skin and in the air, and urine levels, in dental technicians working with tools and alloys that may result in skin and respiratory exposure. METHODS: The metal skin dose was quantified with acid wipe sampling in dental technicians (n = 13). Air exposure was monitored by personal air sampling. Spot urine samples were collected for 24 h. Metals were analysed with inductively coupled plasma mass spectrometry. RESULTS: Before work, Co was detected on the skin of 10 participants (0.00025-0.0039 µg/cm2 ), and Cr (0.00051-0.011 µg/cm2 ) and Ni (0.0062-0.15 µg/cm2 ) on the skin of all participants. After a 2-h period without hand washing, CoCr-exposed participants had more Co on the skin (p = 0.004) than non-CoCr-exposed participants. Co was found in 10 air samples (0.22-155 µg/m3 ), Cr in nine (0.43-71 µg/m3 ), and Ni in four (0.48-3.7 µg/m3 ). Metal urine concentrations were considered to be normal. CONCLUSIONS: Dental technicians were exposed to Co, Cr and Ni on the skin and through the air, which was not reflected in the urine concentrations in this study. Cobalt skin doses may potentially elicit allergic contact dermatitis and cause sensitization.

Realizing Strong Light-Matter Interactions between Single-Nanoparticle Plasmons and Molecular Excitons at Ambient Conditions.

Realizing strong light-matter interactions between individual two-level systems and resonating cavities in atomic and solid state systems opens up possibilities to study optical nonlinearities on a single-photon level, which can be useful for future quantum information processing networks. However, these efforts have been hampered by unfavorable experimental conditions, such as cryogenic temperatures and ultrahigh vacuum, required to study such systems and phenomena. Although several attempts to realize strong light-matter interactions at room temperature using plasmon resonances have been made, successful realizations on the single-nanoparticle level are still lacking. Here, we demonstrate the strong coupling between plasmons confined within a single silver nanoprism and excitons in molecular J aggregates at ambient conditions. Our findings show that deep subwavelength mode volumes V together with quality factors Q that are reasonably high for plasmonic nanostructures result in a strong-coupling figure of merit-Q/sqrt[V] as high as ∼6×10^{3} µm^{-3/2}, a value comparable to state-of-the-art photonic crystal and microring resonator cavities. This suggests that plasmonic nanocavities, and specifically silver nanoprisms, can be used for room temperature quantum optics.

Web-based self-reported height, weight, and body mass index among Swedish adolescents: a validation study.

BACKGROUND: Web-collected height and weight are increasingly used in epidemiological studies; however, the validity has rarely been evaluated. OBJECTIVE: The aim of the study was to validate self-reported height, weight, and corresponding body mass index (BMI) among Swedish adolescents aged approximately 16 years. A secondary aim was to investigate possible prediction factors for validity of self-reported BMI. METHODS: The study included 1698 adolescents from the population-based cohort BAMSE. Height and weight were collected through a Web-based questionnaire and subsequently measured using standard procedures. Differences between reported and measured height, weight, and corresponding BMI were compared by t tests and agreement was evaluated by Pearson correlation and Bland-Altman plots. Multivariable linear regression analysis was used to investigate whether lifestyle and demographic factors predicted validity of self-reported BMI. RESULTS: On average, weight was underestimated by 1.1 kg and height was overestimated by 0.5 cm, leading to an underestimation of BMI by 0.5 kg/m2. Correlation coefficients were .98 for height, .97 for weight, and .94 for BMI, and highly significant. Females underestimated weight to a higher extent than males and overweight and obese participants underestimated weight to a higher extent than normal-weight participants, which resulted in higher underestimation of BMI. Underweight participants, on the contrary, overestimated weight and correspondingly BMI. Overall, a high proportion of participants were classified into the correct BMI category; however, among overweight and obese participants, only 60.2% (139/231) and 46% (20/44) were correctly classified, respectively. In the multivariable prediction model, only gender and BMI status significantly predicted discrepancy between reported and measured BMI. CONCLUSIONS: Web-collected BMI may be used as a valid, quick, and cost-effective alternative to measured BMI among Swedish adolescents. The accuracy of self-reported BMI declines with increasing BMI and self-reported BMI should not be used to estimate the prevalence of overweight or obesity.

Performance of an innovative culture-based digital dipstick for detection of bacteriuria.

IMPORTANCE: In this study, we explore the transformative potential of UTI-lizer, an emerging technology not yet commercially available. Our manuscript shows that UTI-lizer is a promising alternative for detecting the five main pathogens that cause urinary tract infections (UTIs). The results also indicate that digital dipsticks have the potential to uniquely provide UTI diagnostic quality on par with that of gold-standard testing, with the added benefits of ease of testing, rapid test handling time, and simple test equipment. This technology can be helpful in quickly ruling out bacterial infections and reducing the unnecessary use of antibiotics, especially in primary care settings or at the point of care. Moreover, the UTI-lizer test can reduce the number of negative urine samples sent to central laboratories, thus easing the burden of UTI diagnostics on the healthcare system. We believe our study, as well as current and upcoming research based on this technology, is highly relevant for clinical microbiologists, microbiology scientists, general practitioners, and urologists.

Neural network enabled nanoplasmonic hydrogen sensors with 100 ppm limit of detection in humid air.

Environmental humidity variations are ubiquitous and high humidity characterizes fuel cell and electrolyzer operation conditions. Since hydrogen-air mixtures are highly flammable, humidity tolerant H2 sensors are important from safety and process monitoring perspectives. Here, we report an optical nanoplasmonic hydrogen sensor operated at elevated temperature that combined with Deep Dense Neural Network or Transformer data treatment involving the entire spectral response of the sensor enables a 100 ppm H2 limit of detection in synthetic air at 80% relative humidity. This significantly exceeds the <1000 ppm US Department of Energy performance target. Furthermore, the sensors pass the ISO 26142:2010 stability requirement in 80% relative humidity in air down to 0.06% H2 and show no signs of performance loss after 140 h continuous operation. Our results thus demonstrate the potential of plasmonic hydrogen sensors for use in high humidity and how neural-network-based data treatment can significantly boost their performance.

Analysis of binding sites on complement factor I using artificial N-linked glycosylation.

Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors, such as factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein. FI is composed of two chains linked by a disulfide bridge; the light chain comprises only the serine protease (SP) domain, whereas the heavy chain contains the FI membrane attack complex domain (FIMAC), CD5 domain, and low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based three-dimensional models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface-exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished the ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespective of the cofactor used. On the other hand, only a few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI.

Evaluation of CADASTER QSAR models for the aquatic toxicity of (benzo)triazoles and prioritisation by consensus prediction.

QSAR regression models of the toxicity of triazoles and benzotriazoles ([B]TAZs) to an alga (Pseudokirchneriella subcapitata), Daphnia magna and a fish (Onchorhynchus mykiss), were developed by five partners in the FP7-EU Project, CADASTER. The models were developed by different methods - Ordinary Least Squares (OLS), Partial Least Squares (PLS), Bayesian regularised regression and Associative Neural Network (ASNN) - by using various molecular descriptors (DRAGON, PaDEL-Descriptor and QSPR-THESAURUS web). In addition, different procedures were used for variable selection, validation and applicability domain inspection. The predictions of the models developed, as well as those obtained in a consensus approach by averaging the data predicted from each model, were compared with the results of experimental tests that were performed by two CADASTER partners. The individual and consensus models were able to correctly predict the toxicity classes of the chemicals tested in the CADASTER project, confirming the utility of the QSAR approach. The models were also used for the prediction of aquatic toxicity of over 300 (B)TAZs, many of which are included in the REACH pre-registration list, and were without experimental data. This highlights the importance of QSAR models for the screening and prioritisation of untested chemicals, in order to reduce and focus experimental testing.

Electron Beam Induced Enhancement and Suppression of Oxidation in Cu Nanoparticles in Environmental Scanning Transmission Electron Microscopy.

We have investigated the effects of high-energy electron irradiation on the oxidation of copper nanoparticles in environmental scanning transmission electron microscopy (ESTEM). The hemispherically shaped particles were oxidized in 3 mbar of O2 in a temperature range 100-200 °C. The evolution of the particles was recorded with sub-nanometer spatial resolution in situ in ESTEM. The oxidation encompasses the formation of outer and inner oxide shells on the nanoparticles, arising from the concurrent diffusion of copper and oxygen out of and into the nanoparticles, respectively. Our results reveal that the electron beam actively influences the reaction and overall accelerates the oxidation of the nanoparticles when compared to particles oxidized without exposure to the electron beam. However, the extent of this electron beam-assisted acceleration of oxidation diminishes at higher temperatures. Moreover, we observe that while oxidation through the outward diffusion of Cu+ cations is enhanced, the electron beam appears to hinder oxidation through the inward diffusion of O2- anions. Our results suggest that the impact of the high-energy electrons in ESTEM oxidation of Cu nanoparticles is mostly related to kinetic energy transfer, charging, and ionization of the gas environment, and the beam can both enhance and suppress reaction rates.

The Role of Grain Boundary Sites for the Oxidation of Copper Catalysts during the CO Oxidation Reaction.

The oxidation of transition metal surfaces is a process that takes place readily at ambient conditions and that, depending on the specific catalytic reaction at hand, can either boost or hamper activity and selectivity. Cu catalysts are no exception in this respect since they exhibit different oxidation states for which contradicting activities have been reported, as, for example, in the catalytic oxidation of CO. Here, we investigate the impact of low-coordination sites on nanofabricated Cu nanoparticles with engineered grain boundaries on the oxidation of the Cu surface under CO oxidation reaction conditions. Combining multiplexed in situ single particle plasmonic nanoimaging, ex situ transmission electron microscopy imaging, and density functional theory calculations reveals a distinct dependence of particle oxidation rate on grain boundary density. Additionally, we found that the oxide predominantly nucleates at grain boundary-surface intersections, which leads to nonuniform oxide growth that suppresses Kirkendall-void formation. The oxide nucleation rate on Cu metal catalysts was revealed to be an interplay of surface coordination and CO oxidation behavior, with low coordination favoring Cu oxidation and high coordination favoring CO oxidation. These findings explain the observed single particle-specific onset of Cu oxidation as being the consequence of the individual particle grain structure and provide an explanation for widely distributed activity states of particles in catalyst bed ensembles.

Citrullination of C1-inhibitor as a mechanism of impaired complement regulation in rheumatoid arthritis.

Background: Dysregulated complement activation, increased protein citrullination, and production of autoantibodies against citrullinated proteins are hallmarks of rheumatoid arthritis (RA). Citrullination is induced by immune cell-derived peptidyl-Arg deiminases (PADs), which are overactivated in the inflamed synovium. We characterized the effect of PAD2- and PAD4-induced citrullination on the ability of the plasma-derived serpin C1-inhibitor (C1-INH) to inhibit complement and contact system activation. Methods: Citrullination of the C1-INH was confirmed by ELISA and Western blotting using a biotinylated phenylglyoxal probe. C1-INH-mediated inhibition of complement activation was analyzed by C1-esterase activity assay. Downstream inhibition of complement was studied by C4b deposition on heat-aggregated IgGs by ELISA, using pooled normal human serum as a complement source. Inhibition of the contact system was investigated by chromogenic activity assays for factor XIIa, plasma kallikrein, and factor XIa. In addition, autoantibody reactivity to native and citrullinated C1-INH was measured by ELISA in 101 RA patient samples. Results: C1-INH was efficiently citrullinated by PAD2 and PAD4. Citrullinated C1-INH was not able to bind the serine protease C1s and inhibit its activity. Citrullination of the C1-INH abrogated its ability to dissociate the C1-complex and thus inhibit complement activation. Consequently, citrullinated C1-INH had a decreased capacity to inhibit C4b deposition via the classical and lectin pathways. The inhibitory effect of C1-INH on the contact system components factor XIIa, plasma kallikrein, and factor XIa was also strongly reduced by citrullination. In RA patient samples, autoantibody binding to PAD2- and PAD4-citrullinated C1-INH was detected. Significantly more binding was observed in anti-citrullinated protein antibody (ACPA)-positive than in ACPA-negative samples. Conclusion: Citrullination of the C1-INH by recombinant human PAD2 and PAD4 enzymes impaired its ability to inhibit the complement and contact systems in vitro. Citrullination seems to render C1-INH more immunogenic, and citrullinated C1-INH might thus be an additional target of the autoantibody response observed in RA patients.

Competing oxidation mechanisms in Cu nanoparticles and their plasmonic signatures.

Chemical reactions involving nanoparticles often follow complex processes. In this respect, real-time probing of single nanoparticles under reactive conditions is crucial for uncovering the mechanisms driving the reaction pathway. Here, we have captured in situ the oxidation of single Cu nanoparticles to unravel a sequential competitive activation of different mechanisms at temperatures 50-200 °C. Using environmental scanning transmission electron microscopy, we monitor the evolution of oxide formation with sub-nanometre spatial resolution, and show how the prevalence of oxide island nucleation, Cabrera-Mott, Valensi-Carter and Kirkendall mechanisms under different conditions determines the morphology of the particles. Moreover, using in situ electron energy-loss spectroscopy, we probe the localised surface plasmons of individual particles during oxidation, and with the aid of finite-difference time-domain electrodynamic simulations investigate the signature of each mechanism in their plasmonic response. Our results shed light on the rich and intricate processes involved in the oxidation of nanoparticles, and provide in-depth insight into how these processes govern their morphology and optical response, beneficial for applications in catalysis, sensing, nanomedicine and plasmonics.

Time-Resolved Thickness and Shape-Change Quantification using a Dual-Band Nanoplasmonic Ruler with Sub-Nanometer Resolution.

Time-resolved measurements of changes in the size and shape of nanobiological objects and layers are crucial to understand their properties and optimize their performance. Optical sensing is particularly attractive with high throughput and sensitivity, and label-free operation. However, most state-of-the-art solutions require intricate modeling or multiparameter measurements to disentangle conformational or thickness changes of biomolecular layers from complex interfacial refractive index variations. Here, we present a dual-band nanoplasmonic ruler comprising mixed arrays of plasmonic nanoparticles with spectrally separated resonance peaks. As electrodynamic simulations and model experiments show, the ruler enables real-time simultaneous measurements of thickness and refractive index variations in uniform and heterogeneous layers with sub-nanometer resolution. Additionally, nanostructure shape changes can be tracked, as demonstrated by quantifying the degree of lipid vesicle deformation at the critical coverage prior to rupture and supported lipid bilayer formation. In a broader context, the presented nanofabrication approach constitutes a generic route for multimodal nanoplasmonic optical sensing.

Analysis of binding sites on complement factor I that are required for its activity.

The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1, and membrane cofactor protein. FI is a serine protease composed of two chains. The light chain comprises the serine protease domain, whereas the heavy chain contains several domains; that is, the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (K(m)) of all FI mutants toward a small substrate was not altered, whereas some mutants showed increased maximum initial velocity (V(max)). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used, whereas only some mutations in the CD5 and LDLr1/2 domains had a similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.

C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation.

After cell death, via apoptosis or necrosis, the uptake of dead cells by neighboring cells or phagocytes prevents the release of intracellular content. An array of molecules, including initiation molecules of the complement system, are involved in marking dead cells for uptake. After binding of these molecules, complement activation takes place, which when uncontrolled might result in a proinflammatory state. In the current study we demonstrate that complement inhibitor, C4b-binding protein (C4BP), binds strongly to necrotic cells, irrespective of the cell type used or the method of induction. After binding of the C4BP-protein S (PS) complex to necrotic cells via PS-phosphatidylserine and C4BP-DNA interactions, C4BP-PS inhibits complement activation on these cells. C4BP binds DNA via a patch of positively charged amino acids, mainly on the second complement control domain of the C4BP alpha-chain (affinity constant: 190 nM). Furthermore, C4BP limits DNA release from necrotic cells and inhibits DNA-mediated complement activation in solution. The C4BP-necrotic cell interaction also occurs in vivo as necrotic areas of arteriosclerotic plaques and of various cancers stain strongly positive for C4BP. This study describes a novel mechanism in which C4BP limits the inflammatory potential of necrotic cells.