The Headgroup (A)Symmetry Strongly Determines the Aggregation Behavior of Single-Chain Phenylene-Modified Bolalipids and Their Miscibility with Classical Phospholipids

In the present work, we describe the synthesis of two single-chain phenylene-modified bolalipids, namely PC-C17pPhC17-PC and PC-C17pPhC17-OH, with either symmetrical (phosphocholine) or asymmetrical (phosphocholine and hydroxyl) headgroups using a Sonogashira cross-coupling reaction as key step. The temperature-dependent aggregation behavior of both bolalipids in aqueous suspension was studied using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, small angle neutron scattering (SANS), and X-ray scattering. We show that different headgroup symmetries lead to a change in the aggregation behavior: Whereas PC-C17pPhC17-PC forms nanofibers with a diameter of 5.7 nm that transform into small ellipsoidal micelles at 23 °C, the PC-C17pPhC17-OH self-assembles into lamellae with bolalipid molecules in an antiparallel orientation up to high temperatures. Furthermore, the mixing behavior of both bolalipids with bilayer-forming phospholipids (DPPC and DSPC) was studied by means of DSC and TEM. The aim was to stabilize bilayer membranes formed of phospholipids in order to improve these mixed lipid vesicles for drug delivery purposes. We show that the symmetrical PC-C17pPhC17-PC is miscible with DPPC and DSPC; however, closed lipid vesicles are not observed, and elongated micelles and bilayer fragments are found instead. In contrast, the asymmetrical PC-C17pPhC17-OH shows no miscibility with phospholipids at all

Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

AbstractPreviously we reported a broadly HIV-1 neutralizing mini-antibody (Fab 3674) of modest potency that was derived from a human non-immune phage library by panning against the chimeric gp41-derived construct NCCG-gp41. This construct presents the N-heptad repeat of the gp41 ectodomain as a stable, helical, disulfide-linked trimer that extends in helical phase from the six-helix bundle of gp41. In this paper, Fab 3674 was subjected to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtype B and C HIV-1 reference panels. The parental Fab 3674 is 10–20-fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (∼50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (∼120 kDa) across the subtype B and C reference panels

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

Application of Ligninolytic Enzymes in the Production of Biofuels from Cotton Wastes

The application of ligninolytic fungi and enzymes is an option to overcome the issues related with the production of biofuels using cotton wastes. In this dissertation, the ligninolytic fungus and enzymes were evaluated as pretreatment for the biochemical conversion of Cotton Gin Trash (CGT) in ethanol and as a treatment for the transformation of cotton wastes biochar in other substances. In biochemical conversion, seven combinations of three pretreatments (ultrasonication, liquid hot water and ligninolytic enzymes) were evaluated on CGT. The best results were achieved by the sequential combination of ultrasonication, hot water, and ligninolytic enzymes with an improvement of 10% in ethanol yield. To improve these results, alkaline-ultrasonication was evaluated. Additionally, Fourier Transform Infrared (FT-IR) and principal component analysis (PCA) were employed as fast methodology to identify structural differences in the biomass. The combination of ultrasonication-alkali hydrolysis, hot liquid water, and ligninolytic enzymes using 15% of NaOH improved 35% ethanol yield compared with the original treatment. Additionally, FT-IR and PCA identified modifications in the biomass structure after different types of pretreatments and conditions. In thermal conversion, this study evaluated the biodepolymerization of cotton wastes biochar using chemical and biological treatments. The chemical depolymerization evaluated three chemical agents (KMnO4, H2SO4, and NaOH), with three concentrations and two environmental conditions. The sulfuric acid treatments performed the largest transformations of the biochar solid phase; whereas, the KMnO4 treatments achieved the largest depolymerizations. The compounds released into the liquid phase were correlated with fulvic and humic acids and silicon compounds. The biological depolymerization utilized four ligninolytic fungi Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Postia placenta, and Bjerkandera adusta. The greatest depolymerization was obtained by C. subvermispora. The depolymerization kinetics of C. subvermispora evidenced the production of laccase and manganese peroxidase and a correlation between depolymerization and production of ligninolytic enzymes. The modifications obtained in the liquid and solid phases showed the production of humic and fulvic acids from the cultures with C. subvermispora. The results of this research are the initial steps for the development of new processes using the ligninolytic fungus and their enzymes for the production of biofuels from cotton wastes

Regulation der Sekretion und Zelloberflächenassoziation von Cathepsin X durch Interaktionen mit dem Integrin alphavbeta3 und seinem Liganden Vitronektin

Cathepsine sind lysosomale Cysteinproteasen, die neben der allgemeinen Proteindegradation in Lysosomen auch spezifische Funktionen ausüben, die eine limitierte Proteolyse erfordern. Zudem werden Cathepsine sezerniert, weshalb man sie auch im Extrazellulärraum findet, wo sie ebenfalls an verschiedenen biologischen Vorgängen, wie etwa der Zellmigration/Invasion, teilnehmen. Über Cathepsin X, einen relativ neu entdeckten Vertreter dieser Proteinklasse, war zu Beginn der Promotionsarbeit noch wenig bekannt. Die Struktur und das Aktivitätsprofil konnten zwar bereits gelöst werden, über mögliche (patho-)physiologische Funktionen gab es jedoch noch keine Erkenntnisse. Das Hauptziel meiner Untersuchungen war daher, mittels geeigneter Methoden nähere Aufschlüsse über die Rolle von Cathepsin X oder seiner Proform innerhalb und außerhalb der Zelle zu erlangen. Dies sollte vorwiegend durch die Analyse der Expression und Sekretion dieser Protease, sowie durch das Auffinden von Interaktionspartnern erfolgen. Wie sich in Vorversuchen zeigte, wird Cathepsin X in humanen Leukozyten unterschiedlich stark exprimiert. Da eine hohe Expression insbesondere in Monozyten vorlag, wurde für weitere Analysen das Zellmodell THP-1 eingesetzt, das auch für die Differenzierung zu Makrophagen-ähnlichen Zellen durch Stimulation mit Phorbol-12-Myristat-13-Acetat (PMA) oder all-trans Retinsäure (ATRA) verwendet werden kann. Interessanterweise zeigten diese Agenzien unterschiedliche Auswirkungen auf die Expression und Sekretion von Cathepsin X. So wurde mit PMA eine starke intra- und extrazelluläre Erhöhung der Protease verzeichnet, während mit ATRA das Gegenteil der Fall war. Da eine differenzielle Expression von Cathepsin X in Leukozyten auf eine mögliche Funktion in der Entzündungsantwort hindeutet, schien eine Untersuchung der Wirkung von proinflammatorischen Zytokinen und extrazellulären Matrix (EZM)-Proteinen sinnvoll, weil diese Faktoren ebenfalls die Sekretion von Proteasen beeinflussen können. Die untersuchten Zytokine hatten allerdings keinen Effekt auf die Sekretion von Cathepsin X aus THP-1-Zellen, wohingegen mit dem EZM-Protein Vitronektin eine Verdopplung der Cathepsin-X-Konzentration im Medium beobachtet wurde. In diesem Kontext konnte nachgewiesen werden, dass Vitronektin durch die Interaktion mit dem Zelloberflächenrezeptor Integrin avb3 den Sekretionsapparat der Zelle beeinflusst, wobei offensichtlich das Sequenzmotiv Arginin-Glyzin-Aspartat (RGD), welches in Vitronektin enthalten ist, für diesen Vorgang entscheidend ist. Neben Cathepsin X wurde auch für die Cathepsine B und L eine erhöhte Freisetzung nach Inkubation mit Vitronektin gemessen, was zeigt, dass dieser durch das EZM-Protein ausgelöste Mechanismus nicht auf Cathepsin X beschränkt ist. Umgekehrt ließ sich in einem weiteren Zellmodell (HUVEC) durch den Einsatz von „small-interfering RNA“ (siRNA) die Expression von Cathepsin X erniedrigen, was zu einer verminderten Migration der HUVEC in einem Invasionsversuch führte. Dies deutet auf eine Funktion von Cathepsin X in der Zellmotilität hin. Weil Cathepsin X, ähnlich wie Vitronektin, ein exponiertes RGD-Motiv in seiner Proregion aufweist, sollte nun eine mögliche Interaktion mit Integrinen untersucht werden. Tatsächlich ließ sich eine RGD-abhängige Interaktion von Procathepsin X mit dem Integrin avb3 zeigen. Somit werden in dieser Arbeit zwei wesentliche neue Aspekte in der Regulation der Sekretion und seiner Beteiligung an Migrationsvorgängen gezeigt, wobei die Interaktion von Procathepsin X mit dem Integrin avb3 eine besondere Rolle zu spielen scheint. Ob diese beiden Vorgänge miteinander gekoppelt sind, konnte mit den bisherigen Ergebnissen noch nicht bewiesen werden. Insgesamt deuten die Ergebnisse jedoch darauf hin, dass extrazelluläres (Pro)Cathepsin X neben seiner Rolle als Protease auch nicht-proteolytische Funktionen, beispielsweise als Ligand bestimmter Zelloberflächenstrukturen ausüben kann. Dieser Aspekt könnte im Hinblick auf eine therapeutische Inhibition von Angiogenese und Metastasierung von Tumorzellen durch Antikörper gegen Cathepsin X und/oder Integrine von großem Nutzen sein

Plasma copeptin levels are inversely associated with intima-media-thickness in men: the population-based KORA F4 study

BACKGROUND: Elevated plasma preprovasopressin (copeptin) levels are associated with cardiovascular complications as well as with an increased risk for type 2 diabetes (T2D). Here, we studied, whether plasma copeptin is related to carotid intima-media thickness (IMT), a measure of early atherosclerosis, and may thus be one explanation for the high cardiovascular risk in T2D. METHODS: Plasma concentrations of copeptin and IMT of the common carotid artery were determined in 1275 participants of the population-based KORA F4 study. We used linear regression models to investigate associations between copeptin levels and IMT. RESULTS: In the whole study group, copeptin levels were not significantly associated with IMT after adjustment for age and sex. Copeptin and IMT were significantly inversely associated after multivariable adjustment in the total cohort (β = -0.020 mm, 95% CI: -0.037 mm; -0.003 mm), in men (β = -0.035 mm, 95% CI: -0.061 mm; -0.009 mm) and in study participants with prediabetes (β = -0.041 mm, 95% CI: -0.078 mm; -0.005 mm) comparing quartile 4 vs quartile 1. The negative association of copeptin and IMT in men was present after adjustment for age alone. In women and patients with T2D, copeptin was not significantly associated with IMT. CONCLUSIONS: Plasma copeptin was not associated with an increased IMT in our study cohort. In contrast, copeptin levels were related to a lower IMT in men and subjects with prediabetes, suggesting that elevated copeptin concentrations do not exert proatherogenic effects on carotid arteries

Serum uromodulin and decline of kidney function in older participants of the population-based KORA F4/FF4 study

Background: Uromodulin, a tissue-specific tubular glycoprotein, has recently emerged as a promising biomarker for kidney function and tubular integrity. However, the association of serum uromodulin (sUmod) with renal function decline is still unknown in an older general population. Methods: We analysed the association of sUmod with the estimated glomerular filtration rate (eGFR) and albuminuria in 1075 participants of the population-based Cooperative Health Research in the Region of Augsburg (KORA) F4 study, ages 62-81 years, at baseline and prospectively after a mean follow-up time of 6.5 years (n = 605) using logistic and linear regression models as well as receiver operating characteristics (ROC) analyses. Results: Cross-sectionally, sUmod was positively associated with eGFR (β = 0.31 ± 0.02 per higher standard deviation sUmod; P < 0.001) and inversely associated with the urinary albumin:creatinine ratio (β = -0.19 ± 0.04; P < 0.001) after adjustment for sex, age, body mass index, arterial hypertension, prediabetes and diabetes. After multivariable adjustment including baseline eGFR, sUmod was not associated with incident chronic kidney disease (CKD), defined as a decrease in eGFR <60 mL/min/1.73 m2 after 6.5 years of follow-up {odds ratio [OR] 1.02 [95% confidence interval (CI) 0.77-1.36] per higher SD sUmod} but was inversely associated with advanced CKD, defined as incident eGFR <45 mL/min/1.73 m2 [OR 0.64 (95% CI 0.42-0.98)]. The ROC showed no added predictive value of sUmod for kidney function decline in the fully adjusted model. Conclusions: Higher sUmod was inversely associated with progression to advanced kidney disease but does not provide additional predictive value for the development of CKD in elderly participants of the population-based KORA study