944 research outputs found
Hydrogen-Bonding Capacity and Brain Penetration
Brain penetration has been reported to correlate with Δlog P, defined as log P (octan-1-ol/H2O) – log P (alkane/H2O). Another recent development, describing log P as the Sum of a cavity or volume contribution and H-bonding capability, the latter expressed by Asolvent values, prompted us to reinvestigate the properties accounting for brain penetration. It was found that Aalkane and the hydrophilic part of the van der Waals surface both correlate well with brain uptake. These findings offer new opportunities for the design of compounds which either should or should not be active at sites located in the brain
Next-generation outer membrane vesicle vaccines from concept to clinical trials
Only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. The OMV vaccines, however, provide limited coverage and are difficult to produce. This is caused by an obligatory detergent treatment, which removes lipopolysaccharide (LPS), a toxic OMV component. This thesis explored an alternative approach, based on OMV with attenuated lpxL1-LPS and a detergent-free process. The alternative approach is referred to as ‘next-generation OMV’ and provided vaccines with improved immunological and biochemical properties. In addition, quantitative proteomics demonstrated a preferred protein composition. This provided justification for further development towards clinical trials. After optimization of specific process steps, an improved pilot-scale production process was developed. The quality of OMV from this optimized process was stable and within pre-set specifications for nine consecutive batches. Studies in mice and rabbits suggested that next-generation OMV are immunogenic and safe for parenteral use in humans. Therefore these vaccines are now ready for clinical evaluation. Several groups are developing broadly protective OMV vaccines against N. meningitidis serogroup B, but also against other serogroups and other pathogens. OMV therefore have the potential to become a versatile technology platform for prophylactic and therapeutic vaccines. Such a platform requires a reliable production process to generate substantial quantities of high quality product. The process described in this thesis is well-suited for this purpose. The results encourage technology transfer to a commercial partner, with the goal to translate nextgeneration OMV technology into actual vaccines and improve global public health.</p
Continuous production of Neisseria meningitidis outer membrane vesicles
Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.publishedVersionPaid Open Acces
Examining the Heterogeneous Genome Content of Multipartite Viruses BMV and CCMV by Native Mass Spectrometry
Since the concept was first introduced by Brian Chait and co-workers in 1991, mass spectrometry of proteins and protein complexes under non-denaturing conditions (native MS) has strongly developed, through parallel advances in instrumentation, sample preparation, and data analysis tools. However, the success rate of native MS analysis, particularly in heterogeneous mega-Dalton (MDa) protein complexes, still strongly depends on careful instrument modification. Here, we further explore these boundaries in native mass spectrometry, analyzing two related endogenous multipartite viruses: the Brome Mosaic Virus (BMV) and the Cowpea Chlorotic Mottle Virus (CCMV). Both CCMV and BMV are approximately 4.6 megadalton (MDa) in mass, of which approximately 1 MDA originates from the genomic content of the virion. Both viruses are produced as mixtures of three particles carrying different segments of the genome, varying by approximately 0.1 MDA in mass (~2%). This mixture of particles poses a challenging analytical problem for high-resolution native MS analysis, given the large mass scales involved. We attempt to unravel the particle heterogeneity using both Q-TOF and Orbitrap mass spectrometers extensively modified for analysis of very large assemblies. We show that manipulation of the charging behavior can provide assistance in assigning the correct charge states. Despite their challenging size and heterogeneity, we obtained native mass spectra with resolved series of charge states for both BMV and CCMV, demonstrating that native MS of endogenous multipartite virions is feasible. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-016-1348-6) contains supplementary material, which is available to authorized users
Cysteine Depletion Causes Oxidative Stress and Triggers Outer Membrane Vesicle Release by Neisseria meningitidis Implications for Vaccine Development
Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (61500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteinedependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis
Improved Production Process for Native Outer Membrane Vesicle Vaccine against Neisseria meningitidis
An improved detergent-free process has been developed to produce vaccine based on native outer membrane vesicles (NOMV) against Neisseria meningitidis serogroup B. Performance was evaluated with the NonaMen vaccine concept, which provides broad coverage based on nine distinct PorA antigens. Scalable aseptic equipment was implemented, replacing undesirable steps like ultracentrifugation, inactivation with phenol, and the use of preservatives. The resulting process is more consistent and gives a higher yield than published reference processes, enabling NOMV production at commercial scale. Product quality met preliminary specifications for 9 consecutive batches, and an ongoing study confirmed real-time stability up to 12 months after production. As the NOMV had low endotoxic activity and induced high bactericidal titres in mice, they are expected to be safe and effective in humans. The production process is not limited to NonaMen and may be applicable for other N. meningitidis serogroups and other gram-negative pathogens. The current results therefore facilitate the late-stage development and clinical evaluation of NOMV vaccines
kLog: A Language for Logical and Relational Learning with Kernels
We introduce kLog, a novel approach to statistical relational learning.
Unlike standard approaches, kLog does not represent a probability distribution
directly. It is rather a language to perform kernel-based learning on
expressive logical and relational representations. kLog allows users to specify
learning problems declaratively. It builds on simple but powerful concepts:
learning from interpretations, entity/relationship data modeling, logic
programming, and deductive databases. Access by the kernel to the rich
representation is mediated by a technique we call graphicalization: the
relational representation is first transformed into a graph --- in particular,
a grounded entity/relationship diagram. Subsequently, a choice of graph kernel
defines the feature space. kLog supports mixed numerical and symbolic data, as
well as background knowledge in the form of Prolog or Datalog programs as in
inductive logic programming systems. The kLog framework can be applied to
tackle the same range of tasks that has made statistical relational learning so
popular, including classification, regression, multitask learning, and
collective classification. We also report about empirical comparisons, showing
that kLog can be either more accurate, or much faster at the same level of
accuracy, than Tilde and Alchemy. kLog is GPLv3 licensed and is available at
http://klog.dinfo.unifi.it along with tutorials
Global Antifungal Profile Optimization of Chlorophenyl Derivatives against Botrytis cinerea and Colletotrichum gloeosporioides
Twenty-two aromatic derivatives bearing a chlorine atom and a different chain in the para or meta
position were prepared and evaluated for their in vitro antifungal activity against the phytopathogenic
fungi Botrytis cinerea and Colletotrichum gloeosporioides. The results showed that maximum inhibition
of the growth of these fungi was exhibited for enantiomers S and R of 1-(40-chlorophenyl)-
2-phenylethanol (3 and 4). Furthermore, their antifungal activity showed a clear structure-activity
relationship (SAR) trend confirming the importance of the benzyl hydroxyl group in the inhibitory
mechanism of the compounds studied. Additionally, a multiobjective optimization study of the
global antifungal profile of chlorophenyl derivatives was conducted in order to establish a rational
strategy for the filtering of new fungicide candidates from combinatorial libraries. The MOOPDESIRE
methodology was used for this purpose providing reliable ranking models that can be
used later
Structure based inhibitor design targeting glycogen phosphorylase b. Virtual screening, synthesis, biochemical and biological assessment of novel N-acyl-β-d-glucopyranosylamines
Glycogen phosphorylase (GP) is a validated target for the development of new type 2 diabetes treatments. Exploiting the Zinc docking database, we report the in silico screening of 1888 β- D-glucopyranose-NH-CO-R putative GP inhibitors differing only in their R groups. CombiGlide and GOLD docking programs with different scoring functions were employed with the best performing methods combined in a “consensus scoring” approach to ranking of ligand binding affinities for the active site. Six selected candidates from the screening were then synthesized and their inhibitory potency was assessed both in vitro and ex vivo. Their inhibition constants’ values, in vitro, ranged from 5 to 377 µM while two of them were effective at causing inactivation of GP in rat hepatocytes at low µM concentrations. The crystal structures of GP in complex with the inhibitors were defined and provided the structural basis for their inhibitory potency and data for further structure based design of more potent inhibitors
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