Generation of recombinant antibodies against the Venezuelan equine encephalitis virus

Das Venezuelanische Pferdeenzephalitis-Virus (VEEV) gehoert zu der Gattung Alphaviren. Bisher sind nur wenige monoklonale, hochaffine Antikoerper gegen diese Viren fuer die schnelle Diagnostik und Therapie verfuegbar. Ziel dieser Arbeit war die Generierung humaner rekombinanter Antikoerperfragmente gegen VEEV aus universellen Antikoerpergenbibliotheken zur verbesserten Detektion. Mittels der Phagen-Display-Technologie wurden erstmals virusspezifische Antikoerperfragmente durch Selektionen mit kompletten VEEV-Partikeln isoliert. Durch Optimierung der Selektionsstrategien konnten zwoelf VEEV-spezifische scFv-Klone aus zwei naiven Antikoerpergenbibliotheken isoliert werden. Zur Charakterisierung wurden scFv-praesentierende, filamentoese M13-Phagen dieser Klone produziert und zur Detektion verschiedener Viruspraeparationen eingesetzt. Die scFv-praesentierenden Phagen erkannten, eingesetzt als Detektionsantikoerper, verschiedene VEEV-Staemme, jedoch keine anderen Alphaviren. Der vielseitige und hochsensitive Einsatz von scFv-praesentierenden Phagen zur Virusdetektion wurde in dieser Arbeit demonstriert. Mittels Immunfaerbungen wurde gezeigt, dass strukturelle Epitope von den scFv-praesentierenden Phagen erkannt werden. Diese Strukturen konnten im Lysat mittels ELISA und auf der Zelloberflaeche VEEV-infizierter Zellen mittels Immunhistochemie spezifisch nachgewiesen werden. Zur Optimierung der Erkennungseigenschaften von VEEV wurde ein Kettenaustausch der variablen Domaene der leichten Antikoerperkette des generierten humanen Antikoerperfragments MK220-IG12 mittels Phagen-Display durchgefuehrt. Die generierten humanen Antikoerperfragmente koennten die schnelle Identifikation und Detektion von VEEV verbessern, sowie potentiell zur Therapie von VEEV-Infektionen eingesetzt werden.Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus. Monoclonal highly affine antibodies against viruses of this group are rare for rapid diagnostic procedures and therapy. The aim of this work was the generation of recombinant antibody fragments against VEEV from universal antibody gene libraries for an improved detection. Virus specific scFv were isolated with selection strategies on whole VEEV particles using phage display for the first time. By optimisation of the selection strategy twelve VEEV specific scFv could be isolated from two naive libraries. Monoclonal scFv presenting filamentous M13 phage of the obtained antibody fragments were produced for characterisation and used for detection of several virus preparations. Used as detection antibodies, the scFv presenting phage were specific for VEEV strains and did not show any cross-reactivity with other alphaviruses. In this study the broad und sensitive approach of scFv presenting phage for virus detection was demonstrated. Immunostains of VEEV proteins indicated the recognition of structural epitopes by the scFv presenting phage. These structures were detected specifically in the lysate by ELISA and on the surface of VEEV infected Vero cells by immunhistochemistry. For an improved recognition of VEEV a chain shuffling of the variable domain of the human antibody fragment MK220-IG12 light chain was performed using phage display. The generated human antibody fragments could improve the fast identification and diagnosis of VEEV and will be potentially used in the therapy of VEEV infections

The role of anxiety in decision-making

Over the past decade, many studies have shown that individuals with reduced sensitivity for risk due to traumatic brain injury in orbital parts of the prefrontal cortex tend to ignore the long term outcomes of their behavioral actions (the same holds true for individuals with socio-/psychopathy). Instead, these individuals merely base decisions on anticipated immediate gains, similar to impulsive choice in children. The Iowa gambling task has been designed specifically to measure this behavioral tendency. We used this task to investigate a state opposite to that of impulsiveness and carelessness, namely enhanced anxiety and risk intolerance. We expected beneficial effects on decision-making, especially since high anxiety in both healthy populations and patients with anxiety disorders has been linked with enhanced activation of orbitofrontal cortex. Our most important finding is that intolerance towards uncertainty is indeed positively correlated with overall performance on the Iowa gambling task in a sample of adults as well as with anxiety in a sample of children. Results illustrate the protective functions of anxiety and risk aversion, and their positive long-term effects on decision-making. These motives seem to enable individuals to better consider future consequences of their actions, and to switch from previously reinforced behaviors to alternative behaviors when contingencies change

The SyBil-AA real-time fMRI neurofeedback study: protocol of a single-blind randomized controlled trial in alcohol use disorder

Background: Alcohol Use Disorder is a highly prevalent mental disorder which puts a severe burden on individuals, families, and society. The treatment of Alcohol Use Disorder is challenging and novel and innovative treatment approaches are needed to expand treatment options. A promising neuroscience-based intervention method that allows targeting cortical as well as subcortical brain processes is real-time functional magnetic resonance imaging neurofeedback. However, the efficacy of this technique as an add-on treatment of Alcohol Use Disorder in a clinical setting is hitherto unclear and will be assessed in the Systems Biology of Alcohol Addiction (SyBil-AA) neurofeedback study. Methods: N = 100 patients with Alcohol Use Disorder will be randomized to 5 parallel groups in a single-blind fashion and receive real-time functional magnetic resonance imaging neurofeedback while they are presented pictures of alcoholic beverages. The groups will either downregulate the ventral striatum, upregulate the right inferior frontal gyrus, negatively modulate the connectivity between these regions, upregulate, or downregulate the auditory cortex as a control region. After receiving 3 sessions of neurofeedback training within a maximum of 2 weeks, participants will be followed up monthly for a period of 3 months and relapse rates will be assessed as the primary outcome measure. Discussion: The results of this study will provide insights into the efficacy of real-time functional magnetic resonance imaging neurofeedback training in the treatment of Alcohol Use Disorder as well as in the involved brain systems. This might help to identify predictors of successful neurofeedback treatment which could potentially be useful in developing personalized treatment approaches. Trial registration: The study was retrospectively registered in the German Clinical Trials Register (trial identifier: DRKS00010253 ; WHO Universal Trial Number (UTN): U1111–1181-4218) on May 10th, 2016

Social acceptance of green hydrogen in Germany: building trust through responsible innovation

Background Social acceptance presents a major challenge for Germany’s transition to green energy. As a power-to-x technology, green hydrogen is set to become a key component of a future sustainable energy system. With a view to averting conflicts like those surrounding wind energy, we have investigated social acceptance of green hydrogen at an early stage in its implementation, before wider rollout. Our study uses a mixed-method approach, wherein semi-structured interviews (n = 24) and two participatory workshops (n = 51) in a selected region in central Germany serve alongside a representative survey (n = 2054) as the basis for both understanding social attitudes and reaching generalisable conclusions. Results Overall, it is possible to observe both a marked lack of knowledge and a large degree of openness towards green hydrogen and its local use, along with high expectations regarding environmental and climate protection. We reach three key conclusions. First, acceptance of green hydrogen relies on trust in science, government, the media, and institutions that uphold distributive justice, with consideration for regional values playing a vital role in establishing said trust. Second, methodologically sound participatory processes can promote acceptance, and active support in particular. Third, recurrent positive participatory experiences can effectively foster trust. Conclusions Accordingly, we argue that trust should be strengthened on a structural level, and that green hydrogen acceptance should be understood as a matter of responsible innovation. As the first empirical investigation into social acceptance of green hydrogen, and by conceptually interlinking acceptance research and responsible innovation, this study constitutes an important contribution to existing research

Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV)

<p>Abstract</p> <p>Background</p> <p>Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.</p> <p>Results</p> <p>In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different scFvs were identified and their immunological specificity was assessed. The specific detection of the VEEV strains TC83, H12/93 and 230 by the selected antibody fragments was proved. Active as well as formalin inactivated virus particles were recognized by the selected antibody fragments which could be also used for Western blot analysis of VEEV proteins and immunohistochemistry of VEEV infected cells. The anti-VEEV scFv phage clones did not show any cross-reactivity with Alphavirus species of the Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) antigenic complex, nor did they react with Chikungunya virus (CHIKV), if they were used as detection reagent.</p> <p>Conclusion</p> <p>For the first time, this study describes the selection of antibodies against a human pathogenic virus from a human naïve scFv antibody gene library using complete, active virus particles as antigen. The broad and sensitive applicability of scFv-presenting phage for the immunological detection and diagnosis of Alphavirus species was demonstrated. The selected antibody fragments will improve the fast identification of VEEV in case of a biological warfare or terroristic attack or a natural outbreak.</p

Single chain Fab (scFab) fragment

BACKGROUND: The connection of the variable part of the heavy chain (VH) and and the variable part of the light chain (VL) by a peptide linker to form a consecutive polypeptide chain (single chain antibody, scFv) was a breakthrough for the functional production of antibody fragments in Escherichia coli. Being double the size of fragment variable (Fv) fragments and requiring assembly of two independent polypeptide chains, functional Fab fragments are usually produced with significantly lower yields in E. coli. An antibody design combining stability and assay compatibility of the fragment antigen binding (Fab) with high level bacterial expression of single chain Fv fragments would be desirable. The desired antibody fragment should be both suitable for expression as soluble antibody in E. coli and antibody phage display. RESULTS: Here, we demonstrate that the introduction of a polypeptide linker between the fragment difficult (Fd) and the light chain (LC), resulting in the formation of a single chain Fab fragment (scFab), can lead to improved production of functional molecules. We tested the impact of various linker designs and modifications of the constant regions on both phage display efficiency and the yield of soluble antibody fragments. A scFab variant without cysteins (scFabΔC) connecting the constant part 1 of the heavy chain (CH1) and the constant part of the light chain (CL) were best suited for phage display and production of soluble antibody fragments. Beside the expression system E. coli, the new antibody format was also expressed in Pichia pastoris. Monovalent and divalent fragments (DiFabodies) as well as multimers were characterised. CONCLUSION: A new antibody design offers the generation of bivalent Fab derivates for antibody phage display and production of soluble antibody fragments. This antibody format is of particular value for high throughput proteome binder generation projects, due to the avidity effect and the possible use of common standard sera for detection

Lateralized reward-related visual discrimination in the avian entopallium

Abstract In humans and many other animals, the two cerebral hemispheres are partly specialized for different functions. However, knowledge about the neuronal basis of lateralization is mostly lacking. The visual system of birds is an excellent model in which to investigate hemispheric asymmetries as birds show a pronounced left hemispheric advantage in the discrimination of various visual objects. In addition, visual input crosses at the optic chiasm and thus testing of each hemisphere is easily accomplished. We aimed to find a neuronal correlate for three hallmarks of visual lateralization in pigeons: first, the animals learn faster with the right eye-left hemisphere; second, they reach higher performance levels under this condition; third, visually guided behavior is mostly under left hemisphere control. To this end, we recorded from the left and right forebrain entopallium while the animals performed a colour discrimination task. We found that, even before learning, left entopallial neurons were more responsive to visual stimulation. Subsequent discrimination acquisition recruited more neuronal responses in the left entopallium and these cells showed a higher degree of differentiation between the rewarded and the unrewarded stimulus. Thus, differential left-right responses are already present, albeit to a modest degree, before learning. As soon as some cues are associated with reward, however, this asymmetry increases substantially and the higher discrimination ratio of the left hemispheric tectofugal pathway would not only contribute to a higher performance of this hemisphere but could thereby also result in a left hemispheric dominance over downstream motor structures via reward-associated feedback systems

Stereoelectronic effects in the reaction of aromatic substrates catalysed by Halomonas elongata transaminase and its mutants

A transaminase from Halomonas elongata and four mutants generated by an in silico-based design, were recombinantly produced in E. coli, purified and applied to the amination of mono-substituted aromatic carbonyl-derivatives. While benzaldehyde derivatives resulted excellent substrates, only NO2-acetophenones were transformed into the (S)-amine with high enantioselectivity. The different behaviour of wild-type and mutated transaminases was assessed by in silico substrate binding mode studies

Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus fumigatus

BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16) which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs) were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus