Biology subjects

Brand, K.

Publications and source records attributed to Brand, K..

3 recordsLinked to original sources

bioRxiv2024

Herpes Simplex Virus Assembly and Spread in murine skin after infection from the outside

Herpes simplex viruses (HSV) cause many skin diseases, particularly in immunocompromised patients. HSV-1 infection of murine skin recapitulates many aspects of human pathology. However, many protocols rely on mechanical or enzymatic skin disruption to induce lesions, although this can alter skin homeostasis and prime antiviral inflammation before inoculation. To investigate the initial events following HSV-1 primary skin infection before the onset of symptoms, we developed a novel murine ex vivo explant model using gentle depilation but no further scarification and infected keratinocytes from the outside with minimal tissue damage. Two-photon microscopy studies showed that HSV-1 spread exclusively in the epidermis. The infection centers increased in number and size over time and contained hundreds of infected keratinocytes. We investigated the HSV-1 spread at the cellular level, using reporter strains with fluorescently-tagged capsid protein VP26, and monitored the formation of nuclear capsid assembly sites, nuclear capsid egress, and the recruitment of the inner tegument protein pUL37GFP, the outer tegument protein VP11/12GFP, and the envelope protein gDGFP to cytoplasmic capsids. By electron microscopy, the skin appeared intact, and keratinocytes contained many nuclear capsids, primary virions in the nuclear envelope, cytosolic membrane-associated capsids, and enveloped virions. Our protocol provides a robust and reproducible approach to investigate the very early events of HSV-1 spread in the skin, to characterize the phenotypes of HSV-1 mutants in terminally differentiated skin tissues, and to evaluate potentially antiviral small molecules in a preclinical ex vivo infection model. IMPORTANCEThis study describes a novel murine ex vivo skin explant model to investigate early events in HSV-1 infection without causing significant tissue damage. To infect from the outside, via the apical keratinocytes, this method relies on gentle depilation, which maintains skin integrity. HSV-1 spread exclusively within the epidermis, with infection centers growing over time and involving hundreds of keratinocytes. Using advanced microscopy techniques, we tracked HSV-1 spread at the cellular level and intracellular assembly of all intermediate virus structures. This model offers a valuable tool for studying the initial stages of HSV-1 infection, assessing viral mutant phenotypes, and testing antiviral compounds in a more physiological context to provide critical insights into HSV-1 pathogenesis and therapeutic strategies.

microbiology↗

bioRxiv2023

Feasibility of improving manufacturability based on protein engineering

While bioactivity and a favorable safety profile for biotherapeutics is of utmost importance, manufacturability is also worth of consideration to ease the manufacturing process. Many biotherapeutics are typically expressed in mammalian cells. Process-related impurities or biological impurities like viruses and host cell proteins (HCP) are present in the harvest which have mostly acid isoelectric points and need to be removed to ensure safety for the patients. Therefore, during molecule design, an isoelectric point of the target molecule should preferably differ sufficiently from the isoelectric points of the impurities to enable an efficient and straightforward purification strategy. In this feasibility study we have evaluated the possibility to improve manufacturability by increasing the isoelectric point of the target protein. We have generated several variants of a GLP1-receptor-agonist-Fc-domain -FGF21 fusion protein and demonstrate that the critical anion exchange chromatography step can be run at high pH values with maximal product recovery theoretically allowing removal of HCP and viruses. Addressing the isoelectric point can be useful for an efficient process for removing HCP and viruses and this topic should be considered early in the research phase to ensure that other important molecule properties, e.g. safety, efficacy and expression yield are not impacted.

bioengineering↗

bioRxiv2023

Incorporating uncertainty within dynamic interoceptive learning

Interoception, the perception of the internal state of the body, has been shown to be closely linked to emotions and mental health. Of particular interest are interoceptive learning processes that capture associations between environmental cues and body signals as a basis for making homeostatically relevant predictions about the future. Here we extended an interoceptive Breathing Learning Task (BLT) to incorporate continuous measures of prediction certainty, and tested its application using a Rescorla Wagner (RW) associative learning model. Sixteen healthy participants completed the continuous version of the BLT, where they were asked to predict the likelihood of breathing resistances. The task was modified from a previous version and required continuous, rather than binary predictions, in order to include a more precise measure of prediction certainty. The RW model was used to fit a learning rate to each participants continuous and binarised predictions, and was additionally extended to test whether learning rates differed according to stimuli valence. The empirical task data demonstrated excellent replicability compared to previously collected data using binary predictions, and the continuous model fits closely captured participant behaviour at the group level. The model extension to estimate different learning rates for negative (i.e. breathing resistance) and positive (i.e. no breathing resistance) trials indicated that learning rates did not significantly differ according to stimuli nature. Furthermore, examining the relationship between estimates of prediction certainty and learning rates with interoceptive and mental health questionnaires demonstrated that fatigue severity was related to both prediction certainty and learning rate, and anxiety sensitivity was related to prediction certainty. The updated task and model show promise for future investigations into interoceptive learning and potential links to mental health.

neuroscience↗