Victor Krajka

Continuous and long‐term pressure monitoring by devices implanted in the human body promises substantial improvements for diagnosis, health monitoring, surveillance of disease progression, or surveillance of the response to a treatment but also for precision during surgeries. Here, we report on a piezoresistive sensor made entirely of parylene‐C eliminating the need for heterogeneous encapsulation. Material characterization demonstrated that parylene‐C is excellently suited as a construction material. The pressure sensor comprises a parylene‐C membrane, which is functionalized with gold strain sensors for pressure sensitivity. It was bonded to the reference pressure cavity, thereby establishing a long‐time stable hermetic sealing. The presented design and fabrication enable for the first time a monolithic, highly soft and flexible pressure sensor entirely made of biocompatible material. It exhibited high pressure sensitivity of up to 6.2 µV·mmHg ⁻¹ with very good repeatability and negligible hysteresis, made possible by thin‐film strain sensors on a parylene‐C membrane with a gauge factor of 7.5. Simulations show how the design can be easily even further miniaturized. Initial in vitro assay confirmed that parylene‐C is biocompatible on cell culture level. Such biocompatible and chronically implantable pressure sensors are opening new possibilities in medical treatments and long‐term supervision.

Carrier nanoparticles facilitate the encapsulation of drug or mRNA molecules thereby enhancing their bioavailability. Microfluidic mixers provide a unique environment for the precise and continuous generation of nanoparticles by antisolvent precipitation. A major challenge is to understand the influence of microfluidic channel designs and geometries on the continuous production of small, uniform lipid nanoparticles (LNPs) and to identify conditions that ensure effective and controllable mixing of aqueous and organic phases in laminar flows. Another important challenge is that sufficient quantities for preclinical and clinical studies must be produced within a reasonable period of time. With this dual objective, different versions of a low aspect ratio laminar mixer (LARLM) were produced using two-photon polymerization (2PP). In the LARLM the organic phase forms a thin layer a few micrometers with a uniform velocity distribution in the center of the channel, surrounded by the aqueous phase. This concept has three major advantages: Firstly, it keeps all particles centralized in the channel, thus preventing contamination during prolonged particle generation. Secondly, diffusive mixing in the thin central stream occurs very quickly, and thirdly, the growing nanoparticles move at a homogeneous speed, which enables inline measurement of the particles. In systematic experiments with design versions of varied channel dimensions the operational parameters such as lipid concentrations and flow rates and the capability to produce LNPs with desired properties and loading capacities were explored. An interfacial dispersion model (IDM) could explain the surprising reduction of particle sizes with increased productivity. The latter allowed us to produce nanoparticles in the range of 50 nm to 180 nm (with 0.02 < PDI < 0.1) under stable conditions with a productivity of around one liter of LNP suspensions every three hours. Such performance has never been reached before with microfluidics. Moreover, LNPs loaded with coumarine-6 and various drugs were produced in the LARLM. Moreover, in-vitro experiments could confirm an improved bioavailability of coumarin-6 in cell culture experiments when loaded in LNPs by the LARLM. These results highlight the unique capabilities of LARLM devices and their potential to support nanoparticle formulation studies including preclinical and in further developments also clinical studies as required for approval as a marketable medicine.

Laboratory courses are foundational in STEM education, traditionally reinforcing a linear view of scientific inquiry through the validation of pre-tested hypotheses. This perspective tends to overlook the iterative, tentative, and unpredictable nature of scientific research. To address this, we developed the “Designed-to-Fail Laboratory” (DtFL), an interdisciplinary pilot course that strategically employs structured experimental failures to foster student engagement with fundamental principles of Nature of Science (NOS). The DtFL relates inquiry-based learning with failure, challenging students to confront and reflect on failure as an intrinsic component of scientific practice. Using a mixed-methods approach with pre/post-surveys and student learning diaries, we evaluated the course’s impact on fostering NOS understanding and student engagement. Qualitative analyses revealed heightened cognitive and emotional engagement with NOS dimensions, including its empirical, tentative, and subjective aspects. Survey scores showed no statistically significant changes, underscoring the complexity of capturing nuanced shifts in NOS understanding. In their learning diaries, students highlighted the DtFL’s value in reshaping their perceptions of failure, promoting resilience, and bridging theory with authentic scientific inquiry. Our findings suggest that the DtFL provides a novel framework for integrating failure into laboratory pedagogy, and an opportunity for students to gain insights into NOS while fostering critical thinking and scientific literacy.

Two-phase biocatalysis in batch reactions often suffers from inefficient mass transfer, inconsistent reaction conditions, and enzyme inactivation issues. Microfluidics offer uniform and controlled environments ensuring better reproducibility and enable efficient, parallel processing of many small-scale reactions, making biocatalysis more scalable. In particular, the use of microfluidic droplets can increase the interfacial area between the two phases and can therefore also increase reaction rates. For these reasons, slug flow has been extensively used in two-phase biocatalysis in recent years. However, microdroplet flow has been largely neglected for this application despite its great potential. In this work, we performed biphasic biocatalysis in microfluidic droplets, both in microdroplets and slugs, as well as in batch, and systematically investigated the effect of various reaction parameters on the outcome of the reaction. We show that microdroplet flow outperforms the more commonly used batch and slug flow configurations for most reaction conditions by providing shorter substrate diffusion paths and larger interfacial area for the reaction. The potential trade-off between maximized mass transfer and possibly higher enzyme inactivation rates in small droplets with large surface-to-volume ratios was also investigated for the first time, and a pipeline was established to allow evaluation in other reactions. Finally, the effect of surfactant necessary for microdroplet stabilization was also investigated in all reaction setups for the first time, and it was shown that a properly selected surfactant can have a positive effect at low concentrations by creating more stable emulsions and smaller droplets, thus increasing the interfacial area between the two phases.

Mutations in the brain-specific β-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.

Mutations in TUBB4A have been identified to cause a wide phenotypic spectrum of diseases ranging from hereditary generalized dystonia with whispering dysphonia (DYT‐TUBB4A) and hereditary spastic paraplegia (HSP) to leukodystrophy hypomyelination with atrophy of the basal ganglia and cerebellum (H‐ABC). TUBB4A encodes the brain‐specific β‐tubulin isotype, β‐tubulin 4A. To elucidate the pathogenic mechanisms conferred by TUBB4A mutations leading to the different phenotypes, we functionally characterized three pathogenic TUBB4A variants (c.4C > G,p.R2G; c.745G > A,p.D249N; c.811G > A, p.A271T) as representatives of the mutational and disease spectrum) in human neuroblastoma cells and human induced pluripotent stem cell (iPSC)‐derived neurons. We showed that mRNA stability was not affected by any of the TUBB4A variants. Although two mutations (p.R2G and p.D249N) are located at the α/β‐tubulin interdimer interface, we confirmed incorporation of all TUBB4A mutants into the microtubule network. However, we showed that the mutations p.D249N and p.A271T interfered with motor protein binding to microtubules and impaired neurite outgrowth and microtubule dynamics. Finally, TUBB4A mutations, as well as heterozygous knockout of TUBB4A, disrupted mitochondrial transport in iPSC‐derived neurons. Taken together, our findings suggest that functional impairment of microtubule‐associated transport is a shared pathogenic mechanism by which the TUBB4A mutations studied here cause a spectrum of diseases. This article is protected by copyright. All rights reserved

Keeping neural stem cells under proliferation, followed by terminal differentiation, can substantially increase the number of neurons generated. With regard to the usability of proliferating neurospheres (NSPHs) cultures, adherent induction protocols have not yet been studied in comparison to embryoid body (EB)-based protocols. To compare these proctocols, neural induction of human induced pluripotent stem cells was performed by dual SMAD inhibition under both adherent and free-floating EB culture conditions. After 10 days, we transferred cells to low-attachment culture plates and proliferated them as free-floating neurospheres. RNA was collected, transcribed to cDNA and analyzed for sonic hedgehog expression that plays an important role during proliferation process. NSPHs were analyzed by immunofluorescence imaging directly and upon continued differentiation. The EB-based approach yielded in higher numbers of cells expressing the neural stem cell marker Nestin, and showed in contrast to the adherent induction protocol increased expression levels of sonic hedgehog. Although improvements to culture consistency and reliability are desirable, the EB-based protocol appears to be superior to the adherent protocol for both, the proliferation and differentiation capacity.

Impairment of the dopaminergic (DA) system is a common cause of several movement disorders including Parkinson’s disease (PD), however, little is known about the underlying disease mechanisms. The recent development of stem-cell-based protocols for the generation of DA neurons partially solved this issue, however, this technology is costly and time-consuming. Commonly used cell lines, i.e., neuroblastoma (SHSY5Y) and PC12 cells are still widely used to investigate PD and significantly contributed to our understanding of mechanisms involved in development of the disease. However, they either do not express DA at all or require additional, only partially efficient differentiations in order to produce DA. Here we generated and characterized transgenic SH-SY5Y cells, ectopically expressing tyrosine hydroxylase (SH^TH+), that can be used as a homogenous, DA-producing model to study alterations in DA metabolism and oxidative stress. We demonstrated that SH^TH+ produce high levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) making this model suitable to investigate not only alterations in DA synthesis but also its turnover. We also provide evidence for the presence of other enzymes involved in DA synthesis and its turnover in these cells. Finally, we showed that these cells can easily be genetically modified using CRISPR/Cas9 technology in order to study genetically defined forms of movement disorders using DJ1-linked PD as a model.

The hypothalamic–pituitary–thyroid (HPT) axis maintains circulating thyroid hormone levels in a narrow physiological range. As axons containing thyrotropin-releasing hormone (TRH) terminate on hypothalamic tanycytes, these specialized glial cells have been suggested to influence the activity of the HPT axis, but their exact role remained enigmatic. Here, we demonstrate that stimulation of the TRH receptor 1 increases intracellular calcium in tanycytes of the median eminence via Gα_q/11proteins. Activation of Gα_q/11pathways increases the size of tanycyte endfeet that shield pituitary vessels and induces the activity of the TRH-degrading ectoenzyme. Both mechanisms may limit the TRH release to the pituitary. Indeed, blocking TRH signaling in tanycytes by deleting Gα_q/11proteins in vivo enhances the response of the HPT axis to the chemogenetic activation of TRH neurons. In conclusion, we identify new TRH- and Gα_q/11-dependent mechanisms in the median eminence by which tanycytes control the activity of the HPT axis.