Biology subjects

Fink, T.

Publications and source records attributed to Fink, T..

10 recordsLinked to original sources

bioRxiv2026

Restoring Antibiotic Sensitivity via Metabolic Pathway Manipulation in Pseudomonas aeruginosa

Stopping antimicrobial multidrug resistance (AMR) is essential to maintain the efficacy of current treatments and sustain modern medicine, but current control relies on a coordinated One Health approach involving stewardship, infection prevention and other control measures, without direct actionable mechanisms against resistance itself. Here, integrating proteomics and metabolomics with high-throughput Caenorhabditis elegans infection models, we show that unique synergy of biotin biosynthesis inhibition with metformin uncouples virulence from bacterial viability shutting down major Pseudomonas aeruginosa (PA) virulence mechanisms that impair host survival. Under directed evolution induced by central carbon metabolic stress, laboratory and clinical PA strain isolates acquire mutations in the MexAB-OprM multidrug efflux pump, leading to reversal of the AMR phenotype and restored susceptibility to multiple legacy and last-resort antibiotics such as meropenem. Our findings reveal a previously unexploited vulnerability in PA, its co-dependence on biotin biosynthesis and energy metabolism for virulence and resistance. This work establishes a conceptual framework in which metabolic co-targeting can abolish pathogenesis and drive reversal of antimicrobial resistance. It lays the foundation for first-in-class, non-antibiotic PA-selective anti-virulence and resistance-reversing therapies, key to extending the lifesaving duration of existing antibiotics.

microbiology↗

bioRxiv2026

Pharmacologically regulated bioorthogonal stabilization domain for regulation of CAR T cells

Regulation of therapeutic cell response is important for safe and effective therapy, particularly for immunotherapy. Ideally, the regulators should be based on human proteins using compounds that have already been approved for human use. Regulation of protein degradation by small molecules enables fast cellular response and a small genetic footprint of genetic constructs. Here, we present a clinically compatible strategy for reversible pharmacological control of chimeric antigen receptor (CAR) T cell function using human estrogen receptor (ER)-based degron domains and FDA-approved small molecules. By fusing tamoxifen-responsive ER ligand-binding domains to CARs, we generate ligand-inducible ON-switch CARs whose stability, signaling, and effector functions are precisely controlled by 4-hydroxytamoxifen. Furthermore, we demonstrate that ER-tagged CARs can be selectively degraded using the FDA-approved ER-targeting PROTAC ARV-471, establishing a complementary OFF-switch mechanism that suppresses CAR expression and effector function. Notably, these results expand the bioorthogonal ON-OFF switch platform for post-translational control of therapeutic proteins, offering new opportunities to improve the safety and precision of cellular immunotherapies.

synthetic biology↗

bioRxiv2025

Function-specific epistasis shapes evolutionary trajectories towards antibiotic resistance

Pre-existing mutations can influence subsequent evolution by constraining or opening evolutionary pathways through epistatic interactions. In some cases, global epistasis allows evolutionary pathways to be predicted from the fitness of the genetic background alone. In other cases, idiosyncratic epistasis makes evolution less predictable. Here, we show that the evolution of antibiotic resistance is highly repeatable, following a common path across most genetic backgrounds. However, a minority of genetic backgrounds exhibit evolutionary trajectories that significantly deviate from this common path. Rather than being predictable from global epistasis, these deviations are modulated by function-specific epistasis: perturbations to specific cellular functions lead to novel evolutionary trajectories towards resistance. Using tightly controlled robotic evolution experiments, we quantitatively analyzed resistance trajectories for three clinically relevant antibiotics across multiple genetic backgrounds, including hundreds of Escherichia coli gene-deletion strains and several clinical isolates from urinary-tract infections. We show that disrupting distinct sets of cellular functions alters evolutionary trajectories for specific drugs and across different drugs, and we identify genetic changes defining these alternative trajectories. Importantly, this function-specific epistasis often slows down resistance evolution. Some of these effects can also be induced by small-molecule inhibitors of the identified targets, suggesting that function-specific epistasis can be exploited to improve drug treatments.

evolutionary biology↗

bioRxiv2022

Designed allosteric protein logic

Regulation of the activity of proteins enables control of complex cellular processes. Allosteric regulation has been introduced individually into few natural proteins. Here, we present a generally applicable regulation of diverse proteins called INSRTR (inserted peptide structure regulator), based on inserting a short unstructured peptide into a solvent-accessible loop that retains protein function. Function of the target protein can be inactivated by the addition of a peptide that forms a rigid coiled-coil dimer. This platform enables the construction of ON/OFF protein switches, their regulation by small molecules, and Boolean logic functions with a rapid response in mammalian cells. INSRTR can be used to regulate a wide range of proteins, as demonstrated on ten members of protein families with diverse biological activities including enzymes, signaling mediators, DNA binders/transcriptional regulators, fluorescent protein, and antibodies regulating chimeric antigen receptor. INSRTR platform presents an extraordinary potential for regulating biological systems and applications. One sentence summaryAuthors have designed a widely applicable system to activate or inactivate function of diverse proteins or form Boolean logic gates based on formation of a coiled-coil dimer within protein loops and demonstrated its implementation on a range of 10 diverse proteins.

synthetic biology↗

bioRxiv2021

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Protein secretion based on transcriptional control is rather slow, as proteins requires transcription, translation, followed by the transport from the endoplasmic reticulum (ER) through the conventional protein secretion (CPS) pathway towards the plasma membrane. An alternative faster bypass would be valuable. Here we present two genetically encoded orthogonal secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by cytosolic protease) or inside the ER lumen (lumER, released by ER luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables precise combination of signals into logic functions and was demonstrated on a chemically regulated insulin secretion. Regulation of ER escape represents a platform for the design of fast responsive and tightly-controlled modular and scalable protein secretion system. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/464966v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@12beb61org.highwire.dtl.DTLVardef@6a664corg.highwire.dtl.DTLVardef@1621e5borg.highwire.dtl.DTLVardef@b728ac_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO membER and lumER system. By equipping a protein of interest (POI) with an N-terminal signaling sequence, which initiates the transport of proteins into the endoplasmic reticulum (ER), and a C-terminal KDEL ER retention sequence for luminal proteins or a KKXX sequence for transmembrane proteins, we can retain those proteins inside the ER and cis-Golgi apparatus (GA) through retrograde transport. Insertion of a protease cleavage site adjacent to the retention signal allows for the regulated fast secretion through proteolytic cleavage. The membrane bound, ER membrane (membER) and ER-luminal (lumER) systems allow for the controlled secretion of pre-synthesized protein, stored inside the ER. This platform enables release of target proteins several hours faster than systems relying transcription and translation. C_FIG

synthetic biology↗

bioRxiv2021

HCV spread kinetics reveal varying contributions of transmission modes to infection dynamics

Hepatitis C virus (HCV) is capable of spreading within a host by two different transmission modes: cell-free and cell-to-cell. Although viral dissemination and diffusion of viral particles facilitates the infection of distant cells, direct cell-to-cell transmission to uninfected neighboring cells is thought to shield the virus from immune recognition. However, the contribution of each of these transmission mechanisms to HCV spread is unknown. To dissect the contribution of these different transmission modes to HCV spread, we measured HCV lifecycle kinetics and used an in vitro spread assay to monitor HCV spread kinetics after low multiplicity of infection in the absence and presence of a neutralizing antibody that blocks cell-free spread. By analyzing these data with a spatially-explicit mathematical model that describes viral spread on a single-cell level, we quantified the contribution of cell-free and cell-to-cell spread to the overall infection dynamics and show that both transmission modes act synergistically to enhance the spread of infection. Thus, the simultaneous occurrence of both transmission modes likely represents an advantage for HCV that may contribute to the efficient establishment of chronic infection. Notably, the relative contribution of each viral transmission mode appeared to vary dependent on different experimental conditions and suggests that viral spread is optimized according to the environment. Together, our analyses provide insight into the transmission dynamics of HCV and reveal how different transmission modes impact each other. ImportanceHepatitis C Virus can spread within a host by diffusing viral particles or direct cell-to-cell transfer of viral material between infected and uninfected cells. To which extend these cell-free and cell-to-cell transmission modes contribute to HCV spread, establishment of chronicity and antiviral escape is still unknown. By combining in vitro experimental HCV spread data with a multi-scale mathematical model we have disentangled the contribution and interplay of cell-free and cell-to-cell transmission modes during HCV infection. Our analysis revealed synergistic effects between the two transmission modes, with the relative contribution of each transmission mode varying dependent on the experimental conditions. This highlights the adaptability of the virus and suggests that transmission modes might be optimized dependent on the environment, which could contribute to viral persistence.

microbiology↗

bioRxiv2020

Immune response to vaccine candidates based on different types of nanoscaffolded RBD domain of the SARS-CoV-2 spike protein

Effective and safe vaccines against SARS-CoV-2 are highly desirable to prevent casualties and societal cost caused by Covid-19 pandemic. The receptor binding domain (RBD) of the surface-exposed spike protein of SARS-CoV-2 represents a suitable target for the induction of neutralizing antibodies upon vaccination. Small protein antigens typically induce weak immune response while particles measuring tens of nanometers are efficiently presented to B cell follicles and subsequently to follicular germinal center B cells in draining lymph nodes, where B cell proliferation and affinity maturation occurs. Here we prepared and analyzed the response to several DNA vaccines based on genetic fusions of RBD to four different scaffolding domains, namely to the foldon peptide, ferritin, lumazine synthase and {beta}-annulus peptide, presenting from 6 to 60 copies of the RBD on each particle. Scaffolding strongly augmented the immune response with production of neutralizing antibodies and T cell response including cytotoxic lymphocytes in mice upon immunization with DNA plasmids. The most potent response was observed for the 24-residue {beta}-annulus peptide scaffold that forms large soluble assemblies, that has the advantage of low immunogenicity in comparison to larger scaffolds. Our results support the advancement of this vaccine platform towards clinical trials.

immunology↗

bioRxiv2020

Photobleaching step analysis for robust determination of protein complex stoichiometries

The composition of cellular structures on the nanoscale is a key determinant of macroscopic functions in cell biology and beyond. Different fluorescence single-molecule techniques have proven ideally suited for measuring protein copy numbers of cellular structures in intact biological samples. Of these, photobleaching step analysis poses minimal demands on the microscope and its counting range has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost. Here, we present a comprehensive framework for photobleaching step analysis, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness and photostability. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we benchmark the framework with experimental data acquired on DNA origami labeled with defined fluorophore numbers to demonstrate counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis for robust protein counting by counting labelled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

biophysics↗

bioRxiv2020

A new mechanism for a familiar mutation - bovine DGAT1 K232A modulates gene expression through multi-junction exon splice enhancement

The DGAT1 gene encodes an enzyme responsible for catalysing the terminal reaction in mammary triglyceride synthesis, and underpins a well-known pleiotropic quantitative trait locus (QTL) with a large influence on milk composition phenotypes. Since first described over 15 years ago, a protein-coding variant K232A has been assumed as the causative variant underlying these effects, following in-vitro studies that demonstrated differing levels of triglyceride synthesis between the two protein isoforms. In the current study, we used a large RNAseq dataset to re-examine the underlying mechanisms of this large milk production QTL, and hereby report novel expression-based functions of the chr14 g.1802265AA>GC variant that encodes the DGAT1 K232A substitution. Using expression QTL (eQTL) mapping, we demonstrate a highly-significant mammary eQTL for DGAT1, where the K232A mutation appears as one of the top associated variants for this effect. By conducting in vitro expression and splicing experiments in bovine mammary cell culture, we further show modulation of splicing efficiency by this mutation, likely through disruption of an exon splice enhancer as a consequence of the allele encoding the 232A variant. Although the relative contributions of the enzymatic and transcription-based mechanisms now attributed to K232A remain unclear, these results suggest that transcriptional impacts contribute to the diversity of lactation effects observed at this locus.

molecular biology↗

bioRxiv2017

Cooperative Contributions of Structural and Functional Connectivity to Successful Memory and Aging

Understanding the precise relation between functional connectivity and structural (white-matter) connectivity and how these relationships account for cognitive changes in older adults are major challenges for neuroscience. We investigate these issues using a new approach in which structural equation modeling (SEM) is employed to integrate functional and structural connectivity data analyzed with a common framework based on regions connected by canonical tract groups (CTGs). CTGs (e.g., uncinate fasciculus, cingulum, etc.) serve as a common currency between functional and structural connectivity matrices, and ensures that the same amount of data contributing to brain-behavior relationships. We used this approach to investigate the neural mechanisms supporting memory for items and memory for associations, and how they are affected by healthy aging. Our results are threefold. Firstly, structural and functional CTGs made independent contributions to associative memory performance, suggesting that both forms of connectivity underlie age-related changes in associative memory. Secondly, distinct groups of CTGs supported associative versus item memory. Lastly, the relationship between functional and structural connectivity was best explained by the relationship between latent variables describing functional and structural CTGs based on a constrained set of tracts--but no one specific CTG group--suggesting that age effects in connectivity are constrained to specific pathways. These results provide further insights into the interplay between structural and functional connectivity patterns, and help to elucidate their relative contribution to age-related changes in associative memory performance.

neuroscience↗