Biology subjects

Kaiser, J.

Publications and source records attributed to Kaiser, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Real-time measurement of short-chain fatty acids via microwave sensing: A pilot study

BackgroundShort-chain fatty acids (SCFAs) - acetate, propionate, and butyrate - are key microbial metabolites that influence host metabolism, barrier function, and immune tone. Yet, in vivo measurements of SCFAs remain poorly characterized because stool measurements only capture a small, spatially averaged fraction of luminal production. PurposeTo evaluate whether label-free microwave sensing using a vector network analyzer (VNA) can differentiate SCFA identity, concentration, and their ratios via unique dielectric signatures. MethodsSodium acetate, sodium propionate, and sodium butyrate solutions (10 uM - 1M) were prepared by measuring their electrochemical properties (pH and mV). Using a 2-port VNA interfaced with a high-frequency copper plate (1 - 3 GHz), scattering and impedance parameters were measured for SCFAs and their ratios. A per-frequency ANOVA was employed to identify discriminatory frequency bands, and supervised machine learning (random forest) was employed to identify additional features differentiating SCFAs. ResultsSCFAs displayed distinct (ANOVA, p < 0.05) electrochemical signatures for both pH and mV. The 21 transmission spectrum revealed discriminatory frequencies with prominent bands at 1.38 - 1.40 GHz and 1.79 - 1.85 GHz. For SCFA ratios, the random forest model achieved up to 80.6% accuracy (k = 0.72) with S11 (77.7%) and S22 (69.9%) unwrapped phases observed as the top two important features contributing to the models performance. ConclusionVNA-based microwave sensing resolves SCFA-specific, frequency-dependent dielectric signatures across biologically relevant concentrations and classifies SCFA-like mixtures with high accuracy. These findings support microwave sensing as a foundation for real-time, non-invasive monitoring of gut microbial metabolism. IMPORTANCEShort-chain fatty acids (SCFAs) are central to host-microbe interactions, yet conventional stool assays provide only snapshots of microbial metabolism and poorly reflect real-time intestinal production of these microbial metabolites. SCFAs mediate intestinal barrier integrity, immune regulation, energy availability, and energy metabolism. Alterations in SCFA production and composition are increasingly recognized as biomarkers of gut microbial dysbiosis, with strong connections to conditions such as inflammatory bowel disease, metabolic syndrome, obesity, and neurodegenerative disorders. As such, the dynamic and quantitative assessment of SCFA profiles in vivo may provide valuable insight into gut health, disease progression, and therapeutic response. Despite large technological advances in the past decade, there is a limited understanding of in vivo SCFA dynamics within the gut, largely due to the inaccessibility and limitation of developing non-invasive diagnostic tools. Furthermore, the spatiotemporal resolution of microbial-derived metabolites in the gut is also very limited, with most current research methods relying on utilizing stool samples, which only contain a fraction of produced SCFA concentrations. In this pilot study, SCFA solutions of known concentrations and identity were measured and analyzed via their capacitance to use as an emerging diagnostic tool in clinical and/or athletic research and to further practice assessing gut microbial dynamics. We demonstrate that microwave sensing - a label-free, electrical approach - can distinguish among SCFAs and their concentrations by exploiting their dielectric differences and frequency-resolved behavior. Using a Vector Network Analyzer and a low-loss dielectric sensor, we identify reproducible GHz-scale bands that separate acetate, propionate, and butyrate. Results indicated that these metabolites can be differentiated via their S parameter (frequency response) electrical signals and pH values. Additionally, we classify physiological SCFA mixtures with strong performance.The study will be repeated by measuring various fluid media, starting with acidic solutions and testing with fluid mixes such as mock carbohydrate solutions that best mimic gut microbial conditions in hopes of developing a diagnostic tool (i.e., swallowable sensor) that can be used to monitor real-time human gut environments. This establishes a practical path toward dynamic, non-invasive readouts of gut microbial activity with potential applications in clinical monitoring.

bioengineering↗

bioRxiv2026

Origin-1: a generative AI platform for de novo antibody design against novel epitopes

0Generative artificial intelligence has advanced antibody discovery, yet de novo design of therapeutic antibodies against targets with "zero-prior" epitopes remains a fundamental challenge. We define "zero-prior" epitopes as target sites lacking structural data from any reported antibody-antigen or protein-protein complex involving the target. Here we present Origin-1, a generative AI platform that overcomes this by integrating epitope-conditioned all-atom structure generation, paired complementarity determining region sequence design, and a specialized co-folding-based scoring protocol to select antibody designs predicted to be high-confidence, specific binders with favorable developability. We evaluated Origin-1 on a panel of ten targets selected to have no available protein-protein complex structures and minimal homology ([≤]60% sequence identity) to proteins with known complexes, creating stringent design conditions. In fewer than one hundred design attempts per target, we identified developable, specific antibodies, validated across multiple biophysical and developability assays, for four targets: COL6A3, AZGP1, CHI3L2, and IL36RA, with functional inhibition demonstrated for IL36RA. Cryogenic electron microscopy confirmed the atomic accuracy of our designs, revealing complexes that closely matched the computational models with high structural fidelity (3.0-3.3 [A] resolution; 0.83-0.91 DockQ). Furthermore, we employed AI-guided affinity maturation to optimize a de novo antibody binder against IL36RA, producing functional antagonists with sub-nanomolar affinities and a top EC50 of 12.3 nM. These results demonstrate a framework for targeting epitopes without structural precedent, expanding the programmable therapeutic antibody landscape.

molecular biology↗

bioRxiv2025

Ventromedial prefrontal cortex shields value computations from bodily arousal in decision making

Emotional reactions to norm violations influence how people trade-off selfish interests against social norms, but it remains unknown which brain regions causally contribute to integrating physiological arousal associated with norm violations into the decision process. Here, we provide evidence that the ventromedial prefrontal cortex (vmPFC) causally contributes to decoupling value computations in normative choice from physiological arousal. Excitatory brain stimulation over the vmPFC reduced aversion to disadvantageous inequality in an Ultimatum game and increased post-decision confidence after acceptance of unfair monetary splits. Physiological arousal as measured via heart rate changes was increased in response to splits perceived as unfair. Enhancing vmPFC excitability reduced the sensitivity to unfairness not by directly inhibiting physiological arousal but by blocking its influence on the decision process. This suggests that vmPFC causally moderates how physiological responses to norm violations affect trade-offs between self-interests and norm considerations, highlighting its key role for integrating emotional and cognitive processes in decision making. Significance statementEmotions like anger about the violation of social norms are associated with physiological arousal, and the interoceptive awareness of this arousal is thought to influence decision making. Here, we identify the neural mechanism that causally mediates the influence of physiological arousal on the decision process. Increasing the excitability of the ventromedial prefrontal cortex strengthened selfish interests over fairness considerations in social interactions via decoupling the decision process from unfairness-evoked arousal. This provides a neuroscientific explanation for how physiological arousal influences social decisions and suggests that the role of the ventromedial prefrontal cortex for the interaction between emotion and cognition in decision making is based on interoception rather than emotion regulation.

neuroscience↗

bioRxiv2025

Multi-millennial genetic resilience of Baltic diatom populations disturbed in the past centuries

Little is known about the genetic diversity and stability of natural populations over millennial time scales, although the current biodiversity crisis calls for heightened understanding. Marine phytoplankton, the primary producers forming the basis of food webs in the oceans, play a pivotal role in maintaining marine ecosystems health and serve as indicators of environmental change. This study examines the genetic diversity and shifts in allelic composition in the diatom species Skeletonema marinoi over [~] 8000 years in the Baltic Sea by analyzing chloroplast and mitochondrial genomes. Ancient environmental DNA (aeDNA) from sediment cores demonstrates stability and resilience of genetic composition and diversity of this species across millennia in the context of major climate events. Accelerated change in allelic composition is observed from historical periods onwards, coinciding with times of intensifying human activity, like the Roman Empire, the Viking Age, and the Hanseatic Age, suggesting that anthropogenic stressors have profoundly impacted this species for the last two millennia. The data indicate a very high natural stability and resilience of the genomic composition of the species and underscore the importance of uncovering genomic disruptions caused by human impact on organisms, even those not directly exploited, to better predict and manage future biodiversity.

genomics↗

bioRxiv2024

Pleiotropic function of Dlx5/6 in coordinating the development of mammalian vocal and auditory organs

Acoustic communication, essential for mammalian social interactions, involves both effector (vocal) and receptor (auditory) organs that present a wide variety of morphologies between species. The molecular mechanisms supporting the harmonious diversification of effector and receptor systems along with the evolution of species-specific acoustic communication are still poorly understood. It is conceivable that common genetic pathways determine the parallel morphogenesis of vocal and auditory systems. Here, we investigated this question by generating mutant mice with targeted invalidation of Dlx5/6 genes in the otic placode and cephalic neural crest cells, contributing to the formation of the ear and vocal tract components. We show that Dlx5/6 inactivation provokes simultaneous defects of the outer, middle and inner ear and of the jaw, pharynx and larynx musculoskeletal systems. Our findings support the notion that Dlx5/6 genes played a pleiotropic role in the co-adaptation of vocal and auditory systems in mammals.

developmental biology↗

bioRxiv2024

Selective Targeting of a Defined Subpopulation of Corticospinal Neurons using a Novel Klhl14-Cre Mouse Line Enables Molecular and Anatomical Investigations through Development into Maturity

The corticospinal tract (CST) facilitates skilled, precise movements, which necessitates that subcerebral projection neurons (SCPN) establish segmentally specific connectivity with brainstem and spinal circuits. Developmental molecular delineation enables prospective identification of corticospinal neurons (CSN) projecting to thoraco-lumbar spinal segments; however, it remains unclear whether other SCPN subpopulations in developing sensorimotor cortex can be prospectively identified in this manner. Such molecular tools could enable investigations of SCPN circuitry with precision and specificity. During development, Kelch-like 14 (Klhl14) is specifically expressed by a specific SCPN subpopulation, CSNBC-lat, that reside in lateral sensorimotor cortex with axonal projections exclusively to bulbar-cervical targets. In this study, we generated Klhl14-T2A-Cre knock-in mice to investigate SCPN that are Klhl14+ during development into maturity. Using conditional anterograde and retrograde labeling, we find that Klhl14-Cre is specifically expressed by CSNBC-lat only at specific developmental time points. We establish conditional viral labeling in Klhl14-T2A-Cre mice as a new approach to reliably investigate CSNBC-lat axon targeting and confirm that this identifies known molecular regulators of CSN axon targeting. Therefore, Klhl14-T2A-Cre mice can be used as a novel tool for identifying molecular regulators of CST axon guidance in a relatively high-throughput manner in vivo. Finally, we demonstrate that intersectional viral labeling enables precise targeting of only Klhl14-Cre+ CSNBC-lat in the adult central nervous system. Together, our results establish that developmental molecular delineation of SCPN subpopulations can be used to selectively and specifically investigate their development, as well as anatomical and functional organization into maturity. Significance StatementThe cortex connects to brainstem and spinal targets through subcerebral projection neurons (SCPN), which exhibit molecular diversity during development based on their neocortical location and axonal targets. We generated a novel Klhl14-Cre mouse line to utilize this developmental delineation and drive Cre expression in a specific SCPN subpopulation. This developmental specificity enabled investigation of 1) areal locations of Klhl14+ SCPN in mature cortex, 2) their axonal collateralization at maturity, and 3) which genes can control their axon targeting. Using intersectional tools, we can also selectively label these neurons in the adult CNS. Therefore, developmental molecular delineation of SCPN not only provides prospective identification but also enables molecular analysis during development, as well as anatomical and functional investigations in adulthood.

neuroscience↗

bioRxiv2024

Unveiling Vertebrate Development Dynamics in Frog Xenopus laevis using Micro-CT Imaging

BackgroundXenopus laevis, the African clawed frog, is a versatile vertebrate model organism employed across various biological disciplines, prominently in developmental biology to elucidate the intricate processes underpinning body plan reorganization during metamorphosis. Despite its widespread utility, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus late developmental stages. FindingsIn the present study, we harnessed micro-computed tomography (micro-CT), a non-invasive 3D imaging technique utilizing X-rays to examine structures at a micrometer scale, to investigate the developmental dynamics and morphological changes of this crucial vertebrate model. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adult frogs. This extensive dataset enhances our understanding of vertebrate development and is adaptable for various analyses. For instance, we conducted a thorough examination, analyzing body size, shape, and morphological features, with a specific emphasis on skeletogenesis, teeth, and organs like the brain at different stages. Our analysis yielded valuable insights into the morphological changes and structure dynamics in 3D space during Xenopus development, some of which were not previously documented in such meticulous detail. This implies that our datasets effectively capture and thoroughly examine Xenopus specimens. Thus, these datasets hold the solid potential for additional morphological and morphometric analyses, including individual segmentation of both hard and soft tissue elements within Xenopus. ConclusionsOur repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus development and the associated morphological changes. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans have the potential for use in virtual reality, 3D printing, and educational contexts, further expanding their value and impact. Graphical abstract & lay summary3D images of selected developmental stages of X. laevis in a comparison (scale bar = 10 mm). O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/598452v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@1bc4664org.highwire.dtl.DTLVardef@1b1750dorg.highwire.dtl.DTLVardef@17bddaaorg.highwire.dtl.DTLVardef@115de4e_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryX-ray tomography was used to examine the African clawed frog (Xenopus laevis). An extensive data set of specimens from tadpoles to adult frogs provides novel insights into the changes and developmental dynamics of selected structures, which opens avenues to an improved understanding of this crucial animal model.

developmental biology↗

bioRxiv2024

A direct neural signature of serial dependence in working memory

Serial dependence describes the phenomenon that current object representations are attracted to previously encoded and reported representations. While attractive biases have been observed reliably in behavior, a direct neural correlate has not been established. Previous studies have either shown a reactivation of past information without observing a neural signal related to the bias of the current information, or a repulsive distortion of current neural representations contrasting the behavioral bias. The present study recorded neural signals with magnetoencephalography during a working memory task to identify neural correlates of serial dependence. Participants encoded and memorized two sequentially presented motion directions per trial, one of which was later retro-cued for report. Multivariate analyses provided reliable reconstructions of both motion directions. Importantly, the reconstructed directions in the current trial were attractively shifted towards the target direction of the previous trial. This neural bias mirrored the behavioral attractive bias, thus reflecting a direct neural signature of serial dependence. The use of a retro-cue task in combination with magnetoencephalography allowed us to determine that this neural bias emerged at later, post-encoding time points. This timing suggests that serial dependence in working memory affects memorized information during read-out and reactivation processes that happen after the initial encoding. Impact StatementMagnetoencephalographic recordings revealed a direct neural signature of serial dependence, i.e., an attractive bias of current towards previous representations, which emerged at late, post-encoding stages of processing in working memory.

neuroscience↗

bioRxiv2024

Local FK506 delivery induces osteogenesis in in vivo rat bone defect and rabbit spine fusion models

Bone grafting procedures are commonly used for the repair, regeneration, and fusion of bones in in a wide range of orthopaedic surgeries, including large bone defects and spine fusion procedures. Autografts are the clinical gold standard, though recombinant human bone morphogenetic proteins (rhBMPs) are often used, particularly in difficult clinical situations. However, treatment with rhBMPs can have off-target effects and significantly increase surgical costs, adding to patients already high economic and mental burden. Recent studies have identified that FDA-approved immunosuppressant drug, FK506 (Tacrolimus), can also activate the BMP pathway by binding to its inhibitors. This study tested the hypothesis that FK506, as a standalone treatment, could induce osteogenic differentiation of human mesenchymal stromal cells (hMSCs), as well as functional bone formation in a rat segmental bone defect model and rabbit spinal fusion model. FK506 potentiated the effect of low dose BMP-2 to enhance osteogenic differentiation and mineralization of hMSCs in vitro. Standalone treatment with FK506 delivered on a collagen sponge, produced consistent bone bridging of a rat critically-sized femoral defect with functional mechanical properties comparable to naive bone. In a rabbit single level posterolateral spine fusion model, treatment with FK506 delivered on a collagen sponge successfully fused the L5-L6 vertebrae at rates comparable to rhBMP-2 treatment. These data demonstrate the ability of FK506 to induce bone formation in human cells and two challenging in vivo models, and indicate FK506 can be utilized either as a standalone treatment or in conjunction with rhBMP to treat a variety of spine disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/584163v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1b0b690org.highwire.dtl.DTLVardef@a1e032org.highwire.dtl.DTLVardef@1862a55org.highwire.dtl.DTLVardef@1456fc1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Decoding the Baltic Sea's Past and Present: A simple Molecular Index for Ecosystem Assessment

Marginal sea ecosystems, such as the Baltic Sea, are severely affected by anthropogenic pressures, such as climate warming, pollution, and eutrophication, which increased in the course of the past century. Biodiversity monitoring data and assessment of environmental status in such systems have typically been carried out only for the past few decades, if at all, and knowledge on pre-impact stability and good ecological status is limited. An extension of monitoring time series can potentially be achieved through analyses of paleoecological records, e.g. for phytoplankton, which form the base of the food web and are highly susceptible to environmental changes. Within the phytoplankton community, dinoflagellates and diatoms play a significant role as primary producers, and their relative dominance in the spring bloom, calculated as Dia/Dino index, is used as an indicator for the environmental status of the Baltic Sea. To extend time series on the dominance patterns and include non-fossilized dinoflagellates, we here establish a simple droplet digital PCR (ddPCR) reaction on ancient DNA from sediment cores that decodes phytoplankton dynamics. We focus on two common spring bloom species, the diatom Skeletonema marinoi and the dinoflagellate Apocalathium malmogiense, for which we evaluate a DNA based dominance index. It performs very well in comparison to DNA metabarcoding and modern monitoring and can elucidate past species dominance across the past century in three basins of the Baltic and across millennia in two of these basins. For the past century, we see a dominance shift already starting before the mid-20th century in two of the Baltic Sea basins, thus substantially predating current monitoring programs. Shifts are only partly coeval among the cores and the index shows different degrees of stability. This pattern is confirmed across millennia, where a long-term stable relationship between the diatom and the dinoflagellate is observed in the Eastern Gotland Basin, while data from the Gulf of Finland bear testimony to a much more unstable relationship. This confirms that good ecological status based on the dominance pattern of diatoms and dinoflagellates must be established locally and exemplifies how sediment core DNA can be employed to extend monitoring data.

ecology↗

bioRxiv2024

Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising 4-aminopyridinium ion-substituted MB327 analogs (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new 4-aminopyridinium ion-substituted analogs of the aforementioned compounds (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute energetically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

pharmacology and toxicology↗

bioRxiv2024

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations

BackgroundMutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. ResultsWe applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3 splice sites. Our data highlighted a constrained window at canonical 3 splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3 splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E. ConclusionsOur work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases. HighlightsO_LILong-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines. C_LIO_LISF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3 splice site usage. C_LIO_LIMutation effect depends on alternative 3 splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites C_LIO_LIMolecular dynamics simulations predict reduced binding of SF3B1K700E to mRNA at the second binding pocket harboring the polypyrimidine tract. C_LI

cancer biology↗

bioRxiv2023

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

pharmacology and toxicology↗

bioRxiv2023

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR as well. In the present study, MS Binding Assays utilizing UNC0642 as a reporter ligand have been established. Thus, the binding of UNC0642 towards Torpedo-nAChR has been characterized in MS saturation and competition experiments. According to the results, UNC0642 addresses the MB327 binding site of the Torpedo-nAChR. This conclusion is further supported by the outcome of ex vivo studies performed with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of the most affine analog UNC0646 in the recently proposed binding site of MB327 (MB327-PAM-1). The new MS Binding Assays based on the commercially available reporter ligand UNC0642 as one of the most affine ligands for the MB327 binding site are a potent and valuable alternative to established assays.

pharmacology and toxicology↗

bioRxiv2023

Domain-Invariant Brainstem Nuclei Segmentation and Signal Quantification

AO_SCPLOWBSTRACTC_SCPLOWBrainstem nuclei are hard to distinguish due to very few distinctive features which makes detecting them with high accuracy extremely difficult. We introduce StARQ that builds on SeBRe, a deep learning-based framework to segment regions of interest. StARQ provides new functionalities for automated segmentation of brainstem nuclei at high granularity, and quantification of underlying neural features such as axonal tracings, and synaptic punctae. StARQ will serve as a toolbox for generalized brainstem analysis, enabling reliable high-throughput computational analysis with open-source models.

neuroscience↗

bioRxiv2023

Biosynthesis of H-GDGTs linked to ocean oxygen deficiency

Archaeal membrane lipids GDGTs (glycerol dialkyl glycerol tetraethers) are biomarkers used for tracking Earths historical environmental changes. Among these GDGTs, the H-shaped GDGTs (H-GDGTs, or GMGTs) represent a less-explored and often overlooked subset, with its biosynthetic pathway and geological significance remaining elusive. Here, we identified the gene responsible for biosynthesizing H-GDGTs, which encodes to a radical S-adenosyl-L-methionine (SAM) enzyme, named as H-GDGTs bridge synthase (Hbs). Heterologous expression of the gene hbs in a methanogen, as well as in vitro activity assay using the purified Hbs enzyme were performed. Additionally, we found that the genes encoding Hbs are exclusively present in obligate anaerobic archaea genomes and the metagenomes obtained from oxygen-deficient environments, but not in oxic settings. The H-GDGTs lipids were also consistently enriched in the modern oxygen-deficient environments, and remarkably accumulated in ancient sediments during oceanic anoxic event-2 (OAE-2, [~]94 million years ago) period. Our findings indicate H-GDGTs holds significant promise as a novel biomarker for studying historical ocean oxygen deficiency supported by a well-established biological basis.

microbiology↗

bioRxiv2023

InCellCryst - A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by conventional methods extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely unmodified proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, new strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

biophysics↗

bioRxiv2022

Light-dependent Control of Bacterial Expression at the mRNA Level

Sensory photoreceptors mediate numerous light-dependent adaptations across organisms. In optogenetics, photoreceptors achieve the reversible, non-invasive, and spatiotemporally precise control by light of gene expression and other cellular processes. The light-oxygen-voltage receptor PAL binds to small RNA aptamers with sequence specificity upon blue-light illumination. By embedding the responsive aptamer in the ribosome-binding sequence of genes of interest, their expression can be downregulated by light. We developed the pCrepusculo and pAurora optogenetic systems that are based on PAL and allow to down- and upregulate, respectively, bacterial gene expression using blue light. Both systems are realized as compact, single plasmids that exhibit stringent blue-light responses with low basal activity and up to several ten-fold dynamic range. As PAL exerts light-dependent control at the RNA level, it can be combined with other optogenetic circuits that generally control transcription initiation. By integrating regulatory mechanisms operating at the DNA and mRNA levels, optogenetic circuits with emergent properties can thus be devised. As a case in point, the pEnumbra setup permits to upregulate gene expression under moderate blue light whereas strong blue light shuts off expression again. Beyond providing novel signal-responsive expression systems for diverse applications in biotechnology and synthetic biology, our work also illustrates how the light-dependent PAL-aptamer interaction can be harnessed for the control and interrogation of RNA-based processes.

biochemistry↗