Biology subjects

Schmidt, M.

Publications and source records attributed to Schmidt, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Integration of In-Line Light-Mediated Cell Activation in Extrusion-Based Bioprinting: A New Biofabrication Strategy for Bone Tissue Engineering

Controlled activation of cells through photobiomodulation can influence osteogenic outcomes. However, factors such as skin penetration depth limit its clinical relevance. In this study, we present a workflow integrating broadband light stimulation directly into the bioprinting process by equipping a custom, low-cost extrusion bioprinter with a self-made, high-voltage spark-gap stimulation unit, enabling in-line stimulation of cells embedded in hydrogel. Optimal duration of broadband light exposure was screened with SaOS-2 cells in 2D for 5, 10, or 15 min, and cultured under osteogenic and control conditions. In 3D, SaOS-2 or adipose-derived stromal cells (ASCs) were bioprinted in an alginate-gelatin bioink under 10 minutes of pulsatile light stimulation. Cellular responses were assessed after 21 days by cell yield, viability, metabolic activity, apoptosis, DNA damage and osteogenic differentiation through immunofluorescence, histology and qPCR. Additionally, vascularisation capacity was assessed in light-stimulated HUVEC/ASC co-cultures within bioprinted constructs. In 2D, light stimulation did not negatively affect cell yield, viability, metabolic activity, cytotoxicity and DNA damage across all exposure times. 3D cultures showed a 2.7-fold and 2.4-fold increase in calcium and hydroxyapatite, respectively, elevated osteocalcin/collagen type I protein expression and gene expression of ALPL, BGLAP, SPP-1, COL1A1, COL10A1 and no increase in DNA damage after 21 days. In the HUVEC/ASC co-culture, light stimulation promoted endothelial elongation (1.25-fold), network formation (2-fold) and perivascular ASC organisation. This work establishes a proof-of-concept methodology for in-line, broadband light cell activation during bioprinting as a strategy to enhance osteogenic differentiation and vascularisation potential.

bioengineering↗

bioRxiv2026

History of opioid use disorder correlates with altered neuroimmune signatures in the human dorsal root ganglia

Understanding mechanisms for the negative reinforcing effects of opioids are fundamental to improving patient care. Using human dorsal root ganglia (DRG) from donors with and without opioid use disorder (OUD), we set out to examine functional and molecular correlates of chronic opioid use. At a molecular level, we detected disrupted immune signatures throughout cells in the peripheral nervous system (PNS), spanning glial-neuroimmune interactions to changes in non-neuronal senescence. At the physiological level these changes are paired with sensitization of DRG sensory neurons. Our findings reveal a neuro-inflammatory state in the PNS of individuals with OUD. Targeting therapeutics towards these PNS-based mechanisms could be an approach to alleviating the negative reinforcing effects of opioids.

neuroscience↗

bioRxiv2026

The Signaling Pathway of a Bacterial Two-Component System Determined by cryo-EM

Although two-component systems (TCSs) play ubiquitous roles in sensing signals for bacterial survival and virulence, a detailed structural map of the signal transduction pathway is lacking. Bacteriophytochromes (BphPs) are enzymatic photoreceptors with homologs in plants that regulate bacterial physiological responses via TCS signaling. Here, we employ cryo-EM to elucidate the structural basis of a light-regulated TCS by capturing the full-length BphP histidine kinase (HK) in complex with its downstream response regulator (RR). The RR binds as a dimer to the HK but contacts the enzyme only through one subunit. Light absorption perturbs the entire BphP structure, including the HK domain, which disrupts phosphorylation and phosphotransfer reactions and promote TCS complex dissociation. Thus, light controls signaling through both HK activity and reversible binding of the RR.

biophysics↗

bioRxiv2026

Systematic De-Risking of TCR-Mimic Therapeutics Through Proteome-Wide Off-Target Landscaping and a Generalizable Design Rule Framework

TCR-mimic (TCRm) antibodies targeting peptide-human leukocyte antigen (pHLA) complexes enable precision immunotherapy against intracellular antigens, including cancer-testis antigens (CTAs). Achieving high specificity, however, remains challenging because of the vast diversity of the human immunopeptidome and the associated risk of off-target recognition. Here, we introduce ValidaTe, a unified framework for the proteome-scale prediction, validation, and mitigation of off-target liabilities in pHLA-directed therapeutics. ValidaTe integrates rational target prioritization, peptide-centric binder selection, proteome-wide off-target prediction, and therapeutic engineering into a hierarchical de-risking workflow. Using the CTA MAGE-A4 as a proof-of-concept, we identify the TCRm antibodies VR-4 and VR-6 with superior specificity and demonstrate how this workflow enables the discovery of safer pHLA-targeted binders. Furthermore, ValidaTe establishes the basis for the WiFi (Widened Fingerprint) engineering principle, which rationally combines TCRms with complementary off-target fingerprints in trivalent T-cell engagers to minimize unintended interactions while preserving potent target-specific activity. Together, these findings establish a generalizable framework for the rational development of safer and more selective pHLA-targeted therapeutics. We further discuss how orthogonal proteomic characterization may complement this workflow as a final layer of translational safety assessment prior to clinical development. TeaserValidaTe accelerates safe pHLA-targeted immunotherapy through proteome-wide off-target mapping and WiFi design

immunology↗

bioRxiv2026

Diesel exhaust particles disrupt mouse and human iPSC-derived microglial function and Amyloid-beta clearance in Alzheimer's disease models

Alzheimers disease (AD) is one of the most common neurodegenerative disorders, yet the environmental drivers that accelerate its progression remain poorly defined. Traffic-related air pollution is emerging as a modifiable AD risk factor, but how inhaled particles perturb microglial clearance of amyloid beta (A{beta}) is unknown. Microglia are the principal A{beta}-clearing phagocytes of the brain. Here, we showed that exposure of primary mouse microglia and human induced pluripotent stem cell-derived microglia (iMGLs) to 3-100 {micro}g/mL diesel exhaust particles (DEP) disrupted microglial homeostasis, induced morphological abnormalities, increased reactive oxygen species, impaired lysosomal degradation, and led to a concentration-dependent loss of phagocytic capacity. Importantly, DEP markedly reduces A{beta} uptake in both species. Transcriptomic profiling revealed a DEP-induced, non-canonical state characterized by metabolic reprogramming, broad suppression of inflammatory pathways, antigen-presentation, chemokine, and species-specific remodeling during subsequent A{beta} challenge, including defective chemotaxis, cell cycle, and cytoskeletal signatures. These data show that DEP profoundly alters microglial transcriptional and metabolic states, leading to impaired A{beta} clearance, which could, thereby, further contribute to AD progression.

neuroscience↗

bioRxiv2026

A Standardized Methodology for FAIRness Assessment and Multi-Dimensional Scoring in Agrosystem Research Data Infrastructures

The NFDI-consortium FAIRagro has established a systematic framework for evaluating the FAIRness of Research Data Infrastructures (RDI) within the German agrosystem research landscape. While FAIR principles are widely accepted, their practical implementation by RDIs remains challenging. By operationalizing the FAIR principles into a reproducible multi-dimensional scoring methodology, this initiative addresses the critical need for a transparent and citable benchmark of RDIs that moves beyond simple compliance. This paper details the underlying assessment criteria, comprising 20 aggregated core metrics, the iterative community-driven validation process, and the integration of these metrics into the FAIRagro Search Hub. This framework evaluates RDIs, like repositories or databases, instead of sampling hosted data sets, across the four distinct categories of FAIR independently, yielding granular, pillar-specific ratings. Unlike aggregate scoring models, which can inadvertently mask technical deficiencies by averaging performance across categories, this multi-dimensional approach ensures that a repositorys distinct strengths and bottlenecks remain fully visible. Our findings demonstrate that standardized scoring not only clarifies data accessibility for users but also highlights specific operational gaps, allowing repository providers to identify precisely where the service implementation can be enhanced. By establishing this data-driven service in the agronomy domain, we provide a scalable template for the broader NFDI and EOSC ecosystems to foster a culture of excellence in research data stewardship.

bioinformatics↗

bioRxiv2026

Forward Modelling for Magnetospinography: Systematic Comparison of Boundary Element and Finite Element Methods

Magnetospinography (MSG) enables non-invasive measurement of spinal cord electrophysiology, but accurate interpretation of these signals depends critically on forward modelling assumptions. We compared four vertebral bone representations: continuous, homogeneous-toroidal, inhomogeneous-toroidal, and MRI-derived-realistic, implemented within both boundary element method (BEM) and finite element method (FEM) frameworks. Lead-fields were computed across the full spinal cord for three orthogonal source orientations. When comparing matched model pairs, BEM and FEM produced consistent forward solutions, with median relative errors below 3.1% and median squared correlation coefficients exceeding 0.998 across all bone geometries. Vertebral bone geometry exerted a systematic and orientation-dependent influence on predicted lead fields. The dominant distinction was between continuous and segmented bone representations rather than between simplified and anatomically detailed segmented models. For left-right oriented sources, segmented geometries produced substantially higher field amplitudes (fT/nAm) than the continuous model throughout the cord (35-72%), while toroidal and MRI-derived realistic models produced comparable results across both frameworks, with median r2 exceeding 0.97 between segmented models compared to 0.67-0.85 for continuous versus segmented comparisons. Sensor placement further modulated sensitivity profiles, with posterior arrays exhibiting greater sensitivity lower down the cord, but anterior and posterior sensors showing comparable sensitivity in the cervical region.

neuroscience↗

bioRxiv2026

Altered cellular VEGF dynamics in Chronic Obstructive Pulmonary Disease

Pulmonary vascular remodelling is common in patients with chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factors (VEGFs) are key mediators in angiogenesis and vascular remodelling and exist in different isoforms. VEGF-A is the most potent angiogenic member binding to VEGF receptor 2 (VEGFR2). There are, however, few studies on other isoforms, as VEGF-C, and its receptor VEGFR3 in COPD and subsequent impact of cAMP therapies on VEGF isoforms. Our aim was to evaluate the VEGF isoform synthesis in primary distal lung fibroblasts from control subjects (non-smokers (n=6) and ex-smokers (n=4), and COPD subjects with GOLD stage II (n=4) or GOLD stage IV (n=6), and the expression of VEGFR2 and VEGFR3 in human lung tissue. Primary lung fibroblasts were exposed to the cAMP generating therapies formoterol, iloprost, or roflumilast, the adenylyl cyclase activator forskolin or to transforming growth factor (TGF)-b1. VEGF isoforms were evaluated with ELISA. VEGF-C release was not significantly altered by TGF-{beta}1, in contrast to the increased levels of VEGF-A, in all fibroblasts. VEGF-C was significantly decreased by iloprost, forskolin and formoterol, whereas VEGF-A was significantly increased by iloprost and forskolin, with differences in release pattern between and within fibroblasts from control and COPD subjects. Exposure to VEGF-C specifically towards VEGFR3 decreased proliferative rate in human lung fibroblasts and bronchial epithelial cells. VEGFR2 and VEGFR3 were both present in parenchymal lung tissue and VEGFR2 in pulmonary blood vessels. in both healthy and COPD, whereas there was elevated expression of VEGFR3 in bronchial epithelium. In conclusion, TGF-{beta}1 and cAMP generating compounds have significant effects on VEGF-C and VEGF-A synthesis, which appear dysregulated in lung fibroblasts from ex-smokers and patients with COPD. Increased VEGFR3 expression in the bronchial epithelium in lung tissue, and studies into their functional impact, warrants further investigations.

pathology↗

bioRxiv2026

Leaf and cluster spectral signatures reveal trait-dependent prediction performance for grapevine cluster architecture and juice quality

Grapevine cluster architecture is a key selection target in breeding programs because it influences disease susceptibility, yield stability and juice quality. High-throughput phenotyping offers a rapid and non-destructive approach to capture biochemical and structural variation in these traits, yet the influence of plant organ reflectance and data partitioning strategies on trait prediction remains poorly understood. In this study, we evaluated how hyperspectral reflectance from different grapevine organs contributes to the prediction of cluster architecture and juice quality traits in two clonal populations of Riesling and Pinot. Using partial least squares regression (PLSR), we assessed the prediction accuracy of eight cluster architecture and six juice quality traits under two data partitioning strategies. Models based on cluster reflectance outperformed those using dry leaf reflectance for most traits, except for pH. Partitioning the dataset by cluster type increased trait variance and improved predictions for number of berries (R{superscript 2} = 0.53), berry diameter (R{superscript 2} = 0.79), and total acidity (R{superscript 2} = 0.48). Visible, red-edge and NIR spectra were most informative regions to predict the traits studied. Together, our results highlight the importance of organ-specific data and appropriate calibration strategies to improve phenomic models for the development of scalable proxies for grapevine improvement. HighlightSpectral phenomics reveals that prediction accuracy in grapevine depends on organ spectral signatures and traits, with cluster reflectance outperforming leaves, informing new phenotyping strategies for breeding improvement.

plant biology↗

bioRxiv2026

External validation and time-stability analysis of STARE, a blood-free quantification tool for irreversible PET tracers

Rationale"Gold standard" blood-based quantification of dynamic 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) data has limited practical clinical applications due to cost and complexity of data collection and analysis. We previously presented a blood-free quantification alternative, STARE (Source-to-Target Automatic Rotating Estimation), that was validated on 18F-FDG data acquired on a ECAT EXACT HR+ scanner. Here, we extend that initial work by externally validating STARE using within-subject data acquired with both a Siemens Biograph mCT scanner and a portable Brain Biosciences CerePET scanner. MethodsPerformance was assessed by comparing regional net influx rates (Ki) estimated using STARE and the standard blood-based Patlak approach. Twenty participants underwent 60-minute 18F-FDG scans, on two different days, once in each scanner. The time-stability of both STARE- and Patlak-based Ki estimates was evaluated by applying each method to the first 20 (STARE only), 30, 40, and 50 minutes of data. ResultsSTARE demonstrated high correlation with Patlak Ki estimates across both scanner types, particularly in the Biograph mCT (r = 0.93), with lower correlation in the CerePET (r = 0.71). In the Biograph dataset, STARE provided reliable Ki estimates at all evaluated scan durations (20 minutes and above), while in the CerePET dataset, only the 50-minute duration yielded STARE Ki estimates that were not significantly different from the full 60 minutes. The Patlak approach provided Ki estimates at 40 minutes scan duration and above that did not differ from the 60-min scan results in both datasets. ConclusionSTARE is a viable, noninvasive alternative to traditional blood-based quantification of dynamic 18F-FDG PET data, facilitating shorter, blood-free acquisition. This advancement could make dynamic 18F-FDG PET imaging more accessible and comfortable for patients, promoting broader clinical adoption.

neuroscience↗

bioRxiv2026

IL-1α drives a tumor-stroma-neutrophil axis through inflammatory fibroblast activation in head and neck cancer

In head and neck squamous cell carcinoma (HNSCC), high tumor-associated neutrophil (TAN) density is a robust biomarker of poor prognosis. TANs predominantly localize to the stroma and their intratumoral density strongly correlates with adverse outcome. Here, we investigated how tumor-stroma communication regulates TAN recruitment, activation, and spatial organization. We identified interleukin-1 (IL-1), released by viable or necrotic tumor cells, as an upstream signal that induces an inflammatory cancer associated fibroblast (iCAF)-like transcriptional program in patient-derived mesenchymal stromal cells (MSCs) of the oral cavity. IL-1-stimulated MSCs secrete factors associated with neutrophil recruitment, survival, and function, together with mediators of extracellular matrix remodeling and angiogenesis. Notably, the IL-1-induced iCAF-like transcriptional program closely resembles CAF subsets in HNSCC that are associated with poor clinical outcome. Functionally, conditioned media from IL-1-stimulated MSCs promoted tumor growth and enhanced polymorphonuclear neutrophil survival, activation, trans-well migration and infiltration into spheroids, in vitro. In zebrafish xenografts, co-injection of IL-1-overexpressing tumor cells and MSCs markedly amplified neutrophil infiltration. TCGA analysis demonstrated robust correlations between the IL-1-induced MSC gene signature and neutrophil signatures across multiple TAN subsets in human HNSCC. Spatial analysis of HNSCC tissues showed that stromal regions adjacent to IL1A-positive tumor islets were enriched for CXCL8/CSF3 double-positive cells and exhibited increased TAN density, including higher frequencies of NE- and MPO-positive neutrophils. Collectively, these findings define an IL-1-dependent tumor-stroma signaling circuit that links tumor inflammation to stromal remodeling and neutrophil infiltration in HNSCC. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/700440v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@f8d282org.highwire.dtl.DTLVardef@1c4575aorg.highwire.dtl.DTLVardef@1430cfcorg.highwire.dtl.DTLVardef@1098887_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: IL-1 drives tumor-stroma communication and neutrophil recruitment in HNSCC. Here, we describe a mechanism in HNSCC that promotes high tumor-associated neutrophil (TAN) density, a biomarker associated with poor prognosis. Tumor-derived IL-1 activates stromal cells to adopt an inflammatory phenotype, resulting in the release of CXCL8, GM-CSF, and G-CSF to enhance neutrophil recruitment, activation, and survival. IL1A-positive tumor islets are surrounded by CXCL8/CSF3-rich stroma with elevated TAN densities in both tumor and stromal compartments. These TANs exhibit increased frequencies of MPO- and NE-positive cells, revealing a spatially organized inflammatory tumor microenvironment. Figure was created using BioRender. C_FIG

cancer biology↗

bioRxiv2026

Progressive neurodegeneration in human dorsal root ganglion from diabetes to painful neuropathy

Diabetic painful neuropathy (DPN) is characterized by neuropathic pain accompanied by loss of sensory function. We hypothesized that neurodegeneration in the dorsal root ganglion (DRG) could underlie DPN progression. To address this question, we performed multi-omic evaluation on DRGs from otherwise healthy organ donors, donors with diabetes but no neuropathy, and donors with clinically diagnosed DPN. We discovered that the first stages of neurodegeneration begin early in diabetes before the onset of DPN, with Nageotte nodule formation accompanied by apoptotic gene expression and decreased proportion of specific populations of A-fibers in DPN with remodeling of non-neuronal cells. Our findings define DPN as a neurodegenerative disorder of the DRG, identify molecular markers of disease stage, and highlight the need for early intervention to prevent irreversible neurodegeneration.

neuroscience↗

bioRxiv2026

Slow breathing impacts inter-organ dynamics modulating brain function and risk behavior

Successful decision-making requires that external information be interpreted in the context of the bodys state. Within the framework of body-brain interaction, deliberately modifying ones autonomic state can shape how we evaluate the world, ultimately influencing choices. Yet, it remains unclear whether and how intentional autonomic regulation affects human decision-making. In this study, we tested instructed prolonged exhalation, a slow-breathing technique designed to boost parasympathetic activity during risky decision-making. Participants followed distinct breathing protocols while making risky choices, with neural and physiological activity measured using fMRI and multi-channel monitoring. Prolonged exhalation increased risky choices by enhancing reward sensitivity and elevating cardiac parasympathetic activity. Importantly, individuals with greater parasympathetic upregulation also showed stronger reward-related responses in the ventromedial prefrontal cortex and precuneus. Our work reveals a transformative role for breathing-based interventions, demonstrating that breathing-based autonomic regulation can shape value-based decision-making through neuro-cardiac pathways. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/698695v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@190293corg.highwire.dtl.DTLVardef@1835c63org.highwire.dtl.DTLVardef@1267ff3org.highwire.dtl.DTLVardef@9a583e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIProlonged exhalation increases risky decisions by increasing the reward sensitivity C_LIO_LIProlonged exhalation enhances cardiac parasympathetic activity without dampening sympathetic activities C_LIO_LIGreater cardiac parasympathetic activity under prolonged exhalation amplifies neural reward sensitivity in the ventromedial prefrontal cortex and precuneus C_LI

neuroscience↗

bioRxiv2026

A dual genomic-epigenomic map of clonal evolution in grapevine

Grapevine is one of the worlds oldest and most economically important perennial crops and has been vegetatively propagated for centuries to millennia. This long history of clonal propagation has generated substantial intra-varietal diversity, but its molecular basis has remained difficult to resolve with short-read sequencing, which misses large structural variation and DNA base modifications. Here, we generated a high-quality, phased diploid reference genome for the elite cultivar Pinot noir and integrate it with Oxford Nanopore sequencing of 23 distinct clones to build an integrated, genome-wide map of clonal genetic and epigenetic variation in Vitis vinifera. The genome assembly reveals a deep history of ancient inbreeding, with approximately 12% of the genome occurring in extended runs of homozygosity. Across molecular layers, we observe contrasting evolutionary dynamics. Somatic genetic variation (67,277 SNPs and 4,037 SVs) is dominated by rare variants and strongly depleted from coding regions, consistent with strong purifying selection. A substantial fraction of the structural variation among clones is mechanistically linked to instability in repetitive DNA, particularly centromeric repeats. In contrast, CG context epigenetic variation is abundant (250,382 differentially methylated cytosines) and strongly enriched within gene bodies, highlighting it as a major source of clonal divergence. Most importantly, CG methylation patterns alone reconstruct clonal phylogenetic relationships with high fidelity, closely mirroring the SNP-based lineages. This congruence, absent in transient non-CG methylation, indicates that stable CG epialleles are mitotically inherited and preserve a persistent record. Together, our results show that clonal identity in grapevine is jointly encoded by genome and methylome, with stable CG methylation capturing propagation history at near-genetic resolution.

plant biology↗

bioRxiv2025

Discovery of a Novel Chimeric Transposase-Transposon System for Advanced Genome Engineering

Transposases revolutionized the field of genetic engineering, yet the scarcity of functional systems remains a challenge. In response, we conducted a metagenomic screening and identified a novel transposase system from Acyrthosiphon pisum. Through systematic optimization, we enhanced nuclear localization, transposon composition, and created a hyperactive transposase variant to boost transposition efficiency. Intriguingly, the combined application of the newly discovered transposase with inverted terminal repeat sequences from a related pea aphid species, Aphis craccivora, further enhanced transposition activity, resulting in the first chimeric transposase system reported so far. We investigated the genomic integration events following transposition in mammalian cells, to understand the underlying mechanisms and optimize the efficiency of transgene integration. This optimized system can expedite the generation of recombinant protein producing CHO cell lines, even surpassing the hyperactive piggyBac system with regards to cell specific productivity. These findings introduce a significant addition to the field of semi-targeted transgene integration technologies, offering substantial potential for enhancing biologics manufacturing.

bioengineering↗

bioRxiv2025

Respiratory viruses induce ferroptosis-like features in lung epithelial cells

During infection, viruses can modulate various regulated cell death (RCD) mechanisms to evade host immunity and enhance replication. However, less is known about how viruses alter the recently discovered RCD ferroptosis, which is characterized by an iron-dependent accumulation of lipid peroxidation and mitochondrial fragmentation. Influenza A virus (IAV) H1N1 and human adenovirus type C5 (HAdV-C5) are two common causes of respiratory infections of the upper and lower respiratory tract and can lead to severe illness. While IAV has been shown to induce ferroptosis to support its replication, less is known whether this is also true for HAdV-C5. Here we perform a comparative study investigating ferroptosis features during IAV H1N1 and HAdV-C5 infections using alveolar (A549) and bronchial (BEAS-2B) epithelial cells. Our data reveal that HAdV-C5, similar to H1N1, induces lipid peroxidation in a time-dependent manner, which is partially suppressed in the presence of the ferroptosis inhibitor ferrostatin-1 (Fer-1). Strikingly, HAdV-C5 infections do not lead to changes in ferritin protein levels, which is responsible for iron storage. Furthermore, both H1N1 and HAdV-C5 infections trigger a profound mitochondrial network remodeling, comparable to that induced by the ferroptosis inducer RSL3, with effects varying by cell type and infection stage. These findings suggest that HAdV-C5, just like H1N1, can activate ferroptosis-like processes, highlighting a potential role for lipid peroxidation and mitochondrial alterations in adenoviral pathogenesis. Understanding this mechanism may uncover new therapeutic targets for viral respiratory infections.

microbiology↗

bioRxiv2025

iFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework

Standardized methods for the assembly of DNA constructs have become indispensable in synthetic biology, biotechnology and basic research. The majority of frameworks - most prominently based on Golden Gate - have been developed and optimized for the assembly of transcriptional units (TUs) and thereof composed multi-gene expression constructs. This is significantly enabled by the modular organization of functional elements underlying any given TU. In contrast, the assembly of protein coding sequences typically necessitates tailored approaches. Addressing this technological gap, iterative functional linker cloning (iFLinkC) was recently developed to provide a standardized framework for assembling protein coding sequences of arbitrary size and complexity from the ground up based on pairwise ligations of any two DNA fragments via a two-base overlap. Yet, a need for physically purifying DNA fragments imposes significant procedural complexity and operator skill. Overcoming these limitations, a new assembly algorithm, termed iFLinkC-EZ, is presented. Crucially, the DNA assembly products are now purified by genetic means which greatly simplifies the underlying assembly process and also renders it compatible with robotic automation. Further, the potency of iFLinkC-EZ is demonstrated in the assembly of mono- and poly-chromatic poly-fluorescent fusion proteins as well as poly-cistronic expression constructs while examining the effect of poly-PT and poly-GGS linkers as well as ribosome binding sites on the functional expression of poly-fluorescent fusion protein constructs. Further, the utility of poly-fluorescent proteins is demonstrated for the enhanced labeling of surface displayed recombinant binders in yeast display. Given its ease and efficiency, iFLinkC-EZ is anticipated to be applicable to the assembly of many different types of fusion proteins and thereof based multi-gene expression constructs including but not limited to synthetic protein switches, sensors and multi-enzyme complexes as well as thereof composed genetic circuits and metabolic pathways.

synthetic biology↗

bioRxiv2025

Comparative distribution of the hypothalamic neurons activated during Wakefulness and Paradoxical (REM) sleep using TRAP2-red mice: contribution of Orexin, MCH, Lhx6 and a new marker Meis2

Study objectivesParadoxical sleep (PS) is a state involving numerous hypothalamic neuronal subpopulations, many remaining neurochemically uncharacterized. Our goal was to compare hypothalamic neurons active during Wakefulness or PS rebound (PSR) and explore their potential overlap, with a focus on melanin-concentrating-hormone (MCH), Orexin (Orx), Lhx6 and a new contingent of Meis2- expressing neurons. MethodIn the same TRAP2-red mouse, neurons activated during Wakefulness (4h) and PSR (2h) express TdTomato and c-Fos, respectively. Double-labelling and triple immunofluorescence with neurochemical markers were performed to characterize and quantify cell populations in hypothalamic structures. ResultsTwelve hypothalamic structures showed distinct activation patterns. The anterior hypothalamic area (AHA), zona incerta (ZI) and tuberal nucleus contained more activated neurons during PSR than Wakefulness, whereas the paraventricular hypothalamic (PVN) and supraoptic (SO) nuclei were predominantly activated during Wakefulness. MCH and Lhx6 neurons were mainly recruited during PSR, whereas Orx neurons were activated during both. A ventral subpopulation of MCH neurons showed higher activation during PSR than the dorsal subpopulation. Additionally, [~]30% of the c-Fos+ neurons in ZI and AHA express Meis2. A similar proportion of TdTomato+ neurons positive for Meis2 were encountered in PVN and SO. Overall, [~]20% of all hypothalamic neurons activated during PSR are now neurochemically identified. ConclusionOur study identifies new neuronal populations activated during PSR in AHA and tuberal nucleus. We further get evidence that Meis2 delineates novel neuronal populations activated during PSR. In summary, our results using TRAP2-red mice characterize new cell populations activated during Wakefulness or PSR, opening experimental paths for determining their function regarding vigilance states. Statement of significanceWakefulness and paradoxical sleep are very similar at the electroencephalographic level. It remains relevant to determine the potential overlap of the neurons active during each vigilance state. We here took advantage of the powerful transgenic TRAP2-red mice to directly compare in the same animal the brain cell activation during both states, with a focus on the hypothalamus. A deeper knowledge of each individual subpopulation of hypothalamic neurons within complex brain circuits underlying the sleep-waking cycle will help the understanding and validation of treatments of sleep disorders, at least those directly linked to demonstrated hypothalamic dysfunction as Narcolepsy (Orx neurons), Amyotrophic Lateral Sclerosis (MCH and Orx signaling) or neurodegenerative diseases (Parkinsons and Alzheimer diseases).

neuroscience↗