Biology subjects

Schmidt, T.

Publications and source records attributed to Schmidt, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Sphingolipid-Driven Lipotoxicity Promotes Calcific Aortic Valve Disease Through ER-Stress-NF-κB-EndMT Signaling and Is Therapeutically Targetable Through Serine Palmitoyltransferase Inhibition

Background: Calcific aortic valve disease (CAVD) lacks disease-modifying therapy. LDL-cholesterol lowering does not attenuate valvular calcification, suggesting cholesterol-independent drivers. Thus, we tested whether ceramide accumulation drives CAVD and whether inhibiting sphingolipid de novo synthesis is therapeutic. Methods: We integrated lipidomics and proteomics of calcified and non-calcified human aortic valves with matched plasma. Mechanistic effects of ceramides were tested in primary human valvular interstitial cells (VICs). Therapeutic efficacy of serine palmitoyltransferase (SPT) inhibition with Myriocin was evaluated in ApoE-/- mice with PCSK9DY-AAV-driven hyperlipidemia on Western diet using preventive treatment and therapeutic initiation after echocardiographically verified gradient elevation. Myriocin was additionally tested in a normolipidemic wild-type aortic valve wire-injury model. Hemodynamics, valve structure, lipid composition, and cell-state transitions were assessed by echocardiography, histology, lipidomics, and single-cell RNA sequencing. Results: Calcified human valves showed enrichment of long-chain ceramides linked to NF-{kappa}B, endothelial-to-mesenchymal transition (EndMT), and ER-stress/unfolded protein response (UPR) programs, whereas matched plasma sphingolipids were not elevated. In hVICs, long-chain C18 ceramide induced GRP78/BiP, CHOP, and ATF4, activated NF-{kappa}B, and promoted dose-dependent calcification. In vivo, SPT inhibition with Myriocin lowered valvular ceramides, reduced leaflet inflammation and mineralization, and improved cusp separation and transvalvular hemodynamics without lowering plasma LDL-cholesterol. In a therapeutic protocol initiated after gradient elevation, Myriocin halted further hemodynamic progression, providing the first evidence that de novo sphingolipid synthesis can be targeted after established CAVD onset in vivo. Myriocin also attenuated injury-induced stenosis in normolipidemic mice. Single-cell RNA sequencing showed reduced NF-{kappa}B/EndMT and ER-stress signatures and shifted VIC states away from osteogenic phenotypes. Conclusions: Human valve lipidomics and human VIC experiments identify ceramide-driven lipotoxicity as a driver of an ER-stress-NF-{kappa}B-EndMT axis in CAVD. In vivo, de novo sphingolipid synthesis is an actionable, LDL-independent target capable of modifying established hemodynamic disease progression, supporting SPT inhibition as a tractable strategy for medical CAVD therapy.

molecular biology↗

bioRxiv2026

Ovarian cancer-on-chip for patient-specific profiling of treatment responses to sequential chemotherapy and PD-L1 blockade

BackgroundOvarian cancer (OvCa) ranks as the most lethal gynecological malignancy in women worldwide. This complex disease, which can develop independently of a womans age, is characterized by late diagnosis, pronounced tumor heterogeneity, and an immunosuppressive tumor microenvironment (TME). Incremental diagnostic tools that could better inform clinicians on potential therapy resistance or subsets of patients that could benefit from new drug modalities represent a critical unmet need to improve patient care and potentially the identification of new biomarkers. ObjectiveThis study aimed to establish a reconfigurable patient-derived OvCa-on-chip platform for longitudinal functional profiling of tumor cell death, immune activation, and patient-specific responses to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. MethodsPatient-derived OvCa microtumors (PDM) were integrated with sequential integration of autologous tumor-infiltrating lymphocytes (TILs) into a perfusable microfluidic chip in the presence of different single and combination treatment regimens of chemotherapy and immune checkpoint inhibitors (ICIs). Treatment responses were assessed by longitudinal quantification of caspase-cleaved cytokeratin 18 (ccCK18) as marker of apoptotic epithelial tumor cell death, as well as cytokine/chemokine release in chip effluents, and multiplex flow cytometry-based characterization of autologous TIL subsets. ResultsThe perfusable OvCa-on-chip platform supported long-term culture of PDM while maintaining key structural and microenvironmental features of the primary tumor. Multidimensional analyses, including time-resolved assessment of tumor cell death, secretome profiling and correlative analysis of autologous TIL subsets revealed patient-specific tumor-immune response patterns and heterogenous sensitivity to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. Correlation analyses identified treatment-dependent associations between specific TIL phenotypes and functional tumor cell killing. PD-1-expressing CD4 TIL subsets correlated with enhanced tumor cell killing, whereas terminally exhausted CD8PD-1Tcf1- TILs negatively correlated with durvalumab responses. In contrast, tumor-reactive CD8CD39 TILs were associated with improved responses under sequential chemo-immunotherapy conditions. ConclusionCollectively, this OvCa-on-chip system represents a complex in vitro model (CIVM) that combines 3D tumor tissue with autologous immune cells in a microfluidic platform. Resembling a physiologically relevant human preclinical platform, it allows for the time-resolved functional assessment of patient-specific responsiveness to OvCa therapies, with direct implications for personalized treatment stratification.

cancer biology↗

bioRxiv2026

Fecal untargeted metabolomic and short-chain fatty acid analyses in cats with chronic kidney disease

BackgroundThe gut-kidney axis plays a direct role in gastrointestinal and kidney health. Gut-derived metabolites like uremic toxins are associated with the pathophysiology of feline chronic kidney disease (CKD). The aim of the study was to identify novel fecal biomarkers and investigate the roles of gastrointestinal metabolites in feline CKD. ResultsFecal samples from 41 healthy non-CKD (control) and 67 CKD cats, including 5 IRIS stage 1 (CKD1), 37 stage 2a (CKD2a), 18 stage 2b (CKD2b), and 7 stage 3 (CKD3), were subject to fecal untargeted metabolomics and targeted short-chain fatty acid (SCFA) analyses. Multiple linear regression, adjusted for sex, age, body weight and study site, identified 64 differential metabolites between control and across CKD groups (P<0.0001 and FDR<0.10). Approximately 65% of the metabolites were lipids, including polyunsaturated long-chain fatty acids, acylcarnitines, and ceramides. Random Forest algorithm selected N1-methyl-2-pyridone-5-carboxamide (2PY), a uremic toxin from nicotinamide catabolism, as the top fecal marker for classifying feline CKD. Fecal 2PY was increased in CKD1 (P = 0.03), CKD2a, CKD2b, and CKD3 (all P<0.0001) compared to the controls. Data mining revealed serum concentration of 2PY was significantly increased with severity of CKD in cats, possibly due to impaired renal excretion. Cholesterol and arachidonic acid, markers for enterocyte shedding and inflammation, were increased in CKD3 versus control (both P<0.05). In healthy non-CKD cats, evident suggested fecal lipids increased with age (P<0.0001), and were higher in females versus males (P<0.0001). While fecal indole and p-cresol were increased in CKD3 versus control (both P<0.05), no change was observed in indoxyl sulfate (IS) or p-cresol sulfate (PCS). Fecal indole-3-acetic acid (IAA) was decreased in several CKD groups compared to the controls (all P<0.05). Finally, two branched SCFAs, isobutyrate and isovalerate, were increased in CKD3 versus control (both P<0.05). ConclusionsThe study revealed 2PY as a novel marker and unveiled profound alterations in intestinal lipid compositions with a potential link to gut barrier integrity and inflammation in CKD.

molecular biology↗

bioRxiv2026

Strong reproductive isolation among sympatric lineages of an international mosquito pest

Background: Reproductive isolation describes the degree to which genetic differences among populations reduce the level of neutral gene flow between them. Genomic approaches are currently showing great utility for identifying reproductive isolation between sympatric, morphologically indistinct populations, where hybridisation and introgression may yet be common. Here we use population genomic approaches and a chromosome-level reference assembly to uncover three highly differentiated, cryptic lineages of Aedes notoscriptus, an Australian mosquito disease vector that has invaded New Zealand (c. 1920) and California (c. 2014). Results: We identified a single lineage (Noto1) that was the source of the New Zealand and Californian invasions, and this lineage was also distributed across much of Australia including the Greater Melbourne region where it was sympatric with a second lineage (Noto2). Although Victorian Noto1 and Noto2 were frequently sampled co-locally, hybrids were observed less frequently than expected under random mating, and admixture levels were low, suggestive of selection against hybrids. A bottleneck observed in Victorian Noto1 suggests that Noto1 may have recently expanded its range into the Greater Melbourne region. We also identified a massive structural variant (~25 Mbp; ~10% of chromosome size) in Noto2 that was segregating at intermediate frequencies across the Greater Melbourne region but was fixed in Noto1. Conclusion: Together, these results identify key genetic differences between cryptic lineages of this international disease vector that warrant further investigation given potential differences in vector competence.

evolutionary biology↗

bioRxiv2026

Bridgehead invasions of ambrosia beetles are structured by inbreeding and hybridisation

When invasive populations establish in regions far from their origin, they may accumulate deleterious mutations that limit population viability and later expansion. Invasions stemming from such bridgehead populations may experience further sequential bottlenecks. However, deleterious mutations can be masked or eliminated when populations outbreed with other lineages. Here, we analyse global invasions of a species complex of persistently inbreeding ambrosia beetles, using genomic data (N=247) from invasive populations in Africa, North America and Australia, and from native populations in Asia. We mostly focus on one species of this complex (Euwallacea fornicatus) which poses a severe threat to tree species worldwide and is rapidly expanding its global range. We uncover a single lineage of this species across California, South Africa, and Western Australia, involving an invasive bridgehead and containing almost no nuclear genetic variation. In South Africa we identify a second lineage that has repeatedly hybridised with the first lineage. Genetic patterns in the native range indicate that such opportunistic outbreeding may be common. Despite lacking nuclear variation, the first lineage contained two CO1 haplotypes that were also observed in every hybrid lineage, pointing to heteroplasmy and possible hybrid origins of this lineage. Native populations had fewer missense mutations than invasive populations, indicating that opportunistic outbreeding may help purge fixed deleterious mutations when local lineage diversity is high. These findings highlight the importance of outbreeding even when inbreeding is common, and they demonstrate the biosecurity threat posed by subsequent gene flow into invasive populations.

evolutionary biology↗

bioRxiv2025

Understanding PebS-Ferredoxin Recognition: A Structural Perspective on Viral and Host Redox Partners

Cyanophages play a critical role in shaping host metabolism during infection by encoding auxiliary metabolic genes (AMGs), including enzymes involved in phycobilin biosynthesis. PebS (phycoerythrobilin synthase) is a phage-encoded ferredoxin-dependent biliverdin reductase (FDBR) that catalyses the two-step reduction of biliverdin IX (BV) to phycoerythrobilin (PEB). Although high-resolution structures are available for both PebS and its electron donor ferredoxin (Fd), the structural details governing their interaction remain unclear. This study leverages the well-characterised structural models of PebS and a host-like ferredoxin from Thermosynechococcus elongatus (Te-Fd), whose NMR structure provides a reliable basis for probing protein- protein interactions. Using [{superscript 1}N]-labelled, gallium-substituted Te-Fd, we employed NMR spectroscopy to monitor chemical shift perturbations upon binding, enabling us to probe the interaction interface in solution. Based on these data, we conducted protein-protein docking with HADDOCK to predict the interaction interface between Te-Fd and PebS. PebS variants designed to disrupt this interface did indeed show corresponding alterations in enzymatic efficiency and product formation as determined by time resolved UV/Vis spectroscopy and HPLC analyses. Utilizing the Fd encoded in the cyanophage PSSM2-Fd in our assays, we could observe a significantly improved catalytic activity, suggesting an coevolution of phage enzyme and electron donor. A comparison of the two available X-ray structures of Te-Fd and PSSM2-Fd with an alphafold-model of the Fd of the natural host Prochlorococcus NATL1A (NATL1A-Fd) also supports this evolutionary adaptation and the role of both PSSM2-Fd and PebS as AMGs involved in viral infection by PSSM2.

molecular biology↗

bioRxiv2025

Causal Lesion Evidence for Two Motor Speech Coordination Networks in the Brain

Speech production is supported by sensory-to-motor transformations to coordinate activity of the larynx and orofacial muscles. Here, we show that lesions to left temporal lobe areas involved in pitch processing cause reduced neural responses when repeating sentences and when humming piano melodies in a dorsal portion of the left precentral gyrus linked to laryngeal motor control. In contrast, lesions to left inferior parietal areas involved in somatosensory processing of speech cause reduced neural responses when repeating sentences but not when humming piano melodies in a ventral portion of the left precentral gyrus linked to orofacial motor control. Analyses in neurotypical participants converge in showing that the dorsal and ventral portions of the left precentral gyrus exhibit strong functional connectivity to left temporal and inferior parietal regions, respectively. These results provide causal lesion evidence that dissociable networks underlie distinct sensory-to-motor transformations supporting laryngeal and orofacial motor control for speech production.

neuroscience↗

bioRxiv2025

Visual processing of manipulable objects in the ventral stream is modulated by inputs from parietal action systems

Functional object use requires the integration of visuomotor representations processed in the dorsal visual pathway with representations of surface texture and material composition of objects, processed in the ventral visual pathway. Do regions in the ventral visual pathway project outputs to dorsal visual pathway action systems, or are the outputs of the dorsal visual pathway communicated to the ventral visual pathway to modulate processing? And what are the white matter pathways that mediate structural connectivity in support of functional object use? Here we show that the left inferior parietal cortex, a region within the dorsal visual pathway, exerts a direct effect on neural responses in ventral occipital-temporal cortex during visual processing of manipulable objects. We studied a series of consecutively enrolled participants in the pre-operative phase of their neurosurgical care (N = 109) with lesions principally distributed throughout the left hemisphere. Participants completed an object processing category localizer functional MRI experiment in which they viewed images of manipulable objects, animals, faces, and places. We then used Voxel-based Lesion-Activity Mapping (VLAM), a technique in which functional responses in a region-of-interest are used to predict variance in voxel-wise lesion incidence throughout the brain. In the VLAM analyses performed here, we found that lesions to the left anterior intraparietal sulcus and left supramarginal gyrus, two inferior parietal regions known to support object-directed grasping and manipulation, respectively, cause reduced neural responses for manipulable objects (compared to faces, places and animals) in the fusiform gyrus. Parietal lesions do not affect neural responses during visual processing of places in the same region of the fusiform gyrus, even though places elicit stronger responses in the fusiform gyrus than manipulable objects. Seventy-five of 109 participants took part in a common diffusion MRI protocol, permitting a connectome-wide analyses relating white matter fiber integrity to the strength of functional responses for manipulable objects in the left fusiform gyrus. This analysis demonstrated that the descending portion of the left arcuate fasciculus mediates parietal-to-temporal lobe connectivity for manipulable objects, supporting the integration of action representations with conceptual and perceptual attributes of objects. By combining voxel-based and connectome-wide lesion-symptom mapping methods with functional MRI, we have demonstrated that structural connectivity to dorsal visual pathway areas supporting skilled manual action shape category-specific neural responses for manipulable objects within the ventral visual pathway.

neuroscience↗

bioRxiv2025

The differential mechanisms of eIF4A1-mediated translational activation instructed by distinct RNA features

All mRNAs require eukaryotic translation initiation factor (eIF) 4A1 for translation through its different functions: loading of the pre-initiation complex onto mRNAs and unwinding of RNA structure. eIF4A1 is the catalytic subunit of the cap- binding eIF4F complex and presumed to select the mRNAs for translation through these activities, instructed by signalling pathways driving cell fate. The mechanisms underlying activation of the distinct eIF4A1 functions and establish translational selectivity are unknown. Here, we have unravelled the complexity of mRNA selection by eIF4A1. We have mechanistically characterised the biological and atomic basis of inhibition by gain- and loss-of-function eIF4A1-inhibitors eFT226 and hippuristanol, and used machine learning to model the mRNA features associated with specific inhibition. This uncovered the eIF4A1 function - mRNA sequence relationship: 5UTRs containing C/CG-rich require efficient mRNA loading by eIF4F which is specifically targeted by hippuristanol, while 5UTRs harbouring alternatives starts sites together with AG-rich motifs utilise eIF4A1 for start site selection, specifically perturbed by eFT226. Our model is validated through a massively parallel reporter assay using 5UTRs from a distinct evolutionary origin. This prompted us to examine the conservation between mRNA sequence and eIF4A1 function, and revealed their co- development. Our findings highlight opportunities for novel therapeutic strategies targeting eIF4A1 and for improved design of mRNA-based therapeutics.

cell biology↗

bioRxiv2025

Matrix stiffness affects spheroid invasion, collagen remodeling and the effective reach of stress into the ECM.

The extracellular matrix (ECM) provides structural support to cells thereby forming a functional tissue. In cancer, the growth of the tumor creates an internal mechanical stress which, together with remodeling activity of tumor cells and fibroblasts, alters the ECM structure leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness in turn stimulates tumor growth, activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and an increased therapy resistance. The connection between internal tumor stress, ECM stiffness, ECM remodeling, and cell migration are unresolved. Here we used 3D ECM-embedded spheroids and hydrogel-particle stress sensors, to quantify and correlate internal tumor-spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion - described by area, complexity, number of branches and branch area - in a stiffer, cross-linked ECM. On the other hand, changing the ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers.. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

biophysics↗

bioRxiv2025

Oxalotrophic bacteria in desert and drylands: Enzymatic pathways and Carbon sequestration

Oxalotrophy refers to the ability of bacteria to utilize oxalate as a carbon and energy source. This is a critical process with significant implications for the global carbon cycle. Oxalate-degrading bacteria play a key role in carbon sequestration through the oxalate-carbonate pathway (OCP), contributing to stable inorganic carbon pools. In this study, we identified and cataloged 20 enzymes associated with various facets of oxalate metabolism to characterize the oxalotrophic potential of bacteria. Within this group, sets of enzymes were grouped into two functional categories in the context of carbon sequestration: a biomineralization toolkit for converting oxalate to inorganic carbon and an assimilation toolkit for incorporating oxalate into metabolic pathways such as amino acid biosynthesis and energy production. Using bioinformatic approaches, we analyzed a collection of 536 bacterial genomes from desert and dryland strains spanning 81 genera to identify oxalotrophs. To validate our findings, we tested several bacterial strains for growth on media supplemented with exogenous oxalate. Notably, while multiple bacterial strains grew on oxalate media, two Pseudomonas species, namely JZ043 and JZ097, failed to grow despite genomic predictions suggesting otherwise. Further investigation of these strains revealed several non-conservative amino acid substitutions in the glyoxylate carboligase enzyme (EC 4.1.1.47), a key player in oxalate metabolism, suggesting a potential link between these mutations and their inability to metabolize oxalate. Our findings highlight the significance of our approach for identifying oxalotrophic bacteria (OxB) and offer valuable insights into the molecular basis of oxalate metabolism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/637404v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@13d1408org.highwire.dtl.DTLVardef@ee2e5eorg.highwire.dtl.DTLVardef@1265323org.highwire.dtl.DTLVardef@11c56d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Tracing social mechanisms and interregional connections in Early Bronze Age Societies in Lower Austria

In this study, we present the results of archaeogenetic investigations of Early Bronze Age individuals from Lower Austria, specifically associated with the Un[e]tice and Unterwolbling cultural groups. Through analysing newly generated genome-wide data of 138 individuals, we explore the social structure and genetic relationships within and between these communities. Our results reveal a predominantly patrilocal society with non-strict female exogamic practices. Additionally, Identity-by-Descent (IBD) analysis detects long-distance genetic connections, emphasizing the complex network of interactions in Central Europe during this period. Despite shared social dynamics, notable genetic distinctions emerge between the Un[e]tice and Unterwolbling groups. These insights contribute to our understanding of Bronze Age population interconnections and call for a nuanced interpretation of social dynamics in this historical context.

genetics↗

bioRxiv2025

Unprecedented Number of Recurrent Mutations Drive the Rapid Evolution of Pesticide Resistance in a Notorious Pest

The genetic basis of pesticide resistance has been widely studied, but the exact nature of this evolutionary process in the field is often unclear, particularly when a limited number of populations is considered and when there is a lag between the evolutionary event and its investigation. We showed that an unprecedented number of recurrently evolved mutations in an arthropod pest, the two-spotted spider mite Tetranychus urticae, drive the rapid evolution of resistance to a recently commercialized acaricide, cyetpyrafen. We first observed high levels of resistance that appeared and became widespread within three years. Genome scans revealed genetic heterogeneity of resistance among populations and identified 15 target mutations, including six mutations on five amino acid residues of subunit sdhB, and nine mutations on three amino acid residues of subunit sdhD of the pesticide target succinate dehydrogenase, with as many as five substitutions on one residue. No mutations were present in 2666 screened historical specimens, suggesting that mutations arose rapidly through de novo substitutions or from very rare segregating mutations. Identical mutations recurrently appeared in different genetic backgrounds, increasing the likelihood of resistance evolution. The high number of mutational options available for the evolution of target site resistance in this pest challenges resistance management practices.

evolutionary biology↗

bioRxiv2025

The yeast phosphofructokinase β-subunit has ATP-dependent RNA unwinding activity and modulates cell cycle progression

Phosphofructokinase (PFK) is a rate-limiting glycolytic enzyme that also possesses an unexplored RNA binding activity. Here, we show that the - and {beta}-subunits of yeast PFK, encoded by PFK1 and PFK2, respectively, bind hundreds of mRNAs in cells, including ones coding for proteins involved in regulation of mitotic cell cycle. Pkf2p directly binds in a Mg-ATP-dependent manner to short GA-, UC-, AU- and U-rich motifs overrepresented in its mRNA targets. Strikingly, Pfk2p displays directional 5 - 3 double-stranded RNA unwinding activity not seen with Pfk1p. Furthermore, Pfk2p dynamically associates with ribosomes and promotes translation of cell cycle genes. Consequently, pfk2{Delta}, but not pfk1{Delta}, mutant cells show severely delayed G1/S phase transition which is independent of the enzymes glycolytic activity. Our results uncovered a hidden function for the Pfk2 subunit as a translational activator of mitotic cell cycle gene transcripts possibly through energy-dependent RNA unwinding activity.

molecular biology↗

bioRxiv2025

Pre-clinical validation of in vitro engineered but cell-free human tissue grafts for skeletal regeneration

Tissue engineering strategies predominantly consist of the autologous generation of living substitutes capable of restoring damaged body parts. Persisting challenges with patient-specific approaches include inconsistent performance, high costs and delayed graft availability. Towards developing a one-for-all solution, a more attractive paradigm lies in the exploitation of dedicated cell lines for the fabrication of human tissue grafts. Following decellularization, this new class of biomaterials relies on the sole extracellular matrix and embedded growth factors instructing endogenous repair. This conceptual approach was previously validated using a custom mesenchymal line for the manufacturing of human cartilage, exhibiting remarkable osteoinductive capacity following lyophilization. Key missing criteria to envision clinical translation include proper decellularization as well as stringent assessment of both immunogenicity and regenerative performance. Here, we report the engineering and subsequent decellularization of human cartilage tissue with minimal matrix impairment. Ectopic evaluation in immunocompetent and immunocompromised animals reveal preservation of osteoinductivity predicted by macrophage kinetic of polarization. By establishing in vitro human allogeneic co-culture models, we evidenced the immuno-evasive properties of cell-free human cartilages, controlling macrophages and dendritic cells maturation as well as T cell activation. Lastly, regenerative performance was stringently assessed in an immunocompetent rat orthotopic model whereby decellularized human cartilage grafts achieved morphological and mechanical restoration of all critical-sized femoral defects. Taken together, our study compiles robust safety and efficacy pre-requisites prompting a first-in-human trial for engineered and decellularized human tissue grafts.

bioengineering↗

bioRxiv2024

Unifying the analysis of bottom-up proteomics data with CHIMERYS

Proteomic workflows generate vastly complex peptide mixtures that are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), creating thousands of spectra, most of which are chimeric and contain fragment ions from more than one peptide. Because of differences in data acquisition strategies such as data-dependent (DDA), data-independent (DIA) or parallel reaction monitoring (PRM), separate software packages employing different analysis concepts are used for peptide identification and quantification, even though the underlying information is principally the same. Here, we introduce CHIMERYS, a novel, spectrum-centric search algorithm designed for the deconvolution of chimeric spectra that unifies proteomic data analysis. Using accurate predictions of peptide retention time, fragment ion intensities and applying regularized linear regression, it explains as much fragment ion intensity as possible with as few peptides as possible. Together with rigorous false discovery rate control, CHIMERYS accurately identifies and quantifies multiple peptides per tandem mass spectrum in DDA, DIA and PRM experiments.

biochemistry↗

bioRxiv2024

Altered Mechanobiology of PDAC Cells with Acquired Chemoresistance to Gemcitabine and Paclitaxel.

BackgroundPancreatic ductal adenocarcinoma (PDAC) acquired resistance to chemotherapy poses a major limitation to patient survival. Despite understanding of some biological mechanisms of chemoresistance, much of those mechanisms remain to be uncovered. Mechanobiology, which studies physical properties of cells, holds promise as a potential target for addressing challenges of chemoresistance in PDAC. Therefore, we here in an initial step, assessed the altered mechanobiology of PDAC cells with acquired chemoresistance to gemcitabine and paclitaxel. MethodsFive PDAC cell lines and six stably-resistant subclones were assessed for force generation on elastic micropillar arrays. Those measurements of mechanical phenotype were complemented by single-cell motility and invasion in collagen matrix were investigated using 2D models and 3D extracellular matrix-mimetic, respectively. Further the nuclear translocation of Yes-associted protein (YAP), as a measure of active mechanical status, was compared, and biomarkers of the epithelial-to-mesenchymal transition (EMT) were evaluated using RT-PCR. ResultsPDAC cells with acquired chemoresistance exert higher traction forces than their parental/wild-type (WT) cells. In 2D, single-cell motility was altered for all chemoresistant cells, with a cell-type specific pattern. In 3D, spheroids of chemoresistant PDAC cells were able to invade the matrix, and remodel collagen more than their WT clones. However, YAP nuclear translocation and EMT were not significantly altered in relation to changes in other physical parameters. ConclusionThis is the first study to investigate and report on the altered mechanobiological features for PDAC cells that have acquired chemoresistance. A better understanding of mechanical features could help in identifying future targets to overcome chemoresistance in PDAC.

biophysics↗

bioRxiv2024

DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids

Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3 overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.

biochemistry↗