Biology subjects

Pastore, A.

Publications and source records attributed to Pastore, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Controlled Blood-Brain Barrier Modulation by a High-Affinity Claudin-5 Peptide Binder

The blood-brain barrier (BBB) is a specialized interface that tightly regulates the exchange of molecules between the bloodstream and the brain. Its barrier function relies on a monolayer of brain endothelial cells sealed by tight junctions (TJs) that restrict paracellular flux through claudin-5 (CLDN5) multimeric complexes. To improve the delivery of nutrients and drugs to the brain, CLDN5-competitive peptides are promising carriers for creating size-controlled, temporary openings in the paracellular pathway. Here, we combine generative protein design and atomistic simulations to design ST9, a peptide with high nanomolar affinity for CLDN5. Compared with f1-C5C2, a CLDN5-binding peptide that we previously reported, ST9 induces a rapid, transient, size-controlled, and fully reversible increase in paracellular permeability, without altering CLDN5 expression or the proteomic profile of brain endothelial cells, indicating distinct mechanisms of TJ destabilization. This work provides a promising approach for developing next-generation BBB-opening agents to effectively treat neurological diseases.

neuroscience↗

bioRxiv2026

Hydration and H/D exchange-dependent infrared signatures of the GCN4 leucine zipper

Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of proteins in aqueous solution is often limited by water absorption and other optical artifacts. To overcome these limitations, we evaluated the structural features and hydrogen-deuterium exchange (HDX) kinetics of the -helical protein GCN4 in both hydrated (wet) and vacuum-dried (dry) states. While solvent heavily mask the second-derivative spectra of wet samples, vacuum drying yielded a thin, protein-rich film on the ATR crystal, significantly enhancing the signal-to-noise ratio and resolving the protein features without altering the native structure. Dry-state analysis clearly resolved the Amide I, Amide II, and deuterium-shifted Amide II (1450 cm-1) bands. Notably, second-derivative analysis of the dry spectra of the HDX samples revealed a bimodal Amide I distribution consisting of a stationary band at 1653 cm-1 from the solvent-inaccessible regions and an isotopically sensitive band shifting from 1648 cm-1 to 1644 cm-1 from solvent-accessible regions. These results demonstrate that vacuum-dried ATR-FTIR spectroscopy effectively eliminates solvent masking, providing the spectral clarity required to resolve discrete -helical sub-populations after deuteration.

biochemistry↗

bioRxiv2026

DNA supercoiling modulates bZIP transcription factor/DNA interaction

DNA topology is a key regulator of chromatin structure and transcription, yet its direct role in transcription factor recognition remains unclear. Here, we investigate how distinct DNA topological states modulate binding of the Saccharomyces cerevisiae bZIP transcription factor GCN4 using topologically defined plasmids. By combining, complementary biochemical approaches, including Bio-Layer Interferometry applied here for the first time to topology-dependent protein-DNA interactions, we show that DNA supercoiling directly reshapes GCN4-DNA recognition. Positively supercoiled DNA forms more stable and persistent complexes, whereas negatively supercoiled DNA retains greater conformational heterogeneity. To interpret these effects, we performed multiscale molecular simulations. Coarse-grained simulations of plasmids recapitulate the global topology-dependent trends observed experimentally, while matched minicircle models reproduce the same behaviour at the local scale. In strong agreement with experimental data, simulations reveal that DNA topology modulates the conformational ensemble of the GCN4 basic region. Overall, positively supercoiled DNA promotes a more ordered binding mode and localized protein distribution, whereas negatively supercoiled DNA supports increased structural plasticity. These findings identify DNA topology as an active determinant of transcription factor recognition and provide a multiscale framework linking global DNA mechanics to local protein-DNA interactions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/722604v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@18f8ba9org.highwire.dtl.DTLVardef@11a395dorg.highwire.dtl.DTLVardef@ac093borg.highwire.dtl.DTLVardef@923212_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

From Single-Cell Emergent Behaviors to Clinical Outcome: PTEN-driven Migratory Efficiency as a Potential New Vulnerability in Glioblastoma

BackgroundGlioblastoma (GB) is a highly aggressive brain tumor with a median survival of approximately 14 months, primarily due to its ability to infiltrate healthy brain tissue both as single cells and in collectives. A deeper understanding of GB cell motility, both individual and collective, is crucial for developing patient-specific therapies. We aimed to characterize migration in patient-derived GB cells using advanced modeling to identify stratification markers and therapeutic vulnerabilities. MethodsWe developed Single-Cell Behavior Live Imaging (ScBLI), an approach integrating live imaging with computational analysis, applied to 30 GB primary cell cultures. Trajectories and morphological features were tracked and analyzed. Diffusion Entropy Analysis (DEA) was applied to classify trajectories based on the Delta Scaling parameter ({delta} scaling). We evaluated functional responses correlating all findings with clinical outcomes and transcriptomic profiles. ResultsWe analyzed 4,279 cell trajectories. Based on {delta} scaling (range 0.28-0.837), we defined three distinct motility groups: Low (L, {delta} scaling [≤]0.5), Medium (M, 0.5 < {delta} scaling [≤] 0.7), and High (H, {delta} scaling >0.7). Functional assays demonstrated that Group H cells are more performant in both positive and negative chemotaxis. Clinically, the three groups showed a clear linear progression with patient survival: High {delta} scaling correlated with the shortest survival (poorer prognosis), while Low {delta} correlated with the longest survival, suggesting that structured motility drives invasiveness. Integrative multi-omic analysis, encompassing both exome and transcriptome profiling, demonstrated that these groups are defined by distinct molecular landscapes rather than poor behavioral traits. Moreover, exome data revealed that Group H is significantly enriched in PTEN alterations (75% vs. 8% in Group L), with PTEN gain-of-function (GoF) mutations exclusively restricted to this group (100% vs 0% in Group L). Notably, within our extended cohort (n=51) currently characterized by whole-exome sequencing, we observed that specific PTEN GoF mutations were associated with a significantly shorter survival compared to PTEN wild-type cases (median OS 6.4 vs 16.6 months; p=0.02), which typically harbor the canonical loss of chromosome 10q. A similar clinical trend was observed when comparing directly GoF carriers to patients with truncating (Ter) alterations (median OS 6.4 vs 14.3 months; p=0.09). Conversely, no survival difference was found between truncating (Ter) mutations and wild-type cases. ConclusionOur findings demonstrate for the first time that migratory efficiency, quantified through DEA, represents a powerful predictor of glioblastoma aggressiveness. Tumor cells adopting highly efficient exploration strategies are strongly associated with poor clinical outcomes and are characterized by distinct molecular signatures, notably PTEN gain-of-function alterations. Statement of significanceOur multi-scale computational framework elucidates emergent behavioral phenotypes as pivotal drivers of glioblastoma progression. By demonstrating a correlation between enhanced migratory efficiency, PTEN gain-of-function, and significantly reduced overall survival, we establish a foundational paradigm for deciphering the emergent complexity governing tumor invasiveness.

cancer biology↗

bioRxiv2026

DNA binding drives phase separation of the Gcn4 bZIP domain and reveals its conformational ensemble in the diluted and condensate phases

Liquid-liquid phase separation is widely invoked in transcriptional regulation, yet prevailing models attribute condensate formation primarily to intrinsically disordered activation domains rather than structured DNA-binding motifs. Here, we overturn this view by demonstrating that the isolated basic leucine zipper (bZIP) domain of the yeast transcription factor Gcn4 undergoes robust DNA-induced phase separation in the complete absence of its activation domain. Using small-angle X-ray scattering in combination with all-atom molecular dynamics simulations and ensemble optimization, we directly resolve the conformational landscape of the Gcn4 bZIP-DNA complex across coexisting dilute and condensed phases. Beyond the canonical uninterrupted helical conformation captured in crystal structures, we identify a previously unrecognized minor population featuring a pronounced helical kink at the basic region-leucine zipper junction. These findings establish DNA binding as a sufficient physical driver of bZIP phase separation and demonstrate that small-angle scattering can quantitatively interrogate protein conformational ensembles within biomolecular condensates, opening new avenues for the structural chemistry of phase-separated systems.

biophysics↗

bioRxiv2025

Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.

biophysics↗

bioRxiv2024

A comprehensive guide through the fold and stability of cytochrome c with implications for disease

Cytochrome c (Cyt-c), encoded by the CYCS gene, is crucial for electron transport, peroxidase activity, and apoptosis. Mutations in CYCS causes thrombocytopenia 4, a disorder with low platelet counts. We have, for instance, recently described six Italian families with five different heterozygous missense CYCS variants. These mutations likely enhance peroxidase and apoptotic activities, yet the mechanisms causing reduced platelet production and increased apoptosis are unclear. This study investigates clinically-related Cyt-c variants using an integrated bioinformatics approach. Our findings reveal that all variants are at evolutionarily conserved sites, potentially disrupting Cyt-c function and contributing to disease phenotypes. Specific variants are predicted to affect phosphorylation (T20I, V21G, Y49H), and ubiquitination (G42S, A52T, A52V, T103I). Molecular dynamics simulations (500 ns) revealed significant structural differences from the wild-type protein, with mutants showing reduced stability and increased unfolding and flexibility, particularly in the {Omega}-loops. These changes result in the displacement of the {Omega}-loops away from the heme iron, weakening critical hydrogen bonds and consequently opening the heme active site. This open conformation may enhance accessibility to small molecules such as H2O2, thereby promoting peroxidase activity, which may enhance apoptosis and likely impact megakaryopoiesis and platelet homeostasis in THC4.

bioinformatics↗

bioRxiv2024

Understanding the physical determinants of pressure denaturation: Temperature switches the pressure-induced unfolding pathway

Proteins unfold under different environmental insults, among which are heat, cold, high pressure and chaotropic agents. Understanding the mechanisms that determine unfolding under each of these conditions is an important problem that directly relates to the physical forces that determine the three-dimensional structure of a protein. Here, we studied a residue-specific description of the unfolding transitions of the marginally stable yeast protein Yfh1 using high-pressure nuclear magnetic resonance. We compared the cold, heat and pressure unfolded states and demonstrated what has up to now been only a hypothesis: the pressure-unfolded spectrum shares features in common with that at low but not at high temperature and room pressure, suggesting a tighter similarity of the mechanisms and a similar role of hydration in these two processes. By exploring the phase diagram of the protein and mapping unfolding onto the three-dimensional structure of the protein, we also show that the pressure-induced unfolding pathways at low and high temperatures differ, suggesting a synergic mechanism between pressure- and temperature-induced denaturation. Our observations help us to reconstruct the structural events determining unfolding and distinguish the mechanisms that rule the different processes of unfolding.

biophysics↗

bioRxiv2024

Amygdala TDP-43 pathology is associated with behavioural dysfunction and ferritin accumulation in amyotrophic lateral sclerosis.

BackgroundCognitive and behavioural symptoms associated with amyotrophic lateral sclerosis and frontotemporal spectrum disorders (ALSFTSD) are thought to be driven, at least in part, by the pathological accumulation of TDP-43. MethodsHere we examine post-mortem tissue from six brain regions associated with cognitive and behavioural symptoms in a cohort of 30 people with sporadic ALS (sALS), a proportion of which underwent standardized neuropsychological behavioural assessment as part of the Edinburgh Cognitive ALS Screen (ECAS). ResultsOverall, the behavioural screen performed as part of the ECAS predicted accumulation of pathological phosphorylated TDP-43 (pTDP-43) with 100% specificity and 86% sensitivity in behaviour-associated brain regions. Notably, of these regions, pathology in the amygdala was the most predictive correlate of behavioural dysfunction in sALS. In the amygdala of sALS patients, we show variation in morphology, cell type predominance, and severity of pTDP-43 pathology. Further, we demonstrate that the presence and severity of intra-neuronal pTDP-43 pathology, but not astroglial pathology, or phosphorylated Tau pathology, is associated with behavioural dysfunction. Cases were also evaluated using a TDP-43 aptamer (TDP-43APT), which revealed that pathology was not only associated with behavioural symptoms, but also with ferritin levels, a measure of brain iron. ConclusionsIntra-neuronal pTDP-43 and cytoplasmic TDP-43APT pathology in the amygdala is associated with behavioural symptoms in sALS. TDP-43APT staining intensity is also associated with increased ferritin, regardless of behavioural phenotype, suggesting that ferritin increases may occur upstream of clinical manifestation, in line with early TDP-43APT pathology, representing a potential region-specific imaging biomarker of early disease in ALS. Key MessagesO_ST_ABSWhat is already known on this topicC_ST_ABSThe amygdala is a key brain region in regulating behavior and emotional cognition and has been shown recently, through imaging studies, to be affected in ALS and FTD patients. What this study addsHere we examine the underlying pathology driving the association between the amygdala and behavioural symptoms in sporadic ALS demonstrating that region specific TDP-43 pathology and brain iron accumulation could represent potential early biomarkers of dysfunction. How this study might affect research, practice, or policyThe correlation between early TDP-43 pathology (detected by RNA aptamer) and increased ferritin (brain iron accumulation) occurring upstream of clinical manifestation represents a potential, region-specific (amygdala), early imaging biomarker in ALS. This means that people at risk could be identified early and stratified for clinical trials prior to substantial neuronal cell loss and symptom onset.

neuroscience↗

bioRxiv2024

N-acetylcysteine counteracts immune dysfunction and autistic-related behaviors in the Shank3b mouse model of Autism Spectrum Disorders

Autism Spectrum Disorder (ASD) includes a range of neurodevelopmental disabilities characterized by social interaction deficits, communication impairments, and repetitive behaviors. Previous studies have shown that pro-inflammatory conditions play a key role in ASD. Here we reported that increased levels of molecules related to inflammation are present in the cerebellum and peripheral blood (PB) of mice lacking Shank3b, established model of syndromic ASD. In parallel, immune dysfunction was documented in the bone marrow (BM) and spleen of mutant mice. N-acetylcysteine (NAC) treatment rescued inflammation in the cerebellum and PB, as well as impaired production of pro-inflammatory molecules in the BM and spleen. In addition, social impairment was counteracted in NAC-treated Shank3b-/- animals. Taken together, our study further confirms the key role of cerebellar inflammation in the establishment of ASD-related behaviors. Furthermore, our findings underscore the importance of considering ASD as a systemic disorder. Our findings therefore suggest that the interplay between oxidative stress and inflammation may support ASD-related behaviors in mice.

immunology↗

bioRxiv2023

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS

TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, analysis of deeply-phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism, but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 aggregation and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply-phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic aggregation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic aggregation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS. Short AbstractRecent identification of cryptic-splicing events such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism in amyotrophic lateral sclerosis (ALS). However, the temporal nature of TDP-43 loss and its relation to clinical phenotype is not known. Here, we used a novel RNA aptamer to detect TDP-43 aggregation and used single molecule ISH to sensitively reveal TDP-43 loss-of-function, applying these methods in a deeply-phenotyped human post-mortem tissue cohort. We show that nuclear TDP-43 pathology is an early event, that coincides with STMN-2 cryptic splicing. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics and intervention prior to symptom onset in ALS. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/563701v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1e49c3forg.highwire.dtl.DTLVardef@1ce05b0org.highwire.dtl.DTLVardef@d77205org.highwire.dtl.DTLVardef@7ed2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

The interplay between oxidative stress and inflammation supports autistic-related behaviors in mice

Autism Spectrum Disorder (ASD) is a highly prevalent neurodevelopmental condition characterized by social communication deficits and repetitive/restricted behaviors. Several studies showed that inflammation may contribute to ASD. Here we used RT-qPCR, RNA sequencing, immunohistochemistry, and flow cytometry to show that pro-inflammatory molecules were increased in the cerebellum and periphery of mice lacking Cntnap2 (Cntnap2-/-), a robust model of ASD. In parallel, oxidative stress was present in the cerebellum of mutant animals. Systemic treatment with N-acetyl-cysteine (NAC) rescued cerebellar oxidative stress and inflammation as well as motor and social impairments in Cntnap2-/- mice. This was accompanied by improved function of microglia cells in NAC-treated mutant animals. Intriguingly, social deficits, cerebellar inflammation and microglia dysfunction were induced by NAC in Cntnap2+/+animals. Our findings therefore suggest that the interplay between oxidative stress and inflammation may support ASD-related behaviors in mice.

immunology↗

bioRxiv2023

BIRC3: A Prognostic Predictor and Novel Therapeutic Target in TMZ-Resistant Glioblastoma Tumors

BackgroundGlioblastoma (GB) is an incurable malignant tumor of the central nervous system, with a poor prognosis. Robust molecular biomarkers associated with therapeutic response or survival are still lacking in GB. Previously, using NADH-fluorescence lifetime imaging (NADH-FLIM), as a new drug screening precision medicine ex-vivo approach, we categorized patient-derived vital tumors into TMZ responder (Resp) and non-responder (Non-Resp) groups, revealing differentially expressed genes. MethodsExpanding on our previous study, we assessed TMZ response in a larger cohort of primary and recurrent ex-vivo live GB tumors (n=33) using NADH-FLIM. Transcriptome analysis was performed to characterize TMZ Resp and Non-Resp cases, and in-silico and functional cellular investigations were conducted to explore the efficacy of potential biomarkers. ResultsGenes dysregulated in the previous study showed consistent expression patterns. BIRC3, a potent apoptosis inhibitor, was significantly upregulated in TMZ-resistant samples. BIRC3 expression complemented MGMT status as a prognostic factor in multiple TCGA cohorts. BIRC3 functioned as a prognostic factor of survival also in separate European private glioblastoma cohorts. The BIRC3 antagonist, AZD5582, in combination with TMZ, effectively reversed TMZ resistance by restoring apoptosis in glioblastoma cell lines and patient-derived organoids. ConclusionsBIRC3 holds promise as a prognostic biomarker and predictor of TMZ response in GB. Assessing BIRC3 expression could aid in stratifying patients for combined TMZ and AZD5582 therapy. Our study highlights the potential of functional precision medicine and BIRC3 assessment as a standard tool in glioblastoma clinical oncology, improving outcomes. KEYPOINTSO_LIBIRC3, previously overlooked, identified through dynamic precision medicine using TMZ perturbation of glioblastoma tissue as a robust prognostic factor. C_LIO_LIThe gene BIRC3 is an independent prognostic factor associated with shorter survival and TMZ resistance, rigorously validated across various case studies and datasets, including two expansive European case studies. C_LIO_LIProposal of anti-BIRC3 drug, AZD5582, shows promise as a novel therapeutic option to overcome TMZ resistance in GB tumors, providing hope for improved outcomes and personalized treatment strategies for patients with limited treatment options C_LI IMPORTANCE OF THE STUDYGlioblastoma (GB), an aggressive cancer type with a bleak prognosis, lacks dependable biomarkers for treatment prediction. Few markers like MGMT promoter methylation, IDH1 mutation, TERT gene mutations, and EGFR amplification are known, but their predictive consistency varies. Temozolomide (TMZ) resistance, seen in over 50% of GB patients, complicates matters. BIRC3, an apoptosis-inhibiting gene, displays heightened expression in TMZ-resistant tumors. Our study examined BIRC3 in GB patient samples, finding it an independent prognostic factor linked to shorter survival and TMZ resistance. Our research builds upon Wang et al.s 2016 and 2017 findings, delving deeper through TCGA data and European case studies. BIRC3s consistent prominence suggests its significance, with functional experiments confirming its role. We assessed AZD5582, targeting BIRC3, which, when combined with TMZ, curtailed cell growth and induced apoptosis. Notably, AZD5582 countered TMZ resistance in patient-derived GB-EPXs, except for low BIRC3 cases. Our precision medicine approach enhances personalized therapies and outcomes, highlighting BIRC3s potential as a prognostic marker and AZD5582 as a new therapy for TMZ-resistant GB.

cancer biology↗

bioRxiv2023

Robotics-driven manufacturing of cartilaginous microtissues for the bio-assembly of skeletal implants

Automated technologies are attractive for enhancing a robust manufacturing of tissue engineered products for clinical translation. In this work, we present an automation strategy using a robotics platform for media changes of cartilaginous microtissues cultured in static microwell platforms. We use an automated image analysis pipeline to extract microtissue displacements and morphological features, which serve as input for statistical factor analysis. To minimize microtissue displacement and suspension leading to uncontrolled fusion, we performed a mixed factorial DoE on liquid handling parameters for large and small microwell platforms. As a result, 144 images, with 51 471 spheroids could be processed automatically. The automated imaging workflow takes 2 minutes per image, and it can be implemented for on-line monitoring of microtissues, thus allowing informed decision making during manufacturing. We found that time in culture is the main factor for microtissue displacements, explaining 10 % of the displacements. Aspiration and dispension speed were not significant at manual speeds or beyond, with an effect size of 1 %. We defined optimal needle placement and depth for automated media changes and we suggest that robotic plate handling could improve the yield and homogeneity in size of microtissue cultures. After three weeks culture, increased expression of COL2A1 confirmed chondrogenic differentiation and RUNX2 shows no osteogenic specification. Histological analysis showed the secretion of cartilaginous extracellular matrix. Furthermore, microtissue-based implants were capable of forming mineralized tissues and bone after four weeks of ectopic implantation in nude mice. We demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous microtissues. We anticipate the progressive substitution of manual operations with automated solutions for manufacturing of microtissue-based living implants.

bioengineering↗

bioRxiv2021

The disease associated Tau35 fragment has an increased propensity to aggregate compared to full-length tau

Tau35 is a truncated form of tau found in human brain in a subset of tauopathies. Tau35 expression in mice recapitulates key features of human disease, including progressive increase in tau phosphorylation, along with cognitive and motor dysfunction. The appearance of aggregated tau suggests that Tau35 may have structural properties distinct from those of other tau species that could account for its pathological role in disease. To address this hypothesis, we performed a structural characterization of monomeric and aggregated Tau35 and compared the results to those of two longer isoforms, 2N3R and 2N4R tau. We used small angle X-ray scattering to show that Tau35, 2N3R and 2N4R tau all behave as disordered monomeric species but Tau35 exhibits higher rigidity. In the presence of the poly-anion heparin, Tau35 increases thioflavin T fluorescence significantly faster and to a greater extent than full-length tau, demonstrating a higher propensity to aggregate. We used atomic force microscopy, transmission electron microscopy and X-ray fiber diffraction to demonstrate that Tau35 aggregates are morphologically similar to previously reported tau fibrils but they are more densely packed. These data increase our understanding of the aggregation inducing properties of clinically relevant tau fragments and their potentially damaging role in the pathogenesis of human tauopathies.

biophysics↗

bioRxiv2021

Aggregation is a context-dependent constraint on protein evolution

Solubility is a requirement for many cellular processes. Loss of solubility and aggregation can lead to the partial or complete abrogation of protein function. Thus, understanding the relationship between protein evolution and aggregation is an important goal. Here, we analysed two deep mutational scanning experiments to investigate the role of protein aggregation in molecular evolution. In one data set, mutants of a protein involved in RNA biogenesis and processing, human TAR DNA binding protein 43 (TDP-43), were expressed in S. cerevisiae. In the other data set, mutants of a bacterial enzyme that controls resistance to penicillins and cephalosporins, TEM-1 beta-lactamase, were expressed in E. coli under the selective pressure of an antibiotic treatment. We found that aggregation differentiates the effects of mutations in the two different cellular contexts. Specifically, aggregation was found to be associated with increased cell fitness in the case of TDP-43 mutations, as it protects the host from aberrant interactions. By contrast, in the case of TEM-1 beta-lactamase mutations, aggregation is linked to a decreased cell fitness due to inactivation of protein function. Our study shows that aggregation is an important context-dependent constraint of molecular evolution and opens up new avenues to investigate the role of aggregation in different cellular contexts.

bioinformatics↗

bioRxiv2021

Anatomy of unfolding: The site-specific fold stability of Yfh1 measured by 2D NMR

Most techniques allow detection of protein unfolding either by following the behaviour of single reporters or as an averaged all-or-none process. We recently added 2D NMR spectroscopy to the well-established techniques able to obtain information on the process of unfolding using resonances of residues in the hydrophobic core of a protein. Here, we questioned whether an analysis of the individual stability curves from each resonance could provide additional site-specific information. We used the Yfh1 protein that has the unique feature to undergo both cold and heat denaturation at temperatures above water freezing at low ionic strength. We show that stability curves inconsistent with the average NMR curve from hydrophobic core residues mainly comprise exposed outliers that do nevertheless provide precious information. By monitoring both cold and heat denaturation of individual residues we gain knowledge on the process of cold denaturation and convincingly demonstrate that the two unfolding processes are intrinsically different.

biophysics↗

bioRxiv2021

New lessons on TDP-43 from the killifish N. furzeri

Frontotemporal dementia and amyotrophic lateral sclerosis are fatal and incurable neurodegenerative diseases linked to the pathological aggregation of the TDP-43 protein. This is an essential DNA/RNA binding protein involved in transcription regulation, pre-RNA processing and RNA transport. Having suitable animal models to study the mechanisms of TDP-43 aggregation is crucial to develop treatments against disease. We have previously demonstrated that the killifish Nothobranchius furzeri offers the unique advantage as a model system that it is an organism with compressed lifespan and a conserved ageing phenotype that develops within months, making this organism the available shortest-lived vertebrate with a clear ageing phenotype. Here, we show that the two paralogs of TDP-43 from the killifish N. furzeri share high sequence homology with the human protein and recapitulate its cellular and biophysical behaviour. We prove that, during ageing, N. furzeri TDP-43 spontaneously forms insoluble intracellular TDP-43 aggregates that have amyloid characteristics and colocalize with the stress granule core protein G3BP. Our results propose this organism as a valuable model of TDP-43-related pathologies and show that even minute differences between the human and N. furzeri proteins may help to shed new light onto the role of TDP-43 in RNA recognition and granule formation.

neuroscience↗