Biology subjects

Schneider, S.

Publications and source records attributed to Schneider, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis of eIF4E recognition of synthetic mRNA 5' cap analogues links cap architecture to affinity and biological activity

Eukaryotic mRNAs carry a 5'-terminal 7-methylguanosine (m7G) cap that is recognized by multiple cellular proteins and is essential for mRNA self-discrimination, stability, and translation. The eukaryotic translation initiation factor 4E (eIF4E) binds the 5' cap and promotes recruitment of mRNAs to the ribosomal translation initiation complex, a critical step in cap-dependent translation. Beyond naturally occurring cap modifications that modulate mRNA interactions and fate, non-natural 5' cap modifications can be used to finely tune diverse aspects of mRNA biology. This has gained particular relevance with the emergence of in vitro transcribed mRNAs as a powerful therapeutic modality for protein replacement therapies and vaccines. Here, we report binding data for a series of synthetic 5' cap analogues interacting with eIF4E, together with X-ray crystal structures of eIF4E-cap complexes, providing detailed insights into the structural determinants of cap recognition by eIF4E. Furthermore, analysis of gene expression from mRNAs carrying different synthetic cap analogues in human cell lines and mice revealed a relationship between cap architecture and protein expression. Taken together, our structural, biochemical, and functional data establish structure-activity relationships for synthetic cap analogues and identify structural features associated with enhanced translational performance.

biochemistry↗

bioRxiv2026

Harmonized nucleoside mass spectrometry enables reproducible cross-platform RNA modification quantification

RNA modification analysis by LC-MS/MS is central to epitranscriptomics, yet quantitative comparison across laboratories and instrument platforms remains poorly standardized. Here, we performed a community-driven benchmarking study during the first Human RNome Project workshop to systematically evaluate cross-platform reproducibility of ribonucleoside mass spectrometry workflows. Using the same analytical column and gradient, standardized RNA samples, and shared reagents, we compared nucleoside quantification across quadrupole, time-of-flight, and orbitrap-based LC-MS platforms employing distinct acquisition strategies. While chromatographic separation was highly reproducible across systems, nucleoside-specific MS response behavior differed substantially between platforms and limited direct comparability of relative signal intensities. These response differences varied across analytes and concentration ranges, demonstrating that harmonized chromatography alone is insufficient for transferable quantitative analysis. Stable isotope-labeled internal standard (SILIS) normalization substantially reduced platform-and method-dependent response and improved agreement for most evaluated modifications. External calibration improved agreement between qTOF and Orbitrap workflows for a subset of modifications but did not fully resolve residual intersystem differences. Based on these findings, we establish benchmark-derived recommendations for harmonized relative and absolute RNA modification quantification, including guidance for calibration design, quality control, and data reporting. Together, this work provides a methodological framework for reproducible nucleoside LC-MS/MS workflows and establishes a foundation for large-scale comparative epitranscriptomic studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/739095v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@6e4e98org.highwire.dtl.DTLVardef@1ea13beorg.highwire.dtl.DTLVardef@b65858org.highwire.dtl.DTLVardef@1623337_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Prevalent glutamyl-endopeptidases in the commensal skin microbiome have itch-relevant activity

Atopic dermatitis (AD) is frequently accompanied by pruritus, which has predominantly been attributed to skin colonization by Staphylococcus aureus, particularly through cleavage of protease-activated receptor 1 (PAR1) by the glutamyl endopeptidase (GEP) V8 protease. Whether related GEPs from other skin-associated staphylococci contribute to this process remains unclear. Here, we analyzed 273 staphylococcal isolates from skin swabs of 10 AD patients with pruritus, dominated by S. aureus and Staphylococcus epidermidis. Genome mining using a custom hidden Markov model identified 678 candidate GEPs, which were clustered and resolved into five structurally distinct protease architectures. Representative proteases (V8, Esp, SplB, Csp, and Hsp) were expressed and characterized. Esp displayed GEP activity and PAR1 tethered-ligand cleavage comparable to V8, generating noncanonical cleavage products, while Csp cleaved PAR1 with reduced but substantial efficiency. All representative proteases significantly disrupted barrier integrity in an epithelial barrier model. Analysis of isolate genomes and publicly available Staphylococcus genomes showed that V8 and Esp are highly conserved and largely species-restricted, whereas Csp is more broadly distributed across species. These findings identify Esp and Csp as functional GEP virulence factors in S. epidermidis and S. capitis, capable of activating itch signaling and compromising barrier function. Our study suggests that GEP-mediated pruritus and barrier dysfunction in AD may arise not only from pathogens like S. aureus, but also from commensals or opportunistic pathogens such as S. epidermidis and S. capitis. ImportanceStaphylococcus aureus colonization on the skin is closely associated with itch in atopic dermatitis (AD) through a secreted protease that cleaves PAR1 on sensory neurons. However, AD patients can experience pruritus without S. aureus colonization. This raises the question of whether other skin-colonizing staphylococci contribute to this process. We identify glutamyl endopeptidase homologs, including Esp from Staphylococcus epidermidis and Csp from Staphylococcus capitis, that cleave the same itch receptor and disrupt epithelial barrier integrity. These serine proteases are distributed across staphylococcal species that colonize human skin. Therefore, itch and barrier dysfunction in AD may not be restricted to S. aureus but instead arise from the proteolytic activity of multiple staphylococcal species. This functional redundancy means that other staphylococcal species can sustain GEP-driven itch and barrier dysfunction even in the absence of S. aureus, suggesting that therapeutic strategies targeting only S. aureus may overlook these alternative drivers of disease.

microbiology↗

bioRxiv2026

The CD19-4-1BBL antibody fusion protein unleashes the immune system against high-risk chronic lymphocytic leukemia

CD19-4-1BBL is a bispecific antibody fusion protein that targets CD19 and costimulates 4-1BB on T cells and other immune cells. Its antitumor activity has been reported in B-cell non-Hodgkin lymphoma with emphasis on its T-cell mediated cytotoxic activity. Its effect on other 4-1BB expressing immune cells is unexplored. Here, we investigated the molecular mechanisms and the antileukemic effect of CD19-4-1BBL in chronic lymphocytic leukemia (CLL), a B-cell malignancy profoundly marked by the immunosuppressive activity of myeloid-derived suppressor cells, tumor-associated macrophages and CD4+ regulatory T cells. We demonstrated that CD19-4-1BBL simultaneously mitigates the immunosuppressive phenotype and transcriptome machinery of these cells and promotes antitumor CD8+ T-cell immunity. Finally, in a preclinical, patient-derived xenograft model of CLL, we observed a favourable survival impact, especially in mice transplanted with immune cells from patients with high-risk/progressive leukemia. Our findings provide evidence that the CD19-4-1BBL treatment is a multifaceted, immune-based strategy that should be clinically explored in patients with chronic lymphocytic leukemia. KEY POINTSO_LICD19-4-1BBL sharpens the myeloid cell transcriptome and stimulates diverse memory CD8+ T cell clonotypic responses. C_LIO_LICD19-4-1BBL costimulation can be therapeutically exploited in high-risk chronic lymphocytic leukemia. C_LI

immunology↗

bioRxiv2026

Evidence of predictive information compression in latent space in humans during speech listening

Speech perception requires transforming acoustic input into neural representations that support linguistic understanding, yet its underlying computational principles remain unclear. Classical efficient coding theories posit optimal compression of sensory input, whereas alternative accounts propose that neural systems preferentially encode information that supports prediction. A key open question is whether such predictive encoding operates on fixed inputs or on flexible internal representations. We instantiated three hypothesis models of speech processing: (i) optimal compression with deep autoencoders, (ii) predictive reconstruction with predictive autoencoders, and (iii) predictive information representation via latent-space prediction using contrastive learning. We compared resulting speech latent representations to electroencephalographic (EEG) activity during speech listening. Representations learned under the predictive information objective best explained neural latents. Crucially, only representations that selectively compressed predictive information predicted behavioral performance, suggesting that neural speech representations are structured to encode predictive information in latent space rather than to maximize compression or input prediction.

neuroscience↗

bioRxiv2026

Advanced Open-source Experimental-Design Tools for Microplate-Based Assays with Acoustic Liquid Handling

Acoustic droplet ejection (ADE) enables nanoliter-scale liquid handling for complex microplate assays, yet translating experimental designs into validated, instrument-ready instructions remains a bottleneck. We present PickliPy, an open-source framework that converts spreadsheet-based assay designs into validated ADE picklists. PickliPy.Assay supports combinatorial, dose-response, and multi-addition time-course dispensing, while PickliPy.Screen extends to high-throughput workflows, including library reformatting and shortlisting. Across biological contexts, the framework generated reproducible, assay-ready plates and standardized execution in human cohort studies. Acoustic pre-dispensing deepened bioenergetic phenotyping of human skeletal muscle mitochondria, capturing substrate switching and sharpened dose-response precision in human pancreatic {beta}-cells, revealing an age-associated change in succinate dehydrogenase kinetics. We benchmarked a wash-free, live-cell screen of mitochondrial function and morphology, in which deep-learning image analysis widened the assay window, and ADE enabled integrative dose-response co-response analysis. These tools, including their agentic use, make complex ADE experiments easier to design and scale from single benches to screening campaigns.

cell biology↗

bioRxiv2026

Loss of perinuclear theca protein ACTRT2 causes subfertility and acrosome destabilization in mice

Actin-related protein T2 (ACTRT2) localizes to the perinuclear theca (PT) of male germ cells, yet its functional significance remains unclear. ACTRT2 is evolutionarily conserved and exhibits significant sequence similarity to other testis-specific actin-related proteins, with the highest conservation observed within the canonical actin core domain. We generated Actrt2-deficient mice which displayed male subfertility with pronounced acrosomal malformations originating during the Cap phase of acrosome biogenesis. Actrt2-deficient male mice showed reduced fertilization rate and poor blastocysts quality. Co-immunoprecipitation identified ACTRT2 interactions with PT proteins ACTRT1, ACTRT3, ACTL7A, ACTL9, PFN3, SPEM2 and CCIN while the interaction with CYLC1 was not detected. ACTRT2 overexpression in HEK293T cells altered cell morphology and F-actin distribution. Further, cytoskeletal regulator CFL1 was enriched in testis from Actrt2-deficient mice. We propose that ACTRT2 is a structural component of the PT stabilizing the acroplaxome during spermiogenesis and acrosome biogenesis by modulating actin dynamics. Finally, the high degree of sequence conservation and similarity with ACTRT1 and ACTRT3 together with their similar phenotypes when deleted, indicate that ACTRT2 shares a partial functional redundancy and compensatory capacity with other Arp proteins in testis. Taken together, these findings establish ACTRT2 as a structural regulator of sperm head architecture and male fertility in mice.

molecular biology↗

bioRxiv2026

Male mice heterozygous for Protamine-1 and Protamine-2 are infertile displaying sperm damage and retention of Protamine-2 precursors, transition proteins and histones.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

developmental biology↗

bioRxiv2026

Inositol hexakisphosphate Functions as a Cofactor and Modulator of ADAR1 Activity

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn{superscript 2}-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

molecular biology↗

bioRxiv2025

Cortex-Wide Preservation of Multi-Stimulus Information across Degrees of Network Synchronization

Sensory-evoked cortical responses vary with global network dynamics, yet the link between cortical state and stimulus processing remains unclear. Here, we introduce a paradigm that jointly decodes stimulus identity and network state from wide-field calcium imaging in mice undergoing multisensory and optogenetic stimulation across isoflurane-induced transitions from compartmentalized to synchronized activity. Effective dimensionality, a summary measure of network complexity, correlated well with anesthesia depth, while non-linear contrastive learning achieved >97% stimulus decoding accuracy across all states. Individual cortical regions maintained [≥]82.5% accuracy even during deep anesthesia with prominent slow waves. Preservation of stimulus-specific information extended throughout the cortex, demonstrating that distinct representations remain decodable within synchronized networks. Direct optogenetic cortical stimulation exhibited state-invariant decoding performance, contrasting with anesthesia-dependent decline observed for sensory stimuli. Multi-stimulus cortical representations remain decodable across varying levels of network synchronization, with implications for brain-machine interfaces and clinical tools that assess preserved cortical responsiveness under variable arousal conditions.

neuroscience↗

bioRxiv2025

ALKBH1 activity in vitro and human cell lines by isotope dilution mass spectrometry

AlkB homolog 1 (ALKBH1) is a member of the AlkB family of Fe(II) and -ketoglutarate (-KG)-dependent dioxygenases, known for its enzymatic activity on various nucleic acid substrates. Its known targets include N1-methyladenosine (m1A), N6-methyladenosine (m6A), N3-methylcytidine (m3C), and 5-methylcytosine (m5C) in RNA, as well as N6-methyladenine (N6-mA, 6mA) in DNA and the histone protein H2A. Moreover, dysregulation or dysfunction of ALKBH1 has been implicated in a broad spectrum of human diseases. In order to shed further light on the substrate scope and role of ALKBH1, we used quantitative mass spectrometry to assess its activity in vitro and in human cell lines. To study ALKBH1 activity on defined substrates in vitro, we enzymatically generated tRNAs specifically carrying the m3C32 (tRNA-ThrUGU and tRNA-SerUCN) and i6A37 (tRNA-SerUCN) modifications. Here we show that ALKBH1 reduces m3C, m1A and m5C in vitro in total extracts of tRNAs, but does not impact on rRNA modifications in human cell lines. However, upon overexpression or siRNA-mediated knock-down of alkbh1 in human cell lines no impact on the modification of total tRNA extracts or specifically enriched RNAs could be observed. In addition, varying the glucose and fetal bovine serum (FBS) concentration in the growth medium of HEK293T cells, in combination with alkbh1 siRNA-mediated knock-down, also shows no impact on the tRNA modification spectra. Based on our data, we conclude that in human cells lines grown under optimal conditions ALKBH1 does not play an important role in the demethylation of tRNAs.

biochemistry↗

bioRxiv2025

AdiY Acts as a Cytoplasmic pH-Sensor via Histidine Protonation to Regulate Acid Stress Adaptation in Escherichia coli

The arginine-dependent acid resistance (Adi) system is a vital component that enables Escherichia coli and other enterobacteria to withstand the extreme acidity in the human gastrointestinal tract. It consists of the proton-consuming decarboxylation of arginine, catalyzed by AdiA, and the uptake of arginine, as well as the excretion of the more alkaline agmatine, catalyzed by the antiporter AdiC. The corresponding genes adiA and adiC are induced in E. coli under acidic conditions (pH < 5.5), a process that is tightly regulated by the AraC/XylS transcriptional activator AdiY. Here, we show that the pH-sensing mechanism of AdiY functions through the protonation of two histidines (His34 and His60) in the N-terminal domain. Replacing these histidine residues with alanine, glutamine or aspartate abolishes the pH-dependent activation of AdiY, both in vivo, as demonstrated by promoter-reporter assays, and in vitro, as indicated by the loss of DNA-binding activity detected by surface plasmon resonance spectroscopy. Biochemical analyses of purified wild-type AdiY using size-exclusion chromatography and intrinsic tryptophan fluorescence revealed a pronounced and reversible pH-dependent conformational change that does not occur in the pH-sensing-deficient AdiY variant. A model is proposed in which AdiY forms a monomer at physiological pH. At a lower intracellular pH, the protonation of histidine in AdiY causes a conformational change that leads to the binding of AdiY as a tetramer to the DNA. This work elucidates the molecular mechanism of a one-component signal transduction system that combines both sensory and responsive functions. ImportanceThroughout their life, Escherichia coli and other bacteria may encounter acidic environments, for example, when passing through the human stomach. Their chances of survival under these conditions depend on the number and efficiency of acid resistance systems. Although many acid resistance mechanisms have been extensively studied, the molecular mechanism by which bacteria sense low pH is not yet fully understood. This study demonstrates that the transcription factor AdiY acts as a direct pH sensor by using two histidines to detect intracellular acidification in E. coli. When these histidines become protonated, AdiY changes its conformation and activates genes that support cell survival under acid stress. These findings not only reveal a new way in which bacteria can perceive extremely low pH environments but also provide the basis for the development of AdiY as a pH reporter.

microbiology↗

bioRxiv2025

A platform of robust patient-derived leukemia models covering subgroups for which no cell lines exist

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering (*PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, *PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2-mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our *PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/677299v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ead31eorg.highwire.dtl.DTLVardef@1ca702corg.highwire.dtl.DTLVardef@18a7137org.highwire.dtl.DTLVardef@edbe6_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIWe present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. C_LIO_LI*PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. C_LIO_LIOne-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block. C_LI

cancer biology↗

bioRxiv2025

A live tumor fragment platform to assess immunotherapy response in core needle biopsies while addressing challenges of tumor heterogeneity

BackgroundImmune checkpoint inhibitors (ICIs) have revolutionized cancer treatment, providing durable and even curative responses. However, most patients do not respond and current biomarkers (eg, programmed death ligand (PD-L1), mismatch repair deficiency (dMMR)/high microsatellite instability (MSI) and tumor mutational burden) lack predictive accuracy. Ex vivo profiling of patient-derived tumor fragments shows promise as a predictive biomarker but relies on substantial surgical tissue to mitigate intra-specimen heterogeneity. Innovations are needed that address these challenges, particularly where limited tissue is available in core needle biopsies (CNBs). MethodsLive tumor fragments (LTFs) were generated from 59 human tumor resections and 31 CNBs from patients enrolled in observational clinical trials (ClinicalTrials.gov identifiers: NCT05478538, NCT05520099, NCT06349642) to assess cytokine induction following ICI treatment. LTFs were encapsulated in hydrogel and cultured ex vivo for up to 72 hours. A sequential treatment strategy that applies control and treatment within the same well was used with response to ICI or CD3/CD28 assessed using a multiplex secretome assay. Viability was assessed using established metabolic assays and dynamic optical coherence microscopy. ResultsLTFs maintained viability and retained T cells responsive to stimulation throughout ex vivo culture. Multiplex immunofluorescence and immunohistochemistry showed key components of the tumor microenvironment, including relative proportions of CD4+ and CD8+ immune cell populations, were preserved. Specimens positive for PD-L1 or dMMR/MSI-high were enriched for cytokine upregulation, including T-cell response cytokines IFN{gamma} and CXCL10, after PD-1 treatment. To demonstrate clinical applicability of the sequential treatment strategy, CNBs from patients with lung, gastrointestinal or kidney cancer were profiled and differential cytokine induction in response to ICI treatment was observed. ConclusionsThe novel ex vivo platform presented is capable of detecting T-cell response to ICI treatment by using a sequential treatment strategy. This approach addresses challenges associated with cross-well heterogeneity in tissue composition and requires half as much tissue as a cross-well comparison, mitigating tissue limitations typically associated with non-surgical biopsies. Importantly, the platform is compatible with established functional assays as well as non-destructive spatial imaging, enabling researchers to characterize response to ICI longitudinally. Ongoing trials will enable clinicians to assess platform performance in predicting response to immunotherapy.

cancer biology↗

bioRxiv2025

Influence of ULK1 activity on the memory of ATG101 auto-activation dynamics

The initiation of autophagy is marked by the prompt convergence of initiation proteins to form super molecular complexes in spatially-defined autophagic hubs. The recruitment and activation of the ULK-kinase complex is a pivotal event in starting the initiation cascade. ULK complex-component ATG101 is required for initiating autophagy in mammalian cells, but its function is unknown. Its HORMA domain interacts with the HORMA domain of ATG13 and transmembrane protein ATG9A to collectively form the essential autophagy initiation complex ATG9A-ATG13-ATG101. ATG101 has structurally malleable elements, reminiscent of topological conversions used in related metamorphic HORMA domain proteins to control the rate of complex formation. To elucidate the function of ATG101, we monitored the interaction kinetics of ATG101 with ATG13 and ATG9A, and observed that the interaction of ATG101 with ATG9A and ATG13 is exceptionally slow. Dramatic acceleration of complex formation is observed upon a change in the fold of ATG101 induced by its transient homo-dimerization, which in turn is initiated by phosphorylation by the ULK1 kinase. In an auto-catalytic mechanism, ATG101 dimers create a positive feedback to propagate activation to further ATG101 molecules in the absence of ULK1 activity. Despite the competitive nature of the interaction interfaces, homo-dimerization of ATG101 surprisingly accelerates its association to ATG13. Memory of ATG101 activation persists for many hours after dephosphorylation and continues to accelerate the assembly of the ATG9A-ATG13-ATG101 complex. Overall, this work proposes an unusual regulatory mechanism where UKL1 initiates an ATG101 auto activation cascade, whose memory creates a responsive positive feedback that dictates the assembly rate of a key complex in autophagy initiation.

biochemistry↗

bioRxiv2025

Spectral disruption of inter-hemispheric resting-state BOLD coherence at vasomotor frequency (~0.1 Hz) links vascular dysfunction to functional connectivity loss in carotid artery stenosis

In the brain, vasomotor dynamics at infra-slow frequencies ([~]0.1 Hz), driven by synchronized oscillations of smooth muscle cells in vessel walls, are thought to play a crucial role in regulating cerebral perfusion and underlie resting-state functional connectivity (FC), typically measured by correlated time courses of functional signals. In particular, rodent studies have demonstrated that vasomotor activity contributes to the coherence of blood oxygenation level dependent (BOLD) signal fluctuations. However, in humans, detecting this contribution non-invasively remains challenging due to the limited spatiotemporal sensitivity of functional magnetic resonance imaging (fMRI) to vasomotion. Given that prior studies have identified internal carotid artery stenosis (ICAS) as an informative conditional lesion model of vasomotor and hemodynamic impairments in humans, we investigated whether ICAS affects interhemispheric BOLD coherence at [~]0.1 Hz. Using a multi-modal fMRI framework integrating resting-state fMRI with quantitative mapping of cerebral blood volume, blood flow, oxygen metabolism, and BOLD time lag, we compared BOLD coherence between patients with asymptomatic unilateral ICAS and healthy controls. Frequency-specific analysis revealed significantly diminished interhemispheric BOLD coherence at [~]0.1 Hz across canonical resting-state networks in ICAS patients, while ultra-slow (<0.05 Hz) coherence remained largely preserved. This reduction was spatially widespread and particularly pronounced in watershed areas, i.e., border zones between major vascular territories, associated with significantly increased lateralization of cerebral blood volume (p < 0.01). Notably, coherence-based FC patterns at [~]0.1 Hz were heterogeneous within watershed areas but homogeneous outside, suggesting an interplay between compensatory mechanisms and cerebrovascular impairment. Taken together, our findings demonstrate that ICAS induces subtle, frequency-and region-specific alterations in interhemispheric FC, consistent with a model in which impaired vasomotor activity impacts on resting-state FC in the human brain.

neuroscience↗

bioRxiv2025

Perturbed cyclic strain results in a mechanically-induced, premature senescent phenotype in cardiac fibroblasts

The cardiovascular system operates under continuous cyclic mechanical stretch, and changes in mechanics and biochemical responses observed in cardiovascular disorders activate the cardiac fibroblasts (CFs) and increase cellular senescence in the tissue. However, it is unclear if cellular senescence is initiated from biomechanical stimulus alone. Here, we subjected murine CFs to uniaxial stretch and perturbed the mechanical stimulation to examine the impact on induction of a senescent phenotype. Loss of stretch magnitude and increase in frequency, mimicking an injurious hypertrophic or fibrotic response, led to a senescence-like phenotype, including cell cycle, lamin B expression, and DNA damage. Mechanical induction of CF senescence relied on p53/p21, whereas CF senescence induction triggered by reactive oxygen species or involving a mutation in lamin A/C gene occurred through p16. Moreover, mechanical induction of premature senescence was associated with decreases in the nuclear envelope protein emerin. These results demonstrate perturbed mechanical stimulation can initiate a senescent state and altered nuclear integrity may initiate this phenotype. TeaserPerturbed mechanics initiates a premature senescent-like phenotype in cardiac fibroblasts.

biophysics↗

bioRxiv2025

Dietary manipulation of intestinal microbes prolongs survival in a mouse model of Hirschsprung disease

Enterocolitis is a common and potentially deadly manifestation of Hirschsprung disease (HSCR) but disease mechanisms remain poorly defined. Unexpectedly, we discovered that diet can dramatically affect the lifespan of a HSCR mouse model (Piebald lethal, sl/sl) where affected animals die from HAEC complications. In the sl/sl model, diet alters gut microbes and metabolites, leading to changes in colon epithelial gene expression and epithelial oxygen levels known to influence colitis severity. Our findings demonstrate unrecognized similarity between HAEC and other types of colitis and suggest dietary manipulation could be a valuable therapeutic strategy for people with HSCR. AbstractHirschsprung disease (HSCR) is a birth defect where enteric nervous system (ENS) is absent from distal bowel. Bowel lacking ENS fails to relax, causing partial obstruction. Affected children often have "Hirschsprung disease associated enterocolitis" (HAEC), which predisposes to sepsis. We discovered survival of Piebald lethal (sl/sl) mice, a well-established HSCR model with HAEC, is markedly altered by two distinct standard chow diets. A "Protective" diet increased fecal butyrate/isobutyrate and enhanced production of gut epithelial antimicrobial peptides in proximal colon. In contrast, "Detrimental" diet-fed sl/sl had abnormal appearing distal colon epithelium mitochondria, reduced epithelial mRNA involved in oxidative phosphorylation, and elevated epithelial oxygen that fostered growth of inflammation-associated Enterobacteriaceae. Accordingly, selective depletion of Enterobacteriaceae with sodium tungstate prolonged sl/sl survival. Our results provide the first strong evidence that diet modifies survival in a HSCR mouse model, without altering length of distal colon lacking ENS. HighlightsO_LITwo different standard mouse diets alter survival in the Piebald lethal (sl/sl) mouse model of Hirschsprung disease, without impacting extent of distal colon aganglionosis (the region lacking ENS). C_LIO_LIPiebald lethal mice fed the "Detrimental" diet had many changes in colon epithelial transcriptome including decreased mRNA for antimicrobial peptides and genes involved in oxidative phosphorylation. Detrimental diet fed sl/sl also had aberrant-appearing mitochondria in distal colon epithelium, with elevated epithelial oxygen that drives lethal Enterobacteriaceae overgrowth via aerobic respiration. C_LIO_LIElimination of Enterobacteriaceae with antibiotics or sodium tungstate improves survival of Piebald lethal fed the "Detrimental diet". C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/637436v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@d95251org.highwire.dtl.DTLVardef@1ab58caorg.highwire.dtl.DTLVardef@5260b0org.highwire.dtl.DTLVardef@49ce42_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗