Biology subjects

Cramer, J.

Publications and source records attributed to Cramer, J..

8 recordsLinked to original sources

bioRxiv2026

Graph-based modeling of multiparametric MRI deciphers molecular states of high-grade glioma invasion with prognostic implications

AbstractThe infiltrative, non-enhancing margin of IDH wildtype high grade glioma (IDHwt HGG) harbors distinct molecular programs that drive invasion and therapeutic resistance, yet remains largely unevaluable by conventional tissue sampling approaches and by conventional imaging. Here we show that this invasive architecture is encoded within multiparametric MRI (mpMRI) feature relationships and can be decoded using a graph-based framework trained on multiregional image-localized biopsies. Across 134 spatially matched biopsy-imaging pairs from 35 patients with primary IDHwt HGG (29 glioblastomas (GBM) and 6 non-glioblastoma HGGs), unsupervised graph community detection identifies two imaging-defined clusters that localize to invasive tumor regions without molecular supervision. Transcriptomic profiling associates these clusters with neuronal (NEU) and glycolytic-plurimetabolic (GPM) molecular programs. Building on this framework, a graph convolutional network (GCN) accurately predicts NEU and GPM transcriptional states in independent training and validation cohorts and significantly outperforms conventional convolutional neural networks. Applied to whole-tumor mpMRI volumes, the trained GCN generates spatially resolved probability maps that quantify the distribution and relative burden of NEU and GPM programs across both MRI contrast-enhancing and non-enhancing invasive regions. These imaging-derived molecular maps stratify patients by overall survival. Increased GPM burden is associated with poorer survival, consistent with the aggressive behavior associated with mesenchymal-like transcriptional programs in IDHwt HGG. In contrast, increased NEU burden is associated with improved survival, identifying a previously unrecognized imaging-derived prognostic biomarker that was not detected by biopsy-based molecular classification alone. Together, these findings establish a graph-based imaging framework for spatially resolved molecular classification of invasive IDHwt HGG and demonstrate that whole-tumor molecular state architecture carries prognostic information beyond conventional tissue sampling.

cancer biology↗

bioRxiv2026

Dual Carbohydrate Recognition by the Chitinase-like Protein CHI3L1 Through Distinct Glycosaminoglycan and Chitin-Binding Interfaces

CHI3L1 (YKL-40) is a chitinase-like glycoprotein involved in immune regulation, tissue remodeling, and cancer, yet the molecular principles governing its glycan interactions remain incompletely defined. Previous reports suggested that CHI3L1 can bind to chitin oligosaccharides (COS) and glycosaminoglycan (GAG) ligands, however, the molecular basis and binding sites underlying these interactions remain controversial. Here, a combination of biophysical and computational methods is employed to shed light on carbohydrate interactions of the protein and delineate a potential crosstalk between its glycan-binding interfaces. Our results demonstrate that COS and GAGs bind to distinct, non-overlapping sites on CHI3L1. Both ligand classes exhibit a strong dependence of binding affinity on the degree of polymerization. Molecular dynamics simulations, supported by mutational analysis, identify a GAG-binding site centered on residues R144, R145, and K147 and reveal an additional distal interaction site for longer GAG ligands. Biophysical and biochemical assays fail to confirm a previously proposed allo- or orthosteric interaction between both binding sites. However, physiologically relevant protein-protein interactions mediated by the chitin binding site of CHI3L1 are differentially regulated by GAG and COS ligands. COS inhibit binding of galectin-3 to CHI3L1, whereas GAG ligands enhance the affinity between the proteins by ca. 14-fold. Together, these findings establish CHI3L1 as a dual carbohydrate-binding protein with distinct recognition interfaces and reveal a previously unrecognized role for GAGs in modulating CHI3L1-mediated signaling interactions.

biophysics↗

bioRxiv2026

Chitinase-3-like protein 1 decodes chitosan acetylation patterns into toll-like receptor 2 signaling through heparan sulfate

Chitinase-3-like protein 1 (CHI3L1), which is associated with a wide range of inflammatory diseases, lacks chitinase activity but retains the ability to bind chitin and chitosan. Chitin is a major component of fungal cell walls, whereas chitosan is used in biomedicine. In addition to chitosan, CHI3L1 has been proposed to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan on mammalian cell surfaces. Here, we investigated how interactions with chitosan and HS regulate the pro-inflammatory activity of CHI3L1. Mapping of the chitin-binding cleft revealed preferential binding of CHI3L1 to chitosans with a regular acetylation pattern that, together with CHI3L1, promoted toll-like receptor 2 signaling. We further identified a dominant HS-binding site that recognizes a distinct HS sulfation code containing a coherent motif of N- and 6-O-sulfations. Mutation of this HS-binding site or impaired HS biosynthesis prevented CHI3L1 accumulation at the cell surface and abolished CHI3L1-mediated cell activation. Together, our findings establish HS as a critical co-receptor for CHI3L1 and reveal a pro-inflammatory cross-talk between HS, CHI3L1, and chitosan that may contribute to host defense against fungal pathogens and responses to chitosan-based biomaterials. These findings identify HS- and chitosan-dependent CHI3L1 signaling as a potential target for modulating inflammatory responses.

Molecular Biology↗

bioRxiv2026

Therapeutic scheduling of WEE1 inhibition preserves T cell function and promotes immune control of HPV⁺ tumors

Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV OPC) is driven by viral E6 and E7 oncoproteins, which disrupt G1 checkpoint control and impose selective dependency on WEE1-mediated G2/M regulation. While this vulnerability confers sensitivity to WEE1 inhibition, its immunologic consequences remain poorly defined, and the challenge of eliciting antitumor immunity without compromising immune fitness has limited clinical translation. Here, we show that WEE1 inhibition elicits durable antitumor immunity in immunocompetent models of HPV OPC. Using murine and human preclinical systems, we demonstrate that the WEE1 inhibitor azenosertib (ZN-c3) mediates tumor control through both cell-autonomous cytotoxicity and immune-dependent mechanisms requiring T cells and conventional dendritic cells. Mechanistically, HPV tumor cells are deficient in STING signaling and fail to mount canonical type I interferon responses. Instead, tumor cell-intrinsic cGAS drives immune activation through STING-competent host cells within the tumor microenvironment, revealing a non-cell-autonomous relay that circumvents viral immune evasion. Intermittent WEE1 inhibition preserves T cell fitness while maintaining antitumor efficacy, and mice achieving complete responses develop immunologic memory capable of rejecting tumor rechallenge. These findings establish intermittent WEE1 inhibition as an immune-permissive therapeutic strategy that enables antigen-specific T cell responses in HPV-driven malignancies and provides a mechanistic rationale for combination with immunotherapy.

cancer biology↗

bioRxiv2025

Citrullination of TDP-43 is a key post-translation modification associated with structural and functional changes and progressive pathology in TDP-43 mouse models and human proteinopathies

TAR DNA-binding protein 43 (TDP-43) pathology is associated with a spectrum of clinical dementias including limbic-predominant age-related TDP-43 encephalopathy neuropathological changes (LATE-NC). Post-translational modifications (PTM) are linked to TDP-43 toxic gain-of-function and cytoplasmic aggregation1-3. Phosphorylation remains the most investigated PTM and a standard criterion for determining pathology progression and clinical subclassification in TDP-43 proteinopathies4-7. However, full spectrum of PTMs on TDP-43 structure and biology remain unknown. Utilizing mass-spectrometry analysis we identified citrullination as a novel and irreversible "bona-fide" PTM of TDP-43 protein. We recognized peptidyl arginine deiminase 2 and 4 (PAD2 and PAD4) to mediate the conversion of arginine (R) to citrulline (citR) in vitro and demonstrated increased PAD2 and PAD4 expression and TDP-43 citrullination in a human wildtype TDP-43 mouse model (Tg (Thy1-TARDBP4). Transmission electron microscopy imaging analysis revealed citrullination induced vast structural changes while ThT analysis demonstrated altered aggregation kinetics of citrullinated (citR) TDP-43 protein. We further provided mechanistic evidence on reduced electrostatic and pi-pi interactions of citR TDP-43 Low Complexity Domain (LCD) with RNA, favoring liquid-solid phase separation and condensate formation. Generation and validation of several citR TDP-43 specific antibodies against several TDP-43 epitopes revealed epitope and domain-specific effects of citrullination on TDP-43 solubility in vivo. Importantly, we found distinct reactivities of citR TDP-43 antibodies shedding light into the contribution of epitope-specific properties of human citR TDP-43 to novel pathological citR TDP-43 assemblies in human brain tissue from LATE-NC, with or without comorbid Alzheimers disease neuropathologic changes (ADNC). These findings provided a unique look into the temporal citR TDP-43 signatures, and the potential clinical relevance associated with progression of pure LATE-NC and comorbid ADNC + LATE-NC. Collectively, these data reveal the existence of irreversible TDP-43 citrullination at targeted sites via induced PAD2/PAD4 activities, presenting a critical step in TDP-43 proteinopathy.

neuroscience↗

bioRxiv2025

Computationally designed stem-epitope mimetics elicit broadly reactive antibodies

Broad protection against diverse influenza viruses can be conferred by broadly neutralizing antibodies (bnAbs) targeting a conserved site on the hemagglutinin (HA) stem domain. However, the low immunogenicity of this antigenic region hinders the robust induction of such antibodies. Here, we showcase a structure-based immunogen design strategy focusing on the surface mimicry of antigenic sites. By leveraging the structural definition of a stem epitope, we apply computational protein design to develop epitope mimetics to focus the immune response against this site of viral vulnerability. The structurally complex antigenic site is displayed on heterologous protein scaffolds, retaining excellent binding towards known HA stem-specific bnAbs. Our epitope-mimetic induces stem-specific antibodies against highly divergent group 1 and 2 subtypes. The results provide a general framework for the design of novel immunogens eliciting focused immune responses which may be a valuable tool in the development of effective vaccine candidates against other variable pathogens.

biochemistry↗

bioRxiv2020

A bottom-up approach for the de novo design of functional proteins

De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.

bioengineering↗

bioRxiv2019

Trivalent cocktail of de novo designed immunogens enables the robust induction and focusing of functional antibodies in vivo

De novo protein design has been successful in expanding the natural protein repertoire. However, most de novo proteins lack biological function, presenting a major methodological challenge. In vaccinology, the induction of precise antibody responses remains a cornerstone for next-generation vaccines. Here, we present a novel protein design algorithm, termed TopoBuilder, with which we engineered epitope-focused immunogens displaying complex structural motifs. Both in mice and non-human primates, cocktails of three de novo designed immunogens induced robust neutralizing responses against the respiratory syncytial virus. Furthermore, the immunogens refocused pre-existing antibody responses towards defined neutralization epitopes. Overall, our de novo design approach opens the possibility of targeting specific epitopes for vaccine and therapeutic antibody development, and more generally will be applicable to design de novo proteins displaying complex functional motifs.

bioengineering↗