Biology subjects

Hansen, K.

Publications and source records attributed to Hansen, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular matrix context shapes morphogenesis and lactation-associated states in human milk-derived mammary organoids

The mammary gland relies on reciprocal interactions between epithelial cells and their surrounding extracellular matrix (ECM) to form and maintain milk-producing tissue structures. Yet these processes remain difficult to study in human model systems. Mammary epithelial cells (MECs) can be isolated noninvasively from breast milk, but whether they generate three-dimensional organoids and respond to matrix cues has been unclear. Here, human milk-derived MECs (milk MECs) spontaneously form complex organoids, including polarized acinar and terminal duct lobular unit-like structures after isolation. To investigate how matrix composition shapes these organoids, milk MECs were cultured in decellularized mammary ECM (dECM), Matrigel, and collagen I. In dECM, milk MECs formed polarized branched networks with aligned actin organization along collagen fibrils, whereas in Matrigel they adopted a more lactation-associated state, marked by {beta}-casein expression and milk fat globules. Together, these findings establish breast milk-derived MEC organoids as a human model to study how ECM context regulates mammary morphogenesis and lactation biology.

cell biology↗

bioRxiv2026

A CAMKK2-UBR4-19S Proteasome Axis Regulates Chondrocyte Proteostasis and SOX9 Stability

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

cell biology↗

bioRxiv2026

Cohesin bridging as a physical principle of enhancer-promoter communication

Central to genome function, enhancers are non-coding sequences that can control transcription from promoters hundreds of kilobases away. Yet the physical basis of this long-range communication remains unclear. A prevalent view is that enhancers activate promoters when the two elements come into spatial proximity through the 3D folding of chromatin. However, activation by spatial proximity alone has struggled to explain several core features of enhancer function. Here, we propose that the molecular motor cohesin transmits long-range enhancer action by forming bridges between enhancers and promoters during loop extrusion. In this view, rare and transient bridges carry regulatory communication, rather than mere spatial proximity. We develop a quantitative model that predicts transcriptional output from cohesin-bridging dynamics and validate it by engineering cells in which strategically positioned CTCF sites rewire loop extrusion trajectories. The model explains how enhancer action scales with genomic distance, and how it can be either facilitated or insulated by CTCF sites across two orders of magnitude-behaviors incompatible with proximity-based models. Finally, our framework reveals that CTCF sites can block enhancers bidirectionally, by either blocking or releasing cohesin loops, resolving longstanding paradoxes between their effects on transcriptional regulation and genome folding. Together, our results establish cohesin bridging as a mode of enhancer-promoter communication that can be modulated by genomic context to achieve selective and tunable transcriptional control over long genomic distances.

molecular biology↗

bioRxiv2026

A population-scale red blood cell proteome reveals genetically encoded aging clocks predictive of hemolysis and blood donor activity

As the most abundant human cell and the foundation of transfusion medicine, red blood cells (RBCs) offer a unique readout of systemic health, yet they have never been characterized at population scale. We generated a proteome atlas of 13,091 blood donors with multi-omics longitudinal phenotyping, characterizing the influence of demographics and genetic variation on the reproducibility of RBC proteomes across donations. Elastic-net aging clocks captured biological aging with high accuracy and uncovered genetic regulators of {Delta}Age at FN1, C4/IKZF1, CRAT, PFAS, TRIM58. Across independent cohorts, {Delta}Age was accelerated in G6PD deficiency, sickle cell trait/disease, and iron deficiency, reversed by iron repletion, and slowed in high-frequency donors, linking molecular aging to brain iron/myelin and cognitive performance. Molecular aging signatures predicted storage, osmotic, and oxidative hemolysis, hemoglobin increments after transfusion, and long-term donor activity over 12-years. These results establish RBC proteomics as a scalable biomarker of aging, donor healthspan, and transfusion outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/710284v1_ufig1.gif" ALT="Figure 1"> View larger version (96K): org.highwire.dtl.DTLVardef@15bd46eorg.highwire.dtl.DTLVardef@1d7c107org.highwire.dtl.DTLVardef@1c1d870org.highwire.dtl.DTLVardef@168dc6f_HPS_FORMAT_FIGEXP M_FIG Dzieciatkowska et al. generate the first population-scale atlas of the RBC proteome across 13,000 donors and develop proteomic and metabolomic aging clocks that quantify biological age. Molecular {Delta}Age is reproducible across donations, genetically encoded and accelerated in G6PD deficiency, sickle cell trait/disease, and iron deficiency - yet reset by iron repletion, tracking with cognitive function and brain iron/myelin. RBC aging clocks predict hemolytic fragility, transfusion efficacy, and donor activity 12 years later. C_FIG HighlightsO_LIRBC proteome atlas of 13,091 donors reveals demographic and genetic programs C_LIO_LIGenetically encoded RBC aging clocks identify regulators of molecular {Delta}age C_LIO_LIMolecular aging features predict hemolysis and transfusion response across cohorts C_LIO_LIRBC molecular age forecasts long-term donor activity over a 12-year follow-up C_LI

biochemistry↗

bioRxiv2025

Closed-loop acoustic stimulation modified by cathodal tDCS is beneficial for retention

We investigated how combining transcranial direct current stimulation (tDCS) with closed-loop acoustic stimulation (CLAS) during slow-wave sleep (SWS) affects memory consolidation and sleep-related neural oscillations. Cathodal tDCS was used to slightly reduce cortical excitability, thereby simulating a shifted brain state during CLAS and allowing us to compare the effects of standard CLAS to CLAS delivered under altered cortical conditions (CmodCLAS). Twenty-three participants (mean age 21.3 {+/-} 2.6 years) completed two experimental nights: one with CLAS alone and one with CLAS combined with cathodal tDCS (CmodCLAS). Overnight retention on declarative memory tasks and morning learning performance were assessed. Memory outcomes revealed that CmodCLAS, but not CLAS alone, significantly improved overnight retention on the figural paired-associate task. In the EEG, CmodCLAS shifted the time-locked response to acoustic stimulation toward more negative potential values at the frontal region. CmodCLAS also prolonged slow oscillation (SO) duration at frontal sites while shortening SO duration at occipital sites, an effect not observed during standard CLAS. These findings demonstrate that the baseline level of cortical excitability during sleep modulates both the cognitive and electrophysiological effects of CLAS. They highlight the importance of brain state for non-invasive CLAS during sleep and suggest that CmodCLAS may serve as a useful approach for enhancing prefrontal cortical function during SWS. Significance statementNon-invasive brain stimulation (NIBS) is a promising approach to modulate brain activity and function. Unfortunately, NIBS studies are challenged by heterogenous findings. In addition to protocol differences, variability may depend strongly on the brains baseline activity (or brain state). Here, we used transcranial direct current stimulation (tDCS) to simulate a different brain state while applying closed-loop acoustic stimulation (CLAS) during slow-wave sleep (SWS). Sleep has been shown to enhance overnight memory retention. Results demonstrated significantly improved retention performance for cathodal tDCS modulated CLAS (CmodCLAS) as compared to CLAS alone. Furthermore, electrophysiological responses time-locked to the acoustic stimulus and spontaneous electrophysiological rhythms of SWS suggest that hippocampo-cortical function was specifically enhanced by CmodCLAS. Thus, underscoring the potential therapeutic relevance of CmodCLAS.

neuroscience↗

bioRxiv2025

Genetic variation of human G6PD impacts Red Blood Cell transfusion efficacy

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common human enzymopathy, affects 6% of the global population, yet its impact on blood storage and transfusion efficacy remains undefined. We integrated genome-metabolome-proteome analyses of 13,091 blood donors (362 G6PD SNPs), validated in a recalled cohort (n=643), linked donor-recipient databases, humanized mouse models (canonical, African A- [V68M+N126D], Mediterranean [S188F]), and a prospective sickle cell disease study. Common G6PD variants reduced protein abundance, reprogrammed redox metabolism, and increased storage hemolysis. In mice, G6PD-deficient RBCs showed lower post-transfusion recovery, higher oxidative stress, and impaired renal oxygenation. Clinically, recipients of G6PD-deficient units exhibited smaller hemoglobin increments and reduced RBC L{superscript 1}Cr-survival (-8% at 24 h; -12% at 4 weeks). Structural studies revealed kinetic fragility for A- and thermodynamic fragility for Med-, linking genotype to protein instability and transfusion outcome. These findings identify donor G6PD genotype as a determinant of transfusion efficacy, supporting genotype-aware inventory-management strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/689741v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@1897489org.highwire.dtl.DTLVardef@1420587org.highwire.dtl.DTLVardef@178e8ddorg.highwire.dtl.DTLVardef@1003b44_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Development and validation of an ultra-low-cost, open source normothermic ex vivo organ perfusion platform

BackgroundNormothermic ex vivo organ perfusion (NEVOP) promises to catalyze organ preservation, therapeutic discovery, and organ-specific disease modeling. Existing technology platforms remain inaccessible for research due to restricted access to commercial organ perfusion devices, high costs of both devices and proprietary consumables, and steep technical learning curves. Additionally, the available technology is not optimized to perfuse smaller organs such as the kidney. MethodsTo overcome these barriers, a custom NEVOP circuit was developed using recycled, repurposed, and low-cost components. Porcine kidneys and autologous blood were used to iteratively optimize circuit design. A porcine kidney autotransplantation protocol was adapted to evaluate in vivo kidney function after ex vivo perfusion. To pilot the flexibility of this system as a multi-organ platform for ex vivo human biology, non-transplantable human donor kidney, spleen, and pancreas specimens were stably perfused using human blood products and analyzed. ResultsAn ultra low-cost NEVOP system engineered to perfuse porcine kidneys and diverse human organs (kidney, pancreas, and spleen) sustained viable organs for up to 24 hours with evidence of both function and viability. Key innovations included a parallel flow resistor to facilitate low-flow perfusion in non-heparinized organs and a containment bag with adjustable magnets to provide vascular stability and recycling of venous overflow. The circuit costs less than 1,500USD to construct, and porcine kidneys perfused for 24 hours on this platform demonstrated healthy in vivo function upon autotransplantation. ConclusionsCustom NEVOP platforms constitute novel and potentially transformative research platforms which use low-cost and readily available materials. Paired with access to non-transplantable research organs from altruistic donors, this model provides a road map for investigators to advance biomedical discovery and human ex vivo biology.

bioengineering↗

bioRxiv2025

Cardiomyocyte-expressed TGFβ signals to fibroblasts to program early heart maturation and adult myocyte identity

Transforming growth factor {beta} (TGF{beta}) is a secreted growth factor that is sequestered to the extracellular matrix (ECM) as a latent complex. In adult disease TGF{beta} release in the heart transforms fibroblasts into a differentiated state that synthesizes more ECM. However, it is not known how TGF{beta} functions in the early developing heart to impact resident fibroblasts. Here, we observe that deletion of the Tgfb1, Tgfb2, and Tgfb3 genes (TGF{beta} ligands) from cardiomyocytes in the early developing heart results in cardiac dysfunction by 6 weeks of age with altered fibroblast activity and altered ECM content. Early postnatal hearts from Tgfb1/2/3 cardiomyocyte-deleted mice are dysmorphic and cardiac fibroblasts have incorrect activity and produce inappropriate ECM with reduced stiffness. Gene expression profiling of hearts from myocyte-specific Tgfb1/2/3 deleted mice reveal defects in both cardiomyocyte and fibroblast maturation with ectopic expression of multiple skeletal muscle-specific genes beginning at embryonic day 17.5 and progressing with age. However, cardiomyocyte-specific deletion of TGF{beta} receptors I/II encoding genes (Tgfbr1/2) or Smad2/3 encoding genes (Smad2/3) do not recapitulate this phenotype suggesting that TGF{beta} directly programs early heart fibroblast development that in turn specifies cardiomyocyte maturation. Importantly, Col1a2-/-;Col6a2-/- mice with defective cardiac ECM stiffness, mice lacking cardiomyocyte Itgb1 with reduced ECM load sensing, and Tcf21-/- embryos at E17.5 lacking cardiac fibroblasts each fail to generate the same pathologic ECM program with ectopic cardiomyocyte differentiation observed with Tgfb1/2/3 myocyte-specific deletion. These and additional results indicate that TGF{beta} generated by cardiomyocytes in the embryonic heart mediates fibroblast differentiation that co-evolves the ECM environment that in turn programs cardiomyocyte maturation to establish their identity.

developmental biology↗

bioRxiv2025

The ribosome-associated complex regulates cytosolic translation upon mitoprotein-induced stress

The biogenesis of mitochondria relies on the import of newly synthesized precursor proteins from the cytosol. Tom70 is a mitochondrial surface receptor which recognizes precursors and serves as an interface between mitochondrial protein import and the cytosolic proteostasis network. Mitochondrial import defects trigger a complex stress response, in which compromised protein synthesis rates are a characteristic element. The molecular interplay that connects mitochondrial (dys)function to cytosolic translation rates in yeast cells is however poorly understood. Here, we show that the deletion of the two Tom70 paralogs of yeast (TOM70 and TOM71) leads to defects in mitochondrial biogenesis and slow cell growth. Surprisingly, upon heat stress, the deletion of ZUO1, a chaperone of the ribosome-associated complex (RAC), largely prevented the slow growth and the reduced translation rates in the tom70{Delta}/tom71{Delta} double deletion mutant. In contrast, the mitochondrial defects were not cured but even enhanced by ZUO1 deletion. Our study shows that Zuo1 is a critical component in the signaling pathway that mutes protein synthesis upon mitochondrial dysfunction. We propose a novel paradigm according to which RAC serves as a stress-controlled regulatory element of the cytosolic translation machinery.

biochemistry↗

bioRxiv2025

Activation of PP2A-B56α leads to aberrant EGFR signaling and proliferative phenotypes in PDAC

Pancreatic ductal adenocarcinoma (PDAC) stands to become the second most deadly cancer by 2030. The small GTPase, KRAS, is mutated in over 90% of PDAC patients and considered the primary driver mutation. Despite being an almost ubiquitous event, KRAS mutations have been difficult to target therapeutically, particularly KRASG12D, the most common mutation in PDAC. In addition to these pharmacological challenges, KRAS mutations have been shown to drive signaling plasticity and therapeutic resistance through phosphorylation cascades in most cancers. Protein phosphatases are master regulators of kinase signaling, however the contribution of phosphatase deregulation to mutant KRAS cancer phenotypes is poorly understood. Protein phosphatase 2A (PP2A) inhibits effectors downstream of KRAS, placing this family of enzymes as key regulators of PDAC oncogenic signaling. However, our previous studies utilizing small molecule activating compounds of PP2A show a heterogeneous response in PDAC, with some cell lines displaying increased oncogenic signaling despite induction of phosphatase activity. Similarly, specific PP2A subunits exhibit both tumor suppressive and oncogenic functions depending on the cellular context. Therefore, understanding the role of PP2A in regulating cancer phenotypes is critical for the future development of therapeutic strategies that leverage this phosphatase. Here, we determined the impact of the specific PP2A subunit, B56, on PDAC phenotypes using both genetic and pharmacological activation strategies in human PDAC cell lines and genetic mouse models. We demonstrate that while PP2A-B56 suppresses specific oncogenic pathways, B56 activation exacerbates PDAC proliferative phenotypes and decreases overall survival in vivo, potentially through increased epidermal growth factor receptor (EGFR) signaling. EGFR is a critical signaling node in PDAC as inhibition or loss of EGFR prevents KRAS-driven tumorigenesis and increased EGFR activity is associated with poor patient outcome. The activation of EGFR by PP2A-B56 is in part mediated through increased expression and processing of EGFR ligands, specifically amphiregulin, heparin-binding EGF-like growth factor (HB-EGF), and epiregulin. Furthermore, pharmacological PP2A activation in combination with EGFR inhibitors mitigates this signaling and increases cell death. Together, these studies implicate a previously undescribed non-canonical role for PP2A-B56 in EGFR signaling that contributes to PDAC progression.

cancer biology↗

bioRxiv2025

PP2A activation drives aberrant macropinocytosis and cell death in pancreatic ductal adenocarcinoma

Pancreatic cancer is highly aggressive with a five-year survival rate of just 13%. Metabolic rewiring in response to oncogenic signals plays a critical role in pancreatic ductal adenocarcinoma (PDAC) survival, tumor growth, and metastasis. These alterations make PDAC tumors dependent on anabolic metabolism for survival, highlighting a unique vulnerability that can be therapeutically exploited. However, during nutrient deprivation, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids in a process called, macropinocytosis. This process can be induced downstream of oncogenic KRAS expression, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, emphasizing the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process remain poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that regulates a wide variety of cell signaling pathways, including KRAS, and is commonly dysregulated in human PDAC tumors. Here, we show that acute PP2A activation prevents macropinosome processing leading to cell death. Furthermore, we demonstrate that PP2A posttranslationally regulates the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determine that PP2A activating compounds can function synergistically with metabolic inhibitors, supporting a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging.

cancer biology↗

bioRxiv2025

Suppression of PP2A-B56α Drives EMT in EGFR Mutant Non-Small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related deaths in the United States and [~]50% of these patients present with metastatic disease at diagnosis. Epithelial-to-Mesenchymal Transition (EMT) is an important initiating step in the metastatic cascade that allows cells to acquire the migratory and invasive phenotypes necessary for dissemination. The transcriptional reprogramming that takes place during EMT has been well described in multiple cancer types; however, the posttranslational regulatory mechanisms that govern EMT are poorly understood. Protein Phosphatase 2A (PP2A) is serine/threonine (ser/thr) phosphatase that accounts for 50% of cellular ser/thr phosphatase activity and is critically important in regulating signaling homeostasis. PP2A dysregulation has been implicated in cell state regulation, EMT, and metastasis, but the roles of individual PP2A complexes are poorly understood. Our data indicate that suppression of the specific PP2A complex, PP2A-B56, results in decreased expression of epithelial markers and increased expression of mesenchymal markers consistent with EMT. These molecular changes are associated with migratory and invasive phenotypes both in vitro and in vivo. Furthermore, these migratory phenotypes can be rescued with B56 overexpression. Together, these findings implicate B56 as a key regulator of cellular plasticity and highlight the dynamic nature by which PP2A-B56 posttranslationally regulates NSCLC EMT.

cancer biology↗

bioRxiv2025

A stress-NRF2 response axis polarises tumor macrophages and undermines immunotherapy

Tumor-associated macrophages (TAMs) can switch between immune-activating and cancer-promoting states; yet, the stress pathways that lock them into pro-cancerous states remain obscure. In MC38 colon tumors, repeated anti-CD40 or radiotherapy created necrosis that split TAMs into peripheral Cxcl9+ and peri-necrotic Spp1+ subsets. Spatial transcriptomics, single-cell RNA-seq, and Keap1-deficient mice showed that the latter are NRF2high "stress-TAMs", with immunosuppressive and tumor-promoting activity. The same NRF2 activation gradient separates pro-inflammatory CXCL9+ and anti-inflammatory SPP1+ TAMs across diverse human cancers. NRF2high TAMs silence IFN-STAT1 programmes, lose MHC-II and chemokine expression, fail to expand T cells, drive tumor cell invasion in 3D co-cultures, and foster metastasis. Constitutive hematopoietic NRF2 activation accelerated the growth of therapy-naive MMTV-PyMT breast tumors and markedly impaired anti-CD40 efficacy in MC38 subcutaneous and lung-metastasis models. Conversely, macrophage-specific Nrf2 deletion restored immunogenic TAMs and potentiated anti-CD40 and anti-PD-1 treatments. Thus, NRF2 constitutes a stress-response axis that fixes TAMs in a pro-cancer, therapy-resistant state; inhibiting it could revive the macrophage-T-cell amplification loop and broaden immunotherapy responses. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/662726v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@728b03org.highwire.dtl.DTLVardef@4a473org.highwire.dtl.DTLVardef@c8a209org.highwire.dtl.DTLVardef@60681_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Caffeine Impairs Red Blood Cell Storage Quality by Dual Inhibition of ADORA2b Signaling and G6PD Activity

Caffeine is the most widely consumed psychoactive substance globally, yet its peripheral physiological effects remain incompletely understood. Leveraging comprehensive data from 13,091 blood donors in the REDS RBC-Omics study, we identify caffeine as a significant modulator of red blood cell (RBC) storage quality and transfusion outcomes. Elevated caffeine levels were reproducible across multiple donations from 643 recalled donors, selected based on their extremes in hemolytic propensity. Both in the screening and recalled cohorts, higher caffeine levels were associated with disrupted RBC metabolism, characterized by reduced glycolysis, depletion of adenylate pools or 2,3-bisphosphoglycerate, and increased markers of oxidative stress and osmotic fragility, including kynurenine accumulation. These observations were recapitulated in plasma and RBCs of eight volunteers upon consumption of a cup of coffee independently of brewing method (Chemex vs espresso). Clinically, elevated caffeine correlated with increased hemolysis and lower post-transfusion hemoglobin increments, especially pronounced in recipients transfused with RBCs from donors carrying common polymorphisms in the ADORA2b gene, a key regulator of RBC metabolism in hypoxia. These human findings were mechanistically validated using a murine model deficient in ADORA2b, which demonstrated impaired glycolytic flux, compromised antioxidant defenses - including caffeine-dependent direct inhibition of recombinantly-expressed glucose 6-phosphate dehydrogenase, and decreased transfusion efficacy (lower hemoglobin increments, higher bilirubin post-transfusion), effects further exacerbated by caffeine exposure during storage. Our study positions caffeine consumption as a modifiable factor in blood transfusion practice, advocating for precision strategies that integrate genetic and exposome factors, and identifies metabolic interventions to enhance blood quality and clinical outcomes. One sentence summaryCaffeine consumption and genetic variants in the ADORA2b receptor synergistically impair red blood cell metabolism and transfusion efficacy, revealing a modifiable exposome-gene interaction for precision transfusion medicine.

biochemistry↗

bioRxiv2025

Genetic architecture of the red blood cell proteome in genetically diverse mice reveals central role of hemoglobin beta cysteine redox status in maintaining circulating glutathione pools

Red blood cells (RBCs) transport oxygen but accumulate oxidative damage over time, reducing function in vivo and during storage--critical for transfusions. To explore genetic influences on RBC resilience, we profiled proteins, metabolites, and lipids from fresh and stored RBCs obtained from 350 genetically diverse mice. Our analysis identified over 6,000 quantitative trait loci (QTL). Compared to other tissues, prevalence of trans genetic effects over cis reflects the absence of de novo protein synthesis in anucleated RBCs. QTL hotspots at Hbb, Hba, Mon1a, and storage-specific Steap3 linked ferroptosis to hemolysis. Proteasome components clustered at multiple loci, underscoring the importance of degrading oxidized proteins. Post-translational modifications (PTMs) mapped predominantly to hemoglobins, particularly cysteine residues. Loss of reactive C93 in humanized mice (HBB C93A) disrupted redox balance, affecting glutathione pools, protein glutathionylation, and redox PTMs. These findings highlight genetic regulation of RBC oxidation, with implications for transfusion biology and oxidative stress-dependent hemolytic disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/640676v1_ufig1.gif" ALT="Figure 1"> View larger version (98K): org.highwire.dtl.DTLVardef@14a3cc2org.highwire.dtl.DTLVardef@daf8b4org.highwire.dtl.DTLVardef@1fe306forg.highwire.dtl.DTLVardef@11de734_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2024

Metabolic Switch in Endocrine Resistant Estrogen Receptor Positive Breast Cancer

PurposeThe development of endocrine resistance remains a significant challenge in the clinical management of estrogen receptor-positive (ER+) breast cancer. Metabolic reprogramming is a prominent component of endocrine resistance and a potential therapeutic intervention point. However, a limited understanding of which metabolic changes are conserved across the heterogeneous landscape of ER+ breast cancer or how metabolic changes factor into ER DNA binding patterns hinder our ability to target metabolic adaptation as a treatment strategy. This study uses dimethyl fumarate (DMF) to restore tamoxifen (Tam) and fulvestrant (Fulv) sensitivity in endocrine-resistant cell lines and investigates how metabolic changes influence ER DNA-binding patterns. Experimental DesignTo address the challenge of metabolic adaptation in anti-endocrine resistance, we generated Tam and Fulv resistance in six ER+ breast cancer (BC) cell lines, representing ductal (MCF7, T47D, ZR75-1, and UCD12), lobular (MDA-MB-134--VI), and HER2 amplified (BT474) BC molecular phenotypes. Metabolomic profiling, RNA sequencing, proteomics, and CUT&RUN assays were completed to characterize metabolic shifts, transcriptional and protein changes, and ER DNA-binding patterns in resistant cells. Dimethyl fumarate was assessed for its ability to reverse Tam and Fulv resistance, restore tricarboxylic acid cycle (TCA) cycle function, and restore parental cell (endocrine sensitive) ER DNA binding patterns. ResultsTamoxifen-resistant (TamR) and fulvestrant-resistant (FulvR) cells exhibited disrupted TCA cycle activity, reduced glutathione levels, and altered nucleotide and amino acid metabolism. DMF treatment replenished TCA cycle intermediates and reversed resistance in both TamR and FulvR cells. DMF also increased mevalonate pathway enzyme expression in both TamR and FulvR cells, with TamR cells upregulating enzymes in the cholesterol synthesis phase and FulvR enhancing enzymes in the early part of the pathway. DMF restored ER DNA-binding patterns in TamR cells to resemble parental cells, re-sensitizing them to Tam. In FulvR cells, DMF reversed resistance by modulating ER-cofactor interactions but did not restore parental ER DNA-binding signatures. ConclusionsOur findings provide new insights into how metabolic reprogramming affects ER DNA-binding activity in endocrine-resistant breast cancer. We demonstrate how altering metabolism can reprogram ER signaling and influence resistance mechanisms by targeting metabolic vulnerabilities, such as TCA cycle disruptions. Additionally, our data provide a comprehensive metabolomic, RNA-seq, and CUT&RUN data set relevant to tumor metabolic adaptation leading to acquired endocrine resistance in highly utilized ER+ breast cancer cell lines. This study improves our understanding of how metabolic states alter ER function in endocrine-resistant breast cancer.

cancer biology↗

bioRxiv2024

The MUC5B promoter variant results in proteomic changes in the non-fibrotic lung

The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the presence of the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that the epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with the initiation and progression of lung fibrosis.

pathology↗

bioRxiv2024

Anisotropic Articular Cartilage Biofabrication based on Decellularized Extracellular Matrix

Tissue-engineered grafts that mimic articular cartilage show promise for treating cartilage injuries. However, engineering cartilage cell-based therapies to match zonal architecture and biochemical composition remains challenging. Decellularized articular cartilage extracellular matrix (dECM) has gained attention for its chondro-inductive properties, yet dECM-based bioinks have limitations in mechanical stability and printability. This study proposes a rapid light-based bioprinting method using a tyrosine-based crosslinking mechanism, which does not require chemical modifications of dECM and thereby preserves its structure and bioactivity. Combining this resin with Filamented Light (FLight) biofabrication enables the creation of cellular, porous, and anisotropic dECM scaffolds composed of aligned microfilaments. Specifically, we investigate the effects of various biopolymer compositions (i.e., hyaluronic acid, collagen I, and dECM) and inner architecture (i.e., bulk light vs FLight) on immune response and cell morphology, and we investigate their influence on nascent ECM production and long-term tissue maturation. Our findings highlight the importance of FLight scaffolds in directing collagen deposition resembling articular cartilage structure and promoting construct maturation, and they emphasize the superiority of biological-rich dECM over single-component materials for engineering articular cartilage, thereby offering new avenues for the development of effective cartilage tissue engineering strategies.

bioengineering↗