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In an intensifying effort to track ocean change and distinguish between natural and anthropogenic drivers, sustained ocean time series measurements are becoming increasingly important. Advancements in the ocean carbon observation network over the last decade, such as the development and deployment of Moored Autonomous pCO2 (MAPCO2) systems, have dramatically improved our ability to characterize ocean climate, sea–air gas exchange, and biogeochemical processes. The MAPCO2 system provides high-resolution data that can measure interannual, seasonal, and sub-seasonal dynamics and constrain the impact of short-term biogeochemical variability on carbon dioxide (CO2) flux. Overall uncertainty of the MAPCO2 using in situ calibrations with certified gas standards and post-deployment standard operating procedures is < 2 μatm for seawater partial pressure of CO2 (pCO2) and < 1 μatm for air pCO2. The MAPCO2 maintains this level of uncertainty for over 400 days of autonomous operation. MAPCO2 measurements are consistent with shipboard seawater pCO2 measurements and GLOBALVIEW-CO2 boundary layer atmospheric values. Here we provide an open-ocean MAPCO2 data set including over 100 000 individual atmospheric and seawater pCO2 measurements on 14 surface buoys from 2004 through 2011 and a description of the methods and data quality control involved. The climate-quality data provided by the MAPCO2 have allowed for the establishment of open-ocean observatories to track surface ocean pCO2 changes around the globe. Data are available at doi:10.3334/CDIAC/OTG.TSM_NDP092 and http://cdiac.ornl.gov/oceans/Moorings/ndp092.

Atmospheric aerosol vertical distributions were measured above Svalbard, Norway, in April 2011 during the Cooperative Investigation of Climate-Cryosphere Interactions campaign (CICCI). Measurements were made of the particle number concentration and the aerosol light absorption coefficient at three wavelengths. A filter sample was collected on each flight at the altitude of maximum particle number concentration. The filters were analyzed for major anions and cations. The aerosol payload was flown in a NOAA/PMEL MANTA Unmanned Aerial System (UAS). A total of 18 flights were flown during the campaign totaling 38 flight hours. The data show frequent aerosol layers aloft with high particle number concentration (1000 cm−3) and enhanced aerosol light absorption (1 Mm−1). Air mass histories of these aerosol layers were assessed using FLEXPART particle dispersion modeling. The data contribute to an assessment of sources of BC to the Arctic and potential climate impacts.

The abnormally thick glycocalyx of cancer cells can provide a physical barrier to immune cell recognition and effective immunotherapy. Here, we demonstrate an optical method based on Scanning Angle Interference Microscopy (SAIM) for the screening of therapeutic agents that can disrupt the glycocalyx layer as a strategy to improve anti-cancer immune responses. We developed a new membrane labeling strategy utilizing leucine zipper pairs to fluorescently mark the glycocalyx layer boundary for precise and robust measurement of glycocalyx thickness with SAIM. Using this platform, we evaluated the effects of glycosylation inhibitors and targeted enzymatic degraders of the glycocalyx, with particular focus on strategies for cholangiocarcinoma (CCA), a highly lethal malignancy with limited therapeutic options. We found that CCA had the highest mean expression of the cancer-associated mucin, MUC1, across all cancers represented in the cancer cell line encyclopedia. Pharmacological inhibitors of mucin-type O-glycosylation and mucin-specific proteases, such as StcE, could dramatically reduce the glycocalyx layer in the YSCCC model of intrahepatic CCA. Motivated by these findings, we engineered Natural Killer (NK) cells tethered with StcE to enhance NK cell-mediated cytotoxicity against CCA. In a CCA xenograft model, these engineered NK cells demonstrated superior anti-tumor efficacy compared to wild-type NK cells, with no observable adverse effects. Our findings not only provide a reliable imaging-based screening platform for evaluating glycocalyx-targeting pharmacological interventions but also offer mechanistic insights into how CCA may avoid immune elimination through fortification of the glycocalyx layer with mucins. Additionally, this work presents a novel therapeutic strategy for mucin-overexpressing cancers, potentially improving immunotherapy efficacy across various cancer types., Competing Interests: Conflicts of Interest M.J.P. and S.P are inventors on a patent filed by Cornell’s Center for Technology Licensing on the StcE-tethered NK technology (PCT/US2022/080937). All other authors declare no competing interests.

The voluntary recall of textured breast implants due to their association with breast implant-associated anaplastic large cell lymphoma has resulted in the loss of the primary advantage of the textured surface: positional stability. We have engineered a novel soft gel-filled smooth implant with a surface that promotes positional stability without texture, known as the positionally stable smooth implant (PSSI). Miniature anatomically shaped breast implant shells were fabricated from polydimethylsiloxane using 3D-printed molds. The implant shell design incorporates cylindrical wells 1-4 mm in diameter. Implants were filled with commercial breast implant-derived silicone gel. Smooth and textured implants were also fabricated, serving as controls. Six implants per group were implanted subcutaneously into the bilateral rat dorsum. Rotation was measured every 2 weeks for a total of 12 weeks to assess stability. Animals were sacrificed at 4 and 12 weeks, and implant-capsule units were explanted for histological and Micro-computed tomography (MicroCT) analyzes. Four weeks after implantation, PSSI conditions showed tissue ingrowth and conformation to well dimensions, as assessed by histological staining and MicroCT imaging. Twelve weeks post implantation, textured implants and PSSI conditions with larger widths, depths, and well number demonstrated statistically significant increased stability compared to smooth implants ( p < 0.05). Tissue ingrowth into shell features occurred by 4 weeks and remained throughout longer time points. No significant differences were found in capsule thickness or collagen content between groups. These results suggest a promising alternative to textured surfaces for inducing implant positional stability., (© 2024 IOP Publishing Ltd. All rights, including for text and data mining, AI training, and similar technologies, are reserved.)

Augmentation of the nasal dorsum often requires implantation of structural material. Existing methods include autologous, cadaveric or alloplastic materials and injectable hydrogels. Each of these options is associated with considerable limitations. There is an ongoing need for precise and versatile implants that produce long-lasting craniofacial augmentation. Four separate polylactic acid (PLA) dorsal nasal implant designs were 3D-printed. Two implants had internal PLA rebar of differing porosities and two were designed as "shells" of differing porosities. Shell designs were implanted without infill or with either minced or zested processed decellularized ovine cartilage infill to serve as a "biologic rebar", yielding eight total treatment groups. Scaffolds were implanted heterotopically on rat dorsa (N = 4 implants per rat) for explant after 3, 6, and 12 months followed by volumetric, histopathologic, and biomechanical analysis. Low porosity implants with either minced cartilage or PLA rebar infill had superior volume retention across all timepoints. Overall, histopathologic and immunohistochemical analysis showed a resolving inflammatory response with an M1/M2 ratio consistently favoring tissue regeneration over the study course. However, xenograft cartilage showed areas of degradation and pro-inflammatory infiltrate contributing to volume and contour loss over time. Biomechanical analysis revealed all constructs had equilibrium and instantaneous moduli higher than human septal cartilage controls. Biocompatible, degradable polymer implants can induce healthy neotissue ingrowth resulting in guided soft tissue augmentation and offer a simple, customizable and clinically-translatable alternative to existing craniofacial soft tissue augmentation materials. PLA-only implants may be superior to combination PLA and xenograft implants due to contour irregularities associated with cartilage degradation., (© 2024 Wiley Periodicals LLC.)

Cell migration during many fundamental biological processes including metastasis requires cells to traverse tissue with heterogeneous mechanical cues that direct migration as well as determine force and energy requirements for motility. However, the influence of discrete structural and mechanical cues on migration remains challenging to determine as they are often coupled. Here, we decouple the pro-invasive cues of collagen fiber alignment and tension to study their individual impact on migration. When presented with both cues, cells preferentially travel in the axis of tension against fiber alignment. Computational and experimental data show applying tension perpendicular to alignment increases potential energy stored within collagen fibers, lowering requirements for cell-induced matrix deformation and energy usage during migration compared to motility in the direction of fiber alignment. Energy minimization directs migration trajectory, and tension can facilitate migration against fiber alignment. These findings provide a conceptual understanding of bioenergetics during migration through a fibrous matrix., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Breast cancer bone metastasis is a major cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Moreover, mineralized biomaterials are commonly utilized for clinical bone defect repair, but how mineralized biomaterials affect the foreign body response and wound healing is unclear. Here, we investigate how bone mineral affects tumor growth and microenvironmental complexity in vivo by combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to bone mineral content depends on the murine tumor model used. While lack of bone mineral induces tumor-promoting microenvironments in both immunocompromised and immunocompetent animals, these changes are mediated by altered fibroblast phenotype in immunocompromised mice and macrophage polarization in immunocompetent mice. Collectively, our findings suggest that bone mineral density affects tumor growth by impacting microenvironmental complexity in an organism-dependent manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aberrant activation of Wnt signaling results in unregulated accumulation of cytosolic β-catenin, which subsequently enters the nucleus and promotes transcription of genes that contribute to cellular proliferation and malignancy. Here, we sought to eliminate pathogenic β-catenin from the cytosol using designer ubiquibodies (uAbs), chimeric proteins composed of an E3 ubiquitin ligase and a target-binding domain that redirect intracellular proteins to the proteasome for degradation. To accelerate uAb development, we leveraged a protein language model (pLM)-driven algorithm called SaLT&PepPr to computationally design "guide" peptides with affinity for β-catenin, which were subsequently fused to the catalytic domain of a human E3 called C-terminus of Hsp70-interacting protein (CHIP). Expression of the resulting peptide-guided uAbs in colorectal cancer cells led to the identification of several designs that significantly reduced the abnormally stable pool of free β-catenin in the cytosol and nucleus while preserving the normal membrane-associated subpopulation. This selective knockdown of pathogenic β-catenin suppressed Wnt/β-catenin signaling and impaired tumor cell survival and proliferation. Furthermore, one of the best degraders selectively decreased cytosolic but not membrane-associated β-catenin levels in livers of BALB/c mice following delivery as a lipid nanoparticle (LNP)-encapsulated mRNA. Collectively, these findings reveal the unique ability of uAbs to selectively eradicate abnormal proteins in vitro and in vivo and open the door to peptide-programmable biologic modulators of other disease-causing proteins., Competing Interests: Competing Interests Statement. M.P.D. and P.C. have financial interests in UbiquiTx, Inc. M.P.D. also has financial interests in Gauntlet, Inc. Glycobia, Inc., Resilience, Inc. and Versatope Therapeutics, Inc. M.P.D.’s and P.C.’s interests are reviewed and managed by Cornell University and Duke University, respectively, in accordance with their conflict-of-interest policies. All other authors declare no competing interests.

Store-operated calcium entry (SOCE) is essential for cellular signaling. Earlier studies of the pyrazole derivative BTP2, an efficient inhibitor SOCE, identified that SOCE blockade suppresses proinflammatory gene expression. The impact of SOCE blockade on gene expression at the whole transcriptome level, however, is unknown. To fill this gap, we performed RNA sequencing (RNA-seq) and investigated at the whole transcriptome level the effect of BTP2 on gene expression in human peripheral blood mononuclear cells signaled with phytohemagglutinin. Our global gene expression analysis identified that SOCE blockade spares activation-induced expression of anti-inflammatory genes (e.g., IL10, TGFB1, FOXP3, and CTLA4) whereas the induced expression of proinflammatory genes such as IFNG and cytopathic genes such as GZMB are inhibited. We validated the differential expression of immunoregulatory genes identified by RNA-seq using preamplification-enhanced RT-qPCR assays. Because IL-2/IL2RA interaction is essential for T cell clonal expansion, we investigated and confirmed that BTP2 inhibits IL2RA expression at the protein level using multiparameter flow cytometry. Our elucidation that SOCE blockade spares activation-induced expression of anti-inflammatory genes while blocking pro-inflammatory gene expression suggests that SOCE blockers may represent a novel class of immunoregulatory drugs of value for treating autoimmune disease states and organ transplantation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in C. elegans , we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states - an active dimer and an inactive monomer - gates the output of the Inversin complex., Competing Interests: Conflicts of interest: The authors declare no competing interest.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer., (© 2024 Wiley‐VCH GmbH.)

Our understanding of how adipocytes influence metabolic signaling, immune function, and cancer progression remains limited as the culture of primary adipocytes is challenging. Here, we present a protocol to fabricate elastomer microwells for three-dimensional culture of collagen-embedded adipocytes. We describe steps to cure and functionalize elastomer microwells and to isolate and embed primary adipocytes. We then detail how to culture and analyze adipocyte-collagen gels. This protocol provides broad applications to improve our understanding of adipocyte biology in health and disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autologous fat transfer is a common procedure that patients undergo to rejuvenate large soft tissue defects. However, these surgeries are complicated by limited tissue sources, donor-site morbidity, and necrosis. While the biofabrication of fat tissue can serve as a clinical option for reconstructive surgery, the influence of matrix mechanics, specifically stiffness and viscosity, on adipogenesis requires further elucidation. Additionally, the effects of these mechanical parameters on metabolic and thermogenic fat potential have yet to be investigated. In this study, gelatin methacryloyl (GelMA) polymers with varying degrees of methacrylation (DoM) were fabricated to create matrices with different stiffnesses and viscosities. Human adipose-derived mesenchymal stem cells were then encapsulated in mechanically tunable GelMA and underwent adipogenesis to investigate the effects of matrix mechanics on lipid phenotype and fat potential. Mechanical testing confirmed that GelMA stiffness was regulated by DoM and weight composition, whereas viscosity was determined by the latter. Further work revealed that while lipid phenotype became more enriched as matrix stiffness and viscosity declined, the potential toward metabolic and thermogenic fat appeared to be more viscous dependent rather than stiffness dependent. In addition, fatty acid binding protein 4 and uncoupling protein 1 gene expression exhibited viscous-dependent behavior despite comparable levels of peroxisome proliferator-activated receptor gamma. However, despite the superior role of viscosity, lipid quantity and mitochondrial abundance demonstrated stiffness-dependent behavior. Overall, this work revealed that matrix viscosity played a more superior role than stiffness in driving adipogenesis and distinguishing between metabolic and thermogenic fat potential. Ultimately, this differentiation in fat production is important for engineering ideal adipose tissue for large soft tissue defects.

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA- b -PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.

Quercetin, recognized for its antioxidant, anti-inflammatory, and antibacterial properties, faces limited biomedical application due to its low solubility. Cotton, a preferred wound dressing material over synthetic ones, lacks inherent antibacterial and wound-healing attributes and can benefit from quercetin features. This study explores the potential of overcoming these challenges through the inclusion complexation of quercetin with cyclodextrins (CDs) and the development of a nanofibrous coating on a cotton nonwoven textile. Hydroxypropyl-beta-cyclodextrin (HP-β-CD) and hydroxypropyl-gamma-cyclodextrin (HP-γ-CD) formed inclusion complexes of quercetin, with chitosan added to enhance antibacterial properties. Phase solubility results showed that inclusion complexation can enhance quercetin solubility up to 20 times, with HP-γ-CD forming a more stable inclusion complexation compared with HP-β-CD. Electrospinning of the nanofibers from HP-β-CD/Quercetin and HP-γ-CD/Quercetin aqueous solutions without the use of a polymeric matrix yielded a uniform, smooth fiber morphology. The structural and thermal analyses of the HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers confirmed the presence of inclusion complexes between quercetin and each of the CDs (HP-β-CD and HP-γ-CD). Moreover, HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers showed a near-complete loading efficiency of quercetin and followed a fast-releasing profile of quercetin. Both HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers showed significantly higher antioxidant activity compared to pristine quercetin. The HP-β-CD/Quercetin and HP-γ-CD/Quercetin nanofibers also showed antibacterial activity, and with the addition of chitosan in the HP-γ-CD/Quercetin system, the Chitosan/HP-γ-CD/Quercetin nanofibers completely eliminated the investigated bacteria species. The nanofibers were nontoxic and well-tolerated by cells, and exploiting the quercetin and chitosan anti-inflammatory activities resulted in the downregulation of IL-6 and NO secretion in both immune as well as regenerative cells. Overall, CD inclusion complexation markedly enhances quercetin solubility, resulting in a biofunctional antioxidant, antibacterial, and anti-inflammatory wound dressing through a nanofibrous coating on cotton textiles.

Objective . Macrophages and astrocytes play a crucial role in the aftermath of a traumatic spinal cord injury (SCI). Infiltrating macrophages adopt a pro-inflammatory phenotype while resident astrocytes adopt a neurotoxic phenotype at the injury site, both of which contribute to neuronal death and inhibit axonal regeneration. The cytokine interleukin-4 (IL-4) has shown significant promise in preclinical models of SCI by alleviating the macrophage-mediated inflammation and promoting functional recovery. However, its effect on neurotoxic reactive astrocytes remains to be elucidated, which we explored in this study. We also studied the beneficial effects of a sustained release of IL-4 from an injectable biomaterial compared to bolus administration of IL-4. Approach . We fabricated a heparin-based coacervate capable of anchoring and releasing bioactive IL-4 and tested its efficacy in vitro and in vivo. Main results . We show that IL-4 coacervate is biocompatible and drives a robust anti-inflammatory macrophage phenotype in culture. We also show that IL-4 and IL-4 coacervate can alleviate the reactive neurotoxic phenotype of astrocytes in culture. Finally, using a murine model of contusion SCI, we show that IL-4 and IL-4 coacervate, injected intraspinally 2 d post-injury, can reduce macrophage-mediated inflammation, and alleviate neurotoxic astrocyte phenotype, acutely and chronically, while also promoting neuroprotection with significant improvements in hindlimb locomotor recovery. We observed that IL-4 coacervate can promote a more robust regenerative macrophage phenotype in vitro , as well as match its efficacy in vivo, compared to bolus IL-4. Significance . Our work shows the promise of coacervate as a great choice for local and prolonged delivery of cytokines like IL-4. We support this by showing that the coacervate can release bioactive IL-4, which acts on macrophages and astrocytes to promote a pro-regenerative environment following a SCI leading to robust neuroprotective and functional outcomes., (© 2024 IOP Publishing Ltd.)

Alzheimer's disease (AD) is marked by the aggregation of extracellular amyloid-β (Aβ) and astrocyte dysfunction. For Aβ oligomers or aggregates to be formed, there must be Aβ monomers present; however, the roles of monomeric Aβ (mAβ) and oligomeric Aβ (oAβ) in astrocyte pathogenesis are poorly understood. We cultured astrocytes in a brain-mimicking three-dimensional (3D) extracellular matrix and revealed that both mAβ and oAβ caused astrocytic atrophy and hyper-reactivity, but showed distinct Ca 2+ changes in astrocytes. This 3D culture evolved into a microfluidic glymphatics-on-chip model containing astrocytes and endothelial cells with the interstitial fluid (ISF). The glymphatics-on-chip model not only reproduced the astrocytic atrophy, hyper-reactivity, and Ca 2+ changes induced by mAβ and oAβ, but recapitulated that the components of the dystrophin-associated complex (DAC) and aquaporin-4 (AQP4) were properly maintained by the ISF, and dysregulated by mAβ and oAβ. Collectively, mAβ and oAβ cause distinct AD pathophysiological characteristics in the astrocytes.

Cancer immunotherapy focuses on the use of patients' adaptive immune systems to combat cancer. In the past decade, FDA has approved many immunotherapy products for cancer patients who suffer from primary tumors, tumor relapse, and metastases. However, these immunotherapies still show resistance in many patients and often lead to inconsistent responses in patients due to variations in tumor genetic mutations and tumor immune microenvironment. Microfluidics-based organ-on-a-chip technologies or microphysiological systems have opened new ways that can provide relatively fast screening for personalized immunotherapy and help researchers and clinicians understand tumor-immune interactions in a patient-specific manner. They also have the potential to overcome the limitations of traditional drug screening and testing, given the models provide a more realistic 3D microenvironment with better controllability, reproducibility, and physiological relevance. This review focuses on the cutting-edge microphysiological organ-on-a-chip devices developed in recent years for studying cancer immunity and testing cancer immunotherapeutic agents, as well as some of the largest challenges of translating this technology to clinical applications in immunotherapy and personalized medicine., (© 2023 Wiley‐VCH GmbH.)

Spaceflight induces molecular, cellular and physiological shifts in astronauts and poses myriad biomedical challenges to the human body, which are becoming increasingly relevant as more humans venture into space 1-6 . Yet current frameworks for aerospace medicine are nascent and lag far behind advancements in precision medicine on Earth, underscoring the need for rapid development of space medicine databases, tools and protocols. Here we present the Space Omics and Medical Atlas (SOMA), an integrated data and sample repository for clinical, cellular and multi-omic research profiles from a diverse range of missions, including the NASA Twins Study 7 , JAXA CFE study 8,9 , SpaceX Inspiration4 crew 10-12 , Axiom and Polaris. The SOMA resource represents a more than tenfold increase in publicly available human space omics data, with matched samples available from the Cornell Aerospace Medicine Biobank. The Atlas includes extensive molecular and physiological profiles encompassing genomics, epigenomics, transcriptomics, proteomics, metabolomics and microbiome datasets, which reveal some consistent features across missions, including cytokine shifts, telomere elongation and gene expression changes, as well as mission-specific molecular responses and links to orthologous, tissue-specific mouse datasets. Leveraging the datasets, tools and resources in SOMA can help to accelerate precision aerospace medicine, bringing needed health monitoring, risk mitigation and countermeasure data for upcoming lunar, Mars and exploration-class missions., (© 2024. The Author(s).)

Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. Stabilizing improperly ubiquitinated proteins via target-specific deubiquitination is thus a critical therapeutic goal. Building off the major advances in targeted protein degradation (TPD) using bifunctional small-molecule degraders, targeted protein stabilization (TPS) modalities have been described recently. However, these rely on a limited set of chemical linkers and warheads, which are difficult to generate de novo for new targets and do not exist for classically "undruggable" targets. To address the limited reach of small molecule-based degraders, we previously engineered ubiquibodies (uAbs) by fusing computationally-designed "guide" peptides to E3 ubiquitin ligase domains for modular, CRISPR-analogous TPD. Here, we expand the TPS target space by engineering "deubiquibodies" (duAbs) via fusion of computationally-designed guides to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. To demonstrate duAb modularity, we swap in new target-binding peptides designed via our generative language models to stabilize diverse target proteins, including key tumor suppressor proteins such as p53 and WEE1, as well as heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. In total, our duAb system represents a simple, programmable, CRISPR-analogous strategy for TPS., Competing Interests: Competing Interests Statement P.C., L.H., and M.P.D. are listed as inventors on US Patent Application 63/541,921: “Peptide-Guided Protein Stabilizers and Uses Thereof”. P.C. and M.D. are co-founded of UbiquiTx, Inc., which commercializes genetically-encoded proteome editing technologies, and are co-inventors of duAb patents. P.C.’s interests are reviewed and managed by Duke University in accordance with their conflict-of-interest policies. M.P.D.’s interests are reviewed and managed by Cornell University in accordance with their conflict-of-interest policies.

Breast cancer bone metastasis is the leading cause of mortality in patients with advanced breast cancer. Although decreased mineral density is a known risk factor for bone metastasis, the underlying mechanisms remain poorly understood because studying the isolated effect of bone mineral density on tumor heterogeneity is challenging with conventional approaches. Here, we investigate how bone mineral content affects tumor growth and microenvironmental complexity in vivo by combining single-cell RNA-sequencing with mineral-containing or mineral-free decellularized bone matrices. We discover that the absence of bone mineral significantly influences fibroblast and immune cell heterogeneity, promoting phenotypes that increase tumor growth and alter the response to injury or disease. Importantly, we observe that the stromal response to matrix mineral content depends on host immunocompetence and the murine tumor model used. Collectively, our findings suggest that bone mineral density affects tumor growth by altering microenvironmental complexity in an organism-dependent manner., Competing Interests: COMPETING INTERESTS The authors declare no competing interests.

Breast cancer invasion into adipose tissue strongly influences disease progression and metastasis. The degree of cancer cell invasion into adipose tissue depends on both biochemical signaling and the mechanical properties of cancer cells, adipocytes, and other key components of adipose tissue. We model breast cancer invasion into adipose tissue using discrete element method simulations of active, cohesive spherical particles (cancer cells) invading into confluent packings of deformable polyhedra (adipocytes). We quantify the degree of invasion by calculating the interfacial area A t between cancer cells and adipocytes. We determine the long-time value of A t vs the activity and strength of the cohesion between cancer cells, as well as the mechanical properties of the adipocytes and extracellular matrix in which adipocytes are embedded. We show that the degree of invasion collapses onto a master curve as a function of the dimensionless energy scale E c , which grows linearly with the cancer cell velocity persistence time and fluctuations, is inversely proportional to the system pressure, and is offset by the cancer cell cohesive energy. When E c > 1 , cancer cells will invade the adipose tissue, whereas for E c < 1 , cancer cells and adipocytes remain de-mixed. We also show that A t decreases when the adipocytes are constrained by the ECM by an amount that depends on the spatial heterogeneity of the adipose tissue., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

As cells migrate through biological tissues, they must frequently squeeze through micron-sized constrictions in the form of interstitial pores between extracellular matrix fibers and/or other cells. Although it is now well recognized that such confined migration is limited by the nucleus, which is the largest and stiffest organelle, it remains incompletely understood how cells apply sufficient force to move their nucleus through small constrictions. Here, we report a mechanism by which contraction of the cell rear cortex pushes the nucleus forward to mediate nuclear transit through constrictions. Laser ablation of the rear cortex reveals that pushing forces behind the nucleus are the result of increased intracellular pressure in the rear compartment of the cell. The pushing forces behind the nucleus depend on accumulation of actomyosin in the rear cortex and require Rho kinase (ROCK) activity. Collectively, our results suggest a mechanism by which cells generate elevated intracellular pressure in the posterior compartment to facilitate nuclear transit through three-dimensional (3D) constrictions. This mechanism might supplement or even substitute for other mechanisms supporting nuclear transit, ensuring robust cell migrations in confined 3D environments., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N -acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N- glycan, Man 3 GlcNAc 2 (rGALNS opt Gly). The N- glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N- glycans enhanced both protein stability and substrate affinity. rGALNS opt Gly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Urine is a rich source of nucleic acid biomarkers including cell-free DNA (cfDNA) and RNA for monitoring the health of kidney allografts. In this study, we aimed to evaluate whether urine filtration can serve as an alternative to the commonly used method of centrifugation to collect urinary fluid and cell pellets for isolating cfDNA and cellular messenger RNA (mRNA). We collected urine specimens from kidney allograft recipients and obtained the urine supernatant and cell pellet from each specimen using both filtration and centrifugation for paired analyses. We performed DNA sequencing to characterize the origin and properties of cfDNA, as well as quantitative PCR of mRNAs extracted from cell fractions. Our results showed that the biophysical properties of cfDNA, the microbial DNA content, and the tissues of origin of cfDNA were comparable between samples processed using filtration and centrifugation method. Similarly, mRNA quality and quantity obtained using both methods met our criteria for downstream application and the Ct values for each mRNA were comparable between the two techniques.The Ct values demonstrated a high degree of correlation. These findings suggest that urine filtration is a viable alternative to urine centrifugation for isolation of nucleic acid biomarkers from urine specimens., (© 2024. The Author(s).)

Inside the finger-like intestinal projections called villi, strands of smooth muscle cells contract to propel absorbed dietary fats through the adjacent lymphatic capillary, the lacteal, sending fats into the systemic blood circulation for energy production. Despite this vital function, mechanisms of formation, assembly alongside lacteals, and maintenance of villus smooth muscle are unknown. By combining single-cell RNA sequencing and quantitative lineage tracing of the mouse intestine, we identified a local hierarchy of subepithelial fibroblast progenitors that differentiate into mature smooth muscle fibers via intermediate contractile myofibroblasts. This continuum persists as the major mechanism for villus musculature renewal throughout adult life. The NOTCH3-DLL4 signaling axis governs the assembly of smooth muscle fibers alongside their adjacent lacteals and is required for fat absorption. Our studies identify the ontogeny and maintenance of a poorly defined class of intestinal smooth muscle, with implications for accelerated repair and recovery of digestive function following injury., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O -glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general., Competing Interests: CONFLICT OF INTEREST Cornell University has filed patents related to the lubricin sequences and processes described in this manuscript. M.J.C., H.L.R., and M.J.P. are listed as inventors.

Piezo1 regulates multiple aspects of the vascular system by converting mechanical signals generated by fluid flow into biological processes. Here, we find that Piezo1 is necessary for the proper development and function of meningeal lymphatic vessels and that activating Piezo1 through transgenic overexpression or treatment with the chemical agonist Yoda1 is sufficient to increase cerebrospinal fluid (CSF) outflow by improving lymphatic absorption and transport. The abnormal accumulation of CSF, which often leads to hydrocephalus and ventriculomegaly, currently lacks effective treatments. We discovered that meningeal lymphatics in mouse models of Down syndrome were incompletely developed and abnormally formed. Selective overexpression of Piezo1 in lymphatics or systemic administration of Yoda1 in mice with hydrocephalus or Down syndrome resulted in a notable decrease in pathological CSF accumulation, ventricular enlargement and other associated disease symptoms. Together, our study highlights the importance of Piezo1-mediated lymphatic mechanotransduction in maintaining brain fluid drainage and identifies Piezo1 as a promising therapeutic target for treating excessive CSF accumulation and ventricular enlargement., (© 2024. The Author(s).)

Reconstruction of the human auricle remains a formidable challenge for plastic surgeons. Autologous costal cartilage grafts and alloplastic implants are technically challenging, and aesthetic and/or tactile outcomes are frequently suboptimal. Using a small animal "bioreactor", we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimics the size, shape, and biomechanical properties of the native human auricle. The full-scale polylactic acid ear scaffolds were 3D-printed based upon data acquired from 3D photogrammetry of an adult ear. Ovine costal cartilage was processed either through mincing (1 mm 3 ) or zesting (< 0.5 mm 3 ), and then fully decellularized and sterilized. At explantation, both the minced and zested neoears maintained the size and contour complexities of the scaffold topography with steady tissue ingrowth through 6 months in vivo. A mild inflammatory infiltrate at 3 months was replaced by homogenous fibrovascular tissue ingrowth enveloping individual cartilage pieces at 6 months. All ear constructs were pliable, and the elasticity was confirmed by biomechanical analysis. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application. STATEMENT OF SIGNIFICANCE: Accurate reconstruction of the human auricle has always been a formidable challenge to plastic surgeons. In this article, we have bioengineered full-scale ears utilizing decellularized cartilage xenograft placed within a 3D-printed external auricular scaffold that mimic the size, shape, and biomechanical properties of the native human auricle. Longer-term studies of the neoears with faster degrading biomaterials will be warranted for future clinical application., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Nonlinear optical microscopy enables non-invasive imaging in scattering samples with cellular resolution. The spinal cord connects the brain with the periphery and governs fundamental behaviors such as locomotion and somatosensation. Because of dense myelination on the dorsal surface, imaging to the spinal grey matter is challenging, even with two-photon microscopy. Here we show that three-photon excited fluorescence (3PEF) microscopy enables multicolor imaging at depths of up to ~550 μm into the mouse spinal cord, in vivo . We quantified blood flow across vessel types along the spinal vascular network. We then followed the response of neurites and microglia after occlusion of a surface venule, where we observed depth-dependent structural changes in neurites and interactions of perivascular microglia with vessel branches upstream from the clot. This work establishes that 3PEF imaging enables studies of functional dynamics and cell type interactions in the top 550 μm of the murine spinal cord, in vivo ., Competing Interests: Competing interests: The authors declare that they have no competing interests.

Small animal studies in biomedical research often require anesthesia to reduce pain or stress experienced by research animals and to minimize motion artifact during imaging or other measurements. Anesthetized animals must be closely monitored for the safety of the animals and to prevent unintended effects of altered physiology on experimental outcomes. Many currently available monitoring devices are expensive, invasive, or interfere with experimental design. Here, we present MousePZT, a low-cost device based on a simple piezoelectric sensor, with a custom circuit and computer software that allows for measurements of both respiratory rate and heart rate in a non-invasive, minimal contact manner. We find the accuracy of the MousePZT device in measuring respiratory and heart rate matches those of commercial systems. Using the widely-used gas isoflurane and injectable ketamine/xylazine combination, we also demonstrate that changes in respiratory rate are more easily detected and can precede changes in heart rate associated with variations in anesthetic depth. Additional circuitry on the device outputs a respiration-locked trigger signal for respiratory-gating of imaging or other data acquisition and has high sensitivity and specificity for detecting respiratory cycles. We provide detailed instruction documents and all necessary microcontroller and computer software, enabling straightforward construction and utilization of this device., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rivera et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Ovulation is essential for reproductive success, yet the underlying cellular and molecular mechanisms are far from clear. Here, we applied high-resolution spatiotemporal transcriptomics to map out cell type- and ovulation stage-specific molecular programs as function of time during follicle maturation and ovulation in mice. Our analysis revealed dynamic molecular transitions within granulosa cell types that occur in tight coordination with mesenchymal cell proliferation. We identified molecular markers for the emerging cumulus cell fate during the preantral-to-antral transition. We describe transcriptional programs that respond rapidly to ovulation stimulation and those associated with follicle rupture, highlighting the prominent roles of apoptotic and metabolic pathways during the final stages of follicle maturation. We further report stage-specific oocyte-cumulus cell interactions and diverging molecular differentiation in follicles approaching ovulation. Collectively, this study provides insights into the cellular and molecular processes that regulate mouse ovarian follicle maturation and ovulation with important implications for advancing therapeutic strategies in reproductive medicine., Competing Interests: Competing interests statement:The authors declare no competing interest.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising vaccine technology for developing immunity against diverse pathogens. However, antigen display on OMVs can be challenging to control and highly variable due to bottlenecks in protein expression and localization to the bacterial host cell's outer membrane, especially for bulky and complex antigens. Here, we describe methods related to a universal vaccine technology called AvidVax (avidin-based vaccine antigen crosslinking) for rapid and simplified assembly of antigens on the exterior of OMVs during vaccine development. The AvidVax platform involves remodeling the OMV surface with multiple copies of a synthetic antigen-binding protein (SNAP), which is an engineered fusion protein comprised of an outer membrane scaffold protein linked to a biotin-binding protein. The resulting SNAPs enable efficient decoration of OMVs with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, nucleic acids, and short peptides. We detail the key steps in the AvidVax vaccine production pipeline including preparation and isolation of SNAP-OMVs, biotinylation and enrichment of vaccine antigens, and formulation and characterization of antigen-loaded SNAP-OMVs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

Interstitial fluid uptake and retention by lymphatic vessels (LVs) play a role in maintaining interstitial fluid homeostasis. While it is well-established that intraluminal lymphatic valves in the collecting LVs prevent fluid backflow (secondary lymphatic valves), a separate valve system in the initial LVs that only permits interstitial fluid influx into the LVs, preventing fluid leakage back to the interstitium (primary lymphatic valves), remains incompletely understood. Although lymphatic dysfunction is commonly observed in inflammation and autoimmune diseases, how the primary lymphatic valves are affected by acute and chronic inflammation has scarcely been explored and even less so using in vitro lymphatic models. Here, we developed a human initial lymphatic vessel chip where interstitial fluid pressure and luminal fluid pressure are controlled to examine primary lymph valve function. In normal conditions, lymphatic drainage (fluid uptake) and permeability (fluid leakage) in engineered LVs were maintained high and low, respectively, which was consistent with our understanding of healthy primary lymph valves. Next, we examined the effects of acute and chronic inflammation. Under the acute inflammation condition with a TNF-α treatment (2 hours), degradation of fibrillin and impeded lymphatic drainage were observed, which were reversed by treatment with anti-inflammatory dexamethasone. Surprisingly, the chronic inflammation condition (repeated TNF-α treatments during 48 hours) deposited fibrillin to compensate for the fibrillin loss, showing no change in lymphatic drainage. Instead, the chronic inflammation condition led to cell death and disruption of lymphatic endothelial cell-cell junctions, increasing lymphatic permeability and fluid leakage. Our human lymphatic model shows two distinct mechanisms by which primary lymphatic valve dysfunction occurs in acute and chronic inflammation.

Breast cancer metastasis is initiated by invasion of tumor cells into the collagen type I-rich stroma to reach adjacent blood vessels. Prior work has identified that metabolic plasticity is a key requirement of tumor cell invasion into collagen. However, it remains largely unclear how blood vessels affect this relationship. Here, we developed a microfluidic platform to analyze how tumor cells invade collagen in the presence and absence of a microvascular channel. We demonstrate that endothelial cells secrete pro-migratory factors that direct tumor cell invasion toward the microvessel. Analysis of tumor cell metabolism using metabolic imaging, metabolomics, and computational flux balance analysis revealed that these changes are accompanied by increased rates of glycolysis and oxygen consumption caused by broad alterations of glucose metabolism. Indeed, restricting glucose availability decreased endothelial cell-induced tumor cell invasion. Our results suggest that endothelial cells promote tumor invasion into the stroma due, in part, to reprogramming tumor cell metabolism., Competing Interests: The authors have no conflicts to disclose., (© 2023 Author(s).)

Recovering a sufficient amount of microbial DNA from extremely low-biomass specimens, such as human skin, to investigate the community structure of the microbiome remains challenging. We developed a sampling solution containing agar to increase the abundance of recovered microbial DNA. Quantitative PCR targeting the 16S rRNA gene revealed a significant increase in the amount of microbial DNA recovered from the developed sampling solution compared with conventional solutions from extremely low-biomass skin sites such as the volar forearm and antecubital fossa. In addition, we confirmed that the developed sampling solution reduces the contamination rate of probable non-skin microbes compared to the conventional solutions, indicating that the enhanced recovery of microbial DNA was accompanied by a reduced relative abundance of contaminating microbes in the 16S rRNA gene amplicon sequencing data. In addition, agar was added to each step of the DNA extraction process, which improved the DNA extraction efficiency as a co-precipitant. Enzymatic lysis with agar yielded more microbial DNA than conventional kits, indicating that this method is effective for analyzing microbiomes of low-biomass specimens., (© 2023. The Author(s).)

In patients with breast cancer, lower bone mineral density increases the risk of bone metastasis. Although the relationship between bone-matrix mineralization and tumour-cell phenotype in breast cancer is not well understood, mineralization-induced rigidity is thought to drive metastatic progression via increased cell-adhesion forces. Here, by using collagen-based matrices with adjustable intrafibrillar mineralization, we show that, unexpectedly, matrix mineralization dampens integrin-mediated mechanosignalling and induces a less proliferative stem-cell-like phenotype in breast cancer cells. In mice with xenografted decellularized physiological bone matrices seeded with human breast tumour cells, the presence of bone mineral reduced tumour growth and upregulated a gene-expression signature that is associated with longer metastasis-free survival in patients with breast cancer. Our findings suggest that bone-matrix changes in osteogenic niches regulate metastatic progression in breast cancer and that in vitro models of bone metastasis should integrate organic and inorganic matrix components to mimic physiological and pathologic mineralization., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Capsular contracture remains the most common complication following device-based breast reconstruction, occurring in up to 50% of women who also undergo adjuvant radiotherapy either before or after device-based reconstruction. While certain risk factors for capsular contracture have been identified, there remains no clinically effective method of prevention. The purpose of the present study is to determine the effect of coating the implant with the novel small molecule Met-Z2-Y12, with and without delayed, targeted radiotherapy, on capsule thickness and morphologic change around smooth silicone implants placed under the latissimus dorsi in a rodent model., Methods: Twenty-four female Sprague Dawley rats each had 2 mL smooth round silicone breast implants implanted bilaterally under the latissimus dorsi muscle. Twelve received uncoated implants and twelve received implants coated with Met-Z2-Y12. Half of the animals from each group received targeted radiotherapy (20 Gray) on postoperative day ten. At three and 6 months after implantation, the tissue surrounding the implants was harvested for analysis of capsular histology including capsule thickness. Additionally, microCT scans were qualitatively analyzed for morphologic change., Results: Capsules surrounding Met-Z2-Y12-coated implants were significantly thinner (P = 0.006). The greatest difference in capsule thickness was seen in the irradiated 6-month groups, where mean capsule thickness was 79.1 ± 27.3 μm for uncoated versus 50.9 ± 9.6 μm for Met-Z2-Y12-coated implants (P = 0.038). At the time of explant, there were no capsular morphologic differences between the groups either grossly or per microCT., Conclusions: Met-Z2-Y12 coating of smooth silicone breast implants significantly reduces capsule thickness in a rodent model of submuscular breast reconstruction with delayed radiotherapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Impaired lymphatic drainage and lymphedema are major morbidities whose mechanisms have remained obscure. To study lymphatic drainage and its impairment, we engineered a microfluidic culture model of lymphatic vessels draining interstitial fluid. This lymphatic drainage-on-chip revealed that inflammatory cytokines that are known to disrupt blood vessel junctions instead tightened lymphatic cell-cell junctions and impeded lymphatic drainage. This opposing response was further demonstrated when inhibition of rho-associated protein kinase (ROCK) was found to normalize fluid drainage under cytokine challenge by simultaneously loosening lymphatic junctions and tightening blood vessel junctions. Studies also revealed a previously undescribed shift in ROCK isoforms in lymphatic endothelial cells, wherein a ROCK2/junctional adhesion molecule-A (JAM-A) complex emerges that is responsible for the cytokine-induced lymphatic junction zippering. To validate these in vitro findings, we further demonstrated in a genetic mouse model that lymphatic-specific knockout of ROCK2 reversed lymphedema in vivo. These studies provide a unique platform to generate interstitial fluid pressure and measure the drainage of interstitial fluid into lymphatics and reveal a previously unappreciated ROCK2-mediated mechanism in regulating lymphatic drainage.