Abstract Introduction Tertiary lymphoid structures (TLSs) are linked to better patient outcomes and enhanced immunity in some cancers (e.g. ovarian, lung). TLSs support the reprogramming of immune system to attack the tumor and retain a long-term memory. We employed our previously published TLS-on-chip microfluidic device (TOD) and in vivo murine models to test a novel therapeutic formulation that involves engineering in situ TLSs to combat solid tumors. This formulation aims to generate effector T cells, memory B cells, and tumor-antigen-specific antibodies locally. Methods We integrated different cancer cell lines expressing varying levels of PD-L1/PD-L2 with healthy and cancer patients PBMCs in the TODs to quantify TLS formation, lymphocyte activation, and cancer cell killing. Based on the TOD’s composition, we defined a cellularized extracellular matrix gel to create TLS in vivo. To investigate the in vivo efficacy of this gel, we injected it peritumorally in a lung cancer cell line-derived xenograft (CDX) and a syngeneic model to monitor TLS formation and survival benefit measured by tumor volume reduction. Results TOD studies showed that with “hot” cancer cell lines (high PD-L1/PD-L2), our formulation induced TLS formation leading to a robust cytokine response, B cell activation, anti-tumor CD8+ T cell activity, and cancer cell death. In contrast, “cold” tumor cell lines suppressed TLS formation and the immune response. Peri-tumoral administration of the formulation successfully generated TLSs at the tumor site in a lung cancer CDX model in NSG mice. Intriguingly, they syngeneic lung cancer model showed reduced tumor volume and improved survival in the treated cohort. Conclusion Our findings demonstrate the feasibility of an injectable TLS therapeutic to overcome tumor-mediated immune suppression and generate a potent anti-tumor immune response. This approach has the potential to improve patient survival in challenging solid tumors. Further preclinical development of this promising therapy is ongoing. Funding Source ARPA-H Sprint for Women’s Health award Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Sturge-Weber syndrome (SWS), a neurocutaneous disorder, is characterized by capillary malformations (CM) in the skin, brain, and eyes. Patients may suffer from seizures, strokes, and glaucoma, and only symptomatic treatment is available. CM are comprised of enlarged vessels with endothelial cells (ECs) and disorganized mural cells. Our recent finding indicated that the R183Q mutation in ECs leads to heightened signaling through phospholipase Cβ3 and protein kinase C, leading to increased angiopoietin-2 (ANGPT2). Furthermore, knockdown of ANGPT2, a crucial mediator of pro-angiogenic signaling, inflammation, and vascular remodeling, in EC-R183Q rescued the enlarged vessel phenotype in vivo. This prompted us to look closer at the microenvironment in CM-affected vascular beds. We analyzed multiple brain histological sections from patients with GNAQ-R183Q CM and found enlarged vessels devoid of mural cells along with increased macrophage-like cells co-expressing MRC1 (CD206, a mannose receptor), CD163 (a scavenger receptor and marker of the monocyte/macrophage lineage), CD68 (a pan macrophage marker), and LYVE1 (a lymphatic marker expressed by some macrophages). These macrophages were not found in non-SWS control brain sections. To investigate the mechanism of increased macrophages in the perivascular environment, we examined THP1 (monocytic/macrophage cell line) cell adhesion to EC-R183Q versus EC-WT under static and laminar flow conditions. First, we observed increased THP1 cell adhesion to EC-R183Q compared to EC-WT under static conditions. Next, using live cell imaging, we found THP1 cell adhesion to EC-R183Q was dramatically increased under laminar flow conditions and could be inhibited by anti-ICAM1. ICAM1, an endothelial cell adhesion molecule required for leukocyte adhesion, was strongly expressed in the endothelium in SWS brain histological sections, suggesting a mechanism for recruitment of macrophages. In conclusion, our findings demonstrate that macrophages are an important component of the perivascular environment in CM suggesting they may contribute to the CM formation and SWS disease progression.

The fallopian tube serves as a sperm reservoir and it is the site where the oocytes become fertilized. Here, we describe development of an organ-on-a-chip microfluidic model of the fallopian tube (FT Chip) lined by primary human epithelial cells and stromal fibroblasts derived from the FT ampulla. Abundant tissue folds lined by hormone-responsive, epithelial cells resembling those seen in vivo formed on-chip, but not in epithelial organoids cultured in gel cultures. Comparative time-resolved analysis of human sperm versus oocyte-sized microparticles introduced into the epithelial channel in the presence of estradiol revealed that sperm movement was significantly reduced, while the oocyte-sized particles increased, relative to movements in acellular chips. When the non-hormonal contraceptive TDI-11861 was administered to the chip, dose-dependent inhibition of human sperm motility was detected. Thus, this FT Chip may offer a human preclinical tool to study FT physiology and assess the efficacy and mechanism of action of contraceptives.

Predicting the efficacy and toxicity of mRNA vaccines remains challenging. We describe an in vitro human model that replicates immune responses to lipid nanoparticle (LNP)-based mRNA vaccines upon intramuscular injection. Vaccines are administered to human skeletal myoblasts and antigen-presenting cells (APCs). Non-amplifying mRNA (NAM) vaccines directly induce antigen expression in APCs, whereas self-amplifying mRNA (SAM) vaccines require muscle cell-APC contact. The transfer of APCs and soluble factors to a microfluidic human lymphoid follicle chip (LF Chip), to mimic lymphatic drainage, induces LF expansion, de novo immunoglobulin G (IgG) production against a naive antigen, and cytokine release, with responses varying by LNP type. Similar vaccination of LF chips with Moderna vaccine against SARS-CoV-2 spike recall antigen generates neutralizing antibodies and induces somatic hypermutation (SHM). These studies offer an all-human alternative for evaluating vaccine-induced immunity, limiting the need for non-human primates and accelerating vaccine development.

Flavonoids, a diverse class of polyphenols found in many plant-based foods, are increasingly recognized for their health benefits, including antioxidant and anti-inflammatory activities. Recent evidence links higher dietary flavonoid diversity with reduced all-cause mortality and improved outcomes in chronic diseases. Yet, due to concerns about non-specific binding, their exclusion from drug development pipelines has limited deeper mechanistic understanding. Here, we show how flavonoids may promote cellular resilience by forming supramolecular assemblies that interact with proteins. Using molecular dynamics simulations (MDS) and in vitro assays, we found that different flavonoids self-assemble into ordered structures that influence protein structural dynamics and fiber formation. These structures have differential effects on enzyme activity and cell viability under stress. The ability to undergo supramolecular assembly may be important as flavonoids protect human cells against ultraviolet radiation-induced damage through a non-antioxidant mechanism. These findings suggest that supramolecular assembly and structural heterogeneity of flavonoids may underlie their diverse bioactivities and help to explain how the diversity of dietary flavonoids can support adaptive changes in cellular biochemistry, enhance resilience to environmental stressors, and improve human health.

Development of oral, broad-spectrum therapeutics targeting SARS-CoV-2, its variants, and related coronaviruses could curb the spread of COVID-19 and avert future pandemics. We created a novel computational discovery pipeline that employed molecular dynamics simulation (MDS), artificial intelligence (AI)-based docking predictions, and medicinal chemistry to design viral entry inhibitors that target a conserved region in the SARS-CoV-2 spike (S) protein that mediates membrane fusion. DrugBank library screening identified the orally available, FDA-approved AXL kinase inhibitor bemcentinib as binding this site and we demonstrated that it inhibits viral entry in a kinase-independent manner. Novel analogs predicted to bind to the same region and disrupt S protein conformational changes were designed using MDS and medicinal chemistry. These compounds significantly suppressed SARS-CoV-2 infection and blocked the entry of S protein-bearing pseudotyped α,β,γ,δ,ο variants as well as SARS CoV and MERS-CoV in human ACE2-expressing or DPP4-expressing cells more effectively than bemcentinib. When administered orally, the optimized lead compound also significantly inhibited SARS-CoV2 infection in mice. This computational design strategy may accelerate drug discovery for a broad range of applications.

Inflammatory bowel disease (IBD) patients exhibit compromised intestinal barrier function and decreased mucus accumulation, as well as increased inflammation, fibrosis, and cancer risk, with symptoms often being exacerbated in women during pregnancy. Here, we show that these IBD hallmarks can be replicated using human Organ Chips lined by IBD patient-derived colon epithelial cells interfaced with matched fibroblasts cultured under flow. Use of heterotypic tissue recombinants revealed that IBD fibroblasts are the primary drivers of multiple IBD symptoms. Inflammation and fibrosis are accentuated by peristalsis-like motions in IBD Chips and when exposed to pregnancy-associated hormones in female IBD Chips. Carcinogen exposure also increases inflammation, gene mutations, and chromosome duplication in IBD Chips, but not in Healthy Chips. These data enabled by human Organ Chip technology suggest that the intestinal stroma, sex hormones, and peristalsis-associated mechanical deformations play a key role in driving inflammation, fibrosis, and disease progression in male and female IBD patients.

This study explores the protective role of cervicovaginal mucus in maintaining vaginal health, particularly in relation to bacterial vaginosis (BV), using organ chip technology. By integrating human Cervix and Vagina Chips, we demonstrated that cervical mucus significantly reduces inflammation and epithelial damage caused by a dysbiotic microbiome commonly associated with BV. Proteomic analysis of the Vagina Chip, following exposure to mucus from the Cervix Chip, revealed differentially abundant proteins, suggesting potential biomarkers and therapeutic targets for BV management. Our findings highlight the essential function of cervical mucus in preserving vaginal health and underscore the value of organ chip models for studying complex interactions within the female reproductive tract. This research provides new insights into the mechanisms underlying vaginal dysbiosis and opens avenues for developing targeted therapies and diagnostic tools to enhance women’s reproductive health.

CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.

Current SARS-CoV-2 vaccines have demonstrated robust induction of neutralizing antibodies and CD4+ T cell activation, however CD8+ responses are variable, and the duration of immunity and protection against variants are limited. Here we repurposed our DNA origami vaccine nanotechnology, DoriVac, for targeting infectious viruses, namely SARS-CoV-2, HIV, and Ebola. The DNA origami nanoparticle, conjugated with infectious-disease-specific heptad repeat 2 (HR2) peptides, which act as highly conserved antigens, and CpG adjuvant at precise nanoscale spacing, induced neutralizing antibodies, Th1 CD4+ T cells, and CD8+ T cells in naïve mice, with significant improvement over a bolus control. Pre-clinical studies using lymph-node-on-a-chip systems validated that DoriVac, when conjugated with antigenic peptides or proteins, induced promising cellular and humoral immune responses in human cells. Moreover, DoriVac bearing full-length SARS-CoV-2 spike protein achieved immune responses comparable to current mRNA vaccine platforms while potentially reducing storage constraints. These results suggest that DoriVac holds potential as a versatile, modular vaccine platform, capable of inducing both humoral and cellular immunities, underscoring its potential utility in addressing future pandemics.

The regular t4 workshops on biology-inspired microphysiological systems (MPS) have become a reliable benchmark for assessing fundamental scientific, industrial, and regulatory trends in the MPS field. The 2023 workshop participants concluded that MPS technology as used in academia has matured significantly, as evidenced by the steadily increasing number of high-quality research publications, but that broad industrial adoption of MPS has been slow. Academic research using MPS is primarily aimed at accurately recapitulating human biology in MPS-based organ models to enable breakthrough discoveries. Examples of these developments are summarized in the report. In addition, we focus on key challenges identified during the previous workshop. Bridging gaps between academia, regulators, and industry is addressed. We also comment on overcoming barriers to trust and acceptance of MPS-derived data-the latter being particularly important in a regulatory environment. The status of implementation of the recommendations detailed in the 2020 report was reviewed. It is concluded that communication between stakeholders has improved significantly, while the recommendations related to regulatory acceptance still need to be implemented. Participants noted that the remaining challenges for increased translation of these technologies into industrial use and regulatory decision-making will require further efforts on well-defined context of use qualifications, together with increased standardization. This will make MPS data more reliable and ultimately make these novel tools more economically sustainable. The long-term roadmap from the 2015 workshop was critically reviewed and updated. Recommendations for the next period and an outlook conclude the report. Plain language summary The regular t4 workshops on biology-inspired microphysiological systems (MPS) have become a reliable benchmark for assessing trends in the field. Participants at the 2023 workshop concluded that the technology as used in academia has matured significantly, but that broad industry adoption of MPS has been slow. The primary goal of academic research is to accurately recapitulate human biology in MPS-based organ models to enable breakthrough discoveries. Participants commented on overcoming barriers to trust and acceptance of MPS-derived data, the latter being particularly important in a regulatory environment. They reviewed the status of implementation of the recommendations detailed in the 2020 report and conclude that communication between stakeholders has improved significantly, while recommendations related to regulatory acceptance still need to be implemented. Participants highlighted the need for further qualification and standardization. The long-term roadmap from the 2015 workshop was updated. Recommendations for the next period conclude the report.

BACKGROUND:Many neurodevelopmental genetic disorders, such as Rett syndrome, are caused by a single gene mutation but trigger changes in expression of numerous genes. This impairs functions of multiple organs beyond the central nervous system (CNS), making it difficult to develop broadly effective treatments based on a single drug target. This is further complicated by the lack of sufficiently broad and biologically relevant drug screens, and the inherent complexity in identifying clinically relevant targets responsible for diverse phenotypes that involve multiple organs. METHODS:Here, we use computational drug prediction that combines artificial intelligence, human gene regulatory network analysis, and in vivo screening in a CRISPR-edited, Xenopus laevis tadpole model of Rett syndrome to carry out target-agnostic drug discovery. Four-week-old MeCP2-null male mice expressing the Rett phenotype are used to validate the therapeutic efficacy. RESULTS:This approach identifies the FDA-approved drug, vorinostat, which broadly improves both CNS and non-CNS (e.g., gastrointestinal, respiratory, inflammatory) abnormalities in X. laevis and MeCP2-null mice. To our knowledge, this is the first Rett syndrome treatment to demonstrate pre-clinical efficacy across multiple organ systems when dosed after the onset of symptoms. Gene network analysis also reveals a putative therapeutic mechanism for the cross-organ normalizing effects of vorinostat based on its impact on acetylation metabolism and post-translational modifications of microtubules. CONCLUSIONS:Although vorinostat is an inhibitor of histone deacetylases (HDAC), it unexpectedly reverses the Rett phenotype by restoring protein acetylation across hypo- and hyperacetylated tissues, suggesting its activity is based on a previously unknown therapeutic mechanism.

Pathological conditions of the cervix ranging from cervical cancer to structural dysfunction associated with preterm labor all have limited treatment options. Thus, there is a need for physiologically relevant preclinical models that recapitulate the structure and function of this human organ. Here, we describe a protocol for engineering and studying a highly functional in vitro model of the human cervix that is composed of a commercially available, dual-channel, microfluidic, organ-on-a-chip (Organ Chip) device lined by primary cervical epithelial (CE) cells interfaced across a porous membrane with cervical stromal cells. The provision of dynamic and customized media flow through both the epithelial and stromal compartments results in cell growth and differentiation, including the accumulation of a thick mucus layer overlying the epithelium. The resulting model closely mimics the structure, epithelial barrier, mucus composition and structure, and biochemical properties of the in vivo human cervix, as well as its responsiveness to female hormones, pH, and microbiome. This Cervix Chip protocol also includes noninvasive techniques for longitudinal monitoring of the live 3D tissue model. The Cervix Chip offers a powerful preclinical platform for replicating in vivo cervical physiology, studying disease mechanisms, and facilitating the development of new therapeutics and diagnostics. Key features • Creates a functional and physiologically responsive 3D tissue model of the human cervix including a living epithelial-stromal interface. • Enables longitudinal and endpoint analysis of the epithelial and stromal environment and their respective secretions independently. • Allows extended clinically relevant studies, such as assessment of tissue barrier function and mucus production as well as co-culture with microbiome and pathogens. • Uses a commercially available dual-channel microfluidic chip and automated culture system (ZoëTM Culture Module, Emulate Inc., USA).

Cervical dysfunction, a major contributor to preterm labor and neonatal mortality, remains poorly understood due to the absence of physiologically relevant human models. Here, we show that a microfluidic human Endocervix Chip lined by primary endocervical epithelium interfaced with stromal cells and cultured under pregnancy-like hormonal conditions recapitulates key aspects of the biology of the endocervix, including formation of a mucus plug-like structure with antimicrobial properties. Culturing a dysbiotic cervico-vaginal microbiome on-chip increased secretion of pro-inflammatory cytokines observed in patients with preterm labor and enhanced production of matrix metalloproteinases (MMPs) that degrade stromal extracellular matrix (ECM). Perfusion with inflammatory cytokines at clinically relevant concentrations altered cervical mucus composition, upregulated prostaglandin-endoperoxide synthase 2 expression, increased MMP secretion, and reduced collagen production, which together drive dissolution of the stromal ECM and promote cervical ripening. Addition of circulating peripheral blood mononuclear cells (PBMCs) amplified these effects. Administration of a clinically approved drug for prevention of preterm labor that the Food and Drug Administration (FDA) recently deemed ineffective was also found to be inactive in the chip, while an approved therapeutic antagonist of the IL-1 receptor successfully protected against cervical dysfunction in this model. These findings demonstrate that IL-1 acts directly on human cervical tissues to promote changes associated with initiation of labor and that primary human endocervix chips may represent a useful preclinical model for studies on cervical dysfunction associated with preterm birth. One Sentence Summary Human endocervix chip modeling of cervical dysfunction suggests that IL-1 receptor antagonists may prevent preterm labor. ### Competing Interest Statement D.E.I. holds equity in Emulate, chairs its scientific advisory board and is a member of its board of directors. The remaining authors declare no competing interests. Bill & Melinda Gates Foundation, https://ror.org/0456r8d26, INV-035977

Lung manifestations of chronic obstructive pulmonary disease (COPD) are often exacerbated by influenza A virus infections; however, the underlying mechanisms remain largely unknown, and hence therapeutic options are limited. Using a physiologically relevant human lung airway-on-a-chip (Airway Chip) microfluidic culture model lined with human airway epithelium from COPD or healthy donors interfaced with pulmonary microvascular endothelium, we observed that Airway Chips lined with COPD epithelium exhibit an increased sensitivity to influenza virus infection, as is observed clinically in COPD patients. Differentiated COPD airway epithelial cells display increased inflammatory cytokine production, barrier function loss, and mucus accumulation upon virus infection. Transcriptomic analysis revealed gene expression profiles characterized by upregulation of serine proteases that may facilitate viral entry and downregulation of interferon-related genes associated with antiviral immune responses. Importantly, treatment of influenza virus-infected COPD epithelium with a protease inhibitor, nafamostat, ameliorated the disease phenotype, as evidenced by dampened viral replication, reduced mucus accumulation, and improved tissue barrier integrity. These findings suggest that targeting host serine proteases may represent a promising therapeutic avenue against influenza-afflicted COPD exacerbations.

BACKGROUND:Drug delivery to the brain, including to specific diseased brain cells and regions, remains a formidable challenge for the development of effective brain disease treatments. The blood-brain barrier prevents most therapeutics from reaching the brain. We are developing a diverse panel of brain shuttles to enhance brain uptake and allow targeting to specific cell types and regions within the brain. METHODS:We have developed several panels of brain shuttles, each binding a receptor that is highly abundant on brain microvascular endothelial cells. To rank their transcytosis efficiency, the shuttles were fused to trastuzumab or another IgG with no mouse target. We performed brain uptake assays of each shuttle-drug fusion in humanized transcytosis receptor knock-in mice. In each mouse line, the extracellular domain of the mouse TfR, CD98hc, or Target 3 receptor was replaced with its human counterpart. Fusion drug concentrations were assayed in parenchymal brain lysates at time points up to 4 weeks, and their cellular distribution was visualized by immunostaining in brain tissues. Additional shuttle characterizations included human and cynomolgus monkey affinities, epitope binning, sequence liabilities and hematology analysis. RESULTS:The lead anti-TfR and anti-CD98hc brain shuttles increased parenchymal brain uptake of IgGs by 10-fold or more compared to IgG alone, and showed differentiated brain pharmacokinetics and distribution. The anti-hTfR brain shuttles crossed the BBB and reached brain parenchyma in 1 day, while the anti-hCD98hc shuttles showed a slower and more sustained brain exposure for up to 4 weeks, resulting in higher AUC. Anti-hTfR-IgG fusion molecules show a prominent neuronal staining pattern, while anti-hCD98hc-IgG fusions show diffuse staining in the interstitial space. Target 3 shuttles show slow and sustained brain uptake and prominent neuronal staining. In addition, we evaluated the brain uptake of two published anti-IGF1R antibodies. We confirmed binding of anti-IGF-1R-trastuzumab fusion molecules to proteins and cells, but we did not observe significantly increased brain uptake in normal mice. CONCLUSION:We have developed several panels of efficient antibody brain shuttles. The lead anti-hTfR, anti-hCD98hc and anti-Target 3 brain shuttles can be tailored to improve the brain uptake and target engagement of diverse payloads.

BACKGROUND:Esophageal adenocarcinoma (EAC) is the sixth most deadly cancer worldwide, with increasing incidence in North America. As no targeted therapy or immunotherapy has revolutionized the management of EAC, chemotherapy is the only standard of care. Most patients with EAC experience poor outcomes because of the inherent or acquired resistance to chemotherapy. METHODS:Adapting a patient-centered approach, we leveraged a microfluidic cell culture technology platform (Emulate), organoids derived from treatment-naive patient tumors or adjacent normal tissues, and patient-matched cancer-associated or normal fibroblasts respectively, to develop a novel, physiologically relevant, high-fidelity preclinical esophagus-on-a-chip model. H&E, immunofluorescence staining, live/dead assay, LDH assay, and ELISA-based detection of tumor biomarkers were used to assess treatment responses. RESULTS:Each patient-specific stroma-inclusive microfluidic esophageal adenocarcinoma on-a-chip (EAC chip) faithfully recreates the tumor-stroma interface while preserving the full diversity of two cell types (epithelia and fibroblasts), genetic landscapes and histological architecture of the source tumors. EAC chips also accurately predict the response to neoadjuvant chemotherapy (NACT) within a clinically useful timeframe (approx. 12 days). A docetaxel-based triplet chemotherapy regimen matched with the treatment of the source patient was successfully perfused through the interstitial space within this model. Therefore, EAC chips more accurately recapitulate inpatient pathological and objective responses than the corresponding static 3D-organoid-only cultures. CONCLUSIONS:Overall, this model is an effective tool for predicting patients' responses to chemotherapy and testing tumor- or stroma-targeted alternative therapies. Moreover, these high-fidelity, low-throughput EAC chips effectively complement high-throughput PDO culture-based drug testing and provide improved insights into drug efficacy before human studies.

Mass cytometry uses metal-isotope-tagged antibodies to label targets of interest, which enables simultaneous measurements of ~50 proteins or protein modifications in millions of single cells, but its sensitivity is limited. Here, we present a signal amplification technology, termed Amplification by Cyclic Extension (ACE), implementing thermal-cycling-based DNA in situ concatenation in combination with 3-cyanovinylcarbazole phosphoramidite-based DNA crosslinking to enable signal amplification simultaneously on >30 protein epitopes. We demonstrate the utility of ACE in low-abundance protein quantification with suspension mass cytometry to characterize molecular reprogramming during the epithelial-to-mesenchymal transition as well as the mesenchymal-to-epithelial transition. We show the capability of ACE to quantify the dynamics of signaling network responses in human T lymphocytes. We further present the application of ACE in imaging mass cytometry-based multiparametric tissue imaging to identify tissue compartments and profile spatial aspects related to pathological states in polycystic kidney tissues.

Development of coating technologies for electrochemical sensors that consistently exhibit antifouling activities in diverse and complex biological environments over extended time is vital for effective medical devices and diagnostics. Here, we describe a micrometer-thick, porous nanocomposite coating with both antifouling and electroconducting properties that enhances the sensitivity of electrochemical sensors. Nozzle printing of oil-in-water emulsion is used to create a 1 micrometer thick coating composed of cross-linked albumin with interconnected pores and gold nanowires. The layer resists biofouling and maintains rapid electron transfer kinetics for over one month when exposed directly to complex biological fluids, including serum and nasopharyngeal secretions. Compared to a thinner (nanometer thick) antifouling coating made with drop casting or a spin coating of the same thickness, the thick porous nanocomposite sensor exhibits sensitivities that are enhanced by 3.75- to 17-fold when three different target biomolecules are tested. As a result, emulsion-coated, multiplexed electrochemical sensors can carry out simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid, antigen, and host antibody in clinical specimens with high sensitivity and specificity. This thick porous emulsion coating technology holds promise in addressing hurdles currently restricting the application of electrochemical sensors for point-of-care diagnostics, implantable devices, and other healthcare monitoring systems.

ABSTRACT Background The cervicovaginal mucus that coats the upper surface of the vaginal epithelium is thought to serve as a selective barrier that helps to clear pathogens, however, its role in modulating the physiology and pathophysiology of the human vagina is poorly understood. Bacterial vaginosis (BV), a common disease of the female reproductive tract that increases susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and both maternal and neonatal infections is characterized by the presence of a wide array of strict and facultative anaerobes, often including Gardnerella vaginalis . Objective To assess the role of cervical mucus in preventing dysbiosis-associated complications and preserving vaginal health. Study Design To better understand the role of cervicovaginal mucus in vaginal health, we used human organ-on-a-chip (Organ Chip) microfluidic culture technology to analyze the effects of cervical mucus produced in a human Cervix Chip when transferred to a human Vagina Chip BV model. Both chips are lined by primary human organ-specific (cervical or vaginal) epithelium interfaced with organ-specific stromal fibroblasts. Results Our data show that mucus-containing effluents from Cervix Chips protect Vagina Chips from inflammation and epithelial cell injury caused by co-culture with dysbiotic microbiome containing G. vaginalis . Proteomic analysis of proteins produced by the Vagina Chip following treatment with the Cervix Chip mucus also revealed a collection of differentially abundant proteins that may contribute to the vaginal response to dysbiotic microbiome, which could represent potential diagnostic biomarkers or therapeutic targets for management of BV. Conclusions This study highlights the importance of cervical mucus in control of human vaginal physiology and pathophysiology, and demonstrates the potential value of Organ Chip technology for studies focused on health and diseases of the female reproductive tract.