Real-time Confocal Microscopy Research Articles

Abstract 5226: A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology

Abstract We developed a screening platform for immune oncology using an assay based on automated image guided injection of tumoroids in wells of multi-well plates preloaded with a collagen-rich ECM. The identical x-y-z position and size of the ECM-embedded tumoroids in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. The model is used to generate quantitative data for T cell recruitment to tumoroids and killing of tumoroids by T cells. We have applied this to screening a panel of CD3:Her2 bispecific antibodies (BsAb) binding with different affinities to CD3 or Her2 or displaying high affinity interactions with different epitopes on Her2. Exposure to fresh, non-activated peripheral blood mononuclear cell (PBMC) derived T cells shows an initial phase of random T cell movement throughout the ECM followed by a BsAb-dependent phase of active T cell recruitment to tumoroids (day 2-4) and a subsequent phase of tumoroid killing (day 4-6). We show that the wave of T cell recruitment following initial T cell-tumoroid contact involves chemotactic signaling. Decreased affinity at the Her2 or CD3 arm can be compensated for by increasing BsAb concentrations. However, we detect major differences in efficacy for different high affinity epitopes. I.e., of two BsAbs interacting with high affinity with distinct Her2 epitopes and each causing effective tumor cell killing in 2D co-culture, only one was able to trigger a wave of T cell recruitment and subsequent tumoroid killing in 3D. We have applied the same setup to testing the efficacy of T cells expressing engineered TCRs aimed at application in adoptive T-cell therapy. Kinetics of experiments using these activated engineered T cells are considerably shorter (24 hours instead of ~6 days) but show a similar pattern of T cell recruitment and subsequent tumoroid killing. We demonstrate successful generation of quantitative data for T cell recruitment and tumoroid killing for engineered T cells targeting tumor antigens expressed on TNBC and uveal melanoma tumoroids. Citation Format: Erik H. J. Danen. A 3D ECM embedded tumoroid platform for testing antibody drugs and engineered TCRs for immune oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5226.

Abstract 737: Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling

Abstract Chronic fibrosis can lead to organ failure and is also associated with cancer, where the tumor is interpreted as a chronic wound, and the fibrotic tumor environment drives disease progression. Fibrosis occurs in an inflammatory environment where macrophages are activated, and fibroblasts transdifferentiate into myofibroblasts. Activated macrophages stimulate the myofibroblasts to produce excessive amounts of collagen-rich ECM. Changes in proteolytic activity and high contractile forces that myofibroblasts apply onto their tissue environment, further lead to extensive remodeling of the healthy tissue matrix into a scar matrix. Similar activity is displayed by cancer-associated fibroblasts (CAFs) in tumors. Intervening with the excessive ECM remodeling by activated fibroblasts represents a candidate therapeutic strategy to normalize the architecture of fibrotic and malignant tissues. We developed a screening platform using an assay based on automated image guided injection of clusters of fibroblasts in wells of multi-well plates preloaded with a collagen-rich ECM network. The identical x-y-z position, spacing, and size of the ECM-embedded fibroblast clusters in each well facilitates automated real time confocal microscopy and quantitative image analysis algorithms. We use this setup for automated quantitative analysis of ECM remodeling activity of CAFs obtained from early-stage colorectal cancer patients. We find that CAFs from these early-stage lesions display increased ECM remodeling capacity as compared to patient matched normal fibroblasts. We then use the same setup to screen a series of compounds for their ability to attenuate ECM remodeling triggered by TGFb-activated primary human fibroblasts. We detect known and novel pathways, and we identify small molecule inhibitors with a promising efficacy versus toxicity profile. Lastly, we detect a previously unknown concentration-dependent side effect of ALK5 inhibitors that leads to the identification of a novel profibrotic pathway and indicates that caution is warranted for the clinical application of ALK5 inhibitors. Citation Format: Erik H. J. Danen. Arrays of 3D ECM-embedded fibroblast clusters for characterization of ECM remodeling by primary cancer associated fibroblasts and for evaluation of drugs attenuating fibrotic ECM remodeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 737.

Advanced oxidation process/coagulation coupled with membrane distillation (AOP/Coag-MD) for efficient ammonia recovery: Elucidating biofouling control performance and mechanism

The inadequate understanding of the biofouling formation mechanism and the absence of effective control have inhibited the commercial application of membrane distillation (MD). In this study, an advanced oxidation process (AOP)/coagulation-coupled (Coag) membrane distillation system was proposed and exhibited the potential for MD ammonia recovery (recovery rate: 94.1%). Extracellular polymeric substances (EPS) and soluble microbial products (SMP) components such as humic acid and tryptophan-like proteins were disrupted and degraded in the digestate. The curtailment and sterilizing efficiency of AOP on biofilm growth was also verified by optical coherence tomography (OCT) in situ real-time monitoring and confocal laser scanning microscopy (CLSM). Peroxymonosulfate (PMS) was activated to generate sulfate (SO4•-) and hydroxyl radicals (HO•), which altered the microbial community. After oxidative treatment, 16 S rRNA sequencing indicated that the dominant phylum of the microbial community evolved into Firmicutes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that free radicals produced by PMS could disrupt cells' signaling molecules and interactions. In conjunction with these analyses, the mechanisms of response to free radical attack by Gram-negative bacteria, Gram-positive bacteria, and fungi were revealed. This research provided new insights into the field of membrane fouling control for membrane technology resource recovery processes, broadening the impact of AOP applications on microbiological response and fate in the environment.

Prediction of Biliary Complications After Human Liver Transplantation Using Hyperspectral Imaging and Convolutional Neural Networks: A Proof-of-concept Study.

Biliary complications (BCs) negatively impact the outcome after liver transplantation. We herein tested whether hyperspectral imaging (HSI) generated data from bile ducts (BD) on reperfusion and machine learning techniques for data readout may serve as a novel approach for predicting BC. Tissue-specific data from 136 HSI liver images were integrated into a convolutional neural network (CNN). Fourteen patients undergoing liver transplantation after normothermic machine preservation served as a validation cohort. Assessment of oxygen saturation, organ hemoglobin, and tissue water levels through HSI was performed after completing the biliary anastomosis. Resected BD segments were analyzed by immunohistochemistry and real-time confocal microscopy. Immunohistochemistry and real-time confocal microscopy revealed mild (grade I: 1%-40%) BD damage in 8 patients and moderate (grade II: 40%-80%) injury in 1 patient. Donor and recipient data alone had no predictive capacity toward BC. Deep learning-based analysis of HSI data resulted in >90% accuracy of automated detection of BD. The CNN-based analysis yielded a correct classification in 72% and 69% for BC/no BC. The combination of HSI with donor and recipient factors showed 94% accuracy in predicting BC. Deep learning-based modeling using CNN of HSI-based tissue property data represents a noninvasive technique for predicting postoperative BC.

In vitro inhibition of Pseudomonas aeruginosa PAO1 biofilm formation by DZ2002 through regulation of extracellular DNA and alginate production.

Pseudomonas aeruginosa (P. aeruginosa) is a common pathogen associated with biofilm infections, which can lead to persistent infections. Therefore, there is an urgent need to develop new anti-biofilm drugs. DZ2002 is a reversible inhibitor that targets S-adenosylhomocysteine hydrolase and possesses anti-inflammatory and immune-regulatory activities. However, its anti-biofilm activity has not been reported yet. Therefore, we investigated the effect of DZ2002 on P. aeruginosa PAO1 biofilm formation by crystal violet staining (CV), real-time quantitative polymerase chain reaction (RT-qPCR) and confocal laser scanning microscopy (CLSM). The results indicated that although DZ2002 didn't affect the growth of planktonic PAO1, it could significantly inhibit the formation of mature biofilms. During the inhibition of biofilm formation by DZ2002, there was a parallel decrease in the synthesis of alginate and the expression level of alginate genes, along with a weakening of swarming motility. However, these results were unrelated to the expression of lasI, lasR, rhII, rhIR. Additionally, we also found that after treatment with DZ2002, the biofilms and extracellular DNA content of PAO1 were significantly reduced. Molecular docking results further confirmed that DZ2002 had a strong binding affinity with the active site of S-adenosylhomocysteine hydrolase (SahH) of PAO1. In summary, our results indicated that DZ2002 may interact with SahH in PAO1, inhibiting the formation of mature biofilms by downregulating alginate synthesis, extracellular DNA production and swarming motility. These findings demonstrate the potential value of DZ2002 in treating biofilm infections associated with P. aeruginosa.

How to Build Plasmon-Driven Molecular Jackhammers that Disassemble Cell Membranes and Cytoskeletons in Cancer.

Plasmon-driven molecular machines with ultrafast motion at the femtosecond scale are effective for the treatment of cancer and other diseases. It is recently shown that cyanine dyes act as molecular jackhammers (MJH) through vibronic (vibrational and electronic mode coupling) driven activation that causes the molecule to stretch longitudinally and axially through concerted whole molecule vibrations. However, the theoretical and experimental underpinnings of these plasmon-driven motions in molecules are difficult to assess. Here the use of near-infrared (NIR) light-activated plasmons in a broad array of MJH that mechanically disassemble membranes and cytoskeletons in human melanoma A375 cells is described. The characteristics of plasmon-driven molecular mechanical disassembly of supramolecular biological structures are observed and recorded using real-time fluorescence confocal microscopy. Molecular plasmon resonances in MJH are quantified through a new experimental plasmonicity index method. This is done through the measurement of the UV-vis-NIR spectra in various solvents, and quantification of the optical response as a function of the solvent polarity. Structure-activity relationships are used to optimize the synthesis of plasmon-driven MJH, applying them to eradicate human melanoma A375 cells at low lethal concentrations of 75nm and 80mWcm-2 of 730nm NIR-light for 10min.

The Integrin Adaptor Kindlin-3 Is a Gatekeeper for Germinal Center Responses and the Development of Chronic Lymphocytic Leukemia

Background: High affinity antibody production occurs in the germinal centers (GCs), which are segregated into a dark zone (DZ) and a light zone (LZ). GC B cells proliferate in the DZ and encounter follicular dendritic cells in the LZ. Cell trafficking between the two zones is regulated by CXCR4 (DZ) and CXCR5 (LZ) signaling. Integrins may regulate B cell positioning and B cell immune synapse formation. Recently, we have shown that the integrin adaptor kindlin-3 is not only required for integrin activation in B cells, but unexpectedly also acts as a brake on CXCR5 responsiveness. Kindlin-3 thereby prevents B cell hyperactivation and differentiation into GC-like B cells in absence of antigen. 1 Here we investigate the impact of kindlin-3 on GC responses upon antigen challenging and on malignant transformation. Methods: We developed a B cell-specific knockout mouse model (mb1Cre Fermt3fl/fl (K3ΔB)) in which kindlin-3 ( Fermt3) was knocked out in all B cells. K3ΔB and control wild-type (WT) mice were challenged with sheep red blood cells (SRBC). GC formation and immunoglobulin (Ig) production were analyzed by flow cytometry. Ex vivo B cell proliferation and Ig class switching formation assays were performed. We investigated the immune synapse formation of WT and kindlin-3-deficient B cells using a biomimetic model involving a planar artificial lipid bilayer containing GPI-linked integrin ligands and bound fluorescently labeled surrogate antigens. The formation of immune synapse and central antigen aggregation in the immune synapse were analyzed by real-time interference reflection microscopy and laser scanning confocal microscopy. To investigate the contribution of kindlin-3 in a model of mature B cell malignancy, we used TCL1 transgenic (TCL1) mice resembling chronic lymphocytic leukemia (CLL) to generate the mb1Cre Fermt3fl/fl Tcl1 (TCL1-K3ΔB) mouse model. Tumor burden in TCL1-K3ΔB and TCL1 control mice was monitored by flow cytometry over time and at sacrifice using CD19/CD5 antibodies. B cell receptor (BCR) signaling of CLL cells was analyzed using Phosphoflow. Results: K3∆B mice immunized with SRBC exhibited defective GC responses (30% reduction in GC-B cells compared to WT) and antibody production (49% reduction in SRBC-specific IgG2b production in serum compared to WT) on day 7 after immunization. Ex vivo, kindlin-3-deficient B cells were unable to form immune synapses (Figure 1A), to proliferate, and to switch antibody classes. The tumors in TCL1 mice started in the peritoneal cavity and subsequently spread to the spleen, blood and bone marrow. TCL1-K3ΔB mice presented with an almost complete abolished leukemia onset in the peritoneal cavity (92% less tumor burden in TCL1-K3ΔB compared to control TCL1 mice) in 4-month-old mice (Figure 1B i). Moreover, tumor burden in blood (Figure 1B ii), spleen, and bone marrow (Figure 1B iii) was strongly reduced at the late stage of disease (11.5-month-old mice), with an 82% lower tumor burden in spleen and a 90% lower tumor burden in bone marrow of TCL1-K3ΔB mice compared to TCL1 control mice. In addition, kindlin-3-deficient CLL cells exhibited impaired phosphoAkt and phosphoSyk signaling upon BCR engagement. Summary/Conclusion: In summary, kindlin-3 maintains the balance between a state of B cell homeostasis and an effective antigenic response by acting as a gatekeeper of GC formation, and is substantial for leukemic onset in CLL. Reference: 1. Härzschel A, Li L, Krenn PW, et al. Kindlin-3 maintains marginal zone B cells but confines follicular B cell activation and differentiation. J Leukoc Biol. 2022 Apr;111(4):745-758.

Megakaryocyte-induced contraction of plasma clots: cellular mechanisms and structural mechanobiology

AbstractNonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbβ3–fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.

IFP35 aggravates Staphylococcus aureus infection by promoting Nrf2-regulated ferroptosis

IntroductionSerious Staphylococcus aureus (SA) infection is one of the most life-threatening diseases. Interferon-induced protein 35 (IFP35) is a pleiotropic factor that participates in multiple biological functions, however, its biological role in SA infection is not fully understood. Ferroptosis is a new type of regulated cell death driven by the accretion of free iron and toxic lipid peroxides and plays critical roles in tissue damage. Whether ferroptosis is involved in SA-induced immunopathology and its regulatory mechanisms remain unknown. ObjectivesWe aimed to determine the role and underlying mechanisms of IFP35 in SA-induced lung infections. MethodsSA infection models were established using wild-type (WT) and IFP35 knockout (Ifp35−/−) mice or macrophages. Histological analysis was performed to assess lung injury. Quantitative real-time PCR, western blotting, flow cytometry, and confocal microscopy were performed to detect ferroptosis. Co-IP and immunofluorescence were used to elucidate the molecular regulatory mechanisms. ResultsWe found that IFP35 levels increased in the macrophages and lung tissue of SA-infected mice. IFP35 deficiency protected against SA-induced lung damage in mice. Moreover, ferroptosis occurred and contributed to lung injury after SA infection, which was ameliorated by IFP35 deficiency. Mechanically, IFP35 facilitated the ubiquitination and degradation of nuclear factor E2-related factor 2 (Nrf2), aggravating SA-induced ferroptosis and lung injury. ConclusionsOur data demonstrate that IFP35 promotes ferroptosis by facilitating the ubiquitination and degradation of Nrf2 to exacerbate SA infection. Targeting IFP35 may be a promising approach for treating infectious diseases caused by SA.

A structure and knowledge-based combinatorial approach to engineering universal scFv antibodies against influenza M2 protein

BackgroundThe influenza virus enters the host via hemagglutinin protein binding to cell surface sialic acid. Receptor-mediated endocytosis is followed by viral nucleocapsid uncoating for replication aided by the transmembrane viral M2 proton ion channel. M2 ectodomain (M2e) is a potential universal candidate for monoclonal antibody therapy owing to its conserved nature across influenza virus subtypes and its importance in viral propagation.MethodsThe phage-displayed naive human antibody libraries were screened against the short stretch of the N-terminal 10-mer peptide (SLLTEVETPI) of the M2e. ELISA, BLI, and flow cytometry assays were used to examine scFv binding to M2e epitopes. The scFv crystal structures were determined to examine the nature of the interactions. The potencies of the scFvs against the influenza virus were demonstrated by real-time PCR and confocal microscopy imaging.ResultsThe four unique scFv clones were obtained from the scFv phage-display antibody libraries and shown to exhibit binding with the 10-mer conserved part of the M2e and with full-length M2 protein expressed on the HEK293T cells. The crystal structure of scFv AU1 with M2e peptide showed the peptide as a dimer in the parallel beta-sheet conformation bound at the interface of two scFv CDRs. The scFv AU1 significantly restricted the release of H1N1 virus progeny from the infected A549 cells.ConclusionThis structural and biochemical study showcased the binding of antibody scFv molecules with M2e peptide dimer, providing the structural insights for the function effect in terms of recognizing and restricting the release of new viral particles from an infected host cell.

Siglec-9 is an inhibitory receptor on human mast cells in vitro

Mast cell activation is critical for the development of allergic diseases. Ligation of sialic acid-binding immunoglobin-like lectins (Siglecs), such as Siglec-6, -7, and -8 as well as CD33, have been shown to inhibit mast cell activation. Recent studies showed that human mast cells express Siglec-9, an inhibitory receptor also expressed by neutrophils, monocytes, macrophages, and dendritic cells. We aimed to characterize Siglec-9 expression and function in human mast cells invitro. We assessed the expression of Siglec-9 and Siglec-9 ligands on human mast cell lines and human primary mast cells by real-time quantitative PCR, flow cytometry, and confocal microscopy. We used a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing approach to disrupt the SIGLEC9 gene. We evaluated Siglec-9 inhibitory activity on mast cell function by using native Siglec-9 ligands, glycophorin A(GlycA), and high-molecular-weight hyaluronic acid, a monoclonal antibody against Siglec-9, and coengagement of Siglec-9 with the high-affinity receptor for IgE (FcεRI). Human mast cells express Siglec-9 and Siglec-9 ligands. SIGLEC9 gene disruption resulted in increased expression of activation markers at baseline and increased responsiveness to IgE-dependent and IgE-independent stimulation. Pretreatment with GlycA or high-molecular-weight hyaluronic acid followed by IgE-dependent or -independent stimulation had an inhibitory effect on mast cell degranulation. Coengagement of Siglec-9 with FcεRI in human mast cells resulted in reduced degranulation, arachidonic acid production, and chemokine release. Siglec-9 and its ligands play an important role in limiting human mast cell activation invitro.

Dynamics of asymmetric membranes and interleaflet coupling as intermediates in membrane fusion

Membrane fusion is a tool to increase the complexity of model membrane systems. Here, we use silica nanoparticles to fuse liquid-disordered DOPC giant unilamellar vesicles (GUVs) and liquid-ordered DPPC:cholesterol (7:3) GUVs. After fusion, GUVs display large membrane domains as confirmed by fluorescence confocal microscopy. Laurdan spectral imaging of the membrane phases in the fused GUVs shows differences compared with the initial vesicles indicating some lipid redistribution between phase domains as dictated by the tie lines of the phase diagram. Remarkably, using real-time confocal microscopy we were able to record the dynamics of formation of asymmetric membrane domains in hemifused GUVs and detected interleaflet coupling phenomena by which the DOPC-rich liquid-disordered domains in outer monolayer modulates the phase state of the DPPC:cholesterol inner membrane leaflet which transitions from liquid-ordered to liquid-disordered phase. We find that internal membrane stresses generated by membrane asymmetry enhance the efficiency of full fusion compared with our previous studies on symmetric vesicle fusion. Furthermore, under these conditions, the liquid-disordered monolayer dictates the bilayer phase state of asymmetric membrane domains in >90% of observed cases. By comparison to the findings of previous literature, we suggest that the monolayer phase that dominates the bilayer properties could be a mechanoresponsive signaling mechanism sensitive to the local membrane environment.

High-Throughput Biofilm Assay to Investigate Bacterial Interactions with Surface Topographies

The specific topography of biomaterials plays an important role in their biological interactions with cells and thus the safety of medical implants. Antifouling materials can be engineered with topographic features to repel microbes. Meanwhile, undesired topographies of implants can cause complications such as breast implant-associated anaplastic large cell lymphoma (BIA-ALCL). While the cause of BIA-ALCL is not well understood, it is speculated that textured surfaces are prone to bacterial biofilm formation as a contributing factor. To guide the design of safer biomaterials and implants, quantitative screening approaches are needed to assess bacterial adhesion to different topographic surface features. Here we report the development of a high-throughput microplate biofilm assay for such screening. The assay was used to test a library of polydimethylsiloxane (PDMS) textures composed of varying sizes of recessive features and distances between features including those in the range of breast implant textures. Outliers of patterns prone to bacterial adhesion were further studied using real-time confocal fluorescence microscopy. The results from these analyses revealed that surface area itself is a poor predictor for adhesion, while the size and spacing of topographic features play an important role. This high-throughput biofilm assay can be applied to studying bacteria–material interactions and rational development of materials that inhibit bacterial colonization.

Investigating the mechanisms of peritoneal metastasis in gastric adenocarcinoma using a novel ex vivo peritoneal explant model

Gastric adenocarcinoma, commonly known as stomach cancer, has a predilection for metastasis to the peritoneum, which portends limited survival. The peritoneal metastatic cascade remains poorly understood, and existing models fail to recapitulate key elements of the interaction between cancer cells and the peritoneal layer. To explore the underlying cellular and molecular mechanisms of peritoneal metastasis, we developed an ex vivo human peritoneal explant model. Fresh peritoneal tissue samples were suspended, mesothelial layer down but without direct contact, above a monolayer of red-fluorescent dye stained AGS human gastric adenocarcinoma cells for 24 h, then washed thoroughly. Implantation of AGS cells within the explanted peritoneum and invasion beyond the mesothelial layer were examined serially using real-time confocal fluorescence microscopy. Histoarchitecture of the explanted peritoneum was preserved over 5 days ex vivo. Both implantation and invasion were suppressed by restoration of functional E-cadherin through stable transfection of AGS cells, demonstrating sensitivity of the model to molecular manipulation. Thus, our ex vivo human peritoneal explant model permits meaningful investigation of the pathways and mechanism that contribute to peritoneal metastasis. The model will facilitate screening of new therapies that target peritoneal dissemination of gastric, ovarian and colorectal cancer.

Systematic Mapping of Conduction Tissue Regions During Congenital Heart Surgery

PurposeDamage to the cardiac conduction system is a major risk of congenital cardiac surgery. Localization of the conduction system is commonly based on anatomic landmarks, which are variable in congenital heart diseases. We introduce a novel technique for identification of conduction tissue regions based on real-time fiberoptic confocal microscopy. DescriptionWe developed a fiberoptic confocal microscopy–based technique to document conduction tissue regions and deployed it in pediatric patients undergoing repair of common congenital heart defects. The technique applies clockface schematics for intraoperative documentation of the location of conduction tissue regions. EvaluationWe created clockface schematics for 11 patients with ventricular septal defects, 6 with tetralogy of Fallot, and 10 with atrioventricular canal defects. The approach revealed substantial variability in the location of the conduction system in hearts with congenital defects. The clockface schematics were used to create plans for subsequent surgical repair. ConclusionsThe clockface schematic provides a reliable fiducial system to document and communicate variability of conduction tissue regions in the heart and applies this information for decision-making during congenital cardiac surgery.

DNA from macrophages induces fibrosis and vasculopathy through POLR3A/STING/type I interferon axis in systemic sclerosis.

To clarify the role of RNA polymerase III A (POLR3A)/type I IFN in the pathogenesis of SSc. Cytosolic DNA and stimulator of IFN genes (STING) pathway in skin or serum of SSc patients were detected by immunofluorescence, immunohistochemistry and western blotting. DNA from human macrophages was transfected to SSc fibroblasts or human umbilical vein endothelial cells (HUVECs) and then markers of POLR3A/STING pathway were detected by real-time qPCR, western blotting and confocal microscopy. After H151 treatment or knocking down POLR3A/STING, type I IFN response, monocytes adhesion and activation of fibroblasts and HUVECs were evaluated. Regulation of IFN regulatory factor 3 (IRF3) on monocyte chemoattractant protein-1 (MCP-1) was determined by chromatin immunoprecipitation. In bleomycin (BLM)-induced SSc mice, the effect of STING knockout or H151 on vasculopathy and fibrosis was assessed. Cytosolic DNA, colocalization of STING with alpha-smooth muscle actin (α-SMA) or CD31 in the skin, and STING pathway in the serum of SSc patients were increased. Macrophage-derived DNA stimulated the translocation of POLR3A from nucleus to the perinuclear region near STING and activated POLR3A/STING/type I IFN response, monocytes adhesion and MCP-1 expression in fibroblasts/HUVECs and collagen overproduction of fibroblasts. The activated IRF3 bound to the promoter of MCP-1. STING deficiency or H151 administration ameliorated fibrosis and vasculopathy both in vitro and in BLM-induced SSc mice. SSc presented increased DNA leakage and STING pathway activation. DNA from macrophages induced type I IFN signature of fibroblasts and ECs through POLR3A/STING pathway. Blocking POLR3A/STING axis provides a new therapeutic target for SSc.

Actin‐Fence Enhancement Protects against Extra‐Pulmonary Infection of Pseudomonas Aeruginosa

Lung‐endothelial membrane damage underlies acute lung injury (ALI), a major cause of mortality. ALI occurs after extra‐pulmonary infection by Gram‐negative bacteria. However, damage‐inducing bacteria‐endothelial interactions are unclear. Endothelial uptake of blood‐borne bacteria might induce cell‐membrane pores (CMPs), leading to membrane damage. Since the cytoskeletal involvement in this membrane damage remains unclear, we explored the role played by the membrane stabilizing, cell cortical f‐actin layer (PMID: 33749665). We induced extra‐pulmonary infection in mice, by intraperitoneal (i.p.) inoculation of fluorescent (GFP‐labelled) Pseudomonas aeruginosa (GFP‐PA) (3.5x106 cfu). In one group (n=5), after 40 hours we carried out real‐time confocal microscopy (RCM) of the lung to determine GFP‐PA interactions with calcein‐red (CR)‐loaded lung endothelium, per our reported approaches (PMID: 33749665). In another group, we carried out single‐blind assessments of survival in mice given no therapy (n=10), or the actin fence enhancer, TAT‐V12Rac1 (PMID: 33749665) by intravenous injection 7 hours after PA inoculation (n=10). RCM of live lung microvessels revealed GFP‐PA as green particles attached to the lung‐endothelial surface. Therefore, bacteria released from the peritoneum, reached the lung by the blood route. Confocal sections taken through the cell interior revealed internalized bacteria as yellow particles, reflecting the merged fluorescence of green (bacteria) and red (cytosol). Imaging indicated slow loss of CR fluorescence over the time course of an hour. As we reported, this slow loss of cytosolic dye indicates CMP formation (PMID: 22561462). In the survival assay, 80% of the mice died within 3 days of PA inoculation (p<0.01 versus PBS control). By contrast, in the group given TAT‐V12Rac1, mortality was markedly less at 30% (p<0.01 versus bacteria alone). We show for the first time, that within 2 days of i.p. inoculation, GFP‐PA internalized in lung endothelium, causing CMP formation. A day later there was high mortality that however, could be majorly abrogated by enhancing the endothelial actin fence by therapy with intravenous TAT‐V12Rac1. Thus, the CMPs may have promoted endothelial damage, an effect that was mitigated by fence enhancement. We suggest, the actin fence is a major protective factor against bacteria‐induced endothelial damage. Actin fence enhancement might be viable therapy against ALI due to septicemia.

Mechanosensitive Responses of Resident Alveolar

BackgroundThe extent of micromechanical coupling between resident alveolar macrophages (AMs) and the surrounding tissue niche remains unclear. Lung overexpansion due to therapeutic mechanical ventilation imposes distortional stresses on the alveolar epithelium (AE), causing acute lung injury (ALI). Sessile AMs, lying adjacent to the AE, are causally implicated in ALI. However, it is not clear whether micromechanical coupling with the AE induces proinflammatory distortional stresses in sessile AMs. We addressed this question in live mouse lungs by real‐time confocal microscopy (RCM).MethodsFor RCM, we inflated and blood‐perfused mouse lungs at physiological pressures. To detect cytosolic Ca2+ (cCa2+), we microinfused fluo‐4 by alveolar micropuncture. To detect sessile AMs, we microinfused fluorophore‐conjugated anti‐Siglec‐F and anti‐CD11c Abs. To induce transient lung overexpansion, we increased the alveolar pressure from 5 to 15 cmH2O for 15 seconds. We determined alveolar diameter in terms of fluo‐4 fluorescence, which marked the epithelial margins. We determined cell diameter of sessile AMs in terms of Siglec‐F fluorescence, which marked the cell boundary. We detected gap junctional communication (GJC) by fluorescence recovery after photobleaching (FRAP).ResultsSessile AMs were exclusively located in non‐distending microniches of the alveolus and therefore, did not stretch during lung expansion. Nevertheless, a subset of AMs responded to lung overexpansion by transiently increasing cCa2+. The cCa2+ increase initiated in 5 min, reaching peak at two‐times baseline in 20 min, then subsiding to baseline after a further 20 min. Concomitantly, in AE adjacent to AMs, there was marked increase in cCa2+ oscillations, but no increase in mean cCa2+. In lungs with AM‐specific Cx43 deletion, the transient cCa2+ response was blocked in AMs, but not in adjoining AE. FRAP revealed GJC only in cCa2+‐responsive AMs. In paired comparisons, AMs lacking GJC failed to show expansion‐induced Ca2+ responses (p<0.05).ConclusionsWe report the unexpected finding that sessile AMs were exclusively located in immobile microniches of alveoli and therefore did not stretch during lung expansion. Nevertheless, following expansion there was Cx43‐mediated Ca2+communication with the AE, causing cCa2+ increases in sessile AMs. Although further studies are required to evaluate the potentially proinflammatory responses induced by this Ca2+ response, we propose Cx43 of sessile AMs might be a therapeutic target to mitigate lung expansion‐induced ALI.

Systems view of Bacillus subtilis pellicle development

In this study, we link pellicle development at the water–air interface with the vertical distribution and viability of the individual B. subtilis PS-216 cells throughout the water column. Real-time interfacial rheology and time-lapse confocal laser scanning microscopy were combined to correlate mechanical properties with morphological changes (aggregation status, filament formation, pellicle thickness, spore formation) of the growing pellicle. Six key events were identified in B. subtilis pellicle formation that are accompanied by a major change in viscoelastic and morphology behaviour of the pellicle. The results imply that pellicle development is a multifaceted response to a changing environment induced by bacterial growth that causes population redistribution within the model system, reduction of the viable habitat to the water–air interface, cell development, and morphogenesis. The outcome is a build-up of mechanical stress supporting structure that eventually, due to nutrient deprivation, reaches the finite thickness. After prolonged incubation, the formed pellicle collapses, which correlates with the spore releasing process. The pellicle loses the ability to support mechanical stress, which marks the end of the pellicle life cycle and entry of the system into the dormant state.

Two-photon ratiometric carbon dot-based probe for real-time intracellular pH monitoring in 3D environment

• Design and development of novel pH-sensitive two-photon ratiometric carbon dot-based probe was presented. • Probe possessed remarkable thermal stability, photostability, two-photon NIR excitation capability, and biocompatibility. • Probe demonstrated superb reversibility and ratiometric response to pH changes (4–10), upon single wavelength excitation. • Precise real-time monitoring and quantification of the intracellular pH values in 2D and 3D environments was performed. Designing a two-photon ratiometric pH-sensitive nanoprobe for real time monitoring of intracellular pH in biological environment is of great importance for better understanding the pathogenesis of diseases and the design of intracellular drug delivery-based system. However, the development of such probe remains a challenge. Here, we report for the first time the design and development of the two-photon ratiometric carbon dot-based nanoprobe and demonstrate its capability to monitor and quantify the intracellular cytoplasmic pH value, in real time. The nanoprobe comprised of a fluorescent carbon dot functionalized with a pH-responsive fluorescein dye (FACD). With increasing pH, FACD exhibits a clear ratiometric change in the emission intensity ratio, with sensitivity across a wide pH range in both extracellular and intracellular compartments. FACD is non-toxic to adipose stem cells in cell imaging experimental conditions and exhibits remarkable thermal stability, photostability, and two-photon near-infrared excitation capability. Using real-time dual-channel two-photon confocal microscopy we demonstrate the great potential of FACD as an efficient nanoprobe with high accuracy for the intracellular sensing of pH in living adipose stem cells seeded on either cell-culture dishes or on a 3D printed bioactive scaffold.