Confocal Microscopy Research Articles

Protamine-Mediated Tangles Produce Extreme Deoxyribonucleic Acid Compaction.

In sperm cells, protamine replaces histones to compact DNA 10-20 times more than in somatic cells. To characterize the extreme compaction, we employed confocal microscopy and optical tweezers to determine the conformations and stability of protamine-bound λ-DNA. Confocal images show increasing compaction of λ-DNA at increasing protamine concentration. In the presence of protamine, single λ-DNA molecules form tangles that withstand forces strong enough (∼55 pN) for strand separation and shorten the contour length by up to 40% even at high forces, as well as bends and loops that rupture at 10-40 pN forces. Strand separation nucleates tangles, implicating protamine interactions with DNA bases. Molecular dynamics simulations show that Arg sidechains of protamine each form hydrogen bonds with multiple bases, frequently in the form of a wedge between the two strands of DNA. Protamine may participate in both local and higher-order chromatin organization, leading to extreme compaction and global transcription silencing.

Exploring Hidden Connections: Endophytic System and Flower Meristem Development of Pilostyles berteroi (Apodanthaceae) and Interaction with Its Host Adesmia trijuga (Fabaceae).

Pilostyles, an endoparasitic genus within the Apodanthaceae family, grows inside host stems with flowers and fruits being the only external manifestations. Previous studies of P. berteroi growing on Adesmia trijuga provided limited details of the endophyte and omitted the origin of flowers and sinker structure. This study, using classical methods of optical microscopy applied to the analysis with scanning electron microscopy and confocal laser scanning microscopy, expands the understanding of the P. berteroi/A. trijuga complex. We find that P. berteroi develops isophasically with its host, forming endophytic patches between the host's secondary phloem cells. The parasitized Adesmia stem's cambium primarily produces xylem parenchyma, with limited vessel production and halting fiber formation. The radial polarization of endophytic patches led to the formation of floral meristems. Flowers develop endogenously and emerge by the breakthrough of the host stem. Flowers are connected to the host cambium via chimeric sinkers, combining P. berteroi parenchyma and tracheoids with Adesmia vessels. Unlike previous studies that show uniformity among Pilostyles species, our analysis reveals new insights into the structural interaction between P. berteroi and A. trijuga.

Microstructure of confectionery masses revealed by cryo-planing

Here we present a novel, combined cryo-scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) method for imaging of chocolate confectionery product microstructures of several millimeters down to about 100 nm. The SEM part of the method is based on cryo-fixation, cryo-polishing, and scanning electron microscopy, at low vacuum and low temperature using the backscattered electron signal. Starting with cryo-fixation of the chocolate sample in a desired state (cooled, ambient, or melted), the sample is cryo-planed in a cryo-ultramicrotome. Once a polished cut is obtained, the sample is analyzed using a cryo-SEM technique, with the unusual combination of low temperature and low vacuum settings, without heavy metal coating. Imaging is done based on material density contrast. Elemental composition of particles is recorded by energy dispersive x-ray spectroscopy (EDS). The combination of imaging contrast and EDS allows identification and measurement of the four main constituents of chocolate (cocoa solids, fat, sugar, and milk solids). Finally, the same cryo-polished sample sections of solid chocolate products are analyzed using a CLSM imaging technique to reveal complementary microstructural details. An obvious application of the method could be the visualization and quantitative analysis of the size, shape, and composition of chocolate confectionery products.

An In Vitro Comparative Analysis of Physico–Mechanical Properties of Commercial and Experimental Bioactive Endodontic Sealers

This study aimed to evaluate the fracture resistance of root and sealer penetration after obturation using an epoxy resin sealer AH plus (AH+) and two different bioactive endodontic sealers, i.e., Totalfill BC Hiflow (TF BC), and experimental injectable bioactive glass (Exp.BG). A thermo-sensitive injectable sealer was prepared by using a non-ionic triblock copolymer and bioactive glass. The root canals of human extracted teeth were obturated with the respective sealers. The fracture resistance was analyzed at different time intervals, i.e., days 7, 30, and 90. The morphological and elemental analyses of the fractured roots were conducted with a scanning electron microscopy and a electron dispersive spectroscopy. Sealer penetration depth and the percentage of penetrated sealers into the dentinal tubules were assessed with the confocal laser scanning microscope. Statistical analysis was performed using a one-way ANOVA post hoc Tukey’s test. The mean fracture force in AH+ was significantly higher on day 30 (664.08 ± 138.8 N) compared to day 7 (476.07 ± 173.2 N) and day 90 (493.38 ± 120.18 N). There was no statistically significant difference between the TF BC and Exp.BG at different time intervals. The maximum penetration was observed in the middle region compared to coronal and apical for the Exp.BG, followed by the TF BC and AH+ groups; however, a nonsignificant difference in penetration was found over time. It is concluded that the TF BC group showed overall better fracture resistance than AH+ at day 90. Exp.BG showed comparable sealer penetration to those of TF BC and better than those of AH+.

Potential probiotic Lactiplantibacillus plantarum strains alleviate TNF-α by regulating ADAM17 protein and ameliorate gut integrity through tight junction protein expression in in vitro model

BackgroundLactiplantibacillus species are extensively studied for their ability to regulate host immune responses and functional therapeutic potentials. Nevertheless, there is a lack of understanding on the mechanisms of interactions with the hosts during immunoregulatory activities.MethodsTwo Lactiplantibacillus plantarum strains MKMB01 and MKMB02 were tested for probiotic potential following Indian Council of Medical Research (ICMR) guidelines. Human colorectal adenocarcinoma cells such as HT-29, caco-2, and human monocytic cell THP-1 were also used to study the potential of MKMB01 and MKMB02 in regulating the host immune response when challenged with enteric pathogen Salmonella enterica typhimurium. Cells were pre-treated with MKMB01 and MKMB02 for 4 h and then stimulated with Salmonella. qRT-PCR and ELISA were used to analyze the genes and protein expression. Confocal microscopy and field emission scanning electron microscopy (FESEM) were used to visualize the effects. An Agilent Seahorse XF analyzer was used to determine real-time mitochondrial functioning.ResultsBoth probiotic strains could defend against Salmonella by maintaining gut integrity via expressing tight junction proteins (TJPs), MUC-2, and toll-like receptors (TLRs) negative regulators such as single Ig IL-1-related receptor (SIGIRR), toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase (IRAK)-M, A20, and anti-inflammatory transforming growth factor-β and interleukin-10. Both strains also downregulated the expression of pro-inflammatory cytokines/chemokines interleukin-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), interleukin 6, and nitric oxide (NO). Moreover, TNF-α sheddase protein, a disintegrin and metalloproteinase domain 17 (ADAM17), and its regulator iRhom2 were downregulated by both strains. Moreover, the bacteria also ameliorated Salmonella-induced mitochondrial dysfunction by restoring bioenergetic profiles, such as non-mitochondrial respiration, spare respiratory capacity (SRC), basal respiration, adenosine triphosphate (ATP) production, and maximal respiration.ConclusionsMKMB01 and MKMB02 can reduce pathogen-induced gut-associated disorders and therefore should be further explored for their probiotic potential.

Mechanisms of atrial-fibrillation-induced ventricular dysfunction and recovery in the human left ventricular myocardium

Abstract Background Atrial fibrillation (AF) frequently occurs alongside heart failure (HF). Clinical studies indicate improved left ventricular (LV) function following rhythm restoration but the underlying myocardial processes remain unclear. Purpose This study investigated effects of AF and its termination on human LV myocardium as well as potential molecular mechanisms involved in the AF-induced LV dysfunction. Methods Human LV tissue slices obtained from HF patients were long-term cultivated in-vitro. AF was simulated by tachy-arrhythmic culture pacing at 100 bpm with 30% irregularity for 7 days and contractile LV function was compared to slices undergoing sinus rhythm (SR)-simulation (60 bpm/0%). After 7 days, rhythm restoration was simulated by switching to SR-simulation. To elucidate cellular mechanisms involved in AF-induced LV dysfunction, human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were subjected to AF-simulation and examined via epifluorescence microscopy (FURA-2) and confocal microscopy (Fluo-4). Additionally, to investigate the impact of reactive oxygen species (ROS) on cardiomyocytes, iPSC-CM were subjected to treatment with the ROS-scavenger N-acetylcysteine (NAC). Results AF-simulation caused a progressive decline in systolic force of human LV myocardium (n=14 slices) with significantly reduced twitch amplitudes compared to SR-simulation (n=11). Within just 5 days of AF-termination, a significant recovery in LV function was observed, indicated by improved systolic contraction amplitudes. IPSC-CM measurements revealed a significant reduction in systolic Ca2+-transient amplitudes as well as an increased diastolic Ca2+ leak after AF-simulation (n=41) compared to SR-simulation (n=44). However, 5 days after SR-restoration, iPSC-CM exhibited no significant differences anymore, indicating a fast recovery of systolic Ca2+-cycling. We further investigated the impact of ROS on LV function and could show that Ca2+-transient amplitude was preserved after AF-simulation in iPSC-CM when treated with NAC (n=35), suggesting an interplay of ROS with the Ca2+-homeostasis of cardiomyocytes. Conclusions This study demonstrates that AF impairs LV function in human HF myocardium. However, the involved changes in contractility and cardiomyocyte Ca2+ handling are reversible upon AF termination leading to improved LV function already within few days after AF cessation. Moreover, we provided an indication for an interplay of ROS with the Ca2+ homeostasis, endorsing an AF-induced LV-dysfunction, which merits further investigation.

Restoring Colistin Sensitivity in Multidrug-Resistant Pathogenic E. coli Using Cinacalcet Hydrochloride.

Restoring colistin's efficacy is crucial in addressing the resistance crisis of colistin. This study utilized a high-throughput screening method to identify 43 compounds from 800 FDA-approved drugs that exhibited significant antibacterial effects when combined with colistin. Among these, cinacalcet hydrochloride (CH) was selected for its potential synergistic effect with colistin against multidrug-resistant (MDR) E. coli strains, including mcr-1-positive strains. A series of experiments revealed that the combination of CH and colistin showed strong synergy, especially in mcr-1-positive strains, restoring colistin sensitivity. The combination significantly inhibited bacterial growth and reduced CFU counts more effectively than either drug alone. Additionally, CH and colistin together significantly inhibited biofilm formation and eradicated existing biofilms, as visualized through confocal microscopy. Mechanistic studies showed that the combination increased bacterial membrane permeability and disrupted membrane integrity. The treatment also elevated extracellular ATP release and ROS production, indicating oxidative stress-induced bacterial death. Safety evaluations showed that the combination did not increase toxicity in host cells. Finally, animal models further validated the combination's efficacy. Overall, this study showed that the combination of colistin and CH significantly restored colistin sensitivity in mcr-1-positive E. coli, revealing their synergistic antibacterial mechanism involving membrane damage and oxidative stress, with promising clinical applications.

Macrophage-expressed coagulation factor 7 promotes adverse cardiac remodeling

Abstract Background Excess fibrotic remodeling leads to cardiac dysfunction in ischemic heart disease and is driven by MAP kinase-dependent transforming growth factor-ß1 (TGF-ß1) activation by coagulation signaling of myeloid cells. How coagulation-inflammatory circuits can be specifically targeted to achieve beneficial macrophage reprogramming after myocardial infarction (MI) is incompletely understood. Methods Mice with permanent ligation of the proximal left anterior descending artery (LAD) were used to model ischemic heart disease and analyzed by single cell RNA sequencing, protein expression changes, confocal microscopy, and longitudinal monitoring of recovery. We probed the role of the tissue factor (TF)-factor 7 (F7)-integrin ß1-protease activated receptor (PAR) 2 signaling complex by utilizing genetic mouse models and pharmacological intervention. Results Cleavage-insensitive PAR2R38E and myeloid cell integrin ß1-deficient mice had improved cardiac function after MI compared to controls. Proximity ligation assays of monocytic cells in the infarcted myocardium demonstrated that colocalization of F7 with integrin ß1 was diminished in monocyte/macrophage F7-deficient mice. F7fl/fl CX3CR1Cre relative to littermate control mice showed reduced TGF-ß1 and MAP kinase activation, as well as cardiac dysfunction after MI, despite unaltered overall recruitment of myeloid cells into the infarct zone. Single cell mRNA sequencing of CD45+ cells 3 and 7 days after MI uncovered a trajectory from ischemic myocardium-recruited monocytes to inflammatory TF+/F7+/TREM1+ macrophages. As early as 7 days after MI, macrophage F7-deletion led to an expansion of Olfml3+ macrophages with a reparative phenotype and, conversely, to a reduction of TF+/F7+/TREM1+ macrophages, which were also attenuated in activation-resistant PAR2R38E mice. To demonstrate the therapeutic potential of inhibiting the TF-F7-PAR2 signaling complex, we injected a specific monoclonal anti-TF antibody that lacks anticoagulant activity. Short-term treatment from day 1-5 after non-reperfused MI improved cardiac dysfunction, decreased excess fibrosis, attenuated vascular endothelial dysfunction, and increased survival 28 days after MI. Conclusion Extravascular TF-F7-PAR2 complex signaling drives inflammatory macrophage polarization in ischemic heart disease. Targeting this signaling complex for specific therapeutic macrophage reprogramming following MI attenuates cardiac fibrosis and improves cardiovascular function.

Targeting BRD4-HK2 reverses the meta-inflammatory shift in perivascular adipose tissue and rescues cardiometabolic vascular dysfunction

Abstract Background/Introduction Reducing vascular inflammation is crucial to halt and reverse cardiometabolic damage. BRD4, an epigenetic pan-inflammatory activator whose inhibition has shown promising results in cancer, may play an important role in this context. However, its role in cardiometabolic disease remains unclear. Purpose To investigate BRD4-related transcriptional programmes and therapeutic modulation in translational models of cardiometabolic disease. Methods We dissected small arteries (100-300 μM) from visceral fat biopsies in healthy volunteers (n=16) and patients with obesity and hypertension (n=16), a highly prevalent cardiometabolic phenotype. We assessed the ex vivo effects of BRD4 inhibition on vascular function by pressure myography in the presence or absence of perivascular adipose tissue (PVAT), at baseline and after incubation with the BRD4 inhibitor RVX-208 or with anti-inflammatory/anti-metabolic drugs. We used a cardiometabolic mouse model (high-fat diet+L-NAME supplementation) that mirrored our human phenotype and orally administered RVX-208 (150 mg/kg) or vehicle for 5 days (n=6 per group) to confirm the in vivo effect of chronic BRD4 inhibition. Finally, we investigated the molecular pathways involved in the perivascular cross-talk in an in vitro model of human adipocytes (hADSCs) and endothelial cells (HAECs) using gene overexpression/silencing strategies. We assessed ROS and nitric oxide levels (confocal microscopy), protein and gene expressions (Western blot; qPCR), transcriptional (custom qPCR array; chromatin immunoprecipitation (ChIP)) and metabolic changes (metabolomics, lipidomics, mitochondrial swelling). Results Vascular inflammation, remodeling and function were altered in cardiometabolic patients/mice. RVX-208 attenuated ex vivo vascular dysfunction to a greater extent than anti-IL-1β, anti-IL-6 receptor and anti-TNF-α. In vessels with intact PVAT we observed a stronger effect, due to the restoration of healthy PVAT phenotype (Figure 1). In PVAT, Hexokinase-2 (Hk2) - a glycolytic enzyme implicated in mitochondrial dysfunction and inflammation - was the top downregulated gene by RVX-208 treatment; ChIP confirmed increased binding of BRD4 to the Hk2 promoter. In line, metabolomics and lipidomics assays revealed a PVAT glycolytic shift with increased fatty acid storage in disease states, which was reversed by BRD4 inhibition. The in vitro study showed that: i) healthy adipocytes overexpressing HK2 shift to an inflammatory phenotype; ii) their secretome induces an inflammatory phenotype in HAECs; iii) ex vivo PVAT-selective inhibition of HK2 ameliorates vascular dysfunction (Figure 2). Conclusions We identified BRD4-HK2 interaction at the PVAT level as a novel mediator of cardiometabolic damage. Its targeting rescues vascular dysfunction by reversing the PVAT meta-inflammatory shift. Epigenetic modulators of meta-inflammation may represent a promising strategy in patients with obesity and hypertension.

Indoxyl sulfate deteriorates cardiac electrophysiological function and increases the likelihood of arrhythmias in human ventricular myocardial cells

Abstract Background Approximately 10% of the world population is suffering from chronic kidney disease (CKD). In these patients, an increased mortality and morbidity, predominantly derived from cardiovascular diseases, can be observed. Purpose The impact of CKD on myocardial cell mechanisms is only partially understood. This study aims to investigate the impact of the uremic toxin indoxyl sulfate (IS) on human ventricular myocardial cells. Methods Human left ventricular myocardium was obtained from patients with severe aortic stenosis who underwent surgical valve replacement. In addition, human ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) obtained from donors without any known cardiovascular disease were used. Before measurements were conducted, isolated single primary human cardiomyocytes were incubated for 15 minutes with an IS concentration of 120 mg/l (highest measured IS-level in dialysis patients). Further, iPSC-CM were incubated for one week with IS 45 mg/l and IS 120 mg/l, with IS 45 mg/l representing the average dialysis patients IS-level. Epifluorescence and confocal microscopy were performed to investigate Ca2+hemostasis and patch clamp measurements assessed cardiac action potentials and the occurrence of arrythmias. Results Experiments were conducted using human ventricular myocardium from 10 patients with a mean left ventricular ejection fraction of 51.5±12% and a mean glomerular filtration rate of 66.3±22.7 ml/min. Compared to the control group, higher systolic Ca2+ transient amplitudes were observed after acute incubation with IS 120 mg/l in single primary human cardiomyocytes. Measurements after 7 days of IS incubation (IS 45 mg/l and 120 mg/l) in iPSC-CM confirmed these previous results by showing higher Ca2+ transient amplitudes (Figure 1). Additionally, cells showed a diminished diastolic Ca2+ level compared to controls. An increased Ca2+ spark frequency in IS-treated iPSC-CM versus the control group was unveiled by confocal microscopy measurements. While an increased number of delayed afterdepolarizations could be observed, IS seemed to have no effect on cardiac action potential duration in iPSC-CM. Conclusion For the first time, we could reveal the impact of IS on cellular electrophysiology of primary human myocardium cells and iPSC-CM and identify IS as a proarrhythmogenic trigger. Therefore, IS may be of relevance in explaining the high cardiovascular mortality in end-stage CKD patients. To improve the outcome of these patients, new therapeutic approaches will be necessary since IS, a protein-bound uremic toxin, is only insufficiently removed by dialysis.

Non-cell-autonomous effects of arginase-II in macrophages on cardiomyocytes in aging

Abstract Background Aging-associated cardiac structural and functional alterations enhance cardiac susceptibility to ischemic injury. The full elucidation of the underlying molecular mechanisms remains far from complete. The mitochondrial enzyme arginase-II (Arg-II) is implicated in cardiac injury and aging. However, contradictory results have been reported and no systemic analysis of its cellular localization has been performed. The involvement of Arg-II in cardiac aging, as well as the manner in which it participates, remains uncertain. Aim and methods: This project focuses on investigation of Arg-II cellular localization and its functional role in cardiac aging. Experiments are conducted in young (3-4 months) and old (20-22 months) wt and arg-ii-/- mice and in human heart tissues. Cardiac function is assessed by Langendorff apparatus ex vivo. Cellular localization of Arg-II in cardiac tissues is investigated by confocal microscopy. Intercellular interactions are studied using ventricular cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages isolated from wt and arg-ii-/- mice or humans. Results Cardiac Arg-II expression is enhanced with aging. Surprisingly, Arg-II is not found in cardiomyocytes from aged mice and human patients, but upregulated in non-myocytes, including macrophages, fibroblasts, endothelial cells. Arg-ii-/- mice are protected from age-associated cardiac inflammation, cardiomyocyte apoptosis, fibrosis, endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic injury. Further experiments show that Arg-II mediates IL-1b release from macrophages of old mice, contributing to the above-described cardiac aging phenotype. In addition, Arg-II enhances mitochondrial reactive oxygen species (mtROS) and activates cardiac fibroblasts that is inhibited by inhibition of mtROS. Conclusion Our study demonstrates a non-cell-autonomous effect of Arg-II on cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from aging macrophages, which may explain the enhanced vulnerability of aging heart to ischemic injury.Role of Arg-II in cardiac aging

Potent broadly neutralizing antibodies mediate efficient antibody-dependent phagocytosis of HIV-infected cells.

Antibody-dependent cellular phagocytosis (ADCP) has been implicated in protection against HIV-1. However, methods for measuring ADCP currently rely on the phagocytosis of gp120- or gp41-coated beads that do not reflect physiologically relevant conformations of the viral envelope glycoprotein or the size of a virus-infected cell. We therefore developed a novel approach for measuring ADCP of HIV-infected cells expressing natural conformations of Env. A monocytic cell line (THP-1 cells) or primary human monocytes were incubated with a CD4+ T cell line that expresses eGFP upon HIV-1 infection in the presence of antibodies and ADCP was measured as the accumulation of eGFP+ material by flow cytometry. The internalization of HIV-infected cells by monocytes was confirmed visually by image-capture flow cytometry. Cytoskeletal remodeling, pseudopod formation and phagocytosis were also observed by confocal microscopy. We found that potent broadly neutralizing antibodies (bnAbs), but not non-neutralizing antibodies (nnAbs), mediate efficient phagocytosis of cells infected with either primary or lab-adapted HIV-1. A nnAb to a CD4-inducible epitope of gp120 (A32) failed to enable ADCP of HIV-infected cells but mediated efficient phagocytosis of gp120-coated beads. Conversely, a bnAb specific to intact Env trimers (PGT145) mediated potent ADCP of HIV-infected cells but did not facilitate the uptake of gp120-coated beads. These results underscore the importance of measuring ADCP of HIV-infected cells expressing physiologically relevant conformations of Env and show that most antibodies that are capable of binding to Env trimers on virions to neutralize virus infectivity are also capable of binding to Env on the surface of virus-infected cells to mediate ADCP.

Adipose tissue/ heart lipid transport determines cardiac diastolic function

Abstract Introduction Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction and often occurring in obese individuals. Previous research has demonstrated that the modification of lipid metabolism can prevent the onset of diastolic dysfunction. In our project we have investigated how lipid transport influences the onset of diastolic dysfunction utilizing heart- and adipose tissue-specific knockdowns of the fatty acid (FA) transport protein 1 (FatP1) in Drosophila melanogaster. Methods We generated Drosophila with heart-specific (+/+; Hand4.2-GAL4/UAS-FatP1; tdtK/+) (cFatP1-KD) and adipose tissue-specific (+/+; ppl-GAL4/UAS-FatP1; tdtK/+) (atFat-P1-KD) FatP1 knockdowns using the UAS/GAL4 system. The flies' cardiomyocytes endogenously express tdTomato, facilitating fluorescence-based live imaging of the heart by high-resolution video fluorescence microscopy, as previously described. Cardiac morphology and cardiac lipid accumulation was monitored by confocal microscopy. Additionally, mitochondrial function was evaluated using the glycolytic Seahorse assay of whole hearts. Results Blocking FA-uptake into adipose tissue in atFatP1-KD flies resulted in a highly significant reduction in relaxation velocity (p=0.0009, WT vs. atFatP1-KD), indicative of diastolic dysfunction in flies, while contraction velocity and fractional shortening (systolic function) remained unchanged. Diastolic dysfunction was associated with a significant increase of cardiac lipid droplet (LD) quantity and size in flies lacking FatP1 in adipose tissue (p=0.0275 and p<0.0001, respectively, WT vs. atFatP1-KD). Cardiac mitochondrial function was significantly impaired in atFatP1-KD flies compared to WT flies. Reducing cardiac FA uptake in heart-specific FatP1 KD flies led to a decrease in end-diastolic diameter and a significant reduction in relaxation and contraction velocity (p<0.0001 and p=0.0001, respectively, WT vs. cFatP1-KD), suggesting impairment in both diastolic and systolic function. Interestingly, the number of cardiac LDs increased in cFATP1-KD, however their size decreased significantly resulting in an overall unchanged lipid content compared to WT flies (p=0.0475 and p=0.0367, respectively, WT vs. atFatP1-KD). Mitochondrial function showed no significant alterations. Conclusion This study demonstrates that reducing FA-uptake in adipose or cardiac tissue results in cardiac dysfunction in Drosophila melanogaster. Reduction of adipose tissue FA-uptake results exclusively in diastolic dysfunction likely mediated by cardiac lipid overload and subsequent mitochondrial dysfunction. In contrast, blockade of Fatp1-mediated cardiac FA-uptake induces diastolic/ systolic dysfunction which was unrelated to alterations in cardiac lipid content and mitochondrial dysfunction suggesting alternative mechanisms. These study identifies FatP1-mediated FA-uptake in adipose tissue as a potential therapeutic target for the treatment of diastolic dysfunction.

Cationized extracellular vesicles for gene delivery

Last decade, extracellular vesicles (EVs) attracted a lot of attention as potent versatile drug delivery vehicles. We reported earlier the development of EV-based delivery systems for therapeutic proteins and small molecule chemotherapeutics. In this work, we first time engineered EVs with multivalent cationic lipids for the delivery of nucleic acids. Stable, small size cationized EVs were loaded with plasmid DNA (pDNA), or mRNA, or siRNA. Nucleic acid loaded EVs were efficiently taken up by target cells as demonstrated by confocal microscopy and delivered their cargo to the nuclei in triple negative breast cancer (TNBC) cells and macrophages. Efficient transfection was achieved by engineered cationized EVs formulations of pDNA- and mRNA in vitro. Furthermore, siRNA loaded into cationized EVs showed significant knockdown of the reporter gene in Luc-expressing cells. Overall, multivalent cationized EVs represent a promising strategy for gene delivery.

Intronic regulatory sequences regulate epicardial gene expression and EMT during heart development

Abstract Background During organogenesis, cells from the outer layer of the heart, the epicardium, undergo epithelial-to-mesenchymal transition (EMT), contributing essential paracrine signals and different cell types to the growing heart. Epicardial cells are integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties during heart development. Purpose Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is widely accepted, the upstream regulatory mechanisms underpinning its activation remain elusive. Our previous research has led to the identification of two intronic evolutionary conserved regions (ECRs) shared between mouse and human, located within intron 1 of the Wt1 locus, i.e. +4.0kbECR and +5.8kbECR. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Methods & Results Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR (Wt1Δ+4.0kbECR and Wt1Δ+5.8kbECR mice) or a deletion comprising both ECRs (Wt1Δ+4-5.8kbECR mouse). Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation in these models. Mendelian ratios indicated an overall underrepresentation of Wt1ΔECR/ΔECR mutants recovered in the offspring arising from heterozygous inter-crosses. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular or membranous ventricular septum defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal formation/patterning of coronary artery stems in ΔECR/ΔECR hearts. Expression of Wt1 was markedly reduced in ΔECR/ΔECR hearts, but not in the kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue-section immunostaining combined with confocal microscopy revealed abnormal coronary vessel and innervation extension and patterning along the subepicardial space, as well as reduced epicardial EMT and myocardial non-compaction/hyper-trabeculation in Wt1ΔECR/ΔECR hearts. Conclusions Together, we demonstrated a requirement for novel intronic enhancers regulating Wt1 locus activity and impacting on essential epicardial EMT and associated biological processes during normal heart development. Importantly, observation of septum and aortic valve defects in ΔECR mutants suggests an hitherto unrecognised role for WT1/epicardial EMT and opens new avenues of research to improve our understanding of congenital heart disease that affects at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

Neutrophil extracellular traps are an important component of chronic myocardial inflammation in patients with heart failure

Abstract Background and aim We have previously shown that heart failure (HF) is characterized by chronic inflammation. However, the role of neutrophils (N) and extracellular neutrophil traps (NETs) in the myocardium of patients with HF is largely unknown. The present study investigated the number of neutrophils (N) and their proportion that develop NETs in HF. Methods We used quantitative confocal microscopy and NETs markers in the left ventricular biopsies obtained from 5 controls and from patients with HF due to dilated (DCM, n=7), ischemic (ICM, n=7) and inflammatory (infCMP, n=7) cardiomyopathy. We used immunolabeling for CD45 (N) and for NETs (citrullinated histone 3 (CitH3), peptidylarginine deiminase-4 (PAD-4), neutrophil elastase (NE) and myeoloperoxidase (MPO). Results Compared to the control, the number of N was 4.3-5.7 times higher in HF. Using single labeling for NETs marker revealed that 41.1±4.6% of N in DCM, 42.9±4.1% in ICM and 47.6±2.9% in infCMP developed NETs. The use of dual labeling (MPO with CitH3, MPO with PAD-4 and CitH3 with NE) resulted in 54.5±4.7% of N-developing NETs in DCM, 59.5±4.1% in ICM and 77.6%±4.2 in infCMP. The difference between N-undergoing NETs was statistically different in infCMP than in DCM.and ICM. The proportion of N-forming NETs in control tissue was less than 5% and was significantly different from that in HF patients regardless of etiology. Conclusions This is the first study to show the presence of NETs in human hearts in situ and demonstrates that NETs are an important component of chronic inflammation in HF due to cardiomyopathies.

Global deficiency in the inflammatory chemokine receptors 1,2,3 and 5 prevents vascular dysfunction in angiotensin II-induced hypertension and selectively modulates aortic myeloid cell phenotype

Abstract Background Leukocyte migration to the vasculature and the heart plays a critical role in hypertensive immune responses and is a carefully orchestrated process, regulated by the interactions of inflammatory chemokines with their receptors, notably CCR1, CCR2, CCR3, and CCR5 (collectively termed inflammatory chemokine receptors (iCCRs)). However, the inflammatory chemokine/receptor system is characterised by redundancy, ligand sharing and overlapping expression patterns. Consequently, our understanding of the specific and combinatory roles of chemokine receptors in cardiovascular inflammation remains incomplete, thus hindering the development of targeted therapies. To address this challenge, we have utilised two novel mouse models: iREP mice, carrying fluorescent reporters of inflammatory chemokine receptor expression, and TACKO mice, in which the entire iCcr locus containing Ccr1, Ccr2, Ccr3, and Ccr5 has been deleted. Purpose To evaluate the expression pattern of iCCRs in experimental hypertension and understand how the global deletion of iCCRs impacts disease progression. Methods We induced hypertension in mice by subcutaneously implanting osmotic minipumps administering angiotensin II (Ang II) for a duration of 14 days. Single-cell RNA sequencing was utilised for investigating iCCR expression in aortas during hypertension. Additionally, we employed flow cytometry and confocal microscopy to track iCCR expression and localization in aortas and hearts obtained from iREP mice. TACKO mice were utilised to assess the effect of global iCCr deficiency on hypertensive phenotypes. Blood pressure was measured via telemetry and vascular function was assessed using myography. Vascular and heart remodelling were evaluated through histology. Molecular mechanisms were analysed using Western Blotting and RT-qPCR, while immune cell changes in the aorta were evaluated through cytometry of time-of-flight (CyTOF). Results Ang II infusion induced higher iCCRs expression levels in both the aorta and the heart in comparison with sham treatment, particularly in fibrotic areas. After Ang II-treatment, global deletion of iCCRs in TACKO mice resulted in the prevention of endothelial dysfunction (n=5/group, P<0.05) and perivascular fibrosis (n=6-9, P<0.01), and a reduction in cardiomyocyte size (n=12-16, P<0.01) compared to wild type mice. However, no significant improvements were observed in other hypertensive phenotypes, including blood pressure, heart fibrosis, vascular NO synthase, and oxidative stress. CyTOF analysis revealed reductions in inflammatory monocytes and conventional type 2 dendritic cells in TACKO aorta, accompanied by a phenotypic switch in macrophages towards a less inflammatory phenotype. Conclusion Deficiency in iCCRs confers protective effects against vascular dysfunction in experimental hypertension, primarily by selectively reducing inflammatory myeloid cell homing to the aorta.

Immediate Diagnosis of Breast Carcinoma on Core Needle Biopsy Using Ex Vivo Fluorescence Confocal Microscopy: Feasibility in a One-Stop Breast Clinic Workflow

Background: In the one-stop breast clinic setting, breast cytology traditionally provides immediate diagnosis of carcinoma. Fluorescence confocal microscopy (FCM) is an emerging optical technique enabling ex vivo analysis of breast biopsies in real-time. This study represents the first proof of concept for integrating FCM imaging into the routine workflow of breast core needle biopsies (CNB) at Gustave Roussy’s one-stop breast clinic. Methods: Fifty women with breast masses underwent consecutive enrollment. Biopsies were stained with acridine orange and fast green, followed by imaging using the Vivascope 2500M-G4 (FCM). Interpretation was conducted by two pathologists in real time (PT1) or postoperatively (PT2). Concordance with definitive histology, the duration of the FCM protocol, and its impact on conventional histopathology, immunohistochemistry, and FISH analyses were evaluated. Results: In our study of 50 biopsies, a concordant diagnosis of malignancy was performed using FCM on the malignant cases at definitive histology in 93.5% (29/31 cases) and in 90.3% (28/31 cases) according to PT1 and PT2, respectively. When the FCM suspicious cases were added, FCM identified 100% (31/31 cases) and 96.7% (30/31 cases) of the malignant cases according to PT1 and PT2, respectively. A notable false positive case was identified as a complex sclerosing lesion. The median time for sample preparation (including tissue reception) was 5 min, while the median time for imaging acquisition with interpretation was 3 min for PT1, but 1 min required for interpretation alone by PT2. Histopathological alterations were not more prevalent in FCM-imaged biopsies compared to conventionally treated biopsies. The immunophenotyping and molecular assessment of tissue were preserved after FCM protocol. Conclusions: FCM shows promise as a new histological method for the immediate diagnosis of breast carcinoma on core needle biopsies in a one-stop clinic setting, while also preserving tissue specimens for final histology.

Tissue-Specific Immune Transcriptional Signatures in the Bordering Tissues of the Mouse Retina and Brain.

Bordering the central nervous system (CNS) parenchyma are the choroid (underlying the retina) and the leptomeninges (the inner layers of the meninges enveloping the brain). Although near the neural parenchyma, the choroid and leptomeninges are external to the immune privileged environment of the retina and brain and thus are distinct immune compartments. This study aimed to characterize the transcriptomic signatures of immune cells within the choroid and leptomeninges bordering the healthy adult mouse CNS. Eyes and brains were obtained from 7-week-old C57Bl/6J mice. Choroid and leptomeninges were processed for isolation of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ. A total of 3606 cells were sequenced from mouse tissues, including 2125 CD45+ cells from choroid and 1481 CD45+ cells from leptomeninges. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells, and B cells. Whereas some clusters were common to both choroid and leptomeninges, others exhibited tissue-specific gene expression profiles and potential functional specializations. Analysis of 6501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid. This study provides a detailed characterization of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy retina and brain, and their potential roles in immune protection.

Protein redistribution in buccal cells as a prognostic marker for arrhythmogenic cardiomyopathy in paediatric patients

Abstract Background Mutations in genes encoding desmosomal proteins [plakoglobin (JUP), desmoplakin (DSP), plakophilin-2 (PKP2), desmoglein-2 (DSG2) and desmocollin-2 (DSC2)] cause complex genetic heart muscle disorders collectively described as arrhythmogenic cardiomyopathies (ACM). Diagnosis and risk stratification, particularly in the paediatric population, remain challenging owing to heterogeneous phenotypic manifestations and reduced genetic penetrance. Immunohistochemical analysis of heart muscle samples has shown that several junctional and signalling molecules are redistributed in patients with ACM, offering diagnostic information. However, the use of this approach in living patients was limited by need for heart samples. Recent studies in adults with ACM showed that the same set of proteins re-localised in the ACM heart also do so in the buccal epithelium. Purpose Given the ease of analysing serial cheek smears, we sought to establish how protein redistribution in buccal cells relates to disease onset and progression in children bearing desmosomal gene mutations. Methods Serial cheek smears from children with desmosomal variants were subjected to immunocytochemistry and confocal microscopy to study the distribution of JUP, DSP and PKP-1 as well as the major gap junction protein connexin43 (Cx43; GJA1) and the major subunit of the NFκB inflammatory pathway; RelA. Results 37 patients Conclusion These data indicate that shifts in junctional/signalling proteins precede the onset of clinical disease. Indeed, in the majority of pre-clinical children, Cx43 and RelA seem to be redistributed first, suggesting that onset of abnormalities in electrical conductivity and inflammatory pathways occur first in the natural disease progression, in agreement with previous clinical observations. Moreover, the data suggest that additional proteins are shifted as the disease progresses. Although further verification is required, this study suggests that analysis of buccal smears may be used as a prognostic/risk stratification test in children with desmosomal variants.