Confocal Imaging Techniques Research Articles

Quantitative Intracellular Delivery of Anticancer Nanodrugs Via an Immunoassay Employing Pt-SiO2 Janus-Peroxidase Nanozyme.

The accurate and efficient quantification of nanodrug dosage is crucial for early anticancer therapy. The enzyme-linked immunosorbent assay (ELISA) has emerged as a robust tool for detecting anticancer nanodrug dosage; however, the development of sensing elements to quantify anticancer nanodrugs still poses a challenge. To overcome this problem, we utilize polysuccinimide-loaded curcumin (CUR @PSIOAm) as a model to employ an ELISA based on peroxidase nanozyme Pt-SiO2 Janus nanoparticles (Pt-SiO2 JNPs) for the indirect quantitative analysis of intracellular anticancer nanodrug dosage. This novel approach employs an immunoassay to indirectly quantify the dosage of anticancer nanodrugs while preserving its structural integrity. The silica components of Pt-SiO2 JNPs adsorb intermediates, while the Pt NP components exhibit high catalytic activity. Pt-SiO2 JNPs are functionalized with anti-PSIOAm antibody (Pt-SiO2 JNPs-Ab) to serve as an immunosensor capable of specific recognition of CUR @PSIOAm. Additionally, we employed cytotoxicity assays and confocal imaging techniques to demonstrate the excellent biocompatibility of CUR @PSIOAm, as well as its specific uptake by cancer cells. According to the experimental results, the limit of detection (LOD) for the immunoassay of Pt-SiO2 JNPs as a marker for detecting CUR @PSIOAm is approximately 4.5-fold lower than that of horseradish peroxidase. Therefore, by optimizing the conditions, we established a direct competitive ELISA using Pt-SiO2 JNPs as colorimetric indicators for the quantitative detection of intracellular CUR @PSIOAm. The LOD for this ELISA was determined to be 0.01 ng/mL, while the loaded CUR amount calculated from the drug loading capacity was found to be 0.22 pg/mL. Furthermore, the recoveries obtained from this established ELISA ranged between 94.0 and 108%, demonstrating excellent accuracy. Consequently, the peroxidase mimic Pt-SiO2 JNPs-based ELISA exhibits significant potential for precise quantification of intracellular anticancer nanodrug dosages.

Green biosynthesis of selenium and zinc oxide nanoparticles using whole plant extract of Rheum ribes: Characterization, anticancer, and antimicrobial activity

Scientists are becoming interested in nanomedicine as a potential new approach to cancer detection and therapy in the twenty-first century. This paper presents the first investigation of the anticancer and antibacterial properties of selenium (Se) and zinc oxide (ZnO) nanoparticles obtained from Rheum ribes plant extract by a green synthesis method. Morphological and spectroscopic characterization of the synthesized nanoparticles was performed using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible (UV–Vis), which is a useful and straightforward technique for the preliminary characterisation of nanoparticles, dynamic light scattering (DLS) and X-ray diffraction (XRD) analysis. The size of the nanoparticles was determined to be 33 nm for Se-Nps and 32.8 nm for ZnO-Nps. The anticancer activity was assessed by the use of MTT, annexin V, caspase 3/7, and confocal microscopy imaging techniques. ZnO-Nps and Se-Nps were found to have significant antibacterial activity with MIC values for Escherichia coli (0.7 μg/mL, 0.63 μg/mL), and Staphylococcus aureus (1.56 μg/mL and 1.1 μg/mL). Furthermore, the antibacterial activity and the mechanism of action of the nanoparticles on E. coli and S. aureus bacteria were evaluated using microdilution and disc diffusion methods. In addition, the antiproliferative properties of ZnO-Np and Se-Np significantly suppressed the growth of A549 cells during a 24-hour incubation period (IC50 18.89 μg/mL ve 23.88 μg/mL). The results of the anti-cancer and anti-bacterial activity of the present study suggest that certain concentrations of Se-Np and ZnO-Np could be useful for pharmacological applications in cancer treatment and for coating surfaces for sterilization of medical equipment in healthcare settings, particularly in intensive care units.

The NADPH oxidase inhibitor diphenyleneiodonium suppresses Ca2+ signaling and contraction in rat cardiac myocytes.

Diphenyleneiodonium (DPI) has been widely used as an inhibitor of NADPH oxidase (Nox) to discover its function in cardiac myocytes under various stimuli. However, the effects of DPI itself on Ca2+ signaling and contraction in cardiac myocytes under control conditions have not been understood. We investigated the effects of DPI on contraction and Ca2+ signaling and their underlying mechanisms using video edge detection, confocal imaging, and whole-cell patch clamp technique in isolated rat cardiac myocytes. Application of DPI suppressed cell shortenings in a concentration-dependent manner (IC50 of ≅0.17 µM) with a maximal inhibition of ~70% at ~100 µM. DPI decreased the magnitude of Ca2+ transient and sarcoplasmic reticulum Ca2+ content by 20%-30% at 3 µM that is usually used to remove the Nox activity, with no effect on fractional release. There was no significant change in the half-decay time of Ca2+ transients by DPI. The L-type Ca2+ current (ICa) was decreased concentration-dependently by DPI (IC50 of ≅40.3 µM) with ≅13.1%-inhibition at 3 µM. The frequency of Ca2+ sparks was reduced by 3 µM DPI (by ~25%), which was resistant to a brief removal of external Ca2+ and Na+. Mitochondrial superoxide level was reduced by DPI at 3-100 µM. Our data suggest that DPI may suppress L-type Ca2+ channel and RyR, thereby attenuating Ca2+-induced Ca2+ release and contractility in cardiac myocytes, and that such DPI effects may be related to mitochondrial metabolic suppression.

#457 Sparsentan has direct effects on the glomerular capillary wall to attenuate increased permeability after exposure to nephrotic syndrome plasma

Abstract Background and Aims The glomerular endothelial glycocalyx (eGlx), a luminal layer of proteoglycans, glycoproteins and glycolipids, forms the first part of the glomerular filtration barrier (GFB). Nephrotic syndrome (NS) describes a group of pathologies of the renal glomerulus that result in proteinuria and is associated with glomerular endothelial dysfunction. Current treatments are broad and non-specific. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist that has received accelerated approval in the United States for the reduction of proteinuria in adults with IgA nephropathy at high risk of disease progression. Our validated glomerular permeability assay directly measures the albumin permeability (Ps’alb)of capillary loops within individually trapped glomeruli [1]. This ex vivo assay is independent of haemodynamic factors and tubular albumin handling – factors known to affect urine protein concentrations. This work examines whether sparsentan could reduce glomerular albumin permeability in NS, by preserving the eGlx to maintain the GFB. Method Human NS plasma samples were collected under ethical consent from 3 patients that had undergone plasma exchange from periods when they were in relapse (RL), or subsequent remission (RM). Adult male Sprague Dawley rats (175–200 g) were perfused with 4% Ringer BSA solution. Glomeruli were isolated on ice by graded sieving from the cortex of each kidney. Glomeruli were incubated, in the presence of AF488-conjugated BSA (AF488-BSA, 30 μg/ml) and R18 (36.5 μg/ml), with 10% plasma from NS patients for 1 hour, and simultaneously treated with sparsentan (0.1 μM, 1 μM and 10 μM) or vehicle. The glomerular Ps’alb assay was used to measure changes in albumin permeability. We applied a fluorescence profile peak-to-peak confocal imaging technique to treated glomeruli to assess glomerular eGlx thickness [2]. Results Human NS patients in RL had a significantly greater proteinuria compared to RM (RL, 10, 000 ± 1, 354 mg/g; RM, 95.3 ± 59.7 mg/g, P = 0.017). Incubation of rat glomeruli with RL plasma induced a significant increase in Ps’alb (RM+vehicle, 6.0 × 10−7 ± 0.12 × 10−7 cm/s; RL+vehicle, 10.9 × 10−7 ± 0.84 × 10−7 cm/s, P < .001) with a significant reduction in glomerular eGlx thickness (RM+vehicle, 210 ± 21.2 nm; RL+vehicle, 109 ± 7.9 nm, P = .027), compared to rat glomeruli incubated with paired RM plasma. Sparsentan-treated RL incubated glomeruli were protected from both the increase in Ps’alb (RL+spars 10 µM, 6.3 × 10−7 ± 0.19 × 10−7 cm/s, P < .001) and the loss in glomerular eGlx (RL+spars 10 µM, 222 ± 8.5 nm, P = .013), to a level comparable to RM incubated glomeruli. The effect of sparsentan on Ps’alb was dose dependent (RL+spars 1 µM, 8.0 × 10−7 ± 0.46 × 10−7 cm/s; RL+spars 0.1 µM, 9.6 × 10−7 ± 0.22 × 10−7 cm/s) with Ps’alb changes correlating inversely with glomerular eGlx thickness (r² = 0.75, P < .0001). In RM glomeruli, sparsentan alone had no effect on Ps’alb compared with vehicle (RM+spars 10 µM, 6.2 × 10−7 ± 0.25 × 10−7 cm/s, P = .590), suggesting no effect on otherwise healthy capillaries. Conclusion We have shown that dual inhibition of endothelin and angiotensin receptors, with sparsentan, preserves the glomerular eGlx resulting in normalised glomerular permeability in NS. These findings suggest that the direct action of sparsentan on the GFB could help maintain barrier integrity in NS, in particular by glycocalyx protection.

Long-Term Intrauterine Hypoxia Disrupts Calcium Sparks in Middle Cerebral Arterial Myocytes of Fetal Sheep

Local activation of ryanodine receptors (RyRs) on the sarcoplasmic reticulum in vascular smooth muscle cells (VSMCs) generates rapid subcellular changes in cellular Ca2+ that are commonly known as Ca2+ sparks. These Ca2+ sparks are well regarded for their role in the autoregulation of vascular contractility and cerebral blood flow through activation of potassium channels and feedback modulation of the membrane potential. Gestational hypoxia disrupts cerebrovascular development, increasing the risk of intracranial hemorrhage and stroke in the newborn. Due to the role of RyR’s in regulating neonatal cerebrovascular blood flow, understanding intracellular Ca2+ release patterns in VSMCs can offer insight into hypoxia-related perinatal cerebrovascular disease. The central aim of this study is to determine the extent to which gestational hypoxia disrupts RyR activity. This was achieved through examination of RyR activity in VSMCs of fetal ovine middle cerebral arteries (MCAs). MCAs were isolated from term fetal sheep (~140 days of gestation) delivered by cesarean section from ewes held at low- (700 m) and high-altitude conditions (3801 m) for 100+ days of gestation. Arteries were bathed in a physiological buffer with 5 mm K+ (control) or were treated with either 30 mM K+ (30K), to depolarize myocytes and increase Ca2+ spark events, in the presence or absence of 10 μM ryanodine, to block RyR mediated Ca2+ release. Ca2+ spark activity was measured in situ using line scan confocal microscopy fluorescence imaging techniques. Automated analysis of Ca2+ sparks in the records was conducted using SparkLab 5.8. Membrane depolarization with 30K increased Ca2+ spark activity. Long term hypoxia, however, reduced spark activity in the 30K group. Ryanodine attenuated Ca2+ spark activity in both low- and high-altitude groups. Hypoxia’s influence on the spatial and temporal aspects of Ca2+ sparks in fetal myocytes varied. These findings corroborate previous evidence demonstrating that long-term hypoxia attenuates Ca2+ spark activity. The disruption in Ca2+ spark activity could impact brain blood flow in the neonate, potentially impacting brain development and the likelihood of a traumatic event. R01HL155295, R01HL149608, Advanced Imaging and Microscopy Core Loma Linda University School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic Hypoxia during Gestation Disrupts Spontaneous Ca2+ Oscillations in Middle Cerebral Arterial Myocytes of Fetal Sheep

Long Term Hypoxic (LTH) stress is associated with an elevated risk of intracranial hemorrhage and stroke in newborns. Existing evidence illustrates that fetal sheep subjected to LTH conditions exhibit compromised cerebrovascular reactivity and arterial development, leading to perturbations in cerebral blood flow. Given the critical role of whole-cell Ca2+ waveforms in the contraction of middle cerebral arterial (MCA) myocytes and the regulation of cerebral blood flow, a comprehensive understanding of Ca2+ signaling patterns can provide insight into hypoxia-related cerebrovascular disease in neonates. This study was designed to test the hypothesis that intrauterine LTH disrupts whole-cell Ca2+ oscillations in fetal MCA myocytes. The hypothesis was addressed using MCAs isolated from term fetal sheep (approximately 140 days of gestation) delivered by cesarean section. These fetuses were carried by ewes residing at low-altitude (700 m) or high-altitude conditions (3801 m) for over 100 days of gestation. Ca2+ oscillatory activity was recorded using confocal fluorescence imaging techniques in an en face myocyte preparation. Spontaneous Ca2+ oscillations were evaluated in the presence and absence of 30 mM K+ to depolarize the myocytes, with or without 10 μM ryanodine, which blocks ryanodine receptor mediated Ca2+ release. The results of the study reveal that LTH decreases oscillatory activity and restricts the augmentation of activity following membrane depolarization. Furthermore, LTH induces dysregulation in spatial and temporal Ca2+ signals, manifesting as reduced event amplitude, shortened event duration, and decreased area under the curve (AUC). These findings highlight the impact of gestational hypoxia on multiple aspects of Ca2+ signaling in fetal lamb MCA, which may contribute to cerebrovascular dysfunction in the newborn. R01HL155295, R01HL149608, Advanced Imaging and Microscopy Core Loma Linda University School of Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Measuring Presynaptic Calcium Influx at the Drosophila Larval Neuromuscular Junction.

Synaptic transmission plays a critical role in information processing and storage within the nervous system. The triggering of action potentials activates voltage-gated calcium channels at presynaptic active zones, facilitating the calcium-dependent release of synaptic vesicles. Homeostatic mechanisms are crucial in stabilizing synaptic function. At the Drosophila neuromuscular junction, a compensatory increase in presynaptic neurotransmitter release occurs when postsynaptic glutamate receptor function is pharmacologically or genetically impaired, thereby stabilizing synaptic output. This adaptation is known as presynaptic homeostatic potentiation (PHP). Recent advancements, including confocal and super-resolution imaging techniques, have demonstrated an increase in presynaptic calcium influx during both the rapid induction and long-term maintenance of PHP. These observations indicate that the abundance and structural organization of presynaptic calcium channels, along with various active zone components, undergo modifications following the suppression of postsynaptic glutamate receptors. Such findings underscore the critical roles of trafficking and stabilization of presynaptic calcium channels and active zone proteins in homeostatic plasticity. This protocol describes using calcium indicators and confocal imaging methods to measure single-action potential-evoked presynaptic calcium influx during PHP.

Abstract 6624: MYC promotes lobuloalveologenesis and mammary tumorigenesis in male mice

Abstract Male breast cancer is a rare disease that is largely overlooked and clinically regarded to recapitulate female breast cancer biology. The worse overall outcome for male patients compared to females, however, underscores inadequate disease management and a sex-based divergence in disease biology. To gain more insight into male breast biology and pathology, we used mouse models of triple-negative breast cancer and 3D whole-mount confocal imaging techniques to evaluate both in vivo and ex vivo mammary gland development, tumor formation, and hormone signaling dependence. Our results show that Myc overexpression in collaboration with Trp53 deficiency drives mammary tumorigenesis in male mice. We show that MYC overexpression promotes estrogen-independent formation of lobuloalveolar structures, which progress into tumors with a similar histology and immune landscape as female mammary tumors but with significantly more genomic instability. In female mice, MYC overexpression bypasses the dependency on paracrine estrogen signaling in mammary tumorigenesis. These data provide robust evidence that MYC overrides mammary tumor estrogen dependence and abolishes the tumor-preventive effect of a low-estrogen environment. Supporting this notion, in vitro experiments in human female breast cancer cell lines indicate that MYC overexpression may functionally contribute to endocrine therapy resistance and therefore could possibly serve as a prognostic biomarker or clinically targetable pathway for treating both male and female breast cancers. Collectively, this preclinical work presents new insights into male mammary gland biology and mechanisms of cancer progression that might translate to a broader range of breast cancers in the clinic. Citation Format: Emilia M. Pulver, Eline van der Burg, Anne Paulien Drenth, Julia Yemelyanenko, Nils Ravensbergen, Roebi de Bruijn, Sjoerd Klarenbeek, Ji-Ying Song, Manon Boeije, Bjørn Siteur, Natalie Proost, Colinda Scheele, Peter Bouwman, Jos Jonkers. MYC promotes lobuloalveologenesis and mammary tumorigenesis in male mice [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 6624.

Optical path optimization of chromatic line confocal displacement sensor for high resolution and wide range.

This study introduces the optical path-optimized dual-grating chromatic line confocal imaging (DG-LCI) technique for high-resolution and wide-range surface topography measurements. Chromatic line confocal imaging (LCI) finds extensive applications in high-speed 3D imaging of surface morphology, roughness analysis in industrial production, and quality inspection. A key advantage of LCI is its ability to achieve a large depth of focus, enabling the imaging system to measure a wide range in the Z direction. However, the challenge lies in the trade-off between the measurement range and resolution. Increasing the measurement range reduces the resolution, making it unsuitable for precise measurements required in industrial processing. Conversely, enhancing the resolution limits the measurement range, thereby sacrificing the advantage of LCI systems' broad measurement capabilities. Addressing this limitation, we propose a dual optical path dual-grating structure using a simplified and ingenious optical path optimization design. This design overcomes the challenge of sacrificing the millimeter-level measurement range while simultaneously improving the resolution. Rigorous simulations and experiments validate the effectiveness and validity of our proposed method.

"Turn-on" and pinhole-free ultrathin core-shell Au@SiO2 nanoparticle-based metal-enhanced fluorescent (MEF) chemodosimeter for Hg2.

This study reports a metal-enhanced fluorescence chemodosimeter for highly sensitive detection of Hg2+ ions. Silica-coated Au nanoparticles (Au@SiO2 NPs) with a pinhole-free 4-5 nm shell were synthesized and functionalized with a monolayer of turn-on fluorescent probes. Compared to other organic fluorescent probes suffering from poor biocompatibility and detection limits, this design of a monolayer of turn-on fluorescent probes immobilized on the Au@SiO2 NPs with a pinhole-free 4-5 nm shell avoids fluorescence quenching and allows the fluorescent probe within the field of the inner Au NPs to experience metal-enhanced fluorescence. With this design, the chemodosimeter permits fluorescence emission in the presence of Hg2+ ions, because they trigger the ring-opening reaction of the fluorescent probe immobilized on the Au@SiO2 NPs. Additionally, the fluorescent probe is distanced by the thin SiO2 shell from directly attaching to the metallic Au NPs, which not only avoids fluorescence quenching but allows the fluorescent probe within the long-ranged field of the inner Au NPs to experience metal-enhanced fluorescence. As a result, the detection limit for the chemodosimeter can reach up to 5.0 × 10-11 M, nearly two orders of magnitude higher than that achieved for the free fluorescent probe. We also demonstrate the acquisition of images of Hg2+ in HTC116 cells and zebrafish using a simple fluorescence confocal imaging technique. The fluorescence response results for HTC116 cells and zebrafish show that the probes can permeate into cells and organisms. Considering the availability of the many organic fluorescent probes that have been designed, the current designed metal-enhanced fluorescence chemodosimeter holds great potential for fluorescence detection of diverse species and fluorescence imaging.

Assessing pathological features of breast cancer via the multimodal information of multiphoton and Raman imaging

For unveiling the pathological evolution of breast cancer, nonlinear multiphoton microscopic (MPM) and confocal Raman microspectral imaging (CRMI) techniques were both utilized to address the structural and constitutional characteristics of healthy (H), ductal carcinoma in situ (DCIS), and invasive ductal carcinoma (IDC) tissues. MPM-based techniques, including two-photon excited fluorescence (TPEF) and second harmonic generation (SHG), visualized label-free and the fine structure of breast tissue. Meanwhile, CRMI not only presented the chemical images of investigated samples with the K-mean cluster analysis method (KCA), but also pictured the distribution of components in the scanned area through univariate imaging. MPM images illustrated that the cancer cells first arranged around the basement membrane of the duct, then proliferated to fill the lumens of the duct, and finally broke through the basement membrane to infiltrate into the stroma. Although the Raman imaging failed to visualize the cell structure with high resolution, it explained spectroscopically the gradual increase of nucleic acid and protein components inside the ducts as cancer cells proliferated, and displayed the distribution pattern of each biological component during the evolution of breast cancer. Thus, the combination of MPM and CRMI provided new insights into the on-site pathological diagnosis of malignant breast cancer, also ensured technical support for the development of multimodal optical imaging techniques for precise histopathological analysis.

Abstract 4557: 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response

Abstract Introduction: Recent studies showed that patients with deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) tumors, have higher sensitivity to immune checkpointinhibitors (ICIs) compared to patients with microsatellite-stable (MSS)/microsatellite instability-low(MSI-L) tumors. However, the mechanisms of treatment responsiveness and resistance is not wellunderstood. Here, we used a novel 3D-EXpress ex vivo fresh patient tumoroid platform to assess theefficacy of nivolumab/ipilimumab combination therapy in tumors with known MSS/MSI-H status andperformed correlative studies. Materials and Methods: All tumor samples were obtained with patient consent and relevant IRBapproval. Tumoroids measuring 150 µm in size retaining tumor cell heterogeneity, tumor-residentimmune cells, stromal components, and cell-extracellular matrix interaction were prepared frompatients with endometrial and colorectal tumors among others. Tumoroid aliquots were cryopreservedin Nilogen’s tumoroid Biorepository for future studies. For the 3D-EXpress studies cryopreservedtumoroids were selected based on MSS/MSI-H status and treated ex vivo with nivolumab andipilimumab alone and in combinations for 72h. Tumor responses to treatments were evaluated by aproprietary tumor cell killing assay and changes in tumor immune microenvironment. Furthermore,tumor PD-L1 expression levels were analyzed on the associated TMA slides. Results: Treatment-induced tumor cell killing activity in intact tumoroids was assessed by a 3D high-content confocal imaging technique using a proprietary algorithm for data analysis. The impact of exvivo treatment by nivolumab and/or ipilimumab on tumor resident immune cell populations wasmonitored by a multiplex cytokine release assay analyzing release of GM-CSF, sCD137, IFNγ, sFas, sFasL,Granzyme A, Granzyme B, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1α, MIP-1β, TNF-α, and Perforin usingculture supernatants isolated from treated tumoroid samples. Based on the ex vivo responses tumorswere assigned to treatment sensitive and resistant groups and correlative analyses were performed withindividual tumors’ innate and adaptive immune cell populations detected by a 21-color flow cytometrypanel, in addition to tumor MSS/MSI-H and p53 status, and detailed clinicopathologic data readilyavailable for the tumors cryopreserved in the Biorepository. Conclusion: The 3D-Express platform, using cryopreserved 3D tumoroids with intact TME is an effectivetool for the assessment of rational combinations which may prove relevant in the treatment of solidtumors. Furthermore, we believe this platform is a useful tool for the pre-clinical assessment of specifictherapeutic regimens designed for individualized patient care. Citation Format: Brittney Ruedlinger, Seth Currlin, Sharon Camacho, Alliyah Humphrey, Jared Ehrhart, Soner Altiok. 3D-EXpress platform utilizing tumoroids from patients with MSS and MSI-H tumors allows rapid assessment of anti-tumor activity of immune checkpoint inhibitors and development of clinically relevant biomarkers of treatment response. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4557.

Pitx2c deficiency confers cellular electrophysiological hallmarks of atrial fibrillation to isolated atrial myocytes

AimsAtrial fibrillation (AF) has been associated with altered expression of the transcription factor Pitx2c and a high incidence of calcium release-induced afterdepolarizations. However, the relationship between Pitx2c expression and defective calcium homeostasis remains unclear and we here aimed to determine how Pitx2c expression affects calcium release from the sarcoplasmic reticulum (SR) and its impact on electrical activity in isolated atrial myocytes. MethodsTo address this issue, we applied confocal calcium imaging and patch-clamp techniques to atrial myocytes isolated from a mouse model with conditional atrial-specific deletion of Pitx2c. ResultsOur findings demonstrate that heterozygous deletion of Pitx2c doubles the calcium spark frequency, increases the frequency of sparks/site 1.5-fold, the calcium spark decay constant from 36 to 42 ms and the wave frequency from none to 3.2 min−1. Additionally, the cell capacitance increased by 30% and both the SR calcium load and the transient inward current (ITI) frequency were doubled. Furthermore, the fraction of cells with spontaneous action potentials increased from none to 44%. These effects of Pitx2c deficiency were comparable in right and left atrial myocytes, and homozygous deletion of Pitx2c did not induce any further effects on sparks, SR calcium load, ITI frequency or spontaneous action potentials. ConclusionOur findings demonstrate that heterozygous Pitx2c deletion induces defects in calcium homeostasis and electrical activity that mimic derangements observed in right atrial myocytes from patients with AF and suggest that Pitx2c deficiency confers cellular electrophysiological hallmarks of AF to isolated atrial myocytes.

Pericytes and the Control of Blood Flow in Brain and Heart.

Pericytes, attached to the surface of capillaries, play an important role in regulating local blood flow. Using optogenetic tools and genetically encoded reporters in conjunction with confocal and multiphoton imaging techniques, the 3D structure, anatomical organization, and physiology of pericytes have recently been the subject of detailed examination. This work has revealed novel functions of pericytes and morphological features such as tunneling nanotubes in brain and tunneling microtubes in heart. Here, we discuss the state of our current understanding of the roles of pericytes in blood flow control in brain and heart, where functions may differ due to the distinct spatiotemporal metabolic requirements of these tissues. We also outline the novel concept of electro-metabolic signaling, a universal mechanistic framework that links tissue metabolic state with blood flow regulation by pericytes and vascular smooth muscle cells, with capillary KATP and Kir2.1 channels as primary sensors. Finally, we present major unresolved questions and outline how they can be addressed.

Steam explosion pretreatment enhancing enzymatic digestibility of overground tubers of tiger nut (Cyperus esculentus L.).

Tiger nut (TN) is recognized as a high potential plant which can grow in well-drained sandy or loamy soils and provide food nutrients. However, the overground tubers of TN remain unutilized currently, which limits the value-added utilization and large-area cultivation of this plant. In the present study, the overground tubers of TN were subjected to enzymatic hydrolysis to produce fermentable sugars for biofuels production. Steam explosion (SE) was applied to modify the physical-chemical properties of the overground tubers of TN for enhancing its saccharification. Results showed that SE broke the linkages of hemicellulose and lignin in the TN substrates and increased cellulose content through removal of hemicellulose. Meanwhile, SE cleaved inner linkages within cellulose molecules, reducing the degree of polymerization by 32.13-77.84%. Cellulose accessibility was significantly improved after SE, which was revealed visibly by the confocal laser scanning microscopy imaging techniques. As a result, enzymatic digestibility of the overground tubers of TN was dramatically enhanced. The cellulose conversion of the SE treated TN substrates reached 38.18-63.97%, which was 2.5-4.2 times higher than that without a SE treatment. Therefore, SE pretreatment promoted saccharification of the overground tubers of TN, which paves the way for value-added valorization of the TN plants.

Understanding the Influence of Local Physical Stimuli on Chondrocyte Behavior.

Investigating the mechanobiology of chondrocytes is challenging due to the complex micromechanical environment of cartilage tissue. The innate zonal differences and poroelastic properties of the tissue combined with its heterogeneous composition create spatial- and temporal-dependent cell behavior, which further complicates the investigation. Despite the numerous challenges, understanding the mechanobiology of chondrocytes is crucial for developing strategies for treating cartilage related diseases as chondrocytes are the only cell type within the tissue. The effort to understand chondrocyte behavior under various mechanical stimuli has been ongoing over the last 50years. Early studies examined global biosynthetic behavior under unidirectional mechanical stimulus. With the technological development in high-speed confocal imaging techniques, recent studies have focused on investigating real-time individual and collective cell responses to multiple / combined modes of mechanical stimuli. Such efforts have led to tremendous advances in understanding the influence of local physical stimuli on chondrocyte behavior. In addition, we highlight the wide variety of experimental techniques, spanning from static to impact loading, and analysis techniques, from biochemical assays to machine learning, that have been utilized to study chondrocyte behavior. Finally, we review the progression of hypotheses about chondrocyte mechanobiology and provide a perspective on the future outlook of chondrocyte mechanobiology.

Glutathione-capped gold nanoclusters as near-infrared-emitting efficient contrast agents for confocal fluorescence imaging of tissue-mimicking phantoms.

An innovative research has been conducted focused on demonstrating the ability of novel dual-emissive glutathione-stabilized gold nanoclusters (GSH-AuNCs) to perform bright near-infrared (NIR)-emitting contrast agents inside tissue-mimicking agarose-phantoms via two complementary confocal fluorescence imaging techniques. First, using a new and fast microwave-assisted approach, we synthesized photostable dual-emitting GSH-AuNCs with an average size of 3.2 ± 0.4 nm and NIR emission quantum yield of 9.9%. Steady-state fluorescence measurements coupled with fluorescence lifetime imaging microscopy (FLIM) assays performed on lyophilized GSH-AuNCs revealed that the obtained GSH-AuNCs exhibit PL emissions at 610nm (red PL) and, respectively, 800nm (NIR PL) in both solution and powder solid-state. Time-resolved fluorescence measurements showed that the two PL components are characterized by average lifetimes of 407ns (red PL) and 1821ns (NIR PL), respectively. Additionally, due to a partial overlap between the red PL and the absorption of the NIR PL, an energy transfer between the two coexisting emissive centers was discovered and confirmed via steady-state and time-resolved fluorescence measurements. Furthermore, the FLIM analysis performed on powder GSH-AuNCs under 640nm, an excitation more suitable for bioimaging applications, revealed a homogeneous and photostable NIR PL signal from GSH-AuNCs. Finally, the ability of GSH-AuNCs to operate as reliable NIR-emitting contrast agents inside tissue-mimicking agarose-phantoms was demonstrated here for the first time via complementary FLIM and re-scan confocal fluorescence imaging techniques. In consequence, GSH-AuNCs show great promise for future in vivo imaging applications via confocal fluorescence microscopy.

Bioinspired Polysaccharide Derivative with Efficient and Stable Lubrication for Silicon-Based Devices.

It is becoming increasingly important to synthesize efficient biomacromolecule lubricants suitable for medical devices. Even though the development of biomimetic lubricants has made great progress, the current system suitable for hydrophobic silicone-based medical devices is highly limited. In this work, we synthesize one kind of novel polysaccharide-derived macromolecule lubricant of chitosan (CS) grafted polyethylene glycol (PEG) chains and catechol groups (CT) (CS-g-PEG-g-CT). CS-g-PEG-g-CT shows good adsorption ability by applying quantitative analysis of quartz crystal microbalance (QCM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and confocal fluorescence imaging technique, as well as the typical shear-thinning feature. CS-g-PEG-g-CT exhibits low and stable coefficients of friction (COFs) (0.01-0.02) on polydimethylsiloxane (PDMS) surfaces at a wide range of mass concentrations in diverse media including pure water, physiological saline, and PBS buffer solution and is even tolerant to various normal loads and sliding frequencies for complex pressurizing or shearing environments. Subsequently, systematic surface characterizations are used to verify the dynamic attachment ability of the CS-g-PEG-g-CT lubricant on the loading/shearing process. The lubrication mechanism of CS-g-PEG-g-CT can be attributed to the synergy of strong adsorption from catechol groups to form a uniform assembly layer, excellent hydration effect from PEG chains, and typical shear-thinning feature to dissipate viscous resistance. Surprisingly, CS-g-PEG-g-CT exhibits efficient lubricity on silicone-based commercial contact lenses and catheters. The current macromolecule lubricant demonstrates great real application potential in the fields of medical devices and disease treatments.

Acute inhibition of transient receptor potential vanilloid-type 4 cation channel halts cytoskeletal dynamism in microglia.

Microglia, the resident macrophages of the central nervous system, are highly motile cells that support brain development, provision neuronal signaling, and protect brain cells against damage. Proper microglial functioning requires constant cell movement and morphological changes. Interestingly, the transient receptor potential vanilloid 4 (TRPV4) channel, a calcium-permeable channel, is involved in hypoosmotic morphological changes of retinal microglia and regulates temperature-dependent movement of microglial cells both in vitro and in vivo. Despite the broad functions of TRPV4 and the recent findings stating a role for TRPV4 in microglial movement, little is known about how TRPV4 modulates cytoskeletal remodeling to promote changes of microglial motility. Here we show that acute inhibition of TRPV4, but not its constitutive absence in the Trpv4 KO cells, affects the morphology and motility of microglia in vitro. Using high-end confocal imaging techniques, we show a decrease in actin-rich filopodia and tubulin dynamics upon acute inhibition of TRPV4 in vitro. Furthermore, using acute brain slices we demonstrate that Trpv4 knockout microglia display lower ramification complexity, slower process extension speed and consequently smaller surveyed area. We conclude that TRPV4 inhibition triggers a shift in cytoskeleton remodeling of microglia influencing their migration and morphology.

Comparison between Deep-Learning-Based Ultra-Wide-Field Fundus Imaging and True-Colour Confocal Scanning for Diagnosing Glaucoma.

In this retrospective, comparative study, we evaluated and compared the performance of two confocal imaging modalities in detecting glaucoma based on a deep learning (DL) classifier: ultra-wide-field (UWF) fundus imaging and true-colour confocal scanning. A total of 777 eyes, including 273 normal control eyes and 504 glaucomatous eyes, were tested. A convolutional neural network was used for each true-colour confocal scan (Eidon AF™, CenterVue, Padova, Italy) and UWF fundus image (Optomap™, Optos PLC, Dunfermline, UK) to detect glaucoma. The diagnostic model was trained using 545 training and 232 test images. The presence of glaucoma was determined, and the accuracy and area under the receiver operating characteristic curve (AUC) metrics were assessed for diagnostic power comparison. DL-based UWF fundus imaging achieved an AUC of 0.904 (95% confidence interval (CI): 0.861–0.937) and accuracy of 83.62%. In contrast, DL-based true-colour confocal scanning achieved an AUC of 0.868 (95% CI: 0.824–0.912) and accuracy of 81.46%. Both DL-based confocal imaging modalities showed no significant differences in their ability to diagnose glaucoma (p = 0.135) and were comparable to the traditional optical coherence tomography parameter-based methods (all p > 0.005). Therefore, using a DL-based algorithm on true-colour confocal scanning and UWF fundus imaging, we confirmed that both confocal fundus imaging techniques had high value in diagnosing glaucoma.