Live Cell Imaging Research Articles

The ghrelin receptor agonist AC01 improves cardiac function in non-human primates and in mice with heart failure by increasing cardiomyocyte contractility

Abstract Background Current therapy for Heart Failure (HF) includes neurohormonal antagonist and modulator drugs that improve remodelling, symptoms and clinical outcomes. However, these drugs do not target the underlying problem of reduced contractility. Improving cardiac contractility without harmful consequences is an unmet need in heart failure therapy. The human peptide hormone ghrelin increases cardiac output in HF patients and increases contractility in murine cardiomyocytes. Purpose We aim to study the cardiovascular effects of a novel oral small molecule ghrelin receptor agonist, AC01, in in vivo and ex vivo mouse models and in non-human primates. Methods HF mice (N=11) underwent pressure-volume loops analysis of cardiac functional and hemodynamic to assess the effects of intravenous AC01 (N=7) or placebo (N=4). Primary cardiomyocytes isolated from HF (N=3) and SHAM (N=5) mice, were treated with combinations of vehicle, AC01, ghrelin receptor antagonist (D-Lys-3-GHRP-6) and pertussis toxin (PTX, a Gαi signalling inhibitor). Live cell imaging was performed to assess cardiomyocyte fractional shortening and Ca2+ signalling, while biochemistry assays were employed to measure PKA activity, and phosphorylation of cardiac Troponin I (cTnI). Cynomolgus monkeys (N=6) were instrumented with devices to measure pulse contour, central aortic blood pressure and ECG devices. Cardiovascular effects of AC01 following single oral dosing were assessed through pulse contour hemodynamic modelling and analysis of autonomic system activation. Results In HF mice, AC01 significantly improved cardiac output (p< 0.01), stroke volume (p< 0.001), and ejection fraction (p< 0.001), and increased nominally the end-systolic pressure-volume curve relationship. In cardiomyocytes, AC01 dose-dependently improved fractional shortening (p< 0.01) without increasing Ca2+ transient amplitudes. AC01 reduced PKA activity (p< 0.05) and reduced phosphorylation of cTnI at Serine 23-24 (p< 0.01). These effects were blocked by pre-treatment with either D-Lys-3-GHRP-6 or PTX. In monkeys, oral AC01 sustainably increased cardiac output (p< 0.05) and stroke volume (p< 0.05) and reduced heart rate without altering central systolic blood pressure or causing tachycardia or arrhythmia. Conclusions AC01 improved CO, SV, and EF in HF mice by increasing contractility in a load-independent fashion. AC01 increased cardiomyocyte contractility without affecting Ca2+ transient amplitudes, through ghrelin receptor Gαi signalling, leading to reduced phosphorylation of cTnI. AC01 improved left ventricle systolic function in monkeys. AC01 is a first in class novel inotrope, which improves contractility in different species without harmful mobilization of Ca2+. AC01 should be explored for the treatment of patients with heart failure.graphical abstract

Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation.

Actin and microtubules are essential cytoskeletal components and coordinate their dynamics through multiple coupling and decoupling mechanisms. However, how actin and microtubule dynamics are decoupled remains incompletely understood. Here, we identified TBC1D3C as a new regulator that can decouple actin filament assembly from microtubule disassembly. We showed that TBC1D3C induces the release of GEF-H1 from microtubules into the cytosol without perturbing microtubule arrays, leading to RhoA activation and actin filament assembly. Mechanistically, we found that TBC1D3C directly binds to GEF-H1, disrupting its interaction with the Tctex-DIC-14-3-3 complex and thereby displacing GEF-H1 from microtubules independently of microtubule disassembly. Super-resolution microscopy and live-cell imaging further confirmed that TBC1D3C triggers GEF-H1 release and actin filament assembly while maintaining microtubule integrity. Therefore, our findings demonstrated that TBC1D3C functions as a direct GEF activator and a novel regulator in decoupling actin assembly from microtubule disassembly, providing new insights into cytoskeletal regulation.

Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity

Background/Objectives: Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors’ expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated. Methods: CD56⁺ CD3− NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10x magnification for 24 h. Results: Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation. Conclusions: Our findings point to the role of Siglec–sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies.

Label-Free Optical Transmission Tomography for Direct Mycological Examination and Monitoring of Intracellular Dynamics

Live-cell imaging generally requires pretreatment with fluorophores to either monitor cellular functions or the dynamics of intracellular processes and structures. We have recently introduced full-field optical coherence tomography for the label-free live-cell imaging of fungi with potential clinical applications for the diagnosis of invasive fungal mold infections. While both the spatial resolution and technical set up of this technology are more likely designed for the histopathological analysis of tissue biopsies, there is to our knowledge no previous work reporting the use of a light interference-based optical technique for direct mycological examination and monitoring of intracellular processes. We describe the first application of dynamic full-field optical transmission tomography (D-FF-OTT) to achieve both high-resolution and live-cell imaging of fungi. First, D-FF-OTT allowed for the precise examination and identification of several elementary structures within a selection of fungal species commonly known to be responsible for invasive fungal infections such as Candida albicans, Aspergillus fumigatus, or Rhizopus arrhizus. Furthermore, D-FF-OTT revealed the intracellular trafficking of organelles and vesicles related to metabolic processes of living fungi, thus opening new perspectives in fast fungal infection diagnostics.

Development of a microfluidic system with a bearing micropump for applying fluid shear force in an intervertebral disc degeneration model involving mechanical stimulation

Background: The intervertebral disc (IVD) functions as a shock absorber with viscoelastic tissues, including the gelatinous nucleus pulposus and the collagenous annulus fibrosus (AF). External mechanical loading influences IVD homeostasis but can cause damage and inflammatory reactions, contributing to disc degeneration. There is a lack of in vitro platforms for modeling IVD disease with mechanical stimulation.Methods: This study aimed to create a mechanical stimulation-based model by subjecting AF cells to shear stress using a micropump system. A micropump platform was used to measure pulsation, flow rate, and shear stress. AF cells were exposed to fluid shear stress of 0.5 and 1 dyne/cm², and morphological changes, cytotoxicity, and cell viability were analyzed through a live/dead assay. Perimeter, area, diameter, and elongation were assessed using live cell imaging and imaging analysis software.Results: The micropump platform exhibited optimal rotor characteristics for uniform pulsation and shear stress. Fluid shear stress at 0.5 dyne/cm² showed no significant difference from the control group, while 1 dyne/cm² reduced cell adhesion and survival. Comparing the 0.5 dyne/cm² shear stress group and interleukin-1β group to the control, significant decreases in perimeter, area, and diameter were observed.Conclusion: The study successfully developed a micro-peristaltic pump platform for applying fluid shear stress to cells, identifying an optimal rotor for uniform stress application. The platform effectively modeled IVD disease, revealing reduced cell adhesion and survival under specific shear stress conditions. This platform has potential promise for discovering biomarkers and exploring cellular responsiveness to external stimuli.

Human amniotic membrane scaffold enhances adipose mesenchymal stromal cell mitochondrial bioenergetics promoting their regenerative capacities.

The human amniotic membrane (hAM) has been applied as a scaffold in tissue engineering to sustain stem cells and enhance their regenerative capacities. We investigated the molecular and biochemical regulations of mesenchymal stromal cells (MSCs) cultured on hAM scaffold in a three-dimensional (3D) setting. Culture of adipose-MSCs (AMSCs) on decellularized hAM showed significant improvement in their viability, proliferative capacity, resistance to apoptosis, and enhanced MSC markers expression. These cultured MSCs displayed altered expression of markers associated with pro-angiogenesis and inflammation and demonstrated increased potential for differentiation into adipogenic and osteogenic lineages. The hAM scaffold modulated cellular respiration by upregulating glycolysis in MSCs as evidenced by increased glucose consumption, cellular pyruvate and lactate production, and upregulation of glycolysis markers. These metabolic changes modulated mitochondrial oxidative phosphorylation (OXPHOS) and altered the production of reactive oxygen species (ROS), expression of OXPHOS markers, and total antioxidant capacity. They also significantly boosted the urea cycle and altered the mitochondrial ultrastructure. Similar findings were observed in bone marrow-derived MSCs (BMSCs). Live cell imaging of BMSCs cultured in the same 3D environment revealed dynamic changes in cellular activity and interactions with its niche. These findings provide evidence for the favorable properties of hAM as a biomimetic scaffold for enhancing the in vitro functionality of MSCs and supporting their potential usefulness in clinical applications.

Zwitterion Modification in Desalination Membrane for Rejecting Salt and Salt Ions in Brackish Water – A Mini Review

ABSTRACTDesalination membrane using zwitterion modification provides a significant opportunity to enhance excellent properties for rejecting salt and salt ions. The outstanding properties are designed by positive and negative charges attached to the membrane surface, including water permeability, salt permeability, fouling, and hydrophilicity of the membrane surface. The hydrophilic membrane, which is mentioned as one of the types of desalination membrane, presents the ability to repel salt and salt ions and pass the water. The modification of hydrophilic membranes is also affected by several factors, including water permeability, salt permeability, fouling of membrane surfaces, crossflow velocity, transmembrane pressure, and temperature. Regarding membrane modification, zwitterion has been applied for several ranges, such as the hemodialysis process, live cell imaging, antibacterial surfaces, and wound dressing. However, using a zwitterion modification membrane for desalinating water containing salt content, including brackish water, has yet to be developed. In addition, literature reviews related to polymer membranes for brackish water desalination have never been comprehensively documented. Thus, this review article addresses the zwitterion modification for desalination membranes, which can reject salt and salt ions in brackish water. Furthermore, the mechanism of zwitterion modification for desalination membranes has been presented. Finally, the challenge of zwitterion modification for desalination membranes has been evaluated to inspire insight for future studies.

HIV-1 N-myristoylation-dependent hijacking of late endosomes/lysosomes to drive Gag assembly in macrophages.

Macrophages represent an important viral reservoir in HIV-1-infected individuals. Different from T cells, HIV-1 assembly in macrophages occurs at intracellular compartments termed virus-containing compartments (VCCs). Our previous research in HeLa cells - in which assembly resembles that found in infected T cells - suggested that late endosomes/lysosomes (LEL) play a role in HIV-1 trafficking towards its assembly sites. However, LEL's role during assembly at VCCs is not fully understood. Herein, we used the HIV-1-inducible cell line THP-1 GagZip as a model to study HIV-1 Gag intracellular trafficking and assembly in macrophages. We demonstrated LEL involvement at VCCs using various microscopy techniques and biochemical approaches. Live-cell imaging revealed that HIV-1 repositions LEL towards the plasma membrane and modulates their motility. We showed that Arl8bmediated LEL repositioning is not responsible of Gag trafficking to VCCs. Additionally, myristoylation inhibition by PCLX-001 decreased Gag presence on endosomes and inhibited VCCs formation, in both cell-line- and primary macrophages. In conclusion, we presented evidence supporting the idea that HIV-1 manipulates the LEL trajectory to guide Gag to VCCs in an N-myristoylation-dependent manner.

Molecular mechanisms underlying glucose-dependent insulinotropic polypeptide secretion in human duodenal organoids.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by enteroendocrine K cells in the proximal small intestine. This study aimed to explore the function of human K cells at the molecular and cellular levels. CRISPR-Cas9 homology-directed repair was used to insert transgenes encoding a yellow fluorescent protein (Venus) or an Epac-based cAMP sensor (Epac-S-H187) in the GIP locus in human duodenal-derived organoids. Fluorescently labelled K cells were purified by FACS for RNA-seq and peptidomic analysis. GIP reporter organoids were employed for GIP secretion assays, live-cell imaging of Ca2+ using Fura-2 and cAMP using Epac-S-H187, and basic electrophysiological characterisation. The G protein-coupled receptor genes GPR142 and CASR were knocked out to evaluate roles in amino acid sensing. RNA-seq of human duodenal K cells revealed enrichment of several G protein-coupled receptors involved in nutrient sensing, including FFAR1, GPBAR1, GPR119, CASR and GPR142. Glucose induced action potential firing and cytosolic Ca2+ elevation and caused a 1.8-fold increase in GIP secretion, which was inhibited by the sodium glucose co-transporter 1/2 (SGLT1/2) blocker sotagliflozin. Activation of the long-chain fatty acid receptor free fatty acid receptor 1 (FFAR1) induced a 2.7-fold increase in GIP secretion, while tryptophan and phenylalanine stimulated secretion by 2.8- and 2.1-fold, respectively. While CASR knockout blunted intracellular Ca2+ responses, a CASR/GPR142 double knockout was needed to reduce GIP secretory responses to aromatic amino acids. The newly generated human organoid K cell model enables transcriptomic and functional characterisation of nutrient-sensing pathways involved in human GIP secretion. Both calcium-sensing receptor (CASR) and G protein-coupled receptor 142 (GPR142) contribute to protein-stimulated GIP secretion. This model will be further used to identify potential targets for modulation of native GIP secretion in diabetes and obesity.

Three-dimensional isotropic imaging of live suspension cells enabled by droplet microvortices

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells-small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations.

Time-Lapse Live-Cell Imaging Using Fluorescent Protein Sensors in Outflow Pathway Cells Under Fluid Flow Conditions.

The role of shear stress in regulating aqueous humor (AH) outflow and intraocular pressure (IOP) in the trabecular meshwork (TM) and Schlemm's canal (SC) of the eye is an emerging field. Shear stress has been shown to activate mechanosensitive ion channels in TM cells and induce nitric oxide production in SC cells, which can affect outflow resistance and lower IOP. Live-cell imaging using fluorescent protein sensors has provided real-time data to investigate the physiological relationship between fluid flow and shear stress in the outflow pathway cells. The successful application of time-lapse live-cell imaging in primary cultured cells has led to the identification of key cellular and molecular mechanisms involved in regulating AH outflow and IOP, including the role of autophagy and primary cilia as mechanosensors. This chapter presents a detailed protocol for conducting time-lapse live-cell imaging under fluid flow conditions in the outflow pathway cells.

Synthesis and self-assembly of perylene-cored poly(amidoamine) dendrimers with poly[2-(dimethylamino)ethyl methacrylate]-modified arms as fluorescent bio-imaging probes and doxorubicin carriers

A poly(amidoamine) (PAMAM) dendrimer is prepared using a divergent method, with perylene-3,4,9,10-tetracarboxylic diimide (PTCDI) as luminescent core. The peripheral amines are modified using α-bromoisobutyryl bromide, allowing the dendrimers to act as macroinitiators for the synthesis of poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) through atom transfer radical polymerization (ATRP) with three different degrees of polymerization. The successful synthesis is verified by conducting a range of analyses, including Fourier-transform infrared (FT-IR) spectroscopy, proton nuclear magnetic resonance (1H NMR) spectroscopy, and X-ray diffraction (XRD). Additionally, the impact of changes in solution pH on the self-assembly of dendrimers is explored. Field emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS) showed unique morphologies, including spherical micelles and cubic-hexagonal structures, at varying pH levels. The self-assembled dendrimers are utilized to load doxorubicin (DOX) and the drug release kinetics are studied under various conditions. The biocompatibility of the dendrimers is assessed using the MTT assay against SH-SY5Y cells. Additionally, higher dendrimer generations improved the solubility, compatibility, and fluorescent properties of the core. The dendrimers' capacity for cellular uptake and fluorescence imaging is also evaluated using SH-SY5Y cells, demonstrating their effectiveness in live-cell fluorescence imaging.

Nanotubules and Cellular Communication in Trabecular Meshwork Cells.

Glaucoma causes dysfunction to tissues located in the anterior and posterior eye. In the anterior eye, the trabecular meshwork (TM) is the site of pathogenesis, where decreased TM cell numbers and alterations to the amount and composition of extracellular matrix hinder outflow of aqueous humor fluid from the anterior chamber. This causes intraocular pressure (IOP) elevation. Elevated IOP, a main risk factor for primary open-angle glaucoma, damages the axons of retinal ganglion cells in the posterior eye, which ultimately leads to blindness. Thus, clinical treatment paradigms for glaucoma are focused on reducing IOP. Normotensive IOPs are established by balancing the production of aqueous fluid from the ciliary body with drainage through the TM to Schlemm's canal. When IOP becomes elevated, TM cells coordinate a homeostatic response to lower IOP, which requires effective and efficient cellular communication. Tunneling nanotubes (TNTs) are transient specialized structures that allow cells to communicate with one another. Actin-rich tubes allow direct transmission of signals and cargoes between cells. This is important to overcome limitations of diffusion-based signaling in aqueous environments such as the anterior eye. Here, we describe a live-cell imaging method for monitoring TNTs in primary TM cells.

Dual RNA-seq reveals distinct families of co-regulated and structurally conserved effectors in Botrytis cinerea infection of Arabidopsis thaliana

BackgroundBotrytis cinerea is a broad-host-range pathogen causing gray mold disease and significant yield losses of numerous crops. However, the mechanisms underlying its rapid invasion and efficient killing of plant cells remain unclear.ResultsIn this study, we elucidated the dynamics of B. cinerea infection in Arabidopsis thaliana by live cell imaging and dual RNA sequencing. We found extensive transcriptional reprogramming events in both the pathogen and the host, which involved metabolic pathways, signaling cascades, and transcriptional regulation. For the pathogen, we identified 591 candidate effector proteins (CEPs) and comprehensively analyzed their co-expression, sequence similarity, and structural conservation. The results revealed temporal co-regulation patterns of these CEPs, indicating coordinated deployment of effectors during B. cinerea infection. Through functional screening of 48 selected CEPs in Nicotiana benthamiana, we identified 11 cell death-inducing proteins (CDIPs) in B. cinerea.ConclusionsThe findings provide important insights into the transcriptional dynamics and effector biology driving B. cinerea pathogenesis. The rapid infection of this pathogen involves the temporal co-regulation of CEPs and the prominent role of CDIPs in host cell death. This work highlights significant changes in gene expression associated with gray mold disease, underscoring the importance of a diverse repertoire of effectors crucial for successful infection.

Glycosyl Mobile Radical Structures of Folic Acid Receptors Impact the Internalization of Functionalized Folate Amphiphilic Alternating Copolymer in Cancer Cells

Folate receptor alpha (FRα) is a glycosylphosphatidylinositol (GPI) membrane-anchored protein containing three N-glycosylated residues at the N47, N139, and N179 termini. These glycosylation sites have been reported to be crucial for the receptor’s structural integrity and its ability to bind and internalize FA. Here, we investigated the role of FRα glycosylation in the binding and internalization efficacy of FA–DABA–SMA in pancreatic PANC-1 cancer cells. There is a strong association of the FA copolymer with FRα with a Pearson coefficient R-value of 0.7179. PANC-1 cancer cells were pretreated with maackia amurensis lectin II (MAL-2), sambucus Nigra lectin (SNA-1), peanut agglutinin (PNA), and wheat germ agglutinin lectin (WGA) at different doses followed by 20 kDa and 350 kDa FA–DABA–SMA loaded with coumarin 153 (C153). Increasing the dosage of MAL2, SNA-1, PNA, and WGA concomitantly and significantly increased the internalization of C153-loaded FA–DABA–SMA in the cells. The half maximal effective lectin concentrations (EC50) to induce cellular internalization into the cytoplasm of the lectins for MAL-2 were 35.88 µg/mL, 3.051 µg/mL for SNA-1, 7.883 µg/mL for PNA, and 0.898 µg/mL for WGA. Live cell imaging of the internalization of 20 kDa and 350 kDa FA copolymers indicated an aggregation of 350 kDa copolymer with FRα in the cytoplasm. In contrast, the 20 kDa FA copolymer remained in the membrane. The data indicate for the first time that the mobile positions of the glycosyl radical groups and the receptor tilt in generating steric hindrance impacted the individual FRα receptors in the binding and internalization of 350 kDa FA–DABA–SMA in cancer cells.

Profiling Bioactive Components of Natural Eggshell Membrane (NEM) for Cartilage Protection and Its Protective Effect on Oxidative Stress in Human Chondrocytes

The current study aimed to investigate the physicochemical properties of the natural eggshell membrane (NEM) and its protective effects against H2O2-induced oxidative stress in human chondrocytes (SW-1353). Bioactive components from NEM related to cartilage were profiled, consisting of 1.1 ± 0.07% hyaluronic acid, 1.2 ± 0.25% total sulfated glycosaminoglycans as chondroitin sulfate, 3.1 ± 0.33% collagen, and 54.4 ± 2.40% total protein. Protein was hydrolyzed up to 43.72 ± 0.76% using in vitro gastro–intestinal digestive enzymes. Peptides eluted at 9.58, 12.46, and 14.58 min using nano-LC-ESI-MS were identified as TEW, SWVE, and VYL peptides with an M/Z value of 435.1874, 520.2402, and 394.2336, respectively. Radical scavenging activity of NEM at 10 mg/mL using the ABTS assay was revealed to be 2.1 times higher than that of the positive control. NEM treatment significantly enhanced cellular SOD expression (p < 0.05). Pre-treatment with NEM (0.1, 1, and 10 mg/mL) dose-dependently reduced H2O2-induced ROS levels in SW-1353. Cell live imaging confirmed that NEM pre-treatment led to a significant reduction in apoptosis expression compared to control. Results from the present study suggest that NEM rich in cartilage protective components including hyaluronic acid, collagen, and chondroitin antioxidative peptides could be a potential therapeutic agent for osteoarthritis (OA) by scavenging oxidative stress.

GLABRA3-mediated trichome branching requires transcriptional repression of MICROTUBULE-DESTABILIZING PROTEIN25.

Microtubules play pivotal roles in establishing trichome branching patterns, which is a model system for studying cell-shape control in Arabidopsis (Arabidopsis thaliana). However, the signaling pathway that regulates microtubule reorganization during trichome branching remains poorly understood. In this study, we report that MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) is involved in GLABRA3 (GL3)-mediated trichome branching by regulating microtubule stability. Loss of MDP25 function led to excessive trichome branching, and this phenotype in mdp25 could not be rescued by the MDP25 K7A or MDP25 K18A mutated variants. Pharmacological treatment and live-cell imaging revealed increased microtubule stability in the mdp25 mutant. Furthermore, the microtubule collar observed during trichome branching remained more intact in mdp25 compared to the WT under oryzalin treatment. Results of genetic assays further demonstrated that knocking out MDP25 rescued the reduced branching phenotype of gl3 trichomes. In gl3 trichomes, normal microtubule organization was disrupted, and microtubule stability was significantly compromised. Moreover, GL3 physically bound to the MDP25 promoter, thereby inhibiting its expression. Overexpression of GL3 negated the effects of PMDP25-driven MDP25 or its mutant proteins on trichome branching and microtubules in the mdp25 background. Overall, our study uncovers a mechanism by which GL3 inhibits MDP25 transcription, thereby influencing microtubule stability and regulating trichome branching. This mechanism provides a connection between early regulatory components and microtubules during trichome development.

A low-dose pemetrexed-cisplatin combination regimen induces significant nephrotoxicity in mice

BackgroundPemetrexed is combined with cisplatin to treat cancer. Whether pemetrexed-cisplatin combination chemotherapy exacerbates cisplatin nephrotoxicity is unclear. Here, we investigated kidney injury in mice administered a non-lethal low-dose regimen of pemetrexed or cisplatin alone and compared it with a pemetrexed-cisplatin combination.MethodsMice were randomly divided into four groups and administered intraperitoneally the experimental drugs solubilized in captisol (sulfobutylether β-cyclodextrin). Group 1 received captisol, Group 2 pemetrexed (10 mg/kg), Group 3 cisplatin (1 mg/kg), and Group 4 pemetrexed (10 mg/kg) plus cisplatin (1 mg/kg). The mice were treated every other day for two weeks, three times per week. Glomerular filtration rate (GFR) was determined on the third day after the last treatment, followed by a necropsy.ResultsWhereas the relative kidney weight was comparable in the control vs. pemetrexed or cisplatin alone group, it was significantly increased in the combination group. Mice treated with cisplatin and pemetrexed-cisplatin combination exhibited reduced GFR. The pemetrexed-cisplatin combination caused significant increases in the plasma or urinary levels of kidney injury biomarkers, renal lipid peroxidation, and nitrosative stress compared with pemetrexed or cisplatin alone. Histopathology revealed that pemetrexed or cisplatin alone had minimal effects on the kidneys. By contrast, the pemetrexed-cisplatin combination caused tubular degeneration, dilatation, and granular casts. Live-cell imaging showed that the pemetrexed-cisplatin combination caused more severe apoptosis of primary renal epithelial cells than individual concentrations.ConclusionsThese findings suggest that combining pemetrexed and cisplatin causes oxidative kidney damage at individual doses that do not cause significant nephrotoxicity. Hence, the renal function of patients undergoing treatment with the pemetrexed-cisplatin combination needs extensive monitoring.

Imaging of Live Cells by Digital Holographic Microscopy

Imaging of microscopic objects is of fundamental importance, especially in life sciences. Recent fast progress in electronic detection and control, numerical computation, and digital image processing, has been crucial in advancing modern microscopy. Digital holography is a new field in three-dimensional imaging. Digital reconstruction of a hologram offers the remarkable capability to refocus at different depths inside a transparent or semi-transparent object. Thus, this technique is very suitable for biological cell studies in vivo and could have many biomedical and biological applications. A comprehensive review of the research carried out in the area of digital holographic microscopy (DHM) for live-cell imaging is presented. The novel microscopic technique is non-destructive and label-free and offers unmatched imaging capabilities for biological and bio-medical applications. It is also suitable for imaging and modelling of key metabolic processes in living cells, microbial communities or multicellular plant tissues. Live-cell imaging by DHM allows investigation of the dynamic processes underlying the function and morphology of cells. Future applications of DHM can include real-time cell monitoring in response to clinically relevant compounds. The effect of drugs on migration, proliferation, and apoptosis of abnormal cells is an emerging field of this novel microscopic technique.

Glial cell derived pathway directs regenerating optic nerve axons toward the CNS midline.

Several RGC intrinsic signaling pathways have been shown to enhance RGC survival and RGC axonal growth after optic nerve injury. Yet an unresolved challenge for regenerating RGC axons is to properly navigate the optic chiasm located at the Central Nervous System midline. Here, we use live-cell imaging in larval zebrafish to show that regrowing RGC axons initiate growth toward the midline and extend along a trajectory similar to their original projection. From a candidate genetic screen, we identify the glycosyltransferase Lh3 to be required during the process of regeneration to direct regrowing RGC axons toward the midline. Moreover, we find that mutants in collagen 18a1 (col18a1), a putative Lh3 substrate, display RGC axonal misguidance phenotypes similar to those we observe in lh3 mutants, suggesting that lh3 may act through col18a1 during regeneration. Finally, we show that transgenic lh3 expression in sox10+ presumptive olig2+ oligodendrocytes located near the optic chiasm restores directed axonal growth. Combined these data identify lh3 and col18a1 as part of a glial derived molecular pathway critical for guiding in vivo regenerating RGC axons towards and across the optic chiasm.