Confocal Imaging Research Articles

PH-Responsive Rhodamine Nanotube Capable of Self-Reporting the Assembly State.

Nanomaterials that respond to intracellular signals, such as pH, have the potential for many biomedical applications, such as drug delivery, because the assembly/disassembly process can be tailored to respond to a stimulus characteristic of a specific subcellular location. In this work, two rhodamine-peptides that form stable nanotubes at physiological pH but dissociate into highly fluorescent monomers within the acidified interior of endosomal/lysosomal cellular compartments have been developed. The rhodamine dipeptide conjugates, NH2-KK(RhB)-NH2 (RhB-KK) and NH2-EK(RhB)-NH2 (RhB-KE) with rhodamine B chromophores appended at the ε-amino position of a lysine residue, were shown to assemble into well-defined nanotubes at pH values above ∼4-5 and to dissociate into a fluorescent monomer state at lower pH values. The pH dependence of the assembly process was investigated using circular dichroism (CD) and fluorescence spectroscopy along with transmission electron microscopy (TEM), atomic force microscopy (AFM), and confocal imaging. Although the ring opening/closing transition of the rhodamine chromophore took place at pH 4.1 for both peptides, the onset of assembly began at pH 4.6 for RhB-KE and at a comparatively more basic pH (5.8) for RhB-KK. Accordingly, the rhodamine-peptides interconverted between three pH-dependent states: an open-ring, monomeric state (λmax 580 nm, λex 550 nm) at pH values at or below ∼4.6; a closed-ring, nanotube form that exhibits AIEE (λmax 460 nm, λex = 330 nm) at higher pH values; a closed-ring, nonemissive monomeric state that emerged below the critical micelle concentrations (CMC). The pH-responsive features of the peptides were evaluated by live-cell imaging in three cancer cell lines using confocal laser scanning microscopy (CLSM). Visualizing the cells after incubation with either RhB-KE or RhB-KK produced CLSM images with a punctate appearance in the Texas Red channel that colocalized with the lysosomes. These experiments indicate that the nanotubes were rapidly trafficked into the acidic lysosomal compartments within the cells, which induced dissociation into a monomeric, open state. Uptake inhibition studies suggested that cellular uptake was mediated by either caveolae- or clathrin-mediated endocytosis, depending on the cell line studied.

Harnessing forward multiple scattering for optical imaging deep inside an opaque medium

As light travels through a disordered medium such as biological tissues, it undergoes multiple scattering events. This phenomenon is detrimental to in-depth optical microscopy, as it causes a drastic degradation of contrast, resolution and brightness of the resulting image beyond a few scattering mean free paths. However, the information about the inner reflectivity of the sample is not lost; only scrambled. To recover this information, a matrix approach of optical imaging can be fruitful. Here, we report on a de-scanned measurement of a high-dimension reflection matrix R via low coherence interferometry. Then, we show how a set of independent focusing laws can be extracted for each medium voxel through an iterative multi-scale analysis of wave distortions contained in R. It enables an optimal and local compensation of forward multiple scattering paths and provides a three-dimensional confocal image of the sample as the latter one had become digitally transparent. The proof-of-concept experiment is performed on a human opaque cornea and an extension of the penetration depth by a factor five is demonstrated compared to the state-of-the-art.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as β-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Large animal model species in pluripotent stem cell therapy research and development for retinal diseases: a systematic review.

Retinal cell therapy modalities, in the category of advanced therapy medicinal products (ATMPs), are being developed to target several retinal diseases. Testing in large animal models (LAMs) is a crucial step in translating retinal ATMPs into clinical practice. However, challenges including budgetary and infrastructure constraints can hinder LAM research design and execution. Here, to facilitate the comparison of the various LAMs in pluripotent retinal cell therapy research, we aimed to systematically evaluate the species distribution, reported scientific utility, and methodology of a range of LAMs. A systematic search using the words retina, stem cell, transplantation, large animal, pig, rabbit, dog, and nonhuman primate was conducted in the PubMed, Embase, Science Direct and GoogleScholar databases in February 2023. We included 22 studies involving pluripotent stem cells (induced pluripotent stem cells or human embryonic stem cells) in LAMs, including non-human primates (NHP), pigs, dogs, and rabbits. Nearly half of the studies utilized wild-type animal models. In other studies, retinal degeneration features were simulated via laser, chemical, or genetic insult. Transplants were delivered subretinally, either as cell suspensions or pre-formed monolayers (with or without biodegradable scaffolding). The transplanted cells dose per eye varied widely (40,000 - 4,000,000 per dose). Cells were delivered via vitrectomy surgery in 15 studies and by an "ab externo" approach in one study. Structural outcomes were assessed using confocal scanning laser ophthalmoscopy imaging. Functional outcomes included multifocal electroretinogram and, in one case, a measure of visual acuity. Generally, cell suspension transplants exhibited low intraretinal incorporation, while monolayer transplants incorporated more efficiently. Immune responses posed challenges for allogeneic transplants, suggesting that autologous iPSC-derived transplants may be required to decrease the likelihood of rejection. The use of appropriate LAMs helps to advance the development of retinal ATMPs. The anatomical similarity of LAM and human eyes allows the implementation of clinically-relevant surgical techniques. While the FDA Modernization Act 2.0 has provided a framework to consider alternative methods including tissue-on-a-chip and human cell culture models for pharmacologic studies, LAM testing remains useful for cell and tissue replacement studies to inform the development of clinical trial protocols.

Hyperglycemic environments directly compromise intestinal epithelial barrier function in an organoid model and hyaluronan (∼35 kDa) protects via a layilin dependent mechanism

BackgroundMetabolic syndrome and diabetes in obese individuals are strong risk factors for development of inflammatory bowel disease (IBD) and colorectal cancer. The pathogenic mechanisms of low-grade metabolic inflammation, including chronic hyperglycemic stress, in disrupting gut homeostasis are poorly understood. In this study, we sought to understand the impact of a hyperglycemic environment on intestinal barrier integrity and the protective effects of small molecular weight (35 kDa) hyaluronan on epithelial barrier function. MethodsIntestinal organoids derived from mouse colon were grown in normal glucose media (5 mM) or high glucose media (25 mM) to study the impact of hyperglycemic stress on the intestinal barrier. Additionally, organoids were pretreated with 35 kDa hyaluronan (HA35) to investigate the effect of hyaluronan on epithelial barrier under high glucose stress. Immunoblotting as well as confocal imaging was used to understand changes in barrier proteins, quantitative as well as spatial distribution, respectively. Alterations in barrier function were measured using trans-epithelial electrical resistance and fluorescein isothiocyanate flux assays. Untargeted proteomics analysis was performed to elucidate mechanisms by which HA35 exerts a protective effect on the barrier. Intestinal organoids derived from receptor knockout mice specific to various HA receptors were utilized to understand the role of HA receptors in barrier protection under high glucose conditions. ResultsWe found that high glucose stress decreased the protein expression as well as spatial distribution of two key barrier proteins, zona occludens-1 (ZO-1) and occludin. HA35 prevented the degradation or loss of ZO-1 and maintained the spatial distribution of both ZO-1 and occludin under hyperglycemic stress. Functionally, we also observed a protective effect of HA35 on the epithelial barrier under high glucose conditions. We found that HA receptor, layilin, was involved in preventing barrier protein loss (ZO-1) as well as maintaining spatial distribution of ZO-1 and occludin. Additionally, proteomics analysis showed that cell death and survival was the primary pathway upregulated in organoids treated with HA35 under high glucose stress. We found that XIAP associated factor 1 (Xaf1) was modulated by HA35 thereby regulating apoptotic cell death in the intestinal organoid system. Finally, we observed that spatial organization of both focal adhesion kinase (FAK) as well as F-actin was mediated by HA35 via layilin. ConclusionOur results highlight the impact of hyperglycemic stress on the intestinal barrier function. This is of clinical relevance, as impaired barrier function has been observed in individuals with metabolic syndrome. Additionally, we demonstrate barrier protective effects of HA35 through its receptor layilin and modulation of cellular apoptosis under high glucose stress.

Stability of the Dentin-Bonded Interface Using Self-Etching Adhesive Containing Diacrylamide after Bacterial Challenge.

Purpose/Aim: Acrylamides are hydrolytically stable at pH lower than 2, and were shown to preserve bonded interface integrity with two-step, total etch adhesives. The objective of this study was to leverage those two characteristics in self-etching primers containing the acidic monomer 10-MDP and test the microtensile bond strength before and after incubation with S. mutans incubation. Materials and Methods: Acidic primers (10 wt % 10-methacryloyloxydecyl dihydrogen phosphate─10-MDP; 45 wt % N,N-diethyl-1,3-bis(acrylamido)propane─DEBAAP, or 2-hydroxyethyl methacrylate─HEMA; 45 wt %, glycerol-dimethacrylate─GDMA) and adhesives (DEBAAP or HEMA/10-MDP/UDMA 45/10/45 wt %) were made polymerizable by the addition of 0.2 wt % camphorquinone, 0.8 wt % ethyl-4-dimethylaminobenzoate, 0.4 wt % diphenyliodonium hexafluorophosphate, and 0.1 wt % butylhydroxytoluene. Nonsolvated materials were characterized for flexural strength (FS), modulus (E), toughness, water sorption/solubility (WS/SL), contact angle, and vinyl conversion (DC). Viscosity was evaluated after adding 20 and 40 vol % ethanol to the primer and adhesive, respectively. The experimental materials or Clearfil SE Bond (CC─commercial control) were used to bond a commercial composite (Filtek Supreme) to the flat surface of human dentin. Microtensile bond strength (MTBS) was tested in 1 mm2 sticks for the 5 primer/bond combinations: CC (Clearfil Bond Primer and Bond), HH (HEMA/HEMA), DD (DEBAAP/DEBAAP), HD (HEMA/DEBAAP), and DH (DEBAAP/HEMA). Prior to testing, sticks were stored in water or biofilm-inducing culture medium with S. mutans for 1 week. Confocal images and FTIR-ATR evaluation evaluated the hybrid layer of the adhesives. Results were analyzed using Student's t-test (WS, SL, DC, contact angle, FS, E, toughness), one-way ANOVA/Tukey's test for viscosity, and two-way ANOVA/Tukey's test for MTBS (95%). Results: HEMA-based materials had lower contact angle (p = 0.004), higher WS (p < 0.001), and similar SL values compared to DEBAAP (p = 0.126). FS (p = 0.171) and E (p = 0.065) dry values were similar, but after one week of water storage, FS/E dropped more significantly for HEMA materials. Dry and wet toughness was greater for DEBAAP (p < 0.001), but it also had the greatest drop (46%). Clearfil bonds had the highest viscosity, followed by DEBAAP and HEMA, respectively (p = 0.002). For the primers, HEMA had the lowest viscosity (p = 0.003). As far as MTBS, all groups tested in water were statistically different when compared with HH (p < 0.001). After storage in biofilm, DH had the highest MTBS value, being statistically different from HH (p = 0.002), CC (p = 0.015), and DD (p = 0.027). Conclusions: The addition of a diacrylamide and its association with HEMA in self-etching adhesive systems provided greater bonding stability after bacterial challenge.

Confocal structured illumination microscopy for improving the signal-to-noise ratio and depth of fluorescent optical section imaging

Light scattering from the sample is an unavoidable problem in fluorescence imaging. Compared with laser scanning confocal scanning microscopy, although optical-sectioning structured illumination microscopy (OS-SIM) has the advantages of fast imaging speed and low phototoxicity, it faces the challenge of removing the scattering fluorescent noise particularly when imaging thick and densely labeled sampling. To improve the imaging performance of OS-SIM, we introduce the concept of confocal imaging to OS-SIM and propose confocal structured illumination microscopy (CSIM). CSIM exploits the principle of dual imaging to reconstruct a dual image from each camera pixel. The scattered fluorescent noise and the unscattered fluorescent signal recorded by the camera pixel are separated in the reconstructed dual image. By extracting the unscattered fluorescent signal from each dual image based on the conjugate relationship between the camera and the spatial light modulator, we can eliminate the scattered fluorescent noise and reconstruct a confocal image. We have built the theoretical framework of CSIM. Experimental results of fluorescent optical-sectioning demonstrate that CSIM achieves a superior performance in eliminating scattered fluorescent noise and in extending imaging depth compared with existing OS-SIM. CSIM is expected to broaden the application range of OS-SIM.

Nuclear deformation and dynamics of migrating cells in 3D confinement reveal adaptation of pulling and pushing forces.

Eukaryotic cells show an astounding ability to remodel their shape and cytoskeleton and to migrate through pores and constrictions smaller than their nuclear diameter. However, the relation of nuclear deformation and migration dynamics in confinement remains unclear. Here, we study the mechanics and dynamics of mesenchymal cancer cell nuclei transitioning through three-dimensional compliant hydrogel channels. We find a biphasic dependence of migration speed and transition frequency on channel width, peaking at widths comparable to the nuclear diameter. Using confocal imaging and hydrogel bead displacement, we determine nuclear deformations and corresponding forces during confined migration. The nucleus deforms reversibly with a reduction in volume during confinement. With decreasing channel width, the nuclear shape during transmigration changes biphasically, concomitant with the transitioning dynamics. Our proposed physical model explains the observed nuclear shapes and transitioning dynamics in terms of the cytoskeletal force generation adapting from purely pulling-based to a combined pulling- and pushing-based mechanism with increasing nuclear confinement.

Switch to phagocytic microglia by CSFR1 inhibition drives amyloid-beta clearance from glutamatergic terminals rescuing LTP in acute hippocampal slices

Microglia, traditionally regarded as innate immune cells in the brain, drive neuroinflammation and synaptic dysfunctions in the early phases of Alzheimer disease (AD), acting upstream to Aβ accumulation. Colony stimulating factor 1-receptor (CSF-1R) is predominantly expressed on microglia and its levels are significantly increased in neurodegenerative diseases, possibly contributing to the chronic inflammatory microglial response. On the other hand, CSF-1R inhibitors confer neuroprotection in preclinical models of neurodegenerative diseases. Here, we determined the effects of the CSF-1R inhibitor PLX3397 on the Aβ-mediated synaptic alterations in ex vivo hippocampal slices. Electrophysiological findings show that PLX3397 rescues LTP impairment and neurotransmission changes induced by Aβ. In addition, using confocal imaging experiments, we demonstrate that PLX3397 stimulates a microglial transition toward a phagocytic phenotype, which in turn promotes the clearance of Aβ from glutamatergic terminals. We believe that the selective pruning of Aβ-loaded synaptic terminals might contribute to the restoration of LTP and excitatory transmission alterations observed upon acute PLX3397 treatment. This result is in accordance with the mechanism proposed for CSF1R inhibitors, that is to eliminate responsive microglia and replace it with newly generated, homeostatic microglia, capable of promoting brain repair. Overall, our findings identify a connection between the rapid microglia adjustments and the early synaptic alterations observed in AD, possibly highlighting a novel disease-modifying target.

A dual-channel fluorescent probe targeting lysosomes for differential detection of Cys/Hcy and GSH: Applications in food, pharmaceutical analysis and bioimaging

Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.

Pathological Role of High Sugar in Mitochondrial Respiratory Chain Defect-Augmented Mitochondrial Stress.

According to many research groups, high glucose induces the overproduction of superoxide anions, with reactive oxygen species (ROS) generally being considered the link between high glucose levels and the toxicity seen at cellular levels. Respiratory complex anomalies can lead to the production of ROS. Calcium [Ca2+] at physiological levels serves as a second messenger in many physiological functions. Accordingly, mitochondrial calcium [Ca2+]m overload leads to ROS production, which can be lethal to the mitochondria through various mechanisms. F1F0-ATPase (ATP synthase or complex V) is the enzyme responsible for catalyzing the final step of oxidative phosphorylation. This is achieved by F1F0-ATPase coupling the translocation of protons in the mitochondrial intermembrane space and shuttling them to the mitochondrial matrix for ATP synthesis to take place. Mitochondrial complex V T8993G mutation specifically blocks the translocation of protons across the intermembrane space, thereby blocking ATP synthesis and, in turn, leading to Neuropathy, Ataxia, and Retinitis Pigmentosa (NARP) syndrome. This study seeks to explore the possibility of [Ca2+]m overload mediating the pathological roles of high glucose in defective respiratory chain-mediated mitochondrial stress. NARP cybrids are the in vitro experimental models of cells with F1FO-ATPase defects, with these cells harboring 98% of mtDNA T8993G mutations. Their counterparts, 143B osteosarcoma cell lines, are the parental cell lines used for comparison. We observed that NARP cells mediated and enhanced the death of cells (apoptosis) when incubated with hydrogen peroxide (H2O2) and high glucose, as depicted using the MTT assay of cell viability. Furthermore, using fluorescence probe-coupled laser scanning confocal imaging microscopy, NARP cells were found to significantly enable mitochondrial reactive oxygen species (mROS) formation and enhance the depolarization of the mitochondrial membrane potential (ΔΨm). Elucidating the mechanisms of sugar-enhanced toxicity on the mitochondria may, in the future, help to alleviate the symptoms of patients with NARP syndromes and other neurodegenerative diseases.

Dynamic regulation of tissue fluidity controls skin repair during wound healing

During wound healing, different pools of stem cells (SCs) contribute to skin repair. However, how SCs become activated and drive the tissue remodeling essential for skin repair is still poorly understood. Here, by developing a mouse model allowing lineage tracing and basal cell lineage ablation, we monitor SC fate and tissue dynamics during regeneration using confocal and intravital imaging. Analysis of basal cell rearrangements shows dynamic transitions from a solid-like homeostatic state to a fluid-like state allowing tissue remodeling during repair, as predicted by a minimal mathematical modeling of the spatiotemporal dynamics and fate behavior of basal cells. The basal cell layer progressively returns to a solid-like state with re-epithelialization. Bulk, single-cell RNA, and epigenetic profiling of SCs, together with functional experiments, uncover a common regenerative state regulated by the EGFR/AP1 axis activated during tissue fluidization that is essential for skin SC activation and tissue repair.

Inhibition of Botrytis cinerea infection on tomato leaves by submicronization of penthiopyrad particles

If the spray concentration of the pesticide remains constant, submicronization of the active ingredients significantly increase the particle number concentration, thereby substantially enhancing the probability of contact with plant pathogens and is anticipated to result in reduced pesticide usage. In this study, we investigated the effect of submicronization of pesticide active ingredients on the inhibitory effect against plant pathogens. Three types of monodispersed penthiopyrad (pesticide) particles with different median diameters and minimal overlap in the particle size distribution were successfully prepared using a poor solvent dilution method. Using these penthiopyrad particles, we conducted experiments on infection of tomatoes (model plants) with Botrytis cinerea (plant pathogen). The results showed that the submicronization of pesticide particles enhanced the inhibitory effects of the sprayed pesticide on the plant pathogen, while also decreasing the required amount of pesticide sprayed to the plants. Furthermore, confocal microscopy images showed that smaller particles are taken up by B. cinerea hyphae better than larger particles. Additionally, the bacterial flora on the leaf surface was not affected by submicronized pesticide particles larger than 100 nm. These findings imply that submicronization of pesticide active ingredients increases the probability of reaching targeted plant pathogens on the leaf surface, ultimately resulting in an increase in their uptake by the plant pathogens (i.e., significantly improved delivery of pesticides).

The Impact of Low Protein Diet on the Molecular and Cellular Development of the Fetal Kidney.

Low nephron number has a direct impact on the development of hypertension and chronic kidney disease later in life. While intrauterine growth restriction caused by maternal low protein diet (LPD) is thought to be a significant cause of reduced nephron endowment in impoverished communities, its influence on the cellular and molecular processes which drive nephron formation are poorly understood. We conducted a comprehensive characterization of the impact of LPD on kidney development using tomographic and confocal imaging to quantify changes in branching morphogenesis and the cellular and morphological features of nephrogenic niches across development. These analyses were paired with single-cell RNA sequencing to dissect the transcriptional changes that LPD imposes during renal development to affect nephron number. Single cell analysis at E14.5 and P0 revealed differences in the expression of genes and pathways involved in metabolism, cell cycle, epigenetic regulators and reciprocal inductive signals in most cell types analyzed, yielding imbalances and shifts in cellular energy production and cellular trajectories. In the nephron progenitor cells, LPD impeded cellular commitment and differentiation towards pre-tubular and renal vesicle structures. Confocal microscopy revealed a reduction in the number of pre-tubular aggregates and proliferation in nephron progenitor cells. We also found changes in branching morphogenesis, with a reduction in cell proliferation in the ureteric tips as well as reduced tip and tip parent lengths by optical projection tomography which causes patterning defects. This unique profiling demonstrates how a fetal programming defect leads to low nephron endowment which is intricately linked to changes in both branching morphogenesis and the commitment of nephron progenitor cells. The commitment of progenitor cells is pivotal for nephron formation and is significantly influenced by nutritional factors, with a low protein diet driving alterations in this program which directly results in a reduced nephron endowment. While a mother's diet can negatively impact the number of nephrons in the kidneys of her offspring, the root cellular and molecular drivers of these deficits have not been rigorously explored. In this study we use advanced imaging and gene expression analysis in mouse models to define how a maternal low protein diet, analogous to that of impoverished communities, results in reduced nephron endowment. We find that low protein diet has pleiotropic effects on metabolism and the normal developmental programs of gene expression. These profoundly impact the process of branching morphogenesis necessary to establish niches for nephron generation and change cell behaviors which regulate how and when nephron progenitor cells commit to differentiation.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D

Immunohistochemistry is a crucial method for detecting specific proteins within tissue samples, yet constrained to one biomarker per tissue section. Multiplexed immunofluorescence, while allowing simultaneous visualization of multiple proteins, faces limitations in the number of simultaneous fluorescent labels due to spectral overlap. Although cyclic immunofluorescence techniques have successfully broadened antibody staining capacities in a single tissue sample, they are plagued by time-consuming and labor-intensive procedures, sample degradation risks, and inability to scale beyond thin sections. In this study, we introduce the use of 3D confocal Fluorescence Lifetime Imaging Microscopy as a high-throughput, multiplexed immunofluorescence platform that can differentiate 11 or more biomarkers in 3D tissue volumes. Leveraging both spectral and lifetime information, this approach allows for practical spatial biology in thin sections that can readily scale to larger volumes of tissue. We believe that this highly multiplexed and versatile biomarker imaging platform will significantly expedite cancer research and enable new translational approaches in the future.

In Situ Intracellular Autocatalytic Hairpin Assembly of the Y-Shaped DNA Nanostructure for miR-155 Sensing and Gene Silencing.

miR-155 is a class of cancer markers closely related to cancer metastasis and invasion. Combining in situ detection with gene silencing not only helps to analyze the information on the abundance and spatial location of microRNA expression in the cell but also synergizes the therapy. In this work, we prepared HD@CM vesicles with three hairpin DNAs by using MCF-7 cell membranes. The hairpin DNAs can be triggered by endogenous miR-155, which opens the autocatalytic molecular circuit (ACHA) and obtains Y-shaped DNA nanostructures. This nanostructure not only detects endogenous miR-155 with high sensitivity for in situ imaging but also enables gene regulation of intracellular survivin mRNA. The levels of miR-155 in MDA-MB-231, MCF-7, Hela, and HEK-293T cells are found to be 7703, 3978, 1696, and 1229 copies/cell, respectively, as detected by HD@CMs. The fluorescence produced by HD@CM after coincubation with different cells is found to be proportional to the intracellular miR-155 content by confocal imaging. In addition, the gene regulatory function of the Y-shaped DNA structure resulted in significant inhibition of survivin protein expression and apoptosis rates of up to 83%. We look forward to the future application of our HD@CM platform for the precise diagnosis and programmable treatment of clinical cancers.

Muscular expression of pezo-1 differentially contributes to swimming and crawling production in the nematode C. elegans.

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins that are widely expressed in neuronal and muscular tissues. This study explores the role of the mechanoreceptor PEZO-1 in the body wall muscles of Caenorhabditis elegans, focusing on its influence on two locomotor behaviors, swimming and crawling. Using confocal imaging, we reveal that PEZO-1 localizes to the sarcolemma and plays a crucial role in modulating calcium dynamics that are important for muscle contraction. When we knocked down pezo-1 expression in striated muscles with RNA interference, calcium levels in head and tail muscles increased. While heightened, the overall trajectory of the calcium signal during the crawl cycle remained the same. While downregulation of pezo-1 led to an increase in crawling speed, it caused a reduction in swimming speed. Reduction in pezo-1 expression also resulted in the increased activation of the ventral tail muscles, and a disruption of dorsoventral movement asymmetry, a critical feature that enables propulsion in water. These alterations were correlated with impaired swimming posture and path curvature, suggesting that PEZO-1 has different functions during swimming and crawling.

Sustained release of MAPK14-targeting siRNA from polyelectrolyte complex hydrogels mitigates MSC osteogenesis in vitro with potential application in growth plate injury.

The growth plate is a cartilage structure at the end of long bones which mediates growth in children. When fractured, the formation of bony repair tissue known as a "bony bar" can occur and cause limb deformities. There are currently no effective clinical solutions for the prevention of the bony bar formation or regeneration of healthy growth plate cartilage after a fracture. This study employs previously developed alginate/chitosan polyelectrolyte complex (PEC) hydrogels as a sustained release vehicle for the delivery of short-interfering RNA (siRNA). Specifically, the siRNA targets the p38-MAPK pathway in mesenchymal stem cells (MSCs) to prevent their osteogenic differentiation. In vitro experimental findings show sustained release of siRNA from the hydrogels for 6 months. Flow cytometry and confocal imaging indicate that the hydrogels release siRNA to effectively knockdown GFP expression over a sustained period. MAPK-14 targeting siRNA was used to knockdown the expression of MAPK-14 and correspondingly decrease the expression of other osteogenic genes in MSCs in vitro over the span of 21 days. These hydrogels were used in a rat model of growth plate injury to determine whether siMAPK-14 released from the gels could inhibit bony bar formation. No significant reduction of bony bar formation was seen in vivo at the one concentration of siRNA examined. This PEC hydrogel represents a significant advancement for siRNA sustained delivery, and presents an interesting potential therapeutic delivery system for growth plate injuries and other regenerative medicine applications.

Tear neuropeptide Y as a non-invasive marker of peripheral microvascular complications in type 1 diabetes

AimsTo investigate tear neuropeptide Y (NPY) and substance P concentrations in individuals with type 1 diabetes, comparing those with and without both diabetic retinopathy (DR) and peripheral neuropathy. MethodsThis cross-sectional study involved 41 participants with type 1 diabetes and none to moderate DR, and 22 healthy controls. Assessments included clinical ocular surface parameters, quantification of corneal nerve attributes (based on in vivo confocal microscopy imaging), DR grading, and evaluation for small and large fibre neuropathy. Concentrations of NPY and substance P in tear samples were measured using enzyme-linked immunosorbent assay. ResultsMean (± standard deviation) tear NPY concentrations in participants with type 1 diabetes and length-dependent small fibre neuropathy (SFN) was lower than in controls (10.84 ± 4.10 ng/mL vs 14.72 ± 3.12 ng/mL; p=0.004), but not significantly different from type 1 diabetes participants without SFN (13.39 ± 4.66 ng/mL; p=0.11). Tear NPY levels were lower in individuals with type 1 diabetes and mild/moderate non-proliferative DR (10.44 ± 3.46 ng/mL) compared to none/minimal DR (13.79 ± 4.76 ng/mL; p=0.0005) and controls. In separate linear regression models, both the presence of SFN (β = −0.75, p=0.02) and the presence of mild/moderate DR (β = −0.84, p=0.009) were significantly associated with tear NPY levels relative to controls, after adjusting for participant age, sex, and dry eye disease. There were no inter-group differences for tear substance P concentrations. ConclusionsTear NPY has potential utility as an indicator of peripheral microvascular complications associated with type 1 diabetes.

Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.