Cell Biological Studies Research Articles

Single Cell Approaches Reveal Perturbed Brain Vascular Molecules in Alzheimer’s Disease

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, maintenance of the blood‐brain‐barrier (BBB) properties, and brain energy metabolism. Although numerous cell biology studies demonstrate the role of the GVU for BBB breakdown in Alzheimer’s disease (AD), the precise molecular changes contributing to its pathophysiology are unclear.MethodWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in 24 AD and control brains to understand the transcriptional changes in the cells of GVU. We acquired the snRNAseq profile of 79,751 total brain cells that comprise 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as activated pericytes, endothelia and resting pericytes. We identified differentially expressed genes (DEGs) and their enriched pathways in these clusters and detected the most transcriptional changes within activated pericytes. Using our data and a knowledge‐based predictive algorithm, we discovered and prioritized molecular interactions between vascular and astrocyte clusters, the main cell types of the GVU of the BBB.ResultsVascular targets predicted to interact with astrocytic ligands have biological functions in signalling, angiogenesis, amyloid ß metabolism and cytoskeletal structure. Top astrocytic and vascular interacting molecules include both novel and known AD risk genes such as APOE, APP and ECE1. We validated gene expression changes using qPCR. We are in the process of evaluating these gene expression changes in antemortem blood from AD and control participants.ConclusionOur findings provide valuable information into the transcriptional changes in predicted vascular‐astrocytic partners at the GVU, highlighting insights into the molecular mechanisms of BBB breakdown in AD.

Structure-based discovery of a novel small-molecule inhibitor of TEAD palmitoylation with anticancer activity.

The paralogous oncogenic transcriptional coactivators YAP and TAZ are the distal effectors of the Hippo signaling pathway, which plays a critical role in cell proliferation, survival and cell fate specification. They are frequently deregulated in most human cancers, where they contribute to multiple aspects of tumorigenesis including growth, metabolism, metastasis and chemo/immunotherapy resistance. Thus, they provide a critical point for therapeutic intervention. However, due to their intrinsically disordered structure, they are challenging to target directly. Since YAP/TAZ exerts oncogenic activity by associating with the TEAD1-4 transcription factors, to regulate target gene expression, YAP activity can be controlled indirectly by regulating TEAD1-4. Interestingly, TEADs undergo autopalmitoylation, which is essential for their stability and function, and small-molecule inhibitors that prevent this posttranslational modification can render them unstable. In this article we report discovery of a novel small molecule inhibitor of YAP activity. We combined structure-based virtual ligand screening with biochemical and cell biological studies and identified JM7, which inhibits YAP transcriptional reporter activity with an IC50 of 972 nMoles/Ltr. Further, it inhibits YAP target gene expression, without affecting YAP/TEAD localization. Mechanistically, JM7 inhibits TEAD palmitoylation and renders them unstable. Cellular thermal shift assay revealed that JM7 directly binds to TEAD1-4 in cells. Consistent with the inhibitory effect of JM7 on YAP activity, it significantly impairs proliferation, colony-formation and migration of mesothelioma (NCI-H226), breast (MDA-MB-231) and ovarian (OVCAR-8) cancer cells that exhibit increased YAP activity. Collectively, these results establish JM7 as a novel lead compound for development of more potent inhibitors of TEAD palmitoylation for treating cancer.

Rutin-loaded Phyto-Sterosomes as a potential approach for the treatment of hepatocellular carcinoma: In-vitro and in-vivo studies

β-Sitosterol, isolated from Senecio fulgens, was used as a "heart-friendly" alternative to cholesterol to prepare a novel non-phospholipid liposomal nano-carrier "Phyto-Sterosomes". Different saturated fatty acids (Lauric, Stearic, and Palmitic acids) were used, in different ratios with β-sitosterol (1:2 and 1:3), to prepare the Phyto-Sterosomes (P-STs) aiming to enhance the solubility and biological efficacy of the poorly soluble drug under study "Rutin". The in vitro release study performed for the formulations proved that the drug release followed a diffusion-dependent mechanism. Physicochemical investigations showed that P-ST6, composed of β-sitosterol: lauric acid (3:1), was spherical and had the highest drug encapsulation efficiency (95.7%), smallest particle size (250.6 nm) with a homogenous distribution (PDI = 0.2) and a high zeta potential (−51.5 mV). It also attained the highest enhancement of drug release compared to the drug suspension (RE = 66.79% and 26.85%, resp.). Fluorescence spectroscopy and microscopy studies performed using the lipid soluble dye, Oil red O, proved its high affinity towards the selected nanovesicles. After one month of storage at various temperatures (4, 25 and 40 °C), P-ST6 demonstrated good physicochemical stability. Cell biology studies demonstrated that P-ST6 and unmedicated P-ST6 reduced the cell viability of HepG2 cell line in a significant and concentration-dependent manner, with an IC50 of 73.7 and 145 μg/ml, respectively, using Sulforhodamine B assay. Both tested samples had marked anti-proliferative and tumor-suppressive effects by initiating cell cycle arrest as detected by flow-cytometry analysis. Pharmacokinetic study showed a significant increase in the bioavailability of the drug after a single oral administration of P-ST6 compared to the drug suspension. This study proves that the nano-encapsulation of Rutin in the innovated "heart-friendly" vesicular nanoformulation can be a promising therapeutic platform.

A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins.

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not to other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack.

PredML-Site: Predicting Multiple Lysine PTM Sites With Optimal Feature Representation and Data Imbalance Minimization.

Identifying of post-translational modifications (PTM) is crucial in the study of computational proteomics, cell biology, pathogenesis, and drug development due to its role in many bio-molecular mechanisms. Computational methods for predicting multiple PTM at the same lysine residues, often referred to as K-PTM, is still evolving. This paper presents a novel computational tool, abbreviated as predML-Site, for predicting KPTM, such as acetylation, crotonylation, methylation, succinylation from an uncategorized peptide sample involving single, multiple, or no modification. For informative feature representation, multiple sequence encoding schemes, such as the sequence-coupling, binary encoding, k-spaced amino acid pairs, amino acid factor have been used with ANOVA and incremental feature selection. As a core predictor, a cost-sensitive SVM classifier has been adopted which effectively mitigates the effect of class-label imbalance in the dataset. predML-Site predicts multi-label PTM sites with 84.18% accuracy using the top 91 features. It has also achieved 85.34% aiming and 86.58% coverage rate which are much better than the existing state-of-the-art predictors on the same rigorous validation test. This performance indicates that predML-Site can be used as a supportive tool for further K-PTM study. For the convenience of the experimental scientists, predML-Site has been deployed as a user-friendly web-server at http://103.99.176.239/predML-Site.

Functional Applications of Stable Tau Oligomers in Cell Biology and Electrophysiology Studies.

Aggregated tau protein plays a key role in the pathogenesis of neurodegenerative tauopathies including Alzheimer's disease. Soluble, low-molecular-weight tau oligomers are formed early in disease processes and are thought to have toxic functions that disrupt neuronal function. The dynamic and transient nature of tau oligomers complicates in vitro functional studies to explore the mechanistic links between oligomer formation and neurodegeneration. We have previously described a method of producing stable and structurally characterized oligomers that maintain their oligomeric conformation and prevent further aggregation. This method allows for the flexibility of stabilizing tau oligomers by specifically labelling cysteine residues with fluorescent or colorless maleimide conjugates. Here, we describe the functional applications of these preformed stable tau oligomers in cell biology and electrophysiological studies. These investigations allow real-time insights into the cellular uptake of exogenous tau oligomers and their functional effects in the recipient cells.

Septin and actin contributions to endothelial cell-cell junctions and monolayer integrity.

Septins in endothelial cells (ECs) have important roles supporting the integrity of the endothelial monolayer. Cell-cell junctions in EC monolayers are highly dynamic, with continuous retractions and protrusions. Depletion of septins in ECs leads to disruption of cell-cell junctions, which are composed of VE-cadherin and other junctional proteins. In EC monolayers, septins are concentrated at the plasma membrane at sites of cell-cell contact, in curved- and scallop-shaped patterns. These membrane-associated septin accumulations are located in regions of positive membrane curvature, and those regions are often associated with and immediately adjacent to actin-rich protrusions with negative membrane curvature. EC septins associate directly with plasma membrane lipids, based on findings with site-specific mutations of septins in ECs, which is consistent with biochemical and cell biological studies in other systems. Loss of septins leads to disruption of the EC monolayer, and gaps form between cells. The number and breadth of cell-cell contacts and junctions decreases, and the number and frequency of retractions, ruffles, and protrusions at cell edges also decreases. In addition, loss of septins leads to decreased amounts of F-actin at the cortical membrane, along with increased amounts of F-actin in stress fibers of the cytoplasm. Endothelial monolayer disruption from loss of septins is also associated with decreased transendothelial electric resistance (TEER) and increased levels of transendothelial migration (TEM) by immune and cancer cells, owing to the gaps in the monolayer. A current working model is that assembly of septin filaments at regions of positive membrane curvature contributes to a mechanical footing or base for actin-based protrusive forces generated at adjoining regions of the membrane. Specific molecular interactions between the septin and actin components of the cytoskeleton may also be important contributors. Regulators of actin assembly may promote and support the assembly of septin filaments at the membrane, as part of a molecular feedback loop between the assembly of septin and actin filaments.

Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Autophagy‐linked plasma and lysosomal membrane protein PLAC8 is a key host factor for SARS‐CoV‐2 entry into human cells

Better understanding on interactions between SARS‐CoV‐2 and host cells should help to identify host factors that may be targetable to combat infection and COVID‐19 pathology. To this end, we have conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen in human lung cancer cells infected with SARS‐CoV‐2‐pseudotyped lentiviruses. Our results recapitulate many findings from previous screens that used full SARS‐CoV‐2 viruses, but also unveil two novel critical host factors: the lysosomal efflux transporter SPNS1 and the plasma and lysosomal membrane protein PLAC8. Functional experiments with full SARS‐CoV‐2 viruses confirm that loss‐of‐function of these genes impairs viral entry. We find that PLAC8 is a key limiting host factor, whose overexpression boosts viral infection in eight different human lung cancer cell lines. Using single‐cell RNA‐Seq data analyses, we demonstrate that PLAC8 is highly expressed in ciliated and secretory cells of the respiratory tract, as well as in gut enterocytes, cell types that are highly susceptible to SARS‐CoV‐2 infection. Proteomics and cell biology studies suggest that PLAC8 and SPNS1 regulate the autophagolysosomal compartment and affect the intracellular fate of endocytosed virions.

ALIBY: ALFA Nanobody-Based Toolkit for Imaging and Biochemistry in Yeast.

ABSTRACTSpecialized epitope tags continue to be integral components of various biochemical and cell biological applications such as fluorescence microscopy, immunoblotting, immunoprecipitation, and protein purification. However, until recently, no single tag could offer this complete set of functionalities on its own. Here, we present a plasmid-based toolkit named ALIBY (ALFA toolkit for imaging and biochemistry in yeast) that provides a universal workflow to adopt the versatile ALFA tag/NbALFA system within the well-established model organism Saccharomyces cerevisiae. The kit comprises tagging plasmids for labeling a protein of interest with the ALFA tag and detection plasmids encoding fluorescent-protein-tagged NbALFA for live-cell imaging purposes. We demonstrate the suitability of ALIBY for visualizing the spatiotemporal localization of yeast proteins (i.e., the cytoskeleton, nucleus, centrosome, mitochondria, vacuole, endoplasmic reticulum, exocyst, and divisome) in live cells. Our approach has yielded an excellent signal-to-noise ratio without off-target effects or any effect on cell growth. In summary, our yeast-specific toolkit aims to simplify and further advance the live-cell imaging of differentially abundant yeast proteins while also being suitable for biochemical applications.IMPORTANCE In yeast research, conventional fluorescent protein tags and small epitope tags are widely used to study the spatiotemporal dynamics and activity of proteins. Although proven to be efficient, these tags lack the versatility for use across different cell biological and biochemical studies of a given protein of interest. Therefore, there is an urgent need for a unified platform for visualization and biochemical and functional analyses of proteins of interest in yeast. Here, we have engineered ALIBY, a plasmid-based toolkit that expands the benefits of the recently developed ALFA tag/NbALFA system to studies in the well-established model organism Saccharomyces cerevisiae. We demonstrate that ALIBY provides a simple and versatile strain construction workflow for long-duration live-cell imaging and biochemical applications in yeast.

Vectors with a flexible multiple cloning site and modular epitope tags for gene expression studies in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is a widely used eukaryotic model organism for cell biology, genetics and metabolomics studies. S. pombe-specific autonomously replicating shuttle vectors are frequently used for gene expression studies because such vectors can stably replicate, and cells harbouring them can be easily selected. Vectors have been designed with varying promoter strengths and epitope tags, yet higher flexibility and diversity further augmenting their application is desired. Here, we have prepared a set of twenty vectors with auxotrophic markers for leucine and uracil by modifying the widely used pREP plasmid with a thiamine-repressible nmt1 (no message in thiamine) promoter. The vectors offer distinct benefits, including an improved multiple cloning site (MCS) with unique cutting sites for more restriction enzymes and a set of N- and C-terminal 3xMYC, 3xFLAG and EGFP epitope tags for visualization and purification of expressed proteins. Further, different constitutive and thiamine-repressible promoters are used for varying a gene's expression, as verified by western blot detection of vector-borne fusion proteins in S. pombe. Importantly, DNA sequences encoding various elements including the epitope tags are kept modular with respect to their flanking restriction sites. Vectors reported here enhance the repertoire of tools currently available for molecular studies in S. pombe.

Space microgravity improves proliferation of human iPSC-derived cardiomyocytes.

SummaryIn microgravity, cells undergo profound changes in their properties. However, how human cardiac progenitors respond to space microgravity is unknown. In this study, we evaluated the effect of space microgravity on differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiac progenitors compared with 1G cultures on the International Space Station (ISS). Cryopreserved 3D cardiac progenitors were cultured for 3 weeks on the ISS. Compared with 1G cultures, the microgravity cultures had 3-fold larger sphere sizes, 20-fold higher counts of nuclei, and increased expression of proliferation markers. Highly enriched cardiomyocytes generated in space microgravity showed improved Ca2+ handling and increased expression of contraction-associated genes. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cell proliferation, survival, cardiac differentiation, and contraction, consistent with improved microgravity cultures at the late stage. These results indicate that space microgravity increased proliferation of hiPSC-cardiomyocytes, which had appropriate structure and function.

Genetically encoded fluorescent sensing probes

<p indent="0mm"><b/>Genetically encoded fluorescent sensing probes can image various molecular events in living cells and have become powerful tools in studies of cell biology. This paper introduces their categories, designs and applications. We also discuss their advantages, existing problems and solutions, and highlight the future development.

Applications of image analysis in plant chromosome and chromatin structure study

BackgroundThe use of image analysis to understand the structure of chromosome and chromatin is critical to the study of genetic evolution and diversification. Furthermore, a detailed chromosome map and the structure of chromatin in the nucleus may contribute to the plant breeding and the study of fundamental biology and genetics in organisms.ResultsIn plants with a fully annotated genome project, such as the Leguminosae species, the integration of genetic information, including DNA sequence data, a linkage map, and the cytological quantitative chromosome map could further improve their genetic value. The numerical parameters of chromocenters in 3D can provide useful genetic information for phylogenetic studies of plant diversity and heterochromatic markers whose epigenetic changes may explain the developmental and environmental changes in the plant genome. Extended DNA fibers combined with fluorescence in situ hybridization revealed the highest spatial resolution of the obtained genome structure. Moreover, image analysis at the nano‐scale level using a helium ion microscope revealed the surface structure of chromatin, which consists of chromatin fibers compacted into plant chromosomes.ConclusionsThe studies described in this review sought to measure and evaluate chromosome and chromatin using the image analysis method, which may reduce measurement time and improve resolution. Further, we discussed the development of an effective image analysis to evaluate the structure of chromosome and chromatin. An effective application study of cell biology and the genetics of plants using image analysis methods is expected to be a major propeller in the development of new applications.

A Beginner's Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

PHACTR1, a coronary artery disease risk gene, mediates endothelial dysfunction.

Genome-wide association studies (GWAS) have recently identified phosphatase and actin regulator-1 (PHACTR1) as a critical risk gene associated with polyvascular diseases. However, it remains largely unclear how PHACTR1 is involved in endothelial dysfunction. Here, by mining published datasets of human stable and vulnerable/ruptured plaque tissues, we observed upregulated expression of PHACTR1 in vulnerable/ruptured plaques. Congruent with these data, we demonstrated increased Phactr1 gene expression in aortic endothelium from ApoE-/- mice fed a western type diet compared with that in normal C57BL/6J mice. Relevantly, PHACTR1 gene expression was upregulated by pro-inflammatory and pro-atherogenic stimuli, including TNF-α, IL-1β and oxidized LDL (oxLDL). By employing next-generation RNA sequencing, we demonstrate that PHACTR1 overexpression disrupts pathways associated with endothelial homeostasis. Cell biological studies unravel that PHACTR1 mediates endothelial inflammation and monocyte adhesion by activating NF-κB dependent intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) expression. In addition, overexpression of PHACTR1 also reduces the generation of nitric oxide (NO) by inhibiting Akt/eNOS activation. In-house compound screening of vasoprotective drugs identifies several drugs, including lipid-lowering statins, decreases PHACTR1 gene expression. However, PHACTR1 gene expression was not affected by another lipid-lowering drug-fenofibrate. We also performed a proteomic study to reveal PHACTR1 interacting proteins and validated that PHACTR1 can interact with heat shock protein A8 (HSPA8) which was reported to be associated with coronary artery disease and eNOS degradation. Further studies are warranted to confirm the precise mechanism of PHACTR1 in driving endothelial dysfunction. In conclusion, by using systems biology approach and molecular validation, we disclose the deleterious effects of PHACTR1 on endothelial function by inducing endothelial inflammation and reducing NO production, highlighting the potential to prevent endothelial dysfunction and atherosclerosis by targeting PHACTR1 expression. The precise role of endothelial cell PHACTR1 in polyvascular diseases remains to be validated in diseased conditions.

Assay-ready Cryopreserved Cell Monolayers Enabled by Macromolecular Cryoprotectants.

Cell monolayers underpin the discovery and screening of new drugs and allow for fundamental studies of cell biology and disease. However, current cryopreservation technologies do not allow cells to be stored frozen while attached to tissue culture plastic. Hence, cells must be thawed from suspension, cultured for several days or weeks, and finally transferred into multiwell plates for the desired application. This inefficient process consumes significant time handling cells, rather than conducting biomedical research or other value-adding activities. Here, we demonstrate that a synthetic macromolecular cryoprotectant enables the routine, reproducible, and robust cryopreservation of biomedically important cell monolayers, within industry-standard tissue culture multiwell plates. The cells are simply thawed with media and placed in an incubator ready to use within 24 h. Post-thaw cell recovery values were >80% across three cell lines with low well-to-well variance. The cryopreserved cells retained healthy morphology, membrane integrity, proliferative capacity, and metabolic activity; showed marginal increases in apoptotic cells; and responded well to a toxicological challenge using doxorubicin. These discoveries confirm that the cells are “assay-ready” 24 h after thaw. Overall, we show that macromolecular cryoprotectants can address a long-standing cryobiological challenge and offers the potential to transform routine cell culture for biomedical discovery.

High-Resolution Low-Power Hyperspectral Line-Scan Imaging of Fast Cellular Dynamics Using Azo-Enhanced Raman Scattering Probes.

Small-molecule Raman probes for cellular imaging have attracted great attention owing to their sharp peaks that are sensitive to environmental changes. The small cross section of molecular Raman scattering limits dynamic cellular Raman imaging to expensive and complex coherent approaches that acquire single-channel images and lose hyperspectral Raman information. We introduce a new method, dynamic azo-enhanced Raman imaging (DAERI), to couple the new class of azo-enhanced Raman probes with a high-speed line-scan Raman imaging system. DAERI achieved high-resolution low-power imaging of fast cellular dynamics resolved at ∼270 nm along the confocal direction, 75 μW/μm2 and 3.5 s/frame. Based on the azo-enhanced Raman probes with characteristic signals 102-104 stronger than classic Raman labels, DAERI was not restricted to the cellular Raman-silent region as in prior work and enabled multiplex visualization of organelle motions and interactions. We anticipate DAERI to be a powerful tool for future studies in biophysics and cell biology.

Established Yazd human foreskin fibroblast lines (#8, #17, and #18) displaying similar characteristics to mesenchymal stromal cells: A lab resources report.

BackgroundFibroblasts from different parts of the human body have been used in cell biology, drug discovery and cell therapy studies. One of the most available sources of human fibroblasts is neonatal foreskin. Not only do these cells have wound-healing applications, but they are also the most popular source for pluripotent stem cell biotechnology. Moreover, several studies have indicated that different sources of fibroblasts display similar features to mesenchymal stem cells.ObjectiveGeneration and establishment of new human foreskin fibroblast cell lines called Yazd human foreskin fibroblasts (YhFFs).Materials and MethodsIn this lab resources study, the production of 3 YhFF cell lines (YhFF#8, YhFF#17, and YhFF#18) is reported. Their biological features were characterized using immunofluorescence, polymerase chain reaction, and flow-cytometry for mesenchymal markers such as fibronectin, vimentin, CD44, CD73, CD90, CD105, and hematopoietic markers CD34 and CD45.ResultsThe YhFF cell lines were passaged more than 40 times and their normal karyotype was checked using G-binding. Similarly to previous reports, the flow cytometry analysis revealed that the YhFF cell lines displayed mesenchymal stromal cell characteristics.ConclusionThis study will contribute to the development of clinical-grade cell-based products such as micro-vesicles and exosomes for future therapeutic applications in regenerative medicine.

Abstract P1026: Creg1 Inhibits Cardiomyocyte Proliferation And Promotes Myocardial Differentiation And Compaction During Postnatal Cardiac Development

Introduction: Overexpression of cellular repressor of E1A-stimulated genes 1 (CREG1) promotes the formation of compact, contracting myocardium-like structures during cardiomyogenesis from embryonic stem cells. Cell biology studies suggest that CREG1 promotes differentiation and inhibits proliferation of developing cardiomyocytes. This study aims to understand the role of CREG1 during myocardial development in vivo . Methods: Cardiac tissues were harvested from wild-type (WT) and constitutive Creg1 knockout (KO) postnatal day 1.5 and 17 through timed mating of heterozygous Creg1 mice. Creg1 KO mice were compared with Creg1 WT littermates using immunoblotting, immunofluorescence, and methylene blue staining. Results: Postnatal day 1.5 Creg1 KO hearts showed lower levels of expression of slow skeletal muscle troponin I (TnI) compared with WT counterparts. No significant difference in expression was seen between WT and KO Creg1 hearts for myosin light chain 3 (MLC3), myosin heavy chain (MHC), and cardiac troponin I. Immunofluorescence staining revealed increased numbers of PCNA (Proliferating Cell Nuclear Antigen) positive nuclei in postnatal day 1.5 Creg1 KO hearts, suggesting enhanced cardiomyocyte proliferation after loss of CREG1. Methylene blue staining in postnatal day 17 hearts displayed decreased myocardial compaction in Creg1 KO mice. Conclusion: CREG1 may promote cardiomyocyte differentiation and inhibit proliferation during postnatal myocardial development. Ablation of CREG1 results in cardiac compaction defects. Our findings suggest an important role for CREG1 in cardiac development.