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BioRxiv : the Preprint Server For... Jun 2024Endothelial tissues are essential mechanosensors in the vasculature and facilitate adaptation to various blood flow-induced mechanical cues. Defects in endothelial...
Endothelial tissues are essential mechanosensors in the vasculature and facilitate adaptation to various blood flow-induced mechanical cues. Defects in endothelial mechanoresponses can perturb tissue remodelling and functions leading to cardiovascular disease progression. In this context, the precise mechanisms of endothelial mechanoresponses contributing to normal and diseased tissue functioning remain elusive. Here, we sought to uncover how flow-mediated transcriptional regulation drives endothelial mechanoresponses in healthy and atherosclerotic-prone tissues. Using bulk RNA sequencing, we identify novel mechanosensitive genes in response to healthy unidirectional flow (UF) and athero-prone disturbed flow (DF). We find that the transcription as well as protein expression of Four-and-a-half LIM protein 2 (FHL2) are enriched in athero-prone DF both and . We then demonstrate that the exogenous expression of FHL2 is necessary and sufficient to drive discontinuous adherens junction morphology and increased tissue permeability. This athero-prone phenotype requires the force-sensitive binding of FHL2 to actin. In turn, the force-dependent localisation of FHL2 to stress fibres promotes microtubule dynamics to release the RhoGEF, GEF-H1, and activate the Rho-ROCK pathway. Thus, we unravelled a novel mechanochemical feedback wherein force-dependent FHL2 localisation promotes hypercontractility. This misregulated mechanoresponse creates highly permeable tissues, depicting classic hallmarks of atherosclerosis progression. Overall, we highlight crucial functions for the FHL2 force-sensitivity in tuning multi-scale endothelial mechanoresponses.
PubMed: 38948838
DOI: 10.1101/2024.06.16.599227 -
BioRxiv : the Preprint Server For... Jun 2024Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that...
Cochlear hair cell stereocilia bundles are key organelles required for normal hearing. Often, deafness mutations cause aberrant stereocilia heights or morphology that are visually apparent but challenging to quantify. Actin-based structures, stereocilia are easily and most often labeled with phalloidin then imaged with 3D confocal microscopy. Unfortunately, phalloidin non-specifically labels all the actin in the tissue and cells and therefore results in a challenging segmentation task wherein the stereocilia phalloidin signal must be separated from the rest of the tissue. This can require many hours of manual human effort for each 3D confocal image stack. Currently, there are no existing software pipelines that provide an end-to-end automated solution for 3D stereocilia bundle instance segmentation. Here we introduce VASCilia, a Napari plugin designed to automatically generate 3D instance segmentation and analysis of 3D confocal images of cochlear hair cell stereocilia bundles stained with phalloidin. This plugin combines user-friendly manual controls with advanced deep learning-based features to streamline analyses. With VASCilia, users can begin their analysis by loading image stacks. The software automatically preprocesses these samples and displays them in Napari. At this stage, users can select their desired range of z-slices, adjust their orientation, and initiate 3D instance segmentation. After segmentation, users can remove any undesired regions and obtain measurements including volume, centroids, and surface area. VASCilia introduces unique features that measures bundle heights, determines their orientation with respect to planar polarity axis, and quantifies the fluorescence intensity within each bundle. The plugin is also equipped with trained deep learning models that differentiate between inner hair cells and outer hair cells and predicts their tonotopic position within the cochlea spiral. Additionally, the plugin includes a training section that allows other laboratories to fine-tune our model with their own data, provides responsive mechanisms for manual corrections through event-handlers that check user actions, and allows users to share their analyses by uploading a pickle file containing all intermediate results. We believe this software will become a valuable resource for the cochlea research community, which has traditionally lacked specialized deep learning-based tools for obtaining high-throughput image quantitation. Furthermore, we plan to release our code along with a manually annotated dataset that includes approximately 55 3D stacks featuring instance segmentation. This dataset comprises a total of 1,870 instances of hair cells, distributed between 410 inner hair cells and 1,460 outer hair cells, all annotated in 3D. As the first open-source dataset of its kind, we aim to establish a foundational resource for constructing a comprehensive atlas of cochlea hair cell images. Together, this open-source tool will greatly accelerate the analysis of stereocilia bundles and demonstrates the power of deep learning-based algorithms for challenging segmentation tasks in biological imaging research. Ultimately, this initiative will support the development of foundational models adaptable to various species, markers, and imaging scales to advance and accelerate research within the cochlea research community.
PubMed: 38948743
DOI: 10.1101/2024.06.17.599381 -
World Journal of Stem Cells Jun 2024Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by fibroblast proliferation and extracellular matrix formation, causing structural damage...
BACKGROUND
Pulmonary fibrosis (PF) is a chronic interstitial lung disease characterized by fibroblast proliferation and extracellular matrix formation, causing structural damage and lung failure. Stem cell therapy and mesenchymal stem cells-extracellular vesicles (MSC-EVs) offer new hope for PF treatment.
AIM
To investigate the therapeutic potential of MSC-EVs in alleviating fibrosis, oxidative stress, and immune inflammation in A549 cells and bleomycin (BLM)-induced mouse model.
METHODS
The effect of MSC-EVs on A549 cells was assessed by fibrosis markers [collagen I and α-smooth muscle actin (α-SMA), oxidative stress regulators [nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1), and inflammatory regulators [nuclear factor-kappaB (NF-κB) p65, interleukin (IL)-1β, and IL-2]. Similarly, they were assessed in the lungs of mice where PF was induced by BLM after MSC-EV transfection. MSC-EVs ion PF mice were detected by pathological staining and western blot. Single-cell RNA sequencing was performed to investigate the effects of the MSC-EVs on gene expression profiles of macrophages after modeling in mice.
RESULTS
Transforming growth factor (TGF)-β1 enhanced fibrosis in A549 cells, significantly increasing collagen I and α-SMA levels. Notably, treatment with MSC-EVs demonstrated a remarkable alleviation of these effects. Similarly, the expression of oxidative stress regulators, such as Nrf2 and HO-1, along with inflammatory regulators, including NF-κB p65 and IL-1β, were mitigated by MSC-EV treatment. Furthermore, in a parallel manner, MSC-EVs exhibited a downregulatory impact on collagen deposition, oxidative stress injuries, and inflammatory-related cytokines in the lungs of mice with PF. Additionally, the mRNA sequencing results suggested that BLM may induce PF in mice by upregulating pulmonary collagen fiber deposition and triggering an immune inflammatory response. The findings collectively highlight the potential therapeutic efficacy of MSC-EVs in ameliorating fibrotic processes, oxidative stress, and inflammatory responses associated with PF.
CONCLUSION
MSC-EVs could ameliorate fibrosis and by downregulating collagen deposition, oxidative stress, and immune-inflammatory responses.
PubMed: 38948098
DOI: 10.4252/wjsc.v16.i6.670 -
World Journal of Stem Cells Jun 2024Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data...
BACKGROUND
Validation of the reference gene (RG) stability during experimental analyses is essential for correct quantitative real-time polymerase chain reaction (RT-qPCR) data normalisation. Commonly, in an unreliable way, several studies use genes involved in essential cellular functions [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, and β-actin] without paying attention to whether they are suitable for such experimental conditions or the reason for choosing such genes. Furthermore, such studies use only one gene when Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines recommend two or more genes. It impacts the credibility of these studies and causes distortions in the gene expression findings. For tissue engineering, the accuracy of gene expression drives the best experimental or therapeutical approaches.
AIM
To verify the most stable RG during osteogenic differentiation of human dental pulp stem cells (DPSCs) by RT-qPCR.
METHODS
We cultivated DPSCs under two conditions: Undifferentiated and osteogenic differentiation, both for 35 d. We evaluated the gene expression of 10 candidates for RGs [ribosomal protein, large, P0 (), TATA-binding protein (), , actin beta (), tubulin (), aminolevulinic acid synthase 1 (), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta (), eukaryotic translational elongation factor 1 alpha (), succinate dehydrogenase complex, subunit A, flavoprotein (), and beta-2-microglobulin ()] every 7 d (1, 7, 14, 21, 28, and 35 d) by RT-qPCR. The data were analysed by the four main algorithms, ΔCt method, geNorm, NormFinder, and BestKeeper and ranked by the RefFinder method. We subdivided the samples into eight subgroups.
RESULTS
All of the data sets from clonogenic and osteogenic samples were analysed using the RefFinder algorithm. The final ranking showed RPLP0/TBP as the two most stable RGs and TUB/B2M as the two least stable RGs. Either the ΔCt method or NormFinder analysis showed TBP/RPLP0 as the two most stable genes. However, geNorm analysis showed RPLP0/EF1α in the first place. These algorithms' two least stable RGs were B2M/GAPDH. For BestKeeper, ALAS1 was ranked as the most stable RG, and SDHA as the least stable RG. The pair RPLP0/TBP was detected in most subgroups as the most stable RGs, following the RefFinfer ranking.
CONCLUSION
For the first time, we show that RPLP0/TBP are the most stable RGs, whereas TUB/B2M are unstable RGs for long-term osteogenic differentiation of human DPSCs in traditional monolayers.
PubMed: 38948092
DOI: 10.4252/wjsc.v16.i6.656 -
Frontiers in Molecular Biosciences 2024Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present...
Human saliva was used to develop non-invasive liquid biopsy biomarkers to establish saliva as an alternate to blood and plasma in translational research. The present study focused on understanding the impact of sample storage conditions on the extraction of RNA from saliva and the RNA yield, to be applied in clinical diagnosis. In this study, genes related to asthma were used to test the method developed. Salivary RNA was extracted from three subjects using the Qiazol based method and quantified by both spectrophotometric (NanoDrop) and fluorometric (Qubit) methods. RNA integrity was measured using a bioanalyzer. Quantitative PCR was used to monitor the impact of storage conditions on the expression of housekeeping genes: and β-actin, and the asthma related genes: and . In addition, an independent cohort of 38 asthmatics and 10 healthy controls were used to validate the expression of and as mRNA salivary biomarkers. Approximately 2 µg of total RNA was obtained from the saliva stored at 40°C without any preservative for 2 weeks showing consistent gene expression with RNA stored at room temperature (RT) for 48 h with RNA. Although saliva stored with RNA showed a substantial increase in the yield (110 to 234 ng/μL), a similar Cq (15.6 ± 1.4) for the 18s rRNA gene from saliva without preservative showed that the RNA was stable enough. Gene expression analysis from the degraded RNA can be performed by designing the assay using a smaller fragment size spanning a single exon as described below in the case of the and genes in the asthma cohort. This study showed that samples stored at room temperature up to a temperature of 40°C without any preservative for 2 weeks yielded relatively stable RNA. The methodology developed can be employed to transport samples from the point of collection to the laboratory, under non-stringent storage conditions enabling the execution of gene expression studies in a cost effective and efficient manner.
PubMed: 38948078
DOI: 10.3389/fmolb.2024.1363897 -
Journal of Veterinary Research Jun 2024In dairy cattle, oxidative stress is a predominant problem associated with diseases and reproductive health issues. This study aimed to detect the variation in the...
INTRODUCTION
In dairy cattle, oxidative stress is a predominant problem associated with diseases and reproductive health issues. This study aimed to detect the variation in the antioxidant biomarkers by adding different concentrations of β-hydroxybutyric acid (BHBA) and sought to elucidate its effects on the gene expression levels of growth hormone (GH) and antioxidant biomarkers in bovine hepatocytes.
MATERIAL AND METHODS
Four antioxidant biomarkers, namely malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH Px) were evaluated using commercially available bovine ELISA kits. The expression levels of the bovine GH, its receptor (GHR), insulin-like growth factor (IGF), IGF-1, IGF-1 receptor, CAT, SOD, GSH-Px and β-actin (as a reference) genes in liver cell culture were determined by reverse transcriptase-PCR assay.
RESULTS
With the increase of BHBA concentration and culture time, the activities of SOD, CAT, and GSH Px biomarkers in hepatocytes decreased. However, the content of MDA in hepatocytes increased gradually with the increase of hepatocyte culture time and BHBA concentration. The qPCR results revealed that after adding BHBA, gene expression levels of GSH-Px, SOD and IGF biomarkers in hepatocytes began to differ in the culture groups at 12 h, whereas the gene expression level of the CAT and GHR biomarkers in hepatocytes began to differ at 6 h.
CONCLUSION
Quantitative PCR results showed that the BHBA significantly downregulated the expression levels of the GHR gene and CAT, GSH Px and SOD antioxidant biomarker genes.
PubMed: 38947149
DOI: 10.2478/jvetres-2024-0037 -
World Journal of Clinical Oncology Jun 2024Glioma is one of the most common primary intracranial tumors, characterized by invasive growth and poor prognosis. Actin cytoskeletal rearrangement is an essential event...
Glioma is one of the most common primary intracranial tumors, characterized by invasive growth and poor prognosis. Actin cytoskeletal rearrangement is an essential event in tumor cell migration. Scinderin (SCIN), an actin severing and capping protein that regulates the actin cytoskeleton, is involved in the proliferation and migration of certain cancer cells. However, its biological role and molecular mechanism in glioma remain unclear. Lin explored the role and mechanism of SCIN in gliomas. The results showed that SCIN mechanically affected cytoskeleton remodeling and inhibited the formation of lamellipodia RhoA/FAK signaling pathway. This study identifies the cancer-promoting role of SCIN and provides a potential therapeutic target for SCIN in glioma treatment.
PubMed: 38946838
DOI: 10.5306/wjco.v15.i6.687 -
Journal of Cell Communication and... Jun 2024Urethral stricture (US) is a challenging problem in urology and its pathogenesis of US is closely related to the fibrotic process. Previous evidence has indicated the...
Urethral stricture (US) is a challenging problem in urology and its pathogenesis of US is closely related to the fibrotic process. Previous evidence has indicated the downregulation of microRNA (miR)-486 in injured urethral specimens of rats. This study aimed to explore the effects of miR-486-overexpressed bone marrow mesenchymal stem cells (BMSCs) on US. BMSCs were identified by detecting their multipotency and surface antigens. Lentivirus virus expressing miR-486 was transduced into rat BMSCs to overexpress miR-486. Transforming growth factor (TGF)-β1 induced fibrotic phenotypes in urethral fibroblasts (UFs) and rat models. Western blotting showed protein levels of collagen I/III and collagen type XIII alpha 1 chain (Col13a1). Real time quantitative polymerase chain reaction was utilized for messenger RNA level evaluation. Hematoxylin-eosin, Masson's trichrome, and Von Willebrand Factor staining were conducted for histopathological analysis. Immunofluorescence staining was employed for detecting alpha smooth muscle actin (α-SMA) expression. Luciferase reporter assay verified the interaction between miR-486 and Col13a1. The results showed that miR-486-overexpressed BMSCs suppressed collagen I/III and α-SMA expression in TGF-β1-stimulated UFs. miR-486-overexpressed BMSCs alleviated urethral fibrosis, collagen deposition, and epithelial injury in the urethral tissue of US rats. miR-486 targeted and negatively regulated Col13a1 in US rats. In conclusion, overexpression of miR-486 in BMSCs targets Col13a1 and attenuates urethral fibrosis in TGF-β1-triggered UFs and US rats.
PubMed: 38946723
DOI: 10.1002/ccs3.12028 -
Journal of Neurochemistry Jul 2024A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of...
A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three-dimensional-structured illumination microscopy, a type of super-resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z-axis ("z-filopodia") within the growth cone. Z-filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z-filopodia formation and dynamics were regulated by actin-regulatory proteins, such as vasodilator-stimulated phosphoprotein, fascin, and cofilin. Chromophore-assisted laser inactivation of cofilin induced the rapid turnover of z-filopodia. An axon guidance receptor, neuropilin-1, was concentrated in z-filopodia and was transported together with them, whereas its ligand, semaphorin-3A, was selectively bound to them. Membrane domains associated with z-filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin-1. The results suggest that z-filopodia have unique turnover properties, and unlike xy-filopodia, do not function as force-generating structures for axon extension.
PubMed: 38946488
DOI: 10.1111/jnc.16162 -
Developmental Biology Jun 2024The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the...
The Formin protein Daam1 is required for Wnt-induced cytoskeletal changes during gastrulation, though how it accomplishes this remains unresolved. Here we report the characterization of Formin Binding Protein 1 (FNBP1) as a binding partner of Daam1. The interaction of Daam1 with FNBP1 and its domains required for this interaction were delineated. Immunofluorescence studies showed FNBP1 co-localizes with Daam1 and is an integral component of the actin cytoskeletal complex that is responsive to Wnt stimulation. Specifically, FNBP1 can induce intracellular tubule-like structures and localize to focal adhesions suggesting a role for FNBP1 in cell migration. Functional FNBP1 studies in Xenopus embryos uncover a critical role for FNBP1 in regulating vertebrate gastrulation. Additionally, suboptimal doses of Daam1 and FNBP1 synergize to produce severe gastrulation defects, indicating FNBP1 and Daam1 may function within the same signaling pathway. These results together show FNBP1 is an integral component of Daam1-regulated non-canonical Wnt signaling required for vertebrate gastrulation.
PubMed: 38945423
DOI: 10.1016/j.ydbio.2024.06.019