Effects of cytochalasin D on relaxation process of skinned taenia cecum and carotid artery from guinea pig.

Actin linked regulatory mechanisms are known to contribute contraction/relaxation in smooth muscle. In order to clarify whether modulation of polymerization/depolymerization of actin filaments affects relaxation process, we examined the effects of cytochalasin D on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) taenia cecum and carotid artery preparations from guinea pigs. Cytochalasin D, an inhibitor of actin polymerization, significantly suppressed the force during relaxation both in skinned taenia cecum and carotid artery. The data fitting analysis of the relaxation processes indicates that cytochalasin D accelerates slow (latch-like) bridge dissociation. Cytochalasin D seems to directly disrupts actin filament organization or its length, resulting in modulation of actin filament structure that prevents myosin binding.

Calorie restriction mimetic, resveratrol, attenuates hepatic ischemia and reperfusion injury through enhancing efferocytosis of macrophages via AMPK/STAT3/S1PR1 pathway.

Calorie restriction (CR) mimetic, resveratrol (RSV), has the capacity of promoting phagocytosis. However, its role in hepatic ischemia and reperfusion injury (HIRI) remains poorly understood. This study aimed to investigate the effect of RSV on alleviating HIRI and explore the underlying mechanisms. RSV was intraperitoneally injected in mice HIRI model, while RSV was co-incubated with culture medium for 24 h in RAW 264.7 cells and kupffer cells. Macrophage efferocytosis was assessed by immunostaining of PI and F4/80. The clearance of apoptotic neutrophils in the liver was determined by immunostaining of Ly6-G and cleaved-caspase-3. HE staining, Suzuki's score, serum levels of ALT, AST, TNF-α and IL-1ß were analyzed to evaluate HIRI. The efferocytosis inhibitor, Cytochalasin D, was utilized to investigate the effect of RSV on HIRI. Western blot was employed to measure the levels of AMPKα, phospho-AMPKα, STAT3, phospho-STAT3 and S1PR1. SiSTAT3 and inhibitors targeting AMPK, STAT3 and S1PR1, respectively, were used to confirm the involvement of AMPK/STAT3/S1PR1 pathway in RSV-mediated efferocytosis and HIRI. RSV facilitated the clearance of apoptotic neutrophils and attenuated HIRI, which was impeded by Cytochalasin D. RSV boosted macrophage efferocytosis by up-regulating the levels of phospho-AMPKα, phospho-STAT3 and S1PR1, which was reversed by AMPK, STAT3 and S1PR1 inhibitors, respectively. Inhibition of STAT3 suppressed RSV-induced clearance of apoptotic neutrophils and exacerbated HIRI. CR mimetic, RSV, alleviates HIRI by promoting macrophages efferocytosis through AMPK/STAT3/S1PR1 pathway, providing valuable insights into the mechanisms underlying the protective effects of CR on attenuating HIRI.

Actin cytoskeleton aggregation involves the water channel protein aquaporin 1-mediated human chondrocyte degeneration.

Water metabolism and actin cytoskeleton remoulding act as essential characters in the process of osteoarthritis (OA). However, the relation between water channel protein aquaporin 1 (AQP1) and actin filament during chondrocytes (CHs) degeneration is not evident. Therefore, the present study aimed to evaluate the role of actin remoulding in the AQP1 mediated CHs degeneration. Primary CHs were collected from human hip cartilage and were degenerated from long-time monolayer culture or IL-1ß stimulation. Besides, the CHs were transfected with AQP1­specific siRNA or vectors to mediate the AQP1 gene expression. The potent inhibitor of actin polymerization Cytochalasin D was also supplemented during culture. RT-PCR was performed to determine the relative gene expression. AQP1 and F-actin fluorescence staining were performed to determine the AQP1 and F-actin organization. Moreover, the cell area and viability were also analyzed. AQP1 and F-actin organization were both increased during seven days' CHs culture or three days' IL-1ß stimulation. Silencing of AQP1 prevented the cell area spreading and degenerated phenotype of CHs with suppression of F-actin aggregation in both natural or IL-1ß-caused inflammatory-related degeneration. Besides, upregulating the AQP1 in the CHs via gene editing promoted the cell area spreading, and F-actin accumulation, and accelerated the CHs degeneration, which can be alleviated by Cytochalasin D treatment. These findings suggested that AQP1-mediated human CHs degeneration is related to F-actin aggregation.

Enhancing suspended cell transfection by inducing localized distribution of the membrane actin cortex before exposure to electromechanical stimulation.

OBJECTIVES: During physical transfection, an electrical field or mechanical force is used to induce cell transfection. We tested if the disruption of a dense actin layer underneath the membrane of a suspended cell enhances cell transfection. RESULTS: A bubble generator was used to electromechanically stimulate suspended cells. To clarify the influence of the actin layer (the actin cortex) on cell transfection efficiency, we used an actin polymerization inhibitor (cytochalasin D) to disrupt the actin cortex before electromechanical stimulation. Without cytochalasin D treatment, signals from the overall actin cortex decreased after electromechanical stimulation. With cytochalasin D treatment, there was localized F-actin aggregation under static conditions. After electromechanical stimulation, there was a partial loss (localized disruption), but no overall disruption, of the actin cortex. With the pretreatment with cytochalasin D, the transfection efficiency of plasmids (4.7, 8.3, or 11 kbp) into NIH/3T3 or UMR-106 cells increased significantly after exposure to electromechanical stimulation. CONCLUSIONS: Localized distribution of the actin cortex before exposure to electromechanical stimulation is crucial for inducing a partial loss of the cortex, which improves transfection efficiency and large plasmid delivery.

Upregulation of girdin delays endothelial cell apoptosis via promoting engulfment of platelets.

BACKGROUND: Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study. METHODS: Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy. RESULT: Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels. CONCLUSION: Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.

Cytochalasin D restores nuclear size acting on F-actin and IZUMO1 localization in low-quality spermatozoa.

In spermatozoa, the nuclear F-actin supports the acroplaxome, a subacrosomal structure involved in the correct exposure of several acrosomal membrane proteins; among them, the glycoprotein IZUMO1 is the major protein involved in sperm-oocyte fusion. Nuclear F-actin is also involved in sperm head shaping and chromosome compartmentalization. To date, few notions regarding the bivalent role of F-actin on sperm chromatin organization and IZUMO1 positioning have been reported. In our work, we characterized subcellular organization of F-actin in human high- and low-quality spermatozoa (A- and B-SPZ), respectively, showing that F-actin over-expression in sperm head of B-SPZ affected IZUMO1 localization. A correct IZUMO1 repositioning following in vitro induction of F-actin depolymerization, by cytochalasin D treatment, occurred. Interestingly, F-actin depolymerization was also associated with a correct acrosome repositioning, thus to favor a proper acrosome reaction onset, with changes in sperm nuclear size parameters and histone acetylation rate reaching high-quality conditions. In conclusion, the current work shows a key role of F-actin in the control of IZUMO1 localization as well as chromatin remodeling and acetylation events.

The influence of dysfunctional actin on polystyrene-nanotube-mediated mRNA nanoinjection into mammalian cells.

The advancement of nanofabrication technologies has transformed the landscape of engineered nano-bio interfaces, especially with vertically aligned nanoneedles (NNs). This enables scientists to venture into new territories, widening NN applications into increasingly more complex cellular manipulation and interrogation. Specifically, for intracellular delivery application, NNs have been shown to mediate the delivery of various bioactive cargos into a wide range of cells-a physical method termed "nanoinjection". Silicon (Si) nanostructures demonstrated great potential in nanoinjection, whereas the use of polymeric NNs for nanoinjection has rarely been explored. Furthermore, the underlying mechanism of interaction at the cell-NN interface is subtle and multifaceted, and not fully understood-underpinned by the design versatility of the NN biointerface. Recent studies have suggested that actin dynamic plays a pivotal role influencing the delivery efficacy. In this study, we fabricated a new class of NNs-a programmable polymeric nanotubes (NTs)-from polystyrene (PS) cell cultureware, designed to facilitate mRNA delivery into mouse embryonic fibroblast GPE86 cells. The PSNT delivery platform was able to mediate mRNA delivery with high delivery efficiency (∼83%). We also investigated the role of actin cytoskeleton in PSNTs mediated intracellular delivery by introducing two actin inhibitors-cytochalasin D (Cyto D) and jasplakinolide (Jas)-to cause dysfunctional cytoskeleton, via inhibiting actin polymerization and depolymerization, respectively (before and after the establishment of cell-PSNT interface). By inhibiting actin dynamics 12 h before cell-PSNT interfacing (pre-interface treatment), the mRNA delivery efficiencies were significantly reduced to ∼3% for Cyto D-treated samples and ∼1% for Jas-treated sample, as compared to their post-interface (2 h after cell-PSNT interfacing) counterpart (∼46% and ∼68%, respectively). The added flexibility of PSNTs have shown to help withstand mechanical breakage stemming from cytoskeletal forces in contrast to the SiNTs. Such findings will step-change our capacity to use programmable polymeric NTs in fundamental cellular processes related to intracellular delivery.

Intervention time decides the status of autophagy, NLRP3 activity and apoptosis in macrophages induced by ox-LDL.

BACKGROUND: It has been determined through extensive studies that autophagy, the Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome and apoptotic responses in macrophages jointly contribute to atherogenesis and its development in the presence of lipid abnormalities. Few studies have investigated in full-scale if the intervention time for lipids abnormality or NLRP3 activation have a significant effect on autophagy, NLRP3 or the apoptotic status in macrophages. METHODS: Human THP-1 monocyte-derived macrophages were established by challenging THP-1 monocytes with 80 µg/ml oxidized low-density lipoprotein (ox-LDL) for specific durations. Foam cell formation was observed by Oil Red O (ORO) staining. Western blots were employed to determine protein expression. Transmission electron microscope (TEM) and immunofluorescence microscopy were applied to observe the autophagic status of cells. Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). RESULTS: The cells were treated with ox-LDL for 12 h and 36 h, which were considered to represent early and advanced stages of atherogenesis for this study. The results showed that inhibition of ox-LDL phagocytosis by cytochalasin D in the early stage improved autophagic status, reduced NLRP3 activation and the apoptotic response significantly. In contrast, cytochalasin D had little effect on blocking the detrimental effect of ox-LDL at the advanced stage. Moreover, the changes in autophagy, apoptosis and NLRP3 expression after treatment with small interfering (si) RNA targeting NLRP3 in the early and advanced stages of atherogenesis were consistent with the above data. CONCLUSIONS: Interventions against lipid disorders or inflammatory reactions in the early or advanced stages of atherogenesis may have different results depending on when they are applied during the process of atherosclerotic pathogenesis. These results may help improve therapeutic strategies for atherosclerosis prevention. Furthermore, a healthy lifestyle should still be recommended as the most important and inexpensive measure to prevent atherogenesis.

Towards a comprehensive approach for characterizing cell activity in bright-field microscopic images.

When studying physical cellular response observed by light microscopy, variations in cell behavior are difficult to quantitatively measure and are often only discussed on a subjective level. Hence, cell properties are described qualitatively based on a researcher's impressions. In this study, we aim to define a comprehensive approach to estimate the physical cell activity based on migration and morphology based on statistical analysis of a cell population within a predefined field of view and timespan. We present quantitative measurements of the influence of drugs such as cytochalasin D and taxol on human neuroblastoma, SH-SY5Y cell populations. Both chemicals are well known to interact with the cytoskeleton and affect the cell morphology and motility. Being able to compute the physical properties of each cell for a given observation time, requires precise localization of each cell even when in an adhesive state, where cells are not visually differentiable. Also, the risk of confusion through contaminants is desired to be minimized. In relation to the cell detection process, we have developed a customized encoder-decoder based deep learning cell detection and tracking procedure. Further, we discuss the accuracy of our approach to quantify cell activity and its viability in regard to the cell detection accuracy.

Role of actin cytoskeleton in cargo delivery mediated by vertically aligned silicon nanotubes.

Nanofabrication technologies have been recently applied to the development of engineered nano-bio interfaces for manipulating complex cellular processes. In particular, vertically configurated nanostructures such as nanoneedles (NNs) have been adopted for a variety of biological applications such as mechanotransduction, biosensing, and intracellular delivery. Despite their success in delivering a diverse range of biomolecules into cells, the mechanisms for NN-mediated cargo transport remain to be elucidated. Recent studies have suggested that cytoskeletal elements are involved in generating a tight and functional cell-NN interface that can influence cargo delivery. In this study, by inhibiting actin dynamics using two drugs-cytochalasin D (Cyto D) and jasplakinolide (Jas), we demonstrate that the actin cytoskeleton plays an important role in mRNA delivery mediated by silicon nanotubes (SiNTs). Specifically, actin inhibition 12 h before SiNT-cellular interfacing (pre-interface treatment) significantly dampens mRNA delivery (with efficiencies dropping to 17.2% for Cyto D and 33.1% for Jas) into mouse fibroblast GPE86 cells, compared to that of untreated controls (86.9%). However, actin inhibition initiated 2 h after the establishment of GPE86 cell-SiNT interface (post-interface treatment), has negligible impact on mRNA transfection, maintaining > 80% efficiency for both Cyto D and Jas treatment groups. The results contribute to understanding potential mechanisms involved in NN-mediated intracellular delivery, providing insights into strategic design of cell-nano interfacing under temporal control for improved effectiveness.

The role of inflammation in cadmium nephrotoxicity: NF-κB comes into view.

Kidney diseases are major health problem and understanding the underlined mechanisms that lead to kidney diseases are critical research points with a marked potential impact on health. Cadmium (Cd) is a heavy metal that occurs naturally and can be found in contaminated food. Kidneys are the most susceptible organ to heavy metal intoxication as it is the main route of waste excretion. The harmful effects of Cd were previously well proved. Cd induces inflammatory responses, oxidative injury, mitochondrial dysfunction and disturbs Ca2+ homeostasis. The nuclear factor-kappa B (NF-κB) is a cellular transcription factor that regulates inflammation and controls the expression of many inflammatory cytokines. Therefore, great therapeutic benefits can be attained from NF-κB inhibition. In this review we focused on certain compounds including cytochalasin D, mangiferin, N-acetylcysteine, pyrrolidine dithiocarbamate, roflumilast, rosmarinic acid, sildenafil, sinapic acid, telmisartan and wogonin and certain plants as Astragalus Polysaccharide, Ginkgo Biloba and Thymus serrulatus that potently inhibit NF-κB and effectively counteracted Cd-associated renal intoxication. In conclusion, the proposed NF-κB involvement in Cd-renal intoxication clarified the underlined inflammation associated with Cd-nephropathy and the beneficial effects of NF-κB inhibitors that make them the potential to substantially optimize treatment protocols for Cd-renal intoxication.

Assessing Als3 Peptide-Binding Cavity and Amyloid-Forming Region Contributions to Candida albicans Invasion of Human Oropharyngeal Epithelial Cells.

Although it is widely recognized that disruption of ALS3 reduces the invasion of Candida albicans germ tubes into mammalian oral epithelial cells, the mechanism of this interaction was unexplored. C. albicans strains with structurally informed mutations to remove adhesive activity of the peptide-binding cavity (PBC) or aggregative activity mediated by the amyloid-forming region (AFR) were assessed for their ability to invade cultured human oropharyngeal epithelial cells. Initial assays utilized untreated fungal and epithelial cells. Subsequent work used epithelial cells treated with cytochalasin D and C. albicans cells treated with thimerosal to investigate invasion mediated by active penetration of germ tubes and epithelial cell induced endocytosis, respectively. Results demonstrated the importance of the PBC for the invasion process: loss of PBC function resulted in the same reduced-invasion phenotype as a C. albicans strain that did not produce Als3 on its surface. Invasion via active penetration was particularly compromised without PBC function. Loss of AFR function produced a wild-type phenotype in the untreated and thimerosal-treated invasion assays but increased invasion in cytochalasin D-treated epithelial cells. In previous work, reduced AFR-mediated Als3 aggregation increased C. albicans adhesion to cultured epithelial cell monolayers, presumably via increased PBC accessibility for ligand binding. Collectively, results presented here demonstrate that Als3 PBC-mediated adhesion is integral to its invasive function. These new data add to the mechanistic understanding of the role of Als3 in C. albicans invasion into mammalian oral epithelial cells.

Cancer cell-derived exosomal miR-425-3p induces white adipocyte atrophy.

White adipose tissue wasting plays a critical role in the development and progression of cancer cachexia. However, the mechanism behind the loss of adipose tissue remains ill-defined. In this study, we found that cancer cell-derived exosomes highly expressed miR-425-3p. Administration of cancer cell-derived exosomes significantly inhibited proliferation and differentiation of human preadipocytes-viscereal (HPA-v) cells. In mature adipocytes, cancer cell-derived exosomes activated cAMP/PKA signalling and lipophagy, leading to adipocyte lipolysis and browning of white adipocytes. These exosomes-induced alterations were almost abolished by endocytosis inhibitor cytochalasin D (CytoD) and antagomiR-425-3p, or reproduced by miR-425-3p mimics. In addition, bioinformatics analysis and luciferase reporter assay revealed that miR-425-3p directly targeted proliferation-related genes such as GATA2, IGFBP4, MMP15, differentiation-related gene CEBPA, and phosphodiesterase 4B gene (PDE4B). Depletion of PDE4B enhanced cAMP/PKA signalling and lipophagy, but had no effects on HPA-v proliferation and differentiation. Taken together, these results suggested that cancer cell-derived exosomal miR-425-3p inhibited preadipocyte proliferation and differentiation, increased adipocyte lipolysis, and promoted browning of white adipocytes, all of which might contribute to adipocyte atrophy and ultimately the loss of adipose tissue in cancer cachexia.Abbreviations: ADPN: adiponectin; aP2: adipocyte protein 2 or fatty acid binding protein 4 (FABP4); BCA: bicinchoninic acid assay; BFA: bafilomycin A1; BMI: body mass index; C/EBP: CCAAT/enhancer binding protein; CEBPA: CCAAT/enhancer-binding protein-alpha; C-Exo: cancer cell-derived exosomes; CNTL: control; CREB: cAMP-response element binding protein; CytoD: cytochalasin D; ECL: chemiluminescence; GATA2: GATA Binding Protein 2; HFD: high fat diet; HSL: hormone-sensitive lipase; IGFBP4: insulin like growth factor binding protein 4; IRS-1: insulin receptor substrate-1; ISO: isoproterenol hydrochloride; KD: knockdown; KO: knock out; LC3: microtubule-associated protein 1A/1B-light chain 3; LMF: lipid mobilizing factor; LPL: lipoprotein lipase; MMP15: matrix metallopeptidase 15; Mir-Inh-C-Exo: cancer cell-derived exosomes with miR-425-3p inhibition; mTOR: mammalian target of rapamycin; Mut: mutant; N-Exo: normal cell-derived exosomes; NSCLC: non-small cell lung cancer; PBS, phosphate buffered saline; PGC-1: peroxisome proliferator-activated receptor-gamma coactivator-1; PDEs: phosphodiesterases; PKI: PKA inhibitor; PKA: cAMP-dependent protein kinase; PLIN1: Perilipin 1; PTHRP: parathyroid hormone-related protein; PVDF: polyvinylidene difluoride; shRNA: short hairpin RNA; UCP1: uncoupling protein 1; WT: wild type.

Molecular tension microscopy of the LINC complex in live cells.

We present a protocol to measure the effect of pharmacological treatments on the mechanical tension experienced by nesprins at the cytoplasmic surface of the nuclear envelope of mammalian cells in culture. We apply this protocol to MDCK epithelial cells exposed to the actin depolymerization agent cytochalasin D. To do so, we perform confocal spectral imaging of transiently expressed molecular tension sensors of mini-nesprin 2G and analyze the FRET signal from the sensors with a custom-made Fiji script. For complete details on the use and execution of this protocol, please refer to Déjardin et al. (2020).

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D.

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Cancer cell viscoelasticity measurement by quantitative phase and flow stress induction.

Cell viscoelastic properties are affected by the cell cycle, differentiation, and pathological processes such as malignant transformation. Therefore, evaluation of the mechanical properties of the cells proved to be an approach to obtaining information on the functional state of the cells. Most of the currently used methods for cell mechanophenotyping are limited by low robustness or the need for highly expert operation. In this paper, the system and method for viscoelasticity measurement using shear stress induction by fluid flow is described and tested. Quantitative phase imaging (QPI) is used for image acquisition because this technique enables one to quantify optical path length delays introduced by the sample, thus providing a label-free objective measure of morphology and dynamics. Viscosity and elasticity determination were refined using a new approach based on the linear system model and parametric deconvolution. The proposed method allows high-throughput measurements during live-cell experiments and even through a time lapse, whereby we demonstrated the possibility of simultaneous extraction of shear modulus, viscosity, cell morphology, and QPI-derived cell parameters such as circularity or cell mass. Additionally, the proposed method provides a simple approach to measure cell refractive index with the same setup, which is required for reliable cell height measurement with QPI, an essential parameter for viscoelasticity calculation. Reliability of the proposed viscoelasticity measurement system was tested in several experiments including cell types of different Young/shear modulus and treatment with cytochalasin D or docetaxel, and an agreement with atomic force microscopy was observed. The applicability of the proposed approach was also confirmed by a time-lapse experiment with cytochalasin D washout, whereby an increase of stiffness corresponded to actin repolymerization in time.

The Role of Cytoskeleton Revealed by Quartz Crystal Microbalance and Digital Holographic Microscopy.

The connection between cytoskeleton alterations and diseases is well known and has stimulated research on cell mechanics, aiming to develop reliable biomarkers. In this study, we present results on rheological, adhesion, and morphological properties of primary rat cardiac fibroblasts, the cytoskeleton of which was altered by treatment with cytochalasin D (Cyt-D) and nocodazole (Noc), respectively. We used two complementary techniques: quartz crystal microbalance (QCM) and digital holographic microscopy (DHM). Qualitative data on cell viscoelasticity and adhesion changes at the cell-substrate near-interface layer were obtained with QCM, while DHM allowed the measurement of morphological changes due to the cytoskeletal alterations. A rapid effect of Cyt-D was observed, leading to a reduction in cell viscosity, loss of adhesion, and cell rounding, often followed by detachment from the surface. Noc treatment, instead, induced slower but continuous variations in the rheological behavior for four hours of treatment. The higher vibrational energy dissipation reflected the cell's ability to maintain a stable attachment to the substrate, while a cytoskeletal rearrangement occurs. In fact, along with the complete disaggregation of microtubules at prolonged drug exposure, a compensatory effect of actin polymerization emerged, with increased stress fiber formation.

A look into hemostatic characteristics during pediatric liver transplantation using the thromboelastometry (ROTEM® ) test.

There is a paucity of evidence about the coagulation profile regarding the complexity of children undergoing liver transplantation (LT). This study aimed to investigate intraoperative hemostatic changes during pediatric LT according to the etiology for LT and examine the ability of rotational thromboelastometry (ROTEM® , TEM International GmbH, Munich, Germany) as a point-of-care monitoring method. We evaluated 106 patients aged 3 months to 17 years undergoing LT for acute liver failure (ALF) and chronic liver disease, which consists of patients with cholestatic disease, metabolic/genetic disease, and cancer. A total of 731 ROTEM® measurements, including 301 ellagic acid to initiate clotting via the intrinsic pathway, 172 tissue factor to initiate the extrinsic clotting cascade (EXTEM), and 258 cytochalasin D to inhibit platelet activity reflecting fibrinogen (FIBTEM), were analyzed at predetermined time points (the preanhepatic, anhepatic, and postreperfusion phases). We simultaneously conducted conventional coagulation tests. In children with ALF, preanhepatic measurements of conventional coagulation tests and ROTEM® showed a more hypocoagulable state than other diseases. During LT, the coagulation profile was deranged, with a prolonged clotting time and reduced clot firmness, changes that were more profound in the cholestatic disease group. Maximum clot firmness (MCF) on EXTEM and FIBTEM were well correlated with the platelet count and fibrinogen concentration (r = 0.830, p < 0.001 and r = 0.739, p < 0.001, respectively). On the EXTEM, MCF with 30 mm predicted a platelet count <30,000/mm3 (area under the curve, 0.985), and 6 mm predicted a fibrinogen concentration <100 mg/dl on the FIBTEM (area under the curve, 0.876). However, the activated partial thromboplastin time and prothrombin time were significant but only weakly correlated with the clotting time on the ROTEM® . In children undergoing LT, coagulation profiles depend on the etiology for LT. During LT, ROTEM® parameters could help detect thrombocytopenia and hypofibrinogenemia and guide transfusion therapy as a point-of-care monitoring method.

Transcytosis mechanisms of cell-penetrating peptides: Cation-independent CC12 and cationic penetratin.

Cell-penetrating peptides (CPPs) can aid in intracellular and in vivo drug delivery. However, the mechanisms of CPP-mediated penetration remain unclear, limiting the development and further application of CPPs. Flow cytometry and laser confocal fluorescence microscopy were performed to detect the effects of different endocytosis inhibitors on the internalization of CC12 and penetratin in ARPE-19 cells. The co-localization of CPPs with the lysosome and macropinosome was detected via an endocytosis tracing experiment. The flow cytometry results showed that chlorpromazine, wortmannin, cytochalasin D, and the ATP inhibitor oligomycin had dose-dependent endocytosis-inhibitory effects on CC12. The laser confocal fluorescence results showed that oligomycin had the most significant inhibitory effect on CC12 uptake; CC12 was co-located with the lysosome, but not with the macropinosome. For penetratin, cytochalasin D and oligomycin had obvious inhibitory effects. The laser confocal fluorescence results indicated that oligomycin had the most significant inhibitory effect on penetratin uptake; the co-localization of penetratin with the lysosome was higher than that with the macropinosome. Cation-independent CC12 and cationic penetratin may be internalized into cells primarily through caveolae and clathrin-mediated endocytosis, and they are typically dependent on ATP. The transport of penetratin could be partly achieved through the direct transmembrane pathway, as the positive charge of penetratin interacts with the negative charge of the cell membrane, and partly through the endocytic pathway.

Simple methods for quantifying super-resolved cortical actin.

Cortical actin plays a key role in cell movement and division, but has also been implicated in the organisation of cell surface receptors such as G protein-coupled receptors. The actin mesh proximal to the inner membrane forms small fenced regions, or 'corrals', in which receptors can be constrained. Quantification of the actin mesh at the nanoscale has largely been attempted in single molecule datasets and electron micrographs. This work describes the development and validation of workflows for analysis of super resolved fixed cortical actin images obtained by Super Resolved Radial Fluctuations (SRRF), Structured Illumination Microscopy (3D-SIM) and Expansion Microscopy (ExM). SRRF analysis was used to show a significant increase in corral area when treating cells with the actin disrupting agent cytochalasin D (increase of 0.31 µm2 ± 0.04 SEM), and ExM analysis allowed for the quantitation of actin filament densities. Thus, this work allows complex actin networks to be quantified from super-resolved images and is amenable to both fixed and live cell imaging.