Alterations In Cellular Structure Research Articles

Critical contribution of mitochondria in the development of cardiomyopathy linked to desmin mutation.

Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DESE439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DESE439K mutation, and post-mortem heart samples from five control healthy donors. The heterozygous DESE439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient's heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. This work highlights the deleterious effects ofDESE439Kmutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Anisotropic Gold Nanoparticles Synthesized using Litchi chinensis Leaf Extract and their Effect on Breast Cancer

For its medicinal potential, especially in cancer therapy, eco-friendly gold nanoparticles (GNPs) have gained a lot of interest. This investigation used Litchi chinensis leaf extract (LLE) as a reducing agent for the one-step synthesis of regulated-size GNPs. Fourier transform infrared (FTIR) spectrum shows that the bio-molecules act as capping and reducing agents. The formation of GNPs has been confirmed using UV-vis absorption spectra. Effect of various synthesized parameters, i.e., reaction time (5–120 minutes), temperature (25–100°C), solvent quantity (5–45 μL), metal ion concentration (0.25–2 mM) and pH (2–10) has been studied. The size, distribution, and morphology (anisotropic) have been studied using field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) techniques in the range of 10 to 70 nm. X-Ray diffraction (XRD) reveals that the particles were crystalline and had face-centered cubic (FCC) structure, and transmission electron microscopy (TEM) images revealed anisotropic morphology (spherical, hexagonal, and triangular). In-vitro studies of GNPs on breast cancer cell lines show cytotoxicity with cellular structural alterations and caused DNA damage without the micronuclei formation.

Effect of iron on rat serum melatonin levels under different light/dark cycle patterns

Exposure to constant light or darkness for long periods has diverse effects on circadian physiology. Iron (Fe) overloading promotes oxidative stress and causes alterations in cellular structure and function in animals and humans. The aim of this study is to evaluate the interactions among serum melatonin (ML), photoperiod manipulation, and Fe overloading in rats. The results showed that constant darkness exposure for 15 days significantly increased serum ML levels (up to 22%) while the constant light exposure failed to reduce the serum ML level compared to the normal light/dark cycle treated rats. The lost serum ML level usually from the pineal gland under the long term of constant light exposure may be compensated by ML generated by other organs which adapted to the situation. Also, Fe overloading decreased ML production due to this molecule being consumed to scavenge the free radicals induced by the Fe overloading. In addition, we observed interactions among constant light or darkness exposure, Fe overloading and serum ML level. Overall, our results support the hypothesis of ML as scavenging molecule; it may be an effective therapeutic tool in iron-induced oxidative stress.

GEOMETRIC ANALYSIS OF STRUCTURAL CHANGES IN MICROSCOPIC NUCLEI IMAGES FOR DRUG-INDUCED CYTOTOXIC ASSESSMENT

Investigation of drug-induced structural changes in cell lines at different concentrations using microscopic images is essential to understand their cytotoxic effects. In this study, geometric shape descriptors to evaluate the toxicity effects of a particular drug in cell images are formulated. For this, fluorescence microscopic images of drug-untreated and drug-treated mouse cardiac muscle HL1 cells are considered. Ratiometric index of cellular to non-cellular area and, Zernike moment measures are calculated for three different thresholds at different drug concentrations namely 0.6, 1.2, 2.5, 5, and 10[Formula: see text][Formula: see text]M. Statistical analysis is performed to find the significant features. Classification is performed using Support Vector Machine (SVM) to differentiate drug untreated with treated cells at different concentrations. Results demonstrate that the proposed features are able to characterize the shape variations in cell images at different concentrations, and validates the efficacy of segmentation. Mean cellular area ratio is found to decrease from drug-untreated to drug-treated at various concentrations. Significant shape alterations in cellular structures are also obtained using Zernike moment measures for these cases. The machine learning approach using SVM provides better performance in classifying the drug untreated with progressively increasing drug concentrations. Hence, the proposed pipeline of methods could be clinically used to determine the maximum permissible drug tolerance levels during the development of new drugs.

Preparation of biological monolayers for producing high-resolution scanning electron micrographs.

Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, namely., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

Hierarchical Micro-/Nanotopography for Tuning Structures and Mechanics of Cells Probed by Atomic Force Microscopy.

Extracellular matrix plays an important role in regulating the behaviors of cells, and utilizing matrix physics to control cell fate has been a promising way for cell and tissue engineering. However, the nanoscale situations taking place during the topography-regulated cell-matrix interactions are still not fully understood to the best of our knowledge. The invention of atomic force microscopy (AFM) provides a powerful tool to characterize the structures and properties of living biological systems under aqueous conditions with unprecedented spatial resolution. In this work, with the use of AFM, structural and mechanical dynamics of individual cells grown on micro-/nanotopographical surface were revealed. First, the microgroove patterned silicon substrates were fabricated by photolithography. Next, nanogranular topography was formed on microgroove substrates by cell culture medium protein deposition, which was visualized by in situ AFM imaging. The micro-/nanotopographical substrates were then used to grow two types of cells (3T3 cell or MCF-7 cell). AFM morphological imaging and mechanical measurements were applied to characterize the changes of cells grown on the micro-/nanotopographical substrates. The experimental results showed the significant alterations in cellular structures and cellular mechanics caused by micro-/nanotopography. The study provides a novel way based on AFM to unveil the native nanostructures and mechanical properties of cell-matrix interfaces with high spatial resolution in liquids, which will have potential impacts on the studies of topography-tuned cell behaviors.

Effect of Post-mortem Interval and Perfusion on the Biophysical Properties of ex vivo Liver Tissue Investigated Longitudinally by MRE and DWI.

Structural changes of soft tissues on the cellular level can be characterized by histopathology, but not longitudinally in the same tissue. Alterations of cellular structures and tissue matrix are associated with changes in biophysical properties which can be monitored longitudinally by quantitative diffusion-weighted imaging (DWI) and magnetic resonance elastography (MRE). In this work, DWI and MRE examinations were performed in a 0.5-Tesla compact scanner to investigate longitudinal changes in water diffusivity, stiffness and viscosity of ex-vivo rat livers for up to 20 h post-mortem (pm). The effect of blood on biophysical parameters was examined in 13 non-perfused livers (containing blood, NPLs) and 14 perfused livers (blood washed out, PLs). Changes in cell shape, cell packing and cell wall integrity were characterized histologically. In all acquisitions, NPLs presented with higher shear-wave speed (c), higher shear-wave penetration rate (a) and smaller apparent-diffusion-coefficients (ADCs) than PL. Time-resolved analysis revealed three distinct phases: (i) an initial phase (up to 2 h pm) with markedly increased c and a and reduced ADCs; (ii) an extended phase with relatively stable values; and (iii) a degradation phase characterized by significant increases in a (10 h pm in NPLs and PLs) and ADCs (10 h pm in NPLs, 13 h pm in PLs). Histology revealed changes in cell shape and packing along with decreased cell wall integrity, indicating tissue degradation in NPLs and PLs 10 h pm. Taken together, our results demonstrate that the biophysical properties of fresh liver tissue rapidly change within 2 h pm, which seems to be an effect of both cytotoxic edema and vascular blood content. Several hours later, disruption of cell walls resulted in higher water diffusivity and wave penetration. These results reveal the individual contributions of vascular components and cellular integrity to liver elastography and provide a biophysical, imaging-based fingerprint of liver tissue degradation.

CORONAVIRUS- A REVIEW

Coronavirus disease (COVID-19) is an infectious disease caused by a coronavirus. Coronaviruses are a group of enveloped viruses with nonsegmented, single-stranded, and positive-sense RNA genomes. Apart from infecting a variety of economically important vertebrates (such as pigs and chickens), six coronaviruses have been known to infect human hosts and cause respiratory diseases. Among them, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are zoonotic and highly pathogenic coronaviruses that have resulted in regional and global outbreaks Coronaviruses possess a distinctive morphology, the name being derived from the outer fringe, or corona‖ of embedded envelope protein. Members of the family Coronaviridae cause a broad spectrum of animal and human diseases. Uniquely, replication of the RNA genome proceeds through the generation of a nested set of viral mRNA molecules. Human coronavirus (HCoV) infection causes respiratory diseases with mild to severe outcomes. In the last 15 years, we have witnessed the emergence of two zoonotic, highly pathogenic HCoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Replication of HCoV is regulated by a diversity of host factors and induces drastic alterations in cellular structure and physiology. In this review all (as we possible) information about Corona viruses are given.
 Keywords: Corona virus, respiratory, viruses, Hcov, host, RNA.

A Peptide Found in Human Serum, Derived from the C-Terminus of Albumin, Shows Antifungal Activity In Vitro and In Vivo.

The growing problem of antimicrobial resistance highlights the need for alternative strategies to combat infections. From this perspective, there is a considerable interest in natural molecules obtained from different sources, which are shown to be active against microorganisms, either alone or in association with conventional drugs. In this paper, peptides with the same sequence of fragments, found in human serum, derived from physiological proteins, were evaluated for their antifungal activity. A 13-residue peptide, representing the 597–609 fragment within the albumin C-terminus, was proved to exert a fungicidal activity in vitro against pathogenic yeasts and a therapeutic effect in vivo in the experimental model of candidal infection in Galleria mellonella. Studies by confocal microscopy and transmission and scanning electron microscopy demonstrated that the peptide penetrates and accumulates in Candida albicans cells, causing gross morphological alterations in cellular structure. These findings add albumin to the group of proteins, which already includes hemoglobin and antibodies, that could give rise to cryptic antimicrobial fragments, and could suggest their role in anti-infective homeostasis. The study of bioactive fragments from serum proteins could open interesting perspectives for the development of new antimicrobial molecules derived by natural sources.

Assessment of cytotoxicity in gills of fish Labeo rohita reared in untreated and treated sewage water.

The unregulated discharge of untreated municipal sewage water to the natural water bodies is a major threat to the aquatic ecosystems. In the present study, the fingerlings of Labeo rohita were exposed to treated sewage water and 1/10th of LC50 and 1/20th of LC50 of untreated sewage water (UT) obtained from sewage water treatment plant, Ludhiana, India. After determining 96-h LC50 value of UT, fingerlings were divided into four groups: control, treated, 1/10th of LC50 UT and 1/20th of LC50 UT and exposed for a period of 2months. Our study revealed that 1/10th LC50 UT and 1/20th LC50 UT groups had significant reduction in body weight, total body length, standard body length and gills somatic index in comparison with the control and treated groups. Histopathological alterations in cellular structure of gills such as ruptured primary lamellae, broken secondary lamellae, distorted chloride cells and goblet cells, missing lamellae and disintegrated lamellar epithelium were also observed in 1/10th LC50 UT and 1/20th LC50 UT groups. A significantly high frequency of micronucleated (MN) cells was observed in 1/10th LC50 UT and 1/20th LC50 UT groups along with the presence of binucleated cells (BN), elongated nuclei (EN) and nuclear buds in gill cells. Genotoxic nature of UT was further confirmed from significantly high values of genetic damage index (GDI) and percentage (%) DNA damage in gill cells of fingerlings exposed to sub-lethal concentrations of UT. The study concluded that untreated sewage water has potential to induce anatomical and physiological defects in gills cells and the severity of toxicity increases with the increase in duration of exposure.

Human Coronavirus: Host-Pathogen Interaction.

Human coronavirus (HCoV) infection causes respiratory diseases with mild to severe outcomes. In the last 15 years, we have witnessed the emergence of two zoonotic, highly pathogenic HCoVs: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). Replication of HCoV is regulated by a diversity of host factors and induces drastic alterations in cellular structure and physiology. Activation of critical signaling pathways during HCoV infection modulates the induction of antiviral immune response and contributes to the pathogenesis of HCoV. Recent studies have begun to reveal some fundamental aspects of the intricate HCoV-host interaction in mechanistic detail. In this review, we summarize the current knowledge of host factors co-opted and signaling pathways activated during HCoV infection, with an emphasis on HCoV-infection-induced stress response, autophagy, apoptosis, and innate immunity. The cross talk among these pathways, as well as the modulatory strategies utilized by HCoV, is also discussed.

Manipulating Endoplasmic Reticulum-Plasma Membrane Tethering in Plants Through Fluorescent Protein Complementation.

The bimolecular fluorescence complementation (BiFC) assay has been widely used to examine interactions between integral and peripheral proteins within putative plasma membrane (PM) microdomains. In the course of using BiFC assays to examine the co-localization of plasma membrane (PM) targeted receptor-like kinases (RLKs), such as FLS2, with PM micro-domain proteins such as remorins, we unexpectedly observed heterogeneous distribution patterns of fluorescence on the PM of Nicotiana benthamiana leaf cortical cells. These patterns appeared to co-localize with the endoplasmic reticulum (ER) and with ER-PM contact sites, and closely resembled patterns caused by over-expression of the ER-PM tether protein Synaptotagmin1 (SYT1). Using domain swap experiments with SYT1, we inferred that non-specific dimerization between FLS2-VenusN and VenusC-StRem1.3 could create artificial ER-PM tether proteins analogous to SYT1. The same patterns of ER-PM tethering were produced when a representative set of integral membrane proteins were partnered in BiFC complexes with PM-targeted peripheral membrane proteins, including PtdIns(4)P-binding proteins. We inferred that spontaneous formation of mature fluorescent proteins caused the BiFC complexes to trap the integral membrane proteins in the ER during delivery to the PM, producing a PM-ER tether. This phenomenon could be a useful tool to deliberately manipulate ER-PM tethering or to test protein membrane localization. However, this study also highlights the risk of using the BiFC assay to study membrane protein interactions in plants, due to the possibility of alterations in cellular structures and membrane organization, or misinterpretation of protein-protein interactions. A number of published studies using this approach may therefore need to be revisited.

In Vivo Evaluation of Cerebral Hemodynamics and Tissue Morphology in Rats during Changing Fraction of Inspired Oxygen Based on Spectrocolorimetric Imaging Technique.

During surgical treatment for cerebrovascular diseases, cortical hemodynamics are often controlled by bypass graft surgery, temporary occlusion of arteries, and surgical removal of veins. Since the brain is vulnerable to hypoxemia and ischemia, interruption of cerebral blood flow reduces the oxygen supply to tissues and induces irreversible damage to cells and tissues. Monitoring of cerebral hemodynamics and alteration of cellular structure during neurosurgery is thus crucial. Sequential recordings of red-green-blue (RGB) images of in vivo exposed rat brains were made during hyperoxia, normoxia, hypoxia, and anoxia. Monte Carlo simulation of light transport in brain tissue was used to specify relationships among RGB-values and oxygenated hemoglobin concentration (CHbO), deoxygenated hemoglobin concentration (CHbR), total hemoglobin concentration (CHbT), hemoglobin oxygen saturation (StO2), and scattering power b. Temporal courses of CHbO, CHbR, CHbT, and StO2 indicated physiological responses to reduced oxygen delivery to cerebral tissue. A rapid decrease in light scattering power b was observed after respiratory arrest, similar to the negative deflection of the extracellular direct current (DC) potential in so-called anoxic depolarization. These results suggest the potential of this method for evaluating pathophysiological conditions and loss of tissue viability.

Effects of a silver nanomaterial on cellular organelles and time course of oxidative stress in a fish cell line (PLHC-1)

Among the nanomaterials currently in commercial products, those based on silver are the most used, and so there is a high probability that silver nanoparticles (AgNPs) will be released into aquatic environments where they could adversely affect aquatic organisms, including fish. Taking this into account, the aim of the present work was to characterize in depth the mechanisms underlying the toxic action of AgNPs using fish cell lines, determining specifically the contribution of alterations in cellular structures and oxidative stress time course to the cytotoxicity of AgNPs. Since liver plays a key role in detoxification, the hepatoma cell line PLHC-1 was used. Exposure to AgNPs (NM-300K, obtained from the Joint Research Centre Repository) caused alterations at the lysosomal and mitochondrial levels at lower concentrations than those that disrupted plasma membrane (evaluated by means of neutral red, alamarBlue, and 5-carboxyfluorescein diacetate, acetoxymethyl ester assays respectively). AgNO3, used as a control Ag+ ion source, produced similar cytotoxic effects but at lower concentrations than AgNPs. Both silver forms caused oxidative disruption but the initial response was delayed in AgNPs until 6h of exposure. Transmission electron microscopy analysis also evidenced the disruption of mitochondrial structures in cells exposed to cytotoxic concentrations of both forms of silver. At non-cytotoxic concentrations, AgNPs were detected inside the nucleoli and mitochondria, thereby pointing to long-term effects. The present work evidences the mutual interaction between the induction of oxidative stress and the alterations of cellular structures, particularly mitochondria, as cytotoxicity mechanisms not exclusively associated to NPs.

Pathophysiology Associated with Traumatic Brain Injury: Current Treatments and Potential Novel Therapeutics.

Traumatic brain injury (TBI) is one of the leading causes of death of young people in the developed world. In the United States alone, 1.7 million traumatic events occur annually accounting for 50,000 deaths. The etiology of TBI includes traffic accidents, falls, gunshot wounds, sports, and combat-related events. TBI severity ranges from mild to severe. TBI can induce subtle changes in molecular signaling, alterations in cellular structure and function, and/or primary tissue injury, such as contusion, hemorrhage, and diffuse axonal injury. TBI results in blood-brain barrier (BBB) damage and leakage, which allows for increased extravasation of immune cells (i.e., increased neuroinflammation). BBB dysfunction and impaired homeostasis contribute to secondary injury that occurs from hours to days to months after the initial trauma. This delayed nature of the secondary injury suggests a potential therapeutic window. The focus of this article is on the (1) pathophysiology of TBI and (2) potential therapies that include biologics (stem cells, gene therapy, peptides), pharmacological (anti-inflammatory, antiepileptic, progrowth), and noninvasive (exercise, transcranial magnetic stimulation). In final, the review briefly discusses membrane/lipid rafts (MLR) and the MLR-associated protein caveolin (Cav). Interventions that increase Cav-1, MLR formation, and MLR recruitment of growth-promoting signaling components may augment the efficacy of pharmacologic agents or already existing endogenous neurotransmitters and neurotrophins that converge upon progrowth signaling cascades resulting in improved neuronal function after injury.

Impact of RTN3 deficiency on expression of BACE1 and amyloid deposition.

Reticulon 3 (RTN3) has previously been shown to interact with BACE1 and negatively regulate BACE1 activity. To what extent RTN3 deficiency affects BACE1 activity is an intriguing question. In this study, we aimed to address this by generating RTN3-null mice. Mice with complete deficiency of RTN3 grow normally and have no obviously discernible phenotypes. Morphological analyses of RTN3-null mice showed no significant alterations in cellular structure, although RTN3 is recognized as a protein contributing to the shaping of tubular endoplasmic reticulum. Biochemical analysis revealed that RTN3 deficiency increased protein levels of BACE1. This elevation of BACE1 levels correlated with enhanced processing of amyloid precursor protein at the β-secretase site. We also demonstrated that RTN3 deficiency in Alzheimer's mouse models facilitates amyloid deposition, further supporting an in vivo role of RTN3 in the regulation of BACE1 activity. Since it has been shown that RTN3 monomer is reduced in brains of Alzheimer's patients, our results suggest that long-lasting reduction of RTN3 levels has adverse effects on BACE1 activity and may contribute to Alzheimer's pathogenesis.

Differential expression of miRNAs and their target genes in senescing leaves and siliques: insights from deep sequencing of small RNAs and cleaved target RNAs.

MicroRNAs (miRNAs) are a class of small RNAs, which typically function by guiding cleavage of target mRNAs. They are known to play roles in a variety of plant processes including development, responses to environmental stresses and senescence. To identify senescence regulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel analysis of RNA ends libraries, which enable the large-scale examination of miRNA-guided cleavage products, were constructed and sequenced, resulting in over 750 million genome-matched sequences. These large datasets led to the identification a new senescence-inducible small RNA locus, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence.

Comprehensive identification of SUMO2/3 targets and their dynamics during mitosis.

During mitosis large alterations in cellular structures occur rapidly, which to a large extent is regulated by post-translational modification of proteins. Modification of proteins with the small ubiquitin-related protein SUMO2/3 regulates mitotic progression, but few mitotic targets have been identified so far. To deepen our understanding of SUMO2/3 during this window of the cell cycle, we undertook a comprehensive proteomic characterization of SUMO2/3 modified proteins in mitosis and upon mitotic exit. We developed an efficient tandem affinity purification strategy of SUMO2/3 modified proteins from mitotic cells. Combining this purification strategy with cell synchronization procedures and quantitative mass spectrometry allowed for the mapping of numerous novel targets and their dynamics as cells progressed out of mitosis. This identified RhoGDIα as a major SUMO2/3 modified protein, specifically during mitosis, mediated by the SUMO ligases PIAS2 and PIAS3. Our data provide a rich resource for further exploring the role of SUMO2/3 modifications in mitosis and cell cycle regulation.

Stimulative Effects of Hominis Placental Pharmacopuncture Solution Combined with Zinc-oxide Nanoparticles on RAW 264.7 Cells: ZnO HPPS more easily stimulates RAW 264.7 cells.

Objectives:The purpose of this study is to examine whether Hominis Placental pharmacopuncture solution (HPPS) combined with zinc-oxide nanoparticles (ZnO NP) activates RAW 264.7 cells.Methods:We soaked ZnO nanoparticles in the Hominis Placenta pharmacopuncture solution, thereby making a combined form (ZnO NP HPPS). The effect of ZnO NP HPPS on the intracellular reactive oxygen species (ROS) production was measured by 2', 7'-dichlorofluorescin diacetate (DCFH-DA) assay. The effect of ZnO NP HPPS on NF-κ B was measured by using a luciferase assay. The effect of ZnO NP HPPS on the cytokine expression was assessed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The cellular uptake of ZnO NP HPPS was measured by using a flow cytometric analysis, and cellular structural alterations were analyzed by using transmission electron microscopy (TEM).Results:Neither the HPPS nor the ZnO NPs induced intracellular ROS production in RAW 264.7 cells. Neither of the materials activated NF-κ B or it’s dependent genes, such as TNF-α, IL-1, and MCP-1. However, ZnO NP HPPS, the combined form of ZnO NPs and HPPS, did induce the intracellular ROS production, as well as prominently activating NF-κ B and it’s dependent genes. Also, compared to ZnO NPs, it effectively increased the uptake by RAW 264.7 cells. In addition, cellular structural alterations were observed in groups treated with ZnO NP HPPS.Conclusions:Neither ZnO NP nor HPPS activated RAW 264.7 cells, which is likely due to a low cellular uptake. The ZnO NP HPPS, however, significantly activated NF-κ B and up-regulated its dependent genes such as TNF-α, IL-1, and MCP-1. ZnO NP HPPS was also more easily taken into the RAW 264.7 cells than either ZnO NP or HPPS.

Fractal Dimension as a Measure of Altered Actin Cytoskeleton in MC3T3-E1 Cells Under Simulated Microgravity Using 3-D/2-D Clinostats

Osteoblasts, the bone-forming cells, respond to various mechanical forces, such as stretch and fluid shear force in essentially similar ways. The cytoskeleton, as the load-bearing architecture of the cell, is sensitive to altered inertial forces. Disruption of the cytoskeleton will result in alteration of cellular structure and function. However, it is difficult to quantitatively illustrate cytoskeletal rearrangement because of the complexity of cytoskeletal structure. Usually, the morphological changes in actin organization caused by external stimulus are basically descriptive. In this study, fractal dimensions (D) analysis was used to quantify the morphological changes in the actin cytoskeleton of osteoblast-like cells (MC3T3-E1) under simulated microgravity using 3-D/2-D clinostats. The ImageJ software was used to count the fractal dimension of actin cytoskeleton by box-counting methods. Real-time PCR and immunofluroscent assays were used to further confirm the results obtained by fractal dimension analysis. The results showed significant decreases in D value of actin cytoskeleton, β-actin mRNA expression, and the mean fluorescence intensity of F-actin in osteoblast-like cells after 24 or 48 h of incubation under 3-D/2-D clinorotation condition compared with control. The findings indicate that 3-D/2-D clinorotation affects both actin cytoskeleton architecture and mRNA expression, and fractal may be a promising approach for quantitative analysis of the changes in cytoskeleton in different environments.