ABSTRACT
Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.
Subject(s)
Chitin , Microvilli , Microvilli/ultrastructure , Animals , Chitin/metabolism , Chitin/chemistry , Polychaeta/ultrastructure , Actins/metabolism , MorphogenesisABSTRACT
Intra- and extracellular depositions and inclusions occur in a wide range of diseases with exogenous (e.g. infectious, environmental and toxic) or endogenous (e.g. genetic, inflammatory, neoplastic and degenerative) aetiology. The noxious agent and the pathogenesis influence the organ of manifestation, the subcellular localisation and the ultrastructural appearance of the depositions. Whereas some of the inclusions like pathogens, foreign material (e.g. asbestos) or microvilli have an almost pathognomonic morphology, other inclusions are present in lower amounts also under normal conditions (e.g. lipid vacuoles and glycogen). Therefore, the interpretation of ultrastructural findings makes a correlation with the histological features and clinical constellation necessary. Auxiliary investigations by electron energy loss spectroscopy (EELS) or electron spectroscopic imaging (ESI) provide additional information about the chemical composition of the material and are therefore especially helpful for the identification of foreign substances. This review focuses on a selection of deposits and inclusions relevant to diagnostic pathology.
Subject(s)
Inclusion Bodies , Vacuoles , Inclusion Bodies/ultrastructure , Microvilli/ultrastructure , Microscopy, Energy-Filtering Transmission Electron , GlycogenABSTRACT
Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.
Subject(s)
Cell Polarity , Cytoskeletal Proteins/metabolism , Gonorrhea/microbiology , Neisseria gonorrhoeae/genetics , Actins/metabolism , Cervix Uteri/microbiology , Epithelial Cells/microbiology , Epithelial Cells/ultrastructure , Epithelium/microbiology , Female , Humans , Microvilli/ultrastructure , Mucous Membrane/microbiology , Neisseria gonorrhoeae/physiology , PhosphorylationABSTRACT
BACKGROUND: The novel coronavirus SARS-CoV-2 is the etiological agent of COVID-19. This virus has become one of the most dangerous in recent times with a very high rate of transmission. At present, several publications show the typical crown-shape of the novel coronavirus grown in cell cultures. However, an integral ultramicroscopy study done directly from clinical specimens has not been published. METHODS: Nasopharyngeal swabs were collected from 12 Cuban individuals, six asymptomatic and RT-PCR negative (negative control) and six others from a COVID-19 symptomatic and RT-PCR positive for SARS CoV-2. Samples were treated with an aldehyde solution and processed by scanning electron microscopy (SEM), confocal microscopy (CM) and, atomic force microscopy. Improvement and segmentation of coronavirus images were performed by a novel mathematical image enhancement algorithm. RESULTS: The images of the negative control sample showed the characteristic healthy microvilli morphology at the apical region of the nasal epithelial cells. As expected, they do not display virus-like structures. The images of the positive sample showed characteristic coronavirus-like particles and evident destruction of microvilli. In some regions, virions budding through the cell membrane were observed. Microvilli destruction could explain the anosmia reported by some patients. Virus-particles emerging from the cell-surface with a variable size ranging from 80 to 400 nm were observed by SEM. Viral antigen was identified in the apical cells zone by CM. CONCLUSIONS: The integral microscopy study showed that SARS-CoV-2 has a similar image to SARS-CoV. The application of several high-resolution microscopy techniques to nasopharyngeal samples awaits future use.
Subject(s)
COVID-19/pathology , Nasopharynx/ultrastructure , SARS-CoV-2/ultrastructure , Antigens, Viral/metabolism , COVID-19/diagnosis , COVID-19/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , Humans , Image Enhancement , Microscopy , Microvilli/ultrastructure , Nasal Mucosa/ultrastructure , Nasal Mucosa/virology , Nasopharynx/virology , SARS-CoV-2/isolation & purification , Virion/ultrastructureABSTRACT
BACKGROUND: The cellular mechanisms involved in the development of proximal tubules are not only associated with morphogenesis in fetal life, but also with restoration of damaged tubules in adulthood. Knowledge about morphological features of cell differentiation and proliferation along the developing tubule is insufficient, which hinders identification of the cellular origin. OBJECTIVES: This study aimed to investigate ultrastructures of the proximal tubule at different stages of nephrogenesis. METHODS: Electron microscopy was used and guided by computer-assisted tubular tracing to identify the cellular structures. RESULTS: Renal vesicles and S-shaped bodies revealed more proliferative features, such as densely-packed fusiform-shaped cells with numerous protein-producing organelles than membrane specializations typical for mature tubules. At the capillary-loop stage the proximal tubules demonstrated all characteristics of the mature tubules, but not as developed, including shorter but densely packed microvilli, fewer lateral processes with cell-cell contacts, lower basal membrane infoldings, and lower mitochondrial volume density. However, they exhibited an elaborated endocytic system above the nucleus, indicating a membrane transport is being established. Abundant free- and endoplasmic reticulum-adhered ribosomes and Golgi complexes reflected active protein synthesis for cell growth and proliferation. Interestingly, electron dense cells were occasionally intermixed with electron lucent cells characterized by various organelles in less cytosol and a larger nucleus with abundant euchromatin, which is a feature of active proliferation. CONCLUSIONS: These ultrastructures indicate that the morphogenesis of the developing proximal tubule corresponds to the gradually established physiological activities. The two different cellular electron densities may suggest distinctive differentiation of the cells along the tubule.
Subject(s)
Imaging, Three-Dimensional , Kidney Tubules, Proximal , Animals , Imaging, Three-Dimensional/methods , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/ultrastructure , Microscopy, Electron/veterinary , Microvilli/metabolism , Microvilli/ultrastructureABSTRACT
T cells communicate with the environment via surface receptors. Cooperation of surface receptors regulates T-cell responses to diverse stimuli. Recently, finger-like membrane protrusions, microvilli, have been demonstrated to play a role in the organization of receptors and, hence, T-cell activation. However, little is known about the morphogenesis of dynamic microvilli, especially in the cells of immune system. In this review, I focus on the potential role of lipids and lipid domains in morphogenesis of microvilli. Discussed is the option that clustering of sphingolipids with phosphoinositides at the plasma membrane results in dimpling (curved) domains. Such domains can attract phosphoinositide-binding proteins and stimulate actin cytoskeleton reorganization. This process triggers cortical actin opening and bundling of actin fibres to support the growing of microvilli. Critical regulators of microvilli morphogenesis in T cells are unknown. At the end, I suggest several candidates with a potential to organize proteins and lipids in these structures.
Subject(s)
Lipid Metabolism , Microvilli/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/physiology , Animals , Cell Membrane/chemistry , Cell Membrane/metabolism , Humans , Immunomodulation , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Microvilli/ultrastructure , Morphogenesis , Phosphatidylinositols/metabolism , Protein Binding , Signal Transduction , Sphingolipids/metabolism , T-Lymphocytes/ultrastructureABSTRACT
The proteolytic cleavage of histone tails, also termed histone clipping, has been described as a mechanism for permanent removal of post-translational modifications (PTMs) from histone proteins. Such activity has been ascribed to ensure regulatory function in key cellular processes such as differentiation, senescence and transcriptional control, for which different histone-specific proteases have been described. However, all these studies were exclusively performed using cell lines cultured in vitro and no clear evidence that histone clipping is regulated in vivo has been reported. Here we show that histone H3 N-terminal tails undergo extensive cleavage in the differentiated cells of the villi in mouse intestinal epithelium. Combining biochemical methods, 3D organoid cultures and in vivo approaches, we demonstrate that intestinal H3 clipping is the result of multiple proteolytic activities. We identified Trypsins and Cathepsin L as specific H3 tail proteases active in small intestinal differentiated cells and showed that their proteolytic activity is differentially affected by the PTM pattern of histone H3 tails. Together, our findings provide in vivo evidence of H3 tail proteolysis in mammalian tissues, directly linking H3 clipping to cell differentiation.
Subject(s)
Enterocytes/metabolism , Histones/metabolism , Intestine, Small/cytology , Paneth Cells/metabolism , Peptide Hydrolases/metabolism , Protein Processing, Post-Translational , Stem Cells/metabolism , Animals , Cathepsin L/metabolism , Cell Differentiation , Homeostasis , Intestinal Mucosa/cytology , Mice , Microvilli/ultrastructure , Nucleosomes/metabolism , Nucleosomes/ultrastructure , Organoids , Protein Domains , Trypsin/metabolismABSTRACT
The cytoskeleton acts as a scaffold for membrane protrusion, such as microvilli. However, the relationship between the characteristics of microvilli and cytoskeleton remains poorly understood under the physiological state. To investigate the role of the cytoskeleton in regulating microvilli and cellular mechanical properties, atomic force microscopy (AFM) was used to detect the dynamic characteristics of microvillus morphology and elastic modulus of living HeLa cells. First, HeLa and MCF-7 cell lines were stained with Fluor-488-phalloidin and microtubules antibody. Then, the microvilli morphology was analyzed by high-resolution images of AFM in situ. Furthermore, changes in elastic modulus were investigated by the force curve of AFM. Fluorescence microscopy and AFM results revealed that destroyed microfilaments led to a smaller microvilli size, whereas the increase in the aggregation and number of microfilaments led to a larger microvilli size. The destruction and aggregation of microfilaments remarkably affected the mechanical properties of HeLa cells. Microtubule-related drugs induced the change of microtubule, but we failed to note significant differences in microvilli morphology and mechanical properties of cells. In summary, our results unraveled the relationship between microfilaments and the structure of microvilli and Young's modulus in living HeLa cells, which would contribute to the further understanding of the physiological function of the cytoskeleton in vivo.
Subject(s)
Cytoskeleton/metabolism , Microscopy, Atomic Force , Microvilli/metabolism , Microvilli/ultrastructure , Actin Cytoskeleton/metabolism , Biomechanical Phenomena , Cell Survival , Elasticity , HeLa Cells , Humans , MCF-7 CellsABSTRACT
Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation.
Subject(s)
Cell Membrane/immunology , Lymphocyte Activation/physiology , Microvilli/immunology , Signal Transduction/immunology , T-Lymphocytes/ultrastructure , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Animals , Carrier Proteins/pharmacology , Cell Line , Cell Membrane/drug effects , Cell Membrane/ultrastructure , Cell Shape , Cyclodextrins/pharmacology , Cytokines/physiology , Glycocalyx/drug effects , Glycocalyx/immunology , Humans , Lymphocyte Activation/drug effects , Membrane Lipids/immunology , Membrane Proteins/immunology , Mice , Microfilament Proteins/pharmacology , Microscopy, Electron, Scanning , Microvilli/drug effects , Microvilli/ultrastructure , Receptors, Antigen, T-Cell/immunology , Signal Transduction/drug effects , Stress, Mechanical , Surface Properties , Synaptosomes/drug effects , Synaptosomes/immunology , Synaptosomes/ultrastructure , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, the mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the "terminal web." Although classic electron microscopy studies revealed complex ultrastructure, the composition and function of the terminal web remain unclear. Here we identify nonmuscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the subapical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by â¼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades-old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.
Subject(s)
Microvilli/metabolism , Microvilli/ultrastructure , Myosin Heavy Chains/metabolism , Myosin Type II/metabolism , Actins/metabolism , Animals , Cell Membrane/ultrastructure , Cytoskeletal Proteins/metabolism , Cytoskeleton/physiology , Epithelium/ultrastructure , Intestinal Mucosa/metabolism , Intestines/physiology , Mice , Microscopy, Electron , Microvilli/genetics , Muscle Contraction/physiology , Myosin Heavy Chains/physiology , Myosin Type II/physiology , Myosins/metabolismABSTRACT
Necrotizing enterocolitis (NEC) is a devastating disease of premature infants with high mortality rate, indicating the need for precision treatment. NEC is characterized by intestinal inflammation and ischemia, as well derangements in intestinal microcirculation. Remote ischemic conditioning (RIC) has emerged as a promising tool in protecting distant organs against ischemia-induced damage. However, the effectiveness of RIC against NEC is unknown. To address this gap, we aimed to determine the efficacy and mechanism of action of RIC in experimental NEC. NEC was induced in mouse pups between postnatal day (P) 5 and 9. RIC was applied through intermittent occlusion of hind limb blood flow. RIC, when administered in the early stages of disease progression, decreases intestinal injury and prolongs survival. The mechanism of action of RIC involves increasing intestinal perfusion through vasodilation mediated by nitric oxide and hydrogen sulfide. RIC is a viable and non-invasive treatment strategy for NEC.
Subject(s)
Enterocolitis, Necrotizing/pathology , Intestines/blood supply , Intestines/pathology , Ischemia/pathology , Microcirculation , Animals , Enterocytes/pathology , Humans , Hypoxia , Intestinal Mucosa/pathology , Mice, Inbred C57BL , Microvilli/pathology , Microvilli/ultrastructureABSTRACT
The Rab11 apical recycling endosome pathway is a well-established regulator of polarity and lumen formation; however, Rab11-vesicular trafficking also directs a diverse array of other cellular processes, raising the question of how Rab11 vesicles achieve specificity in space, time and content of cargo delivery. In part, this specificity is achieved through effector proteins, yet the role of Rab11 effector proteins in vivo remains vague. Here, we use CRISPR/Cas9 gene editing to study the role of the Rab11 effector Fip5 during zebrafish intestinal development. Zebrafish contain two paralogous genes, fip5a and fip5b, that are orthologs of human FIP5 We find that fip5a- and fip5b-mutant fish show phenotypes characteristic of microvillus inclusion disease, including microvilli defects and lysosomal accumulation. Single and double mutant analyses suggest that fip5a and fip5b function in parallel and regulate trafficking pathways required for assembly of keratin at the terminal web. Remarkably, in some genetic backgrounds, the absence of Fip5 triggers protein upregulation of a closely related family member, Fip1. This compensation mechanism occurs both during zebrafish intestinal development and in tissue culture models of lumenogenesis. In conclusion, our data implicate the Rab11 effectors Fip5 and Fip1 in a trafficking pathway required for apical microvilli formation.
Subject(s)
Carrier Proteins/metabolism , Intestines/embryology , Organogenesis/genetics , Zebrafish/embryology , Zebrafish/genetics , rab GTP-Binding Proteins/metabolism , Animals , Carrier Proteins/genetics , Endosomes , Microvilli/genetics , Microvilli/metabolism , Microvilli/ultrastructure , Mutation , Phenotype , Protein BindingABSTRACT
The mesothelium is a dynamic and specialized tissue layer that covers the somatic cavities (pleural, peritoneal, and pericardial) as well as the surface of the visceral organs such as the lung, heart, liver, bowel and tunica vaginalis testis. The potential therapeutic manipulation of visceral organs has been complicated by the carbohydrate surface layer-here, called the mesopolysaccharide (MPS)-that coats the outer layer of the mesothelium. The traditional understanding of MPS structure has relied upon fixation techniques known to degrade carbohydrates. The recent development of carbohydrate-preserving fixation for high resolution imaging techniques has provided an opportunity to re-examine the structure of both the MPS and the visceral mesothelium. In this report, we used high pressure freezing (HPF) as well as serial section transmission electron microscopy to redefine the structure of the MPS expressed on the murine lung, heart and liver surface. Tissue preserved by HPF and examined by transmission electron microscopy demonstrated a pleural MPS layer 13.01±1.1 um deep-a 100-fold increase in depth compared to previously reported data obtained with conventional fixation techniques. At the base of the MPS were microvilli 1.1±0.35 um long and 42±5 nm in diameter. Morphological evidence suggested that the MPS was anchored to the mesothelium by microvilli. In addition, membrane pits 97±17 nm in diameter were observed in the apical mesothelial membrane. The spatial proximity and surface density (29±4.5%) of the pits suggested an active process linked to the structural maintenance of the MPS. The striking magnitude and complex structure of the MPS indicates that it is an important consideration in studies of the visceral mesothelium.
Subject(s)
Epithelium/ultrastructure , Polysaccharides/ultrastructure , Animals , Epithelium/chemistry , Extracellular Matrix/ultrastructure , Liver/ultrastructure , Lung/ultrastructure , Membrane Glycoproteins/ultrastructure , Mice , Microscopy, Electron, Transmission/methods , Microvilli/ultrastructure , Myocardium/ultrastructureABSTRACT
Brush borders of intestinal epithelial cells are mandatory for nutrient uptake. Yet, which actin nucleators are crucial for forming the F-actin bundles supporting microvilli and the actin filaments of the terminal web, in which microvilli are rooted, is unknown. We show that mice lacking the actin nucleator Cobl surprisingly did not display reduced microvilli densities or changes in microvillar F-actin bundles or microvilli diameter but particularly in the duodenum displayed increased microvillar length. Interestingly, Cobl-deficient mice furthermore showed a significant widening of the terminal web. Quantitative analyses of high-resolution cryo-scanning electron microscopy (EM) of deep-etched duodenum samples revealed that Cobl is specifically important for the formation of fine filaments in the central terminal web that connect the apical structure of the terminal web underlying the plasma membrane, the microvilli rootlets and the basal structure of the terminal web with each other. Thus, the actin nucleator Cobl is critically involved in generating one of the cellular structures of the brush border-decorated apical cortex of enterocytes representing the absorptive intestinal surface.
Subject(s)
Enterocytes/metabolism , Microfilament Proteins/physiology , Actins/metabolism , Animals , Blotting, Western , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cryoelectron Microscopy/methods , Enterocytes/ultrastructure , Intestinal Mucosa/metabolism , Intestinal Mucosa/ultrastructure , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Scanning/methods , Microvilli/physiology , Microvilli/ultrastructure , Real-Time Polymerase Chain ReactionABSTRACT
Paxillin (PXN) is a cytoplasmic protein that plays an important role in regulating focal adhesion, cytoskeletal rearrangements and cell motility. The present study aimed to investigate the role of PXN in the metastasis of human colorectal cancer (CRC) and its possible mechanisms. Immunohistochemical staining of tissues from 102 surgical CRC patients revealed that high PXN expression was positively correlated with tumournodemetastasis (TNM) stage, lymph node metastasis, distant metastasis, and recurrence at distant sites after radical surgery. In 24 cases of stage IV CRC, PXN expression in liver metastasis was higher than that in the matched primary tumour. The knockdown of PXN inhibited the proliferation, migration and invasion potential of SW480 cells in vitro and in vivo. Transmission electron microscopy revealed the effect of PXN on ultrastructural characteristics, observed mainly in microvilli and desmosomes. The downregulation of PXN decreased the activation of extracellular regulated protein kinase (ERK) and suppressed the epithelialmesenchymal transition (EMT) process. Following the downregulation of PXN, the addition of an ERK activator or inhibitor restored or further suppressed EMT, respectively, accompanied by corresponding changes in cell migration and invasion. Collectively, the present results confirmed the important role of PXN in CRC metastasis and revealed that PXN regulated EMT progression via the ERK signalling pathway. PXN may represent a future therapeutic strategy to prevent the EMTassociated progression and invasion of CRC.
Subject(s)
Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Lymphatic Metastasis/pathology , Neoplasm Recurrence, Local/epidemiology , Paxillin/metabolism , Aged , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation/drug effects , Colon/pathology , Colon/surgery , Colorectal Neoplasms/epidemiology , Colorectal Neoplasms/surgery , Desmosomes/pathology , Desmosomes/ultrastructure , Disease Progression , Epithelial-Mesenchymal Transition/drug effects , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Follow-Up Studies , Humans , Intestinal Mucosa/pathology , Intestinal Mucosa/surgery , Liver/pathology , MAP Kinase Signaling System/drug effects , Male , Mice , Microscopy, Electron, Transmission , Microvilli/pathology , Microvilli/ultrastructure , Middle Aged , Neoplasm Invasiveness/pathology , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/prevention & control , Paxillin/genetics , Retrospective Studies , Xenograft Model Antitumor AssaysABSTRACT
Epithelial mesenchymal transition (EMT) in development, tissue repair and carcinogenesis involves cellular plasticity with varying degrees of epithelial and mesenchymal properties. Several recent studies have focused on EMT phenotypic dynamism; however, information on cellular interaction in the context of EMT is inadequate. In our previous study, we investigated EMT phenotypic plasticity and anticipated it as a population driven interactive process. Present study has characterized cellular connectivity as a representative of interactivity during EMT in epithelial normal and cancer cell. It has also explored dynamism of connectivity and phenotype employing Markov model. Further, plasticity was substantiated with cell surface microvilli and molecular marker. The study unveiled interplay between phenotype and connectivity too. Findings have revealed that intercellular connectivity fueled EMT plasticity and its dynamism was more prominent in cancer population. However, normal cells are more vibrant in transition and phenotypic plasticity. We have proposed connectivity plasticity as a hallmark of EMT and needs to be studied in depth. Present study also paves the way in translating in vitro EMT findings in histopathological practices.
Subject(s)
Cell Plasticity , Epithelial-Mesenchymal Transition , Intercellular Junctions/metabolism , Cell Line , Humans , Microvilli/ultrastructure , Phenotype , Time FactorsABSTRACT
The burden of enteric pathogens in poultry is growing after the ban of antibiotic use in animal production. Organic acids gained attention as a possible alternative to antibiotics due to their antimicrobial activities, improved nutrient metabolism and performance. The current study was conducted to evaluate the effectiveness of organic acid blend on broilers cecal microbiota, histomorphometric measurements, and short-chain fatty acid production in Salmonella enterica serovar Typhimurium challenge model. Birds were divided into four treatments, including a negative control, positive control challenged with S. Typhimurium, group supplemented with an organic acid blend, and birds supplemented with organic acid blend and Salmonella challenged. Results illustrate significant differences in feed conversion ratios and production efficiency factor between treatment groups, however, the influence of organic acid supplement was marginal. Organic acid blend significantly increased cecal acetic and butyric acids concentrations when compared to unsupplemented groups and resulted in minor alterations of intestinal bacterial communities.
Subject(s)
Acetates/metabolism , Animal Feed , Butyrates/metabolism , Chickens/microbiology , Dietary Supplements , Fatty Acids/pharmacology , Gastrointestinal Microbiome/drug effects , Poultry Diseases/therapy , Salmonella Infections, Animal/therapy , Salmonella typhimurium/drug effects , Animals , Cecum/microbiology , Chickens/metabolism , Fatty Acids/administration & dosage , Fatty Acids, Volatile/administration & dosage , Fatty Acids, Volatile/pharmacology , Ileum/metabolism , Ileum/ultrastructure , Mannans/administration & dosage , Microvilli/ultrastructure , Poultry Diseases/microbiology , Poultry Diseases/prevention & control , Random Allocation , Salmonella Infections, Animal/microbiology , Salmonella Infections, Animal/prevention & control , Salmonella typhimurium/isolation & purification , Salmonella typhimurium/metabolismABSTRACT
Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC50 = 252.23 µg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata.
Subject(s)
Digestive System/drug effects , Drugs, Chinese Herbal/pharmacology , Epithelial Cells/drug effects , Insecticides/pharmacology , Larva/drug effects , Microvilli/drug effects , Moths/drug effects , Animals , Digestive System/ultrastructure , Epithelial Cells/ultrastructure , Microscopy, Electron, Transmission , Microvilli/ultrastructure , Moths/growth & developmentABSTRACT
This study investigated the effect of Lactobacillus acidophilus-fortified diets on growth performance, antioxidant profiles, and immunity-related gene expressions of common carp, Cyprinus carpio juveniles. Four isonitrogenous diets containing 0, 102, 104, or 106 cfu/mL/kg L. acidophilus were fed to 240 Cyprinus carpio juveniles (mean = 21.34 ± 1.85 g), allotted to 12 rectangular tanks in a completely randomized designed at 3% body weight for 56 days. Growth performance and nutrient utilization were evaluated using standard procedures. Intestinal villi were measured, antioxidant profiles were evaluated from blood sera, and immunity-related gene expressions were evaluated. The results revealed that fish fed dietary 106 had significantly higher weight gain, SGR, feed intake, and lower FCR. Also, villi length, width, and areas of absorption were significantly improved in a dose-dependent manner. Furthermore, innate immune profiles, superoxide dismutase, catalase, respiratory bust activity as well as transforming growth factor beta (TGFß), interleukin 8 (IL-8), and tumor necrosis factor alpha (TNF-alpha) were significantly stimulated. This study evoked that Lactobacillus acidophilus supplementation enhanced better growth performance, improved antioxidant profiles, and modulated expression of immune-related genes of common carp, Cyprinus carpio, than the fish fed control diet. Results show that fish fed fortified diets had better growth performance, improved antioxidant profiles, and modulated expression of immune-related genes.
Subject(s)
Carps/physiology , Lactobacillus acidophilus/physiology , Analysis of Variance , Animals , Antioxidants/metabolism , Carps/growth & development , Carps/immunology , Carps/metabolism , Catalase/metabolism , Dietary Supplements , Eating , Food, Fortified , Gene Expression/immunology , Hydrogen Peroxide/analysis , Hydrogen-Ion Concentration , Intestines/anatomy & histology , Microvilli/ultrastructure , Oxygen/analysis , Random Allocation , Respiratory Burst , Temperature , Water/standards , Weight GainABSTRACT
In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.