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1.
A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24–96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro.  相似文献   

2.
Several experiments have shown that mechanical forces significantly influence the initiation, growth, and retraction of neurites of cultured neurons. A similar role has long been suggested for mechanical forces in vivo, but this hypothesis has remained unverified due to the paucity of in vivo studies of neuronal mechanical behavior. In this study, we used high-resolution micromechanical force sensors to study the mechanical response of motor neurons in live Drosophila embryos. Our experiments showed that Drosophila neurons maintained a rest tension (1-13 nN) and behaved like viscoelastic solids (i.e., with a linear force-deformation response followed by force relaxation to steady state) in response to sustained stretching. More importantly, when the tension was suddenly diminished by a release of the externally applied force, the neurons contracted and actively generated force to restore tension, sometimes to a value close to their rest tension. In addition, axons that were slackened by displacing the neuromuscular junction contracted and became taut in 10-30 min. These observations are remarkably similar to results from in vitro studies and suggest that mechanical tension may also strongly influence neuronal behavior in vivo.  相似文献   

3.
Oxygen tension can exert a significant effect on viral propagation in vitro and possibly in vivo. In general, hypoxia restricts the replication of viruses that naturally infect tissues exposed to ambient oxygen and induces the growth of viruses that naturally target tissues exposed to low oxygen. Some viruses can reprogram cell bioenergetics towards lowering cellular respiration and therefore oxygen consumption in order to support their replication. Aim of this review is to summarize findings on the interplay between viral infection and oxygen levels, highlighting the implicated oxygen tension-sensitive elements and metabolic determinants and concluding with possible therapeutic approaches targeting these mediators.  相似文献   

4.
Aggregation of α‐synuclein is a hallmark of Parkinson's disease and dementia with Lewy bodies. We here investigate the relationship between cytosolic Ca2+ and α‐synuclein aggregation. Analyses of cell lines and primary culture models of α‐synuclein cytopathology reveal an early phase with reduced cytosolic Ca2+ levels followed by a later Ca2+ increase. Aggregated but not monomeric α‐synuclein binds to and activates SERCA in vitro, and proximity ligation assays confirm this interaction in cells. The SERCA inhibitor cyclopiazonic acid (CPA) normalises both the initial reduction and the later increase in cytosolic Ca2+. CPA protects the cells against α‐synuclein‐aggregate stress and improves viability in cell models and in Caenorhabditis elegans in vivo. Proximity ligation assays also reveal an increased interaction between α‐synuclein aggregates and SERCA in human brains affected by dementia with Lewy bodies. We conclude that α‐synuclein aggregates bind SERCA and stimulate its activity. Reducing SERCA activity is neuroprotective, indicating that SERCA and down‐stream processes may be therapeutic targets for treating α‐synucleinopathies.  相似文献   

5.
Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1nu). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.  相似文献   

6.
Mathematical models are extensively employed to understand physicochemical processes in biological systems. In the absence of detailed mechanistic knowledge, models are often based on network inference methods, which in turn rely upon perturbations to nodes by biochemical means. We have discovered a potential pitfall of the approach underpinning such methods when applied to signaling networks. We first show experimentally, and then explain mathematically, how even in the simplest signaling systems, perturbation methods may lead to paradoxical conclusions: for any given pair of two components X and Y, and depending upon the specific intervention on Y, either an activation or a repression of X could be inferred. This effect is of a different nature from incomplete network identification due to underdetermined data and is a phenomenon intrinsic to perturbations. Our experiments are performed in an in vitro minimal system, thus isolating the effect and showing that it cannot be explained by feedbacks due to unknown intermediates. Moreover, our in vitro system utilizes proteins from a pathway in mammalian (and other eukaryotic) cells that play a central role in proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis. This pathway is the perturbation target of contemporary therapies for various types of cancers. The results presented here show that the simplistic view of intracellular signaling networks being made up of activation and repression links is seriously misleading, and call for a fundamental rethinking of signaling network analysis and inference methods.  相似文献   

7.
《Biophysical journal》2020,118(7):1709-1720
Biological tissues contain micrometer-scale gaps and pores, including those found within extracellular matrix fiber networks, between tightly packed cells, and between blood vessels or nerve bundles and their associated basement membranes. These spaces restrict cell motion to a single-spatial dimension (1D), a feature that is not captured in traditional in vitro cell migration assays performed on flat, unconfined two-dimensional (2D) substrates. Mechanical confinement can variably influence cell migration behaviors, and it is presently unclear whether the mechanisms used for migration in 2D unconfined environments are relevant in 1D confined environments. Here, we assessed whether a cell migration simulator and associated parameters previously measured for cells on 2D unconfined compliant hydrogels could predict 1D confined cell migration in microfluidic channels. We manufactured microfluidic devices with narrow channels (60-μm2 rectangular cross-sectional area) and tracked human glioma cells that spontaneously migrated within channels. Cell velocities (vexp = 0.51 ± 0.02 μm min−1) were comparable to brain tumor expansion rates measured in the clinic. Using motor-clutch model parameters estimated from cells on unconfined 2D planar hydrogel substrates, simulations predicted similar migration velocities (vsim = 0.37 ± 0.04 μm min−1) and also predicted the effects of drugs targeting the motor-clutch system or cytoskeletal assembly. These results are consistent with glioma cells utilizing a motor-clutch system to migrate in confined environments.  相似文献   

8.
Bacteria are microscopic, single-celled organisms known for their ability to adapt to their environment. In response to stressful environmental conditions or in the presence of a contact surface, they commonly form multicellular aggregates called biofilms. Biofilms form on various abiotic or biotic surfaces through a dynamic stepwise process involving adhesion, growth, and extracellular matrix production. Biofilms develop on tissues as well as on implanted devices during infections, providing the bacteria with a mechanism for survival under harsh conditions including targeting by the immune system and antimicrobial therapy. Like pathogenic bacteria, members of the human microbiota can form biofilms. Biofilms formed by enteric bacteria contribute to several human diseases including autoimmune diseases and cancer. However, until recently the interactions of immune cells with biofilms had been mostly uncharacterized. Here, we will discuss how components of the enteric biofilm produced in vivo, specifically amyloid curli and extracellular DNA, could be interacting with the host's immune system causing an unpredicted immune response.  相似文献   

9.
Flash-frozen myocardium samples provide a valuable means of correlating clinical cardiomyopathies with abnormalities in sarcomeric contractile and biochemical parameters. We examined flash-frozen left-ventricle human cardiomyocyte bundles from healthy donors to determine control parameters for isometric tension (Po) development and Ca2+ sensitivity, while simultaneously measuring actomyosin ATPase activity in situ by a fluorimetric technique. Po was 17 kN m−2 and pCa50% was 5.99 (28°C, I = 130 mM). ATPase activity increased linearly with tension to 132 μM s−1. To determine the influence of flash-freezing, we compared the same parameters in both glycerinated and flash-frozen porcine left-ventricle trabeculae. Po in glycerinated porcine myocardium was 25 kN m−2, and maximum ATPase activity was 183 μM s−1. In flash-frozen porcine myocardium, Po was 16 kN m−2 and maximum ATPase activity was 207 μM s−1. pCa50% was 5.77 in the glycerinated and 5.83 in the flash-frozen sample. Both passive and active stiffness of flash-frozen porcine myocardium were lower than for glycerinated tissue and similar to the human samples. Although lower stiffness and isometric tension development may indicate flash-freezing impairment of axial force transmission, we cannot exclude variability between samples as the cause. ATPase activity and pCa50% were unaffected by flash-freezing. The lower ATPase activity measured in human tissue suggests a slower actomyosin turnover by the contractile proteins.  相似文献   

10.
Migration-Directing Liquid Properties of Embryonic Amphibian Tissues   总被引:1,自引:0,他引:1  
Deep ectoderm, mesoderm and endoderm excised from gastrulatingamphibian embryos spontaneously undergo liquid-like movementsin organ culture. Cell populations of these tissues on nonadhesivesubstrata will round up into spheres, spread over one anotherand segregate (sort out) from one another just as immiscibleliquid droplets do. In ordinary liquids, movements like theseare controlled by surface tensions; perhaps surface tensionsalso control the similar movements of liquid-like tissues. Onenecessary condition for tissue surface tension analysis is thatthe tissue must be able (just as ordinary liquids are able)to spontaneously relax internal stretching forces (shear stresses).When cellular aggregates of the germ layers were deformed bygentle compression between parallel glass plates, cells withinthe aggregates were initially stretched. However, the cellssoon returned to their original undistorted shapes. Thus, cellstretching forces were gradually relaxed by cell rearrangements.The in vitro spreading movements of the deep germ layers implythat the surface tension of ectoderm should be greater thanthe surface tension of mesoderm which should be greater thanthe surface tension of endoderm. Quantitative measurements oftissue surface tensions made by parallel plate compression confirmprecisely that relationship. Furthermore, the surface tensionsof these tissues remain constant regardless of the amount ofaggregate flattening—another necessary condition for validsurface tension measurements. These results demonstrate thatamphibian deep germ layers possess fundamental liquid propertieswhich are sufficient to direct their liquid-like rearrangementsin organ culture. Furthermore, I also report that one of theseproperties, surface tension, displays a preliminary correlationwith density of cell surface charge (assessed by electrophoreticmobility) and with the onset of in vivo mesodermal involution.  相似文献   

11.
Proton magnetic resonance spectroscopy (1H-MRS) is largely exploited in clinical settings to non-invasively investigate chemical compounds in human tissues. Applications of 1H-MRS in oncology field are connected to the detection of abnormal levels of choline compounds in more active tumours, providing useful information for cancer diagnosis and treatment monitoring. Since benign lesions may also show presence of a choline peak, implementing absolute evaluation will help differentiating benign from malignant tumours. An external reference procedure was described to provide choline quantification in standard unit of measurements. Spectra were acquired on a 1.5 T scanner using both phantoms and healthy volunteers with a PRESS sequence. The implemented quantification procedure used metabolite and noise measurements on the spectrum to remove large part of scanner settings contributing to metabolites of interest. A standard quantification was also used to compare performances of the noise-based method. In vitro quantification had accuracy and precision in the range (95–99)% and (5–13)%, respectively. When applied to in vivo studies on healthy volunteers, the method provided very close values of choline concentration, more exactly (1.73 ± 0.24) mmol/l. The method proposed can quantify the proper choline content in phantoms as well as in human structures, as brain. The method is ease of use, computational costless and it can be rapidly calibrated and implemented in any centre.  相似文献   

12.
Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.Download video file.(79M, mov)  相似文献   

13.
Adhesion of leukocytes and platelets to solid substrates of different surface tensions and hence different wettability is studied from a thermodynamic point of view. A simple thermodynamic model predicts that cellular adhesion should increase with increasing surface tension of the solid substrate if the surface tension of the medium in which the cells are suspended is lower than the surface tension of the cells. If the surface tension of the suspending medium is higher than that of the cells, the opposite behavior is predicted. These predictions are borne out completely by neutrophil adhesion tests, where the surface tension of the aequeous suspending medium is varied by addition of dimethyl sulfoxide (DMSO). Platelet adhesion experiments also confirm these predictions, the only difference being that surface tensions of the suspending medium above that of the platelets cannot be realized, owing to exudation of surface active solutes from the platelets. Utilization of the thermodynamic prediction that cellular adhesion should become independent of the surface tension of the substrate when the surface tensions of the cells and that of the suspending medium are equal leads to a value of the surface tension of neutrophils of 69.0 erg/cm2,† in excellent agreement with the value obtained from contact angles measured on layers of cells.  相似文献   

14.
The effect of carrageenan on the immune response of white shrimp Litopenaeus vannamei, was studied in vitro and in vivo. Shrimp haemocytes receiving carrageenan at 1 mg ml−1 experienced change in cell size, reduction in cell viability, increase in PO activity, serine proteinase activity, and RB in vitro. Shrimp received carrageenan via immersion at 200, 400 and 600 mg L−1 after 3 h and orally at 0.5, 1.0 and 2.0 g kg−1 after 3 weeks showed higher proliferation of haematopoietic tissues (HPTs) together with increases in haemocyte count and other immune parameters. Shrimp that fed a diet containing carrageenan at 0.5 g kg−1 after 3 weeks significantly up-regulated gene expressions of several immune-related proteins. The immune parameters of shrimp that received carrageenan via immersion and orally increased to a plateau after 3 h and after 3 weeks, but decreased after 5 h and 6 weeks, respectively. Phagocytosis and clearance of Vibrio alginolyticus remained high in shrimp that had received carrageenan via immersion after 5 h and orally after 6 weeks, respectively. Resistances of shrimp against V. alginolyticus and white spot syndrome virus were higher over 24–144 h and 72–144 h, respectively in shrimp that received carrageenan at 600 mg L−1 via immersion after 3 and 5 h. It was concluded that carrageenan effectively triggers an innate immunity in vitro, and increases mitotic index of HPT, immune parameters, gene expressions and resistance against pathogens in vivo. Shrimp received carrageenan via immersion and orally exhibited immunocompetence in phagocytosis and clearance of V. alginolyticus, and resistance to pathogen despite the trend in immune parameters to recover to background values.  相似文献   

15.
Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.  相似文献   

16.
As a major component of the extracellular matrix, hyaluronan (HA) plays an important role in defining the biochemical and biophysical properties of tissues. In light of the extremely rapid turnover of HA and the impact of this turnover on HA biology, elucidating the molecular mechanisms underlying HA catabolism is key to understanding the in vivo functions of this unique polysaccharide. Here, we show that TMEM2, a recently identified cell surface hyaluronidase, plays an essential role in systemic HA turnover. Employing induced global Tmem2 knockout mice (Tmem2iKO), we determined the effects of Tmem2 ablation not only on the accumulation of HA in bodily fluids and organs, but also on the process of HA degradation in vivo. Within 3 weeks of tamoxifen-induced Tmem2 ablation, Tmem2iKO mice exhibit pronounced accumulation of HA in circulating blood and various organs, reaching levels as high as 40-fold above levels observed in control mice. Experiments using lymphatic and vascular injection of fluorescent HA tracers demonstrate that ongoing HA degradation in the lymphatic system and the liver is significantly impaired in Tmem2iKO mice. We also show that Tmem2 is strongly expressed in endothelial cells in the subcapsular sinus of lymph nodes and in the liver sinusoid, two primary sites implicated in systemic HA turnover. Our results establish TMEM2 as a physiologically relevant hyaluronidase with an essential role in systemic HA catabolism in vivo, acting primarily on the surface of endothelial cells in the lymph nodes and liver.  相似文献   

17.
Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress‐induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62‐NRF2 axis. We show that TRIM16 is an integral part of the p62‐KEAP1‐NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress‐induced aggregate clearance and protects cells against oxidative/proteotoxic stress‐induced toxicity in vitro and in vivo. Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation‐associated diseases such as neurodegeneration.  相似文献   

18.
The histamine sensitization test (HIST) is a lethal test for batch release of acellular pertussis or its combination vaccines (ACV). Large numbers of animals have been used and it is difficult to standardize. Therefore there is an urgent need to develop an in vitro alternative to HIST.An in vitro test system has been developed as a potential alternative to HIST, to examine both the functional domains of PT based on a combination of enzyme coupled-HPLC (E-HPLC) and carbohydrate binding assays. We describe here an international collaborative study, which involved sixteen laboratories from 9 countries to assess the methodology transferability of the in vitro test system and its suitability for the testing of three different types of ACV products that are currently used worldwide. This study also evaluated further the relationship between the in vivo activity by HIST and the in vitro assay system.The results showed that the methodology of the E-HPLC and carbohydrate binding assays are transferable between laboratories worldwide and is suitable for the three types of ACV products included in the study. Although direct correlation between the in vitro assay system and the in vivo HIST (temperature reduction assay) for each individual vaccine lot cannot be established due to the large variation in the HIST results, the observation that the mean estimates of the in vitro and in vivo activities gave the same rank order of the three vaccine types included in the study is encouraging. The in vitro systems provide reproducible product specific profiles which supports their use as a potential alternative to the HIST.  相似文献   

19.
Wettability of the leaf surface, surface tension of the liquid, and stomatal morphology control penetration of stomata by liquids. The critical surface tension of the lower leaf surface of Zebrina purpusii Brückn. was estimated to be 25 to 30 dyne cm−1. Liquids having a surface tension less than 30 dyne cm−1 gave zero contact angle on the leaf surface and infiltrated stomata spontaneously while liquids having a surface tension greater than 30 dyne cm−1 did not wet the leaf surface and failed to infiltrate stomata. Considering stomata as conical capillaries, we were able to show that with liquids giving a finite contact angle, infiltration depended solely on the relationship between the magnitude of the contact angle and the wall angle of the aperture. Generally, spontaneous infiltration of stomata will take place when the contact angle is smaller than the wall angle of the aperture wall. The degree of stomatal opening (4, 6, 8, or 10 μm) was of little importance. Cuticular ledges present at the entrance to the outer vestibule and between the inner vestibule and substomatal chamber resulted in very small if not zero wall angles, and thus played a major role in excluding water from the intercellular space of leaves. We show why the degree of stomatal opening cannot be assessed by observing spontaneous infiltration of stomata by organic liquids of low surface tension.  相似文献   

20.
Mycobacterium tuberculosis is one of the most successful pathogens known, having infected more than a third of the global population. An important strategy for intracellular survival of pathogenic mycobacteria relies on their capacity to resist delivery to lysosomes, instead surviving within macrophage phagosomes. Several factors of both mycobacterial and host origin have been implicated in this process. However, whether or not this strategy is employed in vivo is not clear. Here we show that in vivo, following intravenous infection, M. tuberculosis and Mycobacterium bovis BCG initially survived by resisting lysosomal transfer. However, after prolonged infection the bacteria were transferred to lysosomes yet continued to proliferate. A M. bovis BCG mutant lacking protein kinase G (PknG), that cannot avoid lysosomal transfer and is readily cleared in vitro, was found to survive and proliferate in vivo. The ability to survive and proliferate in lysosomal organelles in vivo was found to be due to an altered host environment rather than changes in the inherent ability of the bacteria to arrest phagosome maturation. Thus, within an infected host, both M. tuberculosis and M. bovis BCG adapts to infection-specific host responses. These results are important to understand the pathology of tuberculosis and may have implications for the development of effective strategies to combat tuberculosis.  相似文献   

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