Intracellular Particles Research Articles

Mortality Associated with Bushfire Smoke Inhalation in a Captive Population of the Smoky Mouse (Pseudomys fumeus), a Threatened Australian Rodent.

A mortality event of nine threatened smoky mice (Pseudomys fumeus) occurred in January 2020 at a captive breeding facility in southeastern Australia that was affected at the time by hazardous levels of bushfire smoke, despite being more than 20 km from the nearest fire. Pathologic and clinical observations indicated smoke inhalation was the cause of death. All animals had significant pulmonary lesions, notably pulmonary edema and congestion, and moderate amounts of dark brown to black pigmented intracellular and extracellular particles from <0.5-2.5 µm in diameter were observed in the central or hilar region of the lungs of four of six animals examined histologically. Deaths occurred between three and 30 d after exposure to smoke and, for seven animals in outdoor acclimatization enclosures, were associated with very high ambient temperature (>40 C). Similar mortalities did not occur in co-located parrots, suggesting differing species sensitivity to smoke inhalation. Our findings highlight the potential for smoke to be an underdiagnosed cause of mortality in free-ranging wildlife during bushfires and for bushfires to affect wildlife populations outside of burnt areas, including in unburnt refugia. Conservation interventions for wildlife after bushfires should consider and, where possible, mitigate the risk of animals dying due to increased respiratory demand following smoke inhalation injury.

Arthrospira platensis (Cyanobacteria) – a potential biofactory for fluoromagnetic nanoiron production

ABSTRACT Bioconversion of Fe3+ to Fe0 followed by spindle shaped magneto-fluorescent nanoiron production has been investigated using Arthrospira platensis as a potential bio-reagent. The characteristic features, e.g. in situ localisation, magnetic and fluorescent properties of synthesised nanoparticles, were explored. Bioconversion occurred when healthy growing biomass of Arthrospira platensis was exposed to 0.01 M aqueous solution of FeCl3 for 96 h at 25 °C with pH5.2. Spindle-shaped nanoiron was synthesised both inside and outside the cells. The average dry and hydrodynamic sizes for intracellular particles were 24.26 ± 3.40 nm × 5.45 ± 1.66 nm and 175.9 nm, respectively; whereas size variation for extracellular particles was 23.62 ± 3.49 nm × 8.41 ± 4.33 nm and 40.02 nm, respectively, as observed using transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis, respectively. Further characterisations were conducted using scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, energy dispersive X-Ray spectroscopy (EDX), and X-Ray diffraction analysis (XRD). Nonspherical nanoiron particles generated multiple absorption bands at approximately 230, 350, and 480 nm in the UV-Vis spectra. Fluorescence of nanoiron was detected using confocal microscopy both inside and outside cells employing a blue filter (λex–425 nm, λem–475 nm). The magneto-fluorescent nature of synthesised nanoiron was estimated using fluorimetric analysis and dynamic light scattering study using a 0.5-tesla static magnetic field (SMF) at 4 °C. The investigation showed that the static magnetic field induced non-invasive clustering effect of nanoiron directly influence fluorescence intensity and its lifetime. The presence of superparamagnetism was detected in iron nanoparticles using a superconducting quantum interference device-vibrating sample magnetometer (SQUID-VSM).

Effective Antiviral Activity of the Tyrosine Kinase Inhibitor Sunitinib Malate against Zika Virus

IntroductionZika virus (ZIKV), a mosquito-borne flavivirus, causes the outbreaks of Latin America in 2015 - 2016, with the incidence of neurological complications. Sunitinib malate, an orally bioavailable malate salt of the tyrosine kinase inhibitor, is suggested as a broad-spectrum antiviral agent against emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2.Materials and MethodsThis study investigated the antiviral efficacy and antiviral mechanisms of sunitinib malate against ZIKV infection using cytopathic effect reduction, virus yield, and time-of-addition assays.ResultsSunitinib malate concentration-dependently reduced ZIKV-induced cytopathic effect, the expression of viral proteins, and ZIKV yield in supernatant with 50% inhibitory concentration (IC50) value of 0.015 μM, and the selectivity index of greater than 100 against ZIKV infection, respectively. Sunitinib malate had multiple antiviral actions during entry and post-entry stages of ZIKV replication. Sunitinib malate treatment at entry stage significantly reduced the levels of ZIKV RNA replication with the reduction of (+) RNA to (-) RNA ratio and the production of new intracellular infectious particles in infected cells. The treatment at post-entry stage caused a concentration-dependent increase in the levels of ZIKV (+) RNA and (-) RNA in infected cells, along with enlarging the ratio of (+) RNA to (-) RNA, but caused a pointed increase in the titer of intracellular infectious particles by 0.01 and 0.1 μM, and a substantial decrease in the titer of intracellular infectious particles by 1 μM.ConclusionThe study discovered the antiviral actions of sunitinib malate against ZIKV infection, demonstrating a repurposed, host-targeted approach to identify potential antiviral drugs for treating emerging and global viral diseases.

Intracellular Quantification and Localization of Label-Free Iron Oxide Nanoparticles by Holotomographic Microscopy.

BackgroundThe limitations of optical microscopy to determine the cellular localization of label-free nanoparticles prevent a solid prediction of the cellular effect of particles intended for medical applications. To avoid the strong physicochemical changes associated with fluorescent labelling, which often result in differences in cellular uptake, efficiency and toxicity of particles, novel detection techniques are required.MethodsIn the present study, we determined the intracellular content of unlabeled SPIONs by analyzing refractive index (RI)-based images from holotomographic three-dimensional (3D) microscopy and side scatter data measured by flow cytometry. The results were compared with the actual cellular SPION amount as quantified by atomic emission spectroscopy (AES).ResultsLive cell imaging by 3D holotomographic microscopy demonstrated cell-specific differences in intracellular nanoparticle uptake in different pancreatic cell lines. Thus, treatment of PANC-1SMAD4 (1−4) and PANC-1SMAD4 (2−6) with SPIONs resulted in a significant increase in number of areas with higher RI, whereas in PANC-1, SUIT-2 and PaCa DD183, only a minimal increase of spots with high RI was observed. The increase in areas with high RI was in accordance with the SPION content determined by quantitative iron measurements using AES. In contrast, determination of the SPION amount by flow cytometry was strongly cell type-dependent and did not allow the discrimination between intracellular and membrane-bound SPIONs. However, flow cytometry is a very rapid and reliable method to assess the cellular toxicity and allows an estimation of the cell-associated SPION content.ConclusionHolotomographic 3D microscopy is a useful method to distinguish between intracellular and membrane-associated particles. Thus, it provides a valuable tool for scientists to evaluate the cellular localization and the particle load, which facilitates prediction of potential toxicity and efficiency of nanoparticles for medical applications.

Intracellular amorphous Ca-carbonate and magnetite biomineralization by a magnetotactic bacterium affiliated to the Alphaproteobacteria.

Bacteria synthesize a wide range of intracellular submicrometer-sized inorganic precipitates of diverse chemical compositions and structures, called biominerals. Their occurrences, functions and ultrastructures are not yet fully described despite great advances in our knowledge of microbial diversity. Here, we report bacteria inhabiting the sediments and water column of the permanently stratified ferruginous Lake Pavin, that have the peculiarity to biomineralize both intracellular magnetic particles and calcium carbonate granules. Based on an ultrastructural characterization using transmission electron microscopy (TEM) and synchrotron-based scanning transmission X-ray microscopy (STXM), we showed that the calcium carbonate granules are amorphous and contained within membrane-delimited vesicles. Single-cell sorting, correlative fluorescent in situ hybridization (FISH), scanning electron microscopy (SEM) and molecular typing of populations inhabiting sediments affiliated these bacteria to a new genus of the Alphaproteobacteria. The partially assembled genome sequence of a representative isolate revealed an atypical structure of the magnetosome gene cluster while geochemical analyses indicate that calcium carbonate production is an active process that costs energy to the cell to maintain an environment suitable for their formation. This discovery further expands the diversity of organisms capable of intracellular Ca-carbonate biomineralization. If the role of such biomineralization is still unclear, cell behaviour suggests that it may participate to cell motility in aquatic habitats as magnetite biomineralization does.

Deep-learning method for data association in particle tracking.

Biological studies of dynamic processes in living cells often require accurate particle tracking as a first step toward quantitative analysis. Although many particle tracking methods have been developed for this purpose, they are typically based on prior assumptions about the particle dynamics, and/or they involve careful tuning of various algorithm parameters by the user for each application. This may make existing methods difficult to apply by non-expert users and to a broader range of tracking problems. Recent advances in deep-learning techniques hold great promise in eliminating these disadvantages, as they can learn how to optimally track particles from example data. Here, we present a deep-learning-based method for the data association stage of particle tracking. The proposed method uses convolutional neural networks and long short-term memory networks to extract relevant dynamics features and predict the motion of a particle and the cost of linking detected particles from one time point to the next. Comprehensive evaluations on datasets from the particle tracking challenge demonstrate the competitiveness of the proposed deep-learning method compared to the state of the art. Additional tests on real-time-lapse fluorescence microscopy images of various types of intracellular particles show the method performs comparably with human experts. The software code implementing the proposed method as well as a description of how to obtain the test data used in the presented experiments will be available for non-commercial purposes from https://github.com/yoyohoho0221/pt_linking. Supplementary data are available at Bioinformatics online.

Nanoceria distribution and effects are mouse-strain dependent

Prior studies showed nanoparticle clearance was different in C57BL/6 versus BALB/c mice, strains prone to Th1 and Th2 immune responses, respectively. Objective: Assess nanoceria (cerium oxide, CeO2 nanoparticle) uptake time course and organ distribution, cellular and oxidative stress, and bioprocessing as a function of mouse strain. Methods: C57BL/6 and BALB/c female mice were i.p. injected with 10 mg/kg nanoceria or vehicle and terminated 0.5 to 24 h later. Organs were collected for cerium analysis; light and electron microscopy with elemental mapping; and protein carbonyl, IL-1β, and caspase-1 determination. Results: Peripheral organ cerium significantly increased, generally more in C57BL/6 mice. Caspase-1 was significantly elevated in the liver at 6 h, to a greater extent in BALB/c mice, suggesting inflammasome pathway activation. Light microscopy revealed greater liver vacuolation in C57BL/6 mice and a nanoceria-induced decrease in BALB/c but not C57BL/6 mice vacuolation. Nanoceria increased spleen lymphoid white pulp cell density in BALB/c but not C57BL/6 mice. Electron microscopy showed intracellular nanoceria particles bioprocessed to form crystalline cerium phosphate nanoneedles. Ferritin accumulation was greatly increased proximal to the nanoceria, forming core-shell-like structures in C57BL/6 but even distribution in BALB/c mice. Conclusions: BALB/c mice were more responsive to nanoceria-induced effects, e.g. liver caspase-1 activation, reduced liver vacuolation, and increased spleen cell density. Nanoceria uptake, initiation of bioprocessing, and crystalline cerium phosphate nanoneedle formation were rapid. Ferritin greatly increased with a macrophage phenotype-dependent distribution. Further study will be needed to understand the mechanisms underlying the observed differences.

Mechanistic investigation of cellular internalization routes of polymeric particles on breast cancer cells: relevance for drug delivery applications

The tuning of physicochemical properties like particle sizes, surface modifications, etc., is essential to improve delivery efficiency with insignificant toxicity. A quantitative and mechanistic investigation on cellular uptake and intracellular trafficking of fluorescent-labeled polystyrene particles [50 and 500 nm plain and 200 nm and 1 µm carboxylate-coated polystyrene (PPS and CPS)] against breast cancer cells was performed. Examinations of cell proliferation, viability, and cytotoxicity indicated that those applied polymeric particles at concentrations of 4–40 µg/mL are non-toxic to cells except 200 nm CPS (16 µg/mL) which partially reduced cell viability. Quantitative fluorescent microscopy revealed that 200 nm CPS displayed the highest capability to be internalized by MCF-7 cells, in which the average intracellular particles are about 14 and 35 folds higher than 1 µm CPS and 500 nm PPS, respectively. Analysis of the uptake routes using inhibitors provided clear evidence that 200 CPS was mainly ingested via the clathrin-mediated endocytosis, whereas larger particles (> 500 nm) preferentially utilized the macropinocytosis pathway. Fluorescent live-cell imaging suggested that particles were mainly internalized by cells in an individual form instead of large aggregates, despite late occurrence of partial agglomeration in the cytoplasm. Overall, those outcomes enhanced our understanding of the application of polymeric delivery vehicles for the treatment of breast cancer.

Fast field-cycling magnetic resonance detection of intracellular ultra-small iron oxide particles in vitro: Proof-of-concept

PurposeInflammation is central in disease pathophysiology and accurate methods for its detection and quantification are increasingly required to guide diagnosis and therapy. Here we explored the ability of Fast Field-Cycling Magnetic Resonance (FFC-MR) in quantifying the signal of ultra-small superparamagnetic iron oxide particles (USPIO) phagocytosed by J774 macrophage-like cells as a proof-of-principle. MethodsRelaxation rates were measured in suspensions of J774 macrophage-like cells loaded with USPIO (0–200 μg/ml Fe as ferumoxytol), using a 0.25 T FFC benchtop relaxometer and a human whole-body, in-house built 0.2 T FFC-MR prototype system with a custom test tube coil. Identical non-imaging, saturation recovery pulse sequence with 90° flip angle and 20 different evolution fields selected logarithmically between 80 μT and 0.2 T (3.4 kHz and 8.51 MHz proton Larmor frequency [PLF] respectively). Results were compared with imaging flow cytometry quantification of side scatter intensity and USPIO-occupied cell area. A reference colorimetric iron assay was used. ResultsThe T1 dispersion curves derived from FFC-MR were excellent in detecting USPIO at all concentrations examined (0–200 μg/ml Fe as ferumoxytol) vs. control cells, p ≤ 0.001. FFC-NMR was capable of reliably detecting cellular iron content as low as 1.12 ng/µg cell protein, validated using a colorimetric assay. FFC-MR was comparable to imaging flow cytometry quantification of side scatter intensity but superior to USPIO-occupied cell area, the latter being only sensitive at exposures ≥ 10 µg/ml USPIO. ConclusionsWe demonstrated for the first time that FFC-MR is capable of quantitative assessment of intra-cellular iron which will have important implications for the use of USPIO in a variety of biological applications, including the study of inflammation.

Electron Microscopy of In-Plaque Phage T3 Assembly: Proposed Analogs of Neurodegenerative Disease Triggers.

Increased knowledge of virus assembly-generated particles is needed for understanding both virus assembly and host responses to virus infection. Here, we use a phage T3 model and perform electron microscopy (EM) of thin sections (EM-TS) of gel-supported T3 plaques formed at 30 °C. After uranyl acetate/lead staining, we observe intracellular black particles, some with a difficult-to-see capsid. Some black particles (called LBPs) are larger than phage particles. The LBP frequency is increased by including proflavine, a DNA packaging inhibitor, in the growth medium and increasing plaque-forming temperature to 37 °C. Acidic phosphotungstate-precipitate (A-PTA) staining causes LBP substitution by black rings (BRs) that have the size and shape expected of hyper-expanded capsid containers for LBP DNA. BRs are less frequent in liquid cultures, suggesting that hyper-expanded capsids evolved primarily for in-gel (e.g., in-biofilm) propagation. BR-specific A-PTA staining and other observations are explained by α-sheet intense structure of the major subunit of hyper-expanded capsids. We hypothesize that herpes virus triggering of neurodegenerative disease occurs via in-gel propagation-promoted (1) generation of α-sheet intense viral capsids and, in response, (2) host production of α-sheet intense, capsid-interactive, innate immunity amyloid protein that becomes toxic. We propose developing viruses that are therapeutic via detoxifying interaction with this innate immunity protein.

Robust hepatitis E virus infection and transcriptional response in human hepatocytes

Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.

Visualizing intracellular particles and precise control of drug release using an emissive hydrazone photochrome.

The spatiotemporal control over the structure of nanoparticles while monitoring their localization in tumor cells can improve the precision of controlled drug release, thus enhancing the efficiency of drug delivery. Here, we report on a photochromic nanoparticle system (LSNP), assembled from fluorescent bistable hydrazone photoswitch-modified amphiphilic copolymers. The intrinsic emission of the hydrazone switch allows for the visualization of particle uptake, as well as their intracellular distribution. The Z → E photoswitching of the hydrazone switch within the nanoparticle leads to the expansion of the nanoparticles (i.e., drug release) accompanied by emission quenching, the degree of which can function as an internal indicator for the amount of drug released. The bistability of the switch enables the kinetic trapping of particles of different sizes as a function of irradiation time, and allows for the exhibition of light-dependent cell cytotoxicity in MDA-MB-231 cells using LSNP loaded with doxorubicin.

Rilpivirine-associated aggregation-induced emission enables cell-based nanoparticle tracking

Antiretroviral therapy (ART) has improved the quality and duration of life for people living with human immunodeficiency virus (HIV) infection. However, limitations in drug efficacy, emergence of viral mutations and the paucity of cell-tissue targeting remain. We posit that to maximize ART potency and therapeutic outcomes newer drug formulations that reach HIV cellular reservoirs need be created. In a step towards achieving this goal we harnessed the aggregation-induced emission (AIE) property of the non-nucleoside reverse transcriptase inhibitor rilpivirine (RPV) and used it as a platform for drug cell and subcellular tracking. RPV nanocrystals were created with endogenous AIE properties enabling the visualization of intracellular particles in cell and tissue-based assays. The intact drug crystals were easily detected in CD4+ T cells and macrophages, the natural viral target cells, by flow cytometry and ultraperformance liquid chromatography tandem mass spectrometry. We conclude that AIE can be harnessed to monitor cell biodistribution of selective antiretroviral drug nanocrystals.

Migration of Microparticle-Containing Amoeba through Constricted Environments

In many situations, cells migrate through tiny orifices. Examples include the extravasation of immune cells from the bloodstream for fighting infections, the infiltration of cancer cells during metastasis, and the migration of human pathogens. An extremely motile and medically relevant type of human pathogen is Acanthamoeba castellanii. In the study presented here, we investigated how a combination of microparticles and microstructured interfaces controls the migration of A. castellanii trophozoites. The microinterfaces comprised well-defined micropillar arrays, and the trophozoites easily migrated through the given constrictions by adapting the shape and size of their intracellular vacuoles and by adapting intracellular motion. After feeding the trophozoite cells in microinterfaces with synthetic, stiff microparticles of various sizes and shapes, their behavior changed drastically: if the particles were smaller than the micropillar gap, migration was still possible. If the cells incorporated particles larger than the pillar gap, they could become immobilized but could also display remarkable problem-solving capabilities. For example, they turned rod-shaped microparticles such that their short axis fit through the pillar gap or they transported the particles above the structure. As migration is a crucial contribution to A. castellanii pathogenicity and is also relevant to other biological processes in microenvironments, such as cancer metastasis, our results provide an interesting strategy for controlling the migration of cells containing intracellular particles by microstructured interfaces that serve as migration-limiting environments.

Expression of Cytoskeleton Actin in Cancer and Metastasis

Cytoskeleton in Eukaryotic cell is very important for cell to keep the exact shape and supports the plasma membrane and give the mechanical support to cell. Actin is a type of cytoskeleton that is involved in several functions. Actin filaments are made up of indistinguishable actin binding proteins which is set in a lengthy curved chain which performs important function of body. Expresion of some actin binding proteins are regulated during carcinogenesis and metastasis, these proteins include Arp2/3 (Actin related protein 2 and 3 complex), fascin and the tropomyosin’s, WASP/WAVE (Wiskott-Aldrich syndrome protein/WASP and Verprolin homologous protein) complexes. The expansion of cancer cells from the primary tumor to other tissues and the organs is called metastasis. The connection amongst the hyperactivated signaling pathways plays a very important role in the development of metastatic. GPCRs (G Protein Coupled Receptors) through β-arrs (β-arrestins) act as a intracellular signaling particles. In tumor cells the increased and decreased expression of these actin binding proteins are involved in progression of different types of cancer and metastasis.&#x0D; For further insight we have to understand what is actin and the function of actin binding proteins.

Genes A27L and F13L as Genetic Markers for the Isolation of Recombinant Vaccinia Virus

After assembly in the cytosol, some Vaccinia virus particles go through a complex process that leads to virus egress and eventually cell-to-cell transmission. Intracellular particles are fully infectious, and therefore virus mutants lacking essential functions in the exit pathway are unable to form plaques but can multiply intracellularly. We isolated virus mutants in which two of the genes required for virus spread (F13L and A27L) were deleted independently or concurrently. The phenotypes of the mutant viruses were consistent with the need of A27L and F13L for intercellular virus transmission, the effect of the ΔA27L mutation being more severe than that of ΔF13L. Despite their defect in spread, ΔA27L mutant viruses could be expanded by infecting cell cultures at high multiplicity of infection, followed by the release of virions from infected cells by physical means. We developed a novel system for the isolation of recombinant Vaccinia virus in which selection is efficiently achieved by recovering plaque formation capacity after re-introduction of A27L into a ΔA27L virus. This system allowed the insertion of foreign DNA into the viral genome without the use of additional genetic markers. Furthermore, starting with a double mutant (ΔA27L-ΔF13L) virus, A27L selection was used in conjunction with F13L selection to mediate simultaneous dual insertions in the viral genome. This selection system facilitates combined expression of multiple foreign proteins from a single recombinant virus.

Antiviral efficacy of bromo-anilino substituents of 4,5-dihydrofuran-3-carboxylate compound CW-33 against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, occasionally causes severe central nervous system disorders in the risk zone where more than 3 billion people reside. Our prior studies demonstrated antiviral potential of 4,5-dihydrofuran-3-carboxylate compound CW-33 (ethyl 2-(3',5'-dimethylanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) and its derivative CW-33A ((ethyl 2-(2-fluoroanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) against JEV infection ((Int. J. Mol. Sci. 2016, 17: E1386; Sci. Rep. 2018, 8: 16595). This study synthesized six new CW-33 derivatives containing chloro, or bromo groups at the C-2, C-3, or C-4 of anilino ring of CW-33, and assessed the antiviral activity and mechanisms of these chloro- and bromo-anilino substitutedderivatives. CW-33K, CW-33L and CW-33M had the bromo-substituents at the C-2, C-3, or C-4 of anilino ring of CW-33, respectively, showing the higher anti-JEV activity than CW-33 and other derivatives. CW-33K (ethyl 2-(2-bromoanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate) exhibited the highest antiviral efficacy and therapeutic index. The IC50 value of CW-33K was less than 5 μM for reducing JEV-induced cytopathic effect, virus infectivity and virus yield. CW-33K significantly inhibited the JEV replication at the early and late stages, suppressing viral RNA synthesis and intracellular JEV particle production. The study demonstrated that the CW-33 derivative with a bromosubstitutionat the C-2 anilino ring improved the antiviral activity JEV, providing the structure-antiviral activity relationship for the development of anti-JEV agents.

Magnetic Resonance Imaging Tracking of Induced Pluripotent Stem Cells-Induced Neural Stem Cells in Traumatic Brain Injury Rats

Abstract INTRODUCTION Induced pluripotent stem cells (iPS cells) are promising cell source for stem cell replacement strategy applied to brain injury caused by traumatic brain injury (TBI) or stroke. Neural stem cell (NSCs), derived from iPS cells could aid the reconstruction of brain tissue and the restoration of brain function. However, how to trace the fate of iPS cells in host brain is still a challenge. METHODS In our study, iPS cells were derived from skin fibroblasts using the 4 classic factors Oct4, Sox2, Myc, and Klf4. Then these iPS cells were induced to differentiate into NSCs, which were incubated with superparamagnetic iron oxides (SPIOs) in Vitro. Next, 30 TBI rat models were prepared and divided into 3 groups (n = 10). One week after brain injury, group A and B rats received NSCs (labeled with SPIOs) implantation, while group C rats received non-labeled NSCs implantation. After cell implantation, all the rats were performed T2*-weighted magnetic resonance imaging (MRI) scan at day 1, and 1 wk to 4 wk, to track NSCs distribution in rats’ brains. One month after cell implantation, all the rats were performed manganese-enhanced MRI (ME-MRI) scan. In group B, diltiazem was infused during the ME-MRI scan period. RESULTS (1) iPS cells were successfully derived from skin fibroblasts using the 4 classic factors Oct4, Sox2, Myc, and Klf4, expressing the typical antigens including SSEA4, Oct4, Sox2, and Nanog. (2) iPS cells were induced to differentiate into NSCs, which could express Nestin and differentiate into neural cells and glia cells. (3) NSCs were incubated with SPIOs overnight, and prussian blue staining showed intra-cellular particles. (4) After cell implantation, T2*-weighted MRI scan showed these implanted NSCs could migrate to the injury area in chronological order. (5) The subsequent ME-MRI scan detected NSCs function, which could be blocked by diltiazem. CONCLUSION In conclusion, using in Vivo MRI tracking technique to trace the fate of iPS cells-induced NSCs in host brain is feasible.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Wavelength-encoded laser particles for massively multiplexed cell tagging.

Large-scale single-cell analyses have become increasingly important given the role of cellular heterogeneity in complex biological systems. However, no current techniques enable optical imaging of uniquely-tagged individual cells. Fluorescence-based approaches can only distinguish a small number of distinct cells or cell groups at a time because of spectral crosstalk between conventional fluorophores. Here we investigate large-scale cell tracking using intracellular laser particles as imaging probes that emit coherent laser light with a characteristic wavelength. Made of silica-coated semiconductor microcavities, these laser particles have single-mode emission over a broad range from 1170 to 1580 nm with sub-nm linewidths, enabling massive spectral multiplexing. We explore the stability and biocompatibility of these probes in vitro and their utility for wavelength-multiplexed cell tagging and imaging. We demonstrate real-time tracking of thousands of individual cells in a 3D tumour model over several days showing different behavioural phenotypes.