Microplastics (MPs) are present throughout aquatic ecosystems, and can be ingested by a wide variety of organisms. At present, the physical and chemical effects of environmental MPs on aquatic ...organisms are poorly documented. This study aims to examine the physiological and behavioral effects caused by fish consuming environmental microplastics at different life stages. MP samples were collected from beaches on three islands (Easter Island, Guam and Hawaii) located near the North and South gyres of the Pacific Ocean. Larvae and juveniles of Japanese Medaka were fed for 30days with three doses of MPs (0.01, 0.1 and 1% w/w in fish food) approximate to the concentrations measured in moderately and heavily contaminated ocean areas. Ingestion of MPs by medaka larvae caused (variously) death, decreased head/body ratios, increased EROD activity and DNA breaks and, alterations to swimming behavior. A diet of 0.1% MPs was the most toxic. Two-month-old juveniles fed with 0.01% MPs did not exhibit any symptoms except an increase in DNA breaks. Our results demonstrate ingestion and mainly sublethal effects of environmental MPs in early life stages of fish at realistic MP concentrations. The toxicity of microplastics varies from one sample to another, depending on polymer composition, weathering and pollutant content. This study examines the ecological consequences microplastic build-up in aquatic ecosystems, more particularly in coastal marine areas, which serve as breeding and growing grounds for a number of aquatic species.
•Medaka larvae and juveniles were fed with food spiked with environmental microplastics.•Fish at both stages easily ingest microplastics.•Environmental microplastic exposure induces sublethal effects on larval growth and behavior.•Microplastic toxicity varies widely from one sample to another.
During the different steps of bread-making, changes in the microstructure of the dough, particularly in the gas cell walls (GCW), have a major influence on the final bread crumb texture. ...Investigation of the spatial conformation of GCWs is still a challenge because it requires both high resolutions and 3D depth imaging. The originality of the present work lies in the use of label-free non-destructive multiphoton microscopy (NLOM) to image the 3D structure of GCWs, shedding light on their behavior and organization in wheat bread dough. We demonstrated that second and third harmonic generation (SHG, THG) allow imaging, respectively, of starch granules and interfaces in bread dough, while the gluten matrix was detected via two-photon excitation fluorescence (TPEF). Last, a distinction between the gluten network and starch granules was achieved using gluten endogenous fluorescence (EF) imaging, while the position, size, and 3D orientation of starch granules in GCWs were determined from harmonic imaging, made possible by the acquisition of backward and forward SHG with linear polarization. These innovative experiments highlight the strengths of NLOM for a label-free characterization of bread dough microstructure for the first time, in order to understand the role of starch granules in dough stabilization.
Therapeutic gene delivery to the whole spinal cord is a major challenge for the treatment of motor neuron (MN) diseases. Systemic administration of viral gene vectors would provide an optimal means ...for the long-term delivery of therapeutic molecules from blood to the spinal cord but this approach is hindered by the presence of the blood–brain barrier (BBB). Here, we describe the first successful study of MN transduction in adult animals following intravenous (i.v.) delivery of self-complementary (sc) AAV9 vectors (up to 28% in mice). Intravenous MN transduction was achieved in adults without pharmacological disruption of the BBB and transgene expression lasted at least 5 months. Importantly, this finding was successfully translated to large animals, with the demonstration of an efficient systemic scAAV9 gene delivery to the neonate and adult cat spinal cord. This new and noninvasive procedure raises the hope of whole spinal cord correction of MN diseases and may lead to the development of new gene therapy protocols in patients.
In order to assess the therapeutic potential of cell-based strategies, it is of paramount importance to elaborate and validate tools for monitoring the behavior of injected cells in terms of tissue ...dissemination and engraftment properties. Here, we apply bismuth ferrite harmonic nanoparticles (BFO HNPs) to in vitro expanded human skeletal muscle-derived stem cells (hMuStem cells), an attractive therapeutic avenue for patients suffering from Duchenne muscular dystrophy (DMD). We demonstrate the possibility of stem cell labeling with HNPs. We also show that the simultaneous acquisition of second- and third-harmonic generation (SHG and THG) from BFO HNPs helps separate their response from tissue background, with a net increase in imaging selectivity, which could be particularly important in pathologic context that is defined by a highly remodelling tissue. We demonstrate the possibility of identifying <100 nm HNPs in depth of muscle tissue at more than 1 mm from the surface, taking full advantage of the extended imaging penetration depth allowed by multiphoton microscopy in the second near-infrared window (NIR-II). Based on this successful assessment, we monitor over 14 days any modification on proliferation and morphology features of hMuStem cells upon exposure to PEG-coated BFO HNPs at different concentrations, revealing their high biocompatibility. Successively, we succeed in detecting individual HNP-labeled hMuStem cells in skeletal muscle tissue after their intramuscular injection.
Abstract
Methods to test the safety of wood material for hygienically sensitive places are indirect, destructive and limited to incomplete microbial recovery via swabbing, brushing and elution-based ...techniques. Therefore, we chose mCherry
Staphylococcus aureus
as a model bacterium for solid and porous surface contamination. Confocal spectral laser microscope (CSLM) was employed to characterize and use the autofluorescence of Sessile oak (
Quercus petraea
), Douglas fir (
Pseudotsuga menziesii
) and poplar (
Populus euramericana alba
L.) wood discs cut into transversal (RT) and tangential (LT) planes. The red fluorescent area occupied by bacteria was differentiated from that of wood, which represented the bacterial quantification, survival and bio-distribution on surfaces from one hour to one week after inoculation. More bacteria were present near the surface on LT face wood as compared to RT and they persisted throughout the study period. Furthermore, this innovative methodology identified that
S. aureus
formed a dense biofilm on melamine but not on oak wood in similar inoculation and growth conditions. Conclusively, the endogenous fluorescence of materials and the model bacterium permitted direct quantification of surface contamination by using CSLM and it is a promising tool for hygienic safety evaluation.
In addition to important regulatory roles in gene expression through RNA interference, it has recently been shown that microRNAs display immune stimulatory effects through direct interaction with ...receptors of innate immunity of the Toll-like receptor family, aggravating neuronal damage and tumour growth. Yet no evidence exists on consequences of microRNA immune stimulatory actions in the context of an autoimmune disease. Using microRNA analogues, we here show that pancreatic beta cell-derived microRNA sequences induce pro-inflammatory (TNFa, IFNa, IL-12, IL-6) or suppressive (IL-10) cytokine secretion by primary mouse dendritic cells in a sequence-dependent manner. For miR-29b, immune stimulation in RAW264.7 macrophages involved the endosomal Toll-like receptor-7, independently of the canonical RNA interference pathway. In vivo, the systemic delivery of miR-29b activates CD11b+B220- myeloid and CD11b-B220+ plasmacytoid dendritic cells and induces IFNa, TNFa and IL-6 production in the serum of recipient mice. Strikingly, in a murine model of adoptive transfer of autoimmune diabetes, miR-29b reduces the cytolytic activity of transferred effector CD8+ T-cells, insulitis and disease incidence in a single standalone intervention. Endogenous miR-29b, spontaneously released from beta-cells within exosomes, stimulates TNFa secretion from spleen cells isolated from diabetes-prone NOD mice in vitro. Hence, microRNA sequences modulate innate and ongoing adaptive immune responses raising the question of their potential role in the breakdown of tolerance and opening up new applications for microRNA-based immune therapy.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this ...remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.
Beta cell failure and apoptosis following islet inflammation have been associated with autoimmune type 1 diabetes pathogenesis. As conveyors of biological active material, extracellular vesicles (EV) ...act as mediators in communication with immune effectors fostering the idea that EV from inflamed beta cells may contribute to autoimmunity. Evidence accumulates that beta exosomes promote diabetogenic responses, but relative contributions of larger vesicles as well as variations in the composition of the beta cell's vesiculome due to environmental changes have not been explored yet. Here, we made side-by-side comparisons of the phenotype and function of apoptotic bodies (AB), microvesicles (MV) and small EV (sEV) isolated from an equal amount of MIN6 beta cells exposed to inflammatory, hypoxic or genotoxic stressors. Under normal conditions, large vesicles represent 93% of the volume, but only 2% of the number of the vesicles. Our data reveal a consistently higher release of AB and sEV and to a lesser extent of MV, exclusively under inflammatory conditions commensurate with a 4-fold increase in the total volume of the vesiculome and enhanced export of immune-stimulatory material including the autoantigen insulin, microRNA, and cytokines. Whilst inflammation does not change the concentration of insulin inside the EV, specific Toll-like receptor-binding microRNA sequences preferentially partition into sEV. Exposure to inflammatory stress engenders drastic increases in the expression of monocyte chemoattractant protein 1 in all EV and of interleukin-27 solely in AB suggesting selective sorting toward EV subspecies. Functional
assays in mouse dendritic cells and macrophages reveal further differences in the aptitude of EV to modulate expression of cytokines and maturation markers. These findings highlight the different quantitative and qualitative imprints of environmental changes in subpopulations of beta EV that may contribute to the spread of inflammation and sustained immune cell recruitment at the inception of the (auto-) immune response.
Despite widespread usage of β-adrenergic receptor (AR) agonists and antagonists in current clinical practice, our understanding of their interactions with the immune system is surprisingly sparse. ...Among the AR expressed by dendritic cells (DC), β2-AR can modify in vitro cytokine release upon stimulation. Because DC play a pivotal role in CD8(+) T cell immune responses, we examined the effects of β2-AR stimulation on MHC class I exogenous peptide presentation and cross-presentation capacities. We demonstrate that β2-AR agonist-exposed mature DC display a reduced ability to cross-present protein Ags while retaining their exogenous peptide presentation capability. This effect is mediated through the nonclassical inhibitory G (Gαi/0) protein. Moreover, inhibition of cross-presentation is neither due to reduced costimulatory molecule expression nor Ag uptake, but rather to impaired phagosomal Ag degradation. We observed a crosstalk between the TLR4 and β2-AR transduction pathways at the NF-κB level. In vivo, β2-AR agonist treatment of mice inhibits Ag protein cross-presentation to CD8(+) T cells but preserves their exogenous MHC class I peptide presentation capability. These findings may explain some side effects on the immune system associated with stress or β-agonist treatment and pave the way for the development of new immunomodulatory strategies.
An innovative methodology based on non-destructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix and ...topography analysis of food products. Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Fluorescence of starch and oil respectively stained with Safranin O and Nile red was observed from non-linear microscopy. By using sequential scanning and specific emission filters, it was possible to merge fluorescence and harmonic generation signals. Second harmonic generation (SHG) generated by starch granules was superposed with safranin fluorescence, whereas third harmonic generation (THG), not restricted to the superposition with Nile red fluorescent signal, was used to investigate the topography of the fried product. By these experiments, starch granule mapping and topography of the starchy fried product were obtained without any destructive preparation of the sample. This label-free approach using harmonic generation microscopy is a very promising methodology for microstructure investigation of a large panel of starchy food products.
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