Fluorescent Microbeads Research Articles

Bioturbation effects and behavioral changes in buried bivalves after exposure to microplastics

Microplastic pollution has become an increasing concern. Vertical transport of microplastics is one of the major research questions concerning the distribution and fate of microplastics in the marine environment, and biologically mediated vertical transport is particularly significant. However, studies on the effects of different types of benthic organisms on the vertical distribution of microplastics in sediments are still scarce. The results of this study revealed that when exposed to environmentally relevant concentrations of fluorescent polystyrene microbeads (200 μm), Manila clams (Ruditapes philippinarum) exhibited prolonged acclimation period, yet subsequent burrowing behavior (burrowing rate and burrowing velocity) was unaffected. The condition index, clearance rate, and oxygen consumption rate of the clams similarly exhibited no stress response after 14 days of exposure. We determined that microplastics were rapidly transported to deeper layers (6-8cm below the surface) in the sediment under bioturbation. This study elucidates the mechanisms of microplastic transport, showing that clam behaviors such as burrowing, movement, and ingestion contribute to this process. The results suggest that a biologically based management strategy may be a more environmentally friendly means of mitigating microplastic pollution in seawater.

Fe-capsaicin nanozyme attenuates sepsis-induced acute lung injury by regulating the functions of macrophages.

In sepsis, the lung is one of the worst affected organs, often leading to acute lung injury (ALI). More and more evidence suggests that macrophages are also involved in the pathogenesis of ALI. In our previous study, we successfully synthesized Iron-capsaicin-based nanoparticles (Fe-CAP NPs) and found that it could inhibit the secretion of inflammatory cytokines to alleviate ALI. Here, we further explore the anti-inflammatory mechanism of Fe-CAP NPs. Bone marrow-derived macrophages (BMDM) and C57 mice were divided into four groups: control group, lipopolysaccharide (LPS) group, CAP + LPS group and Fe-CAP + LPS group. Western blot and Immunofluorescence were used to detect the expression of macrophage phenotypic markers CD86 and CD206 in BMDM and lung tissue. Fluorescence microbeads, Transwell and ROS kit were used to detect the phagocytosis, migration and ROS clearing capability of BMDM. Western blot was used to detect the expression of JAK2/STAT3 pathway and apoptosis proteins in BMDM. TUNEL kit and H&E staining were used to evaluate apoptosis and pathological changes in lung tissue. In vitro, CD86 expression was increased in LPS, but decreased after Fe-CAP pretreatment. CD206 expression was the opposite. Fe-CAP reduced phagocytosis, migration and scavenged ROS in LPS-treated BMDM. Fe-CAP inhibited P-JAK2 and P-STAT3 expression and reduced apoptosis. In vivo, Fe-CAP improved lung histopathology and reduced apoptosis in lung tissue of LPS group. CD86 expression was increased in lung tissue of LPS group, but decreased in Fe-CAP preconditioning, and CD206 expression was reversed. Fe-CAP NPs could alleviate sepsis-induced ALI by regulating the polarization and function of macrophages, reducing ROS level and apoptosis.

Monolayer-fluorescence counting for ultrasensitive detection of tumour cell-derived extracellular vesicles using a step-wedge microfluidic platform

Extracellular vesicles have emerged as significant noninvasive tumour biomarkers, and ultrasensitive detection methods are important for early cancer diagnosis. Here, a monolayer-fluorescence counting strategy was developed for the ultrasensitive detection of extracellular vesicles using a microfluidic platform. Extracellular vesicles were specially captured and separated from complex matrix by immune magnetic microbeads (IMBs). With an enhancing acoustic wave microfluidic channel, the magnetic-extracellular vesicles immune complex was adequately mixed and reacted with antibody-modified fluorescence microbeads (IFBs). Using a newly designed step-wedge microfluidic structure, micrometer-size particles were trapped in large numbers with monolayer in the step zone, and the fluidic resistance was reduced through the wedge structure. This detection signal of the fluorescence-labelled immune sandwich complex could be read by counting, avoiding background interference and achieving an ultrasensitive detection with a detection limit of 850 particles/mL. Moreover, this method showed strong reliability and specificity in clinic samples. In addition, a miniaturized detection device was developed based on this microfluidic platform, improving this detection automation.

Spiral microchannels with concave cross-section for enhanced cancer cell inertial separation.

Inertial microfluidic technologies have proven effective for particle focusing and separation in many microchannels, typically the channels with the rectangular and trapezoidal shapes. To advance particle focusing in complex channels, we propose a spiral channel combining rectangular and concave cross-sections for high-resolution particle and cell focusing and separation. Numerical simulations were conducted to illustrate the effects of channel geometry on secondary flow distribution and particle focusing positions. The simulation shows the concave cross-section generates two asymmetrical Dean vortices skewing towards the inner and outer channel walls, resulting to stronger flow velocity magnitudes near the walls than the channel center. Consequently, larger particles focus near the inner wall, while smaller particles are trapped closer to the outer wall under the influence of the stronger velocity magnitude near the walls. A microfluidic chip with the proposed channelgeometry, along with a traditional rectangular channel, was fabricated by 3D printing and PDMS casting. Fluorescent microbeads were used to investigate inertial focusing and separation behaviors in the microfluidicchips. Experimental results show that the concave channel facilitates particle focusing or trapping much closer to the walls than the traditional rectangular channel, achieving better separation resolution. Finally, the proposed channel was applied to separate lung cancer A549 cells from human blood, achieving a cancer cell recovery rate of ~ 84.78% (enrichment ratio over 820-fold) and a blood cell rejection rate of ~ 99.88%. This innovative channel design in inertial microfluidics offers new insights for enhanced particle focusing and holds significant promise for cell manipulation with improved separation resolution.

Gold Nanocluster-Based Self-Assembly Fluorescence Microbeads for Sensor Array Discrimination of Multicomponent Metal Ions.

Benefiting from easy visualization and simultaneous detection of multiple targets, fluorescence microbeads are commonly used as fluorescence-sensing elements to detect pollutants in the environment. However, the application of fluorescence microbead-based sensor arrays is still limited because fluorescence dyes always suffer from self-quenching, photobleaching, and spectral overlap. Herein, three kinds of gold nanoclusters (Au NCs) were assembled with polystyrene microspheres (PS NPs) by electrostatic interaction to prepare fluorescence microbeads (PS-Au NCs), developing a sensor array for the simultaneous analysis of multiple metal ions. In this work, different PS-Au NCs showed an enhancing or quenching fluorescence response to various metal ions, owing to distinct binding capacities. Combined with the recognition algorithm from linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA), this sensor assay could realize single-component and multicomponent qualitative detection for 8 kinds of heavy metal ions (HMIs) including Cu2+, Co2+, Pb2+, Hg2+, and Ce3+. Particularly, the large surface area of PS NPs could provide a direct reaction microenvironment to improve the efficiency of the detection process. Meanwhile, the fluorescence property of Au NCs could also be enhanced by a partially effective aggregation-induced emission (AIE) effect to give better fluorescence signal output. Under optimal conditions, 8 kinds of heavy metals and their multicomponent mixtures could be identified at concentrations as low as 0.62 μM. Meanwhile, the analytical performance of this sensor assay in water samples was also verified, meeting the requirement of actual analysis. This study provides a great potential and practical example of single-batch, multicomponent identification for HMIs.

Mitigating Dietary Microplastic Accumulation and Oxidative Stress Response in European Seabass (Dicentrarchus labrax) Juveniles Using a Natural Microencapsulated Antioxidant.

Aquafeed's contamination by microplastics can pose a risk to fish health and quality since they can be absorbed by the gastrointestinal tract and translocate to different tissues. The liver acts as a retaining organ with the consequent triggering of oxidative stress response. The present study aimed to combine the use of natural astaxanthin with natural-based microcapsules to counteract these negative side effects. European seabass juveniles were fed diets containing commercially available fluorescent microplastic microbeads (1-5 μm; 50 mg/kg feed) alone or combined with microencapsulated astaxanthin (AX) (7 g/kg feed; tested for half or whole feeding trial-30 or 60 days, respectively). Fish from the different dietary treatments did not evidence variations in survival and growth performance and did not show pathological alterations at the intestinal level. However, the microplastics were absorbed at the intestinal level with a consequent translocation to the liver, leading, when provided solely, to sod1, sod2, and cat upregulation. Interestingly, the dietary implementation of microencapsulated AX led to a mitigation of oxidative stress. In addition, the microcapsules, due to their composition, promoted microplastic coagulation in the fish gut, limiting their absorption and accumulation in all the tissues analyzed. These results were supported by in vitro tests, which demonstrated that the microcapsules promoted microplastic coagula formation too large to be absorbed at the intestinal level and by the fact that the coagulated microplastics were released through the fish feces.

The protective effects of Kefir extract (KE) on intestinal damage in larval zebrafish induced by Oxytetracycline: Insights into intestinal function, morphology, and molecular mechanisms

The antibiotic oxytetracycline (OTC) can be detected in contemporary natural aquatic environments and has been implicated in causing intestinal damage in humans exposed to OTC-contaminated food or water. The irreversible damage caused by high concentrations of OTC to the intestine suggests that treatment through dietary means could still be necessary. This study proved the effectiveness of kefir extract (KE) in reversing intestinal damage caused by oxytetracycline (OTC) exposure. Following a 24-hour KE treatment subsequent to OTC exposure from 3 to 8 days post-fertilization of zebrafish larvae, molecular-level and microbiomic assessments revealed significant improvements. These included reduced expression of proinflammatory factors (IL-8 and IL-1β), increased antioxidant levels, and reversed unhealthy distribution of intestinal microbiota. Furthermore, KE supplementation showed potential in enhancing intestinal motility in the experiment of Nile red staining and fluorescent microbead transit. However, histological analysis showed that this short-term treatment with KE only partially reversed the intestinal morphological changes induced by OTC, suggesting that a longer treatment period might be necessary for complete restoration.

Fibrillogenesis in collagen hydrogels accelerated by carboxylated microbeads

Collagen type I is a material widely used for 3D cell culture and tissue engineering. Different architectures, such as gels, sponges, membranes, and nanofibers, can be fabricated with it. In collagen hydrogels, the formation of fibrils and fibers depends on various parameters, such as the source of collagen, pH, temperature, concentration, age, etc. In this work, we study the fibrillogenesis process in collagen type I hydrogels with different types of microbeads embedded, using optical techniques such as turbidity assay and confocal reflectance microscopy. We observe that microbeads embedded in the collagen matrix hydrogels modify the fibrillogenesis. Our results show that carboxylated fluorescent microbeads accelerate 3.6 times the gelation, while silica microbeads slow down the formation of collagen fibrils by a factor of 1.9, both compared to pure collagen hydrogels. Our observations suggest that carboxylate microbeads act as nucleation sites and the early collagen fibrils bind to the microbeads.

Short-Term Microplastics Exposure to the Common Mysid Shrimp, Americamysis bahia: Effects on Mortality and DNA Methylation

Primary consumers of microplastics are often zooplankton species such as the mysid shrimp, Americamysis bahia. Ingesting and interacting with these plastics can cause stress and lead to death. In the presence of some environmental stressors, gene expression may be altered without changing DNA sequences via the epigenetic methylation of the DNA. Mysid shrimp were exposed to 5-micrometer fluorescent polystyrene microbeads at different concentrations and different lengths of time. No significant effects were observed on mortality within 72 h, but mortality increased significantly thereafter. Microplastics were consumed by mysids and adhered to the mysid carapace and appendages. An ELISA-like (Enzyme-Linked Imuunosorbent Assay) colorimetric assay was employed to assess mysid DNA for differences in global percent methylation. No significant difference in the average percent methylated DNA nor difference in the number of methylation detections between treatments was found. This is one of few studies that has investigated DNA methylation effects due to microplastics-induced stress and the first study to detect DNA methylation in any member of the order Mysida.

Fluorescent dendritic fibrous nanosilica@chitosan porous composite microbeads: Selective and sensitive probes and removal adsorbents for Hg2+/Hg+ ions

Dendritic fibrous nano-silica (DFNS) possesses unique porous structure, making it an outstanding candidate for heavy metal adsorption and separation. It is interesting to introduce natural polymer (chitosan) into DFNS and produce natural materials-based porous DFNS. Initially, in this work, DFNS underwent modification to enlarge size and enrich with -C≡C bonds. And then, NH2-naphthalimide fluorophore was click-reacted with the modified DFNS, forming fluorescent DFNS (FL-DFNS). Eventually, chitosan was integrated into FL-DFNS using diacyl chloride, generating the organic–inorganic fluorescent composite micro-beads (FL-DFNS@CS-3). FL-DFNS acts as a “turn on” sensor for Hg2+/Hg+ ions, showing up to 5 times fluorescence increase. Also, it exhibited effective mercury adsorption. Due to its porous structure, it demonstrated good performance in adsorbing and separating Hg2+/Hg+ ions, achieving removal ratios of 98.8 % and 96.1 %, and adsorption capacities of 196.2 mg/g and 180.4 mg/g, respectively. It reached effective adsorption equilibrium within 15 min and was reusable up to seven times. Overall, this work confirms the practical applicability of FL-DFNS and FL-DFNS@CS micro-beads for identifying and removing Hg2+/Hg+ ions. Further exploration is worthy to explore its potential in environmental science and engineering.

Abstract WP163: Therapeutic Efficacy and Safety Evaluation of Cilostazol for Intracerebral Hemorrhage Through Enhancing Meningeal Lymphatic Function: A Preclinical Study

Background: Intracerebral hemorrhage (ICH) is the deadliest form of stroke with no effective treatment to date. Toxic byproducts from hematoma-lysis affected ICH-induced brain injury and long-term behavioral impairment. Enhancing hematoma clearance has shown therapeutic potential in reducing brain damage and neurologic deficits. Our previous work shows that cilostazol (CSZ) promotes meningeal lymphangiogenesis and drainage, aiding hematoma resolution and brain recovery after ICH. Here we explore the optimal dosage, therapeutic window, and potential adverse effects of CSZ treatment in preclinical ICH. Methods: A dose of 200 mg/day CSZ is used in clinics which translates to around 40 mg/kg in mice. CSZ (10, 20, and 40 mg/kg) or vehicle was given after ICH induction and was continued for various durations. Pretreatment of CSZ before ICH induction was used to assess its antiplatelet property on intra-parenchymal bleeding. Meningeal lymphatic vessel (mLV) complexity and coverage rate were measured. Lymphatic drainage was evaluated by fluorescent microbead accumulation in the mLV and deep cervical lymph nodes. Neurobehaviors were tested by corner turn test, whisker-evoked tactile responses, hindlimb abduction test, and neurologic deficit scoring. Iron deposition, hematoma, and neuron loss were assessed by immunohistochemistry. Results: ICH mice treated with CSZ showed enhanced mLV complexity and proliferation at the transverse, superior sagittal, and confluence of sinuses in the meninges. In parallel, histological, and behavioral outcomes also improved. Pretreatment with CSZ for 3 days before ICH induction did not increase hematoma volume, suggesting minimal to no adverse anticoagulant effect of CSZ in this setting. Conclusion: In all, our results unfold the effects of CSZ on mLV function regarding ICH. These findings may be applied as outcome parameters and provide a roadmap for sample size estimation in future clinical trials using CSZ as ICH treatment.

Single-shot quantitative phase-fluorescence imaging using cross-grating wavefront microscopy

The article introduces an optical microscopy technique capable of simultaneously acquiring quantitative fluorescence and phase (or equivalently wavefront) images with a single camera sensor, avoiding any delay between both images, or registration of images acquired separately. The method is based on the use of a 2-dimensional diffraction grating (aka cross-grating) positioned at a millimeter distance from a 2-color camera. Fluorescence and wavefront images are extracted from the two color channels of the camera, and retrieved by image demodulation. The applicability of the method is illustrated on various samples, namely fluorescent micro-beads, bacteria and mammalian cells.

Sitagliptin, an anti‐diabetic drug, inhibits the pro‐inflammatory microglia reactivity via dipeptidyl peptidase‐4 (DPP4)

Aims/Purpose: Diabetic retinopathy is characterized by a chronic low‐grade retinal neuroinflammation triggered by diabetes and mediated, at least in part, by microglial cells. We previously showed that sitagliptin, an oral drug for type 2 diabetes, developed to inhibit dipeptidyl peptidase 4 (DPP4) and increase insulin release, can prevent retinal neuroinflammation in diabetic rat models and the LPS‐induced morphological changes of microglial cells in retinal organotypic cultures. However, the mechanisms underlying these effects are not clarified. We hypothesize that sitagliptin is able to directly inhibit microglia reactivity via Dpp4.Methods: BV‐2 microglia cell line was exposed to a non‐toxic dose of lipopolysaccharide (LPS, 0.1 μg/mL) in the presence or absence of 200 μM sitagliptin. The expression of Dpp4, iNOS, IL‐1 beta and TNF were assessed by Western Blot and/or RT‐qPCR. The BV‐2 phagocytic efficiency was assessed using fluorescent microbeads. The metabolic activity and proliferation of BV‐2 cells were assessed by resazurin assay and Edu staining, respectively. Dpp4 deletion in BV‐2 cells was generated using Crispr‐cas9 method.Results: Sitagliptin inhibited the LPS‐induced increase in protein levels of iNOS, IL‐1 beta and TNF to 39.9 ± 6.3%, 47.2 ± 8.1% and 34.0 ± 7.2%, respectively, comparing to LPS. Sitagliptin reduced the LPS‐induced increase in phagocytic efficiency by approximately 40%. Sitagliptin did not affect the metabolism and proliferation of BV‐2. BV‐2 cells express Dpp4. Dpp4 deletion does not affect the LPS‐induced phagocytosis but abrogated the effect of sitagliptin on the reduction of BV‐2 phagocytic efficiency (approx. 320% as in LPS).Conclusions: Sitagliptin exerts direct anti‐inflammatory effects on microglia, mediated by DPP4. These results suggest that sitagliptin can potentially be used in retinal pathologies characterized by microglia‐mediated neuroinflammation.Support.FCT‐Portugal: PEst UID/NEU/04539/2013, UIDB/04539/2020 and UIDP/04539/2020 and PTDC/NEU‐OSD/1113/2012. COMPETE‐FEDER.

A simple dissection method for the isolation of mouse trabecular meshwork cells.

The outflow pathway, especially trabecular meshwork (TM), plays an essential role in glaucoma, and the availability of TM cells is crucial for in vitro research. So far, the isolation of TM cells from mice has been anything but manageable due to the small size of the eye. Direct isolation using a stereomicroscope and forceps requires a high grade of dexterity. Indirect isolation is based on the phagocytic properties of TM cells and involves injecting magnetic microspheres into the anterior chamber of live mice followed by isolation. Therefore, a simpler, less expensive, and nonexperimental strategy for isolating mouse TM cells would be desirable. After enucleation, the eyes were cut in half anterior-to-posteriorly. The lens and posterior segment were removed. Iris and the attached ciliary body were gently pulled backward and disconnected from the remaining tissue to expose the TM. By incising through the cornea anteriorly and posteriorly of the TM, the cornea/TM stripe could be isolated. The cornea/TM stripe was cultured with the pigmented side down in a 6-well. The outgrowing pigmented cells were analyzed by immunocytochemistry and mRNA expression for previously described TM cell markers. The phagocytic properties of the cells were additionally confirmed using fluorescent microspheres. Pigmented phagocytic cells were the first to grow out of the cornea/TM strips after approximately 4-7 days. Cells were positive for Collagen IV, Fibronectin1, Vimentin, and Actin alpha 2 and could phagocytize fluorescent microbeads. Cross-linked actin networks were visible after 9 days of exposure to TGFB2 (transforming growth factor-beta 2). Additionally, treatment with 500 nM Dexamethasone for one week increased myocilin expression, as previously reported for TM cells. In addition, we proved that this method can also be used in albino mice, which lack pigmentation of the trabecular meshwork. The isolated cells show phagocytic properties and specific expression of markers reported in TM cells. Therefore, our dissection-based method is inexpensive and reproducible for isolating TM cells in mice.

Retention and excretion of microplastics by Yellow Mealworm (Tenebrio molitor) larvae reared on an amino formaldehyde polymer microbeads contaminated substrate

Abstract Yellow mealworm (Tenebrio molitor L., TM), one of the main cultured insect species, is used for feed and food. Larval stages of this species can be reared on several substrates, including grains and industrial by-products. However, this species may potentially accumulate contaminants from the substrate, including microplastics (MPs), which may represent a potential hazard for its utilization in food chain. Evidences for plastic degradation by mealworms have been reported, while there are few information about the retention of microplastics in the body and tissues of this species. The aim of the present study was to assess whether TM larvae reared on a MPs-contaminated substrate were able to retain MPs in body tissues and to evaluate the retention and excretion rate after fasting for 24 and 48 hours. For this purpose, fluorescent amino formaldehyde polymer microbeads (1-5 μm) were used to simulate a MPs contamination in the substrate used for larvae rearing. A relevant concentration of MPs were found in the gut and were associated with ingested feed. However, microscopic analyses indicated that the gut acts as a simple transit site and a barrier towards MPs migration in tissues. A marked reduction of MPS content was highlighted in larvae after fasting, even though a 48-hours fasting period was not sufficient to completely depurate insect gut from MPs. Results obtained in the present study suggest that the polymer microbeads used are recalcitrant to digestion by TM larvae, and show the relevant ability of depuration from MPs of this species. Taken together the results showed the suitability of TM to be reared on MPs contaminated substrates since larvae survival and growth resulted not hampered by the MPs presence and open cues on the fasting period able to completely depurate insect body from MPs residues.

Dual optofluidic distributed feedback dye lasers for multiplexed biosensing applications

Integrated optofluidic devices have become subjects of high interest for rapid biosensor devices due to their unique ability to combine the fluidic processing of small volumes of microfluidics with the analysis capabilities of photonic structures. By integrating dynamically reconfigurable optofluidic lasers on-chip, complex coupling can be eliminated while further increasing the capabilities of sensors to detect an increasing number of target biomarkers. Here, we report a polydimethylsiloxane-based device with two on-chip fluidic distributed feedback (DFB) laser cavities that are integrated with an orthogonal analyte channel for multiplexed fluorescence excitation. One DFB grating is filled with 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran dissolved in dimethyl sulfoxide. The second grating is filled with rhodamine 6G dissolved in a diluted ethylene glycol solution. We present characterization of both lasers through analysis of the lasing spectra for spectral narrowing along with a power series to observe threshold behavior. We then demonstrate simultaneous detection of two different fluorescent microbeads as a proof of concept for scalable, single biomarker analysis using on-chip optofluidic lasers.

Macroscopic temporally and spectrally resolved fluorescence imaging enhanced by laser-wavelength multiplexing.

We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.

The effect of morphine on rat microglial phagocytic activity: An in vitro study of brain region-, plating density-, sex-, morphine concentration-, and receptor-dependency

Opioids have long been used for clinical pain management, but also have addictive properties that have contributed to the ongoing opioid epidemic. While opioid activation of opioid receptors is well known to contribute to reward and reinforcement, data now also suggest that opioid activation of immune signaling via toll-like receptor 4 (TLR4) may also play a role in addiction-like processes. TLR4 expression is enriched in immune cells, and in the nervous system is primarily expressed in microglia. Microglial phagocytosis is important for developmental, homeostatic, and pathological processes. To examine how morphine impacts microglial phagocytosis, we isolated microglia from adult male and female rat cortex and striatum and plated them in vitro at 10,000 (10K) or 50,000 cells/well densities. Microglia were incubated with neutral fluorescent microbeads to stimulate phagocytosis in the presence of one of four morphine concentrations. We found that the brain region from which microglia are isolated and plating density, but not morphine concentration, impacts cell survival in vitro. We found that 10−12 M morphine, but not higher concentrations, increases phagocytosis in striatal microglia in vitro independent of sex and plating density, while 10−12 M morphine increased phagocytosis in cortical microglia in vitro independent of sex, but contingent on a plating density. Finally, we demonstrate that the effect of 10−12 M morphine in striatal microglia plated at 10 K density is mediated via TLR4, and not μORs. Overall, our data suggest that in rats, a morphine-TLR4 signaling pathway increases phagocytic activity in microglia independent of sex. This may is useful information for better understanding the possible neural outcomes associated with morphine exposures.

Bone on-a-chip: a 3D dendritic network in a screening platform for osteocyte-targeted drugs

Age-related musculoskeletal disorders, including osteoporosis, are frequent and associated with long lasting morbidity, in turn significantly impacting on healthcare system sustainability. There is therefore a compelling need to develop reliable preclinical models of disease and drug screening to validate novel drugs possibly on a personalized basis, without the need of in vivo assay. In the context of bone tissue, although the osteocyte (Oc) network is a well-recognized therapeutic target, current in vitro preclinical models are unable to mimic its physiologically relevant and highly complex structure. To this purpose, several features are needed, including an osteomimetic extracellular matrix, dynamic perfusion, and mechanical cues (e.g. shear stress) combined with a three-dimensional (3D) culture of Oc. Here we describe, for the first time, a high throughput microfluidic platform based on 96-miniaturized chips for large-scale preclinical evaluation to predict drug efficacy. We bioengineered a commercial microfluidic device that allows real-time visualization and equipped with multi-chips by the development and injection of a highly stiff bone-like 3D matrix, made of a blend of collagen-enriched natural hydrogels loaded with hydroxyapatite nanocrystals. The microchannel, filled with the ostemimetic matrix and Oc, is subjected to passive perfusion and shear stress. We used scanning electron microscopy for preliminary material characterization. Confocal microscopy and fluorescent microbeads were used after material injection into the microchannels to detect volume changes and the distribution of cell-sized objects within the hydrogel. The formation of a 3D dendritic network of Oc was monitored by measuring cell viability, evaluating phenotyping markers (connexin43, integrin alpha V/CD51, sclerostin), quantification of dendrites, and responsiveness to an anabolic drug. The platform is expected to accelerate the development of new drug aimed at modulating the survival and function of osteocytes.

The role of microbe-microplastic associations in marine Nematode feeding behaviors

Fauna across many taxa and trophic levels have been shown to consume microplastics (MPs) in experiments, providing evidence that supports field-based gut content assessments. Multiple explanations exist regarding why fauna consume MPs, one of which posits that microbial growth on MPs may facilitate faunal ingestion. However, laboratory assessments on the reasons why MPs are consumed remain limited. Here, we assessed if the presence of microbes on MPs altered marine nematode feeding behaviors across current and potential future concentrations of MPs in a local system. We used a microcosm experiment in which field-collected sediment was spiked with bacterially treated or untreated fluorescent plastic microbeads (1.0–5.0 μm) in concentrations of 102, 104, and 106 per microcosm, representing local and potential future concentrations of MPs. Ingestion by the dominant interstitial fauna was investigated after 0, 3, and 7 days using bright field microscopy. Nematodes were the only fauna across microcosms that consumed MPs, but this consumption was variable and there were no apparent trends across exposure time, bacterial treatment, or MP concentration. There were also no genera- or feeding-type-specific trends in the number of MPs consumed, though four of the top five nematode genera that consumed MPs were pollution-tolerant genera. Our study demonstrates that microbe-MP associations do not drive marine nematodes to eat MPs, especially at local field concentrations. While there were no trends across any of the nematode genera in our study, we recognize that unrealistic MP concentrations in other studies may provide alternative explanations for nematode consumption of MPs.