Fluorescent Microbeads Research Articles

A Microflow Cytometer with a Rectangular Quasi-Flat-Top Laser Spot.

This work develops a microflow cytometer, based on a microfluidic chip for three-dimensional (3D) hydrodynamic focusing and a binary optical element (BOE) for shaping and homogenizing a laser beam. The microfluidic chip utilizes sheath flows to confine the sample flow along the channel centerline with a narrow cross section. In addition to hydrodynamic focusing, secondary flows are generated to strengthen the focusing in the vertical direction. In experiments, the chip was able to focus the sample flow with cross sections of 15 μm high and 8–30 μm wide at 5 m/s, under the condition of the sample flow rates between 10 and 120 μL/min. Instead of using the conventional elliptical Gaussian spot for optical detection, we used a specially designed BOE and obtained a 50 μm × 10 μm rectangular quasi-flat-top spot. The microflow cytometer combining the chip and the BOE was tested to count 3, 5, and 7 μm fluorescence microbeads, and the experimental results were comparable to or better than those derived from two commercial instruments.

Using binary optical elements (BOEs) to generate rectangular spots for illumination in micro flow cytometer.

This work introduces three rectangular quasi-flat-top spots, which are provided by binary optical elements (BOEs) and utilized for the illumination in a microflow cytometer. The three spots contain, respectively, one, two, and three rectangles (R1, R2, and R3). To test the performance of this mechanism, a microflow cytometer is established by integrating the BOEs and a three-dimensional hydrodynamic focusing chip. Through the experiments of detecting fluorescence microbeads, the three spots present good fluorescence coefficients of variation in comparison with those derived from commercial instruments. Benefiting from a high spatial resolution, when using R1 spot, the micro flow cytometer can perform a throughput as high as 20 000 events per second (eps). Illuminated by R2 or R3 spot, one bead emits fluorescence twice or thrice, thus the velocity can be measured in real time. Besides, the R3 spot provides a long-time exposure, which is conducive to improving fluorescence intensity and the measurement stability. In brief, using the spots shaped and homogenized by BOEs for illumination can increase the performance and the functionality of a micro flow cytometer.

In Vitro Angiogenesis: Co-Culture Model of the Retina in Microfluidic Devices

Background Age-related macular disease (AMD) is a common eye condition and the most severe form of a disease that is the leading cause of vision loss among people age 50 and above. Simple tasks such as driving or reading become impossible without a functional central vision, which is impaired as the result of damages to the macula. Wet form AMD is responsible for 90% of the blindness out of all AMD patients; it is marked by the development of subretinal choroidal neovascularization and degeneration of retinal pigmental epithelial (RPE) cells. Wet macular degeneration occurs when abnormal blood vessels disrupt the retina and leak blood or fluid that blurs the central vision. Recently, MEMS technology which integrates multidisciplinary field of studies offers a new direction in medical investigation and diagnosis. Here we utilized microfabrication technology to reconstruct ocular fundus tissues (OF) in vitro and to simulate neovascularization in the hope of building a disease model for the retina. Method Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelial cells (HUVECs), representing the retinal cells and the choroid, were co-cultured in the microfluidic devices. Two different approaches are developed in fabricating microfluidic devices resulting in 2D and 3D models. 2D model:the microfluidic device was made up of 2 layers, on each a channel (width 500 µm; height 150 µm) was embedded. A porous membrane (thickness 6.5 µm, hole diameter 10 µm) was sandwiched between the layers. Microchannels and membranes were made of poly(dimethylsiloxane) (PDMS). Retinal pigment epithelial (RPE) cells were introduced into the upper channel of the device and umbilical vein endothelial cells (HUVECs) were loaded into the lower channel. 3D model: the wire with a 0.2 mm diameter was pretreated before embedded in collagen (6.5 mg/ml). After the collagen had polymerized, the needle was removed resulting in an empty channel where HUVECs were to be seeded. RPE cells were seeded on the top of the collagen gel to initiate co-culture experiments. Data analysis: RPE cells and HUVEC were dyed respectively with Cell TrackerTM red and green and cell growth was recorded at certain time intervals. RPE cells were under hypoxia and hypoglycemia 24hr prior the start of the co-culture experiment. Before the aforementioned co-culture model, RPE were cultured alone on the upper channel and the VEGF concentration was collected and measured by ELIZA after replacing DME medium High Glucose (20 mM) with DME medium Low Glucose (3 mM). HUVECs were loaded into the lower channel and cultured alone. The upper channel was perfused with 50 ng/ml of VEGF-A165and the number of HUVECs migrating to the upper channel was analyzed using the same monitoring system. Characteristics of cells in the 3D model were characterized following the same methodology. Results Validation of the devices: The devices were validated prior culturing cells inside the devices. Confocal images of the cross-sections of channel length and diameter were taken after perfusion of fluorescent microbeads. ARPE characterization: Results show that RPE cells responded to the lowered glucose microenvironment by increasing VEGF secretion (58.8 % compared to the control). In addition, RPE cells exhibited a polar secretion of VEGF which is consistent to the cell physiology. HUVEC characterization: The increased number of HUVEC migrating through the porous membrane in response to VEGF gradient signifies neovascularization. Comparing to the control, there was a 31.3 % increase of cell migration. Conclusions and Future work The physiology of RPE cells and HUVECs is reproduced in the microfluidic devices. The preliminary results suggest that the alteration of the microenvironment induces responses of RPE cells and the significance of VEGF in neovascularization. In the near future, we anticipate studying responses of the co-culture model to hypoxia to characterize the AMD pathology.

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain.

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 µl of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-µl syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 µl of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

Abstract 3240: Prostate cancer exosomes and their effects on macrophage engulfment and polarization

Abstract The purpose of this study is to explore the effects of prostate cancer-derived exosomes on macrophage engulfment and polarization. Exosomes are small vesicles secreted by virtually all cells, which contain nucleic acids, lipids, proteins, and carbohydrates. Exosomes can be abundantly found in cancer patient secretions (serum, urine, etc.) and contain a high amount of RNA molecules, some of them being miRNA, and parts of the DNA of the cell of origin. One of the functions of exosomes is to mediate cellular communication. This occurs as they are secreted from one cell and then distribute their contents into other cells. Some exosomes specifically target immune cells. The miRNA the exosomes carry can silence genes involved in the activation of these immune cells, leading to immune suppression and tumor growth. Macrophages are often found surrounding the tumor microenvironment, as they are recruited by tumor signals to promote angiogenesis and metastasis. These macrophages, which are often called tumor associated macrophages (TAMs), are exposed to various signals derived from the tumor microenvironment, including exosomes. Previous data generated in our lab suggests that the co-culture of macrophages with PC3 and DU145 cell lines results in a decrease in macrophage phagocytosis and an M2-like polarization state. For this study, we exposed U937 cells to phorbol 12-myristate 13-acetate (PMA) for 24 hours to allow for differentiation. Then, we co-cultured these macrophages with isolated exosomes from spent media from both PC3 and DU145 cell lines at 1, 2, 3, 4, 5, 6, 12, and 24 hours. We then allowed the macrophages to phagocytose fluorescent microbeads for 1 hour. Flow cytometry analysis of these macrophages revealed that the total bead phagocytosis starts decreasing at 3 hours of exosome co-culture compared to control. Interestingly, total bead phagocytosis resumes to normal at 12 and 24 hours of exosome co-culture. Gene expression analysis of these macrophages suggests an M2 phenotype from hours 2-24. These results suggest that prostate cancer-derived exosomes can target macrophages and affect their phagocytosis. Further research is required to elucidate the specific genes these exosomes target. Citation Format: Evita G. Weagel, Wei Meng, Richard A. Robison, Kim L. O’Neill. Prostate cancer exosomes and their effects on macrophage engulfment and polarization. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3240.

Abstract 400: Immunofluorescence chromatographic assay of tumor cells in dried blood spot

Abstract Blood droplet, when desiccated on a surface, forms a ring-like deposit, akin to the coffee ring left after a coffee spill. When blood, coffee, or other aqueous suspensions of the colloidal particles, evaporate with contract line pinned on the underlying hydrophilic surface, a capillary force is generated by the evaporative flux, which drags nearly all the particles to the edge. As the evaporation extends, the volume of the droplet decreases, and the sol-gel transition occurs. The gelation gradually moves inward from the edge to the center, eventually immobilizes all particles, and develops the ring effect. Because smaller sized particles diffuse much more quickly and efficiently than the larger ones, smaller particles tend to preferentially accumulate on the rim, forming multi-layered self-assembly. Contrarily, the majority of the larger particles congregate in the central area. This natural phenomenon simply presents a size-dependent chromatography of variously sized particles in solution. Separation of normal blood cells or break-free tumor cells has been widely utilized for blood disease diagnosis and cancer prognosis. Traditional preparation via blood smear and advanced separation by the external driving forces, e.g. centrifugation and powered microfluidics, could be limited by the technical difficulty, assay complexity, and cell integrity. Coffee-ring based blood cell separation and detection, namely dried spot biopsy, is however a hand-free, cell-harmless, spontaneous process. To perform a dried spot biopsy, 3×1 inch microscope slides were first cleaned to remove surface impurities and residues. 200 uL of freshly collected, anti-coagulated whole blood, bearing fluorescent microbeads (1 and 10 um sized) or pre-stained breast tumor cells (MDA-MB-231, ∼15 um sized), was dispensed onto the slide and allowed to dry statically or acceleratedly on a moving stage at controlled temperature and humidity. Three hydrophilic coatings and five surfactants were examined to balance the outward capillary flow with the inward Marangoni flow for optimal coffee ring effect. It is demonstrated that two sizes of microbeads formed separate rings, a few hundreds of microns in average apart from each other, while tumor cells, acting differently, concentrated in the central area with random dispersion, probably because of the diversified cell sizes. The dried spot biopsy could have a potential for detecting blood borne biomarkers in the form of dried blood specimens that poses longer shelf life, less biohazardous risk, and easier transportation protocol than the liquid form. Additionally, specific cells of interest from microscopic regions of dried blood film can be retrieved by laser microdissection for downstream analyses. Particularly, this method could be valuable for use in low-resourced regions and countries. Citation Format: Ming Zhao, Dakang Ma, Quanxu Shen, Bin Hong. Immunofluorescence chromatographic assay of tumor cells in dried blood spot. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 400.

Feeding Difficulties and Gastrointestinal Tract Morphology and Innervation in ‘Charge’ Syndrome

Abstract BACKGROUND: CHARGE syndrome has been linked to mutations in the CHD7 gene and results in a number of physiological and structural abnormalities. The estimated incidence in the Atlantic Provinces is 1 in 8500 births. Challenges include eating problems, which have a profound impact throughout a child’s life and can lead to complications and even death. OBJECTIVES: To investigate feeding difficulties and model the morphology and innervation of the gastrointestinal tract in CHARGE syndrome. DESIGN/METHODS: Three research studies (two clinical, one basic science) were conducted to assess feeding difficulties in CHARGE syndrome. The first study conducted a qualitative analysis of parent interviews to understand packing and problematic feeding behaviors. The second study investigated general eating and GI motility problems by having parents complete a series of feeding questionnaires. The third study modeled CHARGE syndrome in zebrafish by using a morpholino to knock down the expression of CHD7. RESULTS: Study 1: Twenty parents completed a phone interview, describing their child/adult’s (2-32 years) adverse feeding behaviors. Parents reported food packing most commonly with bread and pasta (33%), and reported that food was held in cheeks for hours after a meal had ended (35%). Packing was reported to prolong mealtimes for over an hour (30%). Parents were worried about choking during eating (30%).Study 2: Sixty-nine parents of children (age 1-18 years) completed the questionnaires. Those who were tube fed had significantly more gastrointestinal symptoms (stomach pain, nausea, etc.) and worse feeding difficulties than those who were orally fed. The CHARGE characteristics of choanal atre-sia/stenosis and cranial nerve IX/X dysfunction were associated with significantly more gastrointestinal symptoms. Parents identified constipation as a major challenge. Study 3: Immunohistochemistry demonstrated changes in the enteric innervation of the gastrointestinal tract in the CHARGE syndrome zebrafish models. There was decreased branching of the gastrointestinal nerve network surrounding the stomach. Use of fluorescent microbeads demonstrated reduced motility and delayed passage of the microbeads through the gastrointestinal tract. CONCLUSION: These three studies provided a comprehensive analysis of feeding and gastrointestinal difficulties, from mouth to anus, in CHARGE syndrome. They provide a deeper understanding of adverse feeding behaviors, feeding difficulties, and the abnormal morphology of the gastrointestinal system. The information from this study can be useful for general pediatricians and feeding therapy teams who are involved in the care of these individuals starting in infancy. The team-based approach of conducting multiple research projects investigating a common issue may be useful in other genetic disorders.

Fluorescent Polystyrene Microbeads as Invisible Security Ink and Optical Vapor Sensor for 4-Nitrotoluene

Color-tunable solid-state emitting polystyrene (PS) microbeads were developed by dispersion polymerization, which showed excellent fluorescent security ink characteristics along with sensitive detection of vapors of nitro aromatics like 4-nitro toluene (4-NT). The fluorophores pyrene and perylenebisimide were incorporated into the PS backbone as acrylate monomer and acrylate cross-linker, respectively. Solid state quantum yields of 94 and 20% were observed for the pyrene and perylenebisimide, respectively, in the PS/Py and PS/PBI polymers. The morphology and solid state fluorescence was measured by SEM, fluorescence microscopy, and absorbance and fluorescence spectroscopy techniques. The ethanol dispersion of the polymer could be used directly as a fluorescent security "invisible" ink, which became visible only under ultraviolet light. The color of the ink could be tuned depending on the amounts of the pyrene and perylenebisimide incorporated with blue and orange-green for pyrene alone or perylenebisimide alone beads respectively and various shades in between including pure white for beads incorporating both the fluorophores. More than 80% quenching of pyrene emission was observed upon exposure of the polymer in the form of powder or as spin-coated films to the vapors of 4-NT while the emission of perylenebisimide was unaffected. The limit of detection was estimated at 10(-5) moles (2.7 ppm) of 4-NT vapors. The ease of synthesis of the material along with its invisible ink characteristics and nitro aromatic vapor detection opens up new opportunities for exploring the application of these PS-based materials as optical sensors and fluorescent ink for security purposes.

Light induced, localized, and abrupt force relaxations in fibroblast cells on soft substrates

Exposure to fluorescent excitation light is known to adversely affect living cells. Here, we provide experimental evidence showing fibroblast cells relax abruptly during illumination with fluorescent excitation light. Cells were plated on polyacrylamide (PA) gels embedded with fluorescent microbeads. The beads served as fiducial markers to track substrate displacements induced by cell force. When the cells adhere, they apply traction forces to their underlying surface, causing the substrate to contract inward with respect to the cell boundary. We find that contractile cell forces are sporadically interrupted by abrupt force relaxation events, characterized by outward “jumps” in displacement (⩾ 30 nm in 0.5 s) of the underlying substrate during exposure to fluorescent light. Jumps occur more frequently on softer substrates. Additionally, the speed and direction of displacements of particles before and after jumps are conserved, suggesting that the jumps do not involve any change in cytoskeletal or adhesion structure of the cell, and are possibly mediated by disengagement and re-engagement of myosin motors with actin filaments. Interestingly, we find that the frequency of jumps is also modulated by the energy introduced by the excitation light source. We propose a mechanistic hypothesis to explain the observations.

Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples

Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses.Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order to assess the influence of breed, sex and age on the expression of CD4 in a larger case series. Based on univariate GLMs, none of these variables influenced CD4 RFI.Normalizing fluorescence data using lymphoid CD4 MFI as a reference would improve the comparison of results obtained by different laboratories, patients or times in diagnostic and research analyses of FI. Further studies are needed to confirm our results with different FC approaches.

Ocean Acidification Affects Hemocyte Physiology in the Tanner Crab (Chionoecetes bairdi).

We used flow cytometry to determine if there would be a difference in hematology, selected immune functions, and hemocyte pH (pHi), under two different, future ocean acidification scenarios (pH = 7.50, 7.80) compared to current conditions (pH = 8.09) for Chionoecetes bairdi, Tanner crab. Hemocytes were analyzed after adult Tanner crabs were held for two years under continuous exposure to acidified ocean water. Total counts of hemocytes did not vary among control and experimental treatments; however, there were significantly greater number of dead, circulating hemocytes in crabs held at the lowest pH treatment. Phagocytosis of fluorescent microbeads by hemocytes was greatest at the lowest pH treatment. These results suggest that hemocytes were dying, likely by apoptosis, at a rate faster than upregulated phagocytosis was able to remove moribund cells from circulation at the lowest pH. Crab hemolymph pH (pHe) averaged 8.09 and did not vary among pH treatments. There was no significant difference in internal pH (pHi) within hyalinocytes among pH treatments and the mean pHi (7.26) was lower than the mean pHe. In contrast, there were significant differences among treatments in pHi of the semi-granular+granular cells. Control crabs had the highest mean semi-granular+granular pHi compared to the lowest pH treatment. As physiological hemocyte functions changed from ambient conditions, interactions with the number of eggs in the second clutch, percentage of viable eggs, and calcium concentration in the adult crab shell was observed. This suggested that the energetic costs of responding to ocean acidification and maintaining defense mechanisms in Tanner crab may divert energy from other physiological processes, such as reproduction.

Mechanics of Adhesion Dependent and Independent Neutrophil Migration in Three-Dimensional Extra-Cellular Matrices

While much research has been dedicated to the identification of the cascade of specific biochemical processes involved in the recruitment of neutrophils, much less is known about the mechanical events driving their migration; in particular, how they generate the necessary traction forces to migrate across three-dimensional (3-D) extravascular spaces and the importance of forming cell-substrate adhesions during this process is unclear. In this study, we investigate the importance of cell-substrate adhesions on the mechanics of 3-D neutrophil motility in collagen gels using Elastographic 3D Force Microscopy (E3DFM). We used wild type neutrophil-like differentiated human promyelocytic leukemia (dHL-60) cells and talin 1 knockout dHL60 cells, which were unable to engage their integrins, as our model systems. Both cell lines were were embedded in collagen matrices containing fluorescent micro-beads. Neutrophil motility was induced via the introduction of the neutrophil chemokine formyl-Methionyl-Leucyl-Phenylalanine (fMLP) in a custom build device. Both Confocal and Fluorescent microscopy techniques were used to image the movement of the embedded micro-beads as well as fluorescently labeled cells. Particle Image Velocimetry (PIV) and Finite Deformation Theory were used to compute displacement fields in the collagen matrices. Stress fields in the matrices were computed using a constitutive relationship with several material parameters. The over determined nature of the problem was used to estimate the aforementioned material parameters. We will present data showing that morphological changes and migratory patterns differed depending on the cell's ability to form cell-substrate adhesions. We will also provide data showing a clear relationship between the aforementioned migratory characteristics and computed displacement and stress fields around migrating neutrophils in collagen matrices. The results from our study show that neutrophils migrating in 3-D environments employ distinct mechanical mechanisms that depend on their ability to form adhesions.

Multi-Color Single Photon Detection With Enhanced Linearity

A new method for multi-color single photon detection with an enhanced linearity is proposed. Due to the use of the multi-channel single photon detection in conjunction with a novel approach to the post processing of the detected signal, the method provides up to an order of magnitude enhancement of the detection linearity and enables an accurate determination of color composition of fluorescence-labeled micro-objects. Application of the new method for the detection of fluorescent microbeads moving in a microfluidic channel is presented.

超薄膜を用いた細胞収縮力の可視化と定量化への試み

Evaluation of cell traction forces is crucial to biological researches. Accordingly, a number of cell traction forces evaluation techniques have been proposed in the past. In contrast to extensive studies on effects of exogenous forces such as tensile stretches and fluid shear stresses, there has been less work on how endogenous contractile forces are generated within cells under various conditions. This may be partly attributed to a lack of experimental techniques for evaluating the cell traction forces. One of the most popular techniques is traction force microscopy (TFM) using fluorescent microbeads embedded within hydrogels on which cells are plated. The cell traction forces are usually estimated from displacement of fluorescent beads and the physical properties of the hydrogel. This technique requires complicated image correlation analyses and sophisticated experimental skills. Hence, it is not practical to use such a technique in large-scale screening programs. An alternative has been developed, which makes it possible to visualize the cell traction forces using a gold ultra-thin film.

Mechanical scanner-less multi-beam confocal microscope with wavefront modulation

We propose a novel full-electronically controlled laser confocal microscope in which a liquid-crystal-on-silicon spatial light modulator and a custom CMOS imaging sensor are synchronized for performing multi-beam confocal imaging. Adaptive wavefront modulation for functional multi-beam excitation can be achieved by displaying appropriate computer generated holograms on the spatial light modulator, in consideration of the numerical aperture of the focusing objective. We also adopted a custom CMOS imaging sensor to realize multi-beam confocal microscopy without any physical pinhole. The confocality of this microscope was verified by improvements in transverse and axial resolutions of fluorescent micro-beads.

Subclinical avian hepatitis E virus infection in layer flocks in the United States

The objective of this study was to determine patterns of avian HEV infection in naturally infected chicken farms. A total of 310 serum samples and 62 pooled fecal samples were collected from 62 chicken flocks on seven commercial in-line egg farms in the Midwestern United States and tested for avian HEV circulation. Serum samples were tested for the presence of anti-avian HEV IgY antibodies by a fluorescent microbead immunoassay (FMIA) which was developed for this study. The FMIA was validated using archived samples of chickens with known exposure (n = 96) and compared to the results obtained with an enzyme-linked immunosorbent assay (ELISA) based on the same capture antigen. There was an overall substantial agreement between the two assays (κ = 0.63) with earlier detection of positive chickens by the FMIA (P = 0.04). On the seven farms investigated, the overall prevalence of anti-avian HEV IgY antibodies in serum samples from commercial chickens was 44.8% (20–82% per farm). Fecal samples were tested for avian HEV RNA by a nested reverse-transcriptase PCR. The overall detection rate of avian HEV RNA in fecal samples was 62.9% (0–100% per farm). Sequencing analyses of partial helicase and capsid genes showed that different avian HEV genotype 2 strains were circulating within a farm. However, no correlation was found between avian HEV RNA detection and egg production, egg weight or mortality. In conclusion, avian HEV infection is widespread among clinically healthy laying hens in the United States.

Particle processing and gut kinematics of planktotrophic bivalve larvae

Although the clearance rates of planktotrophic bivalve larvae have been widely reported, post-oral particle processing is less well understood. Using a series of exposures to differently colored fluorescent polystyrene microbeads, we quantify several post-oral process in the larval gut, including gut fullness, gut passage time, and degree of mixing by modeling larval guts as a continuously stirred tank reactor (CSTR), plug flow reactor (PFR) or combinations of the two in series. We also varied several experimental conditions to understand how these affected estimates of gut kinematic parameters. We found the larval guts of M. galloprovincialis aged 2 and 7 days post-fertilization, had gut exchange time >1 h and were best described either as a CSTR or CSTR in series with a PFR. Mixing stomach contents likely aids post-oral particle selection, physical breakdown of ingested material, and accelerates the diffusion of digestive enzymes in the gut volume. Reactor models also provided estimates of ingestion rates, which were compared to those obtained by other authors who measured rates of bead accumulation. In accordance with reactor theory, ingestion rates were negatively and nonlinearly correlated with gut passage times and positively related to maximal gut fullness. Collectively, these studies provide new insight on the digestive strategy of planktotrophic bivalve larvae.

Bioactive Fibers: Hydrogel Templates for Rapid Manufacturing of Bioactive Fibers and 3D Constructs (Adv. Healthcare Mater. 14/2015).

Hybrid hydrogel fibers were fabricated using hydrogel templates. On page 2146, D. Juncker, A. Khademhosseini, and co-workers show how these fibers containing different fluorescent microbeads have been assembled using textile processes, such as braiding and weaving, to create complex 3D patterns for bioengineering applications. The fabricated constructs with clinically relevant dimensions are mechanically stable.

Evaluation of the impact of polyethylene microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae

Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing the impact of polyethylene (PE 10–45 μM) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 104 and 105 fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48 h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1β) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion.