The fabrication process of the SrHA/Va/GO composite coating on Ti is shown in Graphical Abstract (for review). The following series of experiments were verified: 1) effect of GO on the mechanical ...properties of composite coatings; 2) the surface microstructure, phase composition, roughness, hydrophilicity of the coated implants; 3) the release behavior of Va from the SrHA/Va/GO coating; 4) the in vitro cellular responses of the composite coatings; 5) Effect of vancomycin on antibacterial properties of composite coatings.
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Currently, the clinical application of hydroxyapatite (HA) coatings are limited due to the lack of anti-infection, osteoinductivity and poor mechanical properties. In the present study, the strontium-containing hydroxyapatite (SrHA)/graphene oxide (GO)/vancomycin (Va) nanocomposite coating with drug delivery and antibacterial activity was fabricated by electrochemical deposition (ECD) on titanium substrate. The results showed that by inducing a large number of crystal nucleation points, the oxygen-containing groups on the two-dimensional surface of GO induced the formation of extremely fine SrHA/Va/GO composite coatings. Furthermore, the functional coating exhibited a moderately nanostructured surface with average values of roughness and contact angle of 31.9° and 126 nm, respectively, which provided a suitable contact surface for osteoblast proliferation and differentiation. Moreover, compared with HA coatings without GO, the hardness and elastic modulus of SrHA/Va/GO composite coatings were enhanced by 67.6 % and 121 %, respectively. The release process of Va from the SrHA/Va/GO coating showed a phased release law, which broke out within the first 5 h and then slowly and controlledly released within 14 h, which is due to the physical and chemical adsorption of Va. The inhibition zone and colony count experiments showed that the SrHA/Va/GO coating revealed satisfactory inhibitory effects on S. aureus and S. epidermidis, and the inhibition could last for 3 days. Compared with pure titanium, SrHA/Va/GO composite coating can significantly promote the adhesion, proliferation and differentiation of MC3T3-E1 cells. In conclusion, SrHA/Va/GO exhibited good mechanical properties, excellent osteoinductivity and antibacterial properties, confirming its potential as a new surface modification material for the prevention and treatment of infectious bone defects.
The Atlantic cod's unusual immune system, entirely lacking the Major Histocompatibility class II pathway, has prompted intriguing questions about what mechanisms are used to combat bacterial ...infections and how immunological memory is generated. By single-cell RNA sequencing we here report an in-depth characterisation of cell types found in immune tissues, the spleen and peripheral blood leukocytes of Atlantic cod. Unbiased transcriptional clustering revealed eleven distinct immune cell signatures. Resolution at the single cell level enabled characterisation of the major cell subsets including the cytotoxic T cells, B cells, erythrocytes, thrombocytes, neutrophils, and macrophages. Additionally, to our knowledge we are the first to uncover cell subsets in Atlantic cod which may represent dendritic cells, natural killer-like cells, and a population of cytotoxic cells expressing
, a master transcription factor of T helper 2 cells. We further identify putative gene markers for each cluster and describe the relative proportions of each cell type in the spleen and peripheral blood leukocytes. Of the major haematopoietic cell populations, the lymphocytes make up 55 and 68% of the spleen and peripheral blood leukocytes respectively, while the myeloid cells make up 45 and 32%. By single-cell analysis, this study provides the most detailed molecular and cellular characterisation of the immune system of the Atlantic cod so far.
The neonatal Fc receptor for IgG (FcRn) is a distant member of the MHC class I protein family. It binds IgG and albumin in a pH-dependent manner and protects these from catabolism by diverting them ...from a degradative fate in lysosomes. In addition, FcRn-mediated IgG transport across epithelial barriers is responsible for the transmission of IgG from mother to infant and can also enhance IgG-mediated antigen uptake across mucosal epithelia. We now show a previously undescribed role for FcRn in mediating the presentation of antigens by dendritic cells when antigens are present as a complex with antibody by uniquely directing multimeric immune complexes, but not monomeric IgG, to lysosomes.
Little is known about the repertoire dynamics and persistence of pathogenic T cells in HLA-associated disorders. In celiac disease, a disorder with a strong association with certain HLA-DQ allotypes, ...presumed pathogenic T cells can be visualized and isolated with HLA-DQ:gluten tetramers, thereby enabling further characterization. Single and bulk populations of HLA-DQ:gluten tetramer-sorted CD4+ T cells were analyzed by high-throughput DNA sequencing of rearranged TCR-α and -β genes. Blood and gut biopsy samples from 21 celiac disease patients, taken at various stages of disease and in intervals of weeks to decades apart, were examined. Persistence of the same clonotypes was seen in both compartments over decades, with up to 53% overlap between samples obtained 16 to 28 years apart. Further, we observed that the recall response following oral gluten challenge was dominated by preexisting CD4+ T cell clonotypes. Public features were frequent among gluten-specific T cells, as 10% of TCR-α, TCR-β, or paired TCR-αβ amino acid sequences of total 1813 TCRs generated from 17 patients were observed in 2 or more patients. In established celiac disease, the T cell clonotypes that recognize gluten are persistent for decades, making up fixed repertoires that prevalently exhibit public features. These T cells represent an attractive therapeutic target.
Macroscale additive manufacturing has seen significant advances recently, but these advances are not yet realized for the bottom-up formation of nanoscale polymeric features. We describe a platform ...technology for creating crosslinked polymer features using rapid surface-initiated crosslinking and versatile macrocrosslinkers, delivered by a microfluidic-coupled atomic force microscope known as FluidFM. A crosslinkable polymer containing norbornene moieties is delivered to a catalyzed substrate where polymerization occurs, resulting in extremely rapid chemical curing of the delivered material. Due to the living crosslinking reaction, construction of lines and patterns with multiple layers is possible, showing quantitative material addition from each deposition in a method analogous to fused filament fabrication, but at the nanoscale. Print parameters influenced printed line dimensions, with the smallest lines being 450 nm across with a vertical layer resolution of 2 nm. This nanoscale 3D printing platform of reactive polymer materials has applications for device fabrication, optical systems and biotechnology.
Background & Aims The neonatal Fc receptor for immunoglobulin (Ig)G (FcRn) protects monomeric IgG from catabolism in parenchymal and hematopoietic cells during adult life. In dendritic cells, FcRn ...also promotes presentation of antigens in association with IgG. Because IgGs with anti-bacterial specificity are a hallmark of inflammatory bowel disease, we sought to determine their significance and relationship to FcRn expression in antigen-presenting cells, focusing on IgGs specific for flagellin. Methods Levels of circulating anti-flagellin IgG were induced in wild-type and FcRn−/− mice, followed by induction of colitis with dextran sodium sulfate (DSS). Bone marrow chimera models were used to localize the site of FcRn action. Results Wild-type mice that received anti-flagellin IgG exhibited more severe colitis following administration of DSS, compared with mice that received control IgG. Wild-type mice immunized with flagellin exhibited significantly more severe colitis in response to DSS administration than that observed in similarly treated FcRn−/− mice. In chimera studies, FcRn−/− mice given wild-type bone marrow and immunized with flagellin exhibited significantly more colitis than wild-type mice given FcRn−/− bone marrow and immunized with flagellin. Serum anti-flagellin IgG levels were similar in both sets of chimeric mice, consistent with the equal participation of hematopoietic and nonhematopoeitic cells in FcRn-mediated IgG protection. Conclusions Anti-bacterial IgG antibodies are involved in the pathogenesis of colitis; this pathway requires FcRn in antigen presenting cells, the major subset of hematopoietic cells that express FcRn.
The precision spray of liquid fertilizer and pesticide to plants is an important task for agricultural robots in precision agriculture. By reducing the amount of chemicals being sprayed, it brings in ...a more economic and eco-friendly solution compared to conventional non-discriminated spray. The prerequisite of precision spray is to detect and track each plant. Conventional detection or segmentation methods detect all plants in the image captured under the robotic platform, without knowing the ID of the plant. To spray pesticides to each plant exactly once, tracking of every plant is needed in addition to detection. In this paper, we present LettuceTrack, a novel Multiple Object Tracking (MOT) method to simultaneously detect and track lettuces. When the ID of each plant is obtained from the tracking method, the robot knows whether a plant has been sprayed before therefore it will only spray the plant that has not been sprayed. The proposed method adopts YOLO-V5 for detection of the lettuces, and a novel plant feature extraction and data association algorithms are introduced to effectively track all plants. The proposed method can recover the ID of a plant even if the plant moves out of the field of view of camera before, for which existing Multiple Object Tracking (MOT) methods usually fail and assign a new plant ID. Experiments are conducted to show the effectiveness of the proposed method, and a comparison with four state-of-the-art Multiple Object Tracking (MOT) methods is shown to prove the superior performance of the proposed method in the lettuce tracking application and its limitations. Though the proposed method is tested with lettuce, it can be potentially applied to other vegetables such as broccoli or sugar beat.
Aquaculture is the fastest-growing food sector worldwide but faces sustainability challenges that need to be addressed in many ways, including genetic enhancement. Atlantic cod has re-emerged as an ...aquaculture species and tools for genetic manipulation are needed. Thus, we compared five formats of CRISPR to determine which was most efficient to generate knock outs in Atlantic cod. Cas9 protein was presented in preformed ribonucleoprotein (RNP) complexes with single guide or with duplex guide RNAs or an mRNA encoding Cas9 was used with the same two formats of guide RNAs. Cas12a was tested as RNP complexes with single guide RNAs. We found Cas9 mRNA with single guide RNA to be the most efficient format to knock out both alleles of the slc45a2 gene, which resulted in an albino-like phenotype in up to 75% of surviving larvae. DNA analysis of individual larvae revealed mosaic genotypes with variable indel mutations. The mortality of injected eggs was high, resulting in low overall efficiency. Nevertheless, this study lays the foundation for further genetic and functional research using the CRISPR/Cas9 genome editing system in Atlantic cod.
CD4⁺ T cells that recognize dietary gluten antigens presented by the disease-associated HLA-DQ2 or DQ8 molecules are central players in coeliac disease. Unbiased sequencing of the human TCRα variable ...(TRAV) and humanTCRβ variable (TRBV) genes of 68 HLA-DQ2.5-glia-α2-specific T cells from coeliac disease patients confirmed previous reports of over-usage of the TRBV7-2 gene segment, a conserved Arg residue in the complementarity-determining region (CDR) 3β loop and prevalent usage of the canonical ASSxRxTDTQY CDR3β loop among T cells with this specificity. In 30 clones that had the canonical TCRβ chain, we found a strict usage of the TRAV26-1 gene segment in the TCRα chain. There was variable usage of the TRAJ genes and diverse CDR3α sequences with no apparent conserved motifs. This study extends previous reports on biased TCR usage in both HLA-DQ2.5- and DQ8-restricted gluten-specific TCRs and provides data for further studies on TRAV and TRBV pairing.
By offering high sequencing speed and ultra-high-throughput at a low price, Illumina next-generation sequencing platforms have been widely adopted in recent years. However, an experiment with ...multiplexed library could be at risk of molecular recombination, known as "index switching", which causes a proportion of the reads to be assigned to an incorrect sample. It is reported that a new advance, exclusion amplification (ExAmp) in conjunction with the patterned flow cell technology introduced on HiSeq 3000/HiSeq 4000/HiSeq X sequencing systems, potentially suffers from a higher rate of index switching than conventional bridge amplification. We took advantage of the diverse but highly cell-specific expression of antigen receptors on immune cells to quantify index switching on single cell RNA-seq data that were sequenced on HiSeq 3000 and HiSeq 4000. By utilizing the unique antigen receptor expression, we could quantify the spread-of-signal from many different wells (n = 55 from total of three batches) due to index switching. Based on full-length T cell receptor (TCR) sequences from all samples reconstructed by TraCeR and TCR gene expression quantified by Kallisto, we found index switching in all three batches of experiments investigated. The median percentage of incorrectly detected markers was estimated to be 3.9% (interquartile range (IQR): 1.7%-7.3%). We did not detect any consistent patterns of certain indices to be more prone to switching than others, suggesting that index switching is a stochastic process. Our results confirm that index switching is a problem that affects samples run in multiplexed libraries on Illumina HiSeq 3000 and HiSeq 4000 platforms.
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