Immunodepletion and hypoxia preconditioning of mouse vompact bone cells as a novel protocol to isolate highly immunosuppressive mesenchymal stem cells

Prepublished on Liebert Instant Online December 21, 2016Compact bones (CB) are major reservoirs of mouse mesenchymal stem cells (mMSC). Here, we established a protocol to isolate MSC from CB and tested their immunosuppressive potential. Collagenase type II digestion of BM-flushed CB from C57B/6 mice was performed to liberate mMSC precursors from bone surfaces to establish nondepleted mMSC. CB cells were also immunodepleted based on the expression of CD45 (leukocytes) and TER119 (erythroid cells) to eliminate hematopoietic cells. CD45-TER119- CB cells were subsequently used to generate depleted mMSC. CB nondepleted and depleted mMSC progenitors were cultured under hypoxic conditions to establish primary mMSC cultures. CB depleted mMSC compared to nondepleted mMSC showed greater cell numbers at subculturing and had increased functional ability to differentiate into adipocytes and osteoblasts. CB depleted mMSC had high purity and expressed key mMSC markers (>85% Sca-1, CD29, CD90) with no mature hematopoietic contaminating cells (<5% CD45, CD11b) when subcultured to passage 5 (P5). Nondepleted mMSC cultures, however, were less pure and heterogenous with <72% Sca-1+, CD29+, and CD90+ cells at early passages (P1 or P2), along with high percentages of contaminating CD11b+ (35.6%) and CD45+ (39.2%) cells that persisted in culture long term. Depleted and nondepleted mMSC nevertheless exhibited similar potency to suppress total (CD3+), CD4+ and CD8+ T cell proliferation, in a dendritic cell allostimulatory one-way mixed lymphocyte reaction. CB depleted mMSC, pretreated with proinflammatory cytokines IFN-γ, TNF-α, and IL-17A, showed superior suppression of CD8+ T cell, but not CD4+ T cell proliferation, relative to untreated-mMSC. In conclusion, CB depleted mMSC established under hypoxic conditions and treated with selective cytokines represent a novel source of potent immunosuppressive MSC. As these cells have enhanced immune modulatory function, they may represent a superior product for use in clinical allotransplantation.Kisha Nandini Sivanathan, Stan Gronthos, Shane T. Grey, Darling Rojas-Canales, and Patrick T. Coate

Transcriptome profiling of IL-17A preactivated mesenchymal stem cells: a comparative study to unmodified and IFN-gamma modified mesenchymal stem cells

Published 15 February 2017Human mesenchymal stem cells pretreatment with IL-17A (MSC-17) potently enhances T cell immunosuppression but not their immunogenicity, in addition to avidly promoting the induction of suppressive regulatory T cells. The aim of this study was to identify potential mechanisms by which human MSC-17 mediate their superior immunomodulatory function. Untreated-MSC (UT-MSC), IFN-γ treated MSC (MSC-γ), and MSC-17 were assessed for their gene expression profile by microarray. Significantly regulated genes were identified for their biological functions (Database for Annotation, Visualisation and Integrated Discovery, DAVID). Microarray analyses identified 1278 differentially regulated genes between MSC-γ and UT-MSC and 67 genes between MSC-17 and UT-MSC. MSC-γ were enriched for genes involved in immune response, antigen processing and presentation, humoral response, and complement activation, consistent with increased MSC-γ immunogenicity. MSC-17 genes were associated with chemotaxis response, which may be involved in T cell recruitment for MSC-17 immunosuppression. MMP1, MMP13, and CXCL6 were highly and specifically expressed in MSC-17, which was further validated by real-time PCR. Thus, MMPs and chemokines may play a key role in mediating MSC-17 superior immunomodulatory function. MSC-17 represent a potential cellular therapy to suppress immunological T cell responses mediated by expression of an array of immunoregulatory molecules.Kisha Nandini Sivanathan, Darling Rojas-Canales, Shane T. Grey, Stan Gronthos, and Patrick T. Coate

Immunomodulatory properties of induced pluripotent stem cell-derived mesenchymal cells

Abstract not availableJia Ng, Kim Hynes, Gregory White, Kisha Nandini Sivanathan, Kate Vandyke, Peter Mark Bartold and Stan Grontho

Information exposure from consumer IoT devices: a multidimensional, network-informed measurement approach

Internet of Things (IoT) devices are increasingly found in everyday homes, providing useful functionality for devices such as TVs, smart speakers, and video doorbells. Along with their benefits come potential privacy risks, since these devices can communicate information about their users to other parties over the Internet. However, understanding these risks in depth and at scale is difficult due to heterogeneity in devices' user interfaces, protocols, and functionality. In this work, we conduct a multidimensional analysis of information exposure from 81 devices located in labs in the US and UK. Through a total of 34,586 rigorous automated and manual controlled experiments, we characterize information exposure in terms of destinations of Internet traffic, whether the contents of communication are protected by encryption, what are the IoT-device interactions that can be inferred from such content, and whether there are unexpected exposures of private and/or sensitive information (e.g., video surreptitiously transmitted by a recording device). We highlight regional differences between these results, potentially due to different privacy regulations in the US and UK. Last, we compare our controlled experiments with data gathered from an in situ user study comprising 36 participants

Culture and Patterns of Reciprocity: The Role of Exchange Type, Regulatory Focus, and Emotions

Reciprocity is a fundamental mechanism for sustained social relationships. Escalation-based theories suggest that reciprocity intensifies over time. In contrast, equity-based theories propose that people reciprocate behaviors in kind. We reconcile these conflicting perspectives by examining social exchanges across different cultural contexts. Using three complementary experiments, we investigate when, how, and why individuals in East Asian settings and those in North American settings differentially reciprocate positive versus negative behaviors over time. Study 1 demonstrated that in positively framed exchanges (i.e., giving) Americans escalated their reciprocity, but Singaporeans reciprocated in kind. However, in negatively framed exchanges (i.e., taking), Singaporeans escalated their reciprocity, but Americans reciprocated in kind. Study 2 replicated the results using Hong Kongers and showed that cultural differences in regulatory focus were associated with specific emotions (i.e., anxiety and happiness), which then escalated reciprocity. To establish causality, Study 3 manipulated regulatory focus within one culture and replicated the pattern of results

Sub-6 GHz channel modeling and evaluation in indoor industrial environments

This paper presents sub-6 GHz channel measurements using a directional antenna at the transmitter and a directional or omnidirectional antenna at the receiver at 4.145 GHz in sparse and dense industrial environments for a line-of-sight scenario. Furthermore, the first measured over-the-air error vector magnitude (EVM) results depending on different 5G new radio modulation and coding schemes (MCSs of16 QAM, 64 QAM and 256 QAM) are provided. From the measurement campaigns, the path loss exponents (PLE) using a directional and an omnidirectional antenna at the receiver in the sparse and the dense environment are 1.24/1.39 and 1.35/1.5, respectively. PLE results are lower than the theoretical free space PLE of 2, indicating that indoor industrial environments have rich multipaths. The measured power delay profiles show the maximum root mean square (RMS) delay spreads of 11 ns with a directional antenna and 34 ns with an omnidirectional antenna at the receiver in a sparse industrial environment. However, in a dense industrial environment the maximum RMS delay spreads are significantly increased: maximum RMS delay spreads range from 226 to 282 ns for the omnidirectional and the directional antenna configuration. EVMmeasurements show that to increase coverage and enable higher MCS modes to be used for reliable data transmission, in both industrial environments using a directional antenna at the transmitter and the receiver is required. The large-scale path loss models, multipath time dispersion characteristics and EVM results provide insight into the deployments of 5G networks operating at sub-6 GHz frequency bands in different industrial environment

A Real-Time Olive Fruit Detection for Harvesting Robot Based on Yolo Algorithms

Deep neural network models have become powerful tools of machine learning and artificial intelligence. They can approximate functions and dynamics by learning from examples. This paper reviews the state-of-art of deep learning-based object detection frameworks that are used for fruit detection in general and for olive fruit in particular. A dataset of olive fruit on the tree is built to train and evaluate deep models. The ultimate goal of this work is the capability of on-edge real-time olive fruit detection on the tree from digital videos. Recent work in deep neural networks has led to the development of a state-of-the-art object detector termed You Only Look Once version five (YOLOv5). This paper builds a dataset of 1.2 K source images of olive fruit on the tree and evaluates the latest object detection algorithms focusing on variants of YOLOv5 and YOLOR. The results of the YOLOv5 models show that the YOLOv5 new network models are able to extract rich olive features from images and detect the olive fruit with a high precision of higher than 0.75 mAP_0.5. YOLOv5s performs better for real-time olive fruit detection on the tree over other YOLOv5 variants and YOLOR

Sub-6 GHz channel modelling and evaluation in indoor industrial environments

This paper presents sub-6 GHz channel measurements using a directional antenna at the transmitter and a directional or omnidirectional antenna at the receiver at 4.145 GHz in sparse and dense industrial environments for a line-of-sight scenario. Furthermore, the first measured over-the-air error vector magnitude (EVM) results depending on different 5G new radio modulation and coding schemes (MCSs of16 QAM, 64 QAM and 256 QAM) are provided. From the measurement campaigns, the path loss exponents (PLE) using a directional and an omnidirectional antenna at the receiver in the sparse and the dense environment are 1.24/1.39 and 1.35/1.5, respectively. PLE results are lower than the theoretical free space PLE of 2, indicating that indoor industrial environments have rich multipaths. The measured power delay profiles show the maximum root mean square (RMS) delay spreads of 11 ns with a directional antenna and 34 ns with an omnidirectional antenna at the receiver in a sparse industrial environment. However, in a dense industrial environment the maximum RMS delay spreads are significantly increased: maximum RMS delay spreads range from 226 to 282 ns for the omnidirectional and the directional antenna configuration. EVM measurements show that to increase coverage and enable higher MCS modes to be used for reliable data transmission, in both industrial environments using a directional antenna at the transmitter and the receiver is required. The large-scale path loss models, multipath time dispersion characteristics and EVM results provide insight into the deployments of 5G networks operating at sub-6 GHz frequency bands in different industrial environments

A Defined Terminal Region of the E. coli Chromosome Shows Late Segregation and High FtsK Activity

Background: The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV. Methodology: We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a,400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPSrecognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions. Significance: Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last step

FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process