Context-dependent regulation of feeding behaviour by the insulin receptor, DAF-2, in Caenorhabditis elegans

Insulin signalling plays a significant role in both developmental programmes and pathways modulating the neuronal signalling that controls adult behaviour. Here, we have investigated insulin signalling in food-associated behaviour in adult C. elegans by scoring locomotion and feeding on and off bacteria, the worm’s food. This analysis used mutants (daf-2, daf-18) of the insulin signalling pathway, and we provide evidence for an acute role for insulin signalling in the adult nervous system distinct from its impact on developmental programmes. Insulin receptor daf-2 mutants move slower than wild type both on and off food and showed impaired locomotory responses to food deprivation. This latter behaviour is manifest as a failure to instigate dispersal following prolonged food deprivation and suggests a role for insulin signalling in this adaptive response. Insulin receptor daf-2 mutants are also deficient in pharyngeal pumping on food and off food. Pharmacological analysis showed the pharynx of daf-2 is selectively compromised in its response to 5-HT compared to the excitatory neuropeptide FLP-17. By comparing the adaptive pharyngeal behaviour in intact worms and isolated pharyngeal preparations, we determined that an insulin-dependent signal extrinsic to the pharyngeal system is involved in feeding adaptation. Hence, we suggest that reactive insulin signalling modulates both locomotory foraging and pharyngeal pumping as the animal adapts to the absence of food. We discuss this in the context of insulin signalling directing a shift in the sensitivity of neurotransmitter systems to regulate the worm’s response to changes in food availability in the environment

Elegant Resistance: Dermot Healy’s <em>Fighting with Shadows</em>

Dermot Healy’s Fighting with Shadows (1984) features a broad array of technical innovations: the narrative focus shifts, temporal frames vary, and the inner and outer worlds of the characters frequently interchange, all generating a sense of a world that forever lies just out of sharp focus. Far from being a failure of observation, this registers a way of seeing that extends beyond merely linear modes of representation and is suggestive of a world that is not a neat, easily-observed set of phenomena. In this, Healy’s first novel, a compelling interdependence between complex narrative experiment and deeply-felt social and political engagement with the Northern Troubles is already evident. Healy’s work has always been firmly about resistance to received forms, political fixities, social malaise, and the limits of consciousness itself – and all in a richly-textured Irish landscape. This essay offers detailed analysis of how Fighting with Shadows exemplifies such acts of resistance while seeking to engage with Healy’s provincial Irish context

Elegant Resistance: Dermot Healy’s Fighting with Shadows

Dermot Healy’s Fighting with Shadows (1984) features a broad array of technical innovations: the narrative focus shifts, temporal frames vary, and the inner and outer worlds of the characters frequently interchange, all generating a sense of a world that forever lies just out of sharp focus. Far from being a failure of observation, this registers a way of seeing that extends beyond merely linear modes of representation and is suggestive of a world that is not a neat, easily-observed set of phenomena. In this, Healy’s first novel, a compelling interdependence between complex narrative experiment and deeply-felt social and political engagement with the Northern Troubles is already evident. Healy’s work has always been firmly about resistance to received forms, political fixities, social malaise, and the limits of consciousness itself – and all in a richly-textured Irish landscape. This essay offers detailed analysis of how Fighting with Shadows exemplifies such acts of resistance while seeking to engage with Healy’s provincial Irish context

Peripheral blood derived mononuclear cells enhance osteoarthritic human chondrocyte migration.

INTRODUCTION: A major problem in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into defects. Cartilage is essentially avascular and therefore its healing is not considered to involve mononuclear cells. Peripheral blood derived mononuclear cells (PBMC) offer a readily available autologous cell source for clinical use and therefore this study was designed to evaluate the effects of PBMCs on chondrocytes and cartilage. METHODS: Human primary chondrocytes and cartilage tissue explants were taken from patients undergoing total knee replacement (n = 17). Peripheral blood samples were obtained from healthy volunteers (n = 12) and mononuclear cells were isolated by density-gradient centrifugation. Cell migration and chemokinetic potential were measured using a scratch assay, xCELLigence and CyQuant assay. PCR array and quantitative PCR was used to evaluate mRNA expression of 87 cell motility and/or chondrogenic genes. RESULTS: The chondrocyte migration rate was 2.6 times higher at 3 hour time point (p < 0.0001) and total number of migrating chondrocytes was 9.7 times higher (p < 0.0001) after three day indirect PBMC stimulus and 8.2 times higher (p < 0.0001) after three day direct co-culture with PBMCs. A cartilage explant model confirmed that PBMCs also exert a chemokinetic role on ex vivo tissue. PBMC stimulation was found to significantly upregulate the mRNA levels of 2 chondrogenic genes; collagen type II (COL2A1 600-fold, p < 0.0001) and SRY box 9 (SOX9 30-fold, p < 0.0001) and the mRNA levels of 7 genes central in cell motility and migration were differentially regulated by 24h PBMC stimulation. CONCLUSION: The results support the concept that PBMC treatment enhances chondrocyte migration without suppressing the chondrogenic phenotype possibly via mechanistic pathways involving MMP9 and IGF1. In the future, peripheral blood mononuclear cells could be used as an autologous point-ofcare treatment to attract native chondrocytes from the diseased tissue to aid in cartilage repair.The authors would like to kindly acknowledge the PhD studentship from John Insall Foundation US and thank Dr. Nigel Loveridge for his statistical expertise. Dr. John Wardale acknowledges funding from the Technology Strategy Board and OrthoMimetics and Dr. Roger Brooks acknowledges funding from the National Institute for Health Research.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s13075-015-0709-

Peripheral blood derived mononuclear cells enhance the migration and chondrogenic differentiation of multipotent mesenchymal stromal cells.

A major challenge in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into damaged areas and strategies to promote this should be developed. The aim of this study was to evaluate the effect of peripheral blood derived mononuclear cell (PBMC) stimulation on mesenchymal stromal cells (MSCs) derived from the infrapatellar fat pad of human OA knee. Cell migration was measured using an xCELLigence electronic migration chamber system in combination with scratch assays. Gene expression was quantified with stem cell PCR arrays and validated using quantitative real-time PCR (rtPCR). In both migration assays PBMCs increased MSC migration by comparison to control. In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014). Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold. In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.PhD studentship for Niina Hopper was from charitable trust John Insall Foundation US. Dr. John Wardale acknowledges funding from the Technology Strategy Board and industrial partner OrthoMimetics (currently known as Tigenix) and Dr. Roger Brooks acknowledges funding from the National Institute for Health Research for their salaries.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.013393

Human osteoblasts obtained from distinct periarticular sites demonstrate differences in biological function in vitro.

AIMS: Accumulated evidence indicates that local cell origins may ingrain differences in the phenotypic activity of human osteoblasts. We hypothesized that these differences may also exist in osteoblasts harvested from the same bone type at periarticular sites, including those adjacent to the fixation sites for total joint implant components. METHODS: Human osteoblasts were obtained from the acetabulum and femoral neck of seven patients undergoing total hip arthroplasty (THA) and from the femoral and tibial cuts of six patients undergoing total knee arthroplasty (TKA). Osteoblasts were extracted from the usually discarded bone via enzyme digestion, characterized by flow cytometry, and cultured to passage three before measurement of metabolic activity, collagen production, alkaline phosphatase (ALP) expression, and mineralization. RESULTS: Osteoblasts from the acetabulum showed lower proliferation (p = 0.034), cumulative collagen release (p < 0.001), and ALP expression (p = 0.009), and produced less mineral (p = 0.006) than those from the femoral neck. Osteoblasts from the tibia produced significantly less collagen (p = 0.021) and showed lower ALP expression than those from the distal femur. CONCLUSION: We have demonstrated for the first time an anatomical regional variation in the biological behaviours of osteoblasts on either side of the hip and knee joint. The lower osteoblast proliferation, matrix production, and mineralization from the acetabulum compared to those from the proximal femur may be reflected in differences in bone formation and implant fixation at these sites. Cite this article: Bone Joint Res 2021;10(9):611-618

Peripheral blood derived mononuclear cells enhance the migration and chondrogenic differentiation of multipotent mesenchymal stromal cells.

A major challenge in cartilage repair is the lack of chondrogenic cells migrating from healthy tissue into damaged areas and strategies to promote this should be developed. The aim of this study was to evaluate the effect of peripheral blood derived mononuclear cell (PBMC) stimulation on mesenchymal stromal cells (MSCs) derived from the infrapatellar fat pad of human OA knee. Cell migration was measured using an xCELLigence electronic migration chamber system in combination with scratch assays. Gene expression was quantified with stem cell PCR arrays and validated using quantitative real-time PCR (rtPCR). In both migration assays PBMCs increased MSC migration by comparison to control. In scratch assay the wound closure was 55% higher after 3 hours in the PBMC stimulated test group (P = 0.002), migration rate was 9 times faster (P = 0.008), and total MSC migration was 25 times higher after 24 hours (P = 0.014). Analysis of MSCs by PCR array demonstrated that PBMCs induced the upregulation of genes associated with chondrogenic differentiation over 15-fold. In conclusion, PBMCs increase both MSC migration and differentiation suggesting that they are an ideal candidate for inclusion in regenerative medicine therapies aimed at cartilage repair.Peer Reviewe

Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system