Osteocytes are terminally differentiated bone cells, derived from osteoblasts, which are vital for the regulation of bone formation and resorption. ECM stiffness and cell seeding density have been ...shown to regulate osteoblast differentiation, but the precise cues that initiate osteoblast–osteocyte differentiation are not yet understood. In this study, we cultured MC3T3-E1 cells on (A) substrates of different chemical compositions and stiffnesses, as well as, (B) substrates of identical chemical composition but different stiffnesses. The effect of cell separation was investigated by seeding cells at different densities on each substrate. Cells were evaluated for morphology, alkaline phosphatase (ALP), matrix mineralisation, osteoblast specific genes (Type 1 collagen, Osteoblast specific factor (OSF-2)), and osteocyte specific proteins (dentin matrix protein 1 (DMP-1), sclerostin (Sost)). We found that osteocyte differentiation (confirmed by dendritic morphology, mineralisation, reduced ALP, Col type 1 and OSF-2 and increased DMP-1 and Sost expression) was significantly increased on soft collagen based substrates, at low seeding densities compared to cells on stiffer substrates or those plated at high seeding density. We propose that the physical nature of the ECM and the necessity for cells to establish a communication network contribute substantially to a concerted shift toward an osteocyte-like phenotype by osteoblasts in vitro.
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•MC3T3-E1 cells were cultured on collagen based substrates of different stiffnesses.•Cells display a dendritic morphology on soft collagen based substrates at low seeding density.•Genes indicating osteocyte development (DMP-1, Sost) upregulated under the same conditions.•The physical nature of the ECM may contribute to osteocyte differentiation.•At low seeding densities, MC3T3-E1 cells extend processes to establish a communication network.
Extracellular matrix (ECM) stiffness and cell density can regulate osteoblast differentiation in two dimensional environments. However, it is not yet known how osteoblast-osteocyte differentiation is ...regulated within a 3D ECM environment, akin to that existing in vivo. In this study we test the hypothesis that osteocyte differentiation is regulated by a 3D cell environment, ECM stiffness and cell density. We encapsulated MC3T3-E1 pre-osteoblastic cells at varied cell densities (0.25, 1 and 2 × 106 cells/mL) within microbial transglutaminase (mtgase) gelatin hydrogels of low (0.58 kPa) and high (1.47 kPa) matrix stiffnesses. Cellular morphology was characterised from phalloidin-FITC and 4',6-diamidino-2-phenylindole (DAPI) dilactate staining. In particular, the expression of cell dendrites, which are phenotypic of osteocyte differentiation, were identified. Immunofluorescent staining for the osteocytes specific protein DMP-1 was conducted. Biochemical analyses were performed to determine cell number, alkaline phosphatase activity and mineralisation at 2.5 hours, 3, 21 and 56 days. We found that osteocyte differentiation and the formation of an interconnected network between dendritic cells was significantly increased within low stiffness 3D matrices, compared to cells within high stiffness matrices, at high cell densities. Moreover we saw that this network was interconnected, expressed DMP-1 and also connected with osteoblast-like cells at the matrix surface. This study shows for the first time the role of the 3D physical nature of the ECM and cell density for regulating osteocyte differentiation and the formation of the osteocyte network in vitro. Future studies could apply this method to develop 3D tissue engineered constructs with an osteocyte network in place.
Hybridization and speciation Abbott, R.; Albach, D.; Ansell, S. ...
Journal of evolutionary biology,
February 2013, Letnik:
26, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Hybridization has many and varied impacts on the process of speciation. Hybridization may slow or reverse differentiation by allowing gene flow and recombination. It may accelerate speciation via ...adaptive introgression or cause near‐instantaneous speciation by allopolyploidization. It may have multiple effects at different stages and in different spatial contexts within a single speciation event. We offer a perspective on the context and evolutionary significance of hybridization during speciation, highlighting issues of current interest and debate. In secondary contact zones, it is uncertain if barriers to gene flow will be strengthened or broken down due to recombination and gene flow. Theory and empirical evidence suggest the latter is more likely, except within and around strongly selected genomic regions. Hybridization may contribute to speciation through the formation of new hybrid taxa, whereas introgression of a few loci may promote adaptive divergence and so facilitate speciation. Gene regulatory networks, epigenetic effects and the evolution of selfish genetic material in the genome suggest that the Dobzhansky–Muller model of hybrid incompatibilities requires a broader interpretation. Finally, although the incidence of reinforcement remains uncertain, this and other interactions in areas of sympatry may have knock‐on effects on speciation both within and outside regions of hybridization.
Broadly neutralizing antibodies (bnAbs) can protect against HIV infection but have not been induced by human vaccination. A key barrier to bnAb induction is vaccine priming of rare bnAb-precursor B ...cells. In a randomized, double-blind, placebo-controlled phase 1 clinical trial, the HIV vaccine-priming candidate eOD-GT8 60mer adjuvanted with AS01
had a favorable safety profile and induced VRC01-class bnAb precursors in 97% of vaccine recipients with median frequencies reaching 0.1% among immunoglobulin G B cells in blood. bnAb precursors shared properties with bnAbs and gained somatic hypermutation and affinity with the boost. The results establish clinical proof of concept for germline-targeting vaccine priming, support development of boosting regimens to induce bnAbs, and encourage application of the germline-targeting strategy to other targets in HIV and other pathogens.
Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia ...mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid–structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell–substrate interface were highly stimulated
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ε
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200
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, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo.
In this paper, we introduce the Intervention Mapping (IM) taxonomy of behaviour change methods and its potential to be developed into a coding taxonomy. That is, although IM and its taxonomy of ...behaviour change methods are not in fact new, because IM was originally developed as a tool for intervention development, this potential was not immediately apparent. Second, in explaining the IM taxonomy and defining the relevant constructs, we call attention to the existence of parameters for effectiveness of methods, and explicate the related distinction between theory-based methods and practical applications and the probability that poor translation of methods may lead to erroneous conclusions as to method-effectiveness. Third, we recommend a minimal set of intervention characteristics that may be reported when intervention descriptions and evaluations are published. Specifying these characteristics can greatly enhance the quality of our meta-analyses and other literature syntheses. In conclusion, the dynamics of behaviour change are such that any taxonomy of methods of behaviour change needs to acknowledge the importance of, and provide instruments for dealing with, three conditions for effectiveness for behaviour change methods. For a behaviour change method to be effective: (1) it must target a determinant that predicts behaviour; (2) it must be able to change that determinant; (3) it must be translated into a practical application in a way that preserves the parameters for effectiveness and fits with the target population, culture, and context. Thus, taxonomies of methods of behaviour change must distinguish the specific determinants that are targeted, practical, specific applications, and the theory-based methods they embody. In addition, taxonomies should acknowledge that the lists of behaviour change methods will be used by, and should be used by, intervention developers. Ideally, the taxonomy should be readily usable for this goal; but alternatively, it should be clear how the information in the taxonomy can be used in practice. The IM taxonomy satisfies these requirements, and it would be beneficial if other taxonomies would be extended to also meet these needs.
A high‐fibre diet and one rich in fruit and vegetables have long been associated with lower risk of chronic disease. There are several possible mechanisms underpinning these associations, but one ...likely important factor is the production of bioactive molecules from plant‐based foods by the bacteria in the colon. This links to our growing understanding of the role of the gut microbiome in promoting health. Polyphenolic‐rich plant foods have been associated with potential health effects in many studies, but the bioavailability of polyphenol compounds, as eaten, is often very low. Most of the ingested molecules enter the large intestine where they are catabolised to smaller phenolic acids that may be the key bioactive effectors. Dietary fibres, present in plant foods, are also fermented by the bacteria to short‐chain fatty acids, compounds associated with several beneficial effects on cell turnover, metabolism and eating behaviour. Polyphenols and fibre are often eaten together, but there is a lack of research investigating the interaction between these two groups of key substrates for the colonic bacteria. In a project funded by the Biotechnology and Biological Sciences Research Council Diet and Health Research Industry Club, we are investigating whether combining different fibres and polyphenol sources can enhance the production of bioactive phenolic acids to promote health. This could lead to improved dietary recommendations and to new products with enhanced potential health‐promoting actions.
OBJECTIVE:This meta-analysis (PROSPERO CRD42018100653) uses individual patient data (IPD) to assess the association between recurrence and CTNNB1 mutation status in surgically treated adult ...desmoid-type fibromatosis (DTF) patients.
SUMMARY OF BACKGROUND DATA:The majority of sporadic DTF tumors harbor a CTNNB1 (ß-catenin) mutationT41A, S45F, and S45P or are wild-type (WT). Results are conflicting regarding the recurrence risk after surgery for these mutation types.
METHODS:A systematic literature search was performed on June 6th, 2018. IPD from eligible studies was used to analyze differences in recurrence according to CTNNB1 mutation status using Cox proportional hazards analysis. Predictive factors includedsex, age, mutation type, tumor site, tumor size, resection margin status, and cohort. The PRISMA-IPD guideline was used.
RESULTS:Seven studies, describing retrospective cohorts were included and the IPD of 329 patients were used of whom 154 (46.8%) had a T41A mutation, 66 (20.1%) a S45F mutation, and 24 (7.3%) a S45P mutation, whereas 85 (25.8%) patients had a WT CTNNB1. Eighty-three patients (25.2%) experienced recurrence. Multivariable analysis, adjusting for sex, age, and tumor site yielded a P-value of 0.011 for CTNNB1 mutation. Additional adjustment for tumor size yielded a P-value of 0.082 with hazard ratioʼs of 0.83 95% confidence interval (CI) 0.48–1.42), 0.37 (95% CI 0.12–1.14), and 0.44 (95% CI 0.21–0.92) for T41A, S45P and WT DTF tumors compared to S45F DTF tumors. The effect modification between tumor size and mutation type suggests that tumor size is an important mediator for recurrence.
CONCLUSIONS:Primary sporadic DTFs harboring a CTNNB1 S45F mutation have a higher risk of recurrence after surgery compared to T41A, S45P, and WT DTF, but this association seems to be mediated by tumor size.
Activated$CD8^+ T$cells play a critical role in host defense against viruses, intracellular microbes, and tumors. It is not clear if a key regulatory transcription factor unites the effector ...functions of$CD8^+ T$cells. We now show that Eomesodermin (Eomes), a paralogue of T-bet, is induced in effector$CD8^+ T$cells in vitro and in vivo. Ectopic expression of Eomes was sufficient to invoke attributes of effector$CD8^+ T$cells, including$interferon-\gamma (IFN-\gamma)$, perform, and granzyme B. Loss-of-function analysis suggests Eomes may also be necessary for full effector differentiation of$CD8^+ T$cells. We suggest that Eomesodermin is likely to complement the actions of T-bet and act as a key regulatory gene in the development of cell-mediated immunity.
Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and ...neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.
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