Developmental principles informing human pluripotent stem cell differentiation to cartilage and bone.

Human pluripotent stem cells can differentiate into any cell type given appropriate signals and hence have been used to research early human development of many tissues and diseases. Here, we review the major biological factors that regulate cartilage and bone development through the three main routes of neural crest, lateral plate mesoderm and paraxial mesoderm. We examine how these routes have been used in differentiation protocols that replicate skeletal development using human pluripotent stem cells and how these methods have been refined and improved over time. Finally, we discuss how pluripotent stem cells can be employed to understand human skeletal genetic diseases with a developmental origin and phenotype, and how developmental protocols have been applied to gain a better understanding of these conditions.

SIRT1 activity orchestrates ECM expression during hESC-chondrogenic differentiation.

Epigenetic modification is a key driver of differentiation, and the deacetylase Sirtuin1 (SIRT1) is an established regulator of cell function, ageing, and articular cartilage homeostasis. Here we investigate the role of SIRT1 during development of chondrocytes by using human embryonic stem cells (hESCs). HESC-chondroprogenitors were treated with SIRT1 activator; SRT1720, or inhibitor; EX527, during differentiation. Activation of SIRT1 early in 3D-pellet culture led to significant increases in the expression of ECM genes for type-II collagen (COL2A1) and aggrecan (ACAN), and chondrogenic transcription factors SOX5 and ARID5B, with SOX5 ChIP analysis demonstrating enrichment on the chondrocyte specific -10 (A1) enhancer of ACAN. Unexpectedly, when SIRT1 was activated, while ACAN was enhanced, glycosaminoglycans (GAGs) were reduced, paralleled by down regulation of gene expression for N-acetylgalactosaminyltransferase type 1 (GALNT1) responsible for GAG chain initiation/elongation. A positive correlation between ARID5B and COL2A1 was observed, and co-IP assays indicated association of ARID5B with SIRT1, further suggesting that COL2A1 expression is promoted by an ARID5B-SIRT1 interaction. In conclusion, SIRT1 activation positively impacts on the expression of the main ECM proteins, while altering ECM composition and suppressing GAG content during human cartilage development. These results suggest that SIRT1 activity has a differential effect on GAGs and proteins in developing hESC-chondrocytes and could only be beneficial to cartilage development and matrix protein synthesis if balanced by addition of positive GAG mediators.

Spherulitic Lead Calcium Apatite Minerals in Lead Water Pipes Exposed to Phosphate-Dosed Tap Water.

Phosphate dosing is the principle strategy used in the United Kingdom to reduce the concentration of lead in tap waters supplied by lead water pipes. The mechanisms of phosphate-mediated lead control are not fully understood, but solid solutions of lead calcium apatite are thought to play an important role. This study investigated the microstructure of a lead pipe, supplied with high-alkalinity tap water, in which the lead calcium apatite crystals were spherulitic having rounded and dumb-bell-shaped morphologies. XRD, Fourier transform infrared spectroscopy, optical microscopy, Raman spectroscopy, scanning electron microscopy, and energy-dispersive spectroscopy showed that the lead pipe had a well-established inner layer of litharge; a middle layer containing lead calcium apatite spherulites, plumbonacrite, and some hydrocerussite; and an outer layer containing iron, lead, phosphorus, calcium, silicon, and aluminum. It was found that spherulitic lead calcium apatite could be grown in the laboratory by adding hydrocerussite to synthetic soft and hard water-containing phosphate, chloride, and citrate ions at pH 5.5 but not when the citrate was absent. This suggests that dissolved organic molecules might play a role in spherulite formation on lead water pipes. These molecules might inhibit the formation of lead calcium apatite, reducing the effectiveness of phosphate dosing in lead water pipes.

Pluripotent stem cells for skeletal tissue engineering.

Here, we review the use of human pluripotent stem cells for skeletal tissue engineering. A number of approaches have been used for generating cartilage and bone from both human embryonic stem cells and induced pluripotent stem cells. These range from protocols relying on intrinsic cell interactions and signals from co-cultured cells to those attempting to recapitulate the series of steps occurring during mammalian skeletal development. The importance of generating authentic tissues rather than just differentiated cells is emphasized and enabling technologies for doing this are reported. We also review the different methods for characterization of skeletal cells and constructs at the tissue and single-cell level, and indicate newer resources not yet fully utilized in this field. There have been many challenges in this research area but the technologies to overcome these are beginning to appear, often adopted from related fields. This makes it more likely that cost-effective and efficacious human pluripotent stem cell-engineered constructs may become available for skeletal repair in the near future.

Investigating food insecurity measurement globally to inform practice locally: a rapid evidence review.

The objective of this review was to examine the methods used to measure food insecurity (FI) globally, to inform considerations relating to adopting a novel, or reviewing an existing, FI measurement approach in developed countries. Considerations for measurement are examined with particular applicability to the United Kingdom (UK) which has recently announced adoption of the US Household Food Security Survey Module (HFSSM) as an indicator to facilitate annual FI monitoring. This study uses a Rapid Evidence Assessment (REA) methodological approach to systematically review the literature on FI measurement and considers: geographical jurisdiction, methodological approach, sampling strategy, FI indicator(s) used, and implications for measurement. Results found that the majority of papers reviewed emanate from North America with the US Household Food Security Scale Module (HFSSM) and its various adapted forms being the most commonly reported indicator. FI is becoming a key concern within developed countries with a range of indicators being used to report on the severity of the issue. This paper provides a contribution to knowledge by: (i) identifying various approaches to FI measurement and commonalities of existing measurement approaches; (ii) providing a summation of the methodologies and findings of studies relating to FI measurement, and associated implications for measurement, (iii) providing a justification evidenced by the literature for the adoption of the HFSSM in the UK; and (iv) assessing the methodological usefulness of a REA review. Understanding the components of robust FI indicators and their effectiveness can help inform existing and novel measurement approaches to ensure that data collected on FI are meaningful and thereby useful to inform future policy work in this area.

Knowledge transfer across scientific disciplines.

Thomas Kuhn suggested that symbolic generalizations are applied to concrete systems by a process involving exemplars and analogical reasoning. Using the related concepts of theoretical and formal templates, I argue that the process of applying templates can in some cases be made explicit and that we do not need to rely on similarity relations and tacit knowledge. In so doing I show how some formal models can be transferred from one scientific field to another. Examples include scale-free networks, the Lotka-Volterra model from biology, and the Goodwin model in economics. I also argue that this explicit approach has advantages over the more psychologically oriented approach of Kuhn and explain the sense in which templates do and do not produce unification.

Sustained Bauxite Residue Rehabilitation with Gypsum and Organic Matter 16 years after Initial Treatment.

Bauxite residue is a high volume byproduct of alumina manufacture which is commonly disposed of in purpose-built bauxite residue disposal areas (BRDAs). Natural waters interacting with bauxite residue are characteristically highly alkaline, and have elevated concentrations of Na, Al, and other trace metals. Rehabilitation of BRDAs is therefore often costly and resource/infrastructure intensive. Data is presented from three neighboring plots of bauxite residue that was deposited 20 years ago. One plot was amended 16 years ago with process sand, organic matter, gypsum, and seeded (fully treated), another plot was amended 16 years ago with process sand, organic matter, and seeded (partially treated), and a third plot was left untreated. These surface treatments lower alkalinity and salinity, and thus produce a substrate more suitable for biological colonisation from seeding. The reduction of pH leads to much lower Al, V, and As mobility in the actively treated residue and the beneficial effects of treatment extend passively 20-30 cm below the depth of the original amendment. These positive rehabilitation effects are maintained after 2 decades due to the presence of an active and resilient biological community. This treatment may provide a lower cost solution to BRDA end of use closure plans and orphaned BRDA rehabilitation.

Commensal-derived OMVs elicit a mild proinflammatory response in intestinal epithelial cells.

Under normal physiological conditions, the intestinal immunity remains largely hyporesponsive to the commensal microbiota, yet also retains the inherent ability to rapidly respond to pathogenic antigens. However, immunomodulatory activities of extracellular products from commensal bacteria have been little studied, with previous investigations generally utilizing the live bacterium to study microbiota-epithelial interactions. In this study, we demonstrate that extracellular products of a commensal bacterium, Escherichia coli C25, elicit a moderate release of proinflammatory IL-8 and stimulate transcriptional up-regulation of Toll-like receptors (TLRs) in intestinal epithelial cell lines HT29-19A and Caco-2. Additionally, we show that removal of outer membrane vesicles (OMVs) reduces the proinflammatory effect of secreted products from E. coli C25. Furthermore, we show that isolated OMVs have a dose-dependent proinflammatory effect on intestinal epithelial cells (IECs). Interestingly, a relatively high concentration (40 µg ml-1 protein) of OMVs had no significant regulatory effects on TLR mRNA expression in both cell lines. Finally, we also demonstrate that pre-incubation with E. coli C25-derived OMVs subsequently inhibited the internalization of the bacterium itself in both cell lines. Taken together, our results suggest that commensal-derived extracellular products, in particular OMVs, could significantly contribute to intestinal homeostasis. We also demonstrate a unique interaction between commensal-derived OMVs and host cells.

A Novel Rhamnose-Rich Hetero-exopolysaccharide Isolated from Lactobacillus paracasei DG Activates THP-1 Human Monocytic Cells.

Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of l-rhamnose, d-galactose, and N-acetyl-d-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host. IMPORTANCE: The consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system.

Viability of Lucilia sericata maggots after exposure to wound antiseptics.

After debridement and before dressing a wound with maggots of calliphorid flies, one frequently performed step is the application of antiseptics to the prepared wound bed. However, the concomitant application of antiseptic agents during maggot therapy is regarded controversial as antiseptics may interfere with maggots' viability. In this experimental in vitro study, the viability of fly maggots was investigated after exposure to various antiseptics frequently used in wound care. Here, we show that Lucilia sericata fly maggots can survive up to an hour's exposure to wound antiseptics such as octenidine, povidone-iodine or polihexanide. Concomitant short-term application of wound antiseptics together with maggots on wound beds is tolerated by larvae and does not impair their viability.

Effect of a retinoic acid analogue on BMP-driven pluripotent stem cell chondrogenesis.

Osteoarthritis is the most common degenerative joint condition, leading to articular cartilage (AC) degradation, chronic pain and immobility. The lack of appropriate therapies that provide tissue restoration combined with the limited lifespan of joint-replacement implants indicate the need for alternative AC regeneration strategies. Differentiation of human pluripotent stem cells (hPSCs) into AC progenitors may provide a long-term regenerative solution but is still limited due to the continued reliance upon growth factors to recapitulate developmental signalling processes. Recently, TTNPB, a small molecule activator of retinoic acid receptors (RARs), has been shown to be sufficient to guide mesodermal specification and early chondrogenesis of hPSCs. Here, we modified our previous differentiation protocol, by supplementing cells with TTNPB and administering BMP2 at specific times to enhance early development (referred to as the RAPID-E protocol). Transcriptomic analyses indicated that activation of RAR signalling significantly upregulated genes related to limb and embryonic skeletal development in the early stages of the protocol and upregulated genes related to AC development in later stages. Chondroprogenitors obtained from RAPID-E could generate cartilaginous pellets that expressed AC-related matrix proteins such as Lubricin, Aggrecan, and Collagen II, but additionally expressed Collagen X, indicative of hypertrophy. This protocol could lay the foundations for cell therapy strategies for osteoarthritis and improve the understanding of AC development in humans.

Bacillus-based probiotic cleansers reduce the formation of dry biofilms on common hospital surfaces.

In the absence of liquid suspension, dry biofilms can form upon hard surfaces within a hospital environment, representing a healthcare-associated infection risk. Probiotic cleansers using generally recognized as safe organisms, such as those of the Bacillus genus, represent a potential strategy for the reduction of dry biofilm bioburden. The mechanisms of action and efficacy of these cleaners are, however, poorly understood. To address this, a preventative dry biofilm assay was developed using steel, melamine, and ceramic surfaces to assess the ability of a commercially available Bacillus spp. based probiotic cleanser to reduce the surface bioburden of Escherichia coli and Staphylococcus aureus. Via this assay, phosphate-buffered saline controls were able to generate dry biofilms within 7 days of incubation, with the application of the probiotic cleanser able to prevent >97.7% of dry biofilm formation across both pathogen analogs and surface types. Further to this, surfaces treated with the probiotic mixture alone also showed a reduction in dry biofilm across both pathogen and surface types. Confocal laser scanning microscopy imaging indicated that the probiotic bacteria were able to germinate and colonize surfaces, likely forming a protective layer upon these hard surfaces.

Directed differentiation of hPSCs through a simplified lateral plate mesoderm protocol for generation of articular cartilage progenitors.

Developmentally, the articular joints are derived from lateral plate (LP) mesoderm. However, no study has produced both LP derived prechondrocytes and preosteoblasts from human pluripotent stem cells (hPSC) through a common progenitor in a chemically defined manner. Differentiation of hPSCs through the authentic route, via an LP-osteochondral progenitor (OCP), may aid understanding of human cartilage development and the generation of effective cell therapies for osteoarthritis. We refined our existing chondrogenic protocol, incorporating knowledge from development and other studies to produce a LP-OCP from which prechondrocyte- and preosteoblast-like cells can be generated. Results show the formation of an OCP, which can be further driven to prechondrocytes and preosteoblasts. Prechondrocytes cultured in pellets produced cartilage like matrix with lacunae and superficial flattened cells expressing lubricin. Additionally, preosteoblasts were able to generate a mineralised structure. This protocol can therefore be used to investigate further cartilage development and in the development of joint cartilage for potential treatments.

Optogenetic manipulation of BMP signaling to drive chondrogenic differentiation of hPSCs.

Optogenetics is a rapidly advancing technology combining photochemical, optical, and synthetic biology to control cellular behavior. Together, sensitive light-responsive optogenetic tools and human pluripotent stem cell differentiation models have the potential to fine-tune differentiation and unpick the processes by which cell specification and tissue patterning are controlled by morphogens. We used an optogenetic bone morphogenetic protein (BMP) signaling system (optoBMP) to drive chondrogenic differentiation of human embryonic stem cells (hESCs). We engineered light-sensitive hESCs through CRISPR-Cas9-mediated integration of the optoBMP system into the AAVS1 locus. The activation of optoBMP with blue light, in lieu of BMP growth factors, resulted in the activation of BMP signaling mechanisms and upregulation of a chondrogenic phenotype, with significant transcriptional differences compared to cells in the dark. Furthermore, cells differentiated with light could form chondrogenic pellets consisting of a hyaline-like cartilaginous matrix. Our findings indicate the applicability of optogenetics for understanding human development and tissue engineering.

The evolution of alkaliphilic biofilm communities in response to extreme alkaline pH values.

Extremes of pH present a challenge to microbial life and our understanding of survival strategies for microbial consortia, particularly at high pH, remains limited. The utilization of extracellular polymeric substances within complex biofilms allows micro-organisms to obtain a greater level of control over their immediate environment. This manipulation of the immediate environment may confer a survival advantage in adverse conditions to biofilms. Within the present study alkaliphilic biofilms were created at pH 11.0, 12.0, or 13.0 from an existing alkaliphilic community. In each pH system, the biofilm matrix provided pH buffering, with the internal pH being 1.0-1.5 pH units lower than the aqueous environment. Increasing pH resulted in a reduced removal of substrate and standing biomass associated with the biofilm. At the highest pH investigated (pH 13.0), the biofilms matrix contained a greater degree of eDNA and the microbial community was dominated by Dietzia sp. and Anaerobranca sp.

Development of human cartilage circadian rhythm in a stem cell-chondrogenesis model.

The circadian clock in murine articular cartilage is a critical temporal regulatory mechanism for tissue homeostasis and osteoarthritis. However, translation of these findings into humans has been hampered by the difficulty in obtaining circadian time series human cartilage tissues. As such, a suitable model is needed to understand the initiation and regulation of circadian rhythms in human cartilage. Methods: We used a chondrogenic differentiation protocol on human embryonic stem cells (hESCs) as a proxy for early human chondrocyte development. Chondrogenesis was validated using histology and expression of pluripotency and differentiation markers. The molecular circadian clock was tracked in real time by lentiviral transduction of human clock gene luciferase reporters. Differentiation-coupled gene expression was assessed by RNAseq and differential expression analysis. Results: hESCs lacked functional circadian rhythms in clock gene expression. During chondrogenic differentiation, there was an expected reduction of pluripotency markers (e.g., NANOG and OCT4) and a significant increase of chondrogenic genes (SOX9, COL2A1 and ACAN). Histology of the 3D cartilage pellets at day 21 showed a matrix architecture resembling human cartilage, with readily detectable core clock proteins (BMAL1, CLOCK and PER2). Importantly, the circadian clocks in differentiating hESCs were activated between day 11 (end of the 2D stage) and day 21 (10 days after 3D differentiation) in the chondrogenic differentiation protocol. RNA sequencing revealed striking differentiation coupled changes in the expression levels of most clock genes and a range of clock regulators. Conclusions: The circadian clock is gradually activated through a differentiation-coupled mechanism in a human chondrogenesis model. These findings provide a human 3D chondrogenic model to investigate the role of the circadian clock during normal homeostasis and in diseases such as osteoarthritis.

Conceptualising household food insecurity in Northern Ireland: risk factors, implications for society and the economy, and recommendations for business and policy response.

Household food insecurity in developed nations has been identified as a significant public health concern. Although various research on the topic exists, such as contributors to food insecurity, and implications for individual physical and mental health outcomes; there is currently a lack of consideration as to how individual implications of food insecurity such as poor physical and mental health can consequently impact on business and the wider economy. In addition, there is a lack of conceptual literature related to food insecurity. Stakeholder interviews (n = 19) were conducted, and data were used to inform the conceptual model (risk factors, potential implications for individuals, the economy and business, and opportunities for business and policy response). The main suggested implications related to business and the economy were reduced contribution to the workforce and the economy, and increased cost pressures on the National Health Service. Business responses suggested included the inclusion of initiatives to address food insecurity in corporate social responsibility strategies, and further involvement of food businesses/retailers in redistributing surplus food. Policy responses suggested included policies relating to welfare, wages and work contracts, food redistribution incentives, sustainability, and community interventions in disadvantaged areas. The resulting model is unique in conceptualising food insecurity in the Northern Ireland context, with applicability to the UK and other developed nations.

Regulation of TGFß Signalling by TRPV4 in Chondrocytes.

The growth factor TGFß and the mechanosensitive calcium-permeable cation channel TRPV4 are both important for the development and maintenance of many tissues. Although TRPV4 and TGFß both affect core cellular functions, how their signals are integrated is unknown. Here we show that pharmacological activation of TRPV4 significantly increased the canonical response to TGFß stimulation in chondrocytes. Critically, this increase was only observed when TRPV4 was activated after, but not before TGFß stimulation. The increase was prevented by pharmacological TRPV4 inhibition or knockdown and is calcium/CamKII dependent. RNA-seq analysis after TRPV4 activation showed enrichment for the TGFß signalling pathway and identified JUN and SP1 as key transcription factors involved in this response. TRPV4 modulation of TGFß signalling represents an important pathway linking mechanical signalling to tissue development and homeostasis.

Hydrogenotrophic Methanogenesis Under Alkaline Conditions.

A cement-based geological disposal facility (GDF) is one potential option for the disposal of intermediate level radioactive wastes. The presence of both organic and metallic materials within a GDF provides the opportunity for both acetoclastic and hydrogenotrophic methanogenesis. However, for these processes to proceed, they need to adapt to the alkaline environment generated by the cementitious materials employed in backfilling and construction. Within the present study, a range of alkaline and neutral pH sediments were investigated to determine the upper pH limit and the preferred route of methane generation. In all cases, the acetoclastic route did not proceed above pH 9.0, and the hydrogenotrophic route dominated methane generation under alkaline conditions. In some alkaline sediments, acetate metabolism was coupled to hydrogenotrophic methanogenesis via syntrophic acetate oxidation, which was confirmed through inhibition studies employing fluoromethane. The absence of acetoclastic methanogenesis at alkaline pH values (>pH 9.0) is attributed to the dominance of the acetate anion over the uncharged, undissociated acid. Under these conditions, acetoclastic methanogens require an active transport system to access their substrate. The data indicate that hydrogenotrophic methanogenesis is the dominant methanogenic pathway under alkaline conditions (>pH 9.0).

Optogenetic Control of the BMP Signaling Pathway.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor ß (TGFß) superfamily and have crucial roles during development; including mesodermal patterning and specification of renal, hepatic, and skeletal tissues. In vitro developmental models currently rely upon costly and unreliable recombinant BMP proteins that do not enable dynamic or precise activation of the BMP signaling pathway. Here, we report the development of an optogenetic BMP signaling system (optoBMP) that enables rapid induction of the canonical BMP signaling pathway driven by illumination with blue light. We demonstrate the utility of the optoBMP system in multiple human cell lines to initiate signal transduction through phosphorylation and nuclear translocation of SMAD1/5, leading to upregulation of BMP target genes including Inhibitors of DNA binding ID2 and ID4. Furthermore, we demonstrate how the optoBMP system can be used to fine-tune activation of the BMP signaling pathway through variable light stimulation. Optogenetic control of BMP signaling will enable dynamic and high-throughput intervention across a variety of applications in cellular and developmental systems.