The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels

Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05–0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1–100 μM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1–10 μM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1–100 μM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1–10 μM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone

Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation

Crisis and recovery in the wake of super-salient news: Who moves markets?

Abstract We compare reactions in the prices and trading patterns of common stocks and closed-end funds, which have substantially di¤erent investor clienteles, to the September 11, 2001 terrorist attacks. Even in assets with institutional buying, retail investor trading moved prices signi…cantly lower. Although greater salience has been shown to reduce underreaction to news, we conclude the extreme salience of this event resulted in overreaction. Subsequent reversals were substantially security-speci…c and therefore not due simply to improvement in general sentiment. Consistent with microstructure theory, the speed of reversals depended signi…cantly on the relative quality and availability of information about fundamental values. terrorist attacks. Even in assets with net institutional buying, retail investor trading moved prices signi…cantly lower. Although greater salience has been shown to reduce underreaction to news, we conclude the extreme salience of this event resulted in overreaction. Subsequent reversals were substantially security-speci…c and therefore not due simply to improvement in general sentiment. Consistent with microstructure theory, the speed of reversals depended signi…cantly on the relative quality and availability of information about fundamental values. JEL classi…cations: G12, G1

Tertiary-Treated Municipal Wastewater is a Significant Point Source of Antibiotic Resistance Genes Into Duluth-Superior Harbor

In this study, the impact of tertiary-treated municipal wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline (tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance (16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus Bacteroides). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides, and human-specific Bacteroides were typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect on the quantities of antibiotic resistance genes in otherwise pristine surface waters

Allopurinol and oxypurinol promote osteoblast differentiation and increase bone formation

AbstractAllopurinol and its active metabolite, oxypurinol are widely used in the treatment of gout and hyperuricemia. They inhibit xanthine oxidase (XO) an enzyme in the purine degradation pathway that converts xanthine to uric acid. This investigation examined the effect of allopurinol and oxypurinol on bone formation, cell number and viability, gene expression and enzyme activity in differentiating and mature, bone-forming osteoblasts. Although mRNA expression remained relatively constant, XO activity decreased over time with mature osteoblasts displaying reduced levels of uric acid (20% decrease). Treatment with allopurinol and oxypurinol (0.1–1µM) reduced XO activity by up to 30%. At these concentrations, allopurinol and oxypurinol increased bone formation by osteoblasts ~4-fold and ~3-fold, respectively. Cell number and viability were unaffected. Both drugs increased tissue non-specific alkaline phosphatase (TNAP) activity up to 65%. Osteocalcin and TNAP mRNA expression was increased, 5-fold and 2-fold, respectively. Expression of NPP1, the enzyme responsible for generating the mineralisation inhibitor, pyrophosphate, was decreased 5-fold. Col1α1 mRNA expression and soluble collagen levels were unchanged. Osteoclast formation and resorptive activity were not affected by treatment with allopurinol or oxypurinol. Our data suggest that inhibition of XO activity promotes osteoblast differentiation, leading to increased bone formation in vitro

Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats

The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone‑forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14‑28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular‑shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21‑28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco's modified Eagle's medium (DMEM) after approximately 14 days (although ~3‑fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 µg/ml) for osteogenic differentiation and β‑glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well‑established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1‑100 µM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone‑forming rat osteoblasts

"Who's ready for the cave?" Thailand's Tham Luang Rescue Museum as teaching case study

This article demonstrates how a teaching case study approach can benefit museum studies courses. It takes as its focus the widely reported rescue of a boys’ football team from a flooded cave in Thailand (2018). The success of this multinational effort led to immediate calls for the establishment of a “living museum” dedicated to remembering what became known as the “Tham Luang cave rescue.” Discussion of the potential form and function of this envisaged museum plus its wider ramifications formed a key component of a newly validated international MA program taught in the UK and China by local academics and practitioners. Issues addressed include collections management, interpretation and display as well as ethical considerations relating to funding and forward planning. These were debated through groupwork, student presentations and report writing. The article reflects on this experience and the lessons it has for studying museums. It advocates the use of other topical events as the basis for similar teaching case studies on the grounds that this best equips students with the knowledge and skills required in the workplace

Severe traumatic injury during long duration spaceflight: Light years beyond ATLS

Traumatic injury strikes unexpectedly among the healthiest members of the human population, and has been an inevitable companion of exploration throughout history. In space flight beyond the Earth's orbit, NASA considers trauma to be the highest level of concern regarding the probable incidence versus impact on mission and health. Because of limited resources, medical care will have to focus on the conditions most likely to occur, as well as those with the most significant impact on the crew and mission. Although the relative risk of disabling injuries is significantly higher than traumatic deaths on earth, either issue would have catastrophic implications during space flight. As a result this review focuses on serious life-threatening injuries during space flight as determined by a NASA consensus conference attended by experts in all aspects of injury and space flight

Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1(-/-) mice

Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues

Encountering the Victims of Romanian Communism: Young People and Empathy in a Memorial Museum

Many states in post-communist East-Central Europe have established memorial museums which aim to tell the story of suffering under the communist regime. They also seek to encourage visitors to develop empathy for the victims of communist repression.This paper explores the responses of a group of young people to a memorial museum in Romania (Sighet Memorial Museum), focusing on how these visitors experienced empathy for the victims of communist-era violence. Data were collected using focus groups. Most participants showed a degree of empa- thy for the victims of suffering but this was usually shallow in nature. However some visitors displayed more“active” empathy (characterized by deeper imaginative and cogni- tive engagement). The paper explores how both the design and environment of the museum and the back- ground experiences of visitors influenced the develop- ment of empathy. It argues that empathy is not an automatic response to suffering and instead can be con- sidered as an interaction between the design of the museum and the background knowledge of visitors. The paper argues that empathy is an important means for young people to participate in remembering the commu- nist period, and is a means to make“prosthetic”memories of an authoritarian past which they have not experienced first-hand