Global trends of cropland phosphorus use and sustainability challenges.

To meet the growing food demand while addressing the multiple challenges of exacerbating phosphorus (P) pollution and depleting P rock reserves1-15, P use efficiency (PUE, the ratio of productive P output to P input in a defined system) in crop production needs to be improved. Although many efforts have been devoted to improving nutrient management practices on farms, few studies have examined the historical trajectories of PUE and their socioeconomic and agronomic drivers on a national scale1,2,6,7,11,16,17. Here we present a database of the P budget (the input and output of the crop production system) and PUE by country and by crop type for 1961-2019, and examine the substantial contribution of several drivers for PUE, such as economic development stages and crop portfolios. To address the P management challenges, we found that global PUE in crop production must increase to 68-81%, and recent trends indicate some meaningful progress towards this goal. However, P management challenges and opportunities in croplands vary widely among countries.

Seasonality of temperate forest photosynthesis and daytime respiration.

Terrestrial ecosystems currently offset one-quarter of anthropogenic carbon dioxide (CO2) emissions because of a slight imbalance between global terrestrial photosynthesis and respiration. Understanding what controls these two biological fluxes is therefore crucial to predicting climate change. Yet there is no way of directly measuring the photosynthesis or daytime respiration of a whole ecosystem of interacting organisms; instead, these fluxes are generally inferred from measurements of net ecosystem-atmosphere CO2 exchange (NEE), in a way that is based on assumed ecosystem-scale responses to the environment. The consequent view of temperate deciduous forests (an important CO2 sink) is that, first, ecosystem respiration is greater during the day than at night; and second, ecosystem photosynthetic light-use efficiency peaks after leaf expansion in spring and then declines, presumably because of leaf ageing or water stress. This view has underlain the development of terrestrial biosphere models used in climate prediction and of remote sensing indices of global biosphere productivity. Here, we use new isotopic instrumentation to determine ecosystem photosynthesis and daytime respiration in a temperate deciduous forest over a three-year period. We find that ecosystem respiration is lower during the day than at night-the first robust evidence of the inhibition of leaf respiration by light at the ecosystem scale. Because they do not capture this effect, standard approaches overestimate ecosystem photosynthesis and daytime respiration in the first half of the growing season at our site, and inaccurately portray ecosystem photosynthetic light-use efficiency. These findings revise our understanding of forest-atmosphere carbon exchange, and provide a basis for investigating how leaf-level physiological dynamics manifest at the canopy scale in other ecosystems.

DSFit: a feasibility pilot study of a group exercise programme for adolescents with Down syndrome.

BACKGROUND: While past research has underscored the benefits of physical activity for people with Down syndrome (DS), exercise programming that is customised to and/or accessible for children and adolescents with DS is limited. The objectives of this pilot were to (1) develop and refine an engaging exercise programme for adolescents with DS, called DSFit; (2) assess feasibility over the course of two pilot iterations; and (3) examine participant and parent feedback regarding exercise priorities and the DSFit exercise programme. METHOD: Participants were 12 unique adolescents (ages 11-17 years) with DS. Both pilot iterations of the programme consisted of weekly group exercise sessions and home exercises to complete between sessions. Physical fitness and mood/behaviour were assessed at baseline and at the end of the intervention. Parent and child goal-setting and feedback surveys were collected before and immediately after the intervention, and a 2-month follow-up assessed physical activity and exercise attitudes. Quality improvement methodology and participant/parent feedback were used to modify the second iteration to better meet the needs of our study population. Changes included an expanded age range, modified physical assessments, decreased burden of questionnaires, and video-recorded group sessions for at-home practice. RESULTS: Physical fitness evaluation of core/trunk strength and stability, lower- and upper-body strength, balance, flexibility, and walking was feasible, and the majority of participants in both pilot iterations improved in at least one category of physical assessment between baseline and end of intervention. Assessment of symptoms of anxiety, depression and behavioural concerns was also feasible and results showed slight improvements in some participants. Both parent and participant feedback indicated that participants enjoyed the programme and appreciated the opportunity to start developing sustainable exercise habits. CONCLUSIONS: A group exercise programme with supported at-home components is feasible for adolescents with DS. Future iterations will continue to examine programme efficacy with improved fitness testing and larger sample sizes. Strategies to increase at-home compliance, such as virtual sessions and parent/guardian-guided physical fitness assessments, will also be incorporated.

Increased IL-6 receptor expression and signaling in ageing cartilage can be explained by loss of TGF-ß-mediated IL-6 receptor suppression.

OBJECTIVE: Osteoarthritis (OA) development is strongly associated with ageing, possibly due to age-related changes in transforming growth factor-ß (TGF-ß) signaling in cartilage. Recently, we showed that TGF-ß suppresses interleukin (IL)-6 receptor (IL-6R) expression in chondrocytes. As IL-6 is involved in cartilage degeneration, we hypothesized that age-related loss of TGF-ß signaling results in increased IL-6R expression and signaling in ageing cartilage. DESIGN: Bovine articular cartilage was collected and immediately processed to study age-related changes in IL-6R expression using qPCR and IHC (age-range: 0.5-14 years). Moreover, cartilage from young and aged cows was stimulated with rhIL-6 and/or rhTGF-ß1 to measure IL-6-induced p-STAT3 using Western blot. Expression of STAT3-responsive genes was analyzed using qPCR. RESULTS: Expression of IL-6 receptor (bIL-6R) significantly increased in cartilage upon ageing (slope: 0.32, 95%CI: 0.20-0.45), while expression of glycoprotein 130 (bGP130) was unaffected. Cartilage stimulation with IL-6 showed increased induction of p-STAT3 upon ageing (slope: 0.14, 95%CI: 0.08-0.20). Furthermore, IL-6-mediated induction of STAT3-responsive genes like bSOCS3 and bMMP3 was increased in aged compared to young cartilage. Interestingly, the ability of TGF-ß to suppress bIL6R expression in young cartilage was lost upon ageing (slope: 0.21, 95%CI: 0.13-0.30). Concurrently, an age-related loss in TGF-ß-mediated suppression of IL-6-induced p-STAT3 and bSOCS3 expression was observed. CONCLUSIONS: Ageing results in enhanced IL-6R expression and subsequent IL-6-induced p-STAT3 signaling in articular cartilage. This is likely caused by age-related loss of protective TGF-ß signaling, resulting in loss of TGF-ß-mediated IL-6R suppression. Because of the detrimental role of IL-6 in cartilage, this mechanism may be involved in age-related OA development.

TGF-ß dampens IL-6 signaling in articular chondrocytes by decreasing IL-6 receptor expression.

OBJECTIVE: Transforming growth factor-ß (TGF-ß) is an important homeostatic regulator of cartilage. In contrast, interleukin-6 (IL-6) is a pro-inflammatory cytokine implicated in cartilage degeneration. Cross-talk between TGF-ß and IL-6 is reported in tissues other than articular cartilage. Here, we investigated regulation of IL-6 signaling by TGF-ß in articular chondrocytes. DESIGN: Human primary chondrocytes and the human G6 chondrocyte cell line were stimulated with TGF-ß1 or interleukin-1ß (IL-1ß). Expression of IL-6 and IL-6 receptor (IL-6R) was determined on mRNA and protein level. TGF-ß regulation of IL-6 signaling via phosho-STAT3 (p-STAT3) was determined using Western blot, in presence of inhibitors for IL-6R, and Janus kinase(JAK)- and activin receptor-like kinase ALK)5 kinase activity. Furthermore, induction of STAT3-responsive genes was used as a read-out for IL-6 induced gene expression. RESULTS: TGF-ß1 increased IL-6 mRNA and protein expression in both G6 and primary chondrocytes. Moreover, TGF-ß1 stimulation clearly induced p-STAT3), which was abolished by inhibition of either IL-6R, JAK- or ALK5 kinase activity. However, TGF-ß1 did not increase expression of the STAT3-responsive gene SOCS3 and pre-treatment with TGF-ß1 even inhibited induction of p-STAT3 and SOCS3 by rhIL-6. Interestingly, TGF-ß1 potently decreased IL-6R expression. In contrast, IL-1ß did increase IL-6 levels, but did not affect IL-6R expression. Finally, addition of recombinant IL-6R abolished the inhibitory effect of TGF-ß1 on IL-6-induced p-STAT3 and downstream SOCS3, BCL3, SAA1 and MMP1 expression. CONCLUSIONS: In this study we show that TGF-ß decreases IL-6R expression, thereby dampening IL-6 signaling in chondrocytes. This reveals a novel effect of TGF-ß, possibly important to restrict pro-inflammatory IL-6 effects to preserve cartilage homeostasis.

IL-37 diminishes proteoglycan loss in human OA cartilage: donor-specific link between IL-37 and MMP-3.

OBJECTIVE: A hallmark of osteoarthritis (OA) is degradation of articular cartilage proteoglycans. In isolated human OA chondrocytes, the anti-inflammatory cytokine Interleukin-37 (IL-37) lowers the expression of the proteolytic MMP and ADAMTS enzymes, which mediate this degradation. Therefore, we investigated if IL-37 protects against proteoglycan loss in freshly obtained human OA explants. MATERIAL AND METHODS: Human OA cartilage explants were incubated with IL-37. Release of sulphated proteoglycans (sGAGs) was measured with the dimethylmethylene-blue assay. Production and degradation of newly synthesized proteoglycans was measured using 35S-sulphate. Proteoglycan and proteolytic enzyme expression were analyzed by qPCR and Western Blot. Proteolytic activity was determined by measuring MMP- and ADAMTS-generated aggrecan neo-epitopes with ELISA and by using MMP-3-, MMP-13- or ADAMTS-5-inhibitors. RESULTS: Over time, a linear release of sGAGs from OA cartilage was measured. IL-37 reduced this release by 87 µg/ml (24%) 95%CI [21.04-141.4]. IL-37 did not affect 35S-sulphate incorporation or proteoglycan gene expression. In contrast, IL-37 reduced loss of 35S-sulphate labeled GAGs and reduced MMP-3 protein expression, indicating that IL-37 inhibits proteoglycan degradation. Remarkably, we observed two groups of patients; one group in which MMP-3-inhibition lowered sGAG release, and one group in which ADAMTS5-inhibition had this effect. Remarkably, IL-37 was only functional in the group of patients that responded to MMP-3-inhibition. CONCLUSION: We identified a relationship between IL-37 and reduced sGAG loss in OA cartilage. Most likely, this effect is mediated by inhibition of MMP-3 expression. These results suggest that IL-37 could be applied as therapy in a subgroup of OA patients, in which cartilage degradation is mediated by MMP-3.

Epigenetic marks of in utero exposure to gestational diabetes and childhood adiposity outcomes: the EPOCH study.

AIMS: To identify gestational diabetes mellitus exposure-associated DNA methylation changes and assess whether such changes are also associated with adiposity-related outcomes. METHODS: We performed an epigenome-wide association analysis, using Illumina 450k methylation arrays, on whole blood collected, on average, at 10.5 years of age from 81 gestational diabetes-exposed and 81 unexposed offspring enrolled in the EPOCH (Exploring Perinatal Outcomes in Children) study, and on the cord blood of 31 gestational diabetes-exposed and 64 unexposed offspring enrolled in the Colorado Healthy Start cohort. Validation was performed by pyrosequencing. RESULTS: We identified 98 differentially methylated positions associated with gestational diabetes exposure at a false discovery rate of <10% in peripheral blood, with 51 loci remaining significant (plus additional 40 loci) after adjustment for cell proportions. We also identified 2195 differentially methylation regions at a false discovery rate of <5% after adjustment for cell proportions. We prioritized loci for pyrosequencing validation and association analysis with adiposity-related outcomes based on strengths of association and effect size, network and pathway analysis, analysis of cord blood, and previous publications. Methylation in six out of nine (67%) gestational diabetes-associated genes was validated and we also showed that methylation of SH3PXD2A was significantly (P<0.05) associated with multiple adiposity-related outcomes. CONCLUSIONS: Our findings suggest that epigenetic marks may provide an important link between in utero exposure to gestational diabetes and obesity in childhood, and add to the growing body of evidence that DNA methylation is affected by gestational diabetes exposure.

Developing culturally adapted lifestyle interventions for South Asian migrant populations: a qualitative study of the key success factors and main challenges.

OBJECTIVES: South Asian migrant populations have a high risk of non-communicable diseases, such as type 2 diabetes (T2D). The aim of this study is to provide in-depth insight into key success factors and challenges in developing culturally adapted lifestyle interventions to prevent T2D within South Asian migrant populations. STUDY DESIGN: The study has a qualitative research design. METHODS: In-depth interviews, using a semi-structured interview guide, were conducted with eight researchers and project leaders from five studies of culturally adapted lifestyle interventions for South Asian migrant populations. Data were analysed using a grounded theory approach. RESULTS: Four main themes emerged as key factors for success: 'approaching the community in the right way', 'the intervention as a space for social relations', 'support from public authorities' and 'being reflexive and flexible'. Two themes emerged as challenges: 'struggling with time' and 'overemphasising cultural differences'. CONCLUSIONS: Our findings augment existing research by establishing the importance of cooperation at the organisational and institutional levels, of fostering the creation of social networks through interventions and of acknowledging the multiplicity of identities and resources among individuals of the same ethnic origin.

Osteoarthritis year in review 2016: biology.

This review highlights a selection of literature in the area of osteoarthritis biology published between the 2015 and 2016 Osteoarthritis Research Society International (OARSI) World Congress. Highlights were selected from a pubmed search covering cartilage, bone, inflammation and pain. A personal selection was made based, amongst other things, on topics presented during the 2015 conference. This covers circadian rhythm, TGF-ß signaling, autophagy, SIRT6, exercise, lubricin, TLR's, pain and NGF. Furthermore, in this review we have made an effort to connect these seemingly distant topics into one scheme of connections between them, revealing a theoretical big picture underneath.

An equivalence study: Are patient-completed and telephone interview equivalent modes of administration for the EuroQol survey?

BACKGROUND: To determine if the EuroQol Health Related Quality of Life survey produces equivalent results when administered by phone interview or patient-completed forms. METHODS: People awaiting hip or knee arthroplasty at a major metropolitan hospital participated. They were randomly assigned to receive the EuroQol Health Related Quality of Life survey via telephone, followed by a patient completed form 1 week later, or vice versa. Equivalence was determined using two one-sided tests (TOST) based on minimal clinically-important differences for the visual analogue scale (VAS) and the summary Utility Index. Cohen's Kappa scores were computed to determine agreement for the individual EuroQoL Likert scale items. RESULTS: Seventy-six from 90 (84%) participants completed the survey twice. Based on limits set at ±7 and ±0.11 for the VAS and Utility Index, respectively, equivalence was established between the two methods of administration for both the VAS (mean difference 0.05 [90% CI -3.76-3.67]) and the Utility Index (mean difference 0.06 [90% CI 0.02-0.11]). Varying levels of agreement, ranging from slight to substantial (κ = 0.17-0.67), were demonstrated for the individual health domains. The order of telephone and patient-completed survey administration had no significant effect on results. CONCLUSIONS: Equivalent results are obtained between telephone and patient-completed administration for the VAS and Utility Index of the EuroQol Survey in people with advanced hip or knee osteoarthritis. The limits of agreement for the individual health domains vary which prevents the accurate interpretation of real change in these items across modes.

Ageing is associated with reduction of mechanically-induced activation of Smad2/3P signaling in articular cartilage.

OBJECTIVE: Mechanical signals control key cellular processes in articular cartilage. Previously we have shown that mechanical compression is an important ALK5/Smad2/3P activator in cartilage explants. However, age-related changes in the cartilage are known to affect tissue mechanosensitivity and also ALK5/Smad2/3P signaling. We have investigated whether ageing of cartilage is associated with an altered response to mechanical compression. DESIGN: Articular cartilage explants of two different age groups (young-6-36 months old, aged-6 - 13 years old) were subjected to dynamic mechanical compression with 3 MPa (physiological) or 12 MPa (excessive) load. Subsequently, essential cartilage extracellular matrix (ECM) components and tissue growth factors gene expression was measured in young and aged cartilage by QPCR. Furthermore, the ability of young and aged cartilage, to activate the Smad2/3P signaling in response to compression was analyzed and compared. This was done by immunohistochemical (IH) Smad2P detection and Smad3-responsive gene expression analysis. RESULTS: Aged cartilage showed a highly reduced capacity for mechanically-mediated activation of Smad2/3P signaling when compared to young cartilage. Compression of aged cartilage, induced collagen type II (Col2a1) and fibronectin (Fn1) expression to a far lesser extent than in young cartilage. Additionally, in aged cartilage no mechanically mediated up-regulation of bone morphogenetic protein 2 (Bmp2) and connective tissue growth factor (Ctgf) was observed. CONCLUSIONS: We identified age-related changes in cellular responses to mechanical stimulation of articular cartilage. We propose that these changes might be associated with age-related alterations in cartilage functioning and can underlie mechanisms for development of age-related cartilage diseases like osteoarthritis (OA).

Unloading results in rapid loss of TGFß signaling in articular cartilage: role of loading-induced TGFß signaling in maintenance of articular chondrocyte phenotype?

OBJECTIVE: Recently it was shown that loading of articular cartilage explants activates TGFß signaling. Here we investigated if in vivo chondrocytes express permanently high TGFß signaling, and the consequence of the loss of compressive loading-mediated TGFß signaling on chondrocyte function and phenotype. METHOD: Bovine articular cartilage explants were collected within 10 min post mortem and stained immediately and after 30, 60 and 360 min for phosphorylated-Smad2, indicating active TGFß signaling. Explants were unloaded for 48 h and subsequently repeatedly loaded with a compressive load of 3 MPa. In addition, explants were cultured unloaded for 2 weeks and the effect of loading or exogenous TGFß on proteoglycan level and chondrocyte phenotype (Col10a1 mRNA expression) was analyzed. RESULTS: Unloading of articular cartilage results in rapid loss of TGFß signaling while subsequent compressive loading swiftly restored this. Loading and exogenous TGFß enhanced expression of TGFß1 and ALK5. Unloading of explants for 2 weeks resulted in proteoglycan loss and increased Col10a1 expression. Both loading and exogenous TGFß inhibited elevated Col10a1 expression but not proteoglycan loss. CONCLUSION: Our data might imply that in vivo regular physiological loading of articular cartilage leads to enduring TGFß signaling and TGFß-induced gene expression. We propose a hypothetical model in which loading activates a self-perpetuating system that prevents hypertrophic differentiation of chondrocytes and is crucial for cartilage homeostasis.

Expression of TGFß-family signalling components in ageing cartilage: age-related loss of TGFß and BMP receptors.

OBJECTIVE: Ageing is the main risk factor for osteoarthritis (OA). We investigated if expression of transforming growth factor ß (TGFß)-family components, a family which is crucial for the maintenance of healthy articular cartilage, is altered during ageing in cartilage. Moreover, we investigated the functional significance of selected age-related changes. DESIGN: Age-related changes in expression of TGFß-family members were analysed by quantitative PCR in healthy articular cartilage obtained from 42 cows (age: ¾-10 years). To obtain functional insight of selected changes, cartilage explants were stimulated with TGFß1 or bone morphogenetic protein (BMP) 9, and TGFß1 and BMP response genes were measured. RESULTS: Age-related cartilage thinning and loss of collagen type 2a1 expression (∼256-fold) was observed, validating our data set for studying ageing in cartilage. Expression of the TGFß-family type I receptors; bAlk2, bAlk3, bAlk4 and bAlk5 dropped significantly with advancing age, whereas bAlk1 expression did not. Of the type II receptors, expression of bBmpr2 decreased significantly. Type III receptor expression was unaffected by ageing. Expression of the ligands bTgfb1 and bGdf5 also decreased with age. In explants, an age-related decrease in TGFß1-response was observed for the pSmad3-dependent gene bSerpine1 (P = 0.016). In contrast, ageing did not affect BMP9 signalling, an Alk1 ligand, as measured by expression of the pSmad1/5 dependent gene bId1. CONCLUSIONS: Ageing negatively affects both the TGFß-ALK5 and BMP-BMPR signalling routes, and aged chondrocytes display a lowered pSmad3-dependent response to TGFß1. Because pSmad3 signalling is essential for cartilage homeostasis, we propose that this change contributes to OA development.

Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage.

OBJECTIVES: In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA. METHODS: We cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology. RESULTS: Dox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure. CONCLUSIONS: Our data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.

The high affinity ALK1-ligand BMP9 induces a hypertrophy-like state in chondrocytes that is antagonized by TGFß1.

OBJECTIVE: In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFß1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFß1 signaling was also studied. DESIGN: Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFß1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFß/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line. RESULTS: In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFß1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFß/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFß1. CONCLUSIONS: This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFß1. This observation underlines the importance of TGFß1 in maintenance of chondrocyte phenotype.

TGF-ß is a potent inducer of Nerve Growth Factor in articular cartilage via the ALK5-Smad2/3 pathway. Potential role in OA related pain?

OBJECTIVE: Pain is the main problem for patients with osteoarthritis (OA). Pain is linked to inflammation, but in OA a subset of patients suffers from pain without inflammation, indicating an alternative source of pain. Nerve Growth Factor (NGF) inhibition is very efficient in blocking pain during OA, but the source of NGF is unclear. We hypothesize that damaged cartilage in OA releases Transforming Growth Factor-ß (TGF-ß), which in turn stimulates chondrocytes to produce NGF. DESIGN: Murine and human chondrocyte cell lines, primary bovine and human chondrocytes, and cartilage explants from bovine metacarpal joints and human OA joints were stimulated with TGF-ß1 and/or Interleukin-1 (IL-1)ß. We analyzed NGF expression on mRNA level with QPCR and stained human OA cartilage for NGF immunohistochemically. Cultures were additionally pre-incubated with inhibitors for TAK1, Smad2/3 or Smad1/5/8 signaling to identify the TGF-ß pathway inducing NGF. RESULTS: NGF expression was consistently induced in higher levels by TGF-ß than IL-1 in all of our experiments: murine, bovine and human origin, in cell lines, primary chondrocytes and explants cultures. TAK1 inhibition consistently reduced TGF-ß-induced NGF whereas it fully blocked IL-1ß-induced NGF expression. In contrast, ALK5-Smad2/3 inhibition fully blocked TGF-ß-induced NGF expression. Despite the large variation in basal NGF in human OA samples (mRNA and histology), TGF-ß exposure led to a consistent high level of NGF induction. CONCLUSION: We show for the first time that TGF-ß induces NGF expression in chondrocytes, in a ALK5-Smad2/3 dependent manner. This reveals a potential alternative non-inflammatory source of pain in OA.

The benzene radical anion: A computationally demanding prototype for aromatic anions.

The benzene radical anion is studied with ab initio coupled-cluster theory in large basis sets. Unlike the usual assumption, we find that, at the level of theory investigated, the minimum energy geometry is non-planar with tetrahedral distortion at two opposite carbon atoms. The anion is well known for its instability to auto-ionization which poses computational challenges to determine its properties. Despite the importance of the benzene radical anion, the considerable attention it has received in the literature so far has failed to address the details of its structure and shape-resonance character at a high level of theory. Here, we examine the dynamic Jahn-Teller effect and its impact on the anion potential energy surface. We find that a minimum energy geometry of C2 symmetry is located below one D2h stationary point on a C2h pseudo-rotation surface. The applicability of standard wave function methods to an unbound anion is assessed with the stabilization method. The isotropic hyperfine splitting constants (Aiso) are computed and compared to data obtained from experimental electron spin resonance experiments. Satisfactory agreement with experiment is obtained with coupled-cluster theory and large basis sets such as cc-pCVQZ.

Physiological and excessive mechanical compression of articular cartilage activates Smad2/3P signaling.

OBJECTIVE: Transforming growth factor beta (TGF-ß) in articular cartilage can signal via two routes, the ALK5/Smad2/3P and the ALK1/Smad1/5/8P route, the first being protective and the latter favoring chondrocyte terminal differentiation. Since biomechanical factors are known to play an essential role in osteoarthritis (OA) initiation and progression, we investigated if excessive mechanical compression can alter TGF-ß signaling in cartilage shifting it from ALK5/Smad2/3P to ALK1/Smad1/5/8P pathway, favoring terminal differentiation of chondrocytes. DESIGN: Articular cartilage explants were harvested from bovine metacarpophalangeal joints. After equilibration, explants were subjected to unconfined dynamic mechanical compression (1 Hz) with 3 MPa (physiological) or 12 MPa (excessive) stress. After different time intervals samples were frozen and mRNA levels of selected genes were examined using real-time polymerase chain reaction. RESULTS: In articular cartilage compressed with 3 MPa and also 12 MPa stress the expression of Smad2/3P responsive genes bSerpine1, bSmad7 and bAlk5 was up-regulated, whereas the expression of Smad1/5/8P responsive gene bId1 was down-regulated. Furthermore, the expression of bTgfb1 was significantly up-regulated in both compression groups. When ALK5/Smad2/3P pathway was blocked with a selective ALK4/5/7 inhibitor, the effect of excessive mechanical compression on bSmad7 and bAlk5 expression was prevented. CONCLUSIONS: Here we show that excessive mechanical compression alone is not able to shift TGF-ß signaling toward the ALK1/Smad1/5/8P pathway. In contrast, we show that mechanical compression not only with physiological but also with excessive stress can activate Smad2/3P signaling, which is known to be protective for articular cartilage and to block chondrocyte terminal differentiation.

Evidence for multiple sex-determining loci in Tasmanian Atlantic salmon (Salmo salar).

Phenotypic sex in salmonids is determined primarily by a genetic male heterogametic system; yet, sex reversal can be accomplished via hormonal treatment. In Tasmanian Atlantic salmon aquaculture, to overcome problems associated with early sexual maturation in males, sex-reversed females are crossed with normal females to produce all female stock. However, phenotypic distinction of sex-reversed females (neo-males) from true males is problematic. We set out to identify genetic markers that could make this distinction. Microsatellite markers from chromosome 2 (Ssa02), to which the sex-determining locus (SEX) has been mapped in two Scottish Atlantic salmon families, did not predict sex in a pilot study of seven families. A TaqMan 64 SNP genome-wide scan suggested SEX was on Ssa06 in these families, and this was confirmed by microsatellite markers. A survey of 58 families in total representing 38 male lineages in the SALTAS breeding program found that 34 of the families had SEX on Ssa02, in 22 of the families SEX was on Ssa06, and two of the families had a third SEX locus, on Ssa03. A PCR test using primers designed from the recently published sdY gene is consistent with Tasmanian Atlantic salmon having a single sex-determining gene that may be located on at least three linkage groups.

Enhanced antinociceptive efficacy of epidural compared with i.v. methadone in a rat model of thermal nociception.

BACKGROUND: The properties of methadone suggest a potential advantage for epidural over i.v. administration for pain relief, but little supportive evidence exists. METHODS: To investigate the pharmacokinetic and the pharmacodynamic properties of epidural and i.v. methadone, four doses of methadone (0.1, 0.25, 0.5, and 0.75 mg kg(-1)) were investigated by each route in a rat model. The tail-flick and hot water tail immersion test were used for thermal nociception. The magnitude of antinociceptive efficacy was expressed as per cent maximal possible effect (%MPE) of tail withdrawal latency, and the area under the %MPE vs time curve indicated the cumulative antinociceptive effect. A pharmacokinetic model describing the disposition and elimination of methadone was established. RESULTS: The pharmacokinetic profiles of methadone were not significantly different after epidural and i.v. administration. A two-compartment model with saturable elimination provided a good fit of the experimental data. At equivalent doses, epidural methadone produced higher cumulative antinociceptive effect in both thermal models. Supraspinal opioid effect, assessed by pinna reflex presence, was significantly lower with epidural methadone at equivalent doses. The duration of antinociceptive effect was longer with epidural administration of 0.5 and 0.75 mg kg(-1) doses. CONCLUSIONS: Epidural administration of methadone in rats resulted in systemic exposure similar to that after i.v. administration, but improved thermal antinociceptive efficacy, and reduced supraspinal undesired effects. The findings suggest the presence of local effect at the spinal cord level, in addition to the systemic effect produced by epidural methadone.