Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Sexually dimorphic effects of prenatal alcohol exposure on the murine skeleton.

BACKGROUND: Prenatal alcohol exposure (PAE) can result in lifelong disabilities known as foetal alcohol spectrum disorder (FASD) and is associated with childhood growth deficiencies and increased bone fracture risk. However, the effects of PAE on the adult skeleton remain unclear and any potential sexual dimorphism is undetermined. Therefore, we utilised a murine model to examine sex differences with PAE on in vitro bone formation, and in the juvenile and adult skeleton. METHODS: Pregnant C57BL/6J female mice received 5% ethanol in their drinking water during gestation. Primary calvarial osteoblasts were isolated from neonatal offspring and mineralised bone nodule formation and gene expression assessed. Skeletal phenotyping of 4- and 12-week-old male and female offspring was conducted by micro-computed tomography (µCT), 3-point bending, growth plate analyses, and histology. RESULTS: Osteoblasts from male and female PAE mice displayed reduced bone formation, compared to control (≤ 30%). Vegfa, Vegfb, Bmp6, Tgfbr1, Flt1 and Ahsg were downregulated in PAE male osteoblasts only, whilst Ahsg was upregulated in PAE females. In 12-week-old mice, µCT analysis revealed a sex and exposure interaction across several trabecular bone parameters. PAE was detrimental to the trabecular compartment in male mice compared to control, yet PAE females were unaffected. Both male and female mice had significant reductions in cortical parameters with PAE. Whilst male mice were negatively affected along the tibial length, females were only distally affected. Posterior cortical porosity was increased in PAE females only. Mechanical testing revealed PAE males had significantly reduced bone stiffness compared to controls; maximum load and yield were reduced in both sexes. PAE had no effect on total body weight or tibial bone length in either sex. However, total growth plate width in male PAE mice compared to control was reduced, whilst female PAE mice were unaffected. 4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals. CONCLUSIONS: Evidence herein suggests, for the first time, that PAE exerts divergent sex effects on the skeleton, possibly influenced by underlying sex-specific transcriptional mechanisms of osteoblasts. Establishing these sex differences will support future policies and clinical management of FASD.

Prenatal alcohol exposure (PAE) can lead to a set of lifelong cognitive, behavioural, and physical disabilities known as foetal alcohol spectrum disorder (FASD). FASD is a significant burden on healthcare, justice and education systems, which is set to worsen with rising alcohol consumption rates. FASD children have an increased risk of long bone fracture and adolescents are smaller in stature. However, sex differences and the long-term effects of PAE on the skeleton have not been investigated and was the aim of this study. Using a mouse model of PAE, we examined the function and gene expression of bone-forming cells (osteoblasts). We then analysed the skeletons of male and female mice at 12-weeks-old (adult) and 4-weeks-old (juvenile). PAE reduced osteoblast bone formation in both sexes, compared to control. Differential gene expression was predominantly observed in PAE males and largely involved genes related to blood vessel formation. High resolution x-ray imaging (micro-CT) revealed PAE had a detrimental effect on the inner trabecular bone component in 12-week-old male mice only. Analysis of the outer cortical bone revealed that whilst both male and female PAE mice were negatively affected, anatomical variations were observed. Mechanical testing also revealed differences in bone strength in PAE mice, compared to control. Interestingly, 4-week-old mice did not possess these sex differences observed in our PAE model at 12 weeks of age. Our data suggest PAE has detrimental and yet sex-dependent effects on the skeleton. Establishing these sex differences will support future policies and clinical management of FASD.

Ablation of collagen XII disturbs joint extracellular matrix organization and causes patellar subluxation.

Collagen XII, belonging to the fibril-associated collagens, is a homotrimeric secreted extracellular matrix (ECM) protein encoded by the COL12A1 gene. Mutations in the human COL12A1 gene cause an Ehlers-Danlos/myopathy overlap syndrome leading to skeletal abnormalities and muscle weakness. Here, we studied the role of collagen XII in joint pathophysiology by analyzing collagen XII deficient mice and human patients. We found that collagen XII is widely expressed across multiple connective tissue of the developing joint. Lack of collagen XII in mice destabilizes tendons and the femoral trochlear groove to induce patellar subluxation in the patellofemoral joint. These changes are associated with an ECM damage response in tendon and secondary quadriceps muscle degeneration. Moreover, patellar subluxation was also identified as a clinical feature of human patients with collagen XII deficiency. The results provide an explanation for joint hyperlaxity in mice and human patients with collagen XII deficiency.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

A new method for segmentation and analysis of bone callus in rodent fracture models using micro-CT.

Fracture burden has created a need to better understand bone repair processes under different pathophysiological states. Evaluation of structural and material properties of the mineralized callus, which is integral to restoring biomechanical stability is, therefore, vital. Microcomputed tomography (micro-CT) can facilitate noninvasive imaging of fracture repair, however, current methods for callus segmentation are only semiautomated, restricted to defined regions, time/labor intensive, and prone to user variation. Herein, we share a new automatic method for segmenting callus in micro-CT tomograms that will allow for objective, quantitative analysis of the bone fracture microarchitecture. Fractured and nonfractured mouse femurs were scanned and processed by both manual and automated segmentation of fracture callus from cortical bone after which microarchitectural parameters were analyzed. All segmentation and analysis steps were performed using CTAn (Bruker) with automatic segmentation performed using the software's image-processing plugins. Results showed automatic segmentation reliably and consistently segmented callus from cortical bone, demonstrating good agreement with manual methods with low bias: tissue volume (TV): -0.320 mm3 , bone volume (BV): 0.0358 mm3 , and bone volume/tissue volume (BV/TV): -3.52%, and was faster and eliminated user-bias and variation. Method scalability and translatability across rodent models were verified in scans of fractured rat femora showing good agreement with manual methods with low bias: TV: -3.654 mm3 , BV: 0.830 mm3 , and BV/TV: 7.81%. Together, these data validate a new automated method for segmentation of callus and cortical bone in micro-CT tomograms that we share as a fast, reliable, and less user-dependent tool for application to study bone callus in fracture, and potentially elsewhere.

Increased PHOSPHO1 and alkaline phosphatase expression during the anabolic bone response to intermittent parathyroid hormone delivery.

The administration of intermittent parathyroid hormone (iPTH) is anabolic to the skeleton. Recent studies with cultured osteoblasts have revealed that the expression of PHOSPHO1, a bone-specific phosphatase essential for the initiation of mineralisation, is regulated by PTH. Therefore, this study sought to determine whether the bone anabolic response to iPTH involves modulation of expression of Phospho1 and of other enzymes critical for bone matrix mineralisation. To mimic iPTH treatment, primary murine osteoblasts were challenged with 50 nM PTH for 6 h in every 48 h period for 8 days (4 cycles), 14 days (7 cycles) and 20 days (10 cycles) in total. The expression of both Phospho1 and Smpd3 was almost completely inhibited after 4 cycles, whereas 10 cycles were required to stimulate a similar response in Alpl expression. To explore the in vivo role of PHOSPHO1 in PTH-mediated osteogenesis, the effects of 14- and 28-day iPTH (80 µg/kg/day) administration was assessed in male wild-type (WT) and Phospho1-/- mice. The expression of Phospho1, Alpl, Smpd3, Enpp1, Runx2 and Trps1 expression was enhanced in the femora of WT mice following iPTH administration but remained unchanged in the femora of Phospho1-/- mice. After 28 days of iPTH administration, the anabolic response in the femora of WT was greater than that noted in Phospho1-/- mice. Specifically, cortical and trabecular bone volume/total volume, as well as cortical thickness, were increased in femora of iPTH-treated WT but not in iPTH-treated Phospho1-/- mice. Trabecular bone osteoblast number was also increased in iPTH-treated WT mice but not in iPTH-treated Phospho1-/-  mice. The increased levels of Phospho1, Alpl, Enpp1 and Smpd3 in WT mice in response to iPTH administration is consistent with their contribution to the potent anabolic properties of iPTH in bone. Furthermore, as the anabolic response to iPTH was attenuated in mice deficient in PHOSPHO1, this suggests that the osteoanabolic effects of iPTH are at least partly mediated via bone mineralisation processes.

MCTR3 reprograms arthritic monocytes to upregulate Arginase-1 and exert pro-resolving and tissue-protective functions in experimental arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a progressive degenerative disorder that leads to joint destruction. Available treatments only target the inflammatory component with minimal impact on joint repair. We recently uncovered a previously unappreciated family of pro-resolving mediators, the maresin conjugate in tissue regeneration (MCTR), that display both immunoregulatory and tissue-protective activities. Thus, we queried whether the production of these autacoids is disrupted in RA patients and whether they can be useful in treating joint inflammation and promoting joint repair. METHODS: Using a highly phenotyped RA cohort we evaluated plasma MCTR concentrations and correlated these to clinical markers of disease activity. To evaluate the immunoregulatory and tissue reparative activities we employed both in vivo models of arthritis and organ culture models. FINDINGS: Herein, we observed that plasma MCTR3 concentrations were negatively correlated with joint disease activity and severity in RA patients. Evaluation of the mechanisms engaged by this mediator in arthritic mice demonstrated that MCTR3 reprograms monocytes to confer enduring joint protective properties. Single cell transcriptomic profiling and flow cytometric evaluation of macrophages from mice treated with MCTR3-reprogrammed monocytes revealed a role for Arginase-1 (Arg-1) in mediating their joint reparative and pro-resolving activities. Arg-1 inhibition reversed both the anti-arthritic and tissue reparative actions of MCTR3-reprogrammed monocytes. INTERPRETATION: Our findings demonstrate that circulating MCTR3 levels are negatively correlated with disease in RA. When administered to mice in vivo, MCTR3 displayed both anti-inflammatory and joint reparative activities, protecting both cartilage and bone in murine arthritis. These activities were, at least in part, mediated via the reprogramming of mononuclear phagocyte responses. FUNDING: This work was supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant no: 677542) and the Barts Charity (grant no: MGU0343) to J.D. J.D. is also supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (grant 107613/Z/15/Z).

Risk factors and implications associated with renal mineralization in chronic kidney disease in cats.

BACKGROUND: Nephrocalcinosis is a pathological feature of chronic kidney disease (CKD). Its pathophysiological implications for cats with CKD are unexplored. OBJECTIVES: Identify nephrocalcinosis risk factors and evaluate its influence on CKD progression and all-cause mortality. ANIMALS: Fifty-one euthyroid client-owned cats with International Renal Interest Society (IRIS) stages 2-3 azotemic CKD. METHODS: Retrospective cohort study. Histopathological kidney sections were assessed for nephrocalcinosis (von Kossa stain). Nephrocalcinosis severity was determined by image analysis (ImageJ). Ordinal logistic regressions were performed to identify nephrocalcinosis risk factors. The influence of nephrocalcinosis on CKD progression and mortality risk were assessed using linear mixed model and Cox regression, respectively. Cats were categorized by their owner-reported time-averaged phosphate-restricted diet (PRD) intake, where PRD comprised ≥50%, 10-50%, or none of food intake. RESULTS: Nephrocalcinosis was rated as mild-to-severe in 78.4% and absent-to-minimal in 21.6% of cases. Higher baseline plasma total calcium concentration (tCa; odds ratio [OR] = 3.07 per 1 mg/dL; P = .02) and eating a PRD (10%-50%: OR = 8.35; P = .01; ≥50%: OR = 5.47; P = .01) were independent nephrocalcinosis risk factors. Cats with absent-to-minimal nephrocalcinosis had increasing plasma creatinine (0.250 ± 0.074 mg/dL/month; P = .002), urea (5.06 ± 1.82 mg/dL/month; P = .01), and phosphate (0.233 ± 0.115 mg/dL/month; P = .05) concentrations over a 1-year period, and had shorter median survival times than cats with mild-to-severe nephrocalcinosis. CONCLUSION AND CLINICAL IMPORTANCE: Higher plasma tCa at CKD diagnosis and PRD intake are independently associated with nephrocalcinosis. However, nephrocalcinosis is not associated with rapid CKD progression in cats.

Direct patterning of periodic semiconductor nanostructures using single-pulse nanosecond laser interference.

We demonstrate an effective method for fabricating large area periodic two-dimensional semiconductor nanostructures by means of single-pulse laser interference. Utilizing a pulsed nanosecond laser with a wavelength of 355 nm, precisely ordered square arrays of nanoholes with a periodicity of 300 nm were successfully obtained on UV photoresist and also directly via a resist-free process onto semiconductor wafers. We show improved uniformity using a beam-shaping system consisting of cylindrical lenses with which we can demonstrate highly regular arrays over hundreds of square micrometers. We propose that our novel observation of direct pattern transfer to GaAs is due to local congruent evaporation and subsequent droplet etching of the surface. The results show that single-pulse interference can provide a rapid and highly efficient route for the realization of wide-area periodic nanostructures on semiconductors and potentially on other engineering materials.

Bimodal Whole-Mount Imaging of Tendon Using Confocal Microscopy and X-ray Micro-Computed Tomography.

BACKGROUND: Three-dimensional imaging modalities for optically dense connective tissues such as tendons are limited and typically have a single imaging methodological endpoint. Here, we have developed a bimodal procedure utilising fluorescence-based confocal microscopy and x-ray micro-computed tomography for the imaging of adult tendons to visualise and analyse extracellular sub-structure and cellular composition in small and large animal species. RESULTS: Using fluorescent immunolabelling and optical clearing, we visualised the expression of the novel cross-species marker of tendon basement membrane, laminin-α4 in 3D throughout whole rat Achilles tendons and equine superficial digital flexor tendon 5 mm segments. This revealed a complex network of laminin-α4 within the tendon core that predominantly localises to the interfascicular matrix compartment. Furthermore, we implemented a chemical drying process capable of creating contrast densities enabling visualisation and quantification of both fascicular and interfascicular matrix volume and thickness by x-ray micro-computed tomography. We also demonstrated that both modalities can be combined using reverse clarification of fluorescently labelled tissues prior to chemical drying to enable bimodal imaging of a single sample. CONCLUSIONS: Whole-mount imaging of tendon allowed us to identify the presence of an extensive network of laminin-α4 within tendon, the complexity of which cannot be appreciated using traditional 2D imaging techniques. Creating contrast for x-ray micro-computed tomography imaging of tendon using chemical drying is not only simple and rapid, but also markedly improves on previously published methods. Combining these methods provides the ability to gain spatio-temporal information and quantify tendon substructures to elucidate the relationship between morphology and function.

Theoretical modelling of single-mode lasing in microcavity lasers via optical interference injection.

The effective manipulation of mode oscillation and competition is of fundamental importance for controlling light emission in semiconductor lasers. Here we develop a rate equation model which considers the spatially modulated gain and spontaneous emission, which are inherently governed by the ripple of the vacuum electromagnetic field in a Fabry-Pérot (FP) microcavity. By manipulating the interplay between the spatial oscillation of the vacuum field and external optical injection via dual-beam laser interference, single longitudinal mode operation is observed in a FP-type microcavity with a side mode suppression ratio exceeding 40 dB. An exploration of this extended rate equation model bridges the gap between the classical model of multimode competition in semiconductor lasers and a quantum-optics understanding of radiative processes in microcavities.

Precise Arrays of Epitaxial Quantum Dots Nucleated by In Situ Laser Interference for Quantum Information Technology Applications.

Precisely ordered arrays of InAs quantum dots are formed on a nanoisland-structured GaAs (100) surface using in situ laser interference during self-assembled molecular beam epitaxial growth. Nanoislands induced by single-pulse four-beam laser interference act as preferential nucleation sites for InAs quantum dots and result in site occupation dependent on the size of nanoislands, the InAs coverage, and the laser parameters. By optimizing the growth and interference conditions, regular dense ordering of single dots was obtained for the first time using this in situ noninvasive approach. The photoluminescence spectra of the resulting quantum dot arrays with a period of 300 nm show good optical quality and uniformity. This technique paves the way for the rapid large-scale fabrication of arrays of single dots to enable quantum information technology device platforms.

Broadband, wide-angle antireflection in GaAs through surface nano-structuring for solar cell applications.

We demonstrate broadband and wide-angle antireflective surface nanostructuring in GaAs semiconductors using variable dose electron-beam lithography (EBL). Various designed structures are written with EBL on a positive EB-resist coated GaAs and developed followed by shallow inductively coupled plasma etching. An optimized nanostructured surface shows a reduced surface reflectivity down to less than 2.5% in the visible range of 450-700 nm and an average reflectance of less than 4% over a broad near-infrared wavelength range from 900-1400 nm. The results are obtained over a wide incidence angle of 33.3°. This study shows the potential for anti-reflective structures using a simpler reverse EBL process which can provide optical absorption or extraction efficiency enhancement in semiconductors relevant to improved performance in solar photovoltaics or light-emitting diodes.

Applied mechanical loading to mouse hindlimb acutely increases skeletal perfusion and chronically enhanced vascular porosity.

Blood supply is essential for osteogenesis, yet its relationship to load-related increases in bone mass is poorly defined. Herein, we aim to investigate the link between load-induced osteogenesis and the blood supply (bone perfusion and vascular porosity) using an established osteogenic noninvasive model of axial loading. Accordingly, 12 N mechanical loads were applied to the right tibiae of six male C57BL6 mice at 10-12 wk of age, 3 times/wk for 2 wk. Skeletal perfusion was measured acutely (postloading) and chronically in loaded and contralateral, nonloaded hindlimbs by laser-Doppler imaging. Vascular and lacunar porosity of the cortical bone and tibia load-related changes in trabecular and cortical bone was measured by nanoCT and micro-CT, respectively. We found that the mean skeletal perfusion (loaded: nonloaded limb ratio) increased by 56% immediately following the first loading episode (vs. baseline, P < 0.01), and a similar increase was observed after all loading episodes, demonstrating that these acute responses were conserved for 2 wk of loading. Loading failed, however, to engender any significant chronic changes in mean perfusion between the beginning and the end of the experiment. In contrast, 2 wk of loading engendered an increased vascular canal number in the tibial cortical compartment (midshaft) and, as expected, also increased trabecular and cortical bone volumes and modified tibial architecture in the loaded limb. Our results indicate that each episode of loading both generates acute enhancement in skeletal blood perfusion and also stimulates chronic vascular architectural changes in the bone cortices, which coincide with load-induced increases in bone mass.NEW & NOTEWORTHY This study investigated modifications to the blood supply (bone perfusion and intracortical vascular canals) in mechanoadaptive responses in C57BL6 mice. Each episode of mechanical loading acutely increases skeletal perfusion. Two weeks of mechanical loading increased bone mass and cortical vascular canal number, while there was no chronic increase in hindlimb perfusion. Our findings suggest that the blood supply may participate in the processes that govern load-induced bone formation.

Conditional deletion of E11/Podoplanin in bone protects against ovariectomy-induced increases in osteoclast formation and activity.

E11/Podoplanin (Pdpn) is implicated in early osteocytogenesis and the formation of osteocyte dendrites. This dendritic network is critical for bone modelling/remodelling, through the production of receptor activator of nuclear factor κ B (RANK)-ligand (RANKL). Despite this, the role of Pdpn in the control of bone remodelling is yet to be established in vivo. Here we utilised bone-specific Pdpn conditional knockout mice (cKO) to examine the role of Pdpn in the bone loss associated with ovariectomy (OVX). MicroCT revealed that Pdpn deletion had no significant effect on OVX-induced changes in trabecular microarchitecture. Significant differences between genotypes were observed in the trabecular pattern factor (P<0.01) and structure model index (P<0.01). Phalloidin staining of F-actin revealed OVX to induce alterations in osteocyte morphology in both wild-type (WT) and cKO mice. Histological analysis revealed an expected significant increase in osteoclast number in WT mice (P<0.01, compared with sham). However, cKO mice were protected against such increases in osteoclast number. Consistent with this, serum levels of the bone resorption marker Ctx were significantly increased in WT mice following OVX (P<0.05), but were unmodified by OVX in cKO mice. Gene expression of the bone remodelling markers Rank, Rankl, Opg and Sost were unaffected by Pdpn deletion. Together, our data suggest that an intact osteocyte dendritic network is required for sustaining osteoclast formation and activity in the oestrogen-depleted state, through mechanisms potentially independent of RANKL expression. This work will enable a greater understanding of the role of osteocytes in bone loss induced by oestrogen deprivation.

Compression of morbidity in a progeroid mouse model through the attenuation of myostatin/activin signalling.

BACKGROUND: One of the principles underpinning our understanding of ageing is that DNA damage induces a stress response that shifts cellular resources from growth towards maintenance. A contrasting and seemingly irreconcilable view is that prompting growth of, for example, skeletal muscle confers systemic benefit. METHODS: To investigate the robustness of these axioms, we induced muscle growth in a murine progeroid model through the use of activin receptor IIB ligand trap that dampens myostatin/activin signalling. Progeric mice were then investigated for neurological and muscle function as well as cellular profiling of the muscle, kidney, liver, and bone. RESULTS: We show that muscle of Ercc1Δ/- progeroid mice undergoes severe wasting (decreases in hind limb muscle mass of 40-60% compared with normal mass), which is largely protected by attenuating myostatin/activin signalling using soluble activin receptor type IIB (sActRIIB) (increase of 30-62% compared with untreated progeric). sActRIIB-treated progeroid mice maintained muscle activity (distance travel per hour: 5.6 m in untreated mice vs. 13.7 m in treated) and increased specific force (19.3 mN/mg in untreated vs. 24.0 mN/mg in treated). sActRIIb treatment of progeroid mice also improved satellite cell function especially their ability to proliferate on their native substrate (2.5 cells per fibre in untreated progeroids vs. 5.4 in sActRIIB-treated progeroids after 72 h in culture). Besides direct protective effects on muscle, we show systemic improvements to other organs including the structure and function of the kidneys; there was a major decrease in the protein content in urine (albumin/creatinine of 4.9 sActRIIB treated vs. 15.7 in untreated), which is likely to be a result in the normalization of podocyte foot processes, which constitute the filtration apparatus (glomerular basement membrane thickness reduced from 224 to 177 nm following sActRIIB treatment). Treatment of the progeric mice with the activin ligand trap protected against the development of liver abnormalities including polyploidy (18.3% untreated vs. 8.1% treated) and osteoporosis (trabecular bone volume; 0.30 mm3 in treated progeroid mice vs. 0.14 mm3 in untreated mice, cortical bone volume; 0.30 mm3 in treated progeroid mice vs. 0.22 mm3 in untreated mice). The onset of neurological abnormalities was delayed (by ~5 weeks) and their severity reduced, overall sustaining health without affecting lifespan. CONCLUSIONS: This study questions the notion that tissue growth and maintaining tissue function during ageing are incompatible mechanisms. It highlights the need for future investigations to assess the potential of therapies based on myostatin/activin blockade to compress morbidity and promote healthy ageing.

Sost Haploinsufficiency Provokes Peracute Lethal Cardiac Tamponade without Rescuing the Osteopenia in a Mouse Model of Excess Glucocorticoids.

Glucocorticoid-induced secondary osteoporosis is the most predictable side effect of this anti-inflammatory. One of the main mechanisms by which glucocorticoids achieve such deleterious outcome in bone is by antagonizing Wnt/ß-catenin signaling. Sclerostin, encoded by Sost gene, is the main negative regulator of the proformative and antiresorptive role of the Wnt signaling pathway in the skeleton. It was hypothesized that the partial inactivation of sclerostin function by genetic manipulation will rescue the osteopenia induced by high endogenous glucocorticoid levels. Sost-deficient mice were crossed with an established mouse model of excess glucocorticoids, and the effects on bone mass and structure were evaluated. Sost haploinsufficiency did not rescue the low bone mass induced by high glucocorticoids. Intriguingly, the critical manifestation of Sost deficiency combined with glucocorticoid excess was sporadic, sudden, unprovoked, and nonconvulsive death. Detailed histopathologic analysis in a wide range of tissues identified peracute hemopericardium and cardiac tamponade to be the cause. These preclinical studies reveal outcomes with direct relevance to ongoing clinical trials that explore the use of antisclerostin antibodies as a treatment for osteoporosis. They particularly highlight a potential for increased cardiovascular risk and may inform improved stratification of patients who might otherwise benefit from antisclerostin antibody treatment.

Site-Controlled Single-Photon Emitters Fabricated by Near-Field Illumination.

Many of the most advanced applications of semiconductor quantum dots (QDs) in quantum information technology require a fine control of the QDs' position and confinement potential, which cannot be achieved with conventional growth techniques. Here, a novel and versatile approach for the fabrication of site-controlled QDs is presented. Hydrogen incorporation in GaAsN results in the formation of N-2H and N-2H-H complexes, which neutralize all the effects of N on GaAs, including the N-induced large reduction of the bandgap energy. Starting from a fully hydrogenated GaAs/GaAsN:H/GaAs quantum well, the NH bonds located within the light spot generated by a scanning near-field optical microscope tip are broken, thus obtaining site-controlled GaAsN QDs surrounded by a barrier of GaAsN:H (laterally) and GaAs (above and below). By adjusting the laser power density and exposure time, the optical properties of the QDs can be finely controlled and optimized, tuning the quantum confinement energy over more than 100 meV and resulting in the observation of single-photon emission from both the exciton and biexciton recombinations. This novel fabrication technique reaches a position accuracy <100 nm and it can easily be applied to the realization of more complex nanostructures.

Stable sulforaphane protects against gait anomalies and modifies bone microarchitecture in the spontaneous STR/Ort model of osteoarthritis.

Osteoarthritis (OA), affecting joints and bone, causes physical gait disability with huge socio-economic burden; treatment remains palliative. Roles for antioxidants in protecting against such chronic disorders have been examined previously. Sulforaphane is a naturally occurring antioxidant. Herein, we explore whether SFX-01®, a stable synthetic form of sulforaphane, modifies gait, bone architecture and slows/reverses articular cartilage destruction in a spontaneous OA model in STR/Ort mice. Sixteen mice (n=8/group) were orally treated for 3months with either 100mg/kg SFX-01® or vehicle. Gait was recorded, tibiae were microCT scanned and analysed. OA lesion severity was graded histologically. The effect of SFX-01® on bone turnover markers in vivo was complemented by in vitro bone formation and resorption assays. Analysis revealed development of OA-related gait asymmetry in vehicle-treated STR/Ort mice, which did not emerge in SFX-01®-treated mice. We found significant improvements in trabecular and cortical bone. Despite these marked improvements, we found that histologically-graded OA severity in articular cartilage was unmodified in treated mice. These changes are also reflected in anabolic and anti-catabolic actions of SFX-01® treatment as reflected by alteration in serum markers as well as changes in primary osteoblast and osteoclast-like cells in vitro. We report that SFX-01® improves bone microarchitecture in vivo, produces corresponding changes in bone cell behaviour in vitro and leads to greater symmetry in gait, without marked effects on cartilage lesion severity in STR/Ort osteoarthritic mice. Our findings support both osteotrophic roles and novel beneficial gait effects for SFX-01® in this model of spontaneous OA.

Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.

The transmembrane glycoprotein E11/Podoplanin (Pdpn) has been implicated in the initial stages of osteocyte differentiation. However, its precise function and regulatory mechanisms are still unknown. Due to the known embryonic lethality induced by global Pdpn deletion, we have herein explored the effect of bone-specific Pdpn knockdown on osteocyte form and function in the post-natal mouse. Extensive skeletal phenotyping of male and female 6-week-old Oc-cre;Pdpnflox/flox (cKO) mice and their Pdpnflox/flox controls (fl/fl) has revealed that Pdpn deletion significantly compromises tibial cortical bone microarchitecture in both sexes, albeit to different extents (p < 0.05). Consistent with this, we observed an increase in stiffness in female cKO mice in comparison to fl/fl mice (p < 0.01). Moreover, analysis of the osteocyte phenotype by phalloidin staining revealed a significant decrease in the dendrite volume (p < 0.001) and length (p < 0.001) in cKO mice in which deletion of Pdpn also modifies the bone anabolic loading response (p < 0.05) in comparison to age-matched fl/fl mice. Together, these data confirm a regulatory role for Pdpn in osteocyte dendrite formation and as such, in the control of osteocyte function. As the osteocyte dendritic network is known to play vital roles in regulating bone modeling/remodeling, this highlights an essential role for Pdpn in bone homeostasis.