High-resolution dynamic mapping of the C. elegans intestinal brush border.

The intestinal brush border is made of an array of microvilli that increases the membrane surface area for nutrient processing, absorption and host defense. Studies on mammalian cultured epithelial cells have uncovered some of the molecular players and physical constraints required to establish this apical specialized membrane. However, the building and maintenance of a brush border in vivo has not yet been investigated in detail. Here, we combined super-resolution imaging, transmission electron microscopy and genome editing in the developing nematode Caenorhabditis elegans to build a high-resolution and dynamic localization map of known and new brush border markers. Notably, we show that microvilli components are dynamically enriched at the apical membrane during microvilli outgrowth and maturation, but become highly stable once microvilli are built. This new toolbox will be instrumental for understanding the molecular processes of microvilli growth and maintenance in vivo, as well as the effect of genetic perturbations, notably in the context of disorders affecting brush border integrity.

Differences in bone microarchitecture between genetic and secondary iron-overload mouse models suggest a role for hepcidin deficiency in iron-related osteoporosis.

Iron overload is one of the secondary osteoporosis etiologies. Cellular and molecular mechanisms involved in iron-related osteoporosis are not fully understood. AIM: The aim of the study was to investigate the respective roles of iron excess and hepcidin, the systemic iron regulator, in the development of iron-related osteoporosis. MATERIAL AND METHODS: We used mice models with genetic iron overload (GIO) related to hepcidin deficiency (Hfe-/- and Bmp6-/- ) and secondary iron overload (SIO) exhibiting a hepcidin increase secondary to iron excess. Iron concentration and transferrin saturation levels were evaluated in serum and hepatic, spleen, and bone iron concentrations were assessed by ICP-MS and Perl's staining. Gene expression was evaluated by quantitative RT-PCR. Bone micro-architecture was evaluated by micro-CT. The osteoblastic MC3T3 murine cells that are able to mineralize were exposed to iron and/or hepcidin. RESULTS: Despite an increase of bone iron concentration in all overloaded mice models, bone volume/total volume (BV/TV) and trabecular thickness (Tb.Th) only decreased significantly in GIO, at 12 months for Hfe-/- and from 6 months for Bmp6-/- . Alterations in bone microarchitecture in the Bmp6-/- model were positively correlated with hepcidin levels (BV/TV (ρ = +.481, p < .05) and Tb.Th (ρ = +.690, p < .05). Iron deposits were detected in the bone trabeculae of Hfe-/- and Bmp6-/- mice, while iron deposits were mainly visible in bone marrow macrophages in secondary iron overload. In cell cultures, ferric ammonium citrate exposure abolished the mineralization process for concentrations above 5 µM, with a parallel decrease in osteocalcin, collagen 1, and alkaline phosphatase mRNA levels. Hepcidin supplementation of cells had a rescue effect on the collagen 1 and alkaline phosphatase expression level decrease. CONCLUSION: Together, these data suggest that iron in excess alone is not sufficient to induce osteoporosis and that low hepcidin levels also contribute to the development of osteoporosis.

Sicca syndrome in systemic sclerosis: a narrative review on a neglected issue.

SSc is an auto-immune disease characterized by life-threatening manifestations such as lung fibrosis or pulmonary arterial hypertension. Symptoms with a detrimental impact on quality of life are also reported and sicca syndrome (xerostomia, xeropthalmia) is present in up to 80% of patients with SSc. Sicca syndrome can occur in the absence of overlap with Sjögren's disease and recent studies highlight that fibrosis of minor and major salivary glands, directly linked to the pathogenesis of SSc, could be a major contributor of xerostomia in SSc. This narrative review provides an overview of the clinical presentation, diagnostic strategies, management and future perspectives on sicca syndrome in patients with SSc.

The plakin domain of C. elegans VAB-10/plectin acts as a hub in a mechanotransduction pathway to promote morphogenesis.

Mechanical forces can elicit a mechanotransduction response through junction-associated proteins. In contrast to the wealth of knowledge available for focal adhesions and adherens junctions, much less is known about mechanotransduction at hemidesmosomes. Here, we focus on the C. elegans plectin homolog VAB-10A, the only evolutionary conserved hemidesmosome component. In C. elegans, muscle contractions induce a mechanotransduction pathway in the epidermis through hemidesmosomes. We used CRISPR to precisely remove spectrin repeats (SRs) or a partially hidden Src homology 3 (SH3) domain within the VAB-10 plakin domain. Deleting the SH3 or SR8 domains in combination with mutations affecting mechanotransduction, or just the part of SR5 shielding the SH3 domain, induced embryonic elongation arrest because hemidesmosomes collapse. Notably, recruitment of GIT-1, the first mechanotransduction player, requires the SR5 domain and the hemidesmosome transmembrane receptor LET-805. Furthermore, molecular dynamics simulations confirmed that forces acting on VAB-10 could make the central SH3 domain, otherwise in contact with SR4, available for interaction. Collectively, our data strongly indicate that the plakin domain plays a central role in mechanotransduction and raise the possibility that VAB-10/plectin might act as a mechanosensor.

Patient-reported outcomes and their relation with iron parameters in HFE haemochromatosis during maintenance therapy: A prospective cohort study.

BACKGROUND & AIMS: The standard of care for haemochromatosis is regular phlebotomy in order to maintain low ferritin levels. Many patients report fatigue or joint pain despite serum ferritin within the therapeutic targets. We evaluated Patient-Reported Outcomes, and their relation with iron parameters, in C282Y homozygous patients undergoing maintenance phlebotomy. METHODS: Patients were prospectively enrolled in a French referral care centre. At each phlebotomy, patients completed a numeric fatigue scale, a joint pain questionnaire and SF-36 Mental Component Score (MCS) and Physical Component Score (PCS). Haemoglobin, iron, TS and ferritin were collected concomitantly. RESULTS: About 701 visits were performed in 259 patients. The median fatigue score was 3/10; 171 (66%) patients reported joint pain. Age and worsening of joint pain were associated with fatigue (p < .0001 for both). Female gender (p < .037), age (p < .003), and a decrease of TS (p = .050) were associated with joint pain. Main features associated with PCS <50 were worsening of joint pain and age (p < .001 for both) and TS <20% (p < .02). CONCLUSIONS: Fatigue was independent from iron parameters. The main factor impacting quality of life was joint pain, which was more severe in patients with low TS values. Then, a more precise monitoring of TS should be proposed during haemochromatosis maintenance therapy; while less stringent monitoring of serum ferritin levels could be tested.

Calcium pyrophosphate deposition (CPPD) in a liver transplant patient: are hypomagnesemia, tacrolimus or both guilty? A case-based literature review.

Calcium pyrophosphate deposition (CPPD) can be induced by a persistent hypomagnesemia. Tacrolimus is an immunosuppressive treatment especially used in organ transplant, potentially inducer of hypomagnesemia by renal loss. A 53-year-old man, liver transplant 10 months earlier, developed an acute peripheral oligoarthritis of wrist, hip and elbow with fever, associated with acute low back pain. Synovial fluid was sterile, and revealed calcium pyrophosphate crystals. Spinal imaging showed inflammatory changes. Magnesium blood level was low at 0.51 mmol/l, with high fractional excretion in favor of renal loss. Tacrolimus was changed for everolimus, proton pump inhibitor was stopped, and magnesium oral supplementation was started. After 8 months follow-up and slow prednisone tapering, he did not relapse pain. Persistent hypomagnesemia is a rare secondary cause of CPPD. In this entity, drug liability should be investigated such as tacrolimus in organ transplant patient.

Elaboration of a new synovial predictive score of septic origin for acute arthritis on the native joint (RESAS).

OBJECTIVE: To establish a new predictive score for the diagnosis of septic arthritis (SA) according to different synovial fluid (SF) variables. METHODS: First, we analysed the different clinical, biological and SF variables associated with the diagnosis of SA (according to the Newman's criteria) in a monocentric cohort of acute arthritis (<30 days) (n = 233) (SYNOLACTATE cohort). A new score predictive of SA (RESAS) was created using the independent discriminant variables after multivariate analysis. A value was attributed to each variable of the score according to the weighting based on their likelihood ratio for the diagnosis of SA. RESAS performance was then tested on the first cohort (internal validation) and then checked on a second independent cohort (n = 70) (external validation). RESULTS: After multivariate analysis, four independent variables of the SF were included for RESAS: (i) purulent SF or white blood cells count ≥70 000/mm3; (ii) absence/presence of crystals; (iii) lactate; and (iv) glucose synovial level. RESAS ranged between -4 and +13 points. The performance of RESAS to predicted SA was excellent with area under the curve (AUC)=0.928 (0.877-0.980) in internal validation and AUC=0.986 (0.962-1.00) in external validation. For a RESAS threshold ≥+4, SA was diagnosed with Se=56.0% (0.371-0.733), Sp=98.1% (0.952-0.993), LR+=29.1 (10.4-81.6) in the first cohort and with Se=91.7% (0.646-0.985), Sp=98.3% (0.909-0.997), LR+=53.2 (7.56-373) in the second cohort. CONCLUSION: RESAS is a new composite score of four SF variables with excellent performance to predicted SA in acute arthritis population.

The role of mode switching in a population of actin polymers with constraints.

In this paper, we introduce a stochastic model for the dynamics of actin polymers and their interactions with other proteins in the cellular envelop. Each polymer elongates and shortens, and can switch between several modes depending on whether it is bound to accessory proteins that modulate its behaviour as, for example, elongation-promoting factors. Our main aim is to understand the dynamics of a large population of polymers, assuming that the only limiting quantity is the total amount of monomers, set to be constant to some large N. We first focus on the evolution of a very long polymer, of size [Formula: see text], with a rapid switch between modes (compared to the timescale over which the macroscopic fluctuations in the polymer size appear). Letting N tend to infinity, we obtain a fluid limit in which the effect of the switching appears only through the fraction of time spent in each mode at equilibrium. We show in particular that, in our situation where the number of monomers is limiting, a rapid binding-unbinding dynamics may lead to an increased elongation rate compared to the case where the polymer is trapped in any of the modes. Next, we consider a large population of polymers and complexes, represented by a random measure on some appropriate type space. We show that as N tends to infinity, the stochastic system converges to a deterministic limit in which the switching appears as a flow between two categories of polymers. We exhibit some numerical examples in which the limiting behaviour of a single polymer differs from that of a population of competing (shorter) polymers for equivalent model parameters. Taken together, our results demonstrate that under conditions where the total number of monomers is limiting, the study of a single polymer is not sufficient to understand the behaviour of an ensemble of competing polymers.

Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Hereditary aceruloplasminemia (HA), related to mutations in the ceruloplasmin (Cp) gene, leads to iron accumulation. Ceruloplasmin ferroxidase activity being considered essential for macrophage iron release, macrophage iron overload is expected, but it is not found in hepatic and splenic macrophages in humans. Our objective was to get a better understanding of the mechanisms leading to iron excess in HA. A clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) knockout of the Cp gene was performed on Sprague-Dawley rats. We evaluated the iron status in plasma, the expression of iron metabolism genes, and the status of other metals whose interactions with iron are increasingly recognized. In Cp-/- rats, plasma ceruloplasmin and ferroxidase activity were absent, together with decreased iron concentration and transferrin saturation. Similarly as in humans, the hepatocytes were iron overloaded conversely to hepatic and splenic macrophages. Despite a relative hepcidin deficiency in Cp-/- rats and the loss of ferroxidase activity, potentially expected to limit the interaction of iron with transferrin, no increase of plasma non-transferrin-bound iron level was found. Copper was decreased in the spleen, whereas manganese was increased in the plasma. These data suggest that the reported role of ceruloplasmin cannot fully explain the iron hepatosplenic phenotype in HA, encouraging the search for additional mechanisms.-Kenawi, M., Rouger, E., Island, M.-L., Leroyer, P., Robin, F., Remy, S., Tesson, L., Anegon, I., Nay, K., Derbré, F., Brissot, P., Ropert, M., Cavey, T., Loréal, O. Ceruloplasmin deficiency does not induce macrophagic iron overload: lessons from a new rat model of hereditary aceruloplasminemia.

Hand ultrasound for the diagnosis of scleroderma: a scoring strategy including US items and items from the EULAR/ACR classification.

OBJECTIVES: To evaluate the diagnostic value of hand ultrasound (US) in systemic sclerosis (SSc) and to explore its relevance within a combined diagnostic approach. METHODS: 224 patients with suspected SSc were consecutively included. They all had US evaluation assessing the presence of fibrotic tenosynovitis (fibrotic TS) and ulnar artery occlusion (UAO). The final diagnosis of SSc was based on the clinical evaluation of a board of experts independently of any pre-established classification criteria. RESULTS: 166 patients were finally diagnosed as SSc according to the experts as reference standard. 62 SSc and 8 non-SSc patients had UAO (uni or bilateral) (p=0.001). 23 SSc patients and 1 non-SSc patient had US fibrotic TS (p=0.007). A US SSc-pattern (presence of UAO and/or fibrotic TS) was reported in 73 SSc patients and 9 non-SSc patients (p<0.001). UAO had an area under ROC curve (AUC) for the diagnosis of SSc of 0.618 (95%CI 0.539- 0.697); with Se=0.373 (0.304-0.449) and Sp=0.862 (0.751-0.928). Fibrotic TS had an AUC of 0.561 (0.480-0.643); with Se=0.139 (0.094-0.199) and Sp=0.983 (0.909-0.997). The US-SSc pattern had a AUC of 0.641 (0.563- 0.695), with Se=0.440 (0.367-0.516) and Sp=0.845 (0.731-0.916). A scoring system including these US parameters and items from ACR/EULAR classification criteria had an AUC of 0.979 (0.962-0.996)) and allows the substitution of capillaroscopy by US parameters with similar performances. CONCLUSIONS: The use of hand US parameters may help to refine the diagnostic strategy of SSc and their inclusion in a combined diagnostic approach could be discussed.

Non-centrosomal epidermal microtubules act in parallel to LET-502/ROCK to promote C. elegans elongation.

C. elegans embryonic elongation is a morphogenetic event driven by actomyosin contractility and muscle-induced tension transmitted through hemidesmosomes. A role for the microtubule cytoskeleton has also been proposed, but its contribution remains poorly characterized. Here, we investigate the organization of the non-centrosomal microtubule arrays present in the epidermis and assess their function in elongation. We show that the microtubule regulators γ-tubulin and NOCA-1 are recruited to hemidesmosomes and adherens junctions early in elongation. Several parallel approaches suggest that microtubule nucleation occurs from these sites. Disrupting the epidermal microtubule array by overexpressing the microtubule-severing protein Spastin or by inhibiting the C. elegans ninein homolog NOCA-1 in the epidermis mildly affected elongation. However, microtubules were essential for elongation when hemidesmosomes or the activity of the Rho kinase LET-502/ROCK were partially compromised. Imaging of junctional components and genetic analyses suggest that epidermal microtubules function together with Rho kinase to promote the transport of E-cadherin to adherens junctions and myotactin to hemidesmosomes. Our results indicate that the role of LET-502 in junctional remodeling is likely to be independent of its established function as a myosin II activator, but requires a microtubule-dependent pathway involving the syntaxin SYX-5. Hence, we propose that non-centrosomal microtubules organized by epidermal junctions contribute to elongation by transporting junction remodeling factors, rather than having a mechanical role.

Single-molecule analysis of cell surface dynamics in Caenorhabditis elegans embryos.

We describe a general, versatile and minimally invasive method to image single molecules near the cell surface that can be applied to any GFP-tagged protein in Caenorhabditis elegans embryos. We exploited tunable expression via RNAi and a dynamically exchanging monomer pool to achieve fast, continuous single-molecule imaging at optimal densities with signal-to-noise ratios adequate for robust single-particle tracking (SPT). We introduce a method called smPReSS, single-molecule photobleaching relaxation to steady state, that infers exchange rates from quantitative analysis of single-molecule photobleaching kinetics without using SPT. Combining SPT and smPReSS allowed for spatially and temporally resolved measurements of protein mobility and exchange kinetics. We used these methods to (i) resolve distinct mobility states and spatial variation in exchange rates of the polarity protein PAR-6 and (ii) measure spatiotemporal modulation of actin filament assembly and disassembly. These methods offer a promising avenue to investigate dynamic mechanisms that pattern the embryonic cell surface.

Detection of hypophosphatasia in hospitalised adults in rheumatology and internal medicine departments: a multicentre study over 10 years.

INTRODUCTION: Hypophosphatasia (HPP) is a rare genetic disease caused by loss-of-function mutations in the ALPL gene encoding the tissue non-specific alkaline phosphatase (ALP). Mild HPP is usually misdiagnosed in adult age. While an elevated serum ALP value draws more attention than a low value, low serum ALP should be better recognised and may lead to HPP detection. METHODS: Patients were selected from the records of the biochemistry department of six University Hospitals in France. Patients were hospitalised in the departments of rheumatology and internal medicine between 2007 and 2017. RESULTS: 56 321 hospitalised patients had at least 2 serum ALP dosages and 664 of these patients had at least 2 low serum ALP≤35 UI/L. Among these 664 patients, 482 (72.6%) had fluctuating low values (mean age 62.9 years; 60% of women) and 182 patients (27.4%) had persistent low values below 35 IU/L (mean age 53.4 years; 67% of women). Among patients with persistent hypophosphatasaemia treated with bisphosphonates, 70.8% never had ALP measurement before treatment and 20.8% were treated despite an abnormal decrease of ALP. Genetic testing was performed in 18 patients and was positive in 11. Genetic diagnosis of HPP was at least 6.0% in persistent hypophosphatasaemia and at least 15.9% in patients with at least three symptoms suggestive of HPP. CONCLUSION: In this 10-year retrospective study, 0.32% of adult patients hospitalised in the rheumatology and internal medicine departments had persistently low serum ALP, and among them, 6% had genetically proven HPP. Reported hypophosphatasaemia represented only 3.6% of hospitalised patients.

The ANISEED database: digital representation, formalization, and elucidation of a chordate developmental program.

Developmental biology aims to understand how the dynamics of embryonic shapes and organ functions are encoded in linear DNA molecules. Thanks to recent progress in genomics and imaging technologies, systemic approaches are now used in parallel with small-scale studies to establish links between genomic information and phenotypes, often described at the subcellular level. Current model organism databases, however, do not integrate heterogeneous data sets at different scales into a global view of the developmental program. Here, we present a novel, generic digital system, NISEED, and its implementation, ANISEED, to ascidians, which are invertebrate chordates suitable for developmental systems biology approaches. ANISEED hosts an unprecedented combination of anatomical and molecular data on ascidian development. This includes the first detailed anatomical ontologies for these embryos, and quantitative geometrical descriptions of developing cells obtained from reconstructed three-dimensional (3D) embryos up to the gastrula stages. Fully annotated gene model sets are linked to 30,000 high-resolution spatial gene expression patterns in wild-type and experimentally manipulated conditions and to 528 experimentally validated cis-regulatory regions imported from specialized databases or extracted from 160 literature articles. This highly structured data set can be explored via a Developmental Browser, a Genome Browser, and a 3D Virtual Embryo module. We show how integration of heterogeneous data in ANISEED can provide a system-level understanding of the developmental program through the automatic inference of gene regulatory interactions, the identification of inducing signals, and the discovery and explanation of novel asymmetric divisions.

Should we really include systemic sclerosis specific antibodies in the classification criteria of Sjogren's disease?

We have read with great interest the results from Marketos et al. regarding the positivity of specific systemic sclerosis auto-antibodies in patients with sicca symptoms. Based on complementary data from the literature, we rather believe scleroderma-associated antibodies should be considered either as a yellow flag for an association between scleroderma and Sjogren, or a potential undiagnosed scleroderma, rather than an isolated Sjogren's disease.

Ultrasound Classification of Finger Pulp Blood Flow in Patients With Systemic Sclerosis: A Pilot Study.

OBJECTIVE: To define a semiquantitative classification of finger pulp blood flow (FPBF) and to evaluate whether this classification could be used to assess FPBF in healthy controls and in systemic sclerosis (SSc) patients. METHODS: Thirty controls and 86 SSc patients were consecutively included. A classification of FPBF including 5 grades (from grade 0 [no signal] to 4 [signal detected on the entire finger pulp, including the subepidermal vascular network]) was evaluated. This classification was explored in basal conditions and after hand baths in hot and cold water in controls. Its relevance was also assessed at room temperature in SSc patients. RESULTS: In controls, power Doppler ultrasonography (PDUS) of FPBF was improved after hot challenge (P = 0.024), whereas cold challenge decreased FPBF (P = 0.001). FPBF correlated with the vasodilation status assessed by the resistivity index of radial arteries (Spearman's correlation coefficient = -0.50, P = 0.0049). Grade 0 was more frequent in SSc patients than in controls (22.1% versus 3.3%; P < 0.05). In SSc patients, grade 0 was associated with severity markers of the digital vasculopathy such as digital ulcers (DUs) (current or past) (P < 0.05) or ulnar artery occlusion (P < 0.05). On the other hand, DUs were less frequent in patients with grade 4 (P < 0.05). A pathologic threshold of <2 (grade 0 or 1) was significantly associated with DUs (odds ratio 6.67 [95% confidence interval 2.31-19.21], P < 0.0001). CONCLUSION: PDUS allowed a semiquantitative evaluation of FBPF in SSc patients and controls. Further studies are warranted to validate these results in independent SSc populations and to compare PDUS to existing tools assessing digital blood flow.

Denosumab in the management of Aneurysmal bone cyst.

BACKGROUND: Aneurysmal bone cyst (ABC) is a benign, locally aggressive tumour that arises predominantly in long bones and spine. Following the encouraging results of denosumab use in Giant Cell Tumors (GCT) and the histological similarities between ABC and GCT, the interest on the role of denosumab in the therapeutic arsenal of the most advanced ABC is growing. The purpose of this literature review is to investigate the current state of knowledge about the use of denosumab in ABCs. METHODS: A literature research was conducted through PUBMED, COCHRANE and GOOGLE SCHOLAR using the keywords "aneurysmal bone cyst" AND "denosumab". Seventeen articles were included. RESULTS: A total of 43 cases were reported in the literature. There were 23 males, 20 females. The mean age was 15,9±8,1 year. Pain relief and neurological improvement were rapid and sustained. Radiological assessment showed ossification and/or volume reduction in 36/39 patients. Eight patients (18,6%) presented a recurrence after or during denosumab therapy of whom 7 were adults. Adverse events occurred in 11 patients, 5 of them were admitted to the intensive care unit due to hypercalcemia. CONCLUSION: Denosumab use in non-surgical ABCs has shown a positive impact in pain and neurological symptoms. The oncological outcome remains unclear with a recurrence rate of 18,6% during/after denosumab therapy, mostly in adults. However, regarding the potential clinical benefits, its use might be discussed in the most advanced cases. Further research and clinical trials are mandatory to precise its belonging in the therapeutic arsenal.