An ancient, Antarctic-specific species complex: large divergences between multiple Antarctic lineages of the tardigrade genus Mesobiotus.

Antarctica has been isolated and progressively glaciated for over 30 million years, with only approximately 0.3 % of its area currently ice-free and capable of supporting terrestrial ecosystems. As a result, invertebrate populations have become isolated and fragmented, in some cases leading to speciation. Terrestrial invertebrate species currently found in Antarctica often show multi-million year, and even Gondwanan, heritage, with little evidence of recent colonisation. Mesobiotus is a globally distributed tardigrade genus. It has commonly been divided into two "groups", referred to as harmsworthi and furciger, with both groups currently considered cosmopolitan, with global reports including from both the Arctic and the Antarctic. However, some authors considered that Meb. furciger, as originally described, may represent an Antarctic-specific lineage. Using collections of tardigrades from across the Antarctic continent and publicly available sequences obtained from online databases, we use mitochondrial and nuclear ribosomal sequence data to clarify the relationships of Antarctic Mesobiotus species. Our analyses show that all Antarctic members belong to a single lineage, evolving separately from non-Antarctic representatives. Within this Antarctic lineage there are further deep divisions among geographic regions of the continent, consistent with the presence of a species complex. Based on our data confirming the deep divisions between this Antarctic lineage, which includes representatives of both groups, we recommend that the use of furciger and harmsworthi group terminology is now abandoned, as it leads to systematic and biogeographical confusion.

Vertebral fracture assessment (VFA) for monitoring vertebral reshaping in children and adolescents with osteogenesis imperfecta treated with intravenous neridronate.

PURPOSE: The study was aimed at monitoring vertebral bodies changes with the use of Vertebral Fracture Assessment (VFA) in children and adolescents affected by osteogenesis imperfecta (OI) during treatment with intravenous neridronate. METHODS: 60 children and adolescents (35 males and 25 females; age 1-16 years) with OI type I, III and IV were included in the study. Intravenous neridronate was administered at the dose of 2 mg/kg every 3 months in all patients. Lumbar spine (LS) bone mineral density (BMD) and VFA by dual X-ray absorptiometry (DXA) were assessed every 6 months up to 24 months during treatment. VFA with vertebral morphometry (MXA) was used to calculate the three indices of vertebral deformity: wedging, concavity and crushing. Serum calcium, phosphate, parathyroid hormone (PTH), 25-hydroxy-vitamin D [25(OH)D], total alkaline phosphatase (ALP), bone alkaline phosphatase (BALP) and urinary C-terminal telopeptide of type 1 collagen (CTx) were measured at any time point. RESULTS: Mean LS BMD values significantly increased at 24 months compared to baseline (p < 0.0001); the corresponding Z-score values were -1.28 ± 1.23 at 24 months vs -2.46 ± 1.25 at baseline; corresponding mean Bone Mineral Apparent Density (BMAD) values were 0.335 ± 0.206 vs 0.464 ± 0.216. Mean serum levels of ALP, BALP and CTx significantly decreased from baseline to 24 months. By MXA, we observed a significant 19.1% reduction of the mean wedging index of vertebral reshaping at 12 months, and 38.4% at 24 months (p < 0.0001) and of the mean concavity index (16.3% at 12 months and 35.9% at 24 months; p < 0.0001). Vertebral reshaping was achieved for 66/88 (75%) wedge fractures and 59/70 (84%) concave fractures, but there were 4 incident mild fractures. Finally, VF rate was reduced at 24 months compared to baseline: 37/710 (5.2%) vs 158/710 (22.2%). CONCLUSION: Our study demonstrates the utility of VFA as a safe and alternative methodology in the follow-up of children and adolescents with OI.

Sub-Clinical Effects of Chronic Noise Exposure on Vestibular System.

AIM: to investigate the effect of chronic noise exposure on vestibular function of subjects without clinical evidence of vestibular disorders and with documented cochlear damage from noise. SUBJECTS AND METHODS: 25 patients with chronic noise-induced hearing loss (NIHL) and without vestibular complaints (group A) and 25 matched controls with sensorineural hearing loss without noise exposure (group B), underwent audiological and vestibular test including caloric and cervical vestibular-evoked myogenic potentials tests (cVEMPs). RESULTS: In subjects chronically exposed to noise, similarly to that of the auditory threshold, an increase in the evocation threshold of VEMPs has been documented, statistically significant (p<0,05) and independent of the performance of the auditory threshold. p1-n1 amplitude values showed a significant difference between group A and group B. No significant difference for p1-n1 latencies between the two groups was found. CONCLUSION: We have documented the possibility of vestibular lesion, along with cochlear damage, related to chronic acoustic trauma.

White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways.

OBJECTIVE: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes. METHODS: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a ß3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue. RESULTS: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response. CONCLUSIONS: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

MR appearance of parathyroid adenomas at 3 T in patients with primary hyperparathyroidism: what radiologists need to know for pre-operative localization.

OBJECTIVES: To identify frequent MRI features of parathyroid adenomas (PTAs) in patients with primary hyperparathyroidism (PHPT) using a fast protocol with a 3 T magnet. METHODS: Thirty-eight patients with PHPT underwent a 3 T-MR. All patients had positive US and Tc-99 sestamibi, for a total number of 46 PTAs. T2-weighted IDEAL-FSE and T1 IDEAL-sequences, before and after contrast, were performed. Five features of PTAs were recognised: hyperintensity, homogeneous or "marbled" appearance and elongated morphology on T2-sequences; cleavage plane from thyroid gland on T2-outphase; rapid enhancement in post-contrast T1. Image quality for T2-weighted IDEAL FSE and usefulness for IDEAL post-contrast T1-weighted and T2-outphase sequences were also graded. RESULTS: PTAs were hyperintense in T2-sequences in 44/46 (95.7%), "marbled" in 30/46 (65.2%) and elongated in 38/46 (82.6%) patients. Cleavage plane was observed in 36/46 (78.3%), and rapid enhancement in 20/46 (43.5%) patients. T2-sequences showed both excellent fat suppression and image quality (average scores of 3.2 and 3.1). T2-outphase images demonstrated to be quite useful (score 2.8), whereas, post-contrast T1 images showed a lower degree of utility (score 2.4). CONCLUSIONS: A fast protocol with 3.0-T MRI, recognising most common features of PTAs, may be used as a second-line method in the preoperative detection of PTAs. KEY POINTS: 3 T MRI protocol based on T2-weighted IDEAL FSE sequences was used. T2-hyperintensity and elongated morphology are common features of PTAs. 3 T MRI could be used in the preoperative detection of PTAs.

Mesoderm-specific transcript (MEST) is a negative regulator of human adipocyte differentiation.

BACKGROUND: A growing body of evidence suggests that many downstream pathologies of obesity are amplified or even initiated by molecular changes within the white adipose tissue (WAT). Such changes are the result of an excessive expansion of individual white adipocytes and could potentially be ameliorated via an increase in de novo adipocyte recruitment (adipogenesis). Mesoderm-specific transcript (MEST) is a protein with a putative yet unidentified enzymatic function and has previously been shown to correlate with adiposity and adipocyte size in mouse. OBJECTIVES: This study analysed WAT samples and employed a cell model of adipogenesis to characterise MEST expression and function in human. METHODS AND RESULTS: MEST mRNA and protein levels increased during adipocyte differentiation of human multipotent adipose-derived stem cells. Further, obese individuals displayed significantly higher MEST levels in WAT compared with normal-weight subjects, and MEST was significantly correlated with adipocyte volume. In striking contrast to previous mouse studies, knockdown of MEST enhanced human adipocyte differentiation, most likely via a significant promotion of peroxisome proliferator-activated receptor signalling, glycolysis and fatty acid biosynthesis pathways at early stages. Correspondingly, overexpression of MEST impaired adipogenesis. We further found that silencing of MEST fully substitutes for the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) as an inducer of adipogenesis. Accordingly, phosphorylation of the pro-adipogenic transcription factors cyclic AMP responsive element binding protein (CREB) and activating transcription factor 1 (ATF1) were highly increased on MEST knockdown. CONCLUSIONS: Although we found a similar association between MEST and adiposity as previously described for mouse, our functional analyses suggest that MEST acts as an inhibitor of human adipogenesis, contrary to previous murine studies. We have further established a novel link between MEST and CREB/ATF1 that could be of general relevance in regulation of metabolism, in particular obesity-associated diseases.

Reliability of vertebral fractures assessment (VFA) in children with osteogenesis imperfecta.

The aim of this study is to evaluate the diagnostic accuracy of vertebral fractures assessment (VFA) in comparison with conventional radiography in identifying vertebral fractures in children and adolescents affected by OI. On 58 patients (33 males, 25 females; age range 1-18 years; 41 children and 17 adolescents) with osteogenesis imperfecta (OI type I, n = 44, OI type III, n = 4; OI type IV, n = 10), lateral spine images by radiographs and by dual-energy X-ray absorptiometry (DXA) were acquired. For vertebral fracture diagnosis, plain radiographs were used as "gold standard" and VFA and morphometric X-ray absorptiometry (MXA) were performed. The visualized vertebrae were 738 (97.9%) by radiographs and 685 (90.9%) by DXA of a total of 754 vertebrae from T4 to L4. VFA and MXA identified, respectively, 129 (74%) and 116 (66%) of the 175 vertebral fractures detected by radiographs. Radiographs identified 36 patients with vertebral fractures, VFA 35 and MXA 41 (6 false positives). On a per vertebra basis, radiographs and VFA had elevated agreement (93.9%; k score 0.81, 95% CI 0.76-0.86), that resulted slightly lower for MXA (90.6%; k score 0.72, 95% CI 0.65-0.78). VFA and MXA demonstrated high sensitivity (95.6 and 94.1 %, respectively) while specificity was 100% for VFA and 90.6% for MXA on a per patient basis; the agreement was excellent for VFA (98.3%; k score 0.96, 95% CI 0.89-1.03) and good for MXA (87.9%; k score 0.73, 95% CI 0.55-0.91). The diagnostic performance parameters resulted better for VFA (sensitivity 95.6%; specificity 100%; PPV 100%; NPV 97.2%), than for MXA (sensitivity 94.1%; specificity 85.4%; PPV 72.7%; NPV 97.2%). The results of our study demonstrate the reliability of VFA for diagnosis of vertebral fractures in children with OI suggesting its use as a more safe and practical alternative to conventional radiography.

Vertebral morphometry by dual-energy X-ray absorptiometry (DXA) for osteoporotic vertebral fractures assessment (VFA).

PURPOSE: This study was done to evaluate the diagnostic accuracy of dual-energy X-ray absorptiometry (DXA) compared with conventional radiography for identifying vertebral fractures. MATERIALS AND METHODS: A total of 930 postmenopausal women underwent conventional radiography and DXA imaging of the spine. The images were evaluated by two expert skeletal radiologists using the semiquantitative (SQ) method for conventional radiography and the morphometric vertebral fracture assessment (VFA) for DXA. RESULTS: The SQ method for radiography (SQ-Rx) analysed 99.1% of vertebrae, identifying 442 vertebral fractures; VFA analysed 97.5% vertebrae, detecting 420 vertebral fractures. Agreement between SQ-Rx and VFA reached 98.76%, and the κ-score was 0.96 [95% confidence interval (CI), 0.95-0.98]. Assessing the grading of vertebral fractures, agreement reached 97.5% and the κ-score was 0.841 (95% CI, 0.821-0.891). Considering SQ-Rx method as "gold standard", VFA had a sensitivity of 97.85 % and a specificity of 99.81%. The negative (NPV) and positive (PPV) predictive value for VFA were 99.83 % and 98.15%, respectively. Fractures were identified in 251 (27 %) and 242 (26 %) of patients on SQ-Rx and VFA, respectively. On a per-patient basis, the agreement between the two methods was 97% and the κ-score was 0.95 (95% CI, 0.920-0.968). The diagnostic parameters for VFA were 97.23% sensitivity, 98.86% specificity, 97.60% PPV and 98.84% NPV. CONCLUSIONS: This study demonstrated that VFA with DXA may reach a high level of accuracy for diagnosing vertebral fractures, suggesting that VFA should be introduced in the screening of individuals with a risk of osteoporosis and in the follow-up of osteoporotic patients receiving treatment.

Self-renewal gene tracking to identify tumour-initiating cells associated with metastatic potential.

Tumour-initiating cells (TICs) are rare cancer cells isolated from tumours of different origins including high-grade tumours that sustain neoplasic progression and development of metastatic disease. They harbour deregulated stem cells pathways and exhibit an unchecked ability to self-renew, a property essential for tumour progression. Among the essential factors maintaining embryonic stem (ES) cells properties, OCT-4 (also known as POU5F1) has been detected in tumours of different origins. Although ectopic expression results in dysplasic growth restricted to epithelial tissues, overexpression expands the proportion of immature cells in teratomas. However, OCT-4-expressing cells have not been purified from spontaneously occurring tumours, thus information concerning their properties is rather scant. Here, using p53-/- mice expressing green fluorescent protein and the puromycin resistance gene under the control of the Oct-4 promoter, we show that OCT-4 is expressed in 5% onwards of the undifferentiated tumour cell populations derived from different organs. OCT-4 expression was low as compared with ES cells, but was associated with a 'stemness' signature and expression of the chemokine receptor CXCR4. These cells displayed cancer stem cell features, including increased self-renewal and differentiation ability in vitro and in vivo. They not only formed allografts containing immature bone regions but also disseminated into different organs, including lung, liver and bone. Experiments based on RNA interference revealed that Oct-4 expression drives both their engraftment and metastasis formation. This work points out the crucial contribution of Oct-4-expressing TICs in the hierarchical organization of the malignant potential, leading to metastasis formation. Consequently, it provides an appropriate model to develop novel therapies aiming to strike down TICs by targeting self-renewal genes, therefore efficient to reduce tumour growth and metastatic disease.

Identification and characterization of visual pigments in caecilians (Amphibia: Gymnophiona), an order of limbless vertebrates with rudimentary eyes.

In comparison with the other amphibian orders, the Anura (frogs) and Urodela (salamanders), knowledge of the visual system of the snake-like Gymnophiona (caecilians) is relatively sparse. Most caecilians are fossorial with, as far as is known any surface activity occurring mainly at night. They have relatively small, poorly developed eyes and might be expected to possess detectable changes in the spectral sensitivity of their visual pigments. Microspectrophotometry was used to determine the spectral sensitivities of the photoreceptors in three species of caecilian, Rhinatrema bivittatum, Geotrypetes seraphini and Typhlonectes natans. Only rod opsin visual pigment, which may be associated with scotopic (dim light) vision when accompanied by other 'rod-specific' components of the phototransduction cascade, was found to be present. Opsin sequences were obtained from the eyes of two species of caecilian, Ichthyophis cf. kohtaoensis and T. natans. These rod opsins were regenerated in vitro with 11-cis retinal to give pigments with spectral sensitivity peaks close to 500 nm. No evidence for cone photoreception, associated with diurnal and colour vision, was detected using molecular and physiological methods. Additionally, visual pigments are short-wavelength shifted in terms of the maximum absorption of light when compared with other amphibian lineages.

Where's the glass? Biomarkers, molecular clocks, and microRNAs suggest a 200-Myr missing Precambrian fossil record of siliceous sponge spicules.

The earliest evidence for animal life comes from the fossil record of 24-isopropylcholestane, a sterane found in Cryogenian deposits, and whose precursors are found in modern demosponges, but not choanoflagellates, calcareans, hexactinellids, or eumetazoans. However, many modern demosponges are also characterized by the presence of siliceous spicules, and there are no convincing demosponge spicules in strata older than the Cambrian. This temporal disparity highlights a problem with our understanding of the Precambrian fossil record--either these supposed demosponge-specific biomarkers were derived from the sterols of some other organism and are simply retained in modern demosponges, or spicules do not primitively characterize crown-group demosponges. Resolving this issue requires resolving the phylogenetic placement of another group of sponges, the hexactinellids, which not only make a spicule thought to be homologous to the spicules of demosponges, but also make their first appearance near the Precambrian/Cambrian boundary. Using two independent analytical approaches and data sets--traditional molecular phylogenetic analyses and the presence or absence of specific microRNA genes--we show that demosponges are monophyletic, and that hexactinellids are their sister group (together forming the Silicea). Thus, spicules must have evolved before the last common ancestor of all living siliceans, suggesting the presence of a significant gap in the silicean spicule fossil record. Molecular divergence estimates date the origin of this last common ancestor well within the Cryogenian, consistent with the biomarker record, and strongly suggests that siliceous spicules were present during the Precambrian but were not preserved.

A new predictive index for vertebral fractures: the sum of the anterior vertebral body heights.

UNLABELLED: Evaluation of osteoporotic vertebral fracture risk is currently based on measurement of bone mineral density (BMD), but bone strength depends also on bone quality parameters. Aim of this study was to evaluate the validity of a new vertebral morphometric index, the Anterior Vertebral Heights sum (AHs) in discriminating women at high risk of vertebral fracture, comparing its diagnostic accuracy with that of BMD measured at lumbar spine (LS-BMD) and femoral neck (FN-BMD). MATERIALS AND METHODS: A total of 163 Caucasian post-menopausal women (age range 46-74 years, mean age+/-SD=63.8+/-7.1 years), who did not present prevalent fractures at baseline evaluation, were observed at longitudinal follow-up. X-ray of the thoracic and lumbar spine, LS-BMD and FN-BMD measurements were obtained in all patients at baseline and repeated at the second follow-up visit 18-24 months later (mean 21+/-1.7 months). Radiographs of spine were analysed in order to identify vertebral fractures using a visual semiquantitative method (SQ) and vertebral morphometry as well as by calculating the AHs morphometric index. RESULTS: During follow-up, 21/163 patients (12.9%) sustained a new vertebral fracture; 95.2% (20/21) of fractured patients but only 4.9% (7/142) of non-fractured women had reduced AHs values. As regarding BMD, 66.6% (14/21) and 61.9% (13/21) of women with incident fracture were osteoporotic at lumbar spine and femoral neck baseline evaluation , whereas among non-fractured women, 38% (54/142) at LS-BMD and 33.1% (47/142) at FN-BMD were osteoporotic . Analyses of Receiver Operating Characteristic (ROC) curves showed that AHs discriminated vertebral fractures almost perfectly (AUC 0.97; 95% CI 0.95-0.99). On the other hand, the AUC for LS-BMD was only 0.73 (95% CI 0.64-0.81) and for FN-BMD was 0.72 (95%CI 0.63-0.80), showing that the diagnostic accuracy of AHs was significantly higher compared to that of LS-BMD (p<0.001) or FN-BMD (p<0.001). A modified Poisson regression model for binary data was used to assess the independent role of AHs in predicting vertebral fracture. The effect of AHs remained statistically significant (p<0.001) after adjusting by FN-BMD, age, weight and body height. CONCLUSIONS: Results of this study indicate the validity of this new morphometric index in evaluating the risk of osteoporotic vertebral fractures thus suggesting that AHs should be considered a valid parameter in clinical practice to assess the need for primary prevention of vertebral fractures.

Male osteoporosis.

Osteoporosis is a worldwide major public health problem, defined as "a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fractures". Osteoporosis is diagnosed by bone mineral density measurement (T-score of -2.5 or below) also in men. However, most of the studies carried out in the last decade focused on pathogenesis, diagnosis and treatment of osteoporosis in women. In spite of this, recent epidemiological and observational studies have shown that osteoporosis in men is an increasingly important clinical issue. In part because the world population is aging, it is likely that the total number of hip fractures in men in 2025 will be similar to current estimates in women. Furthermore 25-33% of men in some populations will sustain osteoporotic fractures in their lifetime. Nevertheless, male osteoporosis is still underdiagnosed and further studies are required to clarify the pathogenesis and find out the right therapy. Prevention and early diagnosis are, nowadays, the best ways of treatment.

[Ulcerative colitis and thrombocytopenia: a clinical case of fortuitous association?]. / Rettocolite ulcerosa e piastrinopenia: un caso clinico di associazione fortuita?

Ulcerative colitis (UC) is an inflammatory bowel disease of unknown etiology, involving primarily the rectum with major intestinal symptoms. Additionally, UC is often associated with extraintestinal manifestations, especially arthropathies, as well as with some autoimmune disorders. Vice versa, UC is rarely described in association with hematologic abnormalities, such as autoimmune hemolytic anemia and immune thrombocytopenic purpura with antiplatelet antibodies positive. Usually UC precedes the onset of thrombocytopenia by days or years or coincides with it. We report a case of UC and thrombocytopenia with negative anti-platelet antibodies in which an immunosuppressive therapy with corticosteroids obtained significant remission of intestinal symptoms along with a rapid increase of platelet count.