Observations on population structure and reproductive features of <Emphasis Type="Italic">Laetmonice producta</Emphasis> Grube (Polychaeta,Aphroditidae) in Antarctic waters

The large scale-worm Laetmonice producta Grube 1877 is the most abundant aphroditid polychaete in Antarctic coastal waters. We investigated the demographic structure and some reproductive features of different L. producta populations from high-Antarctic (Weddell Sea) and Antarctic Peninsula (King George Island) shelf bottoms, collected in summer 1996 (ANT-XIII/3, EASIZ I cruise) and autumn 2000 (ANT-XVII/3, EASIZ III cruise). L. producta in the studied geographic areas showed a wide bathymetric range (200-850 m depth), and a different size distribution pattern with depth, characterised by a reduction of large specimens in the deepest stations. The species is gonochoric, with females more abundant in specimens of larger sizes. Eggs at different stages of maturation (ranging from 40 to 320 wm in diameter) were examined in 270 individuals from different stations and size classes. Egg size showed a slightly bimodal trend, with largely overlapping egg cohorts, suggesting a continuous reproduction, and a long-lasting gametogenesis. Significant differences (Kolmogorov-Smirnov test, P<0.05) in egg-size frequency distribution were detected only when data of the two geographic areas were compared (Weddell Sea vs King George Island), and not according to stations within each area, and females' size. The two sets of geographic samples were collected in different seasons and therefore it was not possible to assess if differences observed are due to sampling time or to geographic factors. Mature spermatozoa were recognisable only in autumn male specimens from King George Island, and showed a rounded nucleus and a short conical acrosome. Occurrence of an endosymbiont polychaete, Veneriserva pygoclava meridionalis (new sub-species of Dorvilleidae), was recorded in the coelomic cavity of 163 specimens of L. producta, 125 of which were from the deepest station of the Weddell Sea (stn. 14, 850 m depth). L. producta females with and without the endosymbiont did not show differences in egg-size distribution. The reproductive features of L. producta, together with its large size and slow growth, seem typical of a long-living predator species, and uncoupled from the typical summer environmental conditions and the pulsating system of coastal Antarctic waters.     

Improved in Planta Expression of the Human Islet Autoantigen Glutamic Acid Decarboxylase (GAD65)

The smaller isoform of the enzyme glutamic acid decarboxylase (GAD65) is a major islet autoantigen in autoimmune type 1 diabetes mellitus (T1DM). Transgenic plants expressing human GAD65 (hGAD65) are a potential means of direct oral administration of the islet autoantigen in order to induce tolerance and prevent clinical onset of disease. We have previously reported the successful generation of transgenic tobacco and carrot that express immunoreactive, full-length hGAD65. In the present study, we tested the hypothesis that the expression levels of recombinant hGAD65 in transgenic plants can be increased by targeting the enzyme to the plant cell cytosol and by mediating expression through the potato virus X (PVX) vector. By substituting the NH₂-terminal region of hGAD65 with a homologous region of rat GAD67, a chimeric GAD67_1-87/GAD65_88-585 molecule was expressed in transgenic tobacco plants. Immunolocalization analysis showed that immunoreactive GAD67/65 was found in the plant cell cytosol. By using a radio-immuno assay with human serum from a GAD65 autoantibody-positive T1DM patient, the highest expression level of the recombinant GAD67/65 protein was estimated to be 0.19% of the total soluble protein, compared to only 0.04% of wild-type hGAD65. Transient expression of wild-type, full-length hGAD65 in N. benthamiana mediated by PVX infection was associated with expression levels of immunoreactive protein as high as 2.2% of total soluble protein. This substantial improvement of the expression of hGAD65 in plants paves the way for immunoprevention studies of oral administration of GAD65-containing transgenic plant material in animal models of spontaneous autoimmune diabetes.     

In Central Obesity,Weight Loss Restores Platelet Sensitivity to Nitric Oxide and Prostacyclin

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMA_IR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMA_IR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

Excitability of the Motor Cortex in De Novo Patients with Celiac Disease

Introduction

Celiac disease (CD) may initially present as a neurological disorder or may be complicated by neurological changes. To date, neurophysiological studies aiming to an objective evaluation of the potential central nervous system involvement in CD are lacking.

Objective

To assess the profile of cortical excitability to Transcranial Magnetic Stimulation (TMS) in a group of de novo CD patients.

Materials and methods

Twenty CD patients underwent a screening for cognitive and neuropsychiatric symptoms by means of the Mini Mental State Examination and the Structured Clinical Interview for DSM-IV Axis I Disorders, respectively. Instrumental exams, including electroencephalography and brain computed tomography, were also performed. Cortico-spinal excitability was assessed by means of single and paired-pulse TMS using the first dorsal interosseus muscle of the dominant hand. TMS measures consisted of resting motor threshold, motor evoked potentials, cortical silent period (CSP), intracortical inhibition (ICI) and facilitation (ICF). None of the CD was on gluten-free diet. A group of 20 age-matched healthy controls was used for comparisons.

Results

CD showed a significantly shorter CSP (78.0 vs 125.0 ms, p<0.025), a reduced ICI (0.3 vs 0.2, p<0.045) and an enhanced ICF (1.1 vs 0.7, p<0.042) compared to controls. A dysthymic disorder was identified in five patients. The effect size between dysthymic and non-dysthymic CD patients indicated a low probability of interference with the CSP (Cohen''s d -0.414), ICI (-0.278) and ICF (-0.292) measurements.

Conclusion

A pattern of cortical excitability characterized by “disinhibition” and “hyperfacilitation” was found in CD patients. Immune system dysregulation might play a central role in triggering changes of the motor cortex excitability.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Tree-ring wood anatomy and stable isotopes show structural and functional adjustments in olive trees under different water availability

Background and Aims

Olive tree (Olea europaea L.) is a drought-tolerant tree species cultivated in Mediterranean-type environments. Although it is tolerant to drought, dry conditions decrease its productivity. A thorough analysis of the hydraulic architecture and wood anatomical plasticity, as well as of their physiological significance, is needed to understand how olive trees will adapt to the predicted increase in frequency and severity of drought in the Mediterranean region.

Methods

Dendrochronological, stable isotopic (δ¹³C, δ¹⁸O) and wood anatomical analyses were applied to understand how different water availability can affect wood stem structure and function, in rainfed and irrigated at 100 % of crop evapotranspiration (ETc) olive trees in an experimental orchard close to Benevento (Italy) from 1992 to 2009.

Results

Dendrochronological data indicate that cross-dating and synchronization of ring-width time series in olive tree is possible. After the start of irrigation, significantly more negative δ¹³C and lower δ¹⁸O values were recorded in irrigated trees indicating higher stomatal conductance and transpiration rates. Increased water balance induced the formation of a higher number of vessels with higher diameter.

Conclusions

Water balance variations affected wood anatomy and isotopic composition. Anatomical analyses detected structural and functional adjustments in rainfed trees that produced more vessels with lower diameter to prevent cavitation. Isotopic analyses confirmed that irrigated trees continuously showed enhanced transpiration rates.     

Modeling human osteosarcoma in mice through 3AB-OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer‐related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB‐OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB‐OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB‐OS CSC xenografts lacked crucial regulators of beta‐catenin levels (E‐cadherin, APC, and GSK‐3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB‐OS‐derived tumors expressed high levels of pAKT, beta1‐integrin and pFAK, nuclear beta‐catenin, c‐Myc, cyclin D2, along with high levels of hyperphosphorylated‐inactive pRb and anti‐apoptotic proteins such as Bcl‐2 and XIAP, and matrigel increased the expression of proliferative markers. Thereafter 3AB‐OS tumor xenografts obtained with matrigel co‐injection showed decreased proliferative potential and AKT levels, and undetectable hyperphosphorylated pRb, whereas beta1‐integrin and pFAK levels still increased. Engrafted tumor cells also showed multilineage commitment with matrigel particularly favoring the mesenchymal lineage. Concomitantly, many blood vessels and muscle fibers appeared in the tumor mass. Our findings suggest that matrigel might regulate 3AB‐OS cell behavior providing adequate cues for transducing proliferation and differentiation signals triggered by pAKT, beta1‐integrin, and pFAK and addressed by pRb protein. Our results provide for the first time a mouse model that recapitulates in vivo crucial features of human osteosarcoma CSCs that could be used to test and predict the efficacy in vivo of novel therapeutic treatments. J. Cell. Biochem. 113: 3380–3392, 2012. © 2012 Wiley Periodicals, Inc.