Quantitative Effect of a CNV on a Morphological Trait in Chickens

Copy Number Variation has been associated with morphological traits, developmental defects or disease susceptibility. The autosomal dominant Pea-comb mutation in chickens is due to the massive amplification of a CNV in intron 1 of SOX5 and provides a unique opportunity to assess the effect of variation in the number of repeats on quantitative traits such as comb size and comb mass in Pea-comb chickens. The quantitative variation of comb size was estimated by 2D morphometry and the number of repeats (RQ) was estimated by qPCR, in a total of 178 chickens from 3 experimental lines, two of them showing segregation for the Pea-comb mutation. This study included only Pea-comb chickens. Analysis of variance showed highly significant effects of line and sex on comb measurements. Adult body weight (BW) and RQ were handled as covariates. BW significantly influenced comb mass but not comb size. RQ values significantly influenced comb size, and the linear regression coefficient was highest for heterozygous carriers: the higher the number of repeats, the smaller the comb size. A similar trend was observed for comb mass. The CNV contributed to 3.4% of the phenotypic variance of comb size in heterozygous carriers of the CNV, an order of magnitude frequently encountered for QTLs. Surprisingly, there was no such relationship between RQ values and comb size in the homozygous line. It may be concluded that heterozygosity for a CNV in a non-coding region may contribute to phenotypic plasticity.     

Binding Affinity,Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with ²¹¹At-Rebmab200. Here, the biodistribution of ²¹¹At-Rebmab200 was evaluated, as was the utility of ^99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that ^99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.     

The Role of Edaphic Environment and Climate in Structuring Phylogenetic Pattern in Seasonally Dry Tropical Plant Communities

Seasonally dry tropical plant formations (SDTF) are likely to exhibit phylogenetic clustering owing to niche conservatism driven by a strong environmental filter (water stress), but heterogeneous edaphic environments and life histories may result in heterogeneity in degree of phylogenetic clustering. We investigated phylogenetic patterns across ecological gradients related to water availability (edaphic environment and climate) in the Caatinga, a SDTF in Brazil. Caatinga is characterized by semiarid climate and three distinct edaphic environments – sedimentary, crystalline, and inselberg –representing a decreasing gradient in soil water availability. We used two measures of phylogenetic diversity: Net Relatedness Index based on the entire phylogeny among species present in a site, reflecting long-term diversification; and Nearest Taxon Index based on the tips of the phylogeny, reflecting more recent diversification. We also evaluated woody species in contrast to herbaceous species. The main climatic variable influencing phylogenetic pattern was precipitation in the driest quarter, particularly for herbaceous species, suggesting that environmental filtering related to minimal periods of precipitation is an important driver of Caatinga biodiversity, as one might expect for a SDTF. Woody species tended to show phylogenetic clustering whereas herbaceous species tended towards phylogenetic overdispersion. We also found phylogenetic clustering in two edaphic environments (sedimentary and crystalline) in contrast to phylogenetic overdispersion in the third (inselberg). We conclude that while niche conservatism is evident in phylogenetic clustering in the Caatinga, this is not a universal pattern likely due to heterogeneity in the degree of realized environmental filtering across edaphic environments. Thus, SDTF, in spite of a strong shared environmental filter, are potentially heterogeneous in phylogenetic structuring. Our results support the need for scientifically informed conservation strategies in the Caatinga and other SDTF regions that have not previously been prioritized for conservation in order to take into account this heterogeneity.     

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1 ^-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1 ^-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1 ^-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1 ^-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.     

Treatment of Solitary Painful Osseous Metastases with Radiotherapy,Cryoablation or Combined Therapy: Propensity Matching Analysis in 175 Patients

Purpose

aim of this study was to identify outcomes in pain relief and quality of life in patients with a solitary painful osseous metastasis treated by radiotherapy, cryoablation or the combination using a propensity score matching study design.

Materials and Methods

175 patients with painful bone metastases were included in the study. Twenty-five of them underwent a radiation course (20 Gy in five daily fractions) 15 days after the cryoablation. These subjects were retrospectively matched by propensity analysis with a group of subjects treated by radiotherapy (125 subjects) and with a group treated byCryoablation (25 subjects). The pain relief in terms of complete response, rate of subjects requiring analgesics after treatments and the changes in self-rated quality of life were measured. Informed consent was obtained from the subject and the study was approved by the local Ethical Committee.

Results

An higher proportion of subjects treated by cryoablation (32%) or cryoablation followed by RT (72%;) experienced a complete response compared with patients treated by radiotherapy alone (11.2%). After Bonferroni correction strategy, the addition of radiotherapy to cryoablation significantly improved the rate of complete response compared with cryoablation alone (p = 0.011) and this paralleled with an improved self-rated quality of life. Seventeen subjects (13.6%) of patients in the radiotherapy group, 9 (36%) in the cryoablation group, and 19 (76)% in the cryoablation- radiotherapy group did not require narcotic medications.

Conclusions

The addition of radiotherapy to cryoablation favorably impacts on perceived pain, with a favorable toxicity profile. However, our data should be interpreted with caution and could serve as a framework around which to design future trials.     

Comparison of Humanized IgG and FvFc Anti-CD3 Monoclonal Antibodies Expressed in CHO Cells

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.     

A novel functional role of iduronate-2-sulfatase in zebrafish early development

Sulfated glycosaminoglycan chains of extracellular matrix and cell membrane-tethered proteoglycans exert specific cellular functions by interacting with a broad spectrum of morphogens and growth factors.In humans, a congenital impaired catabolism of sulfated glycosaminoglycans is associated with severe metabolic disorders. Here, we report on the identification and characterization of a zebrafish iduronate sulfatase orthologue. By knocking down its function with antisense morpholino oligos, we demonstrate that iduronate sulfatase plays a critical role during early vertebrate development and its downregulation may be responsible for severe developmental defects, including a misshapen trunk and abnormal craniofacial cartilages. We show that the altered cartilage patterning is mediated by depauperation of sox10-expressing neural crest cell precursors. Through the application of a transactivation reporter assay, we also provide a molecular proof that increased TGFβ (Transforming Growth Factor β) signalling is tightly associated with downregulation of iduronate sulfatase function. Our results provide an insight into the early biological impairments underlying the Hunter syndrome and suggest the use of zebrafish as a novel tool to better understand lysosomal storage disorder pathogenesis.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

Acute exposure to long-chain fatty acids impairs {alpha}2-adrenergic receptor-mediated antilipolysis in human adipose tissue

The acute in vitro and in vivo effects of long-chain fatty acids (LCFAs) on the regulation of adrenergic lipolysis were investigated in human adipose tissue. The effect of a 2 h incubation, without or with LCFA (200 mumol/l), on basal and hormonally induced lipolysis was tested in vitro on isolated fat cells. The lipolytic response to epinephrine was enhanced by suppression of the antilipolytic alpha(2)-adrenergic effect. Then, healthy lean and obese male subjects performed a 45 min exercise bout at 50% of their heart rate reserve either after an overnight fast or 3 h after a high-fat meal (HFM: 95% fat, 5% carbohydrates). Subcutaneous adipose tissue lipolysis was measured by microdialysis in the presence or absence of an alpha-antagonist (phentolamine). In vivo, a HFM increased plasma levels of nonesterified fatty acids in lean and obese subjects. In both groups, the HFM did not alter hormonal responses to exercise. Under fasting conditions, the alpha(2)-adrenergic antilipolytic effect was more pronounced in obese than in lean subjects. The HFM totally suppressed the alpha(2)-adrenergic antilipolytic effect in lean and obese subjects during exercise. LCFAs per se, in vitro as well as in vivo, suppress alpha(2)-adrenergic-mediated antilipolysis in adipose tissue. LCFA-mediated suppression of antilipolytic pathways represents another mechanism whereby a high fat content in the diet might increase adipose tissue lipolysis.