Atomic force spectroscopy-based essay to evaluate oocyte postovulatory aging

Postovulatory aging is a process occurring in the mature (MII) oocyte leading the unfertilized ones to apoptosis. The optimal time window of fertility for different mammalian species after oocytes maturation depends on its timeliness: the higher the time elapsed from the accomplishment of the MII stage, the lower are the chances of fertilization and of development of a viable embryo. In the in vitro fertilization, the selection of competent oocytes for intracytoplasmic sperm injection (ICSI) is mostly made by the visual inspection of the MII oocyte morphology, which does not allow to determine the oocyte postovulatory age. On the other hand, more specific tests usually involve some kind of staining, thus compromising the viability of the oocyte for reproductive purposes. Hence, the need of a noninvasive analysis of oocyte aging to improve the success rate of in vitro fertilization procedures. Here, we exploit atomic force microscopy to examine the evolution of the mechanical properties of mouse oocytes during in vitro postovulatory aging. Three hours before the occurrence of any visual morphological feature related to degradation, we observe a sudden change of the mechanical parameters: the elastic modulus doubles its initial value, while the viscosity decreases significantly. These mechanical variations are temporally correlated with the release of the cortical granules, investigated by fluorescence microscopy. Interestingly, the oocyte mechanics correlates as well with the yield of embryo formation, evaluated up to the blastocyst formation stage. These results demonstrate that minimally invasive mechanical measurements are very sensitive to the aging of the oocyte and can be used as a label-free method to detect the age of the postovulatory oocytes

Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models

<p>Abstract</p> <p>Background</p> <p>Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated.</p> <p>Results</p> <p>We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host.</p> <p>Conclusion</p> <p>In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.</p

Cyanidin 3-glucoside targets a hepatic bilirubin transporter in rats

One of the organ-specific functions of the liver is the excretion of bilirubin into the bile. Membrane transport of bilirubin from the blood to the liver is not only an orphan function, because there is no link to the protein/gene units that perform this function, but also a poorly characterised function. The aim of this study was to investigate the pharmacology of bilirubin uptake in the liver of the female Wistar rat to improve basic knowledge in this neglected area of liver physiology. We treated isolated perfused livers of female rats with repeated single-pass, albumin-free bilirubin boli. We monitored both bilirubin and bilirubin glucuronide in perfusion effluent with a bio-fluorometric assay. We tested the ability of nine molecules known as substrates or inhibitors of sinusoidal membrane transporters to inhibit hepatic uptake of bilirubin. We found that cyanidin 3-glucoside and malvidin 3-glucoside were the only molecules that inhibited bilirubin uptake. These dietary anthocyanins resemble bro-mosulfophthalein (BSP), a substrate of several sinusoidal membrane transporters. The SLCO-specific substrates estradiol-17 beta-glucuronide, pravastatin, and taurocholate inhibited only bilirubin glucuronide uptake. Cya-nidin 3-glucoside and taurocholate acted at physiological concentrations. The SLC22-specific substrates indo-methacin and ketoprofen were inactive. We demonstrated the existence of a bilirubin-glucuronide transporter inhibited by bilirubin, a fact reported only once in the literature. The data suggest that bilirubin and bilirubin glucuronide are transported to the liver via pharmacologically distinct membrane transport pathways. Some dietary anthocyanins may physiologically modulate the uptake of bilirubin into the liver

Conjugated PDT drug: photosensitizing activity and tissue distribution of PEGylated pheophorbide a.

The design of new photosensitizers with enhanced phototoxicity and pharmacokinetic properties remains a central challenge for cancer photodynamic therapy (PDT). In this study, Pheophorbide a (Pba) has been pegylated to methoxypolyethylene glycol (mPE G-Pba) to produce a soluble photosensitizer that exhibits a higher tissue distribution than free Pba. In vitro studies have shown that mPE G-Pba promotes a fairly strong photosensitizing effect in cancer cells, as previously observed for the unpegylated molecule. mPE G-Pba targets the mitochondria where, following photoactivation, ROS are produced which cause a cellular injury by lipid peroxidation. The effect of pegylation on the photosensitizer biodistribution has been examined in different selected organs of female mice, at different time points after intraperitoneal administration of the drug (50 μmol/Kg body weight). Other than free Pba, which showed a low tissue accumulation, mPE G-Pba has been detected in significant amounts (8 to 16 μg/ml) in liver, spleen, duodenum and kidney and, 3-5 hours after intraperitoneal injection, in moderate amounts (3 to 8 μg/ml) in brain and lung. In vivo optical imaging performed on living female C57/BL6 mice bearing a subcutaneous melanoma mass, showed that injected mPEG-Pba distributes all over the body, with an higher uptake in the tumor respect to free Pba. Our results indicate that although pegylation somewhat decreases the phototoxicity, it significantly increases the drug solubility and tissue distribution and tumor uptake of mPE G-Pba, making the conjugate an interesting photosensitizer for PDT

Anti Transglutaminase Antibodies Cause Ataxia in Mice

Background: Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. Methods: We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. Conclusion: The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

An improved understanding of an ovary’s structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin’s solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues

Development, Production and Evaluation of Aerosol Climate Data Records from European Satellite Observations (Aerosol_cci)

Producing a global and comprehensive description of atmospheric aerosols requires integration of ground-based, airborne, satellite and model datasets. Due to its complexity, aerosol monitoring requires the use of several data records with complementary information content. This paper describes the lessons learned while developing and qualifying algorithms to generate aerosol Climate Data Records (CDR) within the European Space Agency (ESA) Aerosol_cci project. An iterative algorithm development and evaluation cycle involving core users is applied. It begins with the application-specific refinement of user requirements, leading to algorithm development, dataset processing and independent validation followed by user evaluation. This cycle is demonstrated for a CDR of total Aerosol Optical Depth (AOD) from two subsequent dual-view radiometers. Specific aspects of its applicability to other aerosol algorithms are illustrated with four complementary aerosol datasets. An important element in the development of aerosol CDRs is the inclusion of several algorithms evaluating the same data to benefit from various solutions to the ill-determined retrieval problem. The iterative approach has produced a 17-year AOD CDR, a 10-year stratospheric extinction profile CDR and a 35-year Absorbing Aerosol Index record. Further evolution cycles have been initiated for complementary datasets to provide insight into aerosol properties (i.e., dust aerosol, aerosol absorption).Peer reviewe

Thoracic cage plasticity in prepubertal New Zealand white rabbits submitted to T1-T12 dorsal arthrodesis: computed tomography evaluation, echocardiographic assessment and cardio-pulmonary measurements.

Purpose: We aimed to describe the morphological changes in the thoracic cage and spinal column induced in New Zealand White (NZW) prepubertal rabbits subjected to dorsal arthrodesis and observed at skeletal maturity by computed tomography (CT) scans. This was done to evaluate the plasticity of the thoracic cage of rabbits with non-deformed spine, by highlighting its modifications after spinal arthrodesis. Emogas data analysis, echocardiographic assessment and cardio-pulmonary measurements completed the evaluation. Methods: Surgery was performed in 16 female rabbits, 6 weeks old. Nine were subjected to T1-T12 dorsal arthrodesis, while seven were sham-operated. Surgery involved the implant of two C-shaped stainless steel bars and heterologous bone graft. CT scans were performed before surgery, 2, 6 and 12 months after surgery. One week after the last CT scan, echocardiographic and emogas evaluations were performed. Results: Chest depth (8 %), thoracic kyphosis (ThK) (23 %), dorsal and ventral length of the thoracic spine (11 %) and sternal length (7 %) were significantly reduced in operated compared to sham-operated rabbits. Mean values \ub1 standard deviation (SD) of PaCO2, PaO2 and sO2 were not significantly different. Mean values \ub1 SD of echocardiographic measurements were not significantly different between the two groups of rabbits, except for thickness of the interventricular septum in systole, contractile capacity of the left ventricle and ejection fraction. Conclusions: T1-T12 dorsal arthrodesis in prepubertal NZW rabbits with non-deformed spine induced changes of the thoracic cage morphology. However, those changes are source of cardio-pulmonary complications not severe enough to reproduce a clinical picture comparable to thoracic insufficiency syndrome in humans. \ua9 2013 Springer-Verlag Berlin Heidelberg

Relocation and Hair Cortisol Concentrations in New Zealand White Rabbits

To investigate how long relocation modified hair cortisol concentrations in New Zealand white rabbits, 19 rabbits were subjected to a change in their breeding facility at the beginning of the trial and then were kept under stable environmental conditions. Hair samples were collected at the time of arrival to the nonhuman animal facility and at 40-day intervals from the same skin area for up to 440 days after the animals' arrival to the facility. A period effect on the hair cortisol concentration was found (p <.01). The transfer of the rabbits to the new facility might have induced an increase in the hypothalamic-pituitary-adrenal axis activity (p <.01). A second increase in hair cortisol concentration (p <.01) occurred at 320 days, after a change of personnel at the facility that occurred at 280 days, which was the only environmental change. The relocation of rabbits to the facility resulted in a stress response leading to elevated cortisol levels. The effect of relocation on mean cortisol concentrations was exhausted within 120 days when all environmental factors were kept stable. \ua9 2016 Taylor & Francis Group, LLC

Dorsal arthrodesis in prepubertal New Zealand white rabbits followed to skeletal maturity: Effect on thoracic dimensions, spine growth and neural elements

Background: Several studies have shown that severe spinal deformity and early arthrodesis can adversely affect the development of the spine and thorax by changing their shape and reducing their normal function. This article analyzes the consequences of posterior fusion on the growth of spine, thorax and neural elements in New Zealand white rabbits and compares with similar human data. Materials and Methods: The first section of the article analyzes the consequences of T1-T6 dorsal arthrodesis on the growth of the spine, sternum, thorax volume and neural elements in 12 prepubertal female New Zealand white rabbits, through a study of CT scans and histology specimens. The second part, evaluates thoracic dimensions in 21 children with spinal arthrodesis for treatment of deformity performed prior to nine years of age. Results: Dorsal arthrodesis in prepubertal rabbits changes thoracic growth patterns. In operated rabbits thoracic depth grows more slowly than thoracic width. The sternum as well as length of thoracic vertebral bodies in the spinal segment T1-T6 show reduced growth. Children undergoing spinal arthrodesis before nine years of age were noted to have shortened height, short trunk and disproportionate body habitus at skeletal maturity. Observed spine height and chest dimension values were reduced compared to the expected norms. The ratio between chest width and chest depth was below normal values. Conclusions: The first part of the study shows that thoracic dorsal arthrodesis in prepubertal New Zealand white rabbit influences thoracic, spine growth and affects the shape of pseudo unipolar neurons of the dorsal root ganglia. The second part demonstrates that children treated before nine years of age have significantly reduced spine height and thoracic dimensions. The thorax becomes elliptical as chest depth grows less than chest width. Both experimental and clinical findings contribute to explain reduced chest growth and subsequent thoracic growth disturbance in patients treated with early arthrodesis