Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures.

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L-1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.

The use of reduced glutathione (GSH) as antioxidant for cryopreserved sperm in dogs / O uso da glutationa reduzida (GSH) como antioxidante na criopreservação seminal em cães

The aim of this study was to evaluate the effect of supplementation with different concentrations of reduced glutathione GSH (0; 5; 7.5; 10mM) in the extender for cryopreservation in dogs with evaluations performed after glycerolization (chilled) and thawing (thawed). For this purpose, we used 8 dogs and two semen collections were performed in a weekly interval, totaling 16 semen samples. The sperm were analyzed by automatic sperm motility (CASA) and flow cytometry analysis of mitochondrial potential (JC1 dye) and membrane/acrosome integrity (FITC-PI dyes). We evaluated subjectively the membrane and acrosome integrity, mitochondrial activity and DNA integrity. Seminal plasma was evaluated for lipid peroxidation (TBARS concentration). Chilled and thawed samples supplemented with 7.5 and 10mM of GSH had lower percentage of sperm with high (DAB - Class I) and medium (DAB - Class II) mitochondrial activity. And 10mM of GSH had higher percentage of low mitochondrial activity (DAB - Class III). Moreover, thawed samples of 10mM of GSH had high DNA fragmentation rates. Probably by a reductive stress effect on mitochondria which lead to an increase in reactive oxygen species, and a mitochondrial malfunction.(AU)

O objetivo deste estudo foi avaliar o efeito da suplementação com diferentes concentrações de glutationa reduzida (GSH - 0; 5; 7,5; 10mM) para criopreservação em cães com avaliações realizadas após glicerolização (refrigeração) e descongelação. Para tal, foram utilizados oito cães e foram realizadas duas coletas de sêmen em intervalo semanal, totalizando 16 amostras de sêmen. Foram avaliadas a motilidade espermática computadorizada (CASA) e a análise de citometria de fluxo do potencial mitocondrial (sonda JC-1) e integridade da membrana/acrossomal (sonda FITC-PI). Subjetivamente foi avaliada a integridade da membrana plasmática e do acrossomal, atividade mitocondrial e integridade do DNA. O plasma seminal foi avaliado quanto à peroxidação lipídica (concentração de TBARS). As amostras refrigeradas e descongeladas suplementadas com 7,5 e 10mM de GSH apresentaram menor porcentagem de espermatozoides com alta atividade mitocondrial (DAB - Classe I) e média (DAB - Classe II). Na concentração de 10mM de GSH, apresentaram maior porcentagem de baixa atividade mitocondrial (DAB - Classe III). Além disso, amostras descongeladas de 10mM de GSH apresentaram taxas de fragmentação de DNA elevadas, provavelmente por efeito de estresse redutivo sobre as mitocôndrias que elevam as espécies reativas de oxigênio e disfunção mitocondrial.(AU)

Produção de sentidos no começo da vida / Making sense in early life

Neste estudo discutiu-se a atividade de produção de sentidos no início da vida apontando-se a operaçãode processos co-regulados. Trata-se de um estudo longitudinal da interação de uma díade mãe-bebê. Apartir de uma análise microgenética foi demonstrada uma trajetória de desenvolvimento da comunicaçãomediada por brinquedos. Nessa trajetória destacaram-se perfis de negociação no compartilhamento deatenção revelados em reorganizações frequentes. A produção de sentidos foi concebida como atos deresponsividade relacionados com experiências passadas e com antecipações de possibilidades deexperiências futuras envolvendo o brinquedo. Concluiu-se que a produção de sentidos na interação sãosituações de resolução momentânea de tensões promovidas por essa dinâmica de ações para ondeconvergem, simultaneamente, as experiências passadas e expectativas de possibilidades futuras(AU)

Transplantation of Human Adipose Mesenchymal Stem Cells in Non-Immunosuppressed GRMD Dogs is a Safe Procedure.

The possibility to treat Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, through cell therapy with mesenchymal stromal cells (MSCs) has been widely investigated in different animal models. However, some crucial questions need to be addressed before starting human therapeutic trials, particularly regarding its use for genetic disorders. How safe is the procedure? Are there any side effects following mesenchymal stem cell transplantation? To address these questions for DMD the best model is the golden retriever muscular dystrophy dog (GRMD), which is the closest model to the human condition displaying a much longer lifespan than other models. Here we report the follow-up of 5 GRMD dogs, which were repeatedly transplanted with human adipose-derived mesenchymal stromal cells (hASC), derived from different donors. Xenogeneic cell transplantation, which was done without immunosuppression, was well tolerated in all animals with no apparent long-term adverse effect. In the present study, we show that repeated heterologous stem-cell injection is a safe procedure, which is fundamental before starting human clinical trials.

A normal life without muscle dystrophin.

Here we summarize the clinical history of Ringo, a golden retriever muscular dystrophy (GRMD) dog, who had a mild phenotype despite the absence of muscle dystrophin. Ringo died of cardiac arrest at age 11 and therefore displayed a normal lifespan. One of his descendants, Suflair, born April 2006, also displays a mild course. Dystrophin analysis confirmed total absence of muscle dystrophin in both dogs. Muscle utrophin expression did not differ from severely affected GRMD dogs. Finding what protects these special dogs from the dystrophic degeneration process is now a great challenge that may open new avenues for treatment. But most importantly, the demonstration that it is possible to have a functional muscle, in a medium-large animal even in the absence of dystrophin, brings new hope for Duchenne patients.

Human adipose-derived mesenchymal stromal cells injected systemically into GRMD dogs without immunosuppression are able to reach the host muscle and express human dystrophin.

Duchenne muscular dystrophy (DMD), a lethal X-linked disorder, is the most common and severe form of muscular dystrophies, affecting 1 in 3,500 male births. Mutations in the DMD gene lead to the absence of muscle dystrophin and a progressive degeneration of skeletal muscle. The possibility to treat DMD through cell therapy has been widely investigated. We have previously shown that human adipose-derived stromal cells (hASCs) injected systemically in SJL mice are able to reach and engraft in the host muscle, express human muscle proteins, and ameliorate the functional performance of injected animals without any immunosuppression. However, before starting clinical trials in humans many questions still need to be addressed in preclinical studies, in particular in larger animal models, when available. The best animal model to address these questions is the golden retriever muscular dystrophy (GRMD) dog that reproduces the full spectrum of human DMD. Affected animals carry a mutation that predicts a premature termination codon in exon 8 and a peptide that is 5% the size of normal dystrophin. These dogs present clinical signs within the first weeks and most of them do not survive beyond age two. Here we show the results of local and intravenous injections of hASCs into GRMD dogs, without immunosuppression. We observed that hASCs injected systemically into the dog cephalic vein are able to reach, engraft, and express human dystrophin in the host GRMD dystrophic muscle up to 6 months after transplantation. Most importantly, we demonstrated that injecting a huge quantity of human mesenchymal cells in a large-animal model, without immunosuppression, is a safe procedure, which may have important applications for future therapy in patients with different forms of muscular dystrophies.

Drifting towards the surface: a shift in newborn pipefish's vertical distribution when exposed to the synthetic steroid ethinylestradiol.

Endocrine disrupting chemical (EDC) effects during early life have the potential to modulate population structure, either directly through increased mortality or by causing inappropriate aggregation events, thus affecting the number of young that will reach adulthood. An alteration in the dispersal and recruitment patterns can also impair the connectivity among geographically distant populations. However, the detection of EDC-induced effects occurring after egg hatch, when newborns increase their chances of contacting with environmentally dispersed contaminants, is not a simple process as effects might be masked by the large natural mortality rates that usually occur during fish early life. Since there is a lack of information regarding the impact of EDCs on fish early life dispersal patterns, particularly on vertical migrations patterns, the effects of environmentally relevant concentrations of EE(2) on the vertical distribution of newborn fish was assessed through an ex situ exposure experiment. Syngnathus abaster newborns were exposed to EE(2) (nominal concentrations of 8, 12 and 36 ng L(-1)) and the dynamics of their vertical distribution was monitored for up to 40 d. No significant differences in overall mortality were observed between treatments or in the dynamics of the registered death curves. Nevertheless, an alteration in the distribution patterns was observed. The commonly benthic newborn tended to shift their vertical distribution towards the surface, in a dose-dependent manner. Curiously, a follow up of the exposed pipefish confirmed that EE(2) effects were also noticeable upon sexual maturity, namely by the alteration of several primary and secondary sexual characters. The observation that vertical distributional patterns, at least in pipefish, are clearly altered at environmentally relevant EE(2) concentrations indicates that EDC's impact in fish larvae behaviour should be considered when addressing the effects of contaminants, given the obvious implications on population connectivity, stability and persistence.

The unpredictable effects of mixtures of androgenic and estrogenic chemicals on fish early life.

Estuarine shallow areas and coastal lagoons are known to receive and concentrate multiple inputs, either from land, rivers or coastal areas, being intensively impacted by chemical contamination, namely endocrine disrupting chemicals (EDCs). Despite the ubiquitous coexistence of several classes of EDCs in most of these aquatic ecosystems, there is still limited information regarding their combined effects. Furthermore, given the immediate implications for population dynamics, the available laboratory studies almost invariably focus on very specific life history stages, such as embryonic development or reproduction, thus creating a gap on our knowledge of what happens in between. During this 'intermediate phase', the newborn larvae and juveniles face numerous challenges whose outcome may impair reproduction or even survival. The black-striped pipefish, Syngnathus abaster, member of the Syngnathidae family (comprising pipefish, seahorses and seadragons), usually breeds in coastal areas such as estuaries, where its newborns are immediately exposed to EDCs. Given the ongoing decline of pipefish populations, together with the observed shrinkage and fragmentation of seagrass meadows, known to be impacted by EDCs, a first reasonable question to address is if pipefish newborns respond to environmentally relevant concentrations of ubiquitous EDCs, either single or in combination. Hence, a seven days exposure experiment to the estrogenic chemical ethinylestradiol (EE(2)) and the androgenic chemical tributyltin (TBT), single and in binary mixtures, was conducted. Selected behavioural (e.g. predator avoidance) and developmental variables (e.g. growth) were monitored in pipefish juveniles after EDCs insult. The obtained results indicate that EE(2), TBT, or their combined exposure, do impact pipefish early life. However, the pattern of results emerging from the measured variables clearly indicates that mixtures significantly modulate newborn responses in distinct ways when compared to individual chemical's exposure. These findings further demonstrate the importance of addressing the issue of chemical mixtures of pollutants acting through dissimilar mode of action. Independently of all the observed response variations, an ultimate conclusion seems certain: EE(2) and TBT, single or in combination, induce disruption patterns able to imbalance pipefish survival. Since these (as well as other) contaminants are present in estuarine areas, profound implications in population structure could be expected, ranging from a decrease in recruitment to a disruption of sexual selection. Inexorably, these stressors simultaneously operate in already declining populations.

Sexing blennies using genital papilla morphology or ano-genital distance.

Information on the genital morphology of male and female Lipophrys pholis is provided, as well as for two other sympatric blenniid species, Coryphoblennius galerita and Lipophrys trigloides. The use of non-invasive sex determination procedures described may be extremely useful not only for ecological studies but also as a proxy for the detection of environmental exposure to endocrine disruptive chemicals, given that blennies have been proposed as potential sentinel species for chemical contamination.

Human multipotent mesenchymal stromal cells from distinct sources show different in vivo potential to differentiate into muscle cells when injected in dystrophic mice.

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Isolation, characterization, and differentiation potential of canine adipose-derived stem cells.

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Mutation analysis in the FKRP gene provides an explanation for a rare cause of intrafamilial clinical variability in LGMD2I.

We report a limb-girdle muscular dystrophy 2I family with three affected sisters and a highly variable clinical course. FKRP gene sequencing showed that all three sisters carried a nonsense paternal mutation (W225X). The two oldest sisters with a severe phenotype carried two maternal mutations V79M and P89A. However, the youngest sister with a milder course carried the paternal and only the V79M maternal mutation, due to an intragenic recombination.

[Frontal astrocytoma with spinal cord metastasis. Report of a case and review of the literature]. / Astrocitoma frontal com metástase medular. Registro de um caso e revisão da literatura.

A case of a right frontal astrocytoma with spinal metastatic lesion in the region of the third dorsal vertebra is reported. The metastatic nodule was removed six months after the craniotomy. In the literature concerning to the dissemination of tumors cells is suggested that there is not a causal relationship between CSF seeding and operative intervention. Access to the ventricular system or basal cisterns is of primary importance in the production of metastases.