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Bone mineral density is an important parameter for the diagnosis of bone diseases, as well as for predicting fractures and treatment monitoring. Thus, the aim of the present study was to evaluate the potential of Quantitative Ultrasound (QUS) to monitor bone changes after calcium, phosphorus, and magnesium loss in rat femurs in vitro during a demineralization process. Four quantitative ultrasound parameters were estimated from bone surface echoes in eight femur diaphysis of rats. The echo signals were acquired during a decalcification process by Ethylenediaminetetraacetic Acid (EDTA). The results were compared to Quantitative Computed Tomography (QCT) and inductively coupled plasma optical emission spectrometry measurements for validation. Integrated Reflection Coefficient (IRC) reflection parameters and Frequency Slope of Reflection Transfer Function (FSRTF) during demineralization tended to decrease, while the backscattering parameter Apparent Integrated Backscatter (AIB) increased and Frequency Slope of Apparent Backscatter (FSAB) showed an oscillatory behavior with no defined trend. Results indicate a clear relation between demineralization and the corresponding decrease in the reflection parameters and increase in the scattering parameters. The trend analysis of the fall curve of the chemical elements showed a better relationship between IRC and QCT. It was possible to monitor bone changes after ions losses, through the QUS. Thus, it is an indication that the proposed protocol has potential to characterize bone tissue in animal models, providing consistent results towards standardization of bone characterization studies by QUS endorsing its use in humans.

Pleistocene climate changes were major historical events that impacted South American biodiversity. Although the effects of such changes are well-documented for several biomes, it is poorly known how these climate shifts affected the biodiversity of the Pantanal floodplain. Fish are one of the most diverse groups in the Pantanal floodplains and can be taken as a suitable biological model for reconstructing paleoenvironmental scenarios. To identify the effects of Pleistocene climate changes on Pantanal's ichthyofauna, we used genetic data from multiple populations of a top-predator long-distance migratory fish, Salminus brasiliensis . We specifically investigated whether Pleistocene climate changes affected the demography of this species. If this was the case, we expected to find changes in population size over time. Thus, we assessed the genetic diversity of S . brasiliensis to trace the demographic history of nine populations from the Upper Paraguay basin, which includes the Pantanal floodplain, that form a single genetic group, employing approximate Bayesian computation (ABC) to test five scenarios: constant population, old expansion, old decline, old bottleneck following by recent expansion, and old expansion following by recent decline. Based on two mitochondrial DNA markers, our inferences from ABC analysis, the results of Bayesian skyline plot, the implications of star-like networks, and the patterns of genetic diversity (high haplotype diversity and low-to-moderate nucleotide diversity) indicated a sudden population expansion. ABC allowed us to make strong quantitative inferences about the demographic history of S . brasiliensis . We estimated a small ancestral population size that underwent a drastic fivefold expansion, probably associated with the colonization of newly formed habitats. The estimated time of this expansion was consistent with a humid and warm phase as inferred by speleothem growth phases and travertine records during Pleistocene interglacial periods. The strong concordance between our genetic inferences and this historical data could represent the first genetic record of a humid and warm phase in the Pantanal in the period since the Last Interglacial to 40 ka.

This study aimed to better understand the role of different microbial groups and the determining fermentation pathways in a biohydrogen (bioH 2 )-producing reactor fed with sugarcane vinasse by using next-generation sequencing and principal component analysis (PCA). Both microbial structure and dynamics were characterized. The highest bioH 2 production levels were associated with the Thermoanaerobacterium genus, whilst lactate-producing bacteria comprised the dominant genera (e.g. Lactobacillus and Leuconostoc) within the reactor at both stable and unstable bioH 2 -producing periods. PCA further revealed that the fermentation of lactate played a dual role in the reactor, as both bioH 2 -producing (acetate+lactate→butyrate+bioH 2 ) and non-bioH 2 -producing (lactate→propionate+acetate) routes could be observed. Overall, the results suggested that lactate is the primary alternative carbon source in vinasse-fed systems subjected to carbohydrate-shortage conditions., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Thermus filiformis is an aerobic thermophilic bacterium isolated from a hot spring in New Zealand. The experimental study of the mechanisms of thermal adaptation is important to unveil response strategies of the microorganism to stress. In this study, the main pathways involved on T. filiformis thermoadaptation, as well as, thermozymes with potential biotechnological applications were revealed based on omics approaches. The strategy adopted in this study disclosed that pathways related to the carbohydrate metabolism were affected in response to thermoadaptation. High temperatures triggered oxidative stress, leading to repression of genes involved in glycolysis and the tricarboxylic acid cycle. During heat stress, the glucose metabolism occurred predominantly via the pentose phosphate pathway instead of the glycolysis pathway. Other processes, such as protein degradation, stringent response, and duplication of aminoacyl-tRNA synthetases, were also related to T. filiformis thermoadaptation. The heat-shock response influenced the carotenoid profile of T. filiformis, favoring the synthesis of thermozeaxanthins and thermobiszeaxanthins, which are related to membrane stabilization at high temperatures. Furthermore, antioxidant enzymes correlated with free radical scavenging, including superoxide dismutase, catalase and peroxidase, and metabolites, such as oxaloacetate and α-ketoglutarate, were accumulated at 77 °C.

The knowledge of spatial pattern and geographic beta-diversity is of great importance for biodiversity conservation and interpreting ecological information. Tropical forests, especially the Amazon Rainforest, are well known for their high species richness and low similarity in species composition between sites, both at local and regional scales. We aimed to determine the effect and relative importance of area, isolation and climate on species richness and turnover in orchid bee assemblages among plateaus in central Brazilian Amazonia. Variance partitioning techniques were applied to assess the relative effects of spatial and environmental variables on bee species richness, phylogeny and composition. We hypothesized that greater abundance and richness of orchid bees would be found on larger plateaus, with a set of core species occurring on all of them. We also hypothesized that smaller plateaus would possess lower phylogenetic diversity. We found 55 bee species distributed along the nine sampling sites (plateaus) with 17 of them being singletons. There was a significant decrease in species richness with decreasing size of plateaus, and a significant decrease in the similarity in species composition with greater distance and climatic variation among sampling sites. Phylogenetic diversity varied among the sampling sites but was directly related to species richness. Although not significantly related to plateau area, smaller or larger PDFaith were observed in the smallest and the largest plateaus, respectively.

B chromosomes, extra elements present in the karyotypes of some eukaryote species, have been described in the grasshopper Xyleus discoideus angulatus. Although some studies have proposed an autosomal origin of the B chromosome in X. d. angulatus, little is known about its repetitive DNA composition and evolutionary dynamics. The aim of the present work was to shed light on the B chromosome evolution in X. d. angulatus by cytogenetic analysis of 27 populations from Pernambuco and Ceará states (Brazil). The frequency of B chromosomes in the different populations was determined, and chromosome measurements and fluorescence in situ hybridization (FISH) with C0t-DNA and telomeric and B chromosome sequences were performed in cells from B-carrying individuals. The results revealed variations in B chromosome prevalence among the populations and showed that some B chromosomes were smaller in certain populations. FISH produced similar patterns for the C0t-DNA probe in all hybridized individuals, whereas telomeric and B chromosome probes, obtained by microdissection, exhibited variations in their distribution. These results indicate the presence of 3 morphotypes of B chromosomes in X. d. angulatus, with variation in repetitive DNA composition during their evolution. In this species, B chromosomes have an intraspecific origin and probably arose from the pericentromeric region of A chromosomes., (© 2017 S. Karger AG, Basel.)

Several signaling molecules that govern development in higher animals have been identified in the parasite Schistosoma mansoni, including the transforming growth factor β, protein tyrosine kinases, nuclear hormone receptors, among others. The Notch pathway is a highly conserved signaling mechanism which is involved in a wide variety of developmental processes including embryogenesis and oogenesis in worms and flies. Here we aimed to provide the molecular reconstitution of the Notch pathway in S. mansoni using the available transcriptome and genome databases. Our results also revealed the presence of the transcripts coded for SmNotch, SmSu(H), SmHes, and the gamma-secretase complex (SmNicastrin, SmAph-1, and SmPen-2), throughout all the life stages analyzed. Besides, it was observed that the viability and separation of adult worm pairs were not affected by treatment with N-[N(3,5)-difluorophenacetyl)-L-Alanyl]-S-phenylglycine t-butyl ester (DAPT), a Notch pathway inhibitor. Moreover, DAPT treatment decreased the production of phenotypically normal eggs and arrested their development in culture. Our results also showed a significant decrease in SmHes transcript levels in both adult worms and eggs treated with DAPT. These results provide, for the first time, functional validation of the Notch pathway in S. mansoni and suggest its involvement in parasite oogenesis and embryogenesis. Given the complexity of the Notch pathway, further experiments shall highlight the full repertoire of Notch-mediated cellular processes throughout the S. mansoni life cycle.

Controlling muscle function is essential for human behaviour and survival, thus, impairment of motor function and muscle paralysis can severely impact quality of life and may be immediately life-threatening, as occurs in many cases of traumatic spinal cord injury (SCI) and in patients with amyotrophic lateral sclerosis (ALS). Repairing damaged spinal motor circuits, in either SCI or ALS, currently remains an elusive goal. Therefore alternative strategies are needed to artificially control muscle function and thereby enable essential motor tasks. This review focuses on recent advances towards restoring motor function, with a particular focus on stem cell-derived neuronal engraftment strategies, optogenetic control of motor function and the potential future translational application of these approaches., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Dance is an embodied activity with benefits for mobility, balance, and quality of life (QoL) of persons affected by Parkinson's Disease (PD). It is enjoyable and likely to support adherence to movement prescriptions. The objective of this study was to assess the feasibility of measuring changes in psychological outcomes, specifically well-being, body self-efficacy, outcome expectations, and experienced beauty after a single Argentine Tango intervention in a workshop format. To anchor experienced beauty in a theory, the article introduces a model of embodied aesthetics featuring active art-making as a central aspect of healing in arts-based interventions., Methods: In a single-group pre-post design, we tested the feasibility of measuring psychological changes of 34 PD patients from Southern Germany after an introductory workshop in Argentine Tango. They participated in a 90 min Tango for PD intervention and completed the Heidelberg State Inventory (HSI-24; (Koch et al., 2007)), the Body Self-Efficacy Scale (BSE; (Fuchs and Koch, 2014)) with a sub-dimension on aesthetic experience, and the Credibility-Expectancy Questionnaire (CEQ; (Devilly and Borkovec, 2000)) before and after the intervention. A subgroup completed the therapeutic factors of arts therapies-scale, a new measure to elaborate on the aesthetic experience. We analyzed pre-post-differences with a t-test for paired samples., Results and Discussion: The study supports the feasibility of measuring health-related psychological changes from a single Argentine Tango intervention for PD patients, as well as acceptance and appropriateness of the intervention for the patient group. After the tango intervention, well-being, body self-efficacy, and outcome expectancies increased. Participants also experienced an increase in beauty of their movements and other aesthetic aspects. We suspect that, in addition to the functional and psychological factors identified so far, the aesthetic experience in dance may be an important therapeutic factor mediating several outcomes of dance and other arts-based interventions. A controlled study for evidence-based testing of targeted variables can now follow to examine the new hypotheses.

Galactanases (endo-β-1,4-galactanases-EC 3.2.1.89) catalyze the hydrolysis of β-1,4 galactosidic bonds in arabinogalactan and galactan side chains found in type I rhamnogalacturan. The aim of this work was to understand the catalytic function, biophysical properties, and use of a recombinant GH53 endo-beta-1,4-galactanase for commercial cocktail supplementation. The nucleotide sequence of the endo-β-1,4-galactanase from Bacillus licheniformis CBMAI 1609 (Bl1609Gal) was cloned and expressed in Escherichia coli, and the biochemical and biophysical properties of the enzyme were characterized. The optimum pH range and temperature of Bl1609Gal activity were 6.5-8 and 40 °C, respectively. Furthermore, Bl1609Gal showed remarkable pH stability, retaining more than 75 % activity even after 24 h of incubation at pH 4-10. The enzyme was thermostable, retaining nearly 100 % activity after 1-h incubation at pH 7.0 at 25-45 °C. The enzymatic efficiency (K cat /K m ) against potato galactan under optimum conditions was 241.2 s(-1) mg(-1) mL. Capillary zone electrophoresis demonstrated that the pattern of galactan hydrolysis by Bl1609Gal was consistent with that of endogalactanases. Supplementation of the commercial cocktail ACCELLERASE(®)1500 with recombinant Bl1609Gal increased hydrolysis of pretreated sugarcane bagasse by 25 %.

In this study, we put forward a new approach to classify early stages of fibrosis based on a multiparametric characterization using backscatter ultrasonic signals. Ultrasonic parameters, such as backscatter coefficient (Bc), speed of sound (SoS), attenuation coefficient (Ac), mean scatterer spacing (MSS), and spectral slope (SS), have shown their potential to differentiate between healthy and pathologic samples in different organs (eye, breast, prostate, liver). Recently, our group looked into the characterization of stages of hepatic fibrosis using the parameters cited above. The results showed that none of them could individually distinguish between the different stages. Therefore, we explored a multiparametric approach by combining these parameters in two and three, to test their potential to discriminate between the stages of liver fibrosis: F0 (normal), F1, F3, and/without F4 (cirrhosis), according to METAVIR Score. Discriminant analysis showed that the most relevant individual parameter was Bc, followed by SoS, SS, MSS, and Ac. The combination of (Bc, SoS) along with the four stages was the best in differentiating between the stages of fibrosis and correctly classified 85% of the liver samples with a high level of significance (p<0.0001). Nevertheless, when taking into account only stages F0, F1, and F3, the discriminant analysis showed that the parameters (Bc, SoS) and (Bc, Ac) had a better classification (93%) with a high level of significance (p<0.0001). The combination of the three parameters (Bc, SoS, and Ac) led to a 100% correct classification. In conclusion, the current findings show that the multiparametric approach has great potential in differentiating between the stages of fibrosis, and thus could play an important role in the diagnosis and follow-up of hepatic fibrosis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Bioheat models have been proposed to predict heat distribution in multilayered biological tissues after therapeutic ultrasound (TUS) stimulation. However, evidence on its therapeutic benefit is still controversial for many clinical conditions. The aim of this study was to evaluate and to compare the TUS heating distribution on commercially available bone phantoms and in vitro femur and tibia human samples, at 1 MHz and several ultrasonic pulse regimens, by means of a thermographic image processing technique., Methods: An infrared camera was used to capture an image after each 5-min 1-MHz TUS stimulation on bone phantoms, as well as in vitro femur and tibia samples (N = 10). An intensity-based processing algorithm was applied to estimate temperature distribution. Sections of five femurs in the coronal plane were also used for the evaluation of heat distribution inside the medullar canal., Results: Temperature increased up to 8.2 and 9.8 °C for the femur and tibia, respectively. Moreover, the temperature increased up to 10.8 °C inside the medullar canal. Although temperature distributions inside the region of interest (ROI) were significantly different (p < 0.001), the average and standard deviation values for bone phantoms were more similar to the femur than to the tibia samples. Pulsed regimens caused lower increments in temperature than continuous sonication, as expected., Conclusions: Commercially available bone phantoms could be used in research focusing on thermal effects of ultrasound. Small differences in mean and standard deviation temperatures were observed between bone samples and phantoms. Temperature can reach more than 10 °C inside the medullar canal on a fixed probe position which may lead to severe cellular damage.

A novel GH1 β-glucosidase (EaBgl1A) from a bacterium isolated from Antarctica soil samples was recombinantly overexpressed in Escherichia coli cells and characterized. The enzyme showed unusual pH dependence with maximum activity at neutral pH and retention of high catalytic activity in the pH range 6 to 9, indicating a catalytic machinery compatible with alkaline conditions. EaBgl1A is also a cold-adapted enzyme, exhibiting activity in the temperature range from 10 to 40°C with optimal activity at 30°C, which allows its application in industrial processes using low temperatures. Kinetic characterization revealed an enzymatic turnover (Kcat) of 6.92s(-1) (cellobiose) and 32.98s(-1) (pNPG) and a high tolerance for product inhibition, which is an extremely desirable feature for biotechnological purposes. Interestingly, the enzyme was stimulated by up to 200 mM glucose, whereas the commercial cocktails tested were found fully inhibited at this concentration. These properties indicate EaBgl1A as a promising biocatalyst for biotechnological applications where low temperatures are required., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: The use of endo-arabinanase from Bacillus licheniformis (ABNase) for sugarcane saccharification has been evaluated by enzyme immobilization and commercial cocktail supplement with the immobilized heterologous protein., Results: Biochemical characterization of the purified ABNase showed that the catalytic activity was strongly inhibited by 5 mM Cu(2+), Zn(2+) or Fe(3+). The optimum pH and temperature for activity were 5.5-6.5 and 35-40 °C, respectively. The enzyme stability increased 128-fold when immobilized with glyoxyl agarose, and the hydrolysis of pretreated sugar cane biomass increased by 15 % when a commercial enzyme cocktail was supplemented with immobilized ABNase., Conclusion: Pectin hydrolysis by recombinant ABNase plays a role in the effective application of enzymatic cocktails for biomass saccharification.

Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-β-xylanase fused to a GH43 β-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the β-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one β-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.

As part of an ongoing directed evolution program, the catalytic performance of the Xylanase A from Bacillus subtilis (XynA), which presents temperature and pH optima of 50°C and 6.0, respectively, has been enhanced to create a highly thermostable and alkali-tolerant enzyme. A library of random XynA mutants generated by error-prone polymerase chain reaction was screened by halo formation on agar containing xylan at pH 8.0. Two mutants showing higher catalytic activity at elevated pH in relation to the wild-type XynA were selected, and pooled with a further 5 XynA variants selected by screening thermostable XynA obtained from a previous directed evolution study for activity at alkaline pH. This pool of variants was used as a template for a further round of error-prone polymerase chain reaction and DNase fragment shuffling, with screening at pH 12.0 at 55°C. Selected mutants were subjected to further DNase shuffling, and a final round of screening at pH 12.0 and 80°C. A XynA variant containing eight mutations was isolated (Q7H/G13R/S22P/S31Y/T44A/I51V/I107L/S179C) that presented a temperature optimum of 80°C, a 3-fold increase in the specific activity compared with the wild-type enzyme at pH 8.0, and a 50% loss of activity (t50) of 60 min at 80°C (wild type <2 min). This directed evolution strategy therefore allows the concomitant adaption of increased thermostability and alkali tolerance of an endo-xylanase., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Central disinhibition is a mechanism involved in the physiopathology of fibromyalgia. Melatonin can improve sleep quality, pain and pain threshold. We hypothesized that treatment with melatonin alone or in combination with amitriptyline would be superior to amitriptyline alone in modifying the endogenous pain-modulating system (PMS) as quantified by conditional pain modulation (CPM), and this change in CPM could be associated with serum brain-derived neurotrophic factor (BDNF). We also tested whether melatonin improves the clinical symptoms of pain, pain threshold and sleep quality., Methods: Sixty-three females, aged 18 to 65, were randomized to receive bedtime amitriptyline (25 mg) (n = 21), melatonin (10 mg) (n = 21) or melatonin (10 mg) + amitriptyline (25 mg) (n = 21) for a period of six weeks. The descending PMS was assessed with the CPM-TASK. It was assessed the pain score on the Visual Analog Scale (VAS 0-100 mm), the score on Fibromyalgia Impact Questionnaire (FIQ), heat pain threshold (HPT), sleep quality and BDNF serum. Delta values (post- minus pre-treatment) were used to compare the treatment effect. The outcomes variables were collected before, one and six weeks after initiating treatment., Results: Melatonin alone or in combination with amitriptyline reduced significantly pain on the VAS compared with amitriptyline alone (P < 0.01). The delta values on the VAS scores were-12.85 (19.93),-17.37 (18.69) and-20.93 (12.23) in the amitriptyline, melatonin and melatonin+amitriptyline groups, respectively. Melatonin alone and in combination increased the inhibitory PMS as assessed by the Numerical Pain Scale [NPS(0-10)] reduction during the CPM-TASK:-2.4 (2.04) melatonin + amitriptyline,-2.65 (1.68) melatonin, and-1.04 (2.06) amitriptyline, (P < 0.05). Melatonin + amitriptyline treated displayed better results than melatonin and amitriptyline alone in terms of FIQ and PPT improvement (P < 0.05, fort both)., Conclusion: Melatonin increased the inhibitory endogenous pain-modulating system as assessed by the reduction on NPS(0-10) during the CPM-TASK. Melatonin alone or associated with amitriptyline was better than amitriptyline alone in improving pain on the VAS, whereas its association with amitriptyline produced only marginal additional clinical effects on FIQ and PPT., Trial Registration: Current controlled trail is registered at clinical trials.gov upon under number NCT02041455. Registered January 16, 2014.

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.

We sampled 11 natural populations of the grasshopper Xyleus discoideus angulatus in Northeastern Brazil to analyze B chromosome frequency and meiotic behavior. We observed a single large B chromosome, resembling the X chromosome, in 29 of the 402 specimens. Eight of the 11 populations had B chromosomes, with a rather broad geographical distribution, suggesting that this is an ancient polymorphism; significant differences were observed in B chromosome prevalence among the populations. Presence of the B chromosome was associated with increased frequency of macrospermatids. Fluorescent in situ hybridization revealed 18S rDNA sites in the pericentromeric regions of the X and L3 chromosomes, although some populations had an additional locus on the M4 chromosome. No variation was found for chromosome location of H4 histone genes, which were always observed in paracentromeric regions of the L2, M4 and X chromosomes, a rather unusual location compared to locations known from the families Acrididae and Proscopiidae. These B chromosomes lacked these two kinds of repetitive DNA, at least in amounts that can be visualized by fluorescent in situ hybridization, suggesting that these B chromosomes did not originate from any of the four chromosomes carrying rDNA or H4 histone genes.

Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.

Vibrations produced in oscillating/vibratory platform generate whole body vibration (WBV) exercises, which are important in sports, as well as in treating diseases, promoting rehabilitation, and improving the quality of life. WBV exercises relevantly increase the muscle strength, muscle power, and the bone mineral density, as well as improving the postural control, the balance, and the gait. An important number of publications are found in the PubMed database with the keyword "flexibility" and eight of the analyzed papers involving WBV and flexibility reached a level of evidence II. The biggest distance between the third finger of the hand to the floor (DBTFF) of a patient with metabolic syndrome (MS) was found before the first session and was considered to be 100%. The percentages to the other measurements in the different sessions were determined to be related to the 100%. It is possible to see an immediate improvement after each session with a decrease of the %DBTFF. As the presence of MS is associated with poorer physical performance, a simple and safe protocol using WBV exercises promoted an improvement of the flexibility in a patient with MS.

PA28γ is a proteasome activator involved in the regulation of the cellular proliferation, differentiation and growth. In the present study, we identified and characterized a cDNA from Schistosoma mansoni exhibiting significant homology to PA28γ of diverse taxa ranging from mammals (including humans) to simple invertebrates. Designated SmPA28γ, this transcript has a 753bp predicted ORF encoding a protein of 250 amino acid residues. Alignment of SmPA28γ with multiple PA28γ orthologues revealed an average similarity of ~40% among the investigated organisms, and 90% similarity with PA28γ from Schistosoma japonicum. In addition, phylogenetic analysis demonstrated a close linkage between SmPA28γ to its sister group that contains well-characterized PA28γ sequences from Drosophila spp., as well as sharing the same branch with PA28γ from S. japonicum. Gene expression profiling of SmPA28γ using real-time quantitative PCR revealed elevated steady-state transcript levels in the eggs, miracidia and paired adult worms compared to other stages. In parallel with gene expression profiles, an affinity-purified anti-SmPA28γ antibody produced against recombinant protein exhibited strongest reactivity in Western blot analyses to endogenous SmPA28γ from miracidia, sporocysts and paired adult worms. Given its known regulatory function in other organisms, we hypothesized that the high level of SmPA28γ transcript and protein in these stages may be correlated with an important role of the PA28γ in the cellular growth and/or development of this parasite. To address this hypothesis, miracidia were transformed in vitro to sporocysts in the presence of SmPA28γ double-stranded RNAs (dsRNAs) and cultivated for 4 days, after which time steady-state transcript and protein levels, and phenotypic changes were evaluated. SmPA28γ dsRNA treatment resulted in gene and protein knockdown of ~60% and ~80%, respectively, which were correlated with a significant decrease in larval length compared to its controls. These findings are consistent with a putative role of SmPA28γ in larval growth/development of the S. mansoni., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background and Purpose: Little information exists on the epidemiology and patterns of treatment of patients admitted to Brazilian hospitals with stroke. Our objective was to describe the frequency of risk factors, patterns of management, and outcome of patients admitted with stroke in Fortaleza, the fifth largest city in Brazil., Methods: Data were prospectively collected from consecutive patients admitted to 19 hospitals in Fortaleza with a diagnosis of stroke or transient ischemic attack from June 2009 to October 2010., Results: We evaluated 2407 consecutive patients (mean age, 67.7±14.4 years; 51.8% females). Ischemic stroke was the most frequent subtype (72.9%) followed by intraparenchymal hemorrhage (15.2%), subarachnoid hemorrhage (6.0%), transient ischemic attack (3%), and undetermined stroke (2.9%). The median time from symptoms onset to hospital admission was 12.9 (3.8-32.5) hours. Hypertension was the most common risk factor. Only 1.1% of the patients with ischemic stroke received thrombolysis. The median time from hospital admission to neuroimaging was 3.4 (1.2-26.5) hours. In-hospital mortality was 20.9% and the frequency of modified Rankin Scale score≤2 at discharge was less than 30%. Older age, prestroke disability, and having a depressed level of consciousness at admission were independent predictors of poor outcome; conversely, male gender was a predictor of good outcome., Conclusions: The prevalence of stroke risk factors and clinical presentation in our cohort were similar to previous series. Treatment with thrombolysis and functional independency after a stroke admission were infrequent. We also found long delays in hospital admission and in evaluation with neuroimaging and high in-hospital mortality.

Ultrasound axial transmission (UAT), a technique using propagation of ultrasound waves along the cortex of cortical bones, has been proposed as a diagnostic technique for the evaluation of fracture healing. Quantitative ultrasound parameters have been reported to be sensitive to callus changes during the regeneration process. The aim of this work was to identify the specific effect of cortical bone mineralization on UAT measurements by means of numerical simulations and experiments using a reverse fracture healing approach. A cortical bovine femur sample was used, in which a 3mm fracture gap was drilled. A 3mm thick cortical bone slice, extracted from another location in the bone sample, was submitted to a progressive demineralization process with EDTA during 12 days. UAT measurements and simulations using a 1MHz probe were performed with the demineralized slice placed into the fracture gap to mimic different stages of mineralization during the healing process. The calcium loss of the slice due to the EDTA treatment was recorded everyday, and its temporal evolution could be modeled by an exponential law. A 50MHz scanning acoustic microscopy was also used to assess the mineralization degree of the bone slice at the end of the intervention. These data were used in the numerical simulations to derive a model of the time evolution of bone slice mechanical properties. From both the experiments and the simulations, a significant and progressive increase in the time of flight (TOF; p<0.001) of the propagating waves measured by UAT was observed during the beginning of the demineralization process (first 4 days). Although the simulated TOF values were slightly larger than the experimental ones, they both exhibited a similar time-dependence, validating the simulation approach. Our results suggest that TOF measured in axial transmission is affected by local changes of speed of sound induced by changes in local mineralization. TOF may be an appropriate indicator to monitor callus maturation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This work aimed at computationally evaluating the compositional factors in fracture healing affecting ultrasound axial transmission (UAT), using four numerical daily-changing healing models, representing more realistic clinical conditions. Using two-dimensional (2-D) simulations, a 1-MHz source and a receiver were positioned parallel to the bone surface to detect the first arriving signal (FAS). The time-of-flight of the FAS (TOF(FAS)) was found to be sensitive only to superficial modifications in the propagation path. It was also shown that callus mature bone better explained alone the variation in TOF(FAS) (R(2) >or= 0.70, p < 0.001). Better TOF(FAS) predictions are obtained when using the callus composition inside cortical fracture gap (R(2) = 0.98, p < 0.01). Callus composition could not well explain the changes in energy attenuation. These results suggest that UAT may be an important clinical tool for fracture healing assessment, identifying callus degree of mineralization and possible consolidation delays and nonunions., (Copyright 2010 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.)

Purpose: To propose a novel model of pseudarthrosis in a small animal and to investigate the ability of backscatter parameters from ultrasound signals in differentiating normal bone from those ones with pseudarthrosis., Methods: Twelve Rattus norvegicus albinus free from pathogenic species (SPF) were randomly divided in two groups, with six animals each. In the Control group a surgical approach to the femur was made, followed by the synthesis of the muscle and skin layers. The Experimental group was submitted to an osteotomy of the femur and a vascularized flap of the fascia lata was interposed in the line of the fractured bone. Then the alignment and bone stabilization were accomplished, by using nylon stitch in U shape introduced in holes made in the proximal and distal fractured bone. Bone samples were scanned with ultrasound and signals were collected for each one to analyze the parameter Apparent Integrated Backscatter - AIB., Results: Radiological and anatomopathologic studies revealed the absence of bone consolidation with persistence of fiber-osteoid tissue. Values of the ultrasound parameter AIB from normal bones were statistically different from those with pseudarthrosis., Conclusion: The experimental model was suitable for pseudarthrosis development in rats and the ultrasound backscatter parameters were able to identify such a bone disease in vitro.

Cartilage tissue has a poor capacity for self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of cartilage tissue. The present work demonstrates that a three-dimensional (3D) chitosan scaffold increases the efficiency of the adhesion and differentiation of mesenchymal stem cells (MSCs) after the addition of a chondrogenic medium. These culture conditions promoted MSC differentiation into chondrocytes during the first 9 weeks of monolayer or 3D culture in a scaffold composed of chitosan or chitosan/gelatin. The results demonstrated that a chitosan scaffold caused a reduction in alkaline phosphatase production and an increase in the collagen concentration indicating phenotypic changes in the cells. In support of these results, the production of collagen type II by the MSCs cultured in the chitosan scaffold increased after 3 weeks of culture, indicating the beginning of differentiation. However, the addition of gelatin to the chitosan scaffold did not improve on the results obtained with chitosan alone. These results suggest that this 3D chitosan scaffold is a promising candidate for biomaterial implants designed to promote MSC colonization and has applications in regenerative medicine., (Copyright 2009 S. Karger AG, Basel.)

The in vitro schistosomicidal activity of curcumin (doses ranging from 5 to 100 microM) was carried out against Schistosoma mansoni adult worms. Curcumin (at 50 and 100 microM) caused death of all worms. When tested at the doses of 5 and 20 microM, it decreased the worm viability in comparison with negative (Roswell Memorial Park Institute (RPMI) 1640 medium alone or RPMI 1640 medium with 10% dimethyl sulfoxide) and positive (heat-killed worms at 56 degrees C or praziquantel 10 microM) control groups. All pairs of coupled adult worms were separated into individual male and female by the action of curcumin at the doses of 20 to 100 microM. When tested at 5 and 10 microM, curcumin reduced egg production by 50% in comparison with the positive control group. It is the first time that the schistosomicidal activity has been reported for curcumin.

A porous 3D scaffold was developed to support and enhance the differentiation process of mesenchymal stem cells (MSC) into osteoblasts in vitro. The 3D scaffold was made with chitosan, gelatin and chondroitin and it was crosslinked by EDAC. The scaffold physicochemical properties were evaluated. SEM revealed the high porosity and interconnection of pores in the scaffold; rheological measurements show that the scaffold exhibits a characteristic behavior of strong gels. The elastic modulus found in compressive tests of the crosslinked scaffold was about 50 times higher than the non-crosslinked one. After 21 days, the 3D matrix submitted to hydrolytic degradation loses above 40% of its weight. MSC were collected from rat bone marrow and seeded in chitosan-gelatin-chondroitin 3D scaffolds and in 2D culture plates as well. MSC were differentiated into osteoblasts for 21 days. Cell proliferation and alkaline phosphatase activity were followed weekly during the osteogenic process. The osteogenic differentiation of MSC was improved in 3D culture as shown by MTT assay and alkaline phosphatase activity. On the 21st day, bone markers, osteopontin and osteocalcin, were detected by the PCR analysis. This study shows that the chitosan-gelatin-chondroitin 3D structure provides a good environment for the osteogenic process and enhances cellular proliferation.

Cartilage tissue has poor capability of self-repair, especially in the case of severe cartilage damage due to trauma or age-related degeneration. Cell-based tissue engineering using scaffolds has provided an option for the repair of defects in adult cartilage tissue. Mesenchymal stem cells (MSC) and chondrocytes are the two major cell sources for cartilage tissue engineering. The present study combined culture conditions of MSC in a chitosan-gelatin matrix in chondrogenic media to evaluate their effects on MSC viability and chondrogenesis for cartilage tissue engineering. MSC were harvested from rabbit bone marrows and cultured in chondrogenic media supplemented, or not, with dexamethasone in a chitosan-gelatin film (C-GF). The association of C-GF and dexamethasone promoted significant increase in cell adhesivity, viability and proliferation when compared to MCS cultured in media without dexamethasone or C-GF. In addition, dexamethasone promoted increase in the collagen concentration of MSC cultures. A reduction of alkaline phosphatase activity after three weeks of culture in chondrogenic media was verified. No influence of the C-GF or of dexamethasone was observed in this matter. Therefore, it is reasonable to suggest that biomaterial-based chitosan-gelatin and chondrogenic media supplemented with dexamethasone may stimulate the proliferation and differentiation of MSC according to the complex environmental conditions. The information presented here should be useful for the development of biomaterials to regulate the chondrogenesis of MSC suitable for cartilage tissue engineering.

This work studied the periodicity of in vitro healthy and pathologic liver tissue, using backscattered ultrasound (US) signals. It utilized the mean scatterer spacing (MSS) as a parameter of tissue characterization, estimated by three methods: the spectral autocorrelation (SAC), the singular spectrum analysis (SSA) and the quadratic transformation method (SIMON). The liver samples were classified in terms of tissue status using the METAVIR scoring system. Twenty tissue samples were classified in four groups: F0, F1, F3 and F4 (five samples for each). The Kolmogorov-Smirnov test (applied on group pairs) resulted as nonsignificant (p > 0.05) for two pairs only: F1/F3 (for SSA) and F3/F4 (for SAC). A discriminant analysis was applied using as parameters the MSS mean (MSS) and standard deviation (sigmaMSS), the estimates histogram mode (mMSS), and the speed of US (mc(foie)) in the medium, to evaluate the degree of discrimination among healthy and pathologic tissues. The better accuracy (Ac) with SAC (80%) was with parameter group (MSS, sigmaMSS, mc(foie)), achieving a sensitivity (Ss) of 92.3% and a specificity (Sp) of 57.1%. For SSA, the group with all four parameters showed an Ac of 75%, an Ss of 78.6% and an Sp of 66.70%. SIMON obtained the best Ac of all (85%) with group (MSS, mMSS, mc(foie)), an Ss of 100%, but with an Sp of 50%.

A new protocol is described for immunization of outbred Swiss mice. The procedure is based on subcutaneous implantation of antigen-coupled polyester-polyurethane sponges cut into disks of 10 mm in diameter vs 2 mm in thickness. Antigen coupling was performed by overnight incubation of the sponge with a solution of ovalbumin (Ova) (2 mg/ml) diluted in sodium carbonate buffer, pH 9.6. The amount of ovalbumin that was taken up by the sponge was between 71.4 to 82.5 micrograms. This was estimated by comparing the Ova absorbance at 280 nm in coating buffer solutions before and after incubation. To compare the efficiency of the proposed method, experimental groups immunized with the antigen in the presence of adjuvants (10 micrograms in Al(OH)3 or 100 micrograms in complete Freund's adjuvant (CFA)) were run in parallel. The data obtained after the 3rd week of immunization indicate that both cellular and humoral immune responses were achieved. These were assayed by antigen-induced footpad swelling and ELISA (specific antibodies), respectively. The levels of both immune responses elicited were similar to the responses observed in mice immunized with ovalbumin in the presence of Al(OH)3. The method might represent an advantage when immunizing with pathogenic antigens. Preliminary experiments have suggested that the antigen remains immobilized or bound to the sponge for a long period of time, since there is an increment on the cell population inside the sponges after boosting the animals. If so, the undesirable effects of immunization would be reduced.