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Chemical and sensory properties of Torrontés Riojano sparkling wines, prepared using second fermentation with Saccharomyces strains EC1118, bayanus C12 and IFI473I, were explored. All sparkling wines showed high levels of several volatile ethyl esters and terpenes associated to fruity and floral aromas. The sensory profiles showed significant differences for the floral aroma descriptor among EC1118, bayanus C12 and IFI473I and for bubble persistence for strain bayanus C12. Our results suggest that the sensory properties of these sparkling wines could be dependent on the chemical and organoleptic properties of the base wine more than the yeast strain used for second fermentation.

Designing and understanding the mechanism of non-stoichiometric materials with enhanced properties is challenging, both experimentally and even computationally, due to the large number of chemical spaces and their distributions through the material. In the current work, it is proposed a Machine Learning approach coupled with the Efficient Global Optimization (EGO) method-an Adaptive Design (AD)-to model local defects in materials from first-principle calculations. Our method takes into account the smallest sample set as possible, envisioning the material defect structure relationship with target properties for new insights. As an example, the AD framework allows us to study the stability and the structure of the modified goethite (Fe 0.875 Al 0.125 OOH) by considering a proper defect distribution, from first-principle calculations. The chemical space search for the modified goethite was evaluated by starting from different sizes and configurations of the samples as well as different surrogate models (ANN and Gaussian Process; GP), acquisition functions, and descriptors. Our results show that the same local solution of several defect arrangements in Fe 0.875 Al 0.125 OOH is found regardless of the initial sample and regression model. This indicates the efficiency of our search method. We also discuss the role of the descriptors in the accelerated global search for defects in material modeling. We conclude that the AD method applied in material defects is a successful approach in automating the search within huge chemical spaces from first-principle calculations by considering small samples. This method can be applied to mechanistic elucidation of non-stoichiometric materials, solid solutions, alloys, and Schottky and Frenkel defects, essential for material design and discovery. Graphical abstract.

Introduction: The aim of this study was to evaluate the in vitro osteogenic potential of osteoblasts from neural crest-derived frontal bone (OB-NC) and mesoderm-derived parietal bone (OB-MS) and the bone formation induced by them when injected into calvarial defects., Materials and Methods: Calvarial bones were collected from newborn Wistar rats (3-day old) and characterized as frontal and parietal prior to OB-NC and OB-MS harvesting. The cells were cultured, and several parameters of osteoblast differentiation were evaluated. These cells, or PBS without cells (control), were locally injected into 5-mm rat calvarial defects (5 × 10 6 cells/defect) and after 4 weeks bone formation was evaluated by morphometric and histological analyses., Results: The characterization of frontal and parietal bones assured the different embryonic origin of both cell populations, OB-NC and OB-MS. The OB-NC presented higher proliferation while the OB-MS presented higher alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of runt-related transcription factor 2, Alp, bone sialoprotein and osteocalcin revealing their high osteogenic potential. µCT analysis indicated that there was higher amount of bone formation in defects injected with both OB-NC and OB-MS compared to the control. Moreover, the bone tissue formed by both cells displayed the same histological characteristics., Conclusions: Despite the distinct in vitro osteogenic potential, OB-NC and OB-MS induced similar bone repair in a rat calvarial defect model. Thus, osteoblasts, irrespective of their in vitro osteogenic potential linked to embryonic origins, seem to be suitable for cell-based therapies aiming to repair bone defects.

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological Saccharomyces cerevisiae strains, CR involving the promoter region of the gene SSU1 lead to a higher sulfite tolerance by enhancing the SO 2 efflux. To date, three different SSU1 associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method ( SSU1 checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 S. cerevisiae strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the SSU1 promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged SSU1 promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of SSU1 promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO 2 concentrations. The SSU1 checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers., (Copyright © 2020 Marullo, Claisse, Raymond Eder, Börlin, Feghali, Bernard, Legras, Albertin, Rosa and Masneuf-Pomarede.)

Yeast microsatellite loci consist of short tandem-repeated DNA sequences of variable length. The high mutational rate at these loci generates a remarkable repertoire of alleles, useful for strain differentiation and population genetic studies. In this work, we analyze the DNA sequences of thirteen alleles from each of ten microsatellite loci described for the yeast Starmerella bacillaris. Our results show that polymorphic variants of some informative alleles are dependent on SNPs and indels rather than on length variation at their originally defined tandem-repeated motifs. The analysis was extended to 55 previously described hypervariable microsatellite loci from a total of 26 sequenced genomes of yeast species that dominate the microbiota of spontaneously fermenting grape musts (i.e., Hanseniaspora uvarum, Saccharomyces cerevisae, Saccharomyces uvarum, and Torulaspora delbrueckii) or lead to wine spoilage (Brettanomyces bruxellensis and Meyerozyma guilliermondii). We found that allelic variants for some microsatellite loci of these yeast species are also dependent on SNPs and/or indels flanking their tandem-repeated motifs. For some loci, the number of units at their tandem repeats was found to be identical among the various characterized alleles, with allelic differences being dependent exclusively on flanking polymorphisms. Our results indicate that allele sizing of microsatellite loci using PCR, although valid for strain differentiation and population genetic studies, does not necessarily score the number of units at their tandem-repeated motifs. Sequence analysis of microsatellite loci alleles could provide relevant information for evolutionary and phylogeny studies of yeast species.

Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs., Competing Interests: Competing interestsThe authors declare no conflict of interest., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

Objective: The present study aimed to investigate the participation of focal adhesion kinases (FAK) in interactions between osteoblastic cells and titanium (Ti) surfaces with three different topographies, namely, untreated (US), microstructured (MS), and nanostructured (NS)., Methodology: Osteoblasts harvested from the calvarial bones of 3-day-old rats were cultured on US, MS and NS discs in the presence of PF-573228 (FAK inhibitor) to evaluate osteoblastic differentiation. After 24 h, we evaluated osteoblast morphology and vinculin expression, and on day 10, the following parameters: gene expression of osteoblastic markers and integrin signaling components, FAK protein expression and alkaline phosphatase (ALP) activity. A smooth surface, porosities at the microscale level, and nanocavities were observed in US, MS, and NS, respectively., Results: FAK inhibition decreased the number of filopodia in cells grown on US and MS compared with that in NS. FAK inhibition decreased the gene expression of Alp, bone sialoprotein, osteocalcin, and ALP activity in cells grown on all evaluated surfaces. FAK inhibition did not affect the gene expression of Fak, integrin alpha 1 ( Itga1 ) and integrin beta 1 ( Itgb1 ) in cells grown on MS, increased the gene expression of Fak in cells grown on NS, and increased the gene expression of Itga1 and Itgb1 in cells grown on US and NS. Moreover, FAK protein expression decreased in cells cultured on US but increased in cells cultured on MS and NS after FAK inhibition; no difference in the expression of vinculin was observed among cells grown on all surfaces., Conclusions: Our data demonstrate the relevance of FAK in the interactions between osteoblastic cells and Ti surfaces regardless of surface topography. Nanotopography positively regulated FAK expression and integrin signaling pathway components during osteoblast differentiation. In this context, the development of Ti surfaces with the ability to upregulate FAK activity could positively impact the process of implant osseointegration.

Introduction: Anticoagulants are widely used in orthopedic surgery to decrease the risk of deep vein thrombosis. While significant bone impairment is induced by long-term heparin therapy, little is known about the effects of direct oral anticoagulants (DOACs). Herein, we investigated the effects of dabigatran etexilate (Pradaxa®), a DOAC inhibitor of thrombin, on bone cells using in vitro and ex vivo cell culture models., Materials and Methods: Osteoblasts and osteoclasts exposed to different concentrations of dabigatran etexilate and untreated cells were assayed for cell differentiation and activity. Favorable osteogenic conditions for osteoblasts were tested using titanium with nanotopography (Ti-Nano). In addition, mice treated with a dabigatran etexilate solution had bone marrow cells analyzed for the ability to generate osteoclasts., Results: Dabigatran etexilate at concentrations of 1 μg/mL and 2 μg/mL did not impact osteoclast or osteoblast viability. The drug inhibited osteoclast differentiation and activity as observed by the reduction of TRAP+ cells, resorption pits and gene and protein expression of cathepsin K. Consistently, osteoclasts from mice treated with dabigatran showed decreased area, resorptive activity, as well as gene and protein expression of cathepsin K. In osteoblast cultures, grown both on polystyrene and Ti-Nano, dabigatran etexilate reduced alkaline phosphatase (ALP) activity, matrix mineralization, gene expression of ALP and osteocalcin., Conclusions: Dabigatran etexilate inhibited osteoclast differentiation in ex vivo and in vitro models in a dose-dependent manner. Moreover, the drug reduced osteoblast activity even under optimal osteogenic conditions. This study provides new evidence regarding the negative overall impact of DOACs on bone cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aim: The aim of this study was to investigate the effect of local injection of osteoblasts differentiated from bone marrow (BM-OB) or adipose tissue (AT-OB) mesenchymal stromal cells on bone tissue formation. Materials & methods: Defects were created in rat calvaria and injected with BM-OB or AT-OB and phosphate-buffered saline without cells were injected as control. Bone formation was evaluated 4 weeks postinjection. Results: Injection of BM-OB or AT-OB resulted in higher bone formation than that obtained with control. The bone tissue induced by cell injections exhibited similar mechanical properties as those of pristine calvarial bone, and its molecular cues suggested the occurrence of a remodeling process. Conclusion: Results of this study demonstrated that cell therapy with osteoblasts induced significant bone formation that exhibited the same quality as that of pre-existent bone.

Wnt/β-catenin signal transduction is involved in the homeostatic control of bone mass. It is well established that a titanium surface with nanotopography (Ti-Nano) favors osteoblast differentiation by modulating different signaling pathways. However, few studies have investigated the participation of the Wnt/β-catenin pathway in the osteogenic effect of nanoscale topographies. In this study, we aimed to determine whether the Wnt/β-catenin signaling pathway is involved in the elevated osteogenic potential of Ti-Nano. MC3T3-E1 cells were cultured on Ti-Nano and machined Ti (Ti-Control) for evaluation of the expression of Wnt/β-catenin signaling pathway-related genes. Based on the results to real-time PCR, the Wnt receptor Fzd4 was selected and silenced by CRISPRi. The resulting cells were cultured on both Ti surfaces, and several events involved in osteoblast differentiation were evaluated. The results revealed that Fzd4 gene silencing, corresponding to negative modulation of Wnt/β-catenin, inhibits expression of the osteoblast phenotype. It is worthy of note that this inhibitory effect on osteoblast differentiation was more pronounced in cells grown on Ti-Nano compared with those grown on Ti-Control. By disrupting Fzd4 gene expression, we have shown that the elevated osteogenic potential of Ti-Nano is due to activation of the Wnt/β-catenin signaling pathway, which reveals a new mechanism to explain osteoblast differentiation induced by nanotopography. Such an understanding of the intracellular machinery involved in surface guiding of osteoblast fate may contribute to the development of smart biomaterials to modulate the process of implant osseointegration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Topographical modifications of titanium (Ti) at the nanoscale level generate surfaces that regulate several signaling pathways and cellular functions, which may affect the process of osseointegration. Here, we investigated the participation of integrin αV in the osteogenic capacity of Ti with nanotopography. Machined titanium discs (untreated) were submitted to treatment with H 2 SO 4 /H 2 O 2 to produce the nanotopography (nanostructured). First, the greater osteogenic capacity of the nanotopography that increased osteoblast differentiation of mesenchymal stem cells compared with untreated topography was shown. Also, the nanostructured surface increased (regulation ≥ 1.9-fold) the gene expression of 6 integrins from a custom array plate utilized to evaluate the gene expression of 84 genes correlated with cell adhesion signaling pathway, including integrin αV, which is involved in osteoblast differentiation. By silencing integrin αV in MC3T3-E1 cells cultured on nanotopography, the impairment of osteoblast differentiation induced by this surface was observed. In conclusion, it was shown that nanotopography regulates the expression of several components of the cell adhesion signaling pathway and its higher osteogenic potential is, at least in part, due to its ability to upregulate the expression of integrin αV. Together with previous data that showed the participation of integrins α1, β1, and β3 in the nanotopography osseoinduction activity, we have uncovered the pivotal role of this family of membrane receptors in the osteogenic potential of this surface., (© 2019 Wiley Periodicals, Inc.)

The Red-cowled Cardinal (Paroaria dominicana) is an endemic passerine of the Caatinga biome in Brazil, and is one of the most traded passerines in the country. Illegal trade can have serious impacts on wild populations, such as reduced population sizes, the introduction of the species to areas outside their historical range or mixing individuals from different populations. Microsatellites constitute an important tool for population genetics and forensics studies, and hold great potential to help authorities manage illegal trafficking and inspect commercial breeders. We developed new microsatellite loci using massive parallel sequencing and characterized them in 23 seized Red-cowled Cardinals with unknown geographic origin. The DNA sequencing generated 2,068,684 paired-reads of which we identified 10,322 tri- to hexanucleotide loci. We selected 30 loci for amplification and polymorphism tests, of which 21 successfully amplified and 19 were polymorphic. The number of alleles ranged from 7 to 18 and the mean expected heterozygosity was 0.863. Six loci deviated from Hardy-Weinberg equilibrium probably due to null alleles and/or the Wahlund effect. Polymorphic loci in Hardy-Weinberg equilibrium showed low identity probability and high paternity exclusion probability. Our results indicate that this new set of microsatellite loci constitutes an important tool for both population genetic and forensic studies, with ultimate potential for assisting authorities in managing animal victims of illegal trafficking and the inspection of commercial breeders of the Red-cowled Cardinal.

Treatment of large bone defects is a challenging clinical situation that may be benefited from cell therapies based on regenerative medicine. This study was conducted to evaluate the effect of local injection of bone marrow-derived mesenchymal stromal cells (BM-MSCs) or adipose tissue-derived MSCs (AT-MSCs) on the regeneration of rat calvarial defects. BM-MSCs and AT-MSCs were characterized based on their expression of specific surface markers; cell viability was evaluated after injection with a 21-G needle. Defects measuring 5 mm that were created in rat calvaria were injected with BM-MSCs, AT-MSCs, or vehicle-phosphate-buffered saline (Control) 2 weeks post-defect creation. Cells were tracked by bioluminescence, and 4 weeks post-injection, the newly formed bone was evaluated by µCT, histology, nanoindentation, and gene expression of bone markers. BM-MSCs and AT-MSCs exhibited the characteristics of MSCs and maintained their viability after passing through the 21-G needle. Injection of both BM-MSCs and AT-MSCs resulted in increased bone formation compared to that in Control and with similar mechanical properties as those of native bone. The expression of genes associated with bone formation was higher in the newly formed bone induced by BM-MSCs, whereas the expression of genes involved in bone resorption was higher in the AT-MSC group. Cell therapy based on local injection of BM-MSCs or AT-MSCs is effective in delivering cells that induced a significant improvement in bone healing. Despite differences observed in molecular cues between BM-MSCs and AT-MSCs, both cells had the ability to induce bone tissue formation at comparable amounts and properties. These results may drive new cell therapy approaches toward complete bone regeneration.

Cell therapy associated with guided bone regeneration (GBR) can be used to treat bone defects under challenging conditions such as osteoporosis. This study aimed to evaluate the effect of mesenchymal stem cells (MSCs) in combination with a poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT) membrane on bone repair in osteoporotic rats. Osteoporosis was induced in female rats by bilateral removal of the ovaries (OVX) or sham surgery (SHAM), and the osteoporotic condition was characterized after 5 months by microtomographic and morphometric analyses. Calvarial defects were created in osteoporotic rats that immediately received the PVDF-TrFE/BT membrane. After 2 weeks, bone marrow-derived MSCs from healthy rats, characterized by the expression of surface markers using flow cytometry, or phosphate-buffered saline (PBS) (Control) were injected into the defects and bone formation was evaluated 4 weeks post-injection by microtomographic, morphometric, and histological analyses. A reduction in the amount of bone tissue in the femurs of OVX compared with SHAM rats confirmed the osteoporotic condition of the experimental model. More bone formation was observed when the defects were injected with MSCs compared to that with PBS. The modification that we are proposing in this study for the classical GBR approach where cells are locally injected after a membrane implantation may be a promising therapeutic strategy to increase bone formation under osteoporotic condition.

The surface of grapes lodges a complex community of yeast species responsible for spontaneous alcoholic fermentation. The study of indigenous Saccharomyces and "non-Saccharomyces" yeasts during grape must fermentation constitutes a major research area in microbial enology. Although there are detailed studies on the microbiota of Vitis vinifera L. grapes, little is known about the diversity of yeast communities present in non-vinifera Vitis ecosystems (i.e., grapes and spontaneously fermenting grape musts). Potentially scientific and/or enological valuable yeast strains from these non-vinifera Vitis ecosystems might never be isolated from V. vinifera L. In this updated review, we summarize relevant aspects of the microbial studies conducted on V. non-vinifera grapes and spontaneously fermenting grape musts., (Copyright © 2018 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.)

Mesenchymal stem cells (MSCs) have been used in therapies for bone tissue healing. The aim of this study was to investigate the effect of cell source and osteoblast differentiation on gene expression profiles of MSCs from bone marrow (BM-MSCs) or adipose tissue (AT-MSCs) to contribute for selecting a suitable cell population to be used in cell-based strategies for bone regeneration. BM-MSCs and AT-MSCs were cultured in growth medium to keep MSCs characteristics or in osteogenic medium to induce osteoblast differentiation (BM-OBs and AT-OBs). The transcriptomic analysis was performed by microarray covering the entire rat functional genome. It was observed that cells from bone marrow presented higher expression of genes related to osteogenesis, whereas cells from adipose tissue showed a higher expression of genes related to angiogenesis and adipocyte differentiation, irrespective of cell differentiation. By comparing cells from the same source, MSCs from both sources exhibited higher expression of genes involved in angiogenesis, osteoblast differentiation, and bone morphogenesis than osteoblasts. The clustering analysis showed that AT-OBs exhibited a gene expression profile closer to MSCs from both sources than BM-OBs, suggesting that BM-OBs were in a more advanced stage of differentiation. In conclusion, our results suggest that in cell-based therapies for bone regeneration AT-MSCs could be considered for angiogenic purposes, whereas BM-MSCs and osteoblasts differentiated from either source could be better for osteogenic approaches., (© 2019 Wiley Periodicals, Inc.)

The major role of integrins is to mediate cell adhesion but some of them are involved in the osteoblasts-titanium (Ti) interactions. In this study, we investigated the participation of integrins in osteoblast differentiation induced by Ti with nanotopography (Ti-Nano) and with microtopography (Ti-Micro). By using a PCR array, we observed that, compared with Ti-Micro, Ti-Nano upregulated the expression of five integrins in mesenchymal stem cells, including integrin β3, which increases osteoblast differentiation. Silencing integrin β3, using CRISPR-Cas9, in MC3T3-E1 cells significantly reduced the osteoblast differentiation induced by Ti-Nano in contrast to the effect on T-Micro. Concomitantly, integrin β3 silencing downregulated the expression of integrin αv, the parent chain that combines with other integrins and several components of the Wnt/β-catenin and BMP/Smad signaling pathways, all involved in osteoblast differentiation, only in cells cultured on Ti-Nano. Taken together, our results showed the key role of integrin β3 in the osteogenic potential of Ti-Nano but not of Ti-Micro. Additionally, we propose a novel mechanism to explain the higher osteoblast differentiation induced by Ti-Nano that involves an intricate regulatory network triggered by integrin β3 upregulation, which activates the Wnt and BMP signal transductions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1303-1313, 2019., (© 2019 Wiley Periodicals, Inc.)

The yeast species Starmerella bacillaris (synonym Candida zemplinina) is widely associated with oenological ecosystems and is frequently isolated from grape and grape must. Previous work showed that the genetic diversity of this species is high in wine environments and it is shaped by geographic location. Most analysed C. zemplinina strains, however, have been isolated from Vitis vinifera, disregarding the existence of other worldwide-distributed Vitis species used in winemaking. In this work, we address the impact of the Vitis species and geographic location on the genetic diversity of C. zemplinina. Microsatellite genotyping analysis was applied to two remarkable populations of C. zemplinina from Argentina and Portugal (Azores Archipelago), isolated from neighbouring V. vinifera and Vitis labrusca vineyards. The study also included a large population of previously characterized worldwide-isolated C. zemplinina strains. Genetic analyses confirmed that geographic localization significantly shaped the genetic diversity of C. zemplinina. No genetic differentiation on the basis of the Vitis species was recorded, indicating that C. zemplinina populations from neighbouring V. vinifera and V. labrusca vineyards are genetically homogeneous. In addition, no impact of the vintage was found on the C. zemplinina populations being both highly diversified and homogeneous during initial stages of alcoholic fermentation. Altogether, these results confirmed that winemaking-related factors (i.e., vintage, Vitis species, and alcoholic fermentation) do not impact the genetic diversity of C. zemplinina and that only geographic localization significantly shapes this yeast species., (© 2019 John Wiley & Sons, Ltd.)

Introduction: Interesting data about the family Asteraceae as a new source of Leishmania major dihydroorotate dehydrogenase (LmDHODH) inhibitors are presented. This key macromolecular target for parasites causing neglected diseases catalyzes the fourth reaction of the de novo pyrimidine biosynthetic pathway, which takes part in major cell functions, including DNA and RNA biosynthesis., Objectives: We aimed to (1) determine LmDHODH inhibitor candidates, revealing the type of chemistry underlying such bioactivity, and (2) predict the inhibitory potential of extracts from new untested plant species, classifying them as active or inactive based on their LC-MS based metabolic fingerprints., Methods: Extracts from 150 species were screened for the inhibition of LmDHODH, and untargeted UHPLC-(ESI)-HRMS metabolomic studies were carried out in combination with in silico approaches., Results: The IC 50 values determined for a subset of 59 species ranged from 148 µg mL -1 to 9.4 mg mL -1 . Dereplication of the metabolic fingerprints allowed the identification of 48 metabolites. A reliable OPLS-DA model (R 2  > 0.9, Q 2  > 0.7, RMSE CV  < 0.3) indicated the inhibitor candidates; nine of these metabolites were identified using data from isolated chemical standards, one of which-4,5-di-O-E-caffeoylquinic acid (IC 50 73 µM)-was capable of inhibiting LmDHODH. The predictive OPLS model was also effective, with 60% correct predictions for the test set., Conclusion: Our approach was validated for (1) the discovery of LmDHODH inhibitors or interesting starting points for the optimization of new leishmanicides from Asteraceae species and (2) the prediction of extracts from untested species, classifying them as active or inactive.

Among bone morphogenetic proteins (BMPs), BMP-9 has been described as one with higher osteogenic potential. Here, we aimed at evaluating the effect of BMP-9 on the osteoblast differentiation of cells grown on titanium (Ti) with nanotopography, a well-known osseoinductive surface. MC3T3-E1 cells were grown either in absence or presence of BMP-9 (20 nM) on Ti with nanotopography (Ti-Nano) or machined Ti (Ti-Machined) for up to 21 days to evaluate the gene expression of RUNX2, osterix, osteocalcin, bone sialoprotein, SMAD6 and SMAD4, protein expression of SMAD4, ALP activity and extracellular matrix mineralization. As expected BMP-9 increased osteoblast differentiation irrespective of Ti surface topography; however, the cells grown on Ti-Nano were more responsible to BMP-9 compared with cells grown on Ti-machined. This could be, at least in part, due to the fact that Ti-Nano may act on both ways, by increasing the activation (SMAD4) and decreasing the inhibition (SMAD6) of the signaling pathway triggered by BMP-9, while Ti-Machined only decrease the inhibition (SMAD6) of this pathway. In conclusion, the combination of the osteogenic potential of BMP-9 with the osseoinductive capacity of Ti-Nano could be a promising strategy to favor the osseointegration of Ti implants., (© 2018 Wiley Periodicals, Inc.)

Background Aims: Regenerative medicine strategies based on cell therapy are considered a promising approach to repair bone defects. The aims of this study were to evaluate the effect of subculturing on the osteogenic potential of osteoblasts derived from newborn rat calvaria and the effect of these osteoblasts on bone repair of rat calvaria defects., Methods: Cells were obtained from 50 newborn rat calvaria, and primary osteoblasts (OB) were compared with first passage (OB-P1) in terms of osteogenic potential by assaying cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of the osteoblastic markers RUNX2, ALP, osteocalcin and bone sialoprotein. Then, 5-mm calvaria defects were created in 24 Wistar rats, and after 2 weeks, they were locally injected with 50 µL of phosphate-buffered saline containing either 5 × 10 6 osteoblasts (OB-P1, n = 12) or no cells (control, n = 12). Four weeks post-injection, the bone formation was evaluated by micro-computed tomography and histological analyses. Data were compared by analysis of variance, followed by the Student-Newman-Keuls's test or Student's t-test (P ≤ 0.05)., Results: OB-P1 showed high proliferation and ALP activity, and despite the reduced gene expression of osteoblastic markers and extracellular matrix mineralization compared with OB, they displayed osteogenic potential, being a good choice for injection into calvaria defects. The micro-tomographic and histological data showed that defects treated with OB-P1 presented higher bone formation compared with control defects., Discussion: Our results indicate that cells derived from newborn rat calvaria retain osteoblastic characteristics after subculturing and that these osteoblasts stimulate bone repair in a rat calvaria defect model., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

[This corrects the article DOI: 10.1128/MRA.00872-18.].

Starmerella bacillaris is an ascomycetous yeast ubiquitously present in grapes and fermenting grape musts. In this report, we present the draft genome sequence of the S. bacillaris type strain CBS 9494, isolated from sweet botrytized wines, which will contribute to the study of this genetically heterogeneous wine yeast species.

Yeast communities associated with Vitis vinifera L. ecosystems have been widely characterized. Less is known, however, about yeast communities present in grapes and fermenting musts from Vitis non- vinifera ecosystems. Moreover, there are no comparative studies concerning yeast communities in grapes from V. vinifera L. and non- vinifera Vitis species in vineyards from a shared terroir . In this work, we have used a culture-dependent strategy, phenotypic analyses, and molecular genotyping, to study the most representative yeast species present in spontaneously fermenting musts of grapes harvested from neighboring V. vinifera L. (cv. Malbec) and V. labrusca L. (cv. Isabella) vineyards. Phenotypic analyses of H 2 S production, ethanol tolerance and carbon utilization, on randomly selected strains of each Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae strains, as well as microsatellite genotyping of S. cerevisiae isolates from each the Malbec and Isabella grape musts, suggest that V. vinifera L. and V. labrusca L. ecosystems could harbor different yeast strain populations. Thus, microbial communities in exotic Vitis species may offer opportunities to look for unique yeast strains that could not be present in conventional V. vinifera L. ecosystems.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman ® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data., (© 2018 Wiley Periodicals, Inc.)

Investigation on functional genome research may contribute to the knowledge of functional roles of different mRNAs and miRNAs in bone cells of osteoporotic animals. Currently, few studies indicate the changes in gene modulation that osteoporosis causes in osteoblastic cells from different sites. Thus, the purpose of this investigation was to evaluate cell viability, alkaline phosphatase activity and modulation of mRNAs/miRNAs in osteoblastic cells from calvaria and bone marrow by means of microarray technology. Wistar female rats were divided in sham operated and ovariectomized groups. After 150 days of ovariectomy, cells were isolated from both sites to perform cell culture. Results showed that calvaria cells from ovariectomized rats had a decrease in viability when compared to control groups and to bone marrow cells from osteoporotic rats after 3 days. Alkaline phosphatase activity decreased in calvaria cells from ovariectomized rats whereas it was increased in bone marrow osteoblastic cells in the same group. Microarray data analysis showed 5447 differentially expressed mRNAs and 82 differentially expressed miRNAs in calvaria cells. The same way, 4399 mRNAs and 54 miRNAs were expressed in bone marrow cells. mRNAs associated with bone metabolism such as Anxa5, Sp7, Spp1, Notch1 were distinctively modulated in both sites, as well as miRNAs such as miR-350, miR-542-3p, miR-204-5p, and miR-30e-3p. The RNA species identified in this study could be further used as targets for treatment or prevention of osteoporosis., (© 2017 International Federation for Cell Biology.)

Pompe's disease (PD) is an infrequent metabolic autosomic recessive disorder produced by the lack or deficiency of the acid alpha-glucosidase lysosomal enzyme in tissues of involved individuals. Delayed-onset PD is considered whenever symptoms onset start after one year of age. We present an update of the recommendations for the management of delayed-onset PD, taking as reference the guidelines from the Argentine Consensus for diagnosis, treatment and follow-up of PD published in 2013. The present consensus gathered several experts in PD in the areas of internal medicine, laboratory diagnosis, neuropathology, pulmonology, nutrition, neurology, metabolic and neuromuscular disorders as well as rehabilitation to perform an update of the literature of delayed-onset PD, with special attention on relevant information published within the last 4 years. The entire working group approved the final version of the consensus. Each participant provided a declaration of conflict of interest. As a result, it is an update of the previous Argentine PD Consensus with focus on the delayed-onset presentation of the disease. Being such infrequent disorder, available data were rather limited and thus, the recommendations represent expert opinions.

Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017., (© 2017 Wiley Periodicals, Inc.)

Osteoblasts and adipocytes coexist in the implantation site and affect the process of titanium (Ti) osseointegration. As extracellular signal-regulated kinases 1/2 (ERK1/2) are involved in osteogenesis and adipogenesis, the aim of our study was to investigate if the effects of Ti surface topography on osteoblast and adipocyte differentiation are modulated by ERK1/2. The experiments were conducted based on the effect of the ERK1/2 inhibitor, PD98059, on mesenchymal stem cells (MSCs) grown under osteogenic and adipogenic conditions on Ti with nanotopography (Ti-Nano) or on machined Ti (Ti-Machined). The results showed that, in general, ERK1/2 inhibition favored osteoblast and adipocyte differentiation of MSCs grown on Ti-Machined. In MSCs grown on Ti-Nano, ERK1/2 inhibition upregulated the expression of alkaline phosphatase and osteocalcin and reduced extracellular matrix mineralization. In terms of adipocyte differentiation, ERK1/2 inhibition elicited similar MSC responses to Ti-Nano and Ti-Machined, upregulating gene expression of adipocyte markers without affecting lipid accumulation. Our results indicate that, under osteogenic and adipogenic conditions, the responses of MSCs to Ti surface topography in terms of osteogenesis and adipogenesis are dependent on ERK1/2. Thus, a precise modulation of ERK1/2 expression and activity induced by surface topography could be a good strategy to drive the process of implant osseointegration., (© 2017 Eur J Oral Sci.)

Latin America (LA) has a population of ~645 million people distributed over 33 countries with marked political, cultural and economic differences. In LA, patients with inherited neuromuscular diseases (NMDs) often do not have access to specialized medical centers and many of them go undiagnosed. General management and care of spinal muscular dystrophy (SMA) patients in the region varies due to heterogeneous health care. An active generation of young clinical neurologists is being trained for the specialized care of SMA and other neuromuscular (NM) patients, both in the private and public sectors. The Euro-Latin-American Summer School of Myology (EVELAM) as well as efforts of professionals at large public centers in the major cities of LA have a leading role in this development. Different regional academic-scientific organizations as well as the expanding number of telethon centers and the creation of parent organizations, mostly concerning SMA, all together are contributing to the increased quality of the management of NMD patients. Over the past years, academic and clinical research, as well as the establishment of qualified centers for the molecular testing of NMD are pushing forward the creation of patient registries and the development of specific clinical trials, with Argentina and Brazil having a major role in this field. Nevertheless, increased awareness and further training of specialized health professionals are necessary to reach patients that are currently lacking care throughout the region.

One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

Aim: To evaluate the effect of a calcium aluminate-based cement (CAC+) on the development of the osteogenic phenotype in vitro., Methodology: Rat calvaria-derived cells were grown on Thermanox ® coverslips for 24 h and then exposed to either samples (4-h set) of CAC+ or mineral trioxide aggregate (MTA) placed on Transwell ® inserts for periods of up to 14 days. Nonexposed cultures were used as the controls. The comparisons were made using the nonparametric Kruskal-Wallis test, followed by the Student-Newman-Keuls post hoc test when appropriate., Results: The results showed that proximity to MTA or CAC+ samples inhibited cell growth, whereas at a distance, viable and proliferative cells adhered to and spread on the Thermanox ® , expressing osteoblast differentiation markers prior to mineralization of the extracellular matrix. Compared with MTA, the osteogenic cell cultures exposed to CAC+ exhibited significantly greater cell viability, alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2, osterix, ALP, bone sialoprotein and osteocalcin (P < 0.05 for all). For the osteogenic cell cultures exposed to CAC+, the quantification of matrix mineralization was not altered (P > 0.05)., Conclusions: CAC+ supported the acquisition of the osteogenic cell phenotype in vitro, rendering this novel material a potential alternative to MTA in endodontic procedures. Further in vivo studies are needed to verify if the beneficial in vitro effects of CAC+ on osteoblastic cells correspond to an increase and/or acceleration of bone repair in the periapical region., (© 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

Grape must harbors a complex community of yeast species responsible for spontaneous alcoholic fermentation. Although there are detailed studies on the microbiota of Vitis vinifera L. grapes, less is known about the diversity and behavior of yeast communities present on fermenting grape must from other species of Vitis . In this work, we used a culture-dependent method to study the identity and dynamics of the indigenous yeast population present during the spontaneous fermentation of Isabella ( Vitis labrusca L.) grape must. Alcoholic fermentation was conducted using standard enological practices, and the associated non- Saccharomyces and S. cerevisiae yeast community was analyzed using selective growth media and 5.8-ITS DNA sequencing. Candida californica, Candida hellenica, Starmerella bacillaris (synonym Candida zemplinina ), Hanseniaspora uvarum , and Hanseniaspora vineae were the main non- Saccharomyces species identified on Isabella fermenting must. Issatchenkia hanoiensis , a yeast species rarely found on Vitis vinifera L. grapes, was also recognized on Isabella grape must. Candida azymoides, Candida californica and Pichia cecembensis , identified in this work on Isabella fermenting must, have not previously been found on Vitis vinifera L. grape must. Interestingly, C. azymoides, I. hanoiensis and P. cecembensis have recently been isolated from the surface of Vitis labrusca L. grapes from vineyards in the Azores archipelago, suggesting that specific Vitis -yeast species associations are formed independently of geographic origin. We suggest that C. azymoides, C. californica , and P. cecembensis are yeast species preferentially associated with Vitis labrusca L. grapes. Specific biological interactions between grapevines and yeast species may underlie the assembly of differential Vitis -microbial communities.

The ability of Biosilicate® with two crystalline phases (BioS-2P) to drive osteoblast differentiation encourages the investigation of the cellular mechanisms involved in this process. Then, the aim of our study was to analyze the large-scale gene expression of osteoblasts grown on BioS-2P compared with Bioglass ® 45S5 (45S5). Osteoblasts differentiated from rat bone marrow mesenchymal stem cells were cultured under osteogenic conditions on BioS-2P, 45S5 and polystyrene (control). After 10 days, the expression of 23,794 genes was analyzed using mRNA Sequencing and the data were validated by real-time PCR. The BioS-2P exhibited 5 genes upregulated and 3 downregulated compared with 45S5. Compared with control, BioS-2P upregulated 15 and downregulated 11 genes, while 45S5 upregulated 25 and downregulated 21 genes. Eight genes were commonly upregulated and 4 downregulated by both bioactive glasses. In conclusion, our results demonstrated that bioactive glasses affect the gene expression profiling of osteoblasts. Most of the regulated genes by both BioS-2P and 45S5 are associated with the process of mineralization highlighting their osteostimulation property that is, at least in part, derived from the ability to modulate the intracellular machinery to promote osteoblast genotype expression. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 419-423, 2017., (© 2016 Wiley Periodicals, Inc.)

Facioscapulohumeral muscular dystrophy (FSHD), a prevalent inherited human myopathy, develops following a complex interplay of genetic and epigenetic events. FSHD1, the more frequent genetic form, is associated with: (1) deletion of an integral number of 3.3 Kb (D4Z4) repeated elements at the chromosomal region 4q35, (2) a specific 4q35 subtelomeric haplotype denominated 4qA, and (3) decreased methylation of cytosines at the 4q35-linked D4Z4 units. FSHD2 is most often caused by mutations at the SMCHD1 (Structural Maintenance of Chromosomes Hinge Domain 1) gene, on chromosome 18p11.32. FSHD2 individuals also carry the 4qA haplotype and decreased methylation of D4Z4 cytosines. Each D4Z4 unit contains a copy of the retrotransposed gene DUX4 (double homeobox containing protein 4). DUX4 gene functionality was questioned in the past because of its pseudogene-like structure, its location on repetitive telomeric DNA sequences (i.e. junk DNA), and the elusive nature of both the DUX4 transcript and the encoded protein, DUX4. It is now known that DUX4 is a nuclear-located transcription factor, which is normally expressed in germinal tissues. Aberrant DUX4 expression triggers a deregulation cascade inhibiting muscle differentiation, sensitizing cells to oxidative stress, and inducing muscle atrophy. A unifying pathogenic model for FSHD emerged with the recognition that the FSHD-permissive 4qA haplotype corresponds to a polyadenylation signal that stabilizes the DUX4 mRNA, allowing the toxic protein DUX4 to be expressed. This working hypothesis for FSHD pathogenesis highlights the intrinsic epigenetic nature of the molecular mechanism underlying FSHD as well as the pathogenic pathway connecting FSHD1 and FSHD2. Pharmacological control of either DUX4 gene expression or the activity of the DUX4 protein constitutes current potential rational therapeutic approaches to treat FSHD., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Osteoporosis is a chronic disease that impairs proper bone remodeling. Guided bone regeneration is a surgical technique that improves bone defect in a particular region through new bone formation, using barrier materials (e.g. membranes) to protect the space adjacent to the bone defect. The polytetrafluorethylene membrane is widely used in guided bone regeneration, however, new membranes are being investigated. The purpose of this study was to evaluate the effect of P(VDFTrFE)/BT [poly(vinylidene fluoride-trifluoroethylene)/barium titanate] membrane on in vivo bone formation. Twenty-three Wistar rats were submitted to bilateral ovariectomy. Five animals were subjected to sham surgery. After 150 days, bone defects were created and filled with P(VDF-TrFE)/BT membrane or PTFE membrane (except for the sham and OVX groups). After 4 weeks, the animals were euthanized and calvaria samples were subjected to histomorphometric and computed microtomography analysis (microCT), besides real time polymerase chain reaction (real time PCR) to evaluate gene expression. The histomorphometric analysis showed that the animals that received the P(VDF-TrFE)/BT membrane presented morphometric parameters similar or even better compared to the animals that received the PTFE membrane. The comparison between groups showed that gene expression of RUNX2, BSP, OPN, OSX and RANKL were lower on P(VDF-TrFE)/BT membrane; the gene expression of ALP, OC, RANK and CTSK were similar and the gene expression of OPG, CALCR and MMP9 were higher when compared to PTFE. The results showed that the P(VDF-TrFE)/BT membrane favors bone formation, and therefore, may be considered a promising biomaterial to support bone repair in a situation of osteoporosis.

Background: Flail chest is managed with mechanical ventilation or inhaloteraphy and analgesia. Little has been published on the use of bioabsorbable material and its evolution in flail chest fixation., Methods: This is a descriptive study of patients with unstable chest undergoing fixation with bioabsorbable plates and screws in a period from February 2009 to December 2011., Results: We report 18 cases, aged 33-74 years (mean 53), three with bilateral involvement; rib fixation was performed between 1-21 days of the accident (mean 1.5). They started walking the next day in 14 cases; postoperative stay was four days (range 3-14). The heart rate of patients prior to surgery was 90 per minute (range 82-100) and lowered to 84 after fixation (range 82-92), preoperative respiratory rate was 26 per minute (range 22-28) and below 22 per minute (range 20 to 26) in postoperative period., Conclusions: The use of bioabsorbable material for osteosynthesis of costal fractures did not show side effects in our period of study.

This study evaluated the temporal expression of osteopontin (OPN) in co-cultures of human osteoblastic cells (SAOS-2) and oral squamous cell carcinoma (OSCC)-derived cells (SCC9) and examined the effects of osteoblast-derived OPN on the neoplastic cell phenotype. Additionally, the effects of these co-cultures on subsequent osteoclastic activity were explored. SCC9 cells were plated on Transwell® membranes that were either coated or not coated with Matrigel and were then co-cultured with SAOS-2 cells during the peak of OPN expression. SCC9 cells exposed to OPN-silenced SAOS-2 cultures and SCC9 cells cultured alone served as controls. SCC9 cells were quantitatively evaluated for cell adhesion, proliferation, migration, and invasion into Matrigel. The impact of co-culturing SAOS-2 and SCC9 cells on the resorptive capacity of U-937-derived osteoclastic cells was also investigated. Furthermore, a reciprocal induction of SAOS-2 and SCC9 cells in terms of OPN expression over the co-culture interval was identified. SAOS-2-secreted OPN altered the SCC9 cell phenotype, leading to enhanced cell adhesion and proliferation and higher Matrigel invasion. This invasion was also enhanced, albeit to a lesser degree, by co-culture with OPN-silenced SAOS-2 cells. Cell migration was not affected. Co-culture with SAOS-2 cells-mainly during the period of peak OPN expression-promoted over-expression of IL-6 and IL-8 by SCC9 cells and enhanced the resorptive capacity of osteoclastic cells. Taken together, these results suggest that osteoblast-derived OPN affects the interactions among OSCC-derived epithelial cells, osteoblasts, and osteoclasts, which could contribute to the process of bone destruction during bone invasion by OSCC.

The poly(vinylidene-trifluoroethylene)/barium titanate (PVDF) membrane enhances in vitro osteoblast differentiation and in vivo bone repair. Here, we hypothesized that this higher bone repair could be also due to bone resorption inhibition mediated by a microRNA (miR)/RANKL circuit. To test our hypothesis, the large-scale miR expression of bone tissue grown on PVDF and polytetrafluoroethylene (PTFE) membranes was evaluated to identify potential RANKL-targeted miRs modulated by PVDF. The animal model used was rat calvarial defects implanted with either PVDF or PTFE. At 4 and 8 weeks, the bone tissue grown on membranes was submitted to a large-scale analysis of miRs by microarray. The expression of miR-34a and some of its targets, including RANKL, were evaluated by real-time polimerase chain reaction and osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. Among more than 250 miRs, twelve, including miR-34a, were simultaneously higher expressed (≥2 fold) at 4 and 8 weeks on PVDF. The higher expression of miR-34a was concomitant with a reduced expression of all its evaluated targets, including RANKL. Additionally, more TRAP-positive cells were observed in bone tissue grown on PTFE compared with PVDF in both time points. In conclusion, our results suggest that the higher bone formation induced by PVDF could be, at least in part, triggered by a miR-34a increase and RANKL decrease, which may inhibit osteoclast differentiation and activity, and bone resorption.

Objective: We investigated the osteoblast and adipocyte differentiation of mesenchymal stem cells (MSCs) from young and aged rats cultured on Ti., Material and Methods: Bone marrow MSCs derived from 1-month and 21-month rats were cultured on Ti discs under osteogenic conditions for periods of up to 21 days and osteoblast and adipocyte markers were evaluated., Results: Cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization and gene expression of RUNX2, osterix, ALP, bone sialoprotein, osteopontin, and osteocalcin were reduced in cultures of 21-month rats compared with 1-month rats grown on Ti. Gene expression of PPAR-γ , adipocyte protein 2, and resistin and lipid accumulation were increased in cultures of 21-month rats compared with 1-month rats grown on the same conditions., Conclusions: These results indicate that the lower osteogenic potential of MSCs derived from aged rats compared with young rats goes along with the higher adipogenic potential in cultures grown on Ti surface. This unbalance between osteoblast and adipocyte differentiation should be considered in dental implant therapy to the elderly population.

Background: Mediastinal tumours in children are rare. Around 25% of them can be malignant. The thymoma is an uncommon neoplasm, and during adulthood it corresponds to 30% of anterior mediastinum tumours. The peak incidence is between 55-65 years., Clinical Case: A case of lymphocytic thymoma case is reported in a 4 year old patient with no previous or associated symptomatology. There was only a volume increase on the anterior neck region. The neck radiography and neck and chest tomography confirmed an anterior mediastinal mass surrounding the aorta and vena cava, as well as multiple mediastinal lymph nodes, Conclusions: Early diagnosis and complete resection are the basis for management and prognosis., (Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.)

Clinical success of implant therapy is directly related to titanium (Ti) surface properties and the quality of bone tissue. The treatment of Ti implants with H2SO4/H2O2 is a feasible, reproducible, and low-cost technique to create surface nanotopography (Ti-Nano). As this nanotopography induces osteoblast differentiation, we hypothesized that it may affect bone response to Ti. Thus, this study was designed to evaluate the bone response to a machined Ti implant treated with H2SO4/H2O2 to generate Ti-Nano and to compare it with a commercially available microtopographic Ti implant (Ti-Porous). Implants were placed in rabbit tibias and evaluated after 2 and 6 weeks, and the bone tissue formed around them was assessed by microtomography to record bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness, and trabecular separation. Undecalcified histological sections were used to determine the percentages of bone-to-implant contact, bone area formed between threads, and bone area formed in the mirror area. At the end of 6 weeks, the removal torque was evaluated using a digital torque gauge. The results showed bone formation in close contact with both Ti-Nano and Ti-Porous implants without relevant morphological and morphometric differences, in addition to a similar removal torque irrespective of surface topography. In conclusion, our results have shown that a simple and low-cost method using H2SO4/H2O2 is highly efficient for creating nanotopography on Ti surfaces, which elicits a similar bone response compared with microtopography presented in a commercially available Ti implant.

Background: Bronchogenic cyst is a malformation of the ventral portion of the intestine, which is limited by bronchial epithelium and produces alterations in the development of the tracheobronchial tree. They may be single or multiple, and are usually confined to one lung or to the mediastinum, rarely to the neck, which is a subcutaneous tissue., Objective: The case of a 9 year old girl is reported, who presented with a clinical picture characterized by a slow-growing, asymptomatic tumour on the left side of the neck of 4 years onset. Chest X-ray, neck ultrasound and computed tomography of the neck and chest ruled out any other injury. A complete resection was performed, and the histopathological study confirmed the diagnosis of bronchogenic cyst., Conclusion: The symptomatology of a bronchogenic cyst is due to the compression of the vascular, digestive or air structures, as well as its size, infection and location. The treatment of choice is a surgical resection, even when asymptomatic., (Copyright © 2015 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.)

Mutations in the WDR45 gene have been identified as causative for the only X-linked type of neurodegeneration with brain iron accumulation (NBIA), clinically characterized by global developmental delay in childhood, followed by a secondary neurological decline with parkinsonism and/or dementia in adolescence or early adulthood. Recent reports suggest that WDR45 mutations are associated with a broader phenotypic spectrum. We identified a novel splice site mutation (c.440-2 A > G) in a 5-year-old Argentinian patient with Rett-like syndrome, exhibiting developmental delay, microcephaly, seizures and stereotypic hand movements, and discuss this finding, together with a review of the literature. Additional patients with a clinical diagnosis of Rett (-like) syndrome were also found to carry WDR45 mutations before (or without) clinical decline or signs of iron accumulation by magnetic resonance imaging (MRI). This information indicates that WDR45 mutations should be added to the growing list of genetic alterations linked to Rett-like syndrome. Further, clinical symptoms associated with WDR45 mutations ranged from early-onset epileptic encephalopathy in a male patient with a deletion of WDR45 to only mild cognitive delay in a female patient, suggesting that analysis of this gene should be considered more often in patients with developmental delay, regardless of severity. The increasing use of next generation sequencing technologies as well as longitudinal follow-up of patients with an early diagnosis will help to gain additional insight into the phenotypic spectrum associated with WDR45 mutations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mesenchymal stem cells from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are attractive tools for cell-based therapies to repair bone tissue. In this study, we investigated the osteogenic and adipogenic potential of BM-MSCs and AT-MSCs as well as the effect of crosstalk between osteoblasts and adipocytes on cell phenotype expression. Rat BM-MSCs and AT-MSCs were cultured either in growth, osteogenic, or adipogenic medium to evaluate osteoblast and adipocyte differentiation. Additionally, osteoblasts and adipocytes were indirectly co-cultured to investigate the effect of adipocytes on osteoblast differentiation and vice versa. BM-MSCs and AT-MSCs exhibit osteogenic and adipogenic potential under non-differentiation-inducing conditions. When exposed to osteogenic medium, BM-MSCs exhibited higher expression of bone markers compared with AT-MSCs. Conversely, under adipogenic conditions, AT-MSCs displayed higher expression of adipose tissue markers compared with BM-MSCs. The presence of adipocytes as indirect co-culture repressed the expression of the osteoblast phenotype, whereas osteoblasts did not exert remarkable effect on adipocytes. The inhibitory effect of adipocytes on osteoblasts was due to the release of tumor necrosis factor alpha (TNF-α) in culture medium by adipocytes. Indeed, the addition of exogenous TNF-α in culture medium repressed the differentiation of BM-MSCs into osteoblasts mimicking the indirect co-culture effect. In conclusion, our study showed that BM-MSCs are more osteogenic while AT-MSCs are more adipogenic. Additionally, we demonstrated the key role of TNF-α secreted by adipocytes on the inhibition of osteoblast differentiation. Thus, we postulate that the higher osteogenic potential of BM-MSCs makes them the first choice for inducing bone repair in cell-based therapies., (© 2015 Wiley Periodicals, Inc.)

This study was designed to investigate the influence of mesenchymal stem cells (MSCs) on osteoblast (OB) differentiation. Rat bone marrow MSCs were cultured either in growth medium that maintained a MSC phenotype or in osteogenic medium that induced differentiation into OBs. Then, cells were grown in two different culture conditions: indirect co-culture of MSCs and OBs and OBs cultured in MSC-conditioned medium. As a control culture condition, OBs were grown in osteogenic medium without the influence of MSCs. We evaluated cell proliferation, the gene expression of key bone markers, alkaline phosphatase (ALP) activity, bone sialoprotein (BSP) expression, and extracellular matrix mineralization. The results showed that, regardless of whether OBs were indirectly co-cultured with MSCs or cultured in MSC-conditioned medium, MSCs repressed OB differentiation, as evidenced by the downregulation of all evaluated bone marker genes, decreased ALP activity, inhibition of BSP protein expression, and reduced extracellular matrix mineralization. Taken together, these results indicate that despite the key role of both MSCs and OBs in the osteogenic process, the repressive effect of MSCs on OB differentiation in an osteogenic environment may represent a barrier to the strategy of using them together in cell-based therapies to induce bone repair., (© 2015 Wiley Periodicals, Inc.)

Unlabelled: The ability of hemostatic agents to promote bone repair has been investigated using in vitro and in vivo models but, up to now, the results are inconclusive. Objective In this context, the aim of this study was to compare the potential of bone repair of collagen sponge with fibrin glue in a rat calvarial defect model., Material and Methods: Defects of 5 mm in diameter were created in rat calvariae and treated with either collagen sponge or fibrin glue; untreated defects were used as control. At 4 and 8 weeks, histological analysis and micro-CT-based histomorphometry were carried out and data were compared by two-way ANOVA followed by Student-Newman-Keuls test when appropriated (p≤0.05)., Results: Three-dimensional reconstructions showed increased bone formation in defects treated with either collagen sponge or fibrin glue compared with untreated defects, which was confirmed by the histological analysis. Morphometric parameters indicated the progression of bone formation from 4 to 8 weeks. Additionally, fibrin glue displayed slightly higher bone formation rate when compared with collagen sponge., Conclusion: Our results have shown the benefits of using collagen sponge and fibrin glue to promote new bone formation in rat calvarial bone defects, the latter being discreetly more advantageous.

The present study evaluated whether the changes in the labeling pattern of cytoskeletal proteins in osteogenic cells cultured on bioactive glass-based materials are due to altered mRNA and protein levels. Primary rat-derived osteogenic cells were plated on Bioglass® 45S5, Biosilicate®, and borosilicate (bioinert control). The following parameters were assayed: (i) qualitative epifluorescence analysis of actin and tubulin; (ii) quantitative mRNA and protein expression for actin and tubulin by real-time PCR and ELISA, respectively, and (iii) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, the cells grown on borosilicate showed typical actin and tubulin labeling patterns, whereas those on the bioactive materials showed roundish areas devoid of fluorescence signals. The cultures grown on bioactive materials showed significant changes in actin and tubulin mRNA expression that were not reflected in the corresponding protein levels. A positive correlation between the mRNA and protein as well as an association between epifluorescence imaging and quantitative data were only detected for the borosilicate. SEM imaging of the cultures on the bioactive surfaces revealed cells partly or totally coated with material aggregates, whose characteristics resembled the substrate topography. The culturing of osteogenic cells on Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA expression but not the corresponding protein levels. Changes in the labeling pattern of these proteins should then be attributed, at least in part, to the presence of a physical barrier on the cell surface as a result of the material surface reactions, thus limiting fluorescence signals., (© 2015 Wiley Periodicals, Inc.)

Objective: Inflammatory biomarkers associated with cardiovascular disease are elevated in HIV-infected persons. These biomarkers improve with antiretroviral therapy (ART) but do not normalize to values observed in HIV-uninfected adults. Little is known regarding biomarkers of inflammation in HIV-infected Peruvians, in whom an increased burden of infectious diseases may exacerbate inflammation, and women, in whom sex difference may alter inflammation compared with men., Methods: Peruvians initiating first-line ART were enrolled in a prospective observational study. Individuals with suppression of HIV RNA plasma loads to less than 30 copies/ml when determined quarterly over 24 months of ART, had biomarkers of inflammation and cellular activation measured pre-ART and at 24-months of ART, and evaluated for associations with sex and clinical parameters., Results: Pre-ART high-sensitivity C-reactive protein (hsCRP) values of men were in the high-risk cardiovascular disease category (>3.0 mg/l) more frequently compared with women (P = 0.02); most women's values were in the low/average-risk categories. At 24 months of suppressive ART, hsCRP concentrations decreased in men (P = 0.03), but tended to increase in women, such that the proportion with high-risk hsCRP did not differ by sex. Pre-ART, soluble CD163 concentrations were higher in women compared with men (P = 0.02), and remained higher after 24 months of suppressive ART (P = 0.02). All other inflammatory biomarkers (P < 0.03) decreased across sexes. Biomarker concentrations were not associated with BMI or coinfections., Conclusion: Elevated inflammatory biomarkers persisted despite 24 months of suppressive ART in a subset of Peruvians, and to a greater extent in women compared with men. These findings suggest that lifestyle or pharmacologic interventions may be required to optimize the health of HIV-infected Peruvians, particularly women.