Modulation of bone marrow mesenchymal stem cell secretome by ECM-like hydrogels

It has been demonstrated that bone marrow mesenchymal stem cell (BM-MSCs) transplantation has beneficial effects on several central nervous system (CNS) debilitating conditions. Growing evidence indicate that trophic factors secreted by these cells are the key mechanism by which they are acting. These cells are frequently used in combination with 3D artificial matrices, for instance hydrogels, in tissue engineering-based approaches. However, so far, no study has been reported on the influence of such matrices, namely the presence or absence of extracellular matrix motifs, on BM-MSCs secretome and its effects in neuronal cell populations. In this sense, we herein studied the impact of a hydrogel, gellan gum, on the behavior and secretome of BM-MSCs, both in its commercial available form (commonly used in tissue engineering) and in a fibronectin peptide-modified form. The results showed that in the presence of a peptide in the gellan gum hydrogel, BM-MSCs presented higher proliferation and metabolic activity than in the regular hydrogel. Moreover, the typical spindle shape morphology of BM-MSCs was only observed in the modified hydrogel. The effects of the secretome of BM-MSCs were also affected by the chemical nature of the extracellular matrix. BM-MSCs cultured in the modified hydrogel were able to secrete factors that induced higher metabolic viabilities and neuronal cell densities, when compared to those of the unmodified hydrogel. Thus adding a peptide sequence to the gellan gum had a significant effect on the morphology, activity, proliferation and secretome of BM-MSCs. These results highlight the importance of mimicking the extracellular matrix when BM-MSCs are cultured in hydrogels for CNS applications.     

Forward head posture is associated with pressure pain threshold and neck pain duration in university students with subclinical neck pain

Abstract

Objective: The aims of this study are to investigate the association between: (i) forward head posture (FHP) and pressure pain thresholds (PPTs); (ii) FHP and maladaptive cognitive processes; and (iii) FHP and neck pain characteristics in university students with subclinical neck pain.

Materials/methods: A total of 140 university students, 90 asymptomatic and 50 with subclinical neck pain, entered the study. Demographic data, anthropometric data, FHP, and PPTs were collected for both groups. In addition, pain characteristics, pain catastrophizing, and fear of movement were assessed for participants with neck pain. FHP was characterized by the angle between C7, the tragus of the ear, and the horizontal line. Correlation analysis and multivariate regression analysis were conducted.

Results: Participants with subclinical neck pain showed significantly lower PPTs than participants without neck pain (p?<?.05), but similar FHP (p?>?.05). No significant association was found between FHP and PPTs in the asymptomatic group. In the group of participants with subclinical neck pain, PPTs at the right trapezius and neck pain duration explained 19% of the variance of FHP (R²?=?0.23; adjusted R²?=?0.19; p?<?.05).

Conclusion: This study suggests that FHP is not associated with PPTs in asymptomatic university students. In university students with subclinical neck pain, increased FHP was associated with right trapezius hypoalgesia and with neck pain of shorter duration. These findings are in contrast with current assumptions on the association between neck pain and FHP.     

Ultrasonication of insulin-loaded microgel particles produced by internal gelation: Impact on particle's size and insulin bioactivity

Alginate-dextran sulfate (ADS) microgel has been used to protect insulin from gastrointestinal attack and as a carrier to promote insulin permeation through intestinal epithelium. The throughput of ADS submicron particles generation by emulsification/internal gelation is limited by its wide size distribution.     

Colony Populations and Biomass in Nests of the Amazonian Forest Termite Anoplotermes banksi Emerson (Isoptera: Termitidae)

The arboreal nests of the termite Anoplotermes banksi are abundant in Central Amazonian primary rain forests. Colony size of 7 nests (weight 92–6891 g) varied between 2,593 and 39,256 individuals/nest (1.5 – 22.1 g termites/nest). Average body fresh weight was 0.9 mg for workers and 2.1 mg for alates. Queens weighed 10–30 mg. No relationship between nest weight and maturity was detected, as the ratio of workers to larvae was 1:1, independent of nest size, and alates were found in nests weighing less than 200 g. Nests of A. banksi (12–18 ha -1) accounted for 12–15% of the nest density of all Isopteran species, but the calculated fresh weight of the termites of this species (15–23 mg/m 2) represented only 0.2–0.4% of the total termite biomass in the study area.     

5′‐ectonucleotidase mediates multiple‐drug resistance in glioblastoma multiforme cells

Glioblastoma multiforme (GBM) cells are characterised by their extreme chemoresistance. The activity of multiple‐drug resistance (MDR) transporters that extrude antitumor drugs from cells plays the most important role in this phenomenon. To date, the mechanism controlling the expression and activity of MDR transporters is poorly understood. Activity of the enzyme ecto‐5′‐nucleotidase (CD73) in tumor cells, which hydrolyses AMP to adenosine, has been linked to immunosuppression and prometastatic effects in breast cancer and to the proliferation of glioma cells. In this study, we identify a high expression of CD73 in surgically resected samples of human GBM. In primary cultures of GBM, inhibition of CD73 activity or knocking down its expression by siRNA reversed the MDR phenotype and cell viability was decreased up to 60% on exposure to the antitumoral drug vincristine. This GBM chemosensitization was caused by a decrease in the expression and activity of the multiple drug associated protein 1 (Mrp1), the most important transporter conferring multiple drug resistance in these cells. Using pharmacological modulators, we have recognized the adenosine A₃ receptor subtype in mediation of the chemoresistant phenotype in these cells. In conclusion, we have determined that the activity of CD73 to trigger adenosine signaling sustains chemoresistant phenotype in GBM cells. J. Cell. Physiol. 228: 602–608, 2013. © 2012 Wiley Periodicals, Inc.     

Somatic embryogenesis in holm oak male catkins

Somatic embryogenesis (SE) of tree species is the most promising method for the implementation of multivarietal forestry and for biotechnological approaches. To date, however, the application of this technology to mature trees is restricted to a few species. This is the first report on the induction of SE from male catkins of 100-year-old holm oaks (Quercus ilex L.). Embryogenic competence was mainly dependent on genotype and restricted to the most advanced catkin developmental stage with distinguishable closed flowers along the axis. Following a three-stage treatment procedure, embryogenic response (frequencies up 3.3 %) was obtained in three [Remedio, Villar del Arzobispo (VA) and Hunde (HU)] out of the five genotypes evaluated. In the culture conditions tested, the preferred protocol to induce SE in holm oak catkins should include: induction on MS medium with 6-benzyladenine and naphthaleneacetic acid, subculture onto medium with a reduced concentration of both plant growth regulators and a final transference to medium without growth regulators. Under these conditions, cotyledonary-stage somatic embryos developed from brown calli with or without nodular structures. Secondary SE, favored by the addition of sorbitol to the manifestation medium, allowed the establishment of 14 embryogenic lines belonging to VA and HU genotypes. Histological observations of the proliferating cultures revealed the presence of globular, torpedo and cotyledonary somatic embryos. Somatic embryos were diploid as verified by flow cytometry analysis, suggesting that they originated from the perianthic tissue of the male flower.     

Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

rpoB gene as a novel molecular marker to infer phylogeny in Planctomycetales

The 16S rRNA gene has been used in the last decades as a gold standard for determining the phylogenetic position of bacteria and their taxonomy. It is a well conserved gene, with some variations, present in all bacteria and allows the reconstruction of genealogies of microorganisms. Nevertheless, this gene has its limitations when inferring phylogenetic relationships between closely related isolates. To overcome this problem, DNA–DNA hybridization appeared as a solution to clarify interspecies relationships when the sequence similarity of the 16S rRNA gene is above 97 %. However, this technique is time consuming, expensive and laborious and so, researchers developed other molecular markers such as sequencing of housekeeping or functional genes for accurate determination of bacterial phylogeny. One of these genes that have been used successfully, particularly in clinical microbiology, codes for the beta subunit of the RNA polymerase (rpoB). The rpoB gene is sufficiently conserved to be used as a molecular clock, it is present in all bacteria and it is a mono-copy gene. In this study, rpoB gene sequencing was applied to the phylum Planctomycetes. Based on the genomes of 19 planctomycetes it was possible to determine the correlation between the rpoB gene sequence and the phylogenetic position of the organisms at a 95–96 % sequence similarity threshold for a novel species. A 1200-bp fragment of the rpoB gene was amplified from several new planctomycetal isolates and their intra and inter-species relationships to other members of this group were determined based on a 96.3 % species border and 98.2 % for intraspecies resolution.     

Biochemical and Functional Studies on the Burkholderia cepacia Complex bceN Gene,Encoding a GDP-D-Mannose 4,6-Dehydratase

This work reports the biochemical and functional analysis of the Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. V_max and K_m values of 1.5±0.2 µmol.min⁻¹.mg⁻¹ and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl₂ per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated K_i of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.     

Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic colon segment and functional intestinal obstruction. The RET proto-oncogene is the major gene associated to HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In addition, many other genes have been described to be associated with this pathology, including the semaphorins class III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) through SNP array analyses and by next-generation sequencing technologies. Semaphorins are guidance cues for developing neurons implicated in the axonal projections and in the determination of the migratory pathway for neural-crest derived neural precursors during enteric nervous system development. In addition, it has been described that increased SEMA3A expression may be a risk factor for HSCR through the upregulation of the gene in the aganglionic smooth muscle layer of the colon in HSCR patients. Here we present the results of a comprehensive analysis of SEMA3A and SEMA3D in a series of 200 Spanish HSCR patients by the mutational screening of its coding sequence, which has led to find a number of potentially deleterious variants. RET mutations have been also detected in some of those patients carrying SEMAs variants. We have evaluated the A131T-SEMA3A, S598G-SEMA3A and E198K-SEMA3D mutations using colon tissue sections of these patients by immunohistochemistry. All mutants presented increased protein expression in smooth muscle layer of ganglionic segments. Moreover, A131T-SEMA3A also maintained higher protein levels in the aganglionic muscle layers. These findings strongly suggest that these mutants have a pathogenic effect on the disease. Furthermore, because of their coexistence with RET mutations, our data substantiate the additive genetic model proposed for this rare disorder and further support the association of SEMAs genes with HSCR.