pRb-dependent cyclin D3 protein stabilization is required for myogenic differentiation

The expression of retinoblastoma (pRb) and cyclin D3 proteins is highly induced during the process of skeletal myoblast differentiation. We have previously shown that cyclin D3 is nearly totally associated with hypophosphorylated pRb in differentiated myotubes, whereas Rb-/- myocytes fail to accumulate the cyclin D3 protein despite normal induction of cyclin D3 mRNA. Here we report that pRb promotes cyclin D3 protein accumulation in differentiating myoblasts by preventing cyclin D3 degradation. We show that cyclin D3 displays rapid turnover in proliferating myoblasts, which is positively regulated through glycogen synthase kinase 3beta (GSK-3beta)-mediated phosphorylation of cyclin D3 on Thr-283. We describe a novel interaction between pRb and cyclin D3 that maps to the C terminus of pRb and to a region of cyclin D3 proximal to the Thr-283 residue and provide evidence that the pRb-cyclin D3 complex formation in terminally differentiated myotubes hinders the access of GSK-3beta to cyclin D3, thus inhibiting Thr-283 phosphorylation. Interestingly, we observed that the ectopic expression of a stabilized cyclin D3 mutant in C2 myoblasts enhances muscle-specific gene expression; conversely, cyclin D3-null embryonic fibroblasts display impaired MyoD-induced myogenic differentiation. These results indicate that the pRb-dependent accumulation of cyclin D3 is functionally relevant to the process of skeletal muscle cell differentiation.     

A dense single-nucleotide polymorphism-based genetic linkage map of grapevine (Vitis vinifera L.) anchoring Pinot Noir bacterial artificial chromosome contigs

The construction of a dense genetic map for Vitis vinifera and its anchoring to a BAC-based physical map is described: it includes 994 loci mapped onto 19 linkage groups, corresponding to the basic chromosome number of Vitis. Spanning 1245 cM with an average distance of 1.3 cM between adjacent markers, the map was generated from the segregation of 483 single-nucleotide polymorphism (SNP)-based genetic markers, 132 simple sequence repeats (SSRs), and 379 AFLP markers in a mapping population of 94 F(1) individuals derived from a V. vinifera cross of the cultivars Syrah and Pinot Noir. Of these markers, 623 were anchored to 367 contigs that are included in a physical map produced from the same clone of Pinot Noir and covering 352 Mbp. On the basis of contigs containing two or more genetically mapped markers, region-dependent estimations of physical and recombinational distances are presented. The markers used in this study include 118 SSRs common to an integrated map derived from five segregating populations of V. vinifera. The positions of these SSR markers in the two maps are conserved across all Vitis linkage groups. The addition of SNP-based markers introduces polymorphisms that are easy to database, are useful for evolutionary studies, and significantly increase the density of the map. The map provides the most comprehensive view of the Vitis genome reported to date and will be relevant for future studies on structural and functional genomics and genetic improvement.     

Heterogeneity of the Y chromosome in Afro-Brazilian populations

Sixteen biallelic markers (SRY10831a, SRY10831b, SRY4064, SRY2627, 92R7, P2, P3, M34, M9, M3, M2, YAP, M60, M89, M213, M216) located in the nonrecombinant region of the Y chromosome were analyzed in 209 individuals belonging to six Brazilian populations: four Afro-Brazilian populations, one population of white European descendants, and one population of Japanese descendants. The results showed that most of the Y chromosomes of the Afro-Brazilians were from sub-Saharan Africa and that the proportion of Y chromosomes of European origin was greater than that of Y chromosomes of Amerindian origin. No typical African or Amerindian haplogroup was detected among Japanese individuals, and only one white individual showed a typical African haplogroup. Haplogroup P-92R7, which is highly frequent in the Portuguese and Italian populations, was the most frequent among whites (54%), and haplogroup K-M9, which shows wide geographic distribution and is absent in Africa, was the most frequent among Japanese individuals (65.6%). The two semi-isolated Afro-Brazilian populations showed the highest and the lowest genetic diversity, respectively. These differences probably reflect the effect of greater or smaller gene flow between a small isolated group and other populations. These findings show that the process of admixture does not occur homogeneously, with a tendency toward preferential marriages within the ethnic group and a clear direction in unions between European men and Amerindian or African women in the past. The results agree with historical and social data about the formation of the Brazilian population and reveal some of the factors that contribute to its heterogeneity.     

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Electro-Acoustic Behavior of the Mitotic Spindle: A Semi-Classical Coarse-Grained Model

The regulation of chromosome separation during mitosis is not fully understood yet. Microtubules forming mitotic spindles are targets of treatment strategies which are aimed at (i) the triggering of the apoptosis or (ii) the interruption of uncontrolled cell division. Despite these facts, only few physical models relating to the dynamics of mitotic spindles exist up to now. In this paper, we present the first electromechanical model which enables calculation of the electromagnetic field coupled to acoustic vibrations of the mitotic spindle. This electromagnetic field originates from the electrical polarity of microtubules which form the mitotic spindle. The model is based on the approximation of resonantly vibrating microtubules by a network of oscillating electric dipoles. Our computational results predict the existence of a rapidly changing electric field which is generated by either driven or endogenous vibrations of the mitotic spindle. For certain values of parameters, the intensity of the electric field and its gradient reach values which may exert a not-inconsiderable force on chromosomes which are aligned in the spindle midzone. Our model may describe possible mechanisms of the effects of ultra-short electrical and mechanical pulses on dividing cells—a strategy used in novel methods for cancer treatment.     

Carbohydrate Depletion in Roots and Leaves of Salt-Stressed Potted <Emphasis Type="Italic">Citrus clementina</Emphasis> L.

In citrus, damage produced by salinity is mostly due to toxic ion accumulation, since this salt-sensitive crop adjusts osmotically with high efficiency. In spite of this observation, the putative role of sugars as osmolites under salinity remains unknown. In this work, we have studied carbohydrate contents (total hexoses, sucrose and starch) in leaves and roots of citrus grown under increasing salinity. The experimental system was characterized through the analyses of several parameters known to be strongly affected by salinity in citrus, such as chloride accumulation, photosynthetic rate, ethylene production and leaf abscission. Three-year-old plants of the Clementina de Nules cultivar grafted on Carrizo citrange rootstock were watered with three different levels of salinity (NaCl was added to the watering solutions to achieve final concentrations of 30, 60 and 90 mM). Data indicate that salt stress caused an accumulation of chloride ions in a way proportional to the external increase in NaCl. The adverse conditions reduced CO₂ assimilation, increased ethylene production and triggered abscission of the injured leaves. Data also show that salinity induced progressive depletions of carbohydrates in leaves and roots of citrus plants. This observation clearly indicates that sugar accumulation is not a main component of the osmotic adjustment machinery in citrus.     

Boat anchoring on Posidonia oceanica beds in a marine protected area (Italy, western Mediterranean): effect of anchor types in different anchoring stages

Seagrasses worldwide are noted for suffering from mechanical damage caused by boat anchoring. This is particularly so in sites highly frequented by boaters (marine protected areas or coastal urbanised areas). In the last decades, different strategies have been put into practice to reduce such impacts on seagrasses (i.e. by anchoring bans or by deploying boat moorings). More recently, in consideration that few marine protected area (MPA) management bodies or local administrations have the resources to enforce their anchorage regulations, the self-regulatory approach based on education and information of boaters has been preferred in several cases. At present, however, very little is known on the correct anchoring practices to ensure the safeguarding of seagrasses. The aim of the present study was to experimentally quantify in the field the damage caused to Posidonia oceanica shoot density by anchoring. A multifactorial experiment was designed to test whether the damage is dependent on (1) different anchor types (Hall, Danforth and Folding grapnel), (2) the use of a chain vs. a rope, (3) the three anchoring stages (anchor fall, dragging/lock-in and weighing), and finally (4) whether the pattern is consistent among different locations of the meadow.As expected, the three anchor types employed in the present study differed in the levels of damage inflicted on the P. oceanica meadows of the Ustica Island MPA. In particular, the use of the Hall type anchor seems to be preferable to minimise this impact in comparison with the other two anchor types. Moreover, the effect on the meadow of the three anchor types is greatly dependent on the anchoring stage. These results confirm that the weighing stage is the critical stage of the anchoring process. The number of damaged shoots of P. oceanica was not affected by the presence of the chain. These patterns were consistent between locations.In the long term, even anchoring on P. oceanica by small boats using low-impact anchors may potentially have detrimental consequences. For this reason, we suggest that in vulnerable sites, it is preferable to implement an educational program based on information of boaters on correct anchoring practices and anchor typology to use, rather than adopting strong restrictions to boat anchoring or deploying mooring buoys. Although the use of these management strategies is still recommended in the case of anchorage frequented by bigger vessels using heavier anchors and chains.     

Structural differences in the hinge region of the glycoprotein hormone receptors: evidence from the sulfated tyrosine residues

Tyrosine sulfation is a late posttranslational modification of proteins that takes place in the Golgi network. In the past few years, this process has been identified as an important modulator of protein-protein interactions. Sulfated tyrosine residues have recently been identified in the C-terminal, so-called hinge region of the ectodomain of glycoprotein hormone receptors [TSH, LH/chorionic gonadotropin (CG), and FSH receptors] and were shown to play an important role in the interaction with their natural ligands. The position of two sulfated tyrosine residues in a Y-D/E-Y motif appears perfectly conserved in the alignment of TSH and LH receptors from different species, and site-directed mutagenesis experiments demonstrated that sulfation of the first residue of this motif was responsible for the functional effect on hormone binding. In contrast, the corresponding motif is not conserved in the FSH receptor, in which the first tyrosine residue is missing: the Y-D/E-Y motif is replaced by F(333)DY(335). We extend here our previous observation that, in this case, it is sulfation of the second sole tyrosine residue in the motif that is functionally important. An LH/CG receptor harboring an F(331)DY(333) motif (i.e. displaying decreased sensitivity to human CG) was used as a backbone in which short portions of the FSH receptor were substituted. Segments from the FSH receptor capable of restoring sensitivity to human CG were identified by transfection of the chimeras in COS-7 cells. These experiments identified key amino acid residues in the hinge region of the FSH receptor associated with the functional role of the second sulfated tyrosine residue in a Y-D/E-Y motif, allowing for efficient hormone binding. The experiments represent strong evidence that structural differences in the hinge regions of FSH and LH/CG receptors play a significant role in hormone-receptor-specific recognition.