Intermediate Stage Of Maturation Research Articles

Cytotoxicity of Quantum Dots in Receptor-Mediated Endocytic and Pinocytic Pathways in Yeast.

Despite the promising applications of the use of quantum dots (QDs) in the biomedical field, the long-lasting effects of QDs on the cell remain poorly understood. To comprehend the mechanisms underlying the toxic effects of QDs in yeast, we characterized defects associated with receptor-mediated endocytosis (RME) as well as pinocytosis using Saccharomyces cerevisiae as a model in the presence of cadmium selenide/zinc sulfide (CdSe/ZnS) QDs. Our findings revealed that QDs led to an inefficient RME at the early, intermediate, and late stages of endocytic patch maturation at the endocytic site, with the prolonged lifespan of GFP fused yeast fimbrin (Sac6-GFP), a late marker of endocytosis. The transit of FM1-43, a lipophilic dye from the plasma membrane to the vacuole, was severely retarded in the presence of QDs. Finally, QDs caused an accumulation of monomeric red fluorescent protein fused carbamoyl phosphate synthetase 1 (mRFP-Cps1), a vacuolar lumen marker in the vacuole. In summary, the present study provides novel insights into the possible impact of CdSe/ZnS QDs on the endocytic machinery, enabling a deeper comprehension of QD toxicity.

Female age at maturity and fecundity in Atlantic Striped Bass

AbstractObjectiveFemale age at maturity and fecundity for the Atlantic stock of Striped Bass Morone saxatilis were estimated using histological methods and image analysis.MethodsOvaries were obtained from surveys encompassing the spring spawning season (March–July; n = 343), primarily from the Chesapeake Bay, and in the fall months (September–December; n = 85), primarily from the Atlantic coast. Histological examination of oocytes revealed some Striped Bass in intermediate stages of maturation during the spawning season. These individuals were identified as undergoing pubertal development, defined as the transition from the juvenile stage to first sexual maturity. Pubertal development was characterized by ovaries containing a population of enlarged, lipid‐filled oocytes but noticeably lacking vitellogenin‐derived yolk globules during the spawning season, and those ovaries were classified as immature. Toward the end of the spawning season, increasing proportions of Striped Bass with unspawned ovaries and oocytes undergoing total atresia were observed.ResultThe female age and length at 50% maturity in Atlantic Striped Bass based on spring samples were 5.5 years and 609 mm total length, respectively. Fecundity was determined gravimetrically via image analysis of ovarian tissue samples from spawning capable individuals (n = 67). Potential annual fecundity was found to exhibit hyperallometric scaling with respect to body size. Specifically, the scaling exponent for the length–fecundity relationship was 3.24, which was greater than the scaling exponent of 3.05 for the length–body mass relationship. This indicates that large females possess a disproportionately greater reproductive capacity with respect to body mass than the equivalent biomass of smaller females.ConclusionCompared with previous studies spanning over a half‐century, age at 50% maturity and fecundity were found to be relatively invariant, although variation found between contemporary studies may represent methodological and interpretive differences. Reproductive‐related life history traits of female Atlantic Striped Bass are apparently robust to long‐term decadal changes in fishing intensity, stock size, habitat alterations, and environmental conditions.

3D-reconstructions of zygospores in Zygnema vaginatum (Charophyta) reveal details of cell wall formation, suggesting adaptations to extreme habitats.

The streptophyte green algal class Zygnematophyceae is the immediate sister lineage to land plants. Their special form of sexual reproduction via conjugation might have played a key role during terrestrialization. Thus, studying Zygnematophyceae and conjugation is crucial for understanding the conquest of land. Moreover, sexual reproduction features are important for species determination. We present a phylogenetic analysis of a field-sampled Zygnema strain and analyze its conjugation process and zygospore morphology, both at the micro- and nanoscale, including 3D-reconstructions of the zygospore architecture. Vegetative filament size (26.18 ± 1.07 μm) and reproductive features allowed morphological determination of Zygnema vaginatum, which was combined with molecular analyses based on rbcL sequencing. Transmission electron microscopy (TEM) depicted a thin cell wall in young zygospores, while mature cells exhibited a tripartite wall, including a massive and sculptured mesospore. During development, cytological reorganizations were visualized by focused ion beam scanning electron microscopy (FIB-SEM). Pyrenoids were reorganized, and the gyroid cubic central thylakoid membranes, as well as the surrounding starch granules, degraded (starch granule volume: 3.58 ± 2.35 μm3 in young cells; 0.68 ± 0.74 μm3 at an intermediate stage of zygospore maturation). Additionally, lipid droplets (LDs) changed drastically in shape and abundance during zygospore maturation (LD/cell volume: 11.77% in young cells; 8.79% in intermediate cells, 19.45% in old cells). In summary, we provide the first TEM images and 3D-reconstructions of Zygnema zygospores, giving insights into the physiological processes involved in their maturation. These observations help to understand mechanisms that facilitated the transition from water to land in Zygnematophyceae.

Molecular dynamics simulation study on Bacillus subtilis EngA: the presence of Mg2+ at the active-sites promotes the functionally important conformation

EngA, a GTPase contains two GTP binding domains [GD1, GD2], and the C-terminal KH domain shown to be involved in the later stages of ribosome maturation. Association of EngA to the ribosomal subunit in the intermediate stage of maturation is essential for complete ribosome maturation. However, this association was shown to be dependent on the nucleotide bound combinations. This nucleotide dependent association tendency is attributed to the conformational changes that occur among different nucleotide bound combinations. Therefore, to explore the conformational changes, all-atom molecular dynamics simulations for Bacillus subtilis EngA in different nucleotide bound combinations along with the presence or absence of Mg2+ in the active-sites were carried out. The presence of Mg2+ along with the bound nucleotide at the GD2 active-site dictates the GD2-Sw-II mobility, but the GD1-Sw-II mobility has not shown any nucleotide or Mg2+ dependent movement. However, the GD1-Sw-II secondary conformations are shown to be influenced by the GD2 nucleotide bound state. This allosteric connection between the GD2 active-site and the GD1-Sw-II is also observed through the dynamic network analysis. Further, the exploration of the GD1-KH interface interactions exhibited a more attractive tendency when GD1 is bound to GTP-Mg2+. In addition, the presence of Mg2+ stabilizes active-site water and also increases the distances between the α- and γ- phosphates of the bound GTP. Curiously, three water molecules in the GD1 active-site and only one water molecule in the GD2 active-site are stabilized. This indicates that the probability of GTP hydrolysis is more in GD1 compared to GD2. Communicated by Ramaswamy H. Sarma

Sorting nexin 24 is required for α-granule biogenesis and cargo delivery in megakaryocytes.

Germline defects affecting the DNA-binding domain of the transcription factor FLI1 are associated with a bleeding disorder that is characterized by the presence of large, fused α-granules in platelets. We investigated whether the genes showing abnormal expression in FLI1-deficient platelets could be involved in platelet α-granule biogenesis by undertaking transcriptome analysis of control platelets and platelets harboring a DNA-binding variant of FLI1. Our analysis identified 2,276 transcripts that were differentially expressed in FLI1-deficient platelets. Functional annotation clustering of the coding transcripts revealed significant enrichment for gene annotations relating to protein transport, and identified Sorting nexin 24 (SNX24) as a candidate for further investigation. Using an induced pluripotent stem cell-derived megakaryocyte model, SNX24 expression was found to be increased during the early stages of megakaryocyte differentiation and downregulated during proplatelet formation, indicating tight regulatory control during megakaryopoiesis. CRISPR-Cas9 mediated knockout (KO) of SNX24 led to decreased expression of immature megakaryocyte markers, CD41 and CD61, and increased expression of the mature megakaryocyte marker CD42b (P=0.0001), without affecting megakaryocyte polyploidisation, or proplatelet formation. Electron microscopic analysis revealed an increase in empty membrane-bound organelles in SNX24 KO megakaryocytes, a reduction in α-granules and an absence of immature and mature multivesicular bodies, consistent with a defect in the intermediate stage of α-granule maturation. Co-localization studies showed that SNX24 associates with each compartment of α-granule maturation. Reduced expression of CD62P and VWF was observed in SNX24 KO megakaryocytes. We conclude that SNX24 is required for α-granule biogenesis and intracellular trafficking of α-granule cargo within megakaryocytes.

Phenolic Composition and Biological Properties of Cynara cardunculus L. var. altilis Petioles: Influence of the Maturity Stage

Hydroethanolic extracts of cardoon petioles collected at sixteen growth stages (P1–P16) were characterized in terms of their phenolic composition and bioactive potential (antioxidant, cytotoxic, anti-inflammatory, and antimicrobial activities). Fifteen phenolic compounds were tentatively identified (i.e., ten phenolic acids and five flavonoid glycosides); the main compounds were 5-O-caffeoylquinic and 1,5-di-O-caffeoylquinic acids. Samples collected at early maturity (P1–P4) presented a weak positive correlation between the higher content in polyphenols (P3: 101-mg/g extract) and better inhibition capacity against thiobarbituric acid reactive substance formation (TBARS; P3: IC50 = 5.0 µg/mL). Samples at intermediate maturation stages (P9) presented higher cytotoxic and anti-inflammatory potential. Moreover, immature petioles showed greater antihemolytic (OxHLIA; P4: IC50 = 65 and 180 µg/mL for Δt of 60 and 120 min, respectively) and antibacterial activity. The antifungal activity varied depending on the maturation stage and the fungi strain. In conclusion, the maturation stage may greatly affect the polyphenols composition and content and the bioactive potential of cardoon petioles.

LIN28B and ZBTB8B Genes Are Highly Expressed in Vitro in a CD34⁺ Cells Subpopulation of β-Thalassemia Major Patients and May be Involved in Increased HbF Production

Patients with homozygous beta thalassemia and the β⁰β⁰ genotype produce almost only fetal hemoglobin and a small proportion of hemoglobin A2. However, in the bone marrow of these patients there are descriptions of two different subpopulations of erythroblasts, one with high production of HbF (High HbF cells) and another with low production of HbF (Low HbF cells). The High F cells are probably those that survive through erythropoiesis and originate the viable red blood cells. The molecular mechanisms that are responsible for this high production of HbF are not clear. Therefore, the central aim of our work was to study, in cultures of CD34⁺ cells from β⁰β⁰ patients, differentiated to erythrocytes in vitro, the molecular characteristics of cells related to high production of HbF (High HbF), compared to those with low production of HbF (Low HbF). For this purpose, peripheral blood was collected from patients and CD34⁺ cells were identified, and further differentiated in vitro and then isolated by flow cytometry (FACS Aria). After separating the High HbF and Low HbF groups, the cells were morphologically evaluated by optical and confocal microscopy and image cytometry. Next RNA was extracted from the pool of isolated cells and the RNA sequencing assay (RNAseq) was performed, so that it was possible to analyze the transcripts that were differentially expressed between each of the groups. Finally, we validated the sequencing data by real-time PCR. The sorting was performed on the 10th day of cell culture, when the cells presented a double labeling profile for anti-transferrin (CD71) and anti-glycophorin (CD235) antibodies, the mean intensity and fluorescence (MIF) of the markers was 1058 and 1365 for CD71 in healthy control and β-thalassemia groups, respectively, and for CD235 was 430 in controls and 516 for patients. The cells were in an intermediate stage of maturation. After the isolation of sub-populations of High HbF and Low HbF cells, confocal microscopy indicated a clear difference in the intracellular HbF levels of the analyzed cells, confirming that the sorting by cytometry had occurred as desired. Imaging cytometry analysis revealed a MIF of 7.8 x 10⁵ for High HbF cells and 1.7 x 10⁵ for Low HbF cells, when we assessed the cell pools and when single cells were studied, these values ranged from 2.2 x 10⁵ for Low HbF cells to 1.9 x 10⁶ of mean intensity and fluorescence for High cells. After all data generated by RNAseq were filtered, we obtained a list of 16 enriched and differentially-expressed genes between High HbF and Low HbF cells. From these genes, it was possible to identify two that were apparently associated with the increased production of fetal hemoglobin, the genes ZBTB8B and LIN28B. ZBTB8B is a protein-coding gene with a zinc finger domain and may be involved in transcriptional regulation, it was included in our analyses since this gene is part of the same family as ZBTB7A, an important transcription factor that represses several genes involved in cell differentiation and proliferation and that has already been described as a regulator of HbF production. The LIN28B gene encodes an RNA-binding protein, which is described as a possible regulator of HbF levels during fetal development, by increasing the expression of gamma globin, culminating in the increase of intracellular levels of fetal hemoglobin through a direct action on the let-7 microRNA family and BCL11A gene.These results not only contribute to a better understanding of the mechanisms of gene regulation involved in the production of HbF, but indicate a potential new therapeutic approach to increase the production of HbF in hemoglobinopathies. DisclosuresCosta: Novartis: Consultancy.

High Throughput Single-Cell Simultaneous Genotyping and Chromatin Accessibility Reveals Genotype to Phenotype Relationship in Human Myeloproliferation

In hematopoiesis, changes in chromatin accessibility define priming and commitment of hematopoietic precursors towards cellular fates. In turn, somatic mutations in hematopoietic stem and progenitor cells (HSPCs) drive the onset and progression of myeloid disorders, such as myeloproliferative neoplasms (MPNs), and reshape differentiation topologies.To chart how somatic mutations disrupt the epigenetic landscape in human clonal outgrowths, we developed Genotyping of Targeted loci with Chromatin Accessibility (GoT-ChA), linking genotypes to chromatin accessibility across thousands of single cells. Crucially, GoT-ChA captures genotypes directly from genomic DNA (Fig. 1a), and thus independently of the genomic location or expression of the target. We tested GoT-ChA via cell line mixing studies targeting either TP53 R248Q (Fig. 1b), or JAK2 V617F (Fig. 1c), assigning cell line identity solely based on chromatin accessibility. Notably, GoT-ChA resulted in genotyping of 54% of cells with 96.9% accuracy for TP53 R248Q and 60% of cells with 99.7% accuracy for JAK2 V617F. These data show the transformative advantage of targeting DNA directly, as prior high-throughput droplet methods to target the lowly expressed JAK2 via cDNA resulted in genotyping of only ~7% of cells (Nam et al, Nature, 2019).Next, we applied GoT-ChA to CD34 + cells from JAK2 V617F-mutant myelofibrosis (MF) samples (Fig. 1d). We clustered cells based on chromatin profiles, revealing the expected cell populations in hematopoiesis (Fig. 1d-e), and then projected genotyping status onto the differentiation map (Fig. 1f). In further validation of genotyping accuracy, copy number inference showed a sample that contained a partial deletion of chromosome 20 (Fig. 1g) concordant with our genotyping. Furthermore, GoT-ChA can be integrated with recent protocols to allow for high mitochondrial genome coverage (Lareau et al, Nature Biotechnology, 2020). We observed mitochondrial mutations that were highly concordant with JAK2 V617F (Fig. 1h), allowing genotyping of >85% of cells.Within MPN samples, wildtype (WT) and mutated (MUT) cells were intermingled across the differentiation topology. Nonetheless, we observed an increase in the mutant fraction within erythroid progenitors (EP; Fig. 1i). Moreover, pseudo-temporal ordering of chromatin accessibility revealed that the mutant cell fraction increased along erythroid or megakaryocyte differentiation in untreated MPN (Fig 1j), in line with clinical phenotypes.Chromatin accessibility profiles can provide clues to the underlying regulatory network through transcription factor (TF) motif accessibility. Uniquely, GoT-ChA enables de novo differential motif accessibility, directly comparing WT and MUT cells co-existing within the same bone marrow. Mutant HSPCs showed increased motif accessibility (FDR < 0.05) for TFs associated with erythropoiesis (Fig. 1k), suggestive of increased erythroid priming. Within EP clusters, we observed increased motif accessibility of STAT5A and STAT5B, downstream targets of JAK2 (Fig. 1l-m). These data demonstrated a cell-type specific effect of the JAK2 V617F mutation.Ruxolitinib is a frontline JAK1/2 inhibitor for MF. Despite improvements in quality of life, ruxolitinib does not clearly target the MPN clone or prevent progression of disease. In ruxolitinib-treated patients the MUT cell fraction was uniformly distributed along the differentiation (Fig.1i-j), demonstrating an abrogation of the fitness advantage of JAK2 V617F in committed progenitors, but not in HSPCs. Consistently, STAT5A motif accessibility remained increased in MUT cells at intermediate stages of erythroid maturation but decreased to similar levels as WT cells at later stages (Fig. 1n-o).Overall, GoT-ChA radically expands the single-cell multi-omics toolkit and obviates limiting dependencies on target gene transcription, allowing high throughput somatic genotype-to-phenotype mapping. Applied to JAK2 V617F-mutated MPN, GoT-ChA uncovered a cell-type specific fitness advantage with erythroid commitment, that was reversed upon JAK2 inhibitor treatment. The reshaping of the differentiation topography traced back to differential transcription factor activity driving uncommitted vs. committed JAK2 V617F progenitors. Thus, single-cell multi-omics with GoT-ChA enables to chart the epigenetic underpinnings of hematopoietic clonal outgrowth. [Display omitted] DisclosuresMimitou: Immunai: Current Employment. Hoffman: Protagonist Therapeutics, Inc.: Consultancy; Kartos Therapeutics, Inc.: Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding. Abdel-Wahab: Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Merck: Consultancy; Foundation Medicine Inc: Consultancy; H3B Biomedicine: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees. Smibert: Immunai: Current Employment.

Super-resolution imaging reveals cytoskeleton-dependent organelle rearrangement within platelets at intermediate stages of maturation

A steady supply of platelets maintains their levels in the blood, and this is achieved by the generation of progeny from platelet intermediates. Using systematic super-resolution microscopy, we examine the ultrastructural organization of various organelles in different platelet intermediates to understand the mechanism of organelle redistribution and sorting in platelet intermediate maturation as the early step of platelet progeny production. We observe the dynamic interconversion between the intermediates and find that microtubules are responsible for controlling the overall shape of platelet intermediates. Super-resolution images show that most of the organelles are located near the cell periphery in oval preplatelets and confined to the bulbous tips in proplatelets. We also find that the distribution of the dense tubular system and α granules is regulated by actin, whereas that of mitochondria and dense granules is governed by microtubules. Altogether, our results call for a reassessment of organelle redistribution in platelet intermediates.

Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates.

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and biodegradability of protein and peptide building blocks, developing sustainable and green bottom up approaches for finely tuning the material properties still remains a challenge. Here, we present an experimental study on the formation of protein microparticles in the form of particulates from the protein α-lactalbumin using bulk mixing in water solution and high temperature. Once formed, the structure and stability of these spherical protein condensates change upon further thermal incubation while the size of aggregates does not significantly increase. Combining advanced microscopy and spectroscopy methods, we prove that this process, named maturation, is characterized by a gradual increase of amyloid-like structure in protein particulates, an enhancement in surface roughness and in molecular compactness, providing a higher stability and resistance of the structure in acidic environments. When dissolved at pH 2, early stage particulates disassemble into a homogeneous population of small oligomers, while the late stage particulates remain unaffected. Particulates at the intermediate stage of maturation partially disassemble into a heterogeneous population of fragments. Importantly, differently matured microparticles show different features when loading a model lipophilic molecule. Our findings suggest conformational transitions localized at the interface as a key step in the maturation of amyloid protein condensates, promoting this phenomenon as an intrinsic knob to tailor the properties of protein microparticles formed via bulk mixing in aqueous solution. This provides a simple and sustainable platform for the design and realization of protein microparticles for tailored applications.

FUZZY LOGIC AND CLUSTER ANALYSIS IN THE QUALITY OF THE BEVERAGE FROM CONILON COFFEE

The quality of coffee beverages has been under study due to the demand of the consumer market for both arabica and conilon coffee. The aim of this work was to study beverage quality from different clones by means of sensory analysis, in 13 clones of the variety Victoria INCAPER 8142 produced at average altitudes of 100.0 m and 528.0 m and with the cherry fruits processed by natural drying or depulping. Fuzzy classification was adopted for the global scores obtained in the sensory analysis, on a scale of 70.0 to 100.0 points, with the Euclidean distance from the cluster analysis being used to define the dissimilarity between the global fuzzified scores for the different clones at the two altitudes and for the two methods of processing the fruit. Clones C4 and C10, at the intermediate maturation stage, presented a mean global score (GS) of 85.0 points for the coffee produced at the altitude of 528.0 m and for the depulped fruit, corresponding to a degree of fuzzy pertinence (FI) of 0.50, and being classified as fine coffee. These same clones presented dissimilarities in the beverage produced by the depulped fruit, with better quality for the coffee at the higher altitude. The fuzzy classification taken together with the cluster analysis to interpret the mean global scores (GS) in the sensory analysis of the beverage for the different treatments under study identified variation in beverage quality

Maturation-related changes in phytochemicals and biological activities of the Brazilian pepper tree (Schinus terebinthifolius Raddi) fruits

Changes in fatty acid profile, lipid quality, essential oil composition, total phenolic (TPC) and flavonoid (TFC) contents, radical scavenging, antibacterial and insecticidal activities were studied during the maturation of Schinus terebinthifolius Raddi fruits. Total lipids showed their maximum at the earlier and intermediate stage (green and half-red) of maturation, whereas, the highest levels of the main components linoleic and oleic acids were found at the late stage (red). Mature fruits were also distinguished by their high lipid quality as revealed by the lower values of n-3/n-6 ratio, calculated oxidizability (Cox), oxidative susceptibility (OS), atherogenic index (AI) and thromobogenic index (TI). The contents of monoterpenes decreased while those of sesquiterpenes increased during maturation. With the exception of α-pinene which dropped remarkably from immature to mature fruits, the content of the other main components (α-phellandrene, β-phellandrene and limonene) showed irregular variations. The TPC in intact and hydrodistilled fruits was higher at the last stage of maturation, while the maximum of TFC was recorded in the intermediate stage. Concomitantly, extract from residual hydrodistilled fruits was found as the most potent radical scavengers, whereas, the oils were found as the least scavengers. Nevertheless, they showed an appreciable antibacterial activity against Enterococcus feacium and Streptococcus agalactiae and exert a strong insecticidal activity against Spodoptera littoralis and Phthorimaea operculella. Collectively, these results suggest that the fruits of S. terebinthifolius could be considered as consolidated source of lipid and essential fatty acids, which may provide some health benefits to consumers. They also showed that the essential oils of S. terebinthifolius fruits can potentially be used in S. littoralis and P. operculella control in association with integrated pest management practices. Moreover, the use of extracts from intact or hydrodistilled residual fruits for the development of functional ingredients and the production of bio-based preservatives is encouraged.

Combined Effects of Temperature and Photoperiod on the Conditioning of the Flat Oyster (Ostrea edulis[Linnaeus, 1758]) in Winter

The flat oyster Ostrea edulis (Linnaeus, 1758) is a commercially valuable species in Europe. The production of O. edulis natural spat has decreased in the past, and it is insufficient to meet demand. Thus, the importance of hatcheries in the production of oyster spat is increasing. The conditioning of O. edulis broodstock may be improved by manipulating several external factors such as temperature, food supply, and photoperiod; however, few studies have reported the effect of these parameters on O. edulis gametogenesis and conditioning. In this report, the combined effects of temperature and photoperiod on broodstock conditioning of O. edulis in winter were studied to provide a protocol that improves flat oyster conditioning. After a resting period in autumn, in winter, oysters' gonads are restructured, and the natural process of gonadal maturation is initiated. Oyster gonads showed follicles in intermediate maturation stages, with the presence of both male and female germinal cells in the same follicles but at different stages of maturation. The combined effect of temperature (TG14-18) and photoperiod on oyster conditioning in winter was evaluated by using direct indexes. Oysters conditioned with a gradient of temperature and a gradient of daylight (PG8-16), a P16 regime or under natural environmental conditions began to spawn 4 wk after the beginning of conditioning. Oysters treated with a P8 regime needed a week more to start spawning. Spawning was a continuous process that lasted for 7 wk. When the total amount of released viable larva was compared, the positive influence of photoperiod was much more evident. Total larval production was two or three times higher in the oysters treated with a gradient of daylight (PG8-16). In summary, using the conditions of temperature and photoperiod described in this study (TG14-18 and PG8-16), it was possible to improve flat oyster conditioning in winter, producing an important amount of viable larvae over a period of 2 mo.

The occurrence and distribution of phenylnaphthalenes, terphenyls and quaterphenyls in selected lacustrine shales and related oils in China

Phenylnaphthalenes (PhNs), terphenyls (TrPs), quaterphenyls (QtPs) and triphenylbenzenes were identified by the use of authentic standards in GC–MS analysis. Phenyl derivatives of fused ring polycyclic aromatic hydrocarbons (PhPAHs) and polyphenyls (PPhs) were observed in selected lacustrine shales and related oils from the Tarim, Liaohe and Bebuwan Basins in China. All isomers of phenylnaphthalene and terphenyl were observed in these oils. The more stable 2-PhN and m-Trp, p-TrP isomers are predominant among phenylnaphthalenes and terphenyls, respectively. The effect of maturity on the distribution patterns of phenylnaphthalenes and terphenyls in a set of lacustrine shales from the Liaohe Basin, East China was investigated. The depth trends of three currently used maturity indicators were investigated. Analysis was based on the relative abundance of phenylnaphthalene and terphenyl isomers, namely PhNR (2-PhN/1-PhN), TrP1 (p-TrP/o-TrP) and TrP2 ((m-TrP+p-TrP)/o-TrP) in lacustrine shales from well S202 in the Liaohe Basin, East China. The indicators increase slightly with relatively great deviations within low to intermediate maturation stages and then increase abruptly in high maturity stages. Therefore, they are not suitable maturity indicators for lacustrine source rocks in this study. An alternative thermal maturity indicator, defined as p-TrP/m-TrP was proposed. It exhibits a uniform increase with increasing maturity and is probably a more suitable indicator for use in maturity assessment in lacustrine source rocks and related oils. This study broadens the current understanding of the occurrence and distribution of PhPAHs and PPhs in sedimentary organic matter of various origins.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10−2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway.

NUTRITIONAL STATUS OF CHILDREN AND ADOLESCENTS: FACTORS ASSOCIATED TO OVERWEIGHT AND FAT ACCUMULATION

Objective: To estimate prevalence and identify factors associated with the nutritional status of individuals aged 8 to 17 years old in schools located at 3 administrative regions in the city of Vitoria, Brazil. Methods: The nutritional status of 400 students was assessed based on a cross-sectional study. Association between overweight and fat accumulation around waist was tested considering the variables: sexual maturaten stage, income level, number of siblings, maternal Body Mass Index (BMI) and physical activity. Individuals considered stunting had height-for-age 25kg/m 2 , and high income level. Fat accumulation around the waist was associated to the following variables: maternal BMI >25kg/m 2 , high income level, age 28 hours/week. Conclusions : Low prevalence of nutritional deficits and high prevalence of overweight and fat accumulation around the waist were found. Higher prevalence rate occurred among individuals showing the following characteristics: higher income level; overweight mother; within initial and intermediate stages of sexual maturation; bellow 14 years old; sedentary.

Antioxidant activity and phenolic composition of Cv. Cobrançosa olives affected through the maturation process

Maturation stage is a critical feature to obtain high quality olive products, with maximized bioactivity. In this study, phenolic composition and antioxidant activity of Cv. Cobrançosa through the maturation process were evaluated. The phenolic profile was assessed by HPLC/DAD, and antioxidant activity was studied through its reducing power and free-radical scavenging activity.Total phenols varied from 34 to 1 g/kg, respectively, in the first and last sampling dates. Oleuropein, the main phenolic in the first stages of maturation, decreased drastically during ripening. At intermediate and high maturation stages hydroxytyrosol was the predominant phenol. Globally, the reducing capacity of Cv. Cobrançosa olive fruits decreased during the maturation process but its radical scavenging activity was only slightly altered. A principal components analysis corroborated the characteristic phenolic profile and changes experienced by the olive fruit during the maturation process. These results are important to maximize Cv. Cobrançosa olive products' quality and biological properties.

The role of androgen hormones in early follicular development.

Background. Although chronic hyperandrogenism, a typical feature of polycystic ovary syndrome, is often associated with disturbed reproductive performance, androgens have been shown to promote ovarian follicle growth in shorter exposures. Here, we review the main effects of androgens on the regulation of early folliculogenesis and the potential of their application in improving follicular in vitro growth. Review. Androgens may affect folliculogenesis directly via androgen receptors (ARs) or indirectly through aromatization to estrogen. ARs are highly expressed in the granulosa and theca cells of early stage follicles and slightly expressed in mature follicles. Short-term androgen exposure augments FSH receptor expression in the granulosa cells of developing follicles and enhances the FSH-induced cAMP formation necessary for the transcription of genes involved in the control of follicular cell proliferation and differentiation. AR activation also increases insulin-like growth factor (IGF-1) and its receptor gene expression in the granulosa and theca cells of growing follicles and in the oocytes of primordial follicles, thus facilitating IGF-1 actions in both follicular recruitment and subsequent development. Conclusion. During the early and intermediate stages of follicular maturation, locally produced androgens facilitate the transition of follicles from the dormant to the growing pool as well as their further development.

Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

Asparaginyl endopeptidase (AEP) is an endo/lysosomal cysteine endopeptidase with a preference for an asparagine residue at the P1 site and plays an important role in the maturation of toll-like receptors 3/7/9. AEP is known to undergo autoproteolytic maturation at acidic pH for catalytic activation. Here, we describe crystal structures of the AEP proenzyme and the mature forms of AEP. Structural comparisons between AEP and caspases revealed similarities in the composition of key residues and in the catalytic mechanism. Mutagenesis studies identified N44, R46, H150, E189, C191, S217/S218 and D233 as residues that are essential for the cleavage of the peptide substrate. During maturation, autoproteolytic cleavage of AEP's cap domain opens up access to the active site on the core domain. Unexpectedly, an intermediate autoproteolytic maturation stage was discovered at approximately pH 4.5 in which the partially activated AEP could be reversed back to its proenzyme form. This unique feature was confirmed by the crystal structure of AEPpH4.5 (AEP was matured at pH 4.5 and crystallized at pH 8.5), in which the broken peptide bonds were religated and the structure was transformed back to its proenzyme form. Additionally, the AEP inhibitor cystatin C could be digested by the fully activated AEP, but could not be digested by activated cathepsins. Thus, we demonstrate for the first time that cystatins may regulate the activity of AEP through substrate competition for the active site.