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[en] Human osteoarthritic chondrocytes (hOACs) are characterized by their “dedifferentiated” and catabolic phenotype and lack the ability for restoring their inherent functions by themselves. Here we investigated whether extrinsically supplemented mechanical signal via compression loading would affect the phenotype of hOACs. Specifically, we applied cyclic compression loading on cultured hOACs-collagen constructs and measured the expression of the major chondrogenic factors, cell-matrix interaction molecules and matrix degradation enzymes. Dynamic compression loading stimulates the expression and nuclear localization of sox9 in hOACs and reduces the catabolic events via downregulated expression of collagenases. These results contribute to better understanding towards mechanoregulation of hOACs.
[en] Metabolic modeling provides the means to define metabolic processes at a systems level; however, genome-scale metabolic models often remain incomplete in their description of metabolic networks and may include reactions that are experimentally unverified. This shortcoming is exacerbated in reconstructed models of newly isolated algal species, as there may be little to no biochemical evidence available for the metabolism of such isolates. The phenotype microarray (PM) technology (Biolog, Hayward, CA, USA) provides an efficient, high-throughput method to functionally define cellular metabolic activities in response to a large array of entry metabolites. The platform can experimentally verify many of the unverified reactions in a network model as well as identify missing or new reactions in the reconstructed metabolic model. The PM technology has been used for metabolic phenotyping of non-photosynthetic bacteria and fungi, but it has not been reported for the phenotyping of microalgae. Here, we introduce the use of PM assays in a systematic way to the study of microalgae, applying it specifically to the green microalgal model species Chlamydomonas reinhardtii. The results obtained in this study validate a number of existing annotated metabolic reactions and identify a number of novel and unexpected metabolites. The obtained information was used to expand and refine the existing COBRA-based C. reinhardtii metabolic network model iRC1080. Over 254 reactions were added to the network, and the effects of these additions on flux distribution within the network are described. The novel reactions include the support of metabolism by a number of d-amino acids, l-dipeptides, and l-tripeptides as nitrogen sources, as well as support of cellular respiration by cysteamine-S-phosphate as a phosphorus source. The protocol developed here can be used as a foundation to functionally profile other microalgae such as known microalgae mutants and novel isolates.
[en] Studies on the adaptability of some soybean mutant lines at four locations. Eight soybean mutant lines and control (Orba variety) were tested for phenotypic, genotypic, environmental and adaptability values at four different locations during the dry season of 1985. According to Chang's formula, the mutant lines no. 63/PsJ, no. 71/PsJ, no. 82/PsJ, no. 91/PsJ, and no. 134/PsJ seem to have a high phenotypic value. While the other mutant no. 62/PsJ, no. 95/PsJ, no. 147/PsJ, and the orba variety seem to be low. All mutant lines and orba variety had the highest environmental value in polmas location but the lowest in the location of Bengkulu utara. The stability of genotypes were measured by Eberhard and Russel method. It was found that the mutant lines no. 62/PsJ, no. 91/PsJ, no. 95/PsJ, no. 147/PsJ, and orba variety could adapt better in infertile soil, while the others no. 63/PsJ, no. 71/PsJ, and no. 82/PsJ in fertile soil. The mutant lines no. 91/PsJ, no. 95/PsJ, no. 134/PsJ were relatively stable compared to the other mutants. (author). 5 refs
[en] In addition to the majority of T cells which carry the αβ T cell receptor (TCR) for antigen, a distinct subset of about 1–5% of human peripheral blood T cells expressing the γδ TCR contributes to immune responses to infection, tissue damage and cancer. T cells with the Vδ2+ TCR, usually paired with Vγ9, constitute the majority of these γδ T cells. Analogous to αβ T cells, they can be sorted into naive (CD27+CD45RA+), central memory (CD27+CD45RA−), effector memory (CD27−CD45RA−), and terminally-differentiated effector memory (CD27−CD45RA+) phenotypes. Here, we found that CD27−CD45RA+ γδ T cells can be further divided into two populations based on the level of expression of CD45RA: CD27−CD45RAint and CD27−CD45RAhi. Those with the CD27−CD45RAhi phenotype lack extensive proliferative capacity, while those with the CD27−CD45RAint phenotype can be easily expanded by culture with zoledronate and IL-2. These CD27-CD45RAhi potentially exhausted γδ T cells were found predominantly in cancer patients but also in healthy subjects. We conclude that γδ T cells can be divided into at least 5 subsets enabling discrimination of γδ T cells with poor proliferative capacity. It was one of our goals to predict the feasibility of γδ T cell expansion to sufficient amounts for adoptive immunotherapy without the necessity for conducting small-scale culture tests. Fulfilling the ≥1.5% criterion for γδ T cells with phenotypes other than CD27−CD45RAhi, may help avoid small-scale culture testing and shorten the preparation period for adoptive γδ T cells by 10 days, which may be beneficial for patients with advanced cancer. - Highlights: • CD27-CD45+ γδ T cells can be divided into two populations, CD27-CD45hi and CD27-CD45int. • CD27-CD45hi γδ T cells are detected in the peripheral blood of both cancer patients and healthy subjects. • CD27-CD45hi γδ T cells are distinct from so-called terminally differentiated CD27-CD45int γδ T cells. • CD27-CD45hi γδ T cells lack vigorous proliferative potential.
[en] Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB tumors and derived cell lines are phenotypically heterogeneous. Cell lines are classified by phenotype, each having distinct differentiation and tumorigenic properties. The neuroblastic phenotype is tumorigenic, has neuronal features and includes stem cells (I-cells) and neuronal cells (N-cells). The non-neuronal phenotype (S-cell) comprises cells that are non-tumorigenic with features of glial/smooth muscle precursor cells. This study identified miRNAs associated with each distinct cell phenotypes and investigated their role in regulating associated differentiation and tumorigenic properties. A miRNA microarray was performed on the three cell phenotypes and expression verified by qRT-PCR. miRNAs specific for certain cell phenotypes were modulated using miRNA inhibitors or stable transfection. Neuronal differentiation was induced by RA; non-neuronal differentiation by BrdU. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21, miR-221 and miR-335 are associated with the non-neuronal phenotype, whereas increased levels of miR-124 and miR-375 are exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1, known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover, miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Thus, miRNAs define distinct NB cell phenotypes. Increased levels of miR-21, miR-221 and miR-335 characterize the non-neuronal, non-malignant phenotype and miR-335 maintains the non-neuronal features possibly by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which, in turn, suppresses HuD. As tumor differentiation state is highly predictive of patient survival, the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer
[en] Chinese wheat cultivar Lantian 9 showed a stable high yield in the field. Together with its other desirable traits such as tolerance to cold, drought, leaf rust and stripe rust, made it a good source for wheat breading. In our preliminary test, Lantian 9 showed a typical adult resistance phenotype (susceptible at seedling stage but high resistance at adult stage) to most of the major Chinese leaf rust pathotypes. To clarify the adult-plant resistance (APR) genes in this cultivar, a population with 197 F2:3 lines was generated by crossing Lantian 9 with susceptible line Huixian Hong. The population was phenotyped in the field over three years (year 2012, 2013 and 2014) by a mixture of three leaf rust pathotypes (THTT, THTS and THTQ). A total of 1232 simple sequence repeat (SSR) markers were used to screen the parental lines. Polymorphic ones were further applied on the population. Linkage mapping analysis showed that one QTL from Lantian 9 was located on chromosome 2BS, which was relative stable among the data from the year 2012 and 2013 with phenotypic variations of 6.0% and 9.1%, respectively. Three other QTLs from Lantian 9 on chromosome 4BS, 3A and 1BL were detected. We also identified one QTL from Huixian Hong on chromosome 1BL. All these identified wheat leaf rust resistance QTLs with their closely linked molecular markers will greatly facilitate genetic improvement of wheat resistance to leaf rust in China. (author)
[en] The correlation of phenotypic outcomes with genetic variation and environmental factors is a core pursuit in biology and biomedicine. Numerous challenges impede our progress: patient phenotypes may not match known diseases, candidate variants may be in genes that have not been characterized, model organisms may not recapitulate human or veterinary diseases, filling evolutionary gaps is difficult, and many resources must be queried to find potentially significant genotype-phenotype associations. Nonhuman organisms have proven instrumental in revealing biological mechanisms. Advanced informatics tools can identify phenotypically relevant disease models in research and diagnostic contexts. Large-scale integration of model organism and clinical research data can provide a breadth of knowledge not available from individual sources and can provide contextualization of data back to these sources. The Monarch Initiative (monarchinitiative.org) is a collaborative, open science effort that aims to semantically integrate genotype-phenotype data from many species and sources in order to support precision medicine, disease modeling, and mechanistic exploration. Our integrated knowledge graph, analytic tools, and web services enable diverse users to explore relationships between phenotypes and genotypes across species.
[en] Aneuploidy is a feature of most cancer cells that is often accompanied by an elevated rate of chromosome mis-segregation termed chromosome instability (CIN). While CIN can act as a driver of cancer genome evolution and tumor progression, recent findings point to the existence of a threshold level beyond which CIN becomes a barrier to tumor growth and therefore can be exploited therapeutically. Drugs known to increase CIN beyond the therapeutic threshold are currently few in number, and the clinical promise of targeting the CIN phenotype warrants new screening efforts. However, none of the existing methods, including the in vitro micronuclei (MNi) assay, developed to quantify CIN, is entirely satisfactory. We have developed a new assay for measuring CIN. This quantitative assay for chromosome mis-segregation is based on the use of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Thus, cells that inherit the HAC display green fluorescence, while cells lacking the HAC do not. This allows the measurement of HAC loss rate by routine flow cytometry. Using the HAC-based chromosome loss assay, we have analyzed several well-known anti-mitotic, spindle-targeting compounds, all of which have been reported to induce micronuclei formation and chromosome loss. For each drug, the rate of HAC loss was accurately measured by flow cytometry as a proportion of non-fluorescent cells in the cell population which was verified by FISH analysis. Based on our estimates, despite their similar cytotoxicity, the analyzed drugs affect the rates of HAC mis-segregation during mitotic divisions differently. The highest rate of HAC mis-segregation was observed for the microtubule-stabilizing drugs, taxol and peloruside A. Thus, this new and simple assay allows for a quick and efficient screen of hundreds of drugs to identify those affecting chromosome mis-segregation. It also allows ranking of compounds with the same or similar mechanism of action based on their effect on the rate of chromosome loss. The identification of new compounds that increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target the CIN phenotype in cancer cells