Selection of Schizochytrium limacinum mutants based on butanol tolerance

Background: Mutation breeding is one of the most important routes to achieving high docosahexaenoic acid (DHA) productivity using Schizochytrium. However, few selection strategies have been reported that aim to generate a high DHA content in Schizochytrium lipids. Results: First, culture temperature altered the butanol tolerance of Schizochytrium limacinum B4D1. Second, S. limacinum E8 was obtained by selecting mutants with high butanol tolerance. This mutant exhibited a 17.97% lower proportion of DHA than the parent strain S. limacinum B4D1. Third, a negative selection strategy was designed in which S. limacinum F6, a mutant with poor butanol tolerance, was obtained. The proportion of DHA in S. limacinum F6 was 11.22% higher than that of parent strain S. limacinum B4D1. Finally, the performances of S. limacinum B4D1, E8 and F6 were compared. These three strains had different fatty acid profiles, but there was no statistical difference in their biomasses and lipid yields. Conclusion: It was feasible to identified the relative DHA content of S. limacinum mutants based on their butanol tolerance.

Selection of Thymocytes Expressing Transgenic TCR Specific for a Minor Histocompatibility Antigen, H60

Minor histocompatibility antigens are MHC-bound peptides and contribute to the generation of allo-responses after allogeneic transplantation. H60 is a dominant minor H antigen that induces a strong CD8 T-cell response in MHC-matched allogeneic transplantation settings. Here, we report establishment of a TCR transgenic mouse line named J15, wherein T cells express TCRs specific for H60 in complex with H-2K(b), and different fates of the thymocytes expressing J15 TCRs in various thymic antigenic environments. Thymocytes expressing the J15 TCRs were positively selected and differentiated into CD8+ single positive (SP) cells in the thymus of C57BL/6 mice, wherein the cognate antigen H60 is not expressed. However, thymocytes were negatively selected in thymus tissue where H60 was transgenically expressed under the control of the actin promoter, with double-positive stages of cells being deleted. Despite the ability of the H60H peptide (LTFHYRNL) variant to induce cytotoxic activity from H60-specific CTL lines at ~50% of the activity induced by normal H60 peptides (LTFNYRNL), J15-expressing thymocytes were positively selected in the thymus where the variant H60H was transgenically expressed. These results demonstrate that a single amino-acid change in the H60 epitope peptide influences the fate of thymocytes expressing the cognate TCR.

The Role of Dendritic Cells in Central Tolerance

Dendritic cells (DCs) play a significant role in establishing self-tolerance through their ability to present self-antigens to developing T cells in the thymus. DCs are predominantly localized in the medullary region of thymus and present a broad range of self-antigens, which include tissue-restricted antigens expressed and transferred from medullary thymic epithelial cells, circulating antigens directly captured by thymic DCs through coticomedullary junction blood vessels, and peripheral tissue antigens captured and transported by peripheral tissue DCs homing to the thymus. When antigen-presenting DCs make a high affinity interaction with antigen-specific thymocytes, this interaction drives the interacting thymocytes to death, a process often referred to as negative selection, which fundamentally blocks the self-reactive thymocytes from differentiating into mature T cells. Alternatively, the interacting thymocytes differentiate into the regulatory T (Treg) cells, a distinct T cell subset with potent immune suppressive activities. The specific mechanisms by which thymic DCs differentiate Treg cells have been proposed by several laboratories. Here, we review the literatures that elucidate the contribution of thymic DCs to negative selection and Treg cell differentiation, and discusses its potential mechanisms and future directions.

Aspectos actuales de la organogénesis. Función e involución del timo / Present aspects of organogenesis. Function and thymic involution

El timo es un órgano linfoide central o primario localizado en la parte anterosuperior del tórax. Constituye el principal sitio de diferenciación y maduración de las células T. En esta revisión se detallan aspectos actuales de la embriología, la histología y la función tímica y en la generación de los diferentes subtipos de timocitos y su diferenciación a células T maduras efectoras, la inducción de las células T tímicas reguladoras involucradas en el mantenimiento de la tolerancia a lo propio y la involución que sufre este órgano durante el proceso de inmunosenescencia

Thymus is a primary organ located in the antesuperior area of the torax. It is the principal place of differentiation and maduration of T cells. In this review present aspects of the embriology, histology and thymic function are detailed, as well as its role in the generation of different kinds of thymic cells; its differentiation to mature cells and of regulator T cells has a crucial role in tolerance induction. Moreover, thymic involution during of immunosenescence process is shown

Detection of circulating tumor cells from peripheral blood in patients with esophageal squamous cell carcinoma and its clinical significance / 中华胸心血管外科杂志

ObjectiveTo explore the relationship between circulating tumor cells(CTCs) in the peripheral blood of patients with esophageal squamous cell carcinoma(ESCC) and the physiopathological characteristics of esophageal neoplasms.MethodsUsing negative selection system,we depleted red blood cells(RBCs) in red blood cell lysis buffer,depleted white blood cells (WBCs) with Miltenyi magnetic beads and enriched the rare cells from ESCC patients'peripheral blood.Immunofluorence staining (IF) was adopted to identify CTCs.ResultsCirculating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma was closely related to cell differentiation grade,the invasion of primary cancer,lymph node status,P-TNM stages,and was rarely related to the sex,age or the location of tumor.ConclusionThe results suggest that circulating tumor cells in the peripheral blood of patients with esophageal squamous cell carcinoma may express the development of esophageal cancer and may be served as a tumor marker to evaluate the biological behavior of esophageal cancer.

Antigen Nonspecific Death of Immature Thymocytes by Corticosteroids and TNF

BACKGROUND: In the thymus, developing thymocytes continually interact with thymic epithelial cell components. Self MHC restriction of mature T cells are imposed in the thymus through interaction of immature double positive thymocytes and thymic cortical epithelial cells. The site of negative selection, however, is a matter of debate. Through systemic injection of anti-TCR antibody or antigenic peptides, investigators suggested that most of the negative selection occurs in the thymic cortex. But the requirements for negative selection, i.e cellular counterparts and costimulatory molecules are more available in the medulla or cortico-medullary junction rather than in the thymic cortex. METHODS: The direct and indirect pathways of thymocyte death after systemic anti-TCR antibody injection were separated through several experimental systems. B6 mice were either adrenalectomized or sham-adrenalectomized to evaluate the role of endogenous glucocorticoids from adrenal gland. Role of TNF were evaluated through using TNF receptor double knockout mice. RESULTS: We found that without indirectly acting mediators such as TNF-alpha or corticosteroid, double positive thymocyte death were minimal by systemic injection of anti-TCR antibody in TNF receptor double knockout neonatal mice. Also by analyzing neonatal wild-type mice with adoptively transferred mature T cells, only peripheral activation of mature T cells could induce extensive double positive thymocyte death. CONCLUSION: Thus, systemically injected anti-TCR antibody mediated thymocyte death are mostly induced through indirect pathway.

Expression of CodA gene from Escherichia coli confering a negative selection phenotype on transgenic Artemisia annua / 中草药

Objective To explore the feasibility of utilizing the cytosine deaminase A (CodA) gene as an effective negative selectable marker in Artemisia annua for gene targeting.Methods The PCR procedure was employed to amplify CodA gene from Escherichia coli.After being cloned and sequenced, the gene was inserted into a plant expression vector, pROKⅡ, and then introduced into Agrobacterium tumefaciens LBA4404 (pAL4404).The leaf disks of A.annua were transformed by the co-cultivation protocol, after which the transformed calli were selected and green shoots of A. annua were regenerated on N6 medium supplemented with 25 ?g/mL Kanamycin (Kan).Then the Kan-resistant transgenic shoots were transplanted onto the MS medium containing 500 ?g/mL 5-fluocytosine (5-FC) plus 25 ?g/mL Kan and continuously cultured for up to two weeks.Results The transgenic shoots have totally died while untransformed shoots still kept normal growth, indicating that A.annua cells introduced into the CodA gene had conferred an expected negative selection phenotype.When detected by RT-PCR, the transgenic shoots displayed a CodA-positive amplified band, but untransformed shoots gave no such CodA-specific amplified pattern.This result suggested that CodA gene had transcribed into corresponding mRNA in A.annua cells with furtherly verifying the result of phenotypic assay.Conclusion The CodA gene can be utilized as an effective negative selectable marker in A.annua for gene targeting.