Investigating the Effect of Heat Treatment on the Microstructure and Hardness of Aluminum-Lithium Alloys.

In this study, the effects of heat treatment on the microstructure and strength (micro-hardness) of an aluminum-lithium (Al-Li) base alloy containing copper (Cu) and scandium (Sc) were investigated, with a view to enhancing the alloy performance for aerospace applications. The heat treatment conditions were investigated to understand the precipitation behavior and the mechanisms involved in strengthening. Aging was carried out at temperatures of 130 °C and 150 °C for aging times of 1 h, 2.5 h, 5 h, 10 h, 15 h, 25 h, 35 h, and 45 h at each temperature for Al-Li alloy and at 160 °C, 180 °C, and 200 °C for aging times of 5 h, 10 h, 15 h, 20 h, 25 h, and 30 h at each temperature for Al-Li-Cu and Al-Li-Cu-Sc alloys. The investigation revealed that both solution heat treatment and artificial aging had a notable impact on strengthening the hardness of the alloy. This effect was attributed to the characteristics of the precipitates, including their type, size, number density, and distribution. The addition of copper (Cu) and scandium (Sc) was observed to have an impact on grain size refinement, while Cu addition specifically affected the precipitation behavior of the alloy. It led to remarkable changes in the number density, size, and distribution of T1 (Al2CuLi) and θ' (Al2Cu) phases. As a result, the hardness of the alloy was significantly improved after the addition of Cu and Sc, in comparison with the base Al-Li alloy. The best heat treatment process was determined as: 580 °C/1 h solution treatment +150 °C/45 h artificial aging for Al-Li alloy and 505 °C/5 h solution treatment +180 °C/20 h artificial aging for Al-Li-Cu and Al-Li-Cu-Sc alloys.

Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δß splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

Microenvironmental regulation of telomerase isoforms in human embryonic stem cells.

Recent evidence points to extra-telomeric, noncanonical roles for telomerase in regulating stem cell function. In this study, human embryonic stem cells (hESCs) were cultured in 20% or 2% O2 microenvironments for up to 5 days and evaluated for telomerase reverse transcriptase (TERT) expression and telomerase activity. Results showed increased cell survival and maintenance of the undifferentiated state with elevated levels of nuclear TERT in 2% O2-cultured hESCs despite no significant difference in telomerase activity compared with their high-O2-cultured counterparts. Pharmacological inhibition of telomerase activity using a synthetic tea catechin resulted in spontaneous hESC differentiation, while telomerase inhibition with a phosphorothioate oligonucleotide telomere mimic did not. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed variations in transcript levels of full-length and alternate splice variants of TERT in hESCs cultured under varying O2 atmospheres. Steric-blocking of Δα and Δß hTERT splicing using morpholino oligonucleotides altered the hTERT splicing pattern and rapidly induced spontaneous hESC differentiation that appeared biased toward endomesodermal and neuroectodermal cell fates, respectively. Together, these results suggest that post-transcriptional regulation of TERT under varying O2 microenvironments may help regulate hESC survival, self-renewal, and differentiation capabilities through expression of extra-telomeric telomerase isoforms.

Mass spectrometry-based proteomic analysis of the matrix microenvironment in pluripotent stem cell culture.

The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis. In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™ pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach.

Proteomic analysis of extracellular matrices used in stem cell culture.

Numerous matrices for the growth of human embryonic stem cells (hESC) in vitro have been described. However, their exact composition is typically unknown. Information on the components of these matrices will aid in the development of a fully defined growth surface for hESCs. These matrices typically consist of mixture of proteins present in a wide range of abundance making their characterization challenging. In this study, we performed the proteomic analysis of five previously uncharacterized matrices: CellStart, Human Basement Membrane Extract (Human BME), StemXVivo, Bridge Human Extracellular Matrix (BridgeECM), and mouse embryonic fibroblast conditioned matrix (MEF-CMTX). Based on a proteomics protocol optimized using lysates from HeLa cells, we undertook the analysis of the five complex extracellular matrix (ECM) samples using a combination of strong anion and cation exchange chromatography and SDS-PAGE. For each of these matrices, we identify numerous proteins, indicating their complex nature. We also compared these results with a similar proteomics analysis of the growth matrix, Matrigel™. From these analyses, we observed that fibronectin is a primary component of nearly all hESC supportive matrices. This observation led to the investigation of the suitability of fibronectin as a defined ECM for the growth of hESCs. We found that fibronectin promotes the maintenance of pluripotent H9 and CA1 hESCs in an undifferentiated state using mTeSR1 medium. This finding validates the utility of characterizing matrices used for hESC growth in revealing ECM components required for culturing hESCs in a universally applicable defined system.