Polystyrene-coated micropallets for culture and separation of primary muscle cells

Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact-printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells

Laser-based directed release of array elements for efficient collection into targeted microwells

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25–400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%

Use of small specimen creep data in component life management: a review

Small specimen creep testing techniques are novel mechanical test techniques that have been developed over the past 25 years. They mainly include the sub-size uniaxial test, the small punch creep test, the impression creep test, the small ring creep test and the two-bar creep test. This paper outlines the current methods in practice for data interpretation as well as the state-of-the-art procedures for conducting the tests. Case studies for the use of impression creep testing and material strength ranking of creep resistant steels are reviewed along with the requirement for the standardisation of the impression creep test method. A database of small specimen creep testing is required to prove the validity of the tests

Pathogen Entrapment by Transglutaminase—A Conserved Early Innate Immune Mechanism

Clotting systems are required in almost all animals to prevent loss of body fluids after injury. Here, we show that despite the risks associated with its systemic activation, clotting is a hitherto little appreciated branch of the immune system. We compared clotting of human blood and insect hemolymph to study the best-conserved component of clotting systems, namely the Drosophila enzyme transglutaminase and its vertebrate homologue Factor XIIIa. Using labelled artificial substrates we observe that transglutaminase activity from both Drosophila hemolymph and human blood accumulates on microbial surfaces, leading to their sequestration into the clot. Using both a human and a natural insect pathogen we provide functional proof for an immune function for transglutaminase (TG). Drosophila larvae with reduced TG levels show increased mortality after septic injury. The same larvae are also more susceptible to a natural infection involving entomopathogenic nematodes and their symbiotic bacteria while neither phagocytosis, phenoloxidase or—as previously shown—the Toll or imd pathway contribute to immunity. These results firmly establish the hemolymph/blood clot as an important effector of early innate immunity, which helps to prevent septic infections. These findings will help to guide further strategies to reduce the damaging effects of clotting and enhance its beneficial contribution to immune reactions

The formation of human populations in South and Central Asia

By sequencing 523 ancient humans, we show that the primary source of ancestry in modern South Asians is a prehistoric genetic gradient between people related to early hunter-gatherers of Iran and Southeast Asia. After the Indus Valley Civilization’s decline, its people mixed with individuals in the southeast to form one of the two main ancestral populations of South Asia, whose direct descendants live in southern India. Simultaneously, they mixed with descendants of Steppe pastoralists who, starting around 4000 years ago, spread via Central Asia to form the other main ancestral population. The Steppe ancestry in South Asia has the same profile as that in Bronze Age Eastern Europe, tracking a movement of people that affected both regions and that likely spread the distinctive features shared between Indo-Iranian and Balto-Slavic languages

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae)

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A.

Extensive chloroplast haplotype variation indicates Pleistocene hybridization and radiation of North American Arabis drummondii, A. x divaricarpa, and A. holboellii (Brassicaceae)

Arabis drummondii, A. holboellii and their hybrid A. x divaricarpa are widespread perennials of open habitats in North America. A phylogenetic analysis based on noncoding chloroplast DNA sequences (trnL intron and trnL/F intergenic spacer) resolved A.