Summary We analyzed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, mRNA arrays, microRNA sequencing and reverse phase protein arrays. Our ability to integrate information across platforms provided key insights into previously-defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at > 10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the Luminal A subtype. We identified two novel protein expression-defined subgroups, possibly contributed by stromal/microenvironmental elements, and integrated analyses identified specific signaling pathways dominant in each molecular subtype including a HER2/p-HER2/HER1/p-HER1 signature within the HER2-Enriched expression subtype. Comparison of Basal-like breast tumors with high-grade Serous Ovarian tumors showed many molecular commonalities, suggesting a related etiology and similar therapeutic opportunities. The biologic finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biologic subtypes of breast cancer.

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A major limitation to yeast aging study has been the inability to track mother cells and observe molecular markers during the aging process. The traditional lifespan assay relies on manual micro-manipulation to remove daughter cells from the mother, which is laborious, time consuming, and does not allow long term tracking with high resolution microscopy. Recently, we have developed a microfluidic system capable of retaining mother cells in the microfluidic chambers while removing daughter cells automatically, making it possible to observe fluorescent reporters in single cells throughout their lifespan. Here we report the development of a new generation of microfluidic device that overcomes several limitations of the previous system, making it easier to fabricate and operate, and allowing functions not possible with the previous design. The basic unit of the device consists of microfluidic channels with pensile columns that can physically trap the mother cells while allowing the removal of daughter cells automatically by the flow of the fresh media. The whole microfluidic device contains multiple independent units operating in parallel, allowing simultaneous analysis of multiple strains. Using this system, we have reproduced the lifespan curves for the known long and short-lived mutants, demonstrating the power of the device for automated lifespan measurement. Following fluorescent reporters in single mother cells throughout their lifespan, we discovered a surprising change of expression of the translation elongation factor TEF2 during aging, suggesting altered translational control in aged mother cells. Utilizing the capability of the new device to trap mother-daughter pairs, we analyzed mother-daughter inheritance and found age dependent asymmetric partitioning of a general stress response reporter between mother and daughter cells.

SummaryBudding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild‐type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long‐lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker – the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.

Background:Cancer stem cells (CSCs) paradigm suggests that CSCs might have important clinical implications in cancer therapy. Previously, we reported that accumulation efficiency of CSCs is different post low- and high-LET irradiation in 48 h.Methods:Cancer stem cells and non-stem cancer cells (NSCCs) were sorted and functionally identified through a variety of assays such as antigen profiles and sphere formation. Inter-conversion between CSCs and NSCCs were in situ visualised. Cancer stem cells proportions were assayed over multiple generations under normal and irradiation surroundings. Supplement and inhibition of TGF-β1, as well as immunofluorescence assay of E-cadherin and Vimentin, were performed.Results:Surface antigen markers of CSCs and NSCCs exist in an intrinsic homoeostasis state with spontaneous and in situ visualisable inter-conversions, irrespective of prior radiations. Supplement with TGF-β1 accelerates the equilibrium, whereas inhibition of TGF-β signalling disturbs the equilibrium and significantly decreases CSC proportion. Epithelial mesenchymal transition (EMT) might be activated during the process.Conclusion:Our results indicate that the intrinsic inter-conversion and dynamic equilibrium between CSCs and NSCCs exist under normal and irradiation surroundings, and TGF-β might have important roles in the equilibrium through activating EMT.

Exercise has been proved to promote the number and activity of circulating endothelial progenitor cells (EPCs) in humans, which contributes to improvement in endothelial function and maintenance of cardiovascular homoeostasis. However, the mechanism underlying the effect of exercise on circulating EPCs in healthy subjects is not completely understood. Here, we investigated whether the regulation of acute exercise on circulating EPCs is associated with nitric oxide (NO), vascular endothelial growth factors (VEGF) and granulocyte macrophage colony stimulating factor (GM-CSF) known to modulate circulating EPCs in healthy subjects. A total of 16 healthy male volunteers underwent a modified Bruce treadmill acute exercise protocol. The number and activity of circulating EPCs, as well as the levels of NO-VEGF and GM-CSF in plasma and culture medium before and after exercise in healthy subjects were measured. The number and activity of circulating EPCs after acute exercise were significantly higher than those before exercise in healthy subjects. In parallel, acute exercise significantly enhanced plasma NO level in healthy subjects. There is a significant linear regression relationship between the enhanced plasma NO level and increased number or activity of circulating EPCs. However, no change of plasma VEGF and GM-CSF level was observed after acute exercise. The secretion of NO-VEGF and GM-CSF by cultured EPCs remained unchanged in response to acute exercise. The present study demonstrates for the first time that acute exercise-induced NO production contributes to upregulation of circulating EPCs in healthy subjects, which suggests that NO plays an important role in the regulation of exercise on circulating EPCs.

Bacterial chemoreceptors mediate chemotactic responses to diverse stimuli. Here, by using an integrated in silico, in vitro, and in vivo approach, we screened a large compound library and found eight novel chemoeffectors for the Escherichia coli chemoreceptor Tar. Six of the eight new Tar binding compounds induce attractant responses, and two of them function as antagonists that can bind Tar without inducing downstream signaling. Comparison between the antagonist and attractant binding patterns suggests that the key interactions for chemotaxis signaling are mediated by the hydrogen bonds formed between a donor group in the attractant and the main-chain carbonyls (Y149 and/or Q152) on the α4 helix of Tar. This molecular insight for signaling is verified by converting an antagonist to an attractant when introducing an N-H group into the antagonist to restore the hydrogen bond. Similar signal triggering effect by an O-H group is also confirmed. Our study suggests that the Tar chemoeffector binding pocket may be separated into two functional regions: region I mainly contributes to binding and region II contributes to both binding and signaling. This scenario of binding and signaling suggests that Tar may be rationally designed to respond to a nonnative ligand by altering key residues in region I to strengthen binding with the novel ligand while maintaining the key interactions in region II for signaling. Following this strategy, we have successfully redesigned Tar to respond to L-arginine, a basic amino acid that does not have chemotactic effect for WT Tar, by two site-specific mutations (R69′E and R73′E).T wo-component signaling pathways are ubiquitous in bacteria. They enable the cells to recognize and respond to different environmental stimuli (1). The control network of bacterial chemotaxis uses such a two-component system to sense the extracellular chemoeffector concentrations (2, 3). Chemoreceptors are the key upstream sensory components in the chemotaxis signaling pathway. They directly interact with specific extracellular chemoeffectors and transfer environmental information to the downstream response regulator, which ultimately controls the cell's motility (4, 5).Tar is one of the major chemoreceptors found in Escherichia coli and Salmonella enterica serovar Typhimurium (6). Attractant and repellent molecules that can induce chemotactic responses of the cells by interacting with Tar were studied (7). In addition to these two types of chemoeffectors, antagonist molecules that can directly bind to chemoreceptors without generating chemotactic responses should also exist. For example, antagonists for the sensor kinase TodS were found in the TodS/TodT two-component system (8). However, so far, antagonist molecules that function by directly binding to E. coli chemoreceptors have not been reported.Much progress has been made in understanding the structural basis of chemoreceptor signaling. Crystal structures show that each monomer (in the Tar homodimer) contains a four-helix bundle (helices α1-α4) structure, of w...

Molecular diffusive membranes or materials are important for biological applications in microfluidic systems. Hydrogels are typical materials that offer several advantages, such as free diffusion for small molecules, biocompatibility with most cells, temperature sensitivity, relatively low cost, and ease of production. With the development of microfluidic applications, hydrogels can be integrated into microfluidic systems by soft lithography, flow-solid processes or UV cure methods. Due to their special properties, hydrogels are widely used as fluid control modules, biochemical reaction modules or biological application modules in different applications. Although hydrogels have been used in microfluidic systems for more than ten years, many hydrogels' properties and integrated techniques have not been carefully elaborated. Here, we systematically review the physical properties of hydrogels, general methods for gel-microfluidics integration and applications of this field. Advanced topics and the outlook of hydrogel fabrication and applications are also discussed. We hope this review can help researchers choose suitable methods for their applications using hydrogels.