CAP1, a target of miR-144/451, negatively regulates erythroid differentiation and enucleation.

The exact molecular mechanism underlying erythroblast enucleation has been a fundamental biological question for decades. In this study, we found that miR-144/451 critically regulated erythroid differentiation and enucleation. We further identified CAP1, a G-actin-binding protein, as a direct target of miR-144/451 in these processes. During terminal erythropoiesis, CAP1 expression declines along with gradually increased miR-144/451 levels. Enforced CAP1 up-regulation inhibits the formation of contractile actin rings in erythroblasts and prevents their terminal differentiation and enucleation. Our findings reveal a negative regulatory role of CAP1 in miR-144/451-mediated erythropoiesis and thus shed light on how microRNAs fine-tune terminal erythroid development through regulating actin dynamics.

Co-expression of Pseudomonas alcaligenes lipase and its specific foldase in Pichia pastoris by a dual expression cassette strategy.

Lipomax is a commercialized foldase-dependent Pseudomonas lipase that was previously expressed only in Pseudomonas strains. Here, using Pichia pastoris as the host, we report a new co-expression method that leads to the successful production of Lipomax. The active Lipomax is extracellularly co-expressed with its cognate foldase (LIM); and the purified enzyme mix has the optimum pH at pH 8.0 and an optimal temperature around 40 °C. N-glycosylation was observed for Pichia produced Lipomax, and its reduction was shown to increase the lipolytic activity. With different p-nitrophenyl esters as the substrates, the substrate profiling analyses further indicate that Lipomax prefers esters with middle-long chain fatty acids, showing the highest specific activity to p-nitrophenyl caprylate (C8). The extracellular co-expression of Lipomax and LIM in Pichia will not only increase our ability to investigate additional eukaryotic hosts for lipase expression, but also be of considerable value in analyzing other foldase-dependent lipases.

In Vitro Modeling of Human Germ Cell Development Using Pluripotent Stem Cells.

Due to differences across species, the mechanisms of cell fate decisions determined in mice cannot be readily extrapolated to humans. In this study, we developed a feeder- and xeno-free culture protocol that efficiently induced human pluripotent stem cells (iPSCs) into PLZF+/GPR125+/CD90+ spermatogonium-like cells (SLCs). These SLCs were enriched with key genes in germ cell development such as MVH, DAZL, GFRα1, NANOS3, and DMRT1. In addition, a small fraction of SLCs went through meiosis in vitro to develop into haploid cells. We further demonstrated that this chemically defined induction protocol faithfully recapitulated the features of compromised germ cell development of PSCs with NANOS3 deficiency or iPSC lines established from patients with non-obstructive azoospermia. Taken together, we established a powerful experimental platform to investigate human germ cell development and pathology related to male infertility.

Molecular Marker Study of Particulate Organic Matter in Southern Ontario Air.

To study the origins of airborne particulate organic matter in southern Ontario, molecular marker concentrations were studied at Hamilton, Simcoe, and York Gateway Tunnel, representing industrial, rural, and heavy traffic sites, respectively. Airborne particulate matter smaller than 10 µm in aerodynamic diameter was collected on quartz filters, and the collected samples were analyzed for total carbons, 5-6 ring PAHs, hopanes, n-alkanes (C20 to C34), and oxygenated aromatic compounds. Results showed that PAH concentrations at all three sites were highly correlated, indicating vehicular emissions as the major source. Meanwhile, in the scatter plots of α,ß-hopane and trisnorhopane, concentrations displayed different trends for Hamilton and Simcoe. The slopes of the linear regressions for Hamilton and the tunnel were statistically the same, while the slope for Simcoe was significantly different from those. Comparison with literature values revealed that the trend observed at Simcoe was explained by the influence from coal combustion. We also found that the majority of oxygenated aromatic compounds at both sites were in the similar level, possibly implying secondary products contained in the southern Ontario air. Regardless of some discrepancies, absolute principal component analysis applied to the datasets could reproduce those findings.

Interplay of transcription factors and microRNAs during embryonic hematopoiesis.

Hematopoietic stem cells (HSCs), which are localized in the bone marrow of adult mammals, come from hematopoietic endothelium during embryonic stages. Although the basic processes of HSC generation and differentiation have been described in the past, the epigenetic regulation of embryonic hematopoiesis remains to be fully described. Here, by utilizing an in vitro differentiation system of mouse embryonic stem cells (ESCs), we identified more than 20 microRNAs that were highly enriched in embryonic hematopoietic cells, including some (e.g. miR-10b, miR-15b, and miR-27a) with previously unknown functions in blood formation. Luciferase and gene expression assays further revealed combinational binding and regulation of these microRNAs by key transcription factors in blood cells. Finally, bioinformatics and functional analyses supported an interactive regulatory control between transcription factors and microRNAs in hematopoiesis.

CD71(high) population represents primitive erythroblasts derived from mouse embryonic stem cells.

The CD71/Ter119 combination has been widely used to reflect dynamic maturation of erythrocytes in vivo. However, because CD71 is expressed on all proliferating cells, it is unclear whether it can be utilized as an erythrocyte-specific marker during differentiation of embryonic stem cells (ESCs). In this study, we revealed that a population expressing high level of CD71 (CD71(high)) during mouse ESC differentiation represented an in vitro counterpart of yolk sac-derived primitive erythroblasts (EryPs) isolated at 8.5days post coitum. In addition, these CD71(high) cells went through "maturational globin switching" and enucleated during terminal differentiation in vitro that were similar to the yolk sac-derived EryPs in vivo. We further demonstrated that the formation of CD71(high) population was regulated differentially by key factors including Scl, HoxB4, Eaf1, and Klf1. Taken together, our study provides a technical advance that allows efficient segregation of EryPs from differentiated ESCs in vitro for further understanding molecular regulation during primitive erythropoiesis.