<i>Arabidopsis</i> ENDO2: Its Catalytic Role and Requirement of N‑Glycosylation for Function

The <i>Arabidopsis thaliana</i> At1g68290 gene encoding an endonuclease was isolated and designated ENDO2, which was cloned into a binary vector to overexpress ENDO2 with a C-terminal 6 × His-tag in <i>A. thaliana</i>. Our <i>Arabidopsis</i> transgenic lines harboring <i>35SP::ENDO2</i> produced stable active enzyme with high yield. The protein was affinity purified from transgenic plants, and its identity was confirmed by liquid chromatography–mass spectrometry and automatic Edman degradation. ENDO2 enzyme digests RNA, ssDNA, and dsDNA, with a substrate preference for ssDNA and RNA. The activity toward ssDNA (361.7 U/mg) is greater than its dsDNase activity (14.1 U/mg) at neutral pH. ENDO2 effectively cleaves mismatch regions in heteroduplex DNA containing single base pair mismatches or insertion/deletion bases and can be applied to high-throughput detection of single base mutation. Our data also validated that the removal of sugar groups from ENDO2 strongly affects its enzymatic stability and activity

Suppressive Effects of Anthrax Lethal Toxin on Megakaryopoiesis

<div><p>Anthrax lethal toxin (LT) is a major virulence factor of <i>Bacillus anthracis</i>. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34⁺ cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.</p> </div

Suppressive effect of LT on CFU-MK formation.

<p>(A) The experimental outline. Human cord blood-derived mononuclear cells (CBMCs) were used in a megakaryocytic colony-forming unit (CFU-MK) assay. This figure shows the morphology (B–C) and quantified numbers (D) of megakaryocytic colonies. Arrowheads in the low magnification images (B-1 to B-6) indicate specific colonies that are highlighted in high-magnification photographs (C-1 to C-6) for each group (C). Scale bar: B, 2 mm; C, 200 µm. The cell-culture medium was used to dilute LT and was used as a treatment control in vehicle-groups. Data are representative of 3 independent experiments. One plate was seeded with 1.1×10⁵ cells. Data are reported as mean ± standard deviation (SD), **<i>p</i><0.01 compared to vehicle groups.</p

TPO pretreatments ameliorated LT-mediated thrombocytopenia in mice.

<p>(A) The experiment outline used in measurement of hematopoietic parameters. (B–D) The platelet, WBC, and RBC counts of mice treated with TPO, LT, or TPO plus LT at 22, 44, and 66 hours, respectively. The measurements of platelet, WBC, and RBC counts right before TPO and/or LT treatments [prior to first TPO treatments of TPO groups in (A)] were served as basal levels (Before exp. groups, C–D). Untreated mice were used as negative controls. **<i>p</i><0.01 compared between indicated groups. Data are reported as mean ± standard deviation (SD) and representative of 2 independent experiments.</p

Characterizations of LT-induced hypoploid cells by apoptosis assay.

<p>The expanded human cord blood-derived CD34⁺ cells (Day 0) were treated with LT on day 12 and then analyzed on day 16 in aforementioned 16-day differentiation courses of megakaryocyte. The cell size (FSC) and cell granularity (SSC) are indicated (A). Propidium iodine (PI) staining revealed the cellular DNA contents, in which the sub-G1 hypoploid cells were increased in LT-treated groups (B). AnnexinV-APC (C) and active caspase-3 antibodies (D) were used to investigate the apoptotic changes of LT-treated cells by flow cytometry. Summarized events were shown on (E). Data are reported as mean ± standard deviation (SD) and represent 4 independent experiments. **<i>p</i><0.01 compared to vehicle-treated groups.</p

Characterizations of LT-induced hypoploid cells by platelet activation agonists.

<p>Human cord blood-derived CD34⁺ cells were treated with LT on day 12 and then analyzed on day 16 in aforementioned 16-day differentiation courses of megakaryocyte. The cell size (FSC) and cell granularity (SSC) are indicated (A). Size (FSC) and granularity (SSC) parameters of cord-blood platelets were plotted as linear scale (b-1) and logarithmic scale (b-2). The cellular DNA contents were revealed by propidium iodine (PI) staining (b-3). Agonist ADP, thrombin, and collagen-induced stimulation of the platelets (b-4) versus cell sorter-enriched R2-cells (day 16, LT-treated groups) (C) was revealed by enhanced surface staining of P-selectin (CD62P), a platelet activation marker. These results obtained from 3 independent experiments.</p

Suppressive effect of LT on mouse bone marrow cells.

<p>(A) The experimental outline. Mononuclear cells were isolated from bone marrow (BMMCs) and induced to become megakaryocytes in the presence of mTPO in a 6-day course. The cell size (FSC), megakaryocytic surface marker (CD41) (B) and DNA content (C) were analyzed by flow cytometry. Summarized events were shown on (D). Data are reported as mean ± standard deviation (SD) and represent 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.01 compared to mTPO-treated groups.</p

TPO pretreatments ameliorated LT-mediated megakaryopoiesis suppression in mice.

<p>(A) The experiment outline used in bone marrow (BM) experiments. Flow cytometry analysis using mouse bone marrow cells was performed at 69 hours after LT treatments. Following a previously described method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059512#pone.0059512-Tomer1" target="_blank">[69]</a>, the population of polyploid megakaryocytes was gated as CD41 high (fluorescent intensity>10²) and large (FSC>400) cells in all groups (B, R1 regions). The DNA content of R1 cells in the untreated group was illustrated (C), and relative R1 cell population (percentage of total 2×10⁵ cells) was quantified (D). **<i>p</i><0.01 compared between indicated groups.</p

TPO treatments reduced LT-mediated mortality in mice.

<p>(A) The experimental outline. (B) The survival rates of mice treated with TPO, LT, or TPO plus LT are shown. Untreated mice were used as negative controls. Asterisk (*) marks in (A) and (B) indicate the starting time point for recording survival rate.</p

Expanding the Product Profile of a Microbial Alkane Biosynthetic Pathway

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of <i>n</i>-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from <i>Bacillus subtilis</i>, an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of <i>Escherichia coli</i>. When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of <i>n</i>-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project