Construction of a cross-species cell landscape at single-cell level.

Individual cells are basic units of life. Despite extensive efforts to characterize the cellular heterogeneity of different organisms, cross-species comparisons of landscape dynamics have not been achieved. Here, we applied single-cell RNA sequencing (scRNA-seq) to map organism-level cell landscapes at multiple life stages for mice, zebrafish and Drosophila. By integrating the comprehensive dataset of > 2.6 million single cells, we constructed a cross-species cell landscape and identified signatures and common pathways that changed throughout the life span. We identified structural inflammation and mitochondrial dysfunction as the most common hallmarks of organism aging, and found that pharmacological activation of mitochondrial metabolism alleviated aging phenotypes in mice. The cross-species cell landscape with other published datasets were stored in an integrated online portal-Cell Landscape. Our work provides a valuable resource for studying lineage development, maturation and aging

Transfection of Eimeria mitis with yellow fluorescent protein as reporter and the endogenous development of the transgenic parasite.

BACKGROUND: Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis. METHODS AND FINDINGS: Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP) expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS) and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain. CONCLUSION: Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector

Rigid versus semi-rigid bis(imidazole) ligands in the assembly of two Co(II) coordination polymers : structural variability, electrochemical properties and photocatalytic behavior

The hydrothermal reactions of 1,2,4,5-cyclohexanetetracarboxylic acid (H4L) with CoCl2 center dot 2H(2)O and rigid or semi-rigid bis(imidazole) ligands were able to generate two Co(II) coordination polymers (CPs), {[Co-5(L)(2)(1,4-bimb)( mu(3)-OH)(2)(H2O)(8)]center dot 2H(2)O}(n) (1), {[Co(L)(0.5)(1,4-bib)]center dot H2O}(n) (2) (1,4-bimb = 1,4-bis(imidazol-1-ylmethyl) benzene, 1,4-bib = 1,4-bis(1H-imidazol-1-yl) benzene). CPs 1 and 2 were structurally characterized by elemental analysis, IR spectroscopy, X-ray powder diffraction and single crystal X-ray diffraction. CP 1 features a 3D 3,3,4,4,5-connected framework with an unprecedented {4(2).8.10(2).12}{4(3).6.8(6)}(2){4(3)}(2){4(6)}(2){6(2).8}(2) topology, which represents the first example of CPs with such a topology. CP 2 possesses a three-fold interpenetration 3D framework with mog topology. The distinct structures of the two CPs may result from diverse coordination modes of the (L)(4-) ligands and different structural characteristics of rigid or semi-rigid N-donor ligands. The thermal stabilities, photoluminescence properties and electrochemical behavior in the solid state for CPs 1 and 2 have been investigated. The photophysical studies indicated that CPs 1 and 2 are potential semiconductive materials. Moreover, both CPs 1 and 2 show high photocatalytic efficiency for the degradation of methylene blue (MB) under UV light irradiation and exhibit good stability and recyclability. A possible photocatalytic mechanism is speculated by introducing t-butyl alcohol (TBA) as a widely used (OH)-O-center dot scavenger

Improvement and Evaluation of Loop-Mediated Isothermal Amplification for Rapid Detection of Toxoplasma gondii Infection in Human Blood Samples.

Loop-mediated isothermal amplification (LAMP), an attractive DNA amplification method, was developed as a valuable tool for the rapid detection of Toxoplasma gondii. In this study, species-specific LAMP primers were designed by targeting the AF146527 sequence, which was a conserved sequence of 200- to 300-fold repetitive 529 bp fragment of T.gondii. LAMP reaction system was optimized so that it could detect the minimal DNA sample such as a single tachyzoite or 10 copies of recombinant plasmid. No cross-reactivity was found when using DNA from other parasites as templates. Subsequently, a total of 200 human blood samples were directly investigated by two diagnostic methods, LAMP and conventional PCR. Fourteen of 200 (7%) samples were positive for Toxoplasma by LAMP (the primers developed in this study), whereas only 5 of 200 (2.5%) were proved positive by conventional PCR. The procedure of the LAMP assay was very simple, as the reaction would be carried out in a single tube under isothermal conditions at 64°C and the result would be read out with 1 h (as early as 35 min with loop primers). Thus, this method has the advantages of rapid amplification, simple operation, and easy detection and would be useful for rapid and reliable clinical diagnosis of acute toxoplasmosis, especially in developing countries

Transfected <i>E. mitis</i> constitutively expressed EYFP molecule throughout the life cycle.

<p>(A) Merogony stage; a-b, trophozoites; c, trophozoites developing into merozont; d, immature merozont; e-j, merozonts and merozoites; (B) Gamogony stage; a, macrogamete at early stage; b, mature macrogamete; c-f, microgametogenesis; g, fertilization of microgametes and macrogametes; h, zygotes; (C) Sporogony stage. a, unsporulated oocysts; b, sporulated oocysts. (D) Flagella of microgamete. Bar = 10 µm. The data represented one of three independent experiments with similar results.</p

Validation of rE.mi stably expressing fused protein and EYFP molecule.

<p>(A) Fluorescent images of the seventh passage of unsporulated and sporulated oocysts of rE.mi;(B) Fluorescence intensity of unsporulated and sporulated oocysts of rE.mi. Flow cytometry was performed with a FACS Calibur analyzer (BD Biosciences, USA). Data was analyzed using Cell Questpro Software (BD Biosciences, USA).</p

Genome analyses of inserted DNA fragments in the rE.mi population.

<p>(A) Detection of HA1chFc and EYFP fragments by PCR. Lane 1 and 4, PCR products from plasmid pHEAAssHA1chFcA (positive control); lanes 2 and 5, DNA extracted from rE.mi; lanes 3 and 5, DNA extracted from wild type of <i>E. mitis</i> (negative control); M, DL plus 2000 molecular weight ladder. (B) Gel electrophoresis of amplified products after three nested genome walking PCRs. 1–3 represent rounds of PCR, AP1-4, random primers from genome walking kit. M, DL plus 2000 marker. (C) Integrated sites of plasmid expression cassettes into <i>E. mitis</i> genome by Genome walking. (D) Detection of recombined HA1chFc protein by Western blotting with mouse HA1 protein polyclonal antibody (upper) or chicken IgY Fc antibody (below). Lane 1, protein from wild type of <i>E. mitis</i> (negative control); lane 2, purified HA1chFc protein expressed in <i>E.coli</i> (positive control); lane 3, protein extracted from rE.mi.</p

Comparison of the oocyst shedding pattern of rE.mi with that of the parent strain.

<p>One group of four chickens were infected with 1000 rE.mi, another group received 1000 parent parasites. Oocyst shedding was measured every 24 h between 5d and 11d post infection. The data represented one of two independent experiments with similar results and were expressed as the mean ± SD.</p

Transfection efficiency of rE.mi of different passages.

<p>Transfection efficiency of rE.mi of different passages.</p

How exposure to ALS-inhibiting gametocide tribenuron-methyl induces male sterility in rapeseed

Abstract Background Acetolactate synthase (ALS)-inhibiting herbicide tribenuron-methyl (TBM) is an efficient gametocide that can cause rapeseed (Brassica napus L.) to become male sterile and outcrossing. To find the reason the TBM treatment leads to male sterility, an integrated study using cytological, physiological, and transcriptomic methods was conducted. Results Some temporary symptoms, including the discoloration of young leaves and a short halt of raceme elongation, were observed in the rapeseed plants exposed to TBM at an application rate of 1 μg per plant. Both chloroplasts in young leaves and plastids in anthers were deformed. TBM also reduced the leaf photosynthetic rate and the contents of chlorophyll, soluble sugar and pyruvate. Both the tapetal cells and uni-nucleate microspores in the treated plants showed large autophagic vacuoles, and the tissue degenerated quickly. A transcriptomic comparison with the control identified 200 upregulated and 163 downregulated differential expression genes in the small flower buds of the TBM treatment. The genes encoding functionally important proteins, including glucan endo-1,3-beta-glucosidase A6, QUARTET3 (QRT3), ARABIDOPSIS ANTHER 7 (ATA7), non-specific lipid-transfer protein LTP11 and LTP12, histone-lysine N-methyltransferase ATXR6, spermidine coumaroyl-CoA acyltransferase (SCT), and photosystem II reaction centre protein psbB, were downregulated by TBM exposure. Some important genes encoding autophagy-related protein ATG8a and metabolic detoxification related proteins, including DTX1, DTX6, DTX35, cytosolic sulfotransferase SOT12, and six members of glutathione S-transferase, were upregulated. In addition, several genes related to hormone stimulus, such as 1-aminocyclopropane-1-carboxylate synthase 8 (ACS8), ethylene-responsive factor ERF1A, ERF1, ERF71, CRF6, and RAP2-3, were also upregulated. The transcriptional regulation is in accordance with the functional abnormalities of pollen wall formation, lipid metabolism, chloroplast structure, ethylene generation, cell cycle, and tissue autophagy. Conclusion The results suggested that except for ALS, the metabolic pathways related to lipid metabolism, pollen exine formation, photosynthesis and hormone response are associated with male sterility induced by TBM. The results provide new insight into the molecular mechanisms of inducing male sterility by sulfonylurea