Lipidomic changes during different growth stages of Nitzschia closterium f. minutissima

Ultra Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight Mass Spectrometry (UPLC-ESI-Q-TOF–MS) is a powerful lipidomic tool. In this study, we developed a UPLC/Q-TOF–MS based method to investigate the lipid metabolomic changes in different growth phases of Nitzschia closterium f. minutissima. The data classification and biomarker selection were carried out by using multivariate statistical analysis, including principal components analysis (PCA), projection to latent structures with discriminant analysis (PLS-DA), and orthogonal projection to latent structures with discriminant analysis (OPLS-DA). We discovered that the intercellular lipid metabolites were significantly different among exponential, early stationary and late stationary phases. Thirty-one lipid molecules were selected and identified as putative biomarkers, including free fatty acid, Harderoporphyrin, phosphatidylglycerol, 1,2-diacyglycerl-3-O-4′-(N,N-trimethy)-homoserine, triacylglycerol, cholesterol, sulfoquinovosyldiacylglycerol, lyso-sulfoquinovosyldiacylglycerol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and lyso-digalactosyldiacylglycerol. These lipids have been shown previously to function in energy storage, membrane stability and photosynthesis efficiency during the growth of diatoms. Further analysis on the putative biomarkers demonstrated that nitrate starvation played critical role in the transition from exponential phase to stationary phase in N. closterium. This study is the first one to explore the lipidomic changes of microalgae in different growth phases, which promotes better understanding of their physiology and ecology.     

Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Heparan sulfate proteoglycans regulate autophagy in Drosophila

Heparan sulfate-modified proteoglycans (HSPGs) are important regulators of signaling and molecular recognition at the cell surface and in the extracellular space. Disruption of HSPG core proteins, HS-synthesis, or HS-degradation can have profound effects on growth, patterning, and cell survival. The Drosophila neuromuscular junction provides a tractable model for understanding the activities of HSPGs at a synapse that displays developmental and activity-dependent plasticity. Muscle cell-specific knockdown of HS biosynthesis disrupted the organization of a specialized postsynaptic membrane, the subsynaptic reticulum (SSR), and affected the number and morphology of mitochondria. We provide evidence that these changes result from a dysregulation of macroautophagy (hereafter referred to as autophagy). Cellular and molecular markers of autophagy are all consistent with an increase in the levels of autophagy in the absence of normal HS-chain biosynthesis and modification. HS production is also required for normal levels of autophagy in the fat body, the central energy storage and nutritional sensing organ in Drosophila. Genetic mosaic analysis indicates that HS-dependent regulation of autophagy occurs non-cell autonomously, consistent with HSPGs influencing this cellular process via signaling in the extracellular space. These findings demonstrate that HS biosynthesis has important regulatory effects on autophagy and that autophagy is critical for normal assembly of postsynaptic membrane specializations.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work     

Detection and Quantification of Fusarium oxysporum f. sp. niveum Race 1 in Plants and Soil by Real-time PCR

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) is the most serious soil-borne disease in the world and has become the main limiting factor of watermelon production. Reliable and quick detection and quantification of Fon are essential in the early stages of infection for control of watermelon Fusarium wilt. Traditional detection and identification tests are laborious and cannot efficiently quantify Fon isolates. In this work, a real-time polymerase chain reaction (PCR) assay has been described to accurately identify and quantify Fon in watermelon plants and soil. The FONRT-18 specific primer set which was designed based on identified specific sequence amplified a specific 172 bp band from Fon and no amplification from the other formae speciales of Fusarium oxysporum tested. The detection limits with primers were 1.26 pg/μl genomic DNA of Fon, 0.2 pg/ng total plant DNA in inoculated plant, and 50 conidia/g soil. The PCR assay could also evaluate the relationships between the disease index and Fon DNA quantity in watermelon plants and soil. The assay was further used to estimate the Fon content in soil after disinfection with CaCN₂. The real-time PCR method is rapid, accurate and reliable for monitoring and quantification analysis of Fon in watermelon plants and soil. It can be applied to the study of disease diagnosis, plant-pathogen interactions, and effective management.     

Risks of Avian Influenza Transmission in Areas of Intensive Free-Ranging Duck Production with Wild Waterfowl

For decades, southern China has been considered to be an important source for emerging influenza viruses since key hosts live together in high densities in areas with intensive agriculture. However, the underlying conditions of emergence and spread of avian influenza viruses (AIV) have not been studied in detail, particularly the complex spatiotemporal interplay of viral transmission between wild and domestic ducks, two major actors of AIV epidemiology. In this synthesis, we examine the risks of avian influenza spread in Poyang Lake, an area of intensive free-ranging duck production and large numbers of wild waterfowl. Our synthesis shows that farming of free-grazing domestic ducks is intensive in this area and synchronized with wild duck migration. The presence of juvenile domestic ducks in harvested paddy fields prior to the arrival and departure of migrant ducks in the same fields may amplify the risk of AIV circulation and facilitate the transmission between wild and domestic populations. We provide evidence associating wild ducks migration with the spread of H5N1 in the spring of 2008 from southern China to South Korea, Russia, and Japan, supported by documented wild duck movements and phylogenetic analyses of highly pathogenic avian influenza H5N1 sequences. We suggest that prevention measures based on a modification of agricultural practices may be implemented in these areas to reduce the intensity of AIV transmission between wild and domestic ducks. This would require involving all local stakeholders to discuss feasible and acceptable solutions.     

Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development

Introduction of selectable marker genes to transgenic animals could create an inconvenience to further research and may exaggerate public concerns regarding biological safety. The objective of the current study was to excise loxP flanked neo^R in transgenic cloned cattle by transient expression of Cre recombinase. Green fluorescent protein gene (GFP) was incorporated to monitor Cre expression; therefore, Cre-expressed cells could be selected indirectly by fluorescence-activated cell sorting (FACS). The neo^R was removed and Cre expressed transiently in GFP-positive colonies; excision of neo^R was confirmed by single-blastocyst PCR in recloned blastocysts, with neo^R-free fibroblast cells as donors. There was no difference (P > 0.05) in rates of cleavage (76.0% vs. 68.8%) or blastocyst formation (56.6% vs. 52.9%) between recloned embryos with neo^R-free or neo^R-included donors. The differential staining of recloned blastocysts were similar (P >0.05) in terms of total cell number (124 vs. 122) and the ratio of ICM (Inner Cell Mass) to the total cell number (38.1% vs. 38.2%). Furthermore, pregnancy and calving rates were not different (P > 0.05) from those of the control. In conclusion, we successfully excised neo^R from transgenic cloned cattle; the manipulation did not affect the developmental competence of recloned preimplantation embryos. This approach should benefit bioreactor and transgenic research in livestock.