The effect of single nucleotide polymorphism identification strategies on estimates of linkage disequilibrium

At present there is tremendous interest in characterizing the magnitude and distribution of linkage disequilibrium (LD) throughout the human genome, which will provide the necessary foundation for genome-wide LD analyses and facilitate detailed evolutionary studies. To this end, a human high-density single-nucleotide polymorphism (SNP) marker map has been constructed. Many of the SNPs on this map, however, were identified by sampling a small number of chromosomes from a single population, and inferences drawn from studies using such SNPs may be influenced by ascertainment bias (AB). Through extensive simulations, we have found that AB is a potentially significant problem in estimating and comparing LD within and between populations. Specifically, the magnitude of AB is a function of the SNP discovery strategy, number of chromosomes used for SNP discovery, population genetic characteristics of the particular genomic region considered, amount of gene flow between populations, and demographic history of the populations. We demonstrate that a balanced SNP discovery strategy (where equal numbers of chromosomes are sampled from multiple subpopulations) is the optimal study design for generating broadly applicable SNP resources. Finally, we validate our theoretical predictions by comparing our results to publicly available data from ten genes sequenced in 24 African American and 23 European American individuals.     

Synthesis and biological evaluation of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-substituted-3-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)pyrazoles 14a-ae, 16a, 16b, and 21a-c has been prepared and evaluated for their ALK5 inhibitory activity in an enzyme assay and in a cell-based luciferase reporter assay. The 4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-(4-methoxyphenyl)-3-(6-methylpyridin-2-yl)-1H-pyrazole-1-carbothioamide (14n) inhibited ALK5 phosphorylation with IC(50) value of 0.57 nM and showed 94% inhibition at 100 nM in a luciferase reporter assay using HaCaT cells permanently transfected with p3TP-luc reporter construct.     

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola , Erwinia carotovora and Pseudomonas syringae , and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2 -inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).     

Slow and persistent increase of [Ca2+]c in response to ligation of surface IgM in WEHI-231 cells

WEHI-231 and Bal 17 B cell lines are representative models for immature and mature B cells, respectively. Their regulation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) was compared using fura-2 fluorescence ratiometry. The ligation of B cell antigen receptor (BCR) by anti-IgM antibody induced a slow but large increase of [Ca(2+)](c) in WEHI-231 cells while not in Bal 17 cells. The thapsigargin-induced store-operated Ca(2+) entry (SOCE) of Bal 17 cells reached a steady state which was blocked by 2-aminoethoxydiphenyl borate (2-APB). On the contrary, the thapsigargin-induced SOCE of WEHI-231 cells increased continuously, which was accelerated by 2-APB. The increase of [Ca(2+)](c) by BCR ligation was also enhanced by 2-APB in WEHI-231 cells while blocked in Bal 17 cells. The Mn(2+) quenching study showed that the thapsigargin-, or the BCR ligation-induced Ca(2+) influx pathway of WEHI-231 was hardly permeable to Mn(2+). The intractable increase of [Ca(2+)](c) may explain the mechanism of BCR-driven apoptosis of WEHI-231 cells, a well-known model of clonal deletion of autoreactive immature B cells.     

Near Neutral pH Redox Flow Battery with Low Permeability and Long‐Lifetime Phosphonated Viologen Active Species

A highly stable phosphonate‐functionalized viologen is introduced as the redox‐active material in a negative potential electrolyte for aqueous redox flow batteries (ARFBs) operating at nearly neutral pH. The solubility is 1.23 m and the reduction potential is the lowest of any substituted viologen utilized in a flow battery, reaching ?0.462 V versus SHE at pH = 9. The negative charges in both the oxidized and the reduced states of 1,1′‐bis(3‐phosphonopropyl)‐[4,4′‐bipyridine]‐1,1′‐diium dibromide ( BPP?Vi ) effect low permeability in cation exchange membranes and suppress a bimolecular mechanism of viologen decomposition. A flow battery pairing BPP?Vi with a ferrocyanide‐based positive potential electrolyte across an inexpensive, non‐fluorinated cation exchange membrane at pH = 9 exhibits an open‐circuit voltage of 0.9 V and a capacity fade rate of 0.016% per day or 0.00069% per cycle. Overcharging leads to viologen decomposition, causing irreversible capacity fade. This work introduces extremely stable, extremely low‐permeating and low reduction potential redox active materials into near neutral ARFBs.     

High-level production of poly (β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) with a new isolated <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> strain

Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l⁻¹) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO₃ in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l⁻¹) and productivity (0.35 g l⁻¹ h⁻¹), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.     

Role of PCK2 in the proliferation of vascular smooth muscle cells in neointimal hyperplasia

Vascular smooth muscle cell (VSMC) proliferation is a hallmark of neointimal hyperplasia (NIH) in atherosclerosis and restenosis post-balloon angioplasty and stent insertion. Although numerous cytotoxic and cytostatic therapeutics have been developed to reduce NIH, it is improbable that a multifactorial disease can be successfully treated by focusing on a preconceived hypothesis. We, therefore, aimed to identify key molecules involved in NIH via a hypothesis-free approach. We analyzed four datasets ({"type":"entrez-geo","attrs":{"text":"GSE28829","term_id":"28829"}}GSE28829, {"type":"entrez-geo","attrs":{"text":"GSE43292","term_id":"43292"}}GSE43292, {"type":"entrez-geo","attrs":{"text":"GSE100927","term_id":"100927"}}GSE100927, and {"type":"entrez-geo","attrs":{"text":"GSE120521","term_id":"120521"}}GSE120521), evaluated differentially expressed genes (DEGs) in wire-injured femoral arteries of mice, and determined their association with VSMC proliferation in vitro. Moreover, we performed RNA sequencing on platelet-derived growth factor (PDGF)-stimulated human VSMCs (hVSMCs) post-phosphoenolpyruvate carboxykinase 2 (PCK2) knockdown and investigated pathways associated with PCK2. Finally, we assessed NIH formation in Pck2 knockout (KO) mice by wire injury and identified PCK2 expression in human femoral artery atheroma. Among six DEGs, only PCK2 and RGS1 showed identical expression patterns between wire-injured femoral arteries of mice and gene expression datasets. PDGF-induced VSMC proliferation was attenuated when hVSMCs were transfected with PCK2 siRNA. RNA sequencing of PCK2 siRNA-treated hVSMCs revealed the involvement of the Akt-FoxO-PCK2 pathway in VSMC proliferation via Akt2, Akt3, FoxO1, and FoxO3. Additionally, NIH was attenuated in the wire-injured femoral artery of Pck2-KO mice and PCK2 was expressed in human femoral atheroma. PCK2 regulates VSMC proliferation in response to vascular injury via the Akt-FoxO-PCK2 pathway. Targeting PCK2, a downstream signaling mediator of VSMC proliferation, may be a novel therapeutic approach to modulate VSMC proliferation in atherosclerosis.     

Metabolism in 1,3‐propanediol fed‐batch fermentation by a D‐lactate deficient mutant of Klebsiella pneumoniae

Klebsiella pneumoniae HR526, a new isolated 1,3‐propanediol (1,3‐PD) producer, exhibited great productivity. However, the accumulation of lactate in the late‐exponential phase remained an obstacle of 1,3‐PD industrial scale production. Hereby, mutants lacking D ‐lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D ‐lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed‐batch fermentation. In experiments using pure glycerol as feedstock, the 1,3‐PD concentrations, conversion, and productivity increased from 95.39 g L^?1, 0.48 and 1.98 g L^?1 h^?1 to 102. 06 g L^?1, 0.52 mol mol^?1 and 2.13 g L^?1 h^?1, respectively. The diol (1,3‐PD and 2,3‐butanediol) conversion increased from 0.55 mol mol^?1 to a maximum of 0.65 mol mol^?1. Lactate would not accumulate until 1,3‐PD exceeded 84 g L^?1, and the final lactate concentration decreased dramatically from more than 40 g L^?1 to <3 g L^?1. Enzymic measurements showed LDH activity decreased by 89–98% during fed‐batch fermentation, and other related enzyme activities were not affected. NADH/NAD⁺ enhanced more than 50% in the late‐exponential phase as the D ‐lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3‐PD industrial production. Biotechnol. Bioeng. 2009; 104: 965–972. © 2009 Wiley Periodicals, Inc.