Synthesis of Six-Membered Nucleoside Analogs. Part 1: Pyrimidine Nucleosides Based on the 1,3-Dioxane Ring System

Abstract

The synthesis of pyrimidine nucleosides, cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]uracil (4) cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]thymine (5) and cis-N-1-[(2-hydroxymethyl)-1,3-dioxan-5-yl]cytosine (6) and their corresponding trans isomers is described. Compound 4 showed modest, selective activity against human immunodeficiency virus in acutely infected primary human lymphocytes.     

Alkyl Polyglucoside (APG) Amendment for Improving the Phytoremediation of Pb-PAH Contaminated Soil by the AquaticPlant Scirpus triqueter

This study assessed the potential abilities of Scirpus triqueter for phytoremediation of soils contaminated with Pb-PAHs, amended with environment-friendly surfactant alkyl polyglucoside (APG). The effects of APG on the removal of PAHs from soil and the plant uptake and translocation of Pb were tested with plant growth and soil enzymatic activities. Experiments demonstrated that APG has an ability to facilitate PAH degradation and Pb uptake in the plant body at appropriate concentrations (20–40mg L^?1). The highest PAH removal rate was observed in 30 mg L^?1 APG treatment, and the highest accumulation of Pb was detected as 40 mg L^?1 APG. Experiments documented the effects of APG on plant growth, soil enzymatic activity, bioaccumulation and translocation of Pb in Scirpus triqueter. Results indicated that the addition of appropriate APG enhanced PAH removal rate and increased plant uptake and translocation of Pb.     

A Fast Workflow for Identification and Quantification of Proteomes

The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.The performance of mass spectrometry has been improved tremendously over the last few years (1–3), making mass spectrometry-based proteomics a viable approach for large-scale protein analysis in biological research. Scientists around the world are striving to fulfill the promise of identifying and quantifying almost all gene products expressed in a cell line or tissue. This would make mass spectrometry-based protein analysis an approach that is compatible to the second-generation mRNA deep-seq technique (4, 5).Two liquid chromatography (LC)-MS strategies have been employed to achieve deep proteome coverage. One is a single run with a long chromatography column and gradient to take advantage of the resolving power of HPLC to reduce the complexity of peptide mixtures; the other is a sequential run with two-dimensional separation (typically ion-exchange and reverse phase) to reduce peptide complexity. It was reported by two laboratories that 2761 and 4500 proteins were identified with a 10 h chromatography gradient on a dual pressure linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap Velos)(6–8). Similarly, 3734 proteins were identified using a 8 h gradient on a 2 m long column with a hybrid triple quadrupole - time of flight (Q-TOF, AB sciex 5600 Q-TOF)(9) mass spectrometer. The two-dimensional approach has yielded more identification with longer time. For example, 10,006 proteins (representing over 9000 gene products, GPs)¹ were identified in U2OS cell (10), and 10,255 proteins (representing 9207 GPs) from HeLa cells (11). It took weeks (for example, 2–3 weeks) of machine running time to achieve such proteome coverage, pushing proteome analysis to the level that is comparable to mRNA-seq. With the introduction of faster machines, human proteome coverage now has reached the level of 7000–8500 proteins (representing 7000–8000 GPs) in 3 days (12). Notwithstanding the impressive improvement, the current approach using long column and long gradient suffers from inherent limitations: it takes long machine running time and it is challenging to keep reproducibility among repeated runs. Thus, current throughput and reproducibility have hindered the application of in-depth proteomics to traditional biological researches. A timesaving approach is in urgent need.In this study, we used the first-dimension (1D) short pH 10 RP prefractionation to reduce the complexity of the proteome (13), followed by sequential 30 min second-dimension (2D) short pH 3 reverse phase-(RP)-LC-MS/MS runs for protein identification (14). The results demonstrated that it is possible to identify 8000 gene products from mammalian cells within 12 h of total MS measurement time by applying this dual-short 2D-RPLC-MS/MS strategy (Fast sequencing, Fast-seq). The robustness of the strategy was revealed by parallel testing on different MS systems including quadrupole orbitrap mass spectrometer (Q-Exactive), hybrid Q-TOF (Triple-TOF 5600), and dual pressure linear ion-trap orbitrap mass spectrometer (LTQ-Orbitrap Velos), indicating the inherent strength of the approach as to merely taking advantage of the better MS instruments. This strategy increases the efficiency of MS sequencing in unit time for the identification of proteins. We achieved identification of 2200 proteins/30 mins on LTQ-Orbitrap Velos, 2800 proteins/30 mins on Q-Exactive and Triple-TOF 5600 respectively. We further optimized Fast-seq and worked out a quantitative-version of the Fast-seq workflow: Fast-quantification (Fast-quan) and applied it for protein abundance quantification in HUVEC cell that was treated with a drug candidate MLN4924 (a drug in phase III clinical trial). We were able to quantify > 6700 GPs in 1 day of MS running time and found 99 proteins were up-regulated with high confidence. We expect this efficient alternative approach for in-depth proteome analysis will make the application of MS-based proteomics more accessible to biological applications.     

Male and female Popillia quadriguttata (Fabricius) and Protaetia brevitarsis (Lewis) (Coleoptera: Scarabaeidae) response to Japanese beetle floral and pheromone lures

Popillia quadriguttata (Fabricius), and Protaetia brevitarsis (Lewis) adults were captured with Japanese beetle, Popillia japonica Newman, sex attractant and floral lures at Changchun, China during July–August 2012. The floral lure (phenethyl propionate:eugenol:geraniol, 3:7:3) was attractive to male and female P. quadriguttata (AV: 1.2 ± 0.9; 1.1 ± 0.3; total: 2.3 ± 0.8), and was similar in attraction to the combination of the sex attractant (SA) [(R, Z)-5-(1-decenyl) dihydro-2(3H)-furanone] plus the floral lure for male (1.60 ± 0.2), female (1.30 ± 1.1) and total captures (2.9 ± 3.0). However, the SA alone captured only males in much higher numbers than when combined with the floral lure (10.0 ± 6.4). In a separate earlier test, the greatest number of P. quadriguttata males (12.5 ± 3.0), female (12.2 ± 1.5) and total captures (24.7 ± 2.5) was in yellow, laboratory-made, bottle traps. The floral lure also attracted female Pro. brevitarsis (10.0 ± 3.4), while the SA attracted only few male beetles (1.0 ± 0.2). The combination SA + floral lure captured similar females (11.0 ± 2.0) and total (14.2 ± 2.2) Pro. brevitarsis as the floral lure alone. Two butterflies, Colias erate poliographus (Motschulsky) and Pieris rapae (Linnaeus), were also attracted to the floral lure. These studies indicate a potential for replacing pesticides by using the Japanese beetle lures for monitoring and control of several insects in China, and that they would be useful in monitoring and eradication of two potential scarab pests, P. quadriguttata and Pro. brevitaris, in the United States and Europe.     

Isolation and molecular analysis of genes Stpk-V2 and Stpk-V3 homologous to powdery mildew resistance gene Stpk-V in a Dasypyrum villosum accession and its derivatives

Wheat-Dasypyrum villosum translocated chromosomes T6V#2S?6AL and T6V#4S?6DL are known to confer excellent resistance to wheat powdery mildew (PM). However, it is difficult to distinguish the two sources of PM resistance genes through multi-pathotype testing because to date no virulence for them has been found. To reveal the relationship between the PM resistance genes from the two translocations, the sequence of the Stpk-V gene, a key member of powdery mildew resistance locus Pm21, was used as a reference to isolate homologous genes from a D. villosum accession No.1026 and its derivatives 6V#4(6D) disomic substitution (DS) line RW15 and T6V#4S?6DL translocation line Pm97033. Two genes Stpk-V2 and Stpk-V3 were cloned from No.1026. Sequence alignment showed that Stpk-V2 and Stpk-V3 shared 98.2 % and 96.2 % of their DNA and 99.3 % and 100 % of their amino acids in identity with Stpk-V. Compared with Stpk-V, a 22-bp direct sequence repeat and a miniature inverted-repeat transposable element (MITE) were found in the intron 4 of Stpk-V2 and Stpk-V3, respectively. However, Stpk-V2 was not present in DS line RW15 and translocation line Pm97033 based on the PCR result, indicating that Stpk-V2 did not contribute to the PM resistance of RW15 and Pm97033. In the promoter region, a 78-bp insertion was found not only in Stpk-V2 and Stpk-V3, but also in its orthologous gene Stpk-A of wheat. In addition, there was a 17 bp/8 bp deletion/insertion in the putative promoter of Stpk-V3 in comparison with that of Stpk-V/Stpk-V2. Real-time quantitative RT-PCR analysis indicated that the expression levels of Stpk-V and Stpk-V3 genes in the translocation lines were induced by the pathogen, but Stpk-V had a higher expression level than Stpk-V3 at 12 h after inoculation with Bgt. The diversity of Stpk-V gene will help to explore new resistance genes to PM in D. villosum for wheat breeding.     

Lipopolysaccharide neutralization by a novel peptide derived from phosvitin

Lipopolysaccharide (LPS), also known as endotoxin, is the primary trigger of sepsis, which is associated with high mortality in patients. No therapeutic agents are currently efficacious enough to protect patients from sepsis characterized by LPS-mediated tissue damage and organ failure. Previously, a phosvitin-derived peptide, Pt5, which consists of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. In this study, we have generated six mutants by site-directed mutagenesis based on the sequence of Pt5, and found that one of the six mutants, Pt5e, showed the strongest bactericidal activities against Escherichia coli and Staphylococcus aureus. We then demonstrated that Pt5e was able to bind to LPS and lipoteichoic acid (LTA). More importantly, we showed that Pt5e significantly inhibited LPS-induced tumor-necrosis factor (TNF)-α and interleukin (IL)-1β release from murine RAW264.7 cells and considerably reduced serum TNF-α and IL-1β levels in mice. Additionally, Pt5e protected the liver from damage by LPS, and remarkably promoted the survival rate of the endotoxemia mice. Furthermore, Pt5e displayed no cytotoxicity to murine RAW264.7 macrophages and no hemolytic activity toward human red blood cells. These data together indicate that Pt5e is an endotoxin-neutralizing agent with a therapeutic potential in clinical treatment of LPS-induced sepsis.     

hPNAS-4 inhibits proliferation through S phase arrest and apoptosis: underlying action mechanism in ovarian cancer cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.