Simultaneous assay of sildenafil and desmethylsildenafil in human plasma using liquid chromatography-tandem mass spectrometry on silica column with aqueous-organic mobile phase

A liquid chromatography-tandem mass spectrometry method was developed for the analysis of sildenafil (SIL) and its metabolite desmethylsildenafil (DMS) in human plasma. Samples were accurately transferred to 96-well plates using a liquid handler (Multiprobe II). Solid-phase extraction was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C8 and cation-exchange mixed-mode sorbent. The extract was injected onto a silica column with an aqueous-organic mobile phase, a combination that was novel for improving the method sensitivity. The low limit of quantitation was 1.0 ng/ml for both SIL and DMS. The method was validated to meet the criteria of current industrial guidance for quantitative bioanalytical methods.     

Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection

Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode. In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope. A cell injector was constructed. Using it, the cell suspension was static, when a voltage for injecting single cells was applied. Histamine in single rat peritoneal mast cells have been identified. Quantitation has been accomplished through the use of calibration curves. The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.     

The crab-eating frog, Rana cancrivora, up-regulates hepatic carbamoyl phosphate synthetase I activity and tissue osmolyte levels in response to increased salinity

The crab-eating frog Rana cancrivora is one of only a handful of amphibians worldwide that tolerate saline waters. They typically inhabit brackish water of mangrove forests of Southeast Asia, but live happily in freshwater and can be acclimated to 75% seawater (25 ppt) or higher. We report here that after transfer of juvenile R. cancrivora from freshwater (1 ppt) to brackish water (10 -->20 or 20 -->25 ppt; 4-8 d) there was a significant increase in the specific activity of the key hepatic ornithine urea cycle enzyme (OUC), carbamoyl phosphate synthetase I (CPSase I). At 20 ppt, plasma, liver and muscle urea levels increased by 22-, 21-, and 11-fold, respectively. As well, muscle total amino acid levels were significantly elevated by 6-fold, with the largest changes occurring in glycine and beta-alanine levels. In liver, taurine levels were 5-fold higher in frogs acclimated to 20 ppt. There were no significant changes in urea or ammonia excretion rates to the environment. As well, the rate of urea influx (J(in) (urea)) and efflux (J(out) (urea)) across the ventral pelvic skin did not differ between frogs acclimated to 1 versus 20 ppt. Taken together, these findings suggest that acclimation to saline water involves the up-regulation of hepatic urea synthesis, which in turn contributes to the dramatic rise in tissue urea levels. The lack of change in urea excretion rates, despite the large increase in tissue-to-water gradients further indicates that mechanisms must be in place to prevent excessive loss of urea in saline waters, but these mechanisms do not include cutaneous urea uptake. Also, amino acid accumulation may contribute to an overall rise in the osmolarity of the muscle tissue, but relative to urea, the contribution is small.     

Atomic contact vectors in protein-protein recognition

The ability to analyze and compare protein-protein interactions on the structural level is critical to our understanding of various aspects of molecular recognition and the functional interplay of components of biochemical networks. In this study, we introduce atomic contact vectors (ACVs) as an intuitive way to represent the physico-chemical characteristics of a protein-protein interface as well as a way to compare interfaces to each other. We test the utility of ACVs in classification by using them to distinguish between homodimers and crystal contacts. Our results compare favorably with those reported by other authors. We then apply ACVs to mine the PDB for all known protein-protein complexes and separate transient recognition complexes from permanent oligomeric ones. Getting at the basis of this difference is important for our understanding of recognition and we achieved a success rate of 91% for distinguishing these two classes of complexes. Although accessible surface area of the interface is a major discriminating feature, we also show that there are distinct differences in the contact preferences between the two kinds of complexes. Illustrating the superiority of ACVs as a basic comparison measure over a sequence-based approach, we derive a general rule of thumb to determine whether two protein-protein interfaces are redundant. With this method, we arrive at a nonredundant set of 209 recognition complexes--the largest set reported so far.     

On the number of genes controlling the grass stage in longleaf pine

The grass stage is an inherent and distinctive developmental trait of longleaf pine (Pinus palustris), in which height growth in the first few years after germination is suppressed. In operational forestry practice the grass stage extends for two to several years and often plays a role in planting failures and decisions to plant alternative species. Interspecies hybrids involving loblolly (P. taeda) and slash (P. elliottii var. elliottii) pines have been investigated as a means to produce planting stock with improved early height growth and to develop backcross populations for advanced generation breeding. We have reevaluated data from several interspecies populations, with the objective of estimating the number of genes contributing to the difference in first-year height growth between longleaf and loblolly pines. Estimates based on means and variances of parental and interspecies hybrid and backcross families suggest a minimum of 4 to 10 genes with standard errors less than half the estimates. These results suggest that the grass stage has evolved through the accumulation of alleles at several loci, each with small effects on various components of first-year height growth. Given the complexity of the grass-stage trait, tree breeders may need to combine genetic marker analysis with recurrent backcross breeding to efficiently develop longleaf pine planting stock for improved reforestation.     

Examining the independent binding assumption for binding of peptide epitopes to MHC-I molecules

MOTIVATION: Various methods have been proposed to predict the binding affinities of peptides to Major Histocompatibility Complex class I (MHC-I) molecules based on experimental binding data. They can be classified into two groups: (1) AIB methods that assume independent contributions of all peptide positions to the binding to MHC-I molecule (e.g. scoring matrices) and (2) general methods which can take into account interactions between different positions (e.g. artificial neural networks). We aim to compare the prediction accuracies of these methods, and quantify the impact of interactions between peptide positions. RESULTS: We compared several previously published and widely used methods and discovered that the best AIB methods gave significantly better predictions than three previously published general methods, possibly due to the lack of a sufficient training data for the general methods. The best results, however, were achieved with our newly developed general method, which combined a matrix describing independent binding with pair coefficients describing pair-wise interactions between peptide positions. The pair coefficients consistently but only slightly improved prediction accuracy, and were much smaller than the matrix entries. This explains why neglecting them-as is done in AIB methods-can still lead to good predictions. AVAILABILITY: The new prediction model is implemented at http://zlab.bu.edu/SMM. The underlying matrix and pair coefficients are also available as supplementary materials.     

Helper-independent, L-selectinlow CD8+ T cells with broad anti-tumor efficacy are naturally sensitized during tumor progression

We recently reported that the CD4(+) T cell subset with low L-selectin expression (CD62L(low)) in tumor-draining lymph nodes (TDLN) can be culture activated and adoptively transferred to eradicate established pulmonary and intracranial tumors in syngeneic mice, even without coadministration of IL-2. We have extended these studies to characterize the small subset of L-selectin(low) CD8(+) T cells naturally present in TDLN of mice bearing weakly immunogenic tumors. Isolated L-selectin(low) CD8(+) T cells displayed the functional phenotype of helper-independent T cells, and when adoptively transferred could consistently eradicate, like L-selectin(low) CD4(+) T cells, both established pulmonary and intracranial tumors without coadministration of exogenous IL-2. Whereas adoptively transferred L-selectin(low) CD4(+) T cells were more potent on a cell number basis for eradicating 3-day intracranial and s.c. tumors, L-selectin(low) CD8(+) T cells were more potent against advanced (10-day) pulmonary metastases. Although the presence of CD4(+) T cells enhanced generation of L-selectin(low) CD8(+) effector T cells, the latter could also be obtained from CD4 knockout mice or normal mice in vivo depleted of CD4(+) T cells before tumor sensitization. Culture-activated L-selectin(low) CD8(+) T cells did not lyse relevant tumor targets in vitro, but secreted IFN-gamma and GM-CSF when specifically stimulated with relevant tumor preparations. These data indicate that even without specific vaccine maneuvers, progressive tumor growth leads to independent sensitization of both CD4(+) and CD8(+) anti-tumor T cells in TDLN, phenotypically L-selectin(low) at the time of harvest, each of which requires only culture activation to unmask highly potent stand-alone effector function.     

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

For some Cas nucleases, trans-cleavage activity triggered by CRISPR/Cas-mediated cis-cleavage upon target nucleic acid recognition has been explored for diagnostic detection. Portable single and multiplex nucleic acid-based detection is needed for crop pathogen management in agriculture. Here, we harnessed and characterized RfxCas13d as an additional CRISPR/Cas nucleic acid detection tool. We systematically characterized AsCas12a, LbCas12a, LwaCas13a, and RfxCas13d combined with isothermal amplification to develop a CRISPR/Cas nucleic acid-based tool for single or multiplex pathogen detection. Our data indicated that sufficient detection sensitivity was achieved with just a few copies of DNA/RNA targets as input. Using this tool, we successfully detected DNA from Fusarium graminearum and Fusarium verticillioides and RNA from rice black-streaked dwarf virus in crude extracts prepared in the field. Our method, from sample preparation to result readout, could be rapidly and easily deployed in the field. This system could be extended to other crop pathogens, including those that currently lack a detection method and have metabolite profiles that make detection challenging. This nucleic acid detection system could also be used for single-nucleotide polymorphism genotyping, transgene detection, and qualitative detection of gene expression in the field.

     

补骨脂素抑制膀胱癌T24细胞增殖和迁移的作用及机制研究

目的探讨补骨脂素对人膀胱癌T24细胞存活率、细胞周期、细胞凋亡和迁移的影响及其分子机制。 方法分别用细胞培养液、3‰二甲基亚砜（DMSO）和不同浓度（10、30、50、100 μg/mL）补骨脂素处理膀胱癌细胞分成对照组、DMSO组和补骨脂素组,CCK-8检测细胞存活率。流式细胞术检测细胞周期和细胞凋亡。划痕实验检测划痕愈合率。RT-qPCR法检测磷脂酰肌醇3激酶（PI3K）和蛋白激酶B （AKT） mRNA表达水平、Western blot法检测PI3K和AKT蛋白的表达及磷酸化情况。多组间比较采用单因素方差分析,组间两两比较采用LSD-t检验。 结果与DMSO组比较,除10 μg/mL补骨脂素作用24 h外,其余浓度补骨脂素作用不同时间的细胞存活率随着补骨脂素浓度增高、作用时间延长而逐渐降低（P < 0.05）。与DMSO组比较,30、100 μg/mL补骨脂素干预24 h后,G1期细胞比例增多,G2/M期比例减少,细胞凋亡率[（9.16±0.97）%、（15.45±1.57）%比（1.02±0.36）%]升高,划痕愈合率[24 h：（45.00±3.44）%、（27.60±2.21）%比（66.10±2.61）%,48 h：（70.00 ± 3.40）%、（45.17±2.44）%比（85.17±3.85）%]降低,PI3K、AKT mRNA表达以及PI3K、AKT蛋白表达水平和磷酸化水平均降低（P均< 0.05）。 结论补骨脂素降低膀胱癌细胞存活率、阻滞细胞周期、诱导细胞凋亡和抑制细胞迁移,其机制可能与下调PI3K、AKT mRNA、蛋白表达及磷酸化水平有关。