Simultaneous quantification of emtricitabine and tenofovir nucleotides in peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Emtricitabine (FTC) and tenofovir (TFV) are widely used antiviral agents that require intracellular phosphorylation to become active. This article describes the development and validation of an assay for the simultaneous quantification of FTC mono-, di- and triphosphate (FTC-MP, -DP and -TP), TFV and TFV mono- and diphosphate (TFV-MP and -DP) in peripheral blood mononuclear cells. Reference compounds and internal standards were obtained by thermal degradation of FTC-TP, TFV-DP, stable isotope-labeled TFV-DP and stable isotope-labeled cytosine triphosphate. Cells were lysed in methanol:water (70:30, v/v) and the extracted nucleotides were analyzed using weak anion-exchange chromatography coupled with tandem mass spectrometry. Calibration ranges in PBMC lysate from 0.727 to 36.4, 1.33 to 66.4 and 1.29 to 64.6 nM for FTC-MP, FTC-DP and FTC-TP and from 1.51 to 75.6, 1.54 to 77.2 and 2.54 to 127 nM for TFV, TFV-MP and TFV-DP, respectively, were validated. Accuracies were within ?10.3 and 16.7% deviation at the lower limit of quantification at which the coefficients of variation were less than 18.2%. At the other tested levels accuracies were within ?14.3 and 9.81% deviation and the coefficients of variation lower than 14.7%. The stability of the compounds was assessed under various analytically relevant conditions. The method was successfully applied to clinical samples.     

The prognostic value of baseline erosions in undifferentiated arthritis

Introduction  

Undifferentiated arthritis (UA) has a variable disease course; 40 to 50% of UA patients remit spontaneously, while 30% develop rheumatoid arthritis (RA). Identifying the UA patients who will develop RA is essential to initiate early disease-modifying anti-rheumatic drug (DMARD) therapy. Although the presence of bone erosions at baseline is predictive for a severe destructive disease course in RA, the prognostic importance of erosive joints for disease outcome in UA is unknown. This study evaluates the predictive value of erosive joints for the disease outcome in UA as measured by RA development and disease persistency.     

Functional screening of willow alleles in Arabidopsis combined with QTL mapping in willow (Salix) identifies SxMAX4 as a coppicing response gene

Willows (Salix spp.) are important biomass crops due to their ability to grow rapidly with low fertilizer inputs and ease of cultivation in short‐rotation coppice cycles. They are relatively undomesticated and highly diverse, but functional testing to identify useful allelic variation is time‐consuming in trees and transformation is not yet possible in willow. Arabidopsis is heralded as a model plant from which knowledge can be transferred to advance the improvement of less tractable species. Here, knowledge and methodologies from Arabidopsis were successfully used to identify a gene influencing stem number in coppiced willows, a complex trait of key biological and industrial relevance. The strigolactone‐related More AXillary growth (MAX) genes were considered candidates due to their role in shoot branching. We previously demonstrated that willow and Arabidopsis show similar response to strigolactone and that transformation rescue of Arabidopsis max mutants with willow genes could be used to detect allelic differences. Here, this approach was used to screen 45 SxMAX1, SxMAX2, SxMAX3 and SxMAX4 alleles cloned from 15 parents of 11 mapping populations varying in shoot‐branching traits. Single‐nucleotide polymorphism (SNP) frequencies were locus dependent, ranging from 29.2 to 74.3 polymorphic sites per kb. SxMAX alleles were 98%–99% conserved at the amino acid level, but different protein products varying in their ability to rescue Arabidopsis max mutants were identified. One poor rescuing allele, SxMAX4D, segregated in a willow mapping population where its presence was associated with increased shoot resprouting after coppicing and colocated with a QTL for this trait.     

Strigolactones are transported through the xylem and play a key role in shoot architectural response to phosphate deficiency in nonarbuscular mycorrhizal host Arabidopsis

The biosynthesis of the recently identified novel class of plant hormones, strigolactones, is up-regulated upon phosphate deficiency in many plant species. It is generally accepted that the evolutionary origin of strigolactone up-regulation is their function as a rhizosphere signal that stimulates hyphal branching of arbuscular mycorrhizal fungi. In this work, we demonstrate that this induction is conserved in Arabidopsis (Arabidopsis thaliana), although Arabidopsis is not a host for arbuscular mycorrhizal fungi. We demonstrate that the increase in strigolactone production contributes to the changes in shoot architecture observed in response to phosphate deficiency. Using high-performance liquid chromatography, column chromatography, and multiple reaction monitoring-liquid chromatography-tandem mass spectrometry analysis, we identified two strigolactones (orobanchol and orobanchyl acetate) in Arabidopsis and have evidence of the presence of a third (5-deoxystrigol). We show that at least one of them (orobanchol) is strongly reduced in the putative strigolactone biosynthetic mutants more axillary growth1 (max1) and max4 but not in the signal transduction mutant max2. Orobanchol was also detected in xylem sap and up-regulated under phosphate deficiency, which is consistent with the idea that root-derived strigolactones are transported to the shoot, where they regulate branching. Moreover, two additional putative strigolactone-like compounds were detected in xylem sap, one of which was not detected in root exudates. Together, these results show that xylem-transported strigolactones contribute to the regulation of shoot architectural response to phosphate-limiting conditions.     

From the Western Alps across Central Europe: Postglacial recolonisation of the tufa stream specialist Rhyacophila pubescens (Insecta,Trichoptera)

Background

Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites.

Results

While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a "Dorylus molestus-like" and a "Dorylus wilverthi-like" form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids.

Conclusions

We conclude that hybridization and gene-flow between the two army ant species D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.     

Edible Lepidoptera in Mexico: Geographic distribution, ethnicity, economic and nutritional importance for rural people

In this paper, we reported the butterflies and moths that are consumed in Mexico. We identified 67 species of Lepidoptera that are eaten principally in their larval stage in 17 states of Mexico. These species belong to 16 families: Arctiidae, Bombycidae, Castniidae, Cossidae, Geometridae, Hepialidae, Hesperiidae, Lasiocampidae, Noctuidae, Nymphalidae, Papilionidae, Pieridae, Pyralidae, Saturniidae, Sesiidae, and Sphingidae. Saturniidae, Pieridae, Noctuidae and Nymphalidae were the more species consumed with 16, 11, 9, and 8 species, respectively. The genera with the largest numbers of species were: Phassus, Phoebis, Hylesia and Spodoptera, with three species. Their local distribution, corresponding to each state of Mexico, is also presented.     

Duodenal mucosal risk markers in patients with familial adenomatous polyposis: effects of celecoxib/ursodeoxycholic acid co-treatment and comparison with patient controls

Background

Familial adenomatous polyposis (FAP) is a disease characterized by the development of hundreds to thousands of adenomatous polyps in the colorectum early in life. Virtually all patients with FAP will develop colorectal cancer before the age of 40 to 50 years, unless prophylactic colectomy is performed, which significantly improves their prognosis. The mortality pattern has changed and duodenal cancer now is one of the main cancer-related causes of death in these patients. Practically all patients with FAP develop premalignant duodenal adenomas, which may develop to duodenal cancer in approximately 3-7% of patients. Duodenal cancer in patients with FAP has a poor prognosis. The clinical challenge is to identify patients at high-risk for duodenal carcinoma. Chemoprevention would be desirable to avoid duodenectomy. The main goal of this study is to identify risk markers in normal duodenal mucosa of patients with FAP, that could help identify patients at increased risk for malignant transformation.

Methods

Messenger RNA (mRNA) levels of glutathione S-transferase A1 (GSTA1), glutathione S-transferase P1 (GSTP1), KIAA1199, E-cadherin, peroxisome proliferative activated receptor δ (PPARδ), caspase-3, cyclin D1, β-catenin, and cyclooxygenase-2 (COX-2) were measured in duodenal mucosa, using the QuantiGene 2.0 Plex assay. Levels in normal appearing mucosa of patients with FAP (n?=?37) were compared with levels in non-FAP patient controls (n?=?16). In addition, levels before and after treatment with either celecoxib & ursodeoxycholic acid (UDCA, n?=?14) or celecoxib & placebo (n?=?13) were evaluated in patients with FAP.

Results

mRNA levels of glutathione S-transferase A1 (28.16% vs. 38.24%, p?=?0.008) and caspase-3 (3.30% vs. 5.31%, p?=?0.001) were significantly lower in patients with FAP vs. non-FAP patient controls, respectively. COX-2 mRNA levels in normal duodenal mucosa of patients with FAP were found to be unexpectedly low. None of the potential risk markers was influenced by celecoxib or celecoxib & UDCA.

Conclusions

Protection against toxins and carcinogens (GSTA1) and apoptosis (caspase-3) is low in patients with FAP, which could contribute to increased susceptibility for malignant transformation of duodenal mucosa.

Trial registration

http://ClinicalTrials.gov number NCT00808743

     

Auxin, cytokinin and the control of shoot branching

BACKGROUND: It has been known for many decades that auxin inhibits the activation of axillary buds, and hence shoot branching, while cytokinin has the opposite effect. However, the modes of action of these two hormones in branching control is still a matter of debate, and their mechanisms of interaction are equally unresolved. SCOPE: Here we review the evidence for various hypotheses that have been put forward to explain how auxin and cytokinin influence axillary bud activity. In particular we discuss the roles of auxin and cytokinin in regulating each other's synthesis, the cell cycle, meristem function and auxin transport, each of which could affect branching. These different mechanisms have implications for the main site of hormone action, ranging from systemic action throughout the plant, to local action at the node or in the bud meristem or leaves. The alternative models have specific predictions, and our increasing understanding of the molecular basis for hormone transport and signalling, cell cycle control and meristem biology is providing new tools to enable these predictions to be tested.