A single‐nucleotide polymorphism‐based approach for rapid and cost‐effective genetic wolf monitoring in Europe based on noninvasively collected samples

Noninvasive genetics based on microsatellite markers has become an indispensable tool for wildlife monitoring and conservation research over the past decades. However, microsatellites have several drawbacks, such as the lack of standardisation between laboratories and high error rates. Here, we propose an alternative single‐nucleotide polymorphism (SNP)‐based marker system for noninvasively collected samples, which promises to solve these problems. Using nanofluidic SNP genotyping technology (Fluidigm), we genotyped 158 wolf samples (tissue, scats, hairs, urine) for 192 SNP loci selected from the Affymetrix v2 Canine SNP Array. We carefully selected an optimised final set of 96 SNPs (and discarded the worse half), based on assay performance and reliability. We found rates of missing data in this SNP set of <10% and genotyping error of ~1%, which improves genotyping accuracy by nearly an order of magnitude when compared to published data for other marker types. Our approach provides a tool for rapid and cost‐effective genotyping of noninvasively collected wildlife samples. The ability to standardise genotype scoring combined with low error rates promises to constitute a major technological advancement and could establish SNPs as a standard marker for future wildlife monitoring.     

CXCR4-A Prognostic and Clinicopathological Biomarker for Pancreatic Ductal Adenocarcinoma: A Meta-Analysis

Adenocarcinomas of the pancreatic duct (PDAC) are characteristically aggressive tumors that are extremely challenging to treat as curative surgical resection, the definitive treatment, is seldom possible. Regretably, most patients are diagnosed with metastatic disease at the time of initial presentation. In addition, current chemotherapeutic concepts that are used for advanced disease stages show frustrating results. Thus, there is an urgent need to identify novel therapeutic molecular targets that are associated with PDAC disease. Recently, the chemokine receptor CXCR4 has been demonstrated to be highly expressed in metastatic PDAC. However, the results of the published data on CXCR4 and its association with clinicopathological variables and prognosis in PDAC seem to be heterogeneous. Consequently, to clarify the relevance of CXCR4 as a biomarker in PDAC we performed a comprehensive literature search by using PubMed and Web of Science databases to identify articles that focused on the expression of CXCR4 in PDAC by using immunohistochemistry. Subsequently, data from nine relevant studies, encompassing 1183 patients were extracted, qualitatively assessed, and entered into a meta-analysis. By using a random effects model, the pooled hazard ratio of the seven studies that reported on patients overall survival revealed a correlation between expression of CXCR4 and poor prognosis (HR 1.49; 95% CI: 1.04-2.14; P = 0.03; I² = 74%). Although heterogeneity became evident, subgroup analyses confirmed the prognostic value of CXCR4 in PDAC, especially in high-quality studies that performed multivariate analysis. In addition, meta-analysis revealed a strong association of CXCR4 expression with the UICC stage (OR: 3.40; 95% CI: 1.67-6.92; P = 0.0007; I² = 0%) and metastatic disease (N-status: OR: 2.55; 95% CI: 1.56-4.15; P = 0.0002; I² = 26%; recurrence to the liver: OR: 2.80; 95% CI: 1.48-5.29; P = 0.001; I² = 0%). Taken together, our meta-analysis suggests that CXCR4 represents a useful prognostic biomarker in PDAC and might therefore be evaluated as a potential therapeutic target in the treatment of metastatic cancer disease of the pancreas.     

Uptake and Distribution of Stable Strontium in 26 Cultivars of Three Crop Species: Oats,Wheat, and Barley for Their Potential Use in Phytoremediation

The main objective of this study was to investigate the accumulation and distribution of strontium (Sr) in 26 cultivars of wheat (Triticum aestivum L.), husk oat (Avena sativa L) and naked oat (Avena nuda), and barley (Hordeum vulgare L.) for their potential use in phytoremediation.Sr levels had no effect on the accumulation of shoot biomass at tillering or at maturity. Mean shoot Sr concentration of naked oat and barley at tillering was significantly (P < 0.05) higher than that of wheat; Neimengkeyimai-1, a naked oat cultivar, had the highest Sr concentrations. At maturity, of four naked oat cultivars, Neimengkeyimai-1 had the highest Sr content at all measured Sr levels. Leaves had the highest Sr concentrations, followed by roots and straw, and then grain with the lowest. Mean enrichment coefficients from soil to shoots ranged from 0.521 to 1.343; the percentage of stable Sr removed from the soil to the shoots at harvest time was more than 1.4% after 120 days. Neimengkeyimai-1 could be used as a model for further research to find more effective cultivars; and naked oat plants could be selected for phytoremediation to clean up contaminated soil.     

Quantitative Measurement of Melanoma Spread in Sentinel Lymph Nodes and Survival

Background

Sentinel lymph node spread is a crucial factor in melanoma outcome. We aimed to define the impact of minimal cancer spread and of increasing numbers of disseminated cancer cells on melanoma-specific survival.

Methods and Findings

We analyzed 1,834 sentinel nodes from 1,027 patients with ultrasound node-negative melanoma who underwent sentinel node biopsy between February 8, 2000, and June 19, 2008, by histopathology including immunohistochemistry and quantitative immunocytology. For immunocytology we recorded the number of disseminated cancer cells (DCCs) per million lymph node cells (DCC density [DCCD]) after disaggregation and immunostaining for the melanocytic marker gp100. None of the control lymph nodes from non-melanoma patients (n = 52) harbored gp100-positive cells. We analyzed gp100-positive cells from melanoma patients by comparative genomic hybridization and found, in 45 of 46 patients tested, gp100-positive cells displaying genomic alterations. At a median follow-up of 49 mo (range 3–123 mo), 138 patients (13.4%) had died from melanoma. Increased DCCD was associated with increased risk for death due to melanoma (univariable analysis; p<0.001; hazard ratio 1.81, 95% CI 1.61–2.01, for a 10-fold increase in DCCD + 1). Even patients with a positive DCCD ≤3 had an increased risk of dying from melanoma compared to patients with DCCD = 0 (p = 0.04; hazard ratio 1.63, 95% CI 1.02–2.58). Upon multivariable testing DCCD was a stronger predictor of death than histopathology. The final model included thickness, DCCD, and ulceration (all p<0.001) as the most relevant prognostic factors, was internally validated by bootstrapping, and provided superior survival prediction compared to the current American Joint Committee on Cancer staging categories.

Conclusions

Cancer cell dissemination to the sentinel node is a quantitative risk factor for melanoma death. A model based on the combined quantitative effects of DCCD, tumor thickness, and ulceration predicted outcome best, particularly at longer follow-up. If these results are validated in an independent study, establishing quantitative immunocytology in histopathological laboratories may be useful clinically. Please see later in the article for the Editors'' Summary     

Frontiers in Neurodegeneration – New Insights and Prospects – 20th HUPO BPP Workshop

The HUPO Brain Proteome Project (HUPO BPP) held its 20th workshop in Yokohama, Japan, September 15, 2013. The focus of the autumn workshop was on new insights and prospects of neurodegenerative diseases.     

Gap-Directed Translesion DNA Synthesis of an Abasic Site on Circular DNA Templates by a Human Replication Complex

DNA polymerase ε (pol ε) is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP) site). We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS) of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged “minicircle” DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC), and the accessory proteins replication protein A (RPA). Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.     

In Search of Glacial Refuges of the Land Snail Orcula dolium (Pulmonata,Orculidae) - An Integrative Approach Using DNA Sequence and Fossil Data

Harboring a large number of endemic species, the Alps and the Western Carpathians are considered as major centers of biodiversity. Nonetheless, the general opinion until the turn of the millennium was that both Central European mountain regions did not provide suitable habitat during the Last Glacial Maximum, but were colonized later from southern refuges. However, recent molecular genetic studies provide new evidence for peripheral Alpine refuges. We studied the phylogeography of the calciphilous land snail O. dolium across its distribution in the Alps and the Western Carpathians to assess the amount of intraspecific differentiation and to detect potential glacial refuges. A partial sequence of the mitochondrial COI was analyzed in 373 specimens from 135 sampling sites, and for a subset of individuals, partial sequences of the mitochondrial 16S and the nuclear histone H3 and H4 were sequenced. A molecular clock analysis was combined with a reconstruction of the species’ geographic range history to estimate how its lineages spread in the course of time. In order to obtain further information on the species’ past distribution, we also screened its extensive Pleistocene fossil record. The reconstruction of geographic range history suggests that O. dolium is of Western Carpathian origin and diversified already around the Miocene-Pliocene boundary. The fossil record supports the species’ presence at more than 40 sites during the last glacial and earlier cold periods, most of them in the Western Carpathians and the Pannonian Basin. The populations of O. dolium display a high genetic diversity with maximum intraspecific p-distances of 18.4% (COI) and 14.4% (16S). The existence of various diverged clades suggests the survival in several geographically separated refuges. Moreover, the sequence patterns provide evidence of multiple migrations between the Alps and the Western Carpathians. The results indicate that the Southern Calcareous Alps were probably colonized only during the Holocene.     

A ternary complex consisting of AICD,FE65, and TIP60 down-regulates Stathmin1

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.     

Nucleolipids as Potential Organogelators

Four different series of nucleolipids or bola-nucleolipids were synthesized or re-synthesized. Most of the compounds were studied with respect to their gelation properties toward either water or aromatic, hetero-aromatic, and aliphatic hydrocarbons. Bola-nucleolipids 6 and 7 do not gelate any solvent tested, neither as sole additive nor by adding up to 10 wt% of a 1:1 mixture. The nucleolipid 22 carrying the antiviral acyclovir as a head group proved to be a potent organogelator for aromatic hydrocarbons such as toluene, but not for hetarenes, aliphatic hydrocarbons or water. The mono-tailed nucleolipid 24 exhibits excellent organogelator properties for both aromatic and aliphatic hydrocarbons. These were studied as a function of concentration and temperature.     

Steric and Stereoelectronic Effects of 7-Deazapurine Bases on the Sugar Conformation of 2′-Deoxynucleosides

Abstract

Conformational analyses of the sugar moieties of a series of 7-(8)- substituted 7-deazapurine-2′-deoxynucleosides on the basis of vicinal [H,H] coupling constants is presented using the PSEUROT 6.0 program.