New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

MMS2plot: An R Package for Visualizing Multiple MS/MS Spectra for Groups of Modified and Non‐Modified Peptides

A large number of post‐translational modifications (PTMs) in proteins are buried in the unassigned mass spectrometric (MS) spectra in shot‐gun proteomics datasets. Because the modified peptide fragments are low in abundance relative to the corresponding non‐modified versions, it is critical to develop tools that allow facile evaluation of assignment of PTMs based on the MS/MS spectra. Such tools will preferably have the ability to allow comparison of fragment ion spectra and retention time between the modified and unmodified peptide pairs or group. Herein, MMS2plot, an R package for visualizing peptide‐spectrum matches (PSMs) for multiple peptides, is described. MMS2plot features a batch mode and generates the output images in vector graphics file format that facilitate evaluation and publication of the PSM assignment. MMS2plot is expected to play an important role in PTM discovery from large‐scale proteomics datasets generated by liquid chromatography‐MS/MS. The MMS2plot package is freely available at https://github.com/lileir/MMS2plot under the GPL‐3 license.     

Over‐Expression of RNA Processing,Heat Shock,and DNA Repair Proteins in Breast Tumor Compared to Normal Tissue

This study identifies the main changes in protein expression in human breast tumors compared to normal breast tissue. Malignant tumors (32) and normal breast tissue samples (23), from formaldehyde‐fixed, paraffin‐embedded specimens are subjected to discovery proteomics using liquid chromatography/tandem mass spectrometry, with spectral counts for quantitation. The dataset contains 1406 proteins. Differential expression is measured using a method that takes advantage of estimates of the percentage of tumor on a slide. This analysis shows that the major classes of proteins over‐expressed by tumors are RNA‐binding, heat shock and DNA repair proteins. RNA‐binding proteins, including heterogeneous nuclear ribonucleoproteins (HNRNPs), SR splice factors (SRSF) and elongation factors form the largest group. Comparison with results from another study demonstrates that the RNA‐binding proteins are associated specifically with malignant transformation, rather than with cell proliferation. HNRNP and SRSF proteins help define splice sites in normal cells. Their over‐expression may dysregulate splicing, which in turn has the potential to promote malignant transformation.     

Functional analysis of RPS27 mutations and expression in melanoma

Next‐generation sequencing has enabled genetic and genomic characterization of melanoma to an unprecedent depth. However, the high mutational background plus the limited depth of coverage of whole‐genome sequencing performed on cutaneous melanoma samples make the identification of novel driver mutations difficult. We sought to explore the somatic mutation portfolio in exonic and gene regulatory regions in human melanoma samples, for which we performed targeted sequencing of tumors and matched germline DNA samples from 89 melanoma patients, identifying known and novel recurrent mutations. Two recurrent mutations found in the RPS27 promoter associated with decreased RPS27 mRNA levels in vitro. Data mining and IHC analyses revealed a bimodal pattern of RPS27 expression in melanoma, with RPS27‐low patients displaying worse prognosis. In vitro characterization of RPS27‐high and RPS27‐low melanoma cell lines, as well as loss‐of‐function experiments, demonstrated that high RPS27 status provides increased proliferative and invasive capacities, while low RPS27 confers survival advantage in low attachment and resistance to therapy. Additionally, we demonstrate that 10 other cancer types harbor bimodal RPS27 expression, and in those, similarly to melanoma, RPS27‐low expression associates with worse clinical outcomes. RPS27 promoter mutation could thus represent a mechanism of gene expression modulation in melanoma patients, which may have prognostic and predictive implications.     

Epidermal keratin 5 expression and distribution is under dermal influence

Human skin melanin pigmentation is regulated by systemic and local factors. According to the type of melanin produced by melanocytes, the transfer and degradation of melanosomes differ, thus accounting for most variations between ethnicities. We made the surprising observation that in a drastically changed environment, white and black phenotypes are reversible since Caucasian skin grafted onto nude mice can become black with all black phenotypic characteristics. Black xenografts differed essentially from other grafts by the levels of epidermal FGF‐2 and keratin 5. In vitro analysis confirmed that FGF‐2 directly regulates keratin 5. Interestingly, this phenomenon may be involved in human pathology. Keratin 5 mutations in Dowling–Degos Disease (DDD) have already been associated with the pheomelanosome–eumelanosome transition. In a DDD patient, keratin 5 was expressed in the basal and spinous layers, as observed in black xenografts. Furthermore, in a common age‐related hyperpigmentation disorder like senile lentigo (SL), keratin 5 distribution is also altered. In conclusion, modulation of keratin 5 expression and distribution either due to mutations or factors may account for the development of pigmentary disorders.     

Analysis of microRNA expression profiles in porcine PBMCs after LPS stimulation

In the present study, we used microRNA (miRNA) sequencing to discover and explore the expression profiles of known and novel miRNAs in 1000 ng/ml LPS stimulated for 8 h vis-à-vis non-stimulated (i.e. control) PBMCs isolated from the blood of healthy pigs. A total of 291 known miRNAs were bio-computationally identified in porcine PBMCs, and 228 novel miRNAs (not enlisted in the swine mirBase) were identified. Among these miRNAs, ssc-miR-148a-3p, ssc-let-7g, ssc-let-7f, 3_8760, ssc-miR-26a, ssc-miR-451, ssc-miR-21, ssc-miR-30d, ssc-miR-99a and ssc-miR-103 were the top 10 most abundant miRNAs in porcine PBMCs. Through miRNA differential analysis combined with quantitative PCR, we found the expressions of ssc-miR-122, ssc-miR-129b, ssc-miR-17-5p and ssc-miR-152 were significantly changed in porcine PBMCs after LPS stimulation. Furthermore, targets prediction and function analysis indicated a significant enrichment in gene ontology functional categories related to diseases, immunity and inflammation. In conclusion, this study on profiling of miRNAs expressed in LPS-stimulated PBMCs provides an important reference point for future studies on regulatory roles of miRNAs in porcine immune system.     

The impacts of warming and nitrogen addition on competitive ability of native and invasive populations of Plantago virginica

全球变化因子(如增温和氮沉降)可能会影响生物入侵,但是这些因子如何影响入侵物种的表现并进一步调节入侵物种与本地竞争者之间的相互作用仍不清楚。本文通过为期五个月的温室实验,研究了增温(开顶式增温箱,+0.62°C)和氮添加(4.2 g N m⁻²)对入侵物种北美 车前(Plantago virginica)原产地和入侵地种群与本地车前草(Plantago asiatica)竞争的影响。实验结果表明,在增温及其与氮添加处理(W × N) 的相互作用下,P. virginica的入侵种群(PV-In)和原产地种群(PV-Na)在与本地竞争者P. asiatica竞争时具有不同的生物量分配策略。其中,PV-Na在与P. asiatica竞争时增加了地下生物量,而PV-In增加了地上生物量。我们还发现,P. virginica对增温和氮添加比P. asiatica的反应更强 烈。增温显著降低了P. virginica的竞争能力,这表明P. virginica比P. asiatica对增温的响应更为敏感。同样,在竞争条件下,氮添加及 其和增温交互作用减少了PV-In地下生物量,但增加了PV-Na地上和总生物量。这些发现表明,P. virginica在入侵过程中改变了生物量分配 策略,PV-In展示出更具弹性的竞争能力以适应环境变化(特别是增温)。这些发现可能有助于我们预测气候变化下的植物入侵并制定相应的 管理策略。     

Variation in the methods leads to variation in the interpretation of biodiversity–ecosystem multifunctionality relationships

生物多样性常常和生态系统多功能性(生态系统同时提供多个生态系统功能的能力)正相关。然而,生物多样性与生态系统多功能性的关系是否依赖于生态系统功能的数目有诸多争议。其中,生物多样性对生态系统多功能性的影响或许不随生态系统功能数目的变化而变化,或者随生态系统功能数目的增多而增强。我们期望通过研究不同生态系统多功能性指数的统计原理来解决这些争议。 我们使用了模型模拟和一系列来自不同空间尺度(从局域到全球)和不同生物群系(温带和高寒草地、森林和干旱地)的经验数据。我们回顾了量化生态系统多功能性的三种方法,包括平均值法、加和法和阈值法。我们发现随着生态系统功能数目的增加,生物多样性与生态系统多功能性的关系要么不变,要么增强。这些结果可由平均和加和的多功能性指数的统计原理来解释。具体来讲,当利用生态系统功能的平均值计算多功能性指数时,由于多样性对多功能性的效应等于多样性对单个生态系统功能效应的平均值,所以不会随生态系统功能数目的变化而变化。同样的道理,当利用单个生态系统的加和值计算多功能性指数时,多样性的效应会随着生态系统功能数目的增加而增强。我们提出了一个改进的多功能性指数,将平均或加和多功能性指数转化为标准化的多功能性指数, 以便于对不同研究的结果进行比较。此外,我们提出了基于变量数值范围的标准化方法来解决阈值法的数学假象问题(多样性效应随生态系统功能数目的增加而增强)。我们的研究结果表明,量化多功能性指数的方法不同,结果也不同。因此,有必要加深对不同方法数理基础的理解。而标准化的多功能性指数为比较不同研究中的生物多样性与生态系统多功能性的关系提供了有效的方法。