Label-free Relative Quantification of Co-eluting Isobaric Phosphopeptides of Insulin Receptor Substrate-1 by HPLC-ESI-MS/MS

Intracellular signal transduction is often regulated by transient protein phosphorylation in response to external stimuli. Insulin signaling is dependent on specific protein phosphorylation events, and analysis of insulin receptor substrate-1 (IRS-1) phosphorylation reveals a complex interplay between tyrosine, serine, and threonine phosphorylation. The phospho-specific antibody-based quantification approach for analyzing changes in site-specific phosphorylation of IRS-1 is difficult due to the dearth of phospho-antibodies compared with the large number of known IRS-1 phosphorylation sites. We previously published a method detailing a peak area-based mass spectrometry approach, using precursor ions for peptides, to quantify the relative abundance of site-specific phosphorylation in the absence or presence of insulin. We now present an improvement wherein site-specific phosphorylation is quantified by determining the peak area of fragment ions respective to the phospho-site of interest. This provides the advantage of being able to quantify co-eluting isobaric phosphopeptides (differentially phosphorylated versions of the same peptide), allowing for a more comprehensive analysis of protein phosphorylation. Quantifying human IRS-1 phosphorylation sites at Ser303, Ser323, Ser330, Ser348, Ser527, and Ser531 shows that this method is linear (n = 3; r2 = 0.85 ± 0.05, 0.96 ± 0.01, 0.96 ± 0.02, 0.86 ± 0.07, 0.90 ± 0.03, 0.91 ± 0.04, respectively) over an approximate 10-fold range of concentrations and reproducible (n = 4; coefficient of variation = 0.12, 0.14, 0.29, 0.30, 0.12, 0.06, respectively). This application of label-free, fragment ion-based quantification to assess relative phosphorylation changes of specific proteins will prove useful for understanding how various cell stimuli regulate protein function by phosphorylation

The Benefits of Family Science Education: The Male Perspective

The majority of university family science courses are predominantly comprised of women. Because family science classes are centered on information and concepts relevant for both men and women, it is important to understand gendered experiences to promote healthy family and romantic relationships. Not only would men benefit from these classes, but increasing male enrollment in family sciences courses will help promote gender diversity in higher education. The current study used qualitative analyses to examine the perceptions of male undergraduate students concerning the benefits of taking family science courses. Male undergraduates from three midsize universities in the Midwestern and Western United States provided open-ended responses via an online survey (N = 64). Three themes emerged: the classes provided students with valuable information; they had a better understanding of themselves and others; and the classes related to their future career path. Results provide support to promote gender diversity in family science classrooms, which is crucial for the interpersonal and educational growth of both men and women. Further implications of participant responses are discussed

Rocaglates as dual-targeting agents for experimental cerebral malaria

Cerebral malaria (CM) is a severe and rapidly progressing complication of infection by Plasmodium parasites that is associated with high rates of mortality and morbidity. Treatment options are currently few, and intervention with artemisinin (Art) has limited efficacy, a problem that is compounded by the emergence of resistance to Art in Plasmodium parasites. Rocaglates are a class of natural products derived from plants of the Aglaia genus that have been shown to interfere with eukaryotic initiation factor 4A (eIF4A), ultimately blocking initiation of protein synthesis. Here, we show that the rocaglate CR-1-31B perturbs association of Plasmodium falciparum eIF4A (PfeIF4A) with RNA. CR-1-31B shows potent prophylactic and therapeutic antiplasmodial activity in vivo in mouse models of infection with Plasmodium berghei (CM) and Plasmodium chabaudi (blood-stage malaria), and can also block replication of different clinical isolates of P. falciparum in human erythrocytes infected ex vivo, including drug-resistant P. falciparum isolates. In vivo, a single dosing of CR-1-31B in P. berghei-infected animals is sufficient to provide protection against lethality. CR-1-31B is shown to dampen expression of the early proinflammatory response in myeloid cells in vitro and dampens the inflammatory response in vivo in P. berghei-infected mice. The dual activity of CR-1-31B as an antiplasmodial and as an inhibitor of the inflammatory response in myeloid cells should prove extremely valuable for therapeutic intervention in human cases of CM.We thank Susan Gauthier, Genevieve Perreault, and Patrick Senechal for technical assistance. This work was supported by a research grant (to P.G.) from the Canadian Institutes of Health Research (CIHR) (Foundation Grant). J.P. and P.G. are supported by a James McGill Professorship salary award. D.L. is supported by fellowships from the Fonds de recherche sante Quebec, the CIHR Neuroinflammation training program. J.P. is supported by CIHR Research Grant FDN-148366. M.S. is supported by a CIHR Foundation grant. J.A.P. is supported by NIH Grant R35 GM118173. Work at the Boston University Center for Molecular Discovery is supported by Grant R24 GM111625. K.C.K. was supported by a CIHR Foundation Grant and the Canada Research Chair program. (Canadian Institutes of Health Research (CIHR); James McGill Professorship salary award; Fonds de recherche sante Quebec; CIHR Neuroinflammation training program; FDN-148366 - CIHR Research Grant; CIHR Foundation grant; R35 GM118173 - NIH; Canada Research Chair program; R24 GM111625

Well-posedness for a model of individual clustering

25 pagesInternational audienceWe study the well-posedness of a model of individual clustering. Given p > N ≥ 1 and an initial condition in W 1,p (Ω), the local existence and uniqueness of a strong solution is proved. We next consider two specific reproduction rates and show global existence if N = 1, as well as, the convergence to steady states for one of these rates

Observation of individual molecules trapped on a nanostructured insulator

For the first time, ordered polar molecules confined in monolayer-deep rectangular pits produced on an alkali halide surface by electron irradiation have been resolved at room temperature by non-contact atomic force microscopy. Molecules self-assemble in a specific fashion inside pits of width smaller than 15 nm. By contrast no ordered aggregates of molecules are observed on flat terraces. Conclusions regarding nucleation and ordering mechanisms are drawn. Trapping in pits as small as 2 nm opens a route to address single molecules

Using exchange bias to extend the temperature range of square loop behavior in [Pt/Co] multilayers with perpendicular anisotropy

This article may be downloaded for personal use only. Any other use requires prior permission of the author and the American Institute of Physics.The temperature dependence of the magnetic properties of [Pt/Co]multilayers (ML), exhibiting perpendicular anisotropy, with and without exchange biasing with an antiferromagnet(AFM) has been investigated. Upon heating, a loss of the out-of-plane anisotropy and, consequently, of the remanence to saturation ratio is observed in these systems. However, such effect occurs at higher temperatures in the [Pt/Co] ML exchange coupled to the AFM than for the unbiased ML. This is attributed to the additional anisotropy induced to the ML by the ferromagnetic-antiferromagnetic exchange coupling

IMPACT OF MOOSE BROWSING ON FOREST REGENERATION IN NORTHEAST VERMONT

Moose (Alces alces) play an important role in the ecological and economic resources of northern New England, a landscape dominated by commercial forests. This study measured the impact of moose browsing on forest regeneration in Wildlife Management Unit E1 in northeastern Vermont where moose density was considered high in the 1990–2000s. We surveyed 37 clearcuts categorized into 4 age classes (3–5, 6–10, 11–15, and 16–20 years old). The stocking rate (stems/plot) of commercial species ranged from 74–76% in the 3–5, 6–10, and 11–15 year age classes, increasing to 86% in the 16–20 year age class. The proportion of plots containing a commercial tree without severe damage was above the accepted threshold stocking level of 40–60% in all age classes. The proportion of plots containing a commercial hardwood stem declined with increasing age class; the opposite occurred with softwood stems indicating a possible shift from hardwood- to softwood-dominated stands from selective browsing pressure. Height of 11–20 year old stems was less than in New Hampshire, indicating that growth was possibly suppressed in Vermont due to higher moose density. Overall, browsing was not considered a major problem based upon stocking rates. Further study is warranted to evaluate whether compensatory growth occurs in response to reduced browsing as forests age and/or moose population density declines

Obesity-induced insulin resistance in human skeletal muscle is characterised by defective activation of p42/p44 MAP kinase

Insulin resistance (IR), an impaired cellular, tissue and whole body response to insulin, is a major pathophysiological defect of type 2 diabetes mellitus. Although IR is closely associated with obesity, the identity of the molecular defect(s) underlying obesity-induced IR in skeletal muscle remains controversial; reduced post-receptor signalling of the insulin receptor substrate 1 (IRS1) adaptor protein and downstream effectors such as protein kinase B (PKB) have previously been implicated. We examined expression and/or activation of a number of components of the insulin-signalling cascade in skeletal muscle of 22 healthy young men (with body mass index (BMI) range, 20–37 kg/m2). Whole body insulin sensitivity (M value) and body composition was determined by the hyperinsulinaemic (40 mU. min−1.m−2.), euglycaemic clamp and by dual energy X-ray absorptiometry (DEXA) respectively. Skeletal muscle (vastus lateralis) biopsies were taken before and after one hour of hyperinsulinaemia and the muscle insulin signalling proteins examined by western blot and immunoprecipitation assay. There was a strong inverse relationship between M-value and BMI. The most striking abnormality was significantly reduced insulin-induced activation of p42/44 MAP kinase, measured by specific assay, in the volunteers with poor insulin sensitivity. However, there was no relationship between individuals' BMI or M-value and protein expression/phosphorylation of IRS1, PKB, or p42/44 MAP kinase protein, under basal or hyperinsulinaemic conditions. In the few individuals with poor insulin sensitivity but preserved p42/44 MAP kinase activation, other signalling defects were evident. These findings implicate defective p42/44 MAP kinase signalling as a potential contributor to obesity-related IR in a non-diabetic population, although clearly multiple signalling defects underlie obesity associated IR