The advantage of laser‐capture microdissection over whole tissue analysis in proteomic profiling studies

Laser‐capture microdissection (LCM) offers a reliable cell population enrichment tool and has been successfully coupled to MS analysis. Despite this, most proteomic studies employ whole tissue lysate (WTL) analysis in the discovery of disease biomarkers and in profiling analyses. Furthermore, the influence of tissue heterogeneity in WTL analysis, nor its impact in biomarker discovery studies have been completely elucidated. In order to address this, we compared previously obtained high resolution MS data from a cohort of 38 breast cancer tissues, of which both LCM enriched tumor epithelial cells and WTL samples were analyzed. Label‐free quantification (LFQ) analysis through MaxQuant software showed a significantly higher number of identified and quantified proteins in LCM enriched samples (3404) compared to WTLs (2837). Furthermore, WTL samples displayed a higher amount of missing data compared to LCM both at peptide and protein levels (p‐value < 0.001). 2D analysis on co‐expressed proteins revealed discrepant expression of immune system and lipid metabolisms related proteins between LCM and WTL samples. We hereby show that LCM better dissected the biology of breast tumor epithelial cells, possibly due to lower interference from surrounding tissues and highly abundant proteins. All data have been deposited in the ProteomeXchange with the dataset identifier PXD002381 ( http://proteomecentral.proteomexchange.org/dataset/PXD002381 ).     

Neurogenetic interactions and aberrant behavioral co-morbidity of attention deficit hyperactivity disorder (ADHD): dispelling myths

Background

Attention Deficit Hyperactivity Disorder, commonly referred to as ADHD, is a common, complex, predominately genetic but highly treatable disorder, which in its more severe form has such a profound effect on brain function that every aspect of the life of an affected individual may be permanently compromised. Despite the broad base of scientific investigation over the past 50 years supporting this statement, there are still many misconceptions about ADHD. These include believing the disorder does not exist, that all children have symptoms of ADHD, that if it does exist it is grossly over-diagnosed and over-treated, and that the treatment is dangerous and leads to a propensity to drug addiction. Since most misconceptions contain elements of truth, where does the reality lie?

Results

We have reviewed the literature to evaluate some of the claims and counter-claims. The evidence suggests that ADHD is primarily a polygenic disorder involving at least 50 genes, including those encoding enzymes of neurotransmitter metabolism, neurotransmitter transporters and receptors. Because of its polygenic nature, ADHD is often accompanied by other behavioral abnormalities. It is present in adults as well as children, but in itself it does not necessarily impair function in adult life; associated disorders, however, may do so. A range of treatment options is reviewed and the mechanisms responsible for the efficacy of standard drug treatments are considered.

Conclusion

The genes so far implicated in ADHD account for only part of the total picture. Identification of the remaining genes and characterization of their interactions is likely to establish ADHD firmly as a biological disorder and to lead to better methods of diagnosis and treatment.

     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

The cytoplasmic domain of MT1-MMP is dispensable for migration augmentation but necessary to mediate viability of MCF-7 breast cancer cells

Membrane-type-1 Matrix Metalloproteinase (MT1-MMP) is a multifunctional protease that regulates ECM degradation, proMMP-2 activation, and varied cellular processes including migration and viability. MT1-MMP is believed to be a central mediator of tumourigenesis whose role is dictated by its functionally distinct protein domains. Both the localization and signal transduction capabilities of MT1-MMP are dependent on its cytoplasmic domain, exemplifying diverse regulatory functions. To further our understanding of the multifunctional contributions of MT1-MMP to cellular processes, we overexpressed cytoplasmic domain altered constructs in MCF-7 breast cancer cells and analyzed migration and viability in 2D culture conditions, morphology in 3D Matrigel culture, and tumorigenic ability in vivo. We found that the cytoplasmic domain was not needed for MT1-MMP mediated migration promotion, but was necessary to maintain viability during serum depravation in 2D culture. Similarly, during 3D Matrigel culture the cytoplasmic domain of MT1-MMP was not needed to initiate a protrusive phenotype, but was necessary to prevent colony blebbing when cells were serum deprived. We also tested in vivo tumorigenic potential to show that cells expressing cytoplasmic domain altered constructs demonstrated a reduced ability to vascularize tumours. These results suggest that the cytoplasmic domain regulates MT1-MMP function in a manner required for cell survival, but is dispensable for cell migration.     

Musical Minds: Attentional Blink Reveals Modality-Specific Restrictions

BackgroundFormal musical training is known to have positive effects on attentional and executive functioning, processing speed, and working memory. Consequently, one may expect to find differences in the dynamics of temporal attention between musicians and non-musicians. Here we address the question whether that is indeed the case, and whether any beneficial effects of musical training on temporal attention are modality specific or generalize across sensory modalities.Conclusion/SignificanceAB magnitude within one modality can generalize to another modality, but this turns out not to be the case for every individual. Formal musical training seems to have a domain-general, but modality-specific beneficial effect on selective attention. The results fit with the idea that a major source of attentional restriction as reflected in the AB lies in modality-specific, independent sensory systems rather than a central amodal system. The findings demonstrate that individual differences in AB magnitude can provide important information about the modular structure of human cognition.     

Microwave fixation of water-cooled insect tissues for immunohistochemistry

Summary In this paper a new technique for microwave-accelerated tissue fixation and washing is described. The temperature of the irradiated specimens is controlled by means of a specially designed water-cooling device. Two types of fixation fluids, one of them containing rather concentrated picric acid and a washing procedure were tested empirically on dissected nervous tissue of the Colorado potato beetle,Leptinotarsa decemlineata (Say). Entire beetles were processed in a similar way. It was shown that specimens could be irradiated as long as was required for good fixation and washing results, without the accumulation of excessive heat. No differences in morphology and immunoreactivity were observed when compared with standard immersion-fixed controls. A considerable reduction in processing time was achieved.     

Monoclonal antibodies obtained through insect brain homogenates: Tools for cell-specific neuroanatomical studies in the Colorado potato beetle

Immunization of mice with crude or purified homogenates of brain and endocrine organs of the Colorado potato beetle, Leptinotarsa decemlineata (Say), generated antibodies against specific antigens in the insect tissue. Hybridomas were prepared and screened immunocytochemically for the production of monoclonal antibodies (MAB) that recognized specific cells and that were useful for neuroanatomical studies. A relatively high proportion of MABs recognized one or more cells or tissues in neuronal, neuroendocrine or nonneural compartments of the brain or in fatbody or haemocytes, using standard immunocytochemical procedures. We could make a clear differentiation in subsets of neurons (both normal and peptidergic) and neuron-specific neuroglial cells. A full account of the obtained responses is given. The preparation of the immunogen and the immunization protocol affects the selection of MABs produced. It is argued that not all possibilities have been exploited and that continued experiments would probably increase the number of selective MABs.

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Résumé L'utilisation de techniques immunocytochimiques lors d'études neuro-anatomiques est encore dans l'enfance par suite de l'absence de données sur la chimie de structures spécifiques et, par conséquent, de l'absence d'anticorps spécifiques d'antigènes et de tissus. Nous apportons la preuve que de nombreuses substances réelles d'homogénats bruts de cerveaux sont de bons antigènes et que les souris peuvent être induites à produire des anticorps contre ceux-ci. Nous avons produit et testé de nombreux anticorps monoclonaux qui reconnaissent ces antigènes sur des lames de microscope, en utilisant des techniques immunocytochimiques courantes ou plus élaborées.Les immunogènes ont été préparés de différentes façons pour examiner si le taux de mélange de tissus du cerveau et/ou des ganglions associés et de glandes endocrines, et si les procédés ultérieurs de purification et d'immunisation, influeraient sur la spécificité des anticorps. L'intégralité de l'ensemble cerveau-glandes endocrines a été le plus immunogénique quand l'homogénat a subi le minimum de manipulations. Une grande diversité d'anticorps a été obtenue après des injections répétées chez la souris. Ces anticorps reconnaissent les épitopes dans toutes les catégories de tissus considérés, c'est-à-dire, les tissus spécifiquement nerveux, neuro-endocrines, et non nerveux. La purification progressive et le cross-linking des constituants de l'homogénat réduisent la variabilité des anticorps. Tout en l'espérant, on ne s'attendait pas à ce que les substances jusqu'alors dans des centres peptidergiques indéfinis fussent parmi les structures les plus souvent identifiées. Les chances de produire de tels anticorps spécifiques de peptides ont augmenté quand les tissus endocrines ont servi d'immunogènes.Nous avons résumé les types de tissus et de cellules mis en évidence par cette sélection d'anticorps monoclonaux. Nous soulignons que l'expérimentateur peut difficilement influencer les préférences du système immunologique de la souris, mais les anticorps produits jusqu'ici sont valables pour les études neuroanatomiques et pour la récolte d'antigènes intéressants à partir de procédés biochimiques de séparation. Il reste à explorer la spécificité sensu stricto des anticorps.