Engineered Mammalian Vector to Express EGFP-Tagged Proteins as Biomarkers

Due to its specialized post-translational machinery, mammalian cells represent an interesting and not fully explored system to express snake toxins. Therefore, in this work, we built up a new mammalian expression vector that enhances the feasibility to use mammalian cells to express proteins as biomarkers. Among the modifications, an Igκ signal peptide and a 6xHis tag were inserted into this vector in order to drive the protein to the supernatant and simplify its purification, respectively. In addition, to facilitate selection of high producing clones and also tag proteins which may function as a biomarker, the sequence of enhanced green fluorescent protein (EGFP) was added. The efficiency of the resulting vector (pToxEGFP) was tested by cloning and expressing the viper venom disintegrin echistatin (Ech) that due to its affinity to integrin αvβ3 was tested as a molecular marker. Expression of EGFP-Ech was achieved in CHO-DXB11 cells resulting in a yield of 22 mg/L. The binding activity of this chimera protein was successfully achieved on human umbilical vein endothelial cells which highly express αvβ3. The results indicate that pToxEGFP may constitute an efficient and versatile expression vector to express tagged proteins with potential biomarker activity.     

Metabotropic glutamate receptors in neurodegeneration/neuroprotection: Still a hot topic?

Moving from early studies, we here review the most recent evidence linking metabotropic glutamate (mGlu) receptors to processes of neurodegeneration/neuroprotection. The use of knockout mice and subtype-selective drugs has increased our knowledge of the precise role played by individual mGlu receptor subtypes in these processes. Activation of mGlu1 and mGlu5 receptors may either amplify or reduce neuronal damage depending on the context and the nature of the toxic insults. In contrast, mGlu1 and mGlu5 receptors antagonists are consistently protective in in vitro and in vivo models of neuronal death. A series of studies suggest that mGlu1 receptor antagonists or negative allosteric modulators (NAMs) are promising candidates for the treatment of ischemic brain damage, whereas mGlu5 receptor NAMs, which have been clinically developed for the treatment of Parkinson's disease (PD) and l-DOPA-induced dyskinesias, protect nigro-striatal dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice and monkeys. Activation of glial mGlu3 receptors promotes the formation of various neurotrophic factors, such as transforming growth factor-β (TGF-β), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Hence, selective mGlu3 receptor agonists or positive allosteric modulators (PAMs) (not yet available) are potentially helpful in the treatment of chronic neurodegenerative disorders such as PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis. Selective mGlu2 receptor PAMs should be used with caution in AD patients because these drugs are shown to amplify β-amyloid neurotoxicity. Finally, mGlu4 receptor agonists/PAMs share with mGlu5 receptor NAMs the ability to improve motor symptoms associated with PD and attenuate nigro-striatal degeneration at the same time. No data are yet available on the role of mGlu7 and mGlu8 receptors in neurodegeneration/neuroprotection.     

Cleptoparasites, social parasites and a common host: Chemical insignificance for visiting host nests, chemical mimicry for living in

Social insect colonies contain attractive resources for many organisms. Cleptoparasites sneak into their nests and steal food resources. Social parasites sneak into their social organisations and exploit them for reproduction. Both cleptoparasites and social parasites overcome the ability of social insects to detect intruders, which is mainly based on chemoreception. Here we compared the chemical strategies of social parasites and cleptoparasites that target the same host and analyse the implication of the results for the understanding of nestmate recognition mechanisms. The social parasitic wasp Polistes atrimandibularis (Hymenoptera: Vespidae), and the cleptoparasitic velvet ant Mutilla europaea (Hymenoptera: Mutillidae), both target the colonies of the paper wasp Polistes biglumis (Hymenoptera: Vespidae). There is no chemical mimicry with hosts in the cuticular chemical profiles of velvet ants and pre-invasion social parasites, but both have lower concentrations of recognition cues (chemical insignificance) and lower proportions of branched alkanes than their hosts. Additionally, they both have larger proportions of alkenes than their hosts. In contrast, post-invasion obligate social parasites have proportions of branched hydrocarbons as large as those of their hosts and their overall cuticular profiles resemble those of their hosts. These results suggest that the chemical strategies for evading host detection vary according to the lifestyles of the parasites. Cleptoparasites and pre-invasion social parasites that sneak into host colonies limit host overaggression by having few recognition cues, whereas post-invasion social parasites that sneak into their host social structure facilitate social integration by chemical mimicry with colony members.     

Chromatographic retention time prediction for posttranslationally modified peptides

Retention time prediction of peptides in liquid chromatography has proven to be a valuable tool for mass spectrometry-based proteomics, especially in designing more efficient procedures for state-of-the-art targeted workflows. Additionally, accurate retention time predictions can also be used to increase confidence in identifications in shotgun experiments. Despite these obvious benefits, the use of such methods has so far not been extended to (posttranslationally) modified peptides due to the absence of efficient predictors for such peptides. We here therefore describe a new retention time predictor for modified peptides, built on the foundations of our existing Elude algorithm. We evaluated our software by applying it on five types of commonly encountered modifications. Our results show that Elude now yields equally good prediction performances for modified and unmodified peptides, with correlation coefficients between predicted and observed retention times ranging from 0.93 to 0.98 for all the investigated datasets. Furthermore, we show that our predictor handles peptides carrying multiple modifications as well. This latest version of Elude is fully portable to new chromatographic conditions and can readily be applied to other types of posttranslational modifications. Elude is available under the permissive Apache2 open source License at http://per-colator.com or can be run via a web-interface at http://elude.sbc.su.se.     

Minibrain-related kinase/dual-specificity tyrosine-regulated kinase 1B implication in stem/cancer stem cells biology

Dual-specificity tyrosine phosphorylation-regulated kinase 1B (DYRK1B), also known as minibrain-related kinase (MIRK) is one of the best functionally studied members of the DYRK kinase family. DYRKs comprise a family of protein kinases that are emerging modulators of signal transduction pathways, cell proliferation and differentiation, survival, and cell motility. DYRKs were found to participate in several signaling pathways critical for development and cell homeostasis. In this review, we focus on the DYRK1B protein kinase from a functional point of view concerning the signaling pathways through which DYRK1B exerts its cell type-dependent function in a positive or negative manner, in development and human diseases. In particular, we focus on the physiological role of DYRK1B in behavior of stem cells in myogenesis, adipogenesis, spermatogenesis and neurogenesis, as well as in its pathological implication in cancer and metabolic syndrome. Thus, understanding of the molecular mechanisms that regulate signaling pathways is of high importance. Recent studies have identified a close regulatory connection between DYRK1B and the hedgehog (HH) signaling pathway. Here, we aim to bring together what is known about the functional integration and cross-talk between DYRK1B and several signaling pathways, such as HH, RAS and PI3K/mTOR/AKT, as well as how this might affect cellular and molecular processes in development, physiology, and pathology. Thus, this review summarizes the major known functions of DYRK1B kinase, as well as the mechanisms by which DYRK1B exerts its functions in development and human diseases focusing on the homeostasis of stem and cancer stem cells.     

Comparative proteomics of Geobacter sulfurreducens PCAT in response to acetate,formate and/or hydrogen as electron donor

Geobacter sulfurreducens is a model bacterium to study the degradation of organic compounds coupled to the reduction of Fe(III). The response of G. sulfurreducens to the electron donors acetate, formate, hydrogen and a mixture of all three with Fe(III) citrate as electron acceptor was studied using comparative physiological and proteomic approaches. Variations in the supplied electron donors resulted in differential abundance of proteins involved in the citric acid cycle (CAC), gluconeogenesis, electron transport, and hydrogenases and formate dehydrogenase. Our results provided new insights into the electron donor metabolism of G. sulfurreducens. Remarkably, formate was the preferred electron donor compared to acetate, hydrogen, or acetate plus hydrogen. When hydrogen was the electron donor, formate was formed, which was associated with a high abundance of formate dehydrogenase. Notably, abundant proteins of two CO₂ fixation pathways (acetyl-CoA pathway and the reversed oxidative CAC) corroborated chemolithoautotrophic growth of G. sulfurreducens with formate or hydrogen and CO₂, and provided novel insight into chemolithoautotrophic growth of G. sulfurreducens.     

Bacterial responses to background organic pollutants in the northeast subarctic Pacific Ocean

Thousands of man-made synthetic chemicals are released to oceans and compose the anthropogenic dissolved organic carbon (ADOC). Little is known about the effects of this chronic pollution on marine microbiome activities. In this study, we measured the pollution level at three sites in the Northeast Subarctic Pacific Ocean (NESAP) and investigated how mixtures of three model families of ADOC at different environmentally relevant concentrations affected naturally occurring marine bacterioplankton communities' structure and metabolic functioning. The offshore northernmost site (North) had the lowest concentrations of hydrocarbons, as well as organophosphate ester plasticizers, contrasting with the two other continental shelf sites, the southern coastal site (South) being the most contaminated. At North, ADOC stimulated bacterial growth and promoted an increase in the contribution of some Gammaproteobacteria groups (e.g. Alteromonadales) to the 16 rRNA pool. These groups are described as fast responders after oil spills. In contrast, minor changes in South microbiome activities were observed. Gene expression profiles at Central showed the coexistence of ADOC degradation and stress-response strategies to cope with ADOC toxicities. These results show that marine microbial communities at three distinct domains in NESAP are influenced by background concentrations of ADOC, expanding previous assessments for polar and temperate waters.     

Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL/6 mice

Cdc42 is a small RhoGTPase regulating multiple functions in eukaryotic cells. The activity of Cdc42 is significantly elevated in several tissues of aged mice, while the Cdc42 gain‐of‐activity mouse model presents with a premature aging‐like phenotype and with decreased lifespan. These data suggest a causal connection between elevated activity of Cdc42, aging, and reduced lifespan. Here, we demonstrate that systemic treatment of aged (75‐week‐old) female C57BL/6 mice with a Cdc42 activity‐specific inhibitor (CASIN) for 4 consecutive days significantly extends average and maximum lifespan. Moreover, aged CASIN‐treated animals displayed a youthful level of the aging‐associated cytokines IL‐1β, IL‐1α, and INFγ in serum and a significantly younger epigenetic clock as based on DNA methylation levels in blood cells. Overall, our data show that systemic administration of CASIN to reduce Cdc42 activity in aged mice extends murine lifespan.     

Gastroduodenal lesions and Helicobacter pylori infection in dyspeptic patients with and without chronic renal failure

BACKGROUND: Patients with chronic renal failure (CRF) often have dyspeptic symptoms and may develop peptic disease or digestive disorders leading to severe gastrointestinal complications. The primary aim of this study was to evaluate the prevalence of peptic lesions and Helicobacter pylori infection, and the severity of dyspeptic symptoms, in dyspeptic patients with and without CRF. Our secondary aim was to investigate whether uremic status may affect the diagnostic efficiency of the [13]C-urea breath test ([13]C-UBT). PATIENTS AND METHODS: We consecutively enrolled in the study 50 dyspeptic patients with chronic kidney failure (mean age 52 +/- 5 years), of whom 11 were on hemodialysis treatment (HD), and 93 subjects (mean age 54 +/- 7 years) with chronic dyspepsia and normal renal function (NRF). All patients completed an oriented and validated questionnaire scoring the severity of nine dyspeptic symptoms (i.e. epigastric pain, epigastric burning, postprandial fullness, early satiety, bloating, belching, nausea and vomiting) and underwent upper endoscopy with multiple bioptic sampling for rapid urease test and histological examination, [13]C-UBT and HpSA test. RESULTS: The prevalences of peptic lesions and H. pylori infection and mean symptom score were 74%, 52% and 3.5 +/- 3, respectively, in dyspeptic patients with CRF and 18%, 36% and 8 +/- 5, respectively, in dyspeptic patients with NRF. The diagnostic accuracy of [13]C-UBT with respect to histological diagnosis was 94% and 97% for dyspeptic patients with and without renal failure, respectively. CONCLUSIONS: 1, A high frequency of peptic lesions and low symptom scores were observed in uremic patients in spite of H. pylori infection; 2, uremic status did not affect the diagnostic accuracy of [13]C-UBT.