Analysis of proteomic profiles and functional properties of human peripheral blood myeloid dendritic cells, monocyte-derived dendritic cells and the dendritic cell-like KG-1 cells reveals distinct characteristics

Important proteomic and functional differences between peripheral blood myeloid dendritic cells, monocyte-derived dendritic cells (moDC) and KG-1 cells have been identified

Role of ROCK isoforms in regulation of stiffness induced myofibroblast differentiation in lung fibrosis

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices

Morphological and molecular identification of brown algae, Padina sp. in Lengeh Port, Persian Gulf

Brown algae, Padina sp., are found across tropical coastal areas as well as inter-tidal and sub-tidal regions. The aim of this research was to identify the morphological and molecular characteristics of the Padina species distributed across Port Lengeh in the Persian Gulf using morphological examinations and rbcL chloroplast gene sequencing. For this purpose, morphological features were undertaken using valid identification keys. For the molecular analysis, genomic DNA was extracted through slightly modified CTAB. The amplification of fragments was carried out using rbcL primers. The analysis of genome sequences was undertaken using Chromas, BioEdit and MEGA6 and the phylogenetic trees were constructed through Neighbor Joining (NJ) and Maximum Likelihood (ML). The results indicated that there was a 99% chance that the two identified Padina species (P. boergessenii and P. australis) belonged to the same cluster and that there were large genetic similarities among the compared Padina species registered in GenBank

Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available

Laminin and Fibronectin Treatment Leads to Generation of Dendritic Cells with Superior Endocytic Capacity

Copyright: 2010 Garcı´a-Nieto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Sampling the microenvironment at sites of microbial exposure by dendritic cells ( DC) and their subsequent interaction with T cells in the paracortical area of lymph nodes are key events for initiating immune responses. Most of our knowledge of such events in human is based on in vitro studies performed in the absence of extracellular matrix (ECM) proteins. ECM in basement membranes and interstitial spaces of different tissues, including lymphoid organs, plays an important role in controlling specific cellular functions such as migration, intracellular signalling and differentiation. The aim of this study was, therefore, to investigate the impact of two abundant ECM components, fibronectin and laminin, on the phenotypical and functional properties of DC and how that might influence DC induced T-cell differentiation. Methodology/Principal Findings: Human monocyte derived DC were treated with laminin and fibronectin for up to 48 hours and their morphology and phenotype was analyzed using scanning electron microscopy, flow cytometry and real time PCR. The endocytic ability of DC was determined using flow cytometry. Furthermore, co-culture of DC and T cells were established and T cell proliferation and cytokine profile was measured using H(3)-thymidine incorporation and ELISA respectively. Finally, we assessed formation of DC-T cell conjugates using different cell trackers and flow cytometry. Our data show that in the presence of ECM, DC maintain a 'more immature' phenotype and express higher levels of key endocytic receptors, and as a result become significantly better endocytic cells, but still fully able to mature in response to stimulation as evidenced by their superior ability to induce antigen-specific T cell differentiation. Conclusion: These studies underline the importance of including ECM components in in vitro studies investigating DC biology and DC-T cell interaction. Within the context of antigen specific DC induced T cell proliferation, inclusion of ECM proteins could lead to development of more sensitive assays.Peer reviewedFinal Published versio

Anti-prion drug mPPIg5 inhibits PrP(C) conversion to PrP(Sc).

Prion diseases, also known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative diseases that include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and Creutzfeldt-Jakob disease (CJD) in humans. The 'protein only hypothesis' advocates that PrP(Sc), an abnormal isoform of the cellular protein PrP(C), is the main and possibly sole component of prion infectious agents. Currently, no effective therapy exists for these diseases at the symptomatic phase for either humans or animals, though a number of compounds have demonstrated the ability to eliminate PrPSc in cell culture models. Of particular interest are synthetic polymers known as dendrimers which possess the unique ability to eliminate PrP(Sc) in both an intracellular and in vitro setting. The efficacy and mode of action of the novel anti-prion dendrimer mPPIg5 was investigated through the creation of a number of innovative bio-assays based upon the scrapie cell assay. These assays were used to demonstrate that mPPIg5 is a highly effective anti-prion drug which acts, at least in part, through the inhibition of PrP(C) to PrP(Sc) conversion. Understanding how a drug works is a vital component in maximising its performance. By establishing the efficacy and method of action of mPPIg5, this study will help determine which drugs are most likely to enhance this effect and also aid the design of dendrimers with anti-prion capabilities for the future

JNK modifies neuronal metabolism to promote proteostasis and longevity.

Aging is associated with a progressive loss of tissue and metabolic homeostasis. This loss can be delayed by single-gene perturbations, increasing lifespan. How such perturbations affect metabolic and proteostatic networks to extend lifespan remains unclear. Here, we address this question by comprehensively characterizing age-related changes in protein turnover rates in the Drosophila brain, as well as changes in the neuronal metabolome, transcriptome, and carbon flux in long-lived animals with elevated Jun-N-terminal Kinase signaling. We find that these animals exhibit a delayed age-related decline in protein turnover rates, as well as decreased steady-state neuronal glucose-6-phosphate levels and elevated carbon flux into the pentose phosphate pathway due to the induction of glucose-6-phosphate dehydrogenase (G6PD). Over-expressing G6PD in neurons is sufficient to phenocopy these metabolic and proteostatic changes, as well as extend lifespan. Our study identifies a link between metabolic changes and improved proteostasis in neurons that contributes to the lifespan extension in long-lived mutants

Production and Initial Characterization of Dad1p, a Component of the Dam1-DASH Kinetochore Complex

In all dividing eukaryotic cells, the mitotic spindle (composed primarily of microtubules) must interact with chromosomes through a complex protein assembly called the kinetochore. In Saccharomyces cerevisiae, the Dam1-DASH complex plays an important role in promoting attachment between the kinetochore and the mitotic spindle. It also actively participates in the physical separation of sister chromatids in anaphase. Understanding the biochemical mechanisms used by Dam1-DASH has been facilitated by bacterial co-expression of the ten Dam1-DASH genes, which results in the production of a heterodecameric protein complex that can be studied in vitro. However, individual protein subunits are not soluble when expressed in E. coli, thus precluding analysis of the nature of the interaction between subunits and an examination of the assembly of the functional complex. In this paper, we describe the expression, solubilization, purification and refolding of Dad1p, one of the Dam1-DASH complex subunits. In addition, we show that Dad1p, when isolated in this manner forms dimers and/or tetramers, dependent upon protein concentration. This work provides an important tool for studying the Dam1-DASH complex that was previously unavailable, and provides an avenue of investigation for understanding how the individual heterodecamers associate with each other to facilitate chromosome segregation

Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g

Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F1FO–ATP synthase (F1FO) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F1FO supercomplexes. Impairment of F1FO oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F1FO oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips