Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.     

Cyclization of the Urokinase Receptor-Derived Ser-Arg-Ser-Arg-Tyr Peptide Generates a Potent Inhibitor of Trans-Endothelial Migration of Monocytes

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration and uPAR_88-92 is the minimal sequence required to induce cell motility. We and others have previously documented that the uPAR_88-92 sequence, even in the form of synthetic linear peptide (SRSRY), interacts with the formyl peptide receptor type 1 (FPR1), henceforth inducing cell migration of several cell lines, including monocytes. FPR1 is mainly expressed by mammalian phagocytic leukocytes and plays a crucial role in chemotaxis. In this study, we present evidence that the cyclization of the SRSRY sequence generates a new potent and stable inhibitor of monocyte trafficking. In rat basophilic leukaemia RBL-2H3/ETFR cells expressing high levels of constitutively activated FPR1, the cyclic SRSRY peptide ([SRSRY]) blocks FPR1 mediated cell migration by interfering with both internalization and ligand-uptake of FPR1. Similarly to RBL-2H3/ETFR cells, [SRSRY] competes with fMLF for binding to FPR1 and prevents agonist-induced FPR1 internalization in human monocyte THP-1 cells. Unlike scramble [RSSYR], [SRSRY] inhibits fMLF-directed migration of monocytes in a dose-dependent manner, with IC₅₀ value of 0.01 nM. PMA-differentiated THP-1 cell exposure to fMLF gradient causes a marked cytoskeletal re-organization with the formation of F-actin rich pseudopodia that are prevented by the addition of [SRSRY]. Furthermore, [SRSRY] prevents migration of human primary monocytes and trans-endothelial migration of monocytes. Our findings indicate that [SRSRY] is a new FPR1 inhibitor which may suggest the development of new drugs for treating pathological conditions sustained by increased motility of monocytes, such as chronic inflammatory diseases.     

Easy to Use Plastic Optical Fiber-Based Biosensor for Detection of Butanal

The final goal of this work is to achieve a selective detection of butanal by the realization of a simple, small-size and low cost experimental approach. To this end, a porcine odorant-binding protein was used in connection with surface plasmon resonance transduction in a plastic optical fiber tool for the selective detection of butanal by a competitive assay. This allows to reduce the cost and the size of the sensing device and it offers the possibility to design a “Lab-on-a-chip” platform. The obtained results showed that this system approach is able to selectively detect the presence of butanal in the concentration range from 20 μM to 1000 μM.     

H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562^shFHC) comparing it with K562 transduced with scrambled RNA (K562^shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.     

Investigation of Repeat Client Drop-Out and Re-Enrolment Cycles in Fourteen Methadone Maintenance Treatment Clinics in Guangdong,China

ObjectiveClient adherence is vital for effective methadone maintenance treatment (MMT). This study explores the pattern and associated factors of client adherence, drop-out and re-enrolment in the Chinese MMT programme over the period of 2006–2013.MethodsThis retrospective study was conducted in 14 MMT clinics in Guangdong Province, China. We employed Kaplan-Meier survival analysis to estimate the rates of drop-out and re-enrolment of MMT clients and multivariate Cox regression to identify associated factors.ResultsAmong 1,512 study participants, 79% have experienced ‘drop-out’ during the 7-year study period. However, 82% ‘dropped-out’ clients resumed treatment at a later time. Low education level (junior high or below versus otherwise, HR = 1.21, 1.05–1.40), low methadone dosage in the first treatment episode (<50 ml versus ≥50 ml, HR = 1.84, 1.64–2.06) and higher proportion of positive urine test (≥50% versus<50%, HR = 3.72, 3.30–4.20) during the first treatment episode were strong predictors of subsequent drop-outs of the participants. Among the ‘dropped-out’ clients, being female (HR = 1.40, 1.23–1.60), being married (HR = 1.19, 1.09–1.30), and having a higher proportion of positive urine tests in the first treatment episode (≥50% versus<50%, HR = 1.35, 1.20–1.51) had greater likelihood of subsequent re-enrolment in MMT. Clients receiving lower methadone dosage (first treatment episode <50 ml versus ≥50 ml, HR = 1.12, 1.03–1.23; the last intake before drop-out <50 ml versus ≥50 ml, HR = 1.16, 1.04–1.30) were also more likely to re-enrol.ConclusionPersistent cycling in-and-out of clients in MMT programmes is common. Insufficient dosage and higher proportion of positive urine samples in the first treatment episode are the key determinants for subsequent client drop-out and re-enrolment. Interventions should target clients in their early stage of treatment to improve retention in the long term.     

Impact of the Uremic Milieu on the Osteogenic Potential of Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs), the precursors of osteoblasts during osteogenesis, play a role in the balance of bone formation and resorption, but their functioning in uremia has not been well defined. To study the effects of the uremic milieu on osteogenic properties, we applied an in vitro assay culturing hMSCs in osteogenic medium supplemented with serum from healthy donors and from uremic patients on hemodialysis. Compared to control, serum from uremic patients induces, in hMSC cultures, a modification of several key regulators of bone remodeling, in particular a reduction of the ratio Receptor Activator of Nuclear factor Kappa B Receptor (RANKL) over osteoprotegerin, indicating an adaptive response of the system to favor osteogenesis over osteoclastosis. However, the levels of osteopontin, osteocalcin, and collagen type I, are increased in cell medium, while BMP-2, and alizarin red staining were decreased, pointing to a reduction of bone formation favoring resorption. Selected uremic toxins, such as p-cresylsulfate, p-cresylglucuronide, parathyroid hormone, indoxyl sulfate, asymmetric dimethylarginine, homocysteine, were able to mimic some of the effects of whole serum from uremic patients. Serum from cinacalcet-treated patients antagonizes these effects. Hydrogen sulfide (H2S) donors as well as hemodialysis treatment are able to induce beneficial effects. In conclusion, bone modifications in uremia are influenced by the capability of the uremic milieu to alter hMSC osteogenic differentiation. Cinacalcet, H2S donors and a hemodialysis session can ameliorate the hampered calcium deposition.