Communication over the Network of Binary Switches Regulates the Activation of A2A Adenosine Receptor

Dynamics and functions of G-protein coupled receptors (GPCRs) are accurately regulated by the type of ligands that bind to the orthosteric or allosteric binding sites. To glean the structural and dynamical origin of ligand-dependent modulation of GPCR activity, we performed total ~ 5 μsec molecular dynamics simulations of A_2A adenosine receptor (A_2AAR) in its apo, antagonist-bound, and agonist-bound forms in an explicit water and membrane environment, and examined the corresponding dynamics and correlation between the 10 key structural motifs that serve as the allosteric hotspots in intramolecular signaling network. We dubbed these 10 structural motifs “binary switches” as they display molecular interactions that switch between two distinct states. By projecting the receptor dynamics on these binary switches that yield 2¹⁰ microstates, we show that (i) the receptors in apo, antagonist-bound, and agonist-bound states explore vastly different conformational space; (ii) among the three receptor states the apo state explores the broadest range of microstates; (iii) in the presence of the agonist, the active conformation is maintained through coherent couplings among the binary switches; and (iv) to be most specific, our analysis shows that W246, located deep inside the binding cleft, can serve as both an agonist sensor and actuator of ensuing intramolecular signaling for the receptor activation. Finally, our analysis of multiple trajectories generated by inserting an agonist to the apo state underscores that the transition of the receptor from inactive to active form requires the disruption of ionic-lock in the DRY motif.     

In vivo Tracking of Dendritic Cell using MRI Reporter Gene,Ferritin

The noninvasive imaging of dendritic cells (DCs) migrated into lymph nodes (LNs) can provide helpful information on designing DCs-based immunotherapeutic strategies. This study is to investigate the influence of transduction of human ferritin heavy chain (FTH) and green fluorescence protein (GFP) genes on inherent properties of DCs, and the feasibility of FTH as a magnetic resonance imaging (MRI) reporter gene to track DCs migration into LNs. FTH-DCs were established by the introduction of FTH and GFP genes into the DC cell line (DC2.4) using lentivirus. The changes in the rate of MRI signal decay (R₂*) resulting from FTH transduction were analyzed in cell phantoms as well as popliteal LN of mice after subcutaneous injection of those cells into hind limb foot pad by using a multiple gradient echo sequence on a 9.4 T MR scanner. The transduction of FTH and GFP did not influence the proliferation and migration abilities of DCs. The expression of co-stimulatory molecules (CD40, CD80 and CD86) in FTH-DCs was similar to that of DCs. FTH-DCs exhibited increased iron storage capacity, and displayed a significantly higher transverse relaxation rate (R₂*) as compared to DCs in phantom. LNs with FTH-DCs exhibited negative contrast, leading to a high R₂* in both in vivo and ex vivo T₂*-weighted images compared to DCs. On histological analysis FTH-DCs migrated to the subcapsular sinus and the T cell zone of LN, where they highly expressed CD25 to bind and stimulate T cells. Our study addresses the feasibility of FTH as an MRI reporter gene to track DCs migration into LNs without alteration of their inherent properties. This study suggests that FTH-based MRI could be a useful technique to longitudinally monitor DCs and evaluate the therapeutic efficacy of DC-based vaccines.     

Bioreactance Is Not Interchangeable with Thermodilution for Measuring Cardiac Output during Adult Liver Transplantation

BackgroundThermodilution technique using a pulmonary artery catheter is widely used for the assessment of cardiac output (CO) in patients undergoing liver transplantation. However, the unclearness of the risk-benefit ratio of this method has led to an interest in less invasive modalities. Thus, we evaluated whether noninvasive bioreactance CO monitoring is interchangeable with thermodilution technique.MethodsNineteen recipients undergoing adult-to-adult living donor liver transplantation were enrolled in this prospective observational study. COs were recorded automatically by the two devices and compared simultaneously at 3-minute intervals. The Bland–Altman plot was used to evaluate the agreement between bioreactance and thermodilution. Clinically acceptable agreement was defined as a percentage error of limits of agreement <30%. The four quadrant plot was used to evaluate concordance between bioreactance and thermodilution. Clinically acceptable concordance was defined as a concordance rate >92%.ResultsA total of 2640 datasets were collected. The mean CO difference between the two techniques was 0.9 l/min, and the 95% limits of agreement were -3.5 l/min and 5.4 l/min with a percentage error of 53.9%. The percentage errors in the dissection, anhepatic, and reperfusion phase were 50.6%, 56.1%, and 53.5%, respectively. The concordance rate between the two techniques was 54.8%.ConclusionBioreactance and thermodilution failed to show acceptable interchangeability in terms of both estimating CO and tracking CO changes in patients undergoing liver transplantation. Thus, the use of bioreactance as an alternative CO monitoring to thermodilution, in spite of its noninvasiveness, would be hard to recommend in these surgical patients.     

Production of butanol and isopropanol with an immobilized <Emphasis Type="Italic">Clostridium</Emphasis>

Clostridium beijerinckii optinoii is a Clostridium species that produces butanol, isopropanol and small amounts of ethanol. This study compared the performances of batch and continuous immobilized cell fermentations, investigating how media flow rates and nutritional modification affected solvent yields and productivity. In 96-h batch cultures, with 80 % of the 30 g L^?1 glucose consumed in synthetic media, solvent concentration was 9.45 g L^?1 with 66.0 % as butanol. In a continuous fermentation using immobilized C. beijerinckii optinoii cells, also with 80 % of 30 g L^?1 glucose utilization, solvent productivity increased to 1.03 g L^?1 h^?1. Solvent concentration reached 12.14 g L^?1 with 63.0 % as butanol. Adjusting the dilution rate from 0.085 to 0.050 h^?1 to allow extended residence time in column was required when glucose concentration in fresh media was increased from 30 to 50 g L^?1. When acetate was used to improve the buffer capacity in media, the solvent concentration reached 12.70 on 50 g L^?1 glucose. This continuous fermentation using immobilized cells showed technical feasibility for solvent production.     

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.