Identification of a collapsed intermediate with non-native long-range interactions on the folding pathway of a pair of Fyn SH3 domain mutants by NMR relaxation dispersion spectroscopy

Recent 15N and 13C spin-relaxation dispersion studies of fast-folding mutants of the Fyn SH3 domain have established that folding proceeds through a low-populated on-pathway intermediate (I) where the central beta-sheet is at least partially formed, but without interactions between the NH2- and COOH-terminal beta-strands that exist in the folded state (F). Initial studies focused on mutants where Gly48 is replaced; in an effort to establish whether this intermediate is a general feature of Fyn SH3 folding a series of 15N relaxation experiments monitoring the folding of Fyn SH3 mutants N53P/V55L and A39V/N53P/V55L are reported here. For these mutants as well, folding proceeds through an on-pathway intermediate with similar features to those observed for G48M and G48V Fyn SH3 domains. However, the 15N chemical shifts extracted for the intermediate indicate pronounced non-native contacts between the NH2 and COOH-terminal regions not observed previously. The kinetic parameters extracted for the folding of A39V/N53P/V55L Fyn SH3 from the three-state folding model F<-->I<-->U are in good agreement with folding and unfolding rates extrapolated to zero denaturant obtained from stopped-flow experiments analyzed in terms of a simplified two-state folding reaction. The folding of the triple mutant was studied over a wide range of temperatures, establishing that there is no difference in heat capacities between F and I states. This confirms a compact folding intermediate structure, which is supported by the 15N chemical shifts of the I state extracted from the dispersion data. The temperature-dependent relaxation data simplifies data analysis because at low temperatures (< 25 degrees C) the unfolded state (U) is negligibly populated relative to I and F. A comparison between parameters extracted at low temperatures where the F<-->I exchange model is appropriate with those from the more complex, three-state model at higher temperatures has been used to validate the protocol for analysis of three-site exchange relaxation data.     

Effects of Radiofrequency Catheter Ablation of Atrial Fibrillation on Soluble P-Selectin,Von Willebrand Factor and IL-6 in the Peripheral and Cardiac Circulation

Background

Catheter ablation (CA) of atrial fibrillation (AF) is associated with inflammatory response, endothelial damage and with increased risk of thrombosis. However, whether these processes differ in peripheral and cardiac circulation is unknown.

Methods

Plasma markers (von Willebrand factor (vWf), soluble P-selectin (sPsel) and interleukin-6 (IL-6)) were measured by ELISA at three time points in 80 patients (62±10 years, 63% males, 41% paroxysmal AF) undergoing CA. These were at baseline – from femoral vein (FV) and left atrium (LA) before ablation; directly after ablation – from the pulmonary vein (PV), LA and FV; and 24 hours after procedure – from a cubital vein (CV).

Results

The levels of vWF and IL6 – but not sP-sel – increased significantly 24h after procedure (p<0.001). Baseline vWF was significantly associated with persistent AF (Beta = .303, p = 0.006 and Beta = .300, p = 0.006 for peripheral and cardiac levels, respectively), while persistent AF (Beta = .250, p = 0.031) and LAA flow pattern (Beta = .386, p<0.001) remained associated with vWF in cardiac blood after ablation. Advanced age was significantly associated with IL6 levels at baseline and after ablation in peripheral and cardiac blood. There were no clinical, procedural or anti-coagulation characteristics associated with sP-sel levels in cardiac blood, while peripheral sP-sel levels were associated with hypertension before (Beta = −.307, p = 0.007) and with persistent AF after ablation (Beta = −.262, p = 0.020).

Conclusions

vWF levels are higher in persistent AF and are associated with LAA rheological pattern after AF ablation. Increase of peripheral vWF and IL6 levels after procedure supports current AF ablation management with careful control of post-procedural anticoagulation to avoid ablation-related thromboembolism.     

Reproducibility of swallow-induced cortical BOLD positive and negative fMRI activity

Functional MRI (fMRI) studies have demonstrated that a number of brain regions (cingulate, insula, prefrontal, and sensory/motor cortices) display blood oxygen level-dependent (BOLD) positive activity during swallow. Negative BOLD activations and reproducibility of these activations have not been systematically studied. The aim of our study was to investigate the reproducibility of swallow-related cortical positive and negative BOLD activity across different fMRI sessions. We studied 16 healthy volunteers utilizing an fMRI event-related analysis. Individual analysis using a general linear model was used to remove undesirable signal changes correlated with motion, white matter, and cerebrospinal fluid. The group analysis used a mixed-effects multilevel model to identify active cortical regions. The volume and magnitude of a BOLD signal within each cluster was compared between the two study sessions. All subjects showed significant clustered BOLD activity within the known areas of cortical swallowing network across both sessions. The cross-correlation coefficient of percent fMRI signal change and the number of activated voxels across both positive and negative BOLD networks were similar between the two studies (r ≥ 0.87, P < 0.0001). Swallow-associated negative BOLD activity was comparable to the well-defined "default-mode" network, and positive BOLD activity had noticeable overlap with the previously described "task-positive" network. Swallow activates two parallel cortical networks. These include a positive and a negative BOLD network, respectively, correlated and anticorrelated with swallow stimulus. Group cortical activity maps, as well as extent and amplitude of activity induced by volitional swallowing in the cortical swallowing network, are reproducible between study sessions.     

Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

The magnetosome membrane protein, MmsF, is a major regulator of magnetite biomineralization in Magnetospirillum magneticum AMB-1

Magnetotactic bacteria (MTB) use magnetosomes, membrane-bound crystals of magnetite or greigite, for navigation along geomagnetic fields. In Magnetospirillum magneticum sp. AMB-1, and other MTB, a magnetosome gene island (MAI) is essential for every step of magnetosome formation. An 8-gene region of the MAI encodes several factors implicated in control of crystal size and morphology in previous genetic and proteomic studies. We show that these factors play a minor role in magnetite biomineralization in vivo. In contrast, MmsF, a previously uncharacterized magnetosome membrane protein encoded within the same region plays a dominant role in defining crystal size and morphology and is sufficient for restoring magnetite synthesis in the absence of the other major biomineralization candidates. In addition, we show that the 18 genes of the mamAB gene cluster of the MAI are sufficient for the formation of an immature magnetosome organelle. Addition of MmsF to these 18 genes leads to a significant enhancement of magnetite biomineralization and an increase in the cellular magnetic response. These results define a new biomineralization protein and lay down the foundation for the design of autonomous gene cassettes for the transfer of the magnetic phenotype in other bacteria.     

Impact of Parameter Variation in Fabrication of Nanostructure by Atomic Force Microscopy Nanolithography

In this letter, we investigate the fabrication of Silicon nanostructure patterned on lightly doped (10¹⁵ cm⁻³) p-type silicon-on-insulator by atomic force microscope nanolithography technique. The local anodic oxidation followed by two wet etching steps, potassium hydroxide etching for silicon removal and hydrofluoric etching for oxide removal, are implemented to reach the structures. The impact of contributing parameters in oxidation such as tip materials, applying voltage on the tip, relative humidity and exposure time are studied. The effect of the etchant concentration (10% to 30% wt) of potassium hydroxide and its mixture with isopropyl alcohol (10%vol. IPA ) at different temperatures on silicon surface are expressed. For different KOH concentrations, the effect of etching with the IPA admixture and the effect of the immersing time in the etching process on the structure are investigated. The etching processes are accurately optimized by 30%wt. KOH +10%vol. IPA in appropriate time, temperature, and humidity.     

Construction of a chimeric antigen receptor bearing a nanobody against prostate a specific membrane antigen in prostate cancer

Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is considered to be a novel anticancer therapy. To date, in most cases, single-chain variable fragments (scFvs) of murine origin have been used in CARs. However, this structure has limitations relating to the potential immunogenicity of mouse antigens in humans and the relatively large size of scFvs. For the first time, we used camelid nanobody (VHH) to construct CAR T cells against prostate specific membrane antigen (PSMA). The nanobody against PSMA (NBP) was used to show the feasibility of CAR T cells against prostate cancer cells. T cells were transfected, and then the surface expression of the CAR T cells was confirmed. Then, the functions of VHH-CAR T cell were evaluated upon coculture with prostate cancer cells. At the end, the cytotoxicity potential of NBPII-CAR in T cells was approximated by determining the cell surface expression of CD107a after encountering PSMA. Our data show the specificity of VHH-CAR T cells against PSMA⁺ cells (LNCaP), not only by increasing the interleukin 2 (IL-2) cytokine (about 400 pg/mL), but also the expression of CD69 by almost 38%. In addition, VHH-CAR T cells were proliferated by nearly 60% when cocultured with LNCaP, as compared with PSMA negative prostate cancer cell (DU-145), which led to the upregulation of CD107a in T cells upto 31%. These results clearly show the possibility of using VHH-based CAR T cells for targeted immunotherapy, which may be developed to target virtually any tumor-associated antigen for adoptive T-cell immunotherapy of solid tumors.     

Graphene oxide/silver nanohybrid: Optimization,antibacterial activity and its impregnation on bacterial cellulose as a potential wound dressing based on GO‐Ag nanocomposite‐coated BC

Recently, bacterial cellulose (BC) based wound dressing have raised significant interests in medical fields. However, to our best knowledge, it is apparent that the BC itself has no antibacterial activity. In this study, we optimized graphene oxide‐silver (GO‐Ag) nanohybrid synthesis using Response Surface Methodology and impregnate it to BC and carefully investigate their antibacterial activities against both the Gram‐negative bacteria Escherichia coli and the Gram‐positive bacteria Staphylococcus aureus. We discover that, compared to silver nanoparticles, GO‐Ag nanohybrid with an optimal GO suspension's pH and ratio is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose. The GO‐Ag nanohybrid is more toxic to E. coli than that to S. aureus. The antibacterial and mechanical properties of BC/GO‐Ag composite are further investigated.