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.     

COVID-19 and male reproductive system: pathogenic features and possible mechanisms

Multiorgan dysfunction is the main characteristic of severe COVID-19 patients and the involvement of male reproductive system may occur among these patients. Although there is a limited evidence to confirm the orchitis and virus presence in the semen of patients, there are concerns about the transmission of virus through the semen. In addition, reduced fertility or infertility can be seen as consequences of severe COVID-19 in recovered subjects. In this study, we aimed to review articles related to COVID-19 and male reproductive system to find the possible underlying mechanisms of SARS-CoV-2 in affecting male fertility. The following keywords of SARS-CoV-2, COVID-19, testis, orchitis, semen, angiotensin-converting enzyme 2 (ACE2), hypothalamic–pituitary–testicular (HPT) axis, Hypothalamus, etc., were defined to find the related publications from standard search engines, e.g., PUBMED, SCOPUS, Google Scholar. According to studies, COVID-19 occurs in severe patients as respiratory disease, along with multi-organ failure. The most important mechanisms are classified as direct and indirect pathogenesis of SARS-CoV-2. The presence of ACE2 on the cell surface of various cells in testis increases the risk of direct infection by this virus. SARS-CoV-2 also affects the testis through the cytokine storm. In addition, the important role of HPT axis dysregulation through impaired Leydig cells and hypothalamus should be considered. Using antiviral and immunomodulatory therapy can be harmful for testis function. Further investigations are required to investigate potential mechanisms of male infertility in survivals of COVID-19. Since involvement of testis is essential for fertility, increasing the knowledge of health system may improve the outcomes.

     

Shedding light on the EpCAM: An overview

The epithelial cell adhesion molecule (EpCAM) is a Type I transmembrane superficial glycoprotein antigen that is expressed on the surface of basolateral membrane of multiple epithelial cells with some exceptions such as epidermal keratinocytes, hepatocytes, thymic cortical epithelial cells, squamous stratified epithelial cells, and myoepithelial cells that do not express the molecule. The molecule plays a pivotal role in the structural integrity, adhesion of the epithelial tissues and their interaction with the underlying layers. EpCAM prevents claudin-7 and claudin-1 molecules from degradation, thereby, decreasing the number of tight junctions and cellular interconnections, and promoting the cells toward carcinogenic transformation. Moreover, the mutations in the EpCAM gene lead to congenital tufting enteropathy, severe intestinal epithelium homeostasis disorders, and Lynch and Lynch syndrome. Overexpression of EpCAM on stem cells of some cancers and the presence of this molecule on circulating tumor cells (CTCs) makes it a promising candidate for cancer diagnosis as well as tracing and isolation of CTCs.     

A review on digestive TAG-lipases of insects

Digestion in insects is a multi-step process to afford nutritional requirements of biological activities. The process starts with nervous stimuli and continues with biochemical activities of digestive enzymes as well as several pumps to digest and absorb the obtained molecules. Carbohydrases, lipases and proteases are the three main digestive enzymes involved in digestion process. Lipases seem to be very important not only for digestive role but also for esteratic activity so that some experts consider lipases as the Class 3 of general esterases. Digestive lipases divided into different groups based on their biological roles namely triacylglycerol lipases, phospholipases and two types of phosphatases. Briefly, triacylglycerol lipases (TAG-lipases) are the hydrolysing enzymes that affect the outer esteric links of triacylglycerols in ingested food. Phospholipases including PLA2 and PLA1 remove phosphatide fatty acids attached to the Position 2 and Position 1. Finally, Alkaline and acid phosphatases are the enzymes that hydrolyse phosphomonoesters under alkaline or acid conditions, respectively. In this review, presence and physiological role of digestive TAG-lipases are explained and their possible importance will be discussed in insect.     

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.