Serum-Free Cultures: Could They Be a Future Direction to Improve Neuronal Differentiation of Mesenchymal Stromal Cells?

Mesenchymal stem/stromal cells (MSCs) are undifferentiated cells with multilinear potential, known for their immunomodulatory and regenerative properties. Although the scientific community is working to improve their application, concerns limit their use to repair tissues following neurological damage. One of these obstacles is represented by the use of culture media supplemented with fetal bovine serum (FBS), which, due to its xenogenic nature and the risk of contamination, has increased scientific, ethical and safety problems. Therefore, the use of serum-free media could improve MSC culture methods, avoiding infectious and immunogenic transmission problems as well as MSC bioprocesses, without the use of animal components. The purpose of our review is to provide an overview of experimental studies that demonstrate that serum-free cultures, along with the supplementation of growth factors or chemicals, can lead to a more defined and controlled environment, enhancing the proliferation and neuronal differentiation of MSCs.     

Single-Step Fast Tissue Clearing of Thick Mouse Brain Tissue for Multi-Dimensional High-Resolution Imaging

Recent advances in optical clearing techniques have dramatically improved deep tissue imaging by reducing the obscuring effects of light scattering and absorption. However, these optical clearing methods require specialized equipment or a lengthy undertaking with complex protocols that can lead to sample volume changes and distortion. In addition, the imaging of cleared tissues has limitations, such as fluorescence bleaching, harmful and foul-smelling solutions, and the difficulty of handling samples in high-viscosity refractive index (RI) matching solutions. To address the various limitations of thick tissue imaging, we developed an Aqueous high refractive Index matching and tissue Clearing solution for Imaging (termed AICI) with a one-step tissue clearing protocol that was easily made at a reasonable price in our own laboratory without any equipment. AICI can rapidly clear a 1 mm thick brain slice within 90 min with simultaneous RI matching, low viscosity, and a high refractive index (RI = 1.466), allowing the imaging of the sample without additional processing. We compared AICI with commercially available RI matching solutions, including optical clear agents (OCAs), for tissue clearing. The viscosity of AICI is closer to that of water compared with other RI matching solutions, and there was a less than 2.3% expansion in the tissue linear morphology during 24 h exposure to AICI. Moreover, AICI remained fluid over 30 days of air exposure, and the EGFP fluorescence signal was only reduced to ~65% after 10 days. AICI showed a limited clearing of brain tissue >3 mm thick. However, fine neuronal structures, such as dendritic spines and axonal boutons, could still be imaged in thick brain slices treated with AICI. Therefore, AICI is useful not only for the three-dimensional (3D) high-resolution identification of neuronal structures, but also for the examination of multiple structural imaging by neuronal distribution, projection, and gene expression in deep brain tissue. AICI is applicable beyond the imaging of fluorescent antibodies and dyes, and can clear a variety of tissue types, making it broadly useful to researchers for optical imaging applications.     

Synthesis and Anti-Cancer Activity of New Pyrazolinyl-Indole Derivatives: Pharmacophoric Interactions and Docking Studies for Identifying New EGFR Inhibitors

Newly designed series of indole-containing pyrazole analogs, pyrazolinylindoles, were synthesized, and their structures were confirmed based on the spectral data of the ¹H NMR, ¹³C NMR, and HR-MS analyses. Preliminary anti-cancer activity testings were carried out by the National Cancer Institute, United States of America (NCI, USA). Compounds HD02, HD05, and HD12 demonstrated remarkable cytotoxic activities against nine categories of cancer types based cell line panels which included leukemia, colon, breast, melanoma, lungs, renal, prostate, CNS, and ovarian cancer cell lines. The highest cytotoxic effects were exhibited by the compounds HD02 [1-(5-(1-H-indol-3-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenylethanone], HD05 [1-(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)-2-phenoxyethanone], and HD12 [(3-(4-chlorophenyl)-5-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-1-yl)(pyridin-4-yl)methanone] against some of the 56 types of NCI-based cell lines in different panels. Compound HD05 showed the maximum range of cancer cell growth inhibitions against all categories of the cell lines in all nine panels. On average, in comparison to the referral standard, imatinib, at a dose level of 10 µM, the HD05 showed significant activity against leukemia in the range of 78.76%, as compared to the imatinib at 9% of cancer cells’ growth inhibitions. Molecular docking simulation studies were performed in silico on the epidermal growth factor receptor (EGFR) tyrosine kinase, in order to validate the activity.     

Peeling dynamics of fluid membranes bridged by molecular bonds: moving or breaking

Biological adhesion is a critical mechanical function of complex organisms. At the scale of cell–cell contacts, adhesion is remarkably tunable to enable both cohesion and malleability during development, homeostasis and disease. It is physically supported by transient and laterally mobile molecular bonds embedded in fluid membranes. Thus, unlike specific adhesion at solid–solid or solid–fluid interfaces, peeling at fluid–fluid interfaces can proceed by breaking bonds, by moving bonds or by a combination of both. How the additional degree of freedom provided by bond mobility changes the mechanics of peeling is not understood. To address this, we develop a theoretical model coupling diffusion, reactions and mechanics. Mobility and reaction rates determine distinct peeling regimes. In a diffusion-dominated Stefan-like regime, bond motion establishes self-stabilizing dynamics that increase the effective fracture energy. In a reaction-dominated regime, peeling proceeds by travelling fronts where marginal diffusion and unbinding control peeling speed. In a mixed reaction–diffusion regime, strengthening by bond motion competes with weakening by bond breaking in a force-dependent manner, defining the strength of the adhesion patch. In turn, patch strength depends on molecular properties such as bond stiffness, force sensitivity or crowding. We thus establish the physical rules enabling tunable cohesion in cellular tissues and in engineered biomimetic systems.     

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.     

钨镍共掺杂V2O5薄膜的光电特性研究

利用溶胶–凝胶旋涂法和后退火工艺在FTO导电玻璃上制备了钨镍共掺杂V₂O₅薄膜,研究了薄膜在不同温度和不同偏压下的光电特性和相变特性。利用X射线衍射仪（XRD）、扫描电子显微镜（SEM） 和X射线光电子能谱仪（XPS） 测试了钨镍共掺杂V₂O₅薄膜的晶体结构、表面形貌和组分,分析了不同钨镍共掺杂浓度对V₂O₅薄膜相变光电特性的影响。结果表明,当钨和镍的掺杂质量分数分别为3 %和1.5 %时,钨镍共掺杂的V₂O₅薄膜的相变温度为218.5 ℃,在可见光范围内有较高的透过率,在近红外1310 nm波长处的光学透过率达48.83%,与未掺杂V₂O₅薄膜的光学透过率相比提高了10.29%,薄膜电阻降低了30.53%,热致回线宽度收窄为15 ℃,说明钨镍共掺杂的V₂O₅薄膜具有良好的可逆相变光电特性,有望在新型光电器件领域得到较好的应用。     

Integrated Analysis of Microarray,Small RNA,and Degradome Datasets Uncovers the Role of MicroRNAs in Temperature-Sensitive Genic Male Sterility in Wheat

Temperature-sensitive genic male sterile (TGMS) line Beijing Sterility 366 (BS366) has been utilized in hybrid breeding for a long time, but the molecular mechanism underlying male sterility remains unclear. Expression arrays, small RNA, and degradome sequencing were used in this study to explore the potential role of miRNA in the cold-induced male sterility of BS366. Microspore observation showed defective cell plates in dyads and tetrads and shrunken microspores at the vacuolated stage. Differential regulation of Golgi vesicle transport, phragmoplast formation, sporopollenin biosynthesis, pollen exine formation, and lipid metabolism were observed between cold and control conditions. Pollen development was significantly represented in the 352 antagonistic miRNA-target pairs in the integrated analysis of miRNA and mRNA profiles. The specific cleavage of ARF17 and TIR1 by miR160 and miR393 were found in the cold-treated BS366 degradome, respectively. Thus, the cold-mediated miRNAs impaired cell plate formation through repression of Golgi vesicle transport and phragmoplast formation. The repressed expression of ARF17 and TIR1 impaired pollen exine formation. The results of this study will contribute to our understanding of the roles of miRNAs in male sterility in wheat.     

Cloning and Characterization of a Thermostable Endolysin of Bacteriophage TP-84 as a Potential Disinfectant and Biofilm-Removing Biological Agent

The obligatory step in the life cycle of a lytic bacteriophage is the release of its progeny particles from infected bacterial cells. The main barrier to overcome is the cell wall, composed of crosslinked peptidoglycan, which counteracts the pressure prevailing in the cytoplasm and protects the cell against osmotic lysis and mechanical damage. Bacteriophages have developed two strategies leading to the release of progeny particles: the inhibition of peptidoglycan synthesis and enzymatic cleavage by a bacteriophage-coded endolysin. In this study, we cloned and investigated the TP84_28 endolysin of the bacteriophage TP-84, which infects thermophilic Geobacillus stearothermophilus, determined the enzymatic characteristics, and initially evaluated the endolysin application as a non-invasive agent for disinfecting surfaces, including those exposed to high temperatures. Both the native and recombinant TP84_28 endolysins, obtained through the Escherichia coli T7-lac expression system, are highly thermostable and retain trace activity after incubation at 100 °C for 30 min. The proteins exhibit strong bacterial wall digestion activity up to 77.6 °C, decreasing to marginal activity at ambient temperatures. We assayed the lysis of various types of bacteria using TP84_28 endolysins: Gram-positive, Gram-negative, encapsulated, and pathogenic. Significant lytic activity was observed on the thermophilic and mesophilic Gram-positive bacteria and, to a lesser extent, on the thermophilic and mesophilic Gram-negative bacteria. The thermostable TP84_28 endolysin seems to be a promising mild agent for disinfecting surfaces exposed to high temperatures.     

Digitoxin Affects Metabolism,ROS Production and Proliferation in Pancreatic Cancer Cells Differently Depending on the Cell Phenotype

Digitoxin has repeatedly shown to have negative effects on cancer cell viability; however, the actual mechanism is still unknown. In this study, we investigated the effects of digitoxin (1–100 nM) in four pancreatic cancer cell lines, BxPC-3, CFPAC-1, Panc-1, and AsPC-1. The cell lines differ in their KRAS/BRAF mutational status and primary tumor or metastasis origin. We could detect differences in the basal rates of cell proliferation, glycolysis, and ROS production, giving the cell lines different phenotypes. Digitoxin treatment induced apoptosis in all four cell lines, but to different degrees. Cells derived from primary tumors (Panc-1 and BxPC-3) were highly proliferating with a high proportion of cells in the S/G2 phase, and were more sensitive to digitoxin treatment than the cell lines derived from metastases (CFPAC-1 and AsPC-1), with a high proportion of cells in G0/G1. In addition, the effects of digitoxin on the rate of glycolysis, ROS production, and proliferation were dependent on the basal metabolism and origin of the cells. The KRAS downstream signaling pathways were not altered by digitoxin treatment, thus the effects exerted by digitoxin were probably disconnected from these signaling pathways. We conclude that digitoxin is a promising treatment in highly proliferating pancreatic tumors.