Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.     

Effects of crocin in reducing DNA damage,inflammation, and oxidative stress in multiple sclerosis patients: A double‐blind,randomized, and placebo‐controlled trial

Multiple sclerosis (MS) is an autoimmune disease in which the immune system attacks the nerve cells, resulting in neurological disorders. Oxidative stress, free radicals, and neuritis have important roles in MS pathogenesis. Here, we aim to evaluate the effect of crocin on inflammatory markers, oxidative damage, and deoxyribonucleic acid (DNA) damage in the blood of patients with MS. A total of 40 patients were divided into two groups, drug and placebo‐treated groups, using random assignment. Participants of the intervention and control groups received two crocin capsules or placebo per day for 28 days, respectively. Findings revealed a significant decrease in the level of important pathogenic factors in MS, including lipid peroxidation, DNA damage, tumor necrosis factor‐alpha, and interleukin 17 as well as a significant increase in the total antioxidant capacity in the serum of patients treated with crocin compared with the placebo group. Our results suggest the beneficial and therapeutic effects of crocin in MS.     

Feeding corn germ instead of corn grain on the performance of Holstein dairy cows fed low-forage diet and human-edible feed conversion efficiency

Using corn germ (CG) instead of corn grain could maintain dairy cow performance and might increase the efficiency of human food production. The primary objective of this study was to evaluate the effects of replacing corn grain with CG on the performance, nutrient intake, and digestibility of dairy cows. It also aimed to investigate the effect of CG on the efficiency of human food production in high-producing Holstein dairy cows in early lactation. Nine multiparous Holstein cows with 65.6 ± 8.5 DIM, milk yield of 55.6 ± 4.5 kg/d, and body weight of 611.3 ± 43.3 kg (mean ± SD) were used in a 3 × 3 Latin square design with 21-d periods. Treatments were (1) control treatment (CT, diet contains corn grain), (2) alternative treatment (AT, diet where corn grain was replaced with CG), and (3) balanced treatment (BT, diet where corn grain was replaced with CG but with the same energy content as CT). Control and balanced diets were isoenergetic (6.61 MJ/kg of DM); however, AT had higher energy (6.77 MJ/kg of DM). Treatments had no effect on dry and organic matter intake. NDF intake, however, was higher in CG diets compared with CT (P = 0.0001). Total-tract digestibility of DM tended to be reduced (P = 0.08), and OM digestibility was reduced (P = 0.05) by the inclusion of CG in diets. Whole and energy-corrected milk production were greater in AT compared with CT and BT (P < 0.05). Milk yield was similar in cows fed CT and BT. Treatments had no effect on milk composition or feed efficiency. Diet CT, when compared with CG diets, had lower efficiency in terms of human-edible feed conversion efficiency (HeFCE) and net food production (P < 0.05). Diet BT had greater HeFCE and net production of human-edible CP than AT (P < 0.05). Plasma BHBA, non-esterified fatty acids, and glucose concentrations were not affected by treatments, but plasma cholesterol was higher in cows that consumed CG diets (P = 0.04). The results indicate that, in high-producing early lactation dairy cows fed high concentrate diets, net food protein production can be substantially improved without lowering milk production through the reduction of dietary starch from 30.2 to 24.8% by replacing corn grain with CG.     

Magnetoelectric nanocomposite scaffold for high yield differentiation of mesenchymal stem cells to neural-like cells

While the differentiation factors have been widely used to differentiate mesenchymal stem cells (MSCs) into various cell types, they can cause harm at the same time. Therefore, it is beneficial to propose methods to differentiate MSCs without factors. Herein, magnetoelectric (ME) nanofibers were synthesized as the scaffold for the growth of MSCs and their differentiation into neural cells without factors. This nanocomposite takes the advantage of the synergies of the magnetostrictive filler, CoFe₂O ₄ nanoparticles (CFO), and piezoelectric polymer, polyvinylidene difluoride (PVDF). Graphene oxide nanosheets were decorated with CFO nanoparticles for a proper dispersion in the polymer through a hydrothermal process. After that, the piezoelectric PVDF polymer, which contained the magnetic nanoparticles, underwent the electrospun process to form ME nanofibers, the ME property of which has the potential to be used in areas such as tissue engineering, biosensors, and actuators.