A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.

Progress in elucidating the molecular and cellular pathophysiology of neuropsychiatric disorders has been hindered by the limited availability of living human brain tissue. The emergence of induced pluripotent stem cells (iPSCs) has offered a unique alternative strategy using patient-derived functional neuronal networks. However, methods for reliably generating iPSC-derived neurons with mature electrophysiological characteristics have been difficult to develop. Here, we report a simplified differentiation protocol that yields electrophysiologically mature iPSC-derived cortical lineage neuronal networks without the need for astrocyte co-culture or specialized media. This protocol generates a consistent 60:40 ratio of neurons and astrocytes that arise from a common forebrain neural progenitor. Whole-cell patch-clamp recordings of 114 neurons derived from three independent iPSC lines confirmed their electrophysiological maturity, including resting membrane potential (-58.2±1.0 mV), capacitance (49.1±2.9 pF), action potential (AP) threshold (-50.9±0.5 mV) and AP amplitude (66.5±1.3 mV). Nearly 100% of neurons were capable of firing APs, of which 79% had sustained trains of mature APs with minimal accommodation (peak AP frequency: 11.9±0.5 Hz) and 74% exhibited spontaneous synaptic activity (amplitude, 16.03±0.82 pA; frequency, 1.09±0.17 Hz). We expect this protocol to be of broad applicability for implementing iPSC-based neuronal network models of neuropsychiatric disorders.

The effect of antioxidant vitamins E and C on cognitive performance of the elderly with mild cognitive impairment in Isfahan, Iran: a double-blind, randomized, placebo-controlled trial.

PURPOSE: This study was carried out to investigate the effect of vitamins E and C on cognitive performance among the elderly in Iran. METHODS: About 256 elderly with mild cognitive impairment, aged 60-75 years, received 300 mg of vitamin E plus 400 mg of vitamin C or placebo daily just for 1 year. BACKGROUND: Demographic characteristics, anthropometric variables food consumption, cognitive function by Mini-Mental State Examination (MMSE), and some of the oxidative stress biomarkers were examined. RESULTS: Antioxidant supplementation reduced malondialdehyde level (P < 0.001) and raised total antioxidant capacity (P < 0.001) and glutathione (P < 0.01). The serum 8-hydroxydeoxyguanosine remained unchanged (P < 0.4). After adjusting for the covariates effects, MMSE scores following 6- (25.88 ± 0.17) and 12-month antioxidant supplementation (26.8 ± 0.17) did not differ from control group (25.86 ± 0.18 and 26.59 ± 0.18, respectively). CONCLUSION: Despite significant improvement in most of the oxidative stress biomarkers, antioxidants' supplementation was not observed to enhance cognitive performance. A large number of kinetic and/or dynamic factors could be suspected.

Electrochemical synthesis of vertically aligned zinc nanowires using track-etched polycarbonate membranes as templates.

In the present paper, vertically aligned arrays of zinc nanowires were synthesized by electrochemical deposition into ion track-etched polycarbonate membranes in the ionic liquid electrolyte 1-ethyl-3-methylimidazolium trifluoromethylsulfonate ([EMIm]TfO)/Zn(TfO)2. Cyclic voltammetry and chronoamperometry were performed to investigate the electrochemical growth of zinc nanowires inside of the membranes. The transport processes and mechanisms of the nanowire growth in the membranes are also discussed. A supporting zinc or copper layer was deposited on the sputtered side in order to make the back layer thick enough to stabilize the wires. Zinc nanowires with a diameter of 90 nm and a length of up to 18 µm were obtained after removing the template. Furthermore, short nanowires with lengths less than 5 µm and a sandwich-like structure with nanowires in the middle were also synthesized. Vertically aligned zinc nanowire structures on such a substrate might be a potential anode candidate for future generation lithium ion batteries.

The essential DNA-binding protein sap1 of Schizosaccharomyces pombe contains two independent oligomerization interfaces that dictate the relative orientation of the DNA-binding domain.

The sap1 gene from Schizosaccharomyces pombe, which is essential for mating-type switching and for growth, encodes a sequence-specific DNA-binding protein with no homology to other known proteins. We have used a reiterative selection procedure to isolate binding sites for sap1, using a bacterially expressed protein and randomized double-strand oligonucleotides. The sap1 homodimer preferentially selects a pentameric motif, TA(A/G)CG, organized as a direct repeat and spaced by 5 nucleotides. Removal of a C-terminal dimerization domain abolishes recognition of the direct repeat and creates a new specificity for a DNA sequence containing the same pentameric motif but organized as an inverted repeat. We present evidence that the orientation of the DNA-binding domain is controlled by two independent oligomerization interfaces. The C-terminal dimerization domain allows a head-to-tail organization of the DNA-binding domains in solution, while an N-terminal domain is involved in a cooperative interaction on the DNA target between pairs of dimers.

Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.

The sequence of a 9.3 kb segment located on the left arm of the yeast chromosome XI reveals five open reading frames including the CCE1 gene and putative products related to MYO2 and to the ribosomal protein L10.

We report here the sequence of a 9.3 kb DNA segment of chromosome XI of Saccharomyces cerevisiae, located between the MAK11 locus and the centromere. This sequence contains four long open reading frames (ORFs), YKL160, YKL162, YKL164, YKL165 and part of another ORF, YKL166, covering altogether 90% of the entire sequence. One of these ORFs, YKL164, corresponds to CCE1. Translation products of two other ORFs, YKL160 and YKL165, exhibit homology with previously known S. cerevisiae proteins: the ribosomal protein L10, and the MYO2 gene product, respectively.

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.