Kv7/KCNQ potassium channels in cortical hyperexcitability and juvenile seizure-related death in Ank2-mutant mice.

Autism spectrum disorders (ASD) represent neurodevelopmental disorders characterized by social deficits, repetitive behaviors, and various comorbidities, including epilepsy. ANK2, which encodes a neuronal scaffolding protein, is frequently mutated in ASD, but its in vivo functions and disease-related mechanisms are largely unknown. Here, we report that mice with Ank2 knockout restricted to cortical and hippocampal excitatory neurons (Ank2-cKO mice) show ASD-related behavioral abnormalities and juvenile seizure-related death. Ank2-cKO cortical neurons show abnormally increased excitability and firing rate. These changes accompanied decreases in the total level and function of the Kv7.2/KCNQ2 and Kv7.3/KCNQ3 potassium channels and the density of these channels in the enlengthened axon initial segment. Importantly, the Kv7 agonist, retigabine, rescued neuronal excitability, juvenile seizure-related death, and hyperactivity in Ank2-cKO mice. These results suggest that Ank2 regulates neuronal excitability by regulating the length of and Kv7 density in the AIS and that Kv7 channelopathy is involved in Ank2-related brain dysfunctions.

Postnatal age-differential ASD-like transcriptomic, synaptic, and behavioral deficits in Myt1l-mutant mice.

Myelin transcription factor 1 like (Myt1l), a zinc-finger transcription factor, promotes neuronal differentiation and is implicated in autism spectrum disorder (ASD) and intellectual disability. However, it remains unclear whether Myt1l promotes neuronal differentiation in vivo and its deficiency in mice leads to disease-related phenotypes. Here, we report that Myt1l-heterozygous mutant (Myt1l-HT) mice display postnatal age-differential ASD-related phenotypes: newborn Myt1l-HT mice, with strong Myt1l expression, show ASD-like transcriptomic changes involving decreased synaptic gene expression and prefrontal excitatory synaptic transmission and altered righting reflex. Juvenile Myt1l-HT mice, with markedly decreased Myt1l expression, display reverse ASD-like transcriptomes, increased prefrontal excitatory transmission, and largely normal behaviors. Adult Myt1l-HT mice show ASD-like transcriptomes involving astrocytic and microglial gene upregulation, increased prefrontal inhibitory transmission, and behavioral deficits. Therefore, Myt1l haploinsufficiency leads to ASD-related phenotypes in newborn mice, which are temporarily normalized in juveniles but re-appear in adults, pointing to continuing phenotypic changes long after a marked decrease of Myt1l expression in juveniles.

Slc6a20a Heterozygous and Homozygous Mutant Mice Display Differential Behavioral and Transcriptomic Changes.

SLC6A20A is a proline and glycine transporter known to regulate glycine homeostasis and NMDA receptor (NMDAR) function in the brain. A previous study found increases in ambient glycine levels and NMDA receptor-mediated synaptic transmission in the brains of Slc6a20a-haploinsufficient mice, but it remained unknown whether Slc6a20a deficiency leads to disease-related behavioral deficits in mice. Here, we report that Slc6a20a heterozygous and homozygous mutant mice display differential behavioral phenotypes in locomotor, repetitive behavioral, and spatial and fear memory domains. In addition, these mice show differential transcriptomic changes in synapse, ribosome, mitochondria, autism, epilepsy, and neuron-related genes. These results suggest that heterozygous and homozygous Slc6a20a deletions in mice lead to differential changes in behaviors and transcriptomes.

On-Chip Gate Electrostatic Discharge Protection Design for 900 V Power Metal Oxide Semiconductor Field Effect Transistor Using Punch-Through Diode Without Degrading Switching Loss.

In order to protect the gate oxide from electrostatic discharge (ESD) in power MOSFET, the on-chip ESD protection circuits are required. Zener diode on poly silicon gate was normally used in power device because Zener diode structure was easy to merge with conditions of vertical structure without adding electrode. However, merged Zener diode can make unnecessary gate capacitance, and then switching characteristics are degraded by added capacitance. In this paper, a stacked punch-through diode (PT diode) with lower capacitance than Zener diode was designed for 900 V power MOSFET. The PT diode was consisted on doped polysilicon gate between the gate pad and the source pad. The electrical characteristics of this device was designed and analyzed by TCAD simulation and experiments. On the basis of this analysis, the stacked PT diode for ESD protection was optimized and compared with conventional Zener diode.

Evaluation of the fit of metal copings fabricated using stereolithography.

STATEMENT OF PROBLEM: Rapid prototyping, including stereolithography (SLA), is a more recent technique for fabricating metal frameworks than the conventional lost-wax technique. However, investigations of the marginal discrepancies and internal spacing of cobalt-chromium (Co-Cr) metal copings fabricated using SLA are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the clinical acceptability of the marginal discrepancies and internal spacing of Co-Cr metal copings fabricated using the SLA technique. MATERIAL AND METHODS: A resin tooth of a maxillary right first premolar was prepared with a deep chamfer margin for a metal-ceramic crown. Titanium master dies were milled after scanning the prepared tooth (n=45). In conventional lost wax group (group LW), the conventional lost-wax technique was used to fabricate Co-Cr metal copings (n=15). In milling group (group MC), a computer-aided design (CAD) system was used to design the metal copings, which were milled from Co-Cr alloy (n=15). The CAD system was also used to design the metal copings in a 3D-printed group (group SL), and Co-Cr metal copings were cast from resin patterns fabricated using the SLA device (n=15). Marginal discrepancies and internal spaces were measured using an optical microscope at ×100 magnification at 11 reference points. The values were analyzed statistically with 1-way analysis of variance (α=.05). RESULTS: The mean (±SD) overall space was 63.2 ±16.6 µm for group LW, 70.2 ±15.5 µm for group SL, and 130.3 ±13.8 µm for group MC. The overall spaces differed significantly between group MC and the other 2 groups (P<.05). The marginal discrepancy and internal spaces were significantly larger in group MC than in groups LW and SL. (P<.05). Occlusal spaces differed significantly among the 3 study groups (P<.05). CONCLUSIONS: Co-Cr metal copings fabricated using an SLA technique showed clinically acceptable marginal discrepancies and internal spaces. These spaces did not differ significantly from those obtained with the conventional lost-wax technique.

Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance.

Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are non-ribosomal peptide and polyketide derived metabolites (NRPs/PKs). Modular non-ribosomal peptide synthetases catalyze main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 Sfp-type 4'-phosphopantetheinyl transferase (Sfp-type PPTase). The inactivation of the gene resulted in loss of NRPs/PKs production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. A26Δsfp biofilm promotion is directly mediated by NRPs/PKs, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their Sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type. Challenges with P. polymyxa genetic manipulation are discussed.

Inactivation of the phosphoglucomutase gene pgm in Paenibacillus polymyxa leads to overproduction of fusaricidin.

Fusaricidin, a lipodepsipeptide isolated from Paenibacillus polymyxa, has high antimicrobial activity against fungi and Gram-positive bacteria. Through mutagenesis, we obtained two mutant strains, N1U7 and N17U7, which produce 6.2- to 7.9-fold more fusaricidin than their parent strain. Causal mutations were identified by whole-genome sequencing, and the two strains each contained at least eleven point mutations, including four common mutations. A mutation in the PPE04441 gene (pgm), encoding an α-phosphoglucomutase, was found to be an important factor in fusaricidin overproduction by complementation experiments. Null mutation of pgm in the parental strain increased fusaricidin production by 5.2-fold. Increased growth and cell viability in stationary phase, reduced exopolysaccharide production, and increased fusA expression were observed in the pgm mutant strains, which might be related to fusaricidin overproduction. This is the first report revealing that PGM deficiency leads to an overproduction of fusaricidin.

Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation.

BACKGROUND: Increased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration. METHODS: The proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of ß1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury. RESULTS: VSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited ß1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited ß1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity. CONCLUSIONS: Magnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation. GENERAL SIGNIFICANCE: This study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis.

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa.

BACKGROUND: Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds. METHODS AND PRINCIPAL FINDINGS: To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and ß-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and ß-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes. CONCLUSIONS: We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and ß-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.

Genome sequence of the polymyxin-producing plant-probiotic rhizobacterium Paenibacillus polymyxa E681.

Paenibacillus polymyxa E681, a spore-forming, low-G+C, Gram-positive bacterium isolated from the rhizosphere of winter barley grown in South Korea, has great potential for agricultural applications due to its ability to promote plant growth and suppress plant diseases. Here we present the complete genome sequence of P. polymyxa E681. Its 5.4-Mb genome encodes functions specialized to the plant-associated lifestyle and characteristics that are beneficial to plants, such as the production of a plant growth hormone, antibiotics, and hydrolytic enzymes.

Identification of a polymyxin synthetase gene cluster of Paenibacillus polymyxa and heterologous expression of the gene in Bacillus subtilis.

Polymyxin, a long-known peptide antibiotic, has recently been reintroduced in clinical practice because it is sometimes the only available antibiotic for the treatment of multidrug-resistant gram-negative pathogenic bacteria. Lack of information on the biosynthetic genes of polymyxin, however, has limited the study of structure-function relationships and the development of improved polymyxins. During whole genome sequencing of Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium, we identified a gene cluster encoding polymyxin synthetase. Here, we report the complete sequence of the gene cluster and its function in polymyxin biosynthesis. The gene cluster spanning the 40.6-kb region consists of five open reading frames, designated pmxA, pmxB, pmxC, pmxD, and pmxE. The pmxC and pmxD genes are similar to genes that encode transport proteins, while pmxA, pmxB, and pmxE encode polymyxin synthetases. The insertional disruption of pmxE led to a loss of the ability to produce polymyxin. Introduction of the pmx gene cluster into the amyE locus of the Bacillus subtilis chromosome resulted in the production of polymyxin in the presence of extracellularly added L-2,4-diaminobutyric acid. Taken together, our findings demonstrate that the pmx gene cluster is responsible for polymyxin biosynthesis.

Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids.

Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C19 and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same Rf value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r2 = 0.997, P < 0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.