Bar graph monitor

Bar graph monitor of pulse position modulation telemetry ground station equipment for sounding rocket

Evaluation of Demand Response on System Reliability and Cable Aging

Emergency loading implementation brings reliability and flexibility benefits to the operator. However, its application results in higher cable temperature, which may lead to increase of aging risk. Furthermore, demand response (DR) option can clip the customer loads and shift them to a different period. The DR clipping process reduces the loading of the network to avoid cable overheating and reduce its aging risk. The study aims to evaluate the impact of DR in increasing the reliability of the system as well as reducing the aging of the network. The proposed DR is implemented in the cable-based transmission network, as a complementary of emergency loading, by taking into account the large thermal inertia of the cable. It uses cable temperature elevation as the trigger as an alternative to demand level trigger, which is the traditional approach. The evaluation uses Sequential Monte Carlo Simulation (SMCS) on the IEEE 14 Bus Reliability Test System. The implementation of temperature-based DR reduces the network aging by 3% as well as expected energy not supplied by 34% compared to the emergency loading only

RNA mutagenesis yields highly diverse mRNA libraries for in vitro protein evolution

BACKGROUND: In protein drug development, in vitro molecular optimization or protein maturation can be used to modify protein properties. One basic approach to protein maturation is the introduction of random DNA mutations into the target gene sequence to produce a library of variants that can be screened for the preferred protein properties. Unfortunately, the capability of this approach has been restricted by deficiencies in the methods currently available for random DNA mutagenesis and library generation. Current DNA based methodologies generally suffer from nucleotide substitution bias that preferentially mutate particular base pairs or show significant bias with respect to transitions or transversions. In this report, we describe a novel RNA-based random mutagenesis strategy that utilizes Qβ replicase to manufacture complex mRNA libraries with a mutational spectrum that is close to the ideal. RESULTS: We show that Qβ replicase generates all possible base substitutions with an equivalent preference for mutating A/T or G/C bases and with no significant bias for transitions over transversions. To demonstrate the high diversity that can be sampled from a Qβ replicase-generated mRNA library, the approach was used to evolve the binding affinity of a single domain V(NAR )shark antibody fragment (12Y-2) against malarial apical membrane antigen-1 (AMA-1) via ribosome display. The binding constant (K(D)) of 12Y-2 was increased by 22-fold following two consecutive but discrete rounds of mutagenesis and selection. The mutagenesis method was also used to alter the substrate specificity of β-lactamase which does not significantly hydrolyse the antibiotic cefotaxime. Two cycles of RNA mutagenesis and selection on increasing concentrations of cefotaxime resulted in mutants with a minimum 10,000-fold increase in resistance, an outcome achieved faster and with fewer overall mutations than in comparable studies using other mutagenesis strategies. CONCLUSION: The RNA based approach outlined here is rapid and simple to perform and generates large, highly diverse populations of proteins, each differing by only one or two amino acids from the parent protein. The practical implications of our results are that suitable improved protein candidates can be recovered from in vitro protein evolution approaches using significantly fewer rounds of mutagenesis and selection, and with little or no collateral damage to the protein or its mRNA

Antisense oligonucleotide therapy for KCNT1 encephalopathy

Published online: 22 November 2022Developmental and epileptic encephalopathies (DEE) are characterized by pharmacoresistant seizures with concomitant intellectual disability. Epilepsy of infancy with migrating focal seizures (EIMFS) is one of the most severe of these syndromes. De novo variants in ion channels, including gain-of-function variants in KCNT1, have been found to play a major role in the etiology of EIMFS. Here, we test a potential precision therapeutic approach in KCNT1-associated DEE using a gene silencing antisense oligonucleotide (ASO) approach. We generated a mouse model carrying the KCNT1 p.P924L pathogenic variant; only the homozygous animals presented with the frequent, debilitating seizures and developmental compromise that are seen in patients. After a single intracerebroventricular bolus injection of a Kcnt1 gapmer ASO in symptomatic mice at postnatal day 40, seizure frequency was significantly reduced, behavioral abnormalities improved, and overall survival was extended compared to mice treated with a control ASO (non-hybridizing sequence). ASO administration at neonatal age was also well-tolerated and effective in controlling seizures and extending the lifespan of treated animals. The data presented here provide proof of concept for ASO-based gene silencing as a promising therapeutic approach in KCNT1-associated epilepsies.Lisseth Estefania Burbano, Melody Li, Nikola Jancovski, Paymaan Jafar-Nejad, Kay Richards, Alicia Sedo, Armand Soriano, Ben Rollo, Linghan Jia, Elena V. Gazina, Sandra Piltz, Fatwa Adikusuma, Paul Q. Thomas, Helen Kopsidas, Frank Rigo, Christopher A. Reid, Snezana Maljevic, Steven Petro

Network Models of TEM β-Lactamase Mutations Coevolving under Antibiotic Selection Show Modular Structure and Anticipate Evolutionary Trajectories

Understanding how novel functions evolve (genetic adaptation) is a critical goal of evolutionary biology. Among asexual organisms, genetic adaptation involves multiple mutations that frequently interact in a non-linear fashion (epistasis). Non-linear interactions pose a formidable challenge for the computational prediction of mutation effects. Here we use the recent evolution of β-lactamase under antibiotic selection as a model for genetic adaptation. We build a network of coevolving residues (possible functional interactions), in which nodes are mutant residue positions and links represent two positions found mutated together in the same sequence. Most often these pairs occur in the setting of more complex mutants. Focusing on extended-spectrum resistant sequences, we use network-theoretical tools to identify triple mutant trajectories of likely special significance for adaptation. We extrapolate evolutionary paths (n = 3) that increase resistance and that are longer than the units used to build the network (n = 2). These paths consist of a limited number of residue positions and are enriched for known triple mutant combinations that increase cefotaxime resistance. We find that the pairs of residues used to build the network frequently decrease resistance compared to their corresponding singlets. This is a surprising result, given that their coevolution suggests a selective advantage. Thus, β-lactamase adaptation is highly epistatic. Our method can identify triplets that increase resistance despite the underlying rugged fitness landscape and has the unique ability to make predictions by placing each mutant residue position in its functional context. Our approach requires only sequence information, sufficient genetic diversity, and discrete selective pressures. Thus, it can be used to analyze recent evolutionary events, where coevolution analysis methods that use phylogeny or statistical coupling are not possible. Improving our ability to assess evolutionary trajectories will help predict the evolution of clinically relevant genes and aid in protein design

Untersuchungen zu Matrix-Metalloproteasen und Metalloproteaseinhibitoren im verletzten und regenerierenden Sehnerv der Ratte:Untersuchungen zu Matrix Metalloproteasen und Metalloproteaseinhibitoren im verletzten und regenerierenden Sehnerv der Ratte

Das Ziel dieser Arbeit ist die immunhistochemische Analyse der Rolle der Matrix-Metalloproteasen (MMP) und der Tissue-Metalloproteaseninhibitoren (TIMP), bei der posttraumatischen Degeneration und bei induzierter Regeneration des Sehnervs. Augen von erwachsenen Ratten wurden als Kontrolle benutzt. Die Degenerations- bzw. die Regenerationsfähigkeit der retinalen Ganglienzellen (RGZ) wurden anhand des Transections- bzw. Transplantationsmodels untersucht. Sowohl die Expression von MMP-1,-2,-3,-14 und TIMP-1,-3, als auch die zellulären Veränderungen sind immunohistochemisch untersucht. MMP-1,-2,-3 werden in den regenerierenden Retinas hochreguliert. MMP-14 ist gleich hochreguliert in beiden experimentellen Gruppen. TIMP-3 ist hochreguliert in den degenerierenden Retinas. TIMP-1 ist in dem regenerierenden Gewebe nachzuweisen. MMPs werden unterschiedlich zwischen De- und Regeneration reguliert. TIMP-1 ist mit dem Überleben von RGZ assoziiert, während TIMP-3 mit Apoptosis assoziiert ist