Quantification of genomic DNA repair capabilities in CHO and identification of genes impacting genomic stability

Genomic instability in CHO cells poses a challenge for biopharmaceutical production because it is associated with decline of productivity, product quality, and culture viability. Chromosome rearrangements are particularly problematic since these can decrease or eliminate transgene expression. These are caused by DNA double-strand breaks (DSBs) that are not adequately repaired by the cell, presumably due to deficiencies in DNA repair genes. In this study we have conducted a genome-wide bioinformatic analysis of single-nucleotide variants (SNVs) in DNA-repair genes in the CHO genome. We implement a reporter system in CHO cells that facilitates the quantification of the cell’s capability to repair DSBs in genomic DNA. This provides a DNA stability assessment that is superior to previous assays since these would merely read out the capability to repair artificial plasmids. By utilizing this genomic DSB repair assay, we can quantify DNA stability in standard CHO cells, various DNA repair-deficient CHO mutants, as well as in primary Chinese hamster cells. Finally, we explore how by targeting defective candidate genes from our bioinformatic analysis, this assay can be used to engineer CHO cell lines with increased genomic stability

Adaptive data acquisition multiplexing system and method

A reconfigurable telemetry multiplexer is described which includes a monitor-terminal and a plurality of remote terminals. The remote terminals each include signal conditioning for a plurality of sensors for measuring parameters which are converted by an analog to digital converter. CPU's in the remote terminals store instructions for prompting system configuration and reconfiguration commands. The measurements, instructions, and the terminal's present configuration and status data are transmitted to the monitor-terminal and displayed. In response to menu-driven prompts generated and displayed at the monitor-terminal, data generation request commands, status and health commands, and the like are input at the monitor-terminal and transmitted to the remote terminals. The CPU in each remote terminal receives the various commands, stores them in electrically alterable memory, and reacts in accordance with the commands to reconfigure a plurality of aspects of the system. The CPU in each terminal also generates parameter measurements, status and health signals, and transmits these signals of the respective terminals to the monitor-terminal for low data rate operator viewing and to higher rate external transmission/monitor equipment. Reconfiguration may be in real time during the general period of parameter measurement acquisition, and may include alteration of the gain, automatic gain rescaling, bias, and or sampling rates associated with one or more of the parameter measurements made by the remote terminals

PinAPL-Py: A comprehensive web-application for the analysis of CRISPR/Cas9 screens.

Large-scale genetic screens using CRISPR/Cas9 technology have emerged as a major tool for functional genomics. With its increased popularity, experimental biologists frequently acquire large sequencing datasets for which they often do not have an easy analysis option. While a few bioinformatic tools have been developed for this purpose, their utility is still hindered either due to limited functionality or the requirement of bioinformatic expertise. To make sequencing data analysis of CRISPR/Cas9 screens more accessible to a wide range of scientists, we developed a Platform-independent Analysis of Pooled Screens using Python (PinAPL-Py), which is operated as an intuitive web-service. PinAPL-Py implements state-of-the-art tools and statistical models, assembled in a comprehensive workflow covering sequence quality control, automated sgRNA sequence extraction, alignment, sgRNA enrichment/depletion analysis and gene ranking. The workflow is set up to use a variety of popular sgRNA libraries as well as custom libraries that can be easily uploaded. Various analysis options are offered, suitable to analyze a large variety of CRISPR/Cas9 screening experiments. Analysis output includes ranked lists of sgRNAs and genes, and publication-ready plots. PinAPL-Py helps to advance genome-wide screening efforts by combining comprehensive functionality with user-friendly implementation. PinAPL-Py is freely accessible at http://pinapl-py.ucsd.edu with instructions and test datasets

Chains of Viscoelastic Spheres

Given a chain of viscoelastic spheres with fixed masses of the first and last particles. We raise the question: How to chose the masses of the other particles of the chain to assure maximal energy transfer? The results are compared with a chain of particles for which a constant coefficient of restitution is assumed. Our simple example shows that the assumption of viscoelastic particle properties has not only important consequences for very large systems (see [1]) but leads also to qualitative changes in small systems as compared with particles interacting via a constant restitution coefficient.Comment: 11 pages, 6 figure

Changes in axonal excitability of primary sensory afferents with general anaesthesia in humans

Background Intraoperative monitoring of neuronal function is important in a variety of surgeries. The type of general anaesthetic used can affect the interpretation and quality of such recordings. Although the principal effects of general anaesthetics are synaptically mediated, the extent to which they affect excitability of the peripheral afferent nervous system is unclear. Methods Forty subjects were randomized in a stratified manner into two groups, anaesthetized with either propofol or sevoflurane. The threshold tracking technique (QTRAC®) was used to measure nerve excitability parameters of the sensory action potential of the median nerve before and after induction of general anaesthesia. Results Several parameters of peripheral sensory afferent nerve excitability changed after induction of general anaesthesia, which were similar for both propofol and sevoflurane. The maximum amplitude of the sensory nerve action potential decreased in both groups (propofol: 25.3%; sevoflurane: 29.5%; both P<0.01). The relative refractory period [mean (sd)] also decreased similarly in both groups [propofol: −0.6 (0.7) ms; sevoflurane: −0.3 (0.5) ms; both P<0.01]. Skin temperature at the stimulation site increased significantly in both groups [propofol: +1.2 (1.0)°C; sevoflurane: +1.7 (1.4)°C; both P<0.01]. Conclusions Small changes in excitability of primary sensory afferents after the induction of anaesthesia with propofol or sevoflurane were detected. These effects, which were non-specific and are possibly explained by changes observed in temperature, demonstrate possible anaesthetic effects on intraoperative neuromonitorin

Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

Biosimilar drugs must closely resemble the pharmacological attributes of innovator products to ensure safety and efficacy to obtain regulatory approval. Glycosylation is one critical quality attribute that must be matched, but it is inherently difficult to control due to the complexity of its biogenesis. This usually implies that costly and time-consuming experimentation is required for clone identification and optimization of biosimilar glycosylation. Here, we describe a computational method that utilizes a Markov model of glycosylation to predict optimal glycoengineering strategies to obtain a specific glycosylation profile with desired properties. The approach uses a genetic algorithm to find the required quantities to perturb glycosylation reaction rates that lead to the best possible match with a given glycosylation profile. Furthermore, the approach can be used to identify cell lines and clones that will require minimal intervention while achieving a glycoprofile that is most similar to the desired profile. Thus, this approach can facilitate biosimilar design by providing computational glycoengineering guidelines that can be generated with a minimal time and cost

A model of ballistic aggregation and fragmentation

A simple model of ballistic aggregation and fragmentation is proposed. The model is characterized by two energy thresholds, Eagg and Efrag, which demarcate different types of impacts: If the kinetic energy of the relative motion of a colliding pair is smaller than Eagg or larger than Efrag, particles respectively merge or break; otherwise they rebound. We assume that particles are formed from monomers which cannot split any further and that in a collision-induced fragmentation the larger particle splits into two fragments. We start from the Boltzmann equation for the mass-velocity distribution function and derive Smoluchowski-like equations for concentrations of particles of different mass. We analyze these equations analytically, solve them numerically and perform Monte Carlo simulations. When aggregation and fragmentation energy thresholds do not depend on the masses of the colliding particles, the model becomes analytically tractable. In this case we show the emergence of the two types of behavior: the regime of unlimited cluster growth arises when fragmentation is (relatively) weak and the relaxation towards a steady state occurs when fragmentation prevails. In a model with mass-dependent Eagg and Efrag the evolution with a cross-over from one of the regimes to another has been detected