Assessment of cloning efficiency and error rates of different single-cell dispensing and high-resolution imaging technologies

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Heavy ion induced DNA-DSB in yeast and mammalian cells

Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes

Generation and characterization of a process-specific HCP reagent using stably transfected Null Cell Pools

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Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation can influence pharmacokinetics and effector functions of therapeutic monoclonal antibodies. Matching glycosylation product quality attributes within defined specification ranges when manufacturing a given antibody at different scales, different sites, or from different host cell lines poses a formidable challenge to the bio-manufacturing industry. One approach to control glycosylation attributes is to supplement cell culture media with additives known to influence glycosylation patterns. Here, we report a two-tier approach for the modulation of nine quality attributes in monoclonal antibodies expressed in CHO cells. In a first step, we tested ten different additives both in univariate and multivariate modes, pursuing a fractional factorial design-of-experiments approach in 24-well deep well format. We found several components to influence one or more quality attributes, either as single agents or in combination with other additives. As a second tier, we designed experiments to manipulate certain aspects of the glycosylation profile in a targeted fashion. We show proof of concept across different lab scale models, including deep wells, shake flasks, and 2L single-use bioreactors. Further, we characterized the extent of product quality modulation across CHO cell lines with different genetic background producing the same IgG1 molecule, and we characterized differences between a canonical IgG1 vs. an isotype-matched mAb with engineered cysteine residues. Taken together, this study provides comprehensive information about the extent and applicability of product quality modulation through media additives, and will enable the implementation of a robust process development platform

Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca2+) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities

An Essential Role for the Tetraspanin LHFPL4 in the Cell-Type-Specific Targeting and Clustering of Synaptic GABAReceptors

Inhibitory synaptic transmission requires the targeting and stabilization of GABAA receptors (GABAARs) at synapses. The mechanisms responsible remain poorly understood, and roles for transmembrane accessory proteins have not been established. Using molecular, imaging, and electrophysiological approaches, we identify the tetraspanin LHFPL4 as a critical regulator of postsynaptic GABAAR clustering in hippocampal pyramidal neurons. LHFPL4 interacts tightly with GABAAR subunits and is selectively enriched at inhibitory synapses. In LHFPL4 knockout mice, there is a dramatic cell-type-specific reduction in GABAAR and gephyrin clusters and an accumulation of large intracellular gephyrin aggregates in vivo. While GABAARs are still trafficked to the neuronal surface in pyramidal neurons, they are no longer localized at synapses, resulting in a profound loss of fast inhibitory postsynaptic currents. Hippocampal interneuron currents remain unaffected. Our results establish LHFPL4 as a synapse-specific tetraspanin essential for inhibitory synapse function and provide fresh insights into the molecular make-up of inhibitory synapses

Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity, with Single-Spike Temporal Resolution

The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals, enabling systematic analysis and engineering of the brain, and quantitative bioengineering of excitable cells

Localizing Brain Regions Associated with Female Mate Preference Behavior in a Swordtail

Female mate choice behavior is a critical component of sexual selection, yet identifying the neural basis of this behavior is largely unresolved. Previous studies have implicated sensory processing and hypothalamic brain regions during female mate choice and there is a conserved network of brain regions (Social Behavior Network, SBN) that underlies sexual behaviors. However, we are only beginning to understand the role this network has in pre-copulatory female mate choice. Using in situ hybridization, we identify brain regions associated with mate preference in female Xiphophorus nigrensis, a swordtail species with a female choice mating system. We measure gene expression in 10 brain regions (linked to sexual behavior, reward, sensory integration or other processes) and find significant correlations between female preference behavior and gene expression in two telencephalic areas associated with reward, learning and multi-sensory processing (medial and lateral zones of the dorsal telencephalon) as well as an SBN region traditionally associated with sexual response (preoptic area). Network analysis shows that these brain regions may also be important in mate preference and that correlated patterns of neuroserpin expression between regions co-vary with differential compositions of the mate choice environment. Our results expand the emerging network for female preference from one that focused on sensory processing and midbrain sexual response centers to a more complex coordination involving forebrain areas that integrate primary sensory processing and reward.This work was funded by research fellowships from the University of Texas (UT) Ecology, Evolution and Behavior graduate program (to RYW), along with a Reeder Fellowship, UT SRA, UT StartUp funds, National Science Foundation SGER IOS-0813742 and IOS-0843000 (to MEC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

A Gap in the Spectrum of the Faltings Height

We show that the minimum h(min) of the stable Faltings height on elliptic curves found by Deligne is followed by a gap. This means that there is a constant C > 0 such that for every elliptic curve E / K with everywhere semistable reduction over a number field K, we either have h(E / K) = h(min) or h(E / K) >= h(min) +C. We determine such an absolute constant explicitly. On the contrary, we show that there is no such gap for elliptic curves with unstable reduction

Linear incongruences for generalized eta-quotients

For a given generalized eta-quotient, we show that linear progressions whose residues fulfill certain quadratic equations do not give rise to a linear congruence modulo any prime. This recovers known results for classical eta-quotients, especially the partition function, but also yields linear incongruences for more general weakly holomorphic modular forms like the Rogers-Ramanujan functions