Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.     

A novel animal-component-free medium for rabies virus production in Vero cells grown on Cytodex 1 microcarriers in a stirred bioreactor

Vero cells growth and rabies production in IPT-AF medium, a property animal-component-free medium are described in this work. Kinetics of cell growth and rabies virus (strain LP 2061) production were first conducted in spinner flasks. Over eight independent experiments, Vero cell growth in IPT-AF medium, on 2 g/l Cytodex 1 was consistent. An average Cd (cell division number) of 3.3 ± 0.4 and a specific growth rate μ of 0.017 ± 0.006 h⁻¹ were achieved. Such performances were comparable to those obtained in serum-containing medium (MEM + 10% FCS). Rabies virus production on Vero cells in IPT-AF medium was also optimised in spinner flasks. The effects of multiplicity of infection (MOI), regulation of glucose level at 1 g/l and cell washing step, were investigated. The highest virus titer was achieved when the cells were infected at an MOI of 0.1; this level was equal to 10⁷ FFU/ml. The step of medium exchange before cell infection can be omitted; nevertheless in this case glucose level should be maintained at 1 g/l to avoid a decrease of specific virus productivity. Process optimisation in a 2-l stirred bioreactor pointed out that the aeration mode was the prominent parameter that affected cell growth in IPT-AF medium and on Cytodex 1 microcarriers. An acceptable level of cell density (cell density level of 1.5 × 10⁶ cells/ml) was achieved when cells were grown in batch mode and using headspace aeration. Nevertheless, this aeration mode is not optimal for large-scale culture. The addition of Pluronic F68 at 0.1% at 24 h post inoculation as well as the switch from surface aeration mode to the sparged mode, 2 days after the start of the culture, had markedly improved cell growth performance. A cell density level of 5.5 × 10⁶ cells/ml was reached when cells were grown in a 2-l bioreactor, on 3 g/l Cytodex 1 in IPT-AF medium and using the recirculation culture mode. Cell infection at an MOI of 0.1 and using perfused culture, resulted in a maximal virus titer of 3.5 × 10⁷ FFU/ml. The activity of the pooled inactivated rabies virus harvests showed a protective activity that meets WHO requirements.     

The H3.3K27M oncohistone affects replication stress outcome and provokes genomic instability in pediatric glioma

While comprehensive molecular profiling of histone H3.3 mutant pediatric high-grade glioma has revealed extensive dysregulation of the chromatin landscape, the exact mechanisms driving tumor formation remain poorly understood. Since H3.3 mutant gliomas also exhibit high levels of copy number alterations, we set out to address if the H3.3K27M oncohistone leads to destabilization of the genome. Hereto, we established a cell culture model allowing inducible H3.3K27M expression and observed an increase in mitotic abnormalities. We also found enhanced interaction of DNA replication factors with H3.3K27M during mitosis, indicating replication defects. Further functional analyses revealed increased genomic instability upon replication stress, as represented by mitotic bulky and ultrafine DNA bridges. This co-occurred with suboptimal 53BP1 nuclear body formation after mitosis in vitro, and in human glioma. Finally, we observed a decrease in ultrafine DNA bridges following deletion of the K27M mutant H3F3A allele in primary high-grade glioma cells. Together, our data uncover a role for H3.3 in DNA replication under stress conditions that is altered by the K27M mutation, promoting genomic instability and potentially glioma development.     

Own song selectivity in the songbird auditory pathway: suppression by norepinephrine

Background

Like human speech, birdsong is a learned behavior that supports species and individual recognition. Norepinephrine is a catecholamine suspected to play a role in song learning. The goal of this study was to investigate the role of norepinephrine in bird''s own song selectivity, a property thought to be important for auditory feedback processes required for song learning and maintenance.

Methodology/Principal Findings

Using functional magnetic resonance imaging, we show that injection of DSP-4, a specific noradrenergic toxin, unmasks own song selectivity in the dorsal part of NCM, a secondary auditory region.

Conclusions/Significance

The level of norepinephrine throughout the telencephalon is known to be high in alert birds and low in sleeping birds. Our results suggest that norepinephrine activity can be further decreased, giving rise to a strong own song selective signal in dorsal NCM. This latent own song selective signal, which is only revealed under conditions of very low noradrenergic activity, might play a role in the auditory feedback and/or the integration of this feedback with the motor circuitry for vocal learning and maintenance.     

Resequencing Three Candidate Genes for Major Depressive Disorder in a Dutch Cohort

Major depressive disorder (MDD) is a psychiatric disorder, characterized by periods of low mood of more than two weeks, loss of interest in normally enjoyable activities and behavioral changes. MDD is a complex disorder and does not have a single genetic cause. In 2009 a genome wide association study (GWAS) was performed on the Dutch GAIN-MDD cohort. Many of the top signals of this GWAS mapped to a region spanning the gene PCLO, and the non-synonymous coding single nucleotide polymorphism (SNP) rs2522833 in the PCLO gene became genome wide significant after post-hoc analysis. We performed resequencing of PCLO, GRM7, and SLC6A4 in 50 control samples from the GAIN-MDD cohort, to detect new genomic variants. Subsequently, we genotyped these variants in the entire GAIN-MDD cohort and performed association analysis to investigate if rs2522833 is the causal variant or simply in linkage disequilibrium with a more associated variant. GRM7 and SLC6A4 are both candidate genes for MDD from literature. We aimed to gather more evidence that rs2522833 is indeed the causal variant in the GAIN-MDD cohort or to find a previously undetected common variant in either PCLO, GRM7, or SLC6A4 with a higher association in this cohort. After next generation sequencing and association analysis we excluded the possibility of an undetected common variant to be more associated. For neither PCLO nor GRM7 we found a more associated variant. For SLC6A4, we found a new SNP that showed a lower P-value (P = 0.07) than in the GAIN-MDD GWAS (P = 0.09). However, no evidence for genome-wide significance was found. Although we did not take into account rare variants, we conclude that our results provide further support for the hypothesis that the non-synonymous coding SNP rs2522833 in the PCLO gene is indeed likely to be the causal variant in the GAIN-MDD cohort.     

7-Azaindole derivatives as potential partial nicotinic agonists

We have investigated a series of 7-azaindoles as potential partial agonists of the alpha4beta2 nicotinic acetylcholine receptor (nAChR). Three series of 7-azaindole derivatives have been synthesized and evaluated for rat brain neuronal nicotinic receptor affinity and functional activity. Compound (+)-51 exhibited the most potent nAChR binding (Ki = 10 nM). Compound 30A demonstrated both moderate binding affinity and partial agonist potency, thus representing a promising lead for the indications of cognition and smoking cessation.     

Effect of Erythropoiesis-Stimulating Agents on Blood Pressure in Pre-Dialysis Patients

Introduction

Erythropoiesis-Stimulating Agents (ESA) are hypothesized to increase cardiovascular mortality in patients with chronic kidney disease. One of the proposed mechanisms is the elevation of blood pressure (BP) by ESA. Therefore, we aimed to determine whether the use of ESA was associated with antihypertensive treatment and higher BP.

Materials and Methods

In this cohort 502 incident pre-dialysis patients were included who started specialized pre-dialysis care in 25 clinics in the Netherlands. Data on medication including ESA use and dose, co-morbidities and BP were routinely collected every 6 months. Antihypertensive treatment and BP were compared for patients with and without ESA at baseline. Differences in antihypertensive medication and BP during pre-dialysis care were estimated with linear mixed models adjusted for age, sex, body mass index, cardiovascular disease, diabetes mellitus and estimated glomerular filtration rate.

Results

At baseline, 95.6% of patients with ESA were treated with antihypertensive medication and 73.1% of patients without ESA. No relevant difference in BP was found. During pre-dialysis care patients with ESA used 0.77 (95% CI 0.63;0.91) more classes of antihypertensive drugs. The adjusted difference in systolic blood pressure (SBP) was −0.3 (95% CI −2.7;2.0) mmHg and in diastolic blood pressure (DBP) was −1.0 (95% CI −2.1;0.3) mmHg for patients with ESA compared to patients without ESA. Adjusted SBP was 3.7 (95% CI −1.6;9.0) mmHg higher in patients with a high ESA dose compared to patients with a low ESA dose.

Conclusions

Our study confirms the hypertensive effect of ESA, since ESA treated patients received more antihypertensive agents. However, no relevant difference in BP was found between patients with and without ESA, thus the increase in BP seems to be controlled for by antihypertensive medication.     

Functional MRI of auditory responses in the zebra finch forebrain reveals a hierarchical organisation based on signal strength but not selectivity

Background

Male songbirds learn their songs from an adult tutor when they are young. A network of brain nuclei known as the ‘song system’ is the likely neural substrate for sensorimotor learning and production of song, but the neural networks involved in processing the auditory feedback signals necessary for song learning and maintenance remain unknown. Determining which regions show preferential responsiveness to the bird''s own song (BOS) is of great importance because neurons sensitive to self-generated vocalisations could mediate this auditory feedback process. Neurons in the song nuclei and in a secondary auditory area, the caudal medial mesopallium (CMM), show selective responses to the BOS. The aim of the present study is to investigate the emergence of BOS selectivity within the network of primary auditory sub-regions in the avian pallium.

Methods and Findings

Using blood oxygen level-dependent (BOLD) fMRI, we investigated neural responsiveness to natural and manipulated self-generated vocalisations and compared the selectivity for BOS and conspecific song in different sub-regions of the thalamo-recipient area Field L. Zebra finch males were exposed to conspecific song, BOS and to synthetic variations on BOS that differed in spectro-temporal and/or modulation phase structure. We found significant differences in the strength of BOLD responses between regions L2a, L2b and CMM, but no inter-stimuli differences within regions. In particular, we have shown that the overall signal strength to song and synthetic variations thereof was different within two sub-regions of Field L2: zone L2a was significantly more activated compared to the adjacent sub-region L2b.

Conclusions

Based on our results we suggest that unlike nuclei in the song system, sub-regions in the primary auditory pallium do not show selectivity for the BOS, but appear to show different levels of activity with exposure to any sound according to their place in the auditory processing stream.     

Growth-Factor-Driven Rescue to Receptor Tyrosine Kinase (RTK) Inhibitors through Akt and Erk Phosphorylation in Pediatric Low Grade Astrocytoma and Ependymoma

Up to now, several clinical studies have been started investigating the relevance of receptor tyrosine kinase (RTK) inhibitors upon progression free survival in various pediatric brain tumors. However, single targeted kinase inhibition failed, possibly due to tumor resistance mechanisms. The present study will extend our previous observations that vascular endothelial growth factor receptor (VEGFR)-2, platelet derived growth factor receptor (PDGFR)β, Src, the epidermal growth factor receptor (ErbB) family, and hepatocyte growth factor receptor (HGFR/cMet) are potentially drugable targets in pediatric low grade astrocytoma and ependymoma with investigations concerning growth-factor-driven rescue. This was investigated in pediatric low grade astrocytoma and ependymoma cell lines treated with receptor tyrosine kinase (RTK) inhibitors e.g. sorafenib, dasatinib, canertinib and crizotinib. Flow cytometry analyses showed high percentage of cells expressing VEGFR-1, fibroblast growth factor receptor (FGFR)-1, ErbB1/EGFR, HGFR and recepteur d’origine nantais (RON) (respectively 52-77%, 34-51%, 63-90%, 83-98%, 65-95%). Their respective inhibitors induced decrease of cell viability, measured with WST-1 cell viability assays. At least this was partially due to increased apoptotic levels measured by Annexin V/Propidium Iodide apoptosis assays. EGF, HGF and FGF, which are normally expressed in brain (tumor) tissue, showed to be effective rescue inducing growth factors resulting in increased cell survival especially during treatment with dasatinib (complete rescue) or sorafenib (partial rescue). Growth-factor-driven rescue was less prominent when canertinib or crizotinib were used. Rescue was underscored by significantly activating downstream Akt and/or Erk phosphorylation and increased tumor cell migration. Combination treatment showed to be able to overcome the growth-factor-driven rescue. In conclusion, our study highlights the extensive importance of environmentally present growth factors in developing tumor escape towards RTK inhibitors in pediatric low grade astrocytoma and ependymoma. It is of great interest to anticipate upon these results for the design of new therapeutic trials with RTK inhibitors in these pediatric brain tumors.