Long term culture promotes changes to growth, gene expression, and metabolism in CHO cells that are independent of production stability.

Phenotypic stability of Chinese hamster ovary (CHO) cells over long term culture (LTC) presents one of the most pressing challenges in the development of therapeutic protein manufacturing processess. However, our current understanding of the consequences of LTC on recombinant (r-) CHO cell lines is still limited, particularly as clonally-derived cell lines present distinct production stability phenotypes. This study evaluated changes of culture performance, global gene expression, and cell metabolism of two clonally-derived CHO cell lines with a stable or unstable phenotype during the LTC (early [EP] vs. late [LP] culture passages). Our findings indicated that LTC altered the behavior of CHO cells in culture, in terms of growth, overall gene expression, and cell metabolism. Regardless whether cells were categorized as stable or unstable in terms of r-protein production, CHO cells at LP presented an earlier decline in cell viability and loss of any observable stationary phase. These changes were parallelled by the upregulation of genes involved in cell proliferation and survival pathways (i.e., MAPK/ERK, PI3K-Akt). Stable and unstable CHO cell lines both showed increased consumption of glucose and amino acids at LP, with a parallel accumulation of greater amounts of lactate and TCA cycle intermediates. In terms of production stability, we found that decreased r-protein production in the unstable cell line directly correlated to the loss in r-gene copy number and r-mRNA expression. Our data revealed that LTC produced ubiquitious effects on CHO cell phenotypes, changes that were rooted in alterations in cell transcriptome and metabolome. Overall, we found that CHO cells adapted their cellular function to proliferation and survival during the LTC, some of these changes may well have limited effects on overall yield or specific productivity of the desired r-product, but they may be critical toward the capacity of cells to handle r-proteins with specific molecular features.

WormQTL--public archive and analysis web portal for natural variation data in Caenorhabditis spp.

Here, we present WormQTL (http://www.wormqtl.org), an easily accessible database enabling search, comparative analysis and meta-analysis of all data on variation in Caenorhabditis spp. Over the past decade, Caenorhabditis elegans has become instrumental for molecular quantitative genetics and the systems biology of natural variation. These efforts have resulted in a valuable amount of phenotypic, high-throughput molecular and genotypic data across different developmental worm stages and environments in hundreds of C. elegans strains. WormQTL provides a workbench of analysis tools for genotype-phenotype linkage and association mapping based on but not limited to R/qtl (http://www.rqtl.org). All data can be uploaded and downloaded using simple delimited text or Excel formats and are accessible via a public web user interface for biologists and R statistic and web service interfaces for bioinformaticians, based on open source MOLGENIS and xQTL workbench software. WormQTL welcomes data submissions from other worm researchers.

VIVID interacts with the WHITE COLLAR complex and FREQUENCY-interacting RNA helicase to alter light and clock responses in Neurospora.

The photoreceptor and PAS/LOV protein VIVID (VVD) modulates blue-light signaling and influences light and temperature responses of the circadian clock in Neurospora crassa. One of the main actions of VVD on the circadian clock is to influence circadian clock phase by regulating levels of the transcripts encoded by the central clock gene frequency (frq). How this regulation is achieved is unknown. Here we show that VVD interacts with complexes central for circadian clock and blue-light signaling, namely the WHITE-COLLAR complex (WCC) and FREQUENCY-interacting RNA helicase (FRH), a component that complexes with FRQ to mediate negative feedback control in Neurospora. VVD interacts with FRH in the absence of WCC and FRQ but does not seem to control the exosome-mediated negative feedback loop. Instead, VVD acts to modulate the transcriptional activity of the WCC.

Cryptic genetic variation of expression quantitative trait locus architecture revealed by genetic perturbation in Caenorhabditis elegans.

Genetic perturbation in different genetic backgrounds can cause a range of phenotypes within a species. These phenotypic differences can be the result of the interaction between the genetic background and the perturbation. Previously, we reported that perturbation of gld-1, an important player in the developmental control of Caenorhabditis elegans, released cryptic genetic variation (CGV) affecting fitness in different genetic backgrounds. Here, we investigated the change in transcriptional architecture. We found 414 genes with a cis-expression quantitative trait locus (eQTL) and 991 genes with a trans-eQTL that were specifically found in the gld-1 RNAi treatment. In total, we detected 16 eQTL hotspots, of which 7 were only found in the gld-1 RNAi treatment. Enrichment analysis of those 7 hotspots showed that the regulated genes were associated with neurons and the pharynx. Furthermore, we found evidence of accelerated transcriptional aging in the gld-1 RNAi-treated nematodes. Overall, our results illustrate that studying CGV leads to the discovery of hidden polymorphic regulators.

Expanding the HPSE2 Genotypic Spectrum in Urofacial Syndrome, A Disease Featuring a Peripheral Neuropathy of the Urinary Bladder.

Urofacial (also called Ochoa) syndrome (UFS) is an autosomal recessive congenital disorder of the urinary bladder featuring voiding dysfunction and a grimace upon smiling. Biallelic variants in HPSE2, coding for the secreted protein heparanase-2, are described in around half of families genetically studied. Hpse2 mutant mice have aberrant bladder nerves. We sought to expand the genotypic spectrum of UFS and make insights into its pathobiology. Sanger sequencing, next generation sequencing and microarray analysis were performed in four previously unreported families with urinary tract disease and grimacing. In one, the proband had kidney failure and was homozygous for the previously described pathogenic variant c.429T>A, p.(Tyr143*). Three other families each carried a different novel HPSE2 variant. One had homozygous triplication of exons 8 and 9; another had homozygous deletion of exon 4; and another carried a novel c.419C>G variant encoding the missense p.Pro140Arg in trans with c.1099-1G>A, a previously reported pathogenic splice variant. Expressing the missense heparanase-2 variant in vitro showed that it was secreted as normal, suggesting that 140Arg has aberrant functionality after secretion. Bladder autonomic neurons emanate from pelvic ganglia where resident neural cell bodies derive from migrating neural crest cells. We demonstrated that, in normal human embryos, neuronal precursors near the developing hindgut and lower urinary tract were positive for both heparanase-2 and leucine rich repeats and immunoglobulin like domains 2 (LRIG2). Indeed, biallelic variants of LRIG2 have been implicated in rare UFS families. The study expands the genotypic spectrum in HPSE2 in UFS and supports a developmental neuronal pathobiology.

A fitness assay for comparing RNAi effects across multiple C. elegans genotypes.

BACKGROUND: RNAi technology by feeding of E. coli containing dsRNA in C. elegans has significantly contributed to further our understanding of many different fields, including genetics, molecular biology, developmental biology and functional genomics. Most of this research has been carried out in a single genotype or genetic background. However, RNAi effects in one genotype do not reveal the allelic effects that segregate in natural populations and contribute to phenotypic variation. RESULTS: Here we present a method that allows for rapidly comparing RNAi effects among diverse genotypes at an improved high throughput rate. It is based on assessing the fitness of a population of worms by measuring the rate at which E. coli is consumed. Critically, we demonstrate the analytical power of this method by QTL mapping the loss of RNAi sensitivity (in the germline) in a recombinant inbred population derived from a cross between Bristol and a natural isolate from Hawaii. Hawaii has lost RNAi sensitivity in the germline. We found that polymorphisms in ppw-1 contribute to this loss of RNAi sensitivity, but that other loci are also likely to be important. CONCLUSIONS: In summary, we have established a fast method that improves the throughput of RNAi in liquid, that generates quantitative data, that is easy to implement in most laboratories, and importantly that enables QTL mapping using RNAi.

Metabolic profiling of Chinese hamster ovary cell cultures at different working volumes and agitation speeds using spin tube reactors.

Culture systems based on spin tube reactors have been consolidated in the development of manufacturing processes based on Chinese hamster ovary (CHO) cells. Despite their widespread use, there is little information about the consequences of varying operational setting parameters on the culture performance of recombinant CHO cell lines. Here, we investigated the effect of varying working volumes and agitation speeds on cell growth, protein production, and cell metabolism of two clonally derived CHO cell lines (expressing an IgG1 and a "difficult-to-express" fusion protein). Interestingly, low culture volumes increased recombinant protein production and decreased cell growth, while high culture volumes had the opposite effect. Altering agitation speeds exacerbated or moderated the differences observed due to culture volume changes. Combining low agitation rates with high culture volumes suppressed growth and recombinant protein production in CHO cells. Meanwhile, high agitation rates narrowed the differences in culture performance between low and high working volumes. These differences were also reflected in cell metabolism, where low culture volumes enhanced oxidative metabolism (linked to a productive phenotype) and high culture volume generated a metabolic profile that was predominately glycolytic (linked to a proliferative phenotype). Our findings indicate that the culture volume influence on metabolism modulates the balance between cell growth and protein production, a key feature that may be useful to adjust CHO cells toward a more productive phenotype.

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma.

BACKGROUND: Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. METHODS: Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. RESULTS: Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. CONCLUSION: These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. KEY POINTS: 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.

Metabolite Profiling of Mammalian Cell Culture Processes to Evaluate Cellular Viability.

Metabolite profiling allows for the identification of metabolites that become limiting during cell culture and/or for finding bottlenecks in metabolic pathways that limit culture growth and proliferation. Here we describe one protocol with two different sampling methodologies for GC-MS-based metabolite profiling. We also highlight an example of the types of datasets that are attainable and how such datasets can be evaluated to identify factors related to cell viability. We also demonstrate, via the same methodology, the accurate quantification of a number of metabolites of interest.

reGenotyper: Detecting mislabeled samples in genetic data.

In high-throughput molecular profiling studies, genotype labels can be wrongly assigned at various experimental steps; the resulting mislabeled samples seriously reduce the power to detect the genetic basis of phenotypic variation. We have developed an approach to detect potential mislabeling, recover the "ideal" genotype and identify "best-matched" labels for mislabeled samples. On average, we identified 4% of samples as mislabeled in eight published datasets, highlighting the necessity of applying a "data cleaning" step before standard data analysis.

Natural Genetic Variation Influences Protein Abundances in C. elegans Developmental Signalling Pathways.

Complex traits, including common disease-related traits, are affected by many different genes that function in multiple pathways and networks. The apoptosis, MAPK, Notch, and Wnt signalling pathways play important roles in development and disease progression. At the moment we have a poor understanding of how allelic variation affects gene expression in these pathways at the level of translation. Here we report the effect of natural genetic variation on transcript and protein abundance involved in developmental signalling pathways in Caenorhabditis elegans. We used selected reaction monitoring to analyse proteins from the abovementioned four pathways in a set of recombinant inbred lines (RILs) generated from the wild-type strains N2 (Bristol) and CB4856 (Hawaii) to enable quantitative trait locus (QTL) mapping. About half of the cases from the 44 genes tested showed a statistically significant change in protein abundance between various strains, most of these were however very weak (below 1.3-fold change). We detected a distant QTL on the left arm of chromosome II that affected protein abundance of the phosphatidylserine receptor protein PSR-1, and two separate QTLs that influenced embryonic and ionizing radiation-induced apoptosis on chromosome IV. Our results demonstrate that natural variation in C. elegans is sufficient to cause significant changes in signalling pathways both at the gene expression (transcript and protein abundance) and phenotypic levels.

Structural characterisation of a perdeuteriomethylated exopolysaccharide by NMR spectroscopy: characterisation of the novel exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus EU23.

The exopolysaccharide (EPS) from Lactobacillus delbrueckii subsp. bulgaricus EU23 was perdeuteriomethylated and the perdeuteriomethylated EPS (pdm-EPS) purified by elution from a C(18) Sep-Pak cartridge. Both 1D and 2D NMR spectra were recorded for the pdm-EPS and these were interpreted to provide assignments for the individual 1H and 13C resonances of the sugar residues of the repeating unit. Using a combination of the results from monomer analysis and linkage analysis of the native EPS and the ROESY and HMBC NMR spectra of the pdm-EPS the following structure has been determined for the repeating unit:A process for characterising polysaccharides having low solubility in aqueous solution is reported.

Temperature-sensitive and circadian oscillators of Neurospora crassa share components.

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.

The PAS/LOV protein VIVID controls temperature compensation of circadian clock phase and development in Neurospora crassa.

Circadian clocks are cellular timekeepers that regulate aspects of temporal organization on daily and seasonal time scales. To allow accurate time measurement, the period lengths of clocks are conserved in a range of temperatures--a phenomenon known as temperature compensation. Temperature compensation of circadian clock period aids in maintaining a stable "target time" or phase of clock-controlled events. Here we show that the Neurospora protein VIVID (VVD) buffers the circadian system against temperature fluctuations. In vvd-null mutants, the circadian period of clock-controlled events such as asexual sporulation (conidiation) is temperature compensated, but the phase of this clock time marker is not. Consistent with delayed conidiation at lower temperatures in vvd(KO) strains, the levels of vvd gene products in the wild type increase with decreasing temperatures. Moreover, vvd(C108A) mutants that lack the light function of VVD maintain a dark activity that transiently influences the phase of conidiation, indicating that VVD influences the time of conidiation downstream from the clock. FREQUENCY (FRQ) phosphorylation is altered in a vvd(KO) strain, suggesting a mechanism by which VVD can influence the timing of clock-controlled processes in the dark. Thus, temperature compensation of clock-controlled output is a key factor in maintaining temperature compensation of the entire circadian system.

The PAS/LOV protein VIVID supports a rapidly dampened daytime oscillator that facilitates entrainment of the Neurospora circadian clock.

A light-entrainable circadian clock controls development and physiology in Neurospora crassa. Existing simple models for resetting based on light pulses (so-called nonparametric entrainment) predict that constant light should quickly send the clock to an arrhythmic state; however, such a clock would be of little use to an organism in changing photoperiods in the wild, and we confirm that true, albeit dampened, rhythmicity can be observed in extended light. This rhythmicity requires the PAS/LOV protein VIVID (VVD) that acts, in the light, to facilitate expression of an oscillator that is related to, but distinguishable from, the classic FREQUENCY/WHITE-COLLAR complex (FRQ/WCC)-based oscillator that runs in darkness. VVD prevents light resetting of the clock at dawn but, by influencing frq RNA turnover, promotes resetting at dusk, thereby allowing the clock to run through the dawn transition and take its phase cues from dusk. Consistent with this, loss of VVD yields a clock whose performance follows the simple predictions of earlier models, and overexpression of VVD restores rhythmicity in the light and sensitivity of phase to the duration of the photoperiod.