The schistosomicidal activity of ethanolic extracts from branches, leaves, flowers and fruits of Handroanthus impetiginosus (Mart. ex DC.) Mattos (Bignoniaceae) plant and metabolic profile characterization by UPLC-ESI-QTOF analysis.

Schistosomiasis, caused by Schistosoma mansoni Sambon, 1907, is a severe and widely distributed parasitic disease, affecting about 200 million people worldwide. The disease is recognized by elevated mortality rates, especially among those living in areas of poor sanitation. Currently, the chemotherapeutic treatment is solely based on using the praziquantel drug. Therefore, there is a need for the discovery of new medicines for the treatment of this parasitosis. Thus, this work aimed to evaluate the schistosomicidal activity of ethanolic crude extracts from the branches, leaves, flowers, and fruits of Handroanthus impetiginosus (Mart ex DC.) Masttos and characterize its metabolic profile by UPLC-ESI-QTOF analysis. Evaluation of plant extract on S. mansoni was carried out in adult worms in vitro, in which the mortality rate was quantified, and the damages in the tegument of the worms were monitored. All extracts induced changes in the viability of adult males of S. mansoni, causing the death of the parasites, which was directly dependent of the concentration.

Heat shock protein 27 overexpression in CHO cells modulates apoptosis pathways and delays activation of caspases to improve recombinant monoclonal antibody titre in fed-batch bioreactors.

CHO cells are major production hosts for recombinant biologics including the rapidly expanding recombinant monoclonal antibodies (mAbs). Heat shock protein 27 (HSP27) expression was observed to be down-regulated towards the late-exponential and stationary phase of CHO fed-batch bioreactor cultures, whereas HSP27 was found to be highly expressed in human pathological cells and reported to have anti-apoptotic functions. These phenotypes suggest that overexpression of HSP27 is a potential cell line engineering strategy for improving robustness of CHO cells. In this work, HSP27 was stably overexpressed in CHO cells producing recombinant mAb and the effects of HSP27 on cell growth, volumetric production titer and product quality were assessed. Concomitantly, HSP27 anti-apoptosis functions in CHO cells were investigated. Stably transfected clones cultured in fed-batch bioreactors displayed 2.2-fold higher peak viable cell density, delayed loss of culture viability by two days and 2.3-fold increase in mAb titer without affecting the N-glycosylation profile, as compared to clones stably transfected with the vector backbone. Co-immunoprecipitation studies revealed HSP27 interactions with Akt, pro-caspase 3 and Daxx and caspase activity profiling showed delayed increase in caspase 2, 3, 8 and 9 activities. These results suggest that HSP27 modulates apoptosis signaling pathways and delays caspase activities to improve performance of CHO fed-batch bioreactor cultures.

28S rRNA is inducibly pseudouridylated by the mTOR pathway translational control in CHO cell cultures.

The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling.

The mammalian target of rapamycin (mTOR) pathway plays essential roles in the regulation of translational activity in many eukaryotes. Thus, from a bioprocessing point of view, understanding its molecular mechanisms may provide potential avenues for improving cell culture performance. Toward this end, the mTOR pathway of CHO cells in batch cultures was subjected to rapamycin treatment (inhibition) or nutrient supplementation (induction) and translational activities of CHO cells producing a monoclonal antibody (mAb) were evaluated with polysome profiling technology. Expectedly, rapamycin induced a shift of mRNAs from polysomes towards monosomes, thus reducing maximum cellular growth rate by 30%, while feeding additional nutrients extended mTOR pathway activity during the stationary growth phase in control batch culture, thereby contributing to an increase in global translation activity by up to 2-fold, and up to 5-fold higher specific translation of the heavy and light chains of the recombinant mAb. These increases in translation activity correlated with a 5-day extension in cellular growth and a 4-fold higher final product titer observed upon nutrient feeding. This first study of the relationship between the mTOR pathway and translational activity in CHO cultures provides key insights into the role of translational control in supporting greater productivity, which will lead to further enhancement of CHO cultures.

Translatome analysis of CHO cells to identify key growth genes.

We report the first investigation of translational efficiency on a global scale, also known as translatome, of a Chinese hamster ovary (CHO) DG44 cell line producing monoclonal antibodies (mAb). The translatome data was generated via combined use of high resolution and streamlined polysome profiling technology and proprietary Nimblegen microarrays probing for more than 13K annotated CHO-specific genes. The distribution of ribosome loading during the exponential growth phase revealed the translational activity corresponding to the maximal growth rate, thus allowing us to identify stably and highly translated genes encoding heterogeneous nuclear ribonucleoproteins (Hnrnpc and Hnrnpa2b1), protein regulator of cytokinesis 1 (Prc1), glucose-6-phosphate dehydrogenase (G6pdh), UTP6 small subunit processome (Utp6) and RuvB-like protein 1 (Ruvbl1) as potential key players for cellular growth. Moreover, correlation analysis between transcriptome and translatome data sets showed that transcript level and translation efficiency were uncoupled for 95% of investigated genes, suggesting the implication of translational control mechanisms such as the mTOR pathway. Thus, the current translatome analysis platform offers new insights into gene expression in CHO cell cultures by bridging the gap between transcriptome and proteome data, which will enable researchers of the bioprocessing field to prioritize in high-potential candidate genes and to devise optimal strategies for cell engineering toward improving culture performance.

Atividade antibacteriana in vitro de quatro espécies vegetais em diferentes graduações alcoólicas / In vitro antibacterial activity of four plant species at different alcoholic contents

Neste trabalho foi realizada a caracterização fitoquímica e avaliada a atividade antibacteriana in vitro dos extratos de Ageratum conyzoides L. (mentrasto), Gossypium hirsutum (algodão), Phyllanthus tenellus (quebra pedra), e Polygonum hydropiperoides (erva de bicho) frente à Staphylococcus aureus e Escherichia coli. Para a avaliação da atividade antibacteriana foi utilizado o método de difusão em ágar. Os testes foram realizados com o extrato nas graduações alcoólicas de 0 a 100% (v/v), na proporção de 20% (m/v - massa/extrator). Os testes fitoquímicos constataram a presença de açucares redutores, compostos fenólicos, flavonoides, taninos, triterpenos, e esteróides nas quatro espécies. O crescimento das culturas de S. aureus foi inibido por todos os extratos, com exceção do extrato de Mentrasto. A maior atividade de inibição foi observada pelo extrato de quebra pedra. Entretanto, nenhum dos extratos foi capaz de inibir o crescimento das cepas de E. coli. Os resultados são promissores, visto que três das quatro plantas selecionadas demonstraram possuir substâncias antibacterianas, o que motiva estudos subsequentes para o isolamento e identificação dos princípios ativos responsáveis por essa atividade, com potencial de uso na indústria farmacêutica.

In this study, phytochemical characterization was conducted and the in vitro antibacterial activity of extracts of Ageratum conyzoides L. (whiteweed), Gossypium hirsutum (cotton), Phyllanthus tenellus (shatterstone) and Polygonum hydropiperoides (swamp smartweed) was evaluated against Staphylococcus aureus and Escherichia coli. To assess the antibacterial activity, the agar diffusion method was used. Tests were performed with the extract at alcoholic contents from 0 to 100% (v/v), at 20% proportion (m/v - mass/extractor). Phytochemical tests indicated the presence of reducing sugars, phenolic compounds, flavonoids, tannins, triterpenes and steroids in all four species. The growth of S. aureus cultures was inhibited by all extracts, except for whiteweed extract. The highest inhibitory activity was observed for shatterstone. However, none of the extracts was capable of inhibiting the growth of E. coli strains. Results are promising since three of the four selected plants showed to have antibacterial substances, which stimulates further studies for the isolation and the identification of active principles responsible for this activity, with potential to be used in the pharmaceutical industry.

Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture.

The increasing demand for recombinant therapeutic proteins highlights the need to constantly improve the efficiency and yield of these biopharmaceutical products from mammalian cells, which is fully achievable only through proper understanding of cellular functioning. Towards this end, the current study exploited a combined metabolomics and in silico modeling approach to gain a deeper insight into the cellular mechanisms of Chinese hamster ovary (CHO) fed-batch cultures. Initially, extracellular and intracellular metabolite profiling analysis shortlisted key metabolites associated with cell growth limitation within the energy, glutathione, and glycerophospholipid pathways that have distinct changes at the exponential-stationary transition phase of the cultures. In addition, biomass compositional analysis newly revealed different amino acid content in the CHO cells from other mammalian cells, indicating the significance of accurate protein composition data in metabolite balancing across required nutrient assimilation, metabolic utilization, and cell growth. Subsequent in silico modeling of CHO cells characterized internal metabolic behaviors attaining physiological changes during growth and non-growth phases, thereby allowing us to explore relevant pathways to growth limitation and identify major growth-limiting factors including the oxidative stress and depletion of lipid metabolites. Such key information on growth-related mechanisms derived from the current approach can potentially guide the development of new strategies to enhance CHO culture performance.

Post-transcriptional regulatory elements for enhancing transient gene expression levels in mammalian cells.

Low yield from transient gene expression in mammalian cells limits its application to areas where large amount of proteins are needed. One effective approach to enhance transient gene expression levels is to use post-transcriptional regulatory elements (PTREs). We have evaluated the effect of five PTREs on the transient gene expression of three proteins in two cell lines. Most of the elements increased expression but exhibited cell-specific and gene-specific effects. The tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer gave the most universal and highest enhancement of gene expression levels. It increased the expression of all three proteins in HEK293 cells and two proteins in CHO K1 cells by 3.6- to 7.6-fold. Combinations of multiple PTREs increased protein expression as much as 10.5-fold.

IRES-mediated Tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines.

A Tricistronic vector utilizing internal ribosome entry site (IRES) elements to express the light chain (LC), heavy chain (HC), and a neomycin phosphotransferase (NPT) selection marker from one transcript is designed for generation of mAb expressing CHO cell lines. As compared to the commonly used vectors, benefits of this design include: (1) minimized non-expressing clones, (2) enhanced stable mAb productivity without gene amplification, (3) control of LC and HC expression at defined ratios, and (4) consistent product quality. After optimization of the LC and HC arrangement and increasing selection stringency by weakening the NPT selection marker, this Tricistronic vector is able to generate stably transfected pools with specific productivity (qmAb) greater than 5pg/cell/day (pcd) and titers over 150mg/L. 5% of clones from these pools have qmAb greater than 20pcd and titers ranging from 300 to more than 500mg/L under non-optimized shake flask batch cultures using commercially available protein-free medium. The mAb produced by these clones have low aggregation and consistent glycosylation profiles. The entire process of transfection to high-expressing clones requires only 6 months. The IRES-mediated Tricistronic vector provides an attractive alternative to commonly used vectors for fast generation of mAb CHO cell lines with high productivity.

Metabolomics-based identification of apoptosis-inducing metabolites in recombinant fed-batch CHO culture media.

A liquid chromatography-mass spectrometry (LC-MS) based metabolomics platform was previously established to identify and profile extracellular metabolites in culture media of mammalian cells. This presented an opportunity to isolate novel apoptosis-inducing metabolites accumulating in the media of antibody-producing Chinese hamster ovary (CHO mAb) fed-batch bioreactor cultures. Media from triplicate cultures were collected daily for the metabolomics analysis. Concurrently, cell pellets were obtained for determination of intracellular caspase activity. Metabolite profiles from the LC-MS data were subsequently examined for their degree of correlation with the caspase activity. A panel of extracellular metabolites, the majority of which were nucleotides/nucleosides and amino acid derivatives, exhibited good (R² > 0.8) and reproducible correlation. Some of these metabolites, such as oxidized glutathione, AMP and GMP, were later shown to induce apoptosis when introduced to fresh CHO mAb cultures. Finally, metabolic engineering targets were proposed to potentially counter the harmful effects of these metabolites.

Simultaneous targeting of Requiem & Alg-2 in Chinese hamster ovary cells for improved recombinant protein production.

Apoptosis is known to be the main cause of cell death in the bioreactor environment, leading to the loss of recombinant protein productivity. In a previous study, transcriptional profiling was used to identify and target four early apoptosis-signaling genes: FADD, FAIM, Alg-2, and Requiem. The resulting cell lines had increased viable cell numbers and extended culture viability, which translated to increased protein productivity. Combinatorial targeting of two genes simultaneously has previously been shown to be more effective than targeting one gene alone. In this study, we sought to determine if targeting Requiem and Alg-2 was more effective than targeting Requiem alone. We found that targeting Requiem and Alg-2 did not result in extended culture viability, but resulted in an increase in maximum viable cell numbers and cumulative IVCD under fed-batch conditions. This in turn led to an approximately 1.5-fold increase in recombinant protein productivity.

DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines.

Production instability currently limits the use of mammalian cells for industrial production of therapeutic proteins. We have previously reported that the loss of productivity in recombinant monoclonal antibody producing Chinese Hamster Ovary (CHO-mAb) cell lines is mainly due to a decrease in heavy chain (HC) and light chain (LC) transcripts. Molecular analysis indicates that the decreased mRNA levels are not due to a loss in gene copies and change of integration sites. In this work, we further demonstrate that impaired trans-acting factors and spontaneous mutations to the DNA are not responsible for the reduced HC and LC transcription. Examination of two CpG sites by methyl-assisted quantitative real-time PCR assay revealed an increase in methylation of the human cytomegalovirus major immediate-early enhancer and promoter (hCMV-MIE) controlling the expression of LC and HC in cells which exhibited loss in productivity. Treatment of these cells with a DNA methylation inhibitor, 5-aza-2'-deoxycytidine, partially restored the lost specific mAb productivity. The increase in productivity correlated to the increase in mRNA levels of HC and LC and the demethylation of hCMV-MIE promoter. This finding, which indicates that DNA methylation contributes to production instability, will be beneficial for generation of high-producing cell lines with stable productivity.

An investigation of intracellular glycosylation activities in CHO cells: effects of nucleotide sugar precursor feeding.

Controlling glycosylation of recombinant proteins produced by CHO cells is highly desired as it can be directed towards maintaining or increasing product quality. To further our understanding of the different factors influencing glycosylation, a glycosylation sub-array of 79 genes and a capillary electrophoresis method which simultaneously analyzes 12 nucleotides and 7 nucleotide sugars; were used to generate intracellular N-glycosylation profiles. Specifically, the effects of nucleotide sugar precursor feeding on intracellular glycosylation activities were analyzed in CHO cells producing recombinant human interferon-gamma (IFN-gamma). Galactose (+/-uridine), glucosamine (+/-uridine), and N-acetylmannosamine (ManNAc) (+/-cytidine) feeding resulted in 12%, 28%, and 32% increase in IFN-gamma sialylation as compared to the untreated control cultures. This could be directly attributed to increases in nucleotide sugar substrates, UDP-Hex ( approximately 20-fold), UDP-HexNAc (6- to 15-fold) and CMP-sialic acid (30- to 120-fold), respectively. Up-regulation of B4gal and St3gal could also have enhanced glycan addition onto the proteins, leading to more complete glycosylation (sialylation). Combined feeding of glucosamine + uridine and ManNAc + cytidine increased UDP-HexNAc and CMP-sialic acid by another two- to fourfold as compared to feeding sugar precursors alone. However, it did not lead to a synergistic increase in IFN-gamma sialylation. Other factors such as glycosyltransferase or glycan substrate levels could have become limiting. In addition, uridine feeding increased the levels of uridine- and cytidine-activated nucleotide sugars simultaneously, which could imply that uridine is one of the limiting substrates for nucleotide sugar synthesis in the study. Hence, the characterization of intracellular glycosylation activities has increased our understanding of how nucleotide sugar precursor feeding influence glycosylation of recombinant proteins produced in CHO cells. It has also led to the optimization of more effective strategies for manipulating glycan quality.

FGF-2 modulates Wnt signaling in undifferentiated hESC and iPS cells through activated PI3-K/GSK3beta signaling.

Fibroblast growth factor-2 (FGF-2) is widely used to culture human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells. Despite its importance in maintaining undifferentiated hESC phenotype, a lack of understanding in the role of FGF-2 still exists. Here, we investigate the signaling events in hESC following the addition of exogenous FGF-2. In this study, we show that hESC express all forms of fibroblast growth factor receptors (FGFRs) which co-localize on Oct3/4 positive cells. Furthermore, downregulation of Oct3/4 in hESC occurs following treatment with an FGFR inhibitor, suggesting that FGF signaling may regulate Oct3/4 expression. This is also observed in iPS cells. Also, downstream of FGF signaling, both mitogen activated protein kinase (MAPK) and phosphoinositide 3-kinase pathways (PI3-K) are activated following FGF-2 stimulation. Notably, inhibition of MAPK and PI3-K signaling using specific kinase inhibitors revealed that activated PI3-K, rather than MAPK, can mediate pluripotent marker expression. To understand the importance of PI3-K activation, activation of Wnt/beta-catenin by FGF-2 was investigated. Wnt signaling had been implicated to have a role in maintaining of pluripotent hESC. We found that upon FGF-2 stimulation, GSK3beta is phosphorylated following which nuclear translocation of beta-catenin and TCF/LEF activation occurs. Interestingly, inhibition of the Wnt pathway with Dikkopf-1 (DKK-1) resulted in only partial suppression of the FGF-2 induced TCF/LEF activity. Prolonged culture of hESC with DKK-1 did not affect pluripotent marker expression. These results suggest that FGF-2 mediated PI3-K signaling may have a direct role in modulating the downstream of Wnt pathway to maintain undifferentiated hESC.

Metabolomics-driven approach for the improvement of Chinese hamster ovary cell growth: overexpression of malate dehydrogenase II.

We have established a liquid chromatography-mass spectrometry based metabolomics platform to identify extracellular metabolites in the medium of recombinant Chinese hamster ovary (CHO) fed-batch reactor cultures. Amongst the extracellular metabolites identified, malate accumulation was the most significant. The contributing factors to malate efflux were found to be the supply of aspartate from the medium, and an enzymatic bottleneck at malate dehydrogenase II (MDH II) in the tricarboxylic acid cycle. Subsequent metabolic engineering to overexpress MDH II in CHO resulted in increases in intracellular ATP and NADH, and up to 1.9-fold improvement in integral viable cell number.

Evaluating regulatory elements of human cytomegalovirus major immediate early gene for enhancing transgene expression levels in CHO K1 and HEK293 cells.

The upstream regulatory sequence (URS), NF1 region, enhancer, promoter, 1st exon, and intron A of human cytomegalovirus major immediate early gene (hCMV MIE) are evaluated for enhancing transient and stable gene expression levels in two industrial cell lines, CHO K1 and HEK293 using firefly luciferase (Fluc) and erythropoietin (EPO). As compared to the control vector which only contains the enhancer and promoter (EP), vectors containing the 1st exon (EPE) and intron A (EPEI) enhance transient expression levels of the two proteins by approximately 2.5- to 4.3-fold in the two cell lines. Addition of NF1 and URS to EP (NEP and UNEP) or EPEI (NEPEI and UNEPEI) results in a lesser effect on the expression. In stable transfections, UNEPEI provides the highest expression level in CHO K1 cells, yielding approximately 4.0-fold increase in Fluc expression and 2.5-fold increase in EPO expression. In HEK293 cells, EPE is the best and enhances Fluc and EPO expression by more than 2.0-fold. Such information is valuable for the development of optimal vectors to enhance transient and stable production of recombinant proteins in CHO K1 and HEK293 cells.

Engineering mammalian cells in bioprocessing - current achievements and future perspectives.

Over the past 20 years, we have seen significant improvements in product titres from 50 mg/l to 5-10 g/l, a more than 100-fold increase. The main methods that have been employed to achieve this increase in product titre have been through the manipulation of culture media and process control strategies, such as the optimization of fed-batch processes. An alternative means to increase productivity has been through the engineering of host cells by altering cellular processes. Recombinant DNA technology has been used to over-express or suppress specific genes to endow particular phenotypes. Cellular processes that have been altered in host cells include metabolism, cell cycle, protein secretion and apoptosis. Cell engineering has also been employed to improve post-translational modifications such as glycosylation. In this article, an overview of the main cell engineering strategies previously employed and the impact of these strategies are presented. Many of these strategies focus on engineering cell lines with more efficient carbon metabolism towards reducing waste metabolites, achieving a biphasic production system by engineering cell cycle control, increasing protein secretion by targeting specific endoplasmic reticulum stress chaperones, delaying cell death by targeting anti-apoptosis genes, and engineering glycosylation by enhancing recombinant protein sialylation and antibody glycosylation. Future perspectives for host cell engineering, and possible areas of research, are also discussed in this review.

Multitagging proteomic strategy to estimate protein turnover rates in dynamic systems.

Current techniques for quantitative proteomics focus mainly on measuring overall protein dynamics, which is the net result of protein synthesis and degradation. Understanding the rate of this synthesis/degradation is essential to fully appreciate cellular dynamics and bridge the gap between transcriptome and proteome data. Protein turnover rates can be estimated through "label-chase" experiments employing stable isotope-labeled precursors; however, the implicit assumption of steady-state in such analyses may not be applicable for many intrinsically dynamic systems. In this study, we present a novel extension of the "label-chase" concept using SILAC and a secondary labeling step with iTRAQ reagents to estimate protein turnover rates in Streptomyces coelicolor cultures undergoing transition from exponential growth to stationary phase. Such processes are of significance in Streptomyces biology as they pertain to the onset of synthesis of numerous therapeutically important secondary metabolites. The dual labeling strategy enabled decoupling of labeled peptide identification and quantification of degradation dynamics at MS and MS/MS scans respectively. Tandem mass spectrometry analysis of these multitagged proteins enabled estimation of degradation rates for 115 highly abundant proteins in S. coelicolor. We compared the rate constants obtained using this dual labeling approach with those from a SILAC-only analysis (assuming steady-state) and show that significant differences are generally observed only among proteins displaying considerable temporal dynamics and that the directions of these differences are largely consistent with theoretical predictions.

Role of Sonic hedgehog signaling and the expression of its components in human embryonic stem cells.

Human embryonic stem cells (hESC) are characterized by their ability to self-renew and differentiate into all cell types of the body, making them a valuable resource for regenerative medicine. Yet, the molecular mechanisms by which hESC retain their capacity for self-renewal and differentiation remain unclear. The Hedgehog signaling pathway plays a pivotal role in organogenesis and differentiation during development, and is also involved in the proliferation and cell-fate specification of neural stem cells and neural crest stem cells. As there has been no detailed study of the Sonic hedgehog (SHH) signaling pathway in hESC, this study examines the expression and functional role of SHH during hESC self-renewal and differentiation. Here, we show the gene and protein expression of key components of the SHH signaling pathway in hESC and differentiated embryoid bodies. Despite the presence of functioning pathway components, SHH plays a minimal role in maintaining pluripotency and regulating proliferation of undifferentiated hESC. However, during differentiation with retinoic acid, a GLI-responsive luciferase assay and target genes PTCH1 and GLI1 expression reveal that the SHH signaling pathway is highly activated. Besides, addition of exogenous SHH to hESC differentiated as embryoid bodies increases the expression of neuroectodermal markers Nestin, SOX1, MAP2, MSI1, and MSX1, suggesting that SHH signaling is important during hESC differentiation toward the neuroectodermal lineage. Our findings provide a new insight in understanding the SHH signaling in hESC and the further development of hESC differentiation for regenerative medicine.

Elucidating the role of requiem in the growth and death of Chinese hamster ovary cells.

Requiem, a hypothesized transcription factor with apoptosis-related activity, was previously shown to be a potential cell engineering gene target for improving recombinant protein production. Requiem suppression has resulted in improved viable cell density and extended culture viability, leading to an overall improvement in recombinant protein productivity. However, not much is known about the function of requiem. We found that requiem is highly conserved at both nucleotide and amino acid levels in Chinese hamster ovary (CHO) cells when compared to human and mouse sequences, suggesting that requiem's functional role is evolutionary well conserved. Upon inducing requiem over-expression, proliferation rates of CHO cells were significantly decreased with doubling times increased by 26%. Interestingly, the over-expression of requiem did not decrease cell viability and could not induce apoptosis. However, requiem sensitized the cells to increased caspase-9 activities under staurosporine-induced apoptosis, suggesting that it has a role to play in mitochondria-mediated apoptosis under staurosporine treatment. The nuclear localization of REQUIEM in CHO cells and its conserved plant homeodomain (PHD) zinc fingers seem to further support the hypothesis that requiem encodes for a potential transcription factor. Upon requiem over-expression, we found that the differentially expressed genes involved in transcriptional regulation and cell proliferation and growth were associated both upstream and downstream of p53.