De novo assembly and annotation of the CHOZN® GS-/- genome supports high-throughput genome-scale screening.

Chinese hamster ovary (CHO) cells have been used as the industry standard for the production of therapeutic monoclonal antibodies for several decades. Despite significant improvements in commercial-scale production processes and media, the CHO cell has remained largely unchanged. Due to the cost and complexity of whole-genome sequencing and gene-editing it has been difficult to obtain the tools necessary to improve the CHO cell line. With the advent of next-generation sequencing and the discovery of the CRISPR/Cas9 system it has become more cost effective to sequence and manipulate the CHO genome. Here, we provide a comprehensive de novo assembly and annotation of the CHO-K1 based CHOZN® GS-/- genome. Using this platform, we designed, built, and confirmed the functionality of a whole genome CRISPR guide RNA library that will allow the bioprocessing community to design a more robust CHO cell line leading to the production of life saving medications in a more cost-effective manner.

Transcriptome Profiling of Porcine Naïve, Intermediate and Terminally Differentiated CD8+ T Cells.

The pig has the potential to become a leading research model for human diseases, pharmacological and transplantation studies. Since there are many similarities between humans and pigs, especially concerning anatomy, physiology and metabolism, there is necessity for a better understanding of the porcine immune system. In adaptive immunity, cytotoxic T lymphocytes (CTLs) are essential for host defense. However, most data on CTLs come from studies in mice, non-human primates and humans, while detailed information about porcine CD8+ CTLs is still sparse. Aim of this study was to analyze transcriptomes of three subsets of porcine CD8ß+ T-cell subsets by using next-generation sequencing technology. Specifically, we described transcriptional profiles of subsets defined by their CD11a/CD27 expression pattern, postulated as naïve (CD8ß+CD27+CD11alow), intermediate differentiated (CD8ß+CD27dimCD11a+), and terminally differentiated cells (CD8ß+CD27-CD11ahigh). Cells were analyzed in ex vivo condition as well as upon in vitro stimulation with concanavalin A (ConA) and PMA/ionomycin. Our analyses show that the highest number of differentially expressed genes was identified between naïve and terminally differentiated CD8+ T-cell subsets, underlining their difference in gene expression signature and respective differentiation stages. Moreover, genes related to early (IL7-R, CCR7, SELL, TCF7, LEF1, BACH2, SATB1, ZEB1 and BCL2) and late (KLRG1, TBX21, PRDM1, CX3CR1, ZEB2, ZNF683, BATF, EZH2 and ID2) stages of CD8+ T-cell differentiation were highly expressed in the naïve and terminally differentiated CD8+ T-cell subsets, respectively. Intermediate differentiated CD8+ T-cell subsets shared a more comparable gene expression profile associated with later stages of T-cell differentiation. Genes associated with cytolytic activity (GNLY, PRF1, GZMB, FASL, IFNG and TNF) were highly expressed in terminally and intermediate differentiated CD8+ T-cell subsets, while naïve CD8+ T cells lacked expression even after in vitro stimulation. Overall, PMA/ionomycin stimulation induced much stronger upregulation of genes compared to stimulation with ConA. Taken together, we provided comprehensive results showing transcriptional profiles of three differentiation stages of porcine CD8+ T-cell subsets. In addition, our study provides a powerful toolbox for the identification of candidate markers to characterize porcine immune cell subsets in more detail.

Vitamin D inhibits the epithelial-mesenchymal transition by a negative feedback regulation of TGF-ß activity.

Vitamin D and TGF-ß exert opposite effects on epithelial-mesenchymal EMT transition. Here we report a novel mechanism of action of TGF-ß that promotes the counteracting activity of vitamin D; in two models of human epithelial-mesenchymal EMT transition we demonstrated for the first time that TGF-ß strongly induced the expression of vitamin D receptor (VDR) and that 1,25(OH)2D3 was able to contrast the TGF-ß-driven EMT transition by transcriptional modulation. In human bronchial epithelial cells the effects of TGF-ß on EMT transition markers (E-Cadherin expression and cell motility) were reversed by pre-treatment and co-treatment with 1,25(OH)2D3, but not when the hormone was given later. Silencing experiments demonstrated that the inhibition of TGF-ß activity was VDR-dependent. 1,25(OH)2D3 abrogated the mitochondrial stimulation triggered by TGF-ß. In fact we showed that 1,25(OH)2D3 repressed the transcriptional induction of respiratory complex, limited the enhanced mitochondrial membrane potential and restrained the increased levels of mitochondrial ATP; 1,25(OH)2D3 also decreased the production of reactive oxygen species promoted by TGF-ß. Overall, our study suggests that the overexpression and activity of VDR may be a regulatory response to TGF-ß signaling that could be exploited in clinical protocols, unraveling the therapeutic potentiality of 1,25(OH)2D3 in the prevention of cancer metastasis.

A thermal gradient modulates the oxidative metabolism and growth of human keratinocytes.

During their spatial and differentiative progression, keratinocytes face a thermal gradient, from 37 °C in the proliferating basal layer to 32 °C found in skin surface. In our study, we hypothesized that this difference in temperature must be balanced by increasing the heat produced during respiratory activity. We demonstrated that at 33 °C human primary keratinocytes and HaCaT cells raised mitochondrial energy metabolism, but not glycolytic activity. At 33 °C, the increased mitochondrial ATP synthesis was associated with a strong induction of the modulator of the respiratory chain estrogen receptor ß, whereas uncoupling protein 1 expression was not changed. The enhanced mitochondrial oxidative metabolism was accompanied by a remarkable reduction in proliferation. These results suggest that environmental temperature can modulate the energy metabolism and proliferation of human keratinocytes.

Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

Extremely low frequency electromagnetic fields affect proliferation and mitochondrial activity of human cancer cell lines.

PURPOSE: To date, the effects of electromagnetic fields on cell metabolism have been overlooked. The objective of the present study was to investigate the influence of extremely low frequency electromagnetic fields (ELF-EMF) over mitochondrial metabolism and the consequent impact on cancer cell growth. MATERIALS AND METHODS: The effects of ELF-EMF on cancer growth were investigated in several human cancer cell lines by crystal violet assay. The modulation of mitochondrial activity was assessed by cytofluorimetric evaluation of membrane potential and by real-time quantification of mitochondrial transcription. Moreover the expression of several mitochondrial proteins and their levels in the organelle were evaluated. RESULTS: The long-term exposure to ELF-EMF reduced the proliferation of several cancer cell lines and the effect was associated to an increased mitochondrial activity without evident changes in ATP levels. The results of our experiments excluded a transcriptional modulation of mitochondrial respiratory complexes, rather suggesting that ELF-EMF increased the energy demand. The altered mitochondrial metabolism led to changes in mitochondrial protein profile. In fact we found a downregulated expression of mitochondrial phospho-ERK, p53 and cytochrome c. CONCLUSION: The results of the present study indicate that ELF-EMF can negatively modulate cancer cell growth increasing respiratory activity of cells and altering mitochondrial protein expression.