Improving transient gene expression in CHO-EBNA1 cells

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly generating recombinant proteins that are highly similar to those produced in stable CHO clones used for biomanufacturing. The higher cost of reagents necessary for TGE, specifically the requirement for large amounts of purified DNA and transfection agent for each production, means that improving the performance of CHO TGE could substantially augment the method’s utility. In the current study, we have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1). Transfection of EBNA-1-expressing cells with plasmid vectors encoding the Epstein-Barr virus OriP sequence boosted TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture in shake flasks, we optimized transfection parameters (plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added at 24 h post-transfection, followed by periodic glucose supplementation. This method gave a maximum yield of 900 mg/L (for the chimeric IgG4 B72-3 mAb), with an average of 570 mg/L (standard deviation: 250 mg/L) for a panel of six mAbs and 320 mg/L (standard deviation: 140 mg/L) for five His-tagged recombinant proteins at 14 days post-transfection. Compared to our current low-density TGE process using CHO-3E7 cells and different culture medium, the new procedure gave on average 3-fold higher yields; purified mAbs produced using the two methods had distinct glycosylation profiles (by HILIC analysis) but showed identical target binding kinetics by SPR. Key advantages of the improved CHO-3E7-based protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change

Double sampling of a faecal immunochemical test is not superior to single sampling for detection of colorectal neoplasia: a colonoscopy controlled prospective cohort study

<p>Abstract</p> <p>Background</p> <p>A single sampled faecal immunochemical test (FIT) has moderate sensitivity for colorectal cancer and advanced adenomas. Repeated FIT sampling could improve test sensitivity. The aim of the present study is to determine whether any of three different strategies of double FIT sampling has a better combination of sensitivity and specificity than single FIT sampling.</p> <p>Methods</p> <p>Test performance of single FIT sampling in subjects scheduled for colonoscopy was compared to double FIT sampling intra-individually. Test positivity of double FIT sampling was evaluated in three different ways: 1) "one of two FITs+" when at least one out of two measurements exceeded the cut-off value, 2) "two of two FITs+" when both measurements exceeded the cut-off value, 3) "mean of two FITs+" when the geometric mean of two FITs exceeded the cut-off value. Receiver operator curves were calculated and sensitivity of single and the three strategies of double FIT sampling were compared at a fixed level of specificity.</p> <p>Results</p> <p>In 124 of 1096 subjects, screen relevant neoplasia (SRN) were found (i.e. early stage CRC or advanced adenomas). At any cut-off, "two of two FITs+" resulted in the lowest and "one of two FITs+" in the highest sensitivity for SRN (range 35-44% and 42%-54% respectively). ROC's of double FIT sampling were similar to single FIT sampling. At specificities of 85/90/95%, sensitivity of any double FIT sampling strategy did not differ significantly from single FIT (p-values 0.07-1).</p> <p>Conclusion</p> <p>At any cut off, "one of two FITs+" is the most sensitive double FIT sampling strategy. However, at a given specificity level, sensitivity of any double FIT sampling strategy for SRN is comparable to single FIT sampling at a different cut-off value. None of the double FIT strategies has a superior combination of sensitivity and specificity over single FIT.</p

Clinical utility and research frontiers of neuroimaging in movement disorders

Neuroimaging in Parkinson’s disease (PD) and other primary Parkinsonian disorders has been increasingly used in the routine clinical work in the last years. The paradigm has changed from an “exclusionary” use, i.e., to rule out causes of secondary Parkinsonism, to an “inclusionary” one, i.e., finding image and network characteristics allowing to identify a specific disease. This is allowed by analyses spanning from the commonly used visual analysis to the most sophisticated postprocessing leading to the identification of covariance patterns both in morphological and functional neuroimaging. However, paralleling the advancement in covariance and connectivity analyses, the issues of standardization and harmonization of data acquisition, and image reconstruction and postprocessing among centers are emerging in the scientific community. Also, the building of scientific evidence still suffers from the lack of large, formal studies and relies on relatively small cohort studies from one or few centers. Joint actions to face these issues are now ongoing in Europe, supported by specific programs, such as the Joint Programming on Neurodegenerative Diseases (JPND). In the present review, some of the most recent and relevant achievements in the field of diffusion tensor magnetic resonance imaging (MRI), functional MRI, fludeoxyglucose-positron-emission tomography, dopamine transporter single-photon emission computed tomography and non-dopaminergic imaging in PD and primary Parkinsonisms are reported

Clinical utility and research frontiers of neuroimaging in movement disorders

Neuroimaging in Parkinson's disease (PD) and other primary Parkinsonian disorders has been increasingly used in the routine clinical work in the last years. The paradigm has changed from an "exclusionary" use, i.e., to rule out causes of secondary Parkinsonism, to an "inclusionary" one, i.e., finding image and network characteristics allowing to identify a specific disease. This is allowed by analyses spanning from the commonly used visual analysis to the most sophisticated postprocessing leading to the identification of covariance patterns both in morphological and functional neuroimaging. However, paralleling the advancement in covariance and connectivity analyses, the issues of standardization and harmonization of data acquisition, and image reconstruction and postprocessing among centers are emerging in the scientific community. Also, the building of scientific evidence still suffers from the lack of large, formal studies and relies on relatively small cohort studies from one or few centers. Joint actions to face these issues are now ongoing in Europe, supported by specific programs, such as the Joint Programming on Neurodegenerative Diseases (JPND). In the present review, some of the most recent and relevant achievements in the field of diffusion tensor magnetic resonance imaging (MRI), functional MRI, fludeoxyglucose-positron-emission tomography, dopamine transporter single-photon emission computed tomography and non-dopaminergic imaging in PD and primary Parkinsonisms are reported

Proenkephalin A gene products activate a new family of sensory neuron\u2013specific GPCRs

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of \u2018orphan\u2019 G protein\u2013 coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron\u2013specific G protein\u2013coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.NRC publication: Ye

Major depressive disorder masking frontotemporal dementia secondary to C9orf72 mutation: A case report

International audienc

Head-to-head comparison among semi-quantification tools of brain FDG-PET to aid the diagnosis of prodromal Alzheimer's disease

Several automatic tools have been implemented for semi-quantitative assessment of brain [18]F-FDG-PET