The complete genome sequence of a single nucleocapsid nucleopolyhedrovirus recently isolated from Chrysodeixis chalcites (ChchNPV) was determined. The viral genome has a size of 149 622 bp and an overall G+C content of 39?1 mol%. The sequence contains 151 predicted open reading frames (ORFs) with a minimal size of 50 codons. The similarity of these ORFs with those of other completely sequenced baculoviruses was calculated using a newly developed database, named GECCO. Phylogenetic analysis of the whole genome confirmed the evolutionary relationship of ChchNPV with group II NPVs, as did the absence of the NPV group I-specific gp64 gene. It is the first group II NPV to encode proliferating cell nuclear antigen. Most noteworthy is the presence of two ORFs encoding a class II cyclobutane pyrimidine dimer DNA photolyase. These two ORFs share only 45 % amino acid identity and have different promoter motifs. Twenty-two additional unique baculovirus genes were identified, including a gene encoding a novel putative RING finger protein with a possible homologue in poxviruses.

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next-Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high-throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813-817, 2016.

Inositol-1,4,5-triphosphate (IP3) kinase B (ITPKB) is a ubiquitously expressed lipid kinase that inactivates IP3, a secondary messenger that stimulates calcium release from the endoplasmic reticulum (ER). Genome-wide association studies have identified common variants in the ITPKB gene locus associated with reduced risk of sporadic Parkinson’s disease (PD). Here, we investigate whether ITPKB activity or expression level impacts PD phenotypes in cellular and animal models. In primary neurons, knockdown or pharmacological inhibition of ITPKB increased levels of phosphorylated, insoluble α-synuclein pathology following treatment with α-synuclein preformed fibrils (PFFs). Conversely, ITPKB overexpression reduced PFF-induced α-synuclein aggregation. We also demonstrate that ITPKB inhibition or knockdown increases intracellular calcium levels in neurons, leading to an accumulation of calcium in mitochondria that increases respiration and inhibits the initiation of autophagy, suggesting that ITPKB regulates α-synuclein pathology by inhibiting ER-to-mitochondria calcium transport. Furthermore, the effects of ITPKB on mitochondrial calcium and respiration were prevented by pretreatment with pharmacological inhibitors of the mitochondrial calcium uniporter complex, which was also sufficient to reduce α-synuclein pathology in PFF-treated neurons. Taken together, these results identify ITPKB as a negative regulator of α-synuclein aggregation and highlight modulation of ER-to-mitochondria calcium flux as a therapeutic strategy for the treatment of sporadic PD.

Comparative LC-MS is a powerful method for detailed quantitative comparison of complex protein mixtures. Dedicated software is required for detection, matching, and alignment of peaks in multiple LC-MS datasets. However, retention time shifts, saturation effects, limitations of experimental accuracy, and possible occurrence of split peaks make it difficult for software to perfectly match all chromatograms. We describe a procedure to assess the above problems and show that dataset quality can be enhanced with the aid of cluster analysis.

Background: Modern omics research involves the application of high-throughput technologies that generate vast volumes of data. These data need to be pre-processed, analyzed and integrated with existing knowledge through the use of diverse sets of software tools, models and databases. The analyses are often interdependent and chained together to form complex workflows or pipelines. Given the volume of the data used and the multitude of computational resources available, specialized pipeline software is required to make high-throughput analysis of large-scale omics datasets feasible.