Search

Background and Objective: Biological medicinal products improve patients' lives, but access is limited, mainly due to high costs. Patents for many existing biological products are expiring, and generic versions, which are referred to as biosimilars, are produced to serve as an alternative to the reference medicinal product (RMP) cutting down the costs and expanding access. The present paper assesses the analytical similarity between Formycon's FYB206 pembrolizumab biosimilar candidate and Keytruda®, an RMP that is approved to treat various types of cancer, with the intention of determining FYB206's suitability to enter clinical biosimilar trials. Methods: Monoclonal antibodies (mAbs) are biological medicinal products that are characterized by a high overall heterogeneity. Due to the complex nature of these molecules, a comprehensive comparative analytical assessment was designed to demonstrate analytical similarity in all clinically relevant quality attributes between RMP and the corresponding biosimilar candidate. This exercise addresses physicochemical, biophysical as well as functional characteristics. Results: The comparative analytical evaluation results demonstrate that the proposed biosimilar is structurally and functionally highly similar to the RMP, showing only minor differences for some quality attributes that are justified to be noncritical for clinical efficacy and safety. Conclusion: Based on physicochemical and biological characteristics, FYB206 is suitable to enter the clinical phase. [ABSTRACT FROM AUTHOR]

Abstract Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.

Abstract Background Honey bee (Apis mellifera) workers are characterized by complex social behavior. Their life-history is dominated by a period of within-nest activity followed by a phase of long-distance flights and foraging. General insights into insect metabolism imply that foraging onset is associated with fundamental metabolic changes, and theory on social evolution suggests metabolic adaptations that are advantageous for the colony as a whole. Results Here we address the life-history characteristics of workers with LC-MS/MS based relative quantification of major proteins. Our approach includes: i. Calculation of a false positive rate for the identifications, ii. Support of relative protein quantification results obtained from spectral count by non-parametric statistics, and iii. Correction for Type 1 error inflation using a bootstrap iteration analysis. Our data are consistent with the use of glucose as the main fuel for honey bee flight. Moreover, the data delivers information on the expression of ATPsynthases/ATPases, and provide new insights into nurse- and forager-specific patterns of protection against oxidative stress. Conclusion The results show the suitability of this approach to investigate fundamental biochemical changes in an insect, and provide new evidence for metabolic specializations that occur during the social ontogeny of worker honey bees.

Abstract Background Protein phosphorylation is accepted as a major regulatory pathway in plants. More than 1000 protein kinases are predicted in the Arabidopsis proteome, however, only a few studies look systematically for in vivo protein phosphorylation sites. Owing to the low stoichiometry and low abundance of phosphorylated proteins, phosphorylation site identification using mass spectrometry imposes difficulties. Moreover, the often observed poor quality of mass spectra derived from phosphopeptides results frequently in uncertain database hits. Thus, several lines of evidence have to be combined for a precise phosphorylation site identification strategy. Results Here, a strategy is presented that combines enrichment of phosphoproteins using a technique termed metaloxide affinity chromatography (MOAC) and selective ion trap mass spectrometry. The complete approach involves (i) enrichment of proteins with low phosphorylation stoichiometry out of complex mixtures using MOAC, (ii) gel separation and detection of phosphorylation using specific fluorescence staining (confirmation of enrichment), (iii) identification of phosphoprotein candidates out of the SDS-PAGE using liquid chromatography coupled to mass spectrometry, and (iv) identification of phosphorylation sites of these enriched proteins using automatic detection of H3PO4 neutral loss peaks and data-dependent MS3-fragmentation of the corresponding MS2-fragment. The utility of this approach is demonstrated by the identification of phosphorylation sites in Arabidopsis thaliana seed proteins. Regulatory importance of the identified sites is indicated by conservation of the detected sites in gene families such as ribosomal proteins and sterol dehydrogenases. To demonstrate further the wide applicability of MOAC, phosphoproteins were enriched from Chlamydomonas reinhardtii cell cultures. Conclusion A novel phosphoprotein enrichment procedure MOAC was applied to seed proteins of A. thaliana and to proteins extracted from C. reinhardtii. Thus, the method can easily be adapted to suit the sample of interest since it is inexpensive and the components needed are widely available. Reproducibility of the approach was tested by monitoring phosphorylation sites on specific proteins from seeds and C. reinhardtii in duplicate experiments. The whole process is proposed as a strategy adaptable to other plant tissues providing high confidence in the identification of phosphoproteins and their corresponding phosphorylation sites.

The vast impact of the replication of the intracellular endosymbiotic bacterium blochmannia floridanus was identified at the time when the formation of larva of the host ant camponotus floridanus took place. Moreover the symbiosis process is required much more by the insects in the early stages of their growth rather than by the adult insects.

The article presents research which analyzed the effect of insulin receptor substrate (IRS) on the development of female caste in honeybees. The authors analyzed the importance of the conservation of insulin/insulin-like signaling (IIS) network to the growth of the bees with the use of the ribonucleic acid interference (RNAi) to the homologue of IRS. They found that by feeding the larvae with a rich double stranded RNA diet, the abundance of IRS in the messenger RNA is decreased.

Background and Objective: Biological medicinal products improve patients' lives, but access is limited, mainly due to high costs. Patents for many existing biological products are expiring, and generic versions, which are referred to as biosimilars, are produced to serve as an alternative to the reference medicinal product (RMP) cutting down the costs and expanding access. The present paper assesses the analytical similarity between Formycon's FYB206 pembrolizumab biosimilar candidate and Keytruda®, an RMP that is approved to treat various types of cancer, with the intention of determining FYB206's suitability to enter clinical biosimilar trials. Methods: Monoclonal antibodies (mAbs) are biological medicinal products that are characterized by a high overall heterogeneity. Due to the complex nature of these molecules, a comprehensive comparative analytical assessment was designed to demonstrate analytical similarity in all clinically relevant quality attributes between RMP and the corresponding biosimilar candidate. This exercise addresses physicochemical, biophysical as well as functional characteristics. Results: The comparative analytical evaluation results demonstrate that the proposed biosimilar is structurally and functionally highly similar to the RMP, showing only minor differences for some quality attributes that are justified to be noncritical for clinical efficacy and safety. Conclusion: Based on physicochemical and biological characteristics, FYB206 is suitable to enter the clinical phase. [ABSTRACT FROM AUTHOR]

N-glycosylation is a common post-translational modification of biopharmaceutical products. Certain types of N-glycans have been shown to influence important properties of monoclonal antibody products including pharmacokinetics and effector functions. Complex biopharmaceuticals e.g. Fc fusion proteins may contain several N- and O-glycosylation sites. Domain specific characterization of two Fc fusion proteins showed an Fc N-glycosylation pattern comparable to IgG molecules. The receptor N-glycosylation was found to contain some larger and more complex N-glycans compared to the Fc part. Analyses of samples from non-clinical studies of the two studied fusion proteins indicate that their N-glycans impact pharmacokinetic properties. Interestingly, besides the type of N-glycan this influence on the pharmacokinetics depends also on the glycosylation site and thus the accessibility on the protein. The same type of N-glycan can influence the clearance of fusion proteins when located at the receptor part, but not if located at the Fc part. In this study, it is shown that N-glycans with terminal galactose or N-acetylglucosamine residues have a negative impact on serum half-life when located at the receptor part. Terminal sialylation of galactose residues prevents this faster clearance even when only one sialic acid is present. O-acetylation, a modification of sialic acids does not impact pharmacokinetics. Thus, type and accessibility of N-glycan moieties of fusion proteins both play an important role in pharmacokinetics. Finally, detailed site specific analysis is critical in the development of biopharmaceuticals. [ABSTRACT FROM AUTHOR]

We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.