Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.     

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

Drug resistance is a major public health challenge in leishmaniasis chemotherapy, particularly in the case of emerging Leishmania/HIV‐1 co‐infections. We have delineated the mechanism of cell death induced by the HIV‐1 protease inhibitor, Nelfinavir, in the Leishmania parasite. In order to further study Nelfinavir–Leishmania interactions, we selected Nelfinavir‐resistant axenic amastigotes in vitro and characterized them. RNA expression profiling analyses and comparative genomic hybridizations of closely related Leishmania species were used as a screening tool to compare Nelfinavir‐resistant and ‐sensitive parasites in order to identify candidate genes involved in drug resistance. Microarray analyses of Nelfinavir‐resistant and ‐sensitive Leishmania amastigotes suggest that parasites regulate mRNA levels either by modulating gene copy numbers through chromosome aneuploidy, or gene deletion/duplication by homologous recombination. Interestingly, supernumerary chromosomes 6 and 11 in the resistant parasites lead to upregulation of the ABC class of transporters. Transporter assays using radiolabelled Nelfinavir suggest a greater drug accumulation in the resistant parasites and in a time‐dependent manner. Furthermore, high‐resolution electron microscopy and measurements of intracellular polyphosphate levels showed an increased number of cytoplasmic vesicular compartments known as acidocalcisomes in Nelfinavir‐resistant parasites. Together these results suggest that Nelfinavir is rapidly and dramatically sequestered in drug‐induced intracellular vesicles.     

Gene Amplification and Point Mutations in Pyrimidine Metabolic Genes in 5-Fluorouracil Resistant Leishmania infantum

Background

The human protozoan parasites Leishmania are prototrophic for pyrimidines with the ability of both de novo biosynthesis and uptake of pyrimidines.

Methodology/Principal Findings

Five independent L. infantum mutants were selected for resistance to the pyrimidine analogue 5-fluorouracil (5-FU) in the hope to better understand the metabolism of pyrimidine in Leishmania. Analysis of the 5-FU mutants by comparative genomic hybridization and whole genome sequencing revealed in selected mutants the amplification of DHFR-TS and a deletion of part of chromosome 10. Point mutations in uracil phosphorybosyl transferase (UPRT), thymidine kinase (TK) and uridine phosphorylase (UP) were also observed in three individual resistant mutants. Transfection experiments confirmed that these point mutations were responsible for 5-FU resistance. Transport studies revealed that one resistant mutant was defective for uracil and 5-FU import.

Conclusion/Significance

This study provided further insights in pyrimidine metabolism in Leishmania and confirmed that multiple mutations can co-exist and lead to resistance in Leishmania.     

Genetic background modifies CNS‐mediated sensorimotor decline in the AD‐BXD mouse model of genetic diversity in Alzheimer's disease

Many patients with Alzheimer's dementia (AD) also exhibit noncognitive symptoms such as sensorimotor deficits, which can precede the hallmark cognitive deficits and significantly impact daily activities and an individual's ability to live independently. However, the mechanisms underlying sensorimotor dysfunction in AD and their relationship with cognitive decline remains poorly understood, due in part to a lack of translationally relevant animal models. To address this, we recently developed a novel model of genetic diversity in Alzheimer's disease, the AD‐BXD genetic reference panel. In this study, we investigated sensorimotor deficits in the AD‐BXDs and the relationship to cognitive decline in these mice. We found that age‐ and AD‐related declines in coordination, balance and vestibular function vary significantly across the panel, indicating genetic background strongly influences the expressivity of the familial AD mutations used in the AD‐BXD panel and their impact on motor function. Although young males and females perform comparably regardless of genotype on narrow beam and inclined screen tasks, there were significant sex differences in aging‐ and AD‐related decline, with females exhibiting worse decline than males of the same age and transgene status. Finally, we found that AD motor decline is not correlated with cognitive decline, suggesting that sensorimotor deficits in AD may occur through distinct mechanisms. Overall, our results suggest that AD‐related sensorimotor decline is strongly dependent on background genetics and is independent of dementia and cognitive deficits, suggesting that effective therapeutics for the entire spectrum of AD symptoms will likely require interventions targeting each distinct domain involved in the disease.     

Use of exogenous hTERT to immortalize primary human cells

A major obstacle to the immortalization of primary human cells and the establishment of human cell lines is telomere-controlled senescence. Telomere-controlled senescence is caused by the shortening of telomeres that occurs each time somatic human cells divide. The enzyme telomerase can prevent the erosion of telomeres and block the onset of telomere-controlled senescence, but its expression is restricted to the early stages of embryonic development, and in the adult, to rare cells of the blood, skin and digestive track. However, we and others have shown that the transfer of an exogenous hTERT cDNA, encoding the catalytic subunit of human telomerase, can be used to prevent telomere shortening, overcome telomere-controlled senescence, and immortalize primary human cells. Most importantly, hTERT alone can immortalize cells without causing cancer-associated changes or altering phenotypic properties. Primary human cells that have so far been established by the forced expression of hTERT alone include fibroblasts, retinal pigmented epithelial cells, endothelial cells, oesophageal squamous cells, mammary epithelial cells, keratinocytes, osteoblasts, and Nestin-positive cells of the pancreas. In this article, we discuss the use of hTERT to immortalize of human cells, the properties of hTERT-immortalized cells, and their applications to cancer research and tissue engineering.     

Nutrient supplementation experiments with saltern microbial communities implicate utilization of DNA as a source of phosphorus

All environments including hypersaline ones harbor measurable concentrations of dissolved extracellular DNA (eDNA) that can be utilized by microbes as a nutrient. However, it remains poorly understood which eDNA components are used, and who in a community utilizes it. For this study, we incubated a saltern microbial community with combinations of carbon, nitrogen, phosphorus, and DNA, and tracked the community response in each microcosm treatment via 16S rRNA and rpoB gene sequencing. We show that microbial communities used DNA only as a phosphorus source, and provision of other sources of carbon and nitrogen was needed to exhibit a substantial growth. The taxonomic composition of eDNA in the water column changed with the availability of inorganic phosphorus or supplied DNA, hinting at preferential uptake of eDNA from specific organismal sources. Especially favored for growth was eDNA from the most abundant taxa, suggesting some haloarchaea prefer eDNA from closely related taxa. The preferential eDNA consumption and differential growth under various nutrient availability regimes were associated with substantial shifts in the taxonomic composition and diversity of microcosm communities. Therefore, we conjecture that in salterns the microbial community assembly is driven by the available resources, including eDNA.Subject terms: Metagenomics, Microbial ecology