Engineering a therapeutic IgG molecule to address cysteinylation,aggregation and enhance thermal stability and expression

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T_m, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.     

Serum Uric Acid and Non-Alcoholic Fatty Liver Disease in Non-Diabetic Chinese Men

Increased serum uric acid (SUA) levels may be involved in the development of non-alcoholic fatty liver disease (NAFLD) in men presenting with metabolic syndrome (MetS) and/or insulin resistance. We aimed to determine the independent relationship between SUA and NAFLD in non-diabetic Chinese male population, and to explore the determinants of SUA levels among indexes of adiposity, lipid, and genotypes pertaining to triglycerides metabolism, inflammation, oxidative stress, and SUA concentrations. A total of 1440 men, classified depending on the presence of ultrasonographically detected NAFLD, underwent a complete healthy checkup program. Genotypes were extracted from our previously established genome-wide association study database. After adjusting for age, smoking, drinking, body mass index, homeostasis model assessment of insulin resistance, C-reactive protein, creatinine, alanine aminotransferase (ALT) and components of metabolic syndrome, the odds ratio for NAFLD, comparing the highest with the lowest SUA quartile, was 2.81 (95% confidence interval 1.66–4.76). A stepwise multivariate linear regression analysis (R² = 0.238, P<0.001) retained age, waist circumference, serum creatinine, triglycerides, the Q141K variant in ABCG2 (rs2231142) and NAFLD as significant predictors of SUA levels (all P<0.001). Besides, ALT and Met196Arg variant in TNFRSF1B (rs1061622) additionally associated with SUA among individuls with NAFLD. Our data suggest that in Chinese men, elevated SUA is significantly associated with NAFLD, independent of insulin resistance and other metabolic disorders, such as central obesity or hypertriglyceridemia. Meanwhile, among subjects with NAFLD, index of liver damage, such as elevated ALT combined with genetic susceptibility to inflammation associated with increased SUA levels.     

CXCR2-Driven Ovarian Cancer Progression Involves Upregulation of Proinflammatory Chemokines by Potentiating NF-κB Activation via EGFR-Transactivated Akt Signaling

Ovarian cancer is an inflammation-associated malignancy with a high mortality rate. CXCR2 expressing ovarian cancers are aggressive with poorer outcomes. We therefore investigated molecular mechanisms involved in CXCR2-driven cancer progression by comparing CXCR2 positive and negative ovarian cancer cell lines. Stably CXCR2 transfected SKOV-3 cells had a faster growth rate as compared to control cells transfected with empty vector. Particularly, tumor necrosis factor (TNF), abundantly expressed in ovarian cancer, enhanced cell proliferation by decreasing the G0-G1 phase in CXCR2 transfected cells. TNF increased nuclear factor-κB (NF-κB) activity to a greater degree in CXCR2 transfected cells than control cells as well as provided a greater activation of IκB. CXCR2 transfected cells expressed higher levels of its proinflammatory ligands, CXCL1/2 and enhanced more proliferation, migration, invasion and colony formation. CXCR2 positive cells also activated more EGFR, which led to higher Akt activation. Enhanced NF-κB activity in CXCR2 positive cells was reduced by a PI3K/Akt inhibitor rather than an Erk inhibitor. CXCL1 added to CXCR2 positive cells led to an increased activation of IκB. CXCL1 also led to a significantly greater number of invasive cells in CXCR2 transfected cells, which was blocked by the NF-κB inhibitor, Bay 11-7082. In addition, enhanced cell proliferation in CXCR2 positive cells was more sensitive to CXCL1 antibody or an NF-κB inhibitor. Finally, CXCR2 transfection of parental cells increased CXCL1 promoter activity via an NF-κB site. Thus augmentation of proinflammatory chemokines CXCL1/2, by potentiating NF-κB activation through EGFR-transactivated Akt, contributes to CXCR2-driven ovarian cancer progression.     

Effect of fermentation inhibitors in the presence and absence of activated charcoal on the growth of Saccharomyces cerevisiae

The acidic hydrolysis of biomass generates numerous inhibitors of fermentation, which adversely affect cell growth and metabolism. The goal of the present study was to determine the effects of fermentation inhibitors on growth and glucose consumption by Saccharomyces cerevisiae. We also conducted in situ adsorption during cell cultivation in synthetic broth containing fermentation inhibitors. In order to evaluate the effect of in situ adsorption on cell growth, five inhibitors, namely 5-hydroxymethylfurfural, levulinic acid, furfural, formic acid, and acetic acid, were introduced into synthetic broth. The existence of fermentation inhibitors during cell culture adversely affects cell growth and sugar consumption. Furfural, formic acid, and acetic acid were the most potent inhibitors in our culture system. The in situ adsorption of inhibitors by the addition of activated charcoal to the synthetic broth increased cell growth and sugar consumption. Our results indicate that detoxification of fermentation media by in situ adsorption may be useful for enhancing biofuel production.     

Rapid identification of potential drought tolerance genes from Solanum tuberosum by using a yeast functional screening method

Identification of major stress tolerance genes of a crop plant is important for the rapid development of its stress-tolerant cultivar. Here, we used a yeast functional screen method to identify potential drought-tolerance genes from a potato plant. A cDNA expression library was constructed from hyperosmotic stressed potato plants. The yeast transformants expressing different cDNAs were selected for their ability to survive in hyperosmotic stress conditions. The relative tolerances of the selected yeast transformants to multiple abiotic stresses were also studied. Specific potato cDNAs expressed in the tolerant yeast transformants were identified. Sixty-nine genes were found capable of enhancing hyperosmotic stress tolerance of yeast. Based on the relative tolerance data generated, 12 genes were selected, which could be most effective in imparting higher drought tolerance to potato with better survival in salt and high-temperature stresses. Orthologues of few genes identified here are previously known to increase osmotic stress tolerance of yeast and plants; however, specific studies are needed to confirm their role in the osmotic stress tolerance of potato.     

Computational and Genetic Reduction of a Cell Cycle to Its Simplest,Primordial Components

What are the minimal requirements to sustain an asymmetric cell cycle? Here we use mathematical modelling and forward genetics to reduce an asymmetric cell cycle to its simplest, primordial components. In the Alphaproteobacterium Caulobacter crescentus, cell cycle progression is believed to be controlled by a cyclical genetic circuit comprising four essential master regulators. Unexpectedly, our in silico modelling predicted that one of these regulators, GcrA, is in fact dispensable. We confirmed this experimentally, finding that ΔgcrA cells are viable, but slow-growing and elongated, with the latter mostly due to an insufficiency of a key cell division protein. Furthermore, suppressor analysis showed that another cell cycle regulator, the methyltransferase CcrM, is similarly dispensable with simultaneous gcrA/ccrM disruption ameliorating the cytokinetic and growth defect of ΔgcrA cells. Within the Alphaproteobacteria, gcrA and ccrM are consistently present or absent together, rather than either gene being present alone, suggesting that gcrA/ccrM constitutes an independent, dispensable genetic module. Together our approaches unveil the essential elements of a primordial asymmetric cell cycle that should help illuminate more complex cell cycles.     

Adipose Vascular Endothelial Growth Factor Regulates Metabolic Homeostasis through Angiogenesis

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Highlights? Two-way modulations of adipose VEGF were generated with aP2-Cre transgene ? Adipose VEGF KO reduces vasculature, increases hypoxia and inflammation in fat ? Adipose VEGF KO accelerates the development of metabolic disease in high-fat diet ? Induced adipose VEGF has opposite effect on fat and restores metabolic homeostasis     

Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated m.14484T>C (MT-ND6) mutation in Chinese families

Mitochondrial m.14484T>C (MT-ND6) mutation has been associated with Leber's hereditary optic neuropathy. Previous investigations revealed that the m.14484T>C mutation is a primary factor underlying the development of optic neuropathy but is not sufficient to produce a clinical phenotype. However, mitochondrial haplogroups have been proposed to modulate the phenotypic manifestation of the m.14484T>C mutation. Here, we performed the clinical, genetic evaluation and complete mitochondrial genome sequence analysis of 41 Han Chinese pedigrees carrying the m.14484T>C mutation. These families exhibited a wide range of penetrances and expressivities of optic neuropathy. The average ratio between affected male/female matrilineal relatives from 41 families was 2:1. The penetrance of optic neuropathy in these Chinese pedigrees ranged from 5.6% to 100%, with the average of 23.8%. Furthermore, the age-of-onset for optic neuropathy varied from 4 to 44 years, with the average of 19.3 years. Sequence analysis of their mitochondrial genomes identified distinct sets of polymorphisms belonging to ten Eastern Asian haplogroups, indicating that the m.14484T>C mutation occurred through recurrent origins and founder events. We showed that mitochondrial haplogroups M9, M10 and N9 increased the penetrance of optic neuropathy in these Chinese families. In particular, these mitochondrial haplogroup specific variants: m.3394T>C (MT-ND1), m.14502T>C (MT-ND4) and m.14693A>G (MT-TE) enhanced the penetrance of visual loss in these Chinese families. These data provided the direct evidence that mitochondrial modifiers modulate the variable penetrance and expressivity of optic neuropathy among Chinese pedigrees carrying the m.14484T>C mutation.     

Formation of the death domain complex between FADD and RIP1 proteins in vitro

Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-κB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD–RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD–RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.     

Antifatigue Effects of Panax ginseng C.A. Meyer: A Randomised,Double-Blind,Placebo-Controlled Trial

The present study investigated the antifatigue effects of Panax ginseng C.A. Meyer in 90 subjects (21 men and 69 women) with idiopathic chronic fatigue (ICF) in a randomised, double-blind, placebo-controlled and parallel designed trial. A bespoke 20% ethanol extract of P. ginseng (1 g or 2 g day^–1) or a placebo was administered to each group for 4 weeks, and then fatigue severity was monitored using a self-rating numeric scale (NRS) and a visual analogue scale (VAS) as a primary endpoint. Serum levels of reactive oxygen species (ROS), malondialdehyde (MDA), total glutathione (GSH) contents and glutathione reductase (GSH-Rd) activity were determined. After 4-week, P. ginseng administration decreased the total NRS score, but they were not statistically significant compared with placebo (P>0.05). Mental NRS score was significantly improved by P. ginseng administrations as 20.4±5.0 to 15.1±6.5 [95% CI 2.3∼8.2] for 1 g and 20.7±6.3 to 13.8±6.2 [95% CI −0.1∼4.2] for 2 g compared with placebo 20.9±4.5 to 18.8±2.9 [95% CI 4.1∼9.9, P<0.01]. Only 2 g P. ginseng significantly reduced the VAS score from 7.3±1.3 to 4.4±1.8 [95% CI 0.7∼1.8] compared with the placebo 7.1±1.0 to 5.8±1.3 [95% CI 2.2 ∼3.7, P<0.01]. ROS and MDA levels were lowered by P. ginseng compared to placebo. P. ginseng 1 g increased GSH concentration and GSH-Rd activity. Our results provide the first evidence of the antifatigue effects of P. ginseng in patients with ICF, and we submit that these changes in antioxidant properties contribute in part to its mechanism.

Trial Registration

Clinical Research Information Service (CRIS) KCT0000048