Tailoring translational strength using Kozak sequence variants improves bispecific antibody assembly and reduces product-related impurities in CHO cells

Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins.     

Theoretical Calculations on Zeolites: The Aluminium Substitution in Mordenite,Ferrierite and ZSM-5

Abstract

The experimental determination of Al siting in zeolites involves the use of multiple techniques. Great effort has been made on both, experimental and theoretical approaches. The present study presents a novel methodology for calculating Al/Si replacement energies. Simple semiempirical calculations were applied on Modenite, Ferrierite and ZSM-5 zeolites, resulting in good agreement with the experimetal evidences. We have found that the favored Al substitution sites are T3 and T4 in Mordenite, while T2 and T4 are in Ferrierite, and only the T9 site is favored in ZSM-5. The method presented is based on an average of partial Al/Si replacement energies, evaluated for all rings belonging to each T site, rather than in the calculation of a total replacement energy evaluated for only one representative aggregate.     

Past and future demographic dynamics of alpine species: limited genetic consequences despite dramatic range contraction in a plant from the Spanish Sierra Nevada

Anthropogenic global climate change is expected to cause severe range contractions among alpine plants. Alpine areas in the Mediterranean region are of special concern because of the high abundance of endemic species with narrow ranges. This study combined species distribution models, population structure analyses and Bayesian skyline plots to trace the past and future distribution and diversity of Linaria glacialis, an endangered narrow endemic species that inhabits summits of Sierra Nevada (Spain). The results showed that: (i) the habitat of this alpine‐Mediterranean species in Sierra Nevada suffered little changes during glacial and interglacial stages of late Quaternary; (ii) climatic oscillations in the last millennium (Medieval Warm Period and Little Ice Age) moderately affected the demographic trends of L. glacialis; (iii) future warming conditions will cause severe range contractions; and (iv) genetic diversity will not diminish at the same pace as the distribution range. As a consequence of the low population structure of this species, genetic impoverishment in the alpine zones of Sierra Nevada should be limited during range contraction. We conclude that maintenance of large effective population sizes via high mutation rates and high levels of gene flow may promote the resilience of alpine plant species when confronted with global warming.     

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.     

TNF-related weak inducer of apoptosis (TWEAK) promotes kidney fibrosis and Ras-dependent proliferation of cultured renal fibroblast

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) regulates apoptosis, proliferation and inflammation in renal epithelial cells and plays a role in acute kidney injury. However, there is little information on the chronic effects of TWEAK. We hypothesized that TWEAK may influence renal fibrosis and regulate kidney fibroblast biology, in part, through Ras pathway.We studied a chronic model of experimental unilateral ureteral obstruction in wild type and TWEAK deficient mice, and a murine model of systemic TWEAK overexpression. TWEAK actions were also explored in cultured renal and embryonic fibroblasts.TWEAK and TWEAK receptor expression was increased in the obstructed kidneys. The absence of TWEAK decreased early kidney tubular damage, inflammatory infiltrates and myofibroblast number. TWEAK deficient mice had decreased renal fibrosis 21 days after obstruction, as assessed by extracellular matrix staining. In mice without prior underlying kidney disease, systemic overexpression of TWEAK induced kidney inflammation and fibrosis. In cultured fibroblasts, TWEAK induced proliferation through activation of the Ras/ERK pathway. TWEAK also activated nuclear factor κB (NFκB)-dependent inflammatory chemokine production in murine renal fibroblasts.In conclusion, lack of TWEAK reduces renal fibrosis in a model of persistent kidney insult and overexpression of TWEAK led to renal fibrosis. TWEAK actions on renal fibroblasts may contribute to the in vivo observations, as TWEAK promotes inflammatory activity and proliferation in fibroblast cultures.     

Increased Learning and Brain Long-Term Potentiation in Aged Mice Lacking DNA Polymerase μ

A definitive consequence of the aging process is the progressive deterioration of higher cognitive functions. Defects in DNA repair mechanisms mostly result in accelerated aging and reduced brain function. DNA polymerase µ is a novel accessory partner for the non-homologous end-joining DNA repair pathway for double-strand breaks, and its deficiency causes reduced DNA repair. Using associative learning and long-term potentiation experiments, we demonstrate that Polµ^−/− mice, however, maintain the ability to learn at ages when wild-type mice do not. Expression and biochemical analyses suggest that brain aging is delayed in Polµ^−/− mice, being associated with a reduced error-prone DNA oxidative repair activity and a more efficient mitochondrial function. This is the first example in which the genetic ablation of a DNA-repair function results in a substantially better maintenance of learning abilities, together with fewer signs of brain aging, in old mice.     

An Optimized Immunohistochemistry Technique Improves NMO-IgG Detection: Study Comparison with Cell-Based Assays

Cell-based assays (CBA) have increased the sensitivity of the neuromyelitis optica (NMO)-IgG/aquaporin-4-antibody detection compared to classical tissue-based indirect assays. We describe the sensitivity of an optimized immunohistochemistry (IHC-o) to detect NMO-IgG/aquaporin-4-antibody in comparison with that of two CBA: an in-house (CBA-ih) and a commercial (CBA-c) assay (Euroimmun, Germany). Coded serum from 103 patients with definite NMO and 122 inflammatory controls were studied by IHC-o, CBA-ih, and CBA-c. IHC-o used the same protocol described to detect antibodies against cell surface antigens. CBA-ih used live cells transfected with the aquaporin-4-M23-isoform. The sensitivity of the IHC-o was 74.8% (95% confidence interval [CI] 65-83) and was similar to that of the CBA-ih 75.7% (95% CI 66-84) and the CBA-c 73.8% (95% CI 64-82). The specificity of the three assays was 100% (95% CI 97-100). Interassay concordance was high, 100 of 103 samples were coincident in all techniques. The optimized immunohistochemistry proves to be as sensitive and specific as the cell-based assays. This assay extends the available tools for NMO-IgG/aquaporin-4-antibody detection.     

Skin autofluorescence predicts cardio-renal outcome in type 1 diabetes: a longitudinal study

Type 1 diabetes is associated with increased cardiovascular disease (CVD). Decreased endothelial progenitor cells (EPCs) number plays a pivotal role in reduced endothelial repair and development of CVD. We aimed to determine if cardioprotective effect of metformin is mediated by increasing circulating endothelial progenitor cells (cEPCs), pro-angiogenic cells (PACs) and decreasing circulating endothelial cells (cECs) count whilst maintaining unchanged glycemic control. This study was an open label and parallel standard treatment study. Twenty-three type 1 diabetes patients without overt CVD were treated with metformin for 8 weeks (treatment group-TG). They were matched with nine type 1 diabetes patients on standard treatment (SG) and 23 age- and sex-matched healthy volunteers (HC). Insulin dose was adjusted to keep unchanged glycaemic control. cEPCs and cECs counts were determined by flow cytometry using surface markers CD45dimCD34+VEGFR-2+ and CD45dimCD133−CD34+CD144+ respectively. Peripheral blood mononuclear cells were cultured to assess changes in PACs number, function and colony forming units (CFU-Hill’s colonies). At baseline TG had lower cEPCs, PACs, CFU-Hills’ colonies and PACs adhesion versus HC (p < 0.001-all variables) and higher cECs versus HC (p = 0.03). Metformin improved cEPCs, PACs, CFU-Hill’s colonies number, cECs and PACs adhesion (p < 0.05-all variables) to levels seen in HC whilst HbA1c (one-way ANOVA p = 0.78) and glucose variability (average glucose, blood glucose standard deviation, mean amplitude of glycaemic excursion, continuous overall net glycaemic action and area under curve) remained unchanged. No changes were seen in any variables in SG. There was an inverse correlation between CFU-Hill’s colonies with cECs. Metformin has potential cardio-protective effect through improving cEPCs, CFU-Hill’s colonies, cECs, PACs count and function independently of hypoglycaemic effect. This finding needs to be confirmed by long term cardiovascular outcome studies in type 1 diabetes. Trial registration ISRCTN26092132     

Effects of co-cropping Bidens pilosa (L.) and Tagetes minuta (L.) on bioaccumulation of Pb in Lactuca sativa (L.) growing in polluted agricultural soils

Polluted agricultural soils are a serious problem for food safety, with phytoremediation being the most favorable alternative from the environmental perspective. However, this methodology is generally time-consuming and requires the cessation of agriculture. Therefore, the purpose of this study was to evaluate two potential phytoextractor plants (the native species Bidens pilosa and Tagetes minuta) co-cropped with lettuce growing on agricultural lead-polluted soils. The concentrations of Pb, as well as of other metals, were investigated in the phytoextractors, crop species, and in soils, with the potential risk to the health of consumers being estimated. The soil parameters pH, EC, organic matter percentage and bioavailable lead showed a direct relationship with the accumulation of Pb in roots. In addition, the concentration of Pb in roots of native species was closely related to Fe (B. pilosa, r = 0.81; T. minuta r = 0.75), Cu (T. minuta, r = 0.93), Mn (B. pilosa, r = 0.89) and Zn (B. pilosa, r = 0.91; T. minuta, r = 0.91). Our results indicate that the interaction between rhizospheres increased the phytoextraction of lead, which was accompanied by an increase in the biomass of the phytoextractor species. However, the consumption of lettuce still revealed a toxicological risk from Pb in all treatments.