Guiding bispecific monovalent antibody formation through proteolysis of IgG1 single-chain

We developed an IgG1 domain-tethering approach to guide the correct assembly of 2 light and 2 heavy chains, derived from 2 different antibodies, to form bispecific monovalent antibodies in IgG1 format. We show here that assembling 2 different light and heavy chains by sequentially connecting them with protease-cleavable polypeptide linkers results in the generation of monovalent bispecific antibodies that have IgG1 sequence, structure and functional properties. This approach was used to generate a bispecific monovalent antibody targeting the epidermal growth factor receptor and the type I insulin-like growth factor receptor that: 1) can be produced and purified using standard IgG1 techniques; 2) exhibits stability and structural features comparable to IgG1; 3) binds both targets simultaneously; and 4) has potent anti-tumor activity. Our strategy provides new engineering opportunities for bispecific antibody applications, and, most importantly, overcomes some of the limitations (e.g., half-antibody and homodimer formation, light chains mispairing, multi-step purification), inherent with some of the previously described IgG1-based bispecific monovalent antibodies.     

Biochar and alternate partial root-zone irrigation greatly enhance the effectiveness of mulberry in remediating lead-contaminated soils

土壤铅污染日益严重,植物修复是一种环保的污染土壤修复技术。本文旨在研究四种土壤铅污染水平下,添加生物碳和分根区交替 灌溉(Alternative Partial Root-zone Irrigation,APRI)对桑树幼苗的生长、铅适应性和铅积累的影响。我们以生物碳(添加与不添加生物碳)、灌溉方式(APRI 与常规灌溉)和土壤铅水平(0、50、200 和800 mg kg⁻¹ Pb)为三因素实施了温室试验。通过测定桑树幼苗的生长性状、渗透物质代谢、抗氧化酶活性、铅的积累和转运等参数,探讨了不同处理对桑树生长发育的影响。结果表明,桑树对土壤铅污染有较强的适应能力;生物碳和APRI 在不同土壤铅水平上协同提高了生物量和吸收根表面积。桑树通过调节谷胱甘肽 (GSH)、脯氨酸代谢和过氧化物酶(POD)活性,增加了渗透和抗氧化调节能力,进而提高了对重度铅污染土壤(800 mg kg⁻¹)的抗性。桑苗中的铅离子主要集中在根中,与土壤铅浓度具有剂量效应。土壤铅、生物碳和ARPI的交互作用影响了叶片和根系中铅的浓度、转运和生物富集系数。综上所述,在桑树栽培中结合外源生物碳和APRI可有效地用于修复土壤铅污染。     

Application of Real-Time PCR To Study Effects of Ammonium on Population Size of Ammonia-Oxidizing Bacteria in Soil

Ammonium oxidation by autotrophic ammonia-oxidizing bacteria (AOB) is a key process in agricultural and natural ecosystems and has a large global impact. In the past, the ecology and physiology of AOB were not well understood because these organisms are notoriously difficult to culture. Recent applications of molecular techniques have advanced our knowledge of AOB, but the necessity of using PCR-based techniques has made quantitative measurements difficult. A quantitative real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) was developed to estimate AOB population size in soil. This assay has a detection limit of 1.3 × 10⁵ cells/g of dry soil. The effect of the ammonium concentration on AOB population density was measured in soil microcosms by applying 0, 1.5, or 7.5 mM ammonium sulfate. AOB population size and ammonium and nitrate concentrations were monitored for 28 days after (NH₄)₂SO₄ application. AOB populations in amended treatments increased from an initial density of approximately 4 × 10⁶ cells/g of dry soil to peak values (day 7) of 35 × 10⁶ and 66 × 10⁶ cells/g of dry soil in the 1.5 and 7.5 mM treatments, respectively. The population size of total bacteria (quantified by real-time PCR with a universal bacterial probe) remained between 0.7 × 10⁹ and 2.2 × 10⁹ cells/g of soil, regardless of the ammonia concentration. A fertilization experiment was conducted in a tomato field plot to test whether the changes in AOB density observed in microcosms could also be detected in the field. AOB population size increased from 8.9 × 10⁶ to 38.0 × 10⁶ cells/g of soil by day 39. Generation times were 28 and 52 h in the 1.5 and 7.5 mM treatments, respectively, in the microcosm experiment and 373 h in the ammonium treatment in the field study. Estimated oxidation rates per cell ranged initially from 0.5 to 25.0 fmol of NH₄⁺ h⁻¹ cell⁻¹ and decreased with time in both microcosms and the field. Growth yields were 5.6 × 10⁶, 17.5 × 10⁶, and 1.7 × 10⁶ cells/mol of NH₄⁺ in the 1.5 and 7.5 mM microcosm treatments and the field study, respectively. In a second field experiment, AOB population size was significantly greater in annually fertilized versus unfertilized soil, even though the last ammonium application occurred 8 months prior to measurement, suggesting a long-term effect of ammonium fertilization on AOB population size.     

Naturally Occurring Bacteria Similar to the Methyl tert-Butyl Ether (MTBE)-Degrading Strain PM1 Are Present in MTBE-Contaminated Groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10³ to 10⁴ cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 μg of MTBE/liter, densities of native PM1 increased to approximately 10⁵ cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties

By simultaneous binding two disease mediators, bispecific antibodies offer the opportunity to broaden the utility of antibody-based therapies. Herein, we describe the design and characterization of Bs4Ab, an innovative and generic bispecific tetravalent antibody platform. The Bs4Ab format comprises a full-length IgG1 monoclonal antibody with a scFv inserted into the hinge domain. The Bs4Ab design demonstrates robust manufacturability as evidenced by MEDI3902, which is currently in clinical development. To further demonstrate the applicability of the Bs4Ab technology, we describe the molecular engineering, biochemical, biophysical, and in vivo characterization of a bispecific tetravalent Bs4Ab that, by simultaneously binding vascular endothelial growth factor and angiopoietin-2, inhibits their function. We also demonstrate that the Bs4Ab platform allows Fc-engineering similar to that achieved with IgG1 antibodies, such as mutations to extend half-life or modulate effector functions.     

Naturally occurring bacteria similar to the methyl tert-butyl ether (MTBE)-degrading strain PM1 are present in MTBE-contaminated groundwater

Methyl tert-butyl ether (MTBE) is a widespread groundwater contaminant that does not respond well to conventional treatment technologies. Growing evidence indicates that microbial communities indigenous to groundwater can degrade MTBE under aerobic and anaerobic conditions. Although pure cultures of microorganisms able to degrade or cometabolize MTBE have been reported, to date the specific organisms responsible for MTBE degradation in various field studies have not be identified. We report that DNA sequences almost identical (99% homology) to those of strain PM1, originally isolated from a biofilter in southern California, are naturally occurring in an MTBE-polluted aquifer in Vandenberg Air Force Base (VAFB), Lompoc, California. Cell densities of native PM1 (measured by TaqMan quantitative PCR) in VAFB groundwater samples ranged from below the detection limit (in anaerobic sites) to 10(3) to 10(4) cells/ml (in oxygen-amended sites). In groundwater from anaerobic or aerobic sites incubated in microcosms spiked with 10 microg of MTBE/liter, densities of native PM1 increased to approximately 10(5) cells/ml. Native PM1 densities also increased during incubation of VAFB sediments during MTBE degradation. In controlled field plots amended with oxygen, artificially increasing the MTBE concentration was followed by an increase in the in situ native PM1 cell density. This is the first reported relationship between in situ MTBE biodegradation and densities of MTBE-degrading bacteria by quantitative molecular methods.     

Casein SNP in Norwegian goats: additive and dominance effects on milk composition and quality

Background

The four casein proteins in goat milk are encoded by four closely linked casein loci (CSN1S1, CSN2, CSN1S2 and CSN3) within 250 kb on caprine chromosome 6. A deletion in exon 12 of CSN1S1, so far reported only in Norwegian goats, has been found at high frequency (0.73). Such a high frequency is difficult to explain because the national breeding goal selects against the variant''s effect.

Methods

In this study, 575 goats were genotyped for 38 Single Nucleotide Polymorphisms (SNP) located within the four casein genes. Milk production records of these goats were obtained from the Norwegian Dairy Goat Control. Test-day mixed models with additive and dominance fixed effects of single SNP were fitted in a model including polygenic effects.

Results

Significant additive effects of single SNP within CSN1S1 and CSN3 were found for fat % and protein %, milk yield and milk taste. The allele with the deletion showed additive and dominance effects on protein % and fat %, and overdominance effects on milk quantity (kg) and lactose %. At its current frequency, the observed dominance (overdominance) effects of the deletion allele reduced its substitution effect (and additive genetic variance available for selection) in the population substantially.

Conclusions

The selection pressure of conventional breeding on the allele with the deletion is limited due to the observed dominance (overdominance) effects. Inclusion of molecular information in the national breeding scheme will reduce the frequency of this deletion in the population.     

Spire and Formin 2 Synergize and Antagonize in Regulating Actin Assembly in Meiosis by a Ping-Pong Mechanism

In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a “ping-pong” mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase–independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.     

Effect of hot water treatment on development of anthracnose (Colletotrichum gloeosporioides) and quality of mango fruit at Jimma southwest Ethiopia

Anthracnose is the major postharvest disease of mango and occurs throughout mango producing areas of the world including Ethiopia. Evaluating effect of hot water treatment on development of anthracnose and quality of mango fruit is imperative. A total of three hot water levels 48, 52 and 56 °C at two time interval (5 and 10 min) were tested with factorial arrangement in completely randomised design. The study indicated that hot water treatment at different temperatures and time interval significantly (p < 0.001) affects disease development and shelf life and postharvest quality of mango fruits. Hot water treatments reduced the incidence and severity of anthracnose disease significantly (p < 0.001) in mango fruits as compared to control. There was a highly significant difference (p < 0.0001) on weight loss, total soluble solids, titratable acidity and fruit firmness of mango fruits due to treatment. The present study reviled that hot water treatment has a potential in reducing the postharvest loss due to anthracnose and improving the shelf life and quality of mango fruits. However, the reduction of disease pressure on fruits was not at applicable level, which call ups future effort on developing on integrated disease management strategies for reduction of postharvest loss of mango fruits.