Combined hydrophobic-metal binding fusion tags for applications in aqueous two-phase partitioning

In this work, we studied the influence of fusion affinity tags containing both hydrophobic and histidines residues on the partitioning of the green fluorescent protein, GFPuv, in aqueous two-phase system. The tags were fused to the N-terminal of GFPuv and tested by immobilized metal affinity partitioning, in a PEG/salt system. The presence of both types of residues in the tag increased the partitioning greatly. Particularly, four engineered tags (H6, FH6, WH6, and YH6) containing a hexa-histidine sequence as well as different hydrophobic residues, all increased partitioning more than twice, reaching K values around 20, as compared to another construct (His6-GFP) containing an isolated hexa-histidine sequence. YH6, also proved be beneficial for protein expression.     

Piezoelectric poly(vinylidene fluoride) microstructure and poling state in active tissue engineering

Tissue engineering strategies rely on suitable membranes and scaffolds, providing the necessary physicochemical stimuli to specific cells. This review summarizes the main results on piezoelectric polymers, in particular poly(vinylidene fluoride), for muscle and bone cell culture. Further, the relevance of polymer microstructure and surface charge on cell response is demonstrated. Together with the necessary biochemical cues, the proper design of piezoelectric polymers can open the way to novel and more reliable tissue engineering strategies for cells in which electromechanical stimuli are present in their environment.     

福寿螺的生物防治现状、问题与对策

福寿螺(Pomacea canaliculata)作为一种世界性入侵生物,引入中国已有30余年,现已在中国南方十多个省市大面积分布,造成不可估量的经济损失和生态灾害。本文系统总结了福寿螺生物防治中的天敌资源种类,涵盖了腹足纲、环带纲、昆虫纲、软甲纲、辐鳍鱼纲、爬行纲、鸟纲、哺乳纲等8个类别。并从天敌的引入、研发、利用等方面以及福寿螺应对天敌的防御策略上指出了目前福寿螺生物防治中面临的主要问题。最后对其未来的研究提出了展望,即重点从原产地天敌、寄生性天敌、微生物、生物防治植物和本地经济动物等方面进行筛选,以期为利用中国丰富的生物资源进行福寿螺的生物防治提供参考。     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Surface plasmon field-enhanced fluorescence spectroscopy apparatus with a convergent optical system for point-of-care testing

Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) is a promising methodology for point-of-care (POC) testing. The SPFS devices that have been reported are equipped with an angle rotating stage to adjust the surface plasmon resonance (SPR) angle. In a clinical setting, however, the SPR angle determination is a tedious and time-consuming process. In this study, we employed an SPFS instrument with a convergent optical system that allows the omission of this procedure. We demonstrated that this instrumentation allowed the sensitive determination of low concentrations of α-fetoprotein in serum and reduced the variation effect caused by the protein concentrations in samples. The SPFS with a convergent optical system is suitable for POC testing.     

Contemporary carbon accumulation in a boreal oligotrophic minerogenic mire – a significant sink after accounting for all C-fluxes

Based on theories of mire development and responses to a changing climate, the current role of mires as a net carbon sink has been questioned. A rigorous evaluation of the current net C-exchange in mires requires measurements of all relevant fluxes. Estimates of annual total carbon budgets in mires are still very limited. Here, we present a full carbon budget over 2 years for a boreal minerogenic oligotrophic mire in northern Sweden (64°11′N, 19°33′E). Data on the following fluxes were collected: land–atmosphere CO₂ exchange (continuous Eddy covariance measurements) and CH₄ exchange (static chambers during the snow free period); TOC (total organic carbon) in precipitation; loss of TOC, dissolved inorganic carbon (DIC) and CH₄ through stream water runoff (continuous discharge measurements and regular C-concentration measurements). The mire constituted a net sink of 27±3.4 (±SD) g C m⁻² yr⁻¹ during 2004 and 20±3.4 g C m⁻² yr⁻¹ during 2005. This could be partitioned into an annual surface–atmosphere CO₂ net uptake of 55±1.9 g C m⁻² yr⁻¹ during 2004 and 48±1.6 g C m⁻² yr⁻¹ during 2005. The annual NEE was further separated into a net uptake season, with an uptake of 92 g C m⁻² yr⁻¹ during 2004 and 86 g C m⁻² yr⁻¹ during 2005, and a net loss season with a loss of 37 g C m⁻² yr⁻¹ during 2004 and 38 g C m⁻² yr⁻¹ during 2005. Of the annual net CO₂-C uptake, 37% and 31% was lost through runoff (with runoff TOC>DIC≫CH₄) and 16% and 29% through methane emission during 2004 and 2005, respectively. This mire is still a significant C-sink, with carbon accumulation rates comparable to the long-term Holocene C-accumulation, and higher than the C-accumulation during the late Holocene in the region.     

A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.     

Aptamer–Au NPs conjugates-enhanced SPR sensing for the ultrasensitive sandwich immunoassay

The goal of this work is to explore the amplification effect of aptamer–gold nanoparticles (Au NPs) conjugates for ultrasensitive detection of large biomolecules by surface plasmon resonance (SPR). A novel sandwich immunoassay is designed to demonstrate the amplification effect of aptamer–Au NPs conjugates by using human immunoglobulin E (IgE) as model analyte. Human IgE, captured by immobilized goat anti-human IgE on SPR gold film, is sensitively detected by SPR spectroscopy with a lowest detection limit of 1 ng/ml after anti-human IgE aptamer–Au NPs conjugates is used as amplification reagent. Meanwhile, the non-specific adsorption of aptamer–Au NPs conjugates on goat anti-human IgE is confirmed by SPR spectroscopy and then it is minimized by treating aptamer–Au NPs conjugates with 6-mercaptohexan-1-ol (MCH). These results confirm that aptamer–Au NPs conjugates is a powerful sandwich element and an excellent amplification reagent for SPR-based sandwich immunoassay.