Expression, purification, and refolding of recombinant collagen alpha1(XI) amino terminal domain splice variants

The amino terminal domain of collagen type XI alpha1 chain is a noncollagenous structure that is essential for the regulation of fibrillogenesis in developing cartilage. The amino terminal domain is alternatively spliced at the mRNA level, resulting in proteins expressed as splice variants. These splice variants, or isoforms, have unique distribution in growing tissues, alluding to distinct roles in development. We report here a rapid and straightforward method for expression, purification and in vitro folding of recombinant collagen XI isoforms alpha1(XI) NTD[p7] and alpha1(XI) NTD[p6b+7]. The recombinant isoforms were expressed in Escherichia coli as bacterial inclusion bodies. Unfolded carboxy terminal polyhistidine tagged proteins were purified via nickel affinity chromatography and refolded with specific protocols optimized for each isoform. Purity was assessed by SDS-PAGE and correct secondary structure by a comparison of circular dichroism data with that obtained for Npp. Protein expression and purification of the recombinant collagen XI splice variants will allow further studies to elucidate the structure and molecular interactions with components of the extracellular matrix. This research will clarify the mechanism of collagen XI mediated regulation of collagen fibrillogenesis.     

Significant increase in natural disturbance impacts on European forests since 1950

Over the last decades, the natural disturbance is increasingly putting pressure on European forests. Shifts in disturbance regimes may compromise forest functioning and the continuous provisioning of ecosystem services to society, including their climate change mitigation potential. Although forests are central to many European policies, we lack the long-term empirical data needed for thoroughly understanding disturbance dynamics, modeling them, and developing adaptive management strategies. Here, we present a unique database of >170,000 records of ground-based natural disturbance observations in European forests from 1950 to 2019. Reported data confirm a significant increase in forest disturbance in 34 European countries, causing on an average of 43.8 million m³ of disturbed timber volume per year over the 70-year study period. This value is likely a conservative estimate due to under-reporting, especially of small-scale disturbances. We used machine learning techniques for assessing the magnitude of unreported disturbances, which are estimated to be between 8.6 and 18.3 million m³/year. In the last 20 years, disturbances on average accounted for 16% of the mean annual harvest in Europe. Wind was the most important disturbance agent over the study period (46% of total damage), followed by fire (24%) and bark beetles (17%). Bark beetle disturbance doubled its share of the total damage in the last 20 years. Forest disturbances can profoundly impact ecosystem services (e.g., climate change mitigation), affect regional forest resource provisioning and consequently disrupt long-term management planning objectives and timber markets. We conclude that adaptation to changing disturbance regimes must be placed at the core of the European forest management and policy debate. Furthermore, a coherent and homogeneous monitoring system of natural disturbances is urgently needed in Europe, to better observe and respond to the ongoing changes in forest disturbance regimes.     

Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation

Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.     

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.     

First is best

We experience the world serially rather than simultaneously. A century of research on human and nonhuman animals has suggested that the first experience in a series of two or more is cognitively privileged. We report three experiments designed to test the effect of first position on implicit preference and choice using targets that range from individual humans and social groups to consumer goods. Experiment 1 demonstrated an implicit preference to buy goods from the first salesperson encountered and to join teams encountered first, even when the difference in encounter is mere seconds. In Experiment 2 the first of two consumer items presented in quick succession was more likely to be chosen. In Experiment 3 an alternative hypothesis that first position merely accentuates the valence of options was ruled out by demonstrating that first position enhances preference for the first even when it is evaluatively negative in meaning (a criminal). Together, these experiments demonstrate a "first is best" effect and we offer possible interpretations based on evolutionary mechanisms of this "bound" on rational behavior and suggest that automaticity of judgment may be a helpful principle in clarifying previous inconsistencies in the empirical record on the effects of order on preference and choice.     

A machine learning approach for the prediction of protein surface loop flexibility

Proteins often undergo conformational changes when binding to each other. A major fraction of backbone conformational changes involves motion on the protein surface, particularly in loops. Accounting for the motion of protein surface loops represents a challenge for protein-protein docking algorithms. A first step in addressing this challenge is to distinguish protein surface loops that are likely to undergo backbone conformational changes upon protein-protein binding (mobile loops) from those that are not (stationary loops). In this study, we developed a machine learning strategy based on support vector machines (SVMs). Our SVM uses three features of loop residues in the unbound protein structures-Ramachandran angles, crystallographic B-factors, and relative accessible surface area-to distinguish mobile loops from stationary ones. This method yields an average prediction accuracy of 75.3% compared with a random prediction accuracy of 50%, and an average of 0.79 area under the receiver operating characteristic (ROC) curve using cross-validation. Testing the method on an independent dataset, we obtained a prediction accuracy of 70.5%. Finally, we applied the method to 11 complexes that involve members from the Ras superfamily and achieved prediction accuracy of 92.8% for the Ras superfamily proteins and 74.4% for their binding partners.     

Streamlined process development procedure incorporating the selection of various stationary phase types established in a mAb aggregate reduction study with different mixed mode ligands

In biopharmaceutical process development time, cost and reliability are the relevant keywords. During the development of chromatographic processes these targets are challenged by many possible scaffolds, ligands and process parameters. The common response to this diversity is the establishment of platform processes in the development of chromatographic unit operations. However, while developing a platform library to simplify and accelerate chromatographic processes, the potential combination of scaffold, ligands and process parameters need to be characterized. This challenge is addressed in a case study on novel mixed mode (MM) adsorber for the removal of monoclonal antibody (mAb) aggregates. We propose a rigorous strategy to reduce the various experimental design space resulting from possible combinations in scaffolds, backbones and ligands. This strategy is based on theoretical considerations, identification of adsorber selectivity and capacity for the identification of a suitable membrane system. For this system, each potential MM membrane adsorber candidate is investigated in its high molecular weight species reduction potential for a given mAb feed stream and referenced to the performance of Capto™ Adhere. The introduced strategy can reduce the developmental effort in an early stage from three to two possible stationary phases. Thereafter, initial examinations at different ionic capacities enlighten one favorable stationary phase. Finalizing the development strategy procedure by studying five different MM ligands by HTS and confirming the study with a 2–3 MV higher dynamic breakthrough capacity in benchtop experiments and provides an insight in the benefits of a living process platform library.     

Vacuoles from Sugarcane Suspension Cultures : I. ISOLATION AND PARTIAL CHARACTERIZATION

Vacuoles were isolated from suspension cultures of sugarcane (Saccharum sp.) cells by centrifugation of protoplasts at high g force against a 12% (w/v) Ficoll solution. Distribution of marker enzymes and Concanavalin A binding showed an 11% contamination of the vacuole preparation by cytoplasmic components, mitochondria, and endoplasmic reticulum, and 18% contamination by plasma membrane. Acid phosphatase, carboxypeptidase, protease, peroxidase, and ribonuclease activities were enriched in isolated vacuoles. Carboxypeptidase was tonoplast-bound, whereas the other enzymes were soluble. Sucrose, reducing sugars, and free amino acids were measured in protoplasts and vacuoles during growth of cells in suspension culture. Sucrose and reducing sugar content of vacuoles increased as the culture aged, while free amino acids decreased sharply.     

Visualization of thrombin receptors on mouse embryo fibroblasts using fluorescein-amine conjugated human α-thrombin

The localization of thrombin receptors on mouse embryo (ME) cells has been examined by direct fluorescence microscopy using a fluorescein aminelabeled thrombin. Two fluorescein amines, 4-(N-6-aminoethyl thioureal)-fluorescein and 4-(N-6-aminohexyl thioureal)-fluorescein, were synthesized and attached to the carbohydrate moiety of highly purified human α-thrombin by periodate oxidation of the carbohydrate and selective reduction of the Schiff's base using sodium cyanoborohydride. Preparations of fluorescent thrombin with from 1 to 4 fluoresceins per molecule of thrombin retained their ability to proteolytically cleave fibrinogin to form fibrin clots, to bind to thrombin receptors on ME cells, and to initiate cell division. After incubating mitogenic concentrations of the fluorescein amine labeled thrombin with ME cells at 4°C, a diffuse fluorescent pattern was observed over the surface of the ME cells. This diffuse pattern was specific: it was not observed on cells from parallel cultures incubated with fluorescent thrombin plus a 20-fold excess of unlabeled thrombin. Thus, thrombin receptors appear to be distributed randomly over the surface of ME cells prior to interaction with thrombin. Increasing the temperature to 37°C following binding at 4° C resulted in a rapid dissociation of the fluorescent pattern from the cells leaving only the autofluorescent vesicles. This result may reflect the unique ability of thrombin to proteolytically cleave its own receptor.