Weighing risk factors associated with bee colony collapse disorder by classification and regression tree analysis

Colony collapse disorder (CCD), a syndrome whose defining trait is the rapid loss of adult worker honey bees, Apis mellifera L., is thought to be responsible for a minority of the large overwintering losses experienced by U.S. beekeepers since the winter 2006-2007. Using the same data set developed to perform a monofactorial analysis (PloS ONE 4: e6481, 2009), we conducted a classification and regression tree (CART) analysis in an attempt to better understand the relative importance and interrelations among different risk variables in explaining CCD. Fifty-five exploratory variables were used to construct two CART models: one model with and one model without a cost of misclassifying a CCD-diagnosed colony as a non-CCD colony. The resulting model tree that permitted for misclassification had a sensitivity and specificity of 85 and 74%, respectively. Although factors measuring colony stress (e.g., adult bee physiological measures, such as fluctuating asymmetry or mass of head) were important discriminating values, six of the 19 variables having the greatest discriminatory value were pesticide levels in different hive matrices. Notably, coumaphos levels in brood (a miticide commonly used by beekeepers) had the highest discriminatory value and were highest in control (healthy) colonies. Our CART analysis provides evidence that CCD is probably the result of several factors acting in concert, making afflicted colonies more susceptible to disease. This analysis highlights several areas that warrant further attention, including the effect of sublethal pesticide exposure on pathogen prevalence and the role of variability in bee tolerance to pesticides on colony survivorship.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

Surface plasmon resonance analysis of the mechanism of binding of apoA-I to high density lipoprotein particles

The partitioning of apolipoprotein A-I (apoA-I) molecules in plasma between HDL-bound and -unbound states is an integral part of HDL metabolism. We used the surface plasmon resonance (SPR) technique to monitor in real time the reversible binding of apoA-I to HDL. Biotinylated human HDL₂ and HDL₃ were immobilized on a streptavidin-coated SPR sensor chip, and apoA-I solutions at different concentrations were flowed across the surface. The wild-type (WT) human and mouse apoA-I/HDL interaction involves a two-step process; apoA-I initially binds to HDL with fast association and dissociation rates, followed by a step exhibiting slower kinetics. The isolated N-terminal helix bundle domains of human and mouse apoA-I also exhibit a two-step binding process, consistent with the second slower step involving opening of the helix bundle domain. The results of fluorescence experiments with pyrene-labeled apoA-I are consistent with the N-terminal helix bundle domain interacting with proteins resident on the HDL particle surface. Dissociation constants (K_d) measured for WT human apoA-I interactions with HDL₂ and HDL₃ are about 10 µM, indicating that the binding is low affinity. This K_d value does not apply to all of the apoA-I molecules on the HDL particle but only to a relatively small, labile pool.Understanding the structure and function of HDL is significant because of the beneficial cardioprotective properties of this lipoprotein (1). The anti-atherogenic effects of HDL arise, in part, from its participation in the reverse cholesterol transport pathway where the principal HDL protein, apolipoprotein A-I (apoA-I), plays a central role (2). As a result, the structure-function relationships of apoA-I have been studied extensively (for reviews, see Refs. 3–5). Perhaps the most important characteristic of the apoA-I molecule is its ability to bind lipids; this interaction is mediated by the amphipathic α-helices present in the protein molecule (6). ApoA-I binds well to phospholipid (PL)-water interfaces and, under appropriate conditions, can solubilize the PL to create discoidal HDL particles (7, 8). The binding of apoA-I to a PL surface involves a two-step mechanism. First, α-helices in the C-terminal domain of the protein interact with the surface, and, second, the N-terminal helix bundle domain opens to allow more helix-lipid interactions to occur (5, 9). Although the binding of apoA-I to model PL particles has been studied extensively, the binding of apoA-I to HDL particles has not been investigated much because of the difficulty of separating free and bound apoA-I in this system. This lack of information about apoA-I/HDL interactions is significant because the cycling of apoA-I molecules on and off HDL particles occurs during the metabolism of HDL particles (10, 11), in particular to release apoA-I molecules into the preβ-HDL pool (10, 12). This recycling is consistent with the well-established ability of apolipoproteins, such as apoA-I, to exchange spontaneously between different populations of lipoprotein particles (13–16) and PL vesicles (17, 18). As a rule, any remodeling event that depletes HDL particles of PL induces particle fusion and dissociation of that fraction of the apoA-I molecules that is in a labile pool (19). At this stage, quantitative understanding of the kinetics of apoA-I interactions with HDL particles is unavailable.Here, we exploit surface plasmon resonance (SPR) to monitor in real time the association and dissociation reactions in the apoA-I/HDL system. SPR has been used to derive quantitative information about the binding of both lipoproteins (20) and apoE (21–23) to proteoglycans. As far as the application of SPR to the HDL system is concerned, the binding of several plasma remodeling factors to HDL immobilized on a sensor chip has been investigated successfully (24–26). Also, the conformation of apoA-I in HDL was explored by comparing the binding of HDL particles to anti-apoA-I monoclonal antibodies immobilized on an SPR chip (27). We have extended these approaches to study the binding of apoA-I to HDL particles. The results show that apoA-I can bind reversibly and with low affinity to HDL particles by a two-step mechanism.     

Intramolecular Movements in EF-G, Trapped at Different Stages in Its GTP Hydrolytic Cycle, Probed by FRET

Elongation factor G (EF-G) is one of several GTP hydrolytic proteins (GTPases) that cycles repeatedly on and off the ribosome during protein synthesis in bacterial cells. In the functional cycle of EF-G, hydrolysis of guanosine 5′-triphosphate (GTP) is coupled to tRNA-mRNA translocation in ribosomes. GTP hydrolysis induces conformational rearrangements in two switch elements in the G domain of EF-G and other GTPases. These switch elements are thought to initiate the cascade of events that lead to translocation and EF-G cycling between ribosomes. To further define the coupling mechanism, we developed a new fluorescent approach that can detect intramolecular movements in EF-G. We attached a fluorescent probe to the switch I element (sw1) of Escherichia coli EF-G. We monitored the position of the sw1 probe, relative to another fluorescent probe anchored to the GTP substrate or product, by measuring the distance-dependent, Förster resonance energy transfer between the two probes. By analyzing EF-G trapped at five different functional states in its cycle, we could infer the cyclical movements of sw1 within EF-G. Our results provide evidence for conformational changes in sw1, which help to drive the unidirectional EF-G cycle during protein synthesis. More generally, our approach might also serve to define the conformational dynamics of other GTPases with their cellular receptors.     

Crystal Structures of Inhibitor Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailed study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.     

Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA,EsxB, and EspA

Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.The WXG100 (WXG; pfam06013) proteins are a class of effector molecules found in gram-positive bacteria (26). WXG proteins are characterized by their small size (∼ 100 amino acids [aa]) and the presence of a WXG motif, or its structural equivalent, near the midpoint of their primary sequence (26). Bioinformatic analyses have shown that one WXG gene is frequently positioned near, or directly adjacent to, a second, related, WXG gene (14). The gene pairs characterized thus far encode proteins that associate to form 1:1 complexes (20, 31). The WXG proteins were once thought to be restricted to the mycobacteria, but homologues have now been detected in species of Bacillus, Listeria, Streptomyces, and Corynebacterium, among others, and the Pfam server lists >89 distinct WXG-encoding species and strains (10).The identification of WXG proteins encoded by the pathogens Mycobacterium tuberculosis (15, 17, 19, 36), Mycobacterium marinum (13), and Staphylococcus aureus (5) has created significant interest in the proteins'' biological activity. Nevertheless, these proteins are not a priori virulence factors (39), since organisms expressing WXG proteins are not necessarily capable of causing disease. In addition to pathogenesis, the WXG proteins are associated with processes as disparate as zinc homeostasis (24) and conjugal gene transfer (9, 11). A model for the mechanism(s) of action of these proteins that includes an explanation for their apparent functional versatility is at present lacking. One reason for this ambiguity may be the near-absence of studies comparing virulence-associated and non-virulence-associated WXG proteins, which is a goal of this study.The M. tuberculosis secreted virulence factors EsxA (also called ESAT-6, or Rv3875) and EsxB (CFP-10; Rv3874) are the founding members of the WXG family, and M. tuberculosis derivatives defective in EsxA and EsxB are attenuated (17, 19, 36). The results of biochemical and structural studies indicate that EsxA and EsxB form a tightly associated heterodimer, EsxAB (25, 30, 31). The M. tuberculosis genome contains 23 WXG genes, named esxA to esxW, and the majority of these are expressed as tandem pairs (26). Of the pairs, five, including esxA and esxB, are contained within larger, highly conserved genetic loci, called esx-1 to esx-5 (Fig. ​(Fig.1).1). These loci have been the focus of much research, since mutants of esx-1 are attenuated, and esx-3 and esx-5 are necessary for in vitro growth of M. tuberculosis and M. marinum (1, 2, 32-34). The esx loci are proposed to encode secretory apparatuses dedicated to the secretion of their cognate WXG proteins (1).Open in a separate windowFIG. 1.Genetic map of the esx-1 loci of M. tuberculosis and M. smegmatis. The M. tuberculosis esx-1 genes discussed in the text are indicated by white arrows, as are their M. smegmatis homologues. The M. tuberculosis map also shows the Rv3884 and Rv3885 genes, which are part of the adjacent esx-2 locus. pRD1-2F9 is the cosmid that was used to create an esx-1-specific prey library. pRD1-2F9 includes the Rv3860 to Rv3885 genes, thus encompassing the entire esx-1 locus and part of esx-2. The four genes below the M. smegmatis map include defective insertion sequences (ISs) inserted into MSMEG_0075.Although the majority of genes required for the secretion of the EsxAB heterodimer are encoded from within esx-1, additional non-esx-1 genes are necessary for secretion. In particular, one M. tuberculosis locus, esp, encodes three proteins essential for EsxAB secretion (12, 23). The first gene of the operon encodes a protein, EspA, that is cosecreted with EsxAB via the ESX-1 apparatus (12). Although no direct physical evidence has been presented, the inference from the interdependent cosecretion of the three proteins is that they likely form a complex, which is secreted by the ESX-1 apparatus. In this paper we provide the first genetic evidence that these three proteins interact.The lack of a genetic assay for the study of ESX-1 activity in M. tuberculosis has hindered the identification of all of the protein components of the apparatus and all of the substrates that it secretes. However, the fast-growing, nonpathogenic organism Mycobacterium smegmatis has a conserved esx-1 locus that is essential for DNA transfer, and we have exploited this requirement for genetic studies (9). These analyses have shown that the M. smegmatis ESX-1 apparatus is functionally related to that of M. tuberculosis (11) and that M. smegmatis encodes non-esx-1 genes necessary for the secretion of the EsxAB heterodimer, including orthologues of EspA (9).Here we have examined whether the secondary and quaternary structures of M. tuberculosis EsxA and EsxB are prototypical for other, functionally distinct and evolutionarily distant members of the WXG family (Fig. ​(Fig.2A).2A). Comparisons were made to homologues encoded by M. smegmatis (esxA and esxB), Corynebacterium diphtheriae (esxA and esxB), and an additional non-virulence-related pair from M. tuberculosis (esxG and esxH, encoded from the esx-3 locus). Structural characterization of these proteins establishes that their secondary and quaternary structures are conserved, with each pair folding into a predominately α-helical structure and associating to form a heterodimer. We next devised and tested the utility of a novel strategy to identify proteins that interact specifically with these WXG heterodimers. This involved fusing EsxB and EsxA to create a biomimetic heterodimer for use in mycobacterial two-hybrid experiments. We reasoned that the use of this unique bait would allow the detection of proteins that interact with both components of the native heterodimer and that these proteins would normally go undetected in the conventional, single-protein two-hybrid screens. Indeed, using this approach, we identified novel protein partners of M. tuberculosis EsxBA (MtbEsxBA). We show for the first time that EspA proteins from M. tuberculosis and M. smegmatis interact with the EsxBA heterodimer (from both species) but not with EsxA or EsxB alone. We also provide evidence for promiscuity between the different M. tuberculosis ESX apparatuses by showing that EsxBA, encoded by esx-1, can interact with Esx proteins encoded by esx-2. Taken together, our studies suggest that the WXG proteins possess similar structures and properties, regardless of the host species and the apparent biological function.Open in a separate windowFIG. 2.Sequence alignment of WXG proteins characterized in this study and the strategy used to facilitate their expression. (A) Amino acid sequence alignment of four pairs of WXG proteins. Conserved sequences are in boldface, and the signature WXG motif is indicated with asterisks. Three residues in Rv3874 (EsxB) and a single residue in Rv3875 (EsxA) are underlined; they are the sites of amino acid substitutions discussed in the text that abrogate Rv3871 interactions. (B) (Bottom) Scheme for coexpression of tandemly arranged WXG genes. (Top) The ribbon cartoon (30) shows how the two monomers are freed from the expressed fusion protein by thrombin cleavage (scissors) at the peptide tether (balls and sticks).     

Highly efficient deletion of FUT8 in CHO cell lines using zinc‐finger nucleases yields cells that produce completely nonfucosylated antibodies

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily α1,6‐fucosylated, a modification that reduces antibody‐dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a α1,6‐fucosyltransferase. FUT8^?/? CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein‐production cell lines has prevented the widespread adoption of FUT8^?/? cells as hosts for antibody production. We have created zinc‐finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8^?/? CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN‐treated cells can be directly selected to give FUT8^?/? cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN‐derived FUT8^?/? cell lines were as transfectable as wild‐type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild‐type and ZFN‐generated FUT8^?/? cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN‐generated FUT8^?/? cell lines was fourfold higher than in the best‐producing clones of FUT8^?/? cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN‐mediated transfer of the Fut8? phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies. Biotechnol. Bioeng. 2010;106: 774–783. © 2010 Wiley Periodicals, Inc.     

Triple light chain antibodies: Factors that influence its formation in cell culture

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species—Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression—evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)—correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production—evaluated as the ratio of the cell‐specific production rate of GSH (q_GSH) to the cell‐specific production rate of THIOMAB (q_p)—corresponded to decreased 3LC levels. In time‐lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q_GSH/q_p ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q_GSH/q_p ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748–760. © 2009 Wiley Periodicals, Inc.     

Freeze-drying of ATP entrapped in cationic,low lipid liposomes

Concerning the instability of ATP liposomes formulated to easily diffuse through the liver (size ～100 nm), this work targets the key parameters that influence the freeze-drying of a preparation that combines cholesterol, DOTAP and phosphatidylcholine (either natural soybean or egg (SPC or EPC) or hydrogenated (HSPC)). After freeze-drying blank liposomes, size increased significantly when initial lipid concentration was lowered from 20 to 5 mM (p = 0.0018). With low lipid concentration preparation (5 mM), SPC limited size increase (SI) more efficiently compared to EPC or HSPC. With SPC and EPC, sucrose showed better size results compared to trehalose (Lyoprotectant/Lipid ratio (w/w) avoiding any SI: ～5 and ～10 (for SPC), ～10 and ～15 (for EPC), for sucrose and trehalose, respectively), but the opposite was evidenced with HSPC liposomes where a Trehalose/Lipid ratio of 25 barely prevented SI. In addition, slow versus quick cooling rate led to limiting SI for HSPC liposomes (p = 0.0035). With sucrose or trehalose at both Lyoprotectant/Lipid ratios ensuring size stabilisation (10:1 and 15:1, respectively), ATP leakage ranged between 38.8 ± 7.9% and 58.2 ± 1.4%. In conclusion, this study emphasizes that using strict size maintenance as the primary objective does not result in drug complete retention inside the liposome core.