Engineering highly stable variants of Corynactis californica green fluorescent proteins.

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

Split green fluorescent protein as a modular binding partner for protein crystallization.

A modular strategy for protein crystallization using split green fluorescent protein (GFP) as a crystallization partner is demonstrated. Insertion of a hairpin containing GFP ß-strands 10 and 11 into a surface loop of a target protein provides two chain crossings between the target and the reconstituted GFP compared with the single connection afforded by terminal GFP fusions. This strategy was tested by inserting this hairpin into a loop of another fluorescent protein, sfCherry. The crystal structure of the sfCherry-GFP(10-11) hairpin in complex with GFP(1-9) was determined at a resolution of 2.6 Å. Analysis of the complex shows that the reconstituted GFP is attached to the target protein (sfCherry) in a structurally ordered way. This work opens the way to rapidly creating crystallization variants by reconstituting a target protein bearing the GFP(10-11) hairpin with a variety of GFP(1-9) mutants engineered for favorable crystallization.

Experimental mapping of soluble protein domains using a hierarchical approach.

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

Conotoxin Prediction: New Features to Increase Prediction Accuracy.

Conotoxins are toxic, disulfide-bond-rich peptides from cone snail venom that target a wide range of receptors and ion channels with multiple pathophysiological effects. Conotoxins have extraordinary potential for medical therapeutics that include cancer, microbial infections, epilepsy, autoimmune diseases, neurological conditions, and cardiovascular disorders. Despite the potential for these compounds in novel therapeutic treatment development, the process of identifying and characterizing the toxicities of conotoxins is difficult, costly, and time-consuming. This challenge requires a series of diverse, complex, and labor-intensive biological, toxicological, and analytical techniques for effective characterization. While recent attempts, using machine learning based solely on primary amino acid sequences to predict biological toxins (e.g., conotoxins and animal venoms), have improved toxin identification, these methods are limited due to peptide conformational flexibility and the high frequency of cysteines present in toxin sequences. This results in an enumerable set of disulfide-bridged foldamers with different conformations of the same primary amino acid sequence that affect function and toxicity levels. Consequently, a given peptide may be toxic when its cysteine residues form a particular disulfide-bond pattern, while alternative bonding patterns (isoforms) or its reduced form (free cysteines with no disulfide bridges) may have little or no toxicological effects. Similarly, the same disulfide-bond pattern may be possible for other peptide sequences and result in different conformations that all exhibit varying toxicities to the same receptor or to different receptors. We present here new features, when combined with primary sequence features to train machine learning algorithms to predict conotoxins, that significantly increase prediction accuracy.

Healthy humans can be a source of antibodies countering COVID-19.

Here, we describe the isolation of 18 unique anti SARS-CoV-2 human single-chain antibodies from an antibody library derived from healthy donors. The selection used a combination of phage and yeast display technologies and included counter-selection strategies meant to direct the selection of the receptor-binding motif (RBM) of SARS-CoV-2 spike protein's receptor binding domain (RBD2). Selected antibodies were characterized in various formats including IgG, using flow cytometry, ELISA, high throughput SPR, and fluorescence microscopy. We report antibodies' RBD2 recognition specificity, binding affinity, and epitope diversity, as well as ability to block RBD2 binding to the human receptor angiotensin-converting enzyme 2 (ACE2) and to neutralize authentic SARS-CoV-2 virus infection in vitro. We present evidence supporting that: 1) most of our antibodies (16 out of 18) selectively recognize RBD2; 2) the best performing 8 antibodies target eight different epitopes of RBD2; 3) one of the pairs tested in sandwich assays detects RBD2 with sub-picomolar sensitivity; and 4) two antibody pairs inhibit SARS-CoV-2 infection at low nanomolar half neutralization titers. Based on these results, we conclude that our antibodies have high potential for therapeutic and diagnostic applications. Importantly, our results indicate that readily available non immune (naïve) antibody libraries obtained from healthy donors can be used to select high-quality monoclonal antibodies, bypassing the need for blood of infected patients, and offering a widely accessible and low-cost alternative to more sophisticated and expensive antibody selection approaches (e.g. single B cell analysis and natural evolution in humanized mice).

Engineering an efficient and bright split Corynactis californica green fluorescent protein.

Split green fluorescent protein (GFP) has been used in a panoply of cellular biology applications to study protein translocation, monitor protein solubility and aggregation, detect protein-protein interactions, enhance protein crystallization, and even map neuron contacts. Recent work shows the utility of split fluorescent proteins for large scale labeling of proteins in cells using CRISPR, but sets of efficient split fluorescent proteins that do not cross-react are needed for multiplexing experiments. We present a new monomeric split green fluorescent protein (ccGFP) engineered from a tetrameric GFP found in Corynactis californica, a bright red colonial anthozoan similar to sea anemones and scleractinian stony corals. Split ccGFP from C. californica complements up to threefold faster compared to the original Aequorea victoria split GFP and enable multiplexed labeling with existing A. victoria split YFP and CFP.

Rational design of antimicrobial peptides targeting Gram-negative bacteria.

Membrane-targeting host antimicrobial peptides (AMPs) can kill or inhibit the growth of Gram-negative bacteria. However, the evolution of resistance among microbes poses a substantial barrier to the long-term utility of the host AMPs. Combining experiment and molecular dynamics simulations, we show that terminal carboxyl capping enhances both membrane insertion and antibacterial activity of an AMP called P1. Furthermore, we show that a bacterial strain with evolved resistance to this peptide becomes susceptible to P1 variants with either backbone capping or lysine-to-arginine substitutions. Our results suggest that cocktails of closely related AMPs may be useful in overcoming evolved resistance.

Comparative analysis of flexible two-parameter models of plant disease epidemics.

ABSTRACT Eleven previously published models of plant disease epidemics, given as differential equations with a rate and a shape parameter, are compared using general model characteristics as well as their usefulness in fitting observed data. Six out of the eleven models can be solved analytically resulting in epidemic growth functions, while the others can be solved only numerically. When all 11 differential equations were fitted to two data sets, all models showed a similar goodness of fit, although the shape parameter in some models could not be estimated very precisely. With respect to useful characteristics (exponential population growth at the beginning, ability to generate monomolecular disease progression, and flexibility of the inflection point), the models of Fleming, Kosman-Levy, Birch, Richards and Waggoner, and Rich are recommended. Formulas were established to calculate the point of inflection as well as the weighted absolute and relative rate, respectively, depending on the shape and rate parameter. These formulas allow transformation of the parameter values of one model into those of another model in many cases. If the two models are required to have the same temporal position of the disease progress curve, then the initial disease level at the start of the epidemic or the time when the inflection point is reached have to be transformed.

Analysis of the fetal placental vascular tree by X-ray micro-computed tomography.

The current understanding of the placental vascular tree largely derives from time-consuming morphometric analyses performed by conventional histology, electron microscopy of corrosion casts and three-dimensional reconstructions based on physical tissue sections. In the present study, we demonstrate for the first time that micro-computed tomography (micro-CT) emerges as a new, non-destructive and fast tool for imaging and quantifying fetoplacental vasculature. Term placentae (n=5) were perfused with contrast agent consisting of barium-sulfate, gelatine and thymol shortly after Caesarean-section-delivery. Samples (1 cm(3)) from eight different regions of the placenta were subsequently scanned in a micro-CT. Using tomographic reconstruction algorithms, three dimensional images were obtained by micro-CT allowing total stereoscopic visualization and continuous quantitative analysis of the vascular structure of the investigated samples. These samples were compared regarding vascular surface (VS) and vascular density (vascular volume fraction, TCVF). Quantitative assessment showed an average vascular density of 16 per cent (SD+/-0.4) and a vascular surface of 475 mm(2)(SD+/-8) per total tissue volume (including intervillous space) of 125 mm(3). Micro-CT image-analysis showed no significant differences in the fetal vascularization among term placentae. Micro-CT imaging is feasible for imaging and analysis of the villous vascular tree, allows further morphologic studies and immunohistochemistry of the placental specimens and may emerge as an additional tool in the investigation of the physiology and pathophysiology of the placental vasculature.

Yield loss in chickpeas in relation to development of fusarium wilt epidemics.

ABSTRACT Development of 108 epidemics of Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris were studied on cvs. P-2245 and PV-61 in field microplots artificially infested with races 0 and 5 of F. oxysporum f. sp. ciceris in 1986 to 1989. Disease progression data were fitted to the Richards model using nonlinear regression. The shape parameter was influenced primarily by date of sowing and, to a lesser extent, by chick-pea cultivars and races of F. oxysporum f. sp. ciceris. Fusarium wilt reduced chickpea yield by decreasing both seed yield and seed weight. These effects were related to sowing date, chickpea cultivar, and virulence of the prevalent F. oxysporum f. sp. ciceris race. Regression models were developed to relate chickpea yield to Fusarium wilt disease intensity with the following independent variables: time to initial symptoms (t(is)), time to inflection point (t(ip)) of the disease intensity index (DII) progress curve, final DII (DII(final)), standardized area under DII progress curve (SAUDPC), and the Richards weighted mean absolute rate of disease progression (rho). Irrespective of the chickpea cultivar x pathogen race combination, the absolute and relative seed yields decreased primarily by delayed sowing. The relative seed yield increased with the delay in t(is) and t(ip) and decreased with increasing DII(final), SAUDPC, and rho. A response surface as developed in which seed yield loss decreased in a linear relationship with the delay in t(is) and increased exponentially with the increase of rho.

Effect of Sowing Date, Host Cultivar, and Race of Fusarium oxysporum f. sp. ciceris on Development of Fusarium Wilt of Chickpea.

ABSTRACT Microplots experiments were carried out at Córdoba, southern Spain, from 1986 to 1989 to determine the effects of sowing date in the management of Fusarium wilt of chickpea as influenced by virulence of the pathogen race and by cultivar susceptibility. A total of 108 epidemics of the disease were described, analyzed, and compared to assess the degree of disease control. The epidemics were characterized by five curve elements: final disease intensity index (DII), standardized area under DII progress curve, time to epidemic onset, time to inflection point (t(ip)), and the DII value at t(ip), the last two parameters being estimates from the Richards function adjusted by nonlinear regression analysis. The structure of Fusarium wilt epidemics was examined by conducting multivariate principal components and cluster analyses. From these analyses, three factors accounting for 98 to 99% of the total variance characterized the DII progress curves and provided plausible epidemiological interpretations. The first factor included the t(ip) and the time to disease onset and can be interpreted as a positional factor over time. This factor accounted for the largest proportion of the total variance and may, therefore, be considered as the main factor for analysis of Fusarium wilt epidemics. The second factor concerns the standardized area under DII progress curves and the final DII of the epidemics. The third factor identified the uniqueness of the estimated value for the point of inflection of the DII progress curve over time. Our results indicate that for each year of experiment epidemic development was related mainly to the date of sowing. Thus, for chickpea crops in southern Spain, advancing the sowing date from early spring to early winter can slow down the development of Fusarium wilt epidemics, delay the epidemic onset, and minimize the final amount of disease. However, the net effect of this disease management practice may also be influenced, though to a lesser extent, by the susceptibility of the chickpea cultivar and the virulence and inoculum density of the Fusarium oxysporum f. sp. ciceris race.

Angular leaf spot of phaseolus beans: relationships between disease, healthy leaf area, and yield.

ABSTRACT Five field experiments were conducted to investigate the relationship between the severity of visible disease (X), area under the disease progress curve (AUDPC), healthy leaf area index on any given day (HLAI), radiation intercepted by healthy leaf area on any given day (HRI), healthy leaf area duration (HAD), total healthy leaf area absorption (HAA), and yield of Phaseolus beans, cultivars Rosinha and Carioca, inoculated with Phaeoisariopsis griseola at several doses. In general, yield was not related to disease severity (X) or AUDPC. In contrast, the highest yields were always related to the highest values of HAD and HAA. The relationship between yield and HAD was linear in each of five trials (29.9 < R(2) < 70.2%, P < 0.001). The relationship between yield and HAA was linear in four of the trials (52.3 < R(2) < 70.3%, P < 0.001) and exponential in one of them (in which the plant canopy was the largest). Singlepoint models using HRI to estimate yield at various times during the crop season were developed. The slope of the yield-HRI relationship proved to be stable (26.8 +/-2.4 g MJ(-1)), regardless of cultivar, locale, planting date, and bean growth stage (from R5 to R8). The yield-HLAI relationship proved to be less consistent. HRI is proposed as a key explanatory variable for a transportable system of disease management; it may be useful in producing precise recommendations at the farm level.

Effects of Angular Leaf Spot and Rust on Yield Loss of Phaseolus vulgaris.

ABSTRACT Three field experiments were conducted in 1997, 1998, and 1999 to investigate the effects of angular leaf spot and rust, separately or combined, on host growth and yield of individual bean plants (Phaseolus vulgaris). In each experiment, three treatments were established by inoculating cv. Carioca with Phaeoisariopsis griseola, Uromyces appendiculatus, or with both pathogens. An additional control treatment was not inoculated, but was sprayed with a fungicide. In the 1997 and 1999 experiments, angular leaf spot reached higher disease levels than rust, whereas in 1998, rust was more severe than angular leaf spot. Host growth, expressed as healthy leaf area duration (HAD), and yield were the highest in 1997 and lowest in 1998. In each experiment, the treatments did not differ significantly to the area under leaf area progress curve, HAD, and healthy leaf area absorption (HAA). All inoculated treatments had significantly more severe disease and less yield than the control treatment. Based on the analysis of 60 plants in each experiment, yield was not related to the areas under disease progress curve for either or both diseases. In 1997 and 1999, yield was related to HAD (R(2) = 0.57 and 0.43) and HAA(R(2) = 0.60 and 0.55). Based on the combined analysis of all 36 plots, angular leaf spot reduced the leaf area because of defoliation, whereas rust did not affect the leaf area. Rust reduced yield more than four times that of angular leaf spot, although the decrease in photosynthesis to angular leaf spot was twice that of rust.

Long-term follow-up results of the treatment of high and intermediate anorectal malformations using a modified technique of posterior sagittal anorectoplasty.

The aim of this study was to evaluate the long-term follow-up result of a modified technique in the operation for high and intermediate anorectal malformations. The operation was performed by a posterior sagittal approach with three modifications: The external sphincter complex was not opened on the posterior side, the rectal pouch was not tapered, the dissection was performed outside the rectal pouch. From January 1984 to December 1992, 70 cases were operated on including 55 boys and 15 girls. There was one postoperative death during the first 48 hours due to malignant hyperthermia and one late death due to an unrelated disease. A follow-up result was obtained in 48 of 68 survivors (71 %) with follow-up ranging from 13 months to 10 years 11 months (mean: 72 months). According to the modified Wingspread criteria, 2 patients (4 %) had very good anorectal function, 26 children (54 %) had good anorectal function, 18 children (38 %) had fair anorectal function, and only two children (4 %) had poor anorectal function. All children had urinary continence and all boys had a normal erection. The long-term follow-up result was satisfactory. Two important factors affecting the anorectal function were the type of malformations and the thickness of the external sphincter complex.

The long-term follow-up result of Swenson's operation in the treatment of Hirschsprung's disease in Vietnamese children.

The aim of this prospective study was to evaluate the long-term follow-up result of Swenson's operation in the treatment of Hirschsprung's disease in Vietnamese children. The surgical intervention was performed in three stages. Swenson's operation was performed by the same surgical team. Hirschsprung's disease was confirmed by an operative rectal biopsy. Regular out-patient attendances were made for follow-up. From January 1987 to January 1990, 69 patients including 61 boys and eight girls underwent Swenson's operation at a mean age of two years and five months (range from seven months to 14 years). There were two postoperative deaths during first 48 hours due to sepsis and two other late deaths due to severe enterocolitis (four months and six months after closure of colostomy respectively). Complications such as anastomotic leakage, anastomotic stenosis, perianal abcess, pelvic abcess did not occur. A follow-up result was obtained in 54 of 65 survivors (83%) with follow-up ranging from two to five years (mean: three years and six months). 51 of 54 children (94%) had a normal evacuation. Constipation persisted in three children (6%). Fecal soiling occurred in four children (8%) and stress urinary incontinence in one child (2%). All boys (47 children) had a normal erection. 53 patients (98%) had a normal weight growth, whereas 48 patients (89%) had a normal height growth. In conclusion, the long-term follow-up result of Swenson's operation in the treatment of Hirschsprung's disease was satisfactory.

A new protein-protein interaction sensor based on tripartite split-GFP association.

Monitoring protein-protein interactions in living cells is key to unraveling their roles in numerous cellular processes and various diseases. Previously described split-GFP based sensors suffer from poor folding and/or self-assembly background fluorescence. Here, we have engineered a micro-tagging system to monitor protein-protein interactions in vivo and in vitro. The assay is based on tripartite association between two twenty amino-acids long GFP tags, GFP10 and GFP11, fused to interacting protein partners, and the complementary GFP1-9 detector. When proteins interact, GFP10 and GFP11 self-associate with GFP1-9 to reconstitute a functional GFP. Using coiled-coils and FRB/FKBP12 model systems we characterize the sensor in vitro and in Escherichia coli. We extend the studies to mammalian cells and examine the FK-506 inhibition of the rapamycin-induced association of FRB/FKBP12. The small size of these tags and their minimal effect on fusion protein behavior and solubility should enable new experiments for monitoring protein-protein association by fluorescence.

A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

1H, 15N and 13C backbone resonance assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis.

We report here the backbone assignment of Rv1567c, an integral membrane protein from Mycobacterium tuberculosis. The backbone resonance assignments were determined based on triple-resonance experiments with uniformly [13C,15N]-labeled protein in LMPG detergent micelles.

Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins.

The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1-4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.