20(R)-Ginsenoside Rf: A new ginsenoside from red ginseng extract

In spite of the general concept that herbal supplements are safe, there is a lack of appropriate quality control measures and regulations that often culminates in serious undesirable effects such as allergic reactions and renal and liver damage. Thus, there is a growing need to establish a suitable methodology that enables authentication and quality assurance of herbal products. The root of Panax ginseng C. A. Meyer (Araliaceae), commonly called ginseng, is traditionally recognized as a prominent herbal medicine in Far East Asia. There are two types of processed ginseng, white and red ginseng, based on processing methods, and these play a significant role in modifying ginsenosides, which are the major bioactive metabolites in these products. Herein we purify and characterize a new ginsenoside, 20(R)-ginsenoside Rf, utilizing NMR, UPLC-ESI-Q-TOF-MS and validate the metabolite is generated from its epimer, 20(S)-ginsenoside Rf during the steaming process to manufacture red ginseng. We further propose a relevant mechanism for the chemical conversion. This finding updates chemical profiling of ginseng products that can be employed in quality assurance and authentication.     

Use of Hangeul Twitter to Track and Predict Human Influenza Infection

Influenza epidemics arise through the accumulation of viral genetic changes. The emergence of new virus strains coincides with a higher level of influenza-like illness (ILI), which is seen as a peak of a normal season. Monitoring the spread of an epidemic influenza in populations is a difficult and important task. Twitter is a free social networking service whose messages can improve the accuracy of forecasting models by providing early warnings of influenza outbreaks. In this study, we have examined the use of information embedded in the Hangeul Twitter stream to detect rapidly evolving public awareness or concern with respect to influenza transmission and developed regression models that can track levels of actual disease activity and predict influenza epidemics in the real world. Our prediction model using a delay mode provides not only a real-time assessment of the current influenza epidemic activity but also a significant improvement in prediction performance at the initial phase of ILI peak when prediction is of most importance.     

Structure prediction of biological assemblies using GALAXY in CAPRI rounds 38-45

We participated in CARPI rounds 38-45 both as a server predictor and a human predictor. These CAPRI rounds provided excellent opportunities for testing prediction methods for three classes of protein interactions, that is, protein-protein, protein-peptide, and protein-oligosaccharide interactions. Both template-based methods (GalaxyTBM for monomer protein, GalaxyHomomer for homo-oligomer protein, GalaxyPepDock for protein-peptide complex) and ab initio docking methods (GalaxyTongDock and GalaxyPPDock for protein oligomer, GalaxyPepDock-ab-initio for protein-peptide complex, GalaxyDock2 and Galaxy7TM for protein-oligosaccharide complex) have been tested. Template-based methods depend heavily on the availability of proper templates and template-target similarity, and template-target difference is responsible for inaccuracy of template-based models. Inaccurate template-based models could be improved by our structure refinement and loop modeling methods based on physics-based energy optimization (GalaxyRefineComplex and GalaxyLoop) for several CAPRI targets. Current ab initio docking methods require accurate protein structures as input. Small conformational changes from input structure could be accounted for by our docking methods, producing one of the best models for several CAPRI targets. However, predicting large conformational changes involving protein backbone is still challenging, and full exploration of physics-based methods for such problems is still to come.     

Pepper osmotin-like protein 1 (CaOSM1) is an essential component for defense response, cell death, and oxidative burst in plants

Osmotin or osmotin-like protein, a PR-5 family member, is differentially induced in plants by abiotic and biotic stresses. Here, we demonstrate that the pepper (Capsicum annuum) osmotin-like protein 1 gene, CaOSM1, was required for the defense and hypersensitive cell death response and oxidative burst signaling during Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaOSM1 protein was localized to the plasma membrane in leaf cells of Nicotiana benthamiana. Agrobacterium-mediated transient expression of CaOSM1 in pepper distinctly induced the hypersensitive cell death response and H₂O₂ accumulation. Knock-down of CaOSM1 in pepper by virus-induced gene silencing increased the susceptibility to Xcv infection, which was accompanied by attenuation of the cell death response and decreased accumulation of H₂O₂. CaOSM1 overexpression in transgenic Arabidopsis conferred reduced susceptibility and accelerated cell death response and H₂O₂ accumulation to infection by Pseudomonas syringe pv. tomato and Hyaloperonospora arabidopsidis. Together, these results suggest that CaOSM1 is involved in cell death and oxidative burst responses during plant defense against microbial pathogens.     

Parameter Sensitivity Analysis of Stochastic Models Provides Insights into?Cardiac Calcium Sparks

We present a parameter sensitivity analysis method that is appropriate for stochastic models, and we demonstrate how this analysis generates experimentally testable predictions about the factors that influence local Ca²⁺ release in heart cells. The method involves randomly varying all parameters, running a single simulation with each set of parameters, running simulations with hundreds of model variants, then statistically relating the parameters to the simulation results using regression methods. We tested this method on a stochastic model, containing 18 parameters, of the cardiac Ca²⁺ spark. Results show that multivariable linear regression can successfully relate parameters to continuous model outputs such as Ca²⁺ spark amplitude and duration, and multivariable logistic regression can provide insight into how parameters affect Ca²⁺ spark triggering (a probabilistic process that is all-or-none in a single simulation). Benchmark studies demonstrate that this method is less computationally intensive than standard methods by a factor of 16. Importantly, predictions were tested experimentally by measuring Ca²⁺ sparks in mice with knockout of the sarcoplasmic reticulum protein triadin. These mice exhibit multiple changes in Ca²⁺ release unit structures, and the regression model both accurately predicts changes in Ca²⁺ spark amplitude (30% decrease in model, 29% decrease in experiments) and provides an intuitive and quantitative understanding of how much each alteration contributes to the result. This approach is therefore an effective, efficient, and predictive method for analyzing stochastic mathematical models to gain biological insight.     

Integrating cell‐free biosyntheses of heme prosthetic group and apoenzyme for the synthesis of functional P450 monooxygenase

Harnessing the isolated protein synthesis machinery, cell‐free protein synthesis reproduces the cellular process of decoding genetic information in artificially controlled environments. More often than not, however, generation of functional proteins requires more than simple translation of genetic sequences. For instance, many of the industrially important enzymes require non‐protein prosthetic groups for biological activity. Herein, we report the complete cell‐free biogenesis of a heme prosthetic group and its integration with concurrent apoenzyme synthesis for the production of functional P450 monooxygenase. Step reactions required for the syntheses of apoenzyme and the prosthetic group have been designed so that these two separate pathways take place in the same reaction mixture, being insulated from each other. Combined pathways for the synthesis of functional P450 monooxygenase were then further integrated with in situ assay reactions to enable real‐time measurement of enzymatic activity during its synthesis. Biotechnol. Bioeng. 2013; 110: 1193–1200. © 2012 Wiley Periodicals, Inc.     

In-gel total protein quantification using a ninhydrin-based method

Precise in-gel quantification of total protein amount of bands or spots in gels is the basis of subsequent biochemical, molecular biological and immunological analyses. Though several methods have been designed to evaluate relative amounts of proteins, these methods are of limited reliability because (semi-) quantifications depend on the amount of protein migrating into the gel and different proteins may lead to different absorptions/intensities of stained bands or spots. In the present study, we described a method to quantify both, hydrophilic and hydrophobic proteins using in-gel digestion with proteinase K, subsequent extraction and acid hydrolysis followed by the use of the ninhydrin reaction. The protocol is accurate and compatible with mass spectrometric characterization of proteins. Reproducible in-gel protein quantification was performed from SDS-PAGE and IEF/SDS-PAGE gels using bovine serum albumin as a standard protein. Bacteriorhodopsin separated on SDS-PAGE gel was quantified in addition in order to show that the method is also suitable for quantification of hydrophobic protein. This protocol for reliable in-gel protein quantification, which not only provides “arbitrary units of optical density”, can also be completed in a minimum of 4 days or maximum 1 week depending on the type of electrophoresis with the disadvantage of being time consuming.     

Sequence polymorphisms in ribosomal RNA genes and variations in chromosomal loci of Oenothera odorata and O. laciniata

Numerous studies on Oenothera species have been investigated for the physiological and ecological characteristics. However, no such an information based on molecular cytogenetic has yet been introduced, in turn, is very essential for identifying sequence polymorphisms of rRNA genes with their loci on mitotic phases for further biological researches. In this study, sequence variations of rRNA genes in Oenothera odorata and O. laciniata were examined to identify informative factors as unique or repeat sequences in intra- and inter-specific variations. Intra-specific variation revealed that the sequences of the rRNA genes including the spacer regions were highly conserved revealing only a few variations. From the inter-specific variation, spacer regions of species were completely different as (1) non-homologous sequences in NTS and (2) different type repeat sequences in ITS 1, 2 and 5.8S rRNA, whereas the remaining coding regions were highly conserved. FISH was carried out on mitotic phases using the 5S rDNA of the analyzed sequences. From the interphase and metaphase chromosomes of the examined species, two loci of 5S rDNA in O. odorata and four loci in O. laciniata were confirmed on the telomeric region of the short arm. Due to the small size and unclear centromere of the chromosomes, karyotype could not be completed. However, we confirmed that the chromosomes are organized by meta- and acrocentric chromosomes and the chromosomes with identified loci were assumed to be paired by the location of loci at the telomeric region on the short arm with relative lengths.     

Frequency of hybridization between Ostrinia nubilalis E‐and Z‐pheromone races in regions of sympatry within the United States

Female European corn borer, Ostrinia nubilalis, produce and males respond to sex pheromone blends with either E‐ or Z‐Δ11‐tetradecenyl acetate as the major component. E‐ and Z‐race populations are sympatric in the Eastern United States, Southeastern Canada, and the Mediterranean region of Europe. The E‐ and Z‐pheromone races of O. nubilalis are models for incipient species formation, but hybridization frequencies within natural populations remain obscure due to lack of a high‐throughput phenotyping method. Lassance et al. previously identified a pheromone gland‐expressed fatty‐acyl reductase gene (pgfar) that controls the ratio of Δ11‐tetradecenyl acetate stereoisomers. We identified three single nucleotide polymorphism (SNP) markers within pgfar that are differentially fixed between E‐ and Z‐race females, and that are ≥98.2% correlated with female pheromone ratios measured by gas chromatography. Genotypic data from locations in the United States demonstrated that pgfar‐z alleles were fixed within historically allopatric Z‐pheromone race populations in the Midwest, and that hybrid frequency ranged from 0.00 to 0.42 within 11 sympatric sites where the two races co‐occur in the Eastern United States (mean hybridization frequency or heterozygosity (H_O) = 0.226 ± 0.279). Estimates of hybridization between the E‐ and Z‐races are important for understanding the dynamics involved in maintaining race integrity, and are consistent with previous estimates of low levels of genetic divergence between E‐ and Z‐races and the presence of weak prezygotic mating barriers.