Hot Air-Assisted Radio Frequency (HARF) Drying on Wild Bitter Gourd Extract.

Wild bitter gourd (Momordica charantia L. var. abbreviata S.) is a kind of Chinese herbal medicine and is also a vegetable and fruit that people eat daily. Wild bitter gourd has many bioactive components, such as saponin, polysaccharide, and protein, and the extract is used to adjust blood sugar in patients with diabetes. The objective of this study was to investigate simultaneous hot air-assisted radio frequency (HARF) drying and pasteurization for bitter gourd extract, and then to evaluate its effects on blood sugar of type II diabetic mice. The results showed that the solid-liquid ratio of the wild bitter gourd powder to water was 1:10 and it was extracted using focused ultrasonic extraction (FUE) for only 10 min with 70 °C water. Then, 1 kg of concentrated bitter gourd extract was mixed with soybean fiber powder at a ratio of 2:1.1. It was dried by HARF, and the temperature of the sample could reach above 80 °C in only 12 min to simultaneously reduce moisture content (wet basis) from 58% to 15% and achieve a pasteurization effect to significantly reduce the total bacterial and mold counts. Type II diabetic mice induced by nicotinamide and streptozocin (STZ) for two weeks and then were fed four-week feeds containing 5% RF-dried wild gourd extract did not raise fasting blood glucose. Therefore, the dried powder of wild bitter gourd extracts by HARF drying had a hypoglycemic effect.

Protein Engineering of a Pyridoxal-5'-Phosphate-Dependent l-Aspartate-α-Decarboxylase from Tribolium castaneum for ß-Alanine Production.

In the present study, a pyridoxal-5'-phosphate (PLP)-dependent L-aspartate-α-decarboxylase from Tribolium castaneum (TcPanD) was selected for protein engineering to efficiently produce ß-alanine. A mutant TcPanD-R98H/K305S with a 2.45-fold higher activity than the wide type was selected through error-prone PCR, site-saturation mutagenesis, and 96-well plate screening technologies. The characterization of purified enzyme TcPanD-R98H/K305S showed that the optimal cofactor PLP concentration, temperature, and pH were 0.04% (m/v), 50 °C, and 7.0, respectively. The 1mM of Na+, Ni2+, Co2+, K+, and Ca2+ stimulated the activity of TcPanD-R98H/K305S, while only 5 mM of Ni2+ and Na+ could increase its activity. The kinetic analysis indicated that TcPanD-R98H/K305S had a higher substrate affinity and enzymatic reaction rate than the wild enzyme. A total of 267 g/L substrate l-aspartic acid was consumed and 170.5 g/L of ß-alanine with a molar conversion of 95.5% was obtained under the optimal condition and 5-L reactor fermentation.

High-Throughput Biochemical Fingerprinting of Oleaginous Aurantiochytrium sp. Strains by Fourier Transform Infrared Spectroscopy (FT-IR) for Lipid and Carbohydrate Productions.

The traditional biochemical methods for analyzing cellular composition of oleaginous microorganisms are time-consuming, polluting, and expensive. In the present study, an FT-IR method was used to analyze the cellular composition of the marine oleaginous protist Aurantiochytrium sp. during various research processes, such as strains screening, medium optimization, and fermentation, and was evaluated as a green, low-cost, high throughput, and accurate method compared with the traditional methods. A total of 109 Aurantiochytrium sp. strains were screened for lipid and carbohydrate production and the best results were found for the strains No. 6 and No. 32. The yields and productivities could reach up to 47.2 g/L and 0.72 g/L/h for lipid, 21.6 g/L and 0.33 g/L/h for docosahexaenoic acid (DHA) in the strain No. 6, and 15.4 g/L and 0.18 g/L/h for carbohydrate in the strain No. 32, under the optimal conditions, respectively. These results confirmed potentials of the two Aurantiochytrium sp. strains for lipid, DHA, and carbohydrate productions at industrial scales. The FT-IR method in this study will facilitate research on the oleaginous Aurantiochytrium sp., and the obtained two strains for lipid and carbohydrate productions will provide the foundations for their applications in medical, food, and feed industries.

Transcriptomic Mechanism of the Phytohormone 6-Benzylaminopurine (6-BAP) Stimulating Lipid and DHA Synthesis in Aurantiochytrium sp.

The phytohormone 6-benzylaminopurine (6-BAP) significantly improves lipid synthesis of oleaginous microorganisms with the great potential applied in lipid production. In the current study, the lipid and DHA productions in oleaginous Aurantiochytrium sp. were found to be improved by 48.7% and 55.3%, respectively, induced by 6-BAP treatments. Then, using high-throughput RNA-seq technology, the overall de novo assembly of the cDNA sequence data generated 53871 unigenes, and 15902 of these were annotated in at least one database. The comparative transcriptomic profiles of cells with and without 6-BAP treatments revealed that a total of 717 were differently expressed genes (DE), with 472 upregulated and 245 downregulated. Further annotation and categorization indicated that some DE genes were involved in pathways crucial to lipid and DHA productions, such as fatty acid synthesis, central carbon metabolism, transcriptional factor, signal transduction, and mevalonate pathway. A regulation mode of 6-BAP, in turn, perception and transduction of 6-BAP signal, transcription factor, expression regulations of the downstream genes, and metabolic changes, respectively, was put forward for the first time in the present study. This research illuminates the transcriptomic mechanism of phytohormone stimulation of lipid and DHA production in an oleaginous microorganism and provides the potential targets modified using genetic engineering for improving lipid and DHA productivity.

[Near infrared spectroscopy synergy interval wavelength selection method using the LSSVM model].

The present paper proposes a wavelength selection algorithm based on nonlinear factors named Synergy interval least squares support vector machines (siLSSVM). siLSSVM combines the interval strategy of wavelength selection method with the idea of synergy interval and overcomes the disadvantages of the traditional wavelength selection methods, i. e. ignoring the nonlinear factors. Taking the near infrared spectrum data of apple sugar as performance verification object of this new algorithm, comparing new algorithm with siPLS, the model performance has been greatly improved. The root-mean-square error (RMSEP) in new algorithm has increased respectively by 37.43% and 47.88% under the model of PLS and LSSVM, with increases of 6.04% and 7.31% in the correlative coefficient (RP). The examples illustrate that siLSSVM can efficiently select the optimum wavelength interval for spectrum data with strong nonlinear factors. This algorithm greatly improves the prediction accuracy and robustness of the model, which provides a new prospect for near infrared spectral with nonlinear factors to select wavelength.

Coordination complexes as molecular glue for immobilization of antibodies on cyclic olefin copolymer surfaces.

A novel metal-based chelating method has been used to provide an order of magnitude increase in immunoassay performance on cyclic olefin copolymer (COC) plastics compared with passive binding. COCs are hydrophobic, and without surface modification they are often unsuitable for applications where protein adhesion is desired. When interacting with the bare plastic, the majority of the bound proteins will be denatured and become nonfunctional. Many of the surface modification techniques reported to date require costly equipment setup or the use of harsh reaction conditions. Here, we have successfully demonstrated the use of a simple and quick metal chelation method to increase the sensitivity, activity, and efficiency of protein binding to COC surfaces. A detailed analysis of the COC surfaces after activation with the metal complexes is presented, and the immunoassay performance was studied using three different antibody pairs.

The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach.

BACKGROUND: Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1-9 post-onset of illness but following seroconversion it can be difficult to detect in serum. AIMS: To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test. METHODOLOGY: The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples. KEY RESULTS: In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers. CONCLUSIONS: This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.

Detection of HSV type-1 and type-2 IgG using an in vitro PCA based homogeneous immunoassay.

Enzyme immunoassays (EIAs) are widely used in the clinical laboratory and research institutes for the detection of biologically relevant analytes. Almost all EIAs are heterogeneous in nature and require multiple steps of process. In contrast, homogeneous immunoassays (HA) offer a simplified one-step approach with a number of potential advantages over contemporary heterogeneous EIAs such as higher throughput and greater clinical utility. Utilizing TEM-1 beta-lactamase as a reporter enzyme, we have developed HAs based on in vitro protein fragment complementation (PCA) for the detection of antibodies and potentially be used for antigens or other biomarkers. In this proof-of-principle study we demonstrate the successful in vitro differentiation of anti-herpes simplex virus (HSV) type-1 and type-2 Immunoglobulin G (IgG) in human serum with high sensitivity and specificity.

Development of homogeneous immunoassays based on protein fragment complementation.

We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1kDa) to large (150kDa) analytes using TEM-1 beta-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.

The discriminative bilateral filter: an enhanced denoising filter for electron microscopy data.

Advances in three-dimensional (3D) electron microscopy (EM) and image processing are providing considerable improvements in the resolution of subcellular volumes, macromolecular assemblies and individual proteins. However, the recovery of high-frequency information from biological samples is hindered by specimen sensitivity to beam damage. Low dose electron cryo-microscopy conditions afford reduced beam damage but typically yield images with reduced contrast and low signal-to-noise ratios (SNRs). Here, we describe the properties of a new discriminative bilateral (DBL) filter that is based upon the bilateral filter implementation of Jiang et al. (Jiang, W., Baker, M.L., Wu, Q., Bajaj, C., Chiu, W., 2003. Applications of a bilateral denoising filter in biological electron microscopy. J. Struc. Biol. 128, 82-97.). In contrast to the latter, the DBL filter can distinguish between object edges and high-frequency noise pixels through the use of an additional photometric exclusion function. As a result, high frequency noise pixels are smoothed, yet object edge detail is preserved. In the present study, we show that the DBL filter effectively reduces noise in low SNR single particle data as well as cellular tomograms of stained plastic sections. The properties of the DBL filter are discussed in terms of its usefulness for single particle analysis and for pre-processing cellular tomograms ahead of image segmentation.

Determination of the disulfide bond arrangement of dengue virus NS1 protein.

The 12 half-cystines of NS1 proteins are absolutely conserved among flaviviruses, suggesting their importance to the structure and function of these proteins. In the present study, peptides from recombinant Dengue-2 virus NS1 were produced by tryptic digestion in 100% H(2)(16)O, peptic digestion in 50% H(2)(18)O, thermolytic digestion in 50% H(2)(18)O, or combinations of these digestion conditions. Peptides were separated by size exclusion and/or reverse phase high performance liquid chromatography and examined by matrix-assisted laser desorption ionization-time of flight mass spectrometry, matrix-assisted laser desorption ionization post-source decay, and matrix-assisted laser desorption ionization tandem mass spectrometry. Where digests were performed in 50% H(2)(18)O, isotope profiles of peptide ions aided in the identification and characterization of disulfide-linked peptides. It was possible to produce two-chain peptides containing C1/C2, C3/C4, C5/C6, and C7/C12 linkages as revealed by comparison of the peptide masses before and after reduction and by post-source decay analysis. However, the remaining four half-cystines (C8, C9, C10, and C11) were located in a three-chain peptide of which one chain contained adjacent half-cystines (C9 and C10). The linkages of C8/C10 and C9/C11 were determined by tandem mass spectrometry of an in-source decay fragment ion containing C9, C10, and C11. This disulfide bond arrangement provides the basis for further refinement of flavivirus NS1 protein structural models.

Bone marrow samples from patients with aplastic anemia are not infected with parvovirus B19 and Mycobacterium tuberculosis.

The majority of patients with aplastic anemia (AA) have an idiopathic form of the disease. The aim of this study was to detect the presence of parvovirus B19 DNA and Mycobacterium tuberculosis (MTB) DNA by nested polymerase chain reaction (N-PCR) assays in the bone marrow biopsy samples from 30 patients with idiopathic AA. Serologic assays for parvovirus B19 were based on indirect antibody capture enzyme-linked immunosorbent assay. Our results indicate that neither parvovirus B19 DNA nor MTB DNA could be demonstrated in any of the bone marrow samples by N-PCR. Moreover, IgM antibody against parvovirus B19 also was undetectable in the serum samples of 17 patients. Thus, our results suggest that parvovirus B19 and MTB are not associated with AA and, consequently, do not have a role in the pathogenesis of this disease.