An innovative molecular approach towards the cost-effective entomological authentication of honey.

Honey authentication and traceability are crucial not only for economic purposes but also for ensuring safety. However, the widespread adoption of cutting-edge technologies in practical applications has been hampered by complex, time-consuming sample pre-treatment processes, the need for skilled personnel, and substantial associated expenses. This study aimed to develop a simple and cost-effective molecular technique to verify the entomological source of honey. By utilizing newly designed primers, we successfully amplified the mitochondrial 16S ribosomal RNA gene of honey bees from honey, confirming the high quality of the extracted DNA. Employing RFLP analysis with AseI endonuclease, species-specific restriction patterns were generated for honey derived from six closely related honey bees of the Apis genus. Remarkably, this method was proven equally effective in identifying heat-treated and aged honey by presenting the same RFLP profiles as raw honey. As far as we know, this is the initial research of the simultaneous differentiation of honey from closely related honey bee species using the restriction endonuclease AseI and mitochondrial 16S rRNA gene fragments. As a result, it holds tremendous potential as a standardized guideline for regulatory agencies to ascertain the insect origins of honey and achieve comprehensive traceability.

GmPLP1 negatively regulates soybean resistance to high light stress by modulating photosynthetic capacity and reactive oxygen species accumulation in a blue light-dependent manner.

High light stress is an important factor limiting crop yield. Light receptors play an important role in the response to high light stress, but their mechanisms are still poorly understood. Here, we found that the abundance of GmPLP1, a positive blue light receptor protein, was significantly inhibited by high light stress and mainly responded to high blue light. GmPLP1 RNA-interference soybean lines exhibited higher light energy utilization ability and less light damage and reactive oxygen species (ROS) accumulation in leaves under high light stress, while the phenotype of GmPLP1:GmPLP1-Flag overexpression soybean showed the opposite characteristics. Then, we identified a protein-protein interaction between GmPLP1 and GmVTC2, and the intensity of this interaction was primarily affected by sensing the intensity of blue light. More importantly, overexpression of GmVTC2b improved soybean tolerance to high light stress by enhancing the ROS scavenging capability through increasing the biosynthesis of ascorbic acid. This regulation was significantly enhanced after interfering with a GmPLP1-interference fragment in GmVTC2b-ox soybean leaves, but was weakened when GmPLP1 was transiently overexpressed. These findings demonstrate that GmPLP1 regulates the photosynthetic capacity and ROS accumulation of soybean to adapt to changes in light intensity by sensing blue light. In summary, this study discovered a new mechanism through which GmPLP1 participates in high light stress in soybean, which has great significance for improving soybean yield and the adaptability of soybean to high light.

A Nuclear Factor Y-B Transcription Factor, GmNFYB17, Regulates Resistance to Drought Stress in Soybean.

Soybean is sensitive to drought stress, and increasing tolerance to drought stresses is an important target for improving the performance of soybean in the field. The genetic mechanisms underlying soybean's drought tolerance remain largely unknown. Via a genome-wide association study (GWAS) combined with linkage analysis, we identified 11 single-nucleotide polymorphisms (SNPs) and 22 quantitative trait locus (QTLs) that are significantly associated with soybean drought tolerance. One of these loci, namely qGI10-1, was co-located by GWAS and linkage mapping. The two intervals of qGI10-1 were differentiated between wild and cultivated soybean. A nuclear factor Y transcription factor, GmNFYB17, was located in one of the differentiated regions of qGI10-1 and thus selected as a candidate gene for further analyses. The analysis of 29 homologous genes of GmNFYB17 in soybean showed that most of the genes from this family were involved in drought stress. The over-expression of GmNFYB17 in soybean enhanced drought resistance and yield accumulation. The transgenic plants grew better than control under limited water conditions and showed a lower degree of leaf damage and MDA content but higher RWC, SOD activity and proline content compared with control. Moreover, the transgenic plants showed a fast-growing root system, especially regarding a higher root-top ratio and more branching roots and lateral roots. The better agronomic traits of yield were also found in GmNFYB17 transgenic plants. Thus, the GmNFYB17 gene was proven to positively regulate drought stress resistance and modulate root growth in soybean. These results provide important insights into the molecular mechanisms underlying drought tolerance in soybean.

GmLecRlk, a Lectin Receptor-like Protein Kinase, Contributes to Salt Stress Tolerance by Regulating Salt-Responsive Genes in Soybean.

Soybean [Glycine max (L.) Merr.] is an important oil crop that provides valuable resources for human consumption, animal feed, and biofuel. Through the transcriptome analysis in our previous study, GmLecRlk (Glyma.07G005700) was identified as a salt-responsive candidate gene in soybean. In this study, qRT-PCR analysis showed that the GmLecRlk gene expression level was significantly induced by salt stress and highly expressed in soybean roots. The pCAMBIA3300-GmLecRlk construct was generated and introduced into the soybean genome by Agrobacterium rhizogenes. Compared with the wild type (WT), GmLecRlk overexpressing (GmLecRlk-ox) soybean lines had significantly enhanced fresh weight, proline (Pro) content, and catalase (CAT) activity, and reduced malondialdehyde (MDA) and H2O2 content under salt stress. These results show that GmLecRlk gene enhanced ROS scavenging ability in response to salt stress in soybean. Meanwhile, we demonstrated that GmLecRlk gene also conferred soybean salt tolerance when it was overexpressed alone in soybean hairy root. Furthermore, the combination of RNA-seq and qRT-PCR analysis was used to determine that GmLecRlk improves the salt tolerance of soybean by upregulating GmERF3, GmbHLH30, and GmDREB2 and downregulating GmGH3.6, GmPUB8, and GmLAMP1. Our research reveals a new mechanism of salt resistance in soybean, which exposes a novel avenue for the cultivation of salt-resistant varieties.

Investigation of the Maturity Evaluation Indicator of Honey in Natural Ripening Process: The Case of Rape Honey.

Honey maturity, a critical factor for quality evaluation, is difficult to detect in the current industry research. The objective of this study was to explore the changes in the composition and find potential maturity indicators of rape honey at different maturity stages through evaluating physicochemical parameters (moisture, sugars, pH, electrical conductivity, total protein, total phenols, total flavonoids, proline, and enzyme activity), the antioxidant capacity, and volatile components. The relevant results are as follows: 1. As the maturity increased, the moisture, sucrose, and maltose content of rape honey gradually decreased, while the glucose, fructose, and total protein content gradually increased. The activities of diastase, invertase, and ß-glucosidase showed a significant increase with the elevation of ripening days, and the activity of glucose oxidase reached the highest before completely capping. 2. The antioxidant capacity of honey increased with the increase in honey maturity. There is a significant and strong correlation between the bioactive components of rape honey and antioxidant capacity (p < 0.01, |r| > 0.857). 3. Thirty-five volatile components have been identified. Nonanal, benzaldehyde monomer, and benzaldehyde dimer can be used as potential indicators for the identification of honey maturity stages. Principal component analysis (PCA) based on antioxidant parameters and volatile components can identify the maturity of honey.

Chemical Analyses and Antimicrobial Activity of Nine Kinds of Unifloral Chinese Honeys Compared to Manuka Honey (12+ and 20+).

Honey has good antimicrobial properties and can be used for medical treatment. The antimicrobial properties of unifloral honey varieties are different. In this study, we evaluated the antimicrobial and antioxidant activities of nine kinds of Chinese monofloral honeys. In addition, headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) technology was used to detect their volatile components. The relevant results are as follows: 1. The agar diffusion test showed that the diameter of inhibition zone against Staphylococcus aureus of Fennel honey (21.50 ± 0.41 mm), Agastache honey (20.74 ± 0.37 mm), and Pomegranate honey (18.16 ± 0.11 mm) was larger than that of Manuka 12+ honey (14.27 ± 0.10 mm) and Manuka 20+ honey (16.52 ± 0.12 mm). The antimicrobial activity of Chinese honey depends on hydrogen peroxide. 2. The total antioxidant capacity of Fennel honey, Agastache honey, and Pomegranate honey was higher than that of other Chinese honeys. There was a significant positive correlation between the total antioxidant capacity and the total phenol content of Chinese honey (r = 0.958). The correlation coefficient between the chroma value of Chinese honey and the total antioxidant and the diameter of inhibition zone was 0.940 and 0.746, respectively. The analyzed dark honeys had better antimicrobial and antioxidant activities. 3. There were significant differences in volatile components among Fennel honey, Agastache honey, Pomegranate honey, and Manuka honey. Hexanal-D and Heptanol were the characteristic components of Fennel honey and Pomegranate honey, respectively. Ethyl 2-methylbutyrate and 3-methylpentanoic acids were the unique compounds of Agastache honey. The flavor fingerprints of the honey samples from different plants can be successfully built using HS-GC-IMS and principal component analysis (PCA) based on their volatile compounds. Fennel honey, Agastache honey, and Pomegranate honey are Chinese honey varieties with excellent antimicrobial properties, and have the potential to be developed into medical grade honey.

Identification and functional prediction of soybean CircRNAs involved in low-temperature responses.

Circular RNAs (circRNAs) are a newly characterized type of noncoding RNA and play important roles in microRNA (miRNA) function and transcriptional control. To unravel the mechanism of soybean circRNAs in low-temperature (LT) stress response, genome-wide identification of soybean circRNAs was conducted under LT (4 °C) treatment via deep sequencing. In this study, the existence of backsplicing sites was validated and circRNAs exhibited specific expression patterns in response to LT. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that circRNAs could participate in LT-responsive processes. Our study revealed a new circRNA-miRNA-mRNA network, which is involved in LT responses. Furthermore, soybean circRNAs were predicted to have potential to encode polypeptides or protein. Taken together, our results indicate that soybean circRNAs might encode proteins and be involved in the regulation of LT responses, providing clues regarding the molecular LT-responsive mechanisms in soybean.

A New Propolis Type from Changbai Mountains in North-east China: Chemical Composition, Botanical Origin and Biological Activity.

Propolis is a bee product with a wide range of biological activities and its chemical compounds depend highly on the type of plant accessible to the bees. The Changbai Mountains are a major mountain range in Northeast China and are one of the major bee product-producing areas in China. In this study, we evaluated the total phenolic acids and flavonoid contents as well as the antioxidant activity of propolis sampled from the Changbai Mountains area (CBM). We identified the major compounds and qualified their contents by HPLC-ESI/MS and HPLC-UV, and found that the content of p-coumaric acid and an unknown peak (CBE) in CBM propolis was higher than in propolis from other parts of China. The unknown compound CBE was isolated, purified, and identified as benzyl p-coumarate by MS and NMR. Possible plant sources of CBM propolis are Populus davidiana dode and Populus simonii Carr, which widely distributed in the Changbai Mountains area. CBM propolis is a new propolis type, that could be an excellent raw material for health foods and pharmaceuticals.

Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera).

The eastern honeybee Apis cerana and the western honeybee Apis mellifera are the two most economically valuable honeybee species used in apiculture. In market, the price of Apis cerana honey (ACH) is usually several times higher than that of Apis mellifera honey (AMH) due to the production limit, resulting in wide adulteration and counterfeiting of ACH by AMH. In the present study, we compared honeybee secretions in these two kinds of honey, and found significant differences in protein profiles and hydrocarbon components. The SDS-PAGE pattern showed three species-specific bands with molecular weights between 15.0 and 29.4 KDa in ACH, and six species-specific bands in AMH with molecular weights between 13.8 and 33.1 KDa. The GC-MS-MS detection of the petroleum ether extracts of the two kinds of honey showed that 17-Pentatriacontene and Hentriacontane were the characteristic constituents of ACH and AMH, respectively. These two methods constitute a system to satisfy different needs for entomological authentication of honey samples.

Authentication of Apis cerana Honey and Apis mellifera Honey Based on Major Royal Jelly Protein 2 Gene.

In Asia, honey is mainly produced by Apis mellifera and Apis cerana. However, the price of A. cerana honey is usually much higher than A. mellifera honey. Seeing considerable profits, some dishonest companies and beekeepers mislabel A. mellifera honey as A. cerana honey or incorporate A. mellifera honey into A. cerana honey. In the present study, we developed methods to discriminate A. cerana honey from A. mellifera honey based on the MRJP2 (major royal jelly protein 2) gene. Two pairs of species-specific primers were designed. The amplification products of A. cerana and A. mellifera were 212 and 560 bp, respectively. As little as one percent incorporation of A. mellifera honey in the mixture can be detected by duplex PCR. Additionally, another method based on the melt curve analysis using the same primers was also developed, allowing a rapid discrimination of real-time PCR product of different species. Our study shows that the entomological authentication of honey samples can be identified by nuclear genes other than mitochondrial genes and this extends the possibility of gene selection in identification. The authentication system we proposed could be a useful tool for discriminating A. cerana honey from A. mellifera honey.

Overexpression of germin-like protein GmGLP10 enhances resistance to Sclerotinia sclerotiorum in transgenic tobacco.

Germin-like proteins (GLPs) are ubiquitous water-soluble glycoproteins that are located in the extracellular matrix. These proteins have been reported to play vital roles in diverse biological processes. In the present study, a GLP in soybean (Glycine max L. Merr.), GmGLP10, was characterized. Sequence analysis revealed that the GmGLP10 gene (GenBank Accession Number EU916258) encodes a 213-amino acid (aa) protein, which contains a N-terminal signal peptide at 1-22 aa and is highly homologous to the members of the GER2 subfamily. GmGLP10 was highly expressed in the leaves, but very faint in the roots. The expression of GmGLP10 was induced by methyl jasmonate (MeJA), ethylene (ET), salicylic acid (SA), oxalate acid (OA), and the infection of Sclerotinia sclerotiorum. Overexpression of GmGLP10 in transgenic tobacco significantly enhanced tolerance to OA and S. sclerotiorum infection. Moreover, higher levels of H2O2 and the upregulated expression of a set of plant defense-related genes and HR (hypersensitive response)-associated genes were detected in the transgenic plants. These results suggest that GmGLP10 functions as a positive regulator of resistance to S. sclerotiorum.

GmPLP1, a PAS/LOV protein, functions as a possible new type of blue light photoreceptor in soybean.

Light is one of the most important environmental factors for the growth and development of plants. To adapt to changes in day length, the photoreception and transmission of the light signals in plants mainly depend on the various light receptor proteins. The PAS/LOV protein (PLP) has a PAS domain in the N-terminal and LOV domain in the C-terminal and has been confirmed as a new type of blue light receptor in Arabidopsis thaliana. However, the role of its counterpart in soybean remains largely unclear. In this study, the expression pattern of the GmPLP1 under different light qualities was determined by real-time RT-PCR analysis using the cultivar 'DongNong 42', a photosensitive soybean cultivar, suggesting that GmPLP1 was affected by the circadian clock and was a dark-induced gene. Moreover, the mRNA abundance increased significantly under blue light. Further analysis revealed that overexpression of GmPLP1 displayed the inhibition of hypocotyl elongation under blue light, and the expression of CRY1, CRY2, CKL3, CKL4, BIT1, and HY5 were simultaneously increased in GmPLP1-transgenic Arabidopsis, suggesting that the shortened hypocotyl was associated with the up-regulation of these genes. Taken together, our results suggest that GmPLP1, which is a new possible type of blue light photoreceptor in soybean, plays an important role in the blue light signaling pathway.

In Vitro Anti-Inflammatory Effects of Three Fatty Acids from Royal Jelly.

Trans-10-hydroxy-2-decenoic acid (10-H2DA), 10-hydroxydecanoic acid (10-HDAA), and sebacic acid (SEA) are the three major fatty acids in royal jelly (RJ). Previous studies have revealed several pharmacological activities of 10-H2DA and 10-HDAA, although the anti-inflammatory effects and underlying mechanisms by which SEA acts are poorly understood. In the present study, we evaluated and compared the in vitro anti-inflammatory effects of these RJ fatty acids in lipopolysaccharide-stimulated RAW 264.7 macrophages. The results showed that 10-H2DA, 10-HDAA, and SEA had potent, dose-dependent inhibitory effects on the release of the major inflammatory-mediators, nitric oxide, and interleukin-10, and only SEA decreased TNF-α production. Several key inflammatory genes have also been modulated by these RJ fatty acids, with 10-H2DA showing distinct modulating effects as compared to the other two FAs. Furthermore, we found that these three FAs regulated several proteins involved in MAPK and NF-κB signaling pathways. Taken together, these findings provide additional references for using RJ against inflammatory diseases.

Sensitive voltammetric determination of baicalein at DNA Langmuir-Blodgett film modified glassy carbon electrode.

The present paper describes to modify a double stranded DNA-octadecylamine (ODA) Langmuir-Blodgett film on a glassy carbon electrode (GCE) surface to develop a voltammetric sensor for the detection of trace amounts of baicalein. The electrode was characterized by atomic force microscopy (AFM) and cyclic voltammetry (CV). Electrochemical behaviour of baicalein at the modified electrode had been investigated in pH 2.87 Britton-Robinson buffer solutions by CV and square wave voltammetry (SWV). Compared with bare GCE, the electrode presented an electrocatalytic redox for baicalein. Under the optimum conditions, the modified electrode showed a linear voltammetric response for the baicalein within a concentration range of 1.0 × 10(-8)-2.0 × 10(-6) mol L(-1), and a value of 6.0 × 10(-9) mol L(-1) was calculated for the detection limit. And the modified electrode exhibited an excellent immunity from epinephrine, dopamine, glucose and ascorbic acid interference. The method was also applied successfully to detect baicalein in the medicinal tablets and spiked human blood serum samples with satisfactory results.

MICA antigens stimulate T cell proliferation and cell-mediated cytotoxicity.

In previous experiments we have found that transplant recipients had specific antibodies against MICA. In the present study, we measured T cell proliferation, cytokine production, and cytotoxicity to investigate whether immunization with MICA can produce a specific cellular immune response. BALB/c mice were immunized with recombinant MICA (rMICA). Lymphoid cell suspensions obtained after immunization were used to measure T cell proliferation. We detected a robust proliferative response in MICA-stimulated cultures as determined by [3H]thymidine uptake. Using carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure proliferation, we found that in MICA-stimulated cultures, 21% of the CD3+ T cells were CD4+ and CFSE-low and 3% of the T cells were proliferating CD8+ T cells. Among CFSE-low CD4+ spleen cells, 25% secreted IL-4 and only 2% produced IFN-gamma, suggesting a predominant Th2-type response. Blocking of MHC class I or class II molecules with monoclonal antibodies resulted in prominent inhibition of CD8+ or CD4+ T cell proliferation, respectively. In addition, we found that blocking the NKG2D receptor did not cause inhibition of the T cell response. MICA-stimulated CD8+ T lymphocytes exerted cytotoxicity against a BALB/c monocyte cell line (RAW 267.4) primed with soluble rMICA. Our results suggested that MICA-activated T cells may have a role in a cellular component of transplant rejection.

Multiplex single nucleotide extension: a robust and high throughput method for HLA-A locus typing.

This report describes a typing method that can identify all known human leukocyte antigen A (HLA-A) alleles by determining nucleotides present at polymorphic sites using single nucleotide extension. Allele specific primers are bound to capture oligonucleotides which allows for a multiplex approach during single nucleotide extension (SNE) reactions. Eleven group-specific polymerase chain reaction amplifications were performed to obtain the templates to be analyzed with sets of primers designed to investigate the polymorphisms. Extension of biotin-labeled ddNTPs onto allele-specific primers was catalyzed by a DNA polymerase and each primer was hybridized to a specific capture oligonucleotide covalently bound to a bead. After staining with streptavidin-PE, incorporated fluorescence was determined with a flow cytometer. Fluorescence intensities were interpreted by computer and the nucleotide sequence was translated into HLA-A genotypes. Group-specific amplification reactions and primer sets for SNE were validated with 42 reference samples of known HLA-A alleles. In addition, 296 samples from three populations (N. A. Caucasian, African-American, Terena S. A. Indian) were analyzed and results compared to previous typing by SSOP. Reproducibility between repeated typings was 100% and ambiguities were quite rare. The method has been found to be accurate, relatively simple to perform and fast. It is our method of choice for high resolution clinical HLA-A typing.

Study of MICA alleles in 201 African Americans by multiplexed single nucleotide extension (MSNE) typing.

We have developed a method for major histocompatibility complex class I chain-related gene A (MICA) genotyping using multiplexed single nucleotide extension (MSNE) and flow cytometric analysis of an array of fluorescent microspheres. This technique employs a polymerase chain reaction-derived target DNA containing all the polymorphic sites of MICA, synthetic complementary primers, biotinylated dideoxynucleotide triphosphate, fluorescent reporter molecules (streptavidin-phycoerythrin), and thermophilic DNA polymerase. Genomic DNA was amplified by MICA locus-specific primers and the MSNE reactions were carried out in the presence of 30 MSNE primers used to assay polymorphisms in exons 2, 3, and 4 of the MICA genes. Thirty-two previously typed cell lines were used as reference material. The MICA gene frequencies among 201 African-American unrelated donors were determined. Of 51 previously known alleles, 18 were observed in African-Americans, compared to 16 that were found in North American Caucasians and 9 in South American Indians, suggesting a more diversified allelic distribution in African-Americans. MICA*00201 and MICA*00801 were the two most frequent alleles in African-Americans. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of human leukocyte antigen-B in the African-American population. The methodology described here offers a powerful new approach to DNA typing of the MICA alleles.

MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies.

The highly polymorphic major histocompatibility class I related chain A (MICA) gene encodes glycoproteins that have been shown to be expressed in epithelial cells, endothelial cells, keratinocytes, monocytes, and tumor cells. In previous experiments, we have studied MICA antigens using rabbit sera obtained by immunization with MICA peptides. We also found that several transplant recipients had specific antibodies against MICA in an ELISA assay with recombinant of MICA (r-MICA). In the present work we produced monoclonal antibodies by immunization of mice with recombinant MICA*008. Based on the different patterns of reactivity observed in ELISA, Western blot, and flow cytometry, mAbs 1.9C2, 2.4F5, 1.7AD, and 2.3D4 only reacted with denatured MICA and mAb 1.7A8 and 3.2H3 reacted also with native MICA as illustrated by flow cytometry with live cells. These monoclonal antibodies were postulated to bind to different sites of the MICA molecule. In order to investigate whether MICA expressed on the cell surface is able to mediate cell killing, antibody absorption, flow cytometry and complement-dependent cytotoxicity (CDC) were performed. We found that mouse monoclonal antibody 3.2H3 was able to kill 70% of HeLa cells. Absorption of a patient serum with pooled human platelets to remove antibodies against class I HLA resulted in a small shift of fluorescence and reduced killing from 100% to 70-75%. Absorption with the platelets and r-MICA produced a remarkable reduction in fluorescence staining and virtually reduced complement-dependent killing to the level of the negative controls. The results suggested that MICA alloantigens may be more immunogenic than could have been previously suspected.

MICA polymorphism in South American Indians.

We have studied the MICA alleles of 196 unrelated subjects from three South American Indian tribes (Toba, Wichi and Terena). They are members of isolated tribes located in the Gran Chaco area in northeastern Argentina and in Mato Grosso do Sul in South Central Brazil. Of 55 previously known alleles, nine were observed in South American Indians, compared with 16 that were found in North American Caucasians, suggesting a more restricted allelic distribution of MICA in these tribes. In South American Indians, MICA*00201 was the most frequent allele, with a gene frequency of 33% in Toba, 47% in Wichi and 44% in Terena. MICA*00201, MICA*027 (external domain sequence like MICA*008/TM allele A5) and MICA*010 accounted for more than 90% of all the MICA genes in South American Indians. In North American Caucasians, MICA*00801 (*008/A5.1) accounted for 42% of the genes and was the most common allele. We observed a high degree of linkage disequilibrium between certain alleles of MICA and of HLA-B in the South American Indian populations. Phylogenetic trees constructed using gene frequencies of the transmembrane short tandem repeats in the populations reported here, and in other populations taken from published reports, suggest that South American Indians are more closely related to Asians than to Europeans.