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Gene editing using clustered regularly interspaced short palindromic repeat/CRISPR-associated proteins (CRISPR/Cas) has revolutionized biotechnology and provides genetic tools for medicine and life sciences. However, the application of this technology to miRNAs, with the function as negative gene regulators, has not been extensively reviewed in plants. Here, we summarize the evolution, biogenesis, and structure of miRNAs, as well as their interactions with mRNAs and computational models for predicting target genes. In addition, we review current advances in CRISPR/Cas for functional analysis and for modulating miRNA genes in plants. Extending our knowledge of miRNAs and their manipulation with CRISPR will provide fundamental understanding of the functions of plant miRNAs and facilitate more sustainable and publicly acceptable genetic engineering of crops.

The shape of an inflorescence varies among cereals, ranging from a highly branched panicle in rice to a much more compact spike in barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). However, little is known about the molecular basis of cereal inflorescence architecture. We profiled transcriptomes at three developmental stages of the barley main shoot apex — spikelet initiation, floral organ differentiation, and floral organ growth — and compared them with those from vegetative seedling tissue. Transcript analyses identified 3688 genes differentially transcribed between the three meristem stages, with a further 1394 genes preferentially expressed in reproductive compared with vegetative tissue. Co-expression assembly and Gene Ontology analysis classified these 4888 genes into 28 clusters, revealing distinct patterns for genes such as transcription factors, histone modification, and cell-cycle progression specific for each stage of inflorescence development. We also compared expression patterns of VRS (SIX-ROWED SPIKE) genes and auxin-, gibberellic acid- and cytokinin-associated genes between two-rowed and six-rowed barley to describe regulators of lateral spikelet fertility. Our findings reveal barley inflorescence phase-specific gene expression, identify new candidate genes that regulate barley meristem activities and flower development, and provide a new genetic resource for further dissection of the molecular mechanisms of spike development. Keywords: Inflorescence meristem, Transcriptome, Gene expression, Hormones, Barley

Understanding the molecular basis of pollen germination in cereals holds great potential to improve yield. Pollen, a highly specialized haploid male gametophyte, transports sperm cells through a pollen tube to the female ovule for fertilization, directly determining grain yield in cereal crops. Although insights into the regulation of pollen germination and gamete interaction have advanced rapidly in the model Arabidopsis thaliana (arabidopsis), the molecular mechanisms in monocot cereals remain largely unknown. Recently, pollen-specific genome-wide and mutant analyses in rice and maize have extended our understanding of monocot regulatory components. We highlight conserved and diverse mechanisms underlying pollen hydration, germination, and tube growth in cereals that provide ideas for translating this research from arabidopsis. Recent developments in gene-editing systems may facilitate further functional genetic research.

Herbal medicines (HMs) usually consist of complex matrices of phytochemical compounds responsible for their efficacy. Supercritical fluid chromatography (SFC) and ultra-high performance SFC (UHPSFC) are efficient techniques that can be advantageously used for the analysis of complex HMs. Here, the comprehensive UHPSFC methods were established for separation of similar flavonoids, chemical profiling and differentiation, and quality assessment of Epimedium species (EPs). An efficient separation of 12 polar flavonoids was enabled in UHPSFC with an important additive, oxalic acid. Then, 51 flavonoids were characterized, and 28 potential markers enabling the differentiation of E. wushanense from other four species were discovered by UHPSFC-QTOF/MS combined pattern recognition multivariate statistical analysis. Additionally, the UHPSFC-PDA was developed and validated for simultaneous quantification of 7 predominant flavonoids in five EPs, and remarkable variation in their contents was observed. This integrated UHPSFC approach has potential applications in systematic analysis and quality evaluation of HMs containing hydrophilic flavonoids.

We performed an international collaborative ring trial to validate gene-specific loop-mediated isothermal amplification (LAMP) assays targeting four exogenous transgenes commonly utilized in the development of genetically modified (GM) crops: bar, pat, cp4-epsps, and Cry1Ac, respectively. A total of 12 laboratories participated in the ring trial and returned their test results. Statistical analysis of the returned results showed high specificity and low limit of detection (LOD) of the four LAMP assays. The LOD for cp4-epsps, Cry1Ac, bar and pat genes assay was 5, 10, 20 and 20 haploid genome equivalents (HGE), respectively. Furthermore, good performance of the four assays was further demonstrated in the analysis of practical samples in which all participating laboratories returned expected and accurate test results. Altogether, the ring trial results confirmed that the four gene-specific LAMP assays can be utilized for quick screening of transgenic traits in routine laboratory analysis.

Goose parvovirus (GPV) usually affects goslings and Muscovy ducks but not Pekin ducks. Earlier works showed that a variant GPV can cause short beak and dwarfism syndrome (SBDS) in Pekin ducks. Here, we investigated the pathogenicity of a variant GPV of Pekin duck-origin (JS1) and a classical GPV of goose-origin (H) in Pekin ducklings. Following intramuscular infection at two days of age, both JS1 and H strains influenced weight gain and development of beaks and bones of wings and legs, and caused microscopic lesions of internal organs of ducks. However, the clinical signs typical of SBDS could only be replicated with the JS1 isolate. The findings suggest that both variant and classical GPVs are pathogenic for Pekin ducklings, while the former is more virulent than the latter. Using a quantitative real-time PCR assay, high levels of viral load were detected from bloods, internal organs, leg muscles, and ileac contents in JS1- and H-infected ducks from 6h to 35days postinfection (DPI). Using a GPV VP3-based ELISA, antibodies in sera of JS1- and H-infected ducks were detectable at 1 DPI and then persistently rose during the subsequent five weeks. These results suggest that both variant and classical GPVs can infect Pekin ducklings. The present work contributes to the understanding of pathogenicity of GPV to Pekin ducks and may provide clues to pathogenesis of GPV-related SBDS.

Allergic diseases are characterized by elevated allergen-specific IgE and excessive inflammatory cell responses. Among the reported plant allergens, grass pollen and grain allergens, derived from agriculturally important members of the Poaceae family such as rice, wheat and barley, are the most dominant and difficult to prevent. Although many allergen homologs have been predicted from species such as wheat and timothy grass, fundamental aspects such as the evolution and function of plant pollen allergens remain largely unclear. With the development of genetic engineering and genomics, more primary sequences, functions and structures of plant allergens have been uncovered, and molecular component-based allergen-specific immunotherapies are being developed. In this review, we aim to provide an update on (i) the distribution and importance of pollen and grain allergens of the Poaceae family, (ii) the origin and evolution, and functional aspects of plant pollen allergens, (iii) developments of allergen-specific immunotherapy for pollen allergy using biotechnology and (iv) development of less allergenic plants using gene engineering techniques. We also discuss future trends in revealing fundamental aspects of grass pollen allergens and possible biotechnological approaches to reduce the amount of pollen allergens in grasses.

Molecular characterization lays a foundation for safety assessment and subsequent monitoring of genetically modified (GM) crops. Due to the target-specific nature, conventional polymerase chain reaction (PCR)-based methods cannot comprehensively detect unintended gene insertions, let alone unknown GM events. As more and more new developed GM crops including new plant breeding technology (NPBT) generated crops are in the pipeline for commercialization, alternative -omics approaches, particularly next generation sequencing, have been developed for molecular characterization of authorized or unauthorized GM (UGM) crops. This review summarizes first those methods, addresses their challenges, and discusses possible strategies for molecular characterization of engineered crops generated by NPBT, highlighting needs for a global information-sharing database and cost-effective, accurate and comprehensive molecular characterization approaches.

Capturing heterosis (hybrid vigor) is a promising way to increase productivity in many crops; hybrid crops often have superior yields, disease resistance, and stress tolerance compared with their parental inbred lines. The full utilization of heterosis faces a number of technical problems related to the specifics of crop reproductive biology, such as difficulties with generating and maintaining male-sterile lines and the low efficiency of natural cross-pollination for some genetic combinations. Innovative technologies, such as development of artificial in vitro systems for hybrid production and apomixis-based systems for maintenance of the resulting heterotic progeny, may substantially facilitate the production of hybrids. Genome editing using specifically targeted nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (CRISPR/Cas9) systems, which recognize targets by RNA:DNA complementarity, has recently become an integral part of research and development in life science. In this review, we summarize the progress of genome editing technologies for facilitating the generation of mutant male sterile lines, applications of haploids for hybrid production, and the use of apomixis for the clonal propagation of elite hybrid lines.

Full-length cDNA of Tembusu virus (TMUV) cloned in a plasmid has been found instable in bacterial hosts. Using a PCR-based protocol, we generated a stable full-length cDNA of TMUV. Different cDNA fragments of TMUV were amplified by reverse transcription (RT)-PCR, and cloned into plasmids. Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates. Subsequently, a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells; infectious viral particles were rescued successfully. Following several passages in BKH-21 cells, the rescued virus was compared with the parental virus by genetic marker checks, growth curve determinations and animal experiments. These assays clearly demonstrated the genetic and biological stabilities of the rescued virus. The present work will be useful for future investigations on the molecular mechanisms involved in replication and pathogenesis of TMUV.

Crop breeding is the focus of global agricultural hi-tech companies and requires phenotypes screening after growing and cultivating. However, rice phenotypic traits are not easily obtained with high-throughput in the field due to the complexity of unstructured environment. This paper presents a design solution of the field robot to construct visual space for measurement of rice traits. The truss system equipped with manipulator modules and vision system locates the object by the position information recorded in the phase of growing seedlings. 3 manipulators including the rice-separating manipulator, the height measuring manipulator and the panicle-expanding manipulator were designed by imitating people’s actions such as pushing adjacent rice, expanding the panicle and rubbing rice long in order to reduce the overlap. Combined with them, 3 imaging sensors including the CCD camera, the structured light sensor and the laser sensor were introduced to measure a phenotype quantitatively. The simulations were designed to obtain workspaces of manipulators. The results showed that the volume of reachable space of clapboards of the rice-separating manipulator was 1.6 × 10-3 cubic meters, the effective movement of the height measuring manipulator was 1300mm and the maximum work area of the panicle-expanding manipulator was 32500 square millimeters. The results are consistent with those of manual operations. In conclusion, this paper describes an effective and reliable approach to acquiring phenotypic traits with high-throughput in the field.

An alternative method for efficient synthesis of urea-functionalized silanes was proposed on the basis of an N, N’-carbonyldiimidazole-mediated acyl-transfer reaction between various amino-containing building blocks. The employment of different parent aminosilanes and alkylamines afforded an array of urea-containing silanes, which were subsequently immobilized onto silica gel to form corresponding urea-embedded alkyl stationary phases for high-performance liquid chromatography. The different substituents on the silicon core of the derivatized silane were found to significantly influence the final chromatographic behaviors. The comparative chromatographic characterization of thus-prepared silica packings with conventional octadecyl (C18) stationary phases revealed that the urea group was beneficial to suppress silanol activity towards basic probes, as well as to increase the water-compatibility of the alkyl stationary phases. The combination of a polar urea moiety and a non-polar long alkyl chain was favorable for an enhanced steric selectivity towards shape-constrained isomers. The polarizability-sensitive feature of such stationary phases made them good candidates for efficient separation of nitro-containing polar substances.

Earlier works identified the second generation (Z8R2) of a resistant Pekin duck line to duck hepatitis A virus genotype 3 (DHAV-3), which displays significantly strong resistance than that of the second generation (Z8S2) of a susceptible Pekin duck line. To understand the genetic mechanisms that determine the different resistance/susceptibility of Z8R2 and Z8S2 to DHAV-3, transcriptome analysis on livers of infected Pekin ducklings was performed to screen differentially expressed genes (DEGs). We found that DHAV-3 infection has a great effect on metabolism of Z8S2 at the transcription level. Using a newly created fourth generation of the resistant Pekin duck line (Z8R4) and an unselected Pekin duck flock (Z7) as models, hypoglycemia and dramatically increased aspartate aminotransferase and alanine aminotransferase were shown to be noticeable signs of fatal cases caused by DHAV-3 infection. These findings, together with expression analysis and verification of DEGs, support the view that DHAV-3 infection results in glucose metabolic abnormalities in susceptible individuals and that there are significant differences in expression patterns of glucose metabolism-related DEGs between susceptible and resistant individuals. Notably, cytokines displayed a negative correlation with glucose synthesis in terms of expression in susceptible individuals following DHAV-3 infection. Mechanism analyses suggests that cytokines will activate PI3K-AKT pathway and/or JAK-STAT pathway by up-regulated expression of JAK2, and thereby causes down-regulated expression of G6PC and/or ACAT1. Cytokines can also cause down-regulated expression of HPGDS by JAK2. The present work contributes to the understanding of pathogenesis of DHAV-3 infection and the resistance breeding project against DHAV-3.

Gene-editing techniques, such as TALEN and CRISPR/Cas9, have been widely used for target DNA editing in many research fields. However, how to rapidly screen and identify the expected gene-edited products with high efficiency and low cost is still a difficult task. Here, we report the development and optimization of one such method that combines quantitative PCR with high-resolution melting analysis (qPCR-HRM) to screen and identify CRISPR/Cas9-edited rice plants. The results showed that gene-edited rice plants with small target DNA in/dels or even single base pair insertion/deletions could be successfully identified. The sensitivity of the qPCR-HRM method is as low as 1%, which is satisfying to be used for quantitative evaluation of gene-editing efficiency. Sanger sequencing results confirmed that the qPCR-HRM method also has high accuracy. It is concluded that the developed qPCR-HRM method is reproducible, accurate, and efficient for quick screening and identification of gene-edited rice plants.

A novel standard reference plasmid (pGEM-PAPAYA3) was constructed as a positive control and reference standard for event-specific qualitative and quantitative detection of genetically modified (GM) papaya (55-1, 16-0-1, and Huanong No.1). The plasmid pGEM-PAPAYA3 contained the specific fragments of papaya endogenous reference gene chymopapain and the 3 GM papaya events. The qualitative PCR assay using pGEM-PAPAYA3 was established with a limit of detection correlating to approximately 5–50 copies of papaya haploid genomes. In the quantitative PCR assay, the square regression coefficients (R2) ranged from 0.993 to 0.997. The standard deviation and relative standard deviation values for repeatability ranged from 0.04 to 0.25 and 0.05%–0.86%, respectively. The method was used to test 11 papaya products purchased from Thailand, China, the Philippines and the USA, and the results revealed 2 varieties of GM papaya in 5 of the 11 samples tested. These results indicate the developed detection methods with the standard reference plasmid are applicable for identifying 3 GM papaya events.

During reproductive development, rice plants develop unique flower organs which determine the final grain yield. OsMADS1, one of SEPALLATA-like MADS-box genes, has been unraveled to play critical roles in rice floral organ identity specification and floral meristem determinacy. However, the molecular mechanisms underlying interactions of OsMADS1 with other floral homeotic genes in regulating flower development remains largely elusive. In this work, we studied the genetic interactions of OsMADS1 with B-, C-, and D-class genes along with physical interactions among their proteins. We show that the physical and genetic interactions between OsMADS1 and OsMADS3 are essential for floral meristem activity maintenance and organ identity specification; while OsMADS1 physically and genetically interacts with OsMADS58 in regulating floral meristem determinacy and suppressing spikelet meristem reversion. We provided important genetic evidence to support the neofunctionalization of two rice C-class genes (OsMADS3 and OsMADS58) during flower development. Gene expression profiling and quantitative RT-PCR analyses further revealed that OsMADS1 affects the expression of many genes involved in floral identity and hormone signaling, and chromatin immunoprecipitation (ChIP)–PCR assay further demonstrated that OsMADS17 is a direct target gene of OsMADS1. Taken together, these results reveal that OsMADS1 has diversified regulatory functions in specifying rice floral organ and meristem identity, probably through its genetic and physical interactions with different floral homeotic regulators.

Ethnopharmacological relevance Paeonia suffruticosa Andrews (PSE) is a well-known Chinese medicine that has been widely used as an anti-tumor, anti-oxidative and anti-inflammatory agent. cis - and trans -gnetin H are two resveratrol oligomers isolated from the seeds of PSE. Although resveratrol is widely considered to be one of the most valuable natural chemopreventive agents and there are numerous studies on the antitumor activities of resveratrol, little is known about the antitumor properties of cis - and trans -gnetin H. Materials and methods The inhibitory effects of cis - and trans -gnetin H in different human cancer cell lines were assessed using fluorescent viability tests. Cytotoxicity in human lung and breast cancer cells was detected via nuclear condensation, cell permeability, and changes in the mitochondrial membrane potential (∆ψm). Apoptosis in human lung and breast cancer cells was assessed by flow cytometry, a luminescence assay and high-content screening analysis. Finally, a xenograft mice model was used to examine the efficacy of cis -gnetin H on lung tumors. Results cis - and trans -gnetin H have superior activity in inhibiting the proliferation of four human cancer cell lines, A549 (lung), BT20 (breast), MCF-7 (breast) and U2OS (osteosarcoma), and promote cell apoptosis, while having a minimal effect on two normal human epithelial cell lines, HPL1A (lung) and HMEC (breast) used as controls. cis - and trans -gnetin H promote apoptosis by releasing mitochondria cytochrome c , activating caspase 3/7 and inhibiting NF-κB activation. Flow cytometry analysis shows that cis - or trans -gnetin H arrested the cell cycle of cancer cells at the G0–G1 phase. Moreover, cis -gnetin H suppressed the growth of xenograft lung tumors in mice. Conclusion Collectively, our findings demonstrate the promise of the natural compounds cis - and trans -gnetin H as candidates for cancer chemotherapy agents.

The birth of new genes in genomes is an important evolutionary event. Several studies reveal that new genes in animals tend to be preferentially expressed in male reproductive tissues such as testis (Betrán et al., 2002; Begun et al., 2007; Dubruille et al., 2012), and thus an "out of testis" hypothesis for the emergence of new genes has been proposed (Vinckenbosch et al., 2006; Kaessmann, 2010). However, such phenomena have not been examined in plant species. Here, by employing a phylostratigraphic method, we dated the origin of protein-coding genes in rice and Arabidopsis thaliana and observed a number of young genes in both species. These young genes tend to encode short extracellular proteins, which may be involved in rapid evolving processes, such as reproductive barriers, species specification, and anti-microbial processes. Further analysis of transcriptome age indexes across different tissues revealed that male reproductive cells express a phylogenetically younger transcriptome than other plant tissues. Compared with sporophytic tissues, the young transcriptomes of the male gametophyte displayed greater complexity and diversity, which included a higher ratio of anti-sense and inter-genic transcripts, reflecting a pervasive transcription state that facilitated the emergence of new genes. Here, we propose that pollen may act as an "innovation incubator" for the birth of de novo genes. With cases of male-biased expression of young genes reported in animals, the "new genes out of the male" model revealed a common evolutionary force that drives reproductive barriers, species specification, and the upgrading of defensive mechanisms against pathogens.

Three divergent groups of duck astroviruses (DAstVs), namely DAstV-1, DAstV-2 (formerly duck hepatitis virus type 3) and DAstV-3 (isolate CPH), and other avastroviruses are known to infect domestic ducks. To provide more data regarding the molecular epidemiology of astroviruses in domestic ducks, we examined the prevalence of astroviruses in 136 domestic duck samples collected from four different provinces of China. Nineteen goose samples were also included. Using an astrovirus-specific reverse transcription-PCR assay, two groups of astroviruses were detected from our samples. A group of astroviruses detected from Pekin ducks, Shaoxing ducks and Landes geese were highly similar to the newly discovered DAstV-3. More interestingly, a novel group of avastroviruses, which we named DAstV-4, was detected in Pekin ducks. Following full-length sequencing and sequence analysis, the variation between DAstV-4 and other avastroviruses in terms of lengths of genome and internal component was highlighted. Sequence identity and phylogenetic analyses based on the amino acid sequences of the three open reading frames (ORFs) clearly demonstrated that DAstV-4 was highly divergent from all other avastroviruses. Further analyses showed that DAstV-4 shared low levels of genome identities (50-58%) and high levels of mean amino acid genetic distances in the ORF2 sequences (0.520-0.801) with other avastroviruses, suggesting DAstV-4 may represent an additional avastrovirus species although the taxonomic relationship of DAstV-4 to DAstV-3 remains to be resolved. The present works contribute to the understanding of epidemiology, ecology and taxonomy of astroviruses in ducks.

This work was supported by funds from the National Key Basic Research Developments Program, Ministry of Science and Technology, China (2013CB126902); National Transgenic Major Program Grant (2016ZX08009003-003-007); National Natural Science Foundation of China (31322040); China Innovative Research Team, Ministry of Education, and the Program of Introducing Talents of Discipline to Universities (111 Project, B14016); the Science and Technology Commission of Shanghai Municipality (grant no. 13JC1408200). Work in the Higgins laboratory is supported by the BBSRC.

Using an ORF1b-based astrovirus-specific reverse transcription (RT)-PCR assay, a duck hepatitis virus type 3 (DHV-3)-like astrovirus was detected from four intestinal samples collected from diseased ducks in China. Complete genome sequencing and comparative sequence analysis showed that the four duck astrovirus (DAstV) isolates were closely related and possessed a typical astrovirus genome organization. Genetic analysis of the complete ORF2 region revealed that mean amino acid genetic distances between the DHV-3-like isolates and previously known avastrovirus species were between 0.579 and 0.721, suggesting that the DHV-3-like isolates could be classified as an additional avastrovirus species. In the ORF1a and ORF1b regions, however, mean amino acid genetic distances between the DHV-3-like viruses and the turkey astrovirus 2 (TAstV-2)-like isolates were substantially less than those between TAstV-2-like isolates and DAstV/C-NGB-like astroviruses belonging to the same species. Pairwise comparisons and phylogenetic analyses demonstrated that the DHV-3-like isolates were most closely related to TAstV-2-like viruses in ORF1a and ORF1b, while showed highest similarity with the chicken astrovirus (CAstV) 612-like viruses in ORF2. These findings provide evidence that recombination events may have occurred during evolution of the avastroviruses and support the view that genomic analysis is required for classification of the avastroviruses.

Flowering plants form male reproductive cells (microsporocytes) during sporophytic generation, which subsequently differentiate into multicellular male gametes in the gametophytic generation. The tapetum is a somatic helper tissue neighboring microsporocytes and supporting gametogenesis. The mechanism controlling the specification of the tapetum and microsporocyte cell fate within the anther has long been a mystery in biology. Recent investigations have revealed molecular switches and signaling pathways underlying the establishment of somatic and reproductive cells in plants. In this review we discuss common and diversified signaling molecules and regulatory pathways including receptor-like protein kinases, redox status, glycoprotein, transcription factors, hormones and microRNA implicated in the specification of tapetum and microsporocytes in plants.

To efficiently identify genetically modified (GM) maize events in foods and feeds, we report here the development of four individual pentaplex PCR analysis systems for event-specific identification of sixteen GM maize events approved in South Korea. In addition to the maize endogenous reference gene, zSSIIb, the four pentaplex PCR assays target group 1 containing TC6275, MON810, T25, and NK603; group 2 with TC1507, MON863, GA21, and DAS-59122-7; group 3 with MIR604, Bt11, Bt176, and MON89034; group 4 with event 3272, LY038, MIR162, and MON88017. These amplicons were designed to be smaller than 200 bp to make the testing system suitable for analyzing processed foods/feeds derived from these 16 GM maize events. After optimizing the reaction condition, the limits of detection of these four assays were approximately 0.25% for all of the 16 GM maize events. This multiplex PCR method for sixteen GM maize events was validated by three operators, and the data confirmed the reliability of the developed assays. Furthermore, 74 food samples containing maize ingredients from the USA, China, Japan, and South Korea were analyzed, and we observed 18 food products containing one or more GM maize events. These results suggest that the developed multiplex system is applicable for use in specific testing of sixteen GM maize events in foods and feeds in South Korean market.

Highlights • We established a method for simultaneous detection of ten viruses that infect ducks. • The method was based on multiplex PCR and MALDI-TOF MS. • The detection limits of the proposed method were 1.3–7.8 copies/μl for ten specific viruses. • The method was specific and sensitive for simultaneous detection., Rapid screening of infectious viral diseases is the key to ensure healthy development of duck livestock industry. Currently routine viral detection methods are primarily used to detect up to 3 viruses. In this study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was used for simultaneous detection and genotyping of ten viruses in duck, including Duck hepatitis A virus 1 (DHAV-1), DHAV-3, Duck astrovirus 1 (DAstV-1), DAstV-2, Duck reovirus 1 (DRV-1), DRV-2, Tembusu virus (TMUV), Avian influenza virus (AIV), Goose parvovirus (GPV) and Duck enteritis virus (DEV). The low detection limits of this proposed method for ten duck viruses ranged from 1.3 copies/μl to 7.8 copies/μl. The novel detection method with high sensitivity, good specificity and high throughput has the potential to be applied for disease diagnosis and surveillance.

Video Objective To demonstrate a novel hysteroscopic surgery for adenomyosis. Setting The benign gynecology department at a university hospital. Interventions We performed a hysteroscopic minimally operation to treat symptomatic myometrial adenomyosis. The operations were performed under transabdominal ultrasound-guide. We used a cutting loop to resect the lesions repeatedly and progressively with the standard electroresection. The operation was considered complete when the pink fasciculate structure of the myometrium appeared. This study was approved by the institutional ethics committee of the International Peace Maternity and Child Health Hospital in Shanghai, China, on 19 April 2016. The approval number is GLW (2015) 19.Following-up were performed for 2 times at 3-months interval. The patient menstruated regularly. The postoperative VAS scores of dysmenorrhea and menstrual blood volume declined significantly after operation. 6 months after the operation the uterine volume evaluated by magnetic resonance imaging (MRI) reduced by about 33%. Conclusion Traditionally, adenomyosis is often an incidental finding in specimens obtained from hysterectomy or uterine biopsies and/or percutaneous ultrasound-based biopsies. The modern diagnostic imaging techniques, such as (MRI), contributing to improving accuracy in the identification of this pathology, results in that the conservative uterine-sparing treatments of adenomyosis appear to be feasible and efficacious. Hysteroscopic excision of uterine adenomyosis has the following benefits: the uterine is reserved and the symptoms of adenomyosis get improvement; the minimally invasive operation is short-time taking and the patients recovers quickly. Therefore, hysteroscopic excision provides an effective and optional conservative technique for the treatment of adenomyosis.

Genetically modified (GM) soybean and derived products make up a large part of the biotech-derived food and feed market. As more GM soybean varieties have been approved for commercialization, labeling requirement by South Korea and other countries needs the technical testing methods. This paper reports the development of a multiplex PCR method for identifying six commercialized GM soybean events using the event-specific fragment. Event specific primers targeting Roundup Ready Soybean (RRS, GTS40-3-2), A2704-12, DP356043-5, MON89788, A5547-127, and DP305423-1 were designed, and a multiplex PCR assay consisting of six event-specific fragments and one endogenous lectin fragment was developed. The specificity of the event-specific PCR method was confirmed using 20 GM events of maize, soybean, cotton, and canola. The limit of detection (LOD) for each event in the multiplex PCR is approximately 0.05%. Intra-lab validation by two different operators confirmed the specificity and LOD of this multiplex PCR method. The method was used to test 30 soybean-derived foods from South Korean and US markets, and results revealed three varieties of GM soybean (RRS, A2704-12, and MON89788) in 19 of the 30 food samples tested. This work provides an efficient and cost-effective approach for event-specific analysis of six commercialized GM soybean varieties and related processed foods in Korea.

Rice ( Oryza sativa ) has unique floral patterns that contribute to grain yield. However, the molecular mechanism underlying the specification of floral organ identities in rice, particularly the interaction among floral homeotic genes, remains poorly understood. Here, we show that the floral homeotic gene OsMADS16 (also called SUPERWOMAN1 , SPW1 , a B-class gene) acts together with the rice C-class genes OsMADS3 and OsMADS58 in specifying floral organ patterning. OsMADS16 and the two C-class genes have an overlapping expression pattern in the third whorl founder cells. Compared with the single mutants, both spw1-1 osmads3-4 and spw1-1 osmads58 double mutants exhibit additional whorls of glume-like organs within the flower, particularly an extra whorl of six glume-like structures formed at the position of the wild-type stamens. These ectopic glume-like structures were shown to have palea identity through cellular observation and in situ hybridization analysis using marker genes. Our results suggest that B- and C-class genes play a key role in suppressing indeterminate growth within the floral meristem, particularly whorl-3 primordia. We also hypothesize that, in contrast to previous assumptions, the specialized spikelet organ in rice, the palea, is the counterpart of the sepal in eudicots, and the lemma is homologous to the bract. SUMMARY The rice floral homeotic B-class gene OsMADS16 interacts with C-class genes in specifying floral organ patterning, particularly suppressing indeterminate growth within whorl-3 primordia. This work provides the evidence of the origin of the specialized organs, the lemma and the palea.

Laccase is one of the most useful green enzymes for cleaner industrial application and environmental protection. It is necessary to produce laccase with known composition of its commercial style at low production cost in an efficient way. In this study, the efficiency of laccase production by Pycnoporus sp. SYBC-L3 in a 65-L air-lift reactor was evaluated and the main extracellular proteins were identified. The highest laccase activity approximated 72,000 U/L after 6 days' cultivation. Using nano-LC–MS/MS protein identification technology, 22 different peptide fragments were identified matching sequences in different proteins with laccase as the predominant extracellular one. The crude laccase exhibited high catalytic activity at temperatures ranging from 0 °C to 100 °C at an acidic condition, with respective relative activity of 40% at 0 °C and 80% at 100 °C. The crude laccase was found to be very thermal stable, with half-lives of 4 h, 6.5 h and 10 h at 70 °C, 60 °C and 50 °C, respectively. The crude laccase still retained approximately 85% of its initial activity after one year of storage at room temperature. These results showed that laccase can be effectively produced by Pycnoporus sp. SYBC-L3 in air-lift reactor at larger scale and has great potential for industrial production and applications.

Currently, the detection methods with lower cost and higher throughput are the major trend in screening genetically modified (GM) food or feed before specific identification. In this study, we developed a quadruplex degenerate PCR screening approach for more than 90 approved GMO events. This assay is consisted of four PCR systems targeting on nine DNA sequences from eight trait genes widely introduced into GMOs, such as CP4-EPSPS derived from Acetobacterium tumefaciens sp. strain CP4, phosphinothricin acetyltransferase gene derived from Streptomyces hygroscopicus ( bar ) and Streptomyces viridochromogenes ( pat ), and Cry1Ab , Cry1Ac , Cry1A ( b / c ), mCry3A , and Cry3Bb1 derived from Bacillus thuringiensis . The quadruplex degenerate PCR assay offers high specificity and sensitivity with the absolute limit of detection (LOD) of approximate 80 target copies. Furthermore, the applicability of the quadruplex PCR assay was confirmed by screening either several artificially prepared samples or samples of Grain Inspection, Packers and Stockyards Administration (GIPSA) proficiency program.

A flavivirus-associated disease of egg-laying ducks was observed in eastern China in 2010, and a novel mosquito-borne flavivirus, Tembusu virus (TMUV), was isolated (Cao et al., 2011). Following up on the earlier study, a virus similar to TMUV was isolated recently from ducklings and characterized. We report that (1) the recently isolated virus, TMUV ZJ-6, replicated in vertebrate cells (DF-1, BHK-21) as well as in mosquito cells (C6/36) and caused cytopathic effect (CPE) in the cell lines tested; (2) extracellular viral particles examined by electron microscopy were approximately 45 nm in diameter and enveloped; (3) the full-length genome of the virus was determined, showing that the TMUV ZJ-6 is more closely related to the Ntaya group of viruses than other members of the Flaviviridae based on the data of phylogenetic analyses. Most importantly, the disease of ducklings was reproducible after administration of plaque-purified virus by intracerebral (i.c.), subcutaneous (s.c.) or intranasal (i.n.) inoculation. This is the first report that TMUV infects not only egg-laying ducks but also 3-21 days-old ducklings. The findings extend our understanding of how the virus spreads and causes disease.

In this study we propose a chromogenic platform for rapid analysis of organophosphate (OP) and carbamate (CM) insecticide residues, based on recombinant Drosophila melanogaster acetylcholinesterase (R- Dm AChE) as enzyme and indoxyl acetate as substrate. The visible chromogenic strip had the advantages identical to those of commonly used lateral flow assays (LFAs) with utmost simplicity in sample loading and result observation. After optimization, depending on the color intensity (CI) values, the well-established assay has the capabilities of both qualitative measurement via naked eyes and quantitative analysis by colorimetric reader with the desirable IC 50 values against the tested six insecticides (0.06 μg mL −1 of carbofuran, 0.28 μg mL −1 of methomyl, 0.03 μg mL −1 of dichlorvos, 31.6 μg mL −1 of methamidophos, 2.0 μg mL −1 of monocrotophos, 6.3 μg mL −1 of omethoate). Acceptable matrix effects and satisfactory detection performance were confirmed by in-parallel LC–MS/MS analysis in different vegetable varieties at various spiked levels of 10 −3 to 10 1 μg g −1 . Overall, the testified suitability and applicability of this novel platform meet the requirements for practical use in food safety management and environmental monitoring, especially in the developing world.

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In this study, to simulate the real industry wastewaters, experiments of laccase-catalyzed decolorization of mixed dyes constituted by azo and anthraquinone dyes were carried out. The results showed that anthraquinone dyes, playing the role of mediators, dramatically promoted the degradation of azo dyes when there was no exogenous mediator in the reaction mixture. This study represents the first attempt to decolorize the mixtures of azo and anthraquinone dyes by purified T. trogii laccase, suggesting great potential for laccase to decolorize textile industry wastewaters.

Three AFPs (AFP-1, AFP-2, AFP-3) of Ligustrum lucidum Ait leaves were purified by using ice-affinity, chromatography separation on a DEAE-cellulose-32 column and a Sephadex G100 column. The ice-affinity proteins were about 1.2 mg/100 g leaves. Their molecular weight was 66.1, 26.3, and 20.2 kDa, respectively. Their thermal hysteresis activity was 0.379, 0.678, and 0.460 °C respectively. Asx was very abundant in these AFPs; its molar ratio was 22.2, 24.9, and 27.8%, respectively. AFP-2 was effective to protect peroxidase, β-glucosidase, and trehalose synthase from freeze–thawing process. Cryoprotection of AFP-2 is better than trehalose.

Duck astrovirus (DAstV) is an important pathogen causing duck viral hepatitis (DVH), a highly contagious and fatal disease in young ducklings. To provide an antigen for a diagnostic serum test, the C-terminus of DAstV ORF2 protein was expressed in Escherichia coli. Four positive and 30 negative sera were used to validate the purified ORF2 protein by developing an indirect enzyme-linked immunosorbent assay (ELISA). No cross-reactions were found against other duck pathogens, including duck hepatitis A virus, duck plague herpesvirus, duck reovirus, Newcastle disease virus, and Riemerella anatipestifer 12/19 (63.2%) and 26/51 (51%) sera samples from two flocks of ducks that survived DAstV infections in commercial duck farms were positive for DAstV by this method, respectively. Interestingly, DAstV-specific antibodies were also detected in 12 (28.6%) of 42 sera samples from a different flock without DVH, indicating a wide distribution of subclinical infections caused by DAstV.

The white-rot fungus Pycnoporus sp. SYBC-L1 produced large amount of laccase in submerged fermentation. Two laccase isozymes (LacI and LacII) were purified using (NH 4 ) 2 SO 4 fractionation, DEAE-cellulose and Sephadex G-100 column chromatography. The molecular masses of LacI and Lac II were 55.89 and 63.07 kDa, respectively by SDS-PAGE. Both the laccases showed acidic pH optima and high catalytic activities at low temperature for oxidations of 2,6-dimethoxyphenol (DMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonate acid) (ABTS), syringaldazine and guaiacol. LacI and LacII were not only with high cold adaptation, but also fairly stable at high temperature. The half-lives of LacI at 50, 60 and 70 °C were 69.31, 2.58 and 0.13 h, respectively, whereas LacII was more stable with half-lives of 256.72, 21.00 and 2.06 h respectively. The best substrates for the enzymes were both found to be ABTS, in which the K m values of LacI and LacII were 0.0166 and 0.0435 mM and the catalytic efficiencies were 19640.36 and 31172.64 S −1 mM −1 , respectively. EDTA and low concentration of Cu 2+ and Mn 2+ almost had non-inhibitions on their activities. LacII with syringaldehyde efficiently decolorized Remazol Brilliant Blue R. The high thermostabilities as well as cold adapted properties made Pycnoporus sp. SYBC-L1 laccases to be excellent candidates in harsh industry.

To understand the molecular mechanism of male reproductive development in the model crop rice, we isolated a complete male sterile mutant post-meiotic deficient anther1 (pda1) from a gamma-ray-treated rice mutant library. Genetic analysis revealed that the pda1 mutant was controlled by a recessive nucleus gene. The pda1 mutant anther seemed smaller with white appearance. Histological analysis demonstrated that the pda1 mutant anther undergoes normal early tapetum development without obvious altered meiosis. However, the pda1 mutant displayed obvious defects in postmeiotic tapetal development, abnormal degeneration occurred in the tapetal cells at stage 9 of anther development. Also we observed abnormal lipidic Ubisch bodies from the tapetal layer of the pda1 mutant, causing no obvious pollen exine formation. RT-PCR analysis indicated that the expression of genes involved in anther development including GAMYB, OsC4 and Wax-deficient anther1 (WDA1) was greatly reduced in the pda1 mutant anther. Using map-based cloning approach, the PDA1 gene was finely mapped between two markers HLF610 and HLF627 on chromosome 6 using 3,883 individuals of F(2) population. The physical distance between HLF610 and HLF627 was about 194 kb. This work suggests that PDA1 is required for post-meiotic tapetal development and pollen/microspore formation in rice.

Molecularly imprinted polymers (MIPs) prepared in water-containing systems are more appropriate as adsorption materials in analyte extraction from biological samples. However, water as a polar solvent involved in the synthesis of MIPs frequently disrupts non-covalent interactions, and causes non-specific binding. In this study Fe(2+) was used as mediator to prepare MIPs, targeting tetracyclines (TCs) of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC), with TC as template molecule and methacrylic acid (MAA) as functional monomer. The subsequent binding assay indicated that Fe(2+) was responsible for substantially improved specific binding in recognition of TCs by decreasing the non-specific binding. Spectrophotometric analysis suggested the existence of the strong interactions among TC, metal ions and MAA in the mixture of methanol and water. Moreover, mass spectrometric measurements verified that Fe(2+) could bridge between TC and MAA to form a ternary complex of one TC, one Fe(2+) and four MAAs with a mass of 844.857. Furthermore, combined with molecularly imprinted solid-phase extraction (MISPE) for sample pretreatment, HPLC-UV analysis data revealed good performance of the obtained MIPs as adsorbents. The recoveries of TC, OTC and CTC in urine samples were 80.1-91.6%, 78.4-89.3% and 78.2-86.2%, respectively. This research strategy provides an example for preparation of desirable water-compatible MIPs extracting target drugs from aqueous samples by introducing metal ion as mediator into conventional polymerization system.

A new hypocrellin A-producing strain, Shiraia sp. SUPER-H168, was isolated from tissues of bamboo, Brachystachyum densiflorum. The morphology of this strain was characterized with a light microscope and a scanning electronic microscope. The mycelia, conidia, pycnidia of fungus were observed. Small pycnidia (10-20 microm in length) full of small conidia appeared on the mycelia, which were first reported in this study. The 18S rDNA region of this strain was amplified and sequenced. Then a neighbor-joining tree of 18S rDNA was constructed. According to the result of analysis, the strain SUPER-H168 was proved to belong to the genus Shiraia. Hypocrellin A was produced by solid-state fermentation, extracted by acetone, isolated by preparative RP-HPLC, and identified by RP-HPLC, ESI-MS and ultraviolet-visible absorbing scanning with diode array detection. The HA production could reach 2.02 mg/g dry solid substrate.

Chlorpromazine is extensively used as a sedative hypnotic in veterinary application, however, improper and over usage of chlorpromazine pose a serious threat to human health and animal production. Due to the difficulty in monitoring trace level of chlorpromazine residues in complicated biological matrices, specific adsorption materials for preconcentration and clean-up of chlorpromazine are indispensable for sample preparation before further determination. In this study, a new molecularly imprinted polymer (MIP) for recognition of chlorpromazine was obtained by bulk polymerization. Then the imprinting effect of MIP was confirmed via chromatographic evaluation by compared with the corresponding non-imprinted polymer (NIP). Furthermore, the adsorption selectivity and capacity analysis revealed that the MIP had high specification for chlorpromazine. Finally, with the optimized protocol of molecularly imprinted solid-phase extraction (MISPE), the target analyte in pig urine at different spiked levels were selectively separated and purified for routine HPLC-UV analysis, with the improved recoveries above 73.3% and the intraday RSD of less than 8.7%, proving that the obtained MIP as specific SPE sorbents for extraction of chlorpromazine is applicable.

As a complex wall system in flowering plants, the pollen outer wall mainly contains aliphatic sporopollenin; however, the mechanism for synthesizing these lipidic precursors during pollen development remains less well understood. Here, we report on the function of the rice tapetum-expressing TDR ( Tapetum Degeneration Retardation ) gene in aliphatic metabolism and its regulatory role during rice pollen development. The observations of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses suggested that pollen wall formation was significantly altered in the tdr mutant. The contents of aliphatic compositions of anther were greatly changed in the tdr mutant revealed by GC–MS (gas chromatography–mass spectrometry) testing, particularly less accumulated in fatty acids, primary alcohols, alkanes and alkenes, and an abnormal increase in secondary alcohols with carbon lengths from C29 to C35 in tdr . Microarray data revealed that a group of genes putatively involved in lipid transport and metabolism were significantly altered in the tdr mutant, indicating the critical role of TDR in the formation of the pollen wall. Also, a wide range of genes (236 in total—154 up-regulated and 82 down-regulated) exhibited statistically significant expressional differences between wild-type and tdr . In addition to its function in promoting tapetum PCD, TDR possibly plays crucial regulatory roles in several basic biological processes during rice pollen development.