Multienzymatic disintegration of DNA-scaffolded magnetic nanoparticle assembly for malarial mitochondrial DNA detection.

Early diagnosis of malaria can prevent the spread of disease and save lives, which, however, remains challenging in remote and less developed regions. Here we report a portable and low-cost optomagnetic biosensor for rapid amplification and detection of malarial mitochondrial DNA. Bioresponsive magnetic nanoparticle assemblies are constructed by using nucleic acid scaffolds containing endonucleolytic DNAzymes and their substrates, which can be activated by the presence of target DNA and self-disintegrated to release magnetic nanoparticles for optomagnetic quantification. Specifically, target molecules can induce padlock probe ligation and subsequent one-pot homogeneous cascade reactions consisting of nicking-enhanced rolling circle amplification, DNAzyme-assisted nucleic acid recycling, and strand-displacement-driven disintegration of the magnetic assembly. With an optimized magnetic actuation process for reaction acceleration, a detection limit of 1 fM can be achieved by the proposed biosensor with a total assay time of ca. 90 min and a dynamic detection range spanning 3 orders of magnitude. The robustness of the system was validated by testing target molecules spiked in 5% serum samples. Clinical sample validation was conducted by testing malaria-positive clinical blood specimens, obtaining quantitative results concordant with qPCR measurements.

OCT4 expression transactivated by GATA protein is essential for non-rodent trophectoderm early development.

Oct4 is exclusively expressed in rodent inner cell mass (ICM) but silenced in its trophectoderm (TE). However, for many non-rodent animals, including pig, cattle, rabbit, goat, and human, OCT4 has a remarkable expression in early TE. This study, applying pig as the main research model, proves that OCT4 expression in TE is supported by a unique GATA motif in the OCT4 upstream conserved regulatory region, and GATA4 is responsible for its activation. Moreover, OCT4 acts as a specific regulator of a narrow range of genes (including BCL2A1 and HNRNP2AB1) that are essential for the first wave of rapid proliferation in early TE. This study describes the regulatory mechanism to direct the OCT4 expression and its significance in TE of porcine preimplantation embryo.

Non-Local Temporal Difference Network for Temporal Action Detection.

As an important part of video understanding, temporal action detection (TAD) has wide application scenarios. It aims to simultaneously predict the boundary position and class label of every action instance in an untrimmed video. Most of the existing temporal action detection methods adopt a stacked convolutional block strategy to model long temporal structures. However, most of the information between adjacent frames is redundant, and distant information is weakened after multiple convolution operations. In addition, the durations of action instances vary widely, making it difficult for single-scale modeling to fit complex video structures. To address this issue, we propose a non-local temporal difference network (NTD), including a chunk convolution (CC) module, a multiple temporal coordination (MTC) module, and a temporal difference (TD) module. The TD module adaptively enhances the motion information and boundary features with temporal attention weights. The CC module evenly divides the input sequence into N chunks, using multiple independent convolution blocks to simultaneously extract features from neighboring chunks. Therefore, it realizes the information delivered from distant frames while avoiding trapping into the local convolution. The MTC module designs a cascade residual architecture, which realizes the multiscale temporal feature aggregation without introducing additional parameters. The NTD achieves a state-of-the-art performance on two large-scale datasets, 36.2% mAP@avg and 71.6% mAP@0.5 on ActivityNet-v1.3 and THUMOS-14, respectively.

AaSPL9 affects glandular trichomes initiation by positively regulating expression of AaHD1 in Artemisia annua L.

Glandular trichomes can secrete and store a large number of secondary metabolites in plants, some of which are of high medicinal and commercial value. For example, artemisinin, isolated from Artemisia annua L. plants, and its derivatives have great high medicinal value. Previous research indicated that artemisinin was synthesized in the glandular trichomes on the leaves of A. annua. It is an important study direction to improve artemisinin yield by promoting the initiation and development of glandular trichome. In this study, SQUAMOSA promoter-binding protein-like 9 (AaSPL9) was identified. In AaSPL9 overexpression transgenic plants, the glandular trichomes density was increased by 45-60 %, and the content of artemisinin was increased by 33-60 %, indicating that AaSPL9 positively regulate the glandular trichomes initiation. Yeast one-hybrid(Y1H), Dual-luciferase (Dual-Luc), Electrophoretic Mobility Shift Assay (EMSA) demonstrated that AaSPL9 activated the expression of AaHD1 by combining directly the GTAC-box of the AaHD1 promoter. Taken together, we identified AaSPL9, a positive transcription factor, regulating the glandular trichome initiation in A. annua, and revealed a novel molecular mechanism by which a SPL protein to promote glandular trichome initiation.

An high-throughput sequencing approach to the preliminary analysis of bacterial communities associated with changes in amino acid nitrogen, organic acid and reducing sugar contents during soy sauce fermentation.

Soy sauce is a traditional fermented soy food for enhancing the umami taste in Asian cuisines. In this study, 16S rRNA gene throughput sequencing analysis showed the bacterial communities and the changes in soy sauce during fermentation. Weissella, Bacillus and Lactococcus were the most abundant at genus level. The uncultured bacterium Weissella and Lactococcus had relatively high abundance at species level. Alpha diversity analysis indicated the bacterial community diversity increased at fermentation initiation, while decreased as fermentation progressed. Based on beta-diversity analysis, four clusters including cluster I (time point A-F), cluster II (G,H), cluster III (I,J) and cluster IV(K) were distinctly separated, indicating the fermentation time significantly affected bacterial community diversity. Also, close associations were found between the bacterial communities in soy sauce and its amino acid nitrogen, organic acid and reducing sugar contents during fermentation. Therefore, it will provide important information for optimization of the soy sauce production process.

Overexpression of blue light receptor AaCRY1 improves artemisinin content in Artemisia annua L. .

Artemisinin, an effective antimalarial compound, is isolated from the medicinal plant Artemisia annua L. However, because of the low content of artemisinin in A. annua, the demand of artemisinin exceeds supply. Previous studies show that the artemisinin biosynthesis is promoted by light in A. annua. Cryptochrome1 (CRY1) is involved in many processes in the light response. In this study, AaCRY1 was cloned from A. annua. Overexpressing AaCRY1 in Arabidopsis thaliana cry1 mutant resulted in blue-light-dependent short hypocotyl phenotype and short coleoptile under blue light. Yeast two-hybrid and subcellular colocalization showed that AaCRY1 interacted with AtCOP1 (ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1). Overexpression of AaCRY1 in transgenic A. annua increased the artemisinin content. When AaCRY1 was overexpressed in A. annua driven by the CYP71AV1 (cytochrome P450 dependent amorpha-4,11-diene 12-hydroxylase) promoter, the artemisinin content was 1.6 times higher than that of the control. Furthermore, we expressed the C terminal of AaCRY1(CCT) involved a GUS-CCT fusion protein in A. annua. The results showed that the artemisinin content was increased to 1.7- to 2.4-fold in GUS-CCT transgenic A. annua plants. These results demonstrate that overexpression of GUS-CCT is an effective strategy to increase artemisinin production in A. annua.

Medical Knowledge Graph to Enhance Fraud, Waste, and Abuse Detection on Claim Data: Model Development and Performance Evaluation.

BACKGROUND: Fraud, Waste, and Abuse (FWA) detection is a significant yet challenging problem in the health insurance industry. An essential step in FWA detection is to check whether the medication is clinically reasonable with respect to the diagnosis. Currently, human experts with sufficient medical knowledge are required to perform this task. To reduce the cost, insurance inspectors tend to build an intelligent system to detect suspicious claims with inappropriate diagnoses/medications automatically. OBJECTIVE: The aim of this study was to develop an automated method for making use of a medical knowledge graph to identify clinically suspected claims for FWA detection. METHODS: First, we identified the medical knowledge that is required to assess the clinical rationality of the claims. We then searched for data sources that contain information to build such knowledge. In this study, we focused on Chinese medical knowledge. Second, we constructed a medical knowledge graph using unstructured knowledge. We used a deep learning-based method to extract the entities and relationships from the knowledge sources and developed a multilevel similarity matching approach to conduct the entity linking. To guarantee the quality of the medical knowledge graph, we involved human experts to review the entity and relationships with lower confidence. These reviewed results could be used to further improve the machine-learning models. Finally, we developed the rules to identify the suspected claims by reasoning according to the medical knowledge graph. RESULTS: We collected 185,796 drug labels from the China Food and Drug Administration, 3390 types of disease information from medical textbooks (eg, symptoms, diagnosis, treatment, and prognosis), and information from 5272 examinations as the knowledge sources. The final medical knowledge graph includes 1,616,549 nodes and 5,963,444 edges. We designed three knowledge graph reasoning rules to identify three kinds of inappropriate diagnosis/medications. The experimental results showed that the medical knowledge graph helps to detect 70% of the suspected claims. CONCLUSIONS: The medical knowledge graph-based method successfully identified suspected cases of FWA (such as fraud diagnosis, excess prescription, and irrational prescription) from the claim documents, which helped to improve the efficiency of claim processing.

Enhancement of ganoderic acid production by constitutively expressing Vitreoscilla hemoglobin gene in Ganoderma lucidum.

The Vitreoscilla hemoglobin (VHb) gene was expressed in Ganoderma lucidum to enhance antitumor ganoderic acid (GA) production. The effects of VHb expression on the accumulation of GAs and lanosterol (intermediate) and the transcription of GA biosynthesis genes were also investigated. In VHb-expressing G. lucidum, the maximum concentrations of four individual GAs (GA-S, GA-T, GA-Mk and GA-Me) were 19.1±1.8, 34.6±2.1, 191.5±13.1 and 45.2±2.8µg/100mg dry weight, respectively, which were 1.4-, 2.2, 1.9- and 2.0-fold higher than those obtained in the wild-type strain. Moreover, the maximum lanosterol concentration in the strain expressing VHb was 1.28-fold lower than that in the wild-type strain. The transcription levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, squalene synthase, and lanosterol synthase genes were up-regulated by 1.6-, 1.5-, and 1.6-fold, respectively, in the strain expressing VHb. This work is beneficial in developing an efficient fermentation process for the hyperproduction of GAs.

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

Ovulation Statuses of Surrogate Gilts Are Associated with the Efficiency of Excellent Pig Cloning.

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technique that can produce multiple copies of excellent livestock. However, low cloning efficiency limits the application of SCNT. In this study, we systematically investigated the major influencing factors related to the overall cloning efficiency in pigs. Here, 13620 cloned embryos derived from excellent pigs were transferred into 79 surrogate gilts, and 119 live cloned piglets were eventually generated. During cloning, group of cloned embryos derived from excellent Landrace or Large white pigs presented no significant differences of cleavage and blastocyst rates, blastocyst cell numbers, surrogate pregnancy and delivery rates, average numbers of piglets born and alive and cloning efficiencies, and group of 101-150, 151-200 or 201-250 cloned embryos transferred per surrogate also displayed a similar developmental efficiency. When estrus stage of surrogate gilts was compared, group of embryo transfer on Day 2 of estrus showed significantly higher pregnancy rate, delivery rate, average number of piglets born, average alive piglet number or cloning efficiency than group on Day 1, Day 3, Day 4 or Day 5, respectively (P<0.05). And, in comparison with the preovulation and postovulation groups, group of surrogate gilts during periovulation displayed a significantly higher overall cloning efficiency (P<0.05). Further investigation of surrogate estrus stage and ovulation status displayed that ovulation status was the real factor underlying estrus stage to determine the overall cloning efficiency. And more, follicle puncture for preovulation, not transfer position shallowed for preovulation or deepened for postovulation, significantly improved the average number of piglets alive and cloning efficiency (P<0.05). In conclusion, our results demonstrated that ovulation status of surrogate gilts was the fundamental factor determining the overall cloning efficiency of excellent pigs, and follicle puncture, not transfer position change, improved cloning efficiency. This work would have important implications in preserving and breeding excellent livestock and improving the overall cloning efficiency.

Rosa26 locus supports tissue-specific promoter driving transgene expression specifically in pig.

Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus.

Development of an expression plasmid and its use in genetic manipulation of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes).

We report the construction of a plasmid, pJW-EXP, designed for the expression of homologous and heterologous genes in Ganoderma lucidum. pJW-EXP was generated from the plasmid pMD19-T by inserting the G. lucidum glyceraldehyde-3-phosphate dehydrogenase gene promoter, the G. lucidum iron-sulfur protein subunit of succinate dehydrogenase gene terminator and the homologous carboxin-resistance gene as selection marker. This expression plasmid can be efficiently transformed into Ganoderma through polyethylene glycol-mediated protoplast transformation. Southern blot analysis showed that most of the integrated DNA appeared as multiple copies in the genome. The applicability of the constructed plasmid was tested by expression of the truncated G. lucidum 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene that encodes the catalytic domain of HMGR. Overexpression of the truncated HMGR gene, which is a key gene in the biosynthetic pathway of the antitumor compounds, ganoderic acids, increased the transcription of the HMGR gene and enhanced ganoderic acid accumulation. pJW-EXP can serve as a useful tool in the genetic improvement and metabolic engineering of Ganoderma.

Muscovy duck reovirus p10.8 protein localizes to the nucleus via a nonconventional nuclear localization signal.

BACKGROUND: It was previously report that the first open reading frame of Muscovy duck reocvirus S4 gene encodes a 95-amino-acid protein, designed p10.8, which has no sequence similarity to other known proteins. Its amino acid sequence offers no clues about its function. RESULTS: Subcellular localization and nuclear import signal of p10.8 were characterized. We found that p10.8 protein localizes to the nucleus of infected and transfected cells, suggesting that p10.8 nuclear localization is not facilitated by viral infection or any other viral protein. A functional non-canonical nuclear localization signal (NLS) for p10.8 was identified and mapped to N-terminus residues 1-40. The NLS has the ability to retarget a large cytoplasmic protein to the nucleus. CONCLUSIONS: p10.8 imported into the nucleus might via a nonconventional signal nuclear signal.

Molecular characterization of a duck Tembusu virus from China.

A new emerging flavivirus caused severe egg-drop in poultry and spread quickly across most duck-producing regions of China in 2010. Complete genome sequencing indicated that the virus genome is 10,989 nucleotides in length and possesses typical flavivirus genome organization, 5' untranslated region (UTR)-Cv-Ci-prM-M-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-3'-UTR. The long open reading frame (ORF) encodes 3,425 amino acids (95-10,372 nt). The 94-nucleotide 5'-UTR is of intermediate size and the 617-nucleotide 3'-UTR is quite long relative to those of other flaviviruses. The polyprotein cleavage sites, potential glycosylation sites, distribution of cysteine residues, and 3'-UTR secondary structure were characterized. Phylogenetic analysis of the polyprotein sequences indicates that the HN isolate is closely related to Tembusu viruses of the Ntaya virus group.