基于ITS 序列和matK 基因序列的中国药用石斛及其混伪品的分子鉴定

为了从分子水平对中国药用石斛及其混伪品进行鉴定，本文选取了核rDNA ITS 序列和叶绿体DNA 的matK 基因序列进行研究。采用改良的CTAB 法提取石斛的基因组DNA，PCR 产物直接测序法对17 种（共32 份）药用石斛的核糖体内转录间隔区ITS 全序列进行测定，克隆测序法对12 种（共22 份）药用石斛的叶绿体的matK 基因序列进行测定，运用BioEd it，MEGA4.0 等生物软件分析了石斛属植物的rDNA ITS 序列及叶绿体的matK 基因序列的特征，比较了石斛属间、种间、种内不同居群（品种）间的序列碱基差异及遗传距离，应用邻接法构建分子系统树。主要研究结果如下： （1）建立了17 种（共32 份）药用石斛rDNA ITS 区碱基全序列数据库，其中，ITS1 的长度为228~234 bp，GC 含量为45.7%~53.0%，变异位点167 个，占总位点67.34%，信息位点106 个，占总位点42.74%，ITS2 长度为241~247 bp，GC含量为44.8%~55.7%，变异位点165 个，占总位点66.27%，信息位点115 个，占总位点46.18%。 （2）建立了12 种（共22 份）药用石斛的叶绿体matK 基因全序列数据库，叶绿体matK 基因长1410 bp，变异位点51 个，信息位点11 个。除了存在碱基替换的遗传变异外，还存在碱基的插入和缺失。 （3）通过ITS 序列比较分析了各材料间的遗传距离和碱基差异，属间的遗传距离为0.295，石斛种间的平均遗传距离为0.142，碱基相差2~156 个，种内各居群间的平均遗传距离为0.002，碱基相差1~2 个。属间的遗传距离大于种间的遗传距离，种间的遗传距离大于种内不同居群（品种）间的遗传距离。 （4）根据分析石斛叶绿体的matK 基因序列得到，外类群（密花石豆兰）与石斛属间最小遗传距离为0.027，石斛种间的平均遗传距离为0.008，种间最大的遗传距离0.014， 最小的遗传距离为0.003，碱基相差8~20 个。种内不同居群（品种）遗传距离为0.001，相差1~5 个碱基。 （5）利用17 种石斛的全序列数据库及遗传分析软件，通过对待检种rDNA I TS区进行序列测定，成功地对10 个待检种进行了鉴定，并且在原植物开花后得到了验证。 （6）运用12 种石斛的matK 基因全序列数据库及遗传分析软件，成功地对4个待检种进行了鉴定，同样在原植物开花后得到了验证。 （7）本文利用石斛的核糖体内转录间隔区ITS 序列和叶绿体的matK 基因序列数据库分别构建了NJ 树，外类群与石斛属间石斛种间以及种内不同居群（品种）间均能在NJ 树中明显分化开来，二者构建的分子系统树一致，为石斛的分子鉴定提供了依据。 In order to identify Chinese Herba Dendrobii and its adulterant species on molecular level, we studied the sequences of rDNA ITS and chloroplast matK gene. Genomic DNA of Dendrobium was extracted using the modified cetyltrimethyl ammonium bromide (CTAB) method. The PCR products of the rDNA ITS sequences of Dendrobium (32 materia ls) were purified and then sequenced. The PCR products of chloroplast matK gene of Dendrobium (22 materia ls) were purified, cloned and then sequenced. The characteristic of the sequences and the genetic dista nce were compared between Bulbophyllum odoratissimum and Dendrobium, Dendrobium interspecies, and different populations. Phylogenetic trees were constructed using the NJ method by the biology softwares including BioEd it, MEGA4.0 etc. The ma in results as follows: (1) It was built up that the database of rDNA ITS sequences of 17 species of Herba Dendrobii (32 materia ls). The ITS1 was 228~234 bp, the GC content accounting for 45.7%~53.0%. Its variable sites were 167, accounting for 67.34%. The Parsim-Informative positions were 106, accounting for 42.74%. The ITS2 was 241~247 bp, the GC accounting for 44.8%~55.7%. The variable sites were 165, accounting for 66.27%. The Parsim-Informative positions were 115, accounting for 46.18%. (2) The database of the chloroplast matK gene sequences was built up, which contained 12 species of Herba Dendrobii (22 materia ls). The matK gene sequences were about 1410bp in length. There were 51 variable sites and 11 Parsim-Informative sites. And there were nucleotides insertions and deletions in some species , in addition to the nucleotides substitutions. (3) The rDNA ITS sequences were compared and analyzed by the biology softwares. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was 0.295. The avera ge genetic dista nce was 0.142 between Dendrobium species, and there were 2~156 variable nucleotides. The avera ge genetic dista nce between different populations was 0.002, and there were 2~156 variable nucleotides. The genetic dista nce between Bulbophyllum odoratissimum and Dendrobium was greater tha n that of Denrobium interspecies. Meanwhile, the genetic dista nce between Denrobium species was also greater tha n that of different populations (variaties). (4) The characteristics of the chloroplast matK gene sequences were obtained after analyzing by the biology softwares. The minima l genetic dista nce was 0.027 between Bulbophyllum odoratissimum and Dendrobium . The ma xima l genetic dista nce was 0.014 between Dendrobium species, and there were 20 variable nucleotides. The minima l genetic dista nce between populations was 0.003, and there were 8 variable nucleotides.The genetic dista nce between populations was 0.001, and there were 1~5 variable nucleotides. (5) The molecular Phylogeny tree was constructed on the database of rDNA ITS the sequences of 17 species of Herba Dendrobii using the biology softwares. Then we authenticated 10 materia ls on molecular level. What’s more, they had been proved when these pla nts flowered. (6) The molecular Phylogeny tree was built up on the database of chloroplast matK gene sequences of 12 species of Herba Dendrobii with the biology softwares.Then 4 materia ls were authenticated on molecular level. Moreover, they had also been proved when these pla nts were in flower. (7) The Phylogenetic trees were separately constructed on the sequences of rDNA ITS and chloroplast matK gene B. odoratissimum and Dendrobium all could be distinguished on the Phylogenetic trees. Meanwhile, the Phylogenetic trees based on two groups of sequences were coincident. rDNA ITS and matK gene sequence could be used as molecular markers for authentication of Herba Dendrobii.

Robust Speaker Recognition in Noisy Conditions

This paper investigates the problem of speaker identi-fication and verification in noisy conditions, assuming that speechsignals are corrupted by environmental noise, but knowledgeabout the noise characteristics is not available. This research ismotivated in part by the potential application of speaker recog-nition technologies on handheld devices or the Internet. Whilethe technologies promise an additional biometric layer of securityto protect the user, the practical implementation of such systemsfaces many challenges. One of these is environmental noise. Due tothe mobile nature of such systems, the noise sources can be highlytime-varying and potentially unknown. This raises the require-ment for noise robustness in the absence of information about thenoise. This paper describes a method that combines multicondi-tion model training and missing-feature theory to model noisewith unknown temporal-spectral characteristics. Multiconditiontraining is conducted using simulated noisy data with limitednoise variation, providing a “coarse” compensation for the noise,and missing-feature theory is applied to refine the compensationby ignoring noise variation outside the given training conditions,thereby reducing the training and testing mismatch. This paperis focused on several issues relating to the implementation of thenew model for real-world applications. These include the gener-ation of multicondition training data to model noisy speech, thecombination of different training data to optimize the recognitionperformance, and the reduction of the model’s complexity. Thenew algorithm was tested using two databases with simulated andrealistic noisy speech data. The first database is a redevelopmentof the TIMIT database by rerecording the data in the presence ofvarious noise types, used to test the model for speaker identifica-tion with a focus on the varieties of noise. The second database isa handheld-device database collected in realistic noisy conditions,used to further validate the model for real-world speaker verifica-tion. The new model is compared to baseline systems and is foundto achieve lower error rates.

Detecção e isolamento de falhas em sistemas dinâmicos baseados em redes neurais

This master dissertation presents the development of a fault detection and isolation system based in neural network. The system is composed of two parts: an identification subsystem and a classification subsystem. Both of the subsystems use neural network techniques with multilayer perceptron training algorithm. Two approaches for identifica-tion stage were analyzed. The fault classifier uses only residue signals from the identification subsystem. To validate the proposal we have done simulation and real experiments in a level system with two water reservoirs. Several faults were generated above this plant and the proposed fault detection system presented very acceptable behavior. In the end of this work we highlight the main difficulties found in real tests that do not exist when it works only with simulation environments

Report of the Stock Identification Methods Working Group (SIMWG) 2016

The Stock Identification Methods Working Group (SIMWG) worked by correspondence in 2016. The working group was chaired by Lisa Kerr (USA). The work plan for SIMWG in 2016 comprised four Terms of Reference (ToR), some of which are continuing goals for SIMWG: a ) Review recent advances in stock identification methods; b ) Build a reference database with updated information on known biological stocks for species of ICES interest; c ) Provide technical reviews and expert opinions on matters of stock identifica-tion, as requested by specific Working Groups and SCICOM; d ) Review and report on advances in mixed stock analysis, and assess their po-tential role in improving precision of stock assessment. ToR a) is an ongoing task of SIMWG in which we provide a comprehensive update on recent applications of stock identification techniques to ICES species of interest, summa-rize new approaches in stock identification, and novel combinations of existing applica-tions. ToR b) is a multi-annual ToR in which SIMWG has taking steps to build a reference data-base consisting of SIMWG reviews of issues of stock identity for ICES species. ToR c) is a key ongoing task by SIMWG in which we addresses specific requests by ICES working groups for technical advice on issues of stock identity. This year we provided advice on mackerel in the Northeast Atlantic as requested by WGWIDE. ToR d) is a multi-annual ToR that is focused on tracking developments in the application of mixed stock analysis and the integration of this information into assessment and management.

Genetic, structural, and molecular insights into the function of ras of complex proteins domains

Ras of complex proteins (ROC) domains were identified in 2003 as GTP binding modules in large multidomain proteins from Dictyostelium discoideum. Research into the function of these domains exploded with their identification in a number of proteins linked to human disease, including leucine-rich repeat kinase 2 (LRRK2) and death-associated protein kinase 1 (DAPK1) in Parkinson’s disease and cancer, respectively. This surge in research has resulted in a growing body of data revealing the role that ROC domains play in regulating protein function and signaling pathways. In this review, recent advances in the structural informa- tion available for proteins containing ROC domains, along with insights into enzymatic function and the integration of ROC domains as molecular switches in a cellular and organismal context, are explored.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes : (Part 10) The infrared and Raman characteristics of phthalocyanine in heteroleptic bis(phthalocyaninato) rare earth complexes with decreased molecular symmetry

The infrared (IR) spectroscopic data for a series of eleven heteroleptic bis(phthalocyaninato) rare earth complexes MIII(Pc)[Pc(α-OC5H11)4] (M = Sm–Lu, Y) [H2Pc = unsubstituted phthalocyanine, H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected with 2 cm−1 resolution. Raman spectroscopic properties in the range of 500–1800 cm−1 for these double-decker molecules have also been comparatively studied using laser excitation sources emitting at 632.8 and 785 nm. Both the IR and Raman spectra for M(Pc)[Pc(α-OC5H11)4] are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues due to the decreased molecular symmetry of these double-decker compounds, namely C4. For this series, the IR Pc√− marker band appears as an intense absorption at 1309–1317 cm−1, attributed to the pyrrole stretching. With laser excitation at 632.8 nm, Raman vibrations derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. In contrast, when excited with laser radiation of 785 nm, the ring radial vibrations of isoindole moieties and dihedral plane deformations between 500 and 1000 cm−1 for M(Pc)[Pc(α-OC5H11)4] intensify to become the strongest scatterings. Both techniques reveal that the frequencies of pyrrole stretching, isoindole breathing, isoindole stretchings, aza stretchings and coupling of pyrrole and aza stretchings depend on the rare earth ionic size, shifting to higher energy along with the lanthanide contraction due to the increased ring-ring interaction across the series. The assignments of the vibrational bands for these compounds have been made and discussed in relation to other unsubstituted and substituted bis(phthalocyaninato) rare earth analogues, such as M(Pc)2 and M(OOPc)2 [H2OOPc = 2,3,9,10,16,17,23,24-octakis(octyloxy)phthalocyanine].

Time-Dependent Density Functional Molecular Orbital and Excited State Calculations on Bis(porphyrinyl)butadiynes in the Monocationic, Neutral, Monoanionic, and Dianionic Oxidation States

We report a theoretical study of the multiple oxidation states (1+, 0, 1−, and 2−) of a meso,meso-linked diporphyrin, namely bis[10,15,20-triphenylporphyrinatozinc(II)-5-yl]butadiyne (4), using Time-Dependent Density Functional Theory (TDDFT). The origin of electronic transitions of singlet excited states is discussed in comparison to experimental spectra for the corresponding oxidation states of the close analogue bis{10,15,20-tris[3‘,5‘-di-tert-butylphenyl]porphyrinatozinc(II)-5-yl}butadiyne (3). The latter were measured in previous work under in situ spectroelectrochemical conditions. Excitation energies and orbital compositions of the excited states were obtained for these large delocalized aromatic radicals, which are unique examples of organic mixed-valence systems. The radical cations and anions of butadiyne-bridged diporphyrins such as 3 display characteristic electronic absorption bands in the near-IR region, which have been successfully predicted with use of these computational methods. The radicals are clearly of the “fully delocalized” or Class III type. The key spectral features of the neutral and dianionic states were also reproduced, although due to the large size of these molecules, quantitative agreement of energies with observations is not as good in the blue end of the visible region. The TDDFT calculations are largely in accord with a previous empirical model for the spectra, which was based simplistically on one-electron transitions among the eight key frontier orbitals of the C4 (1,4-butadiyne) linked diporphyrins.

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

Comparison of molecular markers to detect fresh sewage in environmental waters

Human-specific Bacteroides HF183 (HS-HF183), human-specific Enterococci faecium esp (HS-esp), human-specific adenoviruses (HS-AVs) and human-specific polyomaviruses (HS-PVs) assays were evaluated in freshwater, seawater and distilled water to detect fresh sewage. The sewage spiked water samples were also tested for the concentrations of traditional fecal indicators (i.e., Escherichia coli, enterococci and Clostridium perfringens) and enteric viruses such as enteroviruses (EVs), sapoviruses (SVs), and torquetenoviruses (TVs). The overall host-specificity of the HS-HF183 marker to differentiate between humans and other animals was 98%. However, the HS-esp, HS-AVs and HS-PVs showed 100% hostspecificity. All the human-specific markers showed >97% sensitivity to detect human fecal pollution. E. coli, enterococci and, C. perfringens were detected up to dilutions of sewage 10_5, 10_4 and 10_3 respectively.HS-esp, HS-AVs, HS-PVs, SVs and TVs were detected up to dilution of sewage 10_4 whilst EVs were detected up to dilution 10_5. The ability of the HS-HF183 marker to detect freshsewagewas3–4 orders ofmagnitude higher than that of the HS-esp and viral markers. The ability to detect fresh sewage in freshwater, seawater and distilled water matrices was similar for human-specific bacterial and viral marker. Based on our data, it appears that human-specific molecular markers are sensitive measures of fresh sewage pollution, and the HS-HF183 marker appears to be the most sensitive among these markers in terms of detecting fresh sewage. However, the presence of the HS-HF183 marker in environmental waters may not necessarily indicate the presence of enteric viruses due to their high abundance in sewage compared to enteric viruses. More research is required on the persistency of these markers in environmental water samples in relation to traditional fecal indicators and enteric pathogens.

Molecular architecture of the human sinus node: insights into the function of the cardiac pacemaker.

BACKGROUND: Although we know much about the molecular makeup of the sinus node (SN) in small mammals, little is known about it in humans. The aims of the present study were to investigate the expression of ion channels in the human SN and to use the data to predict electrical activity. METHODS AND RESULTS: Quantitative polymerase chain reaction, in situ hybridization, and immunofluorescence were used to analyze 6 human tissue samples. Messenger RNA (mRNA) for 120 ion channels (and some related proteins) was measured in the SN, a novel paranodal area, and the right atrium (RA). The results showed, for example, that in the SN compared with the RA, there was a lower expression of Na(v)1.5, K(v)4.3, K(v)1.5, ERG, K(ir)2.1, K(ir)6.2, RyR2, SERCA2a, Cx40, and Cx43 mRNAs but a higher expression of Ca(v)1.3, Ca(v)3.1, HCN1, and HCN4 mRNAs. The expression pattern of many ion channels in the paranodal area was intermediate between that of the SN and RA; however, compared with the SN and RA, the paranodal area showed greater expression of K(v)4.2, K(ir)6.1, TASK1, SK2, and MiRP2. Expression of ion channel proteins was in agreement with expression of the corresponding mRNAs. The levels of mRNA in the SN, as a percentage of those in the RA, were used to estimate conductances of key ionic currents as a percentage of those in a mathematical model of human atrial action potential. The resulting SN model successfully produced pacemaking. CONCLUSIONS: Ion channels show a complex and heterogeneous pattern of expression in the SN, paranodal area, and RA in humans, and the expression pattern is appropriate to explain pacemaking.

Interactions of phenyldithioesters with gold nanoparticles (AuNPs): Implications for AuNP functionalization and molecular barcoding of AuNP assemblies

The interactions of phenyldithioesters with gold nanoparticles (AuNPs) have been studied by monitoring changes in the surface plasmon resonance (SPR), depolarised light scattering, and surface enhanced Raman spectroscopy (SERS). Changes in the SPR indicated that an AuNP-phenyldithioester charge transfer complex forms in equilibrium with free AuNPs and phenyldithioester. Analysis of the Langmuir binding isotherms indicated that the equilibrium adsorption constant, Kads, was 2.3 ± 0.1 × 106 M−1, which corresponded to a free energy of adsorption of 36 ± 1 kJ mol−1. These values are comparable to those reported for interactions of aryl thiols with gold and are of a similar order of magnitude to moderate hydrogen bonding interactions. This has significant implications in the application of phenyldithioesters for the functionalization of AuNPs. The SERS results indicated that the phenyldithioesters interact with AuNPs through the C═S bond, and the molecules do not disassociate upon adsorption to the AuNPs. The SERS spectra are dominated by the portions of the molecule that dominate the charge transfer complex with the AuNPs. The significance of this in relation to the use of phenyldithioesters for molecular barcoding of nanoparticle assemblies is discussed.