Double-Tagging Polymerase Chain Reaction with a Thiolated Primer and Electrochemical Genosensing based on Gold Nanocomposite Sensor for Food Safety

A novel material for electrochemical biosensing based on rigid conducting gold nanocomposite (nano-AuGEC) is presented. Islands of chemisorbing material (gold nanoparticles) surrounded by nonreactive, rigid, and conducting graphite epoxy composite are thus achieved to avoid the stringent control of surface coverage parameters required during immobilization of thiolated oligos in continuous gold surfaces. The spatial resolution of the immobilized thiolated DNA was easily controlled by merely varying the percentage of gold nanoparticles in the composition of the composite. As low as 9 fmol (60 pM) of synthetic DNA were detected in hybridization experiments when using a thiolated probe. Moreover, for the first time a double tagging PCR strategy was performed with a thiolated primer for the detection of Salmonella sp., one of the most important foodborne pathogens affecting food safety. Ibis assay was performed by double-labeling the amplicon during the PCR with a -DIG and -SH set of labeled primers. The thiolated end allows the immobilization of the amplicon on the nano-AuGEC electrode, while digoxigenin allows the electrochemical detection with the antiDIG-HRP reporter in the femtomole range. Rigid conducting gold nanocomposite represents a good material for the improved and oriented immobilization of biomolecules with excellent transducing properties for the construction of a wide range of electrochemical biosensors such as immunosensors, genosensors, and enzymosensors.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

State-space modelling of geolocation data reveals sex differences in the use of management areas by breeding northern fulmars

We thank Orkney Islands Council for access to Eynhallow and Talisman Energy (UK) Ltd and Marine Scotland for fieldwork and equipment support. Handling and tagging of fulmars was conducted under licences from the British Trust for Ornithology and the UK Home Office. EE was funded by a Marine Alliance for Science and Technology for Scotland/University of Aberdeen College of Life Sciences and Medicine studentship and LQ was supported by a NERC Studentship. Thanks also to the many colleagues who assisted with fieldwork during the project, and to Helen Bailey and Arliss Winship for advice on implementing the state-space model.

State-space modelling of geolocation data reveals sex differences in the use of management areas by breeding northern fulmars

We thank Orkney Islands Council for access to Eynhallow and Talisman Energy (UK) Ltd and Marine Scotland for fieldwork and equipment support. Handling and tagging of fulmars was conducted under licences from the British Trust for Ornithology and the UK Home Office. EE was funded by a Marine Alliance for Science and Technology for Scotland/University of Aberdeen College of Life Sciences and Medicine studentship and LQ was supported by a NERC Studentship. Thanks also to the many colleagues who assisted with fieldwork during the project, and to Helen Bailey and Arliss Winship for advice on implementing the state-space model.

Materials and methods for tagging purse seine and baitboat caught tunas

ENGLISH: The percentages of return of purse seine-tagged yellowfin and baitboat- tagged yellowfin and skipjack subjected to various combinations of treatments were tested statistically to determine the effects of the treatments. For yellowfin the precentages of return differed among sets or dates of release, and were higher for double-tagged fish, larger fish, and fish released soonest during each set. For skipjack the percentages of return differed among dates of release, and were higher for single-tagged fish and fish tagged in plastic-covered cradles. SPANISH: Los porcentajes de retorno de aleta amarilla marcado en barcos cerqueros y los de aleta amarilla y barrilete marcados en los de carnada sometidos a varias combinaciones de tratamientos fueron probados estadísticamente para determinar los efectos de los tratamientos. Con respecto al aleta amarilla los porcentajes de retorno fueron diferentes entre los lances o fechas de liberación, y fueron más altos para los peces con doble marca, peces de más talla y peces liberados lo más pronto durante cada lance. Los porcentajes de retorno del barrilete fueron diferentes entre las fechas de liberación, y fueron más altos para peces con una sola marca y peces marcados en cunas cubiertas con plástico. (PDF contains 41 pages.)

Growth of skipjack, Katsuwonus pelamis, and yellowfin, Thunnus albacares, tunas in the Eastern Pacific Ocean, as estimated from tagging data

ENGLISH: The average linear growth rate of skipjack in the eastern Pacific is less than 1 mm per day except for fish 375 to 424 mm in length at release. The growth rate shows a decrease with increasing length and increasing time at liberty. The growth rate of fish in the length range of about 43 to 57 cm is apparently more rapid in the eastern Pacific than in the western Pacific. Dsing data for the northeastern and southeastern Pacific combined, K and ~ were estimated to be 0.658 (on an annual basis) and 885 mm, respectively, by the ungrouped method and 0.829 and 846 mm, respectively, by the grouped method. Sensitivity analyses have shown however, that the estimates of these parameters are poorly determined by the sum of squares method used to derive them. Estimates of K and ~ for the eastern Pacific tend to be lower and higher, respectively, than those for the western Pacific. The average linear growth rate of yellowfin in the eastern Pacific is a little less than 1 mm per day for fish between about 25 and 100 cm in length at release. The growth appears to be most rapid in Area 2 (Revillagigedo Islands) and slowest in Areas 1 (Baja California), 5 (Central America- Colombia), and 6 (Ecuador-Peru). There is considerable variation in the growth rates of individual fish. The growth does not show a decrease with increasing length or increasing time at liberty so realistic estimates of the parameters of the von Bertalanffy or other similar equations cannot be calculated from these data. If realistic estimates of these parameters are to be secured larger fish must be tagged and released or many more long-term returns from fish to about 100 cm in length at release must be obtained. The growth patterns for the eastern Pacific, central Pacific and eastern Atlantic found by most other investigators differ from one another and from those found in the present study. Some of these differences may be real and others may be due to deficiencies in the data or the methods of analysis. Estimates obtained from tagging data are believed to be realistic provided the tags do not inhibit the growth of the fish. It appears that the growth rates of single- and double-tagged fish are the same; this indicates, though not unequivocally, that the tags do not inhibit the growth. SPANISH: La tasa media de crecimiento lineal del barrilete en el Pacífico oriental es inferior a lmm/día, excepto en el caso de peces de entre 375y 424mm de longitud de liberación. La tasa de crecimiento disminuye a medida que aumenta la longitud y el tiempo en libertad. La tasa de crecimiento de peces de entre unos 43 y 57 cm de longitud parece ser mayor en el Pacífico oriental que en el occidental. A partir de datos del Pacífico nororiental y suroriental combinados, se estimaron K y loo en 0.658 (anual) y 885mm, respectivamente, usando el método no agrupado, y 0.829 y 846mm, respectivamente, usando el método agrupado. Sin embargo, los análisis de sensitividad han demostrado que el método de suma de cuadrados utilizado para derivar las estimaciones de estos parámetros las determina con poca precisión. Las estimaciones de K y loo para el Pacífico oriental suelen ser inferiores y superiores, respectivamente, a los del Pacífico occidental. La tasa media de crecimiento lineal del aleta amarilla en el Pacífico oriental es ligeramente inferior a lmm/día para los peces de entre unos 25y 100cmde longitud de liberación. El crecimiento parece ser más rápido en el Area 2(Islas Revillagigedo),y más lento en las Areas 1(Baja California), 5 (Centroamérica-Colombia), y 6 (Ecuador-Perú). Las tasas de crecimiento de peces individuales varían considerablemente. El crecimiento no muestra una disminuciónconun aumento en la longitud o en el tiempo en libertad, y por consecuencia no se se pueden calcular estimaciones realistas de los parámetros de la ecuación de von Bertalanffy u otras ecuaciones similares a partir de estos datos. Para obtener estimaciones realistas de estos parámetros sería necesario marcar peces mayores u obtener muchas más devoluciones a largo plazo de marcas de peces de unos 100cm de longitud de liberación. Los patrones de crecimiento correspondientes al Pacífico oriental, Pacífico central, y Atlántico oriental descubiertos por la mayoría de los investigadores son diferentes entre síy también de los del presente estudio. Es posibleque algunas de estas diferencias sean verdaderas, mientras que otras se deban a faltas en los datos on en los métodos analíticos utilizados. Se considera que las estimaciones obtenidas a partir de los datos de marcado son realistas, suponiendo siempre que las marcas no impidan el crecimiento de los peces. Parece ser que las tasas de crecimiento de peces con una marca y con dos son idénticas, lo cual indica, aunque sin certeza total, que las marcas no ejercen tal efecto. (PDF contains 76 pages.)

Comparison of six tag types in sea-trout tagging experiments in the Baltic Sea

In the international Baltic Sea trout tagging experiment 27 753 hatchery reared sea trout smolts were tagged in Denmark, Finland, Poland and Sweden in 1979 and 1980. The fish were tagged with the original Carlin tag, two modified Carlin tag types (Canadian and Finnish polythene), streamer and Floy tags and Polish tags attached with Monel metal wire. The tag returns were affected by the place of release and smolt quality. The best results were obtained in the case of tags attached with double wire or thread -original Carlin, Canadian and Finnish polythene. The poorest results were obtained with streamer tags.

Vibrational spectroscopy of phthalocyanine and naphthalocyanine in sandwich-type (na)phthalocyaninato and porphyrinato rare earth complexes: Part 14. The infrared and Raman characteristics of phthalocyanine in “pinwheel-like” homoleptic bis[1,8,15,22-tetrakis(3-pentyloxy)phthalocyaninato] rare earth(III) double-decker complexes

The infrared (IR) spectroscopic data and Raman spectroscopic properties for a series of 13 “pinwheel-like” homoleptic bis(phthalocyaninato) rare earth complexes M[Pc(α-OC5H11)4]2 [M = Y and Pr–Lu except Pm; H2Pc(α-OC5H11)4 = 1,8,15,22-tetrakis(3-pentyloxy)phthalocyanine] have been collected and comparatively studied. Both the IR and Raman spectra for M[Pc(α-OC5H11)4]2 are more complicated than those of homoleptic bis(phthalocyaninato) rare earth analogues, namely M(Pc)2 and M[Pc(OC8H17)8]2, but resemble (for IR) or are a bit more complicated (for Raman) than those of heteroleptic counterparts M(Pc)[Pc(α-OC5H11)4], revealing the decreased molecular symmetry of these double-decker compounds, namely S8. Except for the obvious splitting of the isoindole breathing band at 1110–1123 cm−1, the IR spectra of M[Pc(α-OC5H11)4]2 are quite similar to those of corresponding M(Pc)[Pc(α-OC5H11)4] and therefore are similarly assigned. With laser excitation at 633 nm, Raman bands derived from isoindole ring and aza stretchings in the range of 1300–1600 cm−1 are selectively intensified. The IR spectra reveal that the frequencies of pyrrole stretching and pyrrole stretching coupled with the symmetrical CH bending of –CH3 groups are sensitive to the rare earth ionic size, while the Raman technique shows that the bands due to the isoindole stretchings and the coupled pyrrole and aza stretchings are similarly affected. Nevertheless, the phthalocyanine monoanion radical Pc′− IR marker band of bis(phthalocyaninato) complexes involving the same rare earth ion is found to shift to lower energy in the order M(Pc)2 > M(Pc)[Pc(α-OC5H11)4] > M[Pc(α-OC5H11)4]2, revealing the weakened π–π interaction between the two phthalocyanine rings in the same order.

Tuning the valence of the cerium center in (Na)phthalocyaninato and porphyrinato cerium double-deckers by changing the nature of the tetrapyrrole ligands

A series of 7 cerium double-decker complexes with various tetrapyrrole ligands including porphyrinates, phthalocyaninates, and 2,3-naphthalocyaninates have been prepared by previously described methodologies and characterized with elemental analysis and a range of spectroscopic methods. The molecular structures of two heteroleptic \[(na)phthalocyaninato](porphyrinato) complexes have also been determined by X-ray diffraction analysis which exhibit a slightly distorted square antiprismatic geometry with two domed ligands. Having a range of tetrapyrrole ligands with very different electronic properties, these compounds have been systematically investigated for the effects of ligands on the valence of the cerium center. On the basis of the spectroscopic (UV−vis, near-IR, IR, and Raman), electrochemical, and structural data of these compounds and compared with those of the other rare earth(III) counterparts reported earlier, it has been found that the cerium center adopts an intermediate valence in these complexes. It assumes a virtually trivalent state in cerium bis(tetra-tert-butylnaphthalocyaninate) as a result of the two electron rich naphthalocyaninato ligands, which facilitate the delocalization of electron from the ligands to the metal center. For the rest of the cerium double-deckers, the cerium center is predominantly tetravalent. The valences (3.59−3.68) have been quantified according to their LIII-edge X-ray absorption near-edge structure (XANES) profiles.

Raman spectroscopic characteristics of phthalocyanine and naphthalocyanine in sandwich-type phthalocyaninato and porphyrinato rare earth complexes Part 5. – Raman spectroscopic characteristics of naphthalocyanine in mixed [tetrakis(4-tert-butylphenyl)porphyrinato]-(naphthalocyaninato) rare earth double-deckers

Raman spectra were recorded in the range 400–1800 cm−1 for a series of 15 mixed \[tetrakis(4-tert-butylphenyl)porphyrinato](2,3-naphthalocyaninato) rare earth double-deckers M(TBPP)(Nc) (M = Y; La–Lu except Pm) using laser excitation at 632.8 and 785 nm. Comparisons with bis(naphthalocyaninato) rare earth counterparts reveal that the vibrations of the metallonaphthalocyanine M(Nc) fragment dominate the Raman features of M(TBPP)(Nc). When excited with radiation of 632.8 nm, the most intense vibration appears at about 1595 cm−1, due to the naphthalene stretching. These complexes exhibit the marker Raman band for Nc•− as a medium-intense band in the range 1496–1507 cm−1, attributed to the coupling of pyrrole and aza stretching, while the marker Raman band of Nc2− in intermediate-valence Ce(TBPP)(Nc) appears as a strong band at 1493 cm−1 and is due to the isoindole stretchings. By contrast, when excited with radiation of 785 nm that is in close resonance with the main Q absorption band of the naphthalocyanine ligand, the ring radial vibrations at ca 680 and 735 cm−1 for MIII(TBPP)(Nc) are selectively intensified and are the most intense bands. For the cerium double-decker, the most intense vibration also acting as the marker Raman band of Nc2− appears at 1497 cm−1 with contributions from both pyrrole CC and aza CN stretches. The same vibrational modes show weak to medium intensity scattering at 1506–1509 cm−1 for MIII(TBPP)(Nc) and this is the marker Raman band of Nc•− when thus excited. The scatterings due to the Nc breathings, ring radial vibration, aza group stretchings, naphthalene stretchings, benzoisoindole stretchings and the coupling of pyrrole CC and aza CN stretchings in MIII(TBPP)(Nc) are all slightly blue shifted along with the decrease in rare earth ionic radius, confirming the effects of increased ring–ring interactions on the Raman characteristics of naphthalocyanine in the mixed ring double-deckers.

Characterization of commercial double-walled carbon nanotube material : composition, structure, and heat capacity

A purified commercial double-walled carbon nanotube (DWCNT) sample was investigated by transmission electron microscopy (TEM), thermogravimetry (TG), and Raman spectroscopy. Moreover, the heat capacity of the DWCNT sample was determined by temperature-modulated differential scanning calorimetry in the range of temperature between -50 and 290 °C. The main thermo-oxidation characterized by TG occurred at 474 °C with the loss of 90 wt% of the sample. Thermo-oxidation of the sample was also investigated by high-resolution TG, which indicated that a fraction rich in carbon nanotube represents more than 80 wt% of the material. Other carbonaceous fractions rich in amorphous coating and graphitic particles were identified by the deconvolution procedure applied to the derivative of TG curve. Complementary structural data were provided by TEM and Raman studies. The information obtained allows the optimization of composites based on this nanomaterial with reliable characteristics.

St. John’s wort and Kava in treating major depressive disorder with comorbid anxiety : a randomised double-blind placebo-controlled pilot trial

Objective - We report the first randomised controlled trial (RCT) using a combination of St. John’s wort (SJW) and Kava for the treatment of major depressive disorder (MDD) with comorbid anxiety. Methods - Twenty-eight adults with MDD and co-occurring anxiety were recruited for a double-blind RCT. After a placebo run-in of 2 weeks, the trial had a crossover design testing SJW and Kava against placebo over two controlled phases, each of 4 weeks. The primary analyses used intention-to-treat and completer analyses. Results - On both intention-to-treat ( p¼0.047) and completer analyses ( p¼0.003), SJW and Kava gave a significantly greater reduction in self-reported depression on the Beck Depression Inventory (BDI-II) over placebo in the first controlled phase. However, in the crossover phase, a replication of those effects in the delayed medication group did not occur. Nor were there significant effects on anxiety or quality of life. Conclusion - There was some evidence of antidepressant effects using SJW and Kava in a small sample with comorbid anxiety. Possible explanations for the absence of anxiolysis may include a potential interaction with SJW, the presence of depression, or an inadequate dose of Kava.