bluuebunny/crossref_metadata_2025 huggingface dataset:
Morphological indicators and the time of transition to flower in Ptilotus nobilis (Amaranthaceae)
Authors: Orzek Sybille , Williams Richard R. , Johnston Margaret E.
Published: October 2012

Floral initiation of Ptilotus nobilis (Lindl.) F.Muell. was investigated by both scanning electron and light microscopy to develop a template for the transition from vegetative to reproductive growth. Four developmental stages were identified, including two vegetative and two reproductive stages. Bract initiation was accompanied by a significant increase in meristem area and diameter, and was defined as the onset of flowering. At the transition, meristem diameters and areas increased 1.5 and 2.3 times, respectively. Most morphological traits, including leaf number, mean leaf area and total leaf area, increased gradually at the time of transition and were further investigated in a subsequent glasshouse trial. Floral initiation occurred very early and at 25 days after sowing all plants had entered the reproductive phase. At the onset of flowering leaf and node number increased significantly with 3.6 and 4.2, respectively. Branching and leaf area expansion were identified as post-initiation processes, and increased by 2.8 (total foliage area), 3.3 (total leaf area) and 1.8 (mean leaf area). During the vegetative phase a discolouration of the shoot apical meristem was noted and underlying reasons are discussed.
Two Arabidopsis thaliana dihydrodipicolinate synthases, DHDPS1 and DHDPS2, are unequally redundant
Authors: Jones-Held Susan , Ambrozevicius Luciana Pimenta , Campbell Michael , Drumheller Bradley , Harrington Emily , Leustek Thomas
Published: October 2012

In Arabidopsis thalinana (L.) Heynh., DHDPS1 and DHDPS2 encode orthologous dihydrodipicolinate synthases (DHDPS), the first enzyme of the lysine (Lys) biosynthesis pathway. A TDNA insertion mutant of dhdps2 was previously reported to be viable and to accumulate free threonine (Thr). Analysis of additional TDNA insertion lines showed that dhdps1 and dhdps2 mutants are both viable and that whereas dhdps2 mutants accumulate Thr, dhdps1 plants do not. Thr-accumulation was complemented by heterologous expression of Escherichia coli DapA, indicating that the phenotype is due to reduced DHDPS activity in dhdps2. DHDPS1 contributes ~30% towards the total DHDPS activity in leaves of young plants and DHDPS2 contributes 70%; therefore, the threshold of activity resulting in Thr accumulation lies within this narrow range. dhdps1–dhdps2 double mutants could not be isolated, even after exogenous feeding with Lys. Segregation analysis indicated that gametes lacking functional DHDPS genes are defective, as are embryos. Plants carrying only a single DHDPS2 gene do not accumulate Thr, but they show a gametophytic defect that is partially rescued by Lys application. Despite the accumulation of Thr, dhdps2 seedlings are no more sensitive than wild-type plants to growth inhibition by Lys or the Lys precursor diaminopimelate. They also are not rescued by methionine at growth-inhibitory Lys concentrations. Exogenous application of Lys and methionine to dhdps2 mutants did not reduce the accumulation of Thr.
Temperature influences the ability of tall fescue to control transpiration in response to atmospheric vapour pressure deficit
Authors: Sermons Shannon M. , Seversike Thomas M. , Sinclair Thomas R. , Fiscus Edwin L. , Rufty Thomas W.
Published: October 2012

Water availability for turfgrass systems is often limited and is likely to become more so in the future. Here, we conducted experiments that examined the ability of tall fescue (Festuca arundinacea Schreb.) to control transpiration with increasing vapour pressure deficit (VPD) and determined whether control was influenced by temperature. The first study was under steady-state conditions at two temperatures (21 and 27°C) and two VPDs (1.2 and 1.8 kPa). At the lower temperature, water use was similar at both VPDs, indicating a restriction of transpiration at high VPD. At 27°C, transpiration control at high VPD was weakened and root growth also declined; both responses increase susceptibility to water-deficit stress. Another series of experiments was used to examine the physiological stability of the transpiration control. Temperature and VPD were adjusted in a stepwise manner and transpiration measured across a range of VPD in the days following environmental shifts. Results indicated that VPD control acclimated to the growth environment, with adjustment to drier conditions becoming evident after ~1 week. Control was again more effective at cool than at hot temperatures. Collectively, the results indicate that transpiration control by this cool season grass is most effective in the temperature range where it is best adapted.
Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle
Authors: Caixeta Ester Siqueira , Machado Mariana Fernandes , Ripamonte Paula , Price Christopher , Buratini José
Published: October 2012

FSH induces expansion of bovine cumulus–oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.
Evaluating the Performance of Vertical Longlines to Survey Reef Fish Populations in the Northern Gulf of Mexico
Authors: Gregalis Kevan C. , Schlenker Lela S. , Drymon J. Marcus , Mareska John F. , Powers Sean P.
Published: October 2012

A common critique of many stock assessments is the lack of fishery‐independent abundance indices and age composition data. Such data streams are essential in evaluating population trajectories that are derived largely from harvest and age composition of landings. For example, high scientific uncertainty in the most recent stock assessment of Gulf of Mexico red snapper Lutjanus campechanus resulted from a conflict between trends in fishery‐dependent and fishery‐independent data. Because sample sizes for the latter data were an order of magnitude lower, resolution of the conflicting trends was even more problematic. Recognizing the need for cost‐effective expansion of fishery‐independent data in the region, we evaluated the performance of vertical longline surveys for sampling reef fish within a large artificial reef zone in the northern Gulf of Mexico. Specifically, we (1) determined species composition and the length frequency of red snapper (the dominant species captured) as a function of hook size and bait type within our survey area during 2010; (2) evaluated the effect of different soak times on catch for various hook types (a combination of hook size and bait type); and (3) utilized our results to test the effect of artificial reef type on red snapper CPUE and mean size. During March–November 2010, we conducted 532 vertical longline sets, capturing 1,217 red snapper that ranged from 184 to 827 mm FL. Mean FL of red snapper differed among hook sizes, with 3/0 and 8/0 hooks sampling smaller fish than 11/0 hooks. Soak time trials revealed a significant effect of soak time on CPUE, with peak catch rates observed at 5 min. As habitat area increased, the mean size and CPUE of red snapper increased. We conclude that our vertical longline is an effective gear for sampling red snapper, and we recommend protocols to maximize its utility and standardize its use.
The genesis of Zelboraf: Targeting mutant B-Raf in melanoma
Authors: Davis Matthew J. , Schlessinger Joseph
Published: October 2012

The protein kinase B-Raf is a critical component of the Ras/MAPK signaling pathway. An oncogenic B-Raf mutation that constitutively activates the kinase was identified in z50% of melanoma patients and in other cancers. A structure-guided drug discovery approach enabled the development of Zelboraf, a targeted inhibitor of oncogenic B-Raf. This drug has been used successfully in the clinic to treat metastatic melanoma patients harboring B-Raf mutations.
The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function
Authors: Engel Benjamin D. , Ishikawa Hiroaki , Wemmer Kimberly A. , Geimer Stefan , Wakabayashi Ken-ichi , Hirono Masafumi , Craige Branch , Pazour Gregory J. , Witman George B. , Kamiya Ritsu , Marshall Wallace F.
Published: October 2012

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.
Progeria: Translational insights from cell biology
Authors: Gordon Leslie B. , Cao Kan , Collins Francis S.
Published: October 2012

Cell biologists love to think outside the box, pursuing many surprising twists and unexpected turns in their quest to unravel the mysteries of how cells work. But can cell biologists think outside the bench? We are certain that they can, and clearly some already do. To encourage more cell biologists to venture into the realm of translational research on a regular basis, we would like to share a handful of the many lessons that we have learned in our effort to develop experimental treatments for Hutchinson-Gilford progeria syndrome (HGPS), an endeavor that many view as a “poster child” for how basic cell biology can be translated to the clinic.
ORCID: a system to uniquely identify researchers
Authors: HAAK Laurel L. , FENNER Martin , PAGLIONE Laura , PENTZ Ed , RATNER Howard
Published: October 2012

The Open Researcher & Contributor ID (ORCID) registry presents a unique opportunity to solve the problem of author name ambiguity. At its core the value of the ORCID registry is that it crosses disciplines, organizations, and countries, linking ORCID with both existing identifier schemes as well as publications and other research activities. By supporting linkages across multiple datasets – clinical trials, publications, patents, datasets – such a registry becomes a switchboard for researchers and publishers alike in managing the dissemination of research findings. We describe use cases for embedding ORCID identifiers in manuscript submission workflows, prior work searches, manuscript citations, and repository deposition. We make recommendations for storing and displaying ORCID identifiers in publication metadata to include ORCID identifiers, with CrossRef integration as a specific example. Finally, we provide an overview of ORCID membership and integration tools and resources.
Book Reviews
Authors: NICHOLAS David
Published: October 2012

Information Users and Usability in the Digital Age Chowdhury and Chowdhury Facet, 2011, 304pp. ISBN: 978‐1‐85604‐597‐1, £44.95 (pbk)
Interactive Information Seeking, Behaviour and Retrieval Ruthven and Kelly Facet, 2011, 320pp. ISBN: 978‐1856047074, £44.95 (pbk)
Book Reviews
Authors: SMART Pippa
Published: October 2012

Books: A Living History Martyn Lyons Thames & Hudson, 2011, 214pp. ISBN 978‐0500251652, £19.95 hbk
Applications of quantum cascade lasers in plasma diagnostics: a review
Authors: Röpcke J , Davies P B , Lang N , Rousseau A , Welzel S
Published: October 2012

Over the past few years mid-infrared absorption spectroscopy based on quantum cascade lasers operating over the region from 3 to 12 µm and called quantum cascade laser absorption spectroscopy or QCLAS has progressed considerably as a powerful diagnostic technique for in situ studies of the fundamental physics and chemistry of molecular plasmas. The increasing interest in processing plasmas containing hydrocarbons, fluorocarbons, nitrogen oxides and organo-silicon compounds has led to further applications of QCLAS because most of these compounds and their decomposition products are infrared active. QCLAS provides a means of determining the absolute concentrations of the ground states of stable and transient molecular species at time resolutions below a microsecond, which is of particular importance for the investigation of reaction kinetics and dynamics. Information about gas temperature and population densities can also be derived from QCLAS measurements. Since plasmas with molecular feed gases are used in many applications such as thin film deposition, semiconductor processing, surface activation and cleaning, and materials and waste treatment, this has stimulated the adaptation of QCLAS techniques to industrial requirements including the development of new diagnostic equipment. The recent availability of external cavity (EC) QCLs offers a further new option for multi-component detection. The aim of this paper is fourfold: (i) to briefly review spectroscopic issues arising from applying pulsed QCLs, (ii) to report on recent achievements in our understanding of molecular phenomena in plasmas and at surfaces, (iii) to describe the current status of industrial process monitoring in the mid-infrared and (iv) to discuss the potential of advanced instrumentation based on EC-QCLs for plasma diagnostics.
High-efficiency dye-sensitized solar cells using ferrocene-based electrolytes and natural photosensitizers
Authors: Sönmezoğlu Savaş , Akyürek Cafer , Akin Seçkin
Published: October 2012

A new and promising dye-sensitized solar cell (DSSC) bilayer design was developed using an Fe2+/Fe3+ (ferrocene) liquid electrolyte and natural dyes extracted from Hypericum perforatum, Rubia tinctorum L. and Reseda luteola. The photovoltaic parameters controlling the device performance were then investigated. A DSSC based on quercetin dye displayed the most efficient solar to electricity conversion efficiency compared with other dyes with a maximum η value of 2.17%. Maximum overall conversion efficiencies under simulated sunlight that was comparable to natural photosynthesis were increased by 15%. The identification of appropriate additives for improving V
OC without causing dye degradation may result in further enhancement of cell performance, making the practical application of such systems more suitable for achieving economically viable solar energy devices.
Plasma chemistry model of surface microdischarge in humid air and dynamics of reactive neutral species
Authors: Sakiyama Yukinori , Graves David B , Chang Hung-Wen , Shimizu Tetsuji , Morfill Gregor E
Published: October 2012

We present a numerical model of a surface microdischarge (SMD) in humid air at atmospheric pressure. Our model includes over 50 species and 600 elementary reactions and consists of two, coupled well-mixed regions: a discharge layer with both charged and neutral species and an afterglow region consisting only of neutral species. Multiple time steps employed in our model enable capturing rapid dynamic behaviour in the discharge layer as well as the relatively slow diffusion and reaction in the afterglow. A short duration, high electric field is assumed to be excited at 10 kHz in the discharge region with power density maintained at 0.05 W cm−2. Among the predicted dominant species in the afterglow are O3, N2O5, N2O, HNO3, H2, NO3, H2O2, HNO2 and NO2. The results are in qualitative agreement with Fourier transform infrared absorption spectroscopy. Our simulation results show that density of those reactive species continues to evolve significantly in time, even after ∼15 min of SMD exposure. This result suggests that SMD treatments on the order of minutes or less may involve significant neutral species concentration and flux transients, potentially affecting interpretation of results.
Instrumented Minimally Invasive Spinal-Transforaminal Lumbar Interbody Fusion (MIS-TLIF)
Authors: Kim Jin-Sung , Jung Byungjoo , Lee Sang-Ho
Published: October 2012

Study Design:

A retrospective study.

Objective:

To determine the clinical and radiologic outcomes of the long-term results of instrumented minimally invasive spinal-transforaminal lumbar interbody fusion (MIS-TLIF) in unstable, single-level, low-grade, isthmic spondylolisthesis (IS) or degenerative spondylosis (DS) including degenerative spondylolisthesis, foraminal stenosis with central stenosis, degenerative disk disease, and recurrent disk herniation.

Summary of Background Data:

MIS-TLIF is a common surgical procedure to treat lumbar spondylolisthesis. However, there are no studies that have documented the long-term results of MIS-TLIF.

Methods:

Forty-four patients who had undergone instrumented MIS-TLIF between July 2003 and January 2005 were retrospectively reviewed. The visual analog scale, Oswestry Disability Index, patient satisfaction rate, and the patient’s return-to-work status were used to assess clinical and functional outcomes. Radiologic follow-up were carried out in patients to check for adjacent segmental degeneration (ASD). The plain radiographs, computed tomography and magnetic resonance imaging, were used in all patients in last follow-up period.

Results:

The mean visual analog scale scores for back and leg pain decreased from 5.8 and 7 to 3.5 and 3.7, respectively, in the DS group (n=19) and from 6.8 and 6.9 to 1.8 and 2.0, respectively, in the IS group (n=25) (P<0.001). The mean Oswestry Disability Index scores improved from 61.7% to 21.5% in the DS group and from 53.9% to 16% in the IS group (P<0.001). Patient satisfaction rate was 80% and 81% in the DS and IS groups, respectively. Evidence of fusion was observed radiologically in 24 (96%) and 19 (100%) of the patients in the IS and the DS group, respectively, giving an overall fusion rate of 97.7% (43/44). The final ASD rate, observed using radiography, was 68.4% (13/19) in the DS and 40% (10/25) in the IS group. However, 15.8% (3/19) in the DS and 4% (1/25) in the IS group had symptoms associated with ASD.

Conclusions:

The long-term clinical and radiologic outcomes after instrumented MIS-TLIF in patients with unstable single-level spine are favorable.
Spectroscopy of H
3
+
based on a new high-accuracy global potential energy surface
Authors: Polyansky Oleg L. , Alijah Alexander , Zobov Nikolai F. , Mizus Irina I. , Ovsyannikov Roman I. , Tennyson Jonathan , Lodi Lorenzo , Szidarovszky Tamás , Császár Attila G.
Published: October 2012

The molecular ion

is the simplest polyatomic and poly-electronic molecular system, and its spectrum constitutes an important benchmark for which precise answers can be obtained
ab initio
from the equations of quantum mechanics. Significant progress in the computation of the ro–vibrational spectrum of

is discussed. A new, global potential energy surface (PES) based on
ab initio
points computed with an average accuracy of 0.01 cm
−1
relative to the non-relativistic limit has recently been constructed. An analytical representation of these points is provided, exhibiting a standard deviation of 0.097 cm
−1
. Problems with earlier fits are discussed. The new PES is used for the computation of transition frequencies. Recently measured lines at visible wavelengths combined with previously determined infrared ro–vibrational data show that an accuracy of the order of 0.1 cm
−1
is achieved by these computations. In order to achieve this degree of accuracy, relativistic, adiabatic and non-adiabatic effects must be properly accounted for. The accuracy of these calculations facilitates the reassignment of some measured lines, further reducing the standard deviation between experiment and theory.
Hydride spectroscopy of the diffuse interstellar medium: new clues on the gas fraction in molecular form and cosmic ray ionization rate in relation to H
3
+
Authors: Gerin M. , Levrier F. , Falgarone E. , Godard B. , Hennebelle P. , Le Petit F. , De Luca M. , Neufeld D. , Sonnentrucker P. , Goldsmith P. , Flagey N. , Lis D. C. , Persson C. M. , Black J. H. , Goicoechea J. R. , Menten K. M.
Published: October 2012

The Herschel-guaranteed time key programme PRobing InterStellar Molecules with Absorption line Studies (PRISMAS)

1

is providing a survey of the interstellar hydrides containing the elements C, O, N, F and Cl. As the building blocks of interstellar molecules, hydrides provide key information on their formation pathways. They can also be used as tracers of important physical and chemical properties of the interstellar gas that are difficult to measure otherwise. This paper presents an analysis of two sight-lines investigated by the PRISMAS project, towards the star-forming regions W49N and W51. By combining the information extracted from the detected spectral lines, we present an analysis of the physical properties of the diffuse interstellar gas, including the electron abundance, the fraction of gas in molecular form, and constraints on the cosmic ray ionization rate and the gas density.
Exploring the central molecular zone of the Galaxy using spectroscopy of H
3
+
and CO
Authors: Geballe T. R.
Published: October 2012

The central 400 parsecs of the Milky Way, a region known as the central molecular zone (CMZ), contains interstellar gas in a wide range of physical environments, from ultra-hot, rarified and highly ionized to warm, dense and molecular. The combination of infrared spectroscopy of

and CO is a powerful way to determine the basic properties of molecular interstellar gas, because the abundance ratio of

to CO in ‘dense’ clouds is quite different from that in ‘diffuse’ clouds. Moreover, the energy-level structure and the radiative properties of

combined with the unusually warm temperatures of molecular gas in the CMZ make

a unique probe of the physical conditions there. This paper describes how, using infrared absorption spectroscopy of

and CO, it has been discovered that a large fraction of the volume of the CMZ is filled with warm, diffuse and partially molecular gas moving at speeds of up to approximately 200 km s
−1
and that the mean cosmic ray ionization rate in the CMZ exceeds by roughly an order of magnitude values found in diffuse molecular clouds elsewhere in the Galaxy.
The Jahn–Teller effect in the 3
pe
′ Rydberg state of H
3
: review of experimental and
ab initio
determinations
Authors: Jungen Ch. , Jungen M. , Pratt S. T.
Published: October 2012

The dissociative recombination (DR) of H

ions with electrons, producing neutral atomic and molecular fragments, is driven primarily by the vibronic Jahn–Teller (JT) interaction between the electronic components of the
pe
′ e
−
–H

collision (Rydberg) channel. The JT parameters characterizing this interaction are therefore of great interest as they are required for the theoretical predictions of the DR cross section. In this contribution, we review various determinations of these quantities that have been made previously, based both on spectroscopic studies of 3
pe
′ Rydberg-excited H
3
states, and on the analysis of the corresponding
ab initio
H
3
Rydberg potential surfaces near the conical intersection (D
3h
symmetry) for
n
=3−5. The highly correlated theoretical 3
pe
′ potential surfaces of Mistrík
et al.
are used for a new determination of both the linear and quadratic JT terms.
Oscillibacter ruminantium sp. nov., isolated from the rumen of Korean native cattle
Authors: Lee Geun-Hye , Rhee Moon-Soo , Chang Dong-Ho , Lee Jonghwan , Kim Seil , Yoon Min Ho , Kim Byoung-Chan
Published: October 2012

A strictly anaerobic, Gram-negative, non-spore-forming bacterium, designated GH1T, was isolated from the rumen of Korean native cattle (HanWoo). Cells were straight to slightly curved rods (2.0–4.5 µm long) and were motile by peritrichous flagella. The isolate grew at 30–45 °C (optimum 40 °C), at pH 5.5–6.5 (optimum pH 6.0) and with up to 3.5 % (w/v) NaCl. Strain GH1Tproduced acid fromd-glucose,d-ribose andd-xylose, with butyric acid being the major end product. The genomic DNA G+C content was 54.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain GH1Twas most closely related toOscillibacter valericigenesSjm18-20T(97.3 % 16S rRNA gene sequence similarity). DNA–DNA hybridization between strain GH1TandO. valericigenesDSM 18026Tshowed 24 % reassociation. The major fatty acids were iso-C13 : 0(13.0 %), iso-C15 : 0(17.6 %), anteiso-C15 : 0(8.4 %) and C14 : 0(4.1 %), and the cellular fatty acid methyl esters as dimethylacetals (DMAs) were C16 : 0DMA (17.8 %), iso-C15 : 0DMA (15.2 %) and C14 : 0DMA (4.52 %). The cell-wall peptidoglycan of strain GH1Tcontainedmeso-diaminopimelic acid and the major cell-wall sugar was galactose. Based on 16S rRNA gene sequence similarity, phylogenetic analysis, DNA G+C content, DNA–DNA relatedness and distinct phenotypic characteristics, strain GH1Tis classified in the genusOscillibacteras a member of a novel species, for which the nameOscillibacterruminantiumsp. nov. is proposed. The type strain is GH1T( = KCTC 15176T = NBRC 108824T = JCM 18333T).
Dissulfuribacter thermophilus gen. nov., sp. nov., a thermophilic, autotrophic, sulfur-disproportionating, deeply branching deltaproteobacterium from a deep-sea hydrothermal vent
Authors: Slobodkin A. I. , Reysenbach A.-L. , Slobodkina G. B. , Kolganova T. V. , Kostrikina N. A. , Bonch-Osmolovskaya E. A.
Published: October 2012

A thermophilic, anaerobic, chemolithoautotrophic bacterium (strain S69T) was isolated from a deep-sea hydrothermal vent chimney located on the Eastern Lau Spreading Center and Valu Fa Ridge, Pacific Ocean, at a depth of 1910 m using anoxic medium with elemental sulfur as the only energy source. Cells of strain S69T were Gram-negative short rods, 0.4–0.6 µm in diameter and 1.0–2.5 µm in length, motile with a single polar flagellum. The temperature range for growth was 28–70 °C, with an optimum at 61 °C. The pH range for growth was 5.6–7.9, with optimum growth at pH 6.8. Growth of strain S69T was observed at NaCl concentrations ranging from 0.9 to 5.0 %, with an optimum at 1.8–2.7 (w/v). Strain S69T grew anaerobically with elemental sulfur as an energy source and bicarbonate/CO2 as a carbon source. Elemental sulfur was disproportionated to sulfide and sulfate. Growth was enhanced in the presence of poorly crystalline Fe(III) oxide (ferrihydrite) as a sulfide-scavenging agent. Strain S69T was also able to grow by disproportionation of thiosulfate and sulfite. Sulfate was not used as an electron acceptor either with H2 or with organic electron donors. Analysis of the 16S rRNA gene sequence revealed that the isolate formed a distinct phylogenetic branch within the

Deltaproteobacteria

. On the basis of its physiological properties and results of phylogenetic analyses, strain S69T is considered to represent a novel species of a new genus, for which the name Dissulfuribacter thermophilus gen. nov., sp. nov. is proposed. The type strain of Dissulfuribacter thermophilus is S69T ( = DSM 25762T = VKM B-2760T).
Promoter-proximal polyadenylation sites reduce transcription activity
Authors: Andersen Pia K. , Lykke-Andersen Søren , Jensen Torben Heick
Published: October 2012

Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.
Autocrine prolactin induced by the Pten–Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways
Authors: Chen Chien-Chung , Stairs Douglas B. , Boxer Robert B. , Belka George K. , Horseman Nelson D. , Alvarez James V. , Chodosh Lewis A.
Published: October 2012

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten–PI3K–Akt pathway. Conditional activation of the PI3K–Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K–Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)–Jak–Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl−/−, Prlr−/−, and Stat5−/− mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1−/−;Akt2+/− mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K–Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K–Akt pathway.
Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
Authors: Golden Molly G. , Dasen Jeremy S.
Published: October 2012

Polycomb repressive complexes (PRCs) establish and maintain gene repression through chromatin modifications, but their specific roles in cell fate determination events are poorly understood. Here we show an essential role for the PRC1 component Bmi1 in motor neuron (MN) subtype differentiation through dose-dependent effects on Hox gene expression. While Bmi1 is dispensable for generating MNs as a class, it has an essential role in specifying and determining the position of Hox-dependent MN columnar and pool subtypes. These actions are mediated through limiting anterior Hox expression boundaries, functions deployed in post-mitotic MNs, temporally downstream from morphogen gradients. Within the HoxC gene cluster, we found a progressive depletion of PRC-associated marks from rostral to caudal levels of the spinal cord, corresponding to major demarcations of MN subtypes. Selective ablation of Bmi1 elicits a derepression of more posterior Hox genes, leading to a switch in MN fates. Unexpectedly, Hox patterns and MN fates appear to be sensitive to absolute PRC1 activity levels; while reducing Bmi1 switches forelimb lateral motor column (LMC) MNs to a thoracic preganglionic (PGC) identity, elevating Bmi1 expression at thoracic levels converts PGC to LMC MNs. These results suggest that graded PRC1 activities are essential in determining MN topographic organization.
ELAV mediates 3′ UTR extension in the Drosophila nervous system
Authors: Hilgers Valérie , Lemke Sandra B. , Levine Michael
Published: October 2012

Post-transcriptional gene regulation is prevalent in the nervous system, where multiple tiers of regulatory complexity contribute to the development and function of highly specialized cell types. Whole-genome studies in Drosophila have identified several hundred genes containing long 3′ extensions in neural tissues. We show that ELAV (embryonic-lethal abnormal visual system) is a key mediator of these neural-specific extensions. Misexpression of ELAV results in the ectopic synthesis of long messenger RNAs (mRNAs) in transgenic embryos. RNA immunoprecipitation assays suggest that ELAV directly binds the proximal polyadenylation signals of many target mRNAs. Finally, ELAV is sufficient to suppress 3′ end formation at a strong polyadenylation signal when tethered to a synthetic RNA. We propose that this mechanism for coordinating 3′ UTR extension may be generally used in a variety of cellular processes.
Filter-Binding Assay for Analysis of RNA–Protein Interactions
Authors: Rio Donald C.
Published: October 2012

One of the oldest and simplest (and still very useful) methods for detecting RNA–protein interactions is the filter-binding assay. If a mixture of RNA and protein is passed through a nitrocellulose filter, the protein will be retained and the RNA will pass through. But if the protein is capable of binding RNA, then RNA will be retained on the filter as well. This protocol requires a purified protein (or chromatographic fractions) of interest and 32P-labeled RNA. To perform the assay, the protein sample is serially diluted to several concentrations. It is then mixed with a fixed amount of radioactive RNA and allowed to bind under desired conditions for 30–60 min. The binding reactions are then applied to a 96-well dot-blot apparatus with low vacuum to trap the complexes on three membranes: The top membrane traps aggregates, the middle membrane (nitrocellulose) binds proteins and RNA–protein complexes, and the bottom membrane (which is charged) collects free RNA. After washing and drying, the membranes are exposed to phosphor-imaging screens for quantitation. Alternatively, single, larger filters (that can be counted in a scintillation counter) and a filter manifold can be used.
Isolation of Total RNA from Yeast Cell Cultures: Figure 1.
Authors: Ares Manuel
Published: October 2012

This article describes two procedures for isolating total RNA from yeast cell cultures. The first allows the convenient isolation of total RNA from early log-phase cultures (vegetative cells). RNA isolated in this way is intact and sufficiently pure for use in microarray experiments, primer extension, and RNase protection mapping. With additional treatment to remove contaminating genomic DNA, the preparation is suitable for reverse transcription–polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), cDNA library construction, high-throughput sequencing of RNA, or other manipulations. However, compared to vegetative cells, the isolation of RNA from cells late in meiosis (asci and ascospores) requires additional effort. This is because a tough cell wall composed of heavily cross-linked polysaccharides and proteins is built around the four spores during meiosis and ascospore development. Therefore, an alternative protocol is presented for extracting RNA from cells late in meiosis. This alternative may also be preferable for cells from stationary cultures or from yeast strains and other fungal species isolated from the environment.
Preparation of Hyaluronan-DNA Matrices and Films
Authors: Chen Weiliam
Published: October 2012

Natural carbohydrate is a class of underexplored polymers for gene delivery. The noninflammatory and nonimmunogenic properties of hyaluronan (hyaluronic acid, HA) are important in clinical situations. It has a role in wound repair and has great lubricating ability. Moreover, the presence of hyaluronidase in vivo enables any vehicle fabricated from HA to be degraded by enzyme-mediated erosion. When DNA is entrapped in a cross-linked HA vehicle, HA-DNA fragments are released on digestion by hyaluronidase. These fragments could serve both as microcarriers of DNA and its protective mechanism. This protocol describes preparation of water-insoluble HA-DNA matrices and films designed for clinical applications, and assays for verification of their bioactivities. Plasmid DNA (pDNA) encoding platelet-derived growth factor (PDGF) is coupled to the matrices that could be implanted into chronic wounds to accelerate their healing. pDNA encoding hyaluronan synthase 2 (HAS2) is coupled to the film that could initially serve as a physical barrier and subsequently a pDNA reservoir for sustaining HAS2 transfection. This would lead to continual HA production for preventing postsurgical adhesion.
Preparing Sample Chambers for Single-Molecule FRET
Authors: Joo Chirlmin , Ha Taekjip
Published: October 2012

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30–80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. To study the conformational changes of individual molecules over extended time periods, molecules must be localized in space. This protocol describes the preparation of sample chambers with either bovine serum albumin (BSA)- or polyethylene glycol (PEG)-coated slides to which single molecules can be tethered for use in FRET studies.
Imaging and Identifying Impurities in Single-Molecule FRET Studies: Figure 1.
Authors: Joo Chirlmin , Ha Taekjip
Published: October 2012

Single-molecule (sm) Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30–80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. To study the conformational changes of individual molecules over extended time periods, the molecules must be immobilized on a coated surface that allows highly specific tethering of DNA, RNA, or protein. Nevertheless, there are always some fluorescent impurities on the surface and, without experience in sm imaging, it is often difficult to distinguish molecules of interest from impurities. This protocol describes the use of an imaging buffer that enhances the photostability of cyanine dyes used for smFRET, with emphasis on distinguishing molecules of interest from impurities.
Preparation of Drosophila Specimens for Examination by Transmission Electron Microscopy
Authors: McDonald Kent L. , Sharp David J. , Rickoll Wayne
Published: October 2012

There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue. This in turn means that fixation takes a relatively long time (measured in minutes or tens of minutes in some cases), during which the cells begin autolysis or are otherwise degraded from their original state. Drosophila requires specific preparation methods for TEM because most fly tissues are surrounded by significant diffusion barriers. In the embryo, it is the vitelline envelope, and in larvae and adults, it is the cuticle. In this article, we discuss methods that have evolved to cope with these barriers to achieve reasonable preservation of ultrastructure.
In Vivo Optical Microendoscopy for Imaging Cells Lying Deep within Live Tissue
Authors: Barretto Robert P.J. , Schnitzer Mark J.
Published: October 2012

Although in vivo microscopy has been pivotal in enabling studies of neuronal structure and function in the intact mammalian brain, conventional intravital microscopy has generally been limited to superficial brain areas such as the olfactory bulb, the neocortex, or the cerebellar cortex. For imaging cells in deeper areas, this article discusses in vivo optical microendoscopy using gradient refractive index (GRIN) microlenses that can be inserted into tissue. Our general methodology is broadly applicable to many deep brain regions and areas of the body. Microendoscopes are available in a wide variety of optical designs, allowing imaging across a range of spatial scales and with spatial resolution that can now closely approach that offered by standard water-immersion microscope objectives. The incorporation of microendoscope probes into portable miniaturized microscopes allows imaging in freely behaving animals. When combined with the broad sets of available fluorescent markers, animal preparations, and genetically modified mice, microendoscopic methods enable sophisticated experimental designs for probing how cellular characteristics may underlie or reflect animal behavior and life experience, in healthy animals and animal models of disease.
Imaging Mouse Embryonic Cardiovascular Development
Authors: Larina Irina V. , Garcia Monica D. , Vadakkan Tegy J. , Larin Kirill V. , Dickinson Mary E.
Published: October 2012

Early development of the mammalian cardiovascular system is a highly dynamic process. Live imaging is an essential tool for analyzing normal and abnormal cardiovascular development and dynamics. This article describes two optical approaches for live dynamic imaging of mouse embryonic cardiovascular development: confocal microscopy and optical coherence tomography (OCT). Confocal microscopy, used in combination with fluorescent protein reporter lines, enables visualization of the developing and remodeling cardiovascular system with submicron resolution and even allows visualization of subcellular details of labeled structures. We describe mouse transgenic lines that can be used to image the developing vasculature and characterize hemodynamics by tracking individual blood cells. Confocal microscopy of vital fluorescent markers reveals unique details about cell morphogenesis and movement; however, the imaging depth of this method is limited to ∼200 µm. This limitation can be addressed by using OCT, which allows three-dimensional (3D) imaging millimeters into tissue, although this is achieved at the expense of lower spatial resolution (2–10 µm). We describe here how OCT can be applied to the structural analysis of developing mouse embryos and hemodynamic analysis in deep embryonic vessels. These complementary approaches can be used to analyze cardiovascular defects in mutant animals to understand genetic signaling pathways regulating human development.
Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants
Authors: Carbonell Alberto , Fahlgren Noah , Garcia-Ruiz Hernan , Gilbert Kerrigan B. , Montgomery Taiowa A. , Nguyen Tammy , Cuperus Josh T. , Carrington James C.
Published: October 2012

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.