bluuebunny/crossref_metadata_2025 huggingface dataset:
Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of ‘Candidatus Liberibacter asiaticus’, Causal Agent of Huanglongbing
Authors: Damsteegt V. D. , Postnikova E. N. , Stone A. L. , Kuhlmann M. , Wilson C. , Sechler A. , Schaad N. W. , Brlansky R. H. , Schneider W. L.
Published: April 2010

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of ‘Candidatus Liberibacter asiaticus’ from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted ‘Ca. L. asiaticus’ into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of ‘Ca. L. asiaticus’ from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of ‘Ca. L. asiaticus’ in infected Murraya.
Outbreak of a New Phytophthora sp. Associated with Severe Decline of Almond Trees in Eastern Spain
Authors: Pérez-Sierra A. , León M. , Álvarez L. A. , Alaniz S. , Berbegal M. , García-Jiménez J. , Abad-Campos P.
Published: April 2010

Since 2007, a decline of young almond trees (Prunus dulcis) has been observed in different field-grown nurseries in Valencia (east-central Spain). Early symptoms in affected trees included chlorosis, wilting, cankers, and profuse stem gumming. A Phytophthora sp. was consistently isolated from cankers, roots, and soil of affected trees. It was a heterothallic species with amphigynous and/or paragynous antheridia, and its morphological features did not conform to any of the described Phytophthora species. Pathogenicity was proved by artificial inoculation, completing Koch's postulates. All isolates were sensitive to the phenylamide fungicides metalaxyl and mefenoxam. Amplification and sequencing of the internal transcribed spacer (ITS) region, translation elongation factor 1 alpha gene (EF-1α), the β-tubulin (β-tub) gene, and the region containing the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene fragment identified the species as Phytophthora taxon “niederhauserii”, and phylogenetic analyses placed it in Phytophthora Clade 7b.
Surprising Results from a Search for Effective Disinfectants for Tobacco mosaic virus–Contaminated Tools
Authors: Lewandowski Dennis J. , Hayes Amanda J. , Adkins Scott
Published: April 2010

Over 100 years after its discovery, Tobacco mosaic virus (TMV) remains an economically important pathogen for producers of many vegetatively propagated crops including petunias (Petunia × hybrida). To directly address this concern, we have developed a robust system to determine efficacy of disinfectants for treating TMV-contaminated cutting tools using a combination of preliminary screens and replicated trials. Contrary to widely held beliefs, wild-type (wt) TMV and four additional tobamovirus species infected four petunia cultivars without producing obvious viral symptoms. In contrast, a petunia isolate of TMV with 99.0% (nucleotide) and 99.4% (amino acid) coat protein sequence identity to wt TMV produced symptoms on all but one tested cultivar. We also show that TMV transmission can occur up to the twentieth petunia plant cut following a single cutting event on a TMV-infected plant. Although many new products are now available, treatment of TMV-contaminated tools with a 20% (wt/vol) solution of nonfat dry milk (NFDM) plus 0.1% Tween 20 or a 1:10 dilution of household bleach (0.6% sodium hypochlorite), two “old standbys”, completely eliminated TMV transmission to petunias. Treatment of contaminated tools with 1% (wt/vol) Virkon S or 20% NFDM also significantly reduced the incidence of infected petunias. Other treatments identified in the preliminary screens are candidates for the second phase of screening that simulates contamination during the process of taking cuttings.
Factors Influencing Epidemiology and Management of Blackberry Rust in Cultivated Rubus laciniatus
Authors: Johnson K. B. , Mahaffee W. F.
Published: April 2010

The blackberry rust pathogen Phragmidium violaceum was first observed in Oregon in spring 2005 on both commercially cultivated Rubus laciniatus (Evergreen blackberry) and naturalized R. armeniacus (Himalayan blackberry). Several commercial plantings suffered severe economic losses. In 2006 to 2008, all five spore stages of this autoecious, macrocyclic rust pathogen were observed annually, and asexual perennation of the pathogen on old leaves or in leaf buds was not evident in the disease cycle. In field experiments, teliospore germination and infection by basidiospores occurred mostly during April. On potted “trap” plants exposed for periods of 1 week under dense collections of dead leaves bearing teliospores, basidiospore infection was associated with wetness durations of >16 h with mean temperatures >8°C. Trap plants placed under the bundles of collected leaves frequently developed spermagonia, whereas only 1 of 630 trap plants placed in a production field of R. laciniatus became diseased, an indication that the effective dispersal distance of basidiospores may be limited. In growth chambers programmed for constant temperatures of 5, 10, 15, 20, 25, and 30°C, a minimum of six continuous hours of leaf wetness was required for infection by urediniospores, with >9 h required for moderate infection (>4 pustules/cm2) at 15 and 20°C. With diurnal temperature regimes averaging 5, 10, 15, 20, or 25°C, urediniospore germination and infection was highest in the range of 5 to 15°C; similarly, in the diurnal environment, >9 h of leaf wetness was required to attain moderate infection. In the field, lime sulfur applied as a delayed dormant treatment significantly suppressed teliospore germination and basidiospore infection. Over two seasons, one application of myclobutanil, a demethylation-inhibitor fungicide, applied in early May near the time of spermagonial appearance provided effective suppression of the summer epidemic.
First Report of Tomato chlorotic dwarf viroid in Tomato in France
Authors: Candresse T. , Marais A. , Tassus X. , Suhard P. , Renaudin I. , Leguay A. , Poliakoff F. , Blancard D.
Published: April 2010

Tomato chlorotic dwarf viroid (TCDVd) is a pospiviroid found naturally infecting tomato (Solanum lycopersicum L.) (3) and several ornamentals such as Brugmansia, petunia (1), and trailing verbena (4). Initially identified in North America (3), it has been reported from India, Europe (the Netherlands and United Kingdom), and Japan. At the end of 2007, 20 to 25% of tomato plants within a group of greenhouses in the Brittany Region of France were observed with top bunching, leaf curling, and epinasty symptoms. Reverse transcription (RT)-PCR with a primer pair specific for several pospiviroids (5′GGGGAAACCTGGAGCGA3′ and 5′GGGGATCCCTGAAGCGC3′) amplified the correctly sized fragment (approximately 360 bp) from total nucleic acid extracts from three symptomatic plants. The sequence of the uncloned amplification product (GenBank Accession No. EU729744) was determined, together with that of five cloned cDNAs. All sequences were highly related with a total of three mutations in these six sequences and they showed 96.9% (GQ169709 and AY372399) to 99.4% (AF162131) identity with TCDVd sequences present in GenBank. Identification of TCDVd was confirmed from the same plant samples by molecular hybridization with a Potato spindle tuber viroid (PSTVd)-specific probe (which cross-hybridizes with TCDVd to a certain extent) and by PCR with the PSTVd/TCDVd-specific 2A-1S primer pair (3) and sequencing of the amplified fragment. The French isolate is most closely related to the original tomato isolate from Canada (GenBank Accession No. AF162131). In a grow-out test involving 2,500 seeds from the original seed lot from which the symptomatic plants were derived, 2 of the 250 pools of 10 plants tested positive for TCDVd infection with the 3H1-2H1 primer pair (2). The sequence of the amplified product proved identical to the isolate detected in the original greenhouse plants, indicating a low level of seed transmission. As with other pospiviroids, which appear to be more and more frequently reported in greenhouse tomatoes, possible sources of infection include contaminated seeds, as seem to be the case in this first outbreak, and also transfer to tomatoes from infected ornamental hosts. This is, to the best of our knowledge, the first report of TCDVd in tomato in France.

References: (1) T. James et al. Plant Pathol. 57:400, 2008. (2) A. M. Shamloul et al. Can. J. Plant Pathol. 19:89, 1997. (3) R. P. Singh et al. J. Gen. Virol. 80:2823, 1999. 4) R. P. Singh et al. Plant Dis. 90:1457, 2006.
First Report of Papaya ringspot virus Infecting Zucchini Plants in Poland
Authors: Hasiów-Jaroszewska B. , Borodynko N. , Rymelska N. , Pospieszny H.
Published: April 2010

Papaya ringspot virus (PRSV), a member of the aphid-transmitted genus Potyvirus, is the cause of a destructive disease and a major limiting factor for papaya and cucurbit cultivation worldwide. The virus occurs in China, France, Germany, India, Italy, Mexico, Taiwan, and the United States. Its P biotype is a devastating pathogen of papaya crops and its W biotype is a pathogen of cucurbits (4). In 2009, zucchini plants with leaf mosaic and marbled fruit were collected from the Kujawsko-Pomorskie Region of Poland. Samples came from the same region where Zucchini yellow mosaic virus (ZYMV) (3) and Watermelon mosaic virus (WMV) (1) have been found previously. Forty leaf and ten fruit samples of zucchini (Cucurbita pepo cv. giromontina) were tested by double-antibody sandwich (DAS)-ELISA with commercial antisera against WMV, ZYMV, and PRSV (DSMZ, Braunschweig, Germany). PRSV was found in two samples tested. Leaf extracts from infected plants were mechanically inoculated onto Carborundum-dusted leaves of the following indicator plants: Cucumis sativus, Chenopodium quinoa, Cucurbita pepo, Nicotiana benthamiana, and N. tabacum cv. Xanthi. After 2 weeks, symptoms of leaf chlorosis on cucumber and chlorotic lesions on zucchini were observed. Total RNA was extracted from infected leaves with a phenol-chloroform based extraction procedure. The presence of PRSV was confirmed by reverse transcription (RT)-PCR reaction using primers 04-02 and 04-04, which amplify the coat protein gene (2). Amplified DNA was gel purified with a Qiaex Kit (Qiagen, Valencia, CA) and cloned into pGEM-T easy (Promega, Madison, WI). Overlapping sequences were obtained using universal M13F and M13R primers. BioEdit software ( http://www.mbio.ncsu.edu/BioEdit/bioedit.html ) was used to assemble the nucleotide consensus sequence. The obtained sequence (861 bp encoding 287 amino acids) was deposited in the GenBank database under Accession No. GQ927328. The comparison with PRSV sequences retrieved from the GenBank database were carried out to investigate the genetic diversity between Polish PRSV isolates and establish their molecular relationships to the previously characterized PRSV isolates from different parts of the world. The sequences of PRSV Polish isolates obtained from two infected plant samples were identical. Comparisons revealed that the Polish isolate designated PRSV-BON shared the highest identity (97%) with three Australian isolates (U14739, U14740, and U14744). To our knowledge, this is the first report of PRSV infecting zucchini plants in Poland. The occurrence of subtropic viruses like PRSV in Poland indicated the introduction of new pathogens that likely affect cucurbit production in this country and beyond.

References: (1) N. Borodynko et al. Plant Pathol. 58:783, 2009. (2) M. Chin et al. Arch. Virol. 152:2101, 2007. (3) H. Pospieszny et al. Plant Dis. 91:639, 2007. (4) D. J. Purcifull et al. CMI/AAB Descriptions of Plant Viruses. No. 292, 1984.
First Report of a Branch Dieback of Olive Trees in Tunisia Caused by a Phoma sp.
Authors: Rhouma A. , Triki M. A. , Krid S. , Tuset J. J. , Msallem M.
Published: April 2010

From 2007 to 2008, a new dieback of branches of olive trees was observed in several orchards in central and southern Tunisia. The appearance of this new syndrome coincided with warm temperatures and frequent rainfall from February to April 2007. Affected trees were observed in seven commercial orchards; disease incidence ranged from 1 to 9% and affected trees were randomly distributed in each orchard. Symptoms included abundant dead branches and wilted leaves remained attached. Distinct brown areas appeared on the bark of current-year shoots as well as on larger branches. Cankers on branches that were >2 years old were difficult to detect but were conspicuous in current-year shoots. To determine the etiology of this new syndrome, a study was carried out on samples of affected branches collected from 2007 to 2008 from different areas of the country. Unidentified species of Chaetomium and Phoma were isolated from the margins of the cankers. Koch's postulates were performed with one isolate each of a Chaetomium sp. and a Phoma sp on 2-year-old olive trees, cv. Chemlali, grown in 13-cm-diameter pots containing a sand/lime/peat mixture. Stems were inoculated by placing 10 μl of conidial suspension (106 conidia/ml) on 1-cm-long longitudinal stem wounds that had been made with a sterile scalpel. Control plants were wounded, but inoculum was replaced with sterile distilled water. Three sets of 10 plants each were wound inoculated with each of the fungi on the same day. Inoculated plants were covered with a polyethylene plastic bag to retain moisture and incubated for 2 months at 30°C with a 12-h photoperiod. After 45 days, only branches inoculated with the Phoma isolate showed brown discoloration areas at the inoculation sites. A Phoma sp. was recovered from necrotic bark from each of the 10 inoculated plants. Conidia were hyaline, unicellular, slightly ellipsoidal, and 4.8 to 6.3 × 1.8 to 2.2 μm. To confirm the identification, DNA extraction was done with hyphae collected from a 7-day-old culture on PDA after incubation at 25°C (1). Fungal primers ITS1 and ITS4 (3) were used for amplification. Purified amplicons were directly sequenced using the ITS1 and ITS4 primers for the internal transcribed spacer region of rDNA. A BLAST search of the GenBank database revealed 96% homology with Phoma sp. isolate (AJ972865.1) and 98% homology with Phoma medicaginis isolate (DQ026014.1). P. incompta has been reported as responsible for branch dieback of olive tree in Italy (2). To our knowledge, this is the first report of a canker disease of olive caused by a Phoma sp. in Tunisia.

References: (1) S. R. Tendulkar et al. Biotechnol. Lett. 22:1941, 2003. (2) L. Tosi and A. Zazzerini. Petria 4:161, 1994. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.
First Report of Beet necrotic yellow vein virus Infecting Spinach in California
Authors: Liu H.-Y. , Mou B. , Richardson K. , Koike S. T.
Published: April 2010

In 2009, plants from two spinach (Spinacia oleracea) experimental fields in Monterey County and one commercial spinach field in Ventura County of California exhibited vein-clearing, mottling, interveinal yellowing, and stunting symptoms. For experimental fields, up to 44% of spinach plants have symptoms. With a transmission electron microscope, rigid rod-shaped particles with central canals were observed from plant sap of the symptomatic spinach. Analysis with a double-antibody sandwich-ELISA assay for Beet necrotic yellow vein virus (BNYVV) showed that all 10 symptomatic plants we tested were positive and 5 asymptomatic plants were negative. Symptomatic spinach from both counties was used for mechanical transmission experiments. Chenopodium quinoa, Tetragonia expansa, and Beta vulgaris (sugar beet) showed chlorotic local lesions and B. macrocarpa and spinach showed vein-clearing, mottling, and systemic infections. To further confirm the presence of BNYVV, reverse transcription (RT)-PCR was conducted. Total RNA was extracted from field- and mechanically inoculated symptomatic spinach plants using an RNeasy Plant Kit (Qiagen Inc., Valencia, CA) and used as a template in RT-PCR. Forward and reverse primers specific to the BNYVV RNA-3 P25 protein gene from the beet isolate were used (2). Amplicons of the expected size (approximately 860 bp) were obtained. Four RT-PCR products were sequenced and the sequences were identical (GenBank Accession No. GU135626). Sequences from the spinach plants had 97 to 99% nucleotide and 94 to 100% amino acid identity with BNYVV RNA-3 P25 protein sequences available in the GenBank. On the basis of the data from electron microscopy, indicator plants, serology, and cDNA sequencing, the virus was identified as BNYVV. BNYVV has been reported from spinach fields in Italy (1). To our knowledge, this is the first report of BNYVV occurring naturally on spinach in California. Since BNYVV is transmitted by the zoospores of the soil-inhabiting plasmodiophorid Polymyxa betae, it could be a new threat to spinach production in the state.

References: (1) C. R. Autonell et al. Inf. Fitopatol. 45:43, 1995. (2) H.-Y. Liu and R. T. Lewellen, Plant Dis. 91:847, 2007.
Population Structure ofRhizoctonia oryzae-sativaein California Rice Fields
Authors: Chaijuckam Patcharavipa , Baek Jong-Min , Greer Christopher A. , Webster Robert K. , Davis R. Michael
Published: April 2010

Six pairs of single-locus microsatellite primers were developed to study the population structure of Rhizoctonia oryzae-sativae, the cause of aggregate sheath spot disease of rice, among and within three rice-growing areas in California over a 3-year period. A high level of gene flow among growing areas was indicated by low population subdivision according to analysis of molecular variance and moderate to no population differentiation between pairs of populations based on the fixation index (FST). Gametic equilibrium of most pairs of microsatellite loci, high numbers of unique multilocus genotypes, and high genotypic diversity indicated extensive sexual recombination within growing areas. Because there was little differentiation among populations in all hierarchical levels, including among growing areas within sampling years, fields within growing areas, and corners within individual fields, a high level of gene flow was revealed in all levels. Basidiospores were likely the main vehicle of gene flow among populations, including short and long distances. Asexual inocula (sclerotia and mycelia) probably overwinter because a few clones were detected over a 2-year period within the same field. A few clones were shared among fields but were not commonly shared among growing areas.
Scaling relationship between tree respiration rates and biomass
Authors: Cheng Dong-Liang , Li Tao , Zhong Quan-Lin , Wang Gen-Xuan
Published: April 2010

The WBE theory proposed by West, Brown and Enquist predicts that larger plant respiration rate,
R
, scales to the three-quarters power of body size,
M
. However, studies on the
R
versus
M
relationship for larger plants (i.e. trees larger than saplings) have not been reported. Published respiration rates of field-grown trees (saplings and larger trees) were examined to test this relationship. Our results showed that for larger trees, aboveground respiration rates
R
A
scaled as the 0.82-power of aboveground biomass
M
A
, and that total respiration rates
R
T
scaled as the 0.85-power of total biomass
M
T
, both of which significantly deviated from the three-quarters scaling law predicted by the WBE theory, and which agreed with 0.81–0.84-power scaling of biomass to respiration across the full range of measured tree sizes for an independent dataset reported by Reich
et al
. (Reich
et al
. 2006
Nature
439
, 457–461). By contrast,
R
scaled nearly isometrically with
M
in saplings. We contend that the scaling exponent of plant metabolism is close to unity for saplings and decreases (but is significantly larger than three-quarters) as trees grow, implying that there is no universal metabolic scaling in plants.
Blindsnake evolutionary tree reveals long history on Gondwana
Authors: Vidal Nicolas , Marin Julie , Morini Marina , Donnellan Steve , Branch William R. , Thomas Richard , Vences Miguel , Wynn Addison , Cruaud Corinne , Hedges S. Blair
Published: April 2010

Worm-like snakes (scolecophidians) are small, burrowing species with reduced vision. Although largely neglected in vertebrate research, knowledge of their biogeographical history is crucial for evaluating hypotheses of snake origins. We constructed a molecular dataset for scolecophidians with detailed sampling within the largest family, Typhlopidae (blindsnakes). Our results demonstrate that scolecophidians have had a long Gondwanan history, and that their initial diversification followed a vicariant event: the separation of East and West Gondwana approximately 150 Ma. We find that the earliest blindsnake lineages, representing two new families described here, were distributed on the palaeolandmass of India+Madagascar named here as Indigascar. Their later evolution out of Indigascar involved vicariance and several oceanic dispersal events, including a westward transatlantic one, unexpected for burrowing animals. The exceptional diversification of scolecophidians in the Cenozoic was probably linked to a parallel radiation of prey (ants and termites) as well as increased isolation of populations facilitated by their fossorial habits.
Nutrient provisioning facilitates homeostasis between tsetse fly (Diptera: Glossinidae) symbionts
Authors: Snyder Anna K. , Deberry Jason W. , Runyen-Janecky Laura , Rio Rita V. M.
Published: April 2010

Host-associated microbial interactions may involve genome complementation, driving-enhanced communal efficiency and stability. The tsetse fly (Diptera: Glossinidae), the obligate vector of African trypanosomes (
Trypanosoma brucei
subspp.), harbours two enteric Gammaproteobacteria symbionts:
Wigglesworthia glossinidia
and
Sodalis glossinidius
. Host coevolution has streamlined the
Wigglesworthia
genome to complement the exclusively sanguivorous tsetse lifestyle. Comparative genomics reveal that the
Sodalis
genome contains the majority of
Wigglesworthia
genes. This significant genomic overlap calls into question why tsetse maintains the coresidence of both symbionts and, furthermore, how symbiont homeostasis is maintained. One of the few distinctions between the
Wigglesworthia
and
Sodalis
genomes lies in thiamine biosynthesis. While
Wigglesworthia
can synthesize thiamine,
Sodalis
lacks this capability but retains a thiamine ABC transporter (
tbp
A
thi
PQ) believed to salvage thiamine. This genetic complementation may represent the early convergence of metabolic pathways that may act to retain
Wigglesworthia
and evade species antagonism. We show that thiamine monophosphate, the specific thiamine derivative putatively synthesized by
Wigglesworthia
, impacts
Sodalis
thiamine transporter expression, proliferation and intracellular localization. A greater understanding of tsetse symbiont interactions may generate alternative control strategies for this significant medical and agricultural pest, while also providing insight into the evolution of microbial associations within hosts.
Chaotic mimic robots
Authors: Buscarino Arturo , Camerano Cristoforo , Fortuna Luigi , Frasca Mattia
Published: April 2010

In this paper, chaos is applied to the control of moving robots in order to generate random-like trajectories needed in tasks such as exploration, scanning natural terrains or mapping of unknown environments. Synchronization between the robots of a team is achieved by exploiting the paradigm of mirror neurons, i.e. a neural structure playing a key role in the process of imitation and behaviour understanding. The experimental results discussed in the paper demonstrate that the introduced approach can be successfully applied to implement an efficient learning system for mobile robots.
MEP and planetary climates: insights from a two-box climate model containing atmospheric dynamics
Authors: Jupp Tim E. , Cox Peter M.
Published: April 2010

A two-box model for equator-to-pole planetary heat transport is extended to include simple atmospheric dynamics. The surface drag coefficient
C
D
is treated as a free parameter and solutions are calculated analytically in terms of the dimensionless planetary parameters
η
(atmospheric thickness),
ω
(rotation rate) and
ξ
(advective capability). Solutions corresponding to maximum entropy production (MEP) are compared with solutions previously obtained from dynamically unconstrained two-box models. As long as the advective capability
ξ
is sufficiently large, dynamically constrained MEP solutions are identical to dynamically unconstrained MEP solutions. Consequently, the addition of a dynamical constraint does not alter the previously obtained MEP results for Earth, Mars and Titan, and an analogous result is presented here for Venus. The rate of entropy production in an MEP state is shown to be independent of rotation rate if the advective capability
ξ
is sufficiently large (as for the four examples in the solar system), or if the rotation rate
ω
is sufficiently small. The model indicates, however, that the dynamical constraint does influence the MEP state when
ξ
is small, which might be the case for some extrasolar planets. Finally, results from the model developed here are compared with previous numerical simulations in which the effect of varying surface drag coefficient on entropy production was calculated.
Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control
Authors: Campos-Guillén Juan , Arvizu-Gómez Jackeline Lizzeta , Jones George H. , Olmedo-Alvarez Gabriela
Published: April 2010

We generated a conditional CCase mutant ofBacillus subtilisto explore the participationin vivoof the tRNA nucleotidyltransferase (CCA transferase or CCase) in the maturation of the single-copy tRNACys, which lacks an encoded CCA 3′ end. We observed that shorter tRNACysspecies, presumably lacking CCA, only accumulated when the inducible Pspac : ccawas introduced into anrnrmutant strain, but not in combination withpnp. We sequenced the tRNA 3′ ends produced in the various mutant tRNACysspecies to detect maturation and decay intermediates and observed that decay of the tRNACysoccurs through the addition of poly(A) or heteropolymeric tails. A few clones corresponding to full-size tRNAs contained either CCA or other C and/or A sequences, suggesting that these are substrates for repair and/or decay. We also observed editing of tRNACysat position 21, which seems to occur preferentially in mature tRNAs. Altogether, our results providein vivoevidence for the participation of theB. subtilis ccagene product in the maturation of tRNAs lacking CCA. We also suggest that RNase R exoRNase inB. subtilisparticipates in the quality control of tRNA.
An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans
Authors: Judelson Howard S. , Ah-Fong Audrey M. V. , Fabritius Anna-Liisa
Published: April 2010

Some strains ofPhytophthora infestans,the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects onP. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness ofP. infestansin nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.
Global survey of escape from X inactivation by RNA-sequencing in mouse
Authors: Yang Fan , Babak Tomas , Shendure Jay , Disteche Christine M.
Published: April 2010

X inactivation equalizes the dosage of gene expression between the sexes, but some genes escape silencing and are thus expressed from both alleles in females. To survey X inactivation and escape in mouse, we performed RNA sequencing in Mus musculus × Mus spretus cells with complete skewing of X inactivation, relying on expression of single nucleotide polymorphisms to discriminate allelic origin. Thirteen of 393 (3.3%) mouse genes had significant expression from the inactive X, including eight novel escape genes. We estimate that mice have significantly fewer escape genes compared with humans. Furthermore, escape genes did not cluster in mouse, unlike the large escape domains in human, suggesting that expression is controlled at the level of individual genes. Our findings are consistent with the striking differences in phenotypes between female mice and women with a single X chromosome—a near normal phenotype in mice versus Turner syndrome and multiple abnormalities in humans. We found that escape genes are marked by the absence of trimethylation at lysine 27 of histone H3, a chromatin modification associated with genes subject to X inactivation. Furthermore, this epigenetic mark is developmentally regulated for some mouse genes.
Preparation of Trojan Horse Liposomes (THLs) for Gene Transfer across the Blood-Brain Barrier
Authors: Pardridge William M.
Published: April 2010

INTRODUCTION

Nonviral plasmid DNA is delivered to the brain via a transvascular route across the blood-brain barrier (BBB) following intravenous administration of DNA encapsulated within Trojan horse liposomes (THLs), also called PEGylated immunoliposomes (PILs). The liposome surface is covered with several thousand strands of polymer (e.g., polyethylene glycol [PEG]), and the tips of 1%-2% of the polymer strands are conjugated with a targeting monoclonal antibody that acts as a molecular Trojan horse (MTH). The MTH binds to a receptor (e.g., for transferrin or insulin) on the BBB and brain cell membrane, triggering receptor-mediated transcytosis of the THL across the BBB in vivo, and receptor-mediated endocytosis into brain cells beyond the BBB. The persistence of transgene expression in the brain is inversely related to the rate of degradation of the episomal plasmid DNA. THL technology enables an exogenous gene to be widely expressed in the majority of cells in adult brain (or other organs) within 1 d of a single intravenous administration. Applications of the THLs include tissue-specific gene expression with tissue-specific promoters, complete normalization of striatal tyrosine hydroxylase in experimental Parkinson’s disease following intravenous tyrosine hydroxylase gene therapy, a 100% increase in survival time of mice with brain tumors following weekly intravenous antisense gene therapy using THLs, and a 90% increase in survival time with weekly intravenous RNA interference (RNAi) gene therapy in mice with intracranial brain tumors. This protocol describes the preparation of THLs for use in gene transfer in vitro or in vivo.
Optimized DNA Preparation from Mycobacteria
Authors: Käser Michael , Ruf Marie-Thérèse , Hauser Julia , Pluschke Gerd
Published: April 2010

INTRODUCTION

Extraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Hence, mycobacterial DNA extraction often results in low DNA yields of unsuitable quality. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for polymerase chain reaction (PCR)-based applications like species identification. However, such DNA quantities and qualities are usually not sufficient for contemporary genomic analyses such as whole genome sequence analysis, single nucleotide polymorphism (SNP) detection, or DNA microarrays, or for investigations of bacterial evolution, virulence, or epidemiology on a world-wide population level. Moreover, most protocols that achieve a high standard in DNA recovery typically employ large reaction volumes and thus require milliliter-scale plasticware and centrifugal equipment as well as large amounts of chemicals, all of which are costly both in purchase and disposal. The DNA extraction method described here was established to address the challenges that result from the slow growth and distinct cell wall composition of mycobacteria, and to greatly enhance both yield and purity of mycobacterial DNA preparations in a small extraction volume. Designed to be performed using 1.5-mL reaction tubes and the corresponding equipment, the method is economical and practical, and reliably yields large amounts of pure genomic DNA--increases of at least 10-fold as compared to earlier protocols.
Calcium Imaging of Neuronal Circuits In Vivo Using a Circuit-Tracing Pseudorabies Virus: Figure 1.
Authors: Granstedt Andrea E. , Kuhn Bernd , Wang Samuel S.-H. , Enquist Lynn W.
Published: April 2010

INTRODUCTION

Pseudorabies virus (PRV) is a neuroinvasive virus of the herpes family that has a broad host range but does not infect higher-order primates. PRV characteristically travels along chains of synaptically connected neurons and has been used extensively for elucidating neural circuits in the peripheral and central nervous system in vivo. The recombinant virus PRV369 is an attenuated retrograde tracer that encodes G-CaMP2, a fluorescent calcium sensor protein that is stable at physiological pH and mammalian temperature. This protocol describes the use of PRV369 to express G-CaMP2 in a neuronal circuit and to monitor its activity in a living animal, specifically in the submandibular ganglia (SMG), the peripheral parasympathetic ganglia that innervate the salivary glands. The procedure describes the delivery of PRV369 to the glands and shows how SMG neurons can then be imaged post-inoculation to explore connectivity and activity.
Live Cell Imaging inDrosophila melanogaster
Authors: Parton Richard M. , Vallés Ana Maria , Dobbie Ian M. , Davis Ilan
Published: April 2010

INTRODUCTION

Although many of the techniques of live cell imaging inDrosophila melanogasterare also used by the greater community of cell biologists working on other model systems, studying living fly tissues presents unique difficulties with regard to keeping the cells alive, introducing fluorescent probes, and imaging through thick, hazy cytoplasm. This article outlines the major tissue types amenable to study by time-lapse cinematography and different methods for keeping the cells alive. It describes various imaging and associated techniques best suited to following changes in the distribution of fluorescently labeled molecules in real time in these tissues. Imaging, in general, is a rapidly developing discipline, and recent advances in imaging technology are able to greatly extend what can be achieved with live cell imaging ofDrosophilatissues. As far as possible, this article includes the latest technical developments and discusses likely future developments in imaging methods that could have an impact on research usingDrosophila.
Green Fluorescent Proteins (GFPs) for Measuring Voltage
Authors: Siegel Micah S. , Isacoff Ehud Y.
Published: April 2010

INTRODUCTION

Measuring signal transduction in large numbers of cells, with high spatial and temporal resolution, is a fundamental problem for studying information processing in the nervous system. To address this problem, a family of detectors that are chimeras between signal transduction proteins and fluorescent proteins has been designed. The prototype sensor is a genetically encoded probe that can be used to measure transmembrane voltage in single cells. This article describes a modified green fluorescent protein (GFP), fused to a voltage-sensitive K+ channel, so that voltage-dependent rearrangements in the K+ channel induce changes in the fluorescence of GFP. The probe has a maximal fractional fluorescence change of 5.1%, making it comparable to some of the best organic voltage-sensitive dyes. Moreover, the fluorescent signal is expanded in time in a manner that makes the signal 30-fold easier to detect than a traditional linear dye. DNA-encoded sensors have the advantage that they may be introduced into an organism noninvasively and targeted to specific brain regions, cell types, and subcellular compartments.
Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death, Phytoalexin Production, and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases, OsCIPK14/15, in Rice Cultured Cells
Authors: Kurusu Takamitsu , Hamada Jumpei , Nokajima Hiroshi , Kitagawa Youichiro , Kiyoduka Masahiro , Takahashi Akira , Hanamata Shigeru , Ohno Ryoko , Hayashi Teruyuki , Okada Kazunori , Koga Jinichiro , Hirochika Hirohiko , Yamane Hisakazu , Kuchitsu Kazuyuki
Published: April 2010

Although cytosolic free Ca2+ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca2+ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca2+ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn2+-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.
PLANT HOMOLOGOUS TO PARAFIBROMIN Is a Component of the PAF1 Complex and Assists in Regulating Expression of Genes within H3K27ME3-Enriched Chromatin
Authors: Park Sunchung , Oh Sookyung , Ek-Ramos Julissa , van Nocker Steven
Published: April 2010

The human Paf1 complex (Paf1C) subunit Parafibromin assists in mediating output from the Wingless/Int signaling pathway, and dysfunction of the encoding gene HRPT2 conditions specific cancer-related disease phenotypes. Here, we characterize the organismal and molecular roles of PLANT HOMOLOGOUS TO PARAFIBROMIN (PHP), the Arabidopsis (Arabidopsis thaliana) homolog of Parafibromin. PHP resides in an approximately 670-kD protein complex in nuclear extracts, and physically interacts with other known Paf1C-related proteins in vivo. In striking contrast to the developmental pleiotropy conferred by mutation in other plant Paf1C component genes in Arabidopsis, loss of PHP specifically conditioned accelerated phase transition from vegetative growth to flowering and resulted in misregulation of a very limited subset of genes that included the flowering repressor FLOWERING LOCUS C. Those genes targeted by PHP were distinguished from the bulk of Arabidopsis genes and other plant Paf1C targets by strong enrichment for trimethylation of lysine-27 on histone H3 (H3K27me3) within chromatin. These findings suggest that PHP is a component of a plant Paf1C protein in Arabidopsis, but has a more specialized role in modulating expression of a subset of Paf1C targets.
Genetic divergence between morphological forms of brown trout Salmo trutta L. in the Balkan region of Macedonia
Authors: Lo Brutto S. , Hristovski N. , Arculeo M.
Published: April 2010

The objective of this study was to characterize the genetic structure of two Balkan brown trout morphotypes, Salmo macedonicus and Salmo pelagonicus, and to test whether molecular traits support the species' status proposed by traditional morphological identification. The mitochondrial DNA 12S‐rDNA, cyt b and control region genes were sequenced in 15 specimens collected from three localities in the Former Yugoslav Republic of Macedonia. The results of these markers did not support the taxonomic category of species but confirmed the existence of two morphotypes, Salmo trutta macedonicus and Salmo trutta pelagonicus, in the Aegean–Adriatic lineages of the Salmo trutta species complex.
Imaging study of pancreatic ductal adenocarcinomas in Syrian hamsters using X‐ray micro‐computed tomography (CT)
Authors: Kitahashi Tsukasa , Mutoh Michihiro , Tsurusaki Masakatsu , Iinuma Gen , Suzuki Masahiro , Moriyama Noriyuki , Yoshimoto Mitsuyoshi , Wakabayashi Keiji , Sugimura Takashi , Imai Toshio
Published: April 2010

X‐ray computed tomography (CT) has been used for diagnoses of human pancreatic cancer. Although micro‐CT is a useful approach to evaluate macromorphology of organs/tissue also in animal models, reports on pancreatic tumors are limited. In this study, the utility of micro‐CT was assessed in characterizing chemically induced pancreatic tumors in Syrian hamsters. Hamsters treated with or without N‐nitrosobis(2‐oxopropyl)amine (BOP) were injected with the antispasmodic agent, scopolamine butylbromide, and contrast agents, 5 or 10 mL/kg body weight of iopamidol or Fenestra VC at 18–38 weeks, then examined by micro‐CT scanning with a respiratory gating system. Both peristaltic and respiratory movements were substantially suppressed by the combination of scopolamine butylbromide treatment and the respiratory gating system, resulting in improvements of image qualities. Iopamidol clearly visualized the pancreatic parenchyma and contrasted the margins among the pancreas and other abdominal organs/tissue. Meanwhile Fenestra VC predominantly contrasted abdominal vascular systems, but the margins among pancreas and other organs/tissue remained obscure. Six pancreatic tumors of 4–13 mm in diameter were detected in four of 15 animals, but not the five tumors of 1–4 mm in diameter. The inner tumor images were heterogeneously or uniformly visualized by iopamidol and Fenestra VC. Overall, iopamidol could clearly contrast between pancreatic parenchyma and the tumors as compared with Fenestra VC. All tumors confirmed were histopathologically diagnosed as pancreatic ductal adenocarcinomas. Thus, micro‐CT could be useful to evaluate the carcinogenic processes and preventive methods of pancreatic cancer in hamsters and to assess the novel contrast agents for detection of small pancreatic cancer in humans. (Cancer Sci 2010)
Membrane phospholipid metabolism during phagocytosis in human neutrophils
Authors: Minakami Reiko , Maehara Yuichi , Kamakura Sachiko , Kumano Osamu , Miyano Kei , Sumimoto Hideki
Published: April 2010

Neutrophils play an essential role via phagocytosis in host defense against microbial infections. However, little is known about molecular mechanisms underlying phagocytosis in neutrophils, because of the difficulty in genetically manipulating these cells. Here, we provide the first comprehensive description of phospholipid metabolism during phagocytosis in human neutrophils, which we have efficiently transfected with cDNAs encoding lipid‐probing protein modules. Phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2), an F‐actin organizer abundant in the plasma membrane, diminishes progressively from phagosomes during phagosome formation and vanishes after phagosome closure with F‐actin disappearance. Diacylglycerol, a metabolite of PtdIns(4,5)P2, appears at phagocytic cups and remains associated with nascent (closed) phagosomes; it may function with phosphatidylserine, present in both plasma and phagosomal membranes, to recruit phagocytosis‐associated proteins. From PtdIns(4,5)P2, PtdIns(3,4,5)P3 is also produced at phagocytic cups but becomes undetectable shortly after phagosome sealing, consistent with its proposed roles in pseudopod extension and phagosome closure. PtdIns(3)P, a putative participant in phagosome maturation, emerges at closed phagosomes as does the class III PtdIns 3‐kinase Vps34. Although the small GTPases Rab5 and Rab7 are thought to contribute to phagosome maturation in macrophages, Rab5 but not Rab7 fails to accumulate at phagosomes in neutrophils, suggesting a difference in phagocytic mechanism between the two phagocytes.
Crossveinless‐c, the Drosophila homolog of tumor suppressor DLC1, regulates directional elongation of dendritic branches via down‐regulating Rho1 activity
Authors: Sato Daichi , Sugimura Kaoru , Satoh Daisuke , Uemura Tadashi
Published: April 2010

Diverse neuronal subtypes develop distinctive morphologies of dendritic arbors that receive synaptic or sensory inputs. Dendritic arbors of many subtypes take on a polarized shape, and one underlying mechanism is unidirectionally biased elongation of dendritic branches. As reported herein, we found that Drosophila Crossveinless‐c (Cv‐c) was a key regulator for such directional growth. In the cv‐c mutant, two subclass of multidendritic sensory neurons examined formed dorsally directed branches; however, dendritic branches had difficulty in growing along the anterior–posterior (A–P) body axis. Cv‐c belongs to the family of Rho GTPase‐activating proteins (RhoGAPs) and is the homolog of human tumor suppressor DLC1. The RhoGAP activity of Cv‐c was required cell‐autonomously for the A–P‐oriented growth, and Cv‐c elevated the GTPase activity of Rho1 and Cdc42 in a cell‐free assay. Our analysis of genetic interactions suggested that Rho1 was the target of Cv‐c in vivo. All of our results suggest that Cv‐c contributes to sprouting and subsequent growth of the A–P‐oriented branches through negative regulation of Rho1. We discuss a role of Cv‐c in dendritic growth in response to environmental cues.