Comparison of High Throughput Sequencing to Standard Protocols for Virus Detection in Berry Crops.

We completed a comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium, and eight Rubus) with known virus profiles. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, reverse transcription (RT) PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared with RT-PCR on a wide spectrum of variants and discovery of novel viruses. More importantly, in most cases in which the two protocols showed parallel virus detection, 11 viruses in 16 selections were not consistently detected by both methods at all sampling points. Based on these data, we propose a testing requirement of four sampling times over two growing seasons for berry and potentially other crops, to ensure that no virus remains undetected independent of titer, distribution, or other virus-virus or virus-host interactions.

Stem cells in the trabecular meshwork: present and future promises.

Primary open-angle glaucoma is recognized as a disease of aging, and studies show a relationship between aging and trabecular meshwork (TM) cell density. Human TM cell division occurs primarily in the anterior, non-filtering region. A commonly used glaucoma treatment, laser trabeculoplasty (LTP), triggers and increases cell division, as well as cell migration of these anterior TM cells. These freshly-divided migrating cells repopulate the burned laser sites, suggesting that they are stem cells. Several studies concerning this putative TM stem cell will be discussed.

Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

The use of reverse transcriptase for efficient first- and second-strand cDNA synthesis from single- and double-stranded RNA templates.

Molecular characterization of eight distinct, difficult-to-clone RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry and several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates, combined with random primers, were denatured with methyl mercuric hydroxide. Reverse transcriptase was added followed by the addition of RNase H. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently into a T-tailed vector after adding an A-overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel-purified dsRNA and with single-stranded RNA templates.

Potyvirus genome-linked protein (VPg) determines pea seed-borne mosaic virus pathotype-specific virulence in Pisum sativum.

The mechanism of Pisum sativum pathotype-specific resistance to pea seed-borne mosaic potyvirus (PSbMV) was investigated and the coding region determinant of PSbMV virulence was defined. Homozygous recessive sbm-1 peas are unable to support replication of PSbMV pathotype 1 (P-1), whereas biochemically and serologically related pathotype 4 (P-4) is fully infectious in the sbm-1/sbm-1 genotype. We were unable to detect viral coat protein or RNA with double antibody sandwich-enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction in sbm-1/sbm-1 P-1-inoculated protoplasts and plants. Lack of viral coat protein or RNA in P-1 transfected sbm-1/sbm-1 protoplasts suggests that sbm-1 resistance is occurring at the cellular level and that inhibition of cell-to-cell virus movement is not the operating form of resistance. In addition, because virus products were not detected at any time post-inoculation, resistance must either be constitutive or expressed very early in the virus infection process. P-1-resistant peas challenged with full-length, infectious P-1/P-4 recombinant clones demonstrated that a specific P-4 coding region, the 21-kDa, genome-linked protein (VPg), was capable of overcoming sbm-1 resistance, whereas clones containing the P-1 VPg coding region were noninfectious to sbm-1/sbm-1 peas. VPg is believed to be involved in potyvirus replication and its identification as the PSbMV determinant of infectivity in sbm-1/sbm-1 peas is consistent with disruption of an early P-1 replication event.

Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.

A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 micrograms/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.

Suppression of potyvirus infection by coexpressed closterovirus protein.

A tobacco etch virus (TEV)-based expression vector has been used for insertion of several ORFs derived from the unrelated beet yellows virus (BYV). Hybrid TEV variants expressing the BYV capsid protein, 20-kDa protein, or HSP70 homolog systemically infected Nicotiana tabacum and stably retained BYV sequences. In contrast, insertion of the ORF encoding BYV leader proteinase (L-Pro) resulted in severely impaired systemic transport and accumulation of recombinant TEV. Progeny of this virus underwent various deletions affecting the L-Pro sequence and mitigating the defects in virus spread. Model experiments involving several spontaneous and engineered mutants indicated that the central domain of BYV L-Pro was responsible for the defect in hybrid virus accumulation, whereas full-size L-Pro was required for maximal debilitation of systemic transport. Strikingly, BYV L-Pro expression did not debilitate systemic infection of hybrid TEV in Nicotiana benthamiana plants. No major defects in replication or encapsidation of recombinant RNA were revealed in N. tabacum protoplasts. These results indicated that BYV L-Pro specifically interfered with TEV systemic transport and accumulation in a host-dependent manner and suggested a potential utility of closterovirus L-Pro as an inhibitor of potyvirus infection. In addition, it was demonstrated that the 107-amino-acid-residues-long N-terminal part of the TEV helper component proteinase is not essential for systemic infection.

Multiple viral determinants affect seed transmission of pea seedborne mosaic virus in Pisum sativum.

Two pea seedborne mosaic potyvirus (PSbMV) isolates, P-1 DPD1 (P-1), which is highly seed-transmitted, and P-4 NY (P-4), which is rarely seed-transmitted, and chimeras between P-1 and P-4 were analysed to map the viral genetic determinants of seed transmission. Infectivity of chimeric viruses was evaluated by inoculating Pisum sativum with RNA transcribed in vitro from recombinant full-length cDNA clones. The chimeric viruses that were used demonstrated that a genomic segment encoding the 49 kDa protease and putative RNA polymerase was responsible for symptom induction. Attempts to determine transmission of the chimeric viruses in P. sativum cultivars known to transmit P1 at high frequencies showed that seed transmission is a quantitative character influenced by multiple viral determinants. Seed transmission frequency did not correlate with accumulation of virus in vegetative tissue. The 5' 2.5 kb of the 10 kb PSbMV genome had a major influence on the seed transmission frequency and was analysed further. This showed that, while the helper-component protease was a major determinant of seed transmission, the potyviral P1 -protease exerted no measurable influence.

Biological and molecular properties of a pathotype P-1 and a pathotype P-4 isolate of pea seed-borne mosaic virus.

Two isolates of pea seed-borne mosaic potyvirus, DPD1 and NY, were identified as pathotypes P-1 and P-4, respectively, by their infectivity on Pisum sativum L. lines homozygous for the recessive resistance genes sbm-1 and sbm-4. The two isolates differed in several biological characteristics. DPD1 induced transient vein clearing, downward rolling of leaflets and internode shortening on P. sativum, whereas NY only caused a slight growth reduction. DPD1 moved systemically in Chenopodium quinoa whereas NY was restricted to inoculated leaves. DPD1 was frequently transmitted by seeds whereas NY was rarely seed-transmitted: 24% and 0.3%, respectively, in P. sativum '549'. Both DPD1 and NY were transmitted by aphids (Myzus persicae), though a DAG triplet was not present in the N terminus of the coat protein. The nucleotide sequence and deduced amino acid sequence of NY were determined and compared to the corresponding sequences of DPD1.

A comparison of the trail making test, symbol digit modalities test, and the Hooper Visual Organization Test in an inpatient substance abuse population.

Ninety inpatients from a substance abuse treatment facility each completed the Symbol Digit Modalities Test, the Trail Making Test and a novel, group-administered form of the Hooper Visual Organization Test. Patients exhibited varying degrees of impairment on all three instruments compared to available norms. The correlations among the tests were all statistically significant. The lowest inter-test correlations were obtained on the Hooper Visual Organization Test. The utility of global neuropsychological screening instruments with inpatient substance abusers' is discussed in light of these findings.

Naproxen in rheumatoid arthritis. A controlled trial.

The efficacy of naproxen in treating rheumatoid arthritis patients was evaluated in a double-blind clinical trial using aspirin as the control drug. The study was conducted at seven centers and involved 80 patients. After an unequivocal increase in disease activity during a drug-free period, patients were randomly assigned to either drug and continued in the trial for 16 weeks. Some patients took low maintenance doses of corticosteroids, or gold salts, or both throughout the trial. Both test drugs significantly decreased disease activity as measured by a number of ways. By objective measurements, naproxen was as effective as aspirin, although patients in the naproxen-treated group entered the trial with more severe disease. By some subjective evaluations, naproxen was considered more effective than aspirin. Although patients taking naproxen had less frequent gastrointestinal side effects and fewer symptoms VIIIth nerve toxicity, the differences were not statistically significant. We conclude that naproxen is a useful addition to the physician's armamentarium for the treatment of rheumatoid arthritis.