Influence of the technique and comorbidities in hypofractionated radiotherapy for prostate cancer.

PURPOSE: To analyze the differences in toxicity and biochemical relapse-free survival with hypofractionated radiotherapy with three-dimensional radiotherapy (3D-CRT) or volumetric arc therapy (VMAT) for prostate cancer taking into account comorbidity measured using the Charlson Comorbidity Index (CCI). METHODS: From January 2011 to June 2016, 451 patients with prostate cancer were treated with 60 Gy (20 daily fractions). VMAT or 3D-CRT was used. Distribution by stage: 17% low-risk, 27.2% intermediate-risk; 39.2% high-risk, 16.6% very high-risk. Mean CCI was 3.4. RESULTS: With a median follow up of 51 months, most patients did not experience any degree of acute GI toxicity (80.9%) compared to 19.1%, who experienced some degree, mainly G-I /II. In the multivariate analysis, only technique was associated with acute GI toxicity ≥ G2. Patients treated with VMAT had greater acute GI toxicity compared with those who received 3D-CRT (23.9% vs. 13.5%, p = 0.005). With respect to acute GU toxicity, 72.7% of patients experienced some degree, fundamentally G-I/II. Neither age, CCI, nor androgen deprivation therapy (ADT) were associated with greater toxicity. Overall survival at 2, 5 and 7 years was 97%, 88% and 83% respectively. The only factor with statistical significance was CCI, with a greater number of events in individuals with a CCI ≥ 4 (p < 0.03). CONCLUSIONS: Hypofractionated radiotherapy for prostate cancer is an effective, well-tolerated treatment even for elderly patients with no associated comorbidity. Longer follow up is needed in order to report data on late toxicity.

The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses.

Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a approximately 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the 'core' region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5'-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5'-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A 'closest match' or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.

A phylogeographical analysis of the bemisia tabaci species complex based on mitochondrial DNA markers

Mitochondrial 16S ( approximately 550 bp) and cytochrome oxidase I (COI) ( approximately 700 bp) sequences were utilized as markers to reconstruct a phylogeography for representative populations or biotypes of Bemisia tabaci. 16S sequences exhibited less divergence than COI sequences. Of the 429 characters examined for COI sequences, 185 sites were invariant, 244 were variable and 108 were informative. COI sequence identities yielded distances ranging from less than 1% to greater than 17%. Whitefly 16S sequences of 456 characters were analysed which consisted of 298 invariant sites, 158 variable sites and 53 informative sites. Phylogenetic analyses conducted by maximum parsimony, maximum-likelihood and neighbour-joining methods yielded almost identical phylogenetic reconstructions of trees that separated whiteflies based on geographical origin. The 16S and COI sequence data indicate that the B-biotype originated in the Old World (Europe, Asia and Africa) and is most closely related to B-like variants from Israel and Yemen, with the next closest relative being a biotype from Sudan. These data confirm the biochemical, genetic and behavioural polymorphisms described previously for B. tabaci. The consideration of all global variants of B. tabaci as a highly cryptic group of sibling species is argued.

Characterization of tobacco geminiviruses in the Old and New World.

Biological differences and molecular variability between six phenotypically distinct tobacco-infecting geminivirus isolates from southern Africa (Zimbabwe) and Mexico were investigated. Host range studies conducted with tobacco virus isolates ZIM H from Zimbabwe and MEX 15 and MEX 32 from Mexico indicated all had narrow host ranges restricted to the Solanaceae. Alignment of coat protein gene (CP) and common region (CR) sequences obtained by PCR, and phylogenetic analysis of the CP sequences indicated Zimbabwean isolates were distantly related to those from Mexico and that geographically proximal isolates shared their closest affinities with Old and New World geminiviruses, respectively. Zimbabwean isolates formed a distinct cluster of closely related variants (> 98% sequence identity) of the same species, while MEX 15 segregated independently from MEX 32, the former constituting a distinct species among New World geminiviruses, and the latter being a variant, Texas pepper virus-Chiapas isolate (TPV-CPS) with 95% sequence identity to TPV-TAM. Results collectively indicated a geographic basis for phylogenetic relationships rather than a specific affiliation with tobacco as a natural host. MEX 15 is provisionally described as a new begomovirus, tobacco apical stunt virus, TbASV, whose closest CP relative is cabbage leaf curl virus, and ZIM isolates are provisionally designated as tobacco leaf curl virus, TbLCV-ZIM, a new Eastern Hemisphere begomovirus, which has as its closest relative, chayote mosaic virus from Nigeria.

First Report of Sinaloa Tomato Leaf Curl Geminivirus in Costa Rica.

In October 1998, geminivirus-like symptoms were widespread in tomato plantings near Turrialba, Costa Rica. Isolates from several fields were experimentally transmitted to tomato seedlings with whiteflies from a Bemisia tabaci (Genn.) colony maintained at CATIE, which resulted in interveinal chlorosis and leaf curling symptoms indistinguishable from those observed in the field. Total DNA was extracted from leaves of 16 of these experimentally inoculated plants and assayed by polymerase chain reaction (PCR) for the presence of begomovirus DNA with the degenerate primers AV324 and AC889 (2) to amplify the core region of the coat protein gene (core Cp). PCR yielded the expected size core Cp fragment (576 bp) from 16 of 16 samples. The core Cp fragments of six samples were cloned and sequenced. A comparison of the core Cp sequences with reference begomovirus sequences indicated all Costa Rican isolates were >95% identical to Sinaloa tomato leaf curl geminivirus reported in 1994 from Sinaloa, Mexico (STLCV-SINALOA). Virus identity was confirmed by multiple sequence alignments of the viral coat protein gene (Cp) and the common region (CR) sequences of A and B components (CR-A and CR-B), respectively, with analogous reference begomovirus sequences. Cp and CRs were obtained by PCR, and amplicons were cloned and sequenced as described (1). The Cp open reading frame (ORF; 756 nucleotides) (AF110515) identified within the A component amplicon shared 92.9% sequence identity with STLCV-SINALOA Cp (AF040635). The CR sequences of the A (AF1150516) and B (AF110517) components (163 nucleotides) shared 98.2% sequence identity with each other, suggesting that they were amplified from the cognate A and B components of the same virus. Further, the CR-A and CR-B components contained the same putative Rep binding site, TGGGGT-AA-TGGGGT, which was also identical to that of STLCV-SINALOA. The mean percent divergences between viral Cp and CR amplicons (n = 6+) ranged from 98 to 100%. Collectively, STLCV-like symptoms in tomato, >92% identity between viral Cp sequences, and identical CR iterons indicate that the Costa Rican tomato virus is STLCV, or a closely related strain. This is the first report of an STLCV-like begomovirus in tomato in Costa Rica (STLCV-CR). References: (1) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Tracing the geminivirus-whitefly transmission pathway by polymerase chain reaction in whitefly extracts, saliva, hemolymph, and honeydew.

ABSTRACT A membrane feeding system and polymerase chain reaction (PCR) were used to track squash leaf curl virus (SLCV) DNA in whole whitefly body extracts and in saliva, honeydew, and hemolymph of its whitefly vector, Bemisia tabaci, and a whitefly nonvector, Trialeurodes vaporariorum. SLCV ingestion was monitored by PCR in whiteflies that were given acquisition access periods (AAPs) ranging from 0.5 to 96 h on virus-infected plants. SLCV detection by PCR in whole body extracts was considered reflective of virus ingestion. As whiteflies were given longer AAPs, the number of whiteflies that ingested SLCV increased. SLCV DNA was detected in honeydew of vector and nonvector whiteflies, indicating that virions, viral DNA, or both passed unimpeded through the digestive system. SLCV DNA was detected in saliva and hemolymph of B. tabaci, but not in these fractions from nonvector whiteflies, despite virus ingestion by both. Although vector and nonvector whiteflies both ingested SLCV, only in the vector, B. tabaci, did virus cross the gut barrier, enter the hemolymph, or pass into the salivary system. These results suggest that digestive epithelia of nonvector whiteflies did not permit SLCV passage from the gut to hemocoel, whereas virus effectively crossed the analogous gut barrier in vector whiteflies.

Partial sequencing and mapping of clones from two maize cDNA libraries.

As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, 'single-pass' sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total 313 clones from the two libraries were submitted to 'single-pass' sequencing from the presumed 5' end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.