Upper extremity limb salvage with microvascular reconstruction in patients with advanced sarcoma

Limb salvage is a viable alternative to amputation in many cases of advanced sarcoma. The authors examined their experience with microvascular reconstruction of upper extremity defects after sarcoma resection, focusing on oncologic and functional outcomes. A retrospective analysis yielded 17 patients who underwent 18 free flap procedures and met the inclusion criteria. Most patients (71 percent, n = 12) had recurrent sarcoma at presentation to the authors' institution. Malignant fibrous histiocytoma was the most common pathologic subtype (n = 6). High-grade tumors were present in 94 percent of patients (n = 16). The free flap survival rate was 100 percent. The rectus abdominis flap was the most common free flap used (39 percent; n = 7). Local recurrence occurred in nine flaps (50 percent), and five patients ultimately required amputations. Six patients (35 percent) had distant recurrence. The mean Enneking score for limb function was 73 percent of the maximum (21.9 of 30). The 5-year disease-specific survival rate was 61.3 percent. In select patients with advanced upper extremity sarcoma undergoing limb salvage, microvascular flap reconstruction can provide reliable, safe coverage with reasonable preservation of function.     

Polymorphisms in detoxification genes CYP1A1, CYP2E1, GSTT1 and GSTM1 in gastric cancer susceptibility

Cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes are involved in activation and detoxification of many potential carcinogens. Genetic polymorphisms in those enzymes have been found to influence the interindividual susceptibility to cancer. Some polymorphisms of those enzymes have been associated specifically with susceptibility to gastric cancer. We conducted a study in a Costa Rican population, where gastric cancer incidence and mortality rates are among the highest in the world. We investigated whether such variations affected the risk of developing gastric cancer. Subjects included 31 with gastric cancer, 58 controls with gastric injures others than cancer and 51 normal controls confirmed by X-rays (double-contrast) or endoscopic diagnostic. DNA from peripheral white blood cell was obtained from all subjects. Deletion of GSTT1 and GSTM1 was assessed by multiplex PCR and genotyping of CYP2E1 was performed using a PCR-based restriction fragment length polymorphism assay with the restriction enzyme PstI and the gene CYP1A1 using the restriction enzyme MspI The prevalence of CYP1A1 Msp1 polymorphism, GSTT1 and GSTM1 null genotype was similar in the three groups of individuals (p = 0.73, p = 0.88 y p = 0.89 respectively). Our findings suggest that the polymorphism CYP2E1 PstI could be associated with a reduced risk of having gastric cancer (OR = 0.09, IC95%:0.01 - 0.83).     

Synthesis of 3-methyl-3-hydroxy-6-oxo-androstane derivatives

3alpha,17beta-Dihydroxy-3beta-methyl-5alpha-androstan-6-one (1) and 3beta,17beta-dihydroxy-3alpha-methyl-5alpha-androstan-6-one (13) were prepared by the reaction of methylmagnesium bromide with the 3-ketosteroids. Structures and configurations in position 3 were determined by NMR spectra. Substitution in the position 6 influences the ratio of the products.     

Efficient targeting of plant disease resistance loci using NBS profiling

The conserved sequences in the nucleotide-binding sites of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance (R) genes have been used for PCR-based R-gene isolation and subsequent development of molecular markers. Here we present a PCR-based approach (NBS profiling) that efficiently targets R genes and R-gene analogs (RGAs) and, at the same time, produces polymorphic markers in these genes. In NBS profiling, genomic DNA is digested with a restriction enzyme, and an NBS-specific (degenerate) primer is used in a PCR reaction towards an adapter linked to the resulting DNA fragments. The NBS profiling protocol generates a reproducible polymorphic multilocus marker profile on a sequencing gel that is highly enriched for R genes and RGAs. NBS profiling was successfully used in potato with several restriction enzymes, and several primers targeted to different conserved motifs in the NBS. Across primers and enzymes, the NBS profiles contained 50–90% fragments that were significantly similar to known R-gene and RGA sequences. The protocol was similarly successful in other crops (including tomato, barley, and lettuce) without modifications. NBS profiling can thus be used to produce markers tightly linked to R genes and R-gene clusters for genomic mapping and positional cloning and to mine for new alleles and new sources of disease resistance in available germplasm.Communicated by H.F. Linskens     

Mapping of AFLP markers linked to seed coat colour loci in<Emphasis Type="Italic"> Brassica juncea</Emphasis> (L.) Czern

Association mapping of the seed-coat colour with amplified fragment length polymorphism (AFLP) markers was carried out in 39 Brassica juncea lines. The lines had genetically diverse parentages and varied for seed-coat colour and other morphological characters. Eleven AFLP primer combinations were used to screen the 39 B. juncea lines, and a total of 335 polymorphic bands were detected. The bands were analysed for association with seed-coat colour using multiple regression analysis. This analysis revealed 15 markers associated with seed-coat colour, obtained with eight AFLP primer combinations. The marker E-ACA/M-CTG₃₅₀ explained 69% of the variation in seed-coat colour. This marker along with markers E-AAC/M-CTC₂₃₅ and E-AAC/M-CTA₂₅₀ explained 89% of the total variation. The 15 associated markers were validated for linkage with the seed-coat colour loci using a recombinant inbred line (RIL) mapping population. Bands were amplified with the eight AFLP primer combinations in 54 RIL progenies. Of the 15 associated markers, 11 mapped on two linkage groups. Eight markers were placed on linkage group 1 at a marker density of 6.0 cM, while the remaining three were mapped on linkage group 2 at a marker density of 3.6 cM. Marker E-ACA/M-CTG₃₅₀ co-segregated with Gene1 controlling seed-coat colour; it was specific for yellow seed-coat colour and mapped to linkage group 1. Marker E-AAC/M-CTC₂₃₅ (AFLP8), which had been studied previously, was present on linkage group 2; it was specific for brown seed-coat colour. Since AFLP markers are not adapted for large-scale applications in plant breeding, it is important to convert these to sequence-characterised amplified region (SCAR) markers. Marker E-AAC/M-CTC₂₃₅ (AFLP8) had been previously converted into a SCAR. Work is in progress to convert the second of the linked markers, E-ACA/M-CTG₃₅₀, to a SCAR. The two linked AFLP markers converted to SCARs will be useful for developing yellow-seeded B. juncea lines by means of marker-assisted selection.Communicated by H.F. Linskens     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

Chloroplast and mitochondrial molecular tests identify European×Japanese larch hybrids

Hybrids between European and Japanese larches combine the properties of both parental species (drought resistance, canker resistance, stem straightness) and exhibit a fast growth rate. They are produced in seed orchards, generally by natural pollination. Seeds are collected and used for afforestation as interspecific hybrids. However, there are no convenient tests to assess the interspecific hybrid proportion. In the present study, we developed diagnostic molecular markers suitable for the individual identification of hybrids, whatever their developmental stage. Our strategy involved testing a combination of maternally inherited markers from the mitochondrial genome (mtDNA) and paternally inherited markers from the chloroplast genome (cpDNA). Hybrids were then identified by the presence of a mitochondrial sequence inherited from one parental species and a chloroplast sequence inherited from the other parental species. To achieve this aim, markers discriminating both parental species were first sought. Amplifications of mitochondrial and chloroplast sequences were performed using specific PCR primers. After testing 33 primer pairs in combination with nine restriction enzymes, we detected one mitochondrial marker, f13 which was amplified in Japanese larch and absent in European larch, and one chloroplast marker, ll-TaqI which showed different restriction patterns depending on the species. A restriction fragment of 601 bp was obtained in Japanese larch while two fragments of 120 bp and 481 bp were observed in European larch. These patterns were found in all 197 individuals tested from the two pure species. These markers were then used for the evaluation of the hybrid proportion in a seed lot produced from seed orchards; this was assessed as between 43% and 53% depending on the parental species. The male and female parental species could be determined for each progeny.Communicated by D.B. Neale     

QTL mapping of <Emphasis Type="Italic">Sclerotinia</Emphasis> midstalk-rot resistance in sunflower

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F₃ families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F₂ genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F₃ families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h²=0.55–0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.     

Ixodes ricinus, transmitted diseases and reservoirs

The tick Ixodes ricinus has been recorded in most Italian regions especially in thermo-mesophilous woods and shrubby habitats where the relative humidity allow the tick to complete its 3 year developmental cycle, as predicted for the European climatic ranges. This tick acts both as vector and reservoir for a series of wildlife zoonotic pathogens, especially the agents of Lyme diseases, Tick borne encephalitis and Human Granulocytic Ehrlichiosis, which are emerging in most of Europe. To assess the spatial distribution of these pathogens and the infection risk for humans and animals within the territory of the Province of Trento, we carried out a long term study using a combination of eco-epidemiological surveys and mathematical modelling. An extensive tick collection with a GIS based habitat suitability analysis allowed us to identify the areas where tick occurs at various density. To identify the areas with higher infection risk, we estimated the values of R0 for Borrelia burgdorferi s.l., TBE virus and Anaplasma phagocytophila under different ecological conditions. We assessed the infection prevalence in the vector and in the wildlife reservoir species that play a central role in the persistence of these infections, ie the small mammals A. flavicollis and C. glareolus. We also considered the double effect of roe deer (Capreolus capreolus) which act as reservoir for A. phagocytophila but is an incompetent host for B. burgdorferi and TBE virus, thus reducing the infection prevalence in ticks of these last two pathogens. Infection prevalence with B. burgdorferi and A. phagocytophila in the vector was assessed by PCR screening 1212 I. ricinus nymphs collected by dragging in six main study areas during 2002. The mean infection prevalence recorded was 1.32% for B. burgdorferi s.l. and 9.84% for A. phagocytophila. Infection prevalence in nymphs with TBE virus, as assessed in a previous study was 0.03%. Infection prevalence in rodents was assessed by screening (with ELISA and PCR) tissues and blood samples collected from 367 rodent individuals trapped extensively during 2002 within 6 main study areas. A. flavicollis (N=238) was found to be infected with all three pathogens investigated, with infection prevalence ranging from 3.3% for TBE virus to 11.7% for A. phagocytophila, and 16.6% with B. burgdorferi s.l. C. glareolus (N=108) showed an infection prevalence of 6.5% with A. phagocytophila and 12.7% with B. burgdorferi s.l., while no individuals were infected with TBE virus. We also screened 98 spleen samples collected from roe deer with PCR, resulting in a mean prevalence of infection with A. phagocytophila of 19.8%. Using a deterministic model we explored the condition for diseases persistence under different rodent and roe deer densities. R0 values resulted largely above 1 for B. burgdorferi s.l. in the vast majority of the areas classified as suitable for I. ricinus occurrence in Trentino, while the condition for TBE persistence appeared to be more restricted by a combination of climatic condition and host densities.