Development of first linkage map for Silphium integrifolium (Asteraceae) enables identification of sporophytic self-incompatibility locus.

Silphium integrifolium (Asteraceae) has been identified as a candidate for domestication as a perennial oilseed crop and is assumed to have sporophytic self-incompatibility system-the genetic basis of which is not well understood in the Asteraceae. To address this gap, we sought to map the genomic location of the self-recognition locus (S-locus) in this species. We used a biparental population and genotyping-by-sequencing to create the first genetic linkage map for this species, which contained 198 SNP markers and resolved into the correct number of linkage groups. Then we developed a novel crossing scheme and set of analysis methods in order to infer S-locus genotypes for a subset of these individuals, allowing us to map the trait. Finally, we evaluated potential genes of interest using synteny analysis with the annual sunflower (Helianthus annuus) and lettuce (Lactuca sativa) genomes. Our results confirm that S. integrifolium does indeed have a sporophytic self-incompatibility system. Our method is effective and efficient, allowed us to map the S. integrifolium S-locus using fewer resources than existing methods, and could be readily applied to other species.

Measurements of lethal and nonlethal inbreeding depression inform the de novo domestication of Silphium integrifolium.

PREMISE: Inbreeding depression, or the reduction in fitness of progeny with related parents, has the potential to adversely affect the long-term viability of both wild and captive plant populations. Silphium integrifolium, a prairie plant native to the central United States, has been identified as a potential candidate for domestication as a perennial oilseed crop. Little is known about the potential for inbreeding depression in this species, but it is expected to be nonnegligible because S. integrifolium is both perennial and self-incompatible. Here, we measure lethal inbreeding depression expressed through embryo deaths, and nonlethal inbreeding depression expressed through changes in vigor and fitness phenotypes of progeny. METHODS: First, we made controlled crosses among related and unrelated individuals to determine the effect of two different levels of inbreeding on seed production. Then, we grew inbred and outbred progeny from this population to reproductive maturity and measured 11 key traits. RESULTS: We found that within an improved S. integrifolium population, individuals carried an average of slightly less than one lethal allele per gamete. In progeny, significant inbreeding depression was observed in at least one family for eight of the 11 measured traits. CONCLUSIONS: Inbreeding depression is likely to be an important challenge to S. integrifolium domestication, reducing overall population fecundity and values for important phenotypes. These effects may grow worse as selection reduces effective population size. We recommend several strategies for S. integrifolium breeding to help mitigate these problems.

Radical excision of vulval disease and multidisciplinary tissue flap reconstruction: the belfast experience.

OBJECTIVE: The aim of this study was to compare Belfast City Hospital's vulval flap reconstructive surgery with recognized standards and published data to facilitate modifications to practice. MATERIALS AND METHODS: Retrospective cohort study involving women who underwent vulval reconstructive skin flap procedures from January 1, 2004, through December 31, 2009. RESULTS: A total of 25 women underwent 28 flaps; age range was 41 to 90 years and mean parity was 2.1. Indications for surgery were recurrence of cancer in 11 (44%) of the 25 women, benign disease in 6 women (24.0%), primary vulval cancer in another 6 women (24.0%), and vulval intraepithelial neoplasia in 2 women. Thirteen women (52.0%) within the study had undergone previous radical vulval excision. Wide local excision was performed in 14 women (56.0%), with 5 undergoing radical vulvectomy and 6 undergoing modified radical vulvectomy. There were 15 (53.6%) of the 28 lotus flaps, 6 (21.4%) rhomboid-type repairs, and 3 (10.7%) gracilus flaps.Intensive care unit admission immediately after operation was required in 2 (8.0%) of the 25 women. Return to the operating theater was required in 5 cases (20.0%). Hospital stay ranged from less than 1 week to greater than 6 weeks, the mode being 8 to 14 days and mean being 21.5 days. A total of 16 (64.0%) of the 25 cases experienced wound infection with or without breakdown, with 12 cases (48.0%) having wound infection. There were 10 cases (40.0%) of partial or complete wound breakdown. Necrosis of wound (5 cases) and lymphocyst (2 cases) were less common. Twelve women (48.0%) experienced some form of medical postoperative complication(s), whereas 15 women (60.0%) had no long-term complications. CONCLUSIONS: The rate of local flap procedure is higher than that published elsewhere, whereas the postoperative complication rate is comparable and adherent to accepted standards. Perioperative protocols have been reported to improve complication rates, and this strategy has been adopted in Belfast.

Serum CA19.9 levels are commonly elevated in primary ovarian mucinous tumours but cannot be used to predict the histological subtype.

AIMS: CA19.9 is a monosialoganglioside secreted by mucinous tumours of the gastrointestinal tract, including the pancreas and biliary tree. Limited studies have shown that this tumour marker may also be elevated in primary ovarian mucinous neoplasms, but no study has assessed whether serum CA19.9 levels can be used to predict if a primary ovarian mucinous tumour is benign, borderline or malignant. The aim of this study was to correlate the serum CA19.9 level with the histological features in a large series of primary ovarian mucinous neoplasms. METHODS: 144 cases of primary ovarian mucinous neoplasm (79 benign, 45 borderline and 20 malignant) were identified in which a preoperative serum CA19.9 level had been performed. The association between the serum levels and the histological subtype and a variety of other parameters was investigated. In a subset of cases, immunohistochemical staining for CA19.9 was performed on tumour blocks. RESULTS: Serum CA19.9 levels were elevated in 27%, 38% and 40% of mucinous cystadenomas, borderline mucinous tumours and mucinous carcinomas, respectively. Markedly elevated levels of serum CA19.9 were observed in each group, with the highest serum CA19.9 measurements being noted in borderline mucinous tumours. There was no relationship between the serum CA19.9 level and whether the tumours were benign, borderline or malignant (Kruskal-Wallis test p value=0.32). A weak but statistically significant correlation was found between tumour maximum dimension and CA19.9 level (Spearman's rank correlation coefficient=0.17, p=0.04). In those cases in which CA19.9 immunohistochemistry was performed, all tumours showed positive staining for CA19.9, with 60% of these cases being associated with an elevated serum CA19.9 level. CONCLUSION: Preoperative CA19.9 levels cannot be used to predict whether a suspected ovarian mucinous tumour is benign, borderline or malignant. Markedly elevated serum levels (>1000 U/ml) may be found in benign mucinous neoplasms as well as in borderline and malignant tumours.

Response of vulval lichen sclerosus and squamous hyperplasia to photodynamic treatment using sustained topical delivery of aminolevulinic acid from a novel bioadhesive patch system.

This study evaluated the clinical and histopathological responses of vulval lichen sclerosus (LS) and squamous hyperplasia (SH) to photodynamic therapy (PDT). A novel bioadhesive patch containing aminolevulinic acid (ALA) at a dose of (38 mg/cm(2)) was used to treat 10 patients before irradiation with light of 630 nm. Clinical, histopathological and pathological responses to treatment were assessed at 6 weeks post-treatment. After 17 cycles of PDT, six patients reported significant symptomatic relief and no cutaneous photosensitivity. Histopathological differences were not demonstrated, but statistically significant induction of apoptosis was seen. It can be concluded that ALA-PDT patch-based formulation is pragmatic and primarily offers symptomatic management of vulval LS and SH.

Germline MSH6 mutations are more prevalent in endometrial cancer patient cohorts than hereditary non polyposis colorectal cancer cohorts.

OBJECTIVE: To determine and compare the prevalence of MSH6 (a mismatch repair gene) mutations in a cohort of families with Hereditary Non-Polyposis Colorectal Cancer (HNPCC), and in an unselected cohort of endometrial cancer patients (EC). DESIGN: Two patient cohorts participated in the study. A cohort of HNPCC families who were known to the Regional Medical Genetics department, and an unselected cohort of patients with a history of EC. All participants received genetic counselling on the implications of molecular testing, and blood was taken for DNA extraction with consent. All samples underwent sequencing and Multiple Ligation probe analysis (MLPA) for mutations in MSH6. POPULATIONS: DNA from one hundred and forty-three probands from HNPCC families and 125 patients with EC were included in the study. METHODS: Molecular analysis of DNA in all participants from both cohorts for mutations in MSH6. OUTCOME MEASURES: Prevalence of pathogenic mutations in MSH6. RESULTS: A truncating mutation in MSH6 was identified in 3.8% (95% CI 1.0-9.5%) of patients in the endometrial cancer cohort, and 2.6% (95% CI 0.5-7.4%) of patients in the HNPCC cohort. A missense mutation was identified in 2.9% and 4.4% of the same cohorts respectively. No genomic rearrangements in MSH6 were identified. CONCLUSION: MSH6 mutations are more common in EC patients than HNPCC families. Genomic rearrangements do not contribute to a significant proportion of mutations in MSH6, but missense variants are relatively common and their pathogenicity can be uncertain. HNPCC families may be ascertained through an individual presenting with EC, and recognition of these families is important so that appropriate cancer surveillance can be put in place.

Novel bioadhesive patch-type system for photodynamic vulvodynia therapy after delivery of 5-aminolevulinic acid: preliminary evaluation.

OBJECTIVE: To assess the applicability of photodynamic therapy (PDT) in the management of vulvodynia whereby a novel, patch-type system, loaded with 5-aminolevulinic acid (ALA), was used to administer PDT to vulvar regions displaying the characteristics of vulvodynia. STUDY DESIGN: Eleven patients underwent PDT using a bioadhesive patch to deliver ALA over 4 hours. A nonlaser light source delivered 100 J cm(-2) to the target area using red light of 630 nm. Fluorescence of protoporphyrin IX was observed under ultraviolet light illumination, with no significant difference found between that produced after the first and second applications of the patch. RESULTS: There was a significant reduction (p= 0.0077) in overall symptoms after completion of treatment. No significant alleviation (p = 0.1088) in pain during intercourse was observed following treatment. Eight patients experienced a symptomatic response, while 3 exhibited no improvements in symptoms. No adverse reactions or worsening of reported symptoms was reported. CONCLUSION: The results suggest that PDT is of value in the management of vulvodynia, most probably as a viable option to conventional approaches. Further studies involving larger numbers of patients are required to confirm the efficacy of PDT in the management of vulvodynia.

Evaluation of a water-soluble bioadhesive patch for photodynamic therapy of vulval lesions.

An innovative bioadhesive patch intended primarily as a vulval drug delivery system and, specifically, as a means to deliver photosensitisers, or their prodrugs, for photodynamic purposes is described. The patch was formulated with a copolymer of methyl vinyl ether and maleic anhydride (PMVE/MA) as a bioadhesive matrix and poly(vinyl chloride) as a drug-impervious backing layer. Adhesive strength to neonate porcine skin, as a model substrate, was evaluated using peel and tensile testing measurements. Acceptabilities of non-drug loaded patches were appraised using human volunteers and visual-analogue scoring devices. An optimal formulation, with water uptake and peel strengths appropriate for vulval drug delivery, was cast from a 20% (w/w) PMVE/MA solution and adhered with a strength of approximately 1.7 Ncm(-2). Patient evaluation demonstrated comfort and firm attachment for up to 4h in mobile patients. Aminolevulinic acid, a commonly used photosensitiser, was formulated into the candidate formulation and applied to vulval intraepithelial neoplastic lesions. Fluorescence under ultraviolet illumination revealed protoporphyrin synthesis. The patch achieves the extended application times obligatory in topical photodynamic therapy of vulval lesions, thereby contributing to potential methods for the eradication of neoplastic lesions in the lower female reproductive tract.

Phototoxicity of 5-aminolevulinic acid in the HeLa cell line as an indicative measure of photodynamic effect after topical administration to gynecological lesions of intraepithelial form.

PURPOSE: The depth-resolved pattern of aminolevulinic acid (ALA) concentration in excised vaginal tissue was determined after in vitro application of an ALA-loaded bioadhesive patch. From this data, the tissue concentration of ALA achievable at a specified depth from the surface could be related to the concentration needed to elicit a photodynamic effect in a model gynecological tumor cell line (HeLa). METHODS: Excised vaginal tissue was mounted in a modified Franz diffusion cell and exposed to a water-soluble, ALA-loaded, bioadhesive patch. After a period of time, the tissue was cryostatically sectioned and the stratal concentration of radiolabeled ALA determined using scintillation spectroscopy. HeLa cells were cultured in media containing specific concentrations of ALA and exposed to standard photodynamic protocols of light exposure. RESULTS: An ALA concentration of 65.6 mM was achievable at 2.375 mm from the tissue surface after application of ALA-loaded patch. The photodynamic effectiveness of this concentration was demonstrated in HeLa with exposure to concentrations exceeding 1.0 mM ALA bringing about reductions in viable cell numbers by 90%. An enhancement of PpIX production using adjunctive EDTA over the clinically relevant 4 h application time interval was shown to be minimal in HeLa. Instead, PpIX production was more closely correlated with ALA concentration, with 100 mM ALA producing approximately 3100 ng PpIX mg(-1) protein in the same time period. CONCLUSIONS: Given that vaginal intraepithelial neoplasias can extend to 2.0 mm from the lesion surface, the ALA permeability derived from a bioadhesive patch is sufficient to induce photosensitization suitable for light induced destruction at deep sites of this type of lesion.