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The stromal compartment surrounding epithelial-derived pancreatic tumors is thought to have a key role in the aggressive phenotype of this malignancy. Emerging evidence suggests that cancer-associated fibroblasts (CAFs), the most abundant cells in the stroma of pancreatic tumors, contribute to the tumor’s invasion, metastasis and resistance to therapy, but the precise molecular mechanisms that regulate CAFs behavior are poorly understood. In this study, we utilized immortalized human pancreatic CAFs to investigate molecular pathways that control the matrix-remodeling and invasion-promoting activity of CAFs. We showed previously that palladin, an actin-associated protein, is expressed at high levels in CAFs of pancreatic tumors and other solid tumors, and also in an immortalized line of human CAFs. In this study, we found that short-term exposure of CAFs to phorbol esters reduced the number of stress fibers and triggered the appearance of individual invadopodia and invadopodial rosettes in CAFs. Molecular analysis of invadopodia revealed that their composition resembled that of similar structures (that is, invadopodia and podosomes) described in other cell types. Pharmacological inhibition and small interfering RNA knockdown experiments demonstrated that protein kinase C, the small GTPase Cdc42 and palladin were necessary for the efficient assembly of invadopodia by CAFs. In addition, GTPase activity assays showed that palladin contributes to the activation of Cdc42. In mouse xenograft experiments using a mixture of CAFs and tumor cells, palladin expression in CAFs promoted the rapid growth and metastasis of human pancreatic tumor cells. Overall, these results indicate that high levels of palladin expression in CAFs enhance their ability to remodel the extracellular matrix by regulating the activity of Cdc42, which in turn promotes the assembly of matrix-degrading invadopodia in CAFs and tumor cell invasion. Together, these results identify a novel molecular signaling pathway that may provide new molecular targets for the inhibition of pancreatic cancer metastasis.

The inheritance of yield-related traits in melon (Cucumis melo L.; 2n = 2x = 24) is poorly understood, and the mapping of quantitative trait loci (QTL) for such traits has not been reported. Therefore, a set of 81 recombinant inbred lines (RIL) was developed from a cross between the monoecious, highly branched line USDA 846-1 and a standard vining, andromonoecious cultivar, 'Top Mark'. The RIL, parental lines, and three control cultivars ('Esteem', 'Sol Dorado', and 'Hales Best Jumbo') were grown at Hancock, WI and El Centro, CA in 2002, and evaluated for primary branch number (PB), fruit number per plant (FN), fruit weight per plant (FW), average weight per fruit (AWF), and percentage of mature fruit per plot (PMF). A 190-point genetic map was constructed using 114 RAPD, 43 SSR, 32 AFLP markers, and one phenotypic trait. Fifteen linkage groups spanned 1,116 cM with a mean marker interval of 5.9 cM. A total of 37 QTL were detected in both locations (PB = 6, FN = 9, FW = 12, AWF = 5, and PMF = 5). QTL analyses revealed four location-independent factors for PB (pb1.1, pb1.2, pb2.3, and pb10.5), five for FN (fn1.1, fn1.2, fn1.3, fn2.4, and fn8.8), four for FW (fw5.8, fw6.10, fw8.11, and fw8.12), two for AWF (awf1.3 and awf8.5), and one for PMF (pmf10.4). The significant (P <= 0.05) positive phenotypic correlations observed among PB, FN, and FW, and negative phenotypic correlations between PB and AWF and between FN and AWF were consistent with the genomic locations and effects (negative vs. positive) of the QTL detected. Results indicate that genes resident in highly branched melon types have potential for increasing yield in US Western Shipping type germplasm via marker-assisted selection.

A set of 171 recombinant inbred lines (RIL) were developed from a narrow cross in cucumber ( Cucumis sativus L.; 2n = 2 x = 14) using the determinate ( de), gynoecious ( F), standard-sized leaf line G421 and the indeterminate, monoecious, little-leaf ( ll) line H-19. A 131-point genetic map was constructed using these RILs and 216 F(2) individuals to include 14 SSRs, 24 SCARs, 27 AFLPs, 62 RAPDs, 1 SNP, and three economically important morphological [ F (gynoecy), de (determinate habit), ll (little leaf)] markers. Seven linkage groups spanned 706 cM with a mean marker interval of 5.6 cM. The location of F and de was defined by genetic linkage and quantitative trait locus (QTL) analysis to be associated with SSR loci CSWCT28 and CSWCTT14 at 5.0 cM and 0.8 cM, respectively. RIL-based QTL analysis of the number of lateral branches in three environments revealed four location-independent factors that cumulatively explained 42% of the observed phenotypic variation. QTLs conditioning lateral branching (mlb1.1), fruit length/diameter ratio (ldr1.2) and sex expression (sex1.2) were associated with de. Sex expression was influenced by three genomic regions corresponding to F and de both on linkage Group 1, and a third locus (sex6.1) on linkage Group 6. QTLs conditioning the number of fruit per plant (fpl1.2), the number of lateral branches (mlb1.4) and fruit length/diameter ratio (ldr1.3) were associated with ll. The potential value of these marker-trait associations (i.e., yield components) for plant improvement is portended by the relatively high LOD scores (2.6 to 13.0) and associated R(2) values (1.5% to 32.4%) that are affiliated with comparatively few genetic factors (perhaps 3 to 10).

Transgenic plants have become attractive systems for production of human therapeutic proteins because of the reduced risk of mammalian viral contaminants, the ability to do large scale-up at low cost, and the low maintenance requirements. Here we report a feasibility study for production of a human therapeutic protein through transplastomic transformation technology, which has the additional advantage of increased biological containment by apparent elimination of the transmission of transgenes through pollen. We show that chloroplasts can express a secretory protein, human somatotropin, in a soluble, biologically active, disulfide-bonded form. High concentrations of recombinant protein accumulation are observed (>7% total soluble protein), more than 300-fold higher than a similar gene expressed using a nuclear transgenic approach. The plastid-expressed somatotropin is nearly devoid of complex post-translational modifications, effectively increasing the amount of usable recombinant protein. We also describe approaches to obtain a somatotropin with a non-methionine N terminus, similar to the native human protein. The results indicate that chloroplasts are a highly efficient vehicle for the potential production of pharmaceutical proteins in plants.

The nutritional value and yield potential of US Western Shipping melon (USWS; Cucumis melo L.) could be improved through the introgression of genes for early fruit maturity (FM) and the enhancement of the quantity of β-carotene (QβC) in fruit mesocarp (i.e., flesh color). Therefore, a set of 116 F₃ families derived from the monoecious, early FM Chinese line 'Q 3-2-2' (no β-carotene, white mesocarp) and the andromonoecious, late FM USWS line 'Top Mark' (possessing β-carotene, orange mesocarp) were examined during 2 years in Wisconsin, USA to identify quantitative trait loci (QTL) associated with FM and QβC. A 171-point F₂₋₃ based map was constructed and used for QTL analysis. Three QTL associated with QβC were detected, which explained a significant portion of the observed phenotypic variation (flesh color; R ² = 4.0-50.0%). The map position of one QTL (β-carM.E.9.1) was uniformly aligned with one carotenoid-related gene (Orange gene), suggesting its likely role in QβC in this melon population and putative relationship with the melon white flesh (wf) gene. Two major (FM.6.1 and FM.11.1; R ² >= 20%) and one minor QTL (FM.2.1; R ² = 8%) were found to be associated with FM. This map was then merged with a previous recombinant inbred line (RIL)-based map used to identify seven QTL associated with QβC in melon fruit. This consensus map [300 molecular markers (187 co-dominant melon and 14 interspecific; 10 LG)] provides a framework for the further dissection and cloning of published QTL, which will consequently lead to more effective trait introgression in melon.

The metabolic precursor of vitamin A, β-carotene, is essential for human health. The gene(s) controlling β-carotene quantity (QβC) has been introgressed from Xishuangbanna gourd (XIS, possessing β-carotene; Cucumis sativus L. var. xishuangbannanesis Qi et Yuan; 2n = 2x = 14) into cultivated cucumber (no β-carotene; Cucumis sativus L.). To determine the inheritance of QβC in cucumber fruit endocarp, F1 progeny and a set of 124 F7 recombinant inbred lines (RILs) derived from the cultivated cucumber line CC3 and XIS line SWCC8 were evaluated for QβC during 2009 and 2010 in Nanjing, China. Segregation analysis revealed that endocarp QβC of greenhouse-grown fruit was controlled by a single recessive gene. Further, marker analysis indicated the gene controlling QβC was linked to seven SSR markers on linkage group 3, where their order was SSR20710–SSR19511–SSR15419–SSR07706–ore–SSR23231–SSR11633–SSR20270. These markers and the putative candidate gene were mapped to cucumber chromosome 3DS. An evaluation of 30 genetically diverse cucumber lines indicated that marker SSR07706 has utility in further genetic analyses of the QβC orange endocarp gene, designated ore. Moreover, the markers defined herein may have utility for marker-assisted selection directed towards the development of cucumber germplasm with high fruit β-carotene content.

To investigate phylogenetic relationships in the genus Cucumis, 9 consensus chloroplast simple sequence repeat (ccSSR) primer pairs (ccSSR3, 9, 11, 13, 14, 17, 20, 21, and 23) were employed for DNA fragment length variation and 5 amplified fragments, ccSSR4, 12, 13, 19, and 20, were sequenced using total DNA from 13 accessions representing 7 African Cucumis species (x = 12), 3 Cucumis melo L. (x = 12) accessions, 2 Cucumis sativus L. (x = 7) accessions, and 1 Cucumis hystrix Chakr. (x = 12) accession. A Citrullus lanatus (Thunb.) Matsum. & Nakai (x = 11) accession was used as an outgroup. While fragment length analysis revealed the existence of 3 major species clusters (i.e., a group of African Cucumis species, a group composed of C. melo accessions, and a group containing C. sativus and C. hystrix species), sequence variation analysis identified 2 major species clusters (i.e., a group of African Cucumis species and a group composed of C. melo, C. sativus, and C. hystrix species). Comparative analysis using nuclear DNA (previous studies) and cpDNA sequence substitution data resulted in the placement of C. melo and C. sativus in different cluster groupings. Thus, both nuclear and cytoplasmic DNA should be employed and compared when a putative progenitor or specimens of an ancestral Cucumis species lineage is investigated. In addition, C. ficifolius (2x) and C. aculeatus (4x) of the African Cucumis species clustered together in this study. This result does not agree with reported isozyme analyses, but does agree with previously characterized chromosome homologies between these 2 species. Although African Cucumis species and C. hystrix do not share a close relationship, genetic affinities between C. sativus and C. hystrix are considerable. Combined evidence from previously published studies and data presented herein lend support to the hypothesis that C. hystrix is either a progenitor species of C. sativus or that they at least share a common ancestral lineage.

At the second visit 3 weeks later and on being questioned again, the patient reported that she was on oral testosterone undecanoat 40 mg/day for 2 months because of a general loss of energy and symptoms of fatigue attributed to a low level of endogenous testosterone. Values on testosterone treatment* Values after cessation of androgen treatment[dagger] Normal range for females Total testosterone 5.30 0.69 0.5-3.0 nmol/l SHBG 37 141 20-118 nmol/l Oestradiol 73 383 40-200 pmol/l DHEA-S 3.6 1.2 3-12 μmol/l LH 60 24 >10 E/l (menopausal) FSH 56 29 >15 E/l (menopausal) DHEA-S, dehydroepiandrosterone sulphate; FSH, follicle-stimulating hormones; LH, luteinising hormone; SHBG, sex-hormone-binding globulin. *Oral testosterone undecanoat (40 mg/day). [dagger]Oral oestradiol (1 mg/day).

Melon (Cucumis melo L.) is highly nutritious vegetable species and an important source of β-carotene (Vitamin A), which is an important nutrient in the human diet. A previously developed set of 81 recombinant inbred lines (RIL) derived from Group Cantalupensis US Western Shipper market type germplasm was examined in two locations [Wisconsin (WI) and California (CA), USA] over 2 years to identify quantitative trait loci (QTL) associated with quantity of beta-carotene (QβC) in mature fruit. A moderately saturated 256-point RIL-based map [104 SSR, 7 CAPS, 4 SNP in putative carotenoid candidate genes, 140 dominant markers and one morphological trait (a) spanning 12 linkage groups (LG)] was used for QβC-QTL analysis. Eight QTL were detected in this evaluation that were distributed across four LG that explained a significant portion of the associated phenotypic variation for QβC (R ² = 8 to 31.0%). Broad sense heritabilities for QβC obtained from RIL grown in WI. and CA were 0.56 and 0.68, respectively, and 0.62 over combined locations. The consistence of QβC in high/low RIL within location across years was confirmed in experiments conducted over 2 years. QTL map positions were not uniformly associated with putative carotenoid genes, although one QTL (β-car6.1) interval was located 10 cM from a β-carotene hydroxylase gene. These results suggest that accumulation of β-carotene in melon is under complex genetic control. This study provides the initial step for defining the genetic control of QβC in melon leading to the development of varieties with enhanced β-carotene content.

To investigate the mechanism by which HSulf-1 expression is downregulated in ovarian cancer, DNA methylation and histone acetylation of HSulf-1 was analysed in ovarian cancer cell lines and primary tumors. Treatment of OV207 and SKOV3 by 5-aza-2′-deoxycytidine resulted in increased transcription of HSulf-1. Sequence analysis of bisulfite-modified genomic DNA from ovarian cell lines and primary tumors without HSulf-1 expression revealed an increase in the frequency of methylation of 12 CpG sites in exon 1A. Chromatin immunoprecipitation assays showed an increase in histone H3 methylation in cell lines without HSulf-1 expression. To assess the significance of HSulf-1 downregulation in ovarian cancer, OV167 and OV202 cells were transfected with HSulf-1 siRNA. Downregulation of HSulf-1 expression in OV167 and OV202 cells lead to an attenuation of cisplatin-induced cytotoxicity. Moreover, patients with ovarian tumors expressing higher levels of HSulf-1 showed a 90% response rate (27/30) to chemotherapy compared to a response rate of 63% (19/30) in those with weak or moderate levels ( P =0.0146, χ 2 test). Collectively, these data indicate that HSulf-1 is epigenetically silenced in ovarian cancer and that epigenetic therapy targeting HSulf-1 might sensitize ovarian tumors to conventional first-line therapies.