An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO₃, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO₃. Moreover, the application of AgNO₃ promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO₃ from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing.

     

Distinct requirement for an intact dimer interface in wild-type, V600E and kinase-dead B-Raf signalling

The dimerisation of Raf kinases involves a central cluster within the kinase domain, the dimer interface (DIF). Yet, the importance of the DIF for the signalling potential of wild-type B-Raf (B-Raf(wt)) and its oncogenic counterparts remains unknown. Here, we show that the DIF plays a pivotal role for the activity of B-Raf(wt) and several of its gain-of-function (g-o-f) mutants. In contrast, the B-Raf(V600E), B-Raf(insT) and B-Raf(G469A) oncoproteins are remarkably resistant to mutations in the DIF. However, compared with B-Raf(wt), B-Raf(V600E) displays extended protomer contacts, increased homodimerisation and incorporation into larger protein complexes. In contrast, B-Raf(wt) and Raf-1(wt) mediated signalling triggered by oncogenic Ras as well as the paradoxical activation of Raf-1 by kinase-inactivated B-Raf require an intact DIF. Surprisingly, the B-Raf DIF is not required for dimerisation between Raf-1 and B-Raf, which was inactivated by the D594A mutation, sorafenib or PLX4720. This suggests that paradoxical MEK/ERK activation represents a two-step mechanism consisting of dimerisation and DIF-dependent transactivation. Our data further implicate the Raf DIF as a potential target against Ras-driven Raf-mediated (paradoxical) ERK activation.     

Effects of temperature, salinity and fish in structuring the macroinvertebrate community in shallow lakes: implications for effects of climate change

Climate warming may lead to changes in the trophic structure and diversity of shallow lakes as a combined effect of increased temperature and salinity and likely increased strength of trophic interactions. We investigated the potential effects of temperature, salinity and fish on the plant-associated macroinvertebrate community by introducing artificial plants in eight comparable shallow brackish lakes located in two climatic regions of contrasting temperature: cold-temperate and Mediterranean. In both regions, lakes covered a salinity gradient from freshwater to oligohaline waters. We undertook day and night-time sampling of macroinvertebrates associated with the artificial plants and fish and free-swimming macroinvertebrate predators within artificial plants and in pelagic areas. Our results showed marked differences in the trophic structure between cold and warm shallow lakes. Plant-associated macroinvertebrates and free-swimming macroinvertebrate predators were more abundant and the communities richer in species in the cold compared to the warm climate, most probably as a result of differences in fish predation pressure. Submerged plants in warm brackish lakes did not seem to counteract the effect of fish predation on macroinvertebrates to the same extent as in temperate freshwater lakes, since small fish were abundant and tended to aggregate within the macrophytes. The richness and abundance of most plant-associated macroinvertebrate taxa decreased with salinity. Despite the lower densities of plant-associated macroinvertebrates in the Mediterranean lakes, periphyton biomass was lower than in cold temperate systems, a fact that was mainly attributed to grazing and disturbance by fish. Our results suggest that, if the current process of warming entails higher chances of shallow lakes becoming warmer and more saline, climatic change may result in a decrease in macroinvertebrate species richness and abundance in shallow lakes.     

Thymidylate synthase polymorphisms are associated to therapeutic outcome of advanced non-small cell lung cancer patients treated with platinum-based chemotherapy

Thymidylate synthase (TYMS) has three polymorphisms that may modulate thymidylate synthase (TS) expression levels: (1) 28 base pairs (bp) variable number tandem repeat (VNTR) (rs34743033); (2) single nucleotide polymorphism (SNP) C>G at the twelfth nucleotide of the second repeat of 3R allele (rs2853542); and (3) 6 bp sequence deletion (1494del6, rs34489327). This study was conducted to evaluate the influence of TYMS polymorphisms on the survival of Portuguese patients with advanced non-small cell lung cancer (NSCLC) undergoing platinum-based chemotherapy. Our results showed no statistically significant differences between VNTR genotypes; although, considering the SNP C>G, homozygotes 3RG presented a better prognostic at 36 months (p = 0.004) and overall survival (p = 0.003) when compared to 2R3RG patients. Patients with “median/high expression genotypes” demonstrated a better survival at 12 months (p = 0.041) when compared to “low expression genotypes”. Furthermore, 6 bp? carriers (p = 0.006) showed a better survival at 12 months when compared to 6 bp+ homozygotes patients. When analyzing TYMS haplotypes, better survival at 12 months was observed for patients carrying haplotypes with the 6 bp? allele (2R6 bp?; p = 0.026 and 3RG6 bp?; p = 0.045). This is the first report that evaluates the three major TYMS polymorphisms in the therapeutic outcome of NSCLC in Portugal. According to our results, the TYMS polymorphisms may be useful tools to predict which advanced NSCLC patients could benefit more from platinum-based chemotherapy regimens.     

鼎湖山南亚热带常绿阔叶林碳素积累和分配特征

研究了鼎湖山南亚热带常绿阔叶林 40 0多年林龄的锥栗 (Castanopsis chinensis)、黄果厚壳桂 (Crypto-carya concinna)和 50多年林龄的黄果厚壳桂、鼎湖钓樟 (Lindera chunii)两个群落碳素积累和分配特征。结果表明 ,两林分间植物碳素含量在不同器官和不同层中的分配格局均十分相似 ,总平均分别为 41 .980 %(锥栗、黄果厚壳桂群落 )和 40 .377% (黄果厚壳桂、鼎湖钓樟群落 )。锥栗、黄果厚壳桂群落生态系统碳总贮量为 2 4 4 .998t/ hm2 ,其中植被部分为 1 54.2 89t/ hm2 ,土壤为 89.1 2 8t/ hm2 ,地表凋落物层为 1 .581 t/ hm2。黄果厚壳桂、鼎湖钓樟群落植被碳总贮量为 84.1 51 t/ hm2。在两林分植被碳总贮量中 ,乔木层分别占了97.47% (锥栗、黄果厚壳桂群落 )和 98.0 4 % (黄果厚壳桂、鼎湖钓樟群落 ) ,而在乔木层碳总贮量中 ,干器官则分别占 47.93% (锥栗、黄果厚壳桂群落 )和 44.66% (黄果厚壳桂、鼎湖钓樟群落 )。锥栗、黄果厚壳桂群落植被碳年积累量为 3.1 4 9t/ (hm2· a) ,黄果厚壳桂、鼎湖钓樟群落植被碳年积累量则为 3.42 5 t/ (hm2· a)。     

Oviposition substrate selection by Florida mosquitoes in response to pathogen‐infected conspecific larvae

The impact of the presence of larval mosquito pathogens with potential for biological control on oviposition choice was evaluated for three mosquito species/pathogen pairs present in Florida. These included Aedes aegypti infected with Edhazardia aedis, Aedes albopictus infected with Vavraia culicis, and Culex quinquefasciatus infected with Culex nigripalpus nucleopolyhedrovirus (CuniNPV). Two‐choice oviposition bioassays were performed on each host and pathogen species with one oviposition cup containing infected larvae and the other cup containing uninfected larvae (control). Both uninfected and E. aedis‐infected female Ae. aegypti laid significantly fewer eggs in oviposition cups containing infected larvae. Uninfected gravid female Ae. albopictus and Cx. quinquefasciatus oviposited equally in cups containing uninfected larvae or containing larvae infected with V. culicis or CuniNPV, respectively. Gravid female Ae. albopictus infected with V. culicis did not display ovarian development and did not lay eggs. The decreased oviposition by gravid Ae. aegypti in containers containing E. aedis‐infected larvae may indicate that the infected larvae produce chemicals deterring oviposition.     

Intraracial harassment on campus: explaining between- and within-group differences

Using the National Longitudinal Survey of Freshmen (NLSF), we examine both between- and within-group differences in the odds of feeling intraracially harassed. Specifically, we investigate the effects of colleges’ and universities’ racial composition as well as the nature of students’ associations with non-group members, including involvement in racially homogeneous campus organizations, ethnoracial diversity of friendship networks, and interracial dating. Our findings suggest that although college racial composition appears to have little effect on experiencing intraracial harassment, the nature of students’ involvement with other-race students matters a great deal. For all groups, interracial dating increased odds of harassment. Among black and white students, more diverse friendship networks did as well. And among Asian and Latino students, involvement in any racially homogeneous campus organization was associated with increases in reports of intraracial harassment. Thus, we propose a baseline theoretical model of intraracial harassment that highlights the nature of students’ associations with outgroups.     

Combined Genetic and Genealogic Studies Uncover a Large BAP1 Cancer Syndrome Kindred Tracing Back Nine Generations to a Common Ancestor from the 1700s

We recently discovered an inherited cancer syndrome caused by BRCA1-Associated Protein 1 (BAP1) germline mutations, with high incidence of mesothelioma, uveal melanoma and other cancers and very high penetrance by age 55. To identify families with the BAP1 cancer syndrome, we screened patients with family histories of multiple mesotheliomas and melanomas and/or multiple cancers. We identified four families that shared an identical BAP1 mutation: they lived across the US and did not appear to be related. By combining family histories, molecular genetics, and genealogical approaches, we uncovered a BAP1 cancer syndrome kindred of ~80,000 descendants with a core of 106 individuals, whose members descend from a couple born in Germany in the early 1700s who immigrated to North America. Their descendants spread throughout the country with mutation carriers affected by multiple malignancies. Our data show that, once a proband is identified, extended analyses of these kindreds, using genomic and genealogical studies to identify the most recent common ancestor, allow investigators to uncover additional branches of the family that may carry BAP1 mutations. Using this knowledge, we have identified new branches of this family carrying BAP1 mutations. We have also implemented early-detection strategies that help identify cancers at early-stage, when they can be cured (melanomas) or are more susceptible to therapy (MM and other malignancies).     

FOXO1 promotes wound healing through the up-regulation of TGF-β1 and prevention of oxidative stress

Keratinocyte mobilization is a critical aspect of wound re-epithelialization, but the mechanisms that control its precise regulation remain poorly understood. We set out to test the hypothesis that forkhead box O1 (FOXO1) has a negative effect on healing because of its capacity to inhibit proliferation and promote apoptosis. Contrary to expectations, FOXO1 is required for keratinocyte transition to a wound-healing phenotype that involves increased migration and up-regulation of transforming growth factor β1 (TGF-β1) and its downstream targets, integrin-α3 and -β6 and MMP-3 and -9. Furthermore, we show that FOXO1 functions in keratinocytes to reduce oxidative stress, which is necessary to maintain cell migration and prevent cell death in a TGF-β1–independent manner. Thus, our studies identify a novel function for FOXO1 in coordinating the response of keratinocytes to wounding through up-regulation of TGF-β1 and other factors needed for keratinocyte migration and protection against oxidative stress, which together promote migration and decrease apoptosis.