SummaryTo minimize interspecific pollination, it has been suggested that pollen is placed on different parts of a pollinator's body corresponding to the conspecific location of pollen pickup by the stigma.Although Pedicularis is regarded as a classic example of pollinator-mediated floral isolation, such reciprocal pollen placement has not been demonstrated experimentally. This leads us to question previous observations of pollen release in Pedicularis species.Here, we show that pollen grains are released from the tip, rather than the basal opening, of the galea (the hoodlike upper lip of the corolla) in eight nectarless Pedicularis species, mimicking pollen release from poricidal anthers. We used safranin-stained pollen within anthers to track pollen placement in three Pedicularis species, and showed that pollen was deposited on numerous parts of the bumblebee's body. However, fluorescent powder placed on the stigmas to detect the contact location on the bumblebee's body was deposited mainly on the major position of pollen placement in each of the three species.Such segregation of pollen placement and pickup between species sharing the same pollinator probably helps to reduce reproductive interference, but the positions of pollen placement and stigma contact on the bumblebee's body were not as precise as previously thought.

Gambogic acid (GA) is a natural compound derived from Chinese herbs that has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients; however, its molecular targets have not been thoroughly studied. Here, we report that GA inhibits tumor proteasome activity, with potency comparable to bortezomib but much less toxicity. First, GA acts as a prodrug and only gains proteasome-inhibitory function after being metabolized by intracellular CYP2E1. Second, GA-induced proteasome inhibition is a prerequisite for its cytotoxicity and anticancer effect without off-targets. Finally, because expression of the CYP2E1 gene is very high in tumor tissues but low in many normal tissues, GA could therefore produce tissue-specific proteasome inhibition and tumor-specific toxicity, with clinical significance for designing novel strategies for cancer treatment.

The Pedicularis species studied in the eastern Himalayan region did not conform with Grant's 'Pedicularis Model' of mechanical reproductive isolation. The specialized flowers of this diverse group of plants seem unlikely to have increased the potential for reproductive isolation or influenced rates of speciation. It is suggested instead that the extreme species richness of the Pedicularis clade was generated in other ways and that specialized flowers and substantial pollination accuracy evolved as a response to selection generated by the diversity of co-occurring congeners.

Summary Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins ( AFP s) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy‐to‐use, open access viral vector based on Tobacco mosaic virus ( TMV ). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFP s in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFP s were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFP s fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market.

PurposeClinical development of cancer drugs has a low success rate. Prognostic and predictive biomarkers using minimally invasive approaches hold promise for increasing the probability of success by enabling disease characterization, patient selection and early detection of drug treatment effect. Enumeration and molecular characterization of circulating tumor cells (CTC) may address some of these needs, and thus were evaluated for utility in a Phase I solid tumor clinical study.Experimental DesignBlood samples for CTC analysis were obtained from 24 cancer patients in a multi-center all-comer Phase I study of MEDI-575, a novel anti-PDGFRα antibody. Samples were taken at screening and analyzed for enumeration of CTC using the CellSearch® platform and for molecular characterization using a novel quantitative RT-PCR assay.ResultsFifty-nine percent of the patients showed at least 1 CTC per 7.5 ml of blood at baseline. Progression-free survival (PFS) and overall survival (OS) of patients with 0 CTCs at baseline were longer than PFS and Os for patients with 1-3 and >3 CTCs (8.8 versus 1.4 and 1.3 months PFS, P = 0.02; 9.0 vs 7.4 and 3.5 months OS, P = 0.20, respectively). Patients with 0 CTC showed a greater percentage of stable disease than the other 2 groups with 1-3 and >3 CTCs (57% vs 29% and 0%). The multimarker qRT-PCR method detected CTC in 40% of the patients, and 80% of these patients were positive for pre-selected drug target genes.ConclusionCTC enumeration of patients in an all-comer study is feasible and may allow for patient stratification for PFS and Os to evaluate the clinical response of investigational agents. Gene expression profiling of isolated CTC may provide a means for molecular characterization of selected tumor targets.

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non‐denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re‐initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA‐binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.

PAFs are short cationic and tryptophan-rich synthetic peptides with cell-penetrating antifungal activity. They show potent and selective killing activity against major fungal pathogens and low toxicity to other eukaryotic and bacterial cells. These properties make them a promising alternative to fulfill the need of novel antifungals with potential applications in crop protection, food preservation, and medical therapies. However, the difficulties of cost-effective manufacturing of PAFs by chemical synthesis or biotechnological production in microorganisms have hampered their development for practical use. This work explores the feasibility of using rice seeds as an economical and safe production system of PAFs. The rationally designed PAF102 peptide with improved antifungal properties was selected for assessing PAF biotechnological production. Two different strategies are evaluated: (1) the production as a single peptide targeted to protein bodies and (2) the production as an oleosin fusion protein targeted to oil bodies. Both strategies are designed to offer stability to the PAF peptide in the host plant and to facilitate its downstream purification. Our results demonstrate that PAF does not accumulate to detectable levels in rice seeds when produced as a single peptide, whereas it is successfully produced as fusion protein to the Oleosin18, up to 20 μg of peptide per gram of grain. We show that the expression of the chimeric Ole18-PAF102 gene driven by the Ole18 promoter results in the specific accumulation of the fusion protein in the embryo and aleurone layer of the rice seed. Ole18-PAF102 accumulation has no deleterious effects on seed yield, germination capacity, or seedling growth. We also show that the Oleosin18 protein serves as carrier to target the fusion protein to oil bodies facilitating PAF102 recovery. Importantly, the recovered PAF102 is active against the fungal phytopathogen Fusarium proliferatum . Altogether, our results prove that the oleosin fusion technology allows the production of PAF bioactive peptides to assist the exploitation of these antifungal compounds.

Human alpha(2)-macroglobulin (alpha(2)M), pregnancy zone protein (PZP), rat alpha(1)M and acute-phase rat alpha(2)M belong to the alpha(2)M gene family of proteins, which can react covalently with nucleophilic monoamines to yield monoamine-activated (MA) macroglobulins. The MA forms of human alpha(2)M, PZP and rat alpha(2)M have been demonstrated previously to inhibit various neurotrophin-promoted neuronal activities, whereas MA-alpha(1)M is neurostimulatory and all native macroglobulins are generally inactive. The mechanism of neuromodulation is unknown, but it has been postulated that MA macroglobulins might inhibit neurons via their binding and sequestration of neurotrophins. This study employed a novel biotinylation-Western blot technique to compare the neurotrophin-binding properties of the four macroglobulins, and to correlate their binding activities with their known neuro-modulatory activities. In comparison with their respective native counterparts, human and rat MA-alpha(2)M bound slightly more NGF, but significantly less BDNF or NT-3. Native human alpha(2)M and PZP in general have no neuro-modulatory activity, but native PZP bound significantly more NGF, BDNF or NT-3 than either native alpha(2)M or MA-alpha(2)M, which is neuro-inhibitory. It is known that MA-PZP is neuro-inhibitory, but it fails to bind more NGF, BDNF, or NT-3 than native PZP. MA-alpha(1)M is the only macroglobulin known to stimulate NGF-promoted neurite outgrowth, but it bound NGF with similar affinities as native alpha(1)M and rat alpha(2)M; in addition, it bound significantly less BDNF or NT-3 than native alpha(1)M. All the bindings were non-covalent and appeared specific. In conclusion, PZP and rat macroglobulins are versatile carriers of neurotrophins with diverse binding capacities, and the neurotrophin-binding property does not appear to mediate the neuro-modulatory activity of these human and rat macroglobulins.