Using extracellular biomarkers for monitoring efficacy of therapeutics in cancer patients: an update

Rapidly detectable and easily accessible markers of tumor cell death are needed for evaluating early therapeutic efficacy for immunotherapy and chemotherapy so that patients and their physicians can decide whether to remain with a given therapeutic strategy. Currently, image-based tests such as computed tomography scans and magnetic resonance imaging are used to visualize the response of a patient’s tumor, but often these evaluations are not conducted for weeks to months after treatment begins. While serum levels of secreted proteins such as carcinoembryonic antigen and prostate specific antigen are commonly monitored to gauge tumor status during therapy and between image evaluations, the levels of these proteins do not always correlate well with the actual tumor response. In laboratory studies, it has been shown that tumor cells undergoing apoptosis can release cellular components into cell culture media such as cytochrome c, nucleosomes, cleaved cytokeratin-18 and E-cadherin. Studies of patient sera have found that these and other macromolecules can be found in circulation during cancer therapy, providing a potential source of material for monitoring treatment efficacy. In the future, analysis of biofluids from severe combined immunodeficiency mice bearing patient tumor specimens treated with a targeted therapy such as Apo2L/tumor necrosis factor-related apoptosis-inducing ligand will be useful in the preclinical identification of therapy response markers. In this review, the current status of the identification of serum markers of tumor cell apoptosis is provided, as well as a discussion of critical research questions that must be addressed and the considerations necessary when identifying a marker that reflects true clinical outcome.     

Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched

The dispatched (disp) gene is required for long-range Hedgehog (Hh) signaling in Drosophila. Here, we demonstrate that one of two murine homologs, mDispA, can rescue disp function in Drosophila and is essential for all Hh patterning activities examined in the early mouse embryo. Embryonic fibroblasts lacking mDispA respond normally to exogenously provided Sonic hedgehog (Shh) signal, but are impaired in stimulation of other responding cells when expressing Shh. We have developed a biochemical assay that directly measures the activity of Disp proteins in release of soluble Hh proteins. This activity is disrupted by alteration of residues functionally conserved in Patched and in a related family of bacterial transmembrane transporters, thus suggesting similar mechanisms of action for all of these proteins.     

A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds

Transgenic plants are attractive biological systems for the large-scale production of pharmaceutical proteins. In particular, seeds offer special advantages, such as ease of handling and long-term stable storage. Nevertheless, most of the studies of the expression of antibodies in plants have been performed in leaves. We report the expression of a secreted (sec-Ab) or KDEL-tagged (Ab-KDEL) mutant of the 14D9 monoclonal antibody in transgenic tobacco leaves and seeds. Although the KDEL sequence has little effect on the accumulation of the antibody in leaves, it leads to a higher antibody yield in seeds. sec-Ab(Leaf) purified from leaf contains complex N-glycans, including Lewis(a) epitopes, as typically found in extracellular glycoproteins. In contrast, Ab-KDEL(Leaf) bears only high-mannose-type oligosaccharides (mostly Man 7 and 8) consistent with an efficient endoplasmic reticulum (ER) retention/cis-Golgi retrieval of the antibody. sec-Ab and Ab-KDEL gamma chains purified from seeds are cleaved by proteases and contain complex N-glycans indicating maturation in the late Golgi compartments. Consistent with glycosylation of the protein, Ab-KDEL(Seed) was partially secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER. This dual targeting may be due to KDEL-mediated targeting to the PSV and to a partial saturation of the vacuolar sorting machinery. Taken together, our results reveal important differences in the ER retention and vacuolar sorting machinery between leaves and seeds. In addition, we demonstrate that a plant-made antibody with triantennary high-mannose-type N-glycans has similar Fab functionality to its counterpart with biantennary complex N-glycans, but the former antibody interacts with protein A in a stronger manner and is more immunogenic than the latter. Such differences could be related to a variable immunoglobulin G (IgG)-Fc folding that would depend on the size of the N-glycan.     

Truncating loss-of-function mutations of <Emphasis Type="Italic">DISP1</Emphasis> contribute to holoprosencephaly-like microform features in humans

Defective function of the Sonic Hedgehog (SHH) signaling pathway is the most frequent alteration underlying holoprosencephaly (HPE) or its various clinical microforms. We performed an extensive mutational analysis of the entire human DISP1 gene, required for secretion of all hedgehog ligand(s) and which maps to the HPE 10 locus of human chromosome 1q41, as a HPE candidate gene. Here, we describe two independent families with truncating mutations in human DISP1 that resemble the cardinal craniofacial and neuro-developmental features of a recently described microdeletion syndrome that includes this gene; therefore, we suggest that DISP1 function contributes substantially to both of these signs in humans. While these clinical features are consistent with common HPE microforms, especially those linked to defective signaling by Sonic Hedgehog, we have insufficient evidence so far that functionally abnormal DISP1 alleles will commonly contribute to the more severe features of typical HPE.     

Genetic engineering of rice to resist rice tungro disease

Rice tungro disease (RTD), caused by the co-infection of rice tungro bacilliform virus (RTBV) and rice tungro spherical virus, is one of the most important viral diseases of rice in South and Southeast Asia. The disease remains one of the major threats to sustainable rice production in many countries. The lack of resistance genes to RTBV—the causal agent of tungro disease—makes it even more difficult to manage RTD. In this review, we summarize previous and current research efforts to genetically engineer rice in order to increase the crop’s resistance to tungro disease, including the use of pathogen-derived resistance and of host genes that confer RTD resistance and/or that restrict feeding by the insect vector. The prospects of developing rice cultivars with durable resistance to RTD are also discussed.     

Plant Resistance to Virus Diseases through Genetic Engineering: Can a Similar Approach Control Plant-parasitic Nematodes?

Genetically engineered resistance against plant virus diseases has been achieved by transforming plants with gene constructs that encode viral sequences. Several successful field trials of virus-resistant transgenic plants have been carried out. Specific features of virus infection make it possible to interfere with different steps of the infection and disease cycle by accumulating products of chimeric genes introduced into transgenic plants. In this paper we describe the most common methods of producing virus-resistant transgenic plants and discuss the possibility of applying the concept of pathogen-derived resistance to non-viral pathogens.     

Foraging behavior of Cerulean Warblers during the breeding and non‐breeding seasons: evidence for the breeding currency hypothesis

Birds require additional resources for raising young, and the breeding currency hypothesis predicts that insectivorous species exploit large soft‐bodied prey during the breeding season, but shift to small, likely hard‐bodied, prey during the non‐breeding season. To test this hypothesis, we examined prey use by Cerulean Warblers (Setophaga cerulea), foliage‐gleaning Nearctic‐Neotropical migrants, during the breeding and non‐breeding seasons. We collected data on foraging behavior during the breeding season (including observations of prey items fed to young) in upland mixed‐oak forest in southeastern Ohio in 2009 and 2010 and, during the non‐breeding season, in shade coffee in the Cordillera de Merida, Venezuela, in 2008–2009. Cerulean Warblers captured 7% more large prey (visible prey extending beyond the bill) during the breeding than the non‐breeding season, but foraged at similar rates during both seasons. Large, soft‐bodied prey appeared to be especially important for feeding young. We found that adults delivered large prey on >50% of provisioning visits to nests and 69% of identifiable large prey fed to nestlings were greenish larvae (likely Lepidoptera or caterpillars) that camouflage against leaves where they would tend to be captured by foliage‐gleaning birds. Availability of specific taxa appeared to influence tree species foraging preferences. As reported by other researchers, we found that Cerulean Warblers selected trees in the genus Carya for foraging and our examination of caterpillar counts from the central Appalachian Mountains (Butler and Strazanac 2000 ) showed that caterpillars with greenish coloration, especially Baileya larvae, may be almost twice as abundant on Carya than Quercus. Our results provide evidence for the breeding currency hypothesis, and highlight the importance of caterpillars to a foliage‐gleaning migrant warbler of conservation concern.     

Foraging behavior of migrant warblers in mixed‐species flocks in Venezuelan shade coffee: interspecific differences,tree species selection,and effects of drought

Shade coffee has been identified as an important habitat for Nearctic‐Neotropical migrants during the non‐breeding season, including species of conservation concern such as Cerulean Warblers (Setophaga cerulea). To better understand habitat features important for migrants in shade coffee, we studied the foraging behavior of migrants in mixed‐species flocks at six shade‐coffee farms in the Cordillera de Merida, Venezuela, in 2008–2009 and the El Niño drought year of 2009–2010. We examined interspecific differences in foraging behavior and tree species selection of three foliage‐gleaning migrants, Blackburnian (Setophaga fusca), Cerulean, and Tennessee (Oreothlypis peregrina) warblers, and aerial‐foraging American Redstarts (Setophaga ruticilla). For morphologically similar Blackburnian and Cerulean warblers, we also examined factors influencing foraging rates (attack and movement rates), capture of large prey, and maneuver/substrate type. We found that aerial‐foraging American Redstarts foraged lower, used more aerial maneuvers, showed no tree species selection, and were less likely to forage in flocks than foliage‐gleaners. Although foraging rates were similar for Blackburnian and Cerulean warblers, the three foliage‐gleaners differed in foraging height and use of maneuvers. Cerulean Warblers foraged lower than the other two species, whereas Blackburnian Warblers used the greatest proportion of woody gleans. All three foliage‐gleaners selected Inga spp. (a commonly planted shade tree in shade‐coffee farms) for foraging, and Blackburnian and Cerulean warblers captured a greater proportion of large prey in Inga spp. than in other tree species. During the drought year, Blackburnian and Cerulean warblers captured half as many large prey and used a greater proportion of woody‐gleans. We found that interactions among behavioral, floristic, and environmental drivers influenced the foraging behavior of migrants wintering in shade coffee. Our results support those of previous studies suggesting that migrants partition resources behaviorally during the non‐breeding season, that foliage‐gleaners may benefit from the presence of shade trees, especially Inga spp., in agroforestry systems, and that drought may influence the foraging behavior of foliage‐gleaning migrants, presumably due to reduced prey availability.     

Antigenic relationship of legume seed proteins to the 7S seed storage protein of soybean

Extracts enriched for globulin proteins were prepared from the seeds of a large number of legume species and were tested for homology to antisera prepared against the glycosylated 7S seed storage protein of the soybean (Glycine max). Electrophoretic identification and subsequent analysis of proteins precipitated with 7S antisera was useful at relatively short taxonomic distances, particularly within the tribe Phaseoleae, to which G. max belongs. Glycine and most other members of the subtribe Glycininae are unusual within the Phaseoleae in having high molecular weight ($\text{> 70 000}$ dalton) subunit polypeptides. Seeds from other plants representing other subtribes of the Phaseoleae also contained proteins that cross-reacted with the G. max antisera; the molecular weights of these proteins varied from 30 000 to nearly 90 000 daltons. Homology was detected across a wider range of legume tribes within the subfamily Papilionoideae by enzyme-linked immunosorbent assay (ELISA). The results of these experiments suggest both that the 7S proteins of these tribes are evolutionarily related and that at least some features of these apparently rapidly-evolving proteins are under relatively strong selectional constraint.