Characterization of abiotic stress-responsive <Emphasis Type="Italic">Arabidopsis thaliana RD29A</Emphasis> and <Emphasis Type="Italic">RD29B</Emphasis> genes and evaluation of transgenes

Abiotic stresses have adverse effects on plant growth and productivity. The homologous RD29A and RD29B genes are exquisitely sensitive to various abiotic stressors. Therefore, RD29A and RD29B gene sequences have potential to confer abiotic stress resistance in crop species grown in arid and semi-arid regions. To our knowledge, no information on the physiological roles of the proteins encoded by RD29A and RD29B are available in the literature. To understand how these proteins function, we used reverse genetic approaches, including identifying rd29a and rd29b T-DNA knockout mutants, and examining the effects of complementing transgenes with the genes under control of their native promoters and chimeric genes with the native promoters swapped. Four binary vectors with the RD29A and RD29B promoters upstream of the cognate RD29A and RD29B cDNAs and as chimeric genes with noncognate promoters were used to transform rd29a and rd29b plants. Cold, drought, and salt induced both genes; the promoter of RD29A was found to be more responsive to drought and cold stresses, whereas the promoter of RD29B was highly responsive to salt stress. Morphological and physiological responses of rd29a and rd29b plants to salt stress were further investigated. Root growth, and photosynthetic properties declined significantly, while solute concentration (Ψπ), water use efficiency (WUE) and δ¹³C ratio increased under salt stress. Unexpectedly, the rd29a and rd29b knockout mutant lines maintained greater root growth, photosynthesis, and WUE under salt stress relative to control. We conclude that the RD29A and RD29B proteins are unlikely to serve directly as protective molecules.     

Major consequences of minor damage: impacts of small grazers on fast-growing kelps

Damage by small herbivores can have disproportionately large effects on the fitness of individual plants if damage is concentrated on valuable tissues or on select individuals within a population. In marine systems, the impact of tissue loss on the growth rates of habitat-forming algae is poorly understood. We quantified the grazing damage by an isopod Amphoroidea typa on two species of large kelps, Lessonia spicata and Macrocystis pyrifera, in temperate Chile to test whether non-lethal grazing damage could reduce kelp growth rates and photosynthetic efficiency. For L. spicata, grazing damage was widespread in the field, unevenly distributed on several spatial scales (among individuals and among tissue types) and negatively correlated with blade growth rates. In field experiments, feeding by A. typa reduced the concentration of photosynthetic pigments and led to large reductions (~80 %) in blade growth rates despite limited loss of kelp biomass (0.5 % per day). For M. pyrifera, rates of damage in the field were lower and high densities of grazers were unable to reduce growth rates in field experiments. These results demonstrate that even low per capita grazing rates can result in large reductions in the growth of a kelp, due the spatial clustering of herbivores in the field and the selective removal of photosynthetically active tissues. The impacts of small herbivores on plant performance are thus not easily predicted from consumption rates or abundance in the field, and vary with plant species due to variation in their ability to compensate for damage.     

Ecophysiological characteristics of Porphyra spp. (Bangiophyceae,Rhodophyta): seasonal and latitudinal variations in northern-central Chile

The red macroalga Porphyra C. Agardh is one of the most ecologically successful genera that lives in the upper intertidal zone. Biochemical, physiological, and morphological acclimation strategies allow their growth and distribution as well as a quick recuperation between tidal regimens. Studies of Porphyra are poorly developed in Chile, and management and exploitation proposals need to be supported by biological and ecophysiological approaches. This study evaluated seasonal and latitudinal physiological performances of Porphyra spp. via maximum quantum yield (F _v?/?F _m), pigments, proteins, phenolic compounds, and antioxidant activity in order to describe how algae can acclimate to their environment and to provide insights to their management and use. Sampling was done at three costal sites in Chile between 25°S and 34°S between winters 2010 and 2011. A total of four different morphotypes were identified (one in the north, one in the center, and two in the south locations) and evaluated separately. Results showed seasonal and latitudinal patterns for all ecophysiological variables studied, with a general tendency of decrease in F _v?/?F _m, pigments, and soluble proteins during spring–summer seasons accompanied by an increase in the antioxidant capacity. Latitudinal differences were observed with a tendency of higher values for ecophysiological traits in central and southern morphotypes. Phenology patterns were different between an annual population in the north location and a perennial one for central-south populations. The taxonomic clarity should be evaluated in order to better understand if there exists intraspecific (dependent on morphology) or interspecific variation.     

Environmental tolerance of the two invasive species Ciona intestinalis and Codium fragile: their invasion potential along a temperate coast

Ciona intestinalis and Codium fragile are among the most successful invasive species in marine systems worldwide, and they are currently in the process of expanding their distributional ranges along the Chilean coast. Herein we evaluated whether their tolerance to a wide range of environmental conditions contributes to the invasion potential of these two species. To examine the environmental tolerance and performance (e.g., growth) of these non-indigenous species, unifactorial experiments (8–10 days) were conducted with different environmental factors (solar radiation, salinity and temperature). Based on the results, the potential risk of invasion along the Chilean coast was evaluated for both species using a mechanistic niche modeling approach. Both species can tolerate extensive ranges of the abiotic factors salinity and temperature, with C. intestinalis being more tolerant to cold-temperate waters. Also, C. intestinalis was more susceptible to high light intensities than C. fragile. These results confirm those of other experiments, and the outcome of the niche modeling shows that both species can potentially invade most regions of the Chilean coast with the exception of the Magellan region. The results suggest that physiological capacity to tolerate and perform in a wide range of physical conditions is a pre-requisite for successful invasions by littoral biota, but predation and possibly competitive exclusion can slow down the invasion success of C. intestinalis, which in contrast to C. fragile, is consumed by many benthic predators. Sexual and asexual reproduction as well as buoyancy of its thalli further contribute to the dispersal and colonization success of C. fragile. Based on these considerations, it is concluded that the invasion risk of C. fragile along the Chilean coast is substantially higher than that of C. intestinalis.     

Properties of baked foams based on cassava starch, sugarcane bagasse fibers and montmorillonite

The objectives of this work were to develop biodegradable trays from cassava starch, sugarcane fibers and Na-montmorillonite (Na-MMT) using a baking process and to study the effects of these components on the microstructure and physicochemical and mechanical properties of the trays. All formulations resulted in well-shaped trays with densities between 0.1941 and 0.2966 g/cm³. The addition of fibers and Na-MMT resulted in less dense and less rigid trays. As observed in the water sorption isotherms, the increase in the equilibrium moisture content was more pronounced when the samples were stored at RH (relative humidity) above 75%. The foams had high water absorption capacities (>50%) when immersed in water (1 min). The studied processing conditions resulted in good nanoclay dispersion, leading to the formation of an exfoliated structure. The trays developed in this study represent an alternative for the packaging of foods with low water contents.     

Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials

Background

Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients.

Findings

Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs present in tumors, draining lymph nodes and spleen and improved long-term survival (50% of mice survived >150 days). No improvement in survival was observed when Tregs were depleted 24 days after tumor implantation, suggesting that tumor burden is an important factor for determining efficacy of Treg depletion in clinical trials. In a T cell dependent model of brain tumor regression elicited by intratumoral delivery of adenoviral vectors (Ad) expressing Fms-like Tyrosine Kinase 3 ligand (Flt3L) and Herpes Simplex Type 1-Thymidine Kinase (TK) with ganciclovir (GCV), we demonstrate that administration of PC61 24 days after tumor implantation (7 days after treatment) inhibited T cell dependent tumor regression and long term survival. Further, depletion with PC61 completely inhibited clonal expansion of tumor antigen-specific T lymphocytes in response to the treatment.

Conclusions

Our data demonstrate for the first time, that although Treg depletion inhibits the progression/eliminates GBM tumors, its efficacy is dependent on tumor burden. We conclude that this approach will be useful in a setting of minimal residual disease. Further, we also demonstrate that Treg depletion, using PC61 in combination with immunotherapy, inhibits clonal expansion of tumor antigen-specific T cells, suggesting that new, more specific targets to block Tregs will be necessary when used in combination with therapies that activate anti-tumor immunity.     

A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus

Background

Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests.

Methodology/Principal Findings

A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011–2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions.

Conclusions

The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.     

Mutations in KPTN Cause Macrocephaly,Neurodevelopmental Delay,and Seizures

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis.