Attenuation of Axonal Degeneration by Calcium Channel Inhibitors Improves Retinal Ganglion Cell Survival and Regeneration After Optic Nerve Crush

Axonal degeneration is one of the initial steps in many traumatic and neurodegenerative central nervous system (CNS) disorders and thus a promising therapeutic target. A focal axonal lesion is followed by acute axonal degeneration (AAD) of both adjacent axon parts, before proximal and distal parts follow different degenerative fates at later time points. Blocking calcium influx by calcium channel inhibitors was previously shown to attenuate AAD after optic nerve crush (ONC). However, it remains unclear whether the attenuation of AAD also promotes consecutive axonal regeneration. Here, we used a rat ONC model to study the effects of calcium channel inhibitors on axonal degeneration, retinal ganglion cell (RGC) survival, and axonal regeneration, as well as the molecular mechanisms involved. Application of calcium channel inhibitors attenuated AAD after ONC and preserved axonal integrity as visualized by live imaging of optic nerve axons. Consecutively, this resulted in improved survival of RGCs and improved axonal regeneration at 28 days after ONC. We show further that calcium channel inhibition attenuated lesion-induced calpain activation in the proximity of the crush and inhibited the activation of the c-Jun N-terminal kinase pathway. Pro-survival signaling via Akt in the retina was also increased. Our data thus show that attenuation of AAD improves consecutive neuronal survival and axonal regeneration and that calcium channel inhibitors could be valuable tools for therapeutic interventions in traumatic and degenerative CNS disorders.     

QTL analysis of cotton fiber length in advanced backcross populations derived from a cross between <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and <Emphasis Type="Italic">G. mustelinum</Emphasis>

Key message

QTLs for fiber length mapped in three generations of advanced backcross populations derived from crossing Gossypium hirsutum and Gossypium mustelinum showed opportunities to improve elite cottons by introgression from wild relatives.

Abstract

The molecular basis of cotton fiber length in crosses between Gossypium hirsutum and Gossypium mustelinum was dissected using 21 BC₃F₂ and 12 corresponding BC₃F_2:3 and BC₃F_2:4 families. Sixty-five quantitative trait loci (QTLs) were detected by one-way analysis of variance. The QTL numbers detected for upper-half mean length (UHM), fiber uniformity index (UI), and short fiber content (SFC) were 19, 20, and 26 respectively. Twenty-three of the 65 QTLs could be detected at least twice near adjacent markers in the same family or near the same markers across different families/generations, and 32 QTLs were detected in both one-way variance analyses and mixed model-based composite interval mapping. G. mustelinum alleles increased UHM and UI and decreased SFC for five, one, and one QTLs, respectively. In addition to the main-effect QTLs, 17 epistatic QTLs were detected which helped to elucidate the genetic basis of cotton fiber length. Significant among-family genotypic effects were detected at 18, 16, and 16 loci for UHM, UI, and SFC, respectively. Six, two, and two loci showed genotype?×?family interaction for UHM, UI and SFC, respectively, illustrating complexities that might be faced in introgression of exotic germplasm into cultivated cotton. Co-location of many QTLs for UHM, UI, and SFC accounted for correlations among these traits, and selection of these QTLs may improve the three traits simultaneously. The simple sequence repeat (SSR) markers associated with G. mustelinum QTLs will assist breeders in transferring and maintaining valuable traits from this exotic source during cultivar development.

     

Biotic Potential of a Short-Horned Grasshopper, <Emphasis Type="Italic">Oxya hyla hyla</Emphasis> Serville (Orthoptera: Acrididae) to Assess Its Biomass Producing Capacity

The growth of fishery and animal husbandry is being hindered by the scarcity of protein rich feed ingredients. Being rich in protein grasshoppers may be an alternative source of protein rich feed ingredients and hence rearing of these insects is essential for sustainable supply of insect biomass to the feed industry. The success of biomass production through insect rearing depends on the reproductive potentiality, rate of survival and growth of that particular insect. This study examines the biotic potential, fecundity, fertility, nymph mortality, life span and biomass production ability of a multivoltine grasshopper, Oxya hyla hyla (Serville, 1931) under semi-controlled condition. The grasshoppers are reared in 35 ± 2 °C temperature and 12L: 12D photoperiod and provided with food as fresh leaves of Cynodon dactylon (L.) Pers. to nymphs and Sorghum halepense (L.) Pers. to the adults. The result shows that a single pair of O. hyla hyla can lay on average 69.87 eggs in 4.96 ± 0.43 egg pods of which 7.37 ± 0.92 % eggs and 8.26 ± 0.99 % nymphs do not survive. The remaining survived nymphs metamorphose to 27.8 ± 3.35 male and 32.6 ± 3.21 female adults which produce a biomass of 18.48 ± 1.67 g wet weight. Thus a mass scale rearing of this insect may produce good amount of insect biomass which may be usable for fish and animal feed.     

Surface display of ACC deaminase on endophytic <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> strains to increase saline resistance of host rice sprouts by regulating plant ethylene synthesis

Background

Most endophytic bacteria in consortia, which provide robust and broad metabolic capacity, are attractive for applications in plant metabolic engineering. The aim of this study was to investigate the effects of engineered endophytic bacterial strains on rice sprout ethylene level and growth under saline stress. A protocol was developed to synthesize engineered strains by expressing bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene on cells of endophytic Enterobacter sp. E5 and Kosakonia sp. S1 (denoted as E5P and S1P, respectively).

Results

Results showed that ACC deaminase activities of the engineered strains E5P and S1P were significantly higher than those of the wild strains E5 and S1. About 32–41% deaminase was expressed on the surface of the engineered strains. Compared with the controls without inoculation, inoculation with the wild and engineered strains increased the deaminase activities of sprouts. Inoculation with the engineered strains increased 15–21% more deaminase activities of sprouts than with the wild strains, and reduced the ethylene concentrations of sprouts more significantly than with wild strains (P < 0.05). Inoculation with the wild and engineered strains promoted the growth of sprouts, while the promoting effects were more profound with the engineered strains than with the wild strains. The engineered strains improved saline resistance of sprouts under salt concentrations from 10 to 25 g L^?1. The engineered strains promoted longer roots and shoots than the wild strains under the salt stresses, indicating that the ACC deaminases on the endophytic bacterial cells could result in plant-produced ACC degradation and inhibit plant ethylene formation.

Conclusions

The protocols of expressing enzymes on endophytic bacterial cells showed greater potentials than those of plant over-expressed enzymes to increase the efficiency of plant metabolic pathways.

     

Defining the importance of landscape metrics for large branchiopod biodiversity and conservation: the case of the Iberian Peninsula and Balearic Islands

The deficiency in the distributional data of invertebrate taxa is one of the major impediments acting on the bias towards the low awareness of its conservation status. The present study sets a basic framework to understand the large branchiopods distribution in the Iberian Peninsula and Balearic Islands. Since the extensive surveys performed in the late 1980s, no more studies existed updating the information for the whole studied area. The present study fills the gap, gathering together all available information on large branchiopods distribution since 1995, and analysing the effect of human population density and several landscape characteristics on their distribution, taking into consideration different spatial scales (100 m, 1 km and 10 km). In overall, 28 large branchiopod taxa (17 anostracans, 7 notostracans and 4 spinicaudatans) are known to occur in the area. Approximately 30% of the sites hosted multiple species, with a maximum of 6 species. Significant positive co-occurring species pairs were found clustered together, forming 4 different associations of large branchiopod species. In general, species clustered in the same group showed similar responses to analysed landscape characteristics, usually showing a better fit at higher spatial scales.     

Detecting Multiple States of Trophic Connectivity Between Mangroves and Salt Marshes

The productivity gradient between adjacent habitats can fluctuate over time due to seasonal cycles and lead to both habitats being alternately subsidized. Although this process is well known for prey subsidies in stream-riparian forest ecotones, few studies are available for other systems or subsidy types. Moreover, the effects of transport intensity on this expected alternate subsidy exchange are still poorly understood. We assessed whether subsidy input and allochthonous carbon assimilation by resident benthic invertebrates alternated between adjacent mangroves and salt marshes during peaks of detritus productivity (summer and winter, respectively) in a subtropical estuary, by using detritus trapping techniques and stable isotope ratios. Sampling was performed simultaneously in the sheltered (inner sector) and exposed (outer sector) regions of the estuary to assess the influence of different physical conditions on the intensity of subsidy flow. Transport of mangrove litter into the salt marsh occurred mainly in the summer in both sectors; however, most of the litter remained trapped in the marsh boundary. The mixing model also showed that there was little influence of allochthonous carbon in the diet of salt marsh benthic invertebrates. Marsh litter supply to mangroves did not vary significantly between seasons but was significantly higher in the outer than in the inner sector. Likewise, the mixing model showed great contribution of salt marsh carbon to the diet of benthic invertebrates from the outer-sector mangroves, whereas autochthonous carbon predominated in those from the inner mangroves. Our findings reinforce the model that trophic connectivity relies on the relative proportion of allochthonous (subsidy) and autochthonous resources rather than only on asymmetric productivity between habitats. Differences in the proportion of resources result from interaction among productivity, permeability, and transport vectors that lead to many states of trophic connectivity.     

Isolation of DNA for RAPD analysis from dry leaf material of some<Emphasis Type="Italic">Hesperis</Emphasis> L. specimens

An improved protocol for the isolation of DNA from dry material of someHesperis specimens is described. The isolated DNA is suitable for random amplification of polymorphic DNA (RAPD) analysis. Different DNA extraction protocols were examined to determine which might yield DNA from dry leaf tissue ofHesperis specimens. The methods examined include the protocols with hexadecyltrimethylammonium bromide (CTAB) described by Doyle and Doyle (1987); sodium dodecyl sulfate (SDS) by Dellaporta et al. (1983); and CTAB and SDS, the modified minipreparation, by Dellaporta et al (1983). None of these procedures yielded DNA of suitable purity for RAPD assay. We established an improved procedure involving CTAB and enzymatic digestion of proteins and RNA. The recovery of DNA with an average yield of 25 mg/g of leaf material was possible with this procedure. RAPD bands, which could be used to distinguish amongHesperis specimens, were generated.     

Microcloning of maize chromosome 9 by using a flow-sorting technique

We constructed a chromosome 9 lambda DNA library from flow-sorted maize chromosomes. Approximately 3 million maize chromosome 9 were collected with high purity by flow cytometric sorting of chromosomes isolated from an oat-maize chromosome 9 addition line based on the cytogram of fluorescent pulse area versus fluorescent pulse width. Chromosome 9 DNA was partially digested withBamH I, dephosphorylated, and ligated with arms ofBamH I-digested lambda DASH vector (Stratagene). A total of 2.0×10⁶ independent recombinants with an average insert size of 15 kb were obtained. For a 99% probability that every sequence of chromosome 9 is represented in at least one chimeric phage, 5.6×10⁴ cloned fragments are needed. This library covers the entire maize chromosome 9. Hybridizing cloned fragments with labeled maize genomic DNA showed that the high, middle, or low copy number DNA sequences presented in the different phage clones. This individual chromosome library is useful in plant genome mapping and gene isolation.     

Cytotoxic T lymphocytes generated by short-term in vitro TCR stimulation in the presence of IL-4 are therapeutically effective against B16 melanoma

P14 TCR transgenic CD8+ T cells (LCMV gp33-specific) were activated by antigen in the presence of either IL-2 or IL-2+IL-4 to generate effector cytotoxic T lymphocytes (CTLs). The therapeutic effectiveness of such IL-2- or IL-2+IL-4-grown CTLs was tested in mice that had received intravenous inoculations of B16.gp33 melanoma cells 7 days previously. Administration of P14 CTLs activated by antigen +IL-2+IL-4 was significantly more effective at reducing melanoma colony formation in the lung than those grown in the presence of antigen +IL-2. Highly significant improvement in survival was observed with 80% of B16.gp33-inoculated mice showing long-term survival after therapy with 10×10⁶ antigen +IL-2+IL-4-activated P14 CTLs. Similar therapeutic effectiveness of antigen +IL-2+IL-4-activated P14 CTLs against subcutaneously inoculated B16.gp33 melanoma cells was also found. There was significant reduction in P14 CD8+ T cells in the peripheral blood of B16.gp33-inoculated mice than in mice that did not receive B16.gp33 melanoma cells, indicating possible homing of P14 CD8+ T cells to the site of tumor growth or antigen-induced apoptotic cell death. These results may have implications in tumor therapy using CTLs grown ex vivo, especially during early stages of tumor formation. They also support the concept that the therapeutic effectiveness of CTLs can be governed by the cytokine context in which they are activated.     

Creation and analysis of a novel chimeric promoter for the complete containment of pollen- and seed-mediated gene flow

Effective containment of gene flow in transgenic plants requires a promoter that is highly specific for male and female gametes or tissues. Here, we report the creation of a novel pollen-, stigma- and carpel-specific (PSC) promoter through the fusion of the pollen-specific LAT52 and carpel-specific AGL5 enhancers to a stigma-specific SLG promoter. Gene expression analysis showed that fusion of the LAT52 enhancer to the SLG promoter enables the latter to gain pollen-specific activity while the acquirement of carpel-specific activity requires the correct orientation of the inserted AGL5 enhancer in the PSC promoter, and only a forward- but not a reverse-oriented one is functional. The resulting fPSC promoter, when fused to DT-A, generated at least three aberrant gynoecium phenotypes. Type I plants exhibited shortened stigmatic tissues, resembling plants containing the DT-A gene controlled by the SLG promoter. However, type II and III plants displayed partial or complete ablation of gynoecia, and were unable to support the reproductive process. Type II and III plants also produced severely perturbed anthers and pollen in comparison to type I or SLG::DT-A plants, and transgenic pollen grains were unable, when out-crossed with control plants, to pass the transgene to the next generation in all plants examined, indicating that they are selectively eliminated. This tissue-specific ablation or perturbation is highly specific, and does not compromise vegetative growth. Evidently, the fPSC promoter faithfully acquires tissue specificity from the incorporated enhancers and promoter, and should have a practical application for transgene containment in non-fruit and -grain producing plant crops.