Two-photon microscopy: shedding light on the chemistry of vision

Two-photon microscopy (TPM) has come to occupy a prominent place in modern biological research with its ability to resolve the three-dimensional distribution of molecules deep inside living tissue. TPM can employ two different types of signals, fluorescence and second harmonic generation, to image biological structures with subcellular resolution. Two-photon excited fluorescence imaging is a powerful technique with which to monitor the dynamic behavior of the chemical components of tissues, whereas second harmonic imaging provides novel ways to study their spatial organization. Using TPM, great strides have been made toward understanding the metabolism, structure, signal transduction, and signal transmission in the eye. These include the characterization of the spatial distribution, transport, and metabolism of the endogenous retinoids, molecules essential for the detection of light, as well as the elucidation of the architecture of the living cornea. In this review, we present and discuss the current applications of TPM for the chemical and structural imaging of the eye. In addition, we address what we see as the future potential of TPM for eye research. This relatively new method of microscopy has been the subject of numerous technical improvements in terms of the optics and indicators used, improvements that should lead to more detailed biochemical characterizations of the eyes of live animals and even to imaging of the human eye in vivo.     

Molecular phylogenetics and the evolution of host plant associations in the nematode genus Fergusobia (Tylenchida: Fergusobiinae)

Fergusobia nematodes (Tylenchida: Fergusobiinae) and Fergusonina flies (Diptera: Fergusoninidae) are putative mutualists that develop together in galls formed in meristematic tissues of many species of the plant family Myrtaceae in Australasia. Fergusobia nematodes were sampled from a variety of myrtaceous hosts and gall types from Australia and one location in New Zealand between 1999 and 2006. Evolutionary relationships of these isolates were inferred from phylogenetic analyses of the DNA sequences of the nuclear ribosomal DNA near-full length small subunit (up to 1689bp for 21 isolates), partial large subunit D2/D3 domain (up to 889bp for 87 isolates), partial mitochondrial cytochrome oxidase subunit I (618 bp for 82 isolates), and combined D2/D3 and mtCOI (up to 1497bp for 66 isolates). The SSU data supported a monophyletic Fergusobia genus within a paraphyletic Howardula. A clade of Drosophila-associated Howardula, including Howardula aoronymphium, was the closest sequenced sister group. Phylogenetic analysis of sequences from D2/D3 and mtCOI, separately and combined, revealed many monophyletic clades within Fergusobia. The relationships inferred by D2/D3 and mtCOI were congruent with some exceptions. Well-supported clades were generally consistent with host plant species and gall type. However, phylogenetic analysis suggested host switching or putative hybridization events in many groups, except the lineage of shoot bud gallers on the broad-leaved Melaleuca species complex.     

An emerging example of tritrophic coevolution between flies (Diptera: Fergusoninidae) and nematodes (Nematoda: Neotylenchidae) on Myrtaceae host plants

A unique obligate mutualism occurs between species of Fergusonina Malloch flies (Diptera: Fergusoninidae) and nematodes of the genus Fergusobia Currie (Nematoda: Neotylenchidae). These mutualists together form different types of galls on Myrtaceae, mainly in Australia. The galling association is species‐specific, and each mutualism in turn displays host specificity. This tritrophic system represents a compelling arena to test hypotheses about coevolution between the host plants, parasitic nematodes and the fergusoninid flies, and the evolution of these intimate mutualisms. We have a basic knowledge of the interactions between the host plant, fly and nematode in this system, but a more sophisticated understanding will require a much more intensive and coordinated research effort. Summaries of the known Fergusonina/Fergusobia species associations and gall type terminology are presented. This paper identifies the key advantages of the system and questions to be addressed, and proposes a number of predictions about the evolutionary dynamics of the system given our understanding of the biology of the mutualists. Future research will profitably focus on (1) gall cecidogenesis and phenology, (2) the interaction between the fly larva and the nematode in the gall, and between the adult female fly and the parasitic nematode, (3) the means by which the fly and nematode life cycles are coordinated, (4) a targeted search of groups in the plant family Myrtaceae that have not yet been identified as gall hosts, and (5) establishment and comparison of the phylogenetic relationships of the host plants, fly species and nematodes. Recently derived phylogenies and divergence time estimation studies of the Diptera and the Myrtaceae show that the fly family Fergusoninidae is less than half the age of the Myrtaceae, discounting the hypothesis of cospeciation and coradiation of the fly/nematode mutualism and the plants at the broadest levels. However, cospeciation may have occurred at shallower levels in the phylogeny, following the establishment of the fly/nematode mutualism on the Myrtaceae. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 699–718.     

A Phase Ia Study to Assess the Safety and Immunogenicity of New Malaria Vaccine Candidates ChAd63 CS Administered Alone and with MVA CS

Background

Plasmodium falciparum (P. falciparum) malaria remains a significant cause of mortality and morbidity throughout the world. Development of an effective vaccine would be a key intervention to reduce the considerable social and economic impact of malaria.

Methodology

We conducted a Phase Ia, non-randomized, clinical trial in 24 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding the circumsporozoite protein (CS) of P. falciparum.

Results

ChAd63-MVA CS administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to CS. With a priming ChAd63 CS dose of 5×10⁹ vp responses peaked at a mean of 1947 SFC/million PBMC (median 1524) measured by ELIspot 7 days after the MVA boost and showed a mixed CD4+/CD8+ phenotype. With a higher priming dose of ChAd63 CS dose 5×10¹⁰ vp T cell responses did not increase (mean 1659 SFC/million PBMC, median 1049). Serum IgG responses to CS were modest and peaked at day 14 post ChAd63 CS (median antibody concentration for all groups at day 14 of 1.3 µg/ml (range 0–11.9), but persisted throughout late follow-up (day 140 median antibody concentration groups 1B & 2B 0.9 µg/ml (range 0–4.7).

Conclusions

ChAd63-MVA is a safe and highly immunogenic delivery platform for the CS antigen in humans which warrants efficacy testing.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01450280","term_id":"NCT01450280"}}NCT01450280     

Breeding for Bio-ethanol Production in Lolium perenne L.: Association of Allelic Variation with High Water-Soluble Carbohydrate Content

Increasing the extractable sugar yield from perennial crops is one strategy to generate renewable fuels such as bio-ethanol. Lolium perenne L. (perennial ryegrass) can contain significant (>30% dry matter) water-soluble sugars in the form of polymeric fructan which is readily extracted, broken down and fermented to bio-ethanol. A population of L. perenne generated from four parents which differed in water-soluble carbohydrate (WSC) content was subjected to multiple rounds of selection and recombination on the basis of early spring WSC content to produce a high WSC, and a low WSC population. A control population was generated by selecting the same number of plants at random. The alleles present at six candidate gene loci were analysed before and after selection and correlated to WSC content. Significant differences in the allele frequency of L. perenne soluble-acid invertase1:4 were observed between the three populations with one haplotype significantly associated with the high WSC C2^S+ population (after three rounds of selection and two rounds of recombination). Moreover, WSC content was also associated with biomass accumulation. Thus, in addition to a 2.84-fold increase in WSC yield, the C2^S+ population also had 1.48-fold more biomass per plant, resulting in 3.9-fold higher WSC yield per plant than the control population.     

Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T))

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.     

High resolution genetic mapping by genome sequencing reveals genome duplication and tetraploid genetic structure of the diploid Miscanthus sinensis

We have created a high-resolution linkage map of Miscanthus sinensis, using genotyping-by-sequencing (GBS), identifying all 19 linkage groups for the first time. The result is technically significant since Miscanthus has a very large and highly heterozygous genome, but has no or limited genomics information to date. The composite linkage map containing markers from both parental linkage maps is composed of 3,745 SNP markers spanning 2,396 cM on 19 linkage groups with a 0.64 cM average resolution. Comparative genomics analyses of the M. sinensis composite linkage map to the genomes of sorghum, maize, rice, and Brachypodium distachyon indicate that sorghum has the closest syntenic relationship to Miscanthus compared to other species. The comparative results revealed that each pair of the 19 M. sinensis linkages aligned to one sorghum chromosome, except for LG8, which mapped to two sorghum chromosomes (4 and 7), presumably due to a chromosome fusion event after genome duplication. The data also revealed several other chromosome rearrangements relative to sorghum, including two telomere-centromere inversions of the sorghum syntenic chromosome 7 in LG8 of M. sinensis and two paracentric inversions of sorghum syntenic chromosome 4 in LG7 and LG8 of M. sinensis. The results clearly demonstrate, for the first time, that the diploid M. sinensis is tetraploid origin consisting of two sub-genomes. This complete and high resolution composite linkage map will not only serve as a useful resource for novel QTL discoveries, but also enable informed deployment of the wealth of existing genomics resources of other species to the improvement of Miscanthus as a high biomass energy crop. In addition, it has utility as a reference for genome sequence assembly for the forthcoming whole genome sequencing of the Miscanthus genus.     

Inference for Proportions in a 2 × 2 Contingency Table: HPD or not HPD?

Summary Highest posterior density intervals are common in Bayesian inference, but as noted by Agresti and Min (2005, Biometrics 61, 515–523) they are not invariant under transformations. Agresti and Min suggested central or “tail” intervals as preferable in the context of the relative risk and odds ratio. A modification to this is proposed for extreme outcomes, as invariance is maintained when replacing central intervals by one‐sided intervals. Bayes–Laplace priors for the binomial parameters appear preferable here, compared to Jeffreys priors, contrary to Agresti and Min's suggestion based on frequentist coverage.     

Structural basis for the lower affinity of the insulin-like growth factors for the insulin receptor

Insulin and the insulin-like growth factors (IGFs) bind with high affinity to their cognate receptor and with lower affinity to the noncognate receptor. The major structural difference between insulin and the IGFs is that the IGFs are single chain polypeptides containing A-, B-, C-, and D-domains, whereas the insulin molecule contains separate A- and B-chains. The C-domain of IGF-I is critical for high affinity binding to the insulin-like growth factor I receptor, and lack of a C-domain largely explains the low affinity of insulin for the insulin-like growth factor I receptor. It is less clear why the IGFs have lower affinity for the insulin receptor. In this study, 24 insulin analogues and four IGF analogues were expressed and analyzed to explore the role of amino acid differences in the A- and B-domains between insulin and the IGFs in binding affinity for the insulin receptor. Using the information obtained from single substituted analogues, four multiple substituted analogues were produced. A "quadruple insulin" analogue ([Phe(A8), Ser(A10), Thr(B5), Gln(B16)]Ins) showed affinity as IGF-I for the insulin receptor, and a "sextuple insulin" analogue ([Phe(A8), Ser(A10), Thr(A18), Thr(B5), Thr(B14), Gln(B16)]Ins) showed an affinity close to that of IGF-II for the insulin receptor, whereas a "quadruple IGF-I" analogue ([His(4), Tyr(15), Thr(49), Ile(51)]IGF-I) and a "sextuple IGF-II" analogue ([His(7), Ala(16), Tyr(18), Thr(48), Ile(50), Asn(58)]IGF-II) showed affinities similar to that of insulin for the insulin receptor. The mitogenic potency of these analogues correlated well with the binding properties. Thus, a small number of A- and B-domain substitutions that map to the IGF surface equivalent to the classical binding surface of insulin weaken two hotspots that bind to the insulin receptor site 1.