Cryptic species of cardinalfish with evidence for old and new divergence

Larval dispersal and limited knowledge of physical boundaries challenge our understanding of the processes that drive genetic divergence and potential speciation in the marine environment. Divergence, both within and between populations of marine taxa, is not uncommon, but spatial and temporal stability of observed genetic structure is not well known. Previously, we detected large genetic differences among populations of the cardinalfish species Ostorhinchus doederleini inhabiting adjacent coral reefs. Here, we determined the spatial and temporal persistence of these genetic structures over the course of ten consecutive generations. Using microsatellite markers, we detected large changes (genetic population distance, D _est, ranged from 0.04 to 0.46) in the genetic structure in some years, but some reefs maintained the same populations for nearly all sampling years. As this species’ life span does not exceed 1 yr, persistence of distinct reef populations suggests natal homing. Mitochondrial identity based on two mtDNA markers corroborates the nuclear genetic evidence for genetic differences large enough to constitute different clades and even cryptic species in O. doederleini, which, based on gross morphology, was thought to be a single taxon. Habitat specialization was observed in one clade that exclusively inhabited reef lagoons, while all clades could be observed on reef slopes. We suggest that local habitat recognition combined with local population recognition and selection against hybrids can form barriers that maintain a cryptic species complex.     

The empowerment of translational research: lessons from laminopathies

The need for a collaborative approach to complex inherited diseases collectively referred to as laminopathies, encouraged Italian researchers, geneticists, physicians and patients to join in the Italian Network for Laminopathies, in 2009. Here, we highlight the advantages and added value of such a multidisciplinary effort to understand pathogenesis, clinical aspects and try to find a cure for Emery-Dreifuss muscular dystrophy, Mandibuloacral dysplasia, Hutchinson-Gilford Progeria and forms of lamin-linked cardiomyopathy, neuropathy and lipodystrophy.     

The Arabidopsis repressor of light signaling SPA1 acts in the phloem to regulate seedling de-etiolation, leaf expansion and flowering time

Plants adjust their growth and development in response to the ambient light environment. These light responses involve systemic signals that coordinate differentiation of different tissues and organs. Here, we have investigated the function of the key repressor of photomorphogenesis SPA1 in different tissues of the plant by expressing GUS-SPA1 under the control of tissue-specific promoters in a spa mutant background. We show that SPA1 expression in the phloem vasculature is sufficient to rescue the spa1 mutant phenotype in dark-grown spa mutant seedlings. Expression of SPA1 in mesophyll, epidermis or root tissues of the seedling, by contrast, has no or only slight effects. In the leaf, SPA1 expression in both the phloem and the mesophyll is required for full complementation of the defect in leaf expansion. SPA1 in phloem and mesophyll tissues affected division and expansion of cells in the epidermal layer, indicating that SPA1 induces non-cell-autonomous responses also in the leaf. Photoperiodic flowering is exclusively controlled by SPA1 expression in the phloem, which is consistent with previous results showing that the direct substrate of the COP1/SPA complex, CONSTANS, also acts in the phloem. Taken together, our results highlight the importance of phloem vascular tissue in coordinating growth and development. Because the SPA1 protein itself is incapable of moving from cell to cell, we suggest that SPA1 regulates the activity of downstream component(s) of light signaling that subsequently act in a non-cell-autonomous manner. SPA1 action in the phloem may also result in mechanical stimuli that affect cell elongation and cell division in other tissues.     

CtBP3/BARS drives membrane fission in dynamin-independent transport pathways

Membrane fission is a fundamental step in membrane transport. So far, the only fission protein machinery that has been implicated in in vivo transport involves dynamin, and functions in several, but not all, transport pathways. Thus, other fission machineries may exist. Here, we report that carboxy-terminal binding protein 3/brefeldin A-ribosylated substrate (CtBP3/BARS) controls fission in basolateral transport from the Golgi to the plasma membrane and in fluid-phase endocytosis, whereas dynamin is not involved in these steps. Conversely, CtBP3/BARS protein is inactive in apical transport to the plasma membrane and in receptor-mediated endocytosis, both steps being controlled by dynamin. This indicates that CtBP3/BARS controls membrane fission in endocytic and exocytic transport pathways, distinct from those that require dynamin.     

One step cloning of defined DNA fragments from large genomic clones

Recently, the nucleotide sequences of entire genomes became available. This information combined with older sequencing data discloses the exact chromosomal location of millions of nucleotide markers stored in the databases at NCBI, EMBO or DDBJ. Despite having resolved the intron/exon structures of all described genes within these genomes with a stroke of a pen, the sequencing data opens up other interesting possibilities. For example, the genomic mapping of the end sequences of the human, murine and rat BAC libraries generated at The Institute for Genomic Research (TIGR), reveals now the entire encompassed sequence of the inserts for more than a million of these clones. Since these clones are individually stored, they are now an invaluable source for experiments which depend on genomic DNA. Isolation of smaller fragments from such clones with standard methods is a time consuming process. We describe here a reliable one-step cloning technique to obtain a DNA fragment with a defined size and sequence from larger genomic clones in less than 48 hours using a standard vector with a multiple cloning site, and common restriction enzymes and equipment. The only prerequisites are the sequences of ends of the insert and of the underlying genome.     

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.     

Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

Methodology/Principal Findings

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

Conclusions/Significance

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.     

Functional annotation by identification of local surface similarities: a novel tool for structural genomics

Background  

Protein function is often dependent on subsets of solvent-exposed residues that may exist in a similar three-dimensional configuration in non homologous proteins thus having different order and/or spacing in the sequence. Hence, functional annotation by means of sequence or fold similarity is not adequate for such cases.