Paternity analysis reveals wide pollen dispersal and high multiple paternity in a small isolated population of the bird-pollinated Eucalyptus caesia (Myrtaceae).

Optimal foraging behaviour by nectavores is expected to result in a leptokurtic pollen dispersal distribution and predominantly near-neighbour mating. However, complex social interactions among nectarivorous birds may result in different mating patterns to those typically observed in insect-pollinated plants. Mating system, realised pollen dispersal and spatial genetic structure were examined in the bird-pollinated Eucalyptus caesia, a species characterised by small, geographically disjunct populations. Nine microsatellite markers were used to genotype an entire adult stand and 181 seeds from 28 capsules collected from 6 trees. Mating system analysis using MLTR revealed moderate to high outcrossing (tm=0.479-0.806) and low estimates of correlated paternity (rp=0.136±s.e. 0.048). Paternity analysis revealed high outcrossing rates (mean=0.72) and high multiple paternity, with 64 different sires identified for 181 seeds. There was a significant negative relationship between the frequency of outcross mating and distance between mating pairs. Realised mating events were more frequent than expected with random mating for plants <40 m apart. The overall distribution of pollen dispersal distances was platykurtic. Despite extensive pollen dispersal within the stand, three genetic clusters were detected by STRUCTURE analysis. These genetic clusters were strongly differentiated yet geographically interspersed, hypothesised to be a consequence of rare recruitment events coupled with extreme longevity. We suggest that extensive polyandry and pollen dispersal is a consequence of pollination by highly mobile honeyeaters and may buffer E. caesia against the loss of genetic diversity predicted for small and genetically isolated populations.

Mismatch in the distribution of floral ecotypes and pollinators: insights into the evolution of sexually deceptive orchids.

Plants are predicted to show floral adaptation to geographic variation in the most effective pollinator, potentially leading to reproductive isolation and genetic divergence. Many sexually deceptive orchids attract just a single pollinator species, limiting opportunities to experimentally investigate pollinator switching. Here, we investigate Drakaea concolor, which attracts two pollinator species. Using pollinator choice tests, we detected two morphologically similar ecotypes within D. concolor. The common ecotype only attracted Zaspilothynnus gilesi, whereas the rare ecotype also attracted an undescribed species of Pogonothynnus. The rare ecotype occurred at populations nested within the distribution of the common ecotype, with no evidence of ecotypes occurring sympatrically. Surveying for pollinators at over 100 sites revealed that ecotype identity was not correlated with wasp availability, with most orchid populations only attracting the rare Z. gilesi. Using microsatellite markers, genetic differentiation among populations was very low (GST = 0.011) regardless of ecotype, suggestive of frequent gene flow. Taken together, these results may indicate that the ability to attract Pogonothynnus has evolved recently, but this ecotype is yet to spread. The nested distribution of ecotypes, rather than the more typical formation of ecotypes in allopatry, illustrates that in sexually deceptive orchids, pollinator switching could occur throughout a species' range, resulting from multiple potentially suitable but unexploited pollinators occurring in sympatry. This unusual case of sympatric pollinators highlights D. concolor as a promising study system for further understanding the process of pollinator switching from ecological, chemical and genetic perspectives.

Avian influenza surveillance in Central and West Africa, 2010-2014.

Avian influenza virus (AIV) is an important zoonotic pathogen, resulting in global human morbidity and mortality and substantial economic losses to the poultry industry. Poultry and wild birds have transmitted AIV to humans, most frequently subtypes H5 and H7, but also different strains and subtypes of H6, H9, and H10. Determining which birds are AIV reservoirs can help identify human populations that have a high risk of infection with these viruses due to occupational or recreational exposure to the reservoir species. To assess the prevalence of AIV in tropical birds, from 2010 to 2014, we sampled 40 099 birds at 32 sites in Central Africa (Cameroon, Central African Republic, Congo-Brazzaville, Gabon) and West Africa (Benin, Côte d'Ivoire, Togo). In Central Africa, detection rates by real-time RT-PCR were 16·6% in songbirds (eight passerine families, n = 1257), 16·4% in kingfishers (family Alcedinidae, n = 73), 8·2% in ducks (family Anatidae, n = 564), and 3·65% in chickens (family Phasianidae, n = 1042). Public health authorities should educate human cohorts that have high exposure to these bird populations about AIV and assess their adherence to biosecurity practices, including Cameroonian farmers who raise small backyard flocks.

MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation.

The gene MID1, the mutation of which causes X-linked Opitz G/BBB syndrome (OS, MIM 300000), encodes a microtubule-associated protein (MAP). We show that mutation of MID1 leads to a marked accumulation of the catalytic subunit of protein phosphatase 2A (PP2Ac), a central cellular regulator. PP2Ac accumulation is caused by an impairment of a newly identified E3 ubiquitin ligase activity of the MID1 protein that normally targets PP2Ac for degradation through binding to its alpha4 regulatory subunit in an embryonic fibroblast line derived from a fetus with OS. Elevated PP2Ac causes hypophosphorylation of MAPs, a pathological mechanism that is consistent with the OS phenotype.

Mutations in the homeobox gene HESX1/Hesx1 associated with septo-optic dysplasia in human and mouse.

During early mouse development the homeobox gene Hesx1 is expressed in prospective forebrain tissue, but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Mice lacking Hesx1 exhibit variable anterior CNS defects and pituitary dysplasia. Mutants have a reduced prosencephalon, anopthalmia or micropthalmia, defective olfactory development and bifurcations in Rathke's pouch. Neonates exhibit abnormalities in the corpus callosum, the anterior and hippocampal commissures, and the septum pellucidum. A comparable and equally variable phenotype in humans is septo-optic dysplasia (SOD). We have cloned human HESX1 and screened for mutations in affected individuals. Two siblings with SOD were homozygous for an Arg53Cys missense mutation within the HESX1 homeodomain which destroyed its ability to bind target DNA. These data suggest an important role for Hesx1/HESX1 in forebrain, midline and pituitary development in mouse and human.

Quality of Life After Severe Burn Injury: A Case Report.

The danger of fire and electric current is underestimated by many people. The associated severe burn injuries are mainly treated in special burn centers because they are challenging and tend to have a strong impact on health-related quality of life. This case report describes a 20-year-old female severe burn victim who suffered second- to third-degree burns to approximately 80% of her total body surface. During in-patient care, we focused on inhalation trauma and anti-infective therapy, surgical management, physiotherapeutic and occupational therapy, in-patient rehabilitation measures, compression therapy and accompanying psychological co-treatment. This interdisciplinary treatment focused on restoring the best possible quality of life for burn victims.

Les dangers du feu et de l'électricité sont largement sous-estimés par la population. Les brûlures graves relèvent des centres spécialisés en raison de la complexité de leur prise en charge et de leur impact majeur sur la qualité de vie. Nous rapportons le cas d'une femme de 20 ans brûlée sur 80% de SCT (2ème et 3ème degrés). La prise en charge initiale a associé le traitement de l'inhalation de fumées, le traitement antiinfectieux, la chirurgie, la rééducation, la pressothérapie et l'accompagnement psychologique, dans le but d'optimiser la qualité de la vie à venir.

In situ multi-level analysis of viscoelastic deformation mechanisms in tendon collagen.

Tendon is a hydrated multi-level fibre composite, in which time-dependent behaviour is well established. Studies indicate significant stress relaxation, considered important for optimising tissue stiffness. However, whilst this behaviour is well documented, the mechanisms associated with the response are largely unknown. This study investigates the sub-structural mechanisms occurring during stress relaxation at both the macro (fibre) and nano (fibril) levels of the tendon hierarchy. Stress relaxation followed a two-stage exponential behaviour, during which structural changes were visible at the fibre and fibril levels. Fibril relaxation and fibre sliding showed a double exponential response, while fibre sliding was clearly the largest contributor to relaxation. The amount of stress relaxation and sub-structural reorganisation increased with increasing load increments, but fibre sliding was consistently the largest contributor to stress relaxation. A simple model of tendon viscoelasticity at the fibril and fibre levels has been developed, capturing this behaviour by serially coupling a Voigt element (collagen fibril), with two Maxwell elements (non-collagenous matrix between fibrils and fibres). This multi-level analysis provides a first step towards understanding how sub-structural interactions contribute to viscoelastic behaviour. It indicates that nano- and micro-scale shearing are significant dissipative mechanisms, and the kinetics of relaxation follows a two-stage exponential decay, well fitted by serially coupled viscoelastic elements.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

Contrasting impacts of pollen and seed dispersal on spatial genetic structure in the bird-pollinated Banksia hookeriana.

In plants, pollen- and seed-dispersal distributions are characteristically leptokurtic, with significant consequences for spatial genetic structure and nearest-neighbour mating. However, most studies to date have been on wind- or insect-pollinated species. Here, we assigned paternity to quantify effective pollen dispersal over 9 years of mating, contrasted this to seed dispersal and examined their effects on fine-scale spatial genetic structure, within the bird-pollinated shrub Banksia hookeriana (Proteaceae). We used 163 polymorphic amplified fragment length polymorphism markers to assess genetic structure and pollen dispersal in a spatially discrete population of 112 plants covering 0.56 ha. Spatial autocorrelation analysis detected spatial genetic structure in the smallest distance class of 0-5 m (r=0.025), with no significant structure beyond 8 m. Experimentally quantified seed-dispersal distances for 337 seedlings showed a leptokurtic distribution around a median of 5 m, reaching a distance of 36 m. In marked contrast, patterns of pollen dispersal for 274 seeds departed strikingly from typical near-neighbour pollination, with a distribution largely corresponding to the spatial distribution of plants. We found very high multiple paternity, very low correlated paternity and an equal probability of siring for the 50 closest potential mates. Extensive pollen carryover was demonstrated by multiple siring in 83 of 86 (96.5%) two-seeded fruits. Highly mobile nectar-feeding birds facilitate this promiscuity through observed movements that were effectively random. As the incidence of bird-pollination is markedly greater in the Southwest Australian Floristic Region than elsewhere, our results have broad and novel significance for the evolution and conservation for many species in Gondwanan lineages.

Regulatory gene expression boundaries demarcate sites of neuronal differentiation in the embryonic zebrafish forebrain.

During development of the zebrafish forebrain, a simple scaffold of axon pathways is pioneered by a small number of neurons. We show that boundaries of expression domains of members of the eph, forkhead, pax, and wnt gene families correlate with the positions at which these neurons differentiate and extend axons. Analysis of genetically or experimentally altered forebrains indicates that if a boundary is maintained, there is appropriate neural differentiation with respect to the boundary. Conversely, in the absence of a boundary, there is concomitant disruption of neural patterning. We also show that a strip of cells within the dorsal diencephalon shares features with ventral midline cells. This strip of cells fails to develop in mutant fish in which specification of the ventral CNS is disrupted, suggesting that its development may be regulated by the same inductive pathways that pattern the ventral midline.

Delayed and fractional use of enzymatic debridement with nexobrid for extensive burn injury: a case report.

Selective enzymatic debridement is increasingly being used in cases of burn wounds. However, until now the use of Nexobrid has been limited to 15% of total body surface area (TBSA) and immediate use on admission day. A 61-year-old Caucasian male suffered a severe burn injury that affected 95% TBSA. After surgical escharotomy and tracheotomy on admission day, we successfully performed a fractional enzymatic debridement of 54% of the TBSA in three different sessions within four days. This case report reveals that a delayed and fractional application of Nexobrid to more than 15% TBSA is possible.

Le débridement enzymatique sélectif des brûlures est de plus en plus utilisé. Cependant, cette technique était jusqu'ici limitée à 15% de la surface corporelle totale (SCT). Nous rapportons le cas d'un homme de 61 ans brûlé sur 95% SCT. Après une trachéotomie et des incisions de décharge le jour de son entrée, nous avons réalisé un débridement enzymatique sur 54% SCT sur 4 j en 3 séances. Cette observation montre que l'utilisation séquentielle de Nexobrid® permet de traiter plus de 15% SCT.

Release of vitamin B12--binding protein by human leukocytes in vitro.

Human granulocytes (G) contain a vitamin B(12)-binding protein (B(12)BP). There is evidence that chronic myelogenous leukemia leukocytes (CMLL) may synthesize B(12)BP. Our prior studies suggested that intact, living intravascular G synthesize and release such protein into extracellular compartments in vivo. In the present study, CMLL were incubated in Trisbuffered Hank's basal salt solution (pH 7.2) containing 0.1% human serum albumin to study release of B(12)BP into the medium. B(12)BP was released continuously and in increasing amounts over a 5 hr period at 37 degrees C; this release was inhibited almost completely when the cells were incubated at 4 degrees C and by about half as much in the presence of N-ethylmaleimide (1 mmole/liter). Cycloheximide (50 mug/ml) had no effect on the release of B(12)BP but significantly inhibited incorporation of leucine-(3)H into leukocyte protein. G incubated with 20 mg/ml of compound 48/80, an experimental histamine-releasing agent, had a 6-fold increase in release of B(12)BP over a 2 hr period. Subcellular fractionation studies of human granulocytes demonstrate that most of the B(12)BP is associated with the granular (20,000 g) layer with an excellent correlation observed between its subcellular distribution and that of acid phosphatase.These findings suggest that the release of B(12)BP from G is mediated by an active process and provide further evidence that granulocytes are secretory as well as phagocytic cells.

Reduced sperm count and normal fertility in male mice with targeted disruption of the ADP-ribosylation factor-like 4 (Arl4) gene.

The ADP-ribosylation factor-like protein 4 (ARL4) is a 22-kDa GTP-binding protein which is abundant in testes of pubertal and adult rodents but absent in testes from prepubertal animals. During testis development, ARL4 expression starts at day 16 when the spermatogenesis proceeds to the late pachytene. In the adult testis, the ARL4 protein was detected in pre- and postmeiotic cells, spermatocytes, and spermatides, but not in spermatogonia and mature spermatozoa. Mouse Arl4-null mutants generated by targeted disruption of the Arl4 gene were viable and grew normally; male as well as female Arl4(-/-) mice were fertile. However, inactivation of the Arl4 gene resulted in a significant reduction of testis weight and sperm count by 30 and 60%, respectively, without reduction of litter size or frequency. It is suggested that the disruption of Arl4 produces a moderate retardation of germ cell development, possibly at the stage of meiosis.

Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

Inhibition of leukocyte adherence by 3-M potassium chloride extracts of human colorectal tumors.

Leukocyte adherence inhibition (LAI) assays were performed to test whether peripheral blood leukocytes (PBL) from patients with colorectal cancer could be inhibited from attachment to a glass surface when tumor-associated antigens (TAA) of human colorectal tumors were present. The assays were performed with PBL from 41 patients with adenocarcinoma of the colon or rectum with 3-M KCl extracts of colorectal tumors. The results demonstrated the presence of colorectal TAA in the 3-M KCl extract of colorectal tumor materials. The reactivity of leukocytes from patients with a favorable prognosis showed an increasing LAI trend; the reactivity of leukocytes from patients with an unfavorable prognosis had a decreasing LAI trend. In individual patients, alterations in the sequential LAI results paralleled changes in the clinical status, which thus strongly indicated that the LAI assay could be of value in the assessment of antitumor immunity.

Versatile, sensitive liquid chromatography mass spectrometry - Implementation of 10 µm OT columns suitable for small molecules, peptides and proteins.

We have designed a versatile and sensitive liquid chromatographic (LC) system, featuring a monolithic trap column and a very narrow (10 µm ID) fused silica open tubular liquid chromatography (OTLC) separation column functionalized with C18-groups, for separating a wide range of molecules (from small metabolites to intact proteins). Compared to today's capillary/nanoLC approaches, our system provides significantly enhanced sensitivity (up to several orders) with matching or improved separation efficiency, and highly repeatable chromatographic performance. The chemical properties of the trap column and the analytical column were fine-tuned to obtain practical sample loading capacities (above 2 µg), an earlier bottleneck of OTLC. Using the OTLC system (combined with Orbitrap mass spectrometry), we could perform targeted metabolomics of sub-µg amounts of exosomes with 25 attogram detection limit of a breast cancer-related hydroxylated cholesterol. With the same set-up, sensitive bottom-up proteomics (targeted and untargeted) was possible, and high-resolving intact protein analysis. In contrast to state-of-the-art packed columns, our platform performs chromatography with very little dilution and is "fit-for-all", well suited for comprehensive analysis of limited samples, and has potential as a tool for challenges in diagnostics.

cDNA sequence of zebrafish (Brachydanio rerio) translation elongation factor-1 alpha: molecular phylogeny of eukaryotes based on elongation factor-1 alpha protein sequences.

We have isolated and determined the nucleotide sequence of a cDNA clone containing the complete coding region for elongation factor-1 alpha (EF-1 alpha) from an embryonic zebrafish cDNA library. A secondary structure model based on all known EF-1 alpha and EF-Tu protein sequences is presented and the presence of conserved putative protein kinase C phosphorylation sites in loop regions of eukaryotic EF-1 alpha is demonstrated. Using distance matrix and maximum parsimony methods we constructed multi-kingdom phylogenetic trees containing 22 different eukaryotic sequences. Strikingly, both tree constructions show Fungi to be the closest relative of Animalia among eukaryotic kingdoms. A 12 amino acid stretch present in all animal and fungal sequences known to date was found to be absent from all plant, protist an archaebacterial EF-1 alpha sequences suggesting that this sequence was inserted following the separation of plants from the lineage leading to fungi and animals. In contrast to our results, molecular phylogenies based on small subunit ribosomal RNA sequences as well as other protein sequences have failed to yield consistent results regarding the branching order among the kingdoms Plantae, Fungi and Animalia. The slow evolutionary rate and universal occurrence of EF-1 alpha (EF-Tu in eubacteria) makes this protein a particularly interesting tool for probing distant evolutionary relationships.

Effects of the mitogen concanavalin A on pathways of thymocyte energy metabolism.

The lectin concanavalin A (Con A) acts as a mitogen that preferentially activates T-cells. It stimulates the energy metabolism of thymocytes within seconds of exposure. We studied short-term effects (<30 min) of Con A on a conceptually simplified model system of rat thymocyte energy metabolism in the concentration range of 0-2 microg Con A per 107 cells, using metabolic control analysis. The model system consisted of three blocks of reactions, linked by the common intermediate mitochondrial membrane potential (Delta[psi]m): the substrate oxidation reactions, which produce the linking intermediate, and the proton conductance (or leak) and ATP turnover pathways which consume Delta[psi]m. Firstly, we used top-down elasticity analysis to establish which subsystems are targeted by Con A. Secondly, we quantitatively analysed the steady-state regulation of the system variables by Con A: how do the subsystem fluxes respond to Con A individually and as a whole? Our results indicate that: (1) steady-state respiration and Delta[psi]m increase as Con A concentration is raised, but at higher concentrations the increase in respiration is less and Delta[psi]m falls; (2) Con A independently changes the kinetics of the reactions that produce and consume Delta[psi]m: the Delta[psi]m-producing reactions are inhibited, and the reactions involved in ATP turnover are stimulated; and (3) the overall effects of Con A are mostly mediated by effects on ATP turnover.

Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).