Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Series resistance compensation for whole-cell patch-clamp studies using a membrane state estimator

Whole-cell patch-clamp techniques are widely used to measure membrane currents from isolated cells. While suitable for a broad range of ionic currents, the series resistance (R(s)) of the recording pipette limits the bandwidth of the whole-cell configuration, making it difficult to measure rapid ionic currents. To increase bandwidth, it is necessary to compensate for R(s). Most methods of R(s) compensation become unstable at high bandwidth, making them hard to use. We describe a novel method of R(s) compensation that overcomes the stability limitations of standard designs. This method uses a state estimator, implemented with analog computation, to compute the membrane potential, V(m), which is then used in a feedback loop to implement a voltage clamp; we refer to this as state estimator R(s) compensation. To demonstrate the utility of this approach, we built an amplifier incorporating state estimator R(s) compensation. In benchtop tests, our amplifier showed significantly higher bandwidths and improved stability when compared with a commercially available amplifier. We demonstrated that state estimator R(s) compensation works well in practice by recording voltage-gated Na(+) currents under voltage-clamp conditions from dissociated neonatal rat sympathetic neurons. We conclude that state estimator R(s) compensation should make it easier to measure large rapid ionic currents with whole-cell patch-clamp techniques.     

Long distance dispersal in the assembly of floras: A review of progress and prospects in North America

Here, we review progress and prospects to explicitly test for long distance dispersal biogeographic events. Long distance dispersal represents a “jump” across some kind of barrier, such as a topographic feature or a zone of unsuitable climate and may include repeated jumps, or stepping‐stone dispersals. Long distance dispersals were considered integral for explaining the organization of biodiversity at large and small scales by early biogeographers, such as Darwin and Wallace. Darwin, Wallace, and others envisioned that long distance dispersals were predictable events because the vectors for dispersal, such as animals, winds, and currents, behaved in non‐random ways. However, these early biogeographers found that dispersal was hard to observe, and, later, with the advent of the theory of Continental Drift, vicariance became regarded as a better scientific explanation for the arrangement of biodiversity, because it represented a falsifiable hypothesis. Thus, long distance dispersal was reduced to a nuisance parameter in biogeography; a random possibility that could never fully be ruled out in a scenario in which evidence supported vicariance. Today, there is strong interest to more fully integrate long distance dispersal into understanding the assembly and organization of biodiversity on earth. In this review, we discuss progress and prospects for explicitly testing long distance dispersal hypotheses including through uses of molecular, morphological, paleontological, and informatics methods. We focus on hypothesis testing of long distance dispersals involved in the assembly of the flora of North America, which is a robust preliminary study system on account of its extant and extinct biodiversity being well‐catalogued.     

A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Proteomic analysis of endothelial cold-adaptation

Background

Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution.

Results

A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed.

Conclusions

The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.