An upgraded comprehensive multilocus phylogeny of the Tardigrada tree of life

Providing accurate animals’ phylogenies rely on increasing knowledge of neglected phyla. Tardigrada diversity evaluated in broad phylogenies (among phyla) is biased towards eutardigrades. A comprehensive phylogeny is demanded to establish the representative diversity and propose a more natural classification of the phylum. So, we have performed multilocus (18S rRNA and 28S rRNA) phylogenies with Bayesian inference and maximum likelihood. We propose the creation of a new class within Tardigrada, erecting the order Apochela (Eutardigrada) as a new Tardigrada class, named Apotardigrada comb. n. Two groups of evidence support its creation: (a) morphological, presence of cephalic appendages, unique morphology for claws (separated branches) and wide‐elongated buccopharyngeal apparatus without placoids, and (b) phylogenetic support based on molecular data. Consequently, order Parachela is suppressed and its superfamilies erected as orders within Eutardigrada, maintaining their current names. We propose a new classification within the family Echiniscidae (Echiniscoidea, Heterotardigrada) with morphological and phylogenetic support: (a) subfamily Echiniscinae subfam. n., with two tribes Echiniscini tribe n. and Bryodelphaxini tribe n.; (b) subfamily Pseudechiniscinae subfam. n., with three tribes Cornechiniscini tribe n., Pseudechiniscini tribe n. and Anthechiniscini tribe n.; and (c) subfamily Parechiniscinae subfam. n., with two tribes Parechiniscini tribe n. and Novechiniscini tribe n. Reliable biodiversity selection for tardigrades in broad phylogenies is proposed due to biased analyses performed up to now. We use our comprehensive molecular phylogeny to evaluate the evolution of claws in the clawless genus Apodibius and claw reduction across the Tardigrada tree of life. Evolutionary consequences are discussed.     

Diffusion Retardation by Binding of Tobramycin in an Alginate Biofilm Model

Microbial cells embedded in a self-produced extracellular biofilm matrix cause chronic infections, e. g. by Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The antibiotic killing of bacteria in biofilms is generally known to be reduced by 100–1000 times relative to planktonic bacteria. This makes such infections difficult to treat. We have therefore proposed that biofilms can be regarded as an independent compartment with distinct pharmacokinetics. To elucidate this pharmacokinetics we have measured the penetration of the tobramycin into seaweed alginate beads which serve as a model of the extracellular polysaccharide matrix in P. aeruginosa biofilm. We find that, rather than a normal first order saturation curve, the concentration of tobramycin in the alginate beads follows a power-law as a function of the external concentration. Further, the tobramycin is observed to be uniformly distributed throughout the volume of the alginate bead. The power-law appears to be a consequence of binding to a multitude of different binding sites. In a diffusion model these results are shown to produce pronounced retardation of the penetration of tobramycin into the biofilm. This filtering of the free tobramycin concentration inside biofilm beads is expected to aid in augmenting the survival probability of bacteria residing in the biofilm.     

Associations between dru Types and SCCmec Cassettes

Molecular typing is an important tool in the investigation of methicillin resistant Staphylococcus aureus (MRSA) outbreaks and in following the evolution of MRSA. The staphylococcal cassette chromosome mec (SCCmec) contains a hypervariable region with a variable number of 40 bp repeats named direct repeat units (dru). The dru region has been suggested as a supplementary typing method for MRSA and an international nomenclature exists. The purpose of this study was to investigate the diversity and variability of the dru region in a diverse collection of MRSA. We studied 302 MRSA isolates harbouring SCCmec types I to VI. The isolates represented a broad genetic background based on Staphylococcal protein A (spa) typing and multilocus sequence typing (MLST) and included 68 isolates (68 patients) from an outbreak with t024-ST8-IVa and 26 isolates from the same patient. Sequencing identified 53 dru types (dt) in 283 isolates, while eighteen isolates contained no dru repeats and one isolate resisted sequencing. The most common dru type, dt10a, was present in 53% of the sequenced isolates and was found in all SCCmec types, except type II. Seven (10%) of the 68 epidemiologically related patients had isolates with dru type variants indicating that dru typing is not useful as a first line epidemiological typing tool. However, MRSA isolates cultured from a single patient over a three year period exhibited a single dru type. The finding of dt10a in most SCCmec types suggests that dru and mecA originate from the same Staphylococcus species.     

Alpha1-AR-mediated activation of NKCC in rat cardiomyocytes involves ERK-dependent phosphorylation of the cotransporter

We studied molecular and functional characteristics as well as hormonal regulation of the Na-K-2Cl cotransporter (NKCC) in the isolated rat heart and cardiomyocytes. NKCC activity was measured as bumetanide-sensitive (86)Rb(+) influx in isolated perfused rat hearts and isolated cardiomyocytes. Stimulation of alpha(1)-adrenoceptors (AR) by phenylephrine (30 microM) increased (86)Rb(+) influx. The NKCC inhibitor bumetanide (50 microM) reduced the response to phenylephrine by 45 +/- 13% (n = 12, P < 0.01). PD-98059 (10 microM), an inhibitor of the activation of the mitogen-activated protein kinases extracellular signal-regulated protein kinase 1 and 2 (ERK1/2), reduced the total response to phenylephrine by 51 +/- 13% (n = 10, P < 0.01) and eliminated the bumetanide-sensitive component, indicating that alpha(1)-AR mediated stimulation of NKCC is dependent on activation of ERK1/2. Inhibitors of protein kinase C or phosphatidylinositol 3-kinase had no effect. The presence of NKCC mRNA and protein was demonstrated in isolated rat cardiomyocytes. Phosphorylation of NKCC after alpha(1)-AR stimulation was shown by immunoprecipitation of the phosphoprotein from (32)P(i) prelabeled cardiomyocytes. Increased phosphorylation of the NKCC protein was also abolished by PD-98059. We conclude that the NKCC is present in rat cardiomyocytes and that ion transport by the cotransporter is regulated by alpha(1)-AR stimulation through phosphorylation of this protein involving the ERK pathway.     

Prevalence of tick-borne encephalitis virus antibodies in dogs from Denmark

Background  

Large regions of central and eastern Europe are recognized as areas where tick-borne encephalitis virus (TBEV) is endemic, including countries neighbouring Denmark. It is therefore timely and relevant to determine if TBEV infections occur in Denmark. This study investigates the presence of antibodies against TBEV in a cross-section of the Danish canine population to assess the level of exposure to TBEV and possibly identify TBEV microfoci in Denmark.     

Biogeochemical cycling of sulfur and iron in sediments of a south-east Asian mangrove,Phuket Island,Thailand

Benthic sulfate reduction and sediment pools of sulfur and iron were examined during January 1992 at 3 stations in the Ao Nam Bor mangrove, Phuket, Thailand. Patterns of sulfate reduction rates (0–53 cm) reflected differences in physical and biological conditions at the 3 stations, and highest rates were found at the vegetated site within the mangrove (Rhizophora apiculata) forest. Due to extended oxidation of mangrove sediments, a large portion of the added³⁵S-label was recovered in the chromium reducible pools (FeS₂ and S⁰) (41–91% of the reduced sulfur). Pyrite was the most important inorganic sulfur component, attaining pool sizes 50–100 times higher than acid volatile pools (FeS). HCl-extractable (0.5 M HCl) iron pools, including Fe(II)_HCl and Fe(III)_HCl, were generally low and Fe(III)_HCl was only present in the upper surface layers (0–5 cm). Maximum concentrations of dissolved Fe²⁺ (35–285 M) occurred just about the depth where dissolved H₂S accumulated. Furthermore Fe²⁺ and H₂S coexisted only where concentrations of both were low. There was an accumulation of organic sulfur in the deep sediment at 2 stations in the inner part of the mangrove. The reoxidation of reduced sulfides was rapid, and storage of sulfur was minor in the upper sediment layers, where factors like bioturbation, the presence of roots, or tidal mixing enhance oxidation processes.Author of correspondence.     

Analysis of cervical smears obtained within three years of the diagnosis of invasive cervical cancer

The cytologic histories of 376 women presenting with invasive carcinoma of the cervix were analyzed. In total, 202 (53.7%) of these women had had 355 smears taken during the three years preceding presentation. All 320 smears with an original cytologic diagnosis of less than cancer were reviewed. The original cytologic diagnosis was low in 95 (30.6%) of 310 adequate smears. Originally, 96 (30.9%) of the adequate smears were evaluated as negative; at review, only 55 (17.5%) of the adequate smears were evaluated as negative. Comparing the review diagnoses to the 355 total smears, the rates of negative smears were 13.5% (42 of 310) for squamous-cell carcinoma, 30.0% (12 of 40) for adenocarcinoma and 20.0% (1 of 5) for adenosquamous carcinoma (P less than .05). The cellular composition of the smear was significantly related to the cytologic detection of abnormalities: endocervical cylindrical and/or metaplastic cells were seen in only 45.5% of smears diagnosed as negative, but in 84.4% and 97.8% of smears diagnosed as atypia and cervical intraepithelial neoplasia, respectively P less than .00001). Smears without endocervical cells should be considered inadequate and should be repeated.     

Mutations in Calmodulin Cause Ventricular Tachycardia and Sudden Cardiac Death

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a devastating inherited disorder characterized by episodic syncope and/or sudden cardiac arrest during exercise or acute emotion in individuals without structural cardiac abnormalities. Although rare, CPVT is suspected to cause a substantial part of sudden cardiac deaths in young individuals. Mutations in RYR2, encoding the cardiac sarcoplasmic calcium channel, have been identified as causative in approximately half of all dominantly inherited CPVT cases. Applying a genome-wide linkage analysis in a large Swedish family with a severe dominantly inherited form of CPVT-like arrhythmias, we mapped the disease locus to chromosome 14q31-32. Sequencing CALM1 encoding calmodulin revealed a heterozygous missense mutation (c.161A>T [p.Asn53Ile]) segregating with the disease. A second, de novo, missense mutation (c.293A>G [p.Asn97Ser]) was subsequently identified in an individual of Iraqi origin; this individual was diagnosed with CPVT from a screening of 61 arrhythmia samples with no identified RYR2 mutations. Both CALM1 substitutions demonstrated compromised calcium binding, and p.Asn97Ser displayed an aberrant interaction with the RYR2 calmodulin-binding-domain peptide at low calcium concentrations. We conclude that calmodulin mutations can cause severe cardiac arrhythmia and that the calmodulin genes are candidates for genetic screening of individual cases and families with idiopathic ventricular tachycardia and unexplained sudden cardiac death.     

Cucurbit phloem serpins are graft-transmissible and appear to be resistant to turnover in the sieve element-companion cell complex

Serpins are unique inhibitors of serine proteases that are located in various plant tissues and organs. An orthologue of the pumpkin (Cucurbita maxima) phloem serpin CmPS-1 was amplified from cucumber (Cucumis sativus) RNA by RT-PCR, cloned, and designated as CsPS-1 (GenBank accession no. AJ866989). Alternative amino acid sequences in the reactive centre loop suggest distinct inhibitory specificity between CmPS-1 and CsPS-1. A difference in the electrophoretic mobility of these serpins was used in heterografts to establish that serpins are phloem-mobile. Immuno light microscopy revealed that the phloem serpins are localized exclusively to sieve elements (SE), while the phloem filament protein CmPP1, used as a reference, is localized to both SEs and companion cells (CCs). Similar to CmPS-1, CsPS-1 accumulates over time in phloem exudates, indicating that serpins differ from other phloem-mobile proteins whose concentrations appear to be stable in phloem exudates. These differences could reflect alternative mechanisms regulating protein turnover and/or inaccessibility of protein degradation. The functionality of the pore/plasmodesma units connecting SEs and CCs was tested with graft-transmitted CmPP1 as a transport marker. The occurrence of CmPP1 in the CCs of the Cucumis graft partner shows that translocated 88 kDa phloem filament protein monomers can symplasmically exit the SE and accumulate in the CC. By contrast, serial sections probed with the serpin antibody demonstrate that the 43 kDa serpin does not enter CCs. Collectively, these data indicate that CCs play a decisive role in homeostasis of exudate proteins; proteins not accessing the CCs accumulate in SEs and display a time-dependent increase in concentration.