Cladistic analysis of the Cirripedia Thoracica

We present a cladistic analysis of the Cirripedia Thoracica using morphological characters and the Acrothoracica and Ascothoracida as outgroups. The list of characters comprised 32 shell and soft body features. The operational taxonomic units (OTUs) comprised 26 well-studied fossil and extant taxa, principally genera, since uncertainty about monophyly exists for most higher ranking taxonomic units. Parsimony analyses using PAUP 3.1.1 and Hennig86 produced 189 trees of assured minimal length. We also examined character evolution in the consensus trees using MacClade and Clados. The monophyly of the Balanomorpha and the Verrucomorpha sensu stricto is confirmed, and all trees featured a sister group relationship between the ‘living fossil Neoverruca and me Brachylepadomorpha. In the consensus trees the sequential progression of ‘pedunculate‘sister groups up to a node containing Neolepas also conforms to current views, but certain well-established taxa based solely on plesiomorphies stand out as paraphyletic, such as Pedunculata (= Lepadomorpha); Eolepadinae, Scalpellomorpha and Chthamaloidea. The 189 trees differed principally in the position of shell-less pedunculates, Neoverruca, the scalpelloid Capitulum, and the interrelationships within the Balanomorpha, although the 50% majority rule consensus tree almost fully resolved the latter. A monophyletic Sessilia comprising both Verrucomorpha and Balanomorpha appeared among the shortest trees, but not in the consensus. A tree with a monophyletic Verrucomorpha including Neoverruca had a tree length two steps longer than the consensus trees. Deletion of all extinct OTUs produced a radically different tree, which highlights the importance of fossils in estimating cirripede phylogeny. Mapping of our character set onto a manually constructed cladogram reflecting die most recent scenario of cirripede evolution resulted in a tree length five steps longer than any of our shortest trees. Our analysis reveals that several key questions in cirripede phylogeny remain unsolved, notably the position of shell-less forms and the transition from ‘pedunculate‘to ‘sessile‘barnacles. The inclusion of more fossil species at this point in our understanding of cirripede phylogeny will only result in even greater levels of uncertainty. When constructing the character list we also identified numerous uncertainties in the homology of traits commonly used in discussing cirripede evolution. Our study highlights larval ultrastructure, detailed studies of early ontogeny, and molecular data as the most promising areas for future research.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

When similar beginnings lead to different ends: Constraints and diversity in cirripede larval development

Summary

Cirripedes are fascinating models for studying both functional constraints and diversity in larval development. Adult cirripedes display an amazing variation in morphology from sessile suspension feeders that still retain many crustacean characters to parasites that have lost virtually all arthropod traits. In contrast, cirripede larval development follows a common scheme with pelagic larvae comprising a series of nauplii followed by a cyprid. Variations are mostly concerned with whether or not the nauplii are feeding and the degree of abbreviation of development, culminating in species where the larvae hatch as cyprids. The cypris larvae are very similar among the ingroups of the Cirripedia, but interesting variations occur in structures used for substrate location and attachment. The cyprid is specialized to both swim through the water and actively explore the substratum by walking on the antennules and using an array of sensory organs in search for a suitable site to attach. This unique morphology and behavior of the cyprid have enabled the Cirripedia to colonize widely different habitats ranging from hard rock to soft animal tissue. Yet, the cyprid can metamorphose into juveniles as different as a setose feeding barnacle and the vermiform stages of the parasitic forms. This emphasizes the importance of the cyprid as one of the key features for the evolutionary success of the Cirripedia.     

An in vivo control map for the eukaryotic mRNA translation machinery

Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non‐intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non‐stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNA_i to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than‐average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.     

Natalizumab Treatment Reduces Fatigue in Multiple Sclerosis. Results from the TYNERGY Trial; A Study in the Real Life Setting

Fatigue is a significant symptom in multiple sclerosis (MS) patients. First-generation disease modifying therapies (DMTs) are at best moderately effective to improve fatigue. Observations from small cohorts have indicated that natalizumab, an antibody targeting VLA-4, may reduce MS-related fatigue. The TYNERGY study aimed to further evaluate the effects of natalizumab treatment on MS-related fatigue. In this one-armed clinical trial including 195 MS patients, natalizumab was prescribed in a real-life setting, and a validated questionnaire, the Fatigue Scale for Motor and Cognitive functions (FSMC), was used both before and after 12 months of treatment to evaluate a possible change in the fatigue experienced by the patients. In the treated cohort all measured variables, that is, fatigue score, quality of life, sleepiness, depression, cognition, and disability progression were improved from baseline (all p values<0.0001). Walking speed as measured by the six-minute walk-test also increased at month 12 (p = 0.0016). All patients were aware of the nature of the treatment agent, and of the study outcomes.

Conclusion

Natalizumab, as used in a real-life setting, might improve MS-related fatigue based on the results from this one-armed un-controlled stud. Also other parameters related to patients'' quality of life seemed to improve with natalizumab treatment.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00884481","term_id":"NCT00884481"}}NCT00884481     

Stable suppression of MDR1 gene expression and function by RNAi in Caco-2 cells

Vector-based RNAi was used to establish a stable Caco-2 cell line with a persistent knockdown of multidrug resistant gene 1 (MDR1) and P-glycoprotein (P-gp). Several positive clones were collected, many of which showed significantly reduced levels of MDR1 mRNA and P-gp compared to wt Caco-2 cells. Selected clones were sub-cultivated for six passages and real-time PCR showed that MDR1 expression remained significantly reduced (up to 96%) over this period of time. RNAi-MDR1 clones frozen long term also kept their low MDR1 expression levels when re-cultured. Permeability studies were performed across RNAi-MDR1 clone cell monolayers, and the efflux of cyclosporine A, digoxin, vinblastine, and vincristine showed 58%, 61%, 91%, and 78% decrease in active transport, respectively, compared to wt Caco-2 cells. This stably modified Caco-2 cell line provides a novel tool for studies on MDR1 and other ABC transporter protein gene cellular functions.     

IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

Importance of human leukocyte antigen (HLA) class I and II alleles on the risk of multiple sclerosis

Multiple sclerosis (MS) is a complex disease of the central nervous system of unknown etiology. The human leukocyte antigen (HLA) locus on chromosome 6 confers a considerable part of the susceptibility to MS, and the most important factor is the class II allele HLA-DRB1*15:01. In addition, we and others have previously established a protective effect of HLA-A*02. Here, we genotyped 1,784 patients and 1,660 healthy controls from Scandinavia for the HLA-A, HLA-B, HLA-C and HLA-DRB1 genes and investigated their effects on MS risk by logistic regression. Several allele groups were found to exert effects independently of DRB1*15 and A*02, in particular DRB1*01 (OR = 0.82, p = 0.034) and B*12 (including B*44/45, OR = 0.76, p = 0.0028), confirming previous reports. Furthermore, we observed interaction between allele groups: DRB1*15 and DRB1*01 (multiplicative: OR = 0.54, p = 0.0041; additive: AP = 0.47, p = 4 × 10(-06)), DRB1*15 and C*12 (multiplicative: OR = 0.37, p = 0.00035; additive: AP = 0.58, p = 2.6 × 10(-05)), indicating that the effect size of these allele groups varies when taking DRB1*15 into account. Analysis of inferred haplotypes showed that almost all DRB1*15 bearing haplotypes were risk haplotypes, and that all A*02 bearing haplotypes were protective as long as they did not carry DRB1*15. In contrast, we found one class I haplotype, carrying A*02-C*05-B*12, which abolished the risk of DRB1*15. In conclusion, these results confirms a complex role of HLA class I and II genes that goes beyond DRB1*15 and A*02, in particular by including all three classical HLA class I genes as well as functional interactions between DRB1*15 and several alleles of DRB1 and class I genes.     

A Longitudinal Study of Disability,Cognition and Gray Matter Atrophy in Early Multiple Sclerosis Patients According to Evidence of Disease Activity

New treatment options may make “no evidence of disease activity” (NEDA: no relapses or disability progression and no new/enlarging MRI lesions, as opposed to “evidence of disease activity” (EDA) with at least one of the former), an achievable goal in relapsing-remitting multiple sclerosis (RRMS). The objective of the present study was to determine whether early RRMS patients with EDA at one-year follow-up had different disability, cognition, treatment and gray matter (GM) atrophy rates from NEDA patients and healthy controls (HC). RRMS patients (mean age 34 years, mean disease duration 2.2 years) were examined at baseline and one-year follow-up with neurological (n = 72), neuropsychological (n = 56) and structural MRI (n = 57) examinations. Matched HC (n = 61) were retested after three years. EDA was found in 46% of RRMS patients at follow-up. EDA patients used more first line and less second line disease modifying treatment than NEDA (p = 0.004). While the patients groups had similar disability levels at baseline, they differed in disability at follow-up (p = 0.010); EDA patients progressed (EDSS: 1.8–2.2, p = 0.010), while NEDA patients improved (EDSS: 2.0–1.7, p<0.001). Cognitive function was stable in both patient groups. Subcortical GM atrophy rates were higher in EDA patients than HC (p<0.001). These results support the relevance of NEDA as outcome in RRMS and indicate that pathological neurodegeneration in RRMS mainly occur in patients with evidence of disease activity.