Temporal Co-Variation between Eye Lens Accommodation and Trapezius Muscle Activity during a Dynamic Near-Far Visual Task

Near work is associated with increased activity in the neck and shoulder muscles, but the underlying mechanism is still unknown. This study was designed to determine whether a dynamic change in focus, alternating between a nearby and a more distant visual target, produces a direct parallel change in trapezius muscle activity. Fourteen healthy controls and 12 patients with a history of visual and neck/shoulder symptoms performed a Near-Far visual task under three different viewing conditions; one neutral condition with no trial lenses, one condition with negative trial lenses to create increased accommodation, and one condition with positive trial lenses to create decreased accommodation. Eye lens accommodation and trapezius muscle activity were continuously recorded. The trapezius muscle activity was significantly higher during Near than during Far focusing periods for both groups within the neutral viewing condition, and there was a significant co-variation in time between accommodation and trapezius muscle activity within the neutral and positive viewing conditions for the control group. In conclusion, these results reveal a connection between Near focusing and increased muscle activity during dynamic changes in focus between a nearby and a far target. A direct link, from the accommodation/vergence system to the trapezius muscles cannot be ruled out, but the connection may also be explained by an increased need for eye-neck (head) stabilization when focusing on a nearby target as compared to a more distant target.     

The FPR2-specific ligand MMK-1 activates the neutrophil NADPH-oxidase,but triggers no unique pathway for opening of plasma membrane calcium channels

Human neutrophils express formyl peptide receptor 1 and 2 (FPR1 and FPR2), two highly homologous G-protein-coupled cell surface receptors important for the cellular recognition of chemotactic peptides. They share many functional as well as signal transduction features, but some fundamental differences have been described. One such difference was recently presented when the FPR2-specific ligand MMK-1 was shown to trigger a unique signal in neutrophils [S. Partida-Sanchez, P. Iribarren, M.E. Moreno-Garcia, et al., Chemotaxis and calcium responses of phagocytes to formyl peptide receptor ligands is differentially regulated by cyclic ADP ribose, J. Immunol. 172 (2004) 1896–1906]. This signal bypassed the emptying of the intracellular calcium stores, a route normally used to open the store-operated calcium channels present in the plasma membrane of neutrophils. Instead, the binding of MMK-1 to FPR2 was shown to trigger a direct opening of the plasma membrane channels. In this report, we add MMK-1 to a large number of FPR2 ligands that activate the neutrophil superoxide-generating NADPH-oxidase. In contrast to earlier findings we show that the transient rise in intracellular free calcium induced by MMK-1 involves both a release of calcium from intracellular stores and an opening of channels in the plasma membrane. The same pattern was obtained with another characterized FPR2 ligand, WKYMVM, and it is also obvious that the two formyl peptide receptor family members trigger the same type of calcium response in human neutrophils.     

Molecular Evolutionary Consequences of Niche Restriction in Francisella tularensis,a Facultative Intracellular Pathogen

Francisella tularensis is a potent mammalian pathogen well adapted to intracellular habitats, whereas F. novicida and F. philomiragia are less virulent in mammals and appear to have less specialized lifecycles. We explored adaptations within the genus that may be linked to increased host association, as follows. First, we determined the genome sequence of F. tularensis subsp. mediasiatica, the only subspecies that had not been previously sequenced. This genome, and those of 12 other F. tularensis isolates, were then compared to the genomes of F. novicida (three isolates) and F. philomiragia (one isolate). Signs of homologous recombination were found in ∼19.2% of F. novicida and F. philomiragia genes, but none among F. tularensis genomes. In addition, random insertions of insertion sequence elements appear to have provided raw materials for secondary adaptive mutations in F. tularensis, e.g. for duplication of the Francisella Pathogenicity Island and multiplication of a putative glycosyl transferase gene. Further, the five major genetic branches of F. tularensis seem to have converged along independent routes towards a common gene set via independent losses of gene functions. Our observations suggest that despite an average nucleotide identity of >97%, F. tularensis and F. novicida have evolved as two distinct population lineages, the former characterized by clonal structure with weak purifying selection, the latter by more frequent recombination and strong purifying selection. F. tularensis and F. novicida could be considered the same bacterial species, given their high similarity, but based on the evolutionary analyses described in this work we propose retaining separate species names.     

Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish

ABSTRACT: BACKGROUND: Prior to this study, relatively few strains of Francisella had been genome-sequenced. Previously published Francisella genome sequences were largely restricted to the zoonotic agent F. tularensis. Only limited data were available for other members of the Francisella genus, including F. philomiragia, an opportunistic pathogen of humans, F. noatunensis, a serious pathogen of farmed fish, and other less well described endosymbiotic species. RESULTS: We determined the phylogenetic relationships of all known Francisella species, including some for which the phylogenetic positions were previously uncertain. The genus Francisella could be divided into two main genetic clades: one included F. tularensis, F. novicida, F. hispaniensis and Wolbachia persica, and another included F. philomiragia and F. noatunensis. Some Francisella species were found to have significant recombination frequencies. However, the fish pathogen F. noatunensis subsp. noatunensis was an exception due to it exhibiting a highly clonal population structure similar to the human pathogen F. tularensis. CONCLUSIONS: The genus Francisella can be divided into two main genetic clades occupying both terrestrial and marine habitats. However, our analyses suggest that the ancestral Francisella species originated in a marine habitat. The observed genome to genome variation in gene content and IS elements of different species supports the view that similar evolutionary paths of host adaptation developed independently in F. tularensis (infecting mammals) and F. noatunensis subsp. noatunensis (infecting fish).     

Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C

Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.     

Delineation of amplification,hybridization and location effects in microarray data yields better-quality normalization

Background  

Oligonucleotide arrays have become one of the most widely used high-throughput tools in biology. Due to their sensitivity to experimental conditions, normalization is a crucial step when comparing measurements from these arrays. Normalization is, however, far from a solved problem. Frequently, we encounter datasets with significant technical effects that currently available methods are not able to correct.     

The FPR2-induced rise in cytosolic calcium in human neutrophils relies on an emptying of intracellular calcium stores and is inhibited by a gelsolin-derived PIP<Subscript>2</Subscript>-binding peptide

Background  

The molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific G-protein-coupled cell surface receptors (GPCRs). Human neutrophils express two pattern recognition GPCRs, FPR1 and FPR2, which belong to the family of formyl peptide receptors. The high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. The aims of this study were to determine whether FPR2 triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived PIP₂-binding peptide, PBP10, selectively inhibits FPR2-mediated transient rise in intracellular Ca²⁺.     

Identification of proteins susceptible to thiol oxidation in endothelial cells exposed to hypochlorous acid and N-chloramines

Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis.     

Reactivation of Desensitized Formyl Peptide Receptors by Platelet Activating Factor: A Novel Receptor Cross Talk Mechanism Regulating Neutrophil Superoxide Anion Production

Neutrophils express different chemoattractant receptors of importance for guiding the cells from the blood stream to sites of inflammation. These receptors communicate with one another, a cross talk manifested as hierarchical, heterologous receptor desensitization. We describe a new receptor cross talk mechanism, by which desensitized formyl peptide receptors (FPR_des) can be reactivated. FPR desensitization is induced through binding of specific FPR agonists and is reached after a short period of active signaling. The mechanism that transfers the receptor to a non-signaling desensitized state is not known, and a signaling pathway has so far not been described, that transfers FPR_des back to an active signaling state. The reactivation signal was generated by PAF stimulation of its receptor (PAFR) and the cross talk was uni-directional. LatrunculinA, an inhibitor of actin polymerization, induced a similar reactivation of FPR_des as PAF while the phosphatase inhibitor CalyculinA inhibited reactivation, suggesting a role for the actin cytoskeleton in receptor desensitization and reactivation. The activated PAFR could, however, reactivate FPR_des also when the cytoskeleton was disrupted prior to activation. The receptor cross talk model presented prophesies that the contact on the inner leaflet of the plasma membrane that blocks signaling between the G-protein and the FPR is not a point of no return; the receptor cross-talk from the PAFRs to the FPR_des initiates an actin-independent signaling pathway that turns desensitized receptors back to a signaling state. This represents a novel mechanism for amplification of neutrophil production of reactive oxygen species.     

The Human Neutrophil Subsets Defined by the Presence or Absence of OLFM4 Both Transmigrate into Tissue In Vivo and Give Rise to Distinct NETs In Vitro

Neutrophil heterogeneity was described decades ago, but it could not be elucidated at the time whether the existence of different neutrophil subsets had any biological relevance. It has been corroborated in recent years that neutrophil subsets, defined by differential expression of various markers, are indeed present in human blood, calling for renewed attention to this question. The expression of the granule protein olfactomedin 4 (OLFM4) has been suggested to define two such neutrophil subsets. We confirm the simultaneous presence of one OLFM4-positive and one OLFM4-negative neutrophil subpopulation as well as the localization of the protein to specific granules. In vitro, these neutrophil subsets displayed equal tendency to undergo apoptosis and phagocytose bacteria. In addition, the subpopulations were recruited equally to inflammatory sites in vivo, and this was true both in an experimental model of acute inflammation and in naturally occurring pathological joint inflammation. In line with its subcellular localization, only limited OLFM4 release was seen upon in vivo transmigration, and release through conventional degranulation required strong secretagogues. However, extracellular release of OLFM4 could be achieved upon formation of neutrophil extracellular traps (NETs) where it was detected only in a subset of the NETs. Although we were unable to demonstrate any functional differences between the OLFM4-defined subsets, our data show that different neutrophil subsets are present in inflamed tissue in vivo. Furthermore, we demonstrate NETs characterized by different markers for the first time, and our results open up for functions of OLFM4 itself in the extracellular space through exposure in NETs.