Postglacial expansion of the arctic keystone copepod calanus glacialis

Calanus glacialis, a major contributor to zooplankton biomass in the Arctic shelf seas, is a key link between primary production and higher trophic levels that may be sensitive to climate warming. The aim of this study was to explore genetic variation in contemporary populations of this species to infer possible changes during the Quaternary period, and to assess its population structure in both space and time. Calanus glacialis was sampled in the fjords of Spitsbergen (Hornsund and Kongsfjorden) in 2003, 2004, 2006, 2009 and 2012. The sequence of a mitochondrial marker, belonging to the ND5 gene, selected for the study was 1249 base pairs long and distinguished 75 unique haplotypes among 140 individuals that formed three main clades. There was no detectable pattern in the distribution of haplotypes by geographic distance or over time. Interestingly, a Bayesian skyline plot suggested that a 1000-fold increase in population size occurred approximately 10,000 years before present, suggesting a species expansion after the Last Glacial Maximum.GAME from the National Science Centre, the Polish Ministry of Science and Higher Education Iuventus Plus [IP2014 050573]; FCT-PT [CCMAR/Multi/04326/2013]; [2011/03/B/NZ8/02876

Cystatin E/M suppresses legumain activity and invasion of human melanoma

<p>Abstract</p> <p>Background</p> <p>High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.</p> <p>Methods</p> <p>A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.</p> <p>Results</p> <p>Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.</p> <p>Conclusions</p> <p>These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.</p

Canagliflozin attenuates the progression of atherosclerosis and inflammation process in APOE knockout mice

Background: Sodium glucose co-transporter2 inhibitors reduce the incidence of cardiovascular events in patients with type 2 diabetes mellitus based on the results of recent cardiovascular outcome studies. Herein, we investigated the efects of long-term treatment with canaglifozin on biochemical and immunohistochemical markers related to atherosclerosis and atherosclerosis development in the aorta of apolipoprotein E knockout (Apo-E(−/−) ) mice. Methods: At the age of 5 weeks, mice were switched from normal to a high-fat diet. After 5 weeks, Apo-E(−/−) mice were divided into control-group (6 mice) treated with 0.5% hydroxypropyl methylcellulose and Cana-group (7 mice) treated with canaglifozin (10 mg/kg per day) per os. After 5 weeks of intervention, animals were sacrifced, and heart and aorta were removed. Sections stained with hematoxylin–eosin (H&E) were used for histomorphometry whereas Masson’s stained tissues were used to quantify the collagen content. Immunohistochemistry to assess MCP-1, CD68, a-smooth muscle actin, MMP-2, MMP-9, TIMP-1 and TIMP-2 expression was carried out and q-PCR experiments were performed to quantify mRNA expression. Results: Canaglifozin-group mice had lower total-cholesterol, triglycerides and glucose levels (P<0.01), while heart rate was signifcantly lower (P<0.05). Histomorphometry revealed that one in seven Cana-group mice versus four in six control mice developed atheromatosis, while aortic root plaque was signifcantly less, and collagen was 1.6 times more intense in canaglifozin-group suggesting increased plaque stability. Immunohistochemistry revealed that MCP-1 was signifcantly less expressed (P<0.05) in the aortic root of canaglifozin-group while reduced expression of a-actin and CD68 was not reaching signifcance (P=0.15). VCAM-1 and MCP-1 mRNA levels were lower (P=0.02 and P=0.07, respectively), while TIMP-1/MMP-2 ratio expression was higher in canaglifozin-group approaching statistical signifcance (P=0.07). Conclusions: Canaglifozin attenuates the progression of atherosclerosis, reducing (1) hyperlipidemia and hyper‑ glycemia, and (2) infammatory process, by lowering the expression of infammatory molecules such as MCP-1 and VCAM-1. Moreover, canaglifozin was found to increase the atherosclerotic plaque stability via increasing TIMP-1/ MMP-2 ratio expression

High power Q-switched thulium doped fibre laser using carbon nanotube polymer composite saturable absorber

We have proposed and demonstrated a Q-switched Thulium doped bre laser (TDFL) with a ‘Yin-Yang’ all- bre cavity scheme based on a combination of nonlinear optical loop mirror (NOLM) and nonlinear ampli ed loop mirror (NALM). Unidirectional lasing operation has been achieved without any intracavity isolator. By using a carbon nanotube polymer composite based saturable absorber (SA), we demonstrated the laser output power of ~197 mW and pulse energy of 1.7 μJ. To the best of our knowledge, this is the highest output power from a nanotube polymer composite SA based Q-switched Thulium doped bre laser

An experimental study of low-level laser therapy in rat Achilles tendon injury

The aim of this controlled animal study was to investigate the effect of low-level laser therapy (LLLT) administered 30 min after injury to the Achilles tendon. The study animals comprised 16 Sprague Dawley male rats divided in two groups. The right Achilles tendons were injured by blunt trauma using a mini guillotine, and were treated with LLLT or placebo LLLT 30 min later. The injury and LLLT procedures were then repeated 15 hours later on the same tendon. One group received active LLLT (λ = 904 nm, 60 mW mean output power, 0.158 W/cm2 for 50 s, energy 3 J) and the other group received placebo LLLT 23 hours after LLLT. Ultrasonographic images were taken to measure the thickness of the right and left Achilles tendons. Animals were then killed, and all Achilles tendons were tested for ultimate tensile strength (UTS). All analyses were performed by blinded observers. There was a significant increase in tendon thickness in the active LLLT group when compared with the placebo group (p < 0.05) and there were no significant differences between the placebo and uninjured left tendons. There were no significant differences in UTS between laser-treated, placebo-treated and uninjured tendons. Laser irradiation of the Achilles tendon at 0.158 W/cm2 for 50 s (3 J) administered within the first 30 min after blunt trauma, and repeated after 15 h, appears to lead to edema of the tendon measured 23 hours after LLLT. The guillotine blunt trauma model seems suitable for inflicting tendon injury and measuring the effects of treatment on edema by ultrasonography and UTS. More studies are needed to further refine this model

Engineered Single-Domain Antibodies with High Protease Resistance and Thermal Stability

The extreme pH and protease-rich environment of the upper gastrointestinal tract is a major obstacle facing orally-administered protein therapeutics, including antibodies. Through protein engineering, several Clostridium difficile toxin A-specific heavy chain antibody variable domains (VHHs) were expressed with an additional disulfide bond by introducing Ala/Gly54Cys and Ile78Cys mutations. Mutant antibodies were compared to their wild-type counterparts with respect to expression yield, non-aggregation status, affinity for toxin A, circular dichroism (CD) structural signatures, thermal stability, protease resistance, and toxin A-neutralizing capacity. The mutant VHHs were found to be well expressed, although with lower yields compared to wild-type counterparts, were non-aggregating monomers, retained low nM affinity for toxin A, albeit the majority showed somewhat reduced affinity compared to wild-type counterparts, and were capable of in vitro toxin A neutralization in cell-based assays. Far-UV and near-UV CD spectroscopy consistently showed shifts in peak intensity and selective peak minima for wild-type and mutant VHH pairs; however, the overall CD profile remained very similar. A significant increase in the thermal unfolding midpoint temperature was observed for all mutants at both neutral and acidic pH. Digestion of the VHHs with the major gastrointestinal proteases, at biologically relevant concentrations, revealed a significant increase in pepsin resistance for all mutants and an increase in chymotrypsin resistance for the majority of mutants. Mutant VHH trypsin resistance was similar to that of wild-type VHHs, although the trypsin resistance of one VHH mutant was significantly reduced. Therefore, the introduction of a second disulfide bond in the hydrophobic core not only increases VHH thermal stability at neutral pH, as previously shown, but also represents a generic strategy to increase VHH stability at low pH and impart protease resistance, with only minor perturbations in target binding affinities. These are all desirable characteristics for the design of protein-based oral therapeutics

Isotopic techniques to measure N2O, N2 and their sources

GHG emissions are usually the result of several simultaneous processes. Furthermore, some gases such as N2 are very difficult to quantify and require special techniques. Therefore, in this chapter, the focus is on stable isotope methods. Both natural abundance techniques and enrichment techniques are used. Especially in the last decade, a number of methodological advances have been made. Thus, this chapter provides an overview and description of a number of current state-of-theart techniques, especially techniques using the stable isotope 15N. Basic principles and recent advances of the 15N gas flux method are presented to quantify N2 fluxes, but also the latest isotopologue and isotopomer methods to identify pathways for N2O production. The second part of the chapter is devoted to 15N tracing techniques, the theoretical background and recent methodological advances. A range of different methods is presented from analytical to numerical tools to identify and quantify pathway-specific N2O emissions. While this chapter is chiefly concerned with gaseous N emissions, a lot of the techniques can also be applied to other gases such as methane (CH4), as outlined in Sect. 5.3