Behavioural Effects of Tourism on Oceanic Common Dolphins,Delphinus sp., in New Zealand: The Effects of Markov Analysis Variations and Current Tour Operator Compliance with Regulations

Common dolphins, Delphinus sp., are one of the marine mammal species tourism operations in New Zealand focus on. While effects of cetacean-watching activities have previously been examined in coastal regions in New Zealand, this study is the first to investigate effects of commercial tourism and recreational vessels on common dolphins in an open oceanic habitat. Observations from both an independent research vessel and aboard commercial tour vessels operating off the central and east coast Bay of Plenty, North Island, New Zealand were used to assess dolphin behaviour and record the level of compliance by permitted commercial tour operators and private recreational vessels with New Zealand regulations. Dolphin behaviour was assessed using two different approaches to Markov chain analysis in order to examine variation of responses of dolphins to vessels. Results showed that, regardless of the variance in Markov methods, dolphin foraging behaviour was significantly altered by boat interactions. Dolphins spent less time foraging during interactions and took significantly longer to return to foraging once disrupted by vessel presence. This research raises concerns about the potential disruption to feeding, a biologically critical behaviour. This may be particularly important in an open oceanic habitat, where prey resources are typically widely dispersed and unpredictable in abundance. Furthermore, because tourism in this region focuses on common dolphins transiting between adjacent coastal locations, the potential for cumulative effects could exacerbate the local effects demonstrated in this study. While the overall level of compliance by commercial operators was relatively high, non-compliance to the regulations was observed with time restriction, number or speed of vessels interacting with dolphins not being respected. Additionally, prohibited swimming with calves did occur. The effects shown in this study should be carefully considered within conservation management plans, in order to reduce the risk of detrimental effects on common dolphins within the region.     

Short-term adaptation during propagation improves the performance of xylose-fermenting <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in simultaneous saccharification and co-fermentation

Background

Inhibitors that are generated during thermochemical pretreatment and hydrolysis impair the performance of microorganisms during fermentation of lignocellulosic hydrolysates. In omitting costly detoxification steps, the fermentation process relies extensively on the performance of the fermenting microorganism. One attractive option of improving its performance and tolerance to microbial inhibitors is short-term adaptation during propagation. This study determined the influence of short-term adaptation on the performance of recombinant Saccharomyces cerevisiae in simultaneous saccharification and co-fermentation (SSCF). The aim was to understand how short-term adaptation with lignocellulosic hydrolysate affects the cell mass yield of propagated yeast and performance in subsequent fermentation steps. The physiology of propagated yeast was examined with regard to viability, vitality, stress responses, and upregulation of relevant genes to identify any links between the beneficial traits that are promoted during adaptation and overall ethanol yields in co-fermentation.

Results

The presence of inhibitors during propagation significantly improved fermentation but lowered cell mass yield during propagation. Xylose utilization of adapted cultures was enhanced by increasing amounts of hydrolysate in the propagation. Ethanol yields improved by over 30 % with inhibitor concentrations that corresponded to ≥2.5 % water-insoluble solids (WIS) load during the propagation compared with the unadapted culture. Adaptation improved cell viability by >10 % and increased vitality by >20 %. Genes that conferred resistance against inhibitors were upregulated with increasing amounts of inhibitors during the propagation, but the adaptive response was not associated with improved ethanol yields in SSCF. The positive effects in SSCF were observed even with adaptation at inhibitor concentrations that corresponded to 2.5 % WIS. Higher amounts of hydrolysate in the propagation feed further improved the fermentation but increased the variability in fermentation outcomes and resulted in up to 20 % loss of cell mass yield.

Conclusions

Short-term adaptation during propagation improves the tolerance of inhibitor-resistant yeast strains to inhibitors in lignocellulosic hydrolysates and improves their ethanol yield in fermentation and xylose-fermenting capacity. A low amount of hydrolysate (corresponding to 2.5 % WIS) is optimal, whereas higher amounts decrease cell mass yield during propagation.

     

Association between Education Level and Prognosis after Esophageal Cancer Surgery: A Swedish Population-Based Cohort Study

Background

An association between education level and survival after esophageal cancer has recently been indicated, but remains uncertain. We conducted a large study with long follow-up to address this issue.

Methods

This population-based cohort study included all patients operated for esophageal cancer in Sweden between 1987 and 2010 with follow-up until 2012. Level of education was categorized as compulsory (≤9 years), intermediate (10–12 years), or high (≥13 years). The main outcome measure was overall 5-year mortality after esophagectomy. Cox regression was used to estimate associations between education level and mortality, expressed as hazard ratios (HRs) with 95% confidence intervals (CIs), with adjustment for sex, age, co-morbidity, tumor stage, tumor histology, and assessing the impact of education level over time.

Results

Compared to patients with high education, the adjusted HR for mortality was 1.29 (95% CI 1.07–1.57) in the intermediate educated group and 1.42 (95% CI 1.17–1.71) in the compulsory educated group. The largest differences were found in early tumor stages (T-stage 0–1), with HRs of 1.73 (95% CI 1.00–2.99) and 2.58 (95% CI 1.51–4.42) for intermediate and compulsory educated patients respectively; and for squamous cell carcinoma, with corresponding HRs of 1.38 (95% CI 1.07–1.79) and 1.52 (95% CI 1.19–1.95) respectively.

Conclusions

This Swedish population-based study showed an association between higher education level and improved survival after esophageal cancer surgery, independent of established prognostic factors. The associations were stronger in patients of an early tumor stage and squamous cell carcinoma.     

Matrix Metalloproteinases -8 and -9 and Tissue Inhibitor of Metalloproteinase-1 in Burn Patients. A Prospective Observational Study

IntroductionMatrix metalloproteinases (MMPs) -8 and -9 are released from neutrophils in acute inflammation and may contribute to permeability changes in burn injury. In retrospective studies on sepsis, levels of MMP-8, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) differed from those of healthy controls, and TIMP-1 showed an association with outcome. Our objective was to investigate the relationship between these proteins and disease severity and outcome in burn patients.MethodsIn this prospective, observational, two-center study, we collected plasma samples from admission to day 21 post-burn, and burn blister fluid samples on admission. We compared MMP-8, -9, and TIMP-1 levels between TBSA<20% (N = 19) and TBSA>20% (N = 30) injured patients and healthy controls, and between 90-day survivors and non-survivors. MMP-8, -9, and TIMP-1 levels at 24-48 hours from injury, their maximal levels, and their time-adjusted means were compared between groups. Correlations with clinical parameters and the extent of burn were analyzed. MMP-8, -9, and TIMP-1 levels in burn blister fluids were also studied.ResultsPlasma MMP-8 and -9 were higher in patients than in healthy controls (P<0.001 and P = 0.016), but only MMP-8 differed between the TBSA<20% and TBSA>20% groups. MMP-8 and -9 were not associated with clinical severity or outcome measures. TIMP-1 differed significantly between patients and controls (P<0.001) and between TBSA<20% and TBSA>20% groups (P<0.002). TIMP-1 was associated with 90-day mortality and correlated with the extent of injury and clinical measures of disease severity. TIMP-1 may serve as a new biomarker in outcome prognostication of burn patients.     

Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.Chondroitin sulfates (CS)¹ are complex polysaccharides present at cell surfaces and in extracellular matrices. The polysaccharides belong to a subclass of glycosaminoglycans (GAGs) and are covalently linked to various core proteins to form CS-proteoglycans (CSPGs), each with differences in the protein structures and/or numbers of CS side chains. Apart from their structural role in cartilage, CSPGs contribute to the regulation of a diverse set of biological processes such as neurogenesis, growth factor signaling, angiogenesis, and morphogenesis (1–5). Although the molecular basis of CSPGs functions remains elusive, accumulating evidence suggests that the underlying activities relate to selective ligand binding to discrete structural variants of the polysaccharides. Thus, the current strategy for understanding the biological role of CSPGs aims to identify selective CS polysaccharide–ligand interactions. However, information on the number of CS-chains and their specific attachment site(s) on any given core protein is often scarce which limits our functional understanding of CSPGs.The biosynthesis of GAGs occurs in the endoplasmic reticulum and Golgi compartments and is initiated by the enzymatic addition of a beta-linked xylose (Xyl) to a Ser residue of the core protein. The sequential addition of two galactose residues (Gal) and a glucuronic acid (GlcA) onto the growing saccharide chain completes the formation of a tetrasaccharide linkage region (GlcAβ3Galβ3Galβ4XylβSer). This part of the biosynthesis is the same for CS and heparan sulfate (HS). However, for CS the biosynthesis continues with the addition of an N-acetylgalactosamine (GalNAcβ3), whereas HS biosynthesis continues with the addition of an N-acetylglucosamine (GlcNAcα4) (6). The CS-chains are thereafter elongated through the addition of repeating units of GlcA and GalNAc and are further modified by the addition of specifically positioned sulfate groups (7). Certain features of the core protein seem to influence if a certain Ser residue is selected for GAG attachment and whether CS or HS will be synthesized, but the selection mechanism is largely unknown. Sequence analysis of previously known GAG-substituted core proteins reveals that the glycosylated serine residues are usually flanked by a glycine residue (-SG-), and are associated with a cluster of acidic residues in close proximity (8). This motif may assist in the prediction of potential GAG-sites of core proteins; however, the use of such strategy is ambiguous because proteoglycans may also contain unoccupied motifs or motifs that are occasionally occupied (9).Glycoproteomics strategies have recently appeared that provide site-specific information of N- and O-glycans. Such strategies are typically based on a specific enrichment of glycopeptides and a subsequent analysis with nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) (10). By further developing this concept for proteoglycans (11), we have now analyzed CSPG linkage region glycopeptides of human samples, which enabled us to identify 13 novel human CSPGs in addition to 13 already established CSPGs. Urine and cerebrospinal fluid (CSF) samples were trypsinized and CS glycopeptides were enriched using strong anion exchange (SAX) chromatography. The CS chains were depolymerized with chondroitinase ABC, generating free disaccharides and a residual hexameric structure composed of the linkage region and a GlcA-GalNAc disaccharide dehydrated on the terminal GlcA residue (12). MS/MS analysis provided the combined sequencing of the residual hexasaccharide and of the core peptide.     

Sprint Conditioning of Junior Soccer Players: Effects of Training Intensity and Technique Supervision

The aims of the present study were to compare the effects of 1) training at 90 and 100% sprint velocity and 2) supervised versus unsupervised sprint training on soccer-specific physical performance in junior soccer players. Young, male soccer players (17 ±1 yr, 71 ±10 kg, 180 ±6 cm) were randomly assigned to four different treatment conditions over a 7-week intervention period. A control group (CON, n=9) completed regular soccer training according to their teams’ original training plans. Three training groups performed a weekly repeated-sprint training session in addition to their regular soccer training sessions performed at A) 100% intensity without supervision (100UNSUP, n=13), B) 90% of maximal sprint velocity with supervision (90SUP, n=10) or C) 90% of maximal sprint velocity without supervision (90UNSUP, n=13). Repetitions x distance for the sprint-training sessions were 15x20 m for 100UNSUP and 30x20 m for 90SUP and 90UNSUP. Single-sprint performance (best time from 15x20 m sprints), repeated-sprint performance (mean time over 15x20 m sprints), countermovement jump and Yo-Yo Intermittent Recovery Level 1 (Yo-Yo IR1) were assessed during pre-training and post-training tests. No significant differences in performance outcomes were observed across groups. 90SUP improved Yo-Yo IR1 by a moderate margin compared to controls, while all other effect magnitudes were trivial or small. In conclusion, neither weekly sprint training at 90 or 100% velocity, nor supervised sprint training enhanced soccer-specific physical performance in junior soccer players.     

Revealing Different Roles of the mTOR-Targets S6K1 and S6K2 in Breast Cancer by Expression Profiling and Structural Analysis

Background

The AKT/mTORC1/S6K pathway is frequently overstimulated in breast cancer, constituting a promising therapeutic target. The benefit from mTOR inhibitors varies, likely as a consequence of tumour heterogeneity, and upregulation of several compensatory feed-back mechanisms. The mTORC1 downstream effectors S6K1, S6K2, and 4EBP1 are amplified and overexpressed in breast cancer, associated with a poor outcome and divergent endocrine treatment benefit. S6K1 and S6K2 share high sequence homology, but evidence of partly distinct biological functions is emerging. The aim of this work was to explore possible different roles and treatment target potentials of S6K1 and S6K2 in breast cancer.

Materials and methods

Whole-genome expression profiles were compared for breast tumours expressing high levels of S6K1, S6K2 or 4EBP1, using public datasets, as well as after in vitro siRNA downregulation of S6K1 and/or S6K2 in ZR751 breast cancer cells. In silico homology modelling of the S6K2 kinase domain was used to evaluate its possible structural divergences to S6K1.

Results

Genome expression profiles were highly different in S6K1 and S6K2 high tumours, whereas S6K2 and 4EBP1 profiles showed significant overlaps, both correlated to genes involved in cell cycle progression, among these the master regulator E2F1. S6K2 and 4EBP1 were inversely associated with IGF1 levels, and their prognostic value was shown to be restricted to tumours positive for IGFR and/or HER2. In vitro, S6K1 and S6K2 silencing resulted in upregulation of genes in the mTORC1 and mTORC2 complexes. Isoform-specific silencing also showed distinct patterns, e.g. S6K2 downregulation lead to upregulation of several cell cycle associated genes. Structural analyses of the S6K2 kinase domain showed unique structure patterns, deviating from those of S6K1, facilitating the development of isoform-specific inhibitors. Our data support emerging proposals of distinct biological features of S6K1 and S6K2, suggesting their importance as separate oncogenes and clinical markers, where specific targeting in different breast cancer subtypes could facilitate further individualised therapies.     

Synthesis and evaluation of 2-pyridylbenzothiazole, 2-pyridylbenzoxazole and 2-pyridylbenzofuran derivatives as 11C-PET imaging agents for β-amyloid plaques

The syntheses and SAR of new series of β-amyloid binding agents are reported. The effort to optimize signal-to-background ratios for these ligands are described. Compounds 8, 21 and 30 displayed desirable lipophilicity and pharmacokinetic properties. Compounds 8 and 21 were evaluated with in vitro autoradiographic studies and in vivo in APP/PS1 transgenic mice. It is shown that it was possible to increase the signal-to-background ratios compared to PIB 1, as demonstrated by compounds 8 and 21.     

Network properties of human disease genes with pleiotropic effects

Background  

The ability of a gene to cause a disease is known to be associated with the topological position of its protein product in the molecular interaction network. Pleiotropy, in human genetic diseases, refers to the ability of different mutations within the same gene to cause different pathological effects. Here, we hypothesized that the ability of human disease genes to cause pleiotropic effects would be associated with their network properties.     

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15): a new marker of all‐cause mortality

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15) is a member of the TGF‐b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC‐1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC‐1/GDF15 may be a marker of all‐cause mortality. To determine whether serum MIC‐1/GDF15 estimation is a predictor of all‐cause mortality, we examined a cohort of 876 male subjects aged 35–80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC‐1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same‐sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL‐6) and C‐reactive protein (CRP) available. Patients were followed for up to 14 years and had cause‐specific and all‐cause mortality determined. Serum MIC‐1/GDF15 levels predicted mortality in the all‐male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38–8.26). This finding was validated in the twin cohort. Serum MIC‐1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL‐6 and CRP. Additionally, serum MIC‐1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC‐1/GDF15 is a novel predictor of all‐cause mortality.