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1.  How Resilient Are Europe’s Inshore Fishing Communities to Change? Differences Between the North and the South 
Ambio  2013;42(8):1037-1046.
One would hypothesize that the Common Fisheries Policy, as the umbrella framework for fisheries management in the EU would have the greatest impact on fishers’ communities across Europe. There are, however, biological, economic, social, and political factors, which vary among fishing communities that can affect how these communities react to changes. This paper explores the links between institutional arrangements and ecological dynamics in two European inshore fisheries socio-ecological systems, using a resilience framework. The Mediterranean small-scale fishers do not seem to have been particularly affected by the Common Fisheries Policy regulations but appear affected by competition with the politically strong recreational fishers and the invasion of the rabbit fish population. The inshore fishers along the East coast of Scotland believe that their interests are not as sufficiently protected as the interests of their offshore counterpart. Decisions and initiatives at global, EU, and sometimes national level, tend to take into account those fisheries sectors which have a national economic importance. A socio-ecological analysis can shift the focus from biological and economic aspects to more sustainable long-term delivery of environmental benefits linked to human wellbeing.
PMCID: PMC3824873  PMID: 24214001
Resilience; Artisanal fisheries; Socio-ecological systems; Governance; Cyprus; Scotland; Mediterranean
2.  Immunisation with ID83 fusion protein induces antigen-specific cell mediated and humoral immune responses in cattle 
Vaccine  2013;31(45):10.1016/j.vaccine.2013.08.051.
In this study we have investigated the potential of mycobacterial proteins as candidate subunit vaccines for bovine tuberculosis. In addition, we have explored the use of TLR-ligands as potential adjuvants in cattle. In vitro screening assays with whole blood from M. bovis-infected and BCG-vaccinated cattle demonstrated that fusion protein constructs were most commonly recognised, and the ID83 fusion protein was selected for further immunisation studies. Furthermore, glucopyranosyl lipid A (GLA) and resiquimod (R848), agonists for TLR4 and TLR7/8 respectively, stimulated cytokine production (IL-12, TNF-α, MIP-1β and IL-10) in bovine dendritic cell cultures, and these were formulated as novel oil-in-water emulsions (GLA-SE and R848-SE) for immunisation studies. Immunisation with ID83 in a water-in-oil emulsion adjuvant (ISA70) induced both cell mediated and humoral immune responses, as characterised by antigen-specific IFN-γ production, cell proliferation, IgG1 and IgG2 antibody production. In comparison, ID83 immunisation with the novel adjuvants induced weaker (ID83/R848-SE) or no (ID83/GLA-SE) antigen-specific IFN-γ production and cell proliferation. However, both did induce ID83-specific antibody production, which was restricted to IgG1 antibody isotype. Overall, these results provide encouraging preliminary data for the further development of ID83 in vaccine strategies for bovine TB.
PMCID: PMC3855245  PMID: 24012566
4.  The effect of an internet option and single-sided printing format to increase the response rate to a population-based study: a randomized controlled trial 
Paper questionnaires are a common means to collect self-reported information in population-based epidemiological studies. Over the past decades, the response rates to epidemiological studies have been decreasing which can affect the selection process of eligible subjects and lead to non-response bias. Hence, research into strategies to increase questionnaire response rates is crucial. The aim of this study was therefore to explore the effectiveness of single-sided questionnaires and an internet option for response in increasing response rates to a population-based study.
A 2×2 factorial experiment was embedded within a large population-based study of pain and pain management. Persons in the study sample were 4600 residents in Grampian (north of Scotland) aged 25 years and over who were randomly selected from health board records. Sampled persons were randomly assigned to either receive a single-sided or double-sided questionnaire with or without an internet option to respond. The study questionnaire was distributed via post.
The overall study response rate was 36.3%. When compared to the reference group that received no intervention (response rate = 35.5%), the response rate changed only marginally when single-sided questionnaires were distributed (35.8%) or when an option to reply via the internet was provided (34.3%). A somewhat higher increase in response rates was achieved when both strategies were employed (39.6%). Overall, no significant effect on response rate was determined for each strategy or their interaction.
Evidence from this study suggests that neither single-sided questionnaires nor the option to reply via the internet resulted in a significant increase in response rates to population-based studies.
PMCID: PMC4236570  PMID: 25204467
Response rate; Randomized controlled trial; Postal questionnaires; Health surveys; Data collection; Internet
5.  Use of Antigen-Specific Interleukin-2 To Differentiate between Cattle Vaccinated with Mycobacterium bovis BCG and Cattle Infected with M. bovis 
We describe here the application of a novel bovine interleukin-2 (IL-2) enzyme-linked immunosorbent assay (ELISA) for the measurement of antigen-specific IL-2 in cattle naturally infected with Mycobacterium bovis and in cattle vaccinated with Mycobacterium bovis BCG and then experimentally challenged with pathogenic M. bovis. Supernatants from whole-blood cultures stimulated with mycobacterial antigen (bovine purified protein derivative [PPDB] or the peptide cocktail ESAT6-CFP10) were assessed using a sandwich ELISA consisting of a new recombinant monoclonal fragment capture antibody and a commercially available polyclonal anti-bovine-IL-2. The production of IL-2 was compared to the production of gamma interferon (IFN-γ) in the same antigen-stimulated whole-blood supernatants. The data show that cattle infected with M. bovis produced quantifiable levels of antigen-specific IL-2, while IL-2 levels in cattle vaccinated with M. bovis BCG did not. Furthermore, cattle vaccinated with M. bovis BCG and then challenged with pathogenic M. bovis displayed a more rapid induction of IL-2 but ultimately had lower levels of infection-induced IL-2 than did unvaccinated challenge control cattle. These data suggest that IL-2 responses are not detectable post-BCG vaccination and that these responses may require infection with virulent M. bovis to develop. This may be useful to differentiate infected cattle from uninfected or BCG-vaccinated cattle, although the overall sensitivity is relatively low, particularly in single intradermal comparative cervical tuberculin (SICCT)-negative infected animals. Furthermore, the strength of the IL-2 response may correlate with pathology, which poses interesting questions on the immunobiology of bovine tuberculosis in contrast to human tuberculosis, which is discussed.
PMCID: PMC3910917  PMID: 24173026
6.  Quantity and Quality of Physical Activity Are Influenced by Outdoor Temperature in People with Knee Osteoarthritis 
Physiotherapy Canada  2013;65(3):248-254.
Purpose: Environmental factors are known to influence physical activity (PA) levels of healthy people, but little work has explored these factors in people with osteoarthritis (OA). The purpose of this study was to explore the influence of outdoor temperature on PA in people with knee OA. Methods: A total of 38 people with knee OA (10 women; mean age 54 y) wore an accelerometer around their waist for 7 consecutive days. Dependent variables from the accelerometers were three PA measures: (1) activity counts/day and time spent at or above moderate levels of PA with (2) a cut-point of 1,041 activity counts/minute (MVPA1041) and (3) a cut-point of 1,952 activity counts/minute (MVPA1952). Independent variables were age, sex, body mass index (BMI), and maximum daily outdoor temperature. Three linear regression analyses were conducted using the three PA dependent variables and independent variables. Results: After controlling for age, sex, and BMI, maximum daily outdoor temperature explained 9% of the variance in activity counts/day (p=0.042), 10% of variance in MVPA1041 (p=0.032), and 14% of variance in MVPA1952 (p=0.016). Participants who engaged in more PA were younger and were exposed to warmer temperatures. Conclusions: Outdoor temperature and age influence the PA levels of people with knee OA. These factors should be considered when designing PA programmes.
PMCID: PMC3740990  PMID: 24403695
knee osteoarthritis; motor activity; temperature; weather; arthrose du genou; activité motrice; temps; température
7.  Relaxed Evolution in the Tyrosine Aminotransferase Gene Tat in Old World Fruit Bats (Chiroptera: Pteropodidae) 
PLoS ONE  2014;9(5):e97483.
Frugivorous and nectarivorous bats fuel their metabolism mostly by using carbohydrates and allocate the restricted amounts of ingested proteins mainly for anabolic protein syntheses rather than for catabolic energy production. Thus, it is possible that genes involved in protein (amino acid) catabolism may have undergone relaxed evolution in these fruit- and nectar-eating bats. The tyrosine aminotransferase (TAT, encoded by the Tat gene) is the rate-limiting enzyme in the tyrosine catabolic pathway. To test whether the Tat gene has undergone relaxed evolution in the fruit- and nectar-eating bats, we obtained the Tat coding region from 20 bat species including four Old World fruit bats (Pteropodidae) and two New World fruit bats (Phyllostomidae). Phylogenetic reconstructions revealed a gene tree in which all echolocating bats (including the New World fruit bats) formed a monophyletic group. The phylogenetic conflict appears to stem from accelerated TAT protein sequence evolution in the Old World fruit bats. Our molecular evolutionary analyses confirmed a change in the selection pressure acting on Tat, which was likely caused by a relaxation of the evolutionary constraints on the Tat gene in the Old World fruit bats. Hepatic TAT activity assays showed that TAT activities in species of the Old World fruit bats are significantly lower than those of insectivorous bats and omnivorous mice, which was not caused by a change in TAT protein levels in the liver. Our study provides unambiguous evidence that the Tat gene has undergone relaxed evolution in the Old World fruit bats in response to changes in their metabolism due to the evolution of their special diet.
PMCID: PMC4019583  PMID: 24824435
8.  Second generation sequencing and morphological faecal analysis reveal unexpected foraging behaviour by Myotis nattereri (Chiroptera, Vespertilionidae) in winter 
Frontiers in Zoology  2014;11:39.
Temperate winters produce extreme energetic challenges for small insectivorous mammals. Some bat species inhabiting locations with mild temperate winters forage during brief inter-torpor normothermic periods of activity. However, the winter diet of bats in mild temperate locations is studied infrequently. Although microscopic analyses of faeces have traditionally been used to characterise bat diet, recently the coupling of PCR with second generation sequencing has offered the potential to further advance our understanding of animal dietary composition and foraging behaviour by allowing identification of a much greater proportion of prey items often with increased taxonomic resolution. We used morphological analysis and Illumina-based second generation sequencing to study the winter diet of Natterer’s bat (Myotis nattereri) and compared the results obtained from these two approaches. For the first time, we demonstrate the applicability of the Illumina MiSeq platform as a data generation source for bat dietary analyses.
Faecal pellets collected from a hibernation site in southern England during two winters (December-March 2009–10 and 2010–11), indicated that M. nattereri forages throughout winter at least in a location with a mild winter climate. Through morphological analysis, arthropod fragments from seven taxonomic orders were identified. A high proportion of these was non-volant (67.9% of faecal pellets) and unexpectedly included many lepidopteran larvae. Molecular analysis identified 43 prey species from six taxonomic orders and confirmed the frequent presence of lepidopteran species that overwinter as larvae.
The winter diet of M. nattereri is substantially different from other times of the year confirming that this species has a wide and adaptable dietary niche. Comparison of DNA derived from the prey to an extensive reference dataset of potential prey barcode sequences permitted fine scale taxonomic resolution of prey species. The high occurrence of non-volant prey suggests that gleaning allows prey capture at low ambient temperatures when the abundance of flying insects may be substantially reduced. Interesting questions arise as to how M. nattereri might successfully locate and capture some of the non-volant prey species encountered in its faeces. The consumption of lepidopteran larvae such as cutworms suggests that M. nattereri eats agricultural pest species.
PMCID: PMC4108090  PMID: 25093034
Echolocation; Ecosystem services; Hibernation; Illumina MiSeq; Metabarcoding; Molecular diet analyses; Natterer’s bat; Sensory ecology; Winter diet
9.  Development of an Unbiased Antigen-Mining Approach To Identify Novel Vaccine Antigens and Diagnostic Reagents for Bovine Tuberculosis 
Clinical and Vaccine Immunology : CVI  2013;20(11):1675-1682.
Previous experiments for the identification of novel diagnostic or vaccine candidates for bovine tuberculosis have followed a targeted approach, wherein specific groups of proteins suspected to contain likely candidates are prioritized for immunological assessment (for example, with in silico approaches). However, a disadvantage of this approach is that the sets of proteins analyzed are restricted by the initial selection criteria. In this paper, we describe a series of experiments to evaluate a nonbiased approach to antigen mining by utilizing a Gateway clone set for Mycobacterium tuberculosis, which constitutes a library of clones expressing 3,294 M. tuberculosis proteins. Although whole-blood culture experiments using Mycobacterium bovis-infected animals and M. bovis BCG-vaccinated controls did not reveal proteins capable of differential diagnosis, several novel immunogenic proteins were identified and prioritized for efficacy studies in a murine vaccination/challenge model. These results demonstrate that Rv3329-immunized mice had lower bacterial cell counts in their spleens following challenge with M. bovis. In conclusion, we demonstrate that this nonbiased approach to antigen mining is a useful tool for identifying and prioritizing novel proteins for further assessment as vaccine antigens.
PMCID: PMC3837790  PMID: 23986315
10.  Integrin linked kinase (ILK) regulates podosome maturation and stability in dendritic cells? 
Podosomes are integrin-based adhesions fundamental for stabilisation of the leading lamellae in migrating dendritic cells (DCs) and for extracellular matrix (ECM) degradation. We have previously shown that soluble factors and chemokines such as SDF 1-a trigger podosome initiation whereas integrin ligands promote podosome maturation and stability in DCs. The exact intracellular signalling pathways that regulate the sequential organisation of podosomal components in response to extracellular cues remain largely undetermined. The Wiskott Aldrich Syndrome Protein (WASP) mediates actin polymerisation and the initial recruitment of integrins and associated proteins in a circular configuration surrounding the core of filamentous actin (F-actin) during podosome initiation. We have now identified integrin linked kinase (ILK) surrounding the podosomal actin core. We report that DC polarisation in response to chemokines and the assembly of actin cores during podosome initiation require PI3K-dependent clustering of the Wiskott Aldrich Syndrome Protein (WASP) in puncta independently of ILK. ILK is essential for the clustering of integrins and associated proteins leading to podosome maturation and stability that are required for degradation of the subjacent extracellular matrix and the invasive motility of DCs across connective tissue barriers.
We conclude that WASP regulates DCs polarisation for migration and initiation of actin polymerisation downstream of PI3K in nascent podosomes. Subsequently, ILK mediates the accumulation of integrin-associated proteins during podosome maturation and stability for efficient degradation of the subjacent ECM during the invasive migration of DCs.
PMCID: PMC3998073  PMID: 24508783
ILK; Dendritic cell; Podosome; WASP; PI3K
11.  Maintained physical activity and physiotherapy in the management of distal upper limb pain – a protocol for a randomised controlled trial (the arm pain trial) 
Distal upper limb pain (pain affecting the elbow, forearm, wrist, or hand) can be non-specific, or can arise from specific musculoskeletal disorders. It is clinically important and costly, the best approach to clinical management is unclear. Physiotherapy is the standard treatment and, while awaiting treatment, advice is often given to rest and avoid strenuous activities, but there is no evidence base to support these strategies. This paper describes the protocol of a randomised controlled trial to determine, among patients awaiting physiotherapy for distal arm pain, (a) whether advice to remain active and maintain usual activities results in a long-term reduction in arm pain and disability, compared with advice to rest; and (b) whether immediate physiotherapy results in a long-term reduction in arm pain and disability, compared with physiotherapy delivered after a seven week waiting list period.
Between January 2012 and January 2014, new referrals to 14 out-patient physiotherapy departments were screened for potential eligibility. Eligible and consenting patients were randomly allocated to one of the following three groups in equal numbers: 1) advice to remain active, 2) advice to rest, 3) immediate physiotherapy. Patients were and followed up at 6, 13, and 26 weeks post-randomisation by self-complete postal questionnaire and, at six weeks, patients who had not received physiotherapy were offered it at this time. The primary outcome is the proportion of patients free of disability at 26 weeks, as determined by the modified DASH (Disabilities of the Arm, Shoulder and Hand) questionnaire.
We hypothesise (a) that advice to maintain usual activities while awaiting physiotherapy will be superior than advice to rest the arm; and (b) that fast-track physiotherapy will be superior to normal (waiting list) physiotherapy. These hypotheses will be examined using an intention-to-treat analysis.
Results from this trial will contribute to the evidence base underpinning the clinical management of patients with distal upper limb pain, and in particular, will provide guidance on whether they should be advised to rest the arm or remain active within the limits imposed by their symptoms.
Trial registration
Registered on (reference number: ISRCTN79085082).
PMCID: PMC3975277  PMID: 24612447
Randomised controlled trial; Arm pain; Physiotherapy; Advice; Rest; Active; Pain management
12.  Identification of Genes Involved in the Biosynthesis of the Third and Fourth Sugars of the Methanococcus maripaludis Archaellin N-Linked Tetrasaccharide 
Journal of Bacteriology  2013;195(18):4094-4104.
N-glycosylation is a protein posttranslational modification found in all three domains of life. Many surface proteins in Archaea, including S-layer proteins, pilins, and archaellins (archaeal flagellins) are known to contain N-linked glycans. In Methanococcus maripaludis, the archaellins are modified at multiple sites with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β-GlcNAc3NAcA-1,3-β-GalNAc, where Sug is the unique sugar (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-l-erythro-hexos-5-ulo-1,5-pyranose. In this study, four genes—mmp1084, mmp1085, mmp1086, and mmp1087—were targeted to determine their potential involvement of the biosynthesis of the sugar components in the N-glycan, based on bioinformatics analysis and proximity to a number of genes which have been previously demonstrated to be involved in the N-glycosylation pathway. The genes mmp1084 to mmp1087 were shown to be cotranscribed, and in-frame deletions of each gene as well as a Δmmp1086Δmmp1087 double mutant were successfully generated. All mutants were archaellated and motile. Mass spectrometry examination of purified archaella revealed that in Δmmp1084 mutant cells, the threonine linked to the third sugar of the glycan was missing, indicating a putative threonine transferase function of MMP1084. Similar analysis of the archaella of the Δmmp1085 mutant cells demonstrated that the glycan lacked the methyl group at the C-5 position of the terminal sugar, indicating that MMP1085 is a methyltransferase involved in the biosynthesis of this unique sugar. Deletion of the remaining two genes, mmp1086 and mmp1087, either singularly or together, had no effect on the structure of the archaellin N-glycan. Because of their demonstrated involvement in the N-glycosylation pathway, we designated mmp1084 as aglU and mmp1085 as aglV.
PMCID: PMC3754732  PMID: 23836872
13.  Determinants and Patterns of Reproductive Success in the Greater Horseshoe Bat during a Population Recovery 
PLoS ONE  2014;9(2):e87199.
An individual's reproductive success will depend on traits that increase access to mates, as well as the number of mates available. In most well-studied mammals, males are the larger sex, and body size often increases success in intra-sexual contests and thus paternity. In comparison, the determinants of male success in species with reversed sexual size dimorphism (RSD) are less well understood. Greater horseshoe bats (Rhinolophus ferrumequinum) exhibit RSD and females appear to exert mate choice when they visit and copulate with males in their underground territories. Here we assessed putative determinants of reproductive success in a colony of greater horseshoe bats during a 19-year period of rapid population growth. We genotyped 1080 bats with up to 40 microsatellite loci and assigned maternity to 99.5% of pups, and paternity to 76.8% of pups. We found that in spite of RSD, paternity success correlated positively with male size, and, consistent with our previous findings, also with age. Female reproductive success, which has not previously been studied in this population, was also age-related and correlated positively with individual heterozygosity, but not with body size. Remarkable male reproductive skew was detected that initially increased steadily with population size, possibly coinciding with the saturation of suitable territories, but then levelled off suggesting an upper limit to a male's number of partners. Our results illustrate that RSD can occur alongside intense male sexual competition, that male breeding success is density-dependent, and that male and female greater horseshoe bats are subject to different selective pressures.
PMCID: PMC3923748  PMID: 24551052
14.  Vinculin Binding Angle in Podosomes Revealed by High Resolution Microscopy 
PLoS ONE  2014;9(2):e88251.
Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.
PMCID: PMC3921150  PMID: 24523880
15.  The importance of invertebrates when considering the impacts of anthropogenic noise 
Anthropogenic noise is now recognized as a major global pollutant. Rapidly burgeoning research has identified impacts on individual behaviour and physiology through to community disruption. To date, however, there has been an almost exclusive focus on vertebrates. Not only does their central role in food webs and in fulfilling ecosystem services make imperative our understanding of how invertebrates are impacted by all aspects of environmental change, but also many of their inherent characteristics provide opportunities to overcome common issues with the current anthropogenic noise literature. Here, we begin by explaining why invertebrates are likely to be affected by anthropogenic noise, briefly reviewing their capacity for hearing and providing evidence that they are capable of evolutionary adaptation and behavioural plasticity in response to natural noise sources. We then discuss the importance of quantifying accurately and fully both auditory ability and noise content, emphasizing considerations of direct relevance to how invertebrates detect sounds. We showcase how studying invertebrates can help with the behavioural bias in the literature, the difficulties in drawing strong, ecologically valid conclusions and the need for studies on fitness impacts. Finally, we suggest avenues of future research using invertebrates that would advance our understanding of the impact of anthropogenic noise.
PMCID: PMC3871318  PMID: 24335986
environmental change; fitness; hearing; insect; noise quantification; pollution
16.  Interleukin-6 Signaling Drives Fibrosis in Unresolved Inflammation 
Immunity  2014;40(1):40-50.
Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.
•Repeated acute resolving inflammation leads to excessive tissue damage•IL-6 regulates profibrotic IFN-γ-secreting T cells•IFN-γ increases detrimental STAT1 signaling in stromal tissue•STAT1 activity alters homeostatic control of extracellular matrix to promote fibrosis
PMCID: PMC3919204  PMID: 24412616
17.  Integrin-Matrix Clusters Form Podosome-like Adhesions in the Absence of Traction Forces 
Cell Reports  2013;5(5):1456-1468.
Matrix-activated integrins can form different adhesion structures. We report that nontransformed fibroblasts develop podosome-like adhesions when spread on fluid Arg-Gly-Asp peptide (RGD)-lipid surfaces, whereas they habitually form focal adhesions on rigid RGD glass surfaces. Similar to classic macrophage podosomes, the podosome-like adhesions are protrusive and characterized by doughnut-shaped RGD rings that surround characteristic core components including F-actin, N-WASP, and Arp2/Arp3. Furthermore, there are 18 podosome markers in these adhesions, though they lack matrix metalloproteinases that characterize invadopodia and podosomes of Src-transformed cells. When nontransformed cells develop force on integrin-RGD clusters by pulling RGD lipids to prefabricated rigid barriers (metal lines spaced by 1–2 μm), these podosomes fail to form and instead form focal adhesions. The formation of podosomes on fluid surfaces is mediated by local activation of phosphoinositide 3-kinase (PI3K) and the production of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) in a FAK/PYK2-dependent manner. Enrichment of PIP3 precedes N-WASP activation and the recruitment of RhoA-GAP ARAP3. We propose that adhesion structures can be modulated by traction force development and that production of PIP3 stimulates podosome formation and subsequent RhoA downregulation in the absence of traction force.
Graphical Abstract
•Nontransformed fibroblasts on RGD membranes form podosome-like protrusions•Nanopatterned RGD membranes enable traction force, suppressing protrusion formation•Local activation of PI3K transforms prepodosomal-like RGD clusters•PIP3-bound RhoA GAP ARAP3 is recruited at the protrusion and downregulates RhoA-GTP
Outside-in integrin activation plays a critical role in cell adhesion and migration. Yu, Sheetz, and colleagues now report that plating cells onto fluid RGD membranes, thus inhibiting the development of traction force, causes the formation of podosome-like protrusions. The podosome-like adhesions on RGD membranes lack matrix metalloproteinases but share many other molecular components with classic podosomes in macrophages. The authors demonstrate that podosome-like protrusion formation is mediated by a PIP3-dependent pathway and is further aided by ARAP3-mediated RhoA inactivation.
PMCID: PMC3898747  PMID: 24290759
18.  Candida nivariensis isolated from a renal transplant patient with persistent candiduria—Molecular identification using ITS PCR and MALDI-TOF☆ 
We report on the isolation of Candida nivariensis from a renal transplant patient with persistent candiduria. Biochemical profiling misidentified isolates as Candida glabrata (3/5) and Candida inconspicua (2/5). All isolates produced white colonies on CHROMagar™ Candida medium. Internal transcribed spacer (ITS) ribosomal gene sequence analysis and MALDI-TOF-MS analysis (Bruker Biotyper™ 2.0) identified all isolates as C. nivariensis, demonstrating the utility of MALDI-TOF as a rapid, accurate approach for the identification of cryptic Candida species.
PMCID: PMC3885950  PMID: 24432244
C. nivariensis; MALDI-TOF; ITS; Candiduria
19.  PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity 
Oncogene  2012;32(16):2114-2120.
Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of over-expression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harboring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the G-loop region of the kinase. Our data suggest E329K mutation leads to a modest increase in kinase activity conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition.
PMCID: PMC3446866  PMID: 22689056
20.  Clinical predictive value of manual muscle strength testing during critical illness: an observational cohort study 
Critical Care  2013;17(5):R229.
Impaired skeletal muscle function has important clinical outcome implications for survivors of critical illness. Previous studies employing volitional manual muscle testing for diagnosing intensive care unit-acquired weakness (ICU-AW) during the early stages of critical illness have only provided limited data on outcome. This study aimed to determine inter-observer agreement and clinical predictive value of the Medical Research Council sum score (MRC-SS) test in critically ill patients.
Study 1: Inter-observer agreement for ICU-AW between two clinicians in critically ill patients within ICU (n = 20) was compared with simulated presentations (n = 20). Study 2: MRC-SS at awakening in an unselected sequential ICU cohort was used to determine the clinical predictive value (n = 94) for outcomes of ICU and hospital mortality and length of stay.
Although the intra-class correlation coefficient (ICC) for MRC-SS in the ICU was 0.94 (95% CI 0.85–0.98), κ statistic for diagnosis of ICU-AW (MRC-SS <48/60) was only 0.60 (95% CI 0.25–0.95). Agreement for simulated weakness presentations was almost complete (ICC 1.0 (95% CI 0.99–1.0), with a κ statistic of 1.0 (95% CI 1.0–1.0)). There was no association observed between ability to perform the MRC-SS and clinical outcome and no association between ICU-AW and mortality. Although ICU-AW demonstrated limited positive predictive value for ICU (54.2%; 95% CI 39.2–68.6) and hospital (66.7%; 95% CI 51.6–79.6) length of stay, the negative predictive value for ICU length of stay was clinically acceptable (88.2%; 95% CI 63.6–98.5).
These data highlight the limited clinical applicability of volitional muscle strength testing in critically ill patients. Alternative non-volitional strategies are required for assessment and monitoring of muscle function in the early stages of critical illness.
PMCID: PMC4057053  PMID: 24112540
21.  Genome-wide association study meta-analysis of chronic widespread pain: evidence for involvement of the 5p15.2 region 
Annals of the rheumatic diseases  2012;72(3):427-436.
Chronic widespread pain (CWP) is a common disorder affecting ~10% of the general population and has an estimated heritability of 48-52%. In the first large-scale genome-wide association study (GWAS) meta-analysis, we aimed to identify common genetic variants associated with CWP.
We conducted a GWAS meta-analysis in 1,308 female CWP cases and 5,791 controls of European descent, and replicated the effects of the genetic variants with suggestive evidence for association in 1,480 CWP cases and 7,989 controls (P<1×10−5). Subsequently, we studied gene expression levels of the nearest genes in two chronic inflammatory pain mouse models, and examined 92 genetic variants previously described associated with pain.
The minor C-allele of rs13361160 on chromosome 5p15.2, located upstream of CCT5 and downstream of FAM173B, was found to be associated with a 30% higher risk of CWP (MAF=43%; OR=1.30, 95%CI=1.19-1.42, P=1.2×10−8). Combined with the replication, we observed a slightly attenuated OR of 1.17 (95%CI=1.10-1.24, P=4.7×10−7) with moderate heterogeneity (I2=28.4%). However, in a sensitivity analysis that only allowed studies with joint-specific pain, the combined association was genome-wide significant (OR=1.23, 95%CI=1.14-1.32, P=3.4×10−8, I2=0%). Expression levels of Cct5 and Fam173b in mice with inflammatory pain were higher in the lumbar spinal cord, not in the lumbar dorsal root ganglions, compared to mice without pain. None of the 92 genetic variants previously described were significantly associated with pain (P>7.7×10−4).
We identified a common genetic variant on chromosome 5p15.2 associated with joint-specific CWP in humans. This work suggests that CCT5 and FAM173B are promising targets in the regulation of pain.
PMCID: PMC3691951  PMID: 22956598
Gene Polymorphism; Fibromyalgia/Pain Syndromes; Epidemiology
22.  WIP Regulates Persistence of Cell Migration and Ruffle Formation in Both Mesenchymal and Amoeboid Modes of Motility 
PLoS ONE  2013;8(8):e70364.
The spatial distribution of signals downstream from receptor tyrosine kinases (RTKs) or G-protein coupled receptors (GPCR) regulates fundamental cellular processes that control cell migration and growth. Both pathways rely significantly on actin cytoskeleton reorganization mediated by nucleation-promoting factors such as the WASP-(Wiskott-Aldrich Syndrome Protein) family. WIP (WASP Interacting Protein) is essential for the formation of a class of polarised actin microdomain, namely dorsal ruffles, downstream of the RTK for PDGF (platelet-derived growth factor) but the underlying mechanism is poorly understood. Using lentivirally-reconstituted WIP-deficient murine fibroblasts we define the requirement for WIP interaction with N-WASP (neural WASP) and Nck for efficient dorsal ruffle formation and of WIP-Nck binding for fibroblast chemotaxis towards PDGF-AA. The formation of both circular dorsal ruffles in PDGF-AA-stimulated primary fibroblasts and lamellipodia in CXCL13-treated B lymphocytes are also compromised by WIP-deficiency. We provide data to show that a WIP-Nck signalling complex interacts with RTK to promote polarised actin remodelling in fibroblasts and provide the first evidence for WIP involvement in the control of migratory persistence in both mesenchymal (fibroblast) and amoeboid (B lymphocytes) motility.
PMCID: PMC3737202  PMID: 23950925
23.  Age-Related Increase in Electromyography Burst Activity in Males and Females 
Journal of Aging Research  2013;2013:720246.
The rapid advancement of electromyography (EMG) technology facilitates measurement of muscle activity outside the laboratory during daily life. The purpose of this study was to determine whether bursts in EMG recorded over a typical 8-hour day differed between young and old males and females. Muscle activity was recorded from biceps brachii, triceps brachii, vastus lateralis, and biceps femoris of 16 young and 15 old adults using portable surface EMG. Old muscles were active 16–27% of the time compared to 5–9% in young muscles. The number of bursts was greater in old than young adults and in females compared to males. Burst percentage and mean amplitude were greater in the flexor muscles compared with the extensor muscles. The greater burst activity in old adults coupled with the unique activity patterns across muscles in males and females provides further understanding of how changes in neuromuscular activity effects age-related functional decline between the sexes.
PMCID: PMC3747463  PMID: 23984069
24.  Independent Losses of Visual Perception Genes Gja10 and Rbp3 in Echolocating Bats (Order: Chiroptera) 
PLoS ONE  2013;8(7):e68867.
A trade-off between the sensory modalities of vision and hearing is likely to have occurred in echolocating bats as the sophisticated mechanism of laryngeal echolocation requires considerable neural processing and has reduced the reliance of echolocating bats on vision for perceiving the environment. If such a trade-off exists, it is reasonable to hypothesize that some genes involved in visual function may have undergone relaxed selection or even functional loss in echolocating bats. The Gap junction protein, alpha 10 (Gja10, encoded by Gja10 gene) is expressed abundantly in mammal retinal horizontal cells and plays an important role in horizontal cell coupling. The interphotoreceptor retinoid-binding protein (Irbp, encoded by the Rbp3 gene) is mainly expressed in interphotoreceptor matrix and is known to be critical for normal functioning of the visual cycle. We sequenced Gja10 and Rbp3 genes in a taxonomically wide range of bats with divergent auditory characteristics (35 and 18 species for Gja10 and Rbp3, respectively). Both genes have became pseudogenes in species from the families Hipposideridae and Rhinolophidae that emit constant frequency echolocation calls with Doppler shift compensation at high-duty-cycles (the most sophisticated form of biosonar known), and in some bat species that emit echolocation calls at low-duty-cycles. Our study thus provides further evidence for the hypothesis that a trade-off occurs at the genetic level between vision and echolocation in bats.
PMCID: PMC3715546  PMID: 23874796
25.  From the ultrasonic to the infrared: molecular evolution and the sensory biology of bats 
Great advances have been made recently in understanding the genetic basis of the sensory biology of bats. Research has focused on the molecular evolution of candidate sensory genes, genes with known functions [e.g., olfactory receptor (OR) genes] and genes identified from mutations associated with sensory deficits (e.g., blindness and deafness). For example, the FoxP2 gene, underpinning vocal behavior and sensorimotor coordination, has undergone diversification in bats, while several genes associated with audition show parallel amino acid substitutions in unrelated lineages of echolocating bats and, in some cases, in echolocating dolphins, representing a classic case of convergent molecular evolution. Vision genes encoding the photopigments rhodopsin and the long-wave sensitive opsin are functional in bats, while that encoding the short-wave sensitive opsin has lost functionality in rhinolophoid bats using high-duty cycle laryngeal echolocation, suggesting a sensory trade-off between investment in vision and echolocation. In terms of olfaction, bats appear to have a distinctive OR repertoire compared with other mammals, and a gene involved in signal transduction in the vomeronasal system has become non-functional in most bat species. Bitter taste receptors appear to have undergone a “birth-and death” evolution involving extensive gene duplication and loss, unlike genes coding for sweet and umami tastes that show conservation across most lineages but loss in vampire bats. Common vampire bats have also undergone adaptations for thermoperception, via alternative splicing resulting in the evolution of a novel heat-sensitive channel. The future for understanding the molecular basis of sensory biology is promising, with great potential for comparative genomic analyses, studies on gene regulation and expression, exploration of the role of alternative splicing in the generation of proteomic diversity, and linking genetic mechanisms to behavioral consequences.
PMCID: PMC3667242  PMID: 23755015
echolocation; hearing; vision; olfaction; taste; perception

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