Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Energy and energy flux in axisymmetric slow and fast waves

Aims: We aim to calculate the kinetic, magnetic, thermal, and total energy densities and the flux of energy in axisymmetric sausage modes. The resulting equations should contain as few parameters as possible to facilitate applicability for different observations. 

Methods: The background equilibrium is a one-dimensional cylindrical flux tube model with a piecewise constant radial density profile. This enables us to use linearised magnetohydrodynamic equations to calculate the energy densities and the flux of energy for axisymmetric sausage modes. 

Results: The equations used to calculate the energy densities and the flux of energy in axisymmetric sausage modes depend on the radius of the flux tube, the equilibrium sound and Alfvén speeds, the density of the plasma, the period and phase speed of the wave, and the radial or longitudinal components of the Lagrangian displacement at the flux tube boundary. Approximate relations for limiting cases of propagating slow and fast sausage modes are also obtained. We also obtained the dispersive first-order correction term to the phase speed for both the fundamental slow body mode under coronal conditions and the slow surface mode under photospheric conditions.

The impact toughness of a nitride-strengthened martensitic heat resistant steel

The nitride-strengthened martensitic heat resistant steel is precipitation strengthened only by nitrides. In the present work, the effect of nitride precipitation behavior on the impact toughness of an experimental steel was investigated. Nitrides could hardly be observed when the steel was tempered at 650°C. When the tempering temperature was increased to 700°C and 750°C, a large amount of nitrides were observed in the matrix. It was surprising to reveal that the impact energy of the half-size samples greatly increased from several Joules to nearly a hundred Joules. The ductile-brittle transition temperature (DBTT) was also discovered to decrease from room temperature to −50°C when the tempering temperature was increased from 650°C to 750°C. The nitride precipitation with increasing tempering temperature was revealed to be responsible for the improved impact toughness.

Investigation of chip formation and fracture toughness in orthogonal cutting of UD-CFRP

Features of chip formation can inform the mechanism of a machining process. In this paper, a series of orthogonal cutting experiments were carried out on unidirectional carbon fiber reinforced polymer (UD-CFRP) under cutting speed of 0.5 m/min. The specially designed orthogonal cutting tools and high-speed camera were used in this paper. Two main factors are found to influence the chip morphology, namely the depth of cut (DOC) and the fiber orientation (angle 휃), and the latter of which plays a more dominant role. Based on the investigation of chip formation, a new approach is proposed for predicting fracture toughness of the newly machined surface and the total energy consumption during CFRP orthogonal cutting is introduced as a function of the surface energy of machined surface, the energy consumed to overcome friction, and the energy for chip fracture. The results show that the proportion of energy spent on tool-chip friction is the greatest, and the proportions of energy spent on creating new surface decrease with the increasing of fiber angle.

A selective antagonist of histamine H₄ receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs:involvement of lipocortin-1

BACKGROUND AND PURPOSE: Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H4 receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

EXPERIMENTAL APPROACH: Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg.kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD2 , LTB4 and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

KEY RESULTS: OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD2 , LTB4 and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD2 , LTB4 and TNF-α in BAL fluid.

CONCLUSIONS AND IMPLICATIONS: Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Performance and Fault Tolerance of Preconditioned Iterative Solvers on Low-Power ARM Architectures

As the complexity of computing systems grows, reliability and energy are two crucial challenges asking for holistic solutions. In this paper, we investigate the interplay among concurrency, power dissipation, energy consumption and voltage-frequency scaling for a key numerical kernel for the solution of sparse linear systems. Concretely, we leverage a task-parallel implementation of the Conjugate Gradient method, equipped with an state-of-the-art pre-conditioner embedded in the ILUPACK software, and target a low-power multi core processor from ARM.In addition, we perform a theoretical analysis on the impact of a technique like Near Threshold Voltage Computing (NTVC) from the points of view of increased hardware concurrency and error rate.

Effects of scalding and dehairing of pig carcasses at abattoirs on the visibility of welfare-related lesions

There is increasing interest in developing abattoir-based welfare measures for pigs.The primary aim of this study was to determine the most appropriate place on theslaughter line to conduct assessments of welfare-related lesions, namely apparentaggression-related skin lesions (hereafter referred to as 'skin lesions'), loin bruising andapparent tail biting damage. The study also lent itself to an assessment of theprevalence of these lesions, and the extent to which they were linked with productionparameters. Finishing pigs processed at two abattoirs on the Island of Ireland (n =1950 in abattoir A, and n = 1939 in abattoir B) were used. Data were collected over 6days in each abattoir in July 2014. Lesion scoring took place at two points on theslaughter line: (1) at exsanguination (Slaughter Stage 1 [SS1]), and (2) followingscalding and dehairing of carcasses (Slaughter Stage 2 [SS2]). At both points, eachcarcass was assigned a skin and tail lesion score ranging from 0 (lesion absent) to 3 or4 (severe lesions), respectively. Loin bruising was recorded as present or absent.Differences in the percentage of pigs with observable lesions of each type werecompared between SS1 and SS2 using McNemar/McNemar-Bowker tests. Theassociations between each lesion type, and both cold carcass weight andcondemnations, were examined at batch level using Pearson's correlations. Batch wasdefined as the group of animals with a particular farm identification code on a givenday. The overall percentage of pigs with a visible skin lesion (i.e. score > 0) decreasedbetween SS1 and SS2 (P<0.001). However, the percentage of pigs with a severe skinlesion increased numerically from SS1 to SS2. The percentage of pigs with a visible taillesion and with loin bruising also increased between SS1 and SS2 (P<0.001). Therewas a positive correlation between the percentage of carcasses that were partiallycondemned, and the percentage of pigs with skin lesions, tail lesions and loin bruising(P<0.05). Additionally, as the batch-level frequency of each lesion type increased,average cold carcass weight decreased (P<0.001). These findings suggest that severeskin lesions, tail lesions and loin bruising are more visible on pig carcasses after theyhave been scalded and dehaired, and that this is when abattoir-based lesion scoringshould take place. The high prevalence of all three lesion types, and the links witheconomically important production parameters, suggests more research into identifying key risk factors is warranted.

The dual EGFR/HER-2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN-38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER-2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN-38, the active metabolite of irinotecan (CPT-11), in colon and gastric cancer cell lines. Concentration-dependent antiproliferative effects of both lapatinib and SN-38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08-11.7 muM; SN-38 range 3.6-256 nM). Lapatinib potently inhibited the growth of a HER-2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER-2 expression. The combination of lapatinib and SN-38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN-38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN-38 when compared to SN-38 treatment alone. Finally, the combination of lapatinib and CPT-11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT-11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas.

HDAC1 and HDAC2 independently predict mortality in hepatocellular carcinoma by a competing risk regression model in a Southeast Asian population

Histone deacetylases (HDACs) are enzymes involved in transcriptional repression. We aimed to examine the significance of HDAC1 and HDAC2 gene expression in the prediction of recurrence and survival in 156 patients with hepatocellular carcinoma (HCC) among a South East Asian population who underwent curative surgical resection in Singapore. We found that HDAC1 and HDAC2 were upregulated in the majority of HCC tissues. The presence of HDAC1 in tumor tissues was correlated with poor tumor differentiation. Notably, HDAC1 expression in adjacent non-tumor hepatic tissues was correlated with the presence of satellite nodules and multiple lesions, suggesting that HDAC1 upregulation within the field of HCC may contribute to tumor spread. Using competing risk regression analysis, we found that increased cancer-specific mortality was significantly associated with HDAC2 expression. Mortality was also increased with high HDAC1 expression. In the liver cancer cell lines, HEP3B, HEPG2, PLC5, and a colorectal cancer cell line, HCT116, the combined knockdown of HDAC1 and HDAC2 increased cell death and reduced cell proliferation as well as colony formation. In contrast, knockdown of either HDAC1 or HDAC2 alone had minimal effects on cell death and proliferation. Taken together, our study suggests that both HDAC1 and HDAC2 exert pro-survival effects in HCC cells, and the combination of isoform-specific HDAC inhibitors against both HDACs may be effective in targeting HCC to reduce mortality.

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.

TH2 and TH17 inflammatory pathways are reciprocally regulated in asthma

Increasing evidence suggests that asthma is a heterogeneous disorder regulated by distinct molecular mechanisms. In a cross-sectional study of asthmatics of varying severity (n = 51), endobronchial tissue gene expression analysis revealed three major patient clusters: TH2-high, TH17-high, and TH2/17-low. TH2-high and TH17-high patterns were mutually exclusive in individual patient samples, and their gene signatures were inversely correlated and differentially regulated by interleukin-13 (IL-13) and IL-17A. To understand this dichotomous pattern of T helper 2 (TH2) and TH17 signatures, we investigated the potential of type 2 cytokine suppression in promoting TH17 responses in a preclinical model of allergen-induced asthma. Neutralization of IL-4 and/or IL-13 resulted in increased TH17 cells and neutrophilic inflammation in the lung. However, neutralization of IL-13 and IL-17 protected mice from eosinophilia, mucus hyperplasia, and airway hyperreactivity and abolished the neutrophilic inflammation, suggesting that combination therapies targeting both pathways may maximize therapeutic efficacy across a patient population comprising both TH2 and TH17 endotypes.

Availability and transfer to grain of As, Cd, Cu, Ni, Pb and Zn in a barley agri-system: Impact of biochar, organic and mineral fertilizers

With biochar becoming an emerging soil amendment and a tool to mitigate climate change, there are only a few studies documenting its effects on trace element cycling in agriculture. Zn and Cu are deficient in many human diets, whilst exposures to As, Pb and Cd need to be decreased. Biochar has been shown to affect many of them mainly at a bench or greenhouse scale, but field research is not available. In our experiment we studied the impact of biochar, as well as its interactions with organic (compost and sewage sludge) and mineral fertilisers (NPK and nitrosulfate), on trace element mobility in a Mediterranean agricultural field (east of Madrid, Spain) cropped with barley. At harvesting time, we analysed the soluble fraction, the available fraction (assessed with the diffusive gradients in thin gels technique, DGT) and the concentration of trace elements in barley grain. No treatment was able to significantly increase Zn, Cu or Ni concentration in barley grain, limiting the application for cereal fortification. Biochar helped to reduce Cd and Pb in grain, whereas As concentration slightly increased. Overall biochar amendments demonstrated a potential to decrease Cd uptake in cereals, a substantial pathway of exposure in the Spanish population, whereas mineral fertilisation and sewage sludge increased grain Cd and Pb. In the soil, biochar helped to stabilise Pb and Cd, while marginally increasing As release/mobilisation. Some of the fertilisation practises or treatments increased toxic metals and As solubility in soil, but never to an extent high enough to be considered an environmental risk. Future research may try to fortify Zn, Cu and Ni using other combinations of organic amendments and different parent biomass to produce enriched biochars.

Address inaccuracy in health registration data: determinants and impacts

Access to demographic data that are complete, accurate and up-to-date is fundamental to many aspects of public health, government and academic work and for accurate interpretation of other databases. Health registration data are the prime source of demographic information for health and social care systems; for example, as an indicator of need, as a source of denominators to convert number of events into rates, or in the case of the residential address information as the basis for generating the call-recall invitation letters that are used for most screening programs (e.g. breast, colo-rectal and AAA screening). However, list inflation (ghosts, duplicates or emigrants) and a degree of address inaccuracy are recognised caveats with the health registration data and a recent NILS-related study on breast screening suggests that improved address accuracy might be a fast and efficient means of increasing screening uptake rates in cities and amongst deprived populations. In NI these data are collated by the BSO who uniquely in the UK also have access to data relating to prescribing, dental registrations and use of A&E services. These can be used to supplement the standard demographic and address information by (i) indicating patients who are alive and resident in NI and (ii) providing an independent source of probably improved address information. This study will use the NI Unique Property Reference Number (UPRN), rather than the addresses per se which are difficult to work with, to compare the addresses registered in the BSO with those addresses in the enumerated 2011 census. Assuming that the census is a more accurate source of address information for individuals, a comparison of the health registration addresses with those recorded at the census, the aim of the proposed study will be to (i) characterise the amount and distributions of these differences, (ii) to see what proportion of those who do not attend for screening did not actually receive an invitation letter because the addresses were incorrect, (iii) to determine how much of the social gradient (and urban/rural differences) in screening uptake are due to address inaccuracies, (iv) a comparison of timing of address changes at the BSO will provide information on the delays in updating of addresses.

Effects of Poly (ε-caprolactone) Coating on the Properties of Three-Dimensional Printed Porous Structures

Powder-based inkjet three-dimensional printing (3DP) to fabricate pre-designed 3D structures has drawn increasing attention. However there are intrinsic limitations associated with 3DP technology due to the weak bonding within the printed structure, which significantly compromises its mechanical integrity. In this study, calcium sulphate ceramic structures demonstrating a porous architecture were manufactured using 3DP technology and subsequently post-processed with a poly (ε-caprolactone) (PCL) coating. PCL concentration, immersion time, and number of coating layers were the principal parameters investigated and improvement in compressive properties was the measure of success. Interparticle spacing within the 3DP structures were successfully filled with PCL material. Consequently the compressive properties, wettability, morphology, and in vitro resorption behaviour of 3DP components were significantly augmented. The average compressive strength, Young’s modulus, and toughness increased 217%, 250%, and 315%, following PCL coating. Addition of a PCL surface coating provided long-term structural support to the host ceramic material, extending the resorption period from less than 7 days to a minimum of 56 days. This study has demonstrated that application of a PCL coating onto a ceramic 3DP structure was a highly effective approach to addressing some of the limitations of 3DP manufacturing and allows this advanced technology to be potentially used in a wider range of applications.

Natural substances and high pressure processing to preserve sea bass

Esta tese teve como objectivo estudar estratégias de conservação de pescado fresco, recorrendo ao uso de extractos e óleos essenciais de plantas e do processamento por alta pressão (HPP), usando filetes de robalo como um caso de estudo modelo. Relativamente aos extractos e óleos essenciais, avaliaram-se as suas propriedades antibacterianas e antioxidantes. Os extractos aquosos quente de poejo e de orégão e o óleo essencial de cravinho apresentaram a maior actividade antioxidante. Os óleos essenciais foram mais eficientes do que os extractos para inibir o crescimento das estirpes bacterianas testadas, tendo-se observado os menores valores de concentração mínima inibitória nos óleos essenciais de orégão, citronela, alho e orégão Espanhol. De seguida, estudou-se o efeito dos óleos essenciais de orégão Espanhol e de limão na conservação de filetes de robalo fresco tendo em conta critérios microbiológicos, químicos, físicos e sensoriais. A aplicação do óleo essencial de orégão Espanhol aumentou o tempo de vida útil dos filetes sob o ponto de vista bacteriano, mas não em termos sensoriais. A combinação dos óleos essenciais de orégão Espanhol e de limão melhorou o efeito antioxidante e reduziu a intensidade do odor e a sua eficácia em relação às Enterobacteriaceae, comparando com o tratamento com óleo essencial de orégão Espanhol per se. No sentido de reduzir o odor conferido pelos óleos essenciais realizou-se um estudo de conservação para avaliar o efeito de películas com óleos essenciais (citronela, alho e tomilho) em filetes de robalo, recorrendo a uma teste de desafio bacteriano. As películas sem óleos essenciais aumentaram o tempo de vida útil sob o ponto de vista bacteriano, mas este efeito não foi observado com a incorporação dos óleos essenciais nas películas. Em relação ao HPP, testaram-se diversas condições (nível de pressão, tempo de pressurização e taxa de pressurização) e avaliaram-se os efeitos na actividade enzimática, na qualidade global e na conservação de filetes de robalo fresco. Em geral, o aumento do nível de pressão e do tempo de pressurização diminuiu a actividade da fosfatase ácida e das enzimas proteolíticas, a carga bacteriana e a capacidade de retenção de água, enquanto que os filetes ficaram mais brancos. O HPP revelou potencial para o desenvolvimento de novos produtos: mais brancos, não translúcidos, mais firmes e com maior tempo de frescura e estabilidade microbiológica. Em conclusão, os óleos essenciais e o HPP têm potencial para conservar pescado fresco, devido aos seus efeitos na qualidade bacteriana. Ainda assim, mais esforços devem ser feitos no sentido de reduzir a transferência de odor dos óleos essenciais para o pescado e os efeitos do HPP no aspecto do pescado e na oxidação lipídica.