Early systemic sclerosis: marker autoantibodies and videocapillaroscopy patterns are each associated with distinct clinical, functional and cellular activation markers

Gabriele Valentini1*, Antonella Marcoccia2, Giovanna Cuomo1, Serena Vettori1, Michele Iudici1,Francesco Bondanini3, Carlo Santoriello, Aldo Ciani5, Domenico Cozzolino6, Giovanni Maria De Matteis7,Salvatore Cappabianca8, Filiberto Vitelli9 and Alberto Spanò

Abstract
Introduction: Early systemic sclerosis (SSc) is characterized by Raynaud’s phenomenon together with scleroderma marker autoantibodies and/or a scleroderma pattern at capillaroscopy and no other distinctive feature of SSc. Patients presenting with marker autoantibodies plus a capillaroscopic scleroderma pattern seem to evolve into definite SSc more frequently than patients with either feature. Whether early SSc patients with only marker autoantibodies or capillaroscopic positivity differ in any aspect at presentation is unclear.

Methods: Seventy-one consecutive early SSc patients were investigated for preclinical cardiopulmonary alterations. Out of these, 44 patients and 25 controls affected by osteoarthritis or primary fibromyalgia syndrome were also investigated for serum markers of fibroblast (carboxyterminal propeptide of collagen I), endothelial (soluble E-selectin) and T-cell (soluble IL-2 receptor alpha) activation.

Results: Thirty-two of the 71 patients (45.1%) had both a marker autoantibody and a capillaroscopic scleroderma pattern (subset 1), 16 patients (22.5%) had only a marker autoantibody (subset 2), and 23 patients (32.4%) had only a capillaroscopic scleroderma pattern (subset 3). Patients with marker autoantibodies (n = 48, 67.6%) had a higher prevalence of impaired diffusing lung capacity for carbon monoxide (P = 0.0217) and increased serum levels of carboxyterminal propeptide of collagen I (P = 0.0037), regardless of capillaroscopic alterations. Patients with a capillaroscopic scleroderma pattern (n = 55, 77.5%) had a higher prevalence of puffy fingers (P = 0.0001) and increased serum levels of soluble E-selectin (P = 0.0003) regardless of marker autoantibodies.

Conclusion: These results suggest that the autoantibody and microvascular patterns in early SSc may each be related to different clinical-preclinical features and circulating activation markers at presentation. Longitudinal studies are warranted to investigate whether these subsets undergo a different disease course over time.

Keywords: Raynaud’s phenomenon, early systemic sclerosis, systemic sclerosis marker autoantibodies, nailfold videocapillaroscopy, preclinical organ involvement, puffy fingerscirculating activation markers, carboxyterminal propeptide of collagen I, soluble E-selectin, soluble IL-2 receptor alpha

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Treatment of Muckle-Wells syndrome: analysis of two IL-1-blocking regimens

Jasmin B Kuemmerle-Deschner1*†, Helmut Wittkowski2†, Pascal N Tyrrell3, Ina Koetter4, Peter Lohse5,Katharina Ummenhofer1, Fabian Reess1, Sandra Hansmann1, Assen Koitschev6, Christoph Deuter7, Anja Bialkowski1,Dirk Foell8† and Susanne M Benseler

Abstract
Objectives: Muckle-Wells syndrome (MWS) is an autoinflammatory disease characterized by excessive interleukin-1 (IL-1) release, resulting in recurrent fevers, sensorineural hearing loss, and amyloidosis. IL-1 inhibition with anakinra, an IL-1 receptor antagonist, improves clinical symptoms and inflammatory markers. Subclinical disease activity is commonly observed. Canakinumab, a fully human IgG1 anti-IL-1b monoclonal antibody, can abolish excess IL-1b. The study aim was to analyze the efficacy and safety of these two anti-IL-1 therapies.

Methods: Two cohorts of patients with severe MWS and confirmed NLRP3 mutation were treated with anakinra and/or canakinumab. Clinical and laboratory features including ESR, CRP, SAA, and the neutrophil marker S100A12 were determined serially. Disease activity was captured by MWS disease activity scores (MWS-DAS). Remission was defined as MWS-DAS ≤5 plus normal CRP and SAA. Treatment efficacy and safety were analyzed.

Results: The study included 12 anakinra- and 14 canakinumab-treated patients; the median age was 33.5 years (3.0 years to 72.0 years); 57% were female patients. Both treatment regimens led to a significant reduction of clinical disease activity and inflammatory markers. At last follow-up, 75% of anakinra-treated and 93% of canakinumabtreated patients achieved remission. During follow-up, S100A12 levels mirrored recurrence of disease activity. Both treatment regimens had favorable safety profiles.

Conclusions: IL-1 blockade is an effective and safe treatment in MWS patients. MWS-DAS in combination with MWS inflammatory markers provides an excellent monitoring tool set. Canakinumab led to a sustained control of disease activity even after secondary failure of anakinra therapy. S100A12 may be a sensitive marker to detect subclinical disease activity.

Keywords: NLRP3, CIAS1, mutation, Muckle-Wells syndrome, autoinflammatory fever syndromes, interleukin-1 inhibition, anakinra, canakinumab, S100A12

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A genetic role for macrophage migration inhibitory factor (MIF) in adult-onset Still’s disease

Fang-Fang Wang1, Xin-Fang Huang1, Nan Shen1, Lin Leng2, Richard Bucala2, Shun-Le Chen1 and Liang-Jing Lu\

Abstract
Introduction: Adult-onset still’s disease (AOSD) is a rare systemic inflammatory disorder in which abnormalities in inflammatory cytokines production appear to play a pathophysiological role. Our previous work has reported increased expression of macrophage migration inhibitory factor (MIF) and revealed its correlation with disease severity and activity in AOSD. A -173 G/C single nucleotide polymorphism (SNP) (rs755622) and a -794 CATT5-8 repeat (rs5844572) in the MIF promoter have been reported. In this study, we sought to explore the relationship between functional MIF promoter polymorphisms and MIF expression in AOSD.

Methods: 100 patients and 200 controls were recruited in the study. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was utilized to analyze the -173 G/C SNP (rs755622) and PCR-based size discrimination assay was applied to detect the -794 CATT5-8 repeat (rs5844572) in the MIF promoter. Plasma MIF levels were measured by ELISA. MIF mRNA levels were quantified by real-time reverse transcription (RT)-PCR. Bisulfate
genomic sequencing was employed to evaluate DNA methylation status within the MIF promoter.

Results: We identified that the frequencies of MIF -794 CATT5 (P = 0.001) allele and the expression of MIF (P <0.001) were increased in patients compared to healthy controls. Plasma levels of MIF in patients with CC genotype were higher than those of patients with GC or GG genotypes (P = 0.05). In patients with established AOSD, a higher frequency of -794 CATT7 containing MIF genotypes was observed in those with liver dysfunction (P = 0.009). Haplotype analysis revealed a higher representation of the MIF haplotype defined by -173*C/-794 CATT5 (C5) in AOSD patients (P = 0.001).

Conclusion: Functional promoter polymorphisms in the MIF gene influence plasma MIF levels in AOSD and may contribute to disease susceptibility or clinical presentation of AOSD.

Keywords: macrophage migration inhibitory factor, adult onset still’s disease, gene polymorphism, gene expression, DNA methylation

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Porphyromonas gingivalis and the pathogenesis of rheumatoid arthritis: analysis of various compartments including the synovial tissue

Michele Ciro Totaro1 , Paola Cattani2, Francesco Ria3 , Barbara Tolusso1, Elisa Gremese1, Anna Laura Fedele1 ,Sara D’Onghia2, Simona Marchetti2, Gabriele DiSante3, Silvia Canestri1 and Gianfranco Ferraccioli1*

Abstract
Introduction: We evaluated the presence of Porphyromonas gingivalis (Pg) DNA in the synovial tissue through synovial biopsy and in other compartments of rheumatoid arthritis (RA) patients in comparison with patients affected by other arthritides. Possible links with clinical, immunologic and genetic features were assessed.

Methods: Peripheral blood (PB), sub-gingival dental plaque, synovial fluid (SF) and synovial tissue samples were collected from 69 patients with active knee arthritis (32 with RA and 37 with other arthritides, of which 14 had undifferentiated peripheral inflammatory arthritis - UPIA). Demographic, clinical, laboratory and immunological data were recorded. The presence of Pg DNA was evaluated through PCR. The HLA-DR haplotype was assessed for 45 patients with RA and UPIA.

Results: No differences arose in the positivity for Pg DNA in the sub-gingival plaque, PB and SF samples between RA and the cohort of other arthritides. Full PB samples showed a higher positivity for Pg DNA than plasma samples (11.8% vs. 1.5%, P = 0.04). Patients with RA showed a higher positivity for Pg DNA in the synovial tissue compared to controls (33.3% vs. 5.9%, P <0.01). UPIA and RA patients carrying the HLA DRB1*04 allele showed a higher positivity for Pg DNA in the synovial tissue compared to patients negative for the allele (57.1% vs. 16.7%, P = 0.04). RA patients positive for Pg DNA in the sub-gingival plaque had a lower disease duration and a higher peripheral blood leucocyte and neutrophil count. The presence of Pg DNA did not influence disease activity, disease disability
or positivity for autoantibodies.

Conclusions: The presence of Pg DNA in the synovial tissue of RA patients suggests a pathogenic role of the bacterium. The higher positivity of Pg DNA in full peripheral blood and synovial tissue samples compared to plasma and synovial fluid suggests a possible intracellular localization of Pg, in particular in patients positive for HLA-DR4.

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Long-term outcome of patients with active ankylosing spondylitis with etanercept-sustained efficacy and safety after seven years

Xenofon Baraliakos1*, Hildrun Haibel2, Claudia Fritz3, Joachim Listing3, Frank Heldmann1, Juergen Braun1 and Joachim Siepe

Abstract
Introduction: Data from clinical studies on the long-term efficacy and safety of anti-tumor necrosHEREis factor (TNF)-a therapy in patients with ankylosing spondylitis (AS) are scarce. This is the first report on continuous treatment with the TNFa fusion protein etanercept over seven years (y).

Methods: Overall, 26 patients with active AS were initially treated with etanercept 2 × 25 mg s.c./week with no concomitant disease modifying anti-rheumatic drugs (DMARDs) or steroids. The clinical response was assessed by standardized parameters. The primary outcome was the proportion of patients in the Spondyloarthritis International Society (ASAS) partial remission at seven years. AS disease activity scores (ASDAS) for status and improvement were
compared to conventional outcome measures.

Results: Overall, 21/26 patients (81%) completed two years of treatment and 16/26 patients (62%) completed seven years. In the completer analysis, 31% patients were in ASAS partial remission at seven years, while 44% patients showed an ASDAS inactive disease status. Mean Bath AS activity index (BASDAI) scores, which were elevated at baseline (6.3 ± 0.9), showed constant improvement and remained low: 3.1 ± 2.5 at two years and 2.5 ± 2.2 at seven years, while ASDAS also improved (3.9 ± 0.7 at baseline, 1.8 ± 0.9 at two years, 1.6 ± 0.8 at seven years), all P <0.001. From the 10 dropouts, only 5 patients discontinued treatment due to adverse events. Patients who completed the study had lower baseline Bath AS function index (BASFI) scores vs. patients who discontinued No other clinical parameter at baseline could predict any long-term outcome.

Conclusions: This study confirms the clinical efficacy and safety of etanercept in patients with active AS over seven years of continuous treatment. After seven years, more than half of the initially treated patients remained on anti-TNF therapy, and one-third were in partial remission.

Trial Registration: ClinicalTrials.gov: NCT01289743

Keywords: ankylosing spondylitis, TNFa, etanercept, ASDAS, BASDAI

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Clinical correlates of a subset of anti-CENP-A antibodies cross-reacting with FOXE3p53-62 in systemic sclerosis

Federico Perosa1*, Elvira Favoino1, Giovanna Cuomo2, Liboria Digiglio1, Franco Dammacco3, Marcella Prete3,Gabriele Valentini2 and Vito Racanelli

 

Abstract
Introduction: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the two immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17.

Methods: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc
sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity.

Results: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI ≥3 (Fisher exact test, P = 0.045) or less restrictive DAI≥2.5 (P = 0.009).

Conclusions: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lc SSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance.

Keywords: Systemic sclerosis, CENP-A, peptide, FOXE-3, disease activity index

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Modulation of monosodium urate crystal-induced responses in neutrophils by the myeloid inhibitory C-type lectin-like receptor: potential therapeutic implications

Valérie Gagné1†, Louis Marois1†, Jean-Michel Levesque1, Hugo Galarneau1, Mireille H Lahoud2, Irina Caminschi2,Paul H Naccache1, Philippe Tessier1 and Maria JG Fernandes

Abstract
Introduction: Monosodium urate crystals (MSU), the etiological agent of gout, are one of the most potent proinflammatory stimuli for neutrophils. The modulation of MSU-induced neutrophil activation by inhibitory receptors remains poorly characterized. The expression of the myeloid inhibitory C-type lectin-like receptor (MICL) in neutrophils is downregulated by several proinflammatory stimuli, suggestive of a role for this receptor in neutrophil function. We thus investigated the potential role of MICL in MSU-induced neutrophil activation.

Methods: The expression of MICL was monitored in human neutrophils by flow cytometry and Western blot analysis after stimulation with MSU. Protein tyrosine phosphorylation was also assessed by Western blot analysis and the production of IL-1 and IL-8 by enzyme-linked immunosorbent assay. Changes in the concentration of cytoplasmic free calcium were monitored with the Fura-2-acetoxymethyl ester calcium indicator. MICL expression was modulated with an anti-MICL antibody in neutrophils and siRNA in the PLB-985 neutrophil-like cell line.

Results: MSU induced the downregulation of MICL expression in neutrophils. A diminution in the expression of MICL induced by antibody cross-linking or siRNA enhanced the MSU-dependent increase in cytoplasmic calcium levels, protein tyrosine phosphorylation and IL-8 but not IL-1 production. Pretreatment of neutrophils with colchicine inhibited the MSU-induced downregulation of MICL expression.

Conclusions: Our findings strongly suggest that MICL acts as an inhibitory receptor in human neutrophils since the downregulation of MICL expression enhances MSU-induced neutrophil activation. Since MSU downregulates the expression of MICL, MICL may play a pathogenic role in gout by enhancing neutrophil effector functions. In support of this notion, colchicine counteracts the MSU-induced loss of MICL expression. Our findings thus also provide further insight into the potential molecular mechanisms behind the anti-inflammatory properties of this drug.

Keywords: Cytokine production, out, Immunoreceptor tyrosine-based inhibitory motif, Monosodium urate crystals,Neutrophil, Signaling

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Comprehensive behavioral characterization of an APP/PS-1 double knock-in mouse model of Alzheimer’s disease

Scott J Webster , Adam D Bachstetter and Linda J Van Eldik

Abstract
Introduction: Despite the extensive mechanistic and pathological characterization of the amyloid precursor protein (APP)/presenilin-1 (PS-1) knock-in mouse model of Alzheimer’s disease (AD), very little is known about the ADrelevant behavioral deficits in this model. Characterization of the baseline behavioral performance in a variety of functional tasks and identification of the temporal onset of behavioral impairments are important to provide a foundation for future preclinical testing of AD therapeutics. Here we perform a comprehensive behavioral characterization of this model, discuss how the observed behavior correlates with the mechanistic and pathological observations of others, and compare this model with other commonly used AD mouse models.

Methods: Four different groups of mice ranging across the lifespan of this model (test groups: 7, 11, 15, and 24 months old) were run in a behavioral test battery consisting of tasks to assess motor function (grip strength, rotor rod, beam walk, open field ambulatory movement), anxiety-related behavior (open field time spent in peripheral zone vs. center zone, elevated plus maze), and cognitive function (novel object recognition, radial armwater maze).

Results: There were no differences in motor function or anxiety-related behavior between APP/PS-1 knock-in mice and wild-type counterpart mice for any age group. Cognitive deficits in both recognition memory (novel object recognition) and spatial reference memory (radial arm water maze) became apparent for the knock-in animals as the disease progressed.

Conclusion: This is the first reported comprehensive behavioral analysis of the APP/PS1 knock-in mouse model of AD. The lack of motor/coordination deficits or abnormal anxiety levels, coupled with the age/disease-related cognitive decline and high physiological relevance of this model, make it well suited for utilization in preclinical testing of AD-relevant therapeutics.

Keywords: Alzheimer’s disease, amyloid precursor protein/presenilin-1, motor behavior, anxiety behavior, cognition,learning and memory, spatial reference memory, recognition memory, transgenic mouse model

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Exploring the potential of the platelet membrane proteome as a source of peripheral biomarkers for Alzheimer’s disease

Laura E Donovan1†, Eric B Dammer2†, Duc M Duong3, John J Hanfelt4, Allan I Levey1, Nicholas T Seyfried1,3* and James J Lah

 

Abstract
Introduction: Peripheral biomarkers to diagnose Alzheimer’s disease (AD) have not been established. Given parallels between neuron and platelet biology, we hypothesized platelet membrane-associated protein changes may differentiate patients clinically defined with probable AD from noncognitive impaired controls.
Methods: Purified platelets, confirmed by flow cytometry were obtained from individuals before fractionation by ultracentrifugation. Following a comparison of individual membrane fractions by SDS-PAGE for general proteome uniformity, equal protein weight from the membrane fractions for five representative samples from AD and five samples from controls were pooled. AD and control protein pools were further divided into molecular weight regions by one-dimensional SDS-PAGE, prior to digestion in gel. Tryptic peptides were analyzed by reverse-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Ionized peptide intensities were averaged for each identified protein in the two pools, thereby measuring relative protein abundance between the two membrane protein pools. Log2 -transformed ratio (AD/control) of protein abundances fit a normal distribution, thereby permitting determination of significantly changed protein abundances in the AD pool.

Results: We report a comparative analysis of the membrane-enriched platelet proteome between patients with mild to moderate AD and cognitively normal, healthy subjects. A total of 144 proteins were determined significantly altered in the platelet membrane proteome from patients with probable AD. In particular, secretory (alpha) granule proteins were dramatically reduced in AD. Of these, we confirmed significant reduction of thrombospondin-1 (THBS1) in the AD platelet membrane proteome by immunoblotting. There was a high protein-protein connectivity of proteins in other pathways implicated by proteomic changes to the proteins that define secretory granules.

Conclusions: Depletion of secretory granule proteins is consistent with a preponderance of post-activated platelets in circulation in AD. Significantly changed pathways implicate additional AD-related defects in platelet glycoprotein synthesis, lipid homeostasis, amyloidogenic proteins, and regulators of protease activity, many of which may be useful plasma membrane-expressed markers for AD. This study highlights the utility of LC-MS/MS to quantify human platelet membrane proteins and suggests that platelets may serve as a source of blood-based biomarkers in neurodegenerative disease.

Keywords: blood biomarkers, platelet activation, mass spectrometry, membrane proteomics, glycoprotein, matrix metalloprotease inhibitor, amyloidogenic protein, alpha granule secretion, coagulation

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Consumer-perceived risks and choices about pharmaceuticals in the environment: a cross-sectional study

Simone Dohle1*, Victoria E A Campbell2,3 and Joseph L Arvai

Abstract
Background: There is increasing concern that pollution from pharmaceuticals used in human medicine and agriculture can be a threat to the environment. Little is known, however, if people are aware that pharmaceuticals may have a detrimental influence on the environment. The present study examines people’s risk perception and choices in regard to environmental risks of pharmaceuticals used in human medicine and for agricultural purposes.

Methods: A representative sample of the U.S. population (N = 640) was surveyed. Respondents completed a hypothetical choice task that involved tradeoffs between human and environmental health. In addition, it was examined how much people would support an environment policy related to drug regulation.

Results: For agricultural pharmaceuticals, respondents reported a high level of satisfaction for a policy requiring farms to limit their use of antibiotics. In the domain of pharmaceuticals used in human medicine, we found that people were willing to consider environmental consequences when choosing a drug, but only when choices were made about treatment options for a rather harmless disease. In contrast, when decisions were made about treatment options for a severe disease, the drug’s effectiveness was the most important criterion.

Conclusions: It can be concluded that the environmental impact of a drug will be hardly considered in decisions about pharmaceuticals for severe diseases like cancer, and this may be due to the fact that these decisions are predominantly affective in nature. However, for less severe health risks, people are willing to balance health and environmental considerations.

Keywords: Affect, Environment, Health effects, Pharmaceuticals, Risk, Survey

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Using PM2.5 concentrations to estimate the health burden from solid fuel combustion, with application to Irish and Scottish homes

Karen S Galea1*, J Fintan Hurley2, Hilary Cowie2, Amy L Shafrir2,3, Araceli Sánchez Jiménez1, Sean Semple4,Jon G Ayres5 and Marie Coggins

Abstract
Background: This study estimates the potential population health burden from exposure to combustion-derived particulate air pollution in domestic settings in Ireland and Scotland.

Methods: The study focused on solid fuel combustion used for heating and the use of gas for cooking. PM2.5 (particulate matter with an aerodynamic diameter < 2.5 μm) was used as the pollutant mixture indicator.Measured PM2.5 concentrations in homes using solid fuels were adjusted for other sources of PM2.5 by
subtracting PM2.5 concentrations in homes using gas for cooking but not solid fuel heating. Health burden was estimated for exposure indoors 6 pm - midnight, or all day (24-hour), by combining estimated attributable annual PM2.5 exposures with (i) selected epidemiological functions linking PM2.5 with mortality and morbidity (involving some re-scaling from PM10 to PM2.5, and adjustments ‘translating’ from concentrations to exposures) and (ii) on the current population exposed and background rates of morbidity and mortality.

Results: PM2.5 concentrations in coal and wood burning homes were similar to homes using gas for cooking, used here as a baseline (mean 24-hr PM2.5 concentrations 8.6 μg/m3 ) and so health impacts were not calculated. Concentrations of PM2.5 in homes using peat were higher (24-hr mean 15.6 μg/m3
); however, health impacts were calculated for the exposed population in Ireland only; the proportion exposed in Scotland was very small. The assessment for winter evening exposure (estimated annual average increase of 2.11 μg/m3 over baseline) estimated 21 additional annual cases of all-cause mortality, 55 of chronic bronchitis, and 30,100 and 38,000 annual lower respiratory symptom days (including cough) and restricted activity days respectively.

Conclusion: New methods for estimating the potential health burden of combustion-generated pollution from solid fuels in Irish and Scottish homes are provided. The methodology involves several approximations and uncertainties but is consistent with a wider movement towards quantifying risks in PM2.5 irrespective of
source. Results show an effect of indoor smoke from using peat (but not wood or coal) for heating and cooking; but they do not suggest that this is a major public health issue.

Keywords: Health burden assessment, Solid fuels, Peat, Scotland, Ireland, PM2.5

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Spatial and temporal estimation of air pollutants in New York City: exposure assignment for use in a birth outcomes study

Zev Ross1*, Kazuhiko Ito2, Sarah Johnson2,Michelle Yee2, Grant Pezeshki2, Jane E Clougherty3, David Savitz4 and Thomas Matt

Abstract
Background: Recent epidemiological studies have examined the associations between air pollution and birth outcomes. Regulatory air quality monitors often used in these studies, however, were spatially sparse and unable to capture relevant within-city variation in exposure during pregnancy.

Methods: This study developed two-week average exposure estimates for fine particles (PM2.5) and nitrogen dioxide (NO2) during pregnancy for 274,996 New York City births in 2008–2010. The two-week average exposures were constructed by first developing land use regression(LUR) models of spatial variation in annual average PM2.5 and NO2 data from 150 locations in the New York City Community Air Survey and emissions source data near monitors. The annual average concentrations from the spatial models were adjusted to account for city-wide temporal trends using time series derived from regulatory monitors. Models were developed using Year 1 data and validated using Year 2 data. Two-week average exposures were then estimated for three buffers of maternal address and were averaged into the last six weeks, the trimesters, and the entire period of gestation. We characterized temporal variation of exposure estimates, correlation between PM2.5 and NO2 , and correlation of exposures across trimesters.

Results: The LUR models of average annual concentrations explained a substantial amount of the spatial variation (R2= 0.79 for PM2.5 and 0.80 for NO2
). In the validation, predictions of Year 2 two-week average concentrations showed strong agreement with measured concentrations (R2= 0.83 for PM2.5 and 0.79 for NO2 ). PM2.5 exhibited greater temporal variation than NO2 . The relative contribution of temporal vs. spatial variation in the estimated exposures varied by time window. The differing seasonal cycle of these pollutants (bi-annual for PM2.5 and annual for NO2) resulted in different patterns of correlations in the estimated exposures across trimesters. The three levels of spatial buffer did not make a substantive difference in estimated exposures.

Conclusions: The combination of spatially resolved monitoring data, LUR models and temporal adjustment using regulatory monitoring data yielded exposure estimates for PM2.5 and NO2 that performed well in validation tests. The interaction between seasonality of air pollution and exposure intervals during pregnancy needs to be considered in future studies.

Keywords: Air pollution, Birth outcomes, Particulate matter, Nitrogen dioxide, Land use regression, NYCCAS,Temporal adjustment

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A Cross-sectional Study of the Impact of Blood Selenium on Blood and Urinary Arsenic Concentrations in Bangladesh

Christine Marie George1*, Mary Gamble2, Vesna Slavkovich3, Diane Levy4, Alauddin Ahmed5, Habibul Ahsan6 and Joseph Graziano

Abstract
Background: Arsenic can naturally occur in the groundwater without an anthropogenic source of contamination.In Bangladesh over 50 million people are exposed to naturally occurring arsenic concentrations exceeding the World Health Organization’s guideline of 10 μg/L. Selenium and arsenic have been shown to facilitate the excretion of each other in bile. Recent evidence suggests that selenium may play a role in arsenic elimination by forming a selenium-arsenic conjugate in the liver before excretion into the bile.

Methods: A cross-sectional study of 1601 adults and 287 children was conducted to assess the relationship between blood selenium and urinary and blood arsenic in a study population residing in a moderately arsenic-contaminated rural area in Bangladesh.

Results: The results of this study indicate a statistically significant inverse relationship between blood selenium and urinary arsenic concentrations in both adult and pediatric populations in rural Bangladesh after adjustment for age, sex, Body Mass Index, plasma folate and B12 (in children), and ever smoking and current betel nut use (in adults). In addition, there appears to be a statistically significant inverse relationship between blood selenium and blood arsenic in children.

Conclusions: Our results suggest that selenium is inversely associated with biomarkers of arsenic burden in both adults and children. These findings support the hypothesis that Se facilitates the biliary elimination of As, possibly via the putative formation of a Se-As conjugate using a glutathione complex. However, laboratory based studies are needed to provide further evidence to elucidate the presence of Se-As conjugate and its role in arsenic elimination in humans.

Keywords: Selenium, Arsenic, Bangladesh, Pediatric populations

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Dispelling urban myths about default uncertainty factors in chemical risk assessment – sufficient protection against mixture effects?

V Martin, Scholze Martin and Andreas Kortenkamp

Abstract
Assessing the detrimental health effects of chemicals requires the extrapolation of experimental data in animals to human populations. This is achieved by applying a default uncertainty factor of 100 to doses not found to be associated with observable effects in laboratory animals. It is commonly assumed that the toxicokinetic and toxicodynamic sub-components of this default uncertainty factor represent worst-case scenarios and that the multiplication of those components yields conservative estimates of safe levels for humans. It is sometimes claimed that this conservatism also offers adequate protection from mixture effects. By analysing the evolution of uncertainty factors from a historical perspective, we expose that the default factor and its sub-components are
intended to represent adequate rather than worst-case scenarios. The intention of using assessment factors for mixture effects was abandoned thirty years ago. It is also often ignored that the conservatism (or otherwise) of uncertainty factors can only be considered in relation to a defined level of protection. A protection equivalent to an effect magnitude of 0.001-0.0001% over background incidence is generally considered acceptable. However, it is impossible to say whether this level of protection is in fact realised with the tolerable doses that are derived by employing uncertainty factors. Accordingly, it is difficult to assess whether uncertainty factors overestimate or underestimate the sensitivity differences in human populations. It is also often not appreciated that the outcome of probabilistic approaches to the multiplication of sub-factors is dependent on the choice of probability distributions. Therefore, the idea that default uncertainty factors are overly conservative worst-case scenarios which can account both for the lack of statistical power in animal experiments and protect against potential mixture effects is ill-founded. We contend that precautionary regulation should provide an incentive to generate better data and recommend adopting a pragmatic, but scientifically better founded approach to mixture risk assessment.

Keywords: Acceptable daily intake, Extrapolation, Hazard assessment, Mixture, Risk assessment

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Dioxin-like compounds and bone quality in Cree women of Eastern James Bay (Canada): a cross-sectional study

Alexandra-Cristina Paunescu, Éric Dewailly, Sylvie Dodin, Evert Nieboer4 and Pierre Ayotte

Abstract
Background: Aboriginal populations living in Canada’s northern regions are exposed to a number of persistent organic pollutants through their traditional diet which includes substantial amounts of predator fish species. Exposure to dioxin-like compounds (DLCs) can cause a variety of toxic effects including adverse effects on bone tissue. This descriptive cross-sectional study was conducted to investigate the relationship between plasma concentrations of DLCs and bone quality parameters in Cree women of Eastern James Bay (Canada).

Methods: Two hundred and forty-nine Cree women from seven communities in Eastern James Bay (Canada), aged 35 to 74 years old, participated in the study. In order to determine the total DLC concentration in plasma samples of participants, we measured the aryl hydrocarbon receptor-mediated transcriptional activity elicited by plasma sample extracts using a luciferase reporter gene assay. Plasma concentrations of mono-ortho-substituted dioxin-like polychlorinated biphenyls (DL-PCBs) 105, 118 and 156 were measured by gas chromatography–mass spectrometry. Bone quality parameters (speed of sound, m/s; broadband ultrasound attenuation, dB/MHz; stiffness index, %) were assessed by quantitative ultrasound at the right calcaneus with the Achilles InSight system. Several factors known to be associated with osteoporosis were documented by questionnaire. Multiple linear regression models were constructed for the three ultrasound parameters.

Results: DL-PCBs 105 and 118 concentrations, but not the global DLC concentration, were inversely associated with the stiffness index, even after adjusting for several confounding factors. The stiffness index (log) decreased by −0.22% (p=0.0414) and −0.04% (p=0.0483) with an increase of one μg/L in plasma concentrations of DL-PCB 105 and DL-PCB 118, respectively. Other factors, including age, height, smoking status, menopausal status and the percentage of omega-6
polyunsaturated fatty acids (PUFAs) in erythrocyte membranes were negatively associated with one of the ultrasound parameters, while the percentage of omega-3 PUFAs in these membranes and levels of physical activity and education were positively associated with them.

Conclusions: Our results show that an increase in plasma concentrations of DL-PCBs 105 and 118 was negatively associated with stiffness index, a measure of bone quality/strength, in women of this population. In addition to environmental contaminants, future studies should also consider PUFA intake as a factor influencing bone quality.

Keywords: Calcaneal Ultrasound Parameters, Dioxin-Like Compounds, Polychlorinated Biphenyls, Cree Women, Canada\

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A time series study on the effects of heat on mortality and evaluation of heterogeneity into European and Eastern-Southern Mediterranean cities: results of EU CIRCE project

Michela Leone1*, Daniela D’Ippoliti1, Manuela De Sario1, Antonis Analitis2, Bettina Menne3, Klea Katsouyanni2,Francesca K de’ Donato1, Xavier Basagana4,5,6, Afif Ben Salah7, Elsa Casimiro8, Zeynep Dörtbudak9,Carmen Iñiguez4,10,11, Chava Peretz12, Tanja Wolf3 and Paola Michelozzi

Abstract
Background: The Mediterranean region is particularly vulnerable to the effect of summer temperature.Within the CIRCE project this time-series study aims to quantify for the first time the effect of summer temperature in Eastern-Southern Mediterranean cities and compared it with European cities around the Mediterranean basin, evaluating city characteristics that explain between-city heterogeneity.

Methods: The city-specific effect of maximum apparent temperature (Tappmax) was assessed by Generalized Estimation Equations, assuming a linear threshold model. Then, city-specific estimates were included in a random effect meta-regression analysis to investigate the effect modification by several city characteristics.

Results: Heterogeneity in the temperature-mortality relationship was observed among cities. Thresholds recorded higher values in the warmest cities of Tunis (35.5°C) and Tel-Aviv (32.8°C) while the effect of Tappmax above threshold was greater in the European cities. In Eastern-Southern Mediterranean cities a higher effect was observed among younger age groups (0–14 in Tunis and 15–64 in Tel-Aviv and Istanbul) in contrast with the European cities where the elderly population was more vulnerable. Climate conditions explained most of the observed heterogeneity and among socio-demographic and economic characteristics only health expenditure and unemployment rate were identified as effect modifiers.

Conclusions: The high vulnerability observed in the young populations in Eastern-Southern Mediterranean cities represent a major public health problem. Considering the large political and economic changes occurring in this region as well future temperature increase due to climate change, it is important to strengthen research and public health efforts in these Mediterranean countries.

Keywords: Hot temperature, Mortality, Mediterranean region, Heterogeneity, Age groups

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Selection of genes for gene-environment interaction studies: a candidate pathway-based strategy using asthma as an example

Marta Rava1,2*†, Ismaïl Ahmed2,3†, Florence Demenais4,5, Margaux Sanchez1,2, Pascale Tubert-Bitter2,3 and Rachel Nadif

Abstract
Background: The identification of gene by environment (GxE) interactions has emerged as a challenging but essential task to fully understand the complex mechanism underlying multifactorial diseases. Until now, GxE interactions have been investigated by candidate approaches examining a small number of genes, or agnostically at the genome wide level.Presentation of the hypothesis: In this paper, we propose a gene selection strategy for investigation of gene-environment interactions. This strategy integrates the information on biological processes shared by genes, the canonical pathways to which they belong and the biological knowledge related to the environment in the gene selection process. It relies on both bioinformatics resources and biological expertise.

Testing the hypothesis: We illustrate our strategy by considering asthma, tobacco smoke as the environmental exposure, and genes sharing the same biological function of “response to oxidative stress”. Our filtering strategy leads to a list of 28 pathways involving 182 genes for further GxE investigation.

Implications of the hypothesis: By integrating the environment into the gene selection process, we expect that our strategy will improve the ability to identify the joint effects and interactions of environmental and genetic factors in disease.

Keywords: Gene by environment interactions, Oxidative stress, Smoking, Pathway-based gene selection

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A biomonitoring study on blood levels of beta-hexachlorocyclohexane among people living close to an industrial area

Daniela Porta1*, Fiorella Fantini2, Elena De Felip3, Francesco Blasetti2, Annalisa Abballe3, Valerio Dell’Orco2,Valeria Fano1,4, Anna Maria Ingelido3 , Silvia Narduzzi1 and Francesco Forastiere

 

Abstract
Background: A chemical plant manufacturing pesticides has been operating since the 1950’s in the Sacco River Valley (Central Italy). In 2005, high beta-hexachlorocyclohexane (Beta-HCH) concentrations were found in milk of cows raised and fed near the river. We report the results of a biomonitoring study conducted in this region to evaluate the body burden of Beta-HCH and to identify the determinants of the human contamination.

Methods: We defined four residential areas by their distance from the chemical plant and the river, and selected a stratified random sample of 626 people aged 25–64 years. We evaluated the association, in terms of the geometric mean ratio (GMR), between several potential determinants and Beta-HCH serum concentrations using multivariate linear regression analysis.

Results: Two hundred forty-six serum samples were analysed to assess Beta-HCH levels (mean concentration: 99ng/g lipid; Standard Deviation: 121; Geometric Mean: 60.6; Geometric Standard Deviation: 2.65). We found a strong association between Beta-HCH and living in the area close to the river (GMR: 2.00; 95%CI: 1.36-2.94). Beta-HCH levels were also associated with age, level of education, use of private wells and consumption of local food.

Conclusions: The results suggest that people living close to the river may have been contaminated by Beta-HCH, most likely through water from private wells and privately grown food. A programme of epidemiological and clinical surveillance is on-going on this population.

Keywords: Human biomonitoring, Beta-HCH, Persistent organic pollutants, Water contamination, Food contamination

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Associations between mortality and meteorological and pollutant variables during the cool season in two Asian cities with sub-tropical climates: Hong Kong and Taipei

William B Goggins1*, Emily YY Chan2, Chunyuh Yang3 and Marc Chong

Abstract
Background: Numerous studies have found associations between extreme temperatures and human mortality but relatively few studies have been done in sub-tropical and tropical cities, especially in Asia. In this study we examine the impact of cold temperatures, cold waves and other meteorological and environmental variables on cool season mortality in 2 subtropical Asian cities.

Methods: Separate analysis of daily mortality time-series from Hong Kong and Taipei using Generalized Additive Models with natural mortality as the outcome daily mean temperature as the main explanatory variable and relative humidity, solar radiation, wind speed, pollutants (nitrogen dioxide (NO2), sulfur dioxide(SO2), respirable suspended particulates (PM10), ozone (O3), seasonality and day of the week controlled as potential confounders. Lags up to 35 days were considered for temperature, and distributed lag models were used to determine the number of lags for final models. Subgroup analyses were also done by gender, age group, cause of death and geographical area of residence.

Results: Cold temperatures were strongly associated with higher mortality with lagged effects persisting up to 3 weeks in Hong Kong and 2 weeks in Taipei. Cold effects were much stronger for deaths among older people and non-cancer deaths. Prolonged cold spells modestly but significantly raised mortality after accounting for the effects of individual cold days. Higher daily ozone levels were also strongly associated with higher short-term mortality in Taipei and Hong
Kong, while relative humidity and solar radiation were weakly and inconsistently associated with mortality.

Conclusions: Cold temperatures and cold spells substantially increase short-term mortality in sub-tropical Asian cities particularly among the elderly. Greater attention needs to be paid to the adverse health effects of cold temperatures. Interventions including provisions of shelters, cold weather warnings and education about the possible health effects of cold temperature should be carried out in sub-tropical areas.

Keywords: Cold wave, Cold spell, Biometeorology, Poisson regression, Mortality

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Meningioma patients diagnosed 2007–2009 and the association with use of mobile and cordless phones: a case–control study

Michael Carlberg1*, Fredrik Söderqvist1,2,3, Kjell Hansson Mild4 and Lennart Hardell

Abstract
Background: To study the association between use of wireless phones and meningioma.

Methods: We performed a case–control study on brain tumour cases of both genders aged 18–75 years and diagnosed during 2007–2009. One population-based control matched on gender and age was used to each case. Here we report on meningioma cases including all available controls. Exposures were assessed by a questionnaire. Unconditional logistic regression analysis was performed.

Results: In total 709 meningioma cases and 1,368 control subjects answered the questionnaire. Mobile phone use in total produced odds ratio (OR) = 1.0, 95% confidence interval (CI) = 0.7-1.4 and cordless phone use gave OR = 1.1, 95% CI = 0.8-1.5. The risk increased statistically significant per 100 h of cumulative use and highest OR was found in the fourth quartile (>2,376 hours) of cumulative use for all studied phone types. There was no statistically significant increased risk for ipsilateral mobile or cordless phone use, for meningioma in the temporal lobe or per year of latency. Tumour volume was not related to latency or cumulative use in hours of wireless phones.

Conclusions: No conclusive evidence of an association between use of mobile and cordless phones and meningioma was found. An indication of increased risk was seen in the group with highest cumulative use but was not supported by statistically significant increasing risk with latency. Results for even longer latency periods of wireless phone use than in this study are desirable.

Keywords: Case–control study, 25 years latency, Benign brain tumour, Meningioma, Wireless phones

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Investigating host dependence of xylose utilization in recombinant Saccharomyces cerevisiae strains using RNA-seq analysis

Xueyang Feng and Huimin Zhao

Abstract
Background: Xylose-based ethanol production by recombinant S. cerevisiae is of great interest to basic and applied bioenergy research. By expressing three different fungal pathways in two S. cerevisiae hosts respectively, we found that the xylose utilization efficiency by recombinant S. cerevisiae depends not only on the choice of xylose pathway but also on the choice of host, exhibiting an obvious host or context dependence. To investigate molecular mechanisms of this context dependence, we applied RNA-seq analysis in this study for a systematic characterization of the xylose utilization via different pathways in different S. cerevisiae hosts.

Results: Based on the RNA-seq analysis, the transcripts that were regulated during xylose utilization have been identified. Three transcription factors involved in regulation of amino acid metabolism, responses to oxidative stresses, and degradation of aggregated proteins, respectively, were found to participate in xylose metabolism regulation regardless of which pathway was expressed and which host the xylose pathway was expressed in. Nine transcription factors, involved in homeostasis, regulation of amino acid metabolism, and stress responses, were identified as the key modules responsible for the host-specific responses to the same xylose pathway. In addition, the transcriptional regulations of xylose utilization in different yeast hosts were compared to two reference  regulation patterns, which indicated that diverse regulation strategies were adopted by different hosts for improved xylose utilization.

Conclusions: This study provides the first transcriptomic study of the host dependence of xylose utilization in S. cerevisiae. Both the conserved regulatory modules for xylose metabolism and the key modules responsible for host dependence were identified. As indicated by the functions of the conserved transcription factors involved in xylose metabolism regulation, the xylose utilization in recombinant S. cerevisiae may be affected by both carbohydrate metabolism regulation and stress responses. Based on the comparison of transcriptional regulation patterns, the metabolic optimizations of xylose utilization in different hosts went toward different directions, which may explain the host dependence observed in this study. The knowledge revealed by this study could provide valuable insights
towards the improvement of metabolic engineering strategies for cellulosic ethanol production.

Keywords: RNA-seq, Xylose metabolism, Transcriptional factors, S. cerevisiae, Pathway switch, Host-specific response

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Freshwater microalgae harvested via flocculation induced by pH decrease

Jiexia Liu1†, Yi Zhu1†, Yujun Tao1, Yuanming Zhang1*, Aifen Li2, Tao Li2, Ming Sang and Chengwu Zhang

 

Abstract
Background: Recent studies have demonstrated that microalga has been widely regarded as one of the most promising raw materials of biofuels. However, lack of an economical, efficient and convenient method to harvest microalgae is a bottleneck to boost their full-scale application. Many methods of harvesting microalgae, including mechanical, electrical, biological and chemical based, have been studied to overcome this hurdle.

Results: A new flocculation method induced by decreasing pH value of growth medium was developed for harvesting freshwater microalgae. The flocculation efficiencies were as high as 90% for Chlorococcum nivale, Chlorococcum ellipsoideum and Scenedesmus sp. with high biomass concentrations (>1g/L). The optimum
flocculation efficiency was achieved at pH 4.0. The flocculation mechanism could be that the carboxylate ions of organic matters adhering on microalgal cells accepted protons when pH decreases and the negative charges were neutralized, resulting in disruption of the dispersing stability of cells and subsequent flocculation of cells. A linear correlation between biomass concentration and acid dosage was observed. Furthermore, viability of flocculated cells was determined by Evans Blue assay and few cells were found to be damaged with pH decrease. After neutralizing pH and adding nutrients to the flocculated medium, microalgae were proved to maintain a similar growth yield in the flocculated medium comparing with that in the fresh medium. The recycling of medium could
contribute to the economical production from algae to biodiesel

Conclusions: The study provided an economical, efficient and convenient method to harvest fresh microalgae. Advantages include capability of treating high cell biomass concentrations (>1g/L), excellent flocculation efficiencies (≥ 90%), operational simplicity, low cost and recycling of medium. It has shown the potential to
overcome the hurdle of harvesting microalgae to promote full-scale application to biofuels from microalgae.

Keywords: Microalgae, Harvest, Flocculation, pH decrease, Recycle

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Inhibition of growth of Zymomonas mobilis by model compounds found in lignocellulosic hydrolysates

Mary Ann Franden, Heidi M Pilath, Ali Mohagheghi, Philip T Pienkos and Min Zhang

Abstract
Background: During the pretreatment of biomass feedstocks and subsequent conditioning prior to saccharification, many toxic compounds are produced or introduced which inhibit microbial growth and in many cases, production of ethanol. An understanding of the toxic effects of compounds found in hydrolysate is critical to improving sugar utilization and ethanol yields in the fermentation process. In this study, we established a useful tool for surveying hydrolysate toxicity by measuring growth rates in the presence of toxic compounds, and examined the effects of selected model inhibitors of aldehydes, organic and inorganic acids (along with various cations), and alcohols on growth of Zymomonas mobilis 8b (a ZM4 derivative) using glucose or xylose as the carbon source.

Results: Toxicity strongly correlated to hydrophobicity in Z. mobilis, which has been observed in Escherichia coli and Saccharomyces cerevisiae for aldehydes and with some exceptions, organic acids. We observed Z. mobilis 8b to be more tolerant to organic acids than previously reported, although the carbon source and growth conditions play a role in tolerance. Growth in xylose was profoundly inhibited by monocarboxylic organic acids compared to growth in glucose, whereas dicarboxylic acids demonstrated little or no effects on growth rate in either substrate. Furthermore, cations can be ranked in order of their toxicity, Ca++ > > Na+
> NH4+> K+ . HMF (5-hydroxymethylfurfural), furfural and acetate which were observed to contribute to inhibition of Z. mobilis growth in dilute acid pretreated corn stover hydrolysate, do not interact in a synergistic manner in combination. We provide further evidence that Z. mobilis 8b is capable of converting the aldehydes furfural, vanillin, 4-hydroxybenzaldehyde and to some extent syringaldehyde to their alcohol forms (furfuryl, vanillyl, 4-hydroxybenzyl and syringyl alcohol) during fermentation.

Conclusions: Several key findings in this report provide a mechanism for predicting toxic contributions of inhibitory components of hydrolysate and provide guidance for potential process development, along with potential future strain improvement and tolerance strategies.

Keywords: Zymomonas mobilis, High-throughput screening, Cell growth assay, Bioscreen C, Inhibitor, Hydrolysate,Lignocellulosic biomass, Ethanol

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Heterotrophic growth of Neochloris oleoabundans using glucose as a carbon source

Daniela Morales-Sánchez, Raunel Tinoco-Valencia, John Kyndt and Alfredo Martinez

Abstract
Background: In comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate).

Results: N. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w).
A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter.

Conclusions: N. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.

Keywords: Neochloris oleoabundans, Heterotrophic growth, Glucose, Glucose transporter, Fed-batch, Nitrate, Lipids, Protein

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Physiological adaptations of Saccharomyces cerevisiae evolved for improved butanol tolerance

Payam Ghiaci, Joakim Norbeck and Christer Larsson

Abstract
Background: Butanol is a chemical with potential uses as biofuel and solvent, which can be produced by microbial fermentation. However, the end product toxicity is one of the main obstacles for developing the production process irrespective of the choice of production organism. The long-term goal of the present project is to produce 2-butanol in Saccharomyces cerevisiae. Therefore, unraveling the toxicity mechanisms of solvents such as butanol and understanding the mechanisms by which tolerant strains of S. cerevisiae adapt to them would be an important contribution to the development of a bio-based butanol production process.

Results: A butanol tolerant S. cerevisiae was achieved through a series of sequential batch cultures with gradual increase of 2-butanol concentration. The final mutant (JBA-mut) tolerates all different alcohols tested at higher concentrations compared to the wild type (JBA-wt). Proteomics analysis of the two strains grown under mild butanol-stress revealed 46 proteins changing their expression by more than 1.5-fold in JBA-mut, 34 of which were upregulated. Strikingly, 21 out of the 34 upregulated proteins were predicted constituents of mitochondria. Among the non-mitochondrial up-regulated proteins, the minor isoform of Glycerol-3-phosphatase (Gpp2) was the most notable, since it was the only tested protein whose overexpression was found to confer butanol tolerance.

Conclusion: The study demonstrates several differences between the butanol tolerant mutant and the wild type. Upregulation of proteins involved in the mitochondrial ATP synthesizing machinery constituents and glycerol biosynthesis seem to be beneficial for a successful adaptation of yeast cells to butanol stress.

Keywords: Saccharomyces cerevisiae, Butanol, Tolerance, Proteomics

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Alkaline twin-screw extrusion pretreatment for fermentable sugar production

Chao Liu1, Evert van der Heide, Haisong Wang1*, Bin Li1, Guang Yu1 and Xindong Mu

Abstract
Background: The inevitable depletion of fossil fuels has resulted in an increasing worldwide interest in exploring alternative and sustainable energy sources. Lignocellulose, which is the most abundant biomass on earth, is widely regarded as a promising raw material to produce fuel ethanol. Pretreatment is an essential step to disrupt the recalcitrance of lignocellulosic matrix for enzymatic saccharification and bioethanol production. This paper established an ATSE (alkaline twin-screw extrusion pretreatment) process using a specially designed twin-screw extruder in the presence of alkaline solution to improve the enzymatic hydrolysis efficiency of corn stover for the production of fermentable sugars.

Results: The ATSE pretreatment was conducted with a biomass/liquid ratio of 1/2 (w/w) at a temperature of 99°C without heating equipment. The results indicated that ATSE pretreatment is effective in improving the enzymatic digestibility of corn stover. Sodium hydroxide loading is more influential factor affecting both sugar yield and lignin degradation than heat preservation time. After ATSE pretreatment under the proper conditions (NaOH loading of 0.06 g/g biomass during ATSE and 1 hour heat preservation after extrusion), 71% lignin removal was achieved and the conversions of glucan and xylan in the pretreated biomass can reach to 83% and 89% respectively via subsequent enzymatic hydrolysis (cellulase loading of 20 FPU/g-biomass and substrate consistency of 2%). About 78% of the original polysaccharides were converted into fermentable sugars.

Conclusions: With the physicochemical functions in extrusion, the ATSE method can effectively overcome the recalcitrance of lignocellulose for the production of fermentable sugars from corn stover. This process can be considered as a promising pretreatment method due to its relatively low temperature (99°C), high biomass/liquid ratio (1/2) and satisfied total sugar yield (78%), despite further study is needed for process optimization and cost reduction.

Keywords: Twin-screw extrusion, Pretreatment, Corn stover, Sugar recovery, Enzymatic hydrolysis

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