Chlamydia pneumoniae and Mycoplasma pneumoniae is controversial. We conducted a cross-sectional study to clarify the association between atypical pneumoniae seropositivity and COPD in a general population. We also investigated genetic polymorphisms conferring susceptibility to a pneumonia titer.Chronic obstructive pulmonary disease (COPD) is a possible risk factor for cardiovascular disease. The association of COPD with the pathogenicity of infection with C pneumoniae were determined using an enzyme-linked immunoassay, and M pneumoniae seropositivity was assessed by a particle agglutination test.The study included 9040 Japanese subjects (54 ± 13 years). COPD was defined as a ratio of forced expiratory volume in 1 second to forced vital capacity of less than 70%. Serum levels of IgA and IgG antibodies to C pneumoniae (26.1%) had a higher prevalence of COPD  after adjustment for age, sex, height, weight, and smoking status. The association between M pneumoniae seropositivity (20.4%) and COPD was also significant in covariate-adjusted analysis (P < 0.001). A genome-wide association analysis of the C pneumoniae IgA index identified a susceptible genotype (rs17634369) near the IKZF1 gene, and the seropositive rate of C pneumoniae significantly differed among genotypes . On multiple regression analysis, seropositivity for both C pneumoniae  and M pneumoniae  was an independent determinant for COPD, while no direct association was found with the rs17634369 genotype.Subjects seropositive for C pneumoniae and M pneumoniae is an independent risk factor for COPD in the general population.Seropositivity for both  COPD has also been associated with increased mortality.6 Although the most important risk factor for COPD is long-term smoking,7 only a small proportion of smokers develop airflow obstruction,8 and some lifelong nonsmokers develop COPD.9 These inconsistent results suggest that other risk factors are also causative in the pathogenesis of COPD.Chronic obstructive pulmonary disease (COPD), as assessed by forced expiratory volume in 1 second (FEV11 Infection with Chlamydia pneumoniae (C pneumoniae), the most common nonviral human respiratory pathogen, has also been proposed as a risk factor for COPD.12 Although the mechanism by which chronic infection with C pneumoniae might trigger the development of COPD is not precisely understood, 1 plausible mechanism may be the release of inflammatory cytokines and subsequent airway inflammation and tissue damage.12 Higher titers of C pneumoniae in blood specimens are commonly observed in patients with respiratory diseases, namely COPD,13 chronic bronchitis,14 and symptomatic respiratory disease,15 and in hospitalized patients with acute exacerbation of COPD.16 Further, approximately 50% of cases of exacerbated COPD are caused by bacterial infection, such as with C pneumoniae.17 Although the prevalence and mortality of COPD are increasing worldwide,18 the involvement of C pneumoniae in the development of COPD in the general population is largely unknown. Mycoplasma pneumoniae (M pneumoniae) is another atypical pathogen that causes community-acquired pneumonia. Infection or colonization of M pneumoniae in the lower airway is associated with the pathogenesis of bronchial asthma,19 suggesting that chronic infection with M pneumoniae may also be a risk factor for COPD.Factors postulated to play a role in the pathogenesis of COPD to date include air pollution, respiratory infection in childhood, bronchial hyperresponsiveness, systemic inflammation, and genetic background.HHIP) and glutathione S-transferase C-terminal domain containing (GSTCD) gene region.20 Despite the possible existence of SNPs which might influence susceptibility to seropositivity for pneumoniae, and the relationship between C pneumoniae and M pneumoniae infection and pulmonary function, no study has yet explored SNPs associated with seropositivity for both strains.Recent advances have enabled the analysis of millions of single nucleotide polymorphisms (SNPs) dispersed throughout the human genome. Genome-wide association studies (GWAS) conducted without a prior hypothesis have successfully identified susceptibility loci for various common diseases and quantitative traits. GWAS of lung function have identified multiple loci such as the hedgehog interacting protein . The Nagahama Study cohort was recruited between 2008 and 2010 from the general population (30–74 years old) living in Nagahama City, a largely suburban city of 125,000 inhabitants in Shiga Prefecture. Nagahama City residents aged 30 to 74 years at recruitment and without serious health problems who agreed to participate in the cohort study of their own accord were recruited via mass communications in the local community, such as public relations magazines and periodical newspapers.M pneumoniae titer (n = 1) or rs17634369 genotype (n = 90). Eventually, a total of 9040 people were included as study subjects.Among a total of 9804 participants, we considered 9237 subjects as the total population in this study, consisting of 3246 subjects whose genome-wide SNP genotype data were available and 5991 other subjects as a subset of the remaining samples available for replication genotyping (Table S1). Among these, participants meeting any of the following conditions were excluded from subsequent association analysis for COPD: pregnancy (n = 40), history of lung cancer (n = 19), unsuccessful assessment of the Brinkman index (n = 23) or spirometric parameters (n = 24), and unavailability of serum All study procedures were approved by the ethics committee of Kyoto University Graduate School of Medicine and the Nagahama Municipal Review Board. Written informed consent was obtained from all participants.Clinical measurements and blood sampling were performed at enrollment. Medical history and smoking status were investigated using a structured questionnaire. The Brinkman index was calculated as the daily number of cigarettes smoked multiplied by the number of years spent smoking.1 to FVC of less than 70%. Predicted normal values for FVC (FVC predicted) (L) and FEV1 (FEV1 predicted) (L) were calculated using the following equations, in accordance with guidelines developed by the Japanese Respiratory Society for the diagnosis and treatment of COPD : FVC male = 0.042 × height (cm) − 0.024 × age − 1.785; FVC female = 0.031 × height − 0.019 × age − 1.105; FEV1 male = 0.036 × height − 0.028 × age − 1.178; FEV1 female = 0.022 × height − 0.022 × age − 0.005.Pulmonary function was measured by an FVC maneuver on a computed spirometer with automated quality checks . Prebronchodilator spirometry was measured by certified medical technologists in accordance with a standardized protocol. COPD was defined by a ratio of FEVC pneumoniae were determined using a specific enzyme-linked immunoassay (ELISA) kit  that detects antibodies to the chlamydial outer membrane complex. IgA and IgG levels in each sample were expressed as the IgA or IgG index, respectively. Seropositivity to C pneumoniae was diagnosed as an IgA and IgG index value of more than 1.1 for both. Details of the ELISA assay and validity of the cut-off point have been described elsewhere.21 Mean intraassay coefficients of variation were 3.3% to 7.7% for the IgA index and 6.3% to 9.0% for the IgG index. Respective mean interassay coefficients of variation were 3.8% to 10.7% and 4.0% to 10.7%.Levels of serum IgA and IgG antibodies to M pneumoniae seropositivity was assessed using a semiquantitative particle agglutination test kit  consisting of gelatin particles coated with cell membrane components of M pneumoniae (Mac strain).22 Serum samples were serially diluted to give final dilutions of 1:40 to 1:20, 480. According to the manufacturer's instructions, antibody titers of 1:40 and higher were considered seropositive.24P < 1.0 × 10−7). This threshold was defined to ensure a concordance ratio of SNPs genotyped by different SNP arrays greater than 99.99%.25 Genotype imputation was performed by a standard 2-step procedure using MACH ver. 1.0.16 software with 1792015 SNPs commonly genotyped in the 192 samples as a reference. Imputed SNPs with a minor allele frequency of less than 0.01 or R2 less than 0.5 were excluded from the following association analysis. Samples with a call rate less than 95% (n = 162) were excluded from analysis. Of the remaining 3548 subjects, 295 individuals were excluded as they showed high degrees of kinship , and 7 individuals were excluded as ancestry outliers, as identified by principal component analysis using the HapMap Phase 2 release 28 JPT dataset as reference (EIGENSTRAT ver. 2.0).DNA was extracted from peripheral blood samples by the phenol–chloroform method. Genome-wide SNP genotyping was performed on 3710 samples of participants who joined the Nagahama cohort from 2008 to 2009. A series of BeadChip DNA arrays were used for analysis, namely HumanHap610 quad (1828 samples), HumanOmni2.5-4 (1616 samples), HumanOmni2.5-8 (378 samples), HumanOmni2.5s (192 samples), and HumanExome (192 samples) . Several samples were repeatedly genotyped using different arrays. As a reference panel, 192 samples were genotyped using HumanOmni2.5-8, HumanOmni2.5s, and HumanExome, and used in the following genotype imputation. Genotyping quality was controlled by excluding SNPs with a call rate below 99%, minor allele frequency below 0.01, or extreme deviation from Hardy–Weinberg equilibrium . Genotypes were analyzed with a TaqMan probe assay using commercially available primer and probe sets purchased from Life Technologies Corporation . Fluorescence level of PCR products was measured using the 7900HT Fast Real-Time PCR System (Life Technologies).C pneumoniae IgA and IgG index values was performed by linear regression analysis under an additive genetic model adjusted for age, age-squared, sex, and body mass index, while analysis of M pneumoniae seropositivity was performed by a Chi-square test (PLINK). Rank-based inverse normal transformation was applied to the C pneumoniae IgA and IgG index values. Population stratification was adjusted using top principal components as covariates. Given an estimation of the appropriate testing burden in a GWAS in Europeans as a million, a P-value less than 5.0 × 10−8 was considered to have genome-wide significance.26 Based on findings that an additive model has reasonable power in detecting both additive and dominant effects,27 we applied only an additive model in the current GWAS.Genome-wide association analysis of the P-value less than 0.05 considered to indicate statistical significance.In other association analyses, differences in numeric variables among subgroups were assessed by analysis of variance, while differences in frequency were assessed by a Chi-square test. Factors independently associated with COPD were identified by multiple logistic regression analysis. All statistical analyses were performed using JMP 9.0.2 software , with a conventional P < 0.001), and mostly male  and current or past smokers . Differences in major clinical parameters by severity of pulmonary function are detailed in Table S2.Table C pneumoniae and M pneumoniae seropositivity was 26.1% and 20.4%, respectively. The number of subjects seropositive for C pneumoniae increased with age, whereas that for M pneumoniae showed the opposite relationship . Associations of seropositivity for both strains with pulmonary function are summarized in Table C pneumoniae exhibited significantly lower pulmonary function and a higher frequency of COPD. Although the values of the C pneumonia e IgA and IgG indices were linearly associated with smoking intensity as assessed by the Brinkman index . Similar results were observed in an analysis which excluded subjects receiving treatment for asthma or COPD (n = 90) (Table S3).The frequency of C pneumoniae IgA index values identified strong association signals at 6p22.3 , 7p12 , and 13q33  . Additional genome-wide significant signals were found in the human leukocyte antigen gene region, but these were lost after adjustment for population stratification . No significant signals were found in GWAS of the C pneumoniae IgG index value , or in analysis of M pneumoniae seropositivity . Statistical power of the GWAS is shown in Figure S8. Additional susceptible SNPs with a lower frequency or smaller effect size might be detectable with a greater number of subjects.GWAS of the P = 1.4 × 10−5), but not rs9460391 (P = 0.414) or rs942102 (P = 0.621) (Table S4). Per-allele effect size of rs17634369 on the C pneumoniae IgA index value calculated from the combined datasets used in the GWAS, and replication analysis was approximately 0.1 (P = 1.3 × 10−11). SNP rs17634369 was located approximately 22 kb upstream of the IKZF1 gene, where no other genes were mapped , and was in strong linkage disequilibrium with the SNP rs4917014 , which was previously identified as conferring susceptibility to systemic lupus erythematosus (SLE).28 There were no significant associations between previously validated SNPs for SLE and C pneumoniae IgA index value (Table S5).Replication analysis of the remaining Nagahama samples confirmed the positive association of rs17634369 , as well as the seropositive rate defined by IgA and IgG indices (P < 0.001), remained significant after adjustment for age, sex, and smoking status.Figure x Figure B and thex Figure C linearlC pneumoniae and M pneumoniae seropositivity were independently associated with COPD (Model 1), even when individuals taking medication for asthma and COPD were excluded (Table S6), while the rs17634369 genotype was not directly associated with COPD (Model 1), even following adjustment for C pneumoniae and M pneumoniae seropositivity (Model 2). In a separate analysis by smoking status, positive associations between pneumoniae seropositivity and COPD were observed only in current or past smokers (Model 3), and not in never-smokers (Model 4). No interaction was observed between the rs17634369 genotype and C pneumoniae seropositivity (P = 0.846).A multiple logistic regression analysis was used to further identify factors independently associated with COPD Table . ResultsC pneumoniae and M pneumoniae was positively associated with COPD. To our knowledge, this is the 1st study to clearly show the risk of C pneumoniae and M pneumoniae seropositivity for COPD in a general population, particularly in smokers. Further, we identified SNPs conferring susceptibility for C pneumoniae seropositivity, although no direct association with COPD was observed.In this study, we demonstrated that seropositivity for C pneumoniae IgA or IgG,15 the East London COPD study in 110 patients with stable COPD reported an inconsistent result.29 A plausible reason for this discrepancy is insufficient statistical power due to the relatively small sample size. For example, von Hertzen et al13 compared IgA and IgG titers in 54 COPD patients and 321 healthy controls, and Brandén et al15 investigated the association of chronic C pneumoniae infection with longstanding airway symptoms in 199 patients, of whom 30 were diagnosed with COPD. In a general population, 1 study investigated the association of C pneumoniae infection with pulmonary function in 1773 men30 and failed to find any association with outcome measures, including baseline spirometric parameters and FEV1 decline during the 5-year follow-up period. However, the frequency of current or past smokers in the previous study (>80%) was more than double that in our present study, and more than double that in the National Survey on Circulatory Disorders of Japan,31 which reported that the frequency of current or past smokers in Japan is approximately 40%. The harmful effects of smoking might therefore render the potential pathogenicity of C pneumoniae infection undetectable. Given that our present study was based on a large population with a ratio of smokers to nonsmokers equivalent to that of the national survey, our observations may provide strong evidence that C pneumoniae seropositivity is a risk factor for reduced pulmonary function in general populations.Although several studies reported that patients with COPD had a significantly higher titer of serum M pneumoniae is another atypical pathogen of community-acquired pneumonia. Although several small studies reported a possible risk of serologically identified M pneumoniae for acute exacerbation of COPD,33 other studies denied this etiological relationship.35 Clinical and epidemiological data on the pulmonary risk of M pneumoniae infection is thus limited and conflicting even in COPD patients. In addition, no data are available on the association between M pneumoniae infection and spirometric parameters. Our present findings are therefore important in helping to elucidate the pulmonary risk of M pneumoniae infection in a general population. Further, the risk of M pneumoniae infection was equal to or somewhat greater than that of C pneumoniae. Of note, however, subjects infected with M pneumoniae first appeared to have better pulmonary function, presumably due to their younger age.5 One potential factor in explaining the possible relationship between COPD and cardiovascular outcomes is atherosclerosis.36 Given that the present and previous reports showed a positive association between C pneumoniae seropositivity and atherosclerotic vascular change,38 as well as cardiovascular and all-cause mortality,39C pneumoniae infection might be a factor underlying the relationship between COPD and cardiovascular outcomes.In general populations, even mild to moderate airflow limitation and COPD have been suggested to confer a risk for the incidence of cardiovascular diseases.C pneumoniae. According to the Encyclopedia of DNA Elements (ENCODE) project datasets (UCSC genome browser: http://genome.ucsc.edu/), this polymorphism lies approximately 22 kb upstream of the IKZF1 gene. A recent systemic expression quantitative trait locus (eQTL) analysis40 found that the T allele of the rs4917014 genotype, a proxy of the G allele of the rs17634369 genotype, was associated with increased expression of the IKZF1 gene (cis-effect). Further, very recently, the rs4917014 genotype was identified as susceptible for cold medicine-related Stevens–Johnson syndrome via a mechanism involving a change in the quantitative ratio of the IKZF1 alternative splicing isoforms Ik1 and Ik2.41 Although we did not perform functional analysis for the relationship between rs17634369 or rs4917014 genotype and C pneumoniae Ig A index, these previous results are sufficient to consider IKZF1 as a factor in C pneumoniae seropositivity.The rs17634369 genotype was significantly associated with the seropositive prevalence of IKZF1 encodes the transcription factor “Ikaros.” Ikaros was reported to be associated with transcriptional regulation of human STAT4,42 which is involved in cell-mediated immune responses via Th1 cell development.43 The STAT4 pathway may therefore help explain the association between IKZF1 genotype and C pneumoniae seropositivity. The eQTL analysis also reported a significant association between the rs4917014 genotype and increased expression of genes involved in the type 1 interferon response (trans-effect).40 Given that increased expression of interferon-α response genes is a distinct expression pattern in SLE patients,44 and that the rs4917014 genotype confers susceptibility to SLE,28 the increased C pneumoniae IgA levels and higher risk of SLE in individuals with the risk allele might partially share a common pathophysiological pathway.C pneumoniae, but interlaboratory variability is an unanswered issue. An ELISA is another convenient method of serodiagnosis. However, discrepancies in the detection ratio between indirect  and direct (PCR) methods remain to be resolved. Our present findings may suggest that the discrepancy in detection ratio among populations, which plays at least some role in the controversy over C pneumoniae infection in chronic inflammatory diseases, may in part be due to genetic heritability.45The microimmunofluorescence (MIF) test is currently the objective standard for the serodiagnosis of pneumoniae by measuring seropositivity. Although the presence of pneumoniae antibodies may reflect the presence of prior infection, this may not be a valid measure of persistent and chronic active reinfection. A guideline from a workshop on the standardization of C pneumoniae diagnostic methods46 suggested that no valid serological marker specific for chronic C pneumoniae infection is available and that any interpretation of infection status on the basis of single titer readings should be done with care. Second, we might not have entirely excluded subjects with bronchial asthma due to a lack of postbronchodilator spirometry values, or subjects with other pulmonary diseases such as diffuse bronchiectasis and bronchiolitis obliterans. Given the positive association between atypical bacterial infection and asthma,47 our findings might have been confounded by interaction with the reversible airflow obstruction. However, our observed positive association between C pneumoniae and M pneumoniae seropositivity and COPD remained significant after the exclusion of subjects with self-reported medication for bronchial asthma or COPD, or with pulmonary disease. Third, as our study subjects were Japanese, the results of this study might not be simply extrapolatable to other populations with different environmental backgrounds. Further, the dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/) shows ethnic differences in the rs17634369 genotype, with the highest frequency of the G allele in Africans, followed by Asians and Caucasians.Several limitations of our study warrant mention. First, we could not distinguish between persistent and past infection of C pneumoniae and M pneumoniae seropositivity is an independent risk factor for pulmonary function. Further investigations are needed to clarify mechanisms regarding the disease pathogenicity of pneumoniae infection.In summary, our study clarified that dual


Chlamydomonas reinhardtii for alterations in oil content. A mutant accumulating five times more oil and twice more starch than wild-type during optimal growth was isolated and named constitutive oil accumulator 1 (coa1). Growth in photobioreactors under highly controlled conditions revealed that the increase in oil and starch content in coa1 was dependent on light intensity. Genetic analysis and DNA hybridization pointed to a single insertional event responsible for the phenotype. Whole genome re-sequencing identified in coa1 a >200 kb deletion on chromosome 14 containing 41 genes. This study demonstrates that, 1), the generation of algal strains accumulating higher reserve amount without compromising biomass accumulation is feasible; 2), light is an important parameter in phenotypic analysis; and 3), a chromosomal region (Quantitative Trait Locus) acts as suppressor of carbon reserve accumulation during optimal growth.Microalgae have emerged as a promising source for biofuel production. Massive oil and starch accumulation in microalgae is possible, but occurs mostly when biomass growth is impaired. The molecular networks underlying the negative correlation between growth and reserve formation are not known. Thus isolation of strains capable of accumulating carbon reserves during optimal growth would be highly desirable. To this end, we screened an insertional mutant library of  With increasing world population and diminishing fossil fuels, alternative fuel sources that are renewable and do not compete with food production for fresh water and arable land are highly sought after11245678nrr1, tar1, and std1) provided regulatory clues as to how cells control oil accumulation in response to nitrogen starvation. Thus, these studies have mostly focused on the transitional changes from a healthy cell to a stressed cell. To our knowledge, no studies have been dedicated to investigate carbon reserve accumulation during optimal growth thus no regulatory gene or protein involved has been reported.The transition from optimal growth to carbon reserve accumulation requires changes at several systems levels including transcriptome, proteome and metabolome, highlighting the tight control that a cell exerts on the repartition of cellular carbon and energy usage8101112131415Chlamydomonas reinhardtii aiming at identifying new regulatory genes from the screening of mutants with altered cellular oil content during optimal growth. We used C. reinhardtii as a model because it has been proven as a suitable alga to study both starch and lipid metabolism2021222324coa1 standing for constitutive oil accumulator 1. This mutant accumulated increased oil and starch relative to the wild-type strain in a light-dependent manner. The isolation of this mutant should provide a molecular basis for our understanding of the control of TAG and starch accumulation during optimal growth.Under environmental conditions favorable for growth, genes of the lipid and starch biosynthetic pathways are expressed at low levelsconstitutive oil accumulator 1 (coa1). No significant difference could be detected in the major membrane lipids  and was maintained at a constant value (OD880nm = 0.4 corresponding to 2 million cells mL−1) by step-wise injection of fresh culture medium. Using this system, photoautotrophic cultures can be maintained at physiological steady states under different light regimes. It should be mentioned here that because PBRs are radially illuminated and cell density maintained at a relatively low level, the light required in this set-up to observe physiological effects of the same magnitude as in flasks are much lower . Increasing light intensity from 40 to 120 μmol photons m−2 s−1 had very little effect on intracellular oil levels in the WT strain, but led to an increase in oil content from 0.5 to 2.5 μg mm−3 in mutant cells  thus resulting in an increase in the chlorophyll a/b (Chl a/b) ratioa/b was observed in both strains under HL, the Chl a/b ratio being however higher in the coa1 mutant than in the WT  antenna protein LHCB5 in the coa1 in comparison to the WT, with LHCB5 decreasing in response to HL acclimation in both strains . After digesting the coa1 genomic DNA with several restriction enzymes independently, a single hybridizing band was observed in all cases using a probe specific to the DNA cassette harboring paromomycin gene AphVIII. This suggested that there is only one insertion of AphVIII cassette in the coa1 genome  and the wild-type strain CC125 (mt+) which is the mt− strain mostly closely related to the coa1’s progenitor (CC124). From the analysis of 3 complete tetrads . Global and local coverage was determined by analysis of BAM output by GATK . The genomic region between 69,200 bp and 315,218 bp compared to the reference Chlamydomonas genome version 4.3 revealed low read coverage in the region of the insertion/deletion . Peaks of high read coverage in the region between 69,200 bp and 317,163 bp on the reference genome were identified as highly repetitive motifs found elsewhere in the Chlamydomonas genome. Furthermore, reads mapped to the 3′ and 5′ end of the gene cassette, and reads paired to those which mapped to the cassette revealed that the genomic loci adjacent to the gene cassette had sequence identity to 69,200 bp and 315,218 bp on chromosome 14.Genomic reads were paired using FASTQ joinerAphVIII cassette was inserted in a 3′ –>5′ orientation between the 69,200 bp and 317,163 bp on chromosome 14, resulting in a substantial genomic deletion of >200 kb in chromosome 14 between the coordinates 69,266 and 317,163 , the cellular homeostasis of carbon reserves is disturbed, thus creating strains that over-accumulate oil and starch simultaneous to growth. Furthermore, it is found that one or more of the genes encoded on the same region may be key in the orchestration of the light response in C. reinhardtii. The reason Chlamydomonas exhibits an increased proportion of TAG and starch during high light growth remains poorly defined, and may remain difficult to analyze without the access to genome editing in Chlamydomonas, however the generation of an indexed genome-wide mutant library for C. reinhardtii (http://jonikaslab.dpb.carnegiescience.edu/chlamy-mutant-library)The link between the lesion on chromosome 14 and the observed increased level of TAG and starch under high light conditions suggest that carbon reserve formation is repressed in optimal growth in wild-type strains. In the absence of this chromosomic region  as transcription factors involved in the response to the physiological pressure of low carbon growthb reductase (Cre14.g608800) is also located in this chromosomic region. Chlorophyllide b reductase catalyzes the first step in the degradation of chlorophyll b in the chlorophyll cyclecoa1 mutant may have perturbed the chlorophyll cycle thus explaining the altered chlorophyll content and chlorophyll a/b ratio observed in the mutant , indicating a higher sensitivity to light that may contribute to an increased overall flux in cellular carbon and an increase in oil content.While there is little information known about the proteins in the locus of interest , some pre mutant . IncreasChlamydomonas, coa1, which accumulates five times more oil and twice more starch than the WT during optimal growth. To our knowledge, this is the first report of such a mutant accumulating higher oil amount in parallel to optimal growth for the green alga C. reinhardtii. Through the study of the mutant coa1, we also pointed out a missing link underlying the regulation of reserve accumulation under high light conditions. Molecular characterization of the coa1 mutant identified a QTL responsible for the observed phenotype. Detailed examination of the genes, via cross-references to published transcriptomic datasets (http://jonikaslab.dpb.carnegiescience.edu/chlamy-mutant-library)1013coa1 phenotype to particular gene(s) located within this region on chromosome 14. Thus the isolation of the coa1 mutant demonstrates that mutants over-accumulating carbon reserves under non-stress conditions can be isolated and further provides a list of candidate genes involved in suppressing carbon reserve accumulation during optimal growth.One of the major obstacles to economically feasible production of algal fuel is the requirement of nitrogen starvation or other stress conditions for higher reserve accumulationdatasets 34, encodChlamydomonas reinhardtii wild-type strain CC124 (mt−nit1 nit2), obtained from the Chlamydomonas resource center (http://chlamycollection.org), was used to generate the insertional mutant library in our laboratory as described previously in Nguyen et al.AphVIII was inserted into the genome of C. reinhardtii via electroporationet al.−2 s−1) with shaking (120 rpm)g, cell pellets were washed twice in TAP-N media, then resuspended into TAP-N for 3 days.TM3 Coulter Counter, Beckman Coulter, USA).Cell numbers, sizes and total cellular volumes were quantified by the use of a Beckman Coulter Multisizer 4 −1 from a stock solution of 1 mg mL−1 in DMSOTo observe lipid droplets, cells were first stained with a solution of Nile red  and operated as turbidostats. Cells were maintained at a constant density (OD880nm = 0.4) throughout experimentation by automated injection of fresh media  and ejection of excess biomass through a waste tube. Culture pH was maintained constant by addition of 0.2 N HCl or 0.2 N KOH using the Biostat module. A gas mixture containing air plus 1.8% CO2 was generated using two mass flow meters  and bubbled into the PBRs at a flow rate of 0.5 L min−1. Eight fluorescent bulbs (Osram Dulux L 18 W) placed around the PBR were used to supply light in a range of light intensities up to 400 μmol photons m−2 s−1.PBR cultures were cultivated in minimal medium et al.g for 2 min at 4 °C in a bench top centrifuge. The lower phase was then isolated and 1 mL of hexane was added to re-extract the lipids, and samples were vortexed again for 10 min and phases were separated. The upper phase containing lipids was then transferred to the chloroform extract using a glass Pasteur pipette, and the rest was discarded. The combined chloroform and hexane extracts were then dried under a flow of nitrogen gas and re-suspended in an appropriate amount of solvent for subsequent analyses. Cellular content of lipid classes were quantified with Thin Layer Chromatography (TLC) using an automated “High-Performance Thin-Layer Chromatography” platform (CAMAG). These procedures were carried out as described previouslyA modified Bligh and Dyer methodg for 10 min) and cell pellets were resuspended in 1 mL methanol. The mixture was kept at −20 °C for at least 1 h before the organic phase was taken for chlorophyll quantification using spectrophotometerA given number of cells (~2 million cells) were harvested by centrifugation (13000 coa1 mutant was back-crossed with the wild-type CC125 (mt+). Genetic crosses were performed following the protocol described previouslyChlamydomonas strains of mt− and mt+ were cultivated in TAP medium until mid-log phase. Cells were gently centrifuged at 360 g for 3 min, washed twice with TAP-N medium and resuspended in the same medium. Cultures were kept in TAP-N for 24 h at 25 °C under shaking. Cells with opposite mating types were mixed together in a glass tube (1 mL:1 mL) and then the tubes were kept in an incline position in a laminar hood for 3 hr. The mixture (1 mL) was then spread into center of TAP plates containing 3% agar (w/v), the plates were dried and kept under low light overnight. Then the plates were wrapped in aluminum foil, kept in the dark for 5–10 days to induce zygote germination upon induction by light. All the vegetative cells were scraped from the plate with a dull scalpel. To further eliminate the vegetative cells, the plates were exposed to chloroform vapor for 20–45 sec. The plates were then placed under low light for 24 h. The tetrads were dissected using a micromanipulator needle connected to a light microscopy under a laminar hood.The Chlamydomonas cells using a Qiagen Plant DNeasy Max kit (with QIAshredder). Briefly, a 200-mL culture of exponentially grown cells in TAP media was centrifuged at 600 g for 5 min at 4 °C. Cells were snap frozen in liquid nitrogen, pellets were briefly pulverised with a sterile mortar and pestle. The rest of the procedure was following that described by the manufacture. Extracted DNA was quantified using a spectrophotometer, and DNA purity was calculated relative to the ratio of the absorbance of A260 nm/A280 nm.Total DNA was extracted and purified from exponentially grown AphVIII gene was cloned by PCR (with primer AphVIII-F: (5′-CGAAGCATGGACGATGCGTT-3′ and AphVIII-R: 5′-CGAGACTGCGATCGAACGGACA-3′) and labelled with Digoxigenin-dUTP  to be used as a hybridization probe. Before hybridization, the probe was denatured by heating at 100 °C for 10 min, and then kept on ice for 5 min. Genomic DNA (~8 μg) was digested overnight by NotI, StuI, or ApaI (NEB Biolabs). Digested DNA was loaded onto a 0.8% agarose gel, and each lane representing DNA cut by one specific enzyme and the gel was run slowly at 50 V to better separate fragmented DNA. Migration, transfer, and hybridization were described in detail inAphVIII insertions in the genome.A fragment of the TM kit (Clontech). In brief, DNA (1 μg) was digested using restriction enzymes FspI, NaeI, PmlI, PvuII, or SmaI (New-England Labs). Digested DNA was then ligated to adapter sequences (5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′). Nested PCRs were carried out using two sets of oligonucleotides, i.e. GWaph8fw1 (5′-CTGGTGCTGCGCGAGCTGGCCCACGAGGAG-3′) or GWaph8rev1 (5′-CCAGCGCGAGATCGGAGTGCCGGTCCG-3′) with AP1 (5′-GTAATACGACTCACTATAGGGC-3′), and GWaph8fw2 (5′-TGGTTCGGGCCGGAGTGTTCCGCGGCGTT-3′) or GWaph8rev2 (5′-CGAGACTGCGATCGAACGGACACCGC-3′) with AP2 (5′-ACTATAGGGCACGCGTGGT-3′), to the cassette, and the adapter sequence in a final volume of 50 μL. PCR product was then cloned and sequenced. The obtained sequence was then put through a BLAST against the genome of C. reinhardtii at PhytozomeTo identify the position of cassette insertion in the genome, genome walking PCR was carried out using a modified protocol of the commercial Genome Walker−1; and two biological replicates were prepared; OD260/280 = 1.66) was sent to Beijing Genome Institute  for whole genome re-sequencing. Two Genomic read files in FASTQ (fq) format obtained from the Beijing Genome Institute  were uploaded to Galaxy (https://main.g2.bx.psu.edu/)Chlamydomonas genome v5 was uploaded as a template genome, the genome was downloaded as C_reinhardtii.110311.tar.gz from http://www.chlamy.org. Joined reads were then mapped using Bowtie with a gap length of 500 bp, and visualized with Trackster53Highly pure DNA .


Speleothem δ18O reveals nine quasi-cyclical events of relatively wet-to-dry climatic conditions during the Holocene. Dynamic Harmonic Regression modelling indicates that changes in precipitation occurred with a ~1500 year frequency during the late Holocene and at a shorter length during the early Holocene. The timing of these cycles coincides with changes in North Atlantic Ocean conditions, indicating a connectivity between ocean conditions and Holocene moisture delivery. Early Holocene climate is potentially dominated by freshwater outburst events, whilst ~1500 year cycles in the late Holocene are more likely driven by changes internal to the ocean system. This is the first continental record of its type that clearly demonstrates millennial scale connectivity between the pulse of the ocean and precipitation over Europe through the entirety of the Holocene.Century-to-millennial scale fluctuations in precipitation and temperature are an established feature of European Holocene climates. Changes in moisture delivery are driven by complex interactions between ocean moisture sources and atmospheric circulation modes, making it difficult to resolve the drivers behind millennial scale variability in European precipitation. Here, we present two overlapping decadal resolution speleothem oxygen isotope (δ Holocene ~11.7–0 ka) climate is characterised by a series of century-to-millennial scale changes which have had varied regional impacts1.7–0 ka 24537910112133611239103618O) record derived from speleothems contained within Cueva de Asiul  situated in the Matienzo depression (Cantabria), N. Spain1920212 = 0.51 p < 0.01), with lower δ18O related to higher rainfall totals242526272 = 0.6; 1948–2008 AD)18O of rainfallHere, we add to this debate by reconstructing precipitation delivery to northern Spain during the last 12.1 ka, using a carbonate oxygen isotope  conditions force winter storm tracks associated with the Atlantic Westerly Jet (AWJ) to migrate northwards29118O composition from the preceding year1918O records provide an almost complete Holocene archive of precipitation amount with a resolution of between 1 and 28 years/ data point  grew just 5 m apart, in the Cueva de Asiul system in a stable cave environment (air temp 13.7 ± 0.5 °C)19ta point , with th18O which occur throughout the Cueva de Asiul data provide timings for reductions in moisture delivery  cycle becomes dominant. The changing cycle amplitudes and periodicities have been estimated using Dynamic Harmonic Regression332 = 0.74) of the variance observed in the remainder of the speleothem record with its residuals not indicating any significant shorter or longer term cycles.The relatively large amplitude (1–2‰) peaks in carbonate δdelivery . FrequenThe beginning of the Cueva de Asiul speleothem record coincides with the latter part of the Younger Dryas  and conf303835The last 2.5 ka of the record broadly agrees with recent high resolution reconstructions of NAO conditions from other European archives124144Variations in the Cueva de Asiul speleothem moisture record are shown to occur on a range of temporal scales. Decadal to centennial fluctuations during the last 2.5 ka broadly agree with other European archives of the NAO33046During the early Holocene, North Atlantic Ocean conditions were significantly influenced by meltwater outburst events48493718O identifies ~6.5 ka as a marker for a change in this millennial cycle and the start of a longer period cycle, often quoted at 1600–1800 years393212At the end of deglaciation the impact of freshwater release into the North Atlantic is reduced, co-inciding with a change in periodicity observed in ocean records3The longer memory effects of the ocean2115222305413310In identifying nine cycles of wetting and drying in northern Iberia during the Holocene, the Cueva de Asiul speleothem data offer a significant contribution to existing terrestrial climate records in Europe. The correlation between these wet and dry events and changes in the North Atlantic Ocean 311 highl32The combined chronology is based upon 10 U-Th ages from speleothem ASR and 12 U-Th ages from ASM .


Melosira arctica showed sign of different stages of degradation and physiological stoichiometries, with carbon to chlorophyll a ratios ranging from 110 to 66700, and carbon to nitrogen molar ratios of 8–35 and 9–40, respectively. Sub-ice algal aggregate densities ranged between 1 and 17 aggregates m−2, maintaining an estimated net primary production of 0.4–40 mg C m−2 d−1, and accounted for 3–80% of total phototrophic biomass and up to 94% of local net primary production. A potential factor controlling the buoyancy of the aggregates was light intensity, regulating photosynthetic oxygen production and the amount of gas bubbles trapped within the mucous matrix, even at low ambient nutrient concentrations. Our data-set was used to evaluate the distribution and importance of Arctic algal aggregates as carbon source for pelagic and benthic communities.Sea-ice diatoms are known to accumulate in large aggregates in and under sea ice and in melt ponds. There is recent evidence from the Arctic that such aggregates can contribute substantially to particle export when sinking from the ice. The role and regulation of microbial aggregation in the highly seasonal, nutrient- and light-limited Arctic sea-ice ecosystem is not well understood. To elucidate the mechanisms controlling the formation and export of algal aggregates from sea ice, we investigated samples taken in late summer 2011 and 2012, during two cruises to the Eurasian Basin of the Central Arctic Ocean. Spherical aggregates densely packed with pennate diatoms, as well as filamentous aggregates formed by  The endPlanktonic diatoms tend to collide and aggregate in nutrient depleted waters after a bloom due to mucus secretion during senescence Many questions remain about aggregate formation, decay, grazing and sinking, as well as their contribution to carbon fluxes in the Central Arctic. Due to the rapid warming of the Arctic leading to a decrease in sea-ice extent and thickness http://doi.pangaea.de/10.1594/PANGAEA.832345).Samples were collected during two cruises to international waters of the Central Arctic Ocean with RV Polarstern in August-September 2011 and 2012 (PS78/3 and PS80/3 respectively) between 81°55′–87°56′N and 31°7′–131°7′E . Six staMelosira were sampled at eight ice stations . Irradiance reaching the aggregates was calculated using the light attenuation coefficients of 1.5 m−1 for sea ice, 10 m−1 for snow −1 for Atlantic influenced Arctic seawater , and the daily average total incoming photosynthetically active radiation (PAR) measured with a pyranometer  mounted on the ship.Replicates of two different algal aggregate types dominated either by pennate diatoms or by stations . For eacMelosira arctica filaments 5 times (a and phaeopigments were measured using high-performance liquid chromatography (HPLC) as described in a measurements was generally below 2%. For transparent exopolymers (TEP), 1–20 ml of the algal slurry  were filtered in triplicate on to 0.4 µm pore size polycarbonate filters , stained with Alcian Blue and stored at −20°C. TEP concentration was measured with the colorimetric method according to Several aggregates (between 2 and 20) were sampled at each station together with some ambient water using a manually operated vacuum pump, when found in melt ponds , or a pl 5 times . AggregaThe qualitative algal aggregate composition was studied directly on board of the ship with a plankton chamber  and an inverted light microscope with phase contrast optics  and an integrated camera . The condition of the diatoms in each sample was defined according to the color and shape of the chloroplasts visible in the interior of the frustules. Green-brownish aggregates contained mostly healthy cells with bright green chloroplasts occupying the entire cytoplasm, while white-yellowish aggregates contained many dead cells with empty frustules or reduced yellowish chloroplasts. To determine bacterial abundance with Acridine Orange direct counts (AODC), sub-samples of the algal slurry were preserved with 2% formalin and sonicated (1×90 s). One milliliter was filtered on to a 0.2 µm pore size polycarbonate filter  previously stained with Irgalan black to eliminate autofluorescence −2.Although green-brownish sub-ice algal aggregates were observed at all ice stations a and POC as for the aggregate slurry. For the phytoplankton community in the water below the ice, samples were taken with Niskin bottles disposed in a rosette attached to the Conductivity-Temperature-Depth sensor (CTD) at discrete depths, the water was filtered, and Chl a and POC was analyzed as above. Water column phytoplankton biomass was integrated over the euphotic zone (1% incoming irradiance) while sea-ice algal biomass accounted for the ice thickness . Temperature was maintained stable at −1.3°C with a thermo bath . Three clear bottles with 10 mL each were incubated in the light and one in the dark. The estimated error of the method from the triplicates incubated in the light was 15% on average. Floating aggregate slurries collected at station PS80/3_224 and PS80/3_237 were incubated at 50 µmol photons m−2 s−1, which was a typical mid-day in situ value. Aggregate slurries from station PS80/3_349, as well as sea ice and water column of all stations were incubated under a range of irradiances  during 24 hours to calculate the in situ NPP from photosynthesis vs irradiance curves (PE curves). Using this incubation time NPP is assessed (gross PP minus respiration) 2>0.5 were retained. In situ NPP was calculated applying the irradiance measurements at the ice station to the PE curve equation.Depth-integrated NPP was estimated from Melosira filamentous biomass (Aggregate M5) was estimated from scaled images of the open melt pond  by the number of aggregates observed per m2 and by the carbon concentration of the aggregate per volume (mg C m−3). The aggregate NPP per m2 was calculated similarly, by multiplying the total aggregate volume per m2 by the measured carbon fixation rate. Chl a was estimated from the C:Chl a ratio measured in the algal slurry from the same aggregate type. A similar procedure was used for the C:N ratios. Since aggregates showed a very patchy distribution, these calculations reflect local estimates at the sampling spot and cannot be directly up scaled to the Arctic-wide ice-cover.To quantify the aggregated biomass and calculate NPP per area, aggregate abundance was quantified from scaled images. At stations PS80/3_224 (Aggregate P4) and PS80/3_237 (Aggregate P5) mean aggregate abundance under the ice was estimated from images taken by the upward looking camera of a Remotely Operated Vehicle (ROV) as described in elt pond . The aggin situ conditions: low light (8 µmol photons m−2 s−1) and cold temperature (0°C). The initial temperature of the melt pond water collected for the experiment was −1°C. After 12 h in the cold lab-container (0°C) under low light intensity (8 µmol photons m−2 s−1), the water reached 0°C and the piece of ice started melting. The vertical position of the aggregates was followed over time and as a function of the salinity gradient and the light intensity, to assess their buoyancy at different environmental conditions. In parallel, sub-samples of the algal slurry from the same type of aggregate were incubated under different conditions to assess oxygen, carbon and nitrogen fluxes.On 18 September 2012, station PS80/3_349 , a piece of ice from the frozen surface of an open melt pond was retrieved with algal aggregates attached . These w14C uptake rates, as described above. In addition, oxygen concentration was followed in the same vials using fiber optic oxygen sensors . Oxygen sensitive sensor spots were glued to the interior of the glass vials previous to the experiment. The sensor spots contain an oxygen quenchable fluorophore (oxygen sensitive dye) which changes its fluorescence properties according to the oxygen concentration 2>0.93) and a photosynthetic quotient of 1.25 was used to convert the oxygen exchange to carbon equivalents Five sub-samples were incubated under different light intensities to estimate NPP from −2 s−1 and nitrate was measured initially and after 1 and 4 days. In parallel, 18 subsamples of the algal slurry were incubated in 12 mL exetainers  to measure the denitrification and nitrification potential. Subsamples of the slurries were spiked either with 15NO3, with 15NH4+ or with a mixture of 14NO3 and 15NH4+ , using a modified version of the method described in −2 s−1, while the remaining sub-samples were placed in darkness. The gradual O2 consumption was followed in both set ups using the optode system described above. Anoxic conditions were reached after two days in the dark. The first time series was ended immediately by injecting 100 µL ZnCl2 into the exetainers, while the remaining samples were terminated as the oxygen level reached 67, 35, and 0%, after 0.8, 1.2 and 2 days respectively. The isotopic composition of the N2 was measured using a gas chromatograph coupled to an isotope ratio mass spectrometer through a ConFlo-III interphase x) were determined To measure the nitrate uptake, a 50 mL slurry sub-sample was incubated at 50 µmol photons mDuring August and September 2011 and 2012, FYI in a late melting stage was the dominant ice type at seven of the eight ice stations investigated . Sea-iceNitzschia sp., Navicula sp., Fragilariopsis sp., and Entomoneis sp.)  , and (2) arctica .−1 nitrate, 0.2 µmol L−1 phosphate, and 2 µmol L−1 silicate at the bottom of melt ponds or below the ice.Both the pennate and centric diatom aggregates were present in different degradation stages . Their ca mass ratios than green-brownish aggregates   of the POC. Greenish-brownish aggregates hosted one order of magnitude more bacteria than white-yellowish aggregates. The DOC concentrations of the algal aggregates ranged between 5 and 35 µmol DOC mg POC−1  to the particulate organic carbon pool of the integrated euphotic zone  were higher than the values for the phytoplankton (∼5) and the sea-ice algae (6–8). Green-brownish Melosira aggregates showed higher C:N ratios (9–28) than all pennate aggregates (8–11). In white-yellowish aggregates C:N ratios were generally higher (11–35 in pennate and 10–40 in Melosira aggregates).At the sampled sites, floating aggregates contributed with 0.5–26% (pennate), and 55–78%   and the sea ice in late September  . In conteptember . The daiMelosira arctica filaments found at station PS80/3_349, both carbon uptake and oxygen production were monitored in homogenized algal slurries at different light intensities. The fitted PE curve of the carbon uptake (R2 = 0.986), showed a photosynthetic maximum (Pmax) of 3.7 mg C L−1 d−1, an initial slope of 120 µg C L−1 d−1 (µmol photons m−2 s−1)−1 and no photoinhibition at irradiances lower than 90 µmol photons m−2 s−1 (−2 s−1). However, no net oxygen production took place at this irradiance, rather oxygen was consumed  anchored to newly formed ice from station PS80/3_349, composed of a mixture of chia sp. , Fig. 5Achia sp. . Once thchia sp. . It was chia sp.  bottom. chia sp. . After 2chia sp. . To testbuoyancy , indicatMelosira slurry showed a bulk oxygen consumption rate of 0.13±0.02 mmol O2 L−1 d−1 at low irradiance (8 µmol photons m−2 s−1). Non-invasive measurements with microsensors on one pennate-diatom also showed net oxygen consumption with almost anoxic conditions (10–120 µmol L−1 Oxygen) in the center of the aggregate (see The gate see .2 production to O2 respiration we transformed the oxygen consumption rate mentioned above into carbon respiration rate. Dividing the POC measured in each sample (Melosira slurry. The turnover of particulate carbon at low irradiance (8 µmol photons m−2 s−1) would be 8–11 days. Net oxygen consumption and carbon remineralization occurred at lower light intensities in the Melosira algal slurry than in the compact pennate-diatom aggregate. Floating green-brownish aggregates incubated for 4–5 days at low irradiances lost their buoyancy and became net heterotrophic. Oxygen production in both aggregate types was probably light regulated. However, this observation is based on single measurements, with the two respective types of aggregates. Further experiments are needed to fully conclude that light-regulated oxygen production is responsible for aggregate buoyancy.Assuming a 1∶1.25 ratio of COh sample  by the cMelosira algal slurry sample incubated under in situ representative light conditions (50 µmol photons m−2 s−1). During the first 24 h, the nitrate consumption rate was 0.94 µmol N L−1 and the ammonium consumption 2.47 µmol N L−1. Denitrification and nitrification potential was measured in slurry samples spiked with labeled nitrate and ammonium, respectively (15NO3 and 15NH4+). During light, nitrate was produced at a rate of 1.7±0.001 µmol L−1 d−1 and ammonium was consumed at a rate of 1.6±0.003 µmol L −1 d−1. In the dark, nitrate was produced at lower rates (0.4–0.5 µmol L−1 d−1) and high net ammonification rates were measured (1.5–5.8 µmol L−1 d−1). A potential for denitrification could be detected (2–5 nmol L−1 d−1) under anoxic conditions. Aggregates appeared to be hot spots for nitrogen cycling with potential for ammonification, nitrification and denitrification, and the rates seemed to be regulated by ambient light availability.Nitrate and ammonium concentrations were monitored in parallel in the Different types of aggregations of algae have been described in Arctic sea ice, mostly by observations made during summer, below pack ice Based on our measurements and a synthesis of previous studies, we propose a conceptual model for aggregate formation and degradation in the Arctic . This coMelosira arctica, grows forming chains and excretes high amounts of mucus that contribute to the formation of filament-shaped aggregates Melosira arctica, build up dense biomass accumulations despite nutrient constrains, that can be exported to the deep sea upon rapid melting of the sea ice Newly formed melt ponds are usually shallow, light blue, have low salinity, and contain little visible life a concentrations (2–6 µg Chl a m−2) when up scaling the contribution of the observed aggregates to the area of the entire ice floe. Here we assessed the standing stock and productivity at a local scale, for two different types of sea-ice algal aggregates. Our study took place at the end of the summer, when nutrient depletion limits pelagic productivity. During this period, we found that algal aggregates contributed significantly to total biomass at a local scale in and below the sea-ice.The spatial and temporal patchiness remains a challenge when estimating the importance of algal aggregates for ecosystem productivity and carbon flux a reported here for spherical aggregates formed by pennate diatoms . The overall relatively low mass ratios of C:Chl a of 110 and 212 for green-brownish pennate and centric diatom aggregates, respectively, indicate a healthy algal community. However, compared to the water column C:N ratio of 5–6, the green-brownish aggregates had an elevated C:N ratio . The white-yellowish aggregates containing many empty frustules even reached ratios of 40. This suggests an important contribution of exudate-carbon to the aggregates.Similar conclusions as to the relative contribution to biomass are reached using the POC values of the aggregates compared to the total sympagic or pelagic community. The contribution to total PON at a local scale was lower (0.3–18%) for pennate aggregates than for in situ integrated NPP at a local scale, and Melosira arctica filaments as much as 83–94% of the total NPP north of 80°N in mid-September, when very little production is taking place in the water column or in the sea-ice. Melosira filaments can be more productive than the integrated euphotic zone in and below the ice in the Central Arctic  than in the ambient water (105–107 cells mg POC−1) suggesting that aggregates are a rich substrate for bacteria. The higher concentrations of DOC in the aggregates compared to the average DOC concentrations of Arctic surface seawater Detrimental conditions to the aggregate community such as very low salinity from melt water, seawater warming, high grazing pressure, or nutrient depletion will eventually lead to buoyancy loss, degradation and sinking Melosira aggregate slurry, suggesting that nitrification might take place as well under photosynthetic conditions at high light intensities. This means that decaying algal aggregates could be sources of nitrate to the surrounding seawater in an otherwise nitrogen-limited system. Previously it has been speculated that internal nutrient regeneration by bacterial remineralization inside aggregates can support growth of at least a fraction of the algal population 2 production, may occur in the anoxic center of intact aggregates Melosira algal slurry were low (0.002–0.005 µmol L−1 d −1). This could indicate that the aggregates might remove considerable amounts of carbon and nitrogen from surface waters by export. Using the aggregate carbon export of 9 g C m−2 estimated previously for 2012 , and the median C:N ratio of 10 of the fresh aggregates (n = 4), approximately 45% of carbon fixed as new production since last winter and 36% of surface nitrogen consumed by sea-ice algae and surface water phytoplankton was exported to depths >4000 m.Net heterotrophic conditions were measured in floating sea-ice algal aggregates during summer indicating a rapid decomposition of the algal biomass in the aggregates . MoreoveMelosira arctica-based ice-algal aggregates contribute substantially to nutrient and carbon cycling and export in the Arctic. Preliminary experiments suggest that their buoyancy is regulated by photosynthetically-produced oxygen. With the current trend of warming of the Arctic causing sea ice retreat as well as thinning of the ice cover that increases drift speed, it is likely that sea-ice algae can grow faster and earlier in the season harvesting nutrients from a wider area of surface waters, but will more often meet unfavorable conditions during summer, e.g. nutrient limitation, freshening and melting of their habitat favoring aggregate formation and degradation. The advantages of the formation of aggregates compared to the single-cell or short-chain life style of pelagic sea ice algae is uncertain. Aggregation into large clumps and filaments may protect diatom species such as Melosira arctica from high irradiances. Trapping of gas bubbles in the aggregates enhancing floatation and eventually serving entrapment into the ice in late autumn might be another advantage. The intense nitrogen cycling observed in the aggregates indicates that they could play a role in nutrient cycling in the oligotrophic Arctic Ocean. It remains methodologically difficult, but important for a better understanding of carbon and nutrient budgets as well as for the ecology of the Arctic Ocean, to quantify temporal and regional variations in aggregate formation, distribution and fate on the ecosystem scale.In conclusion, we suggest that pennate diatom and Figure S1Oxygen profile inside degrading pennate diatom aggregate (P5). Oxygen microprofiles (n = 3) measured using an oxygen microoptode  in a 5 cm diameter spherical pennate-diatom aggregate incubated in a beaker in the lab for 3 days at 50 µmol photons m−2 s−1 and −1.3°C. Oxygen microprofiles across the water-aggregate interface were measured with steps of 0.5 mm and since the original spherical diatom aggregate started to flatten the diffusive oxygen uptake  was calculated using Fick’s first law of diffusion DOU = D0 (dC/dz), where D0 (cm−2 s−1) is the molecular diffusion coefficient in water, C (µmol L−1) is the solute concentration, and z (cm) is the depth within the aggregate. The total oxygen consumption rate of the aggregate was calculated integrating the diffusive flux over the entire aggregate surface area 2 consumption rate of a degrading pennate aggregate is 1.8±0.2 mmol O2 L−1 d−1 (n = 3).(TIFF)Click here for additional data file.Table S1Characteristics of pennate algal aggregates and sea ice stations investigated.(DOCX)Click here for additional data file.Table S2Characteristics of Melosira algal aggregates and sea ice stations investigated.(DOCX)Click here for additional data file.


There are limited published data reporting Australian hospitalized elders’ vulnerability to functional decline to guide best practice interventions. The objectives of this study were to describe the prevalence of vulnerability to functional decline and explore profiles of vulnerability related to the performance of physical activity in a representative group of elders in a single centre in Victoria, Australia.Mean age 82.4, SD 7 years) admitted to a general medical ward of an Australian tertiary-referral metropolitan public hospital from March 2010 to March 2011 (n = 526). Patients were screened using the Vulnerable Elders Survey (VES-13). Distinct typologies of physical difficulties were identified using latent class analysis.A cross-sectional survey of patients aged ≥ 70 years . Four distinct classes of physical difficulty were identified: 1) Elders with higher physical functioning ; 2) Ambulant elders with diminished strength ; 3) Elders with impaired mobility, strength and ability to stoop  and 4) Elders with extensive physical impairment  Vulnerable elders were distributed through all classes.Most elders scored ≥3/10 on the VES-13 and were rated vulnerable to functional decline (Older general medicine patients in Victoria, Australia, are highly vulnerable to functional decline. We identified four distinct patterns of physical difficulties associated with vulnerability to functional decline that can inform health service planning, delivery and education. Hospitalisation is a sentinel event that can precipitate functional decline in older people , 2. FuncNorth American researchers have identified that between 52 and 64 % of hospitalised elders are vulnerable to functional decline on admission to hospital , 10. GriMeasuring vulnerability to functional decline is important in selecting appropriate participants in research and health service evaluation and in targeting and choosing the most appropriate best practice interventions that minimise its onset , 15. ForCross-sectional survey of a convenience sample of 526 of 1380 patients aged 70+ years admitted to a general medical service of a 390 bed tertiary-referral metropolitan public hospital in Victoria, Australia between March 2010 and March 2011. Vulnerability data were collected in order to purposively select a sample of 65 participants for in-depth investigation in an institutional case study designed to evaluate and enhance the management of functional status (FS) in older patients admitted for acute medical care. This involved recruitment of consecutive patients from March to December 2010 and purposive recruitment of patients from January to March 2011. Findings of the institutional case study are not presented in this paper.The present study was approved by The Alfred hospital (Project 177/07) and Deakin University Human Research Ethics Committees [DUHREC] (Project EC-238-2007). Informed written consent was obtained for the 65 participants of the institutional case study. All screened patients, or their proxies, provided verbal consent to participate in screening and for use and publication of their data in a thesis and journal articles. Patients, or their proxies, were interviewed within 48-hours of admission to the ward in the recruitment process to an institutional case study.Patients were excluded from participating in the survey screen if they were: 1) unable to communicate, with no proxy present; 2) incompetent to provide consent, with no proxy; 3) for palliative (end of life) care; or 4) readmitted to the ward within 30 days of discharge or other episode of acute hospitalisation  and specialist nurses. The Acute-Aged Care Assessment Service provided specialist advice to the treating general medical team regarding the suitability of functionally dependent patients for residential aged care placement on discharge. The model of care delivery emphasised patient access to acute services through efficient treatment of patients’ medical issues. Patients with unresolved functional issues were discharged to rehabilitation programs in sub-acute care. The medical team referred potential candidates for aged care placement on discharge to the Acute-Aged Care Assessment Service for assessment.Sociodemographic data were extracted with permission from the hospital database, including: length of stay, discharge destination, incidence of death, admission diagnosis and International Classification of Diseases (ICD-10) category . VulneraThe VES-13 is a validated survey used to identify patients at risk of functional decline. It comprises 13 items that assess age, self-rated health, and difficulty performing 6 physical activities and five functional activities of daily living (ADLs) to give a score from 0 to 10 . The VESTo establish the representativeness of screened patients to the total ward throughput of elders aged 70+ years we compared their sex, age, admission diagnosis, length of stay, discharge destination and incidence of death in hospital. Admission diagnosis was categorised according to the International Classification of Diseases (ICD-10) . Length physical activity they were defined as having substantial difficulty, reflecting increased vulnerability to decline. To reflect scoring rules for items measuring difficulties with functional activities, elders who reported difficulty performing a functional activity, but received help for it, or avoided performing a functional activity due to health were defined as having substantial difficulty.The potential contribution of VES-13 items to overall vulnerability was reported as dichotomous variables that reflected item thresholds for scoring the VES-13. If elders were ‘unable to do’ or identified having ‘a lot of difficulty’ performing a All analyses were undertaken using SPSS version 23. We ran non-parametric and parametric statistical analyses according to the level of data  and distribution of data  to explore associations and differences between patient age group and responses to VES-13 items. Between-group differences in the number of difficulties with physical and functional activities were analysed via one-way Welch’s ANOVA and ANOVA, respectively. Binary logistic regression was used to test associations between age group and the presence of increased vulnerability due to: self-rated health; difficulties with physical activities; and, difficulties with functional activities.stooping, crouching or kneeling, (2) lifting, or carrying objects as heavy as 10 lb, (3) reaching or extending arms above shoulder level, (4) writing, or handling and grasping small objects, (5) walking a quarter of a mile, (6) heavy housework such as scrubbing floors or washing windows. We performed LCA iteratively to identify one through five latent classes. Best model fit was established via parametric bootstrapped likelihood ratio tests of k-1 classes [We used MPLUS 5.3 to identify distinct typologies of physical difficulties using latent class analysis (LCA). Dichotomous variables measuring the potential contribution to total vulnerability due to difficulty with physical activities were included in the model. These were the presence or absence of substantial difficulty: (1)  classes .n = 1380) we compared the demographic characteristics of screened (n = 526) and unscreened (n = 854) patients. No significant differences were found for age (t (1378) = .152, p = .879) or the distribution of sex , admission reason  or discharge destination (Fisher’s exact = 10.223 p = .092) between screened and unscreened elders. However, screened patients had a significantly longer hospital stay  and were significantly less likely to die in hospital  compared with those not screened. These differences are likely to be artefacts of the exclusion of palliative, unwell or confused patients with no proxy present, and, although statistically significant, do not reflect divergence in the core characteristics between groups.Characteristics of all patients on the ward aged 70 years and over are described in Table n = 471, 89.5 %). While prevalence of vulnerability was high across all age groups the VES-13 automatically defines all elders aged 85 years or over as vulnerable because of their advanced age. To investigate VES-13 vulnerability without the contribution of scores from patient age alone, we deducted scores due to age from participants’ total VES-13 score. Adjusted VES-13 scores indicated that the majority of elders were vulnerable to functional decline for reasons other than age alone . This included most patients aged 85+ years .Table n = 304, 56.7 %). Investigation of frequencies indicated that despite their more advanced age, a lower proportion of elders aged 85 years or over, perceived their health as poor or fair compared to elders in other age groups . Elders aged between 70 and 74 years  and 75 to 84 years  had over one and a half the odds of rating their health ‘poor’ or ‘fair’ compared with elders aged 85 years or over.Over half the sample rated their health as ‘poor’ or ‘fair’ . Prevalence of substantial difficulty with physical activities were: stooping, crouching or kneeling ; lifting or carrying objects as heavy as 10 lb, 4.5 kg ; reaching or extending arms above shoulder level , writing or grasping small objects , walking a quarter of a mile, 400 m , heavy housework such as scrubbing floors or washing windows . A one-way Welch’s ANOVA indicated a small, but statistically significant effect of age group on the number of substantial physical difficulties reported by elders  = 4.313, p = .015, η2 = .02). Post hoc analysis with the Games-Howell test indicated that patients aged 85+ years  reported a greater number of substantial physical difficulties than patients aged 70–74 years  but not 75–84 years . There was no significant difference in number of substantial physical difficulties reported between patients aged 70–74 years and patients aged 75–84 years.Most elders reported having at least one substantial difficulty with a physical activity  and p < .001 , respectively. The odds of elders aged 85 years or over reporting substantial difficulty lifting or carrying objects were approximately twice those of elders aged 70 to 74 years. Furthermore, elders aged 85 years or over had approximately three times the odds of reporting substantial difficulty performing heavy housework compared to those of elders aged between 70 and 74 years.Binary logistic regression identified age group as a significant predictor of reporting substantial difficulty lifting or carrying objects as heavy as 10 lb and performing heavy housework ; bathing or showering ; shopping for personal items ; managing money ; and performing light housework . A one-way ANOVA indicated a small, but statistically significant effect of age group on the number of substantial functional difficulties reported by elders . Post-hoc analysis with Tukey’s HSD tests indicated that patients aged 85 years or older  had a significantly greater number of substantial difficulties with functional activities compared with patients aged 70 to 74 years  and patients aged 75 to 84 years . The number of substantial difficulties with functional activities did not significantly differ between patients aged 70 to 74 years and patients aged 75 to 84 years (p = .754).Overall, 411 (78.1 %) screened elders reported having substantial difficulty with at least one functional activity measured by the VES-13. These participants indicated that they had difficulty performing a functional activity, but received help for it, or did not perform a functional activity because of their health. Prevalence of substantial difficulties with functional activities among screened elders was: walking across a room  and p = .001 for shopping for personal items  and managing money . Compared to the odds among elders aged 70 to 74 years, elders aged 85 or over had approximately twice the odds of reporting substantial difficulty with these functional activities compared to patients aged 70 to 74 years.Binary logistic regression revealed that age group was significantly predictive of having substantial difficulty bathing or showering, shopping for personal items and managing money (see Table p = .04), but not the five class model, compared to the four class model (p = .286). Moreover, AIC scores were lowest in the four class model (AIC = 3242.972) compared to the three class (AIC = 3247.139) and five class (AIC = 3245.59) model. This confirmed that the four class model had superior fit. We examined the fit of the bivariate items entered into the model with Pearson’s and likelihood Chi-square tests and item responses did not differ from that expected by the model . Standardised residuals of bivariate Chi-square tests indicated that variables were independent of one another within latent classes (zres <1.96), thus satisfying the assumption of conditional independence.Latent class analysis indicated four distinct classes of difficulty with physical activities see Fig. . First, n = 114, 21.7 %) had the least likelihood of substantial difficulty across the six physical activities and were labelled Elders with higher physical functioning . Members of Class 1 possessed lower likelihood of substantial difficulty stooping (26.5 %), walking (14.7 %) and ability to perform heavy housework (26.2 %) and they had no substantial difficulty lifting. Elders in Class 2  had an absence of substantial difficulty stooping and walking, however, they were very likely to have substantial difficulty lifting (92.2 %) and performing heavy housework (82.9 %). Consequently, Class 2 were categorised as: Ambulant elders with diminished physical strength. Elders in Class 3 comprised half the sample  and were categorised as Elders with impaired mobility, strength and ability to stoop. Class 3 elders had high likelihood of substantial difficulty with stooping (73.2 %), walking (76.3 %) and performing heavy housework (92.2 %). These elders also had very low likelihood of substantial difficulty reaching (6.6 %) and writing (8.5 %). Elders in Class 4  had the greatest likelihood of substantial difficulty with all six physical activities including greater likelihood of substantial difficulty reaching (67.2 %) and writing (42.1 %). These elders were categorised as Elders with extensive physical impairment who may have difficulty even reaching for or handling objects.Elders in Class 1 , Ambulant elders with diminished physical strength , Elders with impaired mobility, strength and ability to stoop  and Elders with extensive physical impairment . The same pattern occurred for the number of substantially impaired functional activities: Class 1 ; Class 2 ; Class 3  and Class 4 .Histograms reporting frequencies of substantial difficulty with physical and functional activities across latent classes are presented in Figs. Elders with extensive physical impairment (Class 4) were significantly more likely than expected to be rated as vulnerable to functional decline (zres = 4.3). Conversely, elders with higher physical functioning (Class 1) were less likely than expected to be rated as vulnerable (zres = −4). A one-way Welch’s ANOVA indicated a significant difference in total VES-13 scores between latent classes F  = 99, p < .001, η2 = .47). Total vulnerability scores were high in all classes except Class 1. Post-hoc analyses with the Games-Howell test indicated that Elders with higher physical functioning  had significantly lower VES-13 vulnerability scores than elders in latent class 2 , class 3  and class 4 . Furthermore, elders with extensive physical impairment (Class 4) had significantly greater VES-13 vulnerability scores than elders with impaired mobility, strength and ability to stoop .Chi-square tests of independence indicated statistically significant associations between latent class membership and vulnerability status  rated as vulnerable to functional decline despite their age. This may be associated with the high level of pre-hospital functional decline in Australian older general medicine patients (64 %) identified in previous research [In this study, we found a much higher rate of vulnerability to functional decline than previously reported , 10 withpatients 4 % identresearch .Clinically and cost effective acute models of geriatric care are available , 24 and The second major finding was that participants’ preexisting difficulties performing physical activities were distributed into 4 distinct profiles of vulnerability see Fig. . This inambulant elders with diminished physical strength had particular difficulty with activities requiring upper body strength. Lean body mass is reduced in age and fat is redistributed [elders with higher physical functioning  had an absence of substantial difficulty lifting. These elders would likely respond best to strategies that promote independence in hospital. For example, evaluation of mobility programs have indicated that daily care that incorporates 2 to 4 walks per day of approximately 10 to 20 min duration can improve functional outcomes in general medicine patients who are capable of ambulating [Future ability to identify new patients within classes of vulnerability to functional decline is needed. For example, tributed  increasitributed . These pbulating –36. Morebulating .Our findings demonstrate that older general medicine patients were likely to require high levels of support to mobilise and complete personal and instrumental ADLs safely in hospital. The identification of four vulnerability profiles provides insight into where best practice interventions can be targeted to address preexisting physical difficulties.This study has various limitations. We acknowledge that some measurement bias could have occurred because patients self-reported their function retrospectively, whereas objective performance based assessments have greater validity . HoweverA high level of vulnerability to functional decline was identified in hospitalised older general medicine patients. Highest difficulty performing ADLs was identified in those aged 85 years and over. Our findings support the need for those involved in health service planning, delivery and education to emphasise provision of 24-hour care that addresses vulnerability to functional decline in older general medicine patients. We identified four patterns of physical impairment associated with vulnerability to functional decline, supporting the need to deliver individualised care. The question of which class a new patient would belong to requires additional clinical investigation. This could be addressed in future research. Despite local government initiatives to guide practice  the probADL, activities of daily living; ICD-10, international classification of diseases 10 – 2010 Version; VES-13, vulnerable elders survey


Here, we developed a novel photo-lithographically fabricated, silicone(PDMS)-based side-view flow chamber to dynamically visualize tether and sling formation. Fluorescently membrane-labeled mouse neutrophils rolled on P-selectin substrate at 10 dyn/cm2. Most rolling cells formed 5 tethers that were 2–30 μm long. Breaking of a single tether caused a reproducible forward microjump of the cell, showing that the tether was load-bearing. About 15% of all tether-breaking events resulted in slings. The tether-to-sling transition was fast (<100 ms) with no visible material extending above the rolling cell, suggesting a very low bending modulus of the tether. The sling downstream of the rolling cell aligned according to the streamlines before landing on the flow chamber. These new observations explain how slings form from tethers and provide insight into their biomechanical properties.Neutrophils rolling at high shear stress (above 6 dyn/cm Most of their immune response activity occurs outside of the blood vessels, requiring extravasation124in vitro experiments, tether tension force is estimated to reach ~80 pN2). Mechanical properties of the tethers allow them to substantially stretch at largely unchanged tension force as the neutrophil rolls forwardTethers are long sub-micron diameter tubes pulled out from neutrophils rolling under hydrodynamic drag. Tethers form behind neutrophils, originating from microvilli, and bind to the vessel wall through P-selectin glycoprotein ligand -1 (PSGL-1) expressed on the tips of the microvilli binding to selectinsTethers were first identified with DIC microscopy in flow chamber experiments96For a neutrophil rolling on a horizontal substrate on a regular vertical (upright or inverted) microscope, capturing an entire tether or sling is difficult, because these structures have large extensions along the vertical (Z-) axis, whereas the imaging is always in a horizontal (XY-) plane. Confocal Z-axis optical sectioning has low temporal and spatial resolutionSide view flow chambers were previously implemented using 45° tilted mirrors1314Here we use a microfluidic flow chamber to study primary mouse neutrophils rolling on a vertical (XZ-plane) wall, such that entire tethers and slings are observed at high resolution in a single XY-plane with high numerical aperture objectives. This setup enables the first direct observation of tether-to-sling transition on well-defined substrate at physiologically relevant WSS values.w = 45 μm, height h = 50 μm, and length of 5 mm , τ, was calculated as τ = 4.3ηυmax/w, where η is the viscosity and 4.3 is a factor derived by numerical simulation.The microfluidic device consisted of a polydimethylsiloxane (PDMS) chip with micro-channels engraved on its surface and a #1.5 cover glass sealing the micro-channels . The chi of 5 mm . In expe+), and the major contaminating cells were monocytes. The neutrophil isolate was resuspended at 2.5 million cells/ml in RPMI supplemented with 10% fetal calf serum  and 0.1% penicillin-streptomycin . To label the cell membrane, 4 μm Vybrant-DiO  or 1 μm Cell Mask Green , or 5 ng/μl (1:100) anti Ly-6G antibody conjugated with Alexa Fluor 488  was added to the cell suspension. After 10 min incubation, with washing (DiO) or without washing , the suspension was perfused through the flow channels. As soon as rolling cells were visible, the perfusion solution was switched to PBS to reduce the background signal by washing out the solution containing free dye.The experimental animal procedures of this study were performed in accordance with approved guidelines. These guidelines were approved by the Institutional Animal Care and Use Committee of La Jolla Institute for Allergy and Immunology, which is an AAALAC international accredited facility. For each experiment, bone marrow from a wild type female mouse was collected, resuspended in PBS and filtered through a 40 μm nylon cell strainer . From that sample, neutrophils were enriched at room temperature via negative selection with a customized neutrophil enrichment kit . FACS measurements indicated that >70% of cells were neutrophils  using a 63× 1.3 glycerol immersion objective. For bottom view images, cells rolling on the bottom surface were imaged, while for side view, cells rolling on the side walls were imaged . A reson2 (n = 28) (Primary mouse bone marrow neutrophils rolled at 5.2 ± 1(SEM) μm/s at WSS = 6–10 dyn/cm(n = 28) . Neutrop2 had 4–10 tethers . The tethers were distributed across most of the apparent neutrophil width  μm; the longest tether length was 30.1 μm and the shortest was 1.2 μm  and the two following frames (frames T + 1 and T + 2). The displacement between T and T + 1 was 1.7 ± 0.1(SEM) times greater than between T + 1 and T + 2 (n = 22), indicating that a rolling neutrophil greatly accelerates, effectively making a jump, immediately after a tether breaks. In some rolling neutrophils, multiple consecutive tether breaks could be observed , and larThis side-view flow chamber allows “for the first time” to visualize the formation of slings. Specifically, we tested the hypothesis that slings derived from tethers, which required detailed observation of the formation of tethers and slings. In side view  and botttether). Consistent with the length of the tethers and the diameter of the neutrophil, the average Φtether was 22.3 ± 1.3(SEM)° (sling) (We measured the angle between the tether and bottom surface (Φ.3(SEM)° , ranging (sling) . CFD sho (sling) . This waWe compared the lengths of tethers immediately before and slings immediately after the tether-to-sling transitions . The aveHere, we introduced a new microfluidic side-view flow chamber compatible with high resolution microscopy that enabled the first direct observation of the tether-to-sling transition, a process of fundamental importance for leukocyte adhesion under high shear stress. In previous studies, tethers were hypothesized to form slings [6], but there was no direct evidence for that. Tether breaks could not be reliably matched to sling formation, because slings are invisible under TIRF microscopy until they are very close to the substrate.Our data indicate that about 15% of all tethers form slings. This might be an underestimate, because the side view flow chamber records only a ~2 μm section in the XZ plane and thus might miss slings that may have moved out from the focal plane in the Y direction. This can be caused by random movement of rolling neutrophils in the Y direction, which is expected whenever the key anchor point is off-center212324The transition from a tether behind a cell to a sling in front of the cell is completed within less than one frame (100 ms). In the 28 tether-to-sling transitions, we could only see slings on the substrate or aligned along the flow streamlines in front of rolling cells and never above the rolling cell. This observation is consistent with the expected physical properties of slings, sub-micron diameter tubes with low resistance to bending72627On most of the side view transition records, the sling can be reliably matched to a broken tether. Slings spend several hundred milliseconds “hanging” in the fluid stream before landing. This explains the delay between tether break and sling formation observed with qDF. The hanging slings enclose an average angle of 6 ± 1(SEM)° with the bottom surface. This is consistent with the notion that the slings follow the flow profile in front of the cell and a previous intravital microscopy study with particle tracking velocimetry30The side view chamber enables more accurate measurements of the tether lengths as compared with the bottom view chamber. However, the apparent lengths of the tethers in the side view flow chamber are still underestimates, because a segment of the tether close to its neck will be obstructed by the brighter cell, when the neck is not in the equatorial plane of the cell. If a tether originates from the cell body in a plane different from the equatorial plane, the part of the tether “behind” or “in front of” the cell equator is invisible and will not be measured. This is significant as shown in , where mThe side view images showed that some tethers have grape-like structures along their length, indicating that the grapes observed by electron microscopy of fixed neutrophils with tethersWe observed that each break of a tether results in a major short-term acceleration of the cell rolling (a microjump). This indicates that, immediately before breaking, each tether is load-bearing, significantly contributing to the forces opposing hydrodynamic drag on the cell, corroborating previous reports6In its current configuration, the side view flow chamber can show many rolling cells from perfusion with 250 μl cell suspension containing 500,000 cells. The volume of cell suspension required can be further reduced in a device with a smaller number of perfusion channels. The PDMS walls can be readily coated with proteins thus making the side view flow chamber useful for studies of neutrophil arrest and interaction of other types of leukocytes3335Here we studied neutrophils rolling on P-selectin with no integrin ligand. Tether and sling formation on more complex substrates has not been studied. Neutrophils rolling on E-selectin also form slings and tethersIn summary, we directly show on neutrophils rolling on P-selectin that some tethers form slings that are aligned along streamlines in front of the cell before landing on the substrate; tethers originate from almost anywhere across the leukocyte width; tethers are load-bearing and thus directly relevant for cell rolling. We conclude that the new side and bottom view flow chamber is a versatile tool for studying cell rolling, adhesion and migration under flow.How to cite this article: Marki, A. et al. Microfluidics-based side view flow chamber reveals tether-to-sling transition in rolling neutrophils. Sci. Rep.6, 28870; doi: 10.1038/srep28870 (2016).