Maternal feeding practices have been proposed to play an important role in early child weight gain and obesogenic eating behaviors. However, to date longitudinal investigations in young children exploring these relationships have been lacking. The aim of the present study was to explore prospective relationships between maternal feeding practices, child weight gain and obesogenic eating behaviors in 2-year-old children. The competing hypothesis that child eating behaviors predict changes in maternal feeding practices was also examined.A sample of 323 mother  and child dyads  were participants. Mothers completed a questionnaire assessing parental feeding practices and child eating behaviors at baseline and again one year later. Child BMI (predominantly objectively measured) was obtained at both time points.z-scores over the follow-up period were predicted by maternal instrumental feeding practices. Furthermore, restriction, emotional feeding, encouragement to eat, weight-based restriction and fat restriction were associated prospectively with the development of obesogenic eating behaviors in children including emotional eating, tendency to overeat and food approach behaviors (such as enjoyment of food and good appetite). Maternal monitoring, however, predicted decreases in food approach eating behaviors. Partial support was also observed for child eating behaviors predicting maternal feeding practices.Increases in child BMI Maternal feeding practices play an important role in the development of weight gain and obesogenic eating behaviors in young children and are potential targets for effective prevention interventions aiming to decrease child obesity. The dramatic rise in overweight and obesity in children in the Western world has led to its identification as a major public health concern. OverweiParental feeding practices aim to influence the amount or type of food a child eats and include monitoring intake, restrictive or controlled feeding, pressure to eat, and instrumental and emotional feeding. These practices, although well-intended, can be associated with both child weight and obesogenic eating behaviors – that iPrevious studies focusing on relationships between maternal feeding practices and child outcomes have produced inconsistent findings and focused on disparate concepts. Maternal monitoring, that is keeping track of one’s child’s eating, could be expected to result in healthy weight and eating behaviors, particularly at a very young age when mothers still strongly direct their child’s eating. While self-reported parent monitoring has been shown in one study to prospectively predict lower BMI, more ofz-scores from 5 to 7 years[decreased BMI[Restrictive feeding practices involve regulating the type and amounts of foods eaten by children. Although parents may attempt to restrict their children’s eating to reduce weight and maintain health, theorists suggest restriction may lead to the opposite effect by encouraging children to seek out restricted foods and failing to help children regulate eating based on satiety. Longitu 7 years. Howeverased BMI,12. Finaased BMI,9. One pParental feeding practices aiming to control when, where and what children eat have also been examined. A distinction has been proposed between overt and covert methods of controlled feeding and their respective influence on child weight and eating behaviors. Overt cAnother feeding practice, pushing to eat, might be expected to predict weight gain over time, although this has not been observed consistently. Among children from birth to 2 years old, as well as in a sample of 5 year olds, maternal pressure to eat has been found to predict lower BMI one year later,10, incrz-scores in the only cross-sectional study that examined this relationship[Feeding practices which promote the use of food for reward or emotional regulation have also been postulated to influence child weight status and obesogenic eating behaviors. One such practice is instrumental feeding which involves using food as a reward for a desired outcome . It has been suggested that reinforcing positive associations of palatable foods through instrumental feeding could strengthen the preference for reward  foods. In one tionship. Althougtionship,20,22.In summary, the existing literature examining parental feeding practices as predictors of changes in child weight and obesogenic eating behaviors is inconsistent and lacks longitudinal studies in very young children. Measurement issues may partially account for these discrepancies. Furthermore, most studies have focused solely on BMI as an outcome, rather than considering child eating behaviors that potentially contribute to overweight.z-score over time, and we expected these maternal feeding practices to be associated positively, both cross-sectionally and prospectively, with obesogenic child eating behaviors. In order to test the competing hypothesis by which child eating behaviors predict changes in maternal feeding practices, we also examined these relationships prospectively.The principle aim of the present study was therefore to explore the role of a wide range of maternal feeding practices in relation to weight gain and obesogenic eating behaviors in a sample of 2 year-old children. To date, a variety of potentially overlapping constructs exist in the literature. Consequently, a preliminary aim was to conduct a principal components analysis to clarify a set of core independent constructs representing maternal feeding styles. Next, we aimed to identify which components of mothers’ feeding practices prospectively predicted the development of obesogenic child eating behaviours and BMI. We hypothesized that restrictive and controlling maternal feeding practices would be associated positively with increases in child BMI Participants were 323 parent–child dyads recruited in 2008 Time 1). A subsample of the first 284 dyads recruited was invited to participate in the second wave of the study a year later (Time 2). Of these, 222 accepted (78% retention rate). Children were aged between 1.5 to 2.5 years  at recruitment. Mothers  were included if they were over the age of 18 years and had a child between 1.5 and 2.5 years old, could read and understand English, and had children with no food allergies, intolerances or deficiencies. Mothers, on average were well educated, and had a medium household income (Table. A subsaz-score[z). Children were lightly clothed for all measurements with shoes removed. All children were weighed by the researcher at Time 1. At Time 2, 64.1% were weighed by researchers but due to resource limitations, 35.9% were not. In the latter cases, maternal health records were used if the child had been measured within the last 3 weeks or parent report was used as last resort.Body Mass Index (BMI). Child height (to the nearest .1 of a centimetre) and weight (to the nearest .1 of a kilogram) were obtained and children’s BMIs were calculated and converted into a standardized z-score adjustinFive main measures assessing maternal feeding behaviors were identified which contained subscales related to monitoring, restriction and control, pressure or prompting to eat, and instrumental and emotional feeding.Child Feeding Questionnaire (CFQ). MaternPreschooler Feeding Questionnaire (PFQ). The fiParent Feeding Style Questionnaire (PFSQ). The foControl Over Eating Questionnaire (COEQ). Overt Comprehensive Feeding Practices Questionnaire (CFPQ). The seThe Child Eating Behavior Questionnaire (CEBQ) was useThe parent version of the Dutch Eating Behaviour Questionnaire (DEBQ-P) was useLa Trobe Human Ethics Committee approval was received. Most participants were recruited from local playgroups within the greater metropolitan area of Melbourne  at which a researcher presented study information and recruited interested mothers. A minority of participants were recruited from maternal and child health centres, leaflets, advertisement in local papers and word of mouth. Following written consent, children’s height and weight were measured at an interview at participants’ homes. Parents completed identical questionnaires at baseline (Time 1) and approximately 52 weeks later (Time 2); questionnaires were returned by reply paid envelope. Mothers received a $10 gift card on each occasion.z change score was computed by subtracting Time 2 BMIz scores from Time 1 values. Relationships between maternal feeding practices and BMIz change, as well as child eating behaviors were explored with correlational analyses at each time point. Prospective relationships between (1) maternal feeding practices at Time 1 and child eating behaviors at Time 2; and (2) between child eating behaviors at Time 1 and maternal feeding practices at Time 2, were investigated using hierarchical multiple regression analyses. Analyses exploring prospective relationships included only the subsample which completed both assessments (N = 222). One outlier was excluded.A square root transformation was conducted on Time 1 Weight-based restriction to improve normality. When transformations were insufficient, non-parametric statistics and robust methods were used. Data reduction was conducted using principal component analysis. A BMIth percentile), 16.3% were overweight (85th percentile ≤ BMI < 95th) and 11.1% were obese (BMI ≥ 95th percentile). At Time 2, 7.2% of children were underweight, 15.3% were overweight and 4.5% were obese. Weight status of both mothers and children was comparable to national data, although at Time 1, our sample was slightly heavier than the national average[z at Time 1 between children who were included in the follow-up and those who were not. Descriptive statistics for eating behaviors and BMIz at Time 1 and Time 2 are shown in Tablez decreased from Time 1 to Time 2, but scores on all eating subscales increased significantly. There was no difference in BMIz change score between children measured by researchers and those who were not (measured mean = −0.18 (SD = 1.16), not measured mean = −0.20 (SD = 1.14), t(218) = −0.12, p = .90).Mean self-reported maternal BMI was 24.21 (± 4.66) with 4.7% of mothers underweight, 60.3% of mothers in the healthy weight range, 24.4% overweight, and 10.6% obese  were conducted. The first PCA was conducted on the 78 Time 1 maternal feeding practice items. Eigenvalues and the scree plot indicated a 9 factor solution explaining 48% of the total variance, with interpretable dimensions after rotation. The first factor, “Instrumental Feeding”, displayed high loadings of the 4 items of the PFSQ Instrumental Feeding subscale, the 3 items of the CFPQ Food as Reward subscale as well as 2 items from the CFQ Restriction subscale. On the second factor, “Encouragement”, the 8 PFSQ Encouragement/Prompting subscale items, 3 items of the CFPQ Encouragement to Variety subscale, and 3 items of the CFPQ Modelling subscale loaded. On the third factor, “Emotional Feeding”, the 5 PFSQ Emotional Feeding subscale items and 4 items of the PFQ Use of Food to Calm subscale loaded highly. The fourth factor, “Control”, revealed high loadings for the 10 items of the PFSQ Control subscale as well as 4 items of the COEQ Overt Control subscale. The fifth factor, “Covert Control”, displayed high loadings for the 5 CORQ Covert Control subscale items. On the sixth factor, “Monitoring”, the 3 CFQ Monitoring subscale items loaded. The seventh factor, “Pushing to Eat More”, revealed high loadings for the 5 PFQ Pushing to eat more subscale items. The eighth factor, “Fat Restriction”, revealed high loadings for 6 items of the CFQ Restriction subscale. Finally the ninth factor, “Weight-based Restriction”, displayed high factor loadings for the 7 CFPQ Restriction for Weight Control subscale items. The standardized regression scores resulting from these factors were used in subsequent analyses.p = .013).An identical PCA was conducted for the Time 2 maternal feeding items in an attempt to replicate and confirm the structure. For the Time 2 items, the eigenvalues and the scree plot indicated an 8 factor solution was the best fit, explaining 48% of the total variance, with interpretable dimensions after rotation. The main differences with the Time 1 factor solution was that the five items from the Preschooler Feeding Questionnaire (PFQ) loaded A second PCA was conducted on the 47 child eating behavior items at Time 1 and Time 2 separately. At Time 1, a 3 factor structure was revealed that explained 44% of the variance, with easily interpretable rotated factors. The first factor, “Emotional Eating”, revealed strong factor loadings for the 13 items of the DEBQ-P Emotional Eating subscale. The second factor, “Food Approach”, revealed strong loadings for the 4 items of the CEBQ Enjoyment of Eating subscale, negative loading of the 9 CEBQ Satiety Responsiveness/Slowness of Eating subscale, and negative loadings of the 6 CEBQ Fussiness items. Finally the third factor, “Tendency to Overeat”, revealed strong loadings for the 8 DEBQ-P External Eating subscale items, and the 3 CEBQ Desire to Drink subscale items and the 5 items of the Food Responsiveness subscale. Repeating the analysis with Time 2 items revealed an almost identical factor structure with the exception of one CEBQ item which loaded moderately strongly on the Tendency to Overeat factor at Time 1 and weakly on the Emotional Eating factor but the reverse was true at Time 2. Again the standardized regression factor scores derived for Time 1 and Time 2 were used in the subsequent analyses. (The PCA summary is available from the authors on request).Findings from the correlation analysis Table revealedProspectively at Time 2, these relationships were somewhat weakened. Time 2 child Emotional Eating was correlated with Time 1 maternal Emotional Feeding and correlated negatively with Monitoring while Encouragement just failed to meet significance. Time 2 child Food Approach was correlated with Time 1 Encouragement and Weight-based Restriction and correlated negatively with Monitoring, while the correlation with Time 1 Instrumental Feeding just failed to meet significance. Time 2 Tendency to Overeat was correlated with Time 1 Encouragement to Eat, Emotional Feeding, Control and Fat Restriction.z change was moderately and positively associated with Time 1 maternal Instrumental Feeding. However, this was the only significant relationship for BMIz change.In relation to our first hypothesis, child BMIA second correlational analysis was conducted exploring the relationship between Time 1 child eating behaviors and Time 2 maternal feeding behaviors Table. Prospecp = .005). Regarding the prediction of child Tendency to Overeat, after controlling for Time 1, maternal Emotional Feeding, Encouragement and Control feeding practices contributed significantly to Time 2 child Tendency to Overeat scores, with both Emotional Feeding  and Encouragement  emerging as significant unique predictors. Regarding child Emotional Eating at Time 2, after controlling for Time 1 levels, maternal Emotional Feeding and Monitoring significantly increased the explained variance in the second step but Emotional Feeding was the only significant unique predictor . No regression analysis was conducted for BMIz change as Instrumental Feeding would have been the only predictor. Hierarchical multiple regression analyses were conducted for each Time 2 child eating behavior, with Time 1 scores for the relevant eating behavior entered in the first step to control for baseline scores, followed by the maternal feeding practices from Time 1 which displayed significant correlations with the Time 2 eating outcome in the second step so as to determine which predictor variables contributed unique variance Table. Regardiβ = −.18, p = .003) was a significant negative predictor of Time 2 Instrumental Feeding and child Time 1 Tendency to Overeat was a significant positive predictor , F  = 26.11, p < .001, R2 = .35. Furthermore, controlling for Time 1 maternal Emotional Feeding, child Time 1 Emotional Eating was a significant predictor of Time 2 maternal Emotional Feeding , F  = 19.99, p < .001, R2 = .31. Finally, controlling for maternal Time 1 Covert Control, child Time 1 Emotional Eating was a significant predictor of maternal Time 2 Covert Control , F  = 52.02, p < .001, R2 = .53.As a final analysis, we conducted hierarchical multiple regression analyses, for each Time 2 maternal feeding practice, with Time 1 scores for the relevant feeding practice entered in the first step to control for baseline scores, followed by the child eating behaviors from Time 1. Findings revealed that, controlling for Time 1 maternal Instrumental Feeding, child Time 1 Approach , and restriction of children’s eating for weight control purposes (weight-based restriction). It could be argued that while the first might be directed towards health goals, the second is directed towards maintaining a low weight, perhaps for appearance considerations. A distinction between pushing to eat more and encouragement and reinforcement to eat more was also revealed. The failure to discriminate between these discrete dimensions may help explain previous discrepant findings regarding correlates of maternal restriction[Our initial aim was to explore the factor structure of different measures of maternal feeding practices including instrumental feeding, encouragement, emotional feeding, control, covert control, monitoring, pushing to eat more, fat restriction, weight-based restriction. Our findings revealed that 9 dimensions best described these practices. Consistent with previous findings, overt and covert control described separate dimensions, suggesttriction,11 and ptriction.Restrictive feeding practices revealed complex relationships with child eating outcomes. Consistent with our hypothesis, mothers who reported restricting their child’s food for purposes of weight were more likely to report that their child tended to approach foods and to overeat at Time 1. Furthermore, restricting food for weight purposes was prospectively associated with food approach behaviors and revealed a similar trend with child emotional eating. In addition, mothers’ restriction of child fat intake and child tendency to overeat were correlated both cross-sectionally and prospectively. Similarly, mothers’ overt control was prospectively associated with children’s tendency to overeat at follow-up. These findings are consistent with previous work among older children,11, and z gains. Furthermore, controlling for Time 1 eating behaviors, maternal emotional feeding practices was a significant predictor of children’s emotional eating and tendency to overeat one year later. Maternal instrumental feeding may lead to increased preference and consumption of high-calorie snacks in response to external cues in children and thus to weight gain. Furthermore, emotional eating in children has been associated with overweight[This study also explored the prospective relationships between instrumental and emotional feeding and children’s weight gain and obesogenic eating behaviors. Findings revealed that maternal instrumental feeding was associated, cross-sectionally with child emotional eating, and tendency to overeat, as well as prospectively with greater child BMIerweight and the erweight and is terweight. FurtherAmong our sample, maternal monitoring of children’s eating was prospectively negatively associated with both child food approach and tendency to overeat behaviors, and was a significant negative predictor of food approach when controlling for Time 1 food approach levels. Consistent with this finding, previous studies have described a negative relationship between monitoring and later BMI. It has z gain. These results are consistent with previous reports that parental pressure to eat was related to decreases in BMI[Mothers’ encouragement feeding practices were associated both cross-sectionally and prospectively with their child’s food approach behaviors, and prospectively with children’s tendency to overeat. Mothers’ pushing to eat more, however, displayed only a weak negative cross-sectional association with food approach, and a negative trend with BMIs in BMI,10, and We also examined the reciprocal relationship in which child eating behaviors predict maternal feeding practices. Our findings provide some partial support for this pathway. Child tendency to overeat predicted increases in maternal instrumental feeding, and child food approach tendencies predicted decreases in maternal instrumental feeding. Furthermore, child emotional eating predicted increases in maternal emotional feeding and maternal covert control. The findings regarding increases in covert control are interesting as this has been suggested to be a particularly helpful feeding practice for helping children to achieve a healthy diet. These fz change and eating behaviors explained by parent feeding practices (2-5%) suggests that other factors not assessed in this study are also likely to predict these outcomes. It has been suggested that other maternal variables including general parenting, family functioning and parental comments[Limitations of this research include the relatively small sample and whilst efforts were made to recruit widely, our sample is not necessarily representative. In addition, use of maternal self-report measures for feeding styles, child eating behaviors and a minority of Time 2 height and weight, may have biased our data. Finally, the somewhat small amount of variance in child BMIcomments,44,45 miOur study explored prospective relationships between a wide range of maternal feeding practices and both child weight gain and obesogenic eating behaviors among children aged 2 to 4 years. Findings suggest that maternal feeding practices contribute to shaping weight and related eating behaviors early in life. In particular, instrumental and emotional feeding as well as restriction and encouragement to eat more may be related to weight gain and obesogenic eating behaviors in young children. Furthermore, maternal monitoring of children’s high-calorie food intake may contribute to shaping healthy eating patterns. While more longitudinal research would help confirm these mechanisms, the present findings suggest the usefulness of engaging parents of young children in obesity prevention interventions and helping parents implement feeding practices which shape healthy eating behaviors and effective strategies for weight control.No author has any competing interests to declare.RFR identified the research question, analysed data and wrote the manuscript. SJP was lead PI on the supporting grant, played a major role in the design of the study, oversaw data collection and cleaning, and assisted in preparation of the manuscript. RM was project manager and co-ordinated data collection and entry. KJC, EHW, HS and KG were PIs on the supporting grant, were closely involved in the design of the study and instrument selection, and assisted in preparation of the manuscript and data interpretation. All authors read and approved the final manuscript.RFR is a Postdoctoral Research Fellow at Northeastern University, Boston. SJP and EHW are Professors in the School of Psychological Science at La Trobe University, Melbourne. RM is a Research Officer in the School of Psychological Science at La Trobe University, Melbourne. KJC is Senior lecturer in Nutrition and Population Health, School of Exercise and Nutrition Science, Deakin University, Melbourne. HS is Associate professor in the School of Psychology, Deakin University, Melbourne. KG is Manager, Nutrition and Food Services, The Royal children’s Hospital, Melbourne.


Avipoxvirus on 226 (6.3%) specimens. Histopathology and viral genotyping of 59 candidate tissue samples from six islands showed that 21 (35.6%) were positive for Avipoxvirus, while alternative diagnoses for some of those testing negative by both methods were feather follicle cysts, non-specific dermatitis, or post mortem fungal colonization. Positive specimens were significantly nonrandomly distributed among islands both for mockingbirds  and for finches , and overall highly significantly distributed toward islands that were inhabited by humans  vs. uninhabited at the time of collection , with only one positive individual on an uninhabited island. Eleven of the positive specimens sequenced successfully were identical at four diagnostic sites to the two canarypox variants previously described in contemporary Galapagos passerines. We conclude that this virus was introduced late in 1890′s and was dispersed among islands by a variety of mechanisms, including regular human movements among colonized islands. At present, this disease represents an ongoing threat to the birds on the Galapagos Islands.The role of disease in regulating populations is controversial, partly owing to the absence of good disease records in historic wildlife populations. We examined birds collected in the Galapagos Islands between 1891 and 1906 that are currently held at the California Academy of Sciences and the Zoologisches Staatssammlung Muenchen, including 3973 specimens representing species from two well-studied families of endemic passerine birds: finches and mockingbirds. Beginning with samples collected in 1899, we observed cutaneous lesions consistent with  Extinction risk associated with disease remains largely hypothetical Avipoxvirus is a pathogen of extreme concern in insular populations of birds Avipoxvirus: Poxviridae). Its recognized strains vary in virulence and host specificity; the best-studied strains are those infecting passerine birds (canarypox virus) and galliform birds (fowlpox virus). The most common lesions associated with infection are epidermal nodules on feet, legs, and tissue surrounding the bill and eyes that may become ulcerated and enlarged to impede sight, feeding, and mobility. The less common diphtheritic form produces lesions inside respiratory and digestive systems, inhibiting breathing and swallowing Avipoxvirus, a Plasmodium blood parasite, and their arthropod vectors Avipoxvirus and Culex quinquefasciatus mosquitoes in the 1800′s, followed by a sharp decline in bird numbers, and the subsequent introduction of the malaria agent Plasmodium relictum, causing yet further drastic declines and extinctions Current understanding of the extinctions of Hawaiian endemic birds involves a complex interaction of Mimus spp.), doves , yellow warblers (Dendroica petechia), and some finches (Geospiza and Camarhynchus spp.). During the 1982–1983 El Niño Southern Oscillation (ENSO) event, Galapagos mockingbirds displaying pox-like lesions suffered significantly higher mortality than asymptomatic birds on Genovesa The Galapagos Islands straddle the equator 1000 km west of Ecuador and comprise 19 major islands; 97 percent of the archipelago has been protected as a National Park and UNESCO World Heritage Site since 1959, and the archipelago retains almost its entire fauna The California Academy of Sciences (CAS) holds 1170 Galapagos avian specimens collected by R.E. Snodgrass and E. Heller in 1898 and 1899, and 7401 collected by R.H. Beck, E.W. Gifford and J.S. Hunter on the second Webster-Harris expedition in 1905 and 1906. Across both expeditions, 5580 specimens represent two passerine taxa in the archipelago: the finches (at least 13 endemic species), and the mockingbirds (four endemic species), from all 19 major islands. In these same taxa, the Zoologisches Staatssammlung Muenchen (ZSM) holds 130 specimens collected during an 1891 expedition by G. Bauer , and another 161 specimens collected in 1897 on the first Webster-Harris Expedition .Avipoxvirus in wild bird populations on Galapagos to assess its contribution to this accelerated population-level extinction rate. Because of the recent arrival and controlled spread of humans on the archipelago, we were able to use this extensive museum collection to ask whether the arrival and distribution of Avipoxvirus on the Galapagos Islands was associated with their inhabitation by humans.Only Floreana (colonized in 1807) was inhabited when Darwin visited in 1835, but the human population on Floreana was intermittent until the 1930′s, since which time it has been inhabited continuously. Today, five islands are inhabited. Floreana, San Cristobal (inhabited continuously since 1837), Isabela (inhabited continuously since 1893), Santa Cruz and its satellite island Baltra (Santa Cruz inhabited continuously since 1920) We  visited the CAS in June 2004 and again in June 2008 and examined 4313 of the 6371 passerine specimens collected between 1898 and 1906, including 2903 finches and 704 mockingbirds for cutaneous nodules consistent with poxvirus. Wearing gloves to prevent cross contamination of specimens, we inspected all skin on legs, feet, and around bills, and lightly ruffled feathers to expose other nodules. RER visited the ZSM in 2007 and similarly inspected the 266 finch and mockingbird specimens collected between 1891 and 1897.Our tissue sampling was restricted by CAS to specimens displaying at least two lesions, so as to not remove evidence of infection from these historically important specimens. In addition, we chose specimens from islands in a manner that maximized the number of samples from each of four islands for each focal taxon to evaluate geographic distribution. For finches, those islands were Floreana, Isabela, San Cristobal, and Santa Cruz, and for mockingbirds the islands sampled were Espanola, San Cristobal, Santa Cruz, and Santa Fe . We exciEach excised lesion was subdivided using aseptic technique and a representative portion of the lesion was processed for histopathology by ELB. The tissues were placed in 10% neutral buffered formalin for three days to re-hydrate and fix the tissue. Samples were then processed routinely, and stained with hematoxylin and eosin for light microscopic examination.PCR primers were designed by NKW for <150 bp segments of the virion core protease gene and the integral membrane protein gene that differed diagnostically for two previously characterized canarypox virus variants in Galapagos Genetic tests:3/isoamyl alcohol (25∶24∶1). The final aqueous phase was dialyzed overnight against TNE2 . A subset of samples was extracted by KP and HF in a different facility dedicated to ancient DNA using similar decontamination procedures. DNA was isolated using the same isolation buffer with 0.1 M DTT added, captured with glass micro-beads (QBiogene GeneClean Ancient DNA kits) following the manufacturer's protocol, then re-eluted in 50 µL H2O.DNA was extracted from the remaining half of each excised lesion in a new lab that had never been used for DNA studies and was on a different floor from any other DNA labs. Poxvirus PCR amplicons from this study were stored in a separate building from the lab used for the CAS extractions and genotyping. We conducted extractions in a fume hood and cleaned all work surfaces with 5% bleach between extractions. We added 250 µL lysis buffer  and used a sterile pipette tip to macerate the lesion, added 20 µL of Proteinase-K , and incubated at 65°C overnight (at least 6 h) before extraction with phenol/CHClACYAGTATTCAGCAATTAATAGGACC and 1586R-AGGGCTGCAGATTTTTCGTAT; numbers correspond to 5′ location of the first base in the primer sequence in the five taxon alignment) and amplified a 115 bp fragment. Gal1 and Gal2 differed from the two canarypox strains at site 1535  and Gal1 and Gal2 differed at site 1563 . The second locus was a 117 bp fragment located within the integral membrane protein (CNPV112) gene (primer sequences: 3521F-TGCTAGATCGTCGTTCGT and 3638R-CACTTTAGATTTCCTTATATATGCTG). Gal1 differs from Gal2 and the canarypox strains at site 3566  and Gal2 differs from Gal1 and the canarypox strains at site 3567 . Gal1 and Gal2 differed from fowlpox at numerous sites at both loci.We designed primers that would: (1) amplify regions <150 bp due to the likelihood that the CAS samples were degraded; and (2) discriminate between previously characterized virus sequences. We created an alignment in Clustal X 2O, 3 µL of each primer (10 µM), 1.5 µL of 25 mM MgCl2, 5 µL of 10× Taq Polymerase Buffer (Sigma), 1 µL Bovine Serum Albumin, 1 µL 10 mM dNTPs, 1 µL of Taq polymerase (Sigma) and 2 µL of template DNA. Alternatively, an antibody-bound Taq buffer system  was used in a 25 µL final volume reaction. The PCR cocktail and template DNA from the CAS samples were added to the PCR tubes in a room that was not used for poxvirus sample or amplicon storage or for poxvirus PCR-amplification. These closed tubes (including a negative control) were then transported on ice to the Parker Lab where template DNA for the positive controls was added to those tubes and the samples were placed in the thermocycler. An annealing temperature of 48°C was used for the first primer pair (1471F and 1586R) and 53°C for the second (3521F and 3638R). Each program ran for 35 cycles under standard reaction conditions with a final 7 minute extension at 72°C. Amplicons were verified on 1–2% TBE agarose gels stained with ethidium bromide and visualized under UV light. If bands of the expected size appeared, we purified those products using a QIAQuick PCR purification kit. All PCRs were rerun at least twice and as many as seven times for samples yielding ambiguous results. To be scored positive, a sample must have amplified at least twice. Ultimately, all samples were either consistently negative or amplified at least twice, in both the UMSL laboratory and the ancient DNA facility.We attempted to PCR-amplify each locus on the extracted CAS lesions and three positive controls  from Direct sequencing was performed on both strands of 17 amplicons using the same primers with ABI PRISM® BigDye Terminator PCR cycling conditions and sequenced on an Applied Biosystems 3100 DNA Analyzer . Raw sequence chromatograms of forward and reverse strands were assembled in Seqman II . The entire length of each strand was evaluated by eye. Poor quality data and primer sequences were trimmed from both strands. Eleven samples yielded good sequence data at the four diagnostic variable sites in the virion core protease and integral membrane protein genes. Comparing against the five-taxon reference alignment, these positive individuals were identified as being infected with Gal1 or Gal2 variants (GenBank accession numbers AY631870 and AY631871).Avipoxvirus, showing raised nodules of smooth or ulcerated surface with well-defined margins, from 1 to 4 mm in diameter. Lesion prevalence by island ranged from 0.011 to 0.18  displayed gross cutaneous lesions consistent with  to 0.18 . The higThe 59 tissue samples taken from finch (from 4 islands) and mockingbird (from 4 islands) specimens represented six major islands overall, three of which were inhabited at time of collection  and three of which were not  of the 59 specimens were scored as positive .Avipoxvirus DNA sequence identical to Gal2 For the 34 mockingbirds sampled, positives were nonrandomly distributed across islands : five from San Cristobal  and one from Santa Fe  were positive , while 1Had avipoxvirus been present in 1891 and 1897 at the apparent prevalences detected in 1898–1906 , the proBeagle anchored and Charles Darwin collected specimens in the Galapagos Islands, Avipoxvirus was present in its endemic birds. At that time the virus was heavily concentrated on the human-inhabited islands, particularly San Cristóbal, which was the most heavily human-populated island at that time, compared to the much smaller settlements on Isabela and Floreana These results indicate that 110 years ago, 64 years after the Avipoxvirus infecting passerine birds in Galapagos initially arrived is unknown. There is no evidence that these infections derived from the poxvirus in domestic farmyard birds; in both Galapagos and Hawaii, the virus infecting the domestic chickens is distinct from that infecting passerines, and the form in passerines clusters with previously described canarypox virus present elsewhere Dolichonyx oryzivorus), the only passerine regularly seen  during its annual migrations between North and South America. We regard arrival with an insect vector as unlikely, partly because the principal mechanical vectors were first recorded relatively recently  is known to take at least occasional blood meals from birds, but its strong population differentiation among islands and habitats suggests very short dispersal distances The other possible mode of movement of the virus is with the arthropod vectors that may mechanically transport virions from one blood meal to the next. The arrival of the bird-biting Today, repeated localized travel occurs regularly among inhabited and uninhabited islands in the form of tourism, Park management visits, scientific research, and permitted and illicit hunting and fishing. And since most of these diverse groups use common landing sites on both inhabited and uninhabited islands, they have likely contributed to the presence of this virus and other pathogens across the archipelago. Of these groups, only scientists undergo rigorous quarantine procedures to minimize or eliminate transport of organisms between islands. In modern times, pox-like symptoms are reported regularly in birds on Santa Cruz, Isabela, San Cristobal, and Floreana Avipoxvirus. The critically small populations of the Floreana Mockingbird  and finches (at least 13 species). Their early colonizations and subsequent diversifications Avipoxvirus should be made carefully, and that visual inspection for cutaneous lesions (e.g., Avipoxvirus. It could be argued that true pox infections may be missed by both PCR and histopathology in such historic samples, but several were given clear alternative diagnoses in histopathology; in other words, causative agents other than Avipoxvirus were provided for the symptoms. At the very least, these preliminary diagnoses should be suggested as “pox-like” (e.g., These results also indicate that diagnosis of ns e.g.,  is not se” e.g., , but in e” e.g.,  that wase” e.g.,  negativeAvipoxvirus; competent vectors in Culex quinquefasciatus and perhaps other mosquitoes; and the recently detected Plasmodium blood parasite Avipoxvirus has been on the islands at least since 1899. Culex quinquefasciatus was first documented in the 1980′s and later confirmed to have breeding populations Plasmodium in Galapagos penguins, we are working hard to understand the transmission dynamics among endemic bird populations for these two pathogens and their associated vector communities. We remain optimistic that the Galapagos avifauna can avoid the declines and extinctions suffered in Hawaii, by effective management practices that require a more thorough understanding of the roles played by each of the three elements, their individual histories and dynamics, and their interactions.Regretfully, the Galapagos Islands are now inhabited by the same three elements that triggered a massive decline of endemic birds in Hawaii: the


Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa , A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types. Nanomedicine is the application of nanotechnology in innovative ways to develop new approaches and therapies for treatment of diseases, including drug delivery and gene therapy Briefly, some aspects of the different uptake pathways are summarised here.Phagocytosis is restricted to specialized mammalian cells such as neutrophils, monocytes and macrophages, which function to engulf and digest cellular debris and pathogens, and are essential to keep the immune system in a clean and sterile state. Interaction of a pathogen with specific cell surface receptors induces signalling cascades mediated by Rho-family GTPases, triggering polymerization of actin membrane protrusions at the site of ingestion. After internalisation, actin is shed from the phagosome, and a series of fusion and fission events, involving components of the endocytic pathway, culminates in the formation of the mature phagolysosome Other well characterised uptake pathways include: (i) Clathrin-mediated endocytosis (CME), which is a process involving specific receptors that recognize and internalize cargo into “coated pits”, formed by the assembly of a cytosolic coat protein, clathrin, which constitutes the main assembly unit. These coated pits invaginate and pinch off to form endocytic vesicles (i.e. early endosomes), that later mature into late endosomes and fuse with lysosomes. The literature on these pathways is extensive and the structure of clathrin and the clathrin coated pits is well resolved, as is the role of other key proteins involved Fundamental questions in the study of the interactions of NPs with cells include whether, and how, NPs map onto these pre-existing cellular uptake mechanisms. As a result of their nanoscale size, which is similar to that of many of the protein-lipid clusters (lipoprotein complexes) routinely transported into and out of cells, and their ability to interact with lipoproteins In the present paper, we seek to investigate by which endocytic mechanism(s) negatively charged carboxylated-modified polystyrene NPs, of two sizes representative of typically endocytotic cargoes , are internalized by a panel of different cell types, including HeLa tumoral epithelial cells from Cervix, commonly used to study cell biology of uptake mechanisms Carboxylated-modified polystyrene (PS-COOH) NPs with nominal sizes of 40 nm and 200 nm were chosen as model particles in order to understand the mechanism of internalisation in different cell lines, due to their wide availability to the scientific community, and the fact that they are fluorescently labeled in a manner that we have previously shown is suitable for uptake studies  2 NPs of several sizes showed the same behavior, with NP uptake being highly energy dependent In order to determine whether NP uptake was an active or passive process, cell cultures were incubated in parallel at 37°C  and at 4°C. It is known that several proteins and enzymes are sensitive to temperature, thus active processes are inhibited by lowered temperatures One important requirement when using endocytosis inhibitors  is that they should not affect the actin cytoskeleton, since reorganization of the actin filaments may, as a side effect, impact on uptake processes which do not directly involve actin and may alter the function of various plasma membrane proteins NP uptake was measured only at short exposure times, since it has been previously reported that blocking one uptake pathway can result in activation of other endocytic mechanisms, which again confounds the data The NP uptake results after treatment with the transport inhibitors are shown in The cytoskeleton plays an important role in multiple cellular events, including endocytosis and trafficking of endocytosis vesicles. For this reason, we have investigated the role of two of the major constituents of the cytoskeleton structure, microtubules and F-actin, in NP uptake and internalisation. Although both microtubules and F-actin are essential in most of the uptake mechanisms described above, it is important to evaluate how strongly NP uptake depends on the presence of these structures. As mentioned above, cytochalasin A can disrupt F-actin polymerization, and as such, has been used to investigate the role of F-actin in cellular uptake of PS-COOH NPs. As shown in et al. (2008) who found that uptake of 120 nm negatively charged polystyrene NPs by HeLa cells was not inhibited by nocodazole.It has been reported that microtubules are also involved in the endocytic process, and thus nocodazole was used as a microtubule disruptor in order to assess the influence of microtubules on NP internalisation by the panel of cells. The effects of this treatment were not as strong as with actin depolymerisation. Nocodazole was found to inhibit endocytosis of larger particles (200 nm PS-COOH) primarily, with the highest inhibition observed in the A549 cell line, where uptake was reduced by ∼40% relative to control cells . In the et al. (2004), using similar negatively charged polystyrene NPs, with sizes between 50 and 200 nm in B16 cells (mouse melanoma cell line), showed instead that chlorpromazine treatment affected more strongly the uptake of the smaller NPs, which is the opposite to what we found in the cell lines used in this study. Moreover, Dausend et al. (2008) showed no significant inhibitory effect of chlorpromazine on the uptake of 120 nm negatively charged polystyrene NPs by HeLa cells.It is well established that transferrin (Tf) is a ligand exclusively internalized via the clathrin-coated-pit endocytosis pathway, and thus its uptake is expected to be affected by treatment with chlorpromazine, an inhibitor of clathrin-mediated endocytosis. For this reason, we used transferrin as a positive control to confirm the inhibition of this particular endocytotic pathway upon treatment with chlorpromazine et al., who suggested that caveolae flask-shaped invaginations are able to internalize larger particles more easily than clathrin coated pits . Although lacking a clear mechanistic explanation as yet, electrostatic interactions and kinetics of internalisation could explain why, despite the size of the caveolae being normally 60–80 nm, genistein has such a big effect in the uptake of 200 nm NPs.As described above, receptor-associated tyrosine-specific protein kinases are reported to be involved in lipid raft-dependent endocytosis, thus genistein, which is a specific inhibitor of these proteins, can be used to study the involvement of lipid raft-dependent uptake mechanisms in the uptake of PS-COOH NPs by our panel of cells. It is noteworthy that the concentration of genistein applied in the present study was not sufficient to significantly inhibit the uptake cholera toxin B  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin (Invitrogen Corp.).Human glial astrocytoma 1321N1 cells (passage 2–10 after defrosting from liquid nitrogen)were cultured at 37°C in 5% CO2 in Minimum Essential Medium  supplemented with 10% foetal bovine serum , 1% penicillin/streptomycin (Invitrogen Corp.), and 1% MEM non-essential amino acids (HyClone).Human lung epithelium A549 cells  were cultured at 37°C in 5% CO2 in Minimum Essential Medium  supplemented with 10% foetal bovine serum  and 1% penicillin/streptomycin (Invitrogen Corp.).Human cervix epithelium HeLa cells (passage 5–10) were cultured at 37°C in 5% COAll cell lines were confirmed to be mycoplasma negative using the mycoAlert kit  and were tested monthly.Red dye-loaded (Excitation/Emission wavelengths: 580–605) carboxylate-modified polystyrene NPs  were used without further modification or purification. Sizes used in this work were 40 nm, 200 nm. All stock solutions were stored at 4°C, and used within 1 year of purchase, in order to ensure their stability.NP dispersions were prepared by diluting the concentrated stock solutions into the complete medium (cMEM) used for cell culture at room temperature, with or without inhibitor drugs, immediately prior to the experiments on cells, with an identical time delay between diluting and introducing to the cells for all experiments. The medium was kept at room temperature and not pre-warmed to 37°C to ensure better NP dispersions. Before sampling, NPs were vigorously mixed by vortexing, as recommended by the company.The mean size and surface charge of NPs were determined using a photon correlation spectrophotometer . Measurements were performed at 25°C in DPBS and in MEM. The particle size distribution and zeta potential data are presented in 5 cells were seeded in individual 30 mm tissue culture dishes (Greiner Bio-one), and incubated for 24 h prior to addition of particles. Each step from NP exposure to cell to sample preparation for flow cytometry has been optimized in order to ensure reproducibility of the data and quantitative information to be obtained. Several artifacts could arise from this kind of experiments without such optimizations and control. Thus, after 24 hrs, cells were pre-incubated for 30 min with the different drugs at the following concentrations: chlorpromazine hydrochloride 10 µg/ml, cytochalasin A 5 µg/ml, genistein 200 µM and nocodazole 20 µM . Energy dependence experiments were performed by pre-incubating the cells at 4°C for 30 min prior to exposure to NPs. After this pre-incubation, NPs  were added and incubated for 120 min, either in the presence of the drugs or at 4°C. Negative controls, i.e., cells without the presence of drugs and/or NPs were also carried out. After this incubation time, the medium was removed and the samples were washed with DPBS, in order to ensure particle removal from the outer cell membrane. As an example of the protocol optimization, 1.25×104 cells/100 µl/well (n = 6) and incubated for 24 h. After 24 hrs, cells were incubated for 120 min with 50 µl of medium containing one of the inhibitors at the concentrations mentioned above. Normal cells were used as the negative control. ATP content was measured in accordance to the protocol of the CellTiter-GloTM luminescent cell viability assay kit (Promega). Briefly, 50 µl of assay reagent was added to the wells and mixed for 2 min at room temperature. After 10 min, intracellular ATP content was measured using a Varioskan Flash plate reader (ThermoFisher Scientific). Normalisation of ATP content was calculated using the following equation: Normalised ATP content (in percentage)  =  [value (cells exposed to drug)/value (cells untreated with drugs)]×100. For the propidium iodide assay, 1.25×105 cells were seeded in individual 30 mm tissue culture dishes (Greiner Bio-one), and incubated for 24 h prior to addition of drugs. After 24 hrs, cells were incubated for 120 min in the presence of one of the drugs at concentration described before. After this incubation time, the medium was removed and the samples were washed thrice with DPBS and harvested with 0.05% trypsin/EDTA 1x. Cell pellets were re-suspended with 0.5 ml of DPBS and kept ice-cold until measurements were performed. 5 µl of propidium iodide was added per sample, and this was vortexed for 1 min and cell-associated fluorescence  was detected using a CyAn ADP (DAKO) flow cytometer. The % of PI positive cells was calculated using the following equation  =  [% of PI positive cells (cells exposed to drugs) - % of PI positive cells (cells untreated with drugs)].Cells were seeded in white, clear bottomed 96 well micro-assay-plates, (Greiner bio-one) at 104 cells were seeded onto glass slides  and incubated for 24 h prior to addition to particles. The cell number was set to ensure a cell density comparable to the flow cytometry experiments and, in order to keep all parameters affecting the experiment constant, the same protocols were used for exposure to particles, sample preparation and cell fixation. Thus, particle dispersions were prepared in cMEM at room temperature just before addition to the cells and, after particle exposure, medium was removed and all samples were washed thrice with DPBS, fixed for 20 min with 4% formalin solution neutral buffered, and the nucleus stained with 4′,6-diamidino-2-phenylindole (DAPI blue). For F-Actin staining, after exposure to one of the inhibitors, and fixation as described above, cells were permeabilised with 0.1% Saponin (Sigma Aldrich) for 5 min before staining the actin filaments by incubation with Texas-red phalloidin (Invitrogen Corp.) for 20 min.For confocal microscopy, 3.0×10A confocal microscope  was used to capture images of the intracellular environment and the sub-cellular localisation of the fluorescent polymeric NPs. For multi-color microscopy, samples were excited with 364 nm (blue channel), 488 nm (green channel), and 543 nm (red channel) laser lines, and images were captured by multi-tracking to avoid bleed-through between the fluorophores. To achieve the necessary signal to noise ratio, while obtaining the thinnest possible optical slices, the pinhole diameters were set to less than 1 airy unit. After adjustment of the pinholes of both lasers to obtain the same optical slices, the optimal optical section that fulfilled our criteria was in the range 0.7–0.8 µm at 63X. The gain and offset for the different channels were kept constant along the full series of experiments in order to allow quantitative comparison of the cell fluorescence intensities.Figure S1Kinetics of uptake of 40 nm and 200 nm PS-COOH NPs by HeLa cell line, as determined using flow cytometry over 24 hrs. NP concentration is 20 µg/ml. Averaged mean values of triplicate experiments are given. Inserts: confocal images, showing 40 nm and 200 nm PS-COOH NPs internalized by HeLa cells . The scale bar corresponds to 20 µm.(TIF)Click here for additional data file.Figure S2Fluorescence emission of the PBS washes of HeLa cells following exposure to NPs. PBS washes were used to remove non internalised nanoparticles adhering on the cell membrane prior to assessment of the cell fluorescence intensity by flow cytometry. Similar data were obtained for the other cell lines. From this data it is clear that 3 washes with PBS are sufficient to ensure that the remaining cell fluorescence is from nanoparticles that have been internalised by the cells.(TIF)Click here for additional data file.Figure S3Confocal images of a) 1321N1 and b) HeLa cells, showing their F-actin morphology, after incubation with the different inhibitors at 4°C for 2 h30 min . The scale bar corresponds to 20 µm.(TIF)Click here for additional data file.Figure S4a) Effect of chlorpromazine on internalization of transferrin into different cells. Cells were pre-treated with chlorpromazine for 30 min, followed by 10 min of exposure to Alexa fluor® 488 labelled transferrin in the presence of chlorpromazine, before being fixed and analyzed by FACS. b) Effect of genistein on internalization of cholera toxin B into A549 cells. Cells were pre-treated with genistein for 30 min, followed by 20 min of exposure to Alexa fluor® 488 labelled cholera toxin B in the presence of genistein, before being fixed and analyzed by FACS. Mean values and standard deviations of duplicate samples are given. Results are reported as % uptake relative to the control cells which were not treated with the inhibitor.(TIF)Click here for additional data file.


Tantalum, as a potential metallic implant biomaterial, is attracting more and more attention because of its excellent anticorrosion and biocompatibility. However, its significantly high elastic modulus and large mechanical incompatibility with bone tissue make it unsuitable for load-bearing implants. In this study, porous tantalum coatings were first successfully fabricated on titanium substrates by vacuum plasma spraying (VPS), which would exert the excellent biocompatibility of tantalum and alleviate the elastic modulus of tantalum for bone tissue. We evaluated cytocompatibility and osteogenesis activity of the porous tantalum coatings using human bone marrow stromal cells (hBMSCs) and its ability to repair rabbit femur bone defects. The morphology and actin cytoskeletons of hBMSCs were observed via electron microscopy and confocal, and the cell viability, proliferation and osteogenic differentiation potential of hBMSCs were examined quantitatively by PrestoBlue assay, Ki67 immunofluorescence assay, real-time PCR technology and ALP staining. For in vivo detection, the repaired femur were evaluated by histomorphology and double fluorescence labeling 3 months postoperation. Porous tantalum coating surfaces promoted hBMSCs adhesion, proliferation, osteogenesis activity and had better osseointegration and faster new bone formation rate than titanium coating control. Our observation suggested that the porous tantalum coatings had good biocompatibility and could enhance osseoinductivity in vitro and promote new bone formation in vivo. The porous tantalum coatings prepared by VPS is a promising strategy for bone regeneration. In the last few years, great interest has been focused on tissue engineering as a potential therapeutic approach for musculoskeletal diseases. The role of metallic implants either for osteo-synthesis or for arthroplasty has been tested in preclinical and clinical settings. An ideal implant material should have appropriate elastic modulus, corrosion resistance, good biocompatibility and favor bone anchorage. However, most medical implant materials do not simultaneously fulfill all these characteristics. Accordingly, various coatings have been developed to improve the biocompatibility and osseoinductivity of load-bearing materials. Hydroxyapatite (HA) coating is one of outstanding examples of such coatings. HA-coated titanium (Ti) implants have the good biological activity of HA and the excellent mechanical properties of Ti alloy, and are thus widely used in orthopedic surgery as bone replacement materials Tantalum (Ta) may be a potential coating material for medical implants. Ta (atomic number 73) is a rare transition metal that is highly corrosion resistant and inert in vivo Bone repair requires a cellular source with the ability of differentiation into bone together with a scaffold that allows the adhesion and proferation of these cells. Bone marrow mesenchymal stem cells (BMSCs) research is currently an exciting area of interest, since these cells have the ability to differentiate into several cell types, including osteoblasts, chondrocytes and adipocytes. In this regard, they have been extensively assessed for bone defects treatment hBMSCs  were donated by patients with written informed consent and this experiment was approved by Independent Ethics Committee of Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM). The rabbits were obtained from the Laboratory Animal Center of Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM) (Certificate number SCXK 2007-0007). Handling of the animals was in accordance with policies of Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM) and approved by the Animal Experimental Ethics Committee, Shanghai Ninth People’s Hospital affiliated to Shanghai Jiao Tong University School of Medicine (SJTUSM) (Permit Number: HKDL [2012]5).Ta and Ti coatings were prepared by a VPS system . Medical Ti-6Al-4V substrate was used to prepare Ta-coated implants and interface samples. The samples for investigating hBMSC proliferation and differentiation in vitro were cuboid with dimensions of 10 mm×10 mm×2 mm and cylindrical with dimensions of Ø 33 mm×2 mm. The samples for implantation in vivo to examine osseointegration and new bone formation rate were cylindrical with dimensions of Ø 2 mm×10 mm.The coating surface was characterized using the following equipment: an X-ray diffraction (XRD) instrument , a contact angle analyzer , a field emission scanning electron microscopy (SEM) system , and an electron probe microanalyzer .2 incubator (MCO-18AIC (UV), SAVYO, Panasonic, Kadoma, Osaka, Japan) was used for cell culture with growth medium .The hBMSCs were isolated and cultured as previously described 4 cells/well. After confluence in growth medium, the cells were treated with osteogenic induction medium , and then replaced every 3 days. After 7 days, the cells were stained using a BCIP/NBT ALP Color Development Kit . Cells cultured in normal growth medium served as control.The hBMSCs were seeded in six-well plates  at a density of 5×105 cells/well. After confluence in growth medium, the cells were treated with adipogenic induction medium  for 2 days. Thereafter, adipogenic maintenance medium  was used and replaced every day for 14 days. Oil Red O staining was performed and then the sample was observed under a microscope . Cells cultured in normal growth medium served as control.The hBMSCs were seeded in six-well plates  at a density of 2×104 cells/well with the growth medium. After 24 h, the samples were transferred to new 24-well plates for further experiment. DRAQ5  was used as fluorescent DNA dye. The samples were scanned with an Odyssey near-infrared scanner  to determine the proliferation rate variation of hBMSCs on porous Ta and Ti coating. PrestoBlue Cell Viability Reagent  was used according to the manufacturer’s instructions to test cell viability, and the absorbance was obtained using a microplate reader  at wavelengths of 570 and 650 nm. LIVE/DEAD Cell Viability Assays  were performed as described in the manual. F-actin was identified by fluorescent phallotoxin . Anti-Ki67 antibody  was used to detect Ki-67 protein expression in hBMSCs, and DAPI  was used to indicate the nucleus of hBMSCs. LIVE/DEAD Cell Viability Assays, fluorescent phallotoxin, and anti-Ki67 antibody/DAPI were observed by confocal laser scanning microscopy .The 10 mm×10 mm×2 mm samples were placed in 24-well plates  and the hBMSCs were seeded at 2×10The CFSE labeling experiment was carried out as described 4 cells/well with the growth medium. After 24 h, the samples were transferred to new six-well plates, and after another 24 h, the growth medium was replaced with the osteogenic induction medium, which was replaced every 3 days. The cells were stained using a BCIP/NBT ALP Color Development Kit , and then scanned with an Odyssey near-infrared scanner  and a normal scanner .The Ø32 mm×2 mm samples with coating were placed in six-well plates  and the hBMSCs were seeded at 5×10In-Cell Western assay was performed following the protocol of CST Ltd. to evaluate the protein expression of ALP in hBMSCs on porous Ta coatings. Paraformaldehyde (4%) was used for fixation. Odyssey Blocking Buffer  was used as a blocking and antibody-diluting buffer. ALP antibody  was used to detect the protein expression of ALP in hBMSCs on the coatings. After incubation, Infra-Red Secondary Antibody IRDye  was used as a secondary antibody and DNA staining dye DRAQ5  was used as normalizing agent Total RNA of cells was isolated using TriPure Isolation Reagent  according to the manufacturer’s instructions. Equivalent amount of RNA samples were reverse transcribed for first-strand cDNA using a RevertAid First Strand cDNA Synthesis Kit . Real-time PCR was performed by Lightcycler480  using SYBR Premix Ex TaqTM  according to the manufacturer’s instructions. GAPDH was used as a reference. The real-time PCR conditions were as follows: denaturation at 95°C for 10 s, 40 cycles at 95°C for 10 s, and 60°C for 20 s. Dissociation was performed for a melting curve analysis to monitor and avoid non-specific amplification as well as primer dimers. The collected data were analyzed by the advanced relative quantification method using Roche Lightcycler480 software.New Zealand white rabbits  were used as the femur implant model. The operation was performed under aseptic conditions by chloral hydrate intramuscular injection. Defect in each femoral condyle was made by a Ø 2 mm drill toward the medial epicondyle orientated perpendicular to the longitudinal and sagittal axes The first labeling was performed by intravenous injection 10 days before sacrificing the rabbits, and the second labeling was performed 3 days before sacrificing. Calcein  was used for both labelings.After cleaning the soft tissue, the samples were fixed in 4% paraformaldehyde buffer in PBS for 10 days and then dehydrated in successive alcohol concentrations . The dehydrated sample was embedded in Technovit 7200VLC  for 2 days using the EXAKT 510 Dehydration and Infiltration System , and then polymerized by the EXAKT 520 Light Polymerization System . Subsequently, 300 µm sections were cut perpendicular to the implants using Saw Microtome Leica SP1600 , ground to 50 µm by the EXAKT 400 CS Micro Grinding System with AW 110 controller , and finally stained by Van Gieson’s Picric–Fuchsin stain for histomorphometry.Different histomorphometric parameters were obtained using a microscope . A semiautomatic image analysis system (BIOQUANT) was used to measure the surfaces of the sections. The area of 50 pixels around the implant was selected as the region of interest (ROI), amounts of bone tissues in the ROI were calculated by using the measurement tools. Bone volume fractions  of different samples were calculated based on Van Gieson’s Picric–Fuchsin staining and compared statistically.Statistical analysis was performed using SAS JMP . Aspin-Welch-Satterthwaite-Student's t-test (t-test works even if variances in the two sample groups are different) were used to compare the means of the two sample groups.The hBMSCs were isolated as previously described ). The oxide layer of Ta (Ta2O5) formed on the surface . The initial surface morphology of Ta and Ti coatings was observed by SME at 300×, 500×, 1000×, and 3000× magnifications , showing that the surface roughness of both Ta and Ti coatings was similar.The XRD results showed that the Ti coating surface was mainly composed of Ti, and the composition of the Ta coating was major Ta and a minor Ta oxide . In n = 3). For day 2, the data were as follows: Ti coating, 0.45268±0.097954; Ta coating, 1.0767±0.495686; P<0.992. For day 3, the data were as follows: Ti coating, 0.56950±0.089554; Ta coating, 1.26228±0.458399; P<0.0620. For day 4, the data were as follows: Ti coating, 0.90275±0.082739; Ta coating, 2.13239±0.699593; P<0.0390*. For day 6, the data were as follows: Ti coating, 0.86752±0.089092; Ta coating 1.68688±0.386935; P<0.0233*. LIVE/DEAD Cell Viability assay and anti-Ki67 immunofluorescence assay  demonstrated not only faster proliferation but also less cell death of hBMSCs on porous Ta coating than on Ti coating in 48 h. The CFSE labeling results (An immunofluorescence experiment was conducted in which the DNA dye Draq5 was applied to monitor cell number (density). The result . These results suggested that the porous Ta coating may be more beneficial for hBMSC osteogenesis than Ti coating.The hBMSCs were cultured on porous Ta and Ti coatings with the osteogenic medium, and then ALP and RUNX2 were detected as osteoplastic differentiation-related markers. Higher ALP activity and expression were detected . The double fluorescence labeling (Van Gieson’s Picric–Fuchsin stain of transverse sections was used to show the osseointegration of the porous Ta and Ti coatings. The mineral apposition rate of new bone formation was also investigated by measuring the distances of double fluorescence labeling. After implantation for 3 months, histomorphometric parameters clearly indicated that more new bone was formed around the porous Ta coating implant than around the Ti coating implant (Over the last two decades, a variety of porous coatings and materials have been used to achieve biological fixation of implants. Ti alloys are found to be very suitable materials for load-bearing in bioimplant applications because of their good and reliable mechanical properties. Unfortunately, like most metals, Ti exhibits poor osteoinductive properties. Among metallic biomaterials, Ta is gaining increased attention as a new biomaterial. Ta has been demonstrated to be corrosion resistant and bioactive in vivo. Although Ta has been used for plates and suture wires in orthopedic, craniofacial surgery, and dentistry The in vitro hBMSC-material interaction results clearly indicated that the porous Ta coating surface significantly improved the adhesion and proliferation of hBMSCs, as assessed by SEM cell morphology, F-actin cytoskeletal staining, immunofluorescence, and PrestoBlue assay. The characteristics and biocompatibility of implant surface material were closely related to cell-material interactions, and can influence cell response and behavior Moreover, the porous Ta coating not only enhanced hBMSC adhesion and proliferation, but also stimulated mesenchymal stem cell osteogenetic differentiation in vitro compared with porous Ti. This finding was confirmed by the enhanced ALP and RUNX2 activity on Ta coating. The possible explanation was that the bone-like elasticity substrates may have been better for bone regeneration and contributed to the differentiation lineage of the hBMSCs These results revealed the ability of porous Ta coating to support the differentiation of mesenchymal stem cells into osteoblasts in vitro and of matured bone cells in vivo. Porous Ta was found to have excellent biocompatibility and be safe to use in vivo Porous Ta coatings were fabricated by VPS with Ti alloy as substrate. The deposition of porous Ta layer effectively alleviated the mechanical incompatibility between metal implant and bone tissue. The biocompatibility, osteoinductivity, and osseointegration of the Ta coated implants were studied to determine their feasibility in clinical applications. In vitro, hBMSCs were used and the results showed that hBMSC adhesion, proliferation, and osteogenic differentiation were enhanced on the Ta coating surface compared with the Ti coating control. Moreover, in vivo implantation in a rabbit femur defect model confirmed the excellent osseointegration and new bone formation of the porous Ta coated implants. These results suggested that porous Ta coating applied by VPS is a promising strategy for bone regeneration.


We explored changes in ocean pH in coastal Washington state, USA, by extending a decadal-scale pH data series, by reporting independent measures of dissolved inorganic carbon (DIC), spectrophotometric pH, and total alkalinity (TA), by exploring pH patterns over larger spatial scales, and by probing for long-term trends in environmental variables reflecting potentially important drivers of pH. We found that pH continued to decline in this area at a rapid rate, that pH exhibited high natural variability within years, that our measurements of pH corresponded well to spectrophotometric pH measures and expected pH calculated from DIC/TA, and that TA estimates based on salinity predicted well actual alkalinity. Multiple datasets reflecting upwelling, including water temperature, nutrient levels, phytoplankton abundance, the NOAA upwelling index, and data on local wind patterns showed no consistent trends over the period of our study. Multiple datasets reflecting precipitation change and freshwater runoff, including precipitation records, local and regional river discharge, salinity, nitrate and sulfate in rainwater, and dissolved organic carbon (DOC) in rivers also showed no consistent trends over time. Dissolved oxygen did not decline over time, indicating that long-term changes did not result from shifts in contributions of respiration to pH levels. These tests of multiple potential drivers of the observed rapid rate of pH decline indicate a primary role for inorganic carbon and suggest that geochemical models of coastal ocean carbon fluxes need increased investigation. There is accumulating direct evidence that ocean pH has declined over the past two decades There has been strong recent interest in the carbon cycle of marine environments, because of the large role oceans play in the carbon cycle. The consequences of the substantial oceanic uptake of anthropogenic releases of CO2, and chemical equilibria of inorganic components of the ocean carbon system predict that ocean pH should decline at a rate of −0.002 to −0.004 units per year, with an acceleration over time as CO2 release increases and buffering capacity of the oceans becomes depleted 2 concentrations in the Strait of Juan de Fuca, which corroborates the data of Wootton et al. 2 delivery to the region. While such space-for-time substitutions can be powerful for identifying potential mechanisms of change 2 are critical to better understand why pH appears to be declining rapidly in high-latitude coastal locales.Global models based on physical models of the ocean, historic measurements and projections of atmospheric CO2 and total alkalinity (TA) to more fully describe the dissolved inorganic carbon (DIC) system at this site. Finally, we probe multiple data series for long-term trends that test several alternative hypotheses to explain rapid pH decline at this site. These hypotheses include:In this paper we temporally and spatially extend the pH data series from the northeastern Pacific off the coast of Washington state Upwelling has increased, bringing water high in DIC derived from respiration Eutrophication has increased, presumably as a result of increasing anthropogenic nutrients, causing increased CO2 content as the products of primary production decay Climate change increases freshwater input, which dilutes the buffering capacity of the ocean Increasing input of dissolved organic carbon (DOC) from land  increases oceanic respiration, lowering pH. This hypothesis predicts declining dissolved oxygen and increasing DOC in rivers in the region.2-derived carbonic acidLow pH arises from input of acids other than CO, such as acid precipitation from increased emissions or changes in input of organic acids from biological processes. This hypothesis predicts declining total alkalinity. In the case of acid precipitation input, the hypothesis predicts long-term increases in sulfate and nitrate concentrations in precipitation.Changes in phytoplankton abundance change pH. Decreasing phytoplankton might reduce organic carbon fixation, reducing inorganic carbon uptake and reducing pH. Phytoplankton has been suggested to be in global decline 2nd = 0.62+0.92 pH1st, where pH1st and pH2nd correspond to the first and second measurements, respectively; linear regression, p(pH2nd = pH1st) <0.0001). Hence at low pH values, subsequent measurements tended to be slightly higher than expected whereas at high pH values, subsequent measurements tended to be lower than expected. Second measurements were made after all first measurements were made; hence, samples could interact for approximately 15–30 min with an air headspace introduced after the first measurement was made. We therefore interpret this deviation as the result of DIC in the water starting to equilibrate with CO2 in the air. Overall, pH measured by the electrochemical probe corresponded to pH measured spectrophotometrically in the lab  = 0.709), indicating that our in-situ electrochemical measurements provided reasonable and unbiased pH estimates, albeit with the larger error for each measurement expected by the method. The pH probe also measured seawater buffer with good accuracy .Sampled pH continued to decline at Tatoosh Island  at a ratThe total alkalinity of our samples measured by the CDQC laboratory was linearly related  were negatively associated with log(DIC) values measured by the CDQC laboratory  = 0.908). Spectrophometric pH measurements from the lab showed a tighter association (spec = 38.16–9.18 log(DIC), r = 0.911). These measurements also exhibited a relationship to pHtotal calculated from DIC and TA that did not differ from the identity relationship (spec = pHDIC) = 0.121).Dissolved CO−1) than predicted by global models of pH (Measurements in transects leading away from Tatoosh Island  showed nls of pH .2 (data not shown), annual means of the variables we probed that should be linked to other key hypothesized drivers of ocean pH generally showed no significant (p<0.05) trends through time, in contrast to the strong trend in ocean pH  fluctuate positively in tandem with the expected stoichiometry as aerobic respiration takes up O2 and generates CO2 (lower pH) while photosynthesis does the opposite  from our study site exhibit a rapid decline in their δ13C values (−0.071°/oo per year) coincident with our measured pH decline but exhibit no temporal patterns in δ18O expected with upwelling associated cold temperature waters Increasing delivery of low pH water from below the surface is an appealing, straightforward hypothesis to explain the declines (hypothesis #1), but examination of multiple lines of evidence provides no clear independent support. There is little doubt that upwelling of subsurface waters with high concentrations of DIC derived from respiration contributes to low pH conditions at some times of the year. Observations in 2007 at transects radiating from the west coast of North America reveal low pH waters lurking close to the surface in near-shore areas characterized by upwelling 2 concentrations that return via upwelling.Although changes in eutrophication (hypothesis #2) have been hypothesized to drive changes in coastal pH in other areas 2 into the atmosphere is predicted to increase precipitation in many areas, including northwestern North America and rising global air temperatures could also increase glacial melting from local mountains 2 into the ocean to generate a strong pH-CO2 relationship. These hypotheses predict that precipitation should increase, river discharge should increase and, importantly, that these increases create an appreciable effect on seawater concentrations of solutes. None of these variables, however, exhibit long-term trends over the course of our pH data series.Reduced buffering capacity via increased freshwater input (hypothesis #3) is another appealing hypothesis that fails to receive any independent support in the data series we have explored. Global warming from anthropogenic input of CO2. Rivers draining into the North Atlantic have shown a general increase in DOC concentrations 2 levels and associated terrestrial plant production lead to greater input of organic carbon into rivers 2 is intriguing, as it would reinforce the strong association observed between our observed pH change and atmospheric CO2. Alternative explanations exist, however, including the effects of declining acid precipitation on soil chemistry 2 concentrations are a global-scale pattern, acid precipitation is a more regional phenomenon that was historically a minor issue along the northeastern Pacific coast, owing to the lack of upwind industrial activity.A variant of the eutrophication hypothesis (hypothesis #4) would be that dissolved organic carbon is being introduced into the coastal ocean at increasing rates, increasing respiration-generated CO3 and NO2 concentrations in our water samples are inconsistent with this variant of the hypothesis, but we know of no SO4 data to more fully test it.Although not historically a factor, increasing industrialization of southeast Asia raises the possibility that acid precipitation input is increasing across the Pacific, which might lead to systematic declines in pH (hypothesis #5). Calculations indicate that this mechanism is not sufficient to generate significant ocean acidification on a global scale, but we address it for completeness here. Data from the nearby station monitoring rainfall chemistry does not support this hypothesis, as concentrations of the major constituents causing acid rain showed no significant trends through time and, if anything, declined. Acid precipitation falling in other areas of the ocean conceivably could be transported via water without being present in local rainfall. The lack of change in NO2 is a central component of several hypotheses to explain rapid pH decline. Because respiration generates CO2 through the use of O2, there should be a tight positive link between DO and pH. Although localized hypoxia events have been reported in central Oregon 2 concentration and pH exists within years, it does not appear among years. The DO data therefore are at odds with the upwelling hypothesis (low pH generated by organic decomposition in the subsurface waters), the eutrophication hypothesis , or the increased DOC input hypothesis .The introduction of organic carbon and its subsequent respiration to generate COTotal alkalinity at our site  tends to2 concentrations. These pH changes have been accompanied by a rapid decline in the δ13C (but no change to δ18O) in mollusk shells that is historically unprecedented over the last 1000 years at Tatoosh Island 13C at Tatoosh is also comparable to that reported for coralline algae in the Gulf of Alaska In sum, our tests of multiple hypotheses for the causes of the rapid pH decline in this region of the California Current have not revealed a definitive driver. Despite our extensive tests of multiple datasets for expected potential drivers, we have been unable to uncover a “smoking gun” of systematically changing environmental parameters through time that are known to be relevant to ocean pH, with the exception of atmospheric CO2 input into the water column during periods of low DIC in the water occurs more rapidly than release of CO2 when water concentrations of DIC are high. More multifaceted scenarios involving spatio-temporal variation in processes driving pH decline in conjunction with differential flux rates into/out of the water and complex coastal currents might also be plausible, if they can explain the decoupling of DO and nutrients from respiration-derived CO2 that would be consistent with our data. For example, O2 has higher flux rates than CO2 into and out of solution Rates of pH decline that are more consistent with model predictions have been observed in series generated in Hawaii All necessary permits were obtained for the described field studies. Specifically, all research was conducted from Makah tribal lands, for which we received the written permission required for access to Tatoosh Island.2 calibration), and then replacing the unit 12–24 hr later. The unit was bolted to the vertical wall of a 5000 l tide pool that is separated from the ocean during the lowest tides (80 cm MLLW) by a 1 m wide wall of rock with a 5 m vertical drop-off into the ocean. Contemporaneous measurements made in the pool and adjacent ocean with handheld probes showed that the two do not differ in temperature, salinity, pH, and DO  4, Si(OH)4, NO32−, NO2−, and NH4+, using standardized methods Focal data sets were collected from Tatoosh Island, Washington state, USA , located in the northeastern Pacific at the mouth of the Strait of Juan de Fuca, 0.7 km from the mainland of the Olympic Peninsula. Tatoosh has been a site of intensive ecological investigation since 1969 Concerns have been raised about the use of electrochemical probes to measure pH in the ocean total = 8.0928 mol/kg, Salinity = 35 at 25°C). We used linear regression to test for deviations in a relationship of the form pH2 = pH1, where pHx represent the x-th measurement of each sample. We also used linear regression to test the strength of correspondence between our spectrophotometric and electrochemical pH measures. To check for possible effects of microbial activity on measured pH, we tested for trends in differences in spectrophotometric- and probe-based measures of pH with time since collection, and found none. We also compared pH measurements of an unfiltered versus a filtered subsample (GF/F glass fiber filter paper) from the same cast on 5 different occasions and found no differences. Second, we collected 500 ml water samples , added 120 µl of HgCl to arrest biological activity, and shipped the samples for analysis of salinity, total alkalinity, and pCO2 at the Carbon Dioxide Quality Control (CDQC) Laboratory at the Scripps Institute of Oceanography, following the methods of 2 measurement to determine the extent to which DIC was associated with variability in pH. We also used the CO2SYS program for Microsoft Excel 2, total alkalinity, salinity, and monthly estimates of nutrient concentrations and compared these predictions to those generated by the our estimates of spectrophotometric pH and the pH probe using linear regression testing the hypothesis observed = predicted . We converted our electrochemical measurements  to the pH total scale to make all pH measurements comparable. To do this, we calculated estimates of both pHtotal and pHNIST from our DIC/TA data with the CO2SYS program, generated the relationship between these estimates with linear regression , and used this relationship to convert all our electrochemical pH measurements to the total scale.To further explore reliability of our measurements, in 2009 and 2010 we collected water samples for lab analysis coincident with times when the probes were sampling the water. First, we analyzed spectrophotometric pH to determine the consistency of our measures, following the general methods of 2 in area. Second, during three dates in 2010  we sampled water offshore aboard the UNOLS vessel R/V Clifford Barnes at four stations along an 85 km transect in the western Strait of Juan de Fuca for pH using a SeaBird SBE-18 sensor calibrated with NIST standards. Third, we analyzed temporal trends from the surface waters (≤2 m) of the inner Strait of Juan de Fuca taken periodically by the Washington Department of Ecology (http://www.ecy.wa.gov/apps/eap/marinewq/mwdataset.asp) using a SeaBird SBE-18 sensor lowered from a float plane. We analyzed the open-water sampling stations in the data set that were sampled for pH over a span of at least 10 years. These were located 105–185 km from Tatoosh over water: Admiralty Inlet, Admiralty Inlet North, Lopez Sound, Strait of Georgia, and Strait of Juan de Fuca near Port Angeles.The pH trends we observed at our focal sampling site http://www.pfeg.noaa.gov/products/las.html) for the closest latitude . To look for potential changes in wind-driven upwelling at the local scale, we also obtained wind information from the NOAA weather station located on Tatoosh Island (http://www.ndbc.noaa.gov/station_page.php?station=ttiw1). We also analyzed two oceanographic indices thought to be associated with changes in currents: the Pacific Decadal Oscillation index (http://jisao.washington.edu/pdo/) and the North Pacific Gyre index (http://www.o3d.org/npgo/data/NPGO.txt).To address alternative hypotheses of environmental drivers of long-term trends in pH we compiled data from a variety of web-based data sources. To assess upwelling hypotheses, we used monthly wind-derived upwelling indices (http://www.ncdc.noaa.gov/oa/climate/climatedata.html) from the closest station available, Quillayute , and annual river discharge data from the closest , the largest local , and the largest regional  gauged rivers from either the United States Geological Survey , or Environment Canada (http://www.wsc.ec.gc.ca/applications/H2O/HydromatD-eng.cfm). We obtained data on dissolved organic carbon (DOC) input from two sources. First, we collected water samples from two sites at each of 9 rivers on the northwestern Olympic Peninsula, Washington, in July 2001 and 2010 for DOC analysis. The sampled rivers flowed through a range of land use levels from relatively pristine , to intensively harvested for timber, and included the following: North Fork Sol Duc, South Fork Sol Duc, Main Stem Sol Duc, North Fork Calawah, South Fork Calawah, Sitkum, Little Hoko, Clallam, and South Fork Pysht. Samples were frozen until analysis, which was done by the University of Washington Ocean Chemistry Laboratory following the procedures in http://waterquality.ec.gc.ca/waterqualityweb/) and the USGS (http://nwis.waterdata.usgs.gov/wa/nwis/qw). To assess potential changes in rain chemistry that might be driving pH trends, we analyzed trends in the long-term precipitation chemistry data  collected from the Hoh River valley , focusing in particular on nitrate and sulfate concentrations that are key components of acid precipitation To address alternative hypotheses relevant to freshwater input and composition, we assembled data from both web-based sources and our own studies of local rivers and precipitation. We obtained monthly precipitation data from the National Weather Service (http://coastwatch.pfel.noaa.gov/coastwatch/CWBrowser.jsp) reported at a pixel scale of 0.125 degrees latitude/longitude, and averaged data from all pixels within 48.365° to 49.445°N and 124.725° to 124.795°W. AquaModis data were not available prior to June 2002; for this period, we used data from the SeaWiFS platform, after checking to insure that no abrupt breaks in mean or trend were present when data series were switched. We analyzed four aspects of the data series: annual mean chlorophyll concentration, mean concentration during the deployment season of our probe (April-September), the standard deviation in monthly chlorophyll concentration, and the peak monthly chlorophyll concentration.We assessed potential effects of changing regional phytoplankton abundance by evaluating satellite-based data of chlorophyll concentration. We obtained monthly data from NOAA for the AquaModis platform (We tested for potential drivers of the observed long-term trend in pH by applying linear regression to different variables through time. Different data are available at different temporal resolutions, ranging from our local measures made at 0.5 hr intervals through spring-summer to annual means. Because statistical power to detect trends can vary with sample size, we carried out analysis on annual means to ensure all data series had comparable power, but also report temporal trends of data series at their highest available sampling resolution for completeness.