Non-motor symptoms (NMS) are important manifestations of Parkinson's disease (PD) that reduce patients' health-related quality of life. Some NMS may also be caused by age-related changes, or manifested as a psychological reaction to a chronic neurological condition. This case-control study compared the NMS burden among PD patients, healthy controls and hemifacial spasm (HFS) patients. In addition, we determined the NMS that discriminated between PD and non-PD subjects.425 subjects were recruited from a tertiary hospital in Singapore . NMS burden in subjects was measured using the Non-Motor Symptoms Scale (NMSS).p<0.0001) and HFS patients (18.0±2.1) (p<0.0001). In addition, NMSS total score was significantly higher in HFS patients compared to healthy controls (p = 0.003). PD patients experienced a higher NMS burden than healthy controls in all domains, and a higher NMS burden than HFS patients in all but attention/memory and urinary domains. NMS burden for HFS and healthy controls differed only in the sleep/fatigue and urinary domains. Using stepwise logistic regression, problems of ‘constipation’, ‘restless legs’, ‘dribbling saliva’, ‘altered interest in sex’ and ‘change in taste or smell’ were found to have significant discriminative power in differentiating between PD patients and healthy controls and between PD patients and HFS patients.NMSS total score was significantly higher in PD patients (37.9±2.6) compared to healthy controls (11.2±0.9) (PD patients experienced a greater overall NMS burden compared to both healthy controls and HFS patients. HFS patients demonstrated a higher NMS burden than controls, and some NMS may be common to chronic neurological conditions while others are more specific to PD. Differentiating patients using NMS domains may help refine the clinical management of NMS in PD patients. Non-motor symptoms (NMS) are important manifestations of Parkinson’s disease (PD) in addition to the cardinal motor symptoms of bradykinesia, tremor, rigidity and postural instability, and some eventually become prominent causes of disability NMS characterization is not straightforward: The range of NMS is vast, and clinical correlation with neuropathological findings is partial and not fully established We utilized the NMSS to compare the NMS burden between non-demented idiopathic PD patients, healthy controls and hemifacial (HFS) patients, and to identify which of the 30 NMS evaluated in the scale best discriminate between PD patients and the two groups of non-PD subjects. HFS was chosen as a comparator because it is a chronic, localized neurologic disease which is not known to have pathology such as that exhibited in a generalized neurodegenerative disorder like PD that could directly cause NMS.200 idiopathic Parkinson's disease (PD) patients, diagnosed according to the UK PD Brain Bank criteria, Demographic data was collected from subjects. Non-motor symptoms (NMS) were assessed using the Non-Motor Symptoms Scale (NMSS) PD patients underwent further clinical assessment for disease severity with Part III (motor section) of the Unified Parkinson's Disease Rating Scale (mUPDRS) Baseline characteristics for continuous variables were compared across all groups using the Kruskal-Wallis test, and pair-wise comparisons were performed using the Wilcoxon rank-sum test. Categorical variables were compared using the chi-square test. Continuous variables for baseline characteristics are summarized as mean ± standard deviation; categorical variables are summarized as percentages.Frequency distribution plots of NMSS scores, whether for each item, domain or total score, consistently exhibited highly right-skewed, heavy tailed distributions resembling those of over-dispersed counting distributions. In the search for an acceptable statistical model, various error distributions and link functions were evaluated in the context of a generalized linear model (GLIM). The best fitting (lowest AIC) model for comparing NMSS scores  between the three groups incorporated the negative binomial error distribution in conjunction with the log link function. An analysis comparing NMSS total score among the three groups incorporated age, sex, presence of hypertension, hyperlipidemia, diabetes mellitus and ischemic heart disease in the model as potential confounders. The only one found to be statistically significant was age. Because an attempt was made to match groups on age and sex, both variables were included in the final model. The Tukey-Kramer approach was used to adjust the type I error for multiple pair-wise comparisons, and tabulated p-values reflect the adjustment. Comparison of NMSS scores between PD patients of the different disease (H&Y) stages was performed using the same methodology. Logistic regression analysis was used to compare prevalence of NMS among groups, controlling for age and sex. In addition to age and sex, diabetes mellitus was a statistically significant factor in the comparison of NMSS scores and prevalence of the symptom of ‘problems having sex’ in the sexual function domain. All reported prevalence values and mean NMSS scores are model-based, predicted proportions and least squares means (± standard errors), respectively, adjusted for covariates. Stepwise logistic regression (significance level to enter  =  significance level to remove  = 0.05) was used to select non-motor symptoms in the NMSS which best discriminated between PD patients and healthy controls, and between PD patients and HFS patients.rho) was used to investigate univariate associations between the NMSS total score and selected variables. Analysis of covariance was used to determine clinical factors associated with NMSS total scores and domain scores–H&Y stage was grouped into 3 classes: stages 1–1.5, 2–2.5, and 3–5.In a subgroup analysis of PD patients, Spearman's rank correlation .Statistical significance was set at The mean age was 64.4±9.7 years in the PD patient group, 60.7±11.3 in the healthy control group, and 62.2±11.1 in the HFS patient group. 54.5% of PD patients, 52.0% of healthy controls and 49.3% of HFS patients were male. Demographic and comorbidity data are summarized in In the PD patient group, mean disease duration was 6.0±4.5 years and mean mUPDRS score was 28.4±13.2. The median H&Y stage was 2.5. 16.5% of PD patients were in H&Y stages 1 – 1.5, 60.5% in stages 2 – 2.5, 21% in stage 3, and 2% in stages 4 – 5. 78.5% of patients were on levodopa and 20.5% were on a dopamine agonist; 11.5% were on both levodopa and a dopamine agonist. 11.0% were on neither levodopa nor a dopamine agonist. The mean levodopa equivalent daily dose (LEDD) was 369.2±295.5 mg/day. For patients on dopamine agonists, the mean dopamine agonist daily dose  was 67.8±48.5 mg/day. The mean disease duration in the HFS patient group was 10.9±5.3 years.p<0.0001) and HFS patients . The adjusted mean NMSS total score in HFS patients was also significantly higher than that of healthy controls (p = 0.003).Least squares (LS) mean NMSS total and domain scores adjusted for age and sex are presented in p = 0.003), ‘forget to do things’  and ‘urinary urgency’ . Domain scores were significantly higher in the PD patient group compared to HFS patients, except for the attention/memory and urinary domains. The item scores for ‘fainting (secondary to orthostatic hypotension)’, ‘flat mood’, ‘delusions’, and ‘problems having sex’ did not differ significantly between the three groups. Symptoms with item scores that were significantly higher in PD patients compared to healthy controls (C) but not when compared to HFS patients included ‘difficulty falling or staying asleep’ , ‘feel nervous’ , ‘forgetting things or events’ , ‘forgetting to do things’ , ‘(urinary) urgency’  and ‘urinary frequency’. The symptoms of ‘fatigue’, ‘difficulty falling or staying asleep’, ‘nocturia’ and ‘pain’ contributed most to NMS burden for all three groups of subjects.All of the domain scores were significantly higher in PD patients than in healthy controls. Sleep/fatigue and urinary domain scores were significantly higher in the HFS group compared to healthy controls. Item scores were higher in HFS patients than in healthy controls for the symptoms of ‘fatigue’ ) and ‘difficulty falling/staying asleep’ ), mood symptoms , ‘lack motivation’ , ‘feel nervous’  and ‘difficulty experiencing pleasure’ ) and the symptom of ‘forget to do things’  were more prevalent in HFS patients compared to controls.The prevalence of NMSS domains (presence of at least one symptom in the respective domain) in our sample are shown in rho  = 0.39, p<0.0001) and LEDD . In a multivariate analysis of covariance investigating age, gender, disease duration, H&Y stage and LEDD, NMSS total score was positively associated with LEDD (p = 0.016) and H&Y stage (p = 0.0012). In the domain analysis, LEDD was positively associated with the sleep/fatigue (p = 0.017) and mood (p = 0.011) domain scores.The NMSS total score  correlated significantly with disease duration , and healthy controls. We demonstrated that the overall NMS burden was significantly greater in non-demented PD patients compared to age- and sex-matched healthy controls, and increased NMS burden in PD applied to all domains of the NMSS, in line with previous studies On the other hand, a challenge that arises in treating generic NMS would be that of discerning the situations where the underlying cause is really intrinsic PD pathology, and early disease-specific treatment would be beneficial. The symptoms of ‘fatigue’, ‘difficulty falling or staying asleep’, ‘nocturia’ and ‘pain’ were found to contribute most to NMS burden on average in subjects regardless of disease status, and investigations and treatments for the common age-related changes or pathologies found in an elderly population may be initiated and fail in PD patients with such symptoms elsewhere before they come to the attention of their movement disorder specialists. Generic NMS resulting from intrinsic PD pathology may nevertheless have distinguishing features, an example being dystonic pain In a specialist outpatient clinic for PD patients, in addition to addressing the non-motor symptoms that are highly prevalent in PD patients and that cause the greatest distress, it is also important to address those that are most specific to PD and may be more likely to be directly caused by its pathology. Such symptoms may be either under-diagnosed or less amenable to treatment from a more accessible general practice. We used a stepwise logistic regression approach to select combinations of NMS to differentiate between i) PD patients and healthy controls, and ii) between PD patients and HFS patients. In both models, area under the ROC curve approached 0.90. In the model differentiating between PD patients and healthy controls, the burden of ‘fatigue’ had the greatest discriminative power. Fatigue may be a consequence of deficient serotonergic function in PD This study had limitations. First, the NMSS scale is not intended for diagnosis and relies on the subjective perception of symptom severity by subjects In conclusion, our study demonstrated a greater burden of non-motor symptoms in non-demented PD patients compared to healthy controls. We showed that certain NMS may be common to chronic neurological diseases and lack specificity for PD, whereas the symptoms of ‘constipation’, ‘restless legs’, ‘dribbling saliva’, ‘altered interest in sex’ and ‘altered sense of taste or smell’ discriminated PD patients from both healthy controls and HFS patients. Clinicians should have a high index of suspicion for NMS in PD patients, and awareness of which symptoms are common to chronic neurological disorders and which are more specific to PD may help in refining treatment strategies. These would enhance the clinical management of PD patients.


Although the World Health Organization (WHO) provides information on the number of TB patients categorised as “other”, there is limited information on treatment regimens or treatment outcomes for “other”. Such information is important, as inappropriate treatment can lead to patients remaining infectious and becoming a potential source of drug resistance. Therefore, using a cohort of TB patients from a large registration centre in Lilongwe, Malawi, our study determined the proportion of all TB re-treatment patients who were registered as “other”, and described their characteristics and treatment outcomes.This retrospective observational study used routine program data to determine the proportion of all TB re-treatment patients who were registered as “other” and describe their characteristics and treatment outcomes between January 2006 and December 2008.1,384 (12%) of 11,663 TB cases were registered as re-treatment cases. Of these, 898 (65%) were categorised as “other”: 707 (79%) had sputum smear-negative pulmonary TB and 191 (21%) had extra pulmonary TB. Compared to the smear-positive relapse, re-treatment after default (RAD) and failure cases, smear-negative “other” cases were older than 34 years and less likely to have their HIV status ascertained. Among those with known HIV status, “other” TB cases were more likely to be HIV positive. Of TB patients categorised as “other”, 462 (51%) were managed on the first-line regimen with a treatment success rate of 63%.A large proportion of re-treatment patients were categorised as “other”. Many of these patients were HIV-infected and over half were treated with a first-line regimen, contrary to national guidelines. Treatment success was low. More attention to recording, diagnosis and management of these patients is warranted as incorrect treatment regimen and poor outcomes could lead to the development of drug resistant forms of TB. With an annual incidence of 260 tuberculosis (TB) cases per 100,000 population The categorization of TB patients as “other” may indicate poor patient management. According to the World Health Organization's (WHO) Global Tuberculosis Control report on relapse, failure, RAD and “other” for Africa, South-East Asia and Western Pacific regions in 2007, about 35% of 581,000 TB re-treatment cases were registered as “other” We therefore carried out a study at the largest TB registration centre in Malawi to determine the numbers and proportions of adult TB re-treatment patients categorised as “other”. We also documented what treatment regimens were used and patient treatment outcomes for “other” TB patients, comparing them to outcomes reported for previously-treated patients registered as relapse, failure and RAD.The study was conducted at Martin Preuss Centre (MPC) clinic in Lilongwe, the largest TB registration centre in Malawi. MPC registers 4,000 TB patients annually. Diagnosis of PTB is based on clinical examination, sputum smear microscopy, and chest radiography. In most cases, diagnosis of EPTB is made based on radiography but it was also diagnosed bacteriogically or histopathologically. For quality control, peripheral laboratories send standard smears with known results to the central laboratory at MPC every month; these smears are rechecked by central laboratory technicians. Once diagnosed with TB, patients are registered by the district TB officer and initiated on standardized treatment regimens Approximately 30% of patients initiated and completed their treatment at MPC; 70% initiated at MPC but chose to complete their treatment at one of 18 peripheral health facilities. Treatment cards for those who completed treatment at MPC remained with the TB officer on site; treatment cards of patients who sought care at peripheral health facilities were sent to, and maintained, at their respective facilities during treatment. After completion of treatment, the cards from peripheral sites should be returned to the initial registration centre (MPC). Every six months, the district TB officer visited peripheral health facilities to collect treatment cards that were not returned. Treatment outcomes  were updated in the TB Register at MPC from treatment cards.This retrospective cohort study of re-treatment adult TB patients (age≥15 years) was conducted using routine program data from TB registers and patient treatment cards collected from January 2006 to December 2008 at MPC. The Malawi National TB control program classifies previously treated patients as follows: 1) “relapse” = completed treatment but subsequently developed active smear-positive pulmonary TB; 2) “failure” = remained sputum smear-positive at five months or more during first-line treatment; 3) “re-treatment after default (RAD)” = interrupted treatment for more than two months and returned with smear-positive TB; and 4) “other” = not fulfilling any of the above categories, including chronic cases Data extracted from registers for each patient included TB registration number, registration date, age, gender, TB classification, patient category, treatment regimen, initial sputum microscopy results and HIV status. The Malawi TB programme does not routinely conduct culture tests and, as such, there were no culture results captured in the national TB register. HIV status was only documented from January 2007. Treatment outcomes were extracted from treatment cards. Missing case registration data in the TB registers were retrieved from treatment cards, if available.a priori in all multivariable models. The log-likelihood ratio test was used to assess the independent contribution of explanatory variables to the models. Statistical significance was determined at the p≤0.05 level, shown with 95% confidence intervals (CI) throughout.Data were double-entered and cleaned in a custom-designed MS Access database and analyzed in STATA 10. Since there was a strong correlation between TB classification and patient retreatment category, only patient category was considered for further analysis. We categorized re-treatment patients in four ways: smear-positive relapse, smear-positive RAD, smear-positive failure and smear-negative “other”. The “other” re-treatment category consisted of smear-negative PTB and EPTB patients except for 3 cases that were registered in 2006. These cases might have been misclassified and were therefore excluded from further analyses. The chi-square test for significance was used to compare patient characteristics and patient re-treatment categories. Because TB treatment outcomes are generally classified as either treatment success or not, we modelled treatment outcome as a binary variable of success versus all other treatment outcomes. Logistic regression was then used to identify patient characteristics that were independently associated with treatment success. The final multivariable model was determined using forward selection, including explanatory variables with a two-sided p-value of ≤0.05. Age was included The study was approved by the Malawi National Health Science Research and the Ethics Advisory Group of The International Union Against Tuberculosis and Lung Disease, Paris, France. The ethics committees waived the need for patient consent because the study used routine programmatic data that did not include any personal identifiers and were analysed anonymously.Between January 2006 and December 2008, 11,663 adult TB cases were registered at MPC. Case registration data were available for all TB patients. Of all TB registrations, 11,653 (99.9%) had smear microscopy: 3,094 (27%) had positive smear results. A total of 1,384 (12%) were registered as ‘re-treatment cases’. Compared to new TB cases, re-treatment cases were more likely to be female , older than 34 years , and less likely to have known HIV status  Table 1.Of 1,384 re-treatment cases, 898 (65%) were classified as smear-negative “other”; 406 (29%) as smear-positive relapse; 46 (4%) as smear-positive RAD; and 34 (3%) as smear-positive failure. Compared to the smear-positive relapse, RAD and failure cases, smear-negative “other” cases were older than 34 years and less likely to have their HIV status ascertained. Among those with known HIV status, “other” TB cases were more likely to be HIV-positive Table 2.Of the 898 smear-negative “other” TB cases, 342 (38%) received the category II re-treatment TB drug regimen while 462 (51%) were inappropriately managed on first-line treatment regimen (2RHZE/4RH). Information on treatment regimen was missing in 94 (10%) patients. A total of 348 (86%) relapse cases, 34(74%) RAD and 30 (88%) failure cases received the category II regimens, indicating that significantly more smear-positive retreatment cases received a category II regimen (p<0.001).Treatment outcomes were recorded for 663 (48%) of 1,384 re-treatment cases. Of the 663, 585 (88%) had PTB and 78 (12%) had EPTB. Treatment cards were missing for 721 (52%); these patients were not included in further analysis. According to the register, re-treatment patients with treatment cards were similar to those without treatment cards in terms of age (p = 0.194), sex (p = 0.557) TB classification (p = 0.092) and place of TB treatment (p = 0.070). Of all outcomes, 66% were treatment success, 20% loss to follow-up, 11% death, 2% transfer-out and 1% treatment failure. Univariable analysis of re-treatment patients with treatment cards showed that sex (p = 0.003), treatment regimen (p = 0.033) and re-treatment category (p = 0.050) were significantly associated with treatment outcome; HIV status (p = 0.683), age (p = 0.248) and year of registration (p = 0.659) were not Table 3.Results from logistic regression Table 4 This is the first study from a routine TB programme setting in sub-Saharan Africa to explore characteristics and treatment outcomes in previously-treated adult TB patients registered as “other”. In this setting, 65% of the re-treatment cases were classified as “other”. Among “other”, 75% had smear-negative PTB, 75% were HIV-positive and most were men older than 34 years. The study also showed a high proportion of all TB cases had smear microscopy results, indicating the TB programme performed well. According to Malawi national TB guidelines, a re-treatment regimen (2SRHZE/1RHZE/5RHE) should have been used for all re-treatment cases. More than half of the patients classified as “other” were inappropriately treated with the first-line TB regimen (2RHZE/4RH). However, overall treatment success rate was low among re-treatment TB patients receiving a category II regimen, suggesting that this regimen may also not be the most appropriate.There are several possible explanations for the large number of “other” patients in this study. Many of these patients had smear-negative TB and were HIV-positive, raising questions about whether these were truly recurrent smear-negative PTB as a result of re-infection or reactivation Similar to other studies Although neither the WHO nor The International Union Against Tuberculosis and Lung Disease TB treatment guidelines provide clear guidance on what regimen should be given to patients classified as “other”, the current Malawi National TB treatment guidelines recommend re-treatment drug regimens for all “other” TB patients. However, less than half of “other” TB patients in our study were treated with a category II regimen, suggesting clinician's uncertainty about this category of TB patients, rather than a general failure to follow guidelines. Other countries treat “other” TB patients with a first-line regimen if their smear is negative because they are considered to be at lower risk of failure and developing resistance. However, some previous studies observed generally low treatment success rates among smear positive re-treatment TB patients treated with the category II regimen Our findings should be viewed with the following limitations. First, 52% of treatment cards for re-treatment patients were missing although all treatment cards should have been returned to the MPC TB registration centre at the time of this study. Some cards might have been lost at the peripheral sites or within the TB registry, introducing bias if patients without treatment cards had different outcomes than those with treatment cards. However, we observed no differences in terms of demographics, TB classification and place of treatment between patients with and without treatment cards. Second, although TB outcome data were supposed to be recorded on treatment cards and in the TB register, a considerable proportion of adverse outcomes were not recorded in the TB registers. As treatment cards are the primary source of outcome information, we based the analysis on outcomes documented on the cards and not the register. Exclusion of patients with missing cards may have resulted in a biased selection of patients with better treatment outcomes, assuming that cards of defaulters were more likely to be lost. Despite these limitations, the study findings are useful to inform policy and programs in Malawi and other comparable settings to improve care for TB patients.In conclusion, our study shows that the “other” TB category constitutes a sizeable population of re-treatment patients and is poorly managed. Accurate definitions and categories from the current “other” category should be created to avoid poor patient management. The high proportion of missing treatment cards suggests that less priority is given to these re-treatment patients. Treatment success may be improved by adoption of more accurate diagnostic tools such as Xpert MTB/RIF and more rigorous training and regular supervision of TB staff. There is also a need to reappraise the category II regimen as our results suggest that use of this regimen is not associated with successful outcomes. Some of these issues are currently under discussion within the Malawi NTP with the aim of improving routine programme management.


ACTN3, the structural gene encoding the skeletal muscle protein α-actinin-3. We compared the ACTN3 R577X genotype/allele frequencies between the following groups of ethnically-matched (Spanish) individuals: centenarians , young healthy controls , and humans who are at the two end-points of exercise capacity phenotypes, i.e. muscle endurance  and muscle power . Although there were no differences in genotype/allele frequencies between centenarians , and controls  or endurance athletes , we observed a significantly higher frequency of the X allele (P = 0.019) and XX genotype (P = 0.011) in centenarians compared with power athletes . Notably, the frequency of the null XX (α-actinin-3 deficient) genotype in centenarians was the highest ever reported in non-athletic Caucasian populations. In conclusion, despite there were no significant differences with the younger, control population, overall the ACTN3 genotype of centenarians resembles that of world-class elite endurance athletes and differs from that of elite power athletes. Our preliminary data would suggest a certain ‘survival’ advantage brought about by α-actinin-3 deficiency and the ‘endurance’/oxidative muscle phenotype that is commonly associated with this condition.Exercise phenotypes have played a key role for ensuring survival over human evolution. We speculated that some genetic variants that influence exercise phenotypes could be associated with exceptional survival (i.e. reaching ≥100years of age). Owing to its effects on muscle structure/function, a potential candidate is the Arg(R)577Ter(X) polymorphism (rs1815739) in  Identifying candidate gene variants associated with ‘longevity assurance’ is possible by studying the genotype of centenarians ACTN3 gene encodes for the synthesis of α-actinin-3 in skeletal-muscle fibres, a sarcomeric protein necessary for producing ‘explosive’ powerful contractions. A premature stop codon polymorphism  in ACTN3 was first described by North et al. The ACTN3 R577X polymorphism between Spanish centenarians (aged 100–108 years), and a group of healthy young adults  of the same ethnic origin. A secondary aim was to compare the data obtained in centenarians with those of two groups of humans who show the two ‘extreme’ phenotypes of the exercise performance continuum, i.e. elite (world-class) athletes excelling in endurance and power sports respectively. We hypothesized that: (i) the XX genotype tends to be overrepresented in centenarians compared with the general population; (ii) the ACTN3 R577X allelic/genotypic frequency distribution of centenarians is similar to that of endurance athletes; and (iii) the XX genotype will be overrepresented in centenarians compared with the power athlete group.The main purpose of the present study was to compare the allelic and genotypic frequency of the NCI-NHGRI Working Group on Replication in Association StudiesThe study was designed and carried out in accordance with the recommendations for the human genotype-phenotype association studies recently published by the Universidad Europea de Madrid (UEM). Spain) and was in accordance with the Declaration of Helsinki for Human Research of 1974 (last modified in 2000).Written consent was obtained from each participant. The study protocol was approved by the institutional ethics committee .All the study participants were of the same Caucasian (Spanish) descent for ≥3 generations. The majority (∼90%) of them lived most of their lives and were born in the same areas of Spain (Universidad Politécnica (N = 200) and UEM (n = 83). Their maximal oxygen uptake (VO2max) was 48.0±4.3 ml·kg−1·min−1. These controls served as subjects in previous research from our group Inclusion criteria for this group were (i) to be woman or a man, aged ≤40 years, (ii) free of any diagnosed cardiometabolic disease, (iii) having no known family history of high longevity (90+ years). During year 2008, we extracted genomic DNA from saliva samples of 283 University students of both genders  who met the aforementioned criteria and were randomly selected from two Universities of the same city : Meseta Castellana). The most prevalent diseases in the centenarians' cohort were osteoarthritis (72%), hypertension (62.5%), dementia (50%) and coronary artery disease (30%). Three centenarians were free of any diagnosed disease.During 2009–2010 we obtained DNA from saliva samples in 64 centenarians of both genders  living in nursing residencies of the Spain central area  road cyclists in the last years, i.e. all Tour de France finishers one or more time, including stage winners and top-3 finishers 2max was 60.3±5.5 ml·kg−1·min−1.This sample included 63 elite power (track and field) Spanish male athletes (27±3 years), including the best Spanish jumpers (n = 18) and sprinters in recent years (n = 45). Thirteen of them were Olympians during the period 2000–2008. Their VOGenotyping was performed specifically for research purposes. The researchers in charge of genotyping were totally blinded to the participants' identities, i.e. saliva samples were tracked solely with bar-coding and personal identities were only made available to the main study researcher who was not involved in actual genotyping.5′- CTGTTGCCTGTGGTAAGTGGG labelled a 5′ with VIC and ACTN3-R 5′- TGGTCACAGTATGCAGGAGGG. The PCR conditions were as follows: initial denaturing at 95°C 5 min; 35 cycles at 95°C 30 s, 60°C 30 s, 72°C 30 s and a final extension at 72°C 10 min. ACTN3 genotypes were established by enzymatic digestion of amplicons with Dde I. The R577X change creates a restriction site resulting in fragments of 108, 97 and 86 bp. Digestion of the R577 allele results in fragments of 205 and 86 bp, and digestion of the 577X allele results in fragments of 108, 97 and 86 bp. The digestion products detected by capillary electrophoresis  were those labelled with VIC, i.e. 108 bp for 577X, and 205 bp for R577.Genotyping was originally performed during 2008–2010 in the genetics laboratory of UEM (Madrid) following previously described methodology ACTN3 R577X polymorphism were replicated in a different laboratory  using a different technology, i.e. newly developed low-density DNA microarray based on allele-specific probes. The design, fabrication, validation and analysis of the arrays were performed following the procedure described elsewhere Following recent recommendations 2 test. All statistical analyses were performed using the PASW . Genotype/allele frequencies were compared among the four study groups  using a χ2 test with α set at 0.05. We used logistic regression analysis to analyse the association between alleles and longevity after adjusting for sex.We tested Hardy-Weinberg equilibrium using a χACTN3 R577X genotype assessment. Parallel genotyping results showed 100% concordance between the two laboratories.There were no failures in sample collection, DNA acquisition or genotyping procedures, except for 5 centenarians, for which the amount of DNA gathered from saliva was insufficient to allow ACTN3 R577X genotype distributions in our healthy controls were overall similar to those previously reported in healthy Spanish adults Genotype distributions met Hardy-Weinberg equilibrium in all study groups . The odds ratio (OR) of being a centenarian if the subject had the X allele was not significant: 1.236 : 0.484–1.803), 1.041 (95% CI: 0.486–2.230), and 1.678 (95% CI: 0.928–3.033) compared to the control, endurance athletes, and power athletes group, respectively, after adjusting for sex. Likewise, the OR of being a centenarian if the subject had the XX genotype did not reach statistical significance: 1.032 : 0.863–1.234), 1.132 (95% CI: 0.784–1.636), and 1.165 (95% CI: 0.945–1.437) compared to the control, endurance athletes, and power athletes group, respectively, after adjusting for sex.ACTN3 R577X polymorphism did not significantly differ between centenarians and the control population, a major finding of our study was that overall, the allelic/genotypic distribution of the ACTN3 R577X polymorphism in centenarians was closest to that of those humans who arguably show the most ‘extreme’ muscle endurance phenotypes, i.e. professional road cyclists able to excel in such demanding events as the Tour de France et al on Australian athletes (30%) Although the allelic/genotypic frequency of the ACE) gene, which has been associated with several exercise phenotypes, e.g. preservation of muscle mass/strength, included in people aged 60 years and over ACTN3 R577X variation is a strong candidate to influence survival over human evolution. Indeed, among the numerous genes that contribute to the ‘fitness’ or ‘performance’ phenotype, the ACTN3 is the first structural skeletal muscle gene for which such an association has been demonstrated ACTN3 deficiency in humans with marked variations in X-allele frequencies among different ethnic groups Though natural selection in the human genome accelerated during the past 40,000 years, probably less than 1% of our genes arose during the late Pleistocene and Holocene epochs , that is, after the Paleolithic era ACTN3 genotype may act as a modifier of disease severity: the X allele has recently been shown to benefit exercise capacity phenotypes (i.e. aerobic capacity) in women with muscle (McArdle) disease Compared with wild-type mice, the muscles of the α-actinin-3 deficient mouse exhibit 33% higher endurance (during treadmill running) and a shift towards more oxidative pathways of metabolism, i.e. increased activity of mitochondrial oxidative metabolism ACTN3 genotypes and muscle phenotypes with regards to ageing are controversial. Compared with the age-matched wild type mouse, the ageing KO mouse shows greater loss of fast muscle force generation and male muscle mass, good maintenance of grip strength, and increased oxidative metabolism and greater force recovery after fatigue ACTN3 genotypes and functional capacity and muscle phenotypes (included 1RM leg press) in women aged 61–80 years; whereas others found that (i) in adults with a mean age ∼65 years, the XX genotype was associated with higher knee extensor concentric peak power compared with RR/RX genotypes, especially in women ACTN3 genotypes on muscle phenotypes  in nonagenarians ACTN3 genotypes on the muscle phenotypes of centenarians is nevertheless a very difficult task: the expected poor physical condition and lack of independence in most of them (which at least was the case in our cohort) precludes the use of valid tools for phenotype assessment in this population.Published data on individually.We believe that a strength from our study comes from our group of cases, i.e. being all centenarians. Indeed, a well accepted approach for identifying those genetic factors that are associated with ‘longevity assurance’ is to study the genotype of centenarians. These people are the survival tail of population, and living 100 or more years is a rare phenotype, i.e. ≤1 every 10,000 people ACTN3 genotype profile of centenarians resembles that of world-class elite endurance athletes and seems to differ from that of elite power athletes. Our preliminary data would suggest a certain ‘survival’ advantage brought about by α-actinin-3 deficiency and the ‘endurance’/oxidative muscle phenotype that is commonly associated with this condition; yet, more data on larger cohorts and on other ethnicities are necessary. Despite its uniqueness, the ‘oldest old’ is the most rapidly growing age group in western countries ACTN3 R577X is currently the best example.In summary, although we did not find significant differences with the younger, control population, overall the


WV) and sympathetic (low frequency power: LFWV and LFWV/HFWV ratio) activity before and after apnea/hypopnea termination. Autonomic cardiac modulations were compared according to sleep stage, apnea/hypopnea type and duration, arterial oxygen saturation, and presence of CA. At apnea/hypopnea termination, RR decreased (p<0.001) while LFWV (p = 0.001) and LFWV/HFWV ratio (p = 0.001) increased. Only RR and LFWV/HFWV ratio changes were higher when apneas/hypopneas produced CA  or deep hypoxia . Multivariate statistical analysis showed that elevated LFWV (p = 0.006) and LFWV/HFWV ratio (p = 0.029) during apneas/hypopneas were independently related to higher CA occurrence. Both the arousal and hypoxia processes may contribute to sympathetic cardiovascular overactivity by recurrent cardiac sympathetic modulation in response to apneas/hypopneas. Sympathetic overactivity also may play an important role in the acute central response to apneas/hypopneas, and in the sleep fragmentation.Autonomic dysfunction is recognized to contribute to cardiovascular consequences in obstructive sleep apnea/hypopnea syndrome (OSAHS) patients who present predominant cardiovascular sympathetic activity that persists during wakefulness. Here, we examined 1) the factors that influence sympathetic cardiac modulation in response to apneas/hypopneas; and 2) the influence of autonomic activity during apneas/hypopneas on CA. Sixteen OSAHS patients underwent in-hospital polysomnography. RR interval (RR) and RR spectral analysis using wavelet transform were used to study parasympathetic (high frequency power: HF Epidemiological studies suggest that obstructive sleep apnea/hypopnea syndrome (OSAHS) is common in the general population OSAHS is characterized by repeated episodes of total (apneas) or partial (hypopneas) upper airway occlusion during sleep, resulting in exaggerated negative intrathoracic pressure and often oxygen desaturation and carbon dioxide retention 2) and sympathetic cardiac modulation The factors that modulate sympathetic cardiac modulation in response to apneas/hypopneas remain unclear, and the research findings are contradictory Sympathetic cardiac activity during sleep was also proposed as a potential contributor to CA occurrence following the observation in animals 2, and apnea/hypopnea duration and type (hypopneas vs. apneas). We hypothesized that 1) sympathetic cardiac activation should be more intense when apneas/hypopneas give rise to CA or 2) when it is accompanied by a deeper hypoxia, but 3) with no difference between sleep stages. We also hypothesized 4) a rise in cardiac sympathetic modulation with longer apnea/hypopnea duration, and 5) with apnea versus hypopnea type. We also assessed the relationship between autonomic activity level during apneas/hypopneas and subsequent CA occurrence. We hypothesized that 6) high sympathetic cardiac activity during apneas/hypopneas should be associated with facilitated CA occurrence.Based on these findings, we first assessed autonomic cardiac modulation at obstructive apnea/hypopnea termination during all-night sleep and its relationship to electroencephalographic (EEG) cortical reactivity, sleep stage, min SaOSixteen untreated OSAHS patients 3 women,  were stu3-F4; C3-C4; O1-O2), three electromyographam (EMG) channels (chin and both legs), two electro-oculogram (EOG) channels , and one EKG channel were used. EEG and EOG data were collected at 100 Hz sampling rate and EKG data were sampled at 200 Hz Standard polysomnographic recordings were performed using an Embla Recorder  according to AASM recommendations To determine in which sleep stage every apneas/hypopneas occurred during the whole night, sleep stages were visually scored according to AASM 2007 criteria Peak-to-peak EKG signals were analyzed to detect R waves in the QRS complex using Matlab . EKG data were first visually selected. All undetected QRS, ectopic beats, and artifacts were corrected on EKG data. If correction was not possible for at least one heartbeat on selected periods, data were eliminated . RR was then analyzed for each apnea/hypopnea on 32 heartbeats before and after apnea/hypopnea termination. Statistical comparisons were performed on the mean of 3 minimal RR between 2 and 7 heartbeats post- apnea/hypopnea termination (mean RR) compared with the mean of the 10 RR pre- apnea/hypopnea termination.Wavelet analysis was performed on the RR signal to analyze both the time and frequency domain, using the mother function Daubechies 4. The wavelet analysis signal allows quantifying temporal changes in the signal frequency contents. Starting from the initial Daubechies 4 function, a family of functions was built by dilatation and translocation to constitute a wavelet frame. A serial list of wavelet coefficients was used to represent the correlation between the signal and the selected wavelet at different analysis levels (or different frequency ranges) along the entire signal. The first levels  correspond to a wavelet analysis conducted with a small dilatation factor applied to 2, 4 and 8 RR, respectively. Thus, these levels represent high-frequency variations in the signal. The last levels  correspond to a wavelet analysis conducted with a large dilatation factor applied to 16, 32, and 64 RR, respectively. Consequently, these levels represent low-frequency variations in the signal.WV) were used to assess parasympathetic modulation. Wavelet power coefficients at levels 16 and 32 (low frequency: LFWV) were used to assess sympathetic modulation. An LFWV/HFWV ratio was calculated as the ratio between the wavelet power coefficient at levels 16 and 32 on the one hand and the sum of the wavelet power coefficients at levels 2, 4, and 8 on the other to obtain a marker of autonomic nervous system balance . The first factor was time (pre- vs. post-apnea/hypopnea termination) in ANOVAs and the second factor was, successively: (a) sleep stage (S2 vs. SWS vs. PS) in the first ANOVA; (b) type (hypopneas vs. apneas); (c) and (d) hypopnea and apnea duration, respectively (short: 10 s≤apneas/hypopneas<20 s vs. medium: 20 s≤apneas/hypopneas<30 s vs. long: apneas/hypopneas≥30 s); (e) min SaO2 ; and (f) CA .Data were analyzed using Statview®  software. Mean RR, HF2, type, sleep stage, interval inter-events, and subject. Risks were expressed as odds ratios (OR) with 95% confidence interval (95% CI), differences were considered significant when p<0.05, and values were expressed as mean ± SEM.To assess whether CA occurrence is independently associated with autonomic activity during apneas/hypopneas, logistic multivariate regressions were performed between each autonomic cardiac index during apneas/hypopneas  and the CA presence, adjusted to minimum and mean SaO2≤85%) and long duration (30 s) in some subjects, statistical analysis was performed on 10 subjects to compare min SaO2 levels, 11 subjects to compare apnea durations, and 13 subjects to compare hypopnea durations.Of a total of 3191 apneas/hypopneas, 2955 were included in the analysis, with 236 excluded for artifacts and ectopic beats. For the whole group, 186±64 events per subject were selected, of which 128±49 were in S2, 19±17 in SWS, and 40±20 in PS. Due to lack of apneas/hypopneas during SWS in some OSAHS patients, statistical analysis was performed on 7 subjects for comparison between sleep stages. Due to lack of apneas/hypopneas with deep hypoxia  produced CA distributed evenly across sleep stages, with no significant effect of sleep stage on their incidence  = 0.857; p = 0.651). However, an effect on CA occurrence was noted according to RE type . RE duration also modulated CA occurrence in apneas  = 11.42; p = 0.003) and hypopneas  = 7.17; p = 0.028). However, hypoxia did not appear to influence CA occurrence  = 2.51; p = 0.285). Thus, the percentage of CA occurrence increased with RE severity and duration, but not according to sleep stage or hypoxia.2 did not differ according to the presence or absence of CA , or according to sleep stage  = 1.455; p = 0.483), type , or RE duration  = 0.676; p = 0.708 and apneas: short: 88.8±2.6%, medium: 89.0±3.5% and long: 88.4±3.9%; Friedman's X(2) = 0.092; p = 0.954).Mini SaOMean RR varied significantly with time  = 58.3, p<0.001, WV varied significantly with time  = 31.4, p<0.001, WV increased at apnea/hypopnea termination, but the LFWV increase did not differ according to sleep stage, type and duration, hypoxia, or CA presence.LFWV varied significantly with time only in the comparisons with apnea/hypopnea type and CA presence  = 16.0, p = 0.001, HFWV/HFWV ratio varied significantly with time  = 14.8, p = 0.001, WV/HFWV ratio did not vary significantly with sleep stage  = 0.3, p = 0.780), type  = 0.2, p = 0.644), or duration  = 0.1, p = 0.961 and apneas: F = 0.1, p = 0.931), and no interaction was observed  = 0.1, p = 0.936; F = 1.0, p = 0.322; F = 0.2, p = 0.814, WV/HFWV ratio also did not vary with hypoxia  = 1.3, p = 0.295, WV/HFWV ratio increased at apnea/hypopnea termination over the level during apneas/hypopneas. The only differences in the increase in LFWV/HFWV ratio were according to the presence of CA and deep oxygen desaturation compared to their absence.LFWV  and LFWV/HFWV ratio , independently of mini and mean SaO2, type and duration, interval inter-events, sleep stage, and subject. No significant results were noted for RR mean  or HFWV .To assess whether autonomic activity during apneas/hypopneas was associated with CA occurrence, multivariate logistic regression analysis was applied to investigate the relationship between the autonomic index of HRV and subsequent CA. Results showed a significant relationship between CA occurrence and LFWV, and LFWV/HFWV ratio remained significant , p = 0.016) to high tertile (OR = 1.92 (1.45–2.57), p<0.001). For LFWV/HFWV ratio, the independent risk increased from medium (OR = 1.55 (1.21–1.98), p<0.001) to high tertile (OR = 1.92 (1.46–2.52), p<0.001). No significant relationship was found between CA occurrence, HFWV, and RR mean during apneas/hypopneas.Using tertiles of HRV data, the independent relationships between CA occurrence, LFnificant . For LFWFirst, the results of this study showed that sympathetic modulation at apnea/hypopnea termination is modulated by the arousal process and hypoxia rather than by sleep stage or apnea/hypopnea type and duration in obstructive sleep apnea/hypopnea syndrome (OSAHS) patients. Greater sympathetic activation in response to apneas/hypopneas is concomitant with the presence of cortical arousal (CA) or deep hypoxia. Second, when cardiac sympathetic indexes of heart rate variability (HRV) were high during apneas/hypopneas, the risk of CA occurrence was magnified (or twice). This indicates that cardiac sympathetic dominance during apneas/hypopneas is related to facilitated CA occurrence. However, the exact relationship between sympathetic activity during apneas/hypopneas and subsequent CA remains unknown.WV and LFWV/HFWV ratio), with a small decrease in parasympathetic activity (HFWV). This parasympathetic withdrawal appeared significant only in comparison with the higher number of subjects, e.g. comparison with and without CA (n = 16), and according to apnea/hypopnea type (n = 16). In addition, using Fourier transform, a less sensitive method to study transit changes in RR, previous studies Spicuzza and collaborators Autonomic dysfunction is recognized to contribute to cardiovascular consequences in these OSAHS patients Of the several factors tested in this study, sleep stage, apnea/hypopnea duration, and respiratory event type showed no effect on sympathetic cardiac modulation: irrespective of sleep stage, type, and duration, apneas/hypopneas produced the same level of cardiac sympathetic modulation –4.The absence of sleep stage difference concurs with previous studies, which showed that exteroceptive stimulations Hierarchy in the arousal process and its close relationship with sympathetic cardiac modulation has been previously reported in several conditions of sleep fragmentation, including spontaneous arousal Then, we hypothesized that severity of respiratory events could be expressed as length or type of respiratory events, but our result rather showed that both type and duration of respiratory events do no bring information on cardiac sympathetic modulation. Our results confirm previous data 2 decrease lesser than 90% was able to induce a significant cardiac sympathetic modulation increase (2 and sympathetic cardiac modulation in response to apneas/hypopneas 2: 88.8±0.4%) and normoxia (mean SaO2: 97.9±0.2%) in cardiac response to auditive stimulations during sleep in healthy volunteers. This discrepancy between their and our results could be explained by the elevated chemoreflex sensitivity in OSAHS patients, which causes cardiac sympathetic modulation in response to decrease in SaO2Finally, recurrent nocturnal hypoxia, which is suspected to be a major factor of cardiac disease in OSAHS patients increase . These rThis cardiac sympathetic modulation in response to respiratory events influenced by the arousal and hypoxia processes could represent a pathway contributing to cardiovascular disease risk in OSAHS. Recurrent hypoxia, known to be a strong factor of cardiac disease in OSAHS patients, can also act by other pathways, such as oxygen free radical production, inflammatory pathways, and impairment in the vascular endothelial function independently of activation of the sympathetic arm WV power and LFWV/HFWV ratio during apneas/hypopneas, sympathetic criteria of HRV, are related to CA occurrence at apnea/hypopnea termination, indicating that cardiac sympathetic dominance during apneas/hypopneas is related to a facilitated CA occurrence.In the last part of this study, we examined the relationship between autonomic indexes of HRV during apneas/hypopneas and subsequent CA. Results showed that the spectral LFThese results are in accord with studies that proposed sympathetic cardiac activity during sleep as a contributing factor to the CA occurrence process, suggested by observations obtained in both animals It therefore appears likely that variations in peripheral afferents information may play an important role in the acute autonomic response as part of a multifactorial process W is related to both sympathetic and parasympathetic activity. It is only by studying the relative changes in LFW and HFW, and normalized indexes as LFW/HFW ratio that an indication of sympathetic activity is obtained. Several studies have showed that LF/HF ratio and LF% allow approaching relative sympathetic activity, in response to tilt test under atropine 2 retention, a factor that was not considered here, could contribute to these autonomic changes. However, a previous study showed that CO2 level did not affect cardiac modulation in response to spontaneous arousals in healthy volunteers Several limitations of this study should be considered. The method is limited for study of sympathetic activity: the LFApneas/hypopneas induce a cardiac sympathetic modulation influenced by the arousal and hypoxia processes rather than by sleep stage, total or partial upper airway occlusion, or occlusion duration. Higher sympathetic activation in response to apneas/hypopneas is concomitant with the presence of cortical arousal or a deep hypoxia compared to no cortical arousal or no hypoxia. Furthermore, cardiac sympathetic dominance during apneas/hypopneas is related to facilitated CA occurrence. Both the recurrent arousal and hypoxia processes by cardiac sympathetic modulation in response to apneas/hypopneas, could contribute to predominant cardiovascular sympathetic activity that persists during wakefulness, increasing the cardiovascular disease risk in OSAHS populations. On the other hand, sympathetic overactivity also may play an important role in the acute central response to apneas/hypopneas, and so in the sleep fragmentation.


Consequently, conversions of ETRs to CO2-fixation requires knowledge of the electron requirement for carbon fixation  and its dependence on environmental gradients. Such knowledge is critical for large scale implementation of active fluorescence to better characterise CO2-uptake. Here we examine the variability of experimentally determined e,CΦ values in relation to key environmental variables with the aim of developing new working algorithms for the calculation of e,CΦ from environmental variables. Coincident FRRf and 14C-uptake and environmental data from 14 studies covering 12 marine regions were analysed via a meta-analytical, non-parametric, multivariate approach. Combining all studies, e,CΦ varied between 1.15 and 54.2 mol e− (mol C)−1 with a mean of 10.9±6.91 mol e− mol C)−1. Although variability of e,CΦ was related to environmental gradients at global scales, region-specific analyses provided far improved predictive capability. However, use of regional Φe,C algorithms requires objective means of defining regions of interest, which remains challenging. Considering individual studies and specific small-scale regions, temperature, nutrient and light availability were correlated with Φe,C albeit to varying degrees and depending on the study/region and the composition of the extant phytoplankton community. At the level of large biogeographic regions and distinct water masses, Φe,C was related to nutrient availability, chlorophyll, as well as temperature and/or salinity in most regions, while light availability was also important in Baltic Sea and shelf waters. The novel Φe,C algorithms provide a major step forward for widespread fluorometry-based NPP estimates and highlight the need for further studying the natural variability of e,CΦ to verify and develop algorithms with improved accuracy.Marine phytoplankton account for about 50% of all global net primary productivity (NPP). Active fluorometry, mainly Fast Repetition Rate fluorometry (FRRf), has been advocated as means of providing high resolution estimates of NPP. However, not measuring CO Following the incorporation of inorganic radio-labelled 14CO2 into algal cells has become a standard method for quantifying primary productivity. However, to this day, there still exists considerable uncertainty as to whether 14CO2 uptake measures gross primary productivity (GPP) or net primary productivity (NPP). Following the traditional view, GPP refers to carbon fixation without accounting for any carbon losses due to respiration and/or excretion, while NPP represents the carbon uptake rate after subtracting out any CO2 lost to oxidation of organic carbon over a diel cycle Accurately evaluating the impact of local environmental and global climate change upon trophic dynamics and biogeochemical nutrient cycling is fundamentally tied to how well primary productivity, defined here as carbon  and reductant (NADPH) used to fix CO2. Thus, measurements of ETR by active fluorescence, at best, provide accurate estimates of the rate of gross O2 evolution from PSII 2 evolution but rather in the photosynthetic currency  with units mol e− (mol C)−1. With ETR and short-term (hours) CO2 fixation incubations representing GPP rather than NPP e,CΦ also captures gross rather than a net efficiency.Active fluorescence can be used to estimate the rate at which electrons flow from water through photosystem II to NADPH , and other electron acceptors. ETR is most closely related to the rate of gross O2 uptake, the latter representing GPP, NPP or something in between depending on incubation times (1 hour to a full diel cycle) 2 fixation can act to consume electrons, ATP and/or reductant; for example, the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) via photorespiration e,CΦe,CΦ data sets suggested the existence of some general taxonomic patterns in the variability of e,CΦe,CΦ.Since the introduction of active fluorescence to marine research, a number of studies have attempted to compare measurements of ETR with (quasi-)simultaneous measurements of COe,CΦ upon routinely measured variables can be established. Therefore, we constructed a database of FRR-based measures of e,CΦ and associated environmental variables known to regulate primary productivity,  from both previously published and unpublished data sets and used a meta-analytical approach to determine the predictability of e,CΦ from environmental variables. Specifically, we addressed the following three questions: (1) To what extent does e,CΦ vary within and between oceanic areas and water masses? (2) Can a ‘global’ e,CΦ ever be applied or should region-specific values always be used instead? (3) How is the variability of e,CΦ related to that of environmental factors or combinations thereof ? Our analysis demonstrated that variability in e,CΦ was strongly correlated with the availability of light and nutrients, as well as temperature and/or salinity, albeit to varying degrees and depending on how data are organized into region-specific subsets. Predictive algorithms based on these relationships are presented. Treatment of data at the regional scale provided much improved predictive capability of these algorithms relative to large scale or global algorithms. Although many of the algorithms still need further verification and revision, this approach demonstrates strong relationships between e,CΦ and environmental gradients in many areas of the ocean.Clearly, active fluorometry could become a much more powerful tool for estimating GPP (or even NPP) in carbon currency if generic relationships describing the dependency of e,CΦ from published literature and previously unpublished data was performed. Web of Science and JSTOR search engines were used to retrieve published data on coincident FRR fluorescence-based ETRs and carbon fixation rates from marine habitats, yielding 17 studies. However, only 7 of these studies reported key environmental variables in addition to e,CΦ or both ETRs and CO2-fixation rates (resembling either NPP or GPP depending on the incubation lengths and growth conditions), from which e,CΦ could be calculated. Previously unpublished data corresponding to parallel ETR and CO2 fixation measurements from an additional 7 field campaigns in 2004–2011, some of which were undertaken as part of PROTOOL (http://www.protool-project.eu/), were also included , the diffuse vertical attenuation coefficient (dK) of photosynthetically active radiation (PAR), optical depth (ζ) and chlorophyll a (chla), with the latter being used as a proxy for phytoplankton biomass. We fully acknowledge that other environmental variables, such as the silicate or iron are often also key in determining phytoplankton community composition and physiological responses and, hence, influence e,CΦ. However, these data were either not collected as part of the studies included here or not available. Methodological variables included differences in FRR protocols and 14C incubation techniques (see below). Locations of samples were characterized by latitude and longitude, while seasonal differences were characterized by converting sampling dates to Julian day. All the environmental data were either kindly provided by the authors of the published work, the British Oceanographic Data Centre  or were taken from the original publications and digitized  from relevant figures.A comprehensive data matrix with 333 different samples and their corresponding physico-chemical and methodological variables was created. Physico-chemical variables included salinity, temperature, nutrient concentrations (NOdK (in units of m−1) were derived from vertical irradiance profiles for the AMT cruises, Bedford Basin, the Celtic Sea (JR98), Baltic Sea (SYNTAX2010), Ariake Bay, the Gulf of Finland Values of eu), here defined as 1% of surface PAR, produced by the Giovanni online data system (NASA Goddard Earth Science Data and Information Services Center) averaged over 8 days were used to calculate dK by solving dK with E0 set to 100%. Multiplying dK by the actual sampling depth then gives ζ (dimensionless), with ζ<4.6 corresponding to irradiance levels >1% eu products were used for calculating dK and subsequently ζ.For the remaining studies, MODIS 4 km satellite products of euphotic zone depth , FIRe benchtop instruments  or a FRRDiving Flash fluorometer  (see FRR fluorescence transients were either measured pan) see . FRR flu0F) and maximal fluorescence (mF) yields measured in the dark, the minimal (0'F), steady state (F') and maximal (m'F) fluorescence yield measured under ambient irradiance, as well as the functional absorption cross section of photosystem II (PSII) in the dark (PSIIσ) and light (PSII'σ) (in units of Å2 quanta−1). For most studies, the photosynthetic electron transfer rate through PSII (units of mol e− (mg chla)−1 h−1) PSIIn the ratio of functional PSII reaction centres to chlorophyll (in units of mol RCII (mol chla)-1), RCΦ an assumed constant of 1 electron yielded from each reaction centre II (RCII) charge separation and 2.43 · 10-5 is the factor that accounts for the conversion of Å2 quantum-1 to m2 (mol RCII)-1, mol chla to mg chla, seconds to hours and μmol quanta to mol quanta PSII'σ account for transient non-photochemical quenching in the antenna bed as a result of exposure to transient light PSII'σ were typically taken from the FRRf dark chamber to increase signal to noise.The biophysical model of Kolber et al. q'/Fv'F /(m'–F0'F) either from fluorescence emissions measured sequentially on dark acclimated samples and then under actinic light, or estimated as the difference in the apparent PSII photochemical efficiency between the FRR light and dark chamber quasi-simultaneously, as in Suggett et al. F’, 0’F or m’F) effectively cancels between light and dark chambers/conditions The PSII efficiency factor, termed v/FmF and PSIIσ are measured in the dark or assumed dark values based on measurements from deeper in the water column, that is, from depths where E<EK and where non-photochemical quenching can be assumed to be negligible q’/Fm’F. This slightly alternative approach was employed to enable ETRs to be estimated in the absence of a 'dark chamber', and consequently, corresponding light and dark acclimated samples from all measurement depths, which would be required in order to apply Eq. 2. Both, Eqns. 1and 2, appear to return consistent estimates for quenching q'/Fv'F requires a measurement to be made following brief dark exposure. Consequently, under circumstances where rapid reversal of certain components of NPQ occurs during the dark measurements, q'/Fv'F may be overestimated potentially causing overestimates of ETR evaluated using PSIInPSIIσ, and (3) differences between ETR-based and CO2-based values of light harvesting efficiency (α) and maximum photosynthesis rates (Pmax). Firstly, PSIIn is known to vary between taxa and environmental conditions by up to a factor of 5 PSIIn. The majority of the other studies assumed values of 0.002 and 0.003 mol RCII (mol chla)−1 for populations dominated by eukaryotes and prokaryotes, respectively PSIIn using a newly developed algorithm, which will also have associated caveats PSIIn in some of our data sets, we compared e,CΦ derived with constant PSIIn to e,CΦ based on measured PSIIn  for the ETR may be lower than that from 14C uptake because of, for example, electron consuming processes K for the ETR may not be wholly indicative of where light is limiting or saturating for CO2 uptake.Although the FRRf approach provides a general estimate of 2 fixation was measured by either in situ incubations in situ incubations 2 fixation in Ariake Bay (Japan) was measured using the stable isotope 13C-labelled NaH13CO3CO2 fixation differed considerably between studies ; these light intensities relative to EK were chosen to yield a measure of e,CΦ corresponding to an irradiance for light-limited and light saturated photosynthesis.Ideally, measurements of photosynthetic carbon fixation and ETR should be made simultaneously on the exact same sample to reduce errors arising from differences in sample treatment and handling  studies . Some of14C-specific PE experiments were performed on discrete water samples whilst FRR data (and hence ETRs) were determined from in situ casts or on deck  was then applied to the 14C-PE equation to yield a measure of the instantaneous 14C uptake for matching with the ETR. For some studies, simulated in situ14C data were collected  also needed to be scaled to the light quality of the actinic source used for the 14C incubations in situ FRR values of PSIIσ and the 14C actinic light source were both spectrally corrected to match the spectral quality corresponding to the sample depth 14C- PE curves were measured on discrete samples, data were spectrally corrected following Moore et al. PSIIσ/1.75 PSIIσ/1.5  based on approximate values from the other studies for the water types in question.Direct comparison of the ETRs and K, as determined from 14C-specific PE curves, i.e. E∶EK (dimensionless). In this way, E can be considered relative to the light history to which cells are acclimated e,CΦ correspond to light limited photosynthesis (E∶EK<1) or light saturated photosynthesis (E∶EK>1). Note, however, that E comes from in situ PAR measurements, while EK was usually based on measurements made in the laboratory. In this case, the spectral quality of E and EK may differ and values of E/EK ratios from different studies must be treated with caution.Taking into account the light history of samples from different studies was the greatest challenge in compiling the database due to considerable inconsistencies in the availability and quality of light data. Thus, irradiances were expressed as optical depths, and instantaneous light was standardised by normalizing E relative to the saturating light intensity, E2 fixation rates (in mg C L−1 h−1) were normalised to the corresponding chla concentration to yield CO2 uptake rates as mol CO2 (mg chla)−1 h−1; thus values of the e,CΦ (in mol electrons mol CO2−1) were determined asAll CO14C uptake and FRR fluorescence ETRs e,CΦ, both, within and between studies. Fortunately, all studies employed similar FRRf protocols (above). Even so, information on core variables associated with the ETR determinations were initially included in our database and examined alongside the environmental variables to verify any role of method upon apparent variability in e,CΦ . Note that the ‘methodological’ data across studies is categorical rather than continuous, ; therefore, the available information was converted into a Boolean code with numbers one and zero representing use and non-use of a particular method, respectively. In total, 3 methodological variables were ultimately included in the initial data set: PSIIn assumed or measured, spectral correction of PSIIσ assumed or measured, and E∶EK>1 or E∶EK<1.Given the large array of data sets and the various differences in approach for determining both e,CΦ. Unfortunately, taxonomic data was not consistently available for the majority of studies. However, we assume that phytoplankton community composition will partially reflect environmental characteristics and some influence of community composition on e,CΦ will likely be implicitly accounted for in our analyses purely based on environmental variables.At this point it should also be noted that apart from environmental factors, differences in phytoplankton community composition may also influence Many of the following analyses were carried out on both the entire dataset or on individual subsets thereof, which, for example, represent different oceanic regions. Although the majority of our samples were collected in the Atlantic Ocean and adjacent shelf waters while other regions  are underrepresented/not represented at all, we still use the term 'global' for analyses carried out on the dataset as a whole.e,CΦ. Correlations, evaluated in SPSS 15.0  were considered significant when p<0.05. These correlations were carried out i) on the individual data sets (i.e. samples grouped by study), ii) on the global dataset, iii) on studies pooled according to regions  and iv) according to shelf/oceanic waters, thus providing an overview of which variables may be important. All data were then combined into one large database to carry out all other statistical analyses using non-parametric multivariate techniques in PRIMER-E version 6  Spearman Rank Order Correlation analysis on the individual data sets (i.e. samples grouped by study) were used to identify key variables that may be associated with a, NO3−, and PO43−) were identified using Draftsman plots and then square-root transformed to stabilise the variance and ensure that Euclidean distance could be used as an appropriate similarity measure (see below). To account for differences in scales and units between different variables, the latter were normalized by subtracting the mean from each entry of a single variable and dividing it by the variable's standard deviation.Inter-correlated and right-skewed physico-chemical variables  was used as a means to map environmental characteristics of samples in a low-dimensional space based on a triangular resemblance matrix that was created by calculating Euclidean distances between every possible pair of samples. Hence, Euclidean distances between samples represented the dissimilarities in the suite of their physico-chemical properties. Principal Component Analysis (PCA) was then used to identify variables accounting for the differences between samples. Variables initially included in the PCA were salinity, chle,CΦ, the results we show in the main text here are based on PCAs without methodological and location variables. Nevertheless, the results of the PCA including these methodological and location variables have been included in e,CΦ.Inclusion of the methodological and location variables  and Julian day) usually increased dissimilarities, i.e. Euclidean distances, between samples of different studies compared to analyses from which methodological and location variables had been excluded. Because the ‘methodological choices’, in particular, cannot be effectively incorporated into predictive algorithms for Both nMDS and PCA generate low dimensional ordinations. Here we only show the nMDS plots, which matched the PCA plots well, because nMDS better preserves the distances between samples when mapping them onto a 2D or 3D space e,CΦ to varying degrees, hierarchical agglomerative cluster analysis and a similarity profile (SIMPROF) test were used to find and define groups of samples with similar physico-chemical properties p-value for the SIMPEROF test was reduced to 0.005. Thus, once a non-significant test result was obtained (p>0.005), samples below that similarity level were no longer partitioned into clusters and could be regarded as homogeneous Because environmental factors showed considerable spatial and temporal variation, which, in turn, influence physiological responses of phytoplankton and subsequently e,CΦe,CΦ  relative to a resemblance matrix generated from subsets of the environmental data matrix searching for high rank correlations between the two and generating a correlation coefficient (ρ). Repeated (99) permutations of randomly ordered environmental data follow to test the significance of the results at a level of p<0.01 All data were then re-grouped according to the results of the cluster analysis and SIMPROF test. The PRIMER-BEST match permutation test was then used to identify variables and variable combinations that best “explain” the variability in e,CΦ from environmental variables for specific regions. Therefore, mathematical relationships between the environmental data and e,CΦ were produced for each cluster that contained at least 5 data points using multiple linear regression (MLR). Only those variables and variable combinations that were significantly correlated with e,CΦ according to the PRIMER-BEST-test were entered into the MLR in SPPS.One of the main goals of this meta-analysis was to generate algorithms to predict e,CΦ in all but four studies were <10 mol e− (mol C)−1, resulting in a global mean (± standard deviation) of 10.9±6.91 mol e− (mol C)−1. Values of e,CΦ less than the theoretical ratio of 5 mol e− (mol C)−1 −1 for all studies combined −1), Massachusetts Bay (8.5–50.1 mol e− (mol C)−1), and the AMT 15 data (1.1–28.2 mol e− (mol C)−1); in contrast, least within-study variance was typically observed for time series' at a single location (Ariake Bay: 5.1–5.7 mol e− (mol C)−1, Bedford Basin: 3.6–10.3 mol e− (mol C)−1, and the Gulf of Finland 2000: 2.0–9.4 mol e− (mol C)−1). As there were no distinct differences in the number and types of PSIIspecσ and PSIIn corrections between the time series studies and the other cruises, it would appear that variability of e,CΦ is generally greater spatially than temporally within the included locations. The time series studies included here were of relatively short duration (1.5–7 months), however, temporal variability may further increase if long-term studies (multiple years) are included.Mean values for C)−1 see  were obscombined . Within-e,CΦ and every environmental variable included in the study, albeit to varying degrees and depending on how data were grouped. On a global scale, i.e. combining the entire data set, e,CΦ exhibited significant positive correlations with Julian day (i.e. time/season) and salinity, while significant negative correlations existed with latitude, longitude, chla concentrations dK and ζ  changes of e,CΦ with depth, which were observed during AMT15, in the Celtic Sea, the North Sea (NS-CEND0811) and the Gulf of Finland/Baltic Sea; 2) a decline in e,CΦ with increasing dK during AMT6 and in the Baltic Sea, and with increasing ζ during AMT15, in the Celtic Sea, Pacific Ocean and in the Gulf of Finland/Baltic Sea; and 3) a change in e,CΦ with nutrient concentrations, during the AMT cruises, one of the Celtic Sea cruises (JR98) and in the Gulf of Finland/Baltic Sea. In the Baltic Sea (SYNTAX2010) and Bedford Basin significant correlations existed between e,CΦ and temperature and salinity, respectively. No correlation of e,CΦ with common environmental variables were found in Massachusetts Bay or during the UK-OA cruise. Note also, that depth-dependent trends in e,CΦ could only be assessed where multiple depths had been sampled at each station across large proportions of the cruise transect/time series, such as in the open ocean studies where data were available up to a depth of ∼200 m  or the Gulf of Finland (SUPREMO11) studies where up to four depths were sampled across the euphotic zone at each station. Thus, a lack of relationship between e,CΦ and depth may simply reflect limited sampling depths or, in shallow waters with rapid vertical mixing, acclimation to an average water column irradiance.At the level of the individual study, different environmental variables showed varying degrees of correlation depending on the region. Multiple notable trends were evident between e,CΦ surface waters with low NO3− and/or PO43− availability  was a pronounced increase of lability , Table 2e,CΦ and ζ for AMT15, the Celtic Sea (JR98), the Pacific Ocean and the Gulf of Finland indicated that e,CΦ often increased towards depths of greater light availability in surface waters or with declining dK values from coastal to offshore regions. Negative correlations of e,CΦ with depth, dK and ζ were also found but only in Baltic Sea and shelf waters.Negative correlations between e,CΦ and environment are dependent upon how data within and between data sets are grouped; therefore ‘choice’ of grouping will inevitably influence the outcome of empirical algorithms generated to predict e,CΦ from physico-chemical variables. A major objective of this study was to identify environmental predictors of e,CΦ; therefore PCA in combination with cluster analysis was subsequently used to 1) identify the principal differences in environmental condition between sites regardless of the study to which they belonged and 2) to form clusters of sites with similar environmental conditions, which could then be analysed further with respect to their association with e,CΦ.The first three principal components of the PCA combined accounted for 86% of the cumulative variation in environmental variables . Temperature, chla and dK had the highest coefficients for the linear combination of variables comprising PC1, i.e. they were associated with the separation of samples along PC1  labelled alphabetically from a-o (p<0.005) across a range of Euclidean distances (representing dissimilarities between samples) from 0–4.4; these clusters often overlapped in the nMDS ordination of the Euclidean distance matrix derived from the environmental variables, indicating that there existed similarities in some of the environmental conditions between samples from different studies  were characterised by almost undetectable nutrient concentrations. Other clusters with samples representative of distinct regions include the North Sea cluster i and cluster k containing Celtic Sea samples and samples from the European shelf edge collected during AMT6. Like in the Baltic Sea, deplete nutrient concentrations in cluster k sets this cluster apart from cluster i.Some of the resulting clusters corresponded to obvious biogeographic regions and/or seasonal (i.e. temperature dependent) groupings, while others represent more or less distinct water masses . All theSamples from Bedford Basin fell into two clusters: cluster d containing samples collected from mid-March to mid-May when chlorophyll concentrations increased and nutrient concentrations and water clarity dropped from previous levels that characterized samples from cluster e, which were collected in late winter (February to mid-March). Interestingly, cluster e also contained almost all of the Massachusetts Bay samples, some North Sea samples from frontal regions as well as AMT 6 and Pacific Ocean samples from the Humboldt and Benguela upwelling areas, respectively. This represents similarities in physico-chemical properties of these different water masses rather than biogeographical regions. During late winter, salinity in the ice covered Bedford Basin was similar to that in off shore waters, while chlorophyll and nutrient concentrations as well as optical properties matched those of upwelling/frontal regions; hence, their grouping together.The remaining clusters  also contained samples from a variety of different cruises/regions. Samples collected from the deep chlorophyll maximum (DCM) in off shore regions during the AMT cruises, the Pacific Ocean cruise and the Celtic Sea (JR98) fell into one cluster (j), which was characterized by high temperatures as well as higher nutrient availability and ζ relative to the other offshore samples. Cluster l contained samples from intermediate depths in temperate waters on the European shelf and the shelf edge, which had relatively low temperatures. What set cluster l apart from cluster n, which also contained samples from the European shelf, was its overall greater optical depth. Open ocean samples with high water clarity and temperatures, as well as extremely low chlorophyll and nutrient concentrations fell into clusters m and o. While cluster m contained samples from intermediate depths collected during AMT6 and AMT11 and from the surface of the South Pacific Gyre (SPG) and HNLC region in the Pacific, cluster o was comprised of surface samples from the open ocean , central North Sea and Celtic Sea (JR98).e,CΦ, grouped according to samples from the surface mixed layer (SML) and DCM against depth and NO3− for each biogeographic province sampled during the Pacific and AMT cruises , and that different variable combinations were driving e,CΦ in different clusters and regions  (2R <0.05) between e,CΦ and environmental variables existed in the Gulf of Finland (cluster b), Bedford Basin (cluster d), European shelf waters (cluster n) and offshore samples from intermediate depths (cluster m), surface waters (cluster n) and the deep chlorophyll maximum (DCM) (cluster j). Surprisingly, PO43−, rather than NO3− availability, was often part of the variable combinations showing the strongest association with e,CΦ, usually in addition to temperature and/or salinity. The strong relationships between e,CΦ and PO43− and lack thereof with NO3− may be due to NO3− concentrations being close to or below the detection limit in open ocean waters and, during the summer months, often also in shelf waters, which compromises the detection of relationships with e,CΦ. Thus, NO3− availability may still be an important determinant of e,CΦ in these waters, but with PO43− being the only macronutrient left in our analysis, only the latter was pulled out. Despite the distinct clustering of samples from the frontal and upwelling regions, the Baltic Sea and European shelf edge, no significant MLR existed between e,CΦ and environmental variables in any of these regions .Clustering all available data based on the inherent environmental characteristics demonstrated that the dependence of e matrix  to furth regions . These ‘ p<0.05) . Signifi2R, they were highly significant. The relationships between e,CΦ and environmental variables were strongest in the Gulf of Finland and the Pacific Ocean (2R<0.05), followed by the Northwest Atlantic shelf samples (i.e. Massachusetts Bay + Bedford Basin). Relationships for the Atlantic were much less pronounced (2R <0.22), albeit highly significant (p<0.01). Once again, PO43− rather than NO3− availability appeared to play a greater role in these MLRs. On a global scale, MLR yielded an 2R of 0.038, but with both NO3− and PO43− contributing to the variability in e,CΦ.To develop region-specific algorithms, we also grouped the clusters according to meaningful water masses and biogeographic regions resulting in 11 significant algorithms covering the Gulf of Finland, the Baltic Sea as a whole (i.e. including the Gulf of Finland), European shelf waters (including the Northeast Atlantic), Northwest Atlantic shelf waters, the equatorial Atlantic and the South Atlantic Ocean. Although some of these region-specific relationships exhibited a low e,CΦ as well as differences between NPP and GPP. Growth rate dependent differential allocation of fixed carbon and varying lifetimes of intermediate products may cause large discrepancies between NPP and GPP, which short term 14CO2 uptake measurements may not capture 14C incubation techniques approximate GPP, Halsey et al. 14C incubations equate NPP or primary productivity rates somewhere between NPP and GPP. Failure to accurately quantify GPP will inevitably affect e,CΦ. Most data published in the past and also those used in our meta-analysis compared ETR to relatively short-term 14CO2 uptake rates, so that most e,CΦ values available to this day would provide a conversion from ETR to GPP or to something between NPP and GPP. Under most conditions, NPP differs from GPP by a factor of 2 to 2.5 e,CΦ values cannot account. Conversion of ETR to NPP and assessments of the potential error in e,CΦ and subsequently NPP due to employing short term 14C incubations will require further investigation and is the focus of ongoing work.While empirical evidence demonstrates that fluorescence-based measures of the PSII photochemical efficiency, and hence ETRs, are linearly related to net or gross carbon fixation rates under many conditions e,CΦ of 4–6 mol e− (mol C)−1− (mol C)−1 to convert ETRs to carbon fixation rates, assuming (i) that at least 4 e− transported through PSII are required per O2 molecule produced and (ii) that 1–1.5 mol of O2 is produced for each mol CO2 fixed, i.e. the photosynthetic quotient takes values of 1–1.5 mol O2 (mol CO2)−1e,CΦ equal (or close to) 4–6 mol e− (mol C)−1). However, the vast majority of estimates for e,CΦ are considerably higher, sometimes reaching extremes of >50 mol e− (mol C)−1 (e.g. BIOSOPE Pacific Ocean Cruise) or alternatively e,CΦ of <5 mol e− (mol C)−1 Gulf of Finland 2000 and AMT 15. Thus, for many cases, application of an assumed value of 4–6 mol e− (mol C)−1 to FRR data would yield erroneous estimates of C-uptake (within the limitations of the 14C-uptake itself for quantifying C-uptake).Under ‘optimal’ growth (where NPP ≈ GPP), and accounting for electron sinks associated with nutrient reduction, the slope of the linear relationship between ETR and NPP should yield values for e,CΦ >5 mol e− (mol C)−1 have been observed previously and potentially reflect processes that act to decouple ETRs from C-fixation, such as photorespiration e,CΦ <5 mol e− (mol C)−1 are more difficult to reconcile with biophysical and physiological processes, potentially indicating the magnitude of remaining methodological discrepancies in deriving e,CΦ, such as incorrect assumptions concerning the value or variability of PSIIn (see below), inadequate spectral correction or remaining non-systematic errors in both carbon fixation and fluorescence estimates. There were two studies with a high proportion of e,CΦ values <5 mol e− (mol C)−1: the AMT15 cruise  and the Gulf of Finland 2000 study. Unlike during the AMT15 cruise where <5 mol e− (mol C)−1 occurred only in samples from the DCM, we could not identify any consistency in the occurrence of such low values that could be related to sample handling and processing in the Gulf of Finland 2000 data. Thus, the high proportion of low e,CΦ values in the Gulf of Finland might point to systematic errors in the ETR calculations, i.e. in the component values for PSII'σ, PSInI and/or E, values that are often assumed (or not well measured). Whilst values of PSII'σ measured by FRRf have been shown to match independent bio-optical measurements well PSII'σ have been appropriately calibrated. In the Gulf of Finland PSII'σ typically range from ca. 150–300 A2 quantum−1, which is on the lower end of the range of values expected for assemblages dominated by diatoms, dinoflagellates, cryptophytes and cyanobacteria (data not shown), but still within the range commonly observed for such species PSII'σ as a result of erroneous instrument calibrations are therefore unlikely a significantly contributing factor. However, in cases when cyanobacteria dominate, ETRs may be underestimated due to the saturating light blue LED pulse being inefficient at driving reaction centre closure, resulting in low e,CΦ values. Furthermore, assumption of a constant PSIIn will introduce errors due to taxonomic variability in physiological traits PSIIn can vary among species by more than a factor of four, from 0.0010 to 0.0042 mol RC (mol Chla)−1PSIIn of 0.002 mol RC (mol Chla)−1, which is representative of the eukaryote phytoplankton community observed in their study −1.Values of e,CΦ due to the use of a constant PSIIn in some of the data sets presented in this study by comparing e,CΦ based on a constant PSInI with e,CΦ values calculated from ETR where PSIIn was either measured via oxygen flash yields (Bedford Basin data) or calculated using a fluorescence based algorithm PSIIn may lead to an underestimate of e,CΦ by 28–47% −1, which is comparable with the range observed for eukaryotic and prokaryotic phytoplankton PSIIn directly measured by Moore et al. PSIIn and validation of algorithms proposed for estimating the value of this variable e,CΦ which is associated with PSIIn can be achieved. Such measurements are the focus of ongoing work.We assessed the potential error in y 28–47% , which cin situ ETR and laboratory 14C-photoynthesis measurements K values derived from ETR and CO2 uptake PSII'σ, PSInI) and 14C incubation techniques, we estimated that, in worst case scenarios, e,CΦ may be over- or underestimated by as much as 53% (data not shown). A detailed assessment of these methodological differences between techniques and studies is beyond the scope of this study, and we refer the reader to a previous comprehensive reviews of these issues e,CΦ was lower than that observed across all studies   studies  for the e,CΦ. Information on phytoplankton community composition could not be routinely included and we assumed that any change in taxonomy was inherently accounted for in the environmental descriptors. Indeed, environment often leads to distinct structural differences of the photosynthetic apparatus and acclimation responses amongst phytoplankton from different biogeographic regions e,CΦ will, of course, be necessary to fully resolve the mechanisms responsible for changes in e,CΦ and the derived algorithms −1 as opposed to estimates of up to 54 mol e− mol C−1 from natural communities  never exceeded 10 mol e− (mol C)−1. The greater variance in the Massachusetts Bay time series study could, on the one hand, have been related to its longer duration (9 month) relative to the other time series (1.5–6 month) and, on the other hand, to its topography; while the Gulf of Finland, Bedford Basin and Ariake Bay, are all surrounded by land forming at least partially land-enclosed basins, Massachusetts Bay is relatively exposed and more strongly influenced by exchange of water with the Atlantic Ocean. Nevertheless, seasonal changes appear to influence the variability in e,CΦ although many areas are still under-sampled.Considerable variability of e,CΦ for the whole region were temperature, salinity and PO43−, resulting in a relatively weak (R2 = 0.266) albeit highly significant relationship. For the Gulf of Finland on its own, however, the relationships were much stronger , indicating that the Gulf of Finland should perhaps be treated as its own region.In the Gulf of Finland, for example, vertical mixing and the depth of the surface mixed layer change across different seasons e,CΦ with salinity, which may have both indirect  and direct (osmotic) effects on e,CΦ. The combination of low nutrients coupled with sudden high light availability due to elevated freshwater discharge after snow melt and ice breakup could cause an up-regulation of electron, ATP and/or reductant-consuming pathways (e.g. photorespiration and Mehler reaction). Conceivably, similar changes in salinity could also directly affect e,CΦ by causing osmotic stress or an increase in respiration of the extant phytoplankton community e,CΦ. Thus, in Bedford Basin multiple environmental factors  were likely at play, potentially exerting light and osmotic stress on the extant, but changing, phytoplankton community e,CΦ in remains a challenge and will require teasing apart daylight hour effects from temperature effects and other physicochemical variables.Distinct temporal changes in environmental gradients also existed in Bedford Basin, where initial correlations  showed se,CΦ and environmental variables. Furthermore, such relationships may not necessarily be linear Many samples from Massachusetts Bay and other open ocean areas fell into one cluster with the Bedford Basin data , Fig. 5.e,CΦ supply rates of the nutrient relative to the demands of the extant community. In the iron and nitrogen deplete SPG e,CΦ declined when NO3− concentrations were above the detection limit . Apart from the extreme outlier of e,CΦ>50 mol e− (mol C)−1, highest e,CΦ values were observed in the upwelling region and the gyre corresponding to samples from a low-NO3− surface water lens with high irradiances. Beneath this surface water lens, NO3− increased while light levels dropped and e,CΦ declined.Most of the other studies, also extended across multiple biogeographic provinces  e,CΦ. Iron limited phytoplankton in HNLC regions, for instance, my express the chlorophyll-binding protein IsiA, which reduces the apparent PSII photosynthetic efficiency and primary productivity rates normalized to chlorophyll e,CΦ to increase. Nutrient limitation in combination with high-light stress in surface waters may hence have been responsible for the highest e,CΦ obtained in these regions, suggesting a high degree of uncoupling between electron transfer and carbon fixation. Similarly, variations in nitrogen assimilation and nitrogen fixation require large amounts of ATP also leading to an uncoupling of electron transport from carbon fixation Iron and nitrogen limitation may be expected to influence the photosynthetic apparatus in different ways, with iron deficiency causing a preferential decline in iron rich-cellular components  regions, while keeping in mind that their boundaries might actually shift in space and time. Hence caution is still required in widespread application of the current algorithms. Even so, the appearance of clear patterns and proof of predictive power of environmental variables in the present article provides strong support and much improved confidence for successful conversion of ETRs to carbon fixation rates at a much greater spatial and temporal resolution than current Figure S1Principal component analysis including the environmental data, location data and methodological information (A) and with the methodological differences excluded from the analysis (B).(TIF)Click here for additional data file.Figure S2Comparison of e,CΦ− (mol C)−1) calculated with an  (in mol ePSIIn = 0.0020 mol RC (mol chla−1 and where )PSIIn was measured with oxygen flash yields (Bedford Basin) or by FRR fluorometry according to Oxborough et al.(UK-OA D366 and North Sea CEND0811). Bold line represents the regression equation for all three studies combined: e,CΦ constant PSIIn = 0.617× +2.765 . Regression coefficients are shown in Table S1 in (TIF)Click here for additional data file.Appendix S1Results of PCA including methodological and location variables and assessment of the effect of variable nPSII on Phie,C.(DOCX)Click here for additional data file.