A new group of arsenolipids based on cell‐membrane phosphatidylcholines has been discovered in herring caviar (fish roe). A combination of HPLC with elemental and molecular mass spectrometry was used to identify five arsenic‐containing phosphatidylcholines; the same technique applied to salmon caviar identified an arsenic‐containing phosphatidylethanolamine. The arsenic group in these membrane lipids might impart particular properties to the molecules not displayed by their non‐arsenic analogues. Additionally, the new compounds have human health implications according to recent results showing high cytotoxicity for some arsenolipids. Arsenic‐containing lipids, so‐called arsenolipids, are natural constituents of marine organisms thought to be involved in arsenic detoxification processes.Research activity over the last eight years, however, has revealed several new groups of naturally occurring arsenolipids, including arsenic‐containing fatty acidsA recent study of a fish oil showed the presence of an unidentified group of arsenolipids thought to be conjugates of arsenic fatty acids because they could be base‐hydrolyzed to known arsenic fatty acids.Clupea harengus; ≈0.8 μg As g−1 dry mass, originating from the Norwegian Sea) with DCM/MeOH and washing the extract with water yielded an organic layer containing approximately 80 % of the initial arsenic. After all traces of DCM were removed, the residue was re‐dissolved in absolute ethanol and analyzed by reversed‐phase HPLC coupled to an elemental mass spectrometer . The HPLC/ICPMS measurements performed in selected‐ion monitoring mode with m/z 75 (As+) indicated the presence of several known arsenic fatty acids and arsenic hydrocarbons, in addition to a cluster of later‐eluting arsenolipids  and one with m/z=123 (C2H8OAs+), when subjected to collision‐induced‐dissociation. Owing to their mass defects, these fragment ions can be uniquely identified by a high‐resolution mass spectrometer even from a complex matrix. Searching the fragmentation spectra for these two fragment ions exposed a multitude of precursor ions, from which six known arsenic fatty acids  and five known arsenic hydrocarbons  could be expressly identified . This procedure similarly implied an ion with [M+H]+ at m/z=529. The accurate mass and fragmentation data of this ion were consistent with a hitherto unknown arsenic fatty acid with eight ethylene groups MexHyO9NAsP, and inspection of the MS/MS (fragmentation) spectra of these ions confirmed similarity as well as a set of prominent fragment ions characteristically observed in the MS/MS spectra of phosphatidylcholines.In the later part of the HPLC/ESIMS chromatogram  did not reveal the presence of AsPCs, but again AsFA 528  and also an arsenic‐containing phosphatidylethanolamine (AsPE) incorporating AsFA 528 were identified , and both contained the five AsPCs described above. The same approach applied to an extract of salmon caviar  PEs and PCs.m/z=585.2919, corresponding to the arsenic fatty acid with part of the glycerol molecule after loss of phosphorylethanolamine and docosahexaenoic acid as the corresponding ketene.The fragmentation behavior of the protonated AsPE 1035  parallels the behavior observed for the protonated AsPCs with the exception that protonated phosphorylethanolamine at The origin of the arsenic phosphatidylcholines in the membrane‐rich fish eggs remains an open question. Phosphatidylcholines are key components of biological membranes. It is possible that infidelity in their biosynthesis results in the arsenic analogues, although a more fundamental role for the arsenic compounds cannot be dismissed. These compounds are likely to be widespread in fatty fish, based on the ubiquity of free arsenic fatty acids and the recent report of unidentified arsenic fatty acid conjugates in fish oil,As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Mycobacterium ulcerans can lead to chronic skin ulceration known as Buruli ulcer. The pathogenesis of this neglected tropical disease is dependent on a lipid‐like toxin, mycolactone, which diffuses through tissue away from the infecting organisms. Since its identification in 1999, this molecule has been intensely studied to elucidate its cytotoxic and immunosuppressive properties. Two recent major advances identifying the underlying molecular targets for mycolactone have been described. First, it can target scaffolding proteins (such as Wiskott Aldrich Syndrome Protein), which control actin dynamics in adherent cells and therefore lead to detachment and cell death by anoikis. Second, it prevents the co‐translational translocation (and therefore production) of many proteins that pass through the endoplasmic reticulum for secretion or placement in cell membranes. These pleiotropic effects underpin the range of cell‐specific functional defects in immune and other cells that contact mycolactone during infection. The dose and duration of mycolactone exposure for these different cells explains tissue necrosis and the paucity of immune cells in the ulcers. This review discusses recent advances in the field, revisits older findings in this context and highlights current developments in structure‐function studies as well as methodology that make mycolactone a promising diagnostic biomarker.Infection of subcutaneous tissue with  Mycobacterium ulcerans (MU) infection can cause Buruli ulcer (BU), a chronic necrotizing skin infection with high prevalence in rural West Africa  encoding the modular type I polyketide synthases as well as accessory genes  and F (m/z 723)  causes a substantial decrease in mycolactone detected in skin biopsies by both cytotoxicity and MS  with an IC50 of 3 µg ml−1. While AT2R was required for this effect, the hyperpolarisation itself was found to be mediated by AT2R‐dependent activation of phospholipase A2, the release of arachidonic acid by COX‐1 and activation of the K+ channel‐TRAAK by prostaglandin E2  and neural WASP (N‐WASP) , something that must occur for about 30–50% of proteins made by mammalian cells, not only cytokines but also membrane receptors and proteins that function inside the ER itself. Normally, most ER‐transiting proteins undergo ‘co‐translational translocation’, i.e. the ribosome becomes attached to the ER membrane via Sec61 accessory proteins and the signal peptide, and the translating peptide is synthesized directly into the ER lumen for folding by chaperones [reviewed by Zimmermann et al. ]. Using  et al.,  in vitro and in vivo by using purified lipid extracts from cultured MU, chemically synthesized mycolactone, or by comparing the effects of wild‐type MU with those of mycolactone‐negative mutant strains with broadly comparable results. Mycolactone seems to diffuse passively across the cell membrane because uptake of fluorescently labelled mycolactone by fibroblasts was both non‐saturable and non‐competitive with excess mycolactone   are vital components of innate and anti‐mycobacterial immunity. Despite the fact that mycolactone has been shown to interfere with phagocytosis by J774 macrophages  mycolactone exposure, but not at lower doses , the transcription or export  of mycolactone leads to massive depletion of T‐cells in peripheral lymph nodes (PLNs)  alters the expression of several microRNAs including let‐7b  and was enhanced at lower doses (1 µg ml−1) after 24 h.Bacterial infection is normally associated with influx of neutrophils into infected tissue. This is notably absent in the necrotic centre of BU lesions, but it is seen in surrounding tissue , mycolactone causes rapid changes in cell permeability leading to cell death by primary necrosis  were associated with rapid generation of reactive oxygen species (ROS) within 45 mins of exposure that could be completely prevented by the ROS inhibiting substance deferoxamine. This Fe2+ chelator could efficiently block mycolactone's cytotoxic effects as assessed by neutral red uptake (an indicator of lysosome numbers) but was less efficient at restoring metabolic activity as assessed by the vital stain WST‐1  is equivalent to 0.27 μM.Because base hydrolysis of mycolactone (separating the lactone core and Northern chain) was shown to result in a molecule with ~1000‐fold reduced cytopathic activity towards L929 fibroblasts  and binds less well to WASP (IC50 97 μM vs. 32 μM). However, tantalisingly, this activity can be restored by removing the Northern chain . In some of the investigations of structure/function, this molecule   is of particular interest because it retains the ability to suppress inflammation while having significantly reduced cytopathic activity. The lowest dose inducing a cytopathic effect in L929 fibroblasts has not been reported, but at doses of 10 μM and 16 μM, it has ~50% cytopathicity . Such resources are expected to be of great utility in the future.The mycolactones are a unique and fascinating class of molecules. Not only are they central to the understanding of BU pathogenesis, they are also potentially important in the diagnosis and monitoring of BU. In the wider context, they are also helping us understand fundamental aspects of cell biology; mycolactone is one of only a handful of molecules that are capable of blocking translocation. However, mycolactones present several challenges in molecular investigations; not least obtaining the chemically pure material because mycolactone production in the laboratory is arduous and has several unstable intermediates. Synthetic mycolactone is preferable over compounds purified from MU becaue difficulties remain in the purification process, and MU itself is a slow‐growing organism requiring high‐level containment for culture. Furthermore, the physical characteristics of this lipid‐like molecule lend it an inherent ‘stickiness’, which can complicate analysis. This, together with its low antigenicity and immunosuppressive activity have hampered generation of mycolactone‐specific antibodies that could demonstrate the location and concentration of mycolactone within BU lesions and be used in biochemical assays. Novel approaches under development include synthesis of recombinant antibodies against mycolactone and mycolactone specific complexes using phage and yeast display methods (Naranjo Despite these challenges, the past 10 years have yielded significant advances in our understanding of the biological activity of mycolactone. Central to this progress is the proposition of molecular mechanisms underpinning its cytotoxic and immunosuppressive properties. We eagerly await ratification of these findings by way of independent confirmation from different laboratories to demonstrate the commonality of the effects. Nevertheless, it is now clear that the interplay between mycolactone's cytotoxic and immunosuppressive effects varies depending on the cell the toxin is interacting with, the duration of exposure and the dose. These pleiotropic effects work together to create a complicated picture in the evolving lesion, whereby different functional effects may be apparent at the core of infection where the mycolactone concentration is highest, compared with the outer edge of biologically relevant mycolactone concentrations furthest from the clusters of MU. Intriguingly, this suggests that mycolactone may be operating via each or all of the described mechanisms in different cells at different times. This is especially true if you consider translocation blockade because of the large range of potentially affected proteins. Around 30–50% of protein coding genes have a signal peptide, but the response would be predicted to be dependent on the specific combination of such proteins expressed by a specific cell/condition and their sensitivity to mycolactone‐dependent blockade.In the face of this complexity, the simple facts remain that mycolactone is the cause of the tissue necrosis, the impaired clearance of MU infection and the painlessness of the ulcers. The recent development of techniques for detection and quantification of mycolactone within host tissues has further deepened our understanding of the relationship between this toxic macrolide and clinical disease and has opened up a new vista for utilising mycolactone as a diagnostic and prognostic biomarker for the management of this neglected tropical disease.


These results suggest that respiratory infection is enhanced during unusually cold conditions and during conditions with unusually low humidity, even in a subtropical location where humidity is typically high.The relationship between weather and influenza and pneumonia mortality was examined retrospectively using daily data from 1980 to 2009 in Auckland, New Zealand, a humid, subtropical location. Mortality events, defined when mortality exceeded 0·95 standard deviation above the mean, followed periods of anomalously cold air ( The strong seasonality evident in mortality records from mid‐latitude locations suggests that climatic factors may amplify cold‐season death rates.Reasons for the seasonal pattern in influenza mortality remain unclear. Recent research suggests a connection to weather, particularly cold and/or dry (low humidity) air.Several studies in the United States have linked the timingDaily mortality counts of pneumonia and influenza (P&I) were tallied from 1980 to 2009 from the Mortality Collection managed by the New Zealand Ministry of Health. Pneumonia and influenza are commonly grouped in retrospective analyses as it is often difficult to ascribe a given mortality event to influenza because of the lack of laboratory confirmation and because pneumonia deaths may occur when the influenza virus is no longer detectable.z‐score by subtracting the mean and dividing by the standard deviation of each period (1980–1998 and 1999–2009). To account for the latency period between infection and death, a 19‐day leading average smoother was applied to the z‐scored time series. This smoother was selected based upon prior researchThe raw mortality time series exhibits significant seasonality and a decline in both mean and variance beginning in 1998 prior to the ICD‐9 to ICD‐10 conversion . Longer mortality “episodes” were defined as three or more consecutive events with no intervening period of more than five non‐event days—13 such episodes were defined, and these varied from 7‐ to 50‐day duration  exhibited a long, positive tail starting at T) and dew point temperature (Td), the latter of which is a measure of atmospheric humidity used in prior influenza research.z‐scores using the mean and standard deviation for each day of the year and were subsequently smoothed with a 3‐day lagging moving average filter. For the weather data, this short filter length was chosen to reflect the high daily variability inherent in Auckland's weather.Weather data were acquired for Auckland International Airport (station #1962) from National Institute of Water and Atmospheric Research archives. Data analyzed included morning and afternoon air temperature . Because the data have been z‐scored to a zero mean, the t‐test is comparing temperature and dew point departures from a zero baseline (P ≤ 0·01). Conversely, we examined P&I mortality on unusually cold days and on days with low humidity. Temporal autocorrelation and potential inflation of the true degrees of freedom was addressed by altering the effective sample size (see Supporting Information).The weather on P&I event and episode days was compared to all other days using a one‐sample P&I mortality is significantly elevated about three weeks after both cold days and dry days Table . Relatioanomalously cold and dry days (relative to the mean for that day of the year) are associated with elevated P&I mortality. Influenza has a very pronounced cold‐season peak (from June to October) in Auckland, where winters tend to be moderately cool and humid  air in this subtropical island location. Unusually cold and dry periods may be associated with conditions that enhance transmission and survival of the influenza virus or other respiratory pathogens, but the atmospheric triggers appear to depend upon relative departures rather than absolute temperature and humidity conditions.The weather data have been standardized to remove seasonality. Therefore, our results indicate that id Table . Similarid Table . Thus, aIndoor heating (without humidification) may lengthen survival times of respiratory viruses and weaken nasal defense mechanisms. Although central heating is not common in Auckland, indoor heating will be used most often during abnormally cool periods in winter when respiratory infection is most prevalent, although fuel poverty is an important problem for deprived groups in New Zealand.As this was a correlative study based on long‐term data archives, several limitations must be considered. New disease coding rules introduced with ICD‐10 reduced the use of pneumonia codes as the underlying cause of death in those cases with other serious chronic illnesses, thereby impacting the time series around the time of the coding transition in 1998–2000. Combining pneumonia and influenza mortality into a single group, while a necessity without laboratory confirmation of influenza, means that the true impact of the influenza virus is unknown .Abnormally cold, dry air tends to precede days and periods with high pneumonia and influenza mortality in Auckland, New Zealand, by up to three weeks. Given the ability to forecast cold and dry periods in advance, such information could potentially be used for “real‐time” respiratory infection forecasting. This could help reduce the impact of pneumonia and influenza in a number of ways: communication to vulnerable groups (particularly the elderly and those with established respiratory disease), deployment of preventive measures , and planning for increased service demands in hospitals. Before doing this, it would be useful to review the various approaches to forecasting, and to calibrate and validate the forecast models.z‐scored P&I mortality in Auckland, New Zealand, 1980–2009.Figure S1. Frequency histogram of smoothed and Table S1. Summary of linear regression results when the second variable is fitted to the residuals of the regression on the first variable.Click here for additional data file.


Integration of knowledge concerning circadian rhythms, metabolic networks, and sleep‐wake cycles is imperative for unraveling the mysteries of biological cycles and their underlying mechanisms. During the last decade, enormous progress in circadian biology research has provided a plethora of new insights into the molecular architecture of circadian clocks. However, the recent identification of autonomous redox oscillations in cells has expanded our view of the clockwork beyond conventional transcription/translation feedback loop models, which have been dominant since the first circadian period mutants were identified in fruit fly. Consequently, non‐transcriptional timekeeping mechanisms have been proposed, and the antioxidant peroxiredoxin proteins have been identified as conserved markers for 24‐hour rhythms. Here, we review recent advances in our understanding of interdependencies amongst circadian rhythms, sleep homeostasis, redox cycles, and other cellular metabolic networks. We speculate that systems‐level investigations implementing integrated multi‐omics approaches could provide novel mechanistic insights into the connectivity between daily cycles and metabolic systems. CLOCKcircadian locomotor output cycles kaputEEGelectroencephalogramsNTOnon‐transcriptional oscillatorPRXperoxiredoxinRBCred blood cellSCNsuprachiasmatic nucleusTTFLtranscription‐translation feedback loopCircadian approx. 24 hour) clocks are believed to exist at almost all levels of life and play a key role in the maintenance of physiological and behavioral processes in accordance with the day/night cycle 4 hour clAdequate sleep is an essential requirement for health. However, a significant proportion of the adult population suffers from trouble sleeping at night, and staying awake during the daytime, most likely due to the aberrations in the switching mechanism that controls transitions between wake and sleep It is clear that there is a rhythmic pattern in cell function and cycles of energy utilization in accordance with a daily rhythm Period (Per) and Cryptochrome (Cry) families Since the 1980s, transcriptional/translational feedback loops (TTFLs), wherein rhythmicity in the expression patterns of specific genes are controlled by the periodic expression of “clock” gene products, were considered as the principal drivers of circadian periodicity in multiple biological systems Information obtained through the TTFL‐based models is undoubtedly essential for understanding various aspects of circadian rhythms, and in particular tissue functions, since transcriptome and proteome alterations are extensive and cyclical in a range of studies Nearly 10 years ago, the major shortcomings of the TTFL‐based mechanism became obvious when circadian rhythms were found to be persisting in cyanobacteria even in the absence of transcription‐translation feedback Another peculiarity about circadian systems previously was the apparent lack of molecular phylogeny of mechanisms controlling circadian rhythms. Virtually all known “clock” genes and proteins are not conserved across the various domains of life, although there are some homologous components in fruit flies and mammals. This previously suggested that the “logic” of the clockwork always involved a TTFL, but the players were different in each model system that has been studied 2O2), peroxinitrite, and hydroperoxides to protect organisms against diverse oxidative stresses. Interestingly, these abundant cellular antioxidant proteins, which are probably evolved from a thioredoxin‐like ancestor 2‐terminal Cys that can become oxidised, while the 2‐Cys type contains both the NH2‐ and COOH‐terminal Cys residues and can therefore form either intermolecular  or intramolecular  disulfide bonds during oxidation. Apart from the detoxification of the various peroxide substrates, eukaryotic 2‐Cys peroxiredoxins (2‐Cys PRXs) play a significant role in hydrogen peroxide‐mediated signal transduction pathways PRXs are thiol‐dependent peroxidases, which serve as an antioxidant in the maintenance of intracellular levels of hydrogen peroxide  by CLOCK and BMAL1 Oscillation of metabolic pathways is not a new concept. In the 1960s, rapid rhythmicity (over minutes rather than hours) in the glycolytic intermediates glucose‐6‐phosphate/fructose‐6‐phosphate and fructose‐1,6‐diphosphate (FDP) levels was first demonstrated in yeast extracts by Ghosh and Chance Intriguingly, several components of central metabolic pathways, such as a number of rate‐limiting enzymes involved in glycolysis and the tricarboxylic acid (TCA) cycle, are redox‐sensitive, indicating the potential of these metabolic pathways to be regulated by the circadian redox oscillations Recently, comprehensive metabolomics analyses of different biological fluids  have provided insights regarding the circadian regulation of various human metabolic pathways In mammalian circadian system, the brain's suprachiasmatic nucleus (SCN), which is considered as the master circadian “clock,” orchestrates synchronization of oscillators in peripheral tissues Bmal1 (Cycle in Drosophila), Clock/Npas2, Per1/Per2, and Cry1/Cry2 result in various phenotypes including increased sleep fragmentation and rebound following deprivation of sleep, in addition to an enhanced tendency to switch between non‐rapid eye movement (NREM) and rapid eye movement (REM) sleep Components of the central circadian pacemaker are known to regulate sleep onset and control the transition to wakefulness, while the quantity of sleep appears to be controlled by homeostatic centers There is a longstanding belief that sleep happens only at the level of the whole organism. However, recent work highlights that even within the brain, there is local and use‐dependent sleep of subsets of neural circuitry, which forces a reappraisal of what sleep is and what is might be for Akin to circadian clocks, it is now imperative to study the self‐sustained mechanism of sleep at the cellular level, which is often difficult to execute in vivo in the mammalian brain due to the presence of the entangled web of neuronal networks that are controlled by signals from both local and global sources. Of note, a recent study on primary mouse cortical cultures demonstrated sleep as a prominent characteristics of simple neuronal networks grown in vitro Although identification of NTOs and metabolic oscillations has enhanced our understanding of circadian rhythmicity, there are still several opaque areas that entail further investigation Fig. . The mosWhile transcriptomic studies have previously provided valuable insights into which gene networks and tissue‐specific programmes are controlled by the circadian clock In recent years, ribosome profiling, which provides genome‐wide information on protein synthesis through deep sequencing of ribosome protected mRNA fragments, is emerging as an efficient technique to track in vivo translation. Circadian clocks play some vital roles in coordinating transcription and translation steps which are essential for ribosome biogenesis. Intriguingly, some very recent studies have demonstrated the promising applications of ribosome profiling for studying translational control in circadian gene expression and for mapping rhythmic translatome Cross‐talk between the cellular redox state and the circadian clocks has been studied extensively in different organisms A complementary set of analyses could also be used to probe specific post‐translational modifications (PTMs) of proteins across the day, and also under sleep restriction paradigms. Such modifications regulate the recruitment, recognition, assembly/disassembly, translocation, and consequently the eventual fates of the majority of eukaryotic proteins http://circadiomics.igb.uci.edu/), which is a computational framework that could serve as a repository for metabolomics and other “omics” level high‐throughput data associated with circadian clocks A recent study by Sassone‐Corsi and colleagues demonstrated synergistic links between oscillations in the circadian transcriptome and metabolic pathways In order to unravel the transient snapshots of dynamic circadian rhythmicity and sleep homeostasis, there is an urgent need to amalgamate the findings obtained from mRNA, protein and metabolite level investigations to get a true understanding of these multiple dynamic facets. In the future, we anticipate that an integrated multi‐omics analysis  of sleep‐deprived models (using different complementary model organisms such as the mouse and fruit fly) will collectively provide a complete representation of bidirectional molecular communication between the sleep centers and the circadian pacemaker. Eventually, this may also lead to the identification of novel conserved markers of circadian rhythms and sleep deprivation Fig. A. ConseqWe now have a vast array of knowledge about the molecular underpinnings of the circadian clock, particularly at the level of transcriptional networks. The influence of the clock is pervasive, with ∼40% of transcripts in the mouse genome displaying daily cycles


It is common practice worldwide for health professionals to study abroad. However, the outcome of such experience has not been rigorously evaluated in China. Our current study aimed to quantify the impact on research of studying abroad among Chinese health professionals.A self-administered structured questionnaire was developed among health professionals in Harbin Medical University and its affiliated hospitals who had studied abroad  and health professionals who did not have experience abroad . 166 ‘returning’ professionals (Group A) and 166 age-, sex- and specialty-matched ‘resident’ professionals (Group B) were included in the study. SPSS software was used for data entry and analysis.P < 0.01) and the number of NSFC was 154 and 34 (P < 0.01), respectively. The total IF of papers published abroad was associated with the duration abroad (P < 0.01) and not with the age of going abroad (P > 0.05). The total IF of papers published at home, and the number of NSFC had no relationship with the duration abroad (both P > 0.05) nor the age of going abroad (both P > 0.05). The total IF of papers published at home and the number of NSFC were positively correlated with the total IF of papers published abroad (both P < 0.01).The total IF of papers published by Group A and Group B was, respectively, 1933.52 and 629.23 (This study reflects the beneficial experience of working overseas. The opportunity for overseas experience should not be limited by age. Overseas study should be prolonged. Globalization and internationalization have become key areas of focus for university educators over the past decade, with an increasing emphasis among universities on global health programs and international experience. Health professional colleges are no exception to the rapid expansion of global health initiatives across university campuses. Many medical programs have incorporated studies on cultural differences and awareness into their curricula, and broadened their opportunities for international training through work, education, and research activities -6. TodayAccording to the National Health Human Resources Survey in China in 2005, only 0.18% of all health professionals had experienced more than 6 months study or training abroad . With thThere are two medical schools in Harbin: Harbin Medical University (HMU) and Heilongjiang University of Chinese Medicine (HUCM). HMU and HUCM are the statutory regulatory and registration authorities, respectively, for Western medicine and Chinese medicine in Harbin. Most medical professionals of HUCM do not have the opportunity to study abroad, as Chinese medicine is only taught in China, and if they do go overseas, they tend to teach techniques such as acupuncture and massage. Thus, we excluded HUCM from our study.HMU is a government-owned institution that offers a 5-year medical program leading to a Bachelor degree, a 3-year program leading to a Master degree and a 3-year program leading to a PhD degree, and has strived to restructure its medical education program to offer comprehensive solutions to national health care problems. There has been a trend of studying or training abroad among health professionals from HMU and its four affiliated hospitals, with around 20% of its approximately 4500 professional staffs having overseas experience.This study was performed from September 2012 to June 2013. Since less than 6-month research training abroad is pretty short and end up insufficient to be a dependent researcher, the 166 ‘returning’ health professionals (Group A) involved in our study had a minimum of 6 months experience abroad including 76 from HMU (Group A1) who are scientific researchers and 90 from the affiliated hospitals (Group A2) who are clinicians. The questionnaire in Table The National Natural Science Foundation of China (NSFC) has implemented an advanced science funding system including peer review and performance evaluation. The system is comprehensive and strict, with the number of NSFC accurately reflecting capability in scientific research. The Science Citation Index (SCI) is internationally recognized as the most authoritative scientific literature search tool, and we used the NSFC evaluation and the SCI impact factor (IF) as good indicators of scientific and research capability. Because authors contribute differently to a research study and there is no system internationally which could eliminate this difference, we ranked the authors to reflect this difference according to ‘The Promotion System of HMU (2013)’: first author and corresponding author of a paper (IF × 100%), second author of a paper (IF × 50%), and third author and later authors of a paper (IF × 25%) .1) and 90 clinicians from the affiliated hospitals (Group B2) for comparison of the total IF of papers published and the number of NSFC between the ‘returning’ and ‘resident’ groups. The control group (Group B) was selected based on the exact match of age, sex and specialty as Group A. When there were more than one match, we randomly selected one before developing questionnaire in case of arbitrarily selecting matching controls who are less capable than the treated individuals. When there were no exact match, we replaced the ‘returning’ professional by another one which had at least one match. Also, we developed questionnaires among these 166 ‘resident’ professionals to collect data and response rates were 100%. All data were anonymized to protect the respondents’ privacy.The following data were collected among 166 ‘returning’ professionals: (1) age; (2) duration of studying or training abroad (months); (3) the age of going abroad; (4) total IF of papers published abroad; (5) total IF of papers published at home; and (6) number of NSFC. Multiple linear regression was used to determine the factors associated with the IF of papers and number of NSFC using SPSS version 19 . To evaluate the impact of studying or training abroad, we randomly selected 166 ‘resident’ age-, sex- and specialty-matched professionals (Group B) including 76 scientific researchers from HMU (Group B1) with an age range of 31–70 years  and 90 clinicians  from affiliated hospitals with an age range of 31–70 years . Similarly, there were 166 ‘resident’ age-, sex-and specialty-matched professionals  with an age range of 31–70 years  and 90 clinicians  with an age range of 31–70 years .Of the 166 ‘returning’ professionals  and the number of NSFC (P < 0.01). There were significant differences between Group A1 and Group B1 in mean scores for total IF of papers (P < 0.01) and the number of NSFC (P < 0.01). The same situation applied to Group A2 and Group B2.The IF of papers published individually at home of ‘returning’ professionals (Group A in Table P < 0.01) and not to the age at which the subjects went abroad (P > 0.05). The total IF of papers published at home and number of NSFC had no association with the duration of studying or training abroad (both P > 0.05) nor with the age of going abroad (both P > 0.05). However, the total IF of papers published at home and the number of NSFC were positively correlated with the total IF of papers published abroad (both P < 0.01).Analyzing the data in Table It is often supposed that studying or training abroad has a positive influence on health professionals’ capacity for scientific research. However, the impact has not been evaluated in Chinese health professionals. This study revealed that there is a wide gap in publishing history between ‘returning’ and ‘resident’ health professionals including both scientific researchers and clinicians. These data strongly suggest that not only scientific researchers but also clinicians could gain much benefit from a period of study or training abroad by broadening their horizons and boosting their capability in scientific research.So what can be done to promote this positive influence? Traditionally, neither legislation nor other measures have been taken to optimize the ‘overseas experience’ of health care . It is iAnother area that requires special attention is the quality of research facilities in universities which professionals attend abroad. It is widely recognized that health professionals cannot publish a paper with a high IF unless the laboratory equipment and facilities are of a high standard. We suggest that a quality control system be developed to ensure that health professionals going abroad have access to institutions offering fully equipped up-to-date laboratories. This would provide ‘returning’ professionals with excellent research facilities and knowledge about the newest procedures.During conversations with ‘returning’ professionals, we realized that there were major differences in the research conditions between China and abroad, and this appeared to be the main factor that motivated health professionals to study abroad. Not only do poor research conditions at home hamper innovative thinking, but they also reduce the capacity for study implementation. This situation can only be improved by very strong political will and greater budget allocations to the health care sector. According to the Ministry of Finance, the total expenditure on health care in 2012 was just 6% of the gross domestic product in China. It is necessary that the political hierarchy take some bold steps in this regard. However, this is not only a governmental responsibility, and the medical workforce working across China should also address all the factors that lead to the poor research capability of health professionals. Legislation should be put in place to ensure that the working environment is made more conducive to medical research. Only then will we be able to improve the national capability in research.Our work represents an initial effort to try to better understand the feasibility and outcomes of studying or training abroad among health professionals in Harbin. This study involved only one medical school and its four affiliated hospitals and thus had a small sample size; therefore it does not represent the entire medical workforce in China. Since authors contribute differently to a research study and there is no author ranking system internationally which could eliminate the difference among authors, we ranked the authors according to ‘The Promotion System of HMU (2013)’. This system could not be applied to all SCI papers worldwide. Moreover, there are some major confounders, for example, language proficiency, funds, facility, equipment, job titles and specialties. Those who get 6 months or more of international training are likely to be selected out of motivated candidates, so they may have higher capability and motivation at baseline . So there may be deviation to compare those with and without international experience unless the authors’ capability to conduct research and write papers at baseline is matched by quantified standards.This study shows the major positive impact on medical research in China of a period of studying or training abroad, and indicates that the opportunity for overseas experience should not be limited by age. The ‘returning’ professionals should focus on making an effort to improve the total IF of papers published abroad by prolonging the duration of study abroad. The study also indicates that there is a strong need for upgrading research facilities in China.


The measurement of progress in maternal and newborn health often relies on data provided by women in surveys on the quality of care they received. The majority of these indicators, however, including the widely tracked “skilled attendance at birth” indicator, have not been validated. We assess the validity of a large set of maternal and newborn health indicators that are included or have the potential to be included in population–based surveys.We compare women’s reports of care received during labor and delivery in two Kenyan hospitals prior to discharge against a reference standard of direct observations by a trained third party (n = 662). We assessed individual–level reporting accuracy by quantifying the area under the receiver operating curve (AUC) and estimated population–level accuracy using the inflation factor (IF) for each indicator with sufficient numbers for analysis.Four of 41 indicators performed well on both validation criteria (AUC>0.70 and 0.75<IF<1.25). These were: main provider during delivery was a nurse/midwife, a support companion was present at birth, cesarean operation, and low birthweight infant (<2500 g). Twenty–one indicators met acceptable levels for one criterion only . The skilled birth attendance indicator met the IF criterion only.Few indicators met both validation criteria, partly because many routine care interventions almost always occurred, and there was insufficient variation for robust analysis. Validity is influenced by whether the woman had a cesarean section, and by question wording. Low validity is associated with indicators related to the timing or sequence of events. The validity of maternal and newborn quality of care indicators should be assessed in a range of settings to refine these findings. Nearly 275 000 maternal deaths occurred globally in 2011, nearly all of which took place in low– and middle–income countries (LMIC) [To measure progress in maternal health, monitoring agencies have relied on tracking indicators proposed as measures of quality of care, such as the proportion of births attended by a skilled birth attendant, that are assumed to be strongly correlated with maternal mortality . Such inA growing, but still limited, body of research has examined the validity of indicators of the quality of care in the intrapartum and early postpartum period. To our knowledge, however, no study has yet reported on how accurately women can recall the skill level of their provider at birth, although there have been some attempts to look at data quality issues . FurtherTo address this gap, this study assessed women’s ability to report on a set of quality of maternal and newborn health care indicators that are either currently in use or have the potential to be included in routine survey–based data collection. In spite of its limitations, it seems likely that the “skilled birth attendance” indicator will continue to be used and so we assess how accurately women report on the skill level of their provider during delivery. We compare women’s self–reports of maternal and newborn care received against third party observations during labor and delivery. Finally, we provide suggestions for modifications to data collection procedures that could improve the measurement of maternal and newborn health care.Validation exercises were conducted in two high volume public hospitals located in Kisumu District and Kiambu District in western and central Kenya, respectively. According to the 2014 Kenya DHS, nationally, 61% of births in the five years preceding the survey were delivered in a health facility; in Kisumu and Kiambu districts the prevalence was 70% and 93%, respectively . FacilitData collection took place from July to September 2013. All pregnant women aged 15 to 44 who were admitted to a study facility maternity unit and in early labor were invited to participate. Participants included eligible women who underwent labor and delivery and were able to provide consent.Our reference standard for validity analysis is data collected by trained researchers who observed providers in the maternity admission room and labor and delivery rooms using a structured checklist–type form. Observers were registered Kenyan nurse/midwives with at least three years of experience in a maternal and newborn health unit and previous research experience. Observations were used as the reference standard as they reflected all facets of caregiving including events related to the birth itself as well as interactions between the women and provider, before, during and up to one hour after delivery. In the few cases in which clarification was needed  observations were supplemented by checking facility records and by asking providers.Exit interviews with women took place prior to hospital discharge. Data collectors who were degree holders in a social science interviewed women using a structured questionnaire. Interview questionnaires were translated into Kiswahili, Dholuo and Kikuyu and were administered in the woman’s language of preference.All data collectors received four days of intensive training on the study procedures, the rationale behind each element of the client questionnaire and observation checklist to ensure full understanding of the instrument components, and how to record responses and observations.Written informed consent was obtained from all participants and their attending providers prior to participation. All women and providers were provided with a description of the study and procedures, including their right to refuse participation at any time. In Kenya, pregnant adolescents between ages 15–17 years are considered “emancipated minors” and their written informed consent was also obtained . Staff wPrior to participant enrollment, the study protocol was approved by the ethical review committees of the Population Council and the Kenya Medical Research Institute (KEMRI).To identify indicators to be validated, a landscaping scan of published and grey literature was conducted from April to July 2012. The scan focused on indicators of the quality and content of care received during labor and delivery and the health outcomes related to this period [Sample size was calculated assuming 50% prevalence for all indicators, given that some harmful practices would rarely occur, and some beneficial practices would almost always occur, at 60% sensitivity ±6% precision, 70% specificity ±6% precision, with type 1 error set at α = 0.05 assuming a normal approximation to a binomial distribution. These specifications imply a minimal sample size of 500, which was increased to 600 women to allow for 20% attrition in a separate study to re–interview women approximately one year following delivery.Statistical analysis was performed using Stata Version 12  to assesMissing pairwise data were excluded. To summarize the accuracy of each indicator, we quantified the area under the receiver operating curve (AUC), which plots the sensitivity  of each indicator against its false positive rate (1–specificity). To measure uncertainty associated with validity, we estimated 95% confidence intervals (CI), assuming a binomial distribution. In practice, the AUC represents the “average accuracy of a diagnostic test” ,24. AUC To assess the population–based validity of indicators, we estimated the inflation factor (IF), also known as the Test to Actual Positives (TAP) ratio . The IF The prevalence of women’s self–report in a survey (Pr) is calculated by applying each indicator’s estimated sensitivity (SE) and specificity (SP) to its true prevalence (P), using the following equation: Pr = P × (SE+SP−1)+(1−SP) . We cautWe categorized individual–level reporting accuracy as high (AUC>0.70), moderate (0.60<AUC<0.70), and low (AUC<0.60)  and the The funders of the study had no role in data collection, analysis, interpretation or writing of the study results, or decision to submit for publication.1039 women admitted to the maternity unit at participating study facilities were recruited to participate. Of those, 676 women were observed . Approximately one–third of women were not observed because they were not in labor but required monitoring on the antenatal ward, did not progress into labor, or they progressed rapidly into labor and full observation was not possible .The full list of indicators selected for validity testing is presented in Of the 41 indicators with at least five cases in each cell of the two–by–two table, four had both high individual reporting accuracy and low population–level bias , 7 had moderate accuracy (AUC>0.60), and 26 had low accuracy (AUC<0.60). Indicators with high AUC results reflected events leading up to  and during the birth itself . Indicators with low value AUC results tended to be related to events immediately following the birth  and postnatal health checks for the mother and newborn. For population–level bias, a total of 13 indicators had low bias (0.75<IF<1.25), 7 had moderate bias (0.5<IF<1.5), and 21 had large bias (IF<0.5 or IF>1.5). Indicators with large bias varied with respect to phase of labor and delivery, but those with the largest bias tended to have a low observed prevalence and be those that may require medical knowledge to report accurately .Who was the main provider assisting you during delivery?” There were sufficient cell counts to assess two provider categories with robust analysis: nurse/midwife and student nurse. The nurse/midwife indicator met both the high AUC and low IF criteria while the student nurse indicator had low individual accuracy (AUC = 0.57) and large bias (IF = 0.45). An indicator constructed in analysis that combines responses of “doctor”, “medical resident” and “nurse/midwife” as “skilled attendants” had low individual accuracy (AUC = 0.55), primarily due to low specificity, and low population–level bias (IF = 1.02) [To assess women’s ability to recall the type of provider who attended them, respondents were asked, “ = 1.02) . Cross–tTo illustrate the implications of indicator properties established in this study for other contexts, we plot the values of the predicted survey prevalence (Pr) of select indicators across all possible levels of intervention coverage (from 0 to 100%). This study provides validity results for 41 indicators of the quality of maternal and immediate newborn health care that are either currently in use or have the potential to be included in household surveys. Across phases of labor and delivery, we found indicators related to concrete, observable aspects of care or which reflected pain or concern were reported with higher accuracy. These results are consistent with previous studies which have found particularly memorable events, such as having a cesarean operation ,22,29 anThat a small number of the initial list of 82 indicators met both validation criteria is partly due to the fact that many preventative care interventions almost always occurred, and there was insufficient variation  for robust analysis. For many preventative care indicators we found that most women accurately reported receiving the care . For example, an indicator that is a proxy for receiving a uterotonic for the prevention of postpartum hemorrhage , was accurately reported by nearly all women. Although the near universal implementation of this practice limited robust analysis, these results suggest some aspects of routine care can be accurately reported. However, given that there were few instances in which standard preventative interventions were not received, unless there was almost perfect negative classification by women, these indicators also tended to have low specificity. An alternate interpretation is that the observed pattern of high sensitivity and low specificity for many preventative practices may reflect “facility reporting bias” among women based on the expectation of receiving appropriate care. This finding has also been described in a study of women’s reporting of maternal and child health care in China .The potential for facility reporting bias may also be relevant for indicators on skilled birth attendance. Indicators measuring the assistance of a skilled provider had high sensitivity and low specificity for both labor and delivery. Women tended to underreport the presence of less skilled providers, such as student nurses, and over–report the presence of a doctor or obstetrician/gynecologist. The positive bias may also be due to differences in how women conceptualized who their “main” provider was. It is possible that women understood their “main” provider to be the attendant with the highest rank and who may have been deemed ‘in–charge’ of her care, while observers identified the primary provider as the attendant who administered the majority of the care to the woman.Study findings also suggest the validity of some indicators may be dependent on context and question wording. Indicators that performed worse on the validity tests tend to be related to the timing or sequence of events, such as whether the newborn was placed skin–to–skin on the mother’s chest immediately after delivery. A two–item question sequence that clarified the precise meaning of “skin–to–skin” greatly reduced women’s overestimation of the practice compared to a one–item indicator (An influential aspect of the birth context was the type of delivery. Women who had a cesarean operation were less likely to be able to report on immediate newborn care than women with normal deliveries. This is reflected in high levels of “Don’t Know” responses. This finding suggests that it may be worth excluding women with cesarean sections from questions about care immediately after birth in routine household surveys.A number of indicators performed well on the IF test only; individual–level misclassification does not inherently signify that measurement at the aggregate level will be inaccurate . In studKnowledge of whether an indicator is likely to be overestimated can also have significant programmatic implications. For example, where skilled birth attendance is over–reported, progress in scaling up the presence of higher cadre providers may not be as great as expected. It is important to recognize that the presence of a skilled provider is one aspect of receiving quality care, one which relies on the assumption that providers have received the necessary training to administer essential interventions and have acquired the competencies to address complications during childbirth. Additionally, even “skilled” providers may not be able to deliver adequate care if they do not have access to necessary equipment and supplies. Information on skilled attendance as reported by women should be corroborated with indicators on the content of care. When possible, we recommend that users also triangulate self–reported data on quality of care with other data sources such as information on stock–outs of essential medicines .While a strength of this study is the use of direct observation as the reference standard, there are some limitations. Validation results are based on women seeking delivery in a large public hospital, and may not be generalizable to women who deliver in other types of facilities or at home. The lack of variation in hospital practices also limited the ability to analyze all of the indicators, which may have otherwise proven to be valid if we had collected data in a range of health institutions. Finally, our results inform a ‘best case’ scenario in terms of recall accuracy because women were interviewed shortly following delivery. To inform how recall changes over time, as well as to investigate women’s understanding of concepts such as who their main provider was, a follow–up study is under way to re–interview women one year after delivery.The measurement of the quality of maternal and newborn health care received in LMIC settings often relies on data from surveys of women. Little research has examined the validity of these indicators. The primary indicator of interest in this study–delivery by a skilled birth attendant–met validation criteria for reporting at the population level only and the results indicate that reporting accuracy may be particularly problematic where skilled birth attendance coverage is low. Indicator properties established here provide insight into contexts where indicator use is appropriate, and where modifications to data collection procedures or question construction may be warranted.


SLR) adaptation planning. However, we know little about to what extent the improved models and data could actually lead to better conservation outcomes under SLR. This is important to know because high‐resolution data are likely to not be available in some data‐poor coastal areas in the world and running more complicated coastal impact models is relatively time‐consuming, expensive, and requires assistance by qualified experts and technicians. We address this research question in the context of identifying conservation priorities in response to SLR. Specifically, we investigated the conservation value of using more accurate light detection and ranging (Lidar)‐based digital elevation data and process‐based coastal land‐cover change models  to identify conservation priorities versus simple “bathtub” models based on the relatively coarse National Elevation Dataset (NED) in a coastal region of northeast Florida. We compared conservation outcomes identified by reserve design software (Zonation) using three different model dataset combinations . The comparisons show that the conservation priorities are significantly different with different combinations of coastal impact models and elevation dataset inputs. The research suggests that it is valuable to invest in more accurate coastal impact models and elevation datasets in SLR adaptive conservation planning because this model–dataset combination could improve conservation outcomes under SLR. Less accurate coastal impact models, including ones created using coarser Digital Elevation Model (DEM) data can still be useful when better data and models are not available or feasible, but results need to be appropriately assessed and communicated. A future research priority is to investigate how conservation priorities may vary among different SLR scenarios when different combinations of model‐data inputs are used.Many studies have explored the value of using more sophisticated coastal impact models and higher resolution elevation data in sea‐level rise ( It is important to accurately identify and delineate conservation priorities as a means of guiding future land‐use planning and decision‐making, and maintaining critical ecosystem services and green infrastructure. However, sea‐level rise (SLR) and other effects of global climate change produce a decision‐making environment marked by high uncertainty Noss . UncertaHigh‐resolution elevation data  and sophisticated coastal impact models (SLAMM) are preferably used in SLR adaptation planning because the modeling results will provide foundations for subsequent analysis relevant to SLR , the Fort Matanzas National Monument, and other Florida conservation management areas including the Matanzas State Forest and the Faver‐Dykes State Park. To incorporate more regional land‐use and ecological considerations , limpkin (Aramus guarauna), painted bunting (Passerina ciris), American oystercatcher , and Marian's marsh wren  were selected as representative species in this study. Habitat for each of these species is considered likely to be significantly affected by future SLR. With species habitat models obtained from the Center for Landscape Conservation Planning at the University of Florida  including soils, streams, ditches, eagle nests. To identify future potential species habitat with different model–dataset combinations, three different land‐use datasets were created incorporating future land‐cover changes identified by each model–dataset combinations. The SLAMM codes were crosswalked to Florida Land Use, Cover and Forms Classification System (FLUCCS) codes , the Cooperative Land Cover dataset  codes , and theZonation software  in SLR adaptive conservation planning and reserve design. Because of timing and funding, this research only compared conservation outcomes with three different model–dataset combinations including Bathtub–NED, Bathtub–Lidar, and SLAMM–Lidar, but the SLAMM–NED combination was not included in the analysis. Including the SLAMM–NED combination in the analysis would add to the existing research and present the conservation outcomes with a complex coastal impact model and coarse elevation data. Compared to conservation outcomes with the SLAMM–Lidar combination, this added analysis would likely demonstrate the value of more detailed elevation data in identifying SLR adaptive conservation priorities while using the complex coastal impact model. Including SLAMM–NED combination in the analysis would be a future research priority for assessing conservation outcomes with different model–dataset combinations. The conservation priorities presented here are purely ecological priority areas that economic concepts were not included in planning process. Given that economic costs could have a large impact on conservation outcomes (Naidoo et al. It is likely not necessary to include different SLR scenarios in the analysis as the aim of this study is to examine conservation outcomes of different model–dataset combinations although this analysis might be useful in the future because differences in predictions and identified priority areas might be significantly different for different SLR levels. Higher rates of SLR will lead to a greater change in vegetation distribution so the bathtub model might have a greater chance of incorrectly predicting this distribution (Runting et al. None declared.


LGM , accessibility to LGM refugia, and contemporary environmental conditions . Functional richness and dispersion exhibited geographic patterns with strong associations to the environmental history of the region. The effect size of accessibility to LGM refugia and climate stability since the LGM was comparable to that of the contemporary predictors. Both functional richness and dispersion increased with temperature stability since the LGM and accessibility to LGM refugia. Functional richness' geographic pattern was primarily associated with accessibility to LGM refugia growing degree‐days, land use heterogeneity, diversity of soil types, and absolute minimum winter temperature. Functional dispersion's geographic pattern was primarily associated with accessibility to LGM refugia growing degree‐days and absolute minimum winter temperature. The high explained variance and model support of historical predictors are consistent with the idea that long‐term variability in environmental conditions supplements contemporary factors in shaping FD patterns at continental scales. Given the importance of FD for ecosystem functioning, future climate change may elicit not just short‐term shifts in ecosystem functioning, but also long‐term functional disequilibria.Numerous studies indicate that environmental changes during the late Quaternary have elicited long‐term disequilibria between species diversity and environment. Despite its importance for ecosystem functioning, the importance of historical environmental conditions as determinants of FD  remains largely unstudied. We quantified the geographic distributions of plant FD (richness and dispersion) across Europe using distribution and functional trait information for 2702 plant species. We then compared the importance of historical and contemporary factors to determine the relevance of past conditions as predictors of current plant FD in Europe. For this, we compared the strength of the relationships between FD with temperature and precipitation stability since the  Contemporary climatic conditions and environmental heterogeneity have been shown to be important drivers of European floristic diversity  that are close to areas considered as refugia at the end of the LGM . These conditions would be as important as those contemporary conditions considered to promote high species diversity  Do current geographic patterns in plant FD across Europe exhibit imprints of environmental changes during the late Quaternary?; and (2) To what extend the influence and importance of historical imprints as predictors of FD patterns are comparable to that of contemporary factors?. We hypothesize that high functional richness and low dispersion would be observed in assemblies where historical conditions show high climatic stability , we characterized the distribution of European angiosperms. We focused on European angiosperms as its current ecology, and historical distribution is relatively well understood and to minimize the effect of different evolutionary trajectories in the region . The AFE maps the distribution of European flora on an equal‐area mapping unit of ~50 × 50 km  listed in AFE as occurring in continental Europe. Forms, varieties, and subspecies were collapsed to species. We are aware of the taxonomic limitations of the AFE (it only covers ~25% of the plant species in Europe) and that it is biased against woody plants. To ensure that our estimates were robust to the used species pool, we evaluated the consistency in the geographic trends in our estimates of FD for seven orders of European angiosperms with a pan‐European distribution (Appendix S1). We observed highly correlated FD patterns across orders, suggesting that analyses based on AFE data can provide a general description of the FD trends in the region.2*g−1), seed mass (mg), maximum stem height (m), wood density for woody species or stem‐specific density for nonwoody species , and growth form . These traits were selected due to their importance for characterizing plant functional strategies  and variability  of the multidimensional functional space of each assemblage. FRich measures the trait spectrum range of the species assemblage, indicating the functional space occupied by a group of co‐occurring species regardless of their abundance  before the estimation FD. By doing this, we ensure that the measuring units or the shape of the distribution has an influence on the FD evaluation. As a complementary analysis and to determine which traits were the main predictors of changes in FRich and FDisp, the range  and dispersion (standard deviation) for each trait were estimated separately as univariate versions of these metrics.Two complementary multivariate FD metrics were used to describe the range . Selected predictors describe some of the most supported ecological mechanisms proposed as drivers of diversity patterns at large spatial scales  and now. As in Sandel et al. , climateρ = 0.92; pDutilleul‐corrected = 0.001) between our climatically derived accessibility and the mean accessibility of 1016 European herbaceous and woody European plants estimated based on statistical models of LGM distribution . A factor known to affect climate velocity estimates is the geographic resolution of the input data  describing all possible noninteracting combinations among linear or unimodal (linear + quadratic) responses of all 12 historical and contemporary predictors evaluated in this study, for each imputed dataset.The relation between environmental and FD estimates was determined using SARR2 values of single‐predictor models with a unimodal response (linear + quadratic terms). Secondly, we estimated variable relative support as WAIC .For all 12 evaluated predictors, we determined its' relative support and importance using two approaches. First, we measured the explained variance of each contemporary and historical factor, using Nagelkerke  pseudo‐RFRich and FDisp while accounting for the effect of contemporary factors. For this, we averaged the standardized regression coefficients across all of the evaluated models, weighting each value by the wAIC for the model that contained it. The use of model averaging based on wAIC has received serious criticisms  and unstandardized FD show similar patterns, our results and discussion are based on raw values only.As a supplementary analysis, we evaluated the relation between historical and contemporary predictors with SES (standardized effect sizes) for n et al. , simulatFRich values were concentrated within the Mediterranean region, most notably in the southern mountainous regions . However, the explained variance of single‐predictor models showed that seed mass and specific leaf area (SLA) range were the main drivers of FRich; and trait dispersion in seed mass, specific leaf area and stem/wood density range determined variability in FDisp.Regions exhibiting high FD values were located primarily in areas south of the 46° latitude , accessibility and precipitation velocity had two of the highest relative importance scores of all 12 evaluated predictors for FRich , NDVI (WAIC 100%), the number of land uses (WAIC 100%), and soil diversity (WAIC 100%). Meanwhile, the contemporary predictors showing the highest multimodel support included for FDisp were growing degree‐days (WAIC 100%), soil diversity (WAIC 99%), annual precipitation (wAIC 99%), topographic variability (WAIC 99%), and NDVI (WAIC 99%).Contemporary predictors with the highest relative importance scores changed between isp Fig. A vs. B. FRich and FDisp decreased as climatic velocities increased, and accessibility to LGM refugia declined , and the fact that historical and contemporary predictors determine FD support or hypothesis that the legacies of Late‐Quaternary climate changes can still be seen today.The influence of contemporary predictors on FD illustrates the well‐known effects of recent environmental conditions as key predictors of species distribution and richness patterns among plants , we show that there is a generalized geographic pattern in the functional space size and dispersion of European plants. Such a consistency across groups with an independent evolutionary history gives us confidence that the reported FD geographic patterns are robust to any AFE taxonomic biases, as also shown when comparing AFE and plot‐based species richness patterns in Europe . Consequently, our results indicate that the importance of historical environmental factors would most likely percolate to the ecosystem functions performed by European plant assemblages. If this is the case, future climate change will not only elicit short‐term shifts in ecosystem functioning, but also induce long‐term impacts on ecosystem functioning. Incorporating the underlying mechanisms behind the importance of historical environmental conditions into predictive models for future vegetation‐related ecosystem functioning will enhance the accuracy of climate change impact and dynamics estimations.None declared.Appendix S1. Robustness of functional diversity estimates to the taxonomic coverage.Appendix S2. Traits sources and description of the trait imputation procedure.Appendix S3. Geographic patterns and association between contemporary and historical environmental variables.Appendix S4. Individual macro‐scale determinants of species richness standardized functional richness and dispersion.Appendix S5. Association between univariate (trait range and dispersion) and multivariate metrics of functional diversity.Click here for additional data file.


Biological systems are composed of numerous components of which proteins are of particularly high functional significance. Network models are useful abstractions for studying these components in context. Network representations display molecules as nodes and their interactions as edges. Because they are difficult to directly measure, functional edges are frequently inferred from suitably structured datasets consisting of the accurate and consistent quantification of network nodes under a multitude of perturbed conditions. For the precise quantification of a finite list of proteins across a wide range of samples, targeted proteomics exemplified by selected/multiple reaction monitoring  mass spectrometry has proven useful and has been applied to a variety of questions in systems biology and clinical studies. Here, we survey the literature of studies using SRM‐MS in systems biology and clinical proteomics. Systems biology studies frequently examine fundamental questions in network biology, whereas clinical studies frequently focus on biomarker discovery and validation in a variety of diseases including cardiovascular disease and cancer. Targeted proteomics promises to advance our understanding of biological networks and the phenotypic significance of specific network states and to advance biomarkers into clinical use. AIMSaccurate inclusion mass scanningCPTACclinical proteomics tumor analysis consortiumESIelectrospray ionizationHPLChigh pressure liquid chromatographyMRMmultiple reaction monitoringMSmass spectrometryMS/MStandem mass spectrometryPTMpost‐translational modificationRPPAreverse‐phase protein arrayRTretention timeSIDstable isotope dilutionSISCAPAstable isotope standards and capture by anti‐peptide antibodiesSRMselected reaction monitoringQQQtriple quadrupole11.1Many observations related to signaling cascades and other biological processes cannot be explained with a simple linear network model. This has led to the realization that the components of biological systems are not simply connected in a linear fashion but through a web of interactions, feedback loops, and crosstalk at multiple spatial and temporal scales and prompted a shift towards considering these processes as dynamic network. Models of such networks aim at increasing our understanding how changes in network state in specific contexts throughout normal development, disease, and in response to perturbations 1.2Proteins are key components of many types of molecular networks, perform most biochemical functions of the cell and are the targets of most current drugs Protein networks pose a number of data analysis challenges that are rooted in the fact that proteins cannot be represented as simple molecular entities. Rather, they operate in a large number of biological contexts, spatial, and temporal scales and functional states exemplified by protein‐specific properties such as reaction mechanisms, substrate/ motif binding and complex formation. In addition, properties of protein networks such as information processing, noise, adaptability, robustness, and even seemingly paradoxical arrangement of components and functions, such as enzyme promiscuity 1.3Saccharomyces cerevisiae strains There are two main ways to detect and quantify proteins: affinity reagent based methods, exemplified by ELISA, Western blotting or immuno histochemistry staining, and MS based peptide identification and quantification, which is mainly used for research and discovery proteomics. However, the dynamic range and number of proteins quantifiable using affinity‐reagent based assays is limited 22.1A targeted MS‐based proteomics experiment consists of multiple steps and is schematically illustrated in Fig. 2.2The target list can be composed of several hundred peptides to be measured in a multiplexed fashion, allowing for a wide and versatile set of hypotheses to be tested. Typically the target list will be chosen from biomarker candidates found in a discovery profiling experiment, previous interest in a pathway, or from computational analysis identifying pathways and networks In general, to test a hypothesis in a basic science model system, a larger set of proteins is typically measured, while quantifying biomarkers in a clinical setting for making treatment decisions usually involves a smaller set of proteins. The selection of a peptide quantification strategy should follow a fit‐for‐purpose approach to achieve the right level of specificity, precision, and quantitative accuracy as described recently in a three‐tiered system in Carr and colleagues All other steps of the SRM‐MS method development have been described in detail elsewhere 33.1Saccharomyces cerevisiaeOne of the earliest studies using targeted proteomics absolutely quantified G‐coupled receptor rhodopsin using chemically synthesized peptides as calibration standard to quantify endogenous levels of membrane bound rhodopsin Following the successful quantification of single proteins or small protein lists, assays for targeted proteomics were established for entire model organisms Systems investigations of the response to perturbations by targeted proteomics are providing numerous insights in many higher eukaryotes. Zulak and coworkers investigated the effects of methyl jasmonate on terpene synthase enzyme induction and activity in protein extracts of Norway spruce, demonstrating a coordinated network of chemical defense response and a prime example of the robustness of biological systems Targeted proteomics is increasingly being used to quantify large numbers of proteins and addressing fundamental questions in biological networks. Sabido and coworkers measured 144 proteins in C57BL/6J and 129Sv mice subjected to various periods of high fat diet, revealing activation of either the peroxisomal beta‐oxidation pathway or the lipogenesis pathway in each strain, respectively 1F0‐ATP synthase super‐assembly in H9c2 cardiomyoblasts undergoing cardiac‐like differentiation Targeted MS is increasingly being used to determine the stoichiometries of protein complexes a topic that is of similar importance to the understanding of biological systems through quantitative modeling. For example, monitoring the functional assembly of the human spliceosomal hPrp19/CDC5L complex under various conditions Saccharomyces cerevisiae 157 proteins and 152 phosphorylated peptides were identified to reflect the status of the cellular signaling activity in a single analysis step, thus providing a broad overview of the state of numerous functional networks of the cell Absolute quantification of proteins using targeted proteomics has considerably matured from single membrane protein quantification to functional stoichiometry determinations of protein complexes and quantification of protein networks. A pioneering approach was recently presented by Soste and coworkers: through literature search and computational prediction methods sentinel proteins were identified which report on the state of signaling pathways in a single SRM‐MS analysis. For 3.2Targeted MS has proven invaluable for the study of PTMs. Glinksi and coworkers examined multisite phosphorylation of trehalose‐6‐phosphate synthase isozymes in vitro in Arabidopsis 44.1Clinical applications aim to translate new discoveries and technologies into improving patient outcomes 4.2The full pipeline of translating biomarker discovery studies into clinical tests with demonstrated health benefits is a multi‐year process that is costly, uncertain, and arduous and, because researchers and projects can easily get stuck in the middle of biomarker development projects, has been likened to a tar pit 4.3The complexity in tissues and body fluids presents an enormous signal‐to‐noise ratio problem 4.4Over the last decade advances in targeted proteomics have lead to assay sensitivity for plasma proteins in the low ng/ml range. In 2004, Kuhn and coworkers used a depletion of abundant proteins strategy and size exclusion fractionation to quantitate C‐reactive protein, a diagnostic marker for rheumatoid arthritis 18O labeling strategy for SID‐MRM‐MS with the ability to produce synthetic peptides for use as internal quantitation standards in only 1 h with excellent stability Technology development has also advanced in labeling incorporation technology and throughput. Zhao and coworkers developed a synthetic peptide strategy using Technology development has gone into using PTMs as handle to enrich for partially low abundant proteins. One such enrichment strategy is the enrichment of N‐glycosylated peptides and the subsequent PNGase F‐catalyzed conversion of Asn to Asp using solid state extraction method Mycobacterium tuberculosis peptides in serum of patients with active or latent M. tuberculosis infection Another strategy is the enrichment microparticles 4.5While immuno‐reagents are commonly used to deplete the most abundant proteins in plasma they can also be used for enrichment of low abundant proteins. A commonly used technique is known as stable isotope standards and capture by anti‐peptide antibodies (SISCAPA) 4 (L‐PHA) for N‐linked glycan capture and a monoclonal anti‐peptide TIMP1 antibody conjugated to magnetic beads to quantitate the cancer candidate biomarker TIMP1, which is present at approximately 0.8 ng/ml in serum using only 1.7 μl of serum from a patient with colorectal cancer Immunoaffinity targeted proteomics can also be used in combination with additional enrichment techniques to reach low limits of quantitation. Ahn and coworkers used a variation of SISCAPA with a combination of phytohemagglutinin‐LImmunoaffinity techniques can also be useful for quantitating proteins in the presence of autoantibodies. Thyroglobulin (Tg) is used to monitor patients after treatment for differentiated thyroid carcinoma and is often accompanied by the presence of auto‐antibodies that interfere with immunoassays. Kushnir and coworkers overcame these challenges by enriching serum samples with a rabbit polyclonal antibody for Tg and immunoaffinity purification of a signature Tg peptide, with a lower limit of quantitation of 0.5 ng/ml Immunoaffinity techniques are also amenable to multiplexing. Using an approach known as mass spectrometric immunoassay (MSIA), Krastins and coworkers rapidly developed assays for 16 different target proteins and their isoforms across seven different clinically important areas and ranging in concentration from pg/ml to ng/ml in bona fide clinical samples 4.6Perhaps the main advantage of targeted MS‐based proteomics over ELISA assays is their multiplexing capability, which is of key importance for biomarker development in disease applications with many putative biomarkers such as cardiovascular disease and cancer. Kuzyk and coworkers developed a mixture of 45 peptide standards in EDTA‐plasma without affinity depletion or enrichment and found that 31 of the 45 are putative markers of cardiovascular disease 4.7The heterogeneity and complexity present in the numerous types of cancer presents an enormous opportunity but significant challenges for targeted proteomics to make transformative contributions. Much technology development has gone into tissue preparation and quantitation of candidate biomarkers addressing the dual challenges of using a limited amount of material in which the candidate biomarker may also be low abundant. DeSouza and coworkers quantified a marker of endometrial cancer, pyruvate kinase M1/M2, in biopsied tissue at 85 nmol/g compared to 21–26 nmol/g in nonmalignant tissue using the mTRAQ labeling method, compared to only a 2x elevation initially determined by discovery iTRAQ proteomics, suggesting that the dynamic range for quantitation may be compressed in discovery scans Clinical proteomics in cancer has also addressed challenges of integrating genomic data such as mutations. Wang and coworkers demonstrated that altered protein products resulting from somatic mutations can be quantified by targeted proteomics by developing assays for Ras proteins and applying them in colorectal and pancreatic tumor tissue and premalignant pancreatic cyst fluids In 2011 a complete verification pipeline of biomarker candidates in plasma was presented in a tour‐de‐force study by Whiteaker and coworkers Hüttenhain and coworkers also completed a tour‐de‐force complete verification pipeline of biomarker candidates by generating assays for 1000 cancer‐related proteins and used a data‐dependent triage strategy by first examining candidate proteins’ detectability in plasma and urine samples Biomarker discovery and verification has also been pursued for post‐translationally modified proteins. Cima and coworkers followed a two‐stage strategy for biomarker discovery, starting with a discovery scan comparing the serum N‐linked glycoproteome of PTEN conditional knockout model of prostate cancer to wild‐type and then developed targeted assays for 39 human orthologs, which were then quantified in the sera of 143 prostate cancer patients and controls over an abundance range of six orders of magnitude Targeted proteomics has been applied successfully to formalin‐fixed, paraffin‐embedded (SFPE) tissue. Sprung and coworkers quantified 114 peptides in FFPE clear cell renal cell carcinomas and Her2 overexpression in FFPE breast cancer samples and determined by comparison with cell lines that lysine‐containing peptides can be used for quantitation and the feasibility of performing targeted proteomics studies on FFPE tissues 5Targeted proteomics is now a well‐established tool for quantitative proteomics and is most useful when researchers can identify a medium‐to‐large target list  takes advantage of a mass spectrometer which first selects precursors using a triple quadrupole and subsequently fragment ions are mass analyzed in an orbitrap The authors have declared no conflict of interest.


Globally, preterm birth affects more than one in every 10 live births. Although the short‐term cardiopulmonary complications of prematurity are well known, long‐term health effects are only now becoming apparent. Indeed, preterm birth has been associated with elevated cardiovascular morbidity and mortality in adulthood. Experimental animal models and observational human studies point toward changes in heart morphology and function from birth to adulthood in people born preterm that may contribute to known long‐term risks. Moreover, recent data support the notion of a heterogeneous cardiac phenotype of prematurity, which is likely driven by various maternal, early, and late life factors. This review aims to describe the early fetal‐to‐neonatal transition in preterm birth, the different structural and functional changes of the preterm human heart across developmental stages, as well as potential factors contributing to the cardiac phenotype of prematurity. Over 15 million neonates are born prematurely each year, which corresponds to more than 10% of live births worldwide  is a fetal condition that occurs in more than 20% of preterm births Gardosi, . It is tAn additional complication and the most common morbidity of prematurity is bronchopulmonary dysplasia (BPD), although incidences vary between regions and institutions ; writing – original draft (lead); writing – review and editing . Adam Lewandowski: Conceptualization ; supervision (lead); writing – original draft (supporting); writing – review and editing .