Biotechnology and Bioengineering published by Wiley Periodicals, Inc.Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors.  Spontaneous integration of non‐viral DNA vectors into the genome of eukaryotic cells is a widely exploited process in research and biotechnology. Its molecular basis, however, remains incompletely understood. It is believed to rely on cellular DNA repair mechanisms, as it is favored by the presence of free DNA ends in the vector resembling double stranded breaks (DSBs). The two major pathways responsible for DSB repair in eukaryotic cells are non‐homologous end‐joining (NHEJ) and homologous recombination (HR) Jackson, . NHEJ isPlasmid integration into the genome of eukaryotic cells is an overall inefficient process, occurring in a minor proportion of cells that take up the exogenous DNA. It was shown to involve two major steps: (i) recombination between vector molecules to form multimeric transgene arrays termed concatemers and (ii) the recombination of the resulting concatemers with the genome, usually at a single or at few chromosomal loci , which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription  and 1× HT (Gibco).Adherent Chinese hamster ovary (CHO) DG44 cells  . The pGEGFP plasmid was transfected in parallel as a positive control of GFP expression.For siRNA‐mediated knock‐downs of DNA repair proteins, adherent CHO DG44 cells were transfected with equimolar mixes of three mRNA‐specific or control siRNA duplexes at a final concentration of 50 nM using Lipofectamine RNAiMAX (Invitrogen), according to manufacturer's instructions . After 2 days, the siRNA‐treated cells were re‐transfected with pGEGFP or p1‐68‐GFP, and with a puromycin resistance plasmid pSVpuro (Clontech), using Lipofectamine 2000 (Invitrogen). Prior to transfection all plasmids were linearized with PvuI and purified by ethanol precipitation. Puromycin (5 μg/mL) was added to the culture medium 24 h after transfection, and stably transfected cells were selected for 2 weeks. Stable GFP expression was analyzed by flow cytometry , whereas aliquots of each sample were used for genomic DNA extraction.To assess the frequency of genomic integration events, CHO DG44 cells were transfected with siRNA duplexes against selected DNA repair proteins, using the protocol described above . Cells were re‐transfected 72 h later with pGEGFP or p1‐68‐GFP, and pSVpuro, using Lipofectamine 2000 (Invitrogen), once the effects of the knock‐down on cell cycle progression had disappeared . For quantitative PCR (qPCR), 6 ng of genomic DNA were analyzed using the SYBR Green I Master kit for the Light Cycler 480 machine (Roche) using AGCAAAGACCCCAACGAGAA and GGCGGCGGTCACGAA as GFP‐specific primers. The beta‐2‐microglobulin (B2M) CHO gene was amplified as a normalization control using ACCACTCTGAAGGAGCCCA and GGAAGCTCTATCTGTGTCAA as primers. The number of integrated transgene (GFP) copies was calculated using the B2M gene as a reference, as previously described Pfaffl, .To assess which CHO genes were expressed in our culture conditions, the transcriptome of the suspension‐adapted parental CHO K1 cells was determined by paired‐end sequencing using the Illumina technology by the Next Generation Sequencing Facility of the University of Lausanne. Expressed coding sequences were annotated using the Annotation Release 101 of the Chinese hamster genome assembly  . After 3 weeks of puromycin selection, total genomic DNA was isolated from polyclonal cells using the Genomic‐tip G/20 kit (Qiagen). The DNA was sequenced using the Single Molecule Real‐Time (SMRT) technology (Pacific Biosciences) at the Next Generation Sequencing Facility of the University of Lausanne. CHO cells transfected with p1‐68‐GFP were sequenced using 20 SMRT cells, and those transfected with pGEGFP required the use of 60 SMRT cells to obtain a similar number of integration site sequences. Transgene integration sites were identified by a custom identification pipeline. PacBio filtered subreads were obtained using the tool DEXTRACTOR  using the standard settings. Plasmid sequences were identified in PacBio filtered subreads with the help of the alignment tool BLASR  . Two days post transfection the cells were electroporated with PvuI‐linearized human immunoglobulin (IgG1) expression vectors containing the MAR 1–68 and a puromycin resistance plasmid (pSVpuro) , using the Neon® transfection system (Invitrogen). After 3 weeks of antibiotic selection the IgG titer in cell culture supernatants was measured by sandwich ELISA and the specific productivity was calculated as described previously  . Efficient reduction of the target mRNA and/or protein levels by siRNA transfection was validated experimentally, to insure decreased levels by at least twofold .To evaluate if the knock‐down of these genes affects DNA recombination, we used previously described HR and NHEJ fluorescent reporter assays based on the repair of transiently transfected plasmids with a I‐SceI‐induced DSB in the GFP coding sequence . The average number of integrated GFP copies was measured in antibiotic‐resistant polyclonal populations, so as to assess the efficiency of plasmid concatemerization prior to genomic integration. Since expression from individual plasmids can be influenced by the surrounding chromatin environment, the level of GFP fluorescence and its normalization to the transgene copy number was used to estimate plasmid integration within transcription permissive or non‐permissive areas of the genome. Finally, we measured the efficiency of plasmid genomic integration by quantifying puromycin‐resistant colonies arising from cells that had successfully integrated transgenes into their genome, focusing on siRNAs that affected GFP expression or plasmid concatemerization, as well as representative targets from each DSB repair pathway.The average GFP expression and plasmid copy number were not affected by the down regulation of NHEJ activities such as DNA‐PKcs, Ligase IV or Xrcc4, nor was the expression per transgene copy or the number of antibiotic resistant colonies Fig. A–D. ThisStable GFP expression and/or transgene copy numbers were increased by the knock‐down of HR proteins, notably MDC1, Rad51, Rad52, Rad54, and Brca1  , which are DNA elements that form chromatin domain boundaries  expression vectors containing the human MAR X‐29 and selected for high stable expression of the therapeutic protein. To do so, we used a whole genome sequencing approach on these clones and devised a software to identify paired sequence reads pertaining to the plasmid and the CHO genome. Six integration sites in one clone (BS01) and two in the other clones (BS03 and Cp33/64) were predicted in silico and validated experimentally by PCR amplification and DNA sequencing. The occurrence of the predicted number of plasmid integration loci was further validated by FISH for two of the analyzed clones .From the five integration sites where the junction sequences were validated experimentally on both sides of the transgenes, two had large deletions (913 bp in BS01 and 320 bp in Cp33/64), as expected from MMEJ‐related mechanisms , suggesting that most integration events had occurred in transcriptionally active genomic loci. These results further suggested that the MAR‐containing plasmids preferably integrate within‐ or in close proximity‐ to expressed CHO genes. To assess whether this indeed resulted from the presence of the MAR in the IgG expression vector or from the selection of highly expressing clones, we directly compared the integration loci of MAR‐containing and MAR‐devoid GFP expression vectors in polyclonal cell populations.P = 0.05), indicating that the MAR may stimulate genomic integration into chromatin regions permissive for transgene expression. In the cells transfected with the MAR‐devoid plasmid, integration in the vicinity of genes was not significantly enriched . Furthermore, these cells required threefold more sequencing reads to identify a comparable number of integration loci as obtained from the cells transfected with the MAR, further indicating that genomic integration events were less frequent in the absence of the MAR.Analysis of the integration sites identified from the whole genome sequencing of these cells revealed that, in presence of the MAR, plasmids indeed often integrated close to cellular genes (10/14 loci) . This result was significantly different from random . This suggested that, in the absence of the MAR, the cells had to integrate the transgenes into transcriptionally active chromatin in order to express the selection gene at a sufficient level to survive antibiotic selection. This may explain the strong decrease in cell survival upon the knock‐down of Rad51, as this protein was recently reported to be primarily responsible for DSB repair in transcriptionally active chromatin , and by the University of Lausanne.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Supplementary FiguresClick here for additional data file.Supplementary TablesClick here for additional data file.


Fixation resulted in significant changes in protein and nucleic acid bands and the spatial distribution of organelles. Raman imaging of Metaphase I and II  and germinal vesicle stage oocytes showed changes in nuclear organisation and cytoplasm macromolecular architecture during these development and maturation stages related to changes in chromosome condensation, mitochondria aggregation and lipid droplet numbers.Confocal Raman spectroscopy (CRS) can provide information about oocyte competency through measurement of changes in the macromolecular architecture during oocyte development and maturation. Hitherto most spectroscopic studies have been limited to fixed oocytes due to the inherent difficulties working with live cells. Here we report the first three-dimensional images of living murine oocytes using CRS. We show that fixation induces significant changes in the macromolecular chemistry compared to living oocytes. A band at 1602 cm The changes involve a highly ordered progression through the meiotic cell cycle from prophase of meiosis I to metaphase of meiosis II (MII). This progression is accompanied by major changes in nuclear architecture as chromatin condenses and the nuclear membrane breaks down. The formation of the first meiotic spindle then takes about 5 hours before it migrates to the cortex and proceeds to separate the chromosomes equally between the oocyte and the first polar body. The maternal chromosomes then reform a second meiotic spindle and the oocyte remains arrested until fertilization triggers the completion of MII and entry into embryogenesis. This progression through meiosis is accompanied by major changes in cellular architecture that extends beyond the cell cycle machinery. The endoplasmic reticulum and mitochondria aggregate around the developing meiotic spindle and then disperse through the oocyte after it reaches metaphase II. The cellular biochemistry of the oocyte also undergoes significant changes because kinase and phosphatase activities drive the cell cycle progression, which is accompanied by changes in oocyte metabolism, energy consumption, redox state and ionic regulation. This knowledge points to a highly dynamic process, the precise details of which remain largely unknown. A deeper understanding of the architectural and biochemical changes will provide an improved understanding of developmental mechanisms as well as being of practical of importance to improving the outcomes of assisted reproductive techniques where better understanding of these processes may allow more efficient identification and selection of developmentally-competent oocytes.The mammalian oocyte undergoes a sequence of nuclear and cytoplasmic changes during the process of oocyte maturation that are essential for fertilization and subsequent development1. A number of approaches including spectroscopic analysis of culture media and metabolomics have been proposed as reliable indicators of oocyte and embryo viability, and although finding crude indicators of embryo viability2, have not yet provided sufficiently reliable predictive tests of oocyte competence.A significant bottleneck in this area is that there are no current methods available for probing the phenotypic changes occurring during oocyte development and maturation. Clinically, this is largely limited to observations of morphological change. Analysis of transcriptome pools has been explored in recent years showing variability in the phenotypes of human oocytes drawn from the same patientin vivo charaterisation of oocyte molecular composition at sub-micron spatial resolution in three dimensions.Recently new molecular imaging approaches based on vibrational spectroscopy have emerged that provide subtle compositional information across a broad range of macromolecules obtained directly through spectroscopy without requiring any contrast agents, and enable simultaneous image reconstruction of multiple components. In this suite of spectroscopic imaging approaches, Raman confocal microspectroscopy is particularly powerful allowing the possibility of in vitro maturation4 and examining the effects of oxidative damage and oocyte age5. None of these works employed the 3D optical sectioning capability of the technique6 and therefore could not assess the cellular molecular architecture of oocytes in an unambiguous manner. More importantly, all previous studies examined fixed oocytes with the possible perturbing effect of fixation on cell chemistry and structure not determined except in the work by Davidson et al.2, who attempted to address this issue comparing two dimensional maps of fixed and living oocytes, but reported little or no changes caused by fixation. In contrast, here we show marked differences between fixed and live oocytes concluding the previous study was limited by measurements with restricted spectral range and sensitivity.There are only a few studies employing Raman spectroscopy to study oocytes including: using the technique to study changes across different maturation stages and after in vivo spectroscopic maker band. The potential of in vivo confocal Raman microspectroscopy to assess oocyte competency in the context of assisted reproductive therapies is discussed.Here we combine Raman three dimensional imaging using optical sectioning and multivariate analysis to investigate fixed and living oocytes in all developmental stages, and assigned an important Raman confocal images of 8 MI, 8 MII and 4 GV murine oocytes were acquired at 1 μm intervals in the x,y plane. K-means clustering was performed to a level of 7 classes. The mean extracted Raman spectra for each of the 7 classes are shown and coded with the same colours in the images  surrounding the light and grey clusters were dominated by protein bands , nucleotides (1200–1600 cm−1) as well as changes in protein features , as well as PO−1)  stretching mode from lipid moieties at 2854 cm−1. In fixed oocytes the lipid rich bodies were clustered around nucleic acid rich regions identified as condensed chromosomes. By contrast lipid droplets in live oocytes were randomly distributed throughout the cytoplasm 19. The nanoparticles observed in the zona pellucida of the fixed MII oocyte are presented in the Fig. −1, tentatively assigned to acetate particles20. Such nanoparticles were previously observed in different Raman studies of biological samples and are recognized as common contamination coming from paints, coatings, plastics and other commonly used products19.A white cluster with a distinct bands at 142, 396, 514 and 639 cmles Fig. ; Table 1−1, the S-S bridging mode from proteins at 514 cm−1 and changes to nucleic acids bands, indicating that these spectroscopic changes are a generalised effect of fixation.Figure −1; Table −1; Fig. Living oocytes from GV, MI and MII stages were compared using Raman microspectroscopy in Fig. 21. Figure −1) corresponding to the concentration of organic matter, and maps of the intensity of the lipid band at 2854 cm−1. These were done at 10 μm intervals in the z-direction, through the central axis of the cells and through positions up to 30 μm above and below the central plane 4 that observed small central lipid deposits superimposed on the nuclear region of GV oocytes using synchrotron FTIR microspectroscopy. The two dimensional nature of the imaging used rendered it impossible for the authors to conclude whether the lipid deposits were in the nucleus or simply in the cytoplasm overlying the nucleus. In this study using confocal Raman microscopy the nucleus of GV nuclei were readily identified in confocal two dimensional maps as regions of lower concentration of organic material, consistent with previous measurements showing low macromolecular concentration in GV nuclei in comparison with surrounding cytoplasm4. By mapping lipid distribution using the intensity of characteristic lipid bands it was clear that the nuclear region was completely devoid of lipid in successive two dimensional optical slices through the nucleus.Visualization of the three dimensional architecture of cells usually requires physical sectioning of the cell, imaging of the two dimensional images and reconstruction of the two dimensional data to form a three dimensional representation. Confocal microscopy, on the other hand, allows information to be gathered from a very narrow and defined depth range in the cell whilst excluding information from other regions, referred to as “optical sectioning”in vivo measurements.We have demonstrated that confocal Raman microspectroscopy can provide high resolution imaging of macromolecular architecture of living oocytes defined in three dimensions. The measurements can be regarded as non-invasive because no contrast agent was applied and the laser power was low and only short acquisition times were required for the measurement. We have demonstrated unequivocally that aldehyde fixation causes marked changes in both macromolecular composition and distribution, drawing into question the conclusions about oocyte macromolecular composition based on Raman spectroscopy of fixed oocytes. Indeed, this work demonstrates clearly that efforts to identify signatures of oocyte competence via oocyte macromolecular composition by Raman spectroscopy must necessarily be done using All research was performed in accordance with national and university guidelines and with approval of the Monash University Animal Ethics Committee.in vitro maturation, GV oocytes were collected in M2 (Sigma) containing 200 µM 3-isobutyly- 1-methylx- anthine (IBMX). Oocyte maturation was performed in drops of M16 medium (Sigma) under mineral oil (Sigma) at 37 °C in a humidified atmosphere of 5% CO2 in air. GV oocytes were allowed to mature for 8 h in IBMX-free M16 media in order to obtain MI samples. For collection of MII stage oocytes, mice were superovulated by sequential intraperitoneal injections of 10IU pregnant mare’s serum gonadotropin  and 10IU human chorionic gonadotropin  at timed intervals before oocyte collection. Oocytes of any stage were fixed using 4% paraformaldehyde made in phosphate-buffered saline (PBS) for 30 min at room temperature. All oocytes, live or fixed were washed into 0.9% saline for spectroscopy.For –1. The monochromator of the spectrometer was calibrated using the Raman scattering line produced by a silicon plate (520.7 cm−1). For Raman map collection water immersive Nikon (x60/1NA) objectives was used. The power for all measurements of the laser at the sample position was ≤10 mW. The average 2D image of the oocyte was measured at ~80 × 80 µm2 with a step of 1 µm and the integration time for each collected spectrum equal to 2 s. In total it took 3.5 h to generate the 2D images. The 2D images presented in Fig. Raman imaging of live and fixed murine oocytes was performed on a WITec confocal CRM alpha 300 Raman microscope. The spectrometer was equipped with an air–cooled solid state laser operating at 532 nm and a CCD detector, which was cooled to −65 °C. The laser was coupled to a microscope via a single mode optical fiber with a diameter of 50 um. The scattered radiation was focused onto a multi–mode fiber (50 um diameter) and a monochromator. The integration time for a single spectrum was 2 s with a spectral resolution of 3 cmTM and WITec softwareTM. Raman maps were generated based on the integration of marker bands and were obtained without pre–processing. Cluster analysis was carried out after cosmic ray spike removal, background subtraction . K–Means Clustering (KMC) results were obtained with the Manhattan distance algorithm and are complementary to the analysis based on the integration of the specific marker bands.Raman data analysis was performed with Opus


Blood pressure (BP) measurement is a central element in clinical practice. According to international recommendations 3 to 5 minutes of resting is needed before blood pressure measurement. Surprisingly, no study has modelled the time course of BP decrease and the minimum resting-time before BP measurement. A cross-sectional bicentric observational study was performed including outpatients addressed for vascular examination. Using two automatic BP monitors we recorded the blood pressure every minute during 11 consecutive minutes. The data was analyzed by non-linear mixed effect regression. Systolic (SBP) and diastolic BPs were studied and we tested the effect of covariates on its evolution through log-likelihood ratio tests. We included 199 patients (66+/−13years old). SBP was found to decrease exponentially. Simulations based on the final model show that only half the population reaches a stabilized SBP (defined as SBP + 5 mmHg) after 5 min of resting-time while it takes 25 min to ensure 90% of the population has a stabilized SBP. In conclusion, our results and simulations suggest that 5 minutes are not enough to achieve a stabilized SBP in most patients and at least 25 minutes are required. This questions whether the diagnosis of hypertension can be reliably made during routine visits in general practitioners’ offices. Various studies have shown that elevated blood pressure increases cardiovascular diseases such as stroke, myocardial infarction, and peripheral artery disease5. In 2010, high blood pressure was recognized as the major risk factor for global disease burden before tobacco smoking and alcohol use6. Yet, lifestyle modifications  and treatments can reduce and control blood pressure, and thus allowing reducing the morbidity and mortality in specific group population11.Hypertension is a highly prevalent disease worldwide affecting currently more than 1 billion people12. These guidelines suggest a resting time varying from 3 min to at least 5 min before blood pressure measurement12. Surprisingly, no study has modelled the time course of blood pressure decrease and the minimum resting time before blood pressure measurement in a population that should be screened for hypertension.The American Heart Association (AHA), the European Society of Cardiology (ESC), the European Society of Hypertension (ESH) and others have proposed recommendations for blood pressure measurement14. Confirming this hypothesis could have important implication regarding hypertension screening. Indeed, following current guidelines could potentially mean that a significant number of individuals are wrongly diagnosed as hypertensive. This potential overdiagnosis could lead to spending money and giving inappropriate medications thus causing adverse events such as falls.The hypothesis is that the resting time before the blood pressure measurement should be longer than 5 minutes to reach the stabilization of the blood pressure in patientsIn this observational study, we evaluated the time course of the blood pressure decrease during the resting time before the blood pressure measurement in patients addressed at our vascular offices and the parameters explaining the blood pressure decrease during the resting time.Between September 2014 and April 2015, 199 patients were recruited. Table Figure Figure An additional analysis was undertaken to reduce the variability in k by defining a covariate reflecting the shape of the decrease. Individual non-linear regression showed that a non-linear model fit the data best in 128 subjects (64%), while 44 (22%) had a linear decrease and 24 (14%) did not exhibit significant variation of BP. This covariate was included in the model, and was found to impact both the systolic Prest and the time to stabilisation, yielding 3 groups of subjects with different values of k: fast, regular and slow stabilisers (further details in the Appendix). Taking into account individual decrease shape allowed reducing the variability in k by 40%.Simulations using the final model showed that only 50% of the population was stabilised to within 5 mmHg of systolic Prest after 5 min resting time, while up to 25 min may be needed to ensure stable BP in 90% of the population (full results in the Appendix). This resting time falls to 15.0 min if a variability of 10 mmHg is accepted.Figure 5mmHg in 90% of the population is 25 min. Refining the analysis by considering individual variations in the evolution of SBP, we found that there were three subgroups of patients. The regular and fast groups had the same estimated systolic Prest suggesting the “fast” group is in fact stable from the start, while slow stabilisers take much longer to reach a stable SBP than the majority of subjects.Our study showed that among outpatients addressed for a vascular examination the minimal resting time before blood pressure measurement to obtain a stable SBP15. AHA scientific statement stated, “ideally, 5 minutes should elapse before the first reading is taken”12. The ESC have shortened this resting time and suggested a time between 3 to 5 minutes15 whereas “The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure” have increased this resting time writing that “at least five minutes” were required5. Therefore, according to our results, the current leading guidelines about the office measurement of blood pressure might contribute to increase the prevalence of hypertension in our societies, and consequently increasing the number of patients receiving antihypertensive drugs.The resting time found in this study is far longer than the resting time proposed by all previous scientific statementsThe other interesting point is that we found 3 different subgroups of patients with three different patterns of pressure evolution within the resting time. The “fast” pressure group hardly needed to rest for pressure to stabilize. To obtain stable pressure for 90% of the patients in the “regular decrease pressure” group, 9 min were needed whereas up to ~53 min were needed in the “slow decrease pressure” group, although this last figure 2. However, applying our results in clinical practice at the GPs office is not realistic due to time constraints. Indeed, the mean duration of GP consultation was evaluated as 10.7 + /−6.7 minutes in six European countries16 and sixteen minutes in France17. Thus in our opinion hypertension diagnosis should be performed in a dedicated place during a specific consultation. At the very least, a suspicion of hypertension in a patient should warrant a second measurement after a longer rest period than the current recommended 5 min. Another option could be to use an out-of-office blood pressure measurement that is currently devoted to specific clinical conditions such as suspicion of white-coat hypertension or masked hypertension for example18. Even in these cases and according to our results, it seems that patients should be counseled that they have to rest at least 25 minutes and always wait the same time before blood pressure measurements. Our findings corroborate recent result showing that BP measured after a 30 mn rest is significantly lower than BP measured according to the current recommendations19.The other point raised by the study concerns the organization of the public health system to determine where and how can we diagnose hypertension. It is widely admitted that office blood pressure measurement remains the gold standard for screening4.Finally, when we extrapolate our results in France where ~15 million patients are treated for a hypertension we can estimate that 696 000 patients could be over diagnosed for an hypertension. This could represent an annual health public cost of 292 million euros only for the treatment cost. Furthermore giving antihypertensive drugs to patients who do not need it might also provoke drug adverse events19, who showed a significantly lower BP measured after a 30 minutes rest compared to BP measured during a routine consultation. On the other hand, the general characteristics of our population are nearly similar to those included in large randomized controlled trials on hypertension10. Furthermore, our outpatients came for a suspected vascular problem and should have benefited from a blood pressure measurement in order to control this cardiovascular risk factor15. Second, in this study, blood pressure measurements were not compared to home or ambulatory blood pressure measurements because it was not the aim of this study, and home blood pressure or ambulatory measurements are not realized in the majority of patients screened for hypertension. Third, the association of blood pressure values at specific times from baseline with cardiovascular events or target organ damage was not evaluated. As our data showed different models of blood pressure decrease upon time, it would be interesting to perform an additional study to determine which value is the best associated with cardiovascular complications. Fourth, the study included all patients addressed for a vascular appointment, and was thus not designed to test for changes in BP evolution or resting BP in populations with specific features such as comorbidities or treatment. However we did find some differences in the analysis, with older patients in particular having both higher BP and larger differences between baseline and resting BP. The model and estimated parameters from the present study could be used to design future studies investigating specific target populations20. Finally, repeated blood pressure measurements that induce repeated short arterial occlusion might have a role in the blood pressure decrease since transient ischemia during cuff inflation and reactive hyperemia after cuff deflation can induce dilation of upper arm artery, which is likely mediated by baroreceptor reflex and endothelium-dependent vasodilation22. However, these physiological responses are seen after occlusions that last 5 minutes22. When measuring blood pressure with the automatic device, the occlusion lasts only 10 s meaning that for 11 measurements the total duration of occlusion is less than 2 minutes. As we performed repeated occlusions, these can also mimic ischemic preconditioning24. However it has been shown that: i) there was no evidence for ischemic preconditioning during a repeated vessel occlusion that lasts 2 minutes25, and ii) a minimal interval of 2 minutes is needed to observe a significant effect26. Therefore it is not likely that such phenomenon occurs. Furthermore, Nikolic et al. measured the ankle SBP in 250 treated hypertensive patients at 5 and 10 mn (taking the average of two measurements 1 mn apart) and found a decrease of 4 mmHg on average between the two measurements (SD 14 mmHg), which is the same as the decrease we observed in our patients, and suggests that the additional measurements taken in our study do not impact the evolution of SBP too strongly27. As a final note, all international recommendations suggest to perform several measurements of blood pressure and to perform a mean of the measurements15.Our study has several limitations. First, our population is a population of outpatients addressed to a vascular examination and not patients issued from GPs’ offices. Because they came for a regular visit, the observation period was limited to the duration of a normal consultation so we could not obtain measurement after 11 minutes, and some of our findings need to be verified using data collected in a longer study. Interestingly, our results are in accordance to a recent study from Bos and BuisOur study suggests that the current recommended practice of measuring SBP after 5 minutes of resting may not allow for adequate stabilization of SBP, which we find could take at least 25 minutes. Public Health Policies should take into account this result to organize the best way to diagnose hypertension in our societies and avoid overdiagnosis.A cross-sectional, observational, bi-center  study was performed in France. The ethics review board of our institution (named “Comité d’éthique du CHU de Rennes”) approved the “Opti-PA study” . All included subjects signed an informed consent. This study has been conducted according to the principles expressed in the declaration of Helsinki.Outpatients (18 years of age or older), arriving on foot for a vascular examination either for a carotid ultrasound exam or suspected peripheral artery disease, or suspected venous disease or a cardiovascular prevention visit. Only patients in whom arm pressure could be measured in both arms were included. Patients with fistula or lymphedema after breast cancer were not included. Several variables were collected such as age, gender, body mass index, comorbidities and medical treatments during the medical interview or retrieved from medical record.Demographic and clinical data were recorded at inclusion. Each patient was invited to participate in this study when coming into the office room, then the patient was invited to lie down on a bed or to seat on a chair in a controlled room temperature for the blood pressure measurements. Two cuffs adapted to the arm circumference were placed at heart level on each arm of the patient. Heart rate as well as systolic and diastolic blood pressures were automatically and simultaneously measured at each arm every minute for 11 minutes, using two Dinamaps CARESCAPE V100 .The primary study outcome was the blood pressure measurements every minute for 11 minutes.The statistical analysis was performed first on Systolic Blood Pressure (SBP) and second on Diastolic Blood Pressure (DBP). We followed for each subject the SBP or DPB for the arm with the highest measurement at the first measurement 1 mn). The main objective was to model the decrease of blood pressure. The secondary objective was to identify the explanatory variables of this decrease, including the general characteristics of patients described in Table  mn. The Baseline pressure was the first pressure measured after 1 minute of resting. A repeated measure ANOVA analysis was first performed to determine whether a significant trend in time could be detected. The data were then analyzed through non-linear mixed effect regression using the Monolix software. We assumed an exponential decrease of blood pressure P(t) with time t from an initial value to an asymptotic resting pressure (Prest), according to the following equation:5mmHg, which was defined as SBP within 5 mmHg of systolic Prest. We also looked at stable SBP10mmHg (SBP within 10 mmHg). The variability of the automatic blood pressure monitor is expected to be 5 mmHg according to the manufacturer.The model and parameter estimates were used to predict the time when most subjects are expected to reach a stable SBP15. We also report the associated prediction intervals.A patient is considered as having hypertension when measured SBP is equal or above 140 mmHg. To investigate the impact of resting time on the proportion of subjects predicted as hypertensive, we simulated 1000 replicates of the original datasets by sampling parameters from the final model estimates, and computed the corresponding proportion of subjects for various measurements (single measurement or mean of two points 1 or 2 mn apart)The datasets generated and analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.Supplementary Information


Evaluating clinician compliance with recommended steps in clinical guidelines provides one measure of quality of process of care but can result in a multiplicity of indicators across illnesses, making it problematic to produce any summative picture of process quality, information that may be most useful to policy‐makers and managers.We set out to develop a clinically logical summative measure of the quality of care provided to children admitted to hospital in Kenya spanning the three diagnoses present in 60% or more of admissions that would provide a patient‐level measure of quality of care in the face of comorbidity.We developed a conceptual model of care based on three domains: assessment, diagnosis and treatment of illnesses. Individual items within domains correspond to recommended processes of care within national clinical practice guidelines. Summative scores were created to reduce redundancy and enable aggregation across illnesses while maintaining a clear link to clinical domains and our conceptual model. The potential application of the score was explored using data from more than 12 000 children from eight hospitals included in a prior intervention study in Kenya.Summative scores obtained from items representing discrete clinical decision points reduced redundancy, aided balance of score contribution across domains and enabled direct comparison of disease‐specific scores and the calculation of scores for children with comorbidity.This work describes the development of a summative Paediatric Admission Quality of Care score measured at the patient level that spans three common diseases. The score may be an efficient tool for assessing quality with an ability to adjust for case mix or other patient‐level factors if needed. The score principles may have applicability to multiple illnesses and settings. Future analysis will be needed to validate the score. Measuring quality of health care is an important aspect of any health system as it provides the information necessary to monitor and improve service delivery. However, quality of care is a multifaceted concept We use recommendations summarised in evidence‐based clinical practice guidelines as standards of technical quality of clinical processes including clinical assessment and treatment. This is a widely adopted approach to assessing process quality In Kenya, standards of inpatient admission care for children are defined in a set of practice guidelines for healthcare workers Aggregating component indicator scores across domains might provide a score for an individual illness episode allowing calculation of a summarised illness score for multiple similar episodes. Aggregating across illnesses might create scores that allow summaries within an individual or for a set of individuals with multiple diagnoses. However, if simply adding itemised steps for different illnesses results in different total scores for specific illnesses then their scores are not directly or intuitively comparable, and creation of a patient‐specific score in the face of comorbidity becomes problematic. Here, we illustrate the problem of using simple additive scores based on all items and propose the PAQC score as a solution to this problem.The data for designing and testing the PAQC score came from a previously published large, pragmatic trial of a multifaceted intervention to improve quality of care for children Component indicators (items) correspond to specific recommendations on the process of care in the guidelines Table . They arThe assessment domain score was the number of signs and symptoms documented by the admitting clinician. The diagnosis domain score was a binary indicator of whether the clinician made a valid classification of the severity of illness recognised in the guidelines. For the treatment domain, indicators based on recommendations on dosages, route and frequency, and durations of treatment were summed up. Deviations of up to 20% of recommended dosages per kilogram of body weight, which are within therapeutically safe dose ranges for all the drugs used, were considered to be correct.There were 19 guideline‐recommended signs and symptoms necessary for identifying and classifying the severity of the three illnesses we focused on. Two of these – ability to drink or breastfeed and level of consciousness – were common across all three diseases. Five treatment indicators were defined to score the treatment of malaria and pneumonia but only three of these – treatment choice, dose and frequency – were applicable to diarrhoea/dehydration. A basic score was created as an arithmetic sum of each item in the assessment, diagnosis and treatment domains. However, a number of problems were observed with the characteristics of this score: they include redundancy between items within domains, domination of the scale by items from one domain (assessment), and non‐equivalent scores across diseases. For these reasons, this approach to creating scores was rejected.To overcome the problems of the simple additive scores, we collapsed all of the original items within domains into new components representing discrete clinical decision points that constitute the desired processes outlined in the guidelines. The resulting domain‐specific components, listed in Table For example, for malaria, the primary assessment sign was fever. Secondary signs depended on severity. According to guidelines, severe malaria was the correct diagnosis for a child who, in addition to fever, presented with at least one danger sign – convulsion, acidotic breathing, inability to drink or breastfeed, altered consciousness or pallor with respiratory distress indicated by grunting or indrawing. Fever in the absence of any danger sign was to be classified as non‐severe malaria. A clinician was required to completely exclude the presence of danger signs to correctly diagnose non‐severe malaria. For this reason, a complete secondary assessment for non‐severe malaria meant documentation of all the danger signs. The assessment domain score thus rewarded identification of danger signs and completion of all assessment tasks as recommended in the guidelines. This approach was also applied to children diagnosed with pneumonia and diarrhoea/dehydration.For the diagnosis domain, the binary indicator of whether a relevant severity classification was made was retained unchanged. However for treatment, two domain‐specific components were generated: (i) selection of a relevant drug for treatment of the disease diagnosed and (ii) correct use of the selected drug which included correct dose, appropriate route of delivery, frequency and duration where applicable. The resulting disease‐specific score was a sum of these domain scores for each individual.Where a child had only one of our three diagnoses, their disease‐specific score became the patient‐level PAQC score. To measure quality of care in an admission episode with more than one illness, a score combining the disease‐specific scores was needed. An intuitive approach would be to use the arithmetic mean of the disease‐specific scores. However, this approach would have created non‐integer score values which no longer represented a count of guideline‐recommended process‐of‐care tasks completed by the clinician. Thus, an alternative approach was used in which a domain‐specific score was 1 if the equivalent items in each of the diagnosed diseases had scored 1 and zero otherwise – an all‐or‐none combination of disease‐specific, domain‐specific scores. For example if a child had malaria and pneumonia, then the primary assessment score was 1 if primary assessment items for both malaria and pneumonia  scored 1; if only one or none of them were documented then the item score was zero. Although this approach made it more difficult to achieve each level of the score, it reflected the clinical reality that multiple diagnoses increase the number of guideline‐recommended tasks required to effectively manage illness thereby increasing the difficulty in providing quality care when there is multimorbidity.In sum therefore, at the patient level, the PAQC score is represented by the disease‐specific score where there is a single diagnosis and by a score that incorporates multimorbidity when the child has more than one diagnosis. It remains possible to report these disease‐specific scores (or scores within their domains) if there is a specific interest in these as outcomes or for monitoring and improvement management purposes. However, it is also now possible to report a patient‐level PAQC score that spans all three diseases and combinations of these diseases.As well as basing score construction on a logical clinical strategy, we also examined score properties. Tetrachoric correlation coefficients were used to flag pairs of items within the same domain that were similar enough to be deemed redundant A simple additive approach across the domains assessment, diagnosis and treatment resulted in a 15‐point score (range 0–14) for malaria and pneumonia and a 13‐point score (range 0–12) for diarrhoea/dehydration. Each had a 9‐point (range 0–8) score for the assessment domain, a 2‐point (binary) score for the diagnosis domain but a 6‐point (range 0–5) score for the treatment domain for malaria and pneumonia and a 4‐point (range 0–3) score for diarrhoea/dehydration. Thus, these simple additive scores were heavily weighted towards the assessment domain. The differences in scale ranges across diseases  also made comparisons of quality of care across diseases less intuitive and presented problems when reporting an overall quality score for a patient with multiple morbidity. Furthermore, there was a high degree of correlation between multiple items in this simple additive score as shown by the tetrachoric correlation coefficients (Tables S2 and S3). Additionally, the malaria assessment and treatment item tetrachoric correlation matrices were not positive (semi)definite, suggesting a high degree of linear dependency between multiple items. These findings imply considerable redundancy and are a justification for rejecting these simple additive scores.For the disease‐specific scores, tetrachoric correlation coefficients of within‐domain components of the score ranged between 0.34 and 0.62 – ‘weak’ to ‘moderate’ according to Evans’ criteria – showing much less of the codependence observed between items in the simple additive score (Table S4). Between‐domain correlation of items was also in this range. However, there was a perfect correlation between the classification indicator and the drug choice indicator in malaria and pneumonia; this was expected because the choice of drug depended on severity classification and for this reason neither indicator was dropped. There is therefore support for retaining the disease‐specific scores as a 7‐point score (range 0–6) across all three diseases, being the sum of six binary items . Although the control group had higher PAQC scores in successive surveys , the intervention group showed a bigger increase . These findings are consistent with previously documented effects of the intervention vs. between 13 and 953 children with disease‐specific and multimorbidity scores. This illustrates the potential gain in efficiency that may result from having a PAQC score that provides a patient‐level measure of quality for multiple diagnoses.The overall PAQC score mirrored the trends observed with disease‐specific and multimorbidity scores, ranging from an 8.7% increase in the documentation of primary signs to a 54.9% improvement in the documentation of illness severity classifications between the two surveys. Furthermore, the distribution of the PAQC scores from baseline through to the post‐intervention survey was also shifted towards higher scores in the intervention group as shown in Figure We have described the development of the Paediatric Admission Quality of Care (PAQC) score to measure the processes of admission care for children with the three commonest diseases resulting in hospitalisation and death. The approach aggregates items representing processes of care recommended in established practice guidelines into a single metric, and items are further grouped into domains representing discrete dimensions of process of care. To reduce redundancy between items, maintain reliability and validity of the measure and to create generic components capable of measuring process of care for a variety of diseases, we have systematically combined similar indicators within the domains. The score itself focuses on a critical period for treatment of acute illnesses when there is sufficient opportunity to intervene and restore health in low‐income countries The PAQC score begins to fulfil the need for a well‐developed quality assessment tool relevant for use in a low‐income setting where less than optimal care provided by health workers Box 1Step 1: Identify the health facilities to be surveyed and assign them unique identity codes. If possible, identify the clinicians responsible for providing admission care to children and assign them unique identity codes too.Step 2: Create a sampling frame of case records to be selected for abstraction. This should include the age range, illnesses of interest, period and number – whether all or a subset – of eligible children whose records will be abstracted.Step 3: Obtain the case records of the children corresponding to the sampling frame and assign them unique identity numbers.Step 4: Use a patient case‐record data abstraction form (electronic or paper‐based) to obtain the data required to calculate the PAQC score, and import the data into any suitable statistical software.Step 5: For each child create binary indicators for each of the disease‐specific and overall PAQC scores. The indicators of what constitutes good care should be based on the most recent guidelines, or published evidence in the absence of guidelines.Step 6: Create the disease‐specific and overall PAQC scores for each child by adding up over their disease‐specific and overall PAQC score indicators.Step 7: Obtain a suitable average  of the individual (child)‐level PAQC scores at the required level of reporting .An advantage of the patient‐level PAQC score is its ability to combine data on quality of care for three common diseases including children with multimorbidity in one summary index. This could make it an efficient endpoint for testing interventions or quality improvement efforts as fewer patients may need to be included in studies. However, the score may be affected by the distribution of diagnoses in the population under study. Patient‐level measurement provides the flexibility to adjust not only for such variations in case mix but also for characteristics such as age, sex, severity of illness and frequency of comorbidity in statistical models. Where data allow it also provides for aggregation at clinician, department and hospital level when contrasting performance across places or time and in response to interventions.Measuring quality of care in terms of process by contrasting what was documented to have been undertaken – or the lack of documentation – with guideline recommendations is a relatively narrow perspective for measuring what is obviously a multifaceted concept; it is nevertheless a very important one considering the central role of clinical processes in providing the means by which health inputs are converted to desirable outcomes and the transparent link it offers between evidence‐based recommendations and practice. This approach to measurement can potentially be extended to other situations – for example, other diseases in childhood or other clinical fields such as surgical admission –as the principle underlying the three domains of process is almost universal in medicine and can be adapted to accommodate variation in guidelines across place or time. The absence of a fourth domain of items relating to diagnostic testing is a weakness worth noting. Perhaps, this reflects the low‐resource nature of this setting where the use of such technologies is not widespread or emphasised as important for the delivery of care This work shows how a quality‐of‐care score, the PAQC score, aimed at childhood admissions to low‐income hospital settings has been derived to provide a clinically logical summative measure of the process of care for common childhood illnesses. Future work will be required to explore the validity of the score, its potential value as a measure used to test interventions or track changes in quality and its acceptance by and value to health systems managers. The approach taken may be of value for other clinical settings, including non‐paediatric care, non‐medical care and non‐communicable diseases.Appendix S1. The paediatric data abstraction form.Click here for additional data file.Appendix S2. Anti‐malarial drug doses.Click here for additional data file.Appendix S3. Oral antibiotic doses.Click here for additional data file.Appendix S4. Intravenous/intramuscular antibiotic doses – ages 7 days and older.Click here for additional data file.Appendix S5. Urgent fluid management – child without severe malnutrition.Click here for additional data file.Appendix S6.Table S1. Number of episodes of each disease/multi‐morbidity across surveys.Table S2. Tetrachoric correlation matrix of assessment items in the basic score.Table S3. Tetrachoric correlation matrix of treatment items in the basic score.Table S4. Tetrachoric correlation matrix of items in the malaria, pneumonia and diarrhoea/dehydration component PAQC scores.Click here for additional data file.


Mental health issues have been increasingly recognized as public health problems globally. Their burden is projected to increase over the next several decades. Additional therapies for mental problems are in urgent need worldwide due to the limitations and costs of existing healthcare approaches. Essential oil aromatherapy can provide a cost‐effective and safe treatment for many mental problems. This pilot study observed the effects of bergamot essential oil inhalation on mental health and well‐being, as measured by the Positive and Negative Affect Scale, in a mental‐health treatment center located in Utah, USA. Fifty‐seven eligible participants  were included for analysis. Fifteen minutes of bergamot essential oil exposure improved participants' positive feelings compared with the control group (17% higher). Unexpectedly, more participants participated in experimental periods rather than control periods, suggesting even brief exposure to essential oil aroma may make people more willing to enroll in clinical trials. This study provides preliminary evidence of the efficacy and safety of bergamot essential oil inhalation on mental well‐being in a mental health treatment center, suggesting that bergamot essential oil aromatherapy can be an effective adjunct treatment to improve individuals' mental health and well‐being. © 2017 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd. Participants were current patients of the center or patients' companions. This study was reviewed and approved by an institutional review board (IRB) before commencement.Both women and men, aged between 18 and 70 years, were included if they could communicate verbally and read and write in English. Women who were pregnant and/or lactating were excluded. People with no sense of smell or with known pre‐existing sensitivities to essential oils were excluded from the study. Patients who, judged by the staff of the treatment center as not good candidates for the study, were also excluded.The trial lasted 8 weeks. Weeks 1, 3, 5, and 7 were essential‐oil diffusion periods; weeks 2, 4, 6, and 8 were distilled‐water diffusion periods. Participants who met the above eligibility criteria and agreed to participate were instructed to sit as still as possible in the waiting room for 15 min. Written consent was waived per an IRB's evaluation. Depending on the week they came to the center, they were exposed to either bergamot or distilled water aromatically. Patients were told the study was intended to assess the waiting time's effect on mental feelings to distract their attention from the smell in the waiting room. After 15 min, participants were asked to complete the PANAS survey and demographic information . Then, they could proceed with their regularly scheduled treatment. Any adverse events were also recorded.The aromatherapy devices were four waterless diffusers, provided by dōTERRA , and placed out of sight of the participants in the waiting room. The gas chromatography–mass spectrometry analysis of BEO showed that its major chemical constituents  are limonene (36%), linalyl acetate (31%), linalool (11%), gamma‐terpinene (8%), and beta‐pinene (7%).The diffusers were turned on 15 min before the first patient arrived and kept running at half speed throughout the day. The speed setup of diffusers and their locations were selected based on previous experiments (unpublished data), ensuring that a comfortable and consistent aroma was maintained through the day. Facility staff routinely checked each diffuser to ensure there was always sufficient essential oil or distilled water, and replaced new bottles of oil or water if needed. This ensured that each participant received an equivalent strength and amount of oil aroma or water vapor during the 15‐min study period, which approximately mimicked the real‐life scenario of aromatic usage of essential oils.et al., very slightly or not at all) to 5 (extremely). Ten items assess respondents' positive feelings, while the other ten assess negative feelings. Scores range from 10 to 50 for either positive or negative affect. The PANAS typically takes less than 5 min to complete.The PANAS has been extensively validated and utilized to assess momentary mental well‐being . The analysis compared the difference between the active group (BEO) and the control group (distilled water) in positive and negative affect scores. In addition, a two‐sample n = 45) than did those in the control group (n = 12)  and meaningful differences  were observed for several items. Participants in the active group reported a 48.77% higher score of feeling ‘proud’ compared with the control group. They also reported a 43.36% higher score of feeling ‘active.’ However, participants in the active group also reported a 60% increase of feeling ‘nervous.’No statistically significant difference was observed for any of the individual items between the active group and control group Table . HoweverParticipants in the waiting room of the mental health treatment center in the BEO group reported higher positive affect scores than did the control group. Participants in the BEO group also showed a smaller increase in negative affect scores than did the control group; however, the reasons why are unclear. It could be due to an effect resulting from the smaller size of the control group. Interestingly, the control group's mean negative affect score was already in the very lower end of the scale. This indicates that there was probably not much room to improve in negative affect, partially because the clear majority of participants (47 out of 57) were patients' companions, who were less likely to have mental disorders than patients. Moreover, the improvement in positive affect scores was greater than the increase in negative affect scores, although neither of them was statistically significant. Collectively, this suggests that BEO aromatherapy might provide beneficial effects on participants' mental well‐being and feelings, as measured by PANAS, in the waiting room of a mental health treatment center.et al., et al., et al., et al., et al., et al., A body of clinical research has provided evidence supporting the therapeutic effects of BEO aromatherapy on mild mental disorders in a variety of settings . This observation appears to be more apparent for patients of the mental health treatment center, who had already been diagnosed with mental health issues: ten patients participated in the BEO periods, while no patient participated in the control periods. To the best of our knowledge, this is unlikely due to any other interventions than the difference between exposure to BEO aroma and water vapor. One explanation would be that even very brief exposure to BEO aroma may somehow make people  become more willing to participate in clinical trial in the current setting. If this is true, it can have profound applications in both clinical trial participation and compliance as well as many other scenarios.This study had several limitations. It was designed as a pilot study to explore the potential benefits of BEO aromatherapy for mental well‐being and feelings. The study did not have sufficient statistical power to make definite conclusions mainly due to the small control group, small overall study size, and study design. The study design was the best possible option at the time the trial commenced. We designed it to mimic the real‐life scenarios of aroma inhalation as much as possible. Furthermore, we intended to minimize the effects that the treatment center had on the trial and its participants. Therefore, only current patients of the center and their companions were invited to participate. Further studies with designs, such as better controlling participation, are recommended.Supplementary Materials _ PANAS and Demographic Information Survey.Click here for additional data file.


Coffee consumption has been shown in some studies to be associated with lower risk of prostate cancer. However, it is unclear if this association is causal or due to confounding or reverse causality. We conducted a Mendelian randomisation analysis to investigate the causal effects of coffee consumption on prostate cancer risk and progression. We used two genetic variants robustly associated with caffeine intake (rs4410790 and rs2472297) as proxies for coffee consumption in a sample of 46,687 men of European ancestry from 25 studies in the PRACTICAL consortium. Associations between genetic variants and prostate cancer case status, stage and grade were assessed by logistic regression and with all‐cause and prostate cancer‐specific mortality using Cox proportional hazards regression. There was no clear evidence that a genetic risk score combining rs4410790 and rs2472297 was associated with prostate cancer risk  or having high‐grade compared to low‐grade disease . There was some evidence that the genetic risk score was associated with higher odds of having nonlocalised compared to localised stage disease . Amongst men with prostate cancer, there was no clear association between the genetic risk score and all‐cause mortality  or prostate cancer‐specific mortality . These results, which should have less bias from confounding than observational estimates, are not consistent with a substantial effect of coffee consumption on reducing prostate cancer incidence or progression. What's new?Does coffee consumption reduce prostate cancer risk? It's biologically plausible that it could, but studies showing a link have relied on observational evidence, which could be affected by confounding factors. These authors set out to isolate coffee's contribution. They focused on two genetic variants that correspond with caffeine intake, and used them as proxies for coffee drinking. Alleles are not affected by behavior or demographic factors, nor can behavior changes after diagnosis change whether a person carries an allele. The authors found no correlation between either of the alleles and prostate cancer risk, suggesting drinking coffee may not protect against prostate cancer. Coffee consumption has been reported to be inversely associated with prostate cancer risk,Mendelian randomization, which uses genetic variants that are associated with exposures of interest as proxies for measured exposures, may help to strengthen causal inference about potentially modifiable exposures.CYP1A1/CYP1A2) and aryl hydrocarbon receptor (AHR) genes, which are known to play a functional role in caffeine metabolism.CYP1A2 is the primary enzyme responsible for metabolizing caffeine, whilst AHR controls transcription of CYP1A2.AHR which increases coffee consumption was associated with lower blood caffeine in a GWAS of blood metabolites.Genetic variants which demonstrate robust associations with caffeine intake have been identified in recent genomewide association studies (GWAS) of coffee consumption.Using variants in these two loci as instruments for coffee consumption, we performed a Mendelian randomization analysis in 46,687 prostate cancer cases and controls from the PRACTICAL consortium to investigate whether coffee consumption is causally associated with prostate cancer risk, stage, grade and mortality. If coffee consumption causes a reduction in prostate cancer risk or progression via compounds other than caffeine, we might expect to see an inverse relationship between number of coffee consumption increasing alleles and these outcomes.http://www.nature.com/ng/journal/v45/n4/extref/ng.2560-S1.pdf. All studies met the appropriate ethical criteria for each country in accordance with the Declaration of Helsinki.We used data on prostate cancer cases and controls from 25 studies in the PRACTICAL Consortium . Men included in the analysis were of European genotypic ancestry. Full details of the individual participating studies have been published previouslyAHR and rs2472297 near CYP1A1/CYP1A2) were imputed using a HapMap 2 CEU reference panel from a Custom Infinium genotyping array (iCOGS). This array was designed for the Collaborative Oncological Gene‐environment Study (COGS) and consisted of 211,155 SNPs .The two caffeine‐related single nucleotide polymorphisms (SNPs) (rs4410790 in P < 1 × 10−7), 201,598 SNPs remained. These SNPs were used to impute 2.6 million SNPs; poorly imputed SNPs (R2 < 0.3) were excluded.Full details of the genotyping and imputation have been published previously.Genetic risk scores were created by summing the number of coffee consumption increasing alleles  for the two SNPs, assuming an additive genetic model. We used allele dosages from imputation  to indicate the number of coffee increasing alleles. This accounts for uncertainty in the imputation of each genotype.Cancers were categorised into low or high grade, according to Gleason score . Cancers were categorised into clinically localised and nonlocalised, using TNM staging  or SEER staging, where TNM staging was not available .Analyses were limited to studies for which mortality follow‐up amongst cases was at least 90% complete and had at least five prostate cancer deaths . Individuals with vital status recorded as “unknown” were excluded from these analyses. Individuals with an unknown cause of death were assumed not to have died of prostate cancer.Data on coffee and tea consumption were available for four of the studies . Out of these studies, information on whether coffee or tea was caffeinated or decaffeinated was only available in UKGPCS. Information about frequency of coffee and tea consumption was collected in categories, but for the purposes of analysis was recoded to number of consumed cups per day using the midpoint of each category. Further details of the coding of these variables and how coffee and tea data were collected in each study are available in Supporting Information (Table S1).metan command in Stata.Analyses were conducted in Stata (version 14). Associations between the genetic risk score and consumption of coffee, tea and coffee and tea combined were assessed using linear regression, adjusting for the top eight principal components that reflect the genetic structure of the population (to control for confounding by population stratification). Robust standard errors were calculated to account for the right skewed nature of the coffee and tea variables. Analyses were conducted within each of the four studies with coffee and tea consumption data available and combined in a random effects meta‐analysis using the N = 23, ProMPT and WUGS excluded). Within prostate cancer cases, we used logistic regression to investigate associations of these SNPs with high grade compared to low grade and nonlocalised compared to localised cancer. For the nonlocalised vs localised analysis, we excluded studies with no nonlocalised cancers (N = 2). In men diagnosed with prostate cancer, we used Cox proportional hazards regression to investigate whether the caffeine‐related SNPs were associated with all‐cause mortality and prostate cancer‐specific mortality. For these analyses, we used age at diagnosis as the start date and age of death or age of last follow up  as the censoring date. All analyses of the associations between the coffee‐related genetic variants and prostate cancer were adjusted for genetic principal components and study and robust standard errors were used to account for clustering by study. To investigate between‐study heterogeneity we calculated estimates separately for each study and combined these in a fixed effects meta‐analysis using the metan command in Stata. Between‐study heterogeneity was low (I2 ≤ 34%), so we report the combined estimates.Associations between the coffee‐related SNPs and prostate cancer risk (case/control status) were assessed using logistic regression. For these analyses, we only included studies contributing both cases and controls . Mean age at prostate cancer diagnosis was 65 years (SD 8) with mean age across the studies ranging from 59 to 72 years. Reflecting the variety of clinical populations across the included studies, the proportion of men with nonlocalised cancer ranged from 0% to 65% and with high grade cancer from 28% to 84%.I2 > 33%).Data on coffee and/or tea consumption were available for 4,722 individuals . Associations between the genetic risk score and tea and coffee consumption were in the expected directions and of similar magnitude to those observed in coffee consumption GWASThere was no clear evidence that the coffee‐related SNPs were associated with prostate cancer case status or having high grade compared to low grade disease Table . The oddThe 15,555 men who contributed to the all‐cause mortality analysis were followed up for an average of 6.8 years, during which 4,081 died. The 14,010 men who contributed to the prostate‐cancer specific analysis were followed up for an average of 7.1 years during which 1,754 died of prostate cancer. There was no clear evidence that the individual coffee related SNPs or the genetic risk score for coffee consumption were associated with all‐cause mortality ) or with prostate cancer mortality: HR 1.03 (95% CI: 0.98 to 1.08) Table . There wWe performed a Mendelian randomization analysis in a large prostate cancer case control study to investigate whether coffee consumption causally influences prostate cancer incidence and progression. We found no clear evidence to suggest that coffee consumption is causally associated with risk of prostate cancer, disease grade or mortality amongst men diagnosed with prostate cancer.Our findings suggest that observational associations indicating that coffee consumption reduce prostate cancer risk and progressionOur finding of a weak positive association between the coffee genetic risk score and increased risk of nonlocalised disease is in the opposite direction to observational evidence suggesting that coffee may reduce risk of disease progression.AHR and CYP1A1/2) have been identified in GWAS of blood pressure, bladder cancer and Parkinson's disease,CYP1A2 metabolises other xenobiotic substrates other than caffeine and although neither of the SNPs used in this analysis were found to associate with blood metabolites (other than caffeine) at genome wide significance level,There are several limitations to these analyses. First, as aforementioned, there is heterogeneity between studies in terms of case definition, treatment received, classification of stage, grade and mortality follow up. Second, as discussed previously and shown by the associations in the four PRACTICAL studies with caffeine consumption data, these genetic instruments are not specific to coffee and associate with consumption of other caffeinated beverages , and even with decaffeinated coffee.In conclusion, our findings do not support a causal role of coffee consumption in prostate cancer incidence or grade and suggest that observational findings that coffee consumption is associated with a reduced risk for prostate cancer may be due to confounding by other lifestyle factors. Further investigation of our finding that the genetic risk score was positively associated with risk of nonlocalised disease is required in samples which also have data on coffee consumption, and which have greater power to investigate a subsequent impact on prostate cancer specific mortality.Supporting InformationClick here for additional data file.


Maternal‐fetal chimerism is miniscule, a testament to the integrity of the uteroplacental interface. The soundness of this border region is potentially altered through cesarean delivery of prior babies with uncertain consequences for the following pregnancies.Using multicolor flow cytometry and quantitative PCR of non‐inherited maternal antigens we performed a retrospective case control pilot study and formulated the null hypothesis that placental implantation over a prior uterine scar does not result in the presence of memory Treg (CD45RO+) in the fetus. We then performed a power calculation and performed a blinded, appropriately powered prospective case control study to test the null hypothesis.p < 0.05 CI:(1.448 to 17.27)]). These memory fetal Tregs are functionally highly suppressive compared to CD45RA‐expressing fetal Tregs, and have specificity for non‐inherited maternal antigens.Fetuses born to mothers with prior uterine scar have a roughly five times higher maternal to fetal microchimerism when the placenta directly interacts with the uterine scar. Unlike exposure to antigens in adult life, in utero antigenic exposure induces tolerogenic (Treg) responses in fetuses and we here report the presence of fetal Treg with a memory phenotype (CD45RO+). However, we only find such CD45RO+ fetal Tregs when the placenta abuts the uterine scar  in the fetus. Thus, our study demonstrates a previously unappreciated role for uterine integrity in limiting fetal antigenic exposure, a key element to avoid the formation of inappropriate tolerances by the fundamentally tolerogenic fetal immune system. How humans are born is changing. Historically, before the advent of safe cesarean delivery, women with contraindications for vaginal birth suffered high rates of morbidity and mortality. More recently, the circumstances that prompt or warrant cesarean delivery have been ever expanding and now include maternal request The human fetal immune system develops the ability to mount adaptive responses by mid‐gestation In this study, we investigate the possibility that a uterine scar may impact the developing fetal immune responses. When the placenta implants in the region of a uterine scar, the potential for a greater degree of invasion that can cause life‐threatening hemorrhage at delivery  increases Healthy women scheduled for repeat cesarean section in the third trimester with singleton pregnancies were recruited for participation between March 2009 and July 2014. Demographic and obstetrical characteristics from the recruited population are provided in Tables Cord blood was collected after delivery of the fetus, but prior to placental separation, into sterile cord blood collection kits containing Citrate Phosphate Dextrose solution .Granulocytes were depleted utilizing the RosetteSep Granulocyte Depletion Cocktail  following manufacturer's recommendations. Granulocyte‐depleted mononuclear cells were isolated by gradient centrifugation over Ficoll‐Paque PLUS from GE Healthcare  following manufacturer's recommendations. Cells were washed twice with sterile PBS and enumerated utilizing an Accuri flow cytometer with Propidium Iodide exclusion of dead cells.132 irradiator  and stained with the antibody panel depicted in Table n = 20) was performed on a BD Accuri C6 flow cytometer  using the antibody panel listed in Table Phenotypic characterization as in Figure Cord blood lymphocytes were isolated as described above. DNA from 2 to 5 ml of maternal saliva and 5 million cord blood lymphocytes was isolated using the DNEasy Blood and Tissue Kit . A quantitative PCR (qPCR) method detecting mother‐fetus polymorphisms in insertion/deletion regions on different chromosomes that show high natural polymorphisms was applied as published previously T testing or analysis of variance (ANOVA). Statistical analyses were accomplished with PRISM .Differences in normally distributed populations were statistically analyzed using unpaired Student's p = 0.0290). With our quantification method, we detected approximately 1 maternal cell per 1000 fetal cells in fetuses with anterior placentas, an amount roughly 1/5th of that in fetuses with posterior placentas .Trafficking of maternal cells into the fetus is a common phenomenon, as much as 0.1–0.5% cord blood lymphocytes are of maternal origin p < 0.05 CI:(1.448 to 17.27)]).The presence of maternal cells in the fetal environment induces the generation of Tregs with specificity for NIMA. These NIMA‐specific Tregs are thought to be peripheral rather than thymic in origin and dependent on TGF‐β for their generation In humans, Treg subtypes are identified by surface expression of CD45RA in combination with intranuclear expression of FoxP3: resting Treg (CD45RAhi FoxP3lo), Cytokine Treg (CD45RA‐FoxP3lo) and Activated Treg (CD45RA‐FoxP3hi) Similar to the maternal immune system, the fetal immune system is “aware” of maternal antigens, but T cell effector responses are subdued by fetal Tregs in an antigen‐specific manner, as demonstrated in Treg depletion experiments The birth of a baby is an important milestone in people's lives; increasingly women choose to undergo cesarean section as a choice informed by concerns other than medical necessity. To date, conversations around elective c‐section largely revolve around concerns for the mother but impacts on the fetuses of future pregnancies have thus far not been included. We found increased trafficking of maternal cells into the fetus and the formation of highly functional memory T reg with specificity for NIMA in those fetuses where the placenta implants over a prior uterine scar. Thus, we have demonstrated that an increasingly common surgical intervention creates conditions that alter the fetal immune repertoire.Plasmodium) was found to elicit Treg and functional, tolerogenic responses toward the parasite in the fetus if this exposure occurred early during pregnancy Plasmodium infection in the study by Malhotra et al displayed a tolerogenic response, or in fact any response, toward Plasmodium antigens indicating that not all exposures result in fetal immune recognition Our findings and reports by others suggest that the fetus can be exposed to antigens from maternal circulation across the placent. For example, in utero exposure to malaria . Each subject provided written informed consent prior to enrollment.Additional supporting information may be found in the online version of this article at the publisher's web‐site.Figure S1. Gating strategy and CD45RA/RO distribution on adult T cells and Treg.Click here for additional data file.


To determine the burden and characteristics of fatal and hospitalised injuries among youth in Fiji.We conducted a cross‐sectional analysis of the Fiji Injury Surveillance in Hospitals database – a prospective population‐based trauma registry – to examine the incidence and epidemiological characteristics associated with injury‐related deaths and hospital admissions among youth aged 15–24 years. The study base was Viti Levu, Fiji, during the 12‐month period concluding on 30 September 2006.n = 515, incidence rate 400/100 000). Injury rates were higher among men, those aged 20–24 years compared with 15‐ to 19‐year‐olds, and indigenous Fijians (iTaukei) compared with Indians. The leading causes among indigenous Fijians were being hit by a person/object (men) and falls (women), whereas for Indians, it was road traffic injuries (men) and intentional poisoning (women). Most injuries occurred at home (39%) or on the road (22%). Of the 63 fatal events, 57% were intentional injuries, and most deaths (73%) occurred prior to hospitalisation. Homicide rates were four times higher among indigenous Fijians than Indians, whereas suicide rates were five times higher among Indians compared with indigenous Fijians.One in four injuries in the Fiji Injury Surveillance in Hospitals database occurred among youth (Important ethnic‐specific differences in the epidemiology of fatal and serious non‐fatal injuries are apparent among youth in Fiji. Efforts to prevent the avoidable burden of injury among Fiji youth thus requires inter‐sectoral cooperation that takes account of important sociocultural, environmental and health system factors such as unmet mental healthcare needs and effective pre‐hospital trauma services. Of the approximately five million injury deaths per annum, over 90% occur in low‐ and middle‐income countries.Youth aged 15 to 24 years account for 16% of injury fatalities worldwide, the commonest causes being road traffic injuries, self‐harm and violence.Contemporary published literature on the overall epidemiology of injuries among youth living in the Pacific Islands is sparse.Youth aged 15–24 years account for a quarter of all injuries resulting in death or hospital admission in Viti Levu, Fiji.Three‐quarters (73%) of the injury deaths in this age group occurred prior to hospital presentation.Injury prevention efforts must take account of sociocultural factors that may account for important differences in the epidemiology of injuries in the two major ethnic groups with indigenous Fijian (iTaukei) youth having higher rates of injury overall and assault/homicide events while Indian youth having five times higher rates of self‐harm.Of the 5.1 million injury‐related deaths reported worldwide, 16% occur in the 15‐ to 24‐year age group.Despite the disproportionate burden of injuries borne by youth in low‐ and middle‐income countries, injury prevention efforts in these settings are hindered by significant gaps in knowledge regarding associated epidemiological characteristics and contextual factors.These are particularly acute challenges for vulnerable economies in the Pacific region where youth make up one‐fifth of the total population (1.6 million) in the 22 Pacific Island countries and territories. With the majority of these Pacific nations categorised as low‐ or middle‐income and a projected rapid increase in the youth population over the next two decades, proactive measures are required to ensure their health and wellbeing.n = 130 000) made up one‐fifth of the Viti Levu population. Indigenous Fijians (iTaukei) and Indians comprised 55% and 39% of the youth population respectively.Research on the burden of injuries among youth in Pacific Island countries and territories has largely focussed on descriptive studies examining self‐harm and its attendant risks.We conducted a secondary analysis of data gathered in a prospective population‐based surveillance system to quantify the incidence‐associated epidemiological characteristics of fatal and hospitalised injuries among youth aged 15 to 24 years in Viti Levu. This analysis complements the information previously published on the epidemiology of childhood injuries in Fiji.The Fiji Injury Surveillance in Hospitals (FISH) database was a study‐specific prospective trauma registry established in all trauma‐admitting hospitals in Viti Levu, Fiji, as part of the traffic‐related injury in the Pacific (TRIP) research project. Using a methodology previously describedWe also sought data on the use of alcohol, kava (a root plant with anxiolytic and sedative properties commonly consumed in Fiji)® Excel Version 2010, and STATA® Version 12 statistical software. Population data from the Fiji census (2007)We analysed data using MicrosoftThe 515 injury events among youth aged 15–24 years accounted for 24% of all injuries recorded in the FISH database, and an annual injury incidence rate for youth of 400/100 000 population. The major causes of injury were being hit by a person or object (27%), falls (18%), road traffic injury (RTI) (17%) and poisoning (13%). Intentional injuries  accounted for 36% of injuries. Common types of injury were fractures (32%), cuts/open wounds (22%) and head injuries (11%). Most injuries occurred in homes (39%), highways/roads (22%) and recreational areas (14%). Although 14% and 3% of youth reported consuming alcohol and kava, respectively within 6 h of the injury, the high rate of missing information (20% and 26% respectively) made it difficult to interpret these results.In general, injury rates were higher among older youth (20 to 24 years) compared with those aged 15 to 19 years and twice as high among men compared with women (Table n = 63) of injury events were fatal, and almost three‐quarters (n = 46) of these deaths occurred before hospital admission. Among indigenous Fijians, fatal injuries were more common among men while no gender difference was apparent among Indians  but Indians had higher rates of fatal injury. Among indigenous Fijians, injured men were most commonly hit by a person/object while women were injured in falls. Among Indians, men were most often injured in road crashes while  poisoning comprised the commonest injury among women. Most fatal injury events occurred at home, and three‐quarters of injury deaths occurred prior to hospital admission. Almost two‐thirds of fatal injuries were intentional, the majority of which were due to self‐harm among Indians. Rates of youth suicide among Indians were five times that of indigenous Fijians.The prospective trauma registry established as part of the wider TRIP project in Viti Levu, Fiji, provided a unique opportunity to investigate the population‐based epidemiology of life‐threatening injuries among youth. By including data from mortuaries as well as all trauma‐admitting hospitals, the study also addressed some common biases in hospital or trauma centre‐based studies, which do not typically capture data on pre‐hospital deaths and deny the opportunity to estimate population‐based incidence of injury. Applying this approach in Fiji helped distinguish ethnic‐specific differences in the epidemiology of serious injuries among Fiji youth.Notwithstanding these strengths, our findings must be interpreted in light of several limitations. The precision of the estimates is limited by the relatively small size of the population base, and the limited period of FISH data collection (12 months). The latter also precluded the ability to examine year to year variations and trends over time. While a standardised quality‐assured data collection method was employed using World Health Organization guidelines for less‐resourced settings,The high rate of interpersonal violence among indigenous Fijians in this study is similar to findings from a population‐based self‐report survey of students aged 14 to 17 years in three Pacific Islands  in which 24 to 62% of participants reported experiencing intentional physical injuries  in the preceding 12 months.The high rates of fatalities by choking or hanging among Indians in this study is consistent with previous studies focussing on suicide in Fiji.Our study was not designed to explore the specific circumstances surrounding injuries, such as those occurring in the context of sport and recreation, violence or self‐harm among Fiji youth. However, our findings alongside the existing literature reveal the need to explore the complex sociocultural, geopolitical, intergenerational and environmental factors that require attention to address this avoidable mortality.Findings regarding RTIs are generally consistent with studies from Papua New Guinea and the Pacific region in terms of the high proportion of young male crash victims and pre‐hospital deaths.Previous studies of RTIs and self‐harm in the Pacific suggest alcohol to be an important contributor, especially among injuries involving young men.Despite the disproportionate burden of injury borne by youth in low‐ and middle‐income countries, there are remarkable gaps in infrastructure and service investment in youth health, social, educational and economic policies in these settings.Although the data were collected a decade ago, our study has added critical injury information not previously available to Fiji and the Pacific region. To our knowledge, there have been no major changes in the epidemiology of injuries because the collection of these data and the rates of fatal injury events for this age group captured by the Fiji Ministry of Health have remained relatively constant. Consequently, this study continues to inform current Pacific Island country road safety initiatives including the Cook Islands Road Safety Strategy 2016–2020 and the World Health Organization Regional Action Plan for Violence and Injury Prevention in the Western Pacific (2016–2020).