Self-assembled metal nanoparticle-polymer nanocomposite particles as nanoreactors are a promising approach for performing liquid phase reactions using water as a bulk solvent. In this work, we demonstrate rapid, scalable self-assembly of metal nanoparticle catalyst-polymer nanocomposite particles via Flash NanoPrecipitation. The catalyst loading and size of the nanocomposite particles can be tuned independently. Using nanocomposite particles as nanoreactors and the reduction of 4-nitrophenol as a model reaction, we study the fundamental interplay of reaction and diffusion. The induction time is affected by the sequence of reagent addition, time between additions, and reagent concentration. Combined, our experiments indicate the induction time is most influenced by diffusion of sodium borohydride. Following the induction time, scaling analysis and effective diffusivity measured using NMR indicate that the observed reaction rate are reaction- rather than diffusion-limited. Furthermore, the intrinsic kinetics are comparable to ligand-free gold nanoparticles. This result indicates that the polymer microenvironment does not de-activate or block the catalyst active sites. Self-assembled amphiphilic molecules, both small molecules and macromolecules, that confine catalysts to micelle, vesicle, and Janus particle nanoreactor systems have proven to offer an efficient approach to perform organic reactions using water as a bulk solvent ,2,3. UsiCore-shell polymer systems have also been considered. Polymeric micelles have been used for several reactions such as asymmetric aldol reactions catalyzed by L-proline , acylatiGenerally, these promising approaches have involved design and synthesis of amphiphiles, block copolymers, or polyelectrolytes that contain catalyst or ligand for covalent attachment of the catalyst. Additionally, nanoreactor properties, such as catalyst loading and nanoreactor size, are related to the molecular properties of the synthesized material. Thus, varying the nanoreactor properties would require additional syntheses. Approaches to metal nanoparticle catalyst-polymer nanocomposite particle fabrication that would facilitate (1) modular material  selection, (2) tunable properties , and (3) rapid, scalable productionwould be beneficial to expanding their potential application.Flash NanoPrecipitation (FNP) is a rapid, scalable method of polymer self-assembly that may be useful for producing nanoreactors. In Flash NanoPrecipitation, an amphiphilic block copolymer and hydrophobic core material are dissolved in a water miscible organic solvent and rapidly mixed against water using a confined impinging jet mixer. Upon mixing, the rapid decrease in solvent quality causes the hydrophobic core material to precipitate and the block copolymer to micellize directing formation of the overall nanocomposite particle. This particle assembly ends when the hydrophobic block of the block copolymer adsorbs on the precipitating core material preventing further growth, while the hydrophilic block sterically stabilizes the nanoparticle. Given the molecular weight of the block copolymer, dynamic exchange of the block copolymer does not occur ,19,20, sHydrophobic, inorganic nanoparticles have been incorporated into nanocomposite particles by dispersing the nanoparticles with the dissolved block copolymer and then mixing with confined impinging jets. Upon mixing, colloidal aggregation and block copolymer self-assembly occur due to the decrease in solvent quality. Nanocomposite particle assembly is complete when sufficient hydrophobic blocks of the block copolymer adsorb to the nanoparticle clusters to prevent further colloidal aggregation. For example, Gindy et al. demonstrated fabrication of polymer nanostructures containing colloidal gold using Flash NanoPrecipitation . More reOther important considerations when using the nanocomposite particles as nanoreactors are the reaction and diffusion within the system. In small molecule micelle systems that are thermodynamically stable, there is constant molecular exchange between the bulk solvent, and the confined hydrophobic mesophase facilitates reaction ,9. In thIn this work, we use Flash NanoPrecipitation for rapid and scalable self-assembly of hybrid metal nanoparticle catalyst-polymer nanocomposite nanoreactors. Independently tuning the nanoreactor properties, namely size and gold loading, is investigated. We focus on fundamental understanding of reaction and diffusion using the reduction of 4-nitrophenol as a model reaction. Kinetic and scaling analysis following the induction time are also discussed.W 800–5000 g/mol) was purchased from Polysciences, Inc. Sodium borohydride and 4-nitrophenol were purchased from Sigma Aldrich . Dodecanethiol (DDT) stabilized 5 nm nanoparticles, tetrahydrofuran (THF), HPLC grade), ethanol (ACS reagent grade), and diethyl ether (ACS reagent grade) were purchased from Fisher Scientific . Environmental Grade Hydrochloric Acid 30-38% and Environmental Grade Nitric Acid 70% were purchased from GFS Chemicals . The 1H-NMR solvent D2O with 4,4-dimethyl-4-silapentane-1-sulfonic acid DSS as an internal standard was purchased from Cambridge Isotope Lab, Inc . These chemicals and materials were used as received. Polystyrene-b-polyethylene glycol  was obtained from Polymer Source (Product No. P13141-SEO). Prior to use, PS-b-PEG was dissolved in THF (500 mg/mL) and precipitated in ether (~1:20 v/v THF:ether). The PS-b-PEG was recovered by centrifuging, decanting, and drying under vacuum at room temperature for 2 days.Citrate stabilized 5 nm gold nanoparticles were purchased from Ted Pella. Polystyrene  were precipitated into ethanol (45 mL) and filtered using a Buchner funnel. The filtered nanoparticles from the filter cake were resuspended in THF and concentrated via evaporation at room temperature overnight to achieve a nominal concentration of around 20 mg/mL. The final concentration was confirmed by inductively coupled plasma optical emission spectroscopy using an Agilent 5110 . UV spectra collected on an Ocean Optics FLAME-S-UV-VIS with a HL-2000-FHSA light source  were compared before and after the solvent switch to confirm processing did not significantly affect gold nanoparticle size.For nanoreactor self-assembly, typically, PS-b-PEG (6 mg), dodecanethiol stabilized 5 nm gold nanoparticles (0.5 mg), and PS homopolymer  were added to 0.5 mL of tetrahydrofuran (THF) and sonicated at 55 °C for 30 min. Using a manually operated confined impinging jet mixer with dilution (CIJ-D) ,24 with v/v aqua regia. Gold concentration of the digested sample was measured using inductively coupled plasma optical emission spectroscopy measurements with an Agilent 5110 .Nanoreactor size was measured after mixing using a Malvern Zetasizer Nano ZS  with a backscatter detection angle of 173°. Size distributions are reported using the average of four measurements of the intensity weight distributed with normal resolution. The reported size is the peak 1 mean intensity. The polydispersity index (PDI) is defined from the moment of the cumulant fit of the autocorrelation function calculated by the instrument software (appropriate for samples with PDI < 0.3) and is reported as a measure of particle size distribution. UV absorbance spectra (300 to 1200 nm) of the nanoparticle dispersions were measured at room temperature with an Ocean Optics FLAME-S-UV-VIS with a HL-2000-FHSA light source . For visualization by TEM, samples were submerged in a dilute dispersion of nanoreactors (10-fold dilution with water) for one hour and dried at ambient conditions overnight. Samples were imaged using a Zeiss Libra 120 TEM  using an accelerating voltage of 120 kV. To determine the gold nanoparticle concentration, nanoreactor dispersions were dissolved in THF and digested in aqua regia (1:3 nitric acid:hydrochloric acid by volume) and diluted to 5% appk) were determined from tracking the absorbance at 425 nm as a function of time. The values of appk and induction time are the averages (± standard deviations) of at least 3 trials of each experiment. Detailed procedures are provided in the The catalytic performance of the nanoreactors was evaluated using the reduction of 4-nitrophenol with sodium borohydride as a model reaction using well established procedures ,26. BrieFor more detailed kinetic analysis, we performed full kinetic analysis considering the two-step reaction mechanism previously established . Full ki1H-NMR spectroscopy and pulsed field gradient (PFG) NMR, combined with saturated transfer difference (STD) spectroscopy, using a Bruker 800 MHz cryo-probe  was performed in accordance with the methods described in the To evaluate effective transport of the 4-nitrophenol, To perform Flash NanoPrecipitation, dodecanethiol stabilized 5 nm gold nanoparticles were dispersed in THF with the molecular dissolved, PS and PS-b-PEG, and rapidly mixed with water using a hand-operated confined impinging jet mixer. The entire formation process was accomplished in less than a second; further, the process can be performed continuously at large scales ,30,31. DWe further characterized the nanoreactors using UV-Vis spectroscopy. Prior to Flash NanoPrecipitation, the dodecanethiol-stabilized nanoparticles dispersed in toluene showed a peak absorbance at 495 nm (as received and after switching solvents). The nanoreactors showed a peak absorbance of 520 nm b. The peThe structure of the nanocomposite particles was visualized using TEM. Based on TEM imaging, clustering of the gold nanoparticles during assembly resulted in multiple catalytic gold nanoparticles per nanoreactor. The majority of the gold nanoparticles appear to be in the nanoreactor core, although multiple polymer layers are not visible on TEM due to low electron density. This result is consistent with previous reports of encapsulated gold nanoparticles via Flash NanoPrecipitation (FNP) ,35. BaseNext, we aimed to independently tune the nanoreactor properties, size and gold loading, using formulation parameters. Nanoreactor assembly depends on the relative time scales of block copolymer micellization, gold nanoparticle clustering, and co-precipitate nucleation and growth. Therefore, the overall nanoreactor size can be affected by the ratio of core material to block copolymer, as well as the total concentration of components in the organic stream .Varying the ratio of block copolymer to core materials has been an effective method for tuning nanostructure size via Flash NanoPrecipitation ,36. To vNext, we aimed to vary the gold loading independently of nanoparticle size. Holding the total core material mass constant and varying the ratio of gold to polymer resulted in a decrease in nanoparticle size with increasing gold concentration. In contrast, with gold nanoparticles and block copolymer without a co-precipitate, Gindy et al. observed that increasing the gold loading results in an increase in nanocomposite particle size that is attributed to the increase in the amount of gold core relative to the block copolymer . The difR is the aggregate radius, K is a constant of proportionality for formation time, Bk is Boltzmann’s constant, T is the absolute temperature, corec is the concentration of core material, BCPc is the concentration of block copolymer, µ is the solvent viscosity, and ρ is the core material density. This model suggests that the nanoreactor size is affected by the volume more than the mass of the core. Thus, as an alternative to holding the mass of the core constant, we held the volume of the core constant, according to:To guide nanoreactor formulation, the Smoluchowski diffusion limited aggregation model has previously been used to formulate inorganic nanoparticle-polymer nanocomposite particles via Flash NanoPrecipitation . Based o ~130 nm b.Overall, nanoreactors were assembled in a rapid, scalable, single-step method using Flash NanoPrecipitation. Nanoreactor size could be tuned independently of gold nanoparticle loading by varying the total solids concentration at a constant ratio of gold to polystyrene. Interestingly, the gold nanoparticle loading was tuned independently of nanoreactor size by varying the ratio of gold to polystyrene at constant total core volume. The constant core volume approach may be useful for formulations of multiple components with disparate densities e.g., inorganic particle-polymer nanocomposite particles.To evaluate the catalytic performance of the nanoreactor, the reduction of 4-nitrophenol by sodium borohydride was used as a model reaction . First, 1 would be slightly underestimated. The conversion of 4-nitrophenol confirmed the gold nanoparticles associated with the nanoreactors were catalytically active , which is a ratio of the reaction rate to the diffusion rate given by:reported ,47. The [−8 m2/s . Using tappk increased; the 2nd order rate constant was on the order of 106 M−1s−1. These values are much lower than the kbm ~ 108 M−1s−1, indicating that neither internal nor external diffusion from the bulk solution to the nanoreactor limited the apparent reaction kinetics.A complementary approach was to consider the bimolecular reaction between 4-nitrophenol and nanoparticle catalyst using the Smoluchowski diffusion limited reaction model ,49. We va and kb observed for the gold encapsulated within the nanoreactors are comparable to ligand-free gold nanoparticles. This result suggests that the reactivity of the gold nanoparticle surface is not significantly affected by self-assembly and their incorporation into the nanoreactors.Since there were no indications of diffusion limitations associated with the reaction following the induction time, we further characterized the reaction kinetics using Langmuir-Hinshelwood kinetics. Based on the previously established two-step reaction model , and fitOverall, diffusion and partitioning of sodium borohydride into the polymer nanoreactor affect the induction time for the reaction. Sufficient equilibration time between adding the sodium borohydride and the 4-nitrophenol (~3 min) for the borohydride to partition and diffuse minimizes induction time. Notably, mass transfer effects are not observed after the induction time and the intrinsic kinetics are comparable to ligand-free gold nanoparticles.Overall, we have presented rapid, scalable self-assembly of metal nanoparticle catalyst-polymer nanocomposite particles as nanoreactors. The size and gold loading of the nanoreactors can be tuned independently, with sizes and nominal loadings ranging from 100–200 nm and 4–50 wt% respectively. Using the 4-nitrophenol reduction as a model reaction, the induction time is affected by sequence or reagent addition, time between addition, and reagent concentration. Combined, our experiments indicate that the induction time is most influenced by diffusion of sodium borohydride. Scaling analysis and effective diffusivity measured using NMR, the observed reaction rate after the induction time are reaction- rather than diffusion-limited. Finally, the intrinsic reaction kinetics of gold associated with the polymer were comparable to ligand-free particles indicating the self-assembly process and resulting polymer microenvironment did not de-activate or block the catalyst active sites. Building on this foundational study, practical considerations such as nanoreactor recycling will be considered in future work.


It is crucial to understand the factors that introduce variability before applying metabolomics to clinical and biomarker research.1H NMR spectroscopy in plasma samples.We quantified technical and biological variability of both fasting and postprandial metabolite concentrations measured using In the Netherlands Epidemiology of Obesity study , 148 metabolite concentrations (101 metabolites belonging to lipoprotein subclasses) were measured under fasting and postprandial states . Technical variability was evaluated among 265 fasting and 851 postprandial samples, with the identical blood plasma sample being measured twice by the same laboratory protocol. Biological reproducibility was assessed by measuring 165 individuals twice across time for evaluation of short- (<6 months) and long-term (>3 years) biological variability. Intra-class coefficients (ICCs) were used to assess variability. The ICCs of the fasting metabolites were compared with the postprandial metabolites using two-sided paired Wilcoxon test separately for short- and long-term measurements.1H NMR spectroscopy (median ICC = 0.99). Postprandial metabolite concentrations revealed slightly higher ICC scores than fasting ones in short-term repeat measures . Variability did not increase further in a long-term repeat measure, with median ICC in postprandial of 0.64 and in fasting metabolite concentrations 0.66.Both fasting and postprandial metabolite concentrations showed high technical reproducibility using Technical reproducibility is excellent. Biological reproducibility of postprandial metabolite concentrations showed a less or equal variability than fasting metabolite concentrations over time. Metabolomics, as one of the pillars within the “omics” technologies, have been widely investigated in the search for disease predictors in clinical and preclinical studies –3. Despi1H Nuclear Magnetic Resonance spectroscopy (NMR)-based platform, Nicholson et.al. showed that nearly 60% of the biological variation could be attributed to stable familial and individual-environment factors in a twin study [1H NMR-based metabolite concentrations [The variability attributable to inter-individual variation is normally defined as reproducibility  and exprin study . Anothertrations . Sampsontrations .Cardiovascular risk prediction and other biomarker research is generally limited to samples taken in the fasting state. However, others and we have also begun to explore the utility of non-fasting measures , 12. NonIn this study, we extend the previous metabolomics reproducibility studies by (1) taking both fasting and postprandial metabolite concentrations into account to evaluate variability and 2) considering both short- and long-term repeat measures  of reproducibility. In short, we aim to estimate how much technical and biological variability there is in metabolite concentrations when sampling an individual in fasting and postprandial states as well as when repeating the experiment in the same individual over two different time intervals.2 or higher living in the greater area of Leiden (in the west of the Netherlands) were eligible to participate in the NEO study. In addition, all inhabitants aged between 45 and 65 years from one municipality (Leiderdorp) were invited irrespective of their BMI. Participants were invited for a baseline visit at the NEO study center in the LUMC after an overnight fast. Prior to their visits, participants completed a questionnaire at home with demographic, lifestyle and clinical data. Fasting blood samples were drawn after an overnight fast. Within the next five minutes after the fasting blood draw, a liquid mixed meal  derived from protein, 50 En% carbohydrates, and 34 En% fat) was consumed and subsequent blood samples were drawn 30 and 150 minutes after the meal, processed within 4 hours and stored at -80 degrees Celsius.The study was embedded in a population-based prospective cohort, the Netherlands Epidemiology of Obesity (NEO) study . All parFrozen serum extracts were shipped to the University of Bristol, UK, in freezer boxes containing 94 samples each. NMR-based metabolomics processing included a single thaw cycle, followed by lipid extractions performed by a single technician, and a subsequent sample preparation carried out by two technicians, with all liquid handling being performed on a Janus liquid handler. All samples of a single sample collection were processed all together. However, the order that the boxes were processed was randomised.Two hundred and sixty-five fasting and 851 postprandial randomly selected technical replicates  were processed at the University of Bristol, UK, in February of 2015 and again in October of 2016. These technical replicates provide a means to evaluate error that may be introduced by the NMR platform when repeating all NMR-processing steps on two aliquots of the same biological sample with a 20-month gap.Biological or experimental repeats, that is repeating the entire experiment—food challenge and all—on a sub-sample of NEO participants, were also collected. From 2011 to 2012, 183 NEO participants were invited for a validation study of the baseline measures, and their blood samples were collected twice across time. Short-term biological reproducibility is specified as those resampled within 6 months, and long-term was determined as an interval longer than 3 years. For the current analyses, individuals were included based on their compliance to (1) overnight fasting ; (2) the meal challenge protocol  and (3) two repeated measures over time after exclusions . After selection, 87 short-term biological repeat and 78 long-term biological repeat participants were included in the analyses of biological repeatability. Since the validation project was conceived in the later phase of the NEO study, most of the revisits occurred in the last year of the NEO baseline collection, i.e. between 2011 and 2012.Metabolomic measurements were performed in both fasting and postprandial  plasma samples using the Nightingale high-throughput NMR metabolomics platform . No metaFor each metabolite, intra-class correlation coefficients (ICC) were calculated to evaluate technical and biological variability separately for fasting and postprandial samples. ICC as the measurement of variability is defined as the ratio of between-subject variance to the total variance composed of sum of between- and within-subject variance, which was calculated by one-way ANOVA random effect model. The corresponding 95% confidence intervals (CIs) were derived from bootstrapping using “ICCest” function in the R package “ICC” . The rep2 vs. 26.1 kg/m2; long-term: 30.4 kg/m2 vs. 30.2 kg/m2).Summaries of baseline characteristics of all the participants in this reproducibility study are presented in In total 1,116  samples were technically repeated, with the same blood plasma sample processed twice with the same laboratory protocols. With the exception of a single metabolite, we observed excellent technical reproducibility with an average ICC score above 0.99, in both fasting and postprandial states. As 101 out of 159 considered metabolites belong to the same category of lipoprotein subclasses and sizes and share high correlations among each other, the high average ICC score reflected the metabolite correlations accordingly. Only the metabolite related to estimated description of fatty acid chain length exhibited lower ICC values of 0.77 and 0.83 in fasting and postprandial states, respectively.−7, 95% CI: -0.037–-0.022). Notably, 3-hydroxybutyrate revealed markedly higher ICC scores in the postprandial state (ICC: 0.26 versus 0.65)  , and simus 0.65) . On the −10, 95% CI of location shift of medians: 0.023–0.041). Based on the four categorical cut-off ICC scores defined in the Methods (excellent to poor reproducibility), 26 metabolite concentrations changed a category between short- and long-term repeat measures, and among these, 20 metabolite concentrations showing excellent reproducibility in the short-term (with ICC≥0.75) decreased to good in the long-term (ICC between 0.51 and 0.74). For the remaining metabolite concentrations with good reproducibility in the long-term, alanine, albumin and lactate displayed merely poor reproducibility in the short-term   .Subsequent to the analysis above, we revisited the question of fasting/postprandial state reproducibility conditioned on the time interval between sampling dates. When evaluating only short-term replicates, we observed an even stronger contrast between fasting and postprandial states than above with the combined full dataset . 3-hydroTo estimate the proportion of variance explained by each of the factors discussed above as well as for sex and age, we fitted each metabolite level to a multifactorial linear mixed model. The total variance of each metabolite level was decomposed into fasting/postprandial state variation, time interval between visits, sex, age, inter-individual variability and remaining intra-individual variability  . On averExcellent technical reproducibility of both fasting and postprandial metabolite concentrations was observed in the current study, indicating limited variation being introduced by laboratory procedures or the technology. Overall, biological reproducibility at both fasting and postprandial states and in the dichotomized short-term and long-term repeatedly measured sub-samples, was good represented by high ICC scores. Interestingly, the liquid meal challenge appeared to reduce the variability among replicates relative to their fasting state. However, this effect is attenuated with increased time interval between the measures.Creatinine levels in serum are commonly used to assess kidney function . From th1H NMR spectroscopy in different types of samples. For plasma metabolite concentrations, a dietary standardization of 24 hours led to very stable inter-individual fasting metabolite concentrations, which did not substantially improve with prolonged diet standardization [Previous studies have shown that both acute (24 hours prior to the visit) and prolonged (two weeks prior to the visit) standardized diet normalized fasting metabolite level measurements using dization . The obsdization .On average, the fasting/postprandial state explained less than 2% of the total variance. Yet, for some of the specific metabolites, particularly amino acids, such as phenylalanine, valine, tyrosine and leucine, more than 30% of the variation was attributable to the fasting/postprandial state. In addition, XXL-, XL- and L-VLDL metabolite concentrations, estimated description of fatty acid chain length and monounsaturated fatty acids had ~10% of their total variation explained by fasting/postprandial state. However, the effect from the liquid meal seems greatly weakened after a repeat measure more than three years later, which might be explained by the individual characteristics changes across time far more significant and the metabolome standardization role from a liquid meal is diluted.There are a number of strengths in the current study. First, the relatively large sample size allowed for the stratification biological reproducibility after short- and long-term intervals. Second, a liquid meal provided insight in the effects of transient diet standardization on reproducibility of metabolite measurements. Several limitations should also be considered. First, the study population is composed of middle-aged individuals, which might have lifestyles different from a younger population, which may have affected the fasting metabolite measurements. Second, BMI was markedly lower in the short-term reproducibility study than the long-term reproducibility, though it is unlikely that this would affect our results. Thirdly, although we observed nearly perfect technical reproducibility in the current study, the sample run scheme was not specifically designed for testing technical variability (such as batch effect). Thus, potential confounding by factors influencing sample collection, preparation, and chemical analysis cannot be excluded.1H NMR spectroscopy showed on average better biological reproducibility than fasting ones after a short-term repeated measure and as good as fasting metabolite concentrations after a long-term repeated measure, which indicates the robustness of applying postprandial metabolites measured after a liquid mixed meal to biomarker research.Postprandial metabolite concentrations after a liquid meal measured using S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.


Insomnia and migraine are closely related; insomnia aggravates migraine symptoms. This study was conducted to investigate the impact of migraine on the clinical presentation of insomnia symptoms.The data of the Korean Headache-Sleep Study (KHSS) were used in the present study. The KHSS is a nation-wide cross-sectional population-based survey regarding headache and sleep in Korean adults aged 19 to 69 years. If a participant’s Insomnia Severity Index (ISI) score ≥ 10, she/he was classified as having insomnia. The clinical presentation of insomnia symptoms was assessed using total and subcomponent scores of the ISI.p <  0.001) and non-migraine headache  were higher among individuals with insomnia compared to those without insomnia. Among participants with insomnia, total ISI scores were not significantly different among participants with migraine, non-migraine, and non-headache . ISI scores for noticeability of sleep problems to others were significantly higher among participants with migraine  and non-migraine headache  compared to those without headache history. Other ISI subcomponent scores did not significantly differ between headache status groups.Of 2695 participants, 290 (10.8%) and 143 (5.3%) individuals were assigned as having insomnia and migraine, respectively. The proportions of migraine (12.8% vs. 4.4%, Participants with insomnia had an increased risk of migraine and non-migraine headache compared to those without insomnia. Among participants with insomnia, overall insomnia severity was not significantly influenced by the headache status. Migraine is a common neurological disorder and affects 5–15% of the general population . Even inInsomnia is another disorder with high prevalence, affecting 10–30% of the general population . InsomniMigraine and insomnia exhibit a strong relationship. Cross-sectional studies have persistently demonstrated a significant comorbidity for these two disorders in clinical and population-based studies . Two lonAccording to previous studies, migraineurs have an increased risk of developing insomnia compared to patients suffering from non-migraine headache and healthy subjects. Furthermore, migraineurs with insomnia present with a higher headache frequency and increased headache intensity compared to those without insomnia . NeverthThe data of the Korean Headache-Sleep Study (KHSS) were used in the present study. The KHSS was a nation-wide, cross-sectional survey regarding headache and sleep disorder among Korean adults aged 19 to 69 years. It also included items regarding symptoms of anxiety and depression. The study design, methods, and process were described in detail previously . Of the 1273 (47.2%) participants, who reported that they experienced at least one attack of headache during the last year, 143 (5.3%) participants were classified as having migraine. Therefore, 1130 (41.9%) were classified as having non-migraine headache (Table 0.8% partp < 0.001) and non-migraine headache  was significantly higher among participants with insomnia compared to that in participants without insomnia , 171 (59.0%), and 82 (28.3%) participants were classified as having migraine, non-migraine headache, and non-headache, respectively. The prevalence of migraine  and non-headache (p < 0.001).Insomnia prevalence among participants with migraine, non-migraine headache, and non-headache was 25.9%, 15.1%, and 5.8%, respectively Fig. . The prep = 0.003) and the Headache Impact Test-6  score were significantly higher in migraineurs with insomnia compared to those in migraineurs without insomnia. Headache frequency per month was discernibly different according to the presence of insomnia without reaching statistical significance . Among participants with non-migraine headache, headache frequency per month, VAS score for headache intensity, and HIT-6 score were significantly higher when insomnia was present  score for headache intensity  The prevalence of migraine and non-migraine headache was significantly higher among participants with insomnia compared to those without insomnia; 2) Among participants with insomnia, the total ISI score was not significantly different among migraine, non-migraine headache, and non-headache groups and 3) Among participants with migraine, the prevalence of insomnia was higher than participants with non-migraine headache and non-headache. Migraine symptoms exacerbated with the presence of insomnia.There is limited information available about the impact of insomnia on the clinical presentation of headache. It has been reported that in individuals with headache or migraine, those with insomnia present an increased symptom severity compared to those without . NeverthWhat could be the underlying mechanism for the difference between the impact of migraine on insomnia severity and the impact of insomnia on migraine severity? It is possible that their distinct anatomy and pathophysiology contribute to the contrast findings. Hypothalamus has been understood to play key regulatory roles both for migraine and sleep controls. The supraoptic nucleus in anterior region plays a key role in the regulation of sleep and arousal. Pain perception was regulated by arcuate nucleus in tuberal region . AlthougAmong the ISI subcomponents, the score for noticeability of sleep problems to others was significantly higher among participants with migraine and non-migraine headache compared to participants without headache. Furthermore, the score for non-refreshing sleep were significantly higher among participants with non-migraine headache compared to participants without headache Table . In contIn the present study, we used the ISI to assess insomnia. This psychometric scale includes items for difficulty in falling asleep, difficulty in staying asleep, and early awakening which enables the classification of insomnia subtypes . AlthougThe present study has some limitations. First, we could not evaluate whether participants of this study underwent pharmacological or non-pharmacological treatment for insomnia. Second, as this is a cross-sectional study, we could not investigate changes in insomnia symptoms before and after migraine treatment. Third, the overall response rate was not high in our study. However, we used a two-stage clustered random sampling, proportional to the population distribution of Korea. Therefore, the distribution of age, sex, size of residential area, and educational level of our participants was similar to those of the Korean general population. In addition, the prevalence of migraine, non-migraine and insomnia in the KHSS were similar to that of previous studies , 33. DesSubjects with insomnia have an increased prevalence of migraine and vice versa in a population-based setting. Although insomnia is associated with increased headache frequency and severity among migraineurs, insomnia severity is, apart from some subcomponents, not significantly influenced by the presence of migraine. Our findings indicate that migraine and insomnia affect each other but their asymmetric causal relationship needs further investigation in future studies.


Major depressive disorder is the main cause of disability worldwide with imperfect treatment options. However, novel therapeutic approaches are currently discussed, from augmentation strategies to novel treatments targeting the immune system or the microbiome-gut-brain axis. Therefore, we examined the potential beneficial effects of minocycline, a tetracycline antibiotic with pleiotropic, immunomodulatory action, alone or as augmentation of escitalopram on behavior, prefrontal microglial density, and the gut microbiome in rats selectively bred for high anxiety-like behavior (HAB). We show that concomitant with their high innate anxiety and depression, HABs have lower microglial numbers in the infralimbic and prelimbic prefrontal cortex and an altered gut microbiota composition compared with controls. Three weeks of minocycline treatment alleviated the depressive-like phenotype, further reduced microglial density, exclusively in male HAB rats, and reduced plasma concentrations of pro-inflammatory cytokines. However, coadministration of escitalopram, which had no effect alone, prevented these minocycline-induced effects. Moreover, minocycline led to a robust shift in cecal microbial composition in both HABs and rats non-selected for anxiety-like behavior. Minocycline markedly increased relative abundance of Lachnospiraceae and Clostridiales Family XIII, families known for their butyrate production, with a corresponding increase and positive correlation in plasma 3-OH-butyrate levels in a trait-dependent manner. Thus, our data suggest that the antidepressant effect of minocycline is sex- and trait-dependent, associated with a reduced microglial number in the prefrontal cortex, and with changes in microbial composition and their metabolites. These results further support the microbiome-gut–brain axis as potential target in the treatment of depression. Numerous treatment options for major depressive disorder (MDD) are available such as the frequently prescribed and highly efficacious selective serotonin reuptake inhibitor (SSRI) escitalopram3. However, antidepressants display a therapeutic lag of 2–4 weeks, and only 70% of patients respond sufficiently4. To achieve therapies with faster onset, two major approaches can be utilized, i.e. optimization of existing options or the discovery of agents with novel mechanisms of action. Treatment regimens move towards augmentation strategies, a combination of conventional antidepressants with non-antidepressant substances to increase efficacy and overcome treatment resistance5. Regarding novel targets, central inflammation and the microbiome-gut–brain axis are two areas receiving growing traction7.According to the World Health Organization, depression has become the leading cause of disability worldwide affecting approximately 300 million people with a lifetime prevalence of 10.7% in men and 18.1% in women in the US11. A significant influence of microbiota on microglia activation and maturation, potentially via bacteria-produced short-chain fatty acids like butyrate, has been reported13. This points towards a complex role of microbiota and microglia in anxiety and depression, suggesting a subtle interplay between behavior, microglia and microbiota. In light of this, one attractive treatment option is the pleiotropic broad-spectrum tetracycline antibiotic minocycline, with neuroprotective and anti-inflammatory properties, e.g. inhibiting microglial activation17, which also affects gut microbiota18. Recent clinical studies have shown that minocycline can augment the clinical efficacy of antidepressants21, an effect that was proposed to be dependent on the presence of an inflammatory state22. These findings are supported by several rodent studies showing that minocycline promotes active stress coping24 and reverses inflammation-induced26 as well as stress-induced depressive-like behavior28. However, substantial evidence is required from appropriate animal models regarding behavioral effects of minocycline and particularly relating to its mechanism(s) of action.Thus, dysregulation in microglia, the immune system of the brain, as well as the gut microbiome have been associated with MDD29. This approach gave rise to Wistar rats with extremely high anxiety-related behavior (HAB) based on their performance on the elevated plus-maze (EPM), a phenotype repeatedly confirmed independent of sex. Moreover, HAB rats consistently show similar characteristics as observed in MDD patients including passive stress-coping, altered serotonin and neuropeptide signaling in the brain and, consequently, dysregulated responses of the hypothalamic–pituitary–adrenal (HPA) axis33. The highly anxious and depressive-like phenotype of male HAB rats has been successfully reversed by chronic citalopram or paroxetine treatment35. However, this required an 8-week treatment regimen, suggesting HAB rats as an animal model for comorbid anxiety and depression, as well as treatment-resistant depression. Amelioration of these behavioral deficits in female HAB rats remains to be studied, an approach urgently needed considering the more frequent diagnosis of MDD in women30.One of the most heuristic approaches to develop appropriate animal models is via selective breeding for a particular trait40. Fittingly, intra-PFC citalopram microinfusion acted as an antidepressant41, whereas glial loss in this region facilitated depressive-like behavior42. Moreover, stress- or inflammation-induced depressive-like behavior paralleled by PFC microglial activation was prevented by minocycline treatment43. Importantly, acute inactivation of the infralimbic cortex in HAB rats attenuated depressive-like behavior, corresponding to the behavioral consequences of deep brain stimulation of Brodmann Area 25 in MDD patients44, indicating the translational relevance of this animal model and the importance of this region.Changes in several cortical and limbic structures like the hippocampus, amygdala and especially the prefrontal cortex (PFC) are documented in the pathophysiology of depressionBased on these prerequisites, we assessed the effects of chronic minocycline treatment on anxiety- and depressive-like behavior in rats in dependence on trait anxiety levels in both sexes. For translational comparison with clinical studies using minocycline as an add-on treatment, the standard SSRI escitalopram and the augmentation of minocycline with escitalopram were included. In order to approach potential mechanisms underlying the differential treatment effects, we further examined microglia density in the PFC, peripheral immune function comprising pro-inflammatory cytokines, and alterations in gut microbiota. Male and female rats non-selected for anxiety (NAB) were included to determine a trait-dependency of treatment.45. All rats were maintained on a 12-h light–dark cycle in a temperature controlled colony  with free access to food and tap water (vehicle) or drug solution  under facility-specific husbandry conditions according to the respective EU regulations (2010/63/EU). All experimental procedures were approved by the Committee on Animal Health and Care of the local government, and performed according to the Guide for the Care and Use of Laboratory Animals of the Government of Oberpfalz and Unterfranken, the ARRIVE guidelines46, and recommendations from the NIH.Male and female NAB rats were either purchased from Charles River  or, like all male and female HAB rats, bred at the University of Regensburg. HAB rats showing <10% of time spent on the open arms of an EPM at the age of 9 weeks were selected43, 10 mg/kg/day escitalopram47, or a combination of both substances with the same concentrations, while in the second set, 80 mg/kg/day minocycline alone, all dissolved in tap water, was applied for 22 days. The treatment duration was chosen based on the fact that antidepressant treatment generally needs 2–3 weeks for the onset of therapeutic efficacy48 and on a previous study showing an antidepressant effect of minocycline already after 7 days28. Minocycline hydrochloride and escitalopram oxalate were kindly provided by the Charité Berlin and Lundbeck A/S , respectively.At the age of 11–12 weeks, rats were group-housed in cages of 3–4 rats according to their experimental group (i.e. sex and trait) and randomly assigned to the treatment. Body weight and water consumption were monitored daily to calculate the respective concentrations needed for oral drug administration via drinking water and to prepare fresh solutions daily. In the first set of experiments, 40 mg/kg/day minocyclineRats were tested in the social preference test , in the light–dark box  and on the EPM (day 19), as well as in the forced swim test  method was developed and optimized for detection of minocycline in liver tissue and fecal boli. The limit of quantitation was 50 ng/ml with an averaged recovery from homogenates of 95%, with a 5–10% intra- and inter-assay coefficient see .Beta-hydroxybutyrate concentrations  in plasma were determined with a commercial assay kit . The enzyme reaction results in a colorimetric product proportional to the presence of the metabolite 3-OH-butyrate see .Luminex xMAP 100 system  according to the manufacturer’s protocol (see Interferon (IFN)-γ and interleukin (IL)-12p40 concentrations were quantified in plasma using a cytokine bead immunoassay  and the ocol see .The methods used for the isolation of DNA from stool specimens, quantification of 16S-rDNA copies by qPCR, PCR amplification of V3-V6 16S-rDNA variable regions, 454 pyrosequencing . Significance was accepted at p ≤ 0.05.Behavioral data, plasma 3-OH-butyrate, and cytokine concentrations were analyzed with the software package SPSS (version 12) using either one-way analysis of variance , two-way ANOVA (factors trait×treatment) or two-way ANOVA for repeated measures (factors trait×treatment×stimulus) followed by a Bonferroni post hoc test for multiple comparisons when appropriate. For analysis of cytokine concentrations, a Mann–Whitney p < 0.001; Fig. p < 0.05 vs. Veh). In both the LDB and EPM, the expected anxious phenotype of HAB rats was visible in both sexes compared to NAB rats .Male and female HAB and NAB rats were treated with either minocycline (40 mg/kg), escitalopram or a combination of both via the drinking water for 22 days, and fluid intake was monitored daily  was performed first, and the most stressful test (FST) last, with a full day of rest between two tests to avoid stress- or experience-induced bias.In the present study, we confirm the antidepressant effect of minocycline and reveal that chronic minocycline treatment reduces depressive-, but not anxiety-like, behavior and affects the gut–brain axis in a sex- and trait anxiety-dependent manner. These findings extend previous reports of antidepressant actions of minocycline59 or citalopram60 treatment has been described to ameliorate chronic stress-induced depressive-like behavior in both rats and mice, and citalopram and paroxetine given over 8 weeks have also been proven to be effective in HAB males35. In the present study we could only reproduce the anxiolytic effect of escitalopram in male NAB rats in the LDB, but not in HAB or female rats, probably due to the short treatment time. Importantly, these results support a sex difference in treatment strategies for MDD and validate the HAB breeding line as a model for innate treatment-resistant depressive-like behavior with minocycline as a potential treatment.Chronic escitalopram5. Fittingly, minocycline treatment, in combination with several conventional antidepressants like SSRIs or antipsychotics, improved MDD symptoms21. However, a study in depressed patients revealed that administration of citalopram in combination with a non-steroidal anti-inflammatory drug inhibited remission61. In keeping, in the present study 3-week treatment with minocycline together with escitalopram abolished the antidepressant effect of minocycline. In agreement with our results, minocycline in combination with the SSRI fluoxetine, but not the tricyclic desipramine, prevented the antidepressant effects of minocycline in rats24. Together, these findings strongly suggest that minocycline specifically counteracts a mechanism responsible for the antidepressant-like effect of SSRIs. Though minocycline interferes with the effectiveness of escitalopram in this case, it does not necessarily target the monoaminergic mode of action, but might target substance-specific secondary mechanisms or elicit local pharmacokinetic interactions.Clinical studies provide growing evidence for an increased efficacy of adjunctive treatment in MDD63 and - as an antibiotic - to modulate the gut microbial composition18. Neuroinflammation and increased microglial activation have been associated with MDD65. However, a dynamic and bi-directional alteration in microglial status has recently been hypothesized to underlie depressive-like behavior, as microglial numbers were found to be initially elevated followed by their decline in the hippocampus and PFC of chronically stressed rodents that displayed depressive-like behavior51. The lower microglial density in the PFC of HAB rats is likely due to their permanent stress state, as shown by elevated trait anxiety and a hyper-reactive HPA axis66. Interestingly, administration of minocycline further reduced microglial numbers, specifically in male HAB rats, which paralleled the antidepressant-like behavioral effect. Similar to a reduction in microglia, acute inhibition of the infralimbic cortex in male HAB rats led to an antidepressant-like phenotype39, indicating the PFC to be involved in depressive-like behavior in male HAB rats. Minocycline has been shown to inhibit microglial proliferation67 and, in view of a constant rate of microglial apoptosis68, should therefore lead to a further reduced microglial density in male HAB rats. Although the underlying mechanisms of the dynamic interplay between proliferation and apoptosis need to be explored in more detail, our results point towards an impaired microglial homeostasis and functioning as an underlying mechanism for the depressive-like behavior in HAB rats, and further support our breeding line as a model for inflammation-associated depressive-like behavior. Of note, the reduction in microglial numbers by minocycline was abolished, when given in combination with escitalopram.Minocycline has been repeatedly demonstrated to inhibit microglial activity and proliferation accompanying its effects on behavior12. Gut microbiota dysbiosis, in turn, can result in neuroinflammation by chronic activation of microglia in the CNS69, potentially contributing to the behavioral phenotype in HAB rats. Supporting the link between the microbiome and emotional behavior70, HAB and NAB rats originating from the same breeding facility show - despite comparable total bacterial numbers in their cecal contents - profound differences in global gut microbiota composition, reflecting the robust behavioral differences between the two lines. As expected, 3 weeks of minocycline treatment reduced bacterial richness similarly in both groups resulting in still persisting, but diminished group differences. This suggests a partial equalization of the microbiota between both lines that might contribute to the observed antidepressant-like effect of minocycline in HAB rats. Interestingly, in patients suffering from MDD, an altered microbial composition was observed that induced similar symptoms in rats upon transplantation72.The microbiome-gut–brain axis has been shown to modulate microglia proliferation and maturation. For example, phenotypical abnormalities of microglia found in germ-free mice were reversed by a microbiota-derived short-chain fatty acid mixture containing butyrate18. We further show that minocycline elevated relative Clostridiales Family XIII levels in both HAB and NAB rats. Both Lachnospiraceae and Clostridiales Family XIII accommodate a large part of butyrate-producing bacterial genera, reflected by increased plasma 3-OH-butyrate levels especially in HAB, but also in NAB rats, after minocycline. In support, a positive correlation between the abundance of Lachnospiraceae, as well as the Clostridiales Family XIII, with 3-OH-butyrate was found in male HAB rats. In contrast, in male NABs a positive correlation was only found with Clostridiales Family XIII. Both butyrate and 3-OH-butyrate have been proposed as anti-inflammatory agents that execute their effects via inhibition of peripheral T cell activity73 and microglia activation74, and even via inducing microglial apoptosis75. In line, minocycline decreased the level of the pro-inflammatory cytokine IFN-γ, produced by IL-12-induced and activated T-helper type 1 cells76, in both HAB and NAB males. Plasma levels of IL-12p40 appeared to be higher in HAB rats and were reduced by minocycline to NAB levels, supporting a regulatory role of minocycline and butyrate on peripheral T cells77 independent of the central immune system that might contribute to the antidepressant effect of minocycline. Although a role of butyrate in the regulation of the central immune system remains to be studied, our results suggest that butyrate-mediated microglial apoptosis75 might contribute to the observed reduction in microglial density in the PFC of male HAB rats. Furthermore, butyrate has been shown to exert antidepressant-like effects in mice79 and rats80. These findings point towards an important role of bacteria-produced butyrate as a mediator of behavioral and microglial alterations in minocycline-treated HAB rats. Thus, our results support the hypothesis of an influence of the gut microbiome on anxiety- and depressive-like behavior in our rodent model of anxiety and depression.Importantly, detailed microbiome comparison between HAB and NAB rats revealed decreased relative abundance of the Lachnospiraceae family in HABs, which expanded after minocycline treatment. Similar dynamic changes in Lachnospiraceae were found in a stress model of depressionIn summary, we show that the depressive- and anxiety-like phenotype of HAB rats is accompanied by reduced microglial numbers in the infralimbic/prelimbic PFC and a distinct shift of the gut-bacterial composition. This emphasizes that HAB rats are a valid model for inflammation-associated depressive-like behavior and points towards a complex interplay between microbiota/microbial metabolites and the immune system, as proposed by the concept of the microbiota-gut–brain axis.In the course of clinical application, the present results indicate that promoting an anti-inflammatory state by control of microglial activation and/or modulation of the gut microbiome and metabolome, as shown after prolonged minocycline treatment, offers promising potential as a therapeutic strategy in treatment-resistant MDD. The present study was designed to detect an early response (3 weeks) to minocycline and did not mimic a clinical antidepressant treatment. Thus, a longer treatment duration might reveal different effects. Nevertheless, caution is needed when considering minocycline as an augmentation strategy of SSRIs like escitalopram, especially in the absence of a pro-inflammatory profile, which may explain some of the recent high-profile clinical failures of minocycline.Supplementary methods and results


With the exception of renal cell carcinoma, studies assessing the association between hypertension and other cancers are inconsistent. We conducted a meta-analysis to assess this evidence. We included observational studies investigating the association between any definition of hypertension or systolic and diastolic blood pressure and risk of any cancer, after searching PubMed until November 2017. We calculated summary relative risks (RR) and 95% confidence intervals (CI) using inverse-variance weighted random effects methods. A total of 148 eligible publications were identified out of 39,891 initially screened citations. Considering only evidence from 85 prospective studies, positive associations were observed between hypertension and kidney, colorectal and breast cancer. Positive associations between hypertension and risk of oesophageal adenocarcinoma and squamous cell carcinoma, liver and endometrial cancer were also observed, but the majority of studies did not perform comprehensive multivariable adjustments. Systolic and diastolic blood pressure were positively associated with risk of kidney cancer but not with other cancers. In addition to the previously well-described association between hypertension and risk of kidney cancer, the current meta-analysis suggested that hypertensive individuals may also be at higher risk of colorectal and breast cancer. However, careful interpretation is required as most meta-analyses included relatively small number of studies, several relative risks had weak or moderate magnitude and maybe affected by residual confounding. It is responsible for almost 50% of heart disease, stroke, and heart failure and about 14% of total deaths in 2015 were related to hypertension2. In addition, an estimated 10% of the healthcare spending is directly related to increased blood pressure and its complications3.Hypertension and cancer are two multifactorial, severe and chronic conditions. Hypertension is a major health problem worldwide, as it affects approximately 3 in 10 adults over age 20, leading to high morbidity and mortality4. Therefore, if blood pressure and/or hypertension are associated with the risk of cancer, this may have important public health consequences.Cancer is also a leading cause of morbidity and mortality. Worldwide, there were approximately 18 million new cases and 9.6 million cancer-related deaths in 2018, and these numbers are expected to rise within the next two decades6, although it is not clear yet whether reverse causation explains part or all of this association7. Claims of association also exist for colorectal, breast, endometrial and prostate cancer12, but the evidence is inconsistent. But recently there has been an influx of new studies investigating the association of metabolic syndrome, which includes hypertension, and cancer risk14, and therefore we conducted a systematic review and meta-analysis to summarize and evaluate this literature.Renal cell carcinoma is the only cancer type that has been consistently associated with hypertension(blood pressure OR hypertension OR “metabolic syndrome”) AND , and also hand-searched for potential missed studies in the references of eligible systematic or narrative reviews24. We considered all cohort and case-control studies conducted in humans assessing the association between systolic blood pressure (SBP) or diastolic blood pressure (DBP) or hypertension and risk of any cancer development or cancer death. Hypertension was defined as any of the following: self-reported or measured SBP or DBP higher than predefined cut-off points (e.g. WHO or NCEP/ATP III criteria), self-reported medical history of hypertension or drug-treated hypertension. When the exposure was metabolic syndrome, we only used the hypertension component. We excluded narrative or systematic reviews, case-series or case-reports, cross-sectional studies, prognostic studies among cancer patients, studies in children, cohort studies with a sample size of less than 100 total individuals, case-control studies with less than 100 cancer cases, studies that did not provide enough information for calculating measures of association, studies in other than the English language and studies of pre-malignant outcomes . Whenever published reports pertained to the same cohort evaluated at different follow-up periods, we retained the publication with the longer follow-up.We searched for eligible published studies in PubMed from inception to November 2017 using the following search algorithm: We recorded information from each eligible study on author name, year and journal of publication, study design, sample size, population characteristics, definition of hypertension, relative risk (RR), 95% confidence interval (CI) and adjustments for confounders. When results from multiple statistical models were reported, we always retained the most adjusted model. The literature search and data extraction were performed independently by two investigators (AS and XT), and data was re-checked for consistency by a third author (SC).25.We used the “Newcastle-Ottawa Scale” to assess the quality of the included studies, and this task was performed independently by two investigators (AS and SC), and disagreements were resolved by a third author (KKT). Based on this tool, every study was judged on eight quality items, grouped into three categories: i) selection, ii) comparability of study groups and iii) exposure or outcome ascertainment for case-control and cohort studies, respectively. A star was awarded for every quality item for a maximum of 9 stars for the highest quality studies27. We summarized RRs and 95% CIs using fixed-effects and random-effects meta-analysis models, if three or more studies were available per exposure and outcome comparison28. Between-study heterogeneity was assessed by the Cochran’s Q test and the I2 statistic29. 95% prediction intervals were also calculated to further assess heterogeneity, which represents the range in which the effect estimates of future studies will lie30. Subgroup meta-analyses were conducted according to sex, study design (prospective vs. case-control) and adjustment factors (at least age vs. age plus further multivariable adjustment). Presence of small-study effects was assessed using the Egger’s regression asymmetry test31. Analyses were performed in STATA 12 . All p-values were two-tailed. The study is reported according to the MOOSE checklist32.Our primary analysis included only prospective studies , whereas analyses including case-control studies are provided in the online supplement. We conducted separate meta-analyses according to systolic or diastolic blood pressure and hypertension. Analyses for systolic and diastolic blood pressure used both a top to bottom category comparison approach and a dose-response approach per 10 mmHg. Studies that reported a dose-response estimate were either pooled directly or after re-scaling to the corresponding increment unit using the generalized weighted least-squares regression model approach116, 72 of which were cohort studies, 11 were nested case-control studies111 and 2 were record-linkage studies79, and 63 case-control studies178. Most studies  investigated associations with cancer incidence. A total of 48 (32%) studies were conducted only in men, whereas 57 (39%) studies were conducted only in women. The majority of the studies  were conducted in the USA followed by Scandinavian countries , Italy , UK and Korea , and finally Japan with 6 studies (4%). Out of the 128 studies that used hypertension as the exposure of interest, this was defined in 20 studies (16%) using the NCEP-ATPIII criteria (≥130/85 mmHg), 17 studies (13%) used the WHO definition (≥140/90 mmHg), 4 studies (3%) used ≥160/95 mmHg, 38 studies (30%) pertained to self-reported hypertension, 16 studies (13%) used self-reported drug treatment for hypertension and 26 studies (20%) used a combination of self-reported disease and treatment, whereas 7 studies used other definitions.Figure Detailed information on the quality assessment for each study, using the Newcastle-Ottawa scale, can be found on Supplementary Table For the primary analysis using only prospective studies, we conducted 30 meta-analyses of hypertension and 16 different cancers, and 29 meta-analyses between SBP or DBP and 11 cancers. Summary random effects relative risk estimates and 95% CIs per cancer site, heterogeneity statistics, 95% prediction intervals and tests of small-study effects are summarized in Fig. 109. We observed large heterogeneity , but no indication for small study effects 179. The association between hypertension and kidney cancer was statistically significant in both women  and men  without between-study heterogeneity within each sex-specific analysis, but considerable larger estimates were observed in women . Similar results were obtained in subgroup meta-analyses of prospective studies that at least adjusted for age vs. studies that adjusted for age plus at least three of the following five risk factors: body mass index, smoking, alcohol, physical activity and family history of kidney cancer  was observed  and DBP  with kidney cancer, but these meta-analyses had evidence of small-study effects , an association that remained significant after excluding one record-linkage study 76 or after meta-analysing only the four studies that performed multivariable adjustments 114. This association was statistically significant in men 114, but not in women 113. No significant associations were identified between SBP or DBP with colorectal cancer risk 110, but the association was not significant for post-menopausal breast cancer risk 87. However, statistically significant associations were observed for total 85, and post-menopausal  breast cancer risk in prospective studies that performed multivariable adjustment  and post-menopausal disease .The meta-analysis yielded a borderline significant association between hypertension and risk of breast cancer . After removing two record linkage studies79, we observed a significant RR of 1.37  but again the heterogeneity was substantial . Only two out of the nine prospective studies used multivariable adjusted models, and the meta-analysis among them yielded null results 116. When 13 case-control studies were meta-analysed together with the prospective studies, a statistically significant association was found . No analysis on SBP or DBP was performed, because there were not enough studies using continuous BP data.Our meta-analysis showed also a statistically significant association between hypertension and risk of endometrial cancer , but four out the five studies did not perform comprehensive multi-variable adjustments 111. We identified only 2 prospective studies on hypertension and esophageal squamous cell carcinoma77, and the meta-analysis yielded a statistically significant estimate , but was based only on 248 cases. Finally, the meta-analysis of all 6 prospective studies on total oesophageal cancer yielded a statistically not significant estimate . Most of these studies however did not perform comprehensive multivariable adjustments . Furthermore, a dose-response approach revealed a 5% and 7% higher risk for kidney cancer per every 10 mmHg higher SBP and DBP, respectively. Similar associations have been reported in previous meta-analyseset al. published another meta-analysis in 2013, which found a 9% relative increase in colorectal cancer due to hypertension using both cohort and case-control studies10. To our knowledge, no clear mechanism has been proposed to link hypertension to colorectal cancer, but hypertension has been shown to increase cancer risk by blocking apoptosis187. Current findings should be approached with caution given the scarcity of well-designed studies in the literature.We also estimated a positive summary association between hypertension and risk of colorectal cancer with a higher risk of 11% for hypertensive individuals. This meta-analysis included data from 13 prospective studies, only four of which performed comprehensive multivariable adjustments, and the potential higher risk was 30% for hypertensive individuals in these studies. Esposito et al. that reported a 7% higher risk for total breast risk in hypertensive individuals regardless of performed adjustments11. Suggestive mechanisms to explain this association involve blocking of apoptosis, adipose tissue related hypoxia and chronic inflammation promoting reactive oxygen species formation188.In a meta-analysis of 13 prospective studies, hypertension was associated with a 7% higher risk of total breast cancer. Only five of these studies performed comprehensive multivariable adjustments, where the risk was 10% for total breast cancer and 38% for post-menopausal disease comparing hypertensive to normotensive individuals. These findings are in agreement with a recent meta-analysis of 12 prospective studies by Han 9.Positive associations between hypertension and risk of esophageal adenocarcinoma and squamous cell carcinoma, liver, and endometrial cancer were also observed in the current meta-analysis, but these meta-analyses included a small number of prospective studies, ranging from 2 for oesophageal squamous cell carcinoma to 9 for endometrial cancer, and the majority of them did not perform comprehensive multivariable adjustments, raising serious concern over the validity of the estimated associations. Previous meta-analyses exist for endometrial cancer, but have in general shaped their conclusions without taking into consideration study design and quality12.We did not observe associations between hypertension or blood pressure and risk of stomach, gallbladder, pancreas, lung, cervix, prostate, bladder and brain cancer. A recent meta-analysis examining the association between hypertension and risk of prostate cancer used 21 cohort and case-control studies and found a statistically significant 8% higher risk, but again this report shaped its conclusions without taking into consideration individual study design and qualityStrengths of the present meta-analysis include the comprehensive search strategy with the inclusion of “metabolic syndrome” in the search algorithm, the wide scope of investigating associations with many individual cancers and the detailed sensitivity and subgroup analyses. There are also limitations in this work. First, the association between hypertension and malignancy may be due to shared risk factors such as age, smoking, alcohol consumption, diet and adiposity. Unfortunately, the majority of the literature in this field contains studies that did not perform comprehensive multivariable adjustments, which raises concerns over the robustness of individual study findings even for the well-described association between hypertension and kidney cancer. Second, most studies did not perform subgroup analyses by potential effect modifiers (e.g. anti-hypertensive medication use), which would have allowed a more accurate assessment of associations in different patient subgroups. Third, detection bias may also account for some of the reported associations, as individuals treated for hypertension are under closer medical surveillance that may lead to easier detection of cancer compared to untreated persons. Future well-designed prospective studies and Mendelian randomization studies should assist in estimating valid measures of association.In conclusion, the currently available published observational evidence across 18 cancer sites showed that hypertensive individuals were at higher risk of kidney, colorectal and breast cancer. However, careful interpretation is required as most meta-analyses included relatively small number of studies, several relative risks had weak or moderate magnitude and maybe affected by residual confounding.Supplementary Information


The pathogenesis of human rhinovirus (HRV) during severe respiratory disease remains undefined; thus, we aimed to explore the relationship between the HRV molecular subtyping results obtained during severe and asymptomatic childhood infections. Nasopharyngeal/oropharyngeal swabs from children (1 to 59 months of age) hospitalized with pneumonia and from age-frequency-matched controls were collected between August 2011 and August 2013. P = 0.693); however, among children 13 to 59 months old, HRV detection was more often case associated , with the results mainly driven by HRV-C . Overall, there were no differences in the results of molecular subtyping of the HRV species prevalence among cases  and controls . Those with pneumonia and HRV-C were older  and more likely to present with wheeze  than those with HRV-A cases. Thus, the rate of HRV detection was high, with similar degrees of genetic diversity among cases and controls, confounding the interpretation of the presence of HRV in nasopharyngeal samples for attribution of a causal role in the pathogenesis of severe pneumonia in infants. However, among children 13 to 59 months of age, HRV detection, in particular, HRV-C detection, was associated with case status, especially among children with wheezing disease.The pathogenesis of human rhinovirus (HRV) during severe respiratory disease remains undefined; thus, we aimed to explore the relationship between the HRV molecular subtyping results obtained during severe and asymptomatic childhood infections. Nasopharyngeal/oropharyngeal swabs from children (1 to 59 months of age) hospitalized with pneumonia and from age-frequency-matched controls were collected between August 2011 and August 2013. Swabs were tested for respiratory pathogens, including HRV, using quantitative real-time PCR assays. HRV-positive samples were sequenced for phylogenetic analysis by targeting the 5′ noncoding region (5′NCR). Our data showed that there were no differences in the prevalence of HRV detection among cases and controls (21% versus 20%,  Pneumonia is the leading cause of childhood morbidity and mortality globally, including approximately 120 million cases annually 24. NevertPicornaviridae family in the Enterovirus genus and is made up of three species, namely, HRV-A, HRV-B, and HRV-C (6HRV was first identified in 1956 in patients presenting with mild upper respiratory tract infection (URTI)  study.The cases were children (1 to 59 months of age) hospitalized with severe or very-severe pneumonia based on World Health Organization definitions , 12, andComprehensive details on case and control enrollment criteria, sample testing, and clinical evaluation in the PERCH study have been described previously , 14. ThiStaphylococcus pneumoniae as previously described (10 transformed. Full blood counts and C-reactive protein (CRP) and blood culture testing using a BacT/Alert microbial system  were conducted for all cases. Where clinically indicated, pleural fluids, gastric aspirates, and lung aspirates were collected and cultured using standard culture and biochemical tests. The pleural fluids and lung aspirates were also tested using FTD-33 respiratory panels and the BinaxNow antigen assay per the instructions of the manufacturer .All cases and controls were sampled for blood and with a flocked nasopharyngeal (NP) swab (flexible minitip)  together with a rayon oropharyngeal (OP) swab (Fisher Scientific) on enrollment into the study. The NP sample was collected by swabbing the posterior nasopharynx, and the OP sample was collected by swabbing the posterior oropharynx and the tonsillar pillars. Both swabs were placed in the same 3 ml of universal transport media (Copan) and processed within 24 h of collection. Samples were stored at 4°C until processing and then archived at −70°C until testing and for long-term storage. A NucliSens EasyMag extraction system was used to extract the total nucleic acids from 400 μl of the NP/OP swab samples using the generic on-board extraction protocol and from 200 μl of blood samples using the specific-B extraction protocol, and the nucleic acids were eluted into a final volume of 110 μl . NP/OP samples were tested in each country using an FTD Resp-33 real-time quantitative PCR kit supplied by Fast-Track Diagnostics  per the instructions of the manufacturer , and bloescribed , and allS. pneumoniae cultured from a normally sterile fluid. In addition, a case was considered to have MCPP if the pleural fluid or lung aspirate was positive for pneumococcus with the BinaxNow or the FTD-33 assay. In another PERCH analysis, it was determined that there is an association between high pneumococcal densities in the nasopharynx or the whole-blood (WB) samples and the presence of MCPP in the pneumonia cases  thresholds were used as markers for likely MCPP in order to analyze the relationship between HRV and pneumococcus disease.Microbiologically confirmed pneumococcal pneumonia (MCPP) was defined as detection of ia cases . It was HRV molecular subtyping was performed as previously described , 20 usint tests and multivariate regression models. Univariate analysis was performed prior to multivariate analysis, and all characteristics with an association corresponding to a P value of <0.2 were included. STATA version 12  was used for all statistical analysis, and a two-sided P value of <0.05 was considered statistically significant.Demographic characteristics of cases and controls were analyzed using chi-square and Wilcoxon tests, and the levels of prevalence of the HRV species were quantified using binary multinomial regression and odds ratio determinations. Continuous variables were tested using MK858576 to MK858936 and MK858937 to MK859375.The sequences for all rhinovirus-positive samples have been deposited in GenBank with accession numbers n = 210/911), followed by Zambia , and was lowest in Mali  among the pneumonia cases. Additionally, 2,284 age-frequency-matched community controls were enrolled across the sites, among whom the levels of prevalence of HRV infection  were similar across the sites, including 22% in South Africa (n = 212/964), 20% in Mali (n = 143/725), and 18% in Zambia .A total of 2,120 hospitalized cases with severe/very-severe pneumonia were enrolled across the three sub-Saharan African sites included in this study. Of these children, 439 (21%) tested positive for HRV and were subtyped. The prevalence of HRV-associated pneumonia was highest in South Africa  and controls . Furthermore, the prevalence of HRV identification, the species distribution, and the viral load did not differ between cases and controls at the individual sites (data not shown). However, among children >12 months of age, the cases had a significantly greater prevalence of HRV detection (21%) than the controls , with the results driven by HRV-C detection   .The seasonal circulation of the three HRV species over the full study period for cases and controls is detailed in n = 446) were successfully amplified, 3% (n = 14) failed to amplify, and 0.5% (n = 2) were of insufficient volume for serotyping. Among the amplified samples, 5% were typed as enterovirus and one as echovirus and 92% as HRV. The dominant HRV species among controls were HRV-C (45%) and HRV-A (45%), and 10% were HRV-B. There were no differences in species distribution between asymptomatic controls  and controls with signs and symptoms of upper respiratory tract infections  .Among the HRV-positive samples from community controls, 97% , there were no differences in demographic characteristics, HRV load, and symptoms of RTI  (P = 0.026)  . However= 0.026) .Further, when the HRV types were compared among the asymptomatic and RTI controls separately, there were no significant differences for any of the demographic, clinical, or molecular markers (Table S1 and S2).n = 11) of the samples failed to amplify, all of which had very low copy numbers  in the real-time PCR assay. Of the 428 amplified samples, 3% (n = 13) were typed as enterovirus and closely related member of HRV. The species of samples was successfully identified in 91% (n = 415) of the HRV-associated cases, among which 48%, 45%, and 7% were found to be infected with HRV-A, HRV-C, and HRV-B (7%), respectively (P = 0.002).Among the HRV-associated pneumonia cases, 97% of the samples were successfully subtyped, while 3% (ectively . The disP = 0.01) and with HRV-C infection   .P = 0.033) cases (P = 0.040) and were more likely to present with concurrent diarrhea . In contrast, cases with HRV-C were more likely to present with wheeze (35%) than cases with HRV-A  (n = 223/415) of the cases, including 55%, 42%, and 54% of HRV-A, HRV-B, and HRV-C infections, respectively (P = 0.742).Further species-specific analyses were limited to comparing HRV-A-associated cases to HRV-C-associated cases, with HRV-A cases being younger (9.4 months) than HRV-C cases  cases . Further= 0.031) . Among tThere were 60 different HRV-A strains, 17 different HRV-B strains, and 28 different HRV-C strains circulating throughout the 2-year period . No discThe HRV sequences for each of the species formed many unique clusters, with mean levels of nucleotide diversity of 82% for HRV-A , 80% for HRV-B , and 74% for HRV-C . The levels of nucleotide diversity did not differ among cases and controls for the different HRV species. The HRV-A and HRV-B sequences clustered with the GenBank sequences of known HRV-A and HRV-B strains, with statistically significant bootstrap support, whereas the HRV-C sequences tended to form numerous subclusters which did not always cluster closely with the GenBank sequences but which did always occur in the same monophyletic groups of known HRV-C strains. The numerous clusters and ranges of nucleotide diversities, especially in the HRV-C population, suggest considerable diversity in the strains present in the population .Among the HRV-A strains, the nucleotide similarities to the closest GenBank prototype reference strains ranged from 82.3% to 99.4% and strain identities with other contemporaneous HRV-A strains ranged from 79.8% to 100%. Among the HRV-B strains, the nucleotide similarities to the closest GenBank prototype reference strains ranged from 87.9% to 99.8% and strain identity with other contemporaneous HRV-B strains ranged from 91.2% to 100%. Among the HRV-C strains, the nucleotide similarities to the closest GenBank prototype reference strains showed much lower levels of relatedness than the other two species and ranged from 74.5% to 98.8%; however, there were high degrees of similarity with the other contemporaneous HRV-C strains, with strain identities ranging from 93.9% to 100%. No novel serotypes were identified in this study.This report presents results of genotyping of 836 HRV-positive samples, including 415 from children hospitalized with severe or very-severe pneumonia and 421 from community controls, representing the largest and most in-depth case-control study reporting on HRV molecular subtyping to date. HRV-A was the dominant species identified among cases (48%) and controls (45%), followed closely by HRV-C (45% each among cases and controls), whereas HRV-B was seen only intermittently and accounted for 7% and 10% of HRV strains among cases and controls, respectively.S. pneumoniae detection in the blood; those levels are comparable to the overall levels of S. pneumoniae detection in the whole of the PERCH study   and controls (20%). However, presence of HRV in the nasopharynx was associated with case status (21% versus 16% among controls) among children in the age group of 12 to 59 months; among those children, a higher percentage of HRV were HRV-C species in cases (12%) than in controls (7%). Furthermore, HRV loads did not differ significantly between the HRV species, and none of the HRV species were more likely to be associated with either more bacterial coinfections or more viral coinfections regardless of case or control status. The HRV species distributions among cases and controls in our study in both hospitalized and control populations are similar to those reported previously by others –28. Nota–Previous smaller studies from Africa, Asia, Europe, America, and Australia –32 have –That HRV-C infection is more common in older children hospitalized with pneumonia has also been reported by others , 33, 34.33–in vivo and has also been associated with increased susceptibility to wheezing and asthma illnesses  of the HRV-positive samples (with similar percentages between cases and controls), which was largely due to PCR failure as a consequence of very low viral loads (TC >37). The possibility cannot be excluded that these PCR failures might have been due to variability in the primer annealing sites; thus, some genetic variants might have been missed by our typing assays. However, the typing failure rate of 3% is lower than that seen in other studies which looked at HRV serotyping by targeting the VP4-VP2 capsid region (15% to 44% failure)  , 26, 27.failure) .Regardless, the considerable genetic diversity of HRV reported in this study highlights the heterogeneity of the HRV strains circulating within the general population and further emphasizes the difficulties in attributing causality of disease to HRV. The similarities in strains among cases and controls also negate the assertion that some strains might be more virulent. Other studies have also reported the diverse nature of the HRV genetic population among cases and controls, including studies from South Africa , Botswan40–Study limitations included the lack of a gold standard for the determination of the actual cause of the pneumonia episode. Additionally, the cross-sectional design of the study means only a single specimen was taken upon admission to the hospital. The viral load is known to vary with time since onset of illness; thus, a longitudinal study would have allowed us to compare peak viral loads between subjects infected with the different HRV species and between cases with different levels of clinical severity. Furthermore, our study was not designed to analyze the role of HRV in the lower respiratory tract. Sampling the upper respiratory tract is more convenient and less invasive for the patient; however, detection of a pathogen and its viral load in these samples might not reflect the viral burden in the lung parenchyma. Direct sampling of the site of infection, namely, the lung, through lung aspirate sampling or bronchoalveolar lavage would provide more-direct evidence of the role of HRV in pneumonia; however, these sampling techniques are more invasive and difficult to perform in infants and young children. Animal models are needed to further study the pathogenicity of HRV infections.In conclusion, this report emphasizes that the HRV populations circulating both in children hospitalized with pneumonia and in community controls are highly divergent, that similar strains are circulating over a large geographical location, and that the strains are similar year to year. Additionally, it further highlights the difficulty in attributing causality of LRTI disease to HRV in infants, as no differences were observed in the levels of prevalence of HRV detection between cases and controls (other than in the >12-month age group) and no differences were found in HRV genotypes between cases and controls, especially among infants. Nevertheless, among the cases, HRV-A tended to be more prevalent among younger children and was associated with more-severe disease and radiographically confirmed pneumonia compared to HRV-C infections, whereas HRV-C was more prevalent among older children with wheezing disease.