Each mycotoxin induced unique set of differential expressed transcripts and proteins, suggesting variable cytotoxicity and biochemical action in cell. We found a weak positive correlation between transcript and protein changes, and the transcript changes were higher than the protein changes. Furthermore, we observed pathway-specific protein homeostasis pattern under mycotoxin stress. Specifically, the “Metabolism”, “Transcription” and “Translation” pathways (housekeeping pathways) showed lower fold changes in protein/mRNA levels than non-housekeeping pathways such as “Cell growth and death” and “Immune system”. Protein molecular weight had a weak negative effect on protein production, and this effect was stronger for non-housekeeping pathways. Overall, we hypothesize housekeeping pathways maintain stable protein production for baseline cellular function, whereas non-housekeeping pathways is associated with the fitness response to environmental stress.Transcript levels are the primary factor determining protein levels, but for the majority of genes, fold changes in transcript levels are larger than the corresponding changes in protein levels, a phenomenon that is termed “protein homeostasis”. However, this phenomenon is not well characterized in the context of environmental changes. In this study, we sequenced the entire transcriptome and proteome of chicken primary hepatocytes administered three mycotoxin treatments Aflatoxin B With next-generation sequencing breakthroughs in recent years, increasing amounts of large-scale transcriptome and proteome data are now available, and researchers must disentangle the general rules governing protein production. Although the concept is still controversial3, several studies have characterized a significant positive correlation between mRNA and protein levels (rho > 0.5) in yeast, Drosophila, Caenorhabditis elegans, zebrafish, human and plants cells in the “steady” states7. Thus, mRNA levels are suggested to be the primary factor determining protein levels. Based on these calculations, mRNA levels possibly accounts for 40% to 80% of protein abundance variance; other factors, such as translation rate, play a relatively minor role in protein production9. However, recent observations from fission yeast, human tumors and evolutionary studies between humans and chimpanzees, revealed that fold changes in mRNA levels are generally larger than fold changes in protein levels12. This phenomenon is termed as protein homeostasis that protein production is maintained at a stable level in the cell despite fluctuations in mRNA1. Two concepts reveal the complicated nature of the protein production process: the first stresses the importance of mRNA quantity in determining protein production, whereas the second indicates that other regulatory mechanisms are also important in determining a stable protein concentration. Thus, further studies, especially in different species or under different cellular conditions such as environmental pressures, are needed to elucidate protein homeostasis.The central dogma that the information flow in a biological system is generally from DNA to RNA to protein is the cornerstone of modern molecular biology. Although this flow appears to be straightforward, there is a very complicated relationship between mRNAs and its encoded proteins. Besides of the regulatory networks in co-/post-transcription processes, protein quantity in cells is influenced by many aspects, including codon composition, ribosomal entry sites, translation rates, protein half-life, mRNA and protein degeneration rate, protein modulation, folding and transport rates13. Approximately 400 mycotoxins have been identified and a dozen have been recognized as important threats to humans and animals, including aflatoxin, citrinin, ergot alkaloids, fumonisin B1, ochoratoxin A (OTA), patulin, trichothecenes, zearalenone (ZEN) and so on14. Mycotoxin contamination in crops is widespread around the world, especially in developing countries due to poor preharvest practice and postharvest storage and transportation15. It is estimated that 25% of world’s crop may be contaminated by mycotoxins. The US Council for Agricultural Science and Technology estimated that the cost of crop losses from aflatoxins, fumonisins and deoxynivalnol is $932 million USD per year and mitigation cost is $466 million per year16. Some mycotoxins have stable molecular properties which are difficult to be removed by common practices such as heating or filtering, and as the consequence, consumptions of mycotoxin-containing foods and feeds lead to various pathologic reactions and can even increase mortality rates18.Mycotoxins are a group of low-molecular-weight secondary metabolites produced by filamentous fungi1 (AFB1), OTA and ZEN are some of the most widely spread mycotoxins in the world. Previous studies reported these mycotoxins have similar toxic actions in cells including prohibiting RNA and protein synthesize, DNA damage and ROS19, but none of them quantified the expression profile at the omics level. In this study, we sequenced the entire transcriptome and proteome of chicken  primary hepatocytes under AFB1, OTA and ZEN treatments. As in steady-state cells, the protein homeostasis pattern was preserved under mycotoxin pressure. However, genes from different pathways showed differing levels of protein homeostasis, indicating protein homeostasis was primarily maintained by housekeeping pathways in the cell.Aflatoxin B1, OTA and ZEN. We used an MTT assay to assess cell viability under mycotoxin administration. The mycotoxin concentration was lower than 0.5 μg/mL, 5 μg/mL and 20 μg/mL  to maintain cell viability >90%  were obtained for each replicate. High-quality scores (Q20 > 95%), low error rates (0.02%) and stable GC content indicated a high-quality transcript dataset Table . To verihttps://www.ebi.ac.uk/GOA/chicken_release, Uniprot_Chicken_24083_20150713.fasta). Peptides were quantified by Proteome Discoverer 2.0  and the false discovery rate (FDR) was calculated based on a Decoy database search with a cutoff of 0.01. The majority of peptide lengths ranged from 5 to 25 amino acids  technology to quantify relative protein levels between three mycotoxin-treated and control samples. Spectra from tandem mass spectrometry were searched using MASCOT engine 2.2  against the UniProt chicken database for peptide identification , including 3,583 up-regulated and 3,411 down-regulated transcripts. For OTA, there were 8,657 DETs , including 4,496 up-regulated and 4,161 down-regulated genes. For ZEN, there were 3,548 DETs , including 1,692 up-regulated and 1,856 down-regulated genes ” pathways. For ZEN treated samples, the up-regulated transcripts were mainly enriched in “Genetic information processing” pathway, including “DNA replication”, “Spliceosome”, “RNA transport”, “Mismatch repair”, “Homologous recombination”, “Base excision repair” and “RNA polymerase” pathways. The down-regulated transcripts were enriched in “Metabolism” and “Cellular processes” pathways for AFB1, “Ribosome” and “Focal adhesion” pathways for OTA and “Valine, leucine and isoleucine degradation” and “PPAR signaling” pathways for ZEN.The set of differentially expressed transcripts (DETs) were first characterized. We got quantitative expression level for 19,948 transcripts in total, and the DETs were identified based on negative binomial distribution with Benjamini and Hochberg procedure adjusted p value < 0.05ges Figs . For AFBes Table ; Fig. 3.es Table . We furt1-, OTA- and ZEN- treated and control samples : only 656 10.3%), 548 (8.6%) and 216 (3.4%) DEPs were between AFBes Table . Similar0.3%, 5481 and OTA, the overall distribution of DETs and DEPs was quite different for the three mycotoxins. Furthermore, the set of DETs were not consistent with the set of DEPs  and ZEN (0.23) were lower compared to previous reports for cells in the “steady” states (rho > 0.5). The transcript level changes were much higher than protein level changes for most genes, thus the protein homeostasis pattern was preserved under mycotoxin stress  and protein expression values for all three mycotoxins, and visualized the expression fold changes Fig. . We foun1 0.9 and ZEN1, OTA and ZEN . In AFB1-treated samples, the overall pattern was the same (p = 0.044). In ZEN-treated samples, we did not observe higher changes in non-housekeeping than housekeeping pathways. The slopes for non-housekeeping pathways were still higher than the slopes of most housekeeping pathways, but the pattern was distorted by the relatively high slope for “Energy metabolism” (0.293). Combining the results obtained with all three mycotoxins, we identified several pathways with high protein changes, including “Cell growth and death” , “Folding, sorting and degradation”, “Replication and repair”, “Membrane transport”, and “Immune system” (>0.2 for two mycotoxin).We further plotted transcript and protein changes for each KEGG pathway for AFBZEN Figs –S7. We fZEN Figs . More spOverall, we found variable protein homeostasis pattern under mycotoxin administration. It is tempting to hypothesize a fitness optimization process in cell that the strong constraint on housekeeping pathways is a baseline requirement for the maintenance of cellular function, and the relaxed constraint on non-housekeeping pathways is primarily related to the functional response to specific mycotoxin pressures.23. Thus, the homeostasis of proteins with high molecular weights should be stronger than the homeostasis of low-molecular-weight proteins. To test this assumption, we calculated the correlation between molecular weight and protein level changes in the three mycotoxin-treated samples, and we found very weak but significant negative correlations for all three samples  and one control sample. For each sample, we collected and sequenced three biological replicates. To detect differentially expressed genes, Benjamini and Hochberg’s approach was used to control the FDR. Pearson’s correlation was used for correlation analysis because the dataset has a normal distribution. We included all data points in the statistical analyses.In this study, we performed transcriptome and proteome sequencing for three mycotoxin-treated samples  containing 10% FBS, 100 U/mL penicillin/streptomycin, 10 nM insulin, and 10 nM dexamethasone (Sigma-Aldrich). We added the medium to each culture plate and cultured cells at 37 °C in a humidified incubator with 5.0% CO2. After 5 h, the medium was removed from adherent cells, and the cells were maintained in Medium 199  supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 10 μg/mL insulin, 1 μg/mL dexamethasone, and 2 mM L-glutamate (Invitrogen). AFB1, ZEN and OTA were purchased from Pribolab . All mycotoxins were dissolved in Dimethyl sulfoxide (DMSO). Williams E medium, Medium 199 and FBS were purchased from Invitrogen .Eighteen-day embryonated specific-pathogen-free (SPF) Arbor Acres Broiler chicken eggs were purchased from the Institute of Animal Science, Guangdong Academy of Agricultural Sciences . All related experiments and methods were performed in accordance with the recommended guidelines and regulations by the Administration of Affairs Concerning Experimental Animals of Guangdong Province, China. This research was approved  by Laboratory Animal Ethics Committee of South China Agricultural University. Hepatocytes were prepared using a collagenase digestion method50. Chicken embryo primary hepatocytes were seeded at a density of 10000 cells/well in 96-well plates and cultured overnight. The hepatocytes were treated with different concentrations of AFB1 (0–10 μg/mL), ZEN (0–120 μg/mL), or OTA(0–10 μg/mL). After 48 h, 0.5 mg/mL MTT  was added to each well. We extracted the total RNA from mycotoxin-treated or untreated chicken primary hepatocytes using Trizol reagent . Total RNA (2 μg) was reverse transcribed using M-MLV reverse transcriptase  with oligo d(T) and random primers . The obtained cDNA products were used as templates for CYP1A4, CYP2D20 and CYP3A37 transcript quantification by RT-PCR. GAPDH was used as an internal reference to normalize gene expression. The expression levels were calculated using the 2−ΔΔCt method51.Cell viability was assessed according to a previously described methodTotal RNA purity, concentration and integrity were assessed using NanoPhotometer® spectrophotometry , Qubit® RNA Assay Kit in a Qubit® 2.0 Fluorometer , and RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system . A total of 3 μg of RNA was extracted per sample. Sequencing libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® , then sequenced on an Illumina Hiseq X Ten platform, with paired-end reads length of 150 bp.52, and paired-end clean reads were aligned to the reference genome using TopHat v2.0.1253. The read numbers mapping to each gene were calculated by HTSeq v0.6.154, and the FPKM of each gene was calculated based on the length of the gene and the reads count mapped to that gene. Differential expression analysis was performed using the DESeq R package (1.18.0)21, and the statistical enrichment of the differential expression genes in KEGG (http://www.genome.jp/kegg/) pathways was calculated by KOBAS56.Raw data were processed through Perl scripts by removing reads that contained adapter, ploy-N and low quality reads. The Q20, Q30 and GC contents for each sample were calculated. All downstream analyses were based on the processed clean data. The reference chicken genome and annotation files were downloaded from NCBI with the GenBank Assembly ID GCA_000002315.3. The index of the reference genome was built using Bowtie v2.2.320. For SDS-PAGE separation, 20 µg of proteins was mixed with 5X loading buffer and boiled for 5 min. The proteins were separated on a 12.5% SDS-PAGE gel  and visualized using Coomassie Blue R-250 staining. The peptide mixture of each sample (100 μg) was labeled with iTRAQ reagent according to the manufacturer’s instructions . The labeled peptides were fractionated by SCX chromatography . LC-MS/MS analysis was performed with a Q Exactive mass spectrometer  and Easy nLC . MS data was collected based on the most abundant precursor ions from the survey scan (300–1800 m/z).SDT buffer was used for the sample preparation as in the universal proteomic sample preparation protocolhttps://www.ebi.ac.uk/GOA/chicken_release, Uniprot_Chicken_24083_20150713.fasta) using NCBI BLAST+ client software (ncbi-blast-2.2.28 + -win32.exe)57 for annotation. Protein ratios were calculated as the median of only the unique peptides for a protein. To exclude experimental bias, we normalized all peptide ratios using the median protein ratio. Protein quantification was calculated using the normalized spectral index (SIN)58. Technical repeats were accessed by performing a multivariate Pearson correlation analysis with R. To categorize genes into specific KEGG pathways, we blasted the annotated proteins against the online KEGG database55. Mapping and ID conversion were facilitated using R package ‘org.Gg.eg.db’ in Bioconductor. Downstream analysis and graphic plotting for transcriptomic and proteomic data were primarily performed using the Rstudio platform59.MS/MS spectra were analyzed using MASCOT engine  with following parameter settings. The search was MS/MS ion search. The protease used to generate peptides was trypsin. Two missed cleavages were permitted. The mass values were monoisotopic. The list of all fixed modifications considered included carbamidomethyl (C), and iTRAQ4PLEX , iTRAQ4plex (K). The list of all variable modifications included oxidation (M) and iTRAQ4plex (Y). The peptide mass tolerance for precursor ions was ±20 ppm. The mass tolerance for fragment ions was 0.1 Da. We used the Decoy database embedded in Proteome Discoverer 2.0 (Thermo Fisher Scientific) to calculate the FDR and set the cutoff to <0.01 for a peptide. The retrieved sequences were locally searched against the UniProt GOA database (chicken) (SupplimentFiguresTables


The aim of this investigation was to assess the association of sedentary behaviour with 12 different sleep problem indicators among rural middle-aged and elderly adults in South Africa. Cross-sectional data were analysed from the “Health and Aging in Africa: A Longitudinal Study of an INDEPTH community in South Africa” (HAALSI) baseline survey. Participants responded to a questionnaire, including sociodemographic, health, anthropometric measures, sedentary behaviour and 12 different sleep problem indicators. The sample included 4782 individuals 40 years and older . Overall, participants engaged <4 h (55.9%), 4–<8 h (34.1%), 8 or more hours a day (9.9%) sedentary time a day. In adjusted multinomial logistic regression, 8 h of more sedentary time was associated with short and long sleep. In adjusted logistic regression analysis, high sedentary time was positively associated with snoring, gasping, breathing stops and restless sleep and negatively associated with insufficient sleep and sleep problems due to a traumatic event. In combined analysis, compared to persons with low or moderate sedentary behaviour and moderate or high physical activity, persons with high sedentary behaviour and low physical activity were more likely to have long sleep, insufficient sleep, snoring, gasping, breathing stops, and restless sleep and less likely to have sleep problems due to traumatic events. Findings show an association between sedentary behaviour and/or combined sedentary behaviour and low physical activity with seven of 12 sleep problem indicators . Sedentary behaviour includes sitting, being in a lying position or reclining needing a very low expenditure of energy , and is Sleep problems or insomnia is one of the most common health problems, affecting approximately 10% to 20% of the world population . “The etwww.haalsi.org). Based on completed home interviews (response rate 85.9%) [Baseline data were analysed from the “Health and Ageing in Africa: A Longitudinal Study of an INDEPTH Community in South Africa (HAALSI) in the INDEPTH Health and Demographic Surveillance System (HDSS) site of Agincourt” in 2015 in rural South Africa . The base 85.9%) , 4782 ine 85.9%) .Sedentary behaviour was assessed with two items “On a usual week day, how many hours/minutes did you spend sitting or reclining (excluding sleep)? This may include time sitting on a chair or bench, visiting friends, reading, sitting in church, sitting down to watch television” on a usual weekday or weekend day [kend day ,13. Sedekend day .Sleep duration was assessed with the question, “Over the past four weeks, how many hours do you think you actually slept each day?” [ch day?” ,16. Respch day?” .Bad sleep quality. “Over the past 4 weeks, how would you rate your sleep quality overall?” (Very good/Good vs. Very bad/Bad) [bad/Bad) ,16.Insufficient sleep. “How often during the past 4 weeks did you get enough sleep to feel rested upon waking up?” (Sometimes/often/very vs. rarely/never) [y/never) ,16.Awakenings. ”During the past 4 weeks, how often did you wake up in the middle of the night or early morning?” (<once per week vs. at least once per week) [er week) ,16.Snoring. “During the past month, have you snored loudly, or ever been told that you were snoring loudly?” (Yes/No) [(Yes/No) ,16.Gasping. “During the last month, have you had, or ever been told that you were snorting or gasping?” (Yes/No) [(Yes/No) ,16.Breathing stops. “During the last month, have you had, or ever been told that your breathing stops or you struggle for breath?” (Yes/No) [(Yes/No) ,16.Sleep latency (>30 min). “During the past 4 weeks, how often could you not get to sleep within 0 min?” (”never or <once a week or once or twice a week” vs. ”three or more times a week”) [a week”) ,16.Restless sleep. ”Much of the time in the past week, your sleep was restless?” (Yes/No) [(Yes/No) .Pain has interfered with your sleep. “On a scale of 0 to 10, where 0 is ‘Does not interfere’ and 10 is ‘Completely interferes’, select the one number that describes how, during the past 24 h, pain has interfered with your sleep?” (zero vs. 1–10 interferes) [erferes) .Sleep problem due to traumatic event. “In your life, have you ever had any experience that was so frightening, horrible, or upsetting that, in the past 30 days you had more trouble than usual falling asleep or staying asleep?” (Yes/No) [(Yes/No) .Sociodemographic information included sex, age, formal education, and a “wealth index in quintiles created from household characteristics and ownership of household items, livestock, and vehicles” [ehicles” ,19.Social cohesion was measured with four questions, e.g., “Most people in this village are willing to help their neighbours” Response options ranged from 1 = strongly agree to 4 = strongly disagree [disagree   questionnaire [ionnaire  ” [ (GPAQ)” ,24. FollBody Mass Index (BMI) was calculated from standardized measures of height and body weight [2), normal weight (18.5–24.9 kg/m2), overweight (25–29.9 kg/m2), and obesity (30+ kg/m2),” using World Health Organization (WHO) criteria [y weight , and clacriteria ,25.Self-reported health status was measured with the item: “In general, how would you rate your health today?” [ today?” . “Respon today?” .p < 0.05 was considered significant. All statistical procedures were conducted with STATA software version 14 .Associations between sedentary behaviour and sleep duration were analysed with multinomial regression, with short (≤6 h) and long sleep (≥10 h) duration as dependent variable, and normal sleep duration (7–9 h) as reference category. Logistic regression was used to estimate the associations between sedentary behaviour and other sleep problem indicators. All models were adjusted for sex, age, education, wealth status, social cohesion, BMI, current tobacco use, problem drinking, and self-rated health status. Moreover, we used multivariable multinomial and logistic regression to estimate the combined relationship between sedentary behaviour and physical activity with problem sleep indicators. For the combined multinomial and logistic regression analysis, the sample was sub-divided based on sedentary and physical activity levels into four groups: (1) low or moderate sedentary time (<8 h) plus moderate or high physical activity group (reference category), (2) low or moderate sedentary time (<8 h) plus low physical activity group, (3) High sedentary time (≥8 h) plus moderate or physical activity group, and (4) high sedentary time (≥8 h) plus low physical activity. The sample included 4782 individuals , 53.3% were female, 93.2% had moderate to high social cohesion, 58.1% overweight or obese, and 42.1% engaged in low physical activity. Overall, 68.8% of respondents reported good self-rated health status, 1.4% alcohol dependence, and 15.6% were current tobacco users.Overall, participants engaged <4 h (55.9%), 4–<8 h (34.1%), and 11 or more hours a day (9.9%) sedentary time a day. Of all participants, 13.1% had short sleep (≤6 h) and 18.2% long sleep (≥10 h). Other sleep problem indicators included, awakening (40.0%), snoring (19.6%), gasping (12.7%), breathing stops (7.1%), bad sleep quality (6.3%), sleep latency (9.0%), restless sleep (32.1%), pain that interferes with sleep (9.5%), problems with sleep due to traumatic experience (15.0%) and insufficient sleep (14.4%) see .In adjusted multinomial logistic regression analysis, 8 h of more sedentary time was associated with short sleep and long sleep. In adjusted logistic regression analysis, 8 h of more sedentary time was associated with snoring, gasping, breathing stops and restless sleep, and 4–<8 h sedentary behaviour was positively associated with snoring and negatively associated with bad sleep quality, breathing stops and restless sleep. Four or more hours sedentary time was negatively associated with insufficient sleep and sleep problems because of a traumatic event. No associations were found between sedentary behaviour and latency, nocturnal awakenings, and pain that interferes with sleep see .In adjusted multinomial logistic regression analysis, compared to persons with low or moderate sedentary behaviour and moderate and high physical activity, persons with high sedentary behaviour and moderate or high physical activity were more likely to have short sleep , and persons with low or moderate sedentary behaviour and low physical activity  as well as persons with high sedentary behaviour and low physical activity  were more likely to have long sleep. In adjusted logistic regression analysis, compared to persons with low or moderate sedentary behaviour and moderate or high physical activity, persons with high sedentary behaviour and low physical activity were more likely to have snoring , gasping , breathing stops , restless sleep  and insufficient sleep  and less likely to have a sleep problem due to a traumatic event . In addition, compared to persons with low or moderate sedentary behaviour and moderate and high physical activity, persons with low or moderate sedentary behaviour and low physical activity were more likely to have bad sleep quality , latency , nocturnal awakenings , insufficient sleep  and pain interfering with sleep , and were less likely to have snoring  and gasping . Further, persons with high sedentary behaviour and moderate or high physical activity were less likely to have nocturnal awakenings  and more likely to have insufficient sleep  and restless sleep  see .This is one of the first studies to investigate the association of sedentary behaviour with 12 different sleep problem indicators in a large sample in South Africa. Results found that high sedentary time (≥8 h), adjusted for relevant confounders such as sociodemographics, body mass index and social cohesion, was associated with six sleep problem indicators: short sleep, long sleep, snoring, gasping, breathing stops, and restless sleep. In addition, a combined association of high sedentary time (≥8 h) and low physical activity was found with six sleep problem indicators: long sleep, snoring, gasping, breathing stops, insufficient and restless sleep. It should be acknowledged that someone who is awake for longer (shorter sleep time) is likely to have higher awake sedentary time just by virtue of being awake for longer. This may explain as to why in the combined analysis the combined association of high sedentary time (≥8 h) and low physical activity was no longer significant on short sleep. Stamtakis et al.  found thSeveral previous studies ,7,8 alsoSedentary time was in this study negatively associated with insufficient sleep and sleep problems due to trauma, and no association was found with bad sleep quality, latency, nocturnal awakenings, and pain that interferes with sleep. In a previous review , also noBoth sedentary and sleep behaviour were assessed by self-report, and future studies should include objective measures. Sedentary time may have been under-reported and physical activity over-reported because of social desirability bias . Self-reFindings show an association between sedentary behaviour and/or combined sedentary behaviour and low physical activity with seven of 12 sleep problem indicators .


A multi-biomarker disease activity (MBDA) score has been validated as an objective measure of disease activity in rheumatoid arthritis (RA) and shown to track response to treatment with several disease-modifying anti-rheumatic drugs (DMARDs). The objective of this study was to evaluate the ability of the MBDA score to track response to treatment with rituximab.Data were used from 57 RA patients from three cohorts treated with rituximab 1000 mg and methylprednisolone 100 mg at days 1 and 15. The MBDA score was assessed in serum samples obtained at baseline and 6 months. Spearman’s rank correlation coefficients were calculated for baseline values, 6-month values, and change from baseline to 6 months (∆), between MBDA score and the following measures: disease activity score assessing 28 joints (DAS28) using erythrocyte sedimentation rate (ESR) or high-sensitivity C-reactive protein (hsCRP), ESR, (hs)CRP, swollen and tender joint counts assessing 28 joints , patient visual analogue scale for general health (VAS-GH), health assessment questionnaire (HAQ), and radiographic progression over 12 months using Sharp/van der Heijde score (SHS), as well as six bone turnover markers. Additionally, multivariable linear regression analyses were performed using these measures as dependent variable and the MBDA score as independent variable, with adjustment for relevant confounders. The association between ∆MBDA score and European League Against Rheumatism (EULAR) response at 6 months was assessed with adjustment for relevant confounders.p = 0.02). Neither baseline MBDA score nor ΔMBDA score correlated statistically significantly with ∆SHS (n = 11) or change in bone turnover markers (n = 23), although ∆SHS ≥ 5 was observed in 5 (56%) of nine patients with high MBDA scores.At baseline, the median MBDA score and DAS28-ESR were 54.0 (IQR 44.3–70.0) and 6.3 (IQR 5.4–7.1), respectively. MBDA scores correlated significantly with DAS28-ESR, DAS28-hsCRP, ESR and (hs)CRP at baseline and 6 months. ∆MBDA score correlated significantly with changes in these measures. ∆MBDA score was associated with EULAR good or moderate response  contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is the most common, chronic inflammatory joint disease, characterised by synovitis, joint damage, and systemic immune and inflammatory manifestations. Achieving remission or low disease activity is the main treatment goal in order to prevent joint damage and disability . The EurThe multi-biomarker disease activity (MBDA) score is based on biochemical markers only. It is thus more objective than the DAS28 and may potentially be a better indicator of systemic inflammation. The MBDA score is calculated with an algorithm that uses the concentrations of 12 serum protein biomarkers to produce a score, on a scale of 1 to 100, that represents the level of disease activity in patients with RA . The MBDThe MBDA score tracks response to a variety of disease-modifying anti-rheumatic drugs (DMARDs), including methotrexate   and TNF. By depleting CD20+ B cells, rituximab treatment leads to a decrease in these pro-inflammatory cytokines , 18, theThe purpose of the current study was to assess the ability of the MBDA score to measure disease activity upon and track response to treatment with rituximab and, if so, if this would be mainly explained by the objective component of the DAS28 (acute phase reactants). Furthermore, we investigated the ability of the MBDA score to predict radiographic progression and change in serum bone turnover markers upon rituximab treatment.We used data from three prospective cohort studies in which adult, refractory RA patients were treated with rituximab because of active disease despite conventional treatment DMARD and/or TNF inhibitor): one cohort from the Leiden University Medical Center (LUMC)  and one Demographics, disease duration, smoking status (no or yes) and serum status for rheumatoid factor (RF) and for autoantibodies against citrullinated peptides (ACPA) were assessed at baseline. Swollen and tender joint counts assessing 28 joints , patient visual analogue scale (VAS) for general health (GH), and health assessment questionnaire (HAQ) were obtained for patients at baseline and 6 months, as were erythrocyte sedimentation rate (ESR), CRP and high-sensitivity (hs)CRP (the latter only in HORUS). The DAS28 was calculated using both ESR and hsCRP. EULAR response at 6 months was determined using DAS28-ESR . RadiogrSerum samples were collected at baseline in all three cohorts, and at 6 months in the UMC Utrecht and HORUS cohorts. Samples were shipped frozen to Crescendo Bioscience, Inc.  for measurement of the 12 MBDA biomarkers. The biomarkers represent inflammatory and destructive processes: vascular cell adhesion molecule-1 (VCAM-1), epidermal growth factor (EGF), vascular endothelial growth factor A (VEGF-A), IL-6, TNF receptor type 1 (TNF-R1), matrix metalloproteinase 1 (MMP-1), MMP-3, human cartilage glycoprotein-39 (YKL-40), leptin, resistin, serum amyloid A (SAA) and CRP. The MBDA biomarkers were measured by electrochemiluminescence-based multiplexed sandwich immunoassays  using the same types of reagents and instrument and the same algorithm as described previously .Baseline characteristics were assessed using descriptive statistics. Differences between the three cohorts were analysed using one-way analysis of variance, Kruskal-Wallis test or chi-square test, as appropriate.Spearman’s rank correlations (r) were analysed for values at baseline, at 6 months and for change from baseline to 6 months (∆) between MBDA score and the following measures: DAS28-ESR, DAS28-hsCRP, ESR, CRP, hsCRP, SJC28, TJC28, VAS-GH, HAQ, SHS (UMC Utrecht cohort), bone turnover markers (HORUS cohort). Multivariable linear regression analyses were performed using these measures as dependent variable and the MBDA score as independent variable, with adjustment by age, gender, smoking status (no or yes), RF status, ACPA status, and cohort. Bone turnover markers were additionally adjusted for menopausal status  . Logistip values < 0.05 were considered statistically significant. All statistical analyses were performed using IBM SPSS Statistics 21 software .Two-sided Baseline characteristics were generally typical of those for patients with established RA starting rituximab treatment and were mostly similar between the three cohorts. SJC28, ESR, CRP and HAQ were statistically significantly different between the three cohorts Table . Overalln = 52), with high (> 44), moderate (30–44) or low , with high, moderate or low scores observed in 26 (62%), 11 (26%) and 5 patients (12%), respectively. The median ∆MBDA score was −7 .At baseline the median MBDA score was 54 (interquartile range (IQR) 44–70, n = 51) and 5.0 , respectively, and the median ∆DAS28-ESR was −1.0 . At baseline and at 6 months, the median values for DAS28-hsCRP were 5.8  and 4.7 , respectively, and the median ∆DAS28-hsCRP was − 0.9 .At baseline and at 6 months, the median values for DAS28-ESR were 6.3  and at 6 months . ∆MBDA score from baseline to 6 months was significantly correlated with ∆DAS28-ESR .Correlations between MBDA score and DAS28 and their changes over time are shown in Fig. r = 0.51, p < 0.01) and at 6 months . ∆MBDA score from baseline to 6 months was significantly correlated with ∆DAS28-hsCRP .Similarly, the MBDA score was significantly correlated with DAS28-hsCRP at baseline  per unit change in MBDA score, Fig. p = 0.02) per unit change in MBDA score).At 6 months, 21 patients (48%) were classified as non-, 19 patients (43%) as moderate and 4 patients (9%) as good EULAR responders. The distribution of values for ∆MBDA score within each EULAR response category is shown in Fig. p = 0.35) per unit MBDA score, even after adjustment by age, gender, smoking status, RF status, ACPA status, and cohort  per unit MBDA score).The MBDA score at baseline was not associated with EULAR response (good or moderate) versus non-response at 6 months, with OR of 1.01 . At baseline, low, moderate and high MBDA scores were observed in 1, 1 and 9 patients, respectively. Radiographic progression (∆SHS ≥ 5) in patients with low, moderate and high MBDA scores was observed in 0 (0%), 0 (0%) and 5 (56%) patients, respectively.No significant Spearman’s correlation was found between MBDA score or ∆MBDA score and ∆SHS over 12 months, nor bone turnover markers Table . SimilarThe MBDA score has been shown to track response to a variety of DMARDs. We found significant correlations between the MBDA score and DAS28-ESR as well as DAS28-hsCRP at baseline and at 6 months, and between ΔMBDA score and ΔDAS28-ESR and ΔDAS28-hsCRP from baseline to 6 months in patients treated with rituximab. Moreover, ΔMBDA score was significantly associated with EULAR response to rituximab treatment. This is the first time it has been shown that the MBDA score can be used to track disease activity in RA patients upon treatment with rituximab and that change in the MBDA score reflects response to rituximab treatment.Our findings on the MBDA score are consistent with several previous studies in RA patients upon treatment with other cs-, biological, or targeted synthetic DMARDs –8, 10–16In our study, we additionally investigated if the MBDA score correlated with the individual components of the DAS28. We found correlations between the MBDA score or ΔMBDA score and ESR, hsCRP or their changes, but found limited correlations between the MBDA score and the other DAS28 components. The correlation between the MBDA score and the DAS28 thus seems predominantly dependent on the biochemical components of the DAS28, the ESR or (hs)CRP. It would be of interest to assess the additional value of the MBDA score above ESR or CRP alone, but the present study was not powered to analyse this. A larger study has reported that an increase in TJC, SJC and patient global assessment was paralleled by an increase in MBDA score; and that, in patients positive for either RF and/or ACPA, an MBDA score excluding CRP was a significant predictor of both DAS28-CRP, and of DAS28 without any CRP or ESR component .In addition, MBDA score appeared to be more sensitive for detecting inflammation than ESR or CRP. A study of 9135 RA patients with active disease found that ESR and CRP were normal in the majority . In othen = 11) and the limited observation period.Previous studies have shown that the MBDA score was a significant predictor of radiographic progression, both in early and established RA , 27–30. B-cell depletion upon rituximab treatment has been shown to be most effective in RF-positive patients , and hasOther studies have shown that rituximab treatment increases bone formation and decreases bone resorption in RA patients , 34. ForIn conclusion, we have shown, for the first time, that the MBDA score correlated with DAS28 following treatment with the B-cell depleting agent rituximab and that ΔMBDA score reflected the treatment response. Our findings are consistent with previous research in RA patients treated with other DMARDs.Additional file 1:Determination of bone turnover markers. (PDF 102 kb)


In particular, we show that if T is an [m]-complex symmetric operator, then m]-complex symmetric operator for any T is an [m]-complex symmetric operator, then m]-complex symmetric operator under perturbation by nilpotent operators commuting with T.In this paper, we introduce the class of [ H be a complex separable Hilbert space, and let H. For T by Let A and B be given operators in k, we have A and B be in B is said to be in Let β), property U in H-valued analytic functions on U and the Fréchet space of all H-valued U, respectively.In operator theory, one of the most important topics is local spectral theory. In the following we consider several properties in local spectral theory such as the single-valued extension property, property (G of T is said to have SVEP if T has SVEP at every point β) at T is said to have Bishop’s property (β) (resp. T has Bishop’s property (β) IfTis invertible, thenTis an [m]-complex symmetric operator if and only if so is(ii)IfTis an [m]-complex symmetric operator, thenis also an [m]-complex symmetric operator for any(iii)Ifis a sequence of [m]-complex symmetric operators such thatthenTis also an [m]-complex symmetric operator.T is an invertible [m]-complex symmetric operator. Since m]-complex symmetric operator.(i) Suppose that m]-complex symmetric operator for any (ii) Sincem]-complex symmetric operators such that m]-complex symmetric operator with conjugation C,T is an [m]-complex symmetric operator. □(iii) Suppose that m]-complex symmetric operator.We examine the nilpotent perturbations of an [LetTbe an [m]-complex symmetric operator with conjugationCandNbe a nilpotent operator of ordernsuch thatThenis an -complex symmetric operator.If T is a [3]-complex symmetric operator.On the other hand, since


Ocimum L.) species are used as medicinal plants due to their essential oils exhibiting specific biological activity. The present work demonstrated that both the variety and season/conditions of cultivation had a significant effect on (i) the produced amount (extraction yield), (ii) qualitative, as well as (iii) quantitative profile of basil essential oil. Among studied basil varieties, a new variety, ‘Mánes’, was characterized for the first time. Based on our quantitative evaluation of GC-MS profiles, the following chemotypes and average concentrations of a main component were detected in the studied basil varieties: ‘Ohře’, ‘Lettuce Leaf’, ‘Purple Opaal’, ‘Dark Green’ , and ‘Mammolo Genovese’, ‘Mánes’, ‘Red Rubin’ . At the same time, when considering other compounds identified in GC-MS profiles, all the studied varieties, except from ‘Lettuce Leaf’, were methyl eugenol-rich with a strong dependence of the eugenol:methyl eugenol ratio on the seasonal changes . More complex and/or variable (depending on the season and cultivation) chemotypes were observed with ‘Lettuce Leaf’ , ‘Dark Green’ , ‘Mammolo Genovese’ , ‘Red Rubin’ , and ‘Mánes’ . When considering superior extraction yield  and consistent amounts (yields) of essential oil when comparing inter-seasonal or inter-year data , this new basil variety is very promising for use in the pharmaceutical, food, and cosmetic industries.Basil ( Ocimum L.) species have been used for centuries as medicinal plants. Basil essential oils are responsible for characteristic aroma and biological activity [Basil  of the basil plants with acceptable content of essential oil. However, only very few works have been published on the study of seasonal variation of basil oil yield during the year . ValuablThe main objectives of our work, in this context, were (i) to characterize for the first time essential oil yield and chemical profile of a new basil variety, namely ‘Mánes’; (ii) to compare qualitative and quantitative composition and extraction yields of seven sweet basil varieties including ‘Mánes’; and (iii) to evaluate an influence of greenhouse conditions of the cultivation in different seasons of one year on the extraction yield and chemical profile of essential oil of seven sweet basil varieties including ‘Mánes’.−1) d. w. (dry weight) in 2015. Similarly, the year 2016 showed the values ranging from 3.7 to 17.7 (mL·kg−1) d. w.The content of the basil essential oil in the aerial part of plant of seven basil varieties was determined by the method of steam distillation according to , withoutStatistical evaluation of the extraction yields obtained from particular varieties and in different years was carried out by means of Tukey’s test. The data presented in The most significant statistical difference, with the highest extraction yield, was observed in case of the new variety ‘Mánes’ compared to the other varieties. ‘Mánes’ provided not only the highest extraction yield but also constant/consistent yields of essential oil over all the pot cultures (seasons) studied. These characteristics make the ‘Mánes’ variety very promising for the use in the pharmaceutical, food, and cosmetic industries.Separation and identification of particular essential oil components in the seven studied basil varieties was carried out by means of the GC-MS method developed for this purpose , detection and quantitation limits , and reproducibility of measurements (standard deviation). The resulting performance and calibration parameters are summarized in The application data in Generally, the pot cultures 1 and 2  exhibited significantly higher concentrations of the particular quantified essential oil components than pot culture 3 (covering darker season including September and October with lower temperature and higher relative humidity), see Eugenol, linalool, eucalyptol, and estragole were purchased as reference standards from Sigma-Aldrich . All the chemicals used were of analytical grade. n-Hexane for gas chromatography was obtained from Merck .Seven basil varieties, namely ‘Ohře’, ‘Lettuce Leaf’, ‘Purple Opaal’, ‘Dark Green’, ‘Mammolo Genovese’, ‘Mánes’, and ‘Red Rubin’, were used in the experiments. The varieties were obtained from Semo Ltd.  and from Seva-Seed Ltd. .Basil plants were cultivated as a pot culture in a greenhouse of the Mendel University in Brno, Faculty of Horticulture in Lednice.Conditions for growing basil  were controlled by the control system LCC 1240 . The control was ensured by PC SuperLink 4.0  with a graphic visualization and archiving capabilities. The average temperature, relative humidity, and solar irradiance in each pot culture during the years 2015–2016 were divided into individual growing weeks and are illustrated in The experimental layout included three individual sowing and harvesting periods (pot cultures 1–3) of the sweet basil in one calendar year. These were following: pot culture 1 (23.3.–1.6. 2015 and 8.2.–25.4. 2016), pot culture 2 (27.4.–14.7. 2015 and 28.4.–20.6. 2016), pot culture 3 (1.9.–26.10. 2015 and 15.8.–17.10. 2016). The greenhouse experiments were carried out in 4 replications for each variety with 30 plants for each variant.Basil was grown in 500 mL plastic pots, at 20 plants per pot. The plants were cultured in medium Horticultural substrate B with active humus , irrigated as needed, protected from pests and diseases, and once fertilized . The basil plants were harvested before their flowering. After drying (at room temperature) the plants were stored in paper bags in a dark place until the time of steam distillation.−1 for 2 h. The volume of isolated essential oil was measured in a proper calibrated tube. The extracted essential oils were stored in sealed vials at 4 °C in the refrigerator until GC-MS analysis. The content of essential oils in the plant material was calculated from 2 parallel distillations and expressed in mL·kg−1.The content of the basil essential oil in the aerial part of plant was determined at the Department of Vegetable Growing and Floriculture of the Mendel University in Brno by the method of steam distillation according to , withoutGas chromatography analyzer Trace GC, Thermo Finnigan  with autosampler AS 2000  was used for the analysis of the extracted basil essential oil samples. Mass spectrometry detector Trace DSQ  was an integral part of the gas chromatographic analyzer. The GC-MS records were processed by means of Thermo Finnigan Xcalibur 1.3 software . The GC-MS instrumental parameters and conditions are given in The distillation apparatus Gerhardt  was used for the extraction of essential oils from the basil plant material. Distilled water used in the experiments was prepared by an Aqua Osmotic type 02  water purification system. The distilled water meets the standards according to ČSN 68 4063, Czech Pharmacopoeia 2005, and ČSN ISO 3696.m/z. Injection conditions were as follows: 10 μL injection syringe, injection volume 1 μL, injection temperature was 240 °C, regime of injection was “split”, split flow was 50 mL/min, split ratio was 100. The content of selected components of the essential oils was calculated from 3 parallel analyses and expressed in mg/mL.The identity and quantity of particular components of the basil essential oil were evaluated by the GC-MS analysis method, using gas chromatograph Trace GC equipped with MS detector Trace DSQ and DB-WAX (highly polar capillary column with 100% polyethylene glycol stationary phase) capillary column (circa 30 m of length with inner diameter of 0.25 mm and a 0.25 μm layer of the inner film). The measurement records started 3 min after the run beginning. Temperature of the GC column was kept at 40 °C for 3 min. Then, the temperature gradually increased with the gradients of 8 °C/min up to 60 °C, 5 °C/min up to 70 °C, and 4 °C/min up to 230 °C. The temperature of 230 °C was kept constant for 1 minute. Subsequent GC working conditions were as follows: carrier gas was He with a constant flow rate of 0.5 mL/min, matrix solvent was n-hexane. MS working conditions were as follows: temperature of ion source was 200 °C, temperature MS transfer line was 200 °C, mass range was 20–300 www.statsoft.cz) was used for the statistical evaluation of the results.Thermo Finnigan NIST 02 Libraries software  was applied to the identification of particular components of basil essential oil. Parameters of calibration lines of the selected components  present in the basil essential oils were calculated by using Microsoft Excel 2007 . The program Statistica Cz v. 12  the content (extraction yield) and the (ii) qualitative as well as (iii) quantitative profile of basil essential oils. Chemotypes of the studied varieties were evaluated by quantification of four main basil essential oil constituents  and comparing the obtained results with data presented in the literature. According to these criteria, ‘Ohře’, ‘Lettuce Leaf’, ‘Purple Opaal’, and ‘Dark Green’ were identified as linalool chemotypes, which was in good agreement with the results by Beatovic et al. , KlimankSurprisingly, the new variety, ‘Mánes’, exhibited considerably higher extraction yield when compared to other examined basil varieties . Here, the extraction yield was enhanced two to five times depending on the compared variety. When considering the superior and stable  extraction yield and favorable essential oil profile of ‘Mánes’ towards the generally important essential oil components, this basil variety is very promising for use in the pharmaceutical, food, and cosmetic industries.


CCR5) polymorphisms, particularly a 32-base pair deletion (∆32) in the open reading frame and −2459G > A in the promoter, are well known for their associations with HIV-1 infection and/or disease progression in a variety of studies. In this era of an HIV cure, where all the emphasis is on ∆32, it seems that −2459G > A has been forgotten or ignored. There is significant importance in the incorporation of the CCR5 −2459G > A genotype information into studies evaluating new immunologic and chemotherapeutic strategies, and those designing and implementing better treatment strategies with current antiretroviral therapy, doing so would enable a better understanding of the response to the intervention, due to a mechanistic or constitutive explanation. Until we find a strategy, whether a stem-cell transplantation or CCR5 editing approach or something else, that delivers a cure to the millions, we should make use of every piece of information that may help curtail HIV/AIDS as a threat to public health.C-C chemokine receptor 5 ( CCR5) polymorphisms  and HIV-1 infection and disease progression. In those studies, the Δ32 allele, compared with the wild-type (wt) allele, was associated with protection against HIV infection and/or delayed disease progression [A variety of studies conducted in the 1990s examined the associations between C-C chemokine receptor 5 , followed by those from the two Baltic states Estonia (15.63%) and Latvia (15.09%). The lowest allele frequency of CCR5 Δ32 was observed in donors from Eritrea (0.26%) and from Ethiopia  [CCR5 Δ32/Δ32  within this data set [CCR5 −2459A ranges from 29% to 59% [According to a recent multipopulation study, donors from Norway had the highest allele frequency of  n = 76) . Donors data set . On the % to 59% ,15,16,17% to 59%  and 98% CCR5 haplotype nomenclature system consists of a total of nine polymorphisms, which include CCR5 ORF wt/∆32 and −2459G > A [CCR2 ORF SNP 190G > A , rs1799864; and six CCR5 promoter SNPs −2773A > G (rs2856758), −2554G > T (rs2734648), −2135T > C (rs1799988), −2132C > T (rs41469351), −2086A > G (rs1800023), and −1835C > T (rs1800024). Using these nine CCR2–CCR5 polymorphisms, haplotypes have been organized into seven evolutionarily distinct human haplogroups (HH) designated HHA, -B, -C, -D, -E, -F (F*1 and F*2), and -G (G*1 and G*2)  .Among these haplotypes, −2459G allele-carrying HHA (frequency range 6–71%) and HHC (frequency range 2–42%), and −2459A allele-carrying HHE (frequency range 12–37%) and HHF*2 (frequency range 5–24%) are highly prevalent ,17,18. ICCR5 haplotypes influence HIV infection and disease outcomes differently [CCR5 haplotypes (HHA–HHG*2), the consistency of the association of HHE homozygosity (E/E diplotype) with an unfavorable outcome across diverse populations is noteworthy: This diplotype was significantly associated with disease acceleration, particularly an accelerated progression to death in Caucasians [CCR5 haplotypes and HIV tropism in Estonian Caucasians found that HHE was associated with the presence of C-X-C chemokine receptor 4-tropic viruses [In the same way that the ORF wt/Δ32 and −2459G/A alleles show differences in phenotypic effects in vitro as well as in HIV/AIDS cohorts, different ferently ,21,22,23ucasians . It was ucasians , and Thaucasians . Even inucasians . A recen viruses . Unfavor viruses ,28,29. TCCR2 ORF 190A (64Ile) allele  allele . ConsistCCR5 Δ32/Δ32 donors for their life-threatening blood cancers. Because CCR5 Δ32/Δ32 donors are rare [CCR5 to engineer cells resistant to HIV-1 infection [CCR5 editing approach is taken, considering the −2459G > A status is, obviously, irrelevant.Given this background, the question is, has −2459G > A been forgotten or ignored in this era of an HIV cure, where all the emphasis is on ∆32? In this regard, I present some examples from the literature, together with my limited personal experiences, which suggest that it may have been. This may be because, recently, we have started to recognize that the effect of host genetic variation in HIV/AIDS is a complex phenomenon , and invare rare , and sucnfection ,32. Whethttps://www.ncbi.nlm.nih.gov/pubmed/) and personal communication/interaction with scientists regarding their work on HIV-1 neutralizing antibodies [However, considering the −2459G > A status may be important in studies where (a) immunologic strategies, such as antibodies for CCR5 , HIV-1 ntibodies ,35 and ttibodies ,41.CCR5 ∆32 and −2459G alleles were associated with improved clinical outcomes in highly active antiretroviral therapy (HAART)-treated patients. This finding was consistent with observations in treatment-naive patients. On the other hand, some studies reported an insignificant trend or no effect of these polymorphisms on response to HAART  are needed in order to elucidate the possible mechanisms underlying this race-specific association. Finally, among the HIV/AIDS treatment studies conducted 10–20 years ago, some showed that the ART  becauseART (see . FurtherCCR5 −2459G > A genotype information into studies evaluating new immunologic and chemotherapeutic strategies is important. In addition, studies designing and implementing better treatment strategies with current HAART should also include this information. Given the significance of ∆32, even when present as a single allele, the ORF wt/∆32 genotype information ought to be included in such studies. If it is included, then why not the information related to CCR5 −2459G > A genotype, if not the entire haplotype? Until we find a strategy that delivers a cure to the millions, we should make use of every piece of information that may help curtail HIV/AIDS as a threat to public health.Thus, in my view, incorporating the


Invadopodia are actin-rich protrusions developed by transformed cells in 2D/3D environments that are implicated in extracellular matrix (ECM) remodeling and degradation. These structures have an undoubted association with cancer invasion and metastasis because invadopodium formation in vivo is a key step for intra/extravasation of tumor cells. Invadopodia are closely related to other actin-rich structures known as podosomes, which are typical structures of normal cells necessary for different physiological processes during development and organogenesis. Invadopodia and podosomes are included in the general term ‘invadosomes,’ as they both appear as actin puncta on plasma membranes next to extracellular matrix metalloproteinases, although organization, regulation, and function are slightly different. Integrins are transmembrane proteins implicated in cell–cell and cell–matrix interactions and other important processes such as molecular signaling, mechano-transduction, and cell functions, e.g., adhesion, migration, or invasion. It is noteworthy that integrin expression is altered in many tumors, and other pathologies such as cardiovascular or immune dysfunctions. Over the last few years, growing evidence has suggested a role of integrins in the formation of invadopodia. However, their implication in invadopodia formation and adhesion to the ECM is still not well known. This review focuses on the role of integrins in invadopodium formation and provides a general overview of the involvement of these proteins in the mechanisms of metastasis, taking into account classic research through to the latest and most advanced work in the field. Invasion is a dynamic and complicated process with critical roles in cell survival and the development of multicellular animals. They play roles in such important processes as cell migration and invasion during embryogenesis, wound healing, and immune response, among others ,2,3. HowIntegrins are the main cellular receptors for ECM components, i.e., collagen, laminin, fibronectin, or vitronectin ,7,8. TheO-(hydroxyl group of threonine or serine residues) glycosylated . Fo. Fo49]. Invadosomes are composed of several actin networks and different actin crosslinking proteins . Together they form actin-rich microdomains and provide the mechanically stable polymerized F-actin necessary to hold up the invadosome structure . InvadosInvadopodia contain many lipid rafts, indicating that molecular clustering, formation of signaling complexes, and molecular recruitment are necessary for invadopodium formation. Cholesterol depletion, which disrupts lipid raft formation, blocks invadopodium degradative activity . Furtherinv). MENAinv seems to induce invadopodium activity, enhancing its sensitivity to EGF as well as the phosphorylated status of cortactin. As a result, the invasiveness of cancer cells is increased [Moreover, other mechanisms of regulation have been reported. Thus, miRNA143 and miRNA145 can negatively regulate podosome formation, although their role in invadopodium formation has not been established . In addincreased .Recently, a new invadosome type has been described in endothelial, embryonic, and tumor cells. It is a structure with ECM remodeling capability, but a different morphology that the classical F-actin dots. These structures present a linear organization and are associated with collagen I fibrils. They are also composed of scaffold elements of invadosomes  and metalloproteinases . Linear invadopodia depend on CDC42 activation and DDR1 , but theCaenorhabditis elegans gonad organization [Podosomes are cellular actin structures characteristic of non-transformed cells, including osteoclasts ,65,66,67nization . Podosomnization . Other nnization ,76,77,78Loss of podosome formation in hematopoietic linages is associated with serious diseases, e.g., WASP mutation is the origin of Wiskott–Aldrich syndrome (WAS) . In zebrThe ultrastructural composition of podosomes is very complex, with different domains, regions, and proteins . PodosomIn macrophages, supervillin and myosin IIA determine the presence of two different podosome subpopulations: the precursors, which are larger and located at both the periphery and the leading edge, and the successors, derived from the precursors and located toward the center of the cell . In addiIn order to expand blood vessels during neo-angiogenesis, endothelial tip cells overexpress VEGF receptors and down-regulate Notch signals to form podosomes ,77. It hOsteoclasts are responsible for bone resorption by attaching to the bone surface while moving through it . In the During first steps of adhesion, fibroblasts form integrin clusters, where F-actin cores start to precede the formation of podosome rings . In the Integrins are proteins present in classical invadosomes, which form the adhesion ring in the upper domain of the structure β2 and β3) and in the podosome core (β1) ,87. Inte and in tIntegrin expression in podosomes depends on both the type of matrix and the cell type. β1-integrin is highly expressed in megakaryocytes and it binds to several collagen isoforms, fibronectin, and laminin. β2-integrin mediates the formation of podosomes in macrophages, binding to fibrinogen, and β3-integrin binds to osteopontin, vitronectin, and fibrinogen, preferentially in osteoclasts ,76. InteThis integrin plays a key role in the assembly of podosome rosettes. Mouse embryonic fibroblasts with constitutively activated SRC form invadosomes associated with β1-integrin expression and PKC regulation . High α6Eosinophils are immune cells associated with allergic responses. These α4β1  integrin-expressing cells form podosome-like structures over endothelial cells in a process dependent on endothelial VCAM1 expression. Unlike adherent fibroblasts, eosinophil podosomes can degrade VCAM-1 from endothelial cells in an ADAM8 metalloproteinase-dependent manner. Eosinophil α4β1 integrin is located in podosome structures, while α4β7 or αDβ2, other VCAM1 partners, present a diffuse distribution . In cultMembers of the β2-integrin family include αLβ2 , αMβ2 , αxβ2  and αDβ2 (CD11d/CD18). They are expressed in most white blood cells (WBC), as well as in smooth muscle cells, and have a crucial role in podosome formation ,91. For Endothelial barriers are critical walls that immune cells must cross. During transcellular diapedesis, leukocytes, lymphocytes, monocytes, and basophils insert their podosomes into endothelial cells to choose the most suitable region to cross. β2-integrin and its partners in endothelial cells are vital in this process, because inhibition of ICAM-1, PECAM, or some podosome regulatory proteins reduces or cancels cell diapedesis ,109. Mat−/− mouse embryonic fibroblasts still form small podosomes in constitutively active SRC conditions [β3-integrin is vital for embryonic development, bone remodelling, platelet function, and angiogenesis. For instance, the interaction of β3-integrin and kindlin-2 to promote podosome formation in endothelial cells has been demonstrated in vitro using optogenetic models . Mice denditions . αvβ3 isnditions ,112. Seanditions . Althougnditions . In factnditions . αvβ3 isnditions . Neverthnditions ,64,67. Tnditions . αII-Spenditions .It has been suggested that the recruitment of β3-integrin, paxilin, and cortactin represents the first step in the assembly of podosomes, and is followed by the recruitment of F-actin in the core and α-actinin, and the increase of β3-integrin in the podosome cloud ,114. PhoEpithelial cells interact with ECM by focal contacts and hemidesmosomes, which contain podosome-like structures. For instance, squamous and transitional epithelial cells have hemidesmosomes with α6β4 integrin in order to bind laminin-5. These podosome-like structures are also present in the actin core, with α3β1 integrin around it. During the assembly of hemidesmosomes, β4-integrin is located at the base of the actin core, but when cells are induced to migrate it becomes co-localized with dynamic actin at the leading edges .Only the α3-integrin subunit has been described individually as a podosome element. It was described in the adhesion ring of podosomes of an oral SCC cell line derived from a primary gingival tumor. It presented in the same location as β1-integrin, and it was proposed that α3β1 is a functional heterodimerization found in the podosomes .Invadopodia are the bad members of the invadosome gang. They are actin-rich tumoral structures with ECM degradative ability associated with metastases and malignant tumors ,39. ThesInvadopodia, like podosomes, are spontaneously formed by tumor cells or as a result of different stimuli: soluble growth factors, cell–cell contact or ECM properties, and signaling. Structurally, invadopodia are described as actin structures in the ventral surface of cells, where they contact with the ECM. They have a central core of polymerized branched actin with many regulatory proteins around the structure, and MMPs for ECM degradation ,89. InteThe first description of integrins and their regulatory proteins in invadopodia was reported twenty years ago . The rolIntegrin ligand binding generates outside-in and inside-out signals which cooperate to regulate cellular process ,142,143,2) derived from fibronectin that inhibits α5β1integrin signaling, and had good toxicity results in a phase I trial against solid tumors [Integrin expression and location are altered in many diseases, therefore, therapies against these proteins have been designed and are in advanced phases of development . Integrid tumors .The β1 subunit is the integrin most often reported to be associated with invadopodium formation in tumors, and of which blockage or silencing reduces invadopodium formation in multiple cancer types ,136,154.β1-integrin performs functions at different stages during invadopodium formation.A. It is associated with ARG phosphorylation of cortactin by direct protein–protein interaction ,49,53. CB. This integrin is also important in NHE1/moesin recruitment to the invadopodium core . An incrINV and α5β1 modulate FAK/SRC interaction and their signaling transduction pathways [C. β1-integrin also regulates SRC activation by indirectly kidnapping FAK at focal adhesion points. MENApathways .D. It has been demonstrated that β1-integrin promotes metalloproteinase secretion at invadopodia . This inE. β1-integrin also participates in mechanosignaling. Cofilin inactivation and β1-integrin over-expression were detected under mechanical stimuli in the HT1080 human fibrosarcoma cell line . KindlinThe role of β3-integrin in invadopodium formation is very controversial, although many cancers show dysregulation of αVβ3 integrin ,161. Somβ3-integrin’s functions during invadopodium formation are also, like β1, associated with different stages.A. αVβ3 integrin interacts with MT1-MMP to promote MMP2 activation in breast cancer cell lines , but it B. In breast tumor cell lines, collagen IV activates Dishevelled-associated activator of morphogenesis 1 (DAAM1) and RHOA to promote haptotaxis. DAAM1 is a formin family member often localized to invadopodia of breast cancer cell lines, where it binds αVβ3 to promote invadopodium formation though the modulation of actin polymerization and free barbed end formation . The αVβC. SNAIL2 is a transcription factor in which silencing is associated with inhibition of invadopodium formation. SNAIL2 is also down-regulated after αVβ3 or fibronectin silencing. Chloride intracellular channel (CLIC1), which is up-regulated in many tumors, interacts with αVβ3 and regulates fibronectin-αVβ3 interactions and fibronectin assembling to promote metastasis ,175. BotD. αVβ3 over-expression correlates with invadopodium formation in renal carcinoma, sarcoma, and glioblastoma cell lines. αVβ3 siRNA reduces invadopodium formation and cell proliferation in these cell lines, and these effects were not detected in β1-silenced cells . β3-inteβ8-integrin subunit heterodimerizes exclusively with αV-integrin, and it binds to latent TGF-β. αVβ8 expression is altered in malignant brain tumors and metastases . β8-inteα3- and αV-integrins have been described individually as invadopodium elements in an oral squamous carcinoma cell line derived from a recurrent gingival tumor. While α3 appeared surrounding the invadopodium membrane, αV was located at the “head” of the invadopodia . GMB areECM and cells present in the surrounding tumor stroma contribute to cancer progression. Non-tumoral cells  and structural aspects of ECM  are factors that have been implicated in tumor malignancy and migration by mechano-transduction signals or ECM remodeling ,179. TheCollagens, laminins, fibronectin, hyaluronic acid, entactin/nidogen or heparin sulfate proteoglycans are extracellular matrix compounds, the interactions of which with the integrin heterodimers modulate their conformational configuration and functionality . These mThese molecules are the main components of ECM and basement membrane. They are ligands for some integrins, and have been demonstrated to regulate invadopodium formation. For instance, megakaryocytes increase podosome formation in collagen matrices, in addition to other matrices such as fibrinogen . Liu obsBoth fibrin and fibrinogen molecules are β2- and β3-integrin ligands associated with podosome formation in macrophages  and otheLaminins are basement membrane glycoproteins that interact with different integrins, such as αVβ3, α5β1, α3β1, α6β1, α7β1, α6β4 and α2β1 . It has This matrix factor has been demonstrated to be associated with development of invasive carcinomas in human and mouse models of breast cancer ,195,196.Matrix crosslinking is a factor related to matrix rigidity that negatively regulates invadopodium formation, penetration, and matrix degradation . Altoget+/H+ exchanger-1 (NHE1) to increase intracellular pH, which releases cofilin from cortactin molecules during invadopodium elongation [2+ and promotes podosome formation, cell adhesion, and bone resorption [pH is a matrix property essential for invadopodium formation. Integrins mediate cellular cues and can act as pH sensors to regulate actin protrusions . α5β1, αongation . In addiongation . Extracesorption .Hypoxia is another microenvironment factor that induces invadosome formation. Thus, the hypoxia-inducible factor (HIF) family modulates metastases in many tumor types . In melaO-GlcNAcylation modification at serine 108 of cofilin is necessary for protein activation, and, subsequently invadopodium formation and matrix degradation in breast cancer cells [Posttranslational modifications of intracellular, membrane, or extracellular proteins are other factors associated with invadopodium formation. Protein glycosylation is a post-transcriptional modification often related to cancer progression and metastasis . The O-Ger cells . Contrarer cells . MUC1 iser cells . Proteiner cells . Lumicaner cells . Lumicaner cells . In osteer cells . Howeverer cells . Lumicaner cells .Stromal cells are located in the surrounding microenvironment of tumor cells. Recently, new roles in tumorigenesis have emerged for these cells ,221,222.Inv and it associated with poor prognosis and invadopodium formation [Many authors have demonstrated that macrophages induce invadopodium formation in tumor cells necessary for trans-endothelial migration. During this process, macrophages, cancer, and endothelial cells form a multicellular structure called tumor microenvironment of metastasis (TMEM) that has been used as prognostic marker of metastasis in some cancer types . This prormation .Although the relationship of fibroblast presence in stroma with cancer development is well demonstrated, its role in invadosome formation remains unclear. It is thought that CAFs are the major ECM producers in tumors , and thaMacrophages are another cell type present in tumor stroma with an important role in tumor regulation. These cells are known as tumor-associated macrophages (TAMs), and it has been reported that they can promote tumor metastasis through paracrine signals . TAMs anDuring recent years, more and more studies have started to unveil the multifaceted roles of integrins in normal tissue remodeling and pathological processes such as tumor progression and metastasis. Crosstalk relationships with growth factor receptors as well as oncogenes have been associated with specific integrins in order to regulate cancer progression and metastasis. Numerous reports have also highlighted the importance of tumor interaction with the microenvironment and stromal factors supporting tumor growth and survival. Furthermore, deregulated integrin-mediated cell adhesion to the ECM could potentiate different stages of tumorigenesis. There is an increasing body of evidence suggesting that integrins are candidate targets for anticancer therapies, among others. In fact, effective anti-integrin therapies, such as vedolizumab, are approved for Crohn’s diseases or ulcerative colitis, and others are in advanced trials against multiple sclerosis, cardiovascular problems, or inflammatory bowel disease. Initial attempts in cancer, however, have not resulted in clinically relevant improvements, probably due to compensatory mechanisms counteracting integrin inhibition. Another possibility may be the in vitro methodological approaches of the studies, i.e., 2D environments and simple matrixes. We think that more complex environments, 3D cultures, or even more intense efforts to use in vivo models could give us a more solid base for therapy design. Apart from individual characteristics of patients, when considering integrins as possible anti-cancer targets, we should also take into account the vast integrin repertoire present in cancer and tumor stroma cells, ECM, and tumor microenvironment composition. Despite this complexity, all these factors may improve clinical stratification, advancing towards personalized medicine and allowing us to select the most effective anti-integrin combinations at appropriate stages.


The article “Maturation of the [Ni–4Fe–4S] active site of carbon monoxide dehydrogenases”, written by Mériem Merrouch, Martino Benvenuti, Marco Lorenzi, Christophe Léger, Vincent Fourmond, Sébastien Dementin was originally published electronically on the publisher’s internet portal (currently SpringerLink) without open access.http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.The copyright of the article changed on 18, May to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (The original article has been corrected.


The other influenza virus surface protein, neuraminidase (referring to brain lipids from which it was first derived) is a virus receptor-destroying enzyme that removes its substrate, sialic acids, from infected cell surfaces so that newly made progeny viruses are released to infect additional cells. At present, 18 hemagglutinin subtypes (H1–H18) and 11 neuraminidase subtypes (N1–N11) are recognized.In 1941, virologist George K. Hirst discovered that adding influenza virus to red blood cells (erythrocytes) in a test tube caused the cells to agglutinate, mediated by one of the virus surface glycoproteins, hemagglutinin (from the Greek  “glue”) . Alfred


The impact of body mass index (BMI) on FPS is also investigated, adjusting for age, sex and under-reporting. Effects of under-reporting on relationships between FPS and BMI; and BMI on consumption frequency (UK only) were explored. OWOB reported larger FPS than normal-weight individuals in many, but not all food subgroups; however, there were only two significant FPS differences. In adjusted analyses, French individuals consumed 1.0 g (99% CI 0.01–2.1 p = 0.01) greater FPS in cakes for 1 point difference in BMI. ‘Other cakes’ and ‘dark chocolate’ were also significantly positively associated with BMI. High-fat bar snacks, but no UK main food groups, were positively associated with BMI. There was limited evidence of links between FPS and BMI in UK and French national cross-sectional data, possibly due to data limitations such as under-reporting. Future work should explore this and relationships between consumed FPS and on-pack suggested serving sizes to provide evidence to assist obesity-prevention policies.Evidence links consumed food portion size (FPS) and excess weight via increased energy intake. Policies to regulate on-pack serving sizes may be needed; determining consumed FPS of popular energy-dense foods for normal weight and overweight or obese (OWOB) adults, as reported here, may provide evidence to assist this. Data were analysed from national cross-sectional surveys, the French Étude Individuelle Nationale des Consommations Alimentaires2 2005–2007 ( Europe is the World Health Organization (WHO) region most severely affected by non-communicable diseases (NCDs), which, alongside related conditions including overweight and obesity, have significant and growing economic and social costs. It is accepted that obesity is one of the biggest health problems facing the European population; it accounts for 2–8% of WHO European health costs  and is eEvidence indicates an indirect link between food portion size (FPS) and excess weight via increased energy intake, and suggests that limiting FPS contributes to reduced energy intake and, therefore, reduced weight gain ,7,8. BotThe positive association between FPS and food intake is known as the ‘portion size effect’ . In theiEvidence suggests that UK commercially available FPS have increased in some energy-dense food categories in the 20 years since government-based FPS guidelines  were relNational diet survey (NDS) methodologies are developed primarily to assess the nutritional status of a population , so are Using their respective NDS ,26,27, tTwo government-funded NDS were obtained and analysed for consumed FPS for selected food groups in relation to BMI in adults aged 19–64 years. Consumed FPS was defined as the total weight (in grams) of a particular food consumed in one eating occasion.Data collected for the French Étude Individuelle Nationale des Consommations Alimentaires 2 (INCA2) from 2005–2007 were used in the analyses . The stuUK data were analysed from the National Diet and Nutrition Survey (NDNS) Rolling Programme (RP) years 1–4 and 5–6 (2008–2012 and 2012–2014) ,27. The Data from 2117 French and 3413 UK adults aged 19–64 years were used for the overall analyses. Underweight adults with a BMI <18.5 were excluded from analyses; this constituted 98 individuals in the French INCA2 and 46 in the UK NDNS. In addition, 25 adults with missing BMI values were excluded from analyses in the INCA2 and 203 adults with missing BMI or infeasible BMR:energy intake values excluded from the NDNS.The NDNS 2008–2014 dataset was used to identify popular energy, fat and sugar-dense foods in the UK. The main NDNS food groups were listed by total number of eating occasions for adults aged 19–64 years and the tertile with the lowest number of eating occasions were excluded. The average energy, fat and total sugar density was then determined for each remaining food groups and those with above average density in all three nutrients were selected for analysis—these were biscuits, buns, cakes, pastries and fruit pies (hereby referred to as ‘Cakes’) and chocolate confectionary (hereby referred to as ‘Chocolate’). Although crisps and savoury snacks (hereby referred to as ‘Crisps’) did not have above average sugar density, it was also selected due to a high salt content, which is a WHO Europe nutrient of concern ) and ‘pastries’ (130 g [93 g]). For OWOB individuals this was ‘tart’ (182 g [SD 58 g]), ‘pastries’ (155 g [85 g]) and ‘fruit pie’ (154 g [44 g]). For Biscuits and Crisps in normal weight individuals this was ‘filled chocolate’ (61 g [40 g]), ‘unfilled coated biscuits with inclusions’ (52 g [51 g]) and ‘filled non-chocolate’ (47 g [32 g]) and for OWOB individuals this was ‘filled chocolate’ (60 g [39 g]), ‘unfilled coated biscuits with inclusions’ (43 g [29 g]) and both ‘cookies’ (42 g [23 g]) and ‘other’ (42 g [26 g]). For the Chocolate group in normal weight individuals this was ‘Mars-type bar’ (52 g [40 g]), ‘wafer bar’ (49 g [22 g]) and ‘chocolate spread’ (41 g [42 g]) and for OWOB individuals this was ‘wafer bar’ (46 g [19 g]), ‘Mars-type bar’ (38 g [15 g]) and ‘chocolate spread’ (31 g [25 g]) see .In the UK NDNS the three food subgroups with the highest mean FPS for normal weight adults 19–64 years in Cakes were ‘pastries’ (93 g [30 g]), ‘fruit pie’ (91 g [38 g]) and ‘éclairs’ (89 g [46 g]) and for OWOB individuals this was ‘pastries’ (110 g [58 g]), ‘fruit pie’ (92 g [46 g]) and ‘doughnut’ (84 g [48 g]). For Biscuits in normal weight individuals this was ‘cookies and flapjack’ (47 g [37 g]), and ‘unfilled coated/inclusions’ (33 g [18 g]), ‘filled non-chocolate’ (33 g [24 g]) and ‘cereal bars’ (33 g [13 g]). For OWOB individuals this was ‘cookies and flapjack’ (43 g [30 g]), ‘jaffa cakes’ (42 g [49 g]) and ‘filled non-chocolate’ (35 g [19 g]). For Crisps in normal weight individuals this was ‘nuts’ (63 g [28 g]), ‘popcorn’ (50 g [31 g]) and ‘tortilla chips’ (44 g [39 g]) and for OWOB individuals this was ‘popcorn’ (123 g [98 g]), was ‘nuts’ (76 g [34 g]) and ‘tortilla chips’ (40 g [32 g]). For Chocolate in normal weight individuals this was ‘coated nuts/fruit’ (81 g [64 g]), ‘Mars-type bar’ (49 g [17 g]) and ‘other’ (48 g [28 g]) and for OWOB individuals this was ‘coated nuts/fruit’ (66 g [69 g]), ‘Mars-type bar’ (47 g [17 g]) and ‘honeycomb/crunch’ (44 g [37 g]) see . Food suThere was little evidence of statistically significant FPS differences by BMI status . Howeverp =< 0.001) in their consumed FPS of ‘other cakes’ and 1.0 g (99% CI 0.1 to 1.9 p = 0.004) in ‘dark chocolate’ was observed in the same direction. In addition, the Cakes main food group FPS was significantly associated with BMI, where FPS increased by 1 g (99% CI 0.01 to 2.1 p = 0.01) with every BMI point increase. When adjusting for under-reporting, age and sex (model 2) FPS was positively associated with BMI in the same subgroups (see When adjusting both for sex and age (model 1) and under-reporting, age and sex (model 2) in the regression analyses, there were just two French food subgroups with significant associations between FPS and BMI used as continuous variables, whereas there were none in the unadjusted analyses. Only total Cakes was statistically significant in both the unadjusted and the adjusted analyses and retained the same direction of association. Overall, after adjusting for sex and age (model 1), for every point increase in BMI between individuals, a difference of 3.1 g (99% CI 1.0 to 5.2 p = 0.01) with every point increase in BMI after adjusting for sex and age. The same was true when adjusting for under-reporting, age and sex (model 2), where ‘high-fat bar snacks’ increased by 3.2 g  with every point increase in BMI. Unlike the French analyses, none of the main UK food groups were significantly associated with BMI , FPS was significantly associated with BMI in high-fat bar snacks only, which increased in FPS by 3.3 g  under-reported overall energy intake and were excluded in the sensitivity analysis. In Biscuits and Crisps this was 12% (n = 247) and in Chocolate 15% (n = 211). In the adjusted analyses ‘other cakes’ lost significance after excluding under-reporters, likely due to lower number of individuals consuming foods in this subgroup and loss of power, but ‘dark chocolate’ remained significantly associated with higher FPS in OWOB individuals (data not shown). The Cakes main food group also retained and strengthened its association, with FPS increasing by a greater amount after excluding under-reporters.Of those who reported consuming Cakes in the French INCA2, 16% (n = 635 unweighted) Cakes consumers as under-reporters of overall energy intake. In Biscuits consumers this was 31% (n = 1170), in Crisps 30% (n = 661) and in Chocolate 22% (n = 551) of consumers. ‘High fat bar snacks’ retained a higher FPS in OWOB individuals after excluding under-reporters, and like in the French INCA2, there was no change in the direction of association (data not shown).In the UK NDNS, sensitivity analysis identified 23% . When adjusting for age and sex after excluding under-reporters (model 2) Cakes was no longer significant, but for every point increase in BMI, consumption frequency per day still decreased in Chocolate −0.008 99% CI −0.01 to −0.001 p = 0.003)  for every point increase in BMI, consumption frequency per day of Cakes and Chocolate decreased (Cakes: −0.006 99% CI −0.01 to −0.003 = 0.003) . So althThese analyses constitute a detailed examination of FPS and how these might vary with BMI in two large, nationally representative groups in energy, fat and sugar-dense snack foods. Of the main food groups analysed, only Cakes in the French INCA2 had a significant association between FPS and BMI, where FPS increased with each BMI point increase. There were very few significant associations between FPS and BMI in the energy-dense food subgroups analysed from the French and UK national dietary surveys, and these categories differed between the countries. The lack of UK and French similarities between the types of foods with significant associations suggests that French and UK diet preferences of OWOB individuals may differ across the selected main food groups. ‘Pastries’, ‘fruit pie’ and ‘Mars-type’ chocolate bars had the largest FPS for both normal and OWOB adults aged 19–64 years in both the French and UK analyses . The conOn-pack serving sizes are not currently regulated or standardised and, unlike the requirement to state pack-size, providing serving-size information is not currently mandatory in the UK . WithoutMean FPS in the high FPS subgroups varied in their proximity to UK government FPS guidance, which was last updated in 1993 ,44 as thThe few significant associations present were positive, showing that adults with higher BMIs reported consuming larger FPS. The extent to which this is a valid finding or one resulting from methodological limitations and misreporting cannot be fully determined. Studies have variously found few associations between FPS and adiposity , concludHerman et al.  claim evUnder-reporting clearly presents a limitation in using NDS to assess FPS, affecting the number and variety of food main and subgroups with associations between FPS and BMI. Using the Goldberg method, under-reporting in the present analysis was estimated to be 1.5 times higher in the UK than in France, at 35% and 23% respectively. This is similar to levels previously reported, at 32% and 22.5% respectively ,55. ThisVanrullen et al.  also fouA limitation of our current study is the low number of consumers in some of the food subgroups, which may mean the analyses are underpowered to find associations at this level. An additional limitation is that it uses cross-sectional survey data, which cannot demonstrate the direction of association and, therefore, a causal relationship . NeverthIn terms of strengths, the categorisation of foods by product type broadly matching the UK and French subgroups and examining FPS of similar products as they would be sold in a commercial environment and subsequently consumed by individuals, allows a more meaningful assessment of FPS than previous categorisations. Previous studies tend to use broader or incompatible food groups ,44,45,46A further strength is that there are few studies exploring FPS across Europe, and the literature focuses primarily on America. However, as only two Western European countries were investigated, the ability to make Europe-wide assertions is limited; quality raw NDS data (as distinct from summary reports) with the necessary food code variables were not readily available to analyse from other countries. Nevertheless, the French and UK NDS used similar dietary methodologies of consecutive, self-reported food diaries  with estimated FPS, making them good comparative cases to report together.Energy density and consumption frequency averaged to one day was not available for the French INCA2, so the main food groups selected for analysis were based on those chosen in the UK NDNS, and consumption frequency analysis limited to the UK. This made comparisons easier. Additionally, they are both developed Western European countries with some similarities in foods consumed and with similar dietary methodologies; however, different French and UK dietary patterns may result in differences in the most frequently consumed main food groups. This has implications for future extension of the analyses to other countries if their NDS lack consumption frequency data or methodologies differ. There is a need for better quality, harmonised NDS methodology and implementation across Europe, potentially achievable with new technologies, in order to generate robust data on which policy can be based. Further methodological limitations include the use of a 1.55 PAL across all individuals rather than being estimated individually, which limits the accuracy with which under-reporters can be identified, although 1.55 is an accepted value for a sedentary lifestyle in the populations used ,40,41. TFPS for commonly consumed high-energy, -fat and -sugar foods were generated for two countries, which could provide important information for food policy work. There was limited evidence of associations between FPS and BMI of energy dense foods, and although the few significant associations found were positive, the subgroups with significant associations differed across the two countries. The limited evidence may be due to the cross-sectional nature and other limitations of the NDS data, such as the high estimates of under-reporting and the small number of individuals in some food subgroups. Excluding under-reporters impacted upon results; levels were higher in the UK, potentially due to the higher number of OWOB individuals previously associated with under-reporting. Future work should further explore data limitations like under-reporting, and investigate relationships between consumed FPS and on-pack serving sizes, which could help to inform future obesity-prevention policies.