Dronedarone is recommended for the treatment of atrial fibrillation. However, we do not know its effect on vascular remodeling. This study was designed to assess whether dronedarone has the potential to improve the intramyocardial artery remodeling induced by chronic hypertension. Ten-month-old male spontaneously hypertensive rats (SHR) were randomly assigned to receive dronedarone (100 mg/kg) or vehicle. Age-matched male Wistar-Kyoto rats served as controls. After 14 days of treatment, we studied the structure (geometry and fibrosis) of the intramyocardial artery using histological analysis. Nitric oxide (NO) in plasma was analyzed. In the untreated SHR, we observed a significant increase in external diameter, lumen diameter, wall width, cross-sectional area, and collagen volume density, as was expected in the experimental model. Dronedarone induced a significant decrease in wall width, cross-sectional area, and collagen volume density in SHR-D in comparison with untreated SHR. The values obtained in SHR-D were similar in the WKY control group. We found significantly higher NO levels in plasma in SHR-D than in untreated SHR. Dronedarone improves the intramyocardial artery remodeling induced by chronic hypertension in SHR through increased nitric oxide bioavailability. Hypertension leads to adverse remodeling  in the coronary artery wall and therefore increases the incidence of cardiovascular events , 2. AntiDronedarone is a novel antiarrhythmic drug for atrial fibrillation, acting as a multichannel blocker . DronedaThis study was designed to evaluate the efficacy of dronedarone in attenuating the intramyocardial branch of the obtuse marginal artery remodeling in spontaneously hypertensive rats (SHR), the most widely used animal models for human essential hypertension, left ventricular hypertrophy, and coronary damage.The study was performed in accordance with European Union Guidelines to use experimental animals (Directive 2010/63/EU and Spanish Law RD 53/2013) and was approved by the Ethics Committee of Hospital Gregorio Marañón, Madrid, Spain.Ten-month-old male SHR were randomly assigned to receive oral dronedarone (100 mg/kg once daily)  for a period of 14 days or to receive vehicle . A third group of normotensive control rats  was also added. Once the treatment was finished, the rats were killed by decapitation after sedation with diazepam 4 mg/kg and ketamine 10 mg/kg .As described in detail in previous studies  systolicμm) and stained with orcein [π/4)×(ED2−LD2) [μm sections (3 sections/rat) of paraffin blocks were stained with picrosirius red, and the collagen volume density (CD) was determined using a stereological method [We analyzed 6 animals for each group as described in detail previously –14. Hearh orcein . The inth orcein . Wall wiED2−LD2) . For colμL of plasma or nitrite standard was mixed with 10 μL of N-ethylenediamine. The protein from samples was precipitated by addition of 110 μL of trichloroacetic acid. 50 μL of supernatant (obtained by centrifugation) was mixed with 50 μL of saturated vanadium chloride, 25 μL of sulfanilamide, and 25 μL of N-naphthyl-ethylenediamine. The mixture was incubated at 37°C for 1 hour, and absorbance was read at 540 nm.We analyzed 5 animals from each group. The same protocols described in previous studies  were fol U test . The results were expressed as mean ± SEM and analyzed using repeated-measures analysis of variance . Statistical significance was set at P ≤ 0.05. The analysis was performed using IBM SPSS Statistics for Windows, version 20.0.The results were expressed as median and 25th and 75th percentiles and analyzed with the Mann-Whitney P<0.01, and 460.40±1.12 vs. 380.51±6.21 g, P<0.001, respectively); however, the difference was not statistically different between SHR and SHR-D.Rat weight was significantly higher in WKY than in SHR and SHR-D  used for the maintenance of sinus rhythm and control of ventricular rate . DronedaIn the present study, the mechanism underlying dronedarone improving the intramyocardial artery remodeling could be related in part to an increase in the bioavailability of NO. Coronary artery remodeling in the presence of hypertension implies structural changes , but alsOur results provide preclinical data on the beneficial effects of dronedarone on intramyocardial artery remodeling, which is associated with an increased incidence of cardiovascular events , 2. DuriThe present study is subject to limitations. This study was designed to evaluate the efficacy of dronedarone in attenuating the intramyocardial branch of the obtuse marginal artery remodeling in SHR. Our results show that dronedarone improves structural intramyocardial artery remodeling; however, vascular remodeling in the presence of hypertension implies not only alterations in the structure of vessels, but also endothelial dysfunction , 27. ThuIn conclusion, this study suggests that dronedarone improves the intramyocardial artery remodeling induced by chronic hypertension in SHR through increased nitric oxide bioavailability.


Phakopsora pachyrhizi, is a devastating disease with an estimated crop yield loss of up to 90%. Yet, there is a nerf of information on the metabolic response of soybean plants to the pathogen Untargeted metabolomics and Global Natural Products Social Molecular Networking platform approach was used to explore soybean metabolome modulation to P. pachyrhizi infection. Soybean plants susceptible to ASR was inoculated with P. pachyrhizi spore suspension and non-inoculated plants were used as controls. Leaves from both groups were collected 14 days post-inoculation and extracted using different extractor solvent mixtures. The extracts were analyzed on an ultra-high performance liquid chromatography system coupled to high-definition electrospray ionization-mass spectrometry. There was a significant production of defense secondary metabolites  when P. pachyrhizi infected soybean plants, such as putatively identified liquiritigenin, coumestrol, formononetin, pisatin, medicarpin, biochanin A, glyoceollidin I, glyoceollidin II, glyoceollin I, glyoceolidin II, glyoceolidin III, glyoceolidin IV, glyoceolidin VI. Primary metabolites  also were putatively identified. This is the first report using untargeted metabolomics and GNPS-Molecular Networking approach to explore ASR in soybean plants. Our data provide insights into the potential role of some metabolites in the plant resistance to ASR, which could result in the development of resistant genotypes of soybean to P. pachyrhizi, and effective and specific products against the pathogen.Asian Soybean Rust (ASR), caused by the biotrophic fungus  Glycine max (L.) Merrill) is prominent among crops due to its agro-economic and nutritional value, used mainly as a source of proteins and oils for human consumption, in animal feeds and for biofuel production2. However, estimates indicate a necessity to double global agricultural output by 2050 to feed a rising population, hence soybean productivity needs to be increased by 2.4% per annum3. Yet, soybean diseases were estimated to reduce crop yield by 11% in the United States4 and 50% or greater in the southeastern United States5.The soybean  was registered for the first time in Brazil during the 2001/2002 harvestPhakopsora pachyrhizi is the causal agent of the ASR. The phytopathogen P. pachyrhizi survives all-year-round whenever a soybean host plant is available and when conditions favor soybean development, thus supporting epidemic outbreaks8. Fungicides, such as demethylation and quinone outside inhibitors are largely used to prevent infection by P. pachyrhizi9. However, the Fungicide Resistance Action Committee (FRAC) recently reported regions in Brazil with intensive use of chemical fungicides where the efficiency of those products was reduced10, which can be caused by intensive use of a single chemical and excessive application. Management strategies to address ASR can range from planting early ripening varieties, field monitoring, eliminating secondary hosts, and even imposing soybean-free growth periods (60–90 days) in the threatened areas9. Thus, no single solution is available to address P. pachyrhizi infection. Therefore, the use of several management strategies must be associated to reduce yield losses and to ensure the crop sustainability, which is time and resource consuming.The fungus 12. The signaling cascade can be mediated by plant hormones such as salicylic and jasmonic acid, and ethylene. These hormones can then generate secondary metabolites with antimicrobial properties and modulate plant defenses against pathogens16. During a pathogen attack, plants recognize pathogens or microbe-associated molecular patterns (PAMPs) and increase its immune defense systems17. Nonetheless, some pathogens can reduce plant immunity18, which triggers a second immune system known as effector-triggered (ETI). The ETI is activated by the recognition of microbial molecules from cell-surface-membrane proteins19. However, P. pachyrhizi can bypass both mechanisms and alter the host metabolism20.Perception of plants by invaders rapidly activates ion flow mechanisms; modulates the production of reactive oxygen species (ROS), primary metabolite modification, and biosynthesis of secondary metabolites; and favors expression of defense genes21. Untargeted metabolomics has already been employed to improve our understanding of the metabolism of inter-kingdom interactions, including plant-pathogen interactions22. The analysis of the whole metabolic profile of a raw plant extract is challenging due to the diversity of chemical classes23. Therefore, high efficiency liquid chromatography coupled to mass spectrometry can be used to explore complex matrix, allowing separation and detection of metabolites in a single chromatographic analysis24. In addition, tandem mass spectrometry, such as the Q-TOF (Quadrupole - Time of Flight) geometry, enables sequential experiments (MS/MS) to provide more discriminating data for the metabolic identification process25.The vast majority of studies exploring plant metabolites have focused on the prospection of bioactive metabolites. However, with recent advances in analytical methods and annotation tools, metabolic modulation in plants as a result of abiotic and biotic stresses can now be systematically addressed27. Therefore, the association of analytical techniques with molecular annotation tools is essential during the identification stage of the vast metabolic chemical diversity present in biological systems. Recently, Dorrestein and colleagues introduced a tandem mass spectrometry (MS/MS) data organizational approach, the Molecular Networking29. Chemical entities can be arranged, visualized, and compared to databases available in the literature30. Furthermore, fragmentation patterns similarities can be explored, thus enabling the discovery of novel compounds. Molecular Networking has led to the development of Global Natural Products Social Molecular Networking (GNPS) (http://gnps.ucsd.edu), a data-sharing platform that allows the research community to perform data-driven, crowd-sourced analysis and, it has been widely used for the annotation of MS-based chemical signatures30.Metabolomics analysis of complex samples by liquid chromatography - mass spectrometry (LC-MS/MS) generate a large amount of data and the identification process is challengingP. pachyrhizi were inoculated or not, and to identify metabolites actively produced by the soybean plant to cope with pathogen infection.In this context, the objective of this study was to use untargeted metabolomics approach to evaluate differences in the metabolic profile of soybean leaves when 31. Two daughter seeds were sown per pot (5 L) containing substrate .The genotype PI636463 (susceptible to ASR), provided by the Germplasm Active Bank of Embrapa Soybean was used. The get seeds (daughters seeds) having homogeneous physiological characteristics were obtained from a single seed (mother seed). The experiment using the get seeds was conducted in a greenhouse under the following growth conditions: temperature = 28 ± 2 °C; ultraviolet radiation = 70%, ±10%; and 12 h/12 h light/dark photoperiod, up to vegetative stage V6-V7P. pachyrhizi were supplied by the Embrapa Soybean Phytopathology Laboratory , obtained from cultivar BRS 284 (susceptible standard), with 92% of germination viability. A total of 500 mL of a spore suspension (3.6 × 104 U mL−1) was prepared with sterile water as a vehicle to be used as an inoculant. 250 μL of 70% ethanol solution was added into the suspension as the dispersing agent. For the control group , inoculation with the same excipients was carried out, however, without the presence of spores.The spores of P. pachyrhizi. The inoculum suspension containing P. pachyrhizi spores was distributed in each plant individually using a spray bottle for group II, and without spores for group I. Both groups were submitted to automatic nebulization (40 seconds) every two hours for a period of 14 hours to ensure increased levels of humidity. Increased humidity and absence of light simulates the dew or night breeze conditions of the field, which favors spore germination. After nebulization, control plants were transferred to another greenhouse kept under similar conditions of temperature, relative humidity and photoperiod to avoid contamination. After 14 days, it was possible to visually observe the sporulation of the pathogen and formation of lesions on soybean leaves (data not shown). Thus, leaves from groups I and II were collected, wrapped in aluminum foil, immediately immersed in liquid nitrogen and transported to the laboratory. The samples were stored at −80 °C until metabolites extraction.The plants were divided into two groups: (I) control (non-inoculated) and (II) inoculated with 32. Control and inoculated soybean leaves (5 whole leaves) were macerated separately under liquid nitrogen. Then, about 100 mg of plant material was transferred to 2 ml glass vials and 1.5 ml of the solvent mixture was added. The solution was shaken in vortex for 10 seconds  and submitted to ultrasound  for 25 minutes. The solution was centrifuged at 12,000 rpm for 10 minutes at 4 °C. The supernatant was collected and filtered using a 0.22 μm PVDF syringe filter (Millex-GV Durapore). The extraction procedures were performed in three replicates for each solvent system and for each group (I and II). The samples were concentrated in nitrogen flow and stored at −80 °C until further analysis.The solvent mixtures ternary 1 , ternary 2  and binary  were used to evaluate the fingerprint of soybean leaves from both groups−1 33. The gradient mixture of solvents A  and B  was as follow: 5% B 0–1 min, 70% B 1–10 min, 98% B 12–20 min and maintained at 5% B 20–25 min at 40 °C, the final five minutes being intended for reconstitution of the column for the next analysis. The instrument was calibrated using a solution of sodium formate  containing 50 µL concentrated formic acid. The ionization source was operated in the positive ionization mode and adjusted to 4500 V, with a potential plate end of −500 V. The dry gas parameters were set to 8 L min−1 at 180 °C with a nebulization gas pressure of 4 bar. The data were obtained in a range of m/z 50 to 1800 with an acquisition rate of 5 Hz. The 5 most intense ions were selected for automatic fragmentation (AutoMS/MS). The data were acquired by the Hystar Application software version 3.2 and Otof Control  and were converted to the mzXML file format34.Extracts were resuspended in 600 μl water:acetonitrile  and 3 μL of each extract were injected and analyzed ultra-high performance liquid chromatograph  coupled to a hybrid quadrupole time-of-flight high resolution mass spectrometer  equipped with an electrospray ionization source. Chromatographic separation was performed using an Acquity UPLC HSS T3 C18 column  at flow rate of 0.250 mL min30. Solvent and injection blanks were subtracted to generate the chemical map. The molecular network (MN) was generated so that the mass tolerance of precursor ions was 0.02 Da, as this value influences the clustering of almost identical fragmentation spectra (MS/MS). The mass variances of ion fragments for each group of acquired MS/MS spectra were stipulated as ±0.02 Da for clustering (consensus spectrum creation). The lines (connections between nodes) were formed only if the cosine score was above 0.7 and with a minimum correspondence of 4 peaks in the fragmentation spectrum. The MN spectra were then compared to the spectra of the GNPS spectral libraries, such as ReSpect, Massbank and HMDB37 where the same data parameters were applied to the sample spectra. MN data were visualized in the Cytoscape software38. Ion fragmentation spectra with similarities to the mass spectral libraries had the fragmentation spectra manually verified and the mass error calculated, using a mass error tolerance of less than 10 ppm. Area-proportional Venn diagrams from exported molecular features data using GNPS were constructed using meta-chart (https://www.meta-chart.com/).The MS/MS data was transferred to the GNPS Molecular Networking server to generate the chemical map (ID = e26e17c3ffca42dc9968f260c2b01cee), according to the GNPS documentationP. pachyrhizi (group II) using extraction solvent mixtures ternary 1, ternary 2, and binary were compiled in Venn diagrams  soybean leaves or when soybean leaves were inoculated with A total of 1101 chemical entities were observed in group I , a total of 1282 chemical entities were observed  were obtained from the control group; and 886 consensus spectra (gray nodes) were obtained by samples of both groups. Fifty-two consensus spectra (black bold border nodes) had hits with the GNPS library . Furthermore, the MN had 97 clusters of nodes interconnected. Clusters containing at least one flavonoid/isoflavonoid, saponins, lipid, amino acid, chlorophyll, carboxylic acids or peptides were identified  were obtained in the MN Fig. . Of thesied Fig. .+m/z 447.125, [M + H]+m/z 463.121, [M + H]+m/z 503.116 and [M + H]+m/z 519.112, putatively identified as biochanin A 7-O-D-glucoside, 6-methoxyluteolin-7-rhamnoside, malonyldaidzin and malonylgenistin, respectively, was explored to highlight the potential of the GNPS approach . Using this approach, other ions belonging to the same cluster could be putatively identified. Furthermore, a pie chart layout39 was generated using the peak ion area in each sample group (control and inoculated) for qualitative evaluation.Metabolites putatively identification was confirmed manually by verification of fragmentation spectra (data not shown). Mass errors ranged from 0 to 3.9 ppm. Part of one cluster Fig.  containiP. pachyrhizi, and one metabolite was putatively identified in the group I. Of the ions exclusively putatively identified in group II, liquiritigenin, coumestrol, formononetin, pisatin, medicarpin, biochanin A . Using one solvent, or a solvent mixture can be a useful to explore a given sample when there is prior knowledge of the chemical class to be prospected, but with limitations. Conversely, the use of different solvent mixtures is needed to comprehensively explore the chemical ecology of a given matrix. Metabolites which were not known previously can be detected . Trans-cinnamic acid can then be incorporated into several phenolic compounds , which are present in the formation of esters, coumarins, flavonoids and lignins45.In this study, we putatively identified four amino acids present in both groups: proline, phenylalanine, tyrosine and tryptophan. Phenylalanine is one of the main precursors of plant defense metabolitesP. pachyrhizi was reported in transcriptome studies of susceptible and resistant soybean genotypes48. We putatively identified 16 phenylpropanoids , one of the major isoflavones belonging to soybean grains and leaves. We putatively identified glyoceollidin I, glyoceollidin II, glyoceollin I, glyoceolidin II, glyoceolidin III, glyoceolidin IV, glyoceolidin VI when ves Fig. . Simons 52. Some polyunsaturated fatty acids, such as linoleic and linolenic acids are precursors of oxylipins in plants53. Oxylipins correspond to a wide range of products generated by self-oxidation or enzymatic oxidation of polyunsaturated fatty acids. Jasmonic acid was putatively identified for both groups in this study. Considered as a signaling substance, jasmonic acid is able to recognize plant infection by the pathogen and activate biosynthesis of defensive metabolites such as phytoalexins, terpenoids and flavonoids19.Lipids and fatty acids are essential constituents of plant cells, providing structural integrity and energy for various metabolic processes. They can act as signal transduction mediators, playing both defense and signaling roles in response to biotic and abiotic stresses54. Indeed, Avena strigosa saponin-deficient genotypes had their resistance compromised to a variety of fungal pathogens55. The medicagenic acid saponins have been reported to have antifungal activity for Sclerotium rolfsii, Rhizoctonia solani, Trichoderma viride, Aspergillus niger and Fusarium oxysporum56. In this study, this class of compounds was putatively identified in control and inoculated leaves (Supplementary Table P. pachyrhizi is still unknown.Other putatively identified compounds were saponins, which are part of the terpenes class. Saponins can regulate plant growth and defense against insects and pathogens. They are present in high concentration in healthy plants, as they act as barriers to chemical reactions from fungi attackP. pachyrhizi. Identification of metabolites associated with plant defenses is powerful information on the development of resistant genotype to resist P. pachyrhizi, as well as in the development of more effective and specific products against the disease.Asian Soybean Rust is an important disease and efficient pathogen control is challenging. Using untargeted metabolomics, we proposed a novel approach to evaluate the soybean metabolome modulation when affected by the Asian Soybean Rust disease. The combination of mass spectrometry and the Global Natural Products Social Molecular network was successful to identify exclusive metabolites present when soybean leaves were inoculated with Supplementary information.


Symmetry of ictal HVSs may predict favourable outcomes following CC for ES/TS.We aimed to analyse the ictal electrographic changes on scalp electroencephalography (EEG), focusing on high-voltage slow waves (HVSs) in children with epileptic spasms (ES) and tonic spasms (TS) and then identified factors associated with corpus callosotomy (CC) outcomes. We enrolled 17 patients with ES/TS who underwent CC before 20 years of age. Post-CC Engel’s classification was as follows: I in 7 patients, II in 2, III in 4, and IV in 4. Welch’s t-test was used to analyse the correlation between ictal HVSs and CC outcomes based on the following three symmetrical indices: (1) negative peak delay: interhemispheric delay between negative peaks; (2) amplitude ratio: interhemispheric ratio of amplitude values for the highest positive peaks; and (3) duration ratio: interhemispheric ratio of slow wave duration. Ages at CC ranged from 17–237 months. Four to 15 ictal EEGs were analysed for each patient. The negative peak delay, amplitude ratio and duration ratio ranged from 0–530 ms, 1.00–7.40 and 1.00–2.74, respectively. The negative peak delay, amplitude ratio and duration ratio were significantly higher in the seizure residual group ( The prolonged muscle contractions following typical ES are defined as tonic spasms (TS)2. Previous studies have identified three contiguous patterns of ictal electroencephalography (EEG) findings during spasms: (1) fast waves or high-frequency oscillations (HFOs) preceding the ES, (2) high-voltage slow waves (HVSs) and (3) desynchronization5. Among these, HVSs coincide with the phasic motor component of the ES in almost 100% of cases5. The pathological mechanisms giving rise to ES remain largely unknown. Previous research has indicated that both the cortex and subcortical structures including the brainstem, thalamus and basal ganglia play a role in the emergence of ES7. Researchers have hypothesised that ES are derived from cortical-subcortical interactions wherein the cortex triggers the activation of subcortical structures, thereby leading to the generation of ES7.Epileptic spasms (ES) are epileptic seizures characterised by brief muscle contractions that typically involve the axial muscles and proximal limb segments9. The pathophysiologic basis of CC is the hypothesis that the corpus callosum is the most important pathway for the spread of epileptic activity between the two hemispheres of the brain10. Some reports have indicated that CC exerts beneficial effects in patients with ES13. In these studies, ES were eliminated after CC in 42 of 87 patients. Ono et al. proposed that interhemispheric recruitment of the epileptogenic state via the corpus callosum accounts for bilateral synchrony14, and that disconnection of the transcallosal volleys may lead to seizure elimination in such cases.Corpus callosotomy (CC) is a valuable palliative surgical option for patients with diffuse or multifocal epileptic discharge resulting in generalised seizureset al. demonstrated that activity in the corpus callosum is significantly correlated with maximal slow wave activity, suggesting that callosal connections play a role in the synchronization of slow cortical activity in the bilateral hemispheres15. Based on this finding, we hypothesised that greater corpus callosum involvement would be associated with increased synchrony of HVSs and disconnection of the corpus callosum would resolve ES/TS especially in patients exhibiting symmetrical HVSs. In the present study, we retrospectively analysed the ictal EEGs of patients with ES/TS, focusing on symmetrical indices in patients exhibiting HVSs, in order to identify prognostic factors for CC.Buchmann We recruited 17 patients  who fulfilled the inclusion criteria. Their clinical profiles are described in Table Nine patients were diagnosed with West syndrome, six with symptomatic generalised epilepsy (SGE) and one with symptomatic localization-related epilepsy. All but patients 4, 8 and 17, who developed only ES/TS, experienced more than two types of seizures, including tonic seizures, complex partial seizures, atonic seizures and myoclonic seizures. The mean frequency of ES/TS was 40.1 times per day (range: 5–200).MECP2 duplication syndrome and acute encephalopathy. Three patients had no neurologic history prior to the onset of epilepsy.Symptomatic aetiologies were identified in 16 patients. Patient 3 had both TSC and acute encephalopathy, whereas patient 7 had both EEG data were reviewed for 201 ES/TS cases. In total, 46 ES/TS cases were excluded due to non-negligible artifacts or misplacement of EEG electrodes, following which we analysed the remaining 155 ES/TS cases. Four to fifteen ictal EEGs were reviewed for each patient (mean: 9.1). Main ictal slow waves tended to present in the occipital area for patients 1, 3, 4, 7, 12 and 13; in the temporal area for patients 2, 5, 6, 8, 9 and 16; in the frontal area for patients 11 and 15; and in the central-parietal area for patients 10, 14 and 17. Among all 155 ictal EEGs, seven were analysed between both front polar electrodes, whereas nine were frontal, 13 were central, 14 were parietal, 50 were occipital, 24 were anterior-temporal, 14 were mid-temporal and 24 were posterior-temporal.The negative peak delay ranged from 0–530 ms (mean: 77.0 ms), the amplitude ratios ranged from 1.00–7.40 (mean: 1.61) and the duration ratios ranged from 1.00–2.74 (mean: 1.24). The entire data of EEG and electromyography (EMG) analyses can be found in Supplementary Table p = 0.042) in the patients free from ES/TS than in the patients with residual ES/TS.Data on clinical information were also collected from the patients. The analyses revealed that the age at CC was higher (p = 0.017), amplitude ratio (p < 0.001) and duration ratio (p < 0.001) were significantly higher in the ES/TS residual group than in the ES/TS free group  curves for the negative peak delay, amplitude ratio, duration ratio and age at CC had an area under the curve (AUC) of 0.97, 1.00, 1.00 and 0.84, respectively Table . The optAccording to the power analysis, the necessary sample size was 10 with a power of 0.989. The sample size in this study was above 10 and hence was thought to be sufficient for the investigation.In this retrospective study, we investigated the correlations between the symmetry indices of ictal HVSs and CC outcomes in patients with ES/TS. The negative peak delay, amplitude ratio and duration ratio among the bilateral hemispheres were significantly higher in the patients with residual ES/TS than in those with ES/TS free outcome. Our findings revealed that asymmetry of ictal HVSs in ES/TS is correlated with insufficient efficacy of CC for seizures.5. Some authors have focused on the association between ES genesis and fast waves or HFOs, revealing that the distribution of HFOs is correlated with epileptogenic brain lesions20. In contrast, few studies have investigated ictal HVS, focusing primarily on their morphology and distribution22.Previous studies have identified the characteristics of ictal EEG during ES, which consist of three contiguous components: fast waves or HFOs preceding the ES, HVSs and desynchronization of electrical activity5. Some researchers have hypothesised that HVSs are generated in the subcortical structures1, while others have suggested that they are generated in the cortex23. Kobayashi et al. proposed that neocortical activation of HVSs occurs according to their complex and localised distribution21. Given the intimate relationship between ictal HVSs and ES, we focused on their characteristics and sites of origin in order to elucidate the pathophysiology of ES. Thus, we also investigated the association between HVSs and CC outcomes in patients with ES/TS.Previous studies have demonstrated that changes in HVSs represent the most essential aspects of EEG alteration associated with ES and that such waves coincide with the phasic motor component in almost 100% of cases13. Pinard et al. reported that total CC was associated with greater seizure reduction than was anterior two-thirds CC in patients with West syndrome11. Conversely, other authors have reported that CC may be ineffective for reducing ES24, suggesting that some forms of ES are less affected by the corpus callosum. Furthermore, in one case report, a patient with West syndrome whose ES were initially bilateral and synchronous developed bilaterally independent ES originating in either hemisphere after total CC25. ES are speculated to represent a heterogeneous group of seizures that derive from a peculiar age-related cortical-subcortical interaction: Vigevano et al. expressed that ES appear to represent a sort of “final common manifestation”3.Some previous studies have demonstrated the efficacy of CC for alleviating ES28. These studies described that intracranial EEG findings could help determine resection planning and predict outcomes. In contrast, limited studies have investigated prognostic factors of CC. In patients with SGE and frontal lobe epilepsy who experience tonic seizures, atypical absences, drop attacks, atonic seizures, generalised tonic-clonic seizures and myoclonic seizures, the degree of the morphologic similarity of the bilateral spike-waves in presurgical scalp EEG has been reported to correlate with excellent outcomes following anterior CC29. Although the efficacy of CC for ES/TS has been clarified in recent years13, no studies have investigated prognostic factors that can be used to predict outcomes. Thus, our findings are the first to demonstrate that presurgical electrophysiological parameters can be used as prognostic indicators for CC in patients with ES/TS. Additionally, another advantage of our study is the low invasiveness of scalp EEG. Since CC occasionally causes serious complications such as haemorrhage, appropriate selection of patients using presurgical non-invasive data is essential.Previous studies have demonstrated prognostic factors of resection surgery for neocortical epilepsy and temporal lobe epilepsyet al. reported that interhemispheric coherence of fast activity was low prior to ES and that such activity increases following ES, suggestive of signal transfer through the corpus callosum during the sequential ES state6. Ono et al. proposed greater involvement of the corpus callosum, suggesting that an interhemispheric recruitment via the corpus callosum precedes cortical spike discharges, maintaining cortical epileptogenic susceptibility in both hemispheres14.Avanzini 15. The contribution of the corpus callosum for coherent activity during NREM sleep is further illustrated by the reduced interhemispheric coherence observed in children with congenital agenesis of the corpus callosum30. Among three symmetrical indices in the present study, the negative peak delay may highly reflect the extent of interhemispheric synchrony, as it may indicate the time difference of cortical-subcortical activation between both hemispheres. The low amplitude ratio and the duration ratio may reflect the approximation of epileptic excitations among bilateral hemispheres via the transcallosal volleys. Further studies involving larger numbers of cases are required to determine the index that is the best prognostic factor for CC outcomes.However, the functional roles of the corpus callosum in generating ictal HVSs associated with ES remain to be identified. Previous report has indicated that the corpus callosum is significantly correlated with maximal slow wave activity during non-rapid eye movement (NREM) sleep, indicating that callosal connections may play a role in the synchronization of slow cortical activity in the bilateral hemispheresGiven that ES are completely eliminated by callosal disconnection in some patients, the corpus callosum may play a role in the generation of symmetrical ictal HVSs, which are regarded to be the most strictly associated with motor phenomena. The functional roles of the corpus callosum that intensify epileptogenesis may be more prominent in patients who respond favourably to CC. Interhemispheric recruitment via the corpus callosum may occur in patients with ES/TS who present with symmetrical HVSs due to bihemispheric synchrony, which may be eliminated via CC.The present study has some limitations of note. Since the number of patients were not enough to perform multivariate analyses in this study, we could not evaluate the effect of age at CC for the symmetrical indices or identify the best prognostic factor among the indices. We visually selected the main ictal HVSs using AP-bipolar and average references. For more objective analysis, further studies should utilise additional approaches, such as mechanical frequency analysis.Outcomes following CC for ES/TS are diverse among patients. We hypothesised that the patients presenting with favourable outcomes following CC may exhibit intense involvement of the corpus callosum in the generation of ES/TS. Thus, we focused on the synchrony of the ictal HVSs. Our findings indicated that the symmetry of the ictal HVSs for ES/TS was correlated with favourable CC outcomes. These findings indicated that the corpus callosum may be involved in the generation of symmetrical HVSs associated with ES/TS. Further studies regarding the pathophysiology of ES are required in order to develop appropriate treatment strategies.Clinical data and video EEG recordings were retrospectively collected from each patient’s medical records. Inclusion criteria were as follows: (1) CC performed in patients at age of < 20 years between 2008 and 2017 at Seirei-Hamamatsu General Hospital, (2) main seizure type of ES and/or TS, (3) availability of ictal EEG recordings prior to CC, (4) ictal EEG containing polyphasic HVS and (5) follow-up period after CC greater than 6 months. Patients with inappropriate EEG recordings  or serious surgical complications were excluded from the analysis.The present study was approved by the institutional review boards of Seirei-Hamamatsu General Hospital and Tottori University. In accordance with approved guidelines, patient information including age, seizure type, aetiology, EEG data and postsurgical seizure outcome was anonymised prior to our analysis. Informed consent was obtained from each subject’s guardian.31.For each patient, we reviewed clinical data including sex, age at epilepsy onset, number of antiepileptic drugs (AEDs) prescribed prior to CC, frequency of ES/TS, classification of epilepsy syndrome, aetiology, age at CC, CC procedure and follow-up period. We defined the patients with ES/TS and developmental delay as having SGE or symptomatic localization-related epilepsy in accordance with seizure types and the distribution of EEG discharge, based on the 1989 International Classification of Epileptic Syndromes32.Seizure outcomes following CC were evaluated for the remission of ES/TS based on Engel’s classification at the last follow-upScalp video-EEG data were recorded using a NicoletOne or BMSI6000 system  for patients 1–4, 6–9 and 11–16 and a Neurofax system  for patients 5, 10 and 17. EEG electrodes were placed in accordance with the international 10–20 scalp-electrode position. The sampling rate was set at 256 Hz , 400 Hz , 500 Hz , 512 Hz (patients 6 and 13), or 1,024 Hz (patient 9). Low-cut and high-cut filters were set at 0.5 Hz and 70 Hz for NicoletOne or BMSI6000 and 1.6 Hz and 60 Hz for Neurofax. EMG electrodes were placed on both deltoid muscles.5. Main ictal HVSs were analysed for each ES/TS based on the montages of AP-bipolar and average reference : time delay of initial negative peaks among bilateral hemispheres [(preceding peak) – (following peak)]Amplitude ratio: amplitude ratio of the highest positive peaks of polyphasic slow waves among bilateral hemispheres [(higher amplitude)/(lower amplitude)]Duration ratio: duration ratio of positive slow waves among bilateral hemispheres [(longer duration)/(shorter duration)].In this study, we defined three symmetrical indices as follows Fig. :NegativeIn addition, we measured EMG latency  and EMG delay . Each value was measured for individual seizures, following which average values were obtained for each patient.We divided the patients into two outcome groups of free from ES/TS (Engel’s I) or residual ES/TS (Engel’s II–IV). We statistically analysed the differences in clinical information using Fisher’s exact probability test, Welch’s t-test and chi-square test, appropriately.We also statistically analysed the differences in three symmetrical indices of negative peak delay, amplitude ratio and duration ratio, using Welch’s t-test. Thereafter, we examined the prognostic values regarding covariates that were significantly associated with the outcomes, by analysis of ROC curves. The optimal operating point was determined via YI. The sensitivity and specificity of each operating point in the ROC curve and each index were conditionally calculated, followed by calculation of YI at each point (YI = sensitivity + specificity - 1).We also examined whether the sample size of this study was enough. The power (z test with arc sin transformation) was calculated based on a hypothesis test of binomial probability. We set the alpha error as 0.05, beta error as 0.20, the power as 0.95 and effect size as 0.65, considering that CC is the last resort for pharmacologically intractable epilepsy with ES/TS.p < 0.05.The analyses were performed using JMP  and R.3.6.1. The level of statistical significance was set at Supplementary information


Bordetella pertussis isolates has been found in these countries.Pertussis outbreaks have occurred in several industrialised countries using acellular pertussis vaccines (ACVs) since the 1990s. High prevalence of pertactin (PRN)-deficient B. pertussis clinical isolates; (ii) if the frequency of PRN-deficient strains in countries correlated with the time since ACV introduction; (iii) the presence of pertussis toxin (PT)-, filamentous haemagglutinin (FHA)- or fimbriae (Fim)-deficient isolates.To evaluate in Europe: (i) whether proportions of PRN-deficient strains increased in consecutive collections of B. pertussis clinical isolates were obtained from different European countries during four periods (EUpert I–IV studies): 1998 to 2001 (n = 102), 2004 to 2005 (n = 154), 2007 to 2009 (n = 140) and 2012 to 2015 (n = 265). The isolates’ selection criteria remained unchanged in all periods. PRN, PT, FHA and Fim2 and Fim3 expression were assessed by ELISA.In each period 1.0% (1/102), 1.9% (3/154), 6.4% (9/140) and 24.9% (66/265) of isolates were PRN-deficient. In EUpert IV, PRN-deficient isolates occurred in all countries sampled and in six countries their frequency was higher than in EUpert III . Sweden and Italy which used ACVs since the mid 1990s had the highest frequencies  while Finland, where primary immunisations with ACV containing PRN dated from 2009 had the lowest (3.6%). Throughout the study, no PT- or FHA-deficient isolate and one Fim2/3-deficient was detected.Results suggest that the longer the period since the introduction of ACVs containing PRN, the higher the frequency of circulating PRN-deficient isolates. Between 2010 and 2012, nationwide pertussis outbreaks occurred in many countries including Australia, the Netherlands, the United Kingdom (UK) and the United States (US). In all of these countries acellular pertussis vaccines (ACVs) had been introduced for booster or primary immunisations in the late 1990s or the early 2000s . CurrentBordetella pertussis circulating strains have been investigated in relation to pertussis vaccine components [B. pertussis PRN-deficient strains can cause typical symptoms of pertussis [B. pertussis not expressing FHA nor PT and PRN was reported in France, Sweden and the US [The genetic characteristics of mponents . It seemertussis ,8. To daertussis -12. PRN-ertussis . Furtherd the US -16.B. pertussis populations in Europe, panels of B. pertussis isolates have been serially collected during four periods in a total of 11 European countries. The EUpert I panel was collected during 1999–2001 and included 102 isolates from five countries , the EUpert II in 2004–05 included 154 isolates from eight countries , and the EUpert III in 2007–09 included 140 isolates from seven countries . Four countries  participated in all collections [B. pertussis isolates were included. Selection criteria have remained unchanged for all four collections, which allows a unique opportunity to study and compare changes in bacterial populations over the past 15 years. Detailed vaccination programmes and vaccination coverage of the EUpert IV study countries are presented in In Europe, vaccines and vaccination programmes vary in different countries . To studlections ,18,19. IB. pertussis isolate collections in Europe over time. We also assessed if the proportions of PRN-deficient isolates detected in given study countries in 2012–15 were related to the time since these countries had introduced ACVs containing PRN. Furthermore, the presence of PT-, FHA- or Fim-deficient isolates in all collected isolates was investigated.In this study, we aimed to evaluate whether there has been an increase in the proportion of PRN-deficient isolates among the clinical B. pertussis isolates, which were collected during the four study periods (EUpert I–IV) from 1998 to 2015. The genotyping results of EUpert I–III studies have been previously published [The total material comprised 661 ublished ,18,19. IIn this study, the data from EUpert I–III were completed, by screening for PRN expression in EUpert I and II panels (n = 256). Moreover, FHA expression in EUpert I, II and III panels (n = 396) was tested (country-based data not shown). The study also included the full analysis of EUpert IV isolates (n = 265 isolates), which were investigated for the presence of PT, FHA, PRN and Fim2 and Fim3. The recommended number of isolates was 30 per country and the selection criteria for isolates in the EUpert IV study were the same as those used in the previous EUpert I–III studies . These were as follows:B. pertussis isolates should be selected from different geographical regions in a given country and be epidemiologically unrelated.(i) (ii) An equal number of isolates from vaccinated (n = 15) and unvaccinated individuals (n = 15) should be collected. Isolates are preferred to be selected from individuals younger than 5 years of age.(iii) For those countries with large numbers of isolates in their collections, isolates should be randomly selected in addition to the above criteria.The following numbers of isolates were received per country: Belgium (n = 38), Denmark (n = 27), Finland (n = 28), France (n = 29), Italy (n = 20), the Netherlands (n = 32), Norway (n = 32), Sweden (n = 29), and the UK (n = 30). For Italy, all isolates were collected from the Rome area as no other isolates were available. Data collection included original code of isolate, country, date of collection, city and available characteristics of patients. The latter included sex, age, vaccination status, number of doses received and hospitalisations due to pertussis.B. pertussis isolates were shipped in frozen storage tubes to the University of Turku (UTU), Finland. Isolates were cultured on Regan-Lowe medium  at + 35 °C for 48h to determine production of PRN, FHA, Fim2 and Fim3. To determine production of PT, isolates were cultured at + 35 °C with 5% CO2 for 48h [ for 48h . The subDetection of PRN, PT, FHA and Fim2 and Fim3 expression was performed by specific enzyme-linked immunosorbent assay (ELISA) as described previously ,21,22. Iprn gene was first amplified and sequenced afterwards. Only isolates with detected PRN-deficiency were sequenced. All primers are presented in To investigate the mechanisms for PRN-deficiency of the EUpert IV collection, the Fisher’s exact and chi-squared tests to assess differences between vaccinated and unvaccinated patients and frequency of PRN-deficient isolates between EUpert I-IV collections were calculated using GraphPad prism 4·0 version , US). Two-tailed p values < 0·05 were considered significant. To analyse the trend in PRN-deficiency over time, the proportions of PRN-deficient isolates per country were estimated using logistic regression with the year of diphtheria-tetanus-acellular pertussis  implementation in the country as the independent variable. Results were given as odds ratios (OR) between start of ACV and frequency of PRN-deficient isolates. The analysis was conducted using the R software (R foundation).In EUpert I, one ) PRN-deficient isolate was found. In EUpert II three , Germany (n=1), the Netherlands (n=1)) and in EUpert III nine , Norway (n=3), Sweden (n=1)) such isolates were detected. In EUpert IV, 66  isolates were deficient for PRN . SignifiThe distribution of the PRN-deficient isolates and their frequencies in the EUpert IV-study countries is shown in prn gene was sequenced. We found six different mechanisms causing PRN expression to be suppressed (IS481 element at position 1613^1614 (from the beginning of the prn gene) either in forward or reverse orientation and one had it at position 2735^2736. IS481 element is found in multiple copies in the genome of B. pertussis and helps it to adapt to different immunised populations [prn promoter area. In one isolate, a nucleotide mutation of a G to a T at position 2077 leading to a stop codon, which was not previously described, was found. Other causes were single point mutation and deletion, and deletion of the entire prn gene. In addition, the mechanism behind PRN-deficiency for one isolate was not located within the prn gene, although repeated sequencing was performed.The entire ppressed . Of the ulations . The secOf 265 patients in the EUpert IV study, 130 (49.1%) were vaccinated and 135 (50.9%) unvaccinated. PRN-deficient isolates were isolated from 66 patients. Of them, 16 (24.2%) were partially vaccinated, 10 (15.2%) had received full primary vaccination, six (9.1%) had full primary and booster vaccination, 32 (48.5%) were unvaccinated and for two patients (3.0%) the vaccination status was unknown. No significant difference in proportion of PRN-deficient isolates was found between vaccinated and unvaccinated patients among study countries, except in the UK where these isolates were found more in vaccinated patients (p = 0.0348). PRN-deficient isolates were found mostly from infants less than one year of age , but the age ranged from < 1 month to 55 years. Hospitalisation data were available only for 41 patients and showed a range from 0 to 28 days. Complications data were only available for 11 patients with one having thrombocytopenia and one bradycardia and hypoxia, for the rest no severe complications were notified. From these patients, only one was vaccinated.Among isolates collected during the four studies (n=661), one (0.15%) did not produce Fim2 or Fim3. This isolate was found in Norway and originated from the EUpert IV collection. No difference in bacterial growth and colony formation on charcoal agar was noticed between Fim-positive and -negative isolates. In the EUpert IV study, 120 (45.3%), 141 (53.2%) and three (1.1%) isolates carried Fim2, Fim3 and Fim2,3 serotypes, respectively. No other Fim-deficient isolate was found.No FHA-deficient isolates were found among EUpert I–IV collections. No PT-deficient isolates were found among the EUpert IV collection.B. pertussis isolates were investigated from 1998 to 2015 in European countries where different vaccines and vaccination strategies are used. The countries submitting isolates varied between collections, however Finland, France, the Netherlands and Sweden contributed to all of them [B. pertussis PRN-deficient isolates was low  during the period 1998–2009 but this had increased in 2012–2015 with a prevalence of 24.9% , suggesting that there is a selective pressure for All study countries in the two last EUpert collections have implemented either ACV booster vaccination or changed to ACV primary vaccination in the 1990s or at the beginning of 2000  and 3 2,20. TherIn Europe, Poland is the only country using whole cell vaccines for primary vaccination. Unfortunately Polish isolates were not available in the latest EUpert III and IV studies. In EUpert II study, 13 isolates were available . None waB. pertussis isolates. One possible explanation might be that the patients in Denmark were infected with PRN-deficient strains from other neighbouring countries like Sweden and Norway where the frequency of PRN-deficient isolates is high. Indeed, the four PRN-deficient isolates observed in Denmark were caused by three different mechanisms  ca 60% of Belgian children (less than 17 years of age) received ACV in 1999 and 40% in 2002. This may explain the low frequency of PRN-deficient isolates found in Belgium in this study. Previously, it has been reported that PRN-deficient isolates are more common in vaccinated than in unvaccinated individuals . In thisB. pertussis. Five mechanisms have been reported previously [prn gene.In this study, seven different molecular mechanisms were found to mediate PRN deficiency for eviously ,12,28. AB. pertussis isolates is increasing constantly 10–15 years after the introduction of ACV vaccination containing PRN.The appearance of PRN-deficient isolates was first described in Italy and France with only a few isolates in 2007 ,11. DuriB. pertussis and plays an essential role in pathogenesis of pertussis. However, the number of isolates from Denmark was limited so we cannot conclude that PT-deficient isolates are not present in Denmark. We did not detect any FHA-deficient isolates either. As FHA is dominant in adhesion and is needed for tracheal colonisation, it seems to be conserved in B. pertussis [In this study we did not find any PT-deficient isolates, even from Denmark, although the selective pressure from the monocomponent PT vaccine is considered to be high. Moreover, only a few PT-deficient isolates have been reported previously ,14,16. Tertussis . For fimertussis . HoweverThere are both strengths and limitations in this study. The strengths of this study are that (i) we have a serial collection of isolates during the past 15 years, (ii) selection criteria have been the same for all collections, (iii) we have isolates from countries with different vaccination programmes and therefore, the impact of ACV introduction could be assessed, (iv) the place of origin is known for all isolates and shows that these isolates were not collected from local outbreaks and (v) all analyses were done by one laboratory with the same protocol so the results are comparable. The limitations included are as follows: (i) clinical information, including e.g. length of hospitalisation and severe complications were not available from many study patients (159 of 265), (ii) the number of isolates included from each country was very limited and not proportional to population size. However, they do comprise almost all isolates available in many countries such as Denmark, Finland and Italy, where the use of culture for diagnosis of pertussis is diminishing. For those countries with limited number of isolates, it would be beneficial if DNA from clinical specimens could be used directly for detection of PRN-deficient isolates e.g. through PCR and sequencing. (iii) The epidemiological pressure of pertussis varies in European countries which could have an effect on the spread of new emerging strains, like in Finland the incidence was continuously decreasing and was very low during the period of EUpert IV study .B. pertussis isolates has increased in Europe and the increase seems to be associated with the time since the introduction of ACVs. PRN-deficient isolates were found in all nine study countries during the latest collection and the isolates were isolated from both vaccinated and unvaccinated individuals. The highest number of these isolates was found in Sweden and Italy, which were the earliest adopters of ACV among the study countries. Our study also underscores the importance of long-term surveillance of B. pertussis antigen production because of its possible impact on the effectiveness of vaccines.In conclusion, the prevalence of PRN-deficient


LCR upper limit of 1.0 × 10−4. While the AC was below the Chinese maximum contaminant level of 0.2 mg/kg, study results indicated that Kunming adolescents may be at increased risk for iAs-related cancers.Inorganic arsenic (iAs) is carcinogenic and highly concentrated in rice. Dietary exposure to iAs is concerning among adolescents due to their developmental stage and iAs’s long-latency effects. This paper aimed to assess iAs exposure from rice and related lifetime cancer risks (LCR) among adolescents in Kunming, China. A comprehensive literature review of iAs levels in rice and LCR in humans was also conducted. Average daily consumption of rice (ADC) was estimated from 267 adolescents (15–18 years). Rice samples obtained from 6 markets were analyzed for iAs concentration (AC). Estimated daily intake (EDI) of iAs was calculated using ADC, AC, and average body weight (BW). Lifetime Cancer Risk (LCR) was calculated using EDI and U.S. EPA derived iAs oral slope factor. The AC was 0.058 mg/kg and the average BW and ADC were 67.5 kg and 410 g/day for males and 55.5 kg and 337 g/day for females. The EDI and LCR were 3.52 × 10 Exposure to toxic contaminants in food is presumed to be one of the major public health challenges for the 21st century ,4.Arsenic has no single major mode of action in the human body . A few oFlooded rice fields and the anaerobic nature of paddy soils facilitate the buildup of arsenic in the rice crop compared to other agricultural crops . Rice caUntil recently, official food monitoring across the world considered only total As in rice (not inorganic As species) and lately iAs exposure and its health effects have become national and international concerns . LikewisTherefore, the aims of this study are to: (1) conduct a comprehensive literature review of recent studies published after 2010 that measured arsenic concentrations in rice and/or arsenic exposure levels in humans, or conducted arsenic-related cancer risk assessments; (2) estimate lifetime cancer risk among a cohort of adolescents living in Kunming, China using directly measured iAs levels in locally sampled rice and individual-level rice consumption data of Kunming adolescents; and (3) compare this study cohort’s iAs exposure rates and lifetime cancer risks to that of other populations in the published literature.A comprehensive literature review was conducted using Google Scholar and PubMed with key search terms including arsenic, rice, concentration, cancer, exposure, diet, risk assessment, China, Asia, and adolescents. Papers excluded in this manuscript’s tables were reviews, papers published before 2010 in order to provide a more concise review of more current As levels in rice and exposure levels in populations, and arsenic risk assessments with outcomes other than cancer. However, papers with an outcome other than cancer that had measurements of arsenic concentrations in rice, as well as having China or Asia as the study locations, were included in the assessment in order to account for those Asian risk assessment studies that measured As levels in rice.Short-grain white rice samples were collected from six Kunming markets, varying by location and market type, to represent foods purchased in different Kunming neighborhoods and by different financial preferences, such as from a high-end market selling organic food and at a Carrefour (hypermarket). The collected rice samples were sourced from growing regions in Northern China, as is most of the rice sold in Kunming.n-octanol, diluted to volume with water, and settled for 10 min to determine iAs concentration. Standard series of iAs determination used 1 μg/mL As3+ standard solution [Samples were sent to the commercial laboratory Merieux Nutrisciences located in Ningbo, China for analysis. The laboratory received seven labeled samples from seven different locations, two of which were from the same location in order to assess the reliability of the lab’s measurement procedures. The lab used the 2009 updated version of the GB/T 5009.11-2003 method for determination of abio-arsenic (iAs) in the received rice samples. By means of hydride generation atomic fluorescence spectrophotometry, iAs was extracted in the form of chloride and separated from organic arsenic during water bathing with HCL, and its concentration was measured . Specifisolution .Adolescents  aged 15–18 years old and all of Han ethnicity were recruited from two local Kunming high schools in 2015 using convenience sampling methods. The schools had a wide catchment of students who lived in various regions of the city. Study participants completed 72-h dietary recalls which captured their diets over two weekdays and a weekend day . Participants were trained on estimating portion sizes and how to fill out a 72-h dietary recall. Participants’ weights were also measured in light clothing without shoes to the nearest 0.1 km using an electronic scale. All human subject data collection procedures were approved by the Institutional Review Boards of UC Berkeley and Kunming Medical University (2014-03-6097).Average daily consumption rates (ADC) of white rice (g/day) were estimated using the 72-h recalls for both males and females separately. Estimated daily intake (EDI) of iAs from rice consumption (mg/kg-BW/day) was calculated using the ADC, average concentration (AC) of iAs in sampled rice, and average body weight of the adolescents (BW) :Estimated daily intake:Lifetime Cancer Risk (LCR), the probability of excess lifetime cancer risk, was calculated using the EDI and the U.S. EPA derived iAs oral slope factor (SF), 1.5 (mg/kg)/day . LifetimLifetime cancer risk:t-test was conducted to compare the ADCs and BWs between males and females. A p-value of less than or equal to 0.05 was selected to indicate statistical significance.Stata version 15.0  was used to calculate the average daily consumption rates of rice and mean body weights of males and females in the study cohort. A Fifty four papers were screened and 38 fitted the table inclusion criteria. Only seven studies focused on Southern China and the other eight Chinese studies focused on other regions or multiple provinces throughout China. It was estimated that Southern China has 2.5 times higher rice consumption rates than Northern China and higher concentrations of arsenic in rice ,18. Of t−3 mg/kg-BW/day recommended by the WHO [0.5 of 3.0 μg/kg-BW/day (lower 95% confidence limit on the benchmark dose for a 0.5% increased incidence of lung cancer) residing in its range (2 × 10−3–7 × 10−3 mg/kg-BW/day) [−4 and it is apparent that many Asian countries and even adolescents in those populations greatly exceeded the upper limit [ the WHO . Notably-BW/day) . We noteThe iAs concentrations measured in the Kunming samples ranged from 0.045–0.076 mg/kg and are below the Chinese MCL. Of the two samples from the same location , total arsenic concentrations were 0.080 mg/kg for both samples and iAs concentrations were 0.054 mg/kg and 0.050 mg/kg. This provided assurance of the reliability of the lab measurement procedures. Interestingly, the sample from the high-end market selling organic food had the second highest level of iAs (0.067), which demonstrates that price and quality of products may not be a factor that reduces arsenic contamination in food products. These seven measurements averaged to be 0.058 mg/kg . We appt-test results (p < 0.001) indicated that males and females differed significantly in their average daily consumption rate of rice. Due to uncertainties in assessing rice content in popular rice products such as rice cakes and rice snacks, calculated consumption rates did not take into account these products. Thus, we may have underestimated the g/day of rice consumption in the study participants.Among the study participants, males consumed on average 409.9 g/day of rice, 22% higher than that of females (336.6 g/day). t-test results (p < 0.001) indicating that males and females differed significantly in their body weights. The EDI for males was estimated to be 3.52 × 10−4 mg/kg-BW/day (max. 5.00 × 10−4 mg/kg-BW/day) and for females was estimated to be 3.52 × 10−4 mg/kg-BW/day (max. 5.35 × 10−4 mg/kg-BW/day).−4 (max. 7.51 × 10−4), which is 5 times above the upper limit of the U.S. EPA LCR of 1.0 × 10−4. Due to differences in body weight and ADC, the calculated maximum LCR for females was 8.02 × 10−4, which is eight times above the upper limit of the LCR.The calculated average LCR for both males and females using the cancer slope factor of 1.5 (mg/kg)/day was 5.28 × 10−4. Even though measured iAs concentrations in the sampled rice in this study were below the Chinese MCL, the high consumption rates of rice in this cohort are driving LCR values beyond the LCR acceptable upper limit. Our cohort’s LCR was over three times greater than the LCR of adolescents in Jiang et al., which used consumption data from the Survey of the Nutrition and Health Status (NHS) of the Chinese People in 2002 to estimate iAs exposure from all foods measured, not just rice. In that study, the authors did not directly measure iAs, but used conversion factor of 20.3% to estimate iAs from total arsenic [−5 mg/kg-BW/day, which is 4.4 times lower than that of our study cohort [The mean iAs concentration in this study’s rice samples was 0.058 mg/kg and the lifetime cancer risk for both males and females in this study cohort was estimated to be 5.28 × 10 arsenic . Awata ey cohort . This ma−4 and 18.2 × 10−4 mg/kg-BW/day for both males and females, respectively. The estimated LCR would be 18 times higher than the upper limit of the U.S. EPA LCR of 1.0 ×10−4.The current Chinese MCL of 0.2 mg/kg, reflective of the maximum level set by the Codex Alimentarius Commission, was raised from the 2005 Chinese MCL of 0.15 mg/kg . Given t−3 among children (ages 6–16) and 8.0 × 10−4 among adults (ages 16–67). By using ADAFs, 0–2 year olds acquire 10 times the cancer risk and 2–16 year olds acquire 3 times the cancer risk compared to adults [−4, which is approximately 16 times higher than the U.S. EPA LCR limit.Our cancer risk estimations accounted for “lifetime” exposure to iAs from rice. Although humans may not be consuming rice during infancy, many infant foods are rice-based and contain As. For example, infant rice cereal products sold in the U.S. were shown to have As levels ranging from 0.050 to 0.72 mg/kg, with the lower end of this range being similar to the mean level of iAs found in this study’s rice samples . Importao adults . If we aAs with most of the previously published studies, the limitations of this study include the cross-sectional nature of the study and the lack of long-term exposure history, which restrain our confidence in any causal predictions of arsenic-attributable health risks. Another limitation is the small sample size in both the rice samples collected and number of study participants, which can decrease study power and generalizability to the adolescents living in other parts of China. Additionally, although LCR values across studies could not be confidently compared given that exposure estimation methods are not uniform, the graph in The bioavailability of arsenic after ingestion of white rice has been measured to be about 40% from animal models and between 55–71% from in vitro studies . DespiteAs exposure rates from rice can vary day-to-day due to inconsistent diet consumptions, different As concentrations and speciation in rice, rice from different geographical sources, and different rinsing and cooking methods . CookingDespite these limitations, this study has distinctive strengths. As displayed in N > 40,000) and the use of a control group that had no evidence of skin lesions or black foot disease for a sensitive endpoint. This is considered superior for developing a reference dose for oral exposure (RfD). Moreover, this data from Taiwan is of value because it removes potential exposure misclassification due to villagers residing in the same location most of their lives, therefore reflecting long-term exposure to regional As levels [The current U.S. EPA iAs oral slope factor of 1.5 (mg/kg)/day was last revised in 1995 and is based on old studies ,51 usings levels . Lastly s levels .Because China is the highest rice producing and consuming country in the world and accounts for a third of the global supply of rice , As-moni−4 is 5.28 times higher than the U.S. EPA upper limit of 1 in 10,000, with the upper limit being 100 times higher than the universally accepted cancer risk for an environmental carcinogen of one case/one million people [The results of our literature review and analysis reveal that exposure rates and lifetime cancer risk estimations can vary widely between countries, between populations within countries, and between age groups within specific populations. Our study results also suggest that there should be ongoing risk assessments in specific regions, especially in Southern China where residents may be at risk for high exposure levels. Younger populations may be particularly at risk for long-term health effects of chronic iAs exposure from rice because they are highly exposed during earlier years of life and while they are still developing and they have the potential for long-term exposure. In addition, future studies should assess biomarkers of exposure to As such as in participants’ blood, urine, hair, skin, or nails . There an people .


IL6 genotype, the Mediterranean diet or the central European diet for 16 weeks affect biomarkers of inflammation in centrally obese postmenopausal women, was tested in a randomized controlled trial. Dietary intake was assessed using a three-day food diary. Lipid parameters were measured using a Beckman Coulter AU analyzer. Transcription of TNF and IL6 genes was analyzed in peripheral blood mononuclear cells using real-time PCR. Concentrations of tumor necrosis factor alpha (TNFα) and interleukin 6 (IL6) were measured with ELISA. rs1800795 polymorphism of IL6 was analyzed using hydrolyzing probes. Higher energy intake from fat was associated with higher IL6 levels (p < 0.05). Significantly (p < 0.01) lower total cholesterol (T-C) and low-density lipoprotein cholesterol (LDL-C) concentrations were observed in the GG IL6 rs1800795 genotype group. Both diets significantly (p < 0.001) decreased TNFα concentrations. Neither IL6 gene transcription levels nor blood IL6 concentrations were affected by them. Our findings confirm that habitual fat intake may affect inflammation. The rs1800795 IL6 polymorphism alone did not significantly affect body weight or body composition in aimed group, but C-allele carriers had higher levels of T-C and LDL-C. This polymorphism did not affect inflammation. Both diets may lead to a decrease in TNFα concentration.The hypothesis that habitual fat intake, the  IL6 gene is expressed in many tissues, including adipose tissue, skeletal muscles, and the hypothalamus—all of which are involved in the regulation of energy balance [IL6 gene, with rs1800795 being the most frequently studied. This single nucleotide polymorphism (SNP) is located in the promoter region, 174 bp upstream of the transcription start site, and it affects gene activity. The C allele is associated with a lower expression level [TNF and IL6 genes, and blood concentrations of IL6 and TNFα; (2) to analyze the effect of the IL6 polymorphism on anthropometric parameters and biomarkers of inflammation of targeted population before dietary intervention; and (3) to assess how dietary interventions differing in fat, carbohydrate, MUFAs and dietary fiber content may affect these parameters in targeted population. CRP was not analyzed as its concentrations correlate with IL6 in a general population, and in postmenopausal women.Obesity is associated with systemic, low-grade inflammation, which may play an important role in the pathogenesis of obesity-related insulin resistance, dyslipidemia, and the atherosclerotic process ,2. White balance . Higher  balance ,9,10. Bo balance . There aon level . Althougon level ,13. Studon level . Whetherhttps://www.drks.de/drks_web/) with a parallel arm design and 1:1 allocation. The study methods and reporting were conducted in accordance with the CONSORT 2010 guidelines. The participants were randomized to the CED group or the MED group for 16 weeks. The details composition of the diets are presented in The study design and participants have been described in detail elsewhere . Shortlyn = 47 in the CED and n = 48 in the MED out of the 130 subjects who completed the CED-MED study. We excluded 35 subjects because we had no access to peripheral blood mononuclear cells (PBMC) samples  derived mostly (20% of energy) from MUFAs, where the main source of fat was virgin olive oil and nuts. The CED had lower than 10 percentage points of energy from fat , and moderate levels of carbohydrates  derived mainly from high-fiber locally available and traditional food items . Therefore, the CED was characterized by higher dietary fiber content than the MED, mostly in terms of soluble dietary fiber . Moreove samples .Nutrient intake was assessed using a three-day food diary in which participants were clearly instructed to record information on nonconsecutive days (two weekdays and one weekend day) on their food and beverage intake, using household measures. The average intake of macronutrients and micronutrients at baseline and during the dietary intervention was calculated using the Dietetyk software .Height was measured to the nearest 0.1 cm . Body weight was measured, using a calibrated scale included in the Bod Pod technology, to the nearest 0.1 kg. Waist circumference was measured at the midpoint between the lowest rib and the top of the iliac crest using nonelastic tape. The BodPod was used in line with the manufacturer’s instructions to measure body volume. All subjects wore tightly fitting bathing suits and swim caps.g for 20 min. The PBMC buffy coats were collected, washed twice with PBS, and immediately proceeded to an RNA isolation procedure. Total RNA was extracted from PBMCs using Tripure Isolation Reagent , according to the manufacturer’s protocol. Approximately 1 μg RNA was taken for cDNA synthesis. Samples of RNA were incubated with a set of random hexamers  and oligodT(15)  at 70 °C for 10 min. A mixture of dNTP , 1 U reverse transcriptase AMV , and 20 U Protector RNase Inhibitor (Roche) was added. After two hours of incubation at 37 °C, the AMV enzyme was inactivated at 94 °C for 5 min. The cDNA was then diluted 3 × and stored at −20 °C. The real-time PCR reactions were performed on a Light Cycler 480 (Roche), based on the Universal Probe Library detection system . Whole blood was diluted with sterile PBS, layered over Lymphoprep, and centrifuged at 400× m Roche, .IL6 and TNF gene transcripts was then normalized to a geometric mean of two reference genes using glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-actin (ACTB) [Each 10 μL reaction mixture consisted of 1 μg cDNA, 5 μL LightCycler 480 Probe Master kit (Roche), 0.2 μL of the respective of the Universal Probe Library (UPL), and 0.3 μM forward and reverse primers. The real-time PCR was performed according to the manufacturer’s protocol. The relative quantification of the mRNA level was performed in duplicate based on a Second Derivative Maximum Method (Roche). Standard curves were designed as tenfold dilutions of the appropriate PCR product in the range of 10–0.0001 aM. The abundance of n (ACTB) . IL6 gene was selected for analysis. The blood was collected into tubes containing EDTA and DNA was isolated from fresh blood using a NucleoSpin Blood kit . Genotyping was performed with the use of hybridizing probes designed and synthesized by TibMolBiol, Germany , according to the manufacturer’s directions. Concentrations of total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), and glucose in plasma were measured using a Beckman Coulter AU analyzer. Low-density lipoprotein cholesterol LDL-C concentrations were calculated using the Friedewald formula . rs18007 Germany . PCR wast test. Adjustments for PA and BMI were made by linear regression. To analyze the effects of nutrient intake at baseline on the parameters, the study group was divided into subgroups based on median values of energy derived from fat, protein, or carbohydrates, as well as total intake of fat, saturated fatty acids (SFAs), MUFAs, and PUFAs. p < 0.05 was taken to be statistically significant. Data were analyzed using Statistica software .Normality of the data was tested using the Shapiro–Wilk test with α = 0.05. For data with non-normal distribution (gene expression data), the differences between the parameters before and after the DI were assessed using the Wilcoxon test. Between-group differences were tested using the Mann–Whitney test. Data with normal distribution were analyzed using Student’s IL6 and TNF in PBMCs before the DI were not affected by baseline energy intake from fat, total fat intake, PUFA, or MUFA intake. However, lower intake of SFA was associated with an over 500 times lower TNF transcription (p < 0.05). Specifically, the median values of the TNF relative transcription levels were 0.000576 and 0.000001, in the low and high SFA groups, respectively. The amount of baseline energy intake from fat affected the initial IL6 concentrations (p < 0.05). Specifically, the high energy intake from fat was associated with higher IL6 levels (see p < 0.01). The intake of SFA, MUFA or PUFA did not affect IL6 or TNFα levels , 0.56 (GC), and 0.19 (CC), and the allele frequencies were 0.53 (G) and 0.47 (C). The IL6 genotype groups differed in transcription levels at baseline, but only when SFA intake was considered (p < 0.05). Specifically, when SFA intake was high, the transcription level was over three times higher in the C-allele carriers group than in the GG genotype group. There was no effect of IL6 polymorphism on the anthropometric parameters . TNF and IL6 genes expression in PBMCs, and the transcription levels of the TNF gene decreased in both groups after the dietary intervention, but it was statistically significant only in the CED group . Both types of dietary intervention also led to a decrease in blood TNFα concentration—in the CED by 9% and in the MED group by 17.5% (p < 0.001). We measured by 17.5% b. HoweveIL6 gene transcription levels were not significantly affected by any of the diets .he diets a. We alshe diets b. The onWe hypothesized that habitual dietary fat intake, the IL6 genotype, and MED or the CED for 16 weeks would affect selected biomarkers of inflammation (TNFα and IL6) in postmenopausal women with central obesity; the obtained results partly confirm this hypothesis. We observed that, before the dietary intervention, lower habitual intake of SFA was associated with lower TNF transcription, but not with blood TNFα concentrations. Although no associations between fat or fatty acid (FA) intake and IL6 transcription in PBMCs were detected, blood IL6 concentrations were higher in postmenopausal women whose percentage energy from fat was higher. Our results are convergent with a review presented by Telle-Hansen et al. in 2017, where it was stated that most dietary interventions showed either no or minor effects of dietary fat intake on inflammatory markers in overweight and obese subjects . We alsoTNF transcription in PBMCs and decreased blood TNFα concentrations; the effects of both diets were similar, suggesting that observed result was mainly due to weight loss. Bianchi in his 2018 review concluded that in obese and overweight subjects weight loss, induced both by energy-restricted diet or surgery, is a determinant factor for reducing the level of pro-inflammatory markers [Moreover, the dietary intervention led to decreased  markers . Also, S markers ,25.IL6 transcription or blood IL6 concentrations, which indicated that this inflammatory pathway remained activated even after weight loss. It has previously been shown that the Nordic diet downregulates the expression of genes involved in inflammation in subcutaneous adipose tissue in subjects with MS, but not TNF and IL6 [IL6 was not among these genes [We did not observe any effect of either analyzed diet on  and IL6 , while ase genes . Blomqvise genes . There wse genes , it seemse genes . On the se genes .IL6 gene expression, we hypothesized that it may significantly affect blood IL6 concentrations, but the differences in the transcription levels in different genotype groups were not statistically significant. The blood IL6 concentration was also unaffected by the IL6 genotype, which is in agreement with previous studies that suggest that IL6 expression regulation does not only depend on this polymorphism [IL6 genotype group [IL6 genotype groups could be observed when comparisons are made between lean and obese subjects. Moreover, Joffe et al. showed that, with increasing PUFA, docosahexaenoic acid and eicosapentaenoic acid intake, BMI lowered in subjects with the C allele, also with increasing MUFA intake, TG concentrations decreased in the C allele carriers [IL6 genotype-dependent [IL6 genotype was associated with T-C and LDL-C concentrations, while GG subjects had lower values of these parameters. Surprisingly, Henningsson et al. observed associations between the IL6 genotype and lipid profile, but in the opposite direction—women with the CC genotype had lower levels of cholesterol and LDL-C [Since the rs1800795 polymorphism regulates morphism . Similarpe group . A meta-pe group . In the pe group . Possiblependent . This sund LDL-C . AltogetOur study has several limitations, which include lack of analysis of how gene × diet interactions might have affected the effectiveness of the intervention. The study was based on two different diets, and the total number of participants was too small to this type of analysis. Moreover, although adipose tissue is an important source of TNFα and IL6, especially in obese patients, we did not have access to samples of adipose tissue for gene expression analysis. However, this approach has been successfully applied earlier by other groups ,34.IL6 polymorphism alone does not affect body weight or composition in targeted population. Most likely, this is due to very small gene effects that cannot be detected in a relatively small group with low inter-individual variability. The C-allele carriers have higher levels of T-C and LDL-C. This polymorphism does not significantly affect blood inflammation biomarkers. Both diets, regardless of macronutrient contents lead to reducing inflammation biomarkers in postmenopausal women with central obesity.Blood TNFα and IL6 concentrations are not significantly affected by the transcription levels of their respective genes in PMBCs. Higher energy intake from total fat or protein may be associated with elevated concentrations of circulating plasma IL6 and TNFα, respectively. The rs1800795


Strict glycaemic control early in the treatment process has been shown to reduce the occurrence of micro- and macro- vascular complications of diabetes in the long-term. Thus, treatment guidelines advise early intensification of treatment to achieve glycaemic control goals. However, evidence in Greece suggests that, despite guideline recommendations, glycaemic control among patients with T2DM remains challenging. This study presents the demographic and clinical characteristics of patients with T2DM in Greece using data from an electronic registry designed specifically for this treatment category and investigates the factors that are independently associated with glycaemic control.This is a multi-center, observational, cross-sectional study to investigate epidemiological and clinical factors affecting glycaemic control among patients with T2DM in Greece. Data was collected via a web-based disease registry, the Diabetes Registry, which operated from January 1st to December 31st, 2017. Five large specialized diabetes centers operating in Greek hospitals participated in the study.Data for 1141 patients were retrieved . Glycaemic control (Hb1Ac < 7%) was not achieved in 57.1% of patients. Factors independently associated with poor glycaemic control were: family history of diabetes , BMI score between 25 to 30  or over 30 , elevated LDL levels  and low HDL levels . Lastly, use of injectable antidiabetic agents (in monotherapy or in combination) was less likely to be associated with poor glycaemic control versus treatment with combination of oral and injectable agents . This association was found to be marginally statistically significant.2) were associated with poor glycaemic control among study sample, whereas use of injectable antidiabetic agents was less likely to be associated with poor glycaemic control. These findings indicate how complex optimal glycaemic control is, highlighting the need for tailored interventions in high-risk subpopulations with T2DM.Inadequate lipid control, family history of diabetes and presence of obesity (ΒΜΙ ≥ 30 kg/m Diabetes Mellitus (DM), a metabolic disorder primarily characterized by hyperglycemia, has constituted one of the most critical challenges to health systems all over the world –3. AccorIn Greece, the prevalence of T2DM has been the subject of various epidemiological studies. According to recent estimations based on real-world data, 694,357 patients received prescribed medication for T2DM in 2015, accounting for 6.8% of the country’s population . FurtherStrict glycaemic control early in the treatment process has been shown to reduce the occurrence of micro- and macro- vascular complications of diabetes in the long-term –22. ThusA disease registry integrates a variety of information such as demographic characteristics, laboratory test results, clinical data, comorbidities and follow-up information to gather valuable information on the trends and management of chronic diseases . DiabeteIn the present study, five major diabetes units operating in large, public hospitals enrolled patients in a web-based disease registry, designed and developed to prospectively monitor and report on key disease indicators. The main objective was to explore current epidemiologic trends of the disease in Greece. Data regarding patient characteristics, co-morbidities and glycaemic control of participants were also collected. In addition, the study explored factors affecting the degree of glycaemic control among patient population to further recognize patient groups in need of intensive monitoring.This was a multi-center, cross-sectional observational study conducted in Greece. Participating centers were chosen as follows: out of 13 operating specialized diabetes centers in Attica, Piraeus, Macedonia and Thrace regions , six werAll outpatient subjects, who were older than 18 years, diagnosed with T2DM and scheduled for a routine office visit during the time period between January 2017 and December 2017, were eligible to participate. Diagnosis of T2DM was based on the criteria proposed by World Health Organization (WHO) . PatientAll eligible patients signed informed consent forms prior to enrolment. The study was approved by the Research Committee board of the University of Peloponnese.All data was collected with the use of a web-based diabetes registry that was specifically designed and developed to support this study. The Diabetes Registry was developed through a collaboration of the University of Peloponnese and the Medical School of the University of Athens. The research tool used was developed on the basis of international best practice, published, disease risk indices , 34 as vDemographic characteristics: Gender, age, educational level, place of residence, family and employment status.Clinical characteristics: Duration and family history of T2DM , treatment modalities, presence of established main comorbidities (macro-vascular disorders), severe risk factors for Cardiovascular disorders (CVD) , metabolic disorders (hypothyroidism and hyperthyroidism) and diabetes related complications . Presence of concomitant diseases was self-reported. Data regarding Body Mass Index (BMI) and waist circumference were also recorded. Patients were categorized based on their BMI score as follows: participants with BMI score less than 25 kg/m2 were defined as normal and with BMI score between 25 kg/m2 and 30 kg/m2 as overweight. Patients with obesity were defined as having BMI score equal/over 30 kg/m2. Waist circumference target was set at 80 cm for women and 90 cm for men.Laboratory test results: Levels of HbA1c, blood pressure (BP), high-density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG) and estimated Glomerular Filtration Rate (e-GFR) were assessed. EPI equation was used for the calculation of e-GFR. Glycaemic control was defined as adequate if HbA1c was less than 7%. Only patients who had a recent (during the last 12 months) laboratory test for HbA1c were included. For BP control target was set at less than 130/80 mm/Hg. Regarding lipid control, HDL levels of more than 40 mg/dl, LDL levels of less than 100 mg/dl and TG levels of less than 150 mg/dl were set as optimal. Participants were classified as having dyslipidemia if their serum lipids levels were other than optimal as described above and/or if they were on treatment with lipid lowering medications. Moreover, participants were classified as suffering from hypertension if they reported a previous diagnosis of hypertension or/and were receiving anti-hypertensive medication.Lifestyle behaviors: Alcohol and tobacco consumption, physical activity and dietary habits were recorded. The dietary and physical activity factors were selected from the Diabetes Prevention toolkit [ toolkit , 34. IteThe registry recorded:The diabetes registry was piloted to 100 patients to assess its validity. Pilot study results were evaluated by a group of experts. Patients who participated in the pilot study were excluded from the final analysis.A descriptive and inferential analysis was performed on study data. Absolute and relative frequencies were used to describe categorical variables. Continuous variables were expressed using mean ± SD if normally distributed. Kolmogorov Smirnov criterion was used to test the normality of distributions.p-values are presented. All statistical analyses were conducted using SPSS v. 25 .All socio-demographic and clinical characteristics, laboratory measurements and lifestyle behaviors were univariately associated with glycaemic control (a cut-off point of HbA1c ≥ 7% was used). Patients diagnosed 6 or less months ago were excluded from further analyses. Chi-square tests were performed to test the association amongst categorical variables and independent t-test criterion was used to test for the association between a continuous and a categorical variable. All variables that yield statistically significance at the 5% level were entered into a multiple logistic regression model. Age, sex, BMI and duration of T2DM were entered into the final model as these are factors of pertinent clinical importance to this disease area and were considered to act as confounders. Adjusted Odds Ratios (OR) along with 95% Confidence Intervals and Study population consisted of 1141 patients with T2DM. Mean age was 63.02 ± 12.65 years. Most of the patients were male (56.9%), living in urban areas (95.8%), had completed mandatory education (77.5%) and were economically inactive (i.e. pensioners or students) at the time of the study (63.5%). 81.1% were married and almost half (45.4%) of them had children. Participants’ socio-demographic characteristics are presented in Table Most patients had a T2DM diagnosis for more than 10 years (40.7%) and were categorized as obese based on their Body Mass Index score . The majority of patients were on oral antidiabetic agents only (67.7%) and used 1 antidiabetic agent (66.3%). Family history of diabetes was reported by almost 1 in 2 participants (48.6%).61.5 and 58.9% out of total study sample reported previous diagnosis of dyslipidemia and hypertension, respectively. Moreover, 43.6% of sample reported suffering from both of these conditions. 247 (21.6%) patients had a personal history of coronary artery disease, defined as stable or unstable angina, NSTEMI or STEMI. 94 (8.2%) had been diagnosed with heart failure. 53 (4.6%) had suffered from an ischemic or hemorrhagic stroke and another 50 (4.4%) from a transient ischemic attack (TIA). Hypothyroidism was present in 182 (16%) of the patients, while hyperthyroidism in 21 (1.8%). Sample’s clinical characteristics are presented in detail in Table Almost 1 in 3 patients were smoking and 1 in 5 had ceased smoking during the last 12 months (20.5%). Only 14.7% of total sample had been engaging in a physical activity for at least 30 min per day or 3 times per week. The vast majority of participants did not follow a nutritious diet, as only 10.9 and 13.5% of responders reported consuming 3 or more servings of whole grain cereals daily and vegetable oil most of the days, respectively. Almost one in two (47.8%) reported daily consumption of 3 servings of processed starch. Only 9.7% of study participants reported drinking one or more alcoholic drinks per day, with the majority consuming zero alcohol on a daily basis  HbA1c measurement. Only 42.9% of patients assessed achieved HbA1c levels of less than 7%.p = 0.045) and the type of pharmacological treatment (p = 0.010). In addition, the following laboratory measurements were found to be associated with glycaemic control: LDL (p = 0.047), HDL (p < 0.001) and TG (p < 0.001)  were associated with poor glycaemic control among study sample, whereas use of injectable antidiabetic agents was less likely to be associated with poor glycaemic control. These findings indicate how complex optimal management for patients with T2DM is, highlighting the need for tailored interventions in high-risk subpopulations with T2DM.Prevalence of risk factors for CVD and other comorbidities deemed high among the study population. Furthermore, a small proportion of the sample was engaged in healthy lifestyle behaviors. Inadequate lipid control, family history of diabetes and presence of obesity (ΒΜΙ ≥ 30 kg/m