In osteoarthritis (OA), cartilage matrix is lost gradually despite enhanced matrix synthesis by chondrocytes. This paradox may be explained, at least partly, by reduced chondrocyte anabolism in degenerated area of OA cartilage. However, to date, it is not known why chondrocyte anabolism is suppressed in those areas.Cartilage was obtained from control knees and end-stage OA knees in macroscopically preserved areas and degenerated areas, and gene expression was analyzed in respective regions of cartilage using laser capture microdissection and qPCR. For the cartilage protein analysis, cartilage was obtained from preserved areas and degenerated areas of OA knees in pairs, and proteins were extracted using urea buffer. Protein concentrations were determined by Luminex and compared between the areas. Cartilage explants prepared from preserved areas and degenerated areas of OA knees were cultured in the presence or absence of an AKT inhibitor, and the gene expression was evaluated by qPCR. Finally, the expression of SP1 was evaluated in OA and control cartilage, and the significance of Sp1 on the expression of IGF1R and IRS1 was investigated in experiments using primary cultured chondrocytes.Within OA cartilage, the expression of IGF-1, IGF-2, IGF1R and IRS1 was reduced in degenerated areas compared to preserved areas, while the expression of all six IGF-binding protein genes examined was enhanced in the former areas. Consistent results were obtained by a protein analysis. In explant culture, the inhibition of AKT signaling abrogated the abundant matrix gene expression in the preserved areas over the degenerated areas, indicating that suppressed matrix synthesis in degenerated areas may be ascribed, at least partly, to attenuated IGF signaling. Within OA cartilage, the expression of Sp1 was considerably reduced in severely degenerated areas compared to preserved areas, which correlated well with the expression of IGF1R and IRS1. In experiments using primary cultured chondrocytes, the expression of IGF1R and IRS1 was enhanced by the induction of Sp1 expression and reduced by the suppression of Sp1 expression.The results of this study suggest that attenuated IGF signaling may be responsible, at least partly, for the reduced matrix synthesis in degenerated areas of OA cartilage. Osteoarthritis (OA) is the most prevalent joint disease in developed countries and primarily affects the elderly. The loss of cartilage matrix over an extended period of time is a hallmark of the disease. In OA, cartilage matrix is lost gradually by enhanced proteolysis of induced proteinases , 2. HoweIn OA, chondrocyte metabolism is regulated by various growth factors and cytokines. Among them, insulin-like growth factor (IGF)-1 is known to have potent anabolic actions on chondrocytes –10. The In this study, we explored the mechanisms underlying the regional differences in chondrocyte anabolism within OA cartilage, focusing on IGFs and related molecules. In the analysis, we employed laser capture microdissection (LCM), which is a technology that enables the isolation of a specific area of tissue by its histologic features . CombineThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Committee of the National Hospital Organization Sagamihara Hospital. Prior to the acquisition of cartilage samples, informed consent was obtained in writing from each patient or family of the donor. OA cartilage was harvested from a total of 45 end-stage OA knee joints of 45 patients  who underwent prosthetic surgery within 4 h after the operation. All knees had medial disease involvement, and the diagnosis of OA was based on the established criteria for knee OA .Control cartilage was obtained from 9 non-arthritic knee joints from 9 donors  within 24 h after death. The donors had no known history of joint disease, and the normality of the joint was confirmed macroscopically at the time of harvest.In the analysis, human cartilage tissues were precisely divided into cartilage zones using an LCM device , and the gene expression was determined in respective zones. Cartilage obtained from 16 OA knees and 9 control knees was used for this analysis. In OA knees, cartilage samples were harvested from both macroscopically intact areas (preserved areas) and areas showing various degrees of cartilage degeneration (degenerated areas). In control knees, cartilage samples were obtained at 1–3 sites in each knee from the femoral condyles, confirming that the areas showed little sign of cartilage degeneration. At each site, approximately 20 mm × 5 mm of cartilage was obtained in full thickness above the tide mark. The tissue was immediately embedded in OCT compound , snap-frozen in liquid nitrogen, and stored at -80 °C.GAPDH.For analyses, cryosections were prepared from the OCT-embedded cartilage tissues, and cartilage zones were separated by LCM as described previously , 20. In Cartilage tissues from 20 end-stage OA knees were used for this analysis. In each knee, cartilage tissues were obtained in pairs from preserved areas and degenerated areas on the tibial plateau. At each area, 400–700 mg wet-weight of tissue was harvested above the tide mark with a scalpel, which was rinsed thoroughly in ice-cold phosphate-buffered saline (PBS). Protein extraction was performed following a previously described method with some modifications , 23. In Protein concentrations in the supernatant were determined by a BioPlex 200 system  using commercially available kits . For the measurement, 25 µl of the dialyzed extracts was incubated with a suspension of capture antibody-conjugated magnetic microspheres following the manufacturer’s protocols. The microspheres were then incubated with biotinylated detection antibody and R-phycoerythrin (PE)-conjugated streptavidin, and the fluorescence intensity was measured. Protein concentrations were compared after normalization by the total protein concentration of the extract, which was determined by a Pierce BCA Protein Assay Kit  using bovine serum albumin as a standard.For explant culture, cartilage tissues were obtained from 5 OA knees in pairs from preserved areas and degenerated areas on the tibial plateaus, as described for the cartilage protein analysis. After being rinsed in sterilized PBS, each piece of cartilage tissue was diced into 30–50 cubes of 2–3 mm. The diced cartilage, or explants, were equally divided into two, which were placed onto respective wells of a 12-well plastic plate. These explants were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 supplemented with ITS Liquid Media Supplement  and 25 µg/ml ascorbic acid overnight, and then the media was replaced with those containing Akt inhibitor IV  or DMSO alone (vehicle control). After 48 h of culture, the explants were recovered, embedded in OCT compound, snap frozen in liquid nitrogen and stored at -80 °C until analysis. Extraction of RNA from the explants was performed following a previously described method [5/cm2, and maintained in the culture media described above for the explant culture. Chondrocytes were used for experiments starting two days after plating.To obtain primary culture human chondrocytes, cartilage tissue was obtained from macroscopically intact areas of four OA knee joints, which were subjected to serial enzymatic digestion using Pronase (Sigma Aldrich) and Collagenase P (Sigma Aldrich) , 25. FolSP1 complementary DNA (cDNA) were cloned into the adenoviral-generating constructs [Recombinant adenoviruses were constructed using a ViraPower Adenoviral Expression System (Thermo Fisher Scientific). For this, human nstructs . The con6 chondrocytes were suspended in 100 µl of electroporation buffer containing 20 pmoles of siRNA, and the siRNAs were introduced into the cells by electroporation. Chondrocytes were then plated and cultured in DMEM/F-12 containing 20 % fetal bovine serum (FBS), which was replaced with DMEM/F‐12 containing 10 % FBS the next day. Based on the result of our preliminary experiments, the effect of RNAi was evaluated three days after electroporation. In this study, 2 siRNAs with respective sequences were used to suppress the Sp1 expression, which were confirmed to reduce the expression by 43 % and 56 %, respectively, as evaluated by qPCR.All small interfering RNAs (siRNAs) were purchased from Qiagen. The siRNAs were introduced into primary cultured chondrocytes by electroporation using a Nucleofector , according to the manufacturer’s protocols with some modifications . In briet-tests. For multiple comparisons, data were compared using a one‐way factorial analysis of variance (ANOVA), and when necessary, Fisher’s protected least significant difference was used as a post hoc test. The correlation of the expression was evaluated by a linear regression analysis. P values less than 0.05 were considered significant.Data were compared by paired or unpaired IGF-1 and IGF-2 require IGF1R and IRS1 to initiate IGF signaling. Therefore, we first investigated the expression of these four genes in OA and control cartilage together with type II procollagen and aggrecan, focusing on regional differences within cartilage. For this analysis, OA cartilage was obtained from macroscopically preserved areas and degenerated areas with various degrees of degeneration, and the gene expression was determined in respective cartilage zones using LCM coupled with qPCR  than in those from degenerated areas (Ex-D) after three days of culture Fig. . The AKTWe next wished to clarify the mechanism underlying the decline in the expression of IGF1R and IRS1 in degenerated areas of OA cartilage. Based on the results of previous studies , 29, we Finally, we investigated the relationship in gene expression among IGF1R, IRS1 and Sp1 within OA cartilage. Previous studies showed that the gene expression may differ considerably among cartilage zones , 4. TherTo our surprise, strong positive correlations were observed in all pairs of the three genes, in all three cartilage zones Fig. . This reAlthough an increasing number of studies have been published concerning the pathology of OA, few have focused on the mechanisms underlying the regional difference in chondrocyte metabolism under OA conditions. In this study, we explored this mechanism and obtained data suggesting that a change in the IGF signaling might be involved in these regional differences. Since most of our results were obtained through an analysis of human cartilage samples, they would directly reflect the changes that actually occur in OA patients’ knees. We believe that our findings may provide a new basis for understanding the cartilage pathology in OA.Several investigators have evaluated the expression of IGF-1 and IGF-2 in OA and control cartilage. An elevated IGF-1 expression in OA cartilage was reported repeatedly by previous studies , 30, 31.The expression of IGF1R in OA cartilage has been examined by several investigators, but the results are somewhat conflicting. While one study reported that the expression in OA cartilage was similar to that in control cartilage , others A similar trend was observed for IRS1. Although the expression of IRS1 did not change markedly in preserved areas of OA cartilage compared to the control, its expression was substantially reduced in degenerated areas. This regional difference within OA cartilage was, again, confirmed by a protein analysis. Upon engagement with IGFs, IGF1R phosphorylates IRS1, which then activates the PI3K/AKT/mTOR pathway to promote cartilage matrix synthesis , 27. TheIn 1994, Dore et al. first reported that the chondrocytes from OA cartilage respond poorly to IGF-1 due to an increased IGFBP expression . Since tGiven that all of the above results indicated attenuated IGF signaling in degenerated areas of OA cartilage, we next conducted an explant culture experiment to examine that possibility. We found that matrix gene expression in the explants from preserved areas was considerably reduced by the inhibition of AKT signaling, while such reduction was not apparent with the explants from degenerated areas. As mentioned earlier, AKT signaling is responsible for the promotion of chondrocyte matrix synthesis by IGFs , 27. TheSince IGF1R plays pivotal roles in various biological events, the regulatory mechanisms concerning its expression have been the focus of intense research. Thus far, several transcription factors have been found to regulate the IGF1R expression , 34. AmoTaken together, our findings suggested that at least three changes in the IGF system, namely, a reduced IGF expression, increased IGFBP expression, and reduced IGF1R and IRS1 expression might underlie the reduced cartilage matrix synthesis in degenerated areas of OA cartilage. Of course, changes in chondrocyte metabolism within OA cartilage are likely a very complex event and cannot be explained simply by the modulation of the IGF system. Nonetheless, we believe that our current findings will help those who are engaged in the study of this common, but tenacious disease.Taken together, our findings suggested that at least three changes in the IGF system, namely, a reduced IGF expression, increased IGFBP expression, and reduced IGF1R and IRS1 expression might underlie the reduced cartilage matrix synthesis in degenerated areas of OA cartilage. Although these results seem convincing, this study has several limitations as well. First, though enhanced IGFBP expression in degenerated areas has been shown, to what extent IGF signaling is inhibited by each of the proteins remains unclear. Again, although the suppression of SP1 expression was shown to be responsible for the reduced IGF1R and IRS1 expression in degenerated areas, whether or not their diminished expression can be solely ascribed to the decline in SP1 expression has not been clarified. However, despite these and other possible limitations, we believe that our current findings will help those who are engaged in the study of this common, but tenacious disease.To determine the mechanisms underlying the reduced matrix synthesis in degenerated areas of OA cartilage, we performed gene expression and protein analyses of human cartilage samples, and revealed that the expression of IGF-1, IGF-2, IGF1R and IRS1 was suppressed, while that of six IGFBP genes was elevated in degenerated areas of OA cartilage compared to macroscopically intact areas. This result suggests that the reduction in cartilage matrix synthesis in degenerated areas may be ascribed, at least in part, to attenuated IGF signaling in these areas. We further investigated the mechanisms underlying the reduced IGF1R and IRS1 expression in degenerated areas, and found that their expression was regulated by Sp1 in OA cartilage and that a decline in the Sp1 expression might be responsible for the reduction in the IGF1R and IRS1 expression in those areas. These findings may be worth noting when exploring therapies to inhibit the loss of cartilage matrix in OA.


The slope of the resulting relationship was used to calculate system resistance. Poiseuille’s law of resistance was used to investigate significant contributors to resistance. Resistance ranged from 0.05 to 1.40 Respiratory distress due to preterm birth is a significant cause of death in low-resource settings. The introduction of continuous positive airway pressure (CPAP) systems to treat respiratory distress significantly reduced mortality in high-resource settings, but CPAP was only recently introduced in low-resource settings due to cost and infrastructure limitations. We evaluated pressure stability and imposed work of breathing (iWOB) of five CPAP systems used in low resource settings: the Fisher and Paykel bubble CPAP, the Diamedica baby CPAP, the Medijet nCPAP generator, and the first (2015) and second (2017) generation commercially available Pumani CPAPs. Pressure changes due to fresh gas flow were evaluated for each system by examining the relationship between flow and pressure at the patient interface for four pressures generated at the bottle (0, 3, 5, and 7 cm H Nearly two million infants die each year in the first week of life; nearly half of these deaths arise from complications of preterm birth . RespiraThe World Health Organization and European Guidelines for Management of RDS recommend that respiratory distress in preterm newborns be treated with surfactants and continuous positive airway pressure (CPAP) ,5. In hiLow-resource settings present unique challenges for the design of medical devices, including lack of financial resources to purchase equipment, weak supply chains for consumables, inconsistent power, and harsh environmental conditions ,11. For The purpose of this study was to compare pressures generated by fresh gas flow and imposed work of breathing (iWOB) of five CPAP devices used in low-resource settings.We measured pressure generated by fresh gas flow and imposed work of breathing in a laboratory setting for five CPAP systems: Fisher and Paykel bubble CPAP , Diamedica baby CPAP , Medijet disposable nCPAP generator , first generation commercially available Pumani bCPAP , and second generation commercially available Pumani bubble CPAP .2 concentration) of the Fluke VT650 using a silicone Y-connector  which was factory calibrated to meet manufacturer specifications (±0.5%) prior to data collection. Work of breathing was measured using an ASL 5000 Breathing Simulator with 3L cylinder . The ASL system is calibrated annually to ensure pressure and volume accuracy. Pumani prongs (size 4) were used for all CPAP systems except the Medijet; for the Medijet, the interface was connected directly to both measurement devices. Prongs and the Medijet device were connected to the airway inlet  at intervals of 1 L/min. For the Medijet nCPAP generator, pressure is provided solely through resistance, and therefore, there is no submersion of the expiratory limb. End expiratory pressures were recorded for each pressure/flow combination for one minute each. Pressures were recorded at a sampling rate of 1 Hz.System resistance was evaluated by systematically increasing the fresh gas flow from 5–10 L/min and measuring the pressure at the patient interface. For all but the Medijet nCPAP system, the expiratory limb was submerged to 0, 3, 5, and 7 cm H2O of submersion depth. An F-test of significance was used to determine whether the slope of each line was significantly different than 0.The first 10 and last 10 seconds of each recording were discarded and the mean and standard deviation of the remaining pressure measurements are reported. Resistance was calculated by determining the slope and 95% confidence interval of the best-fit line (least-squares method) of measured pressure vs. flow at 0 cm H−6 Pa-s [Resistances were compared against theoretical values calculated by Poiseulle’s law of resistance 0−6 Pa-s ; length 2O, while flows were set at 6, 7, and 8 liters per minute (L/min). Each test lasted one minute. Breath-by-breath measurements (including imposed work of breathing) were calculated using the ASL 5000 software to determine the integral of the pressure-volume loop for each breath .A sinusoidal breath profile was used to simulate breathing of an infant (5.5 kg), a term neonate (4 kg), and a late preterm neonate (2.5 kg). Tidal volumes were estimated at 6 mL/kg body weight . Infant Imposed work of breathing measurements for the first ten and last ten breaths were discarded, resulting in 40 breaths remaining for the infant model and 47 breaths each for the term neonate and preterm neonate models. Mean and standard deviations of iWOB values were calculated. A paired t-test (α<0.05) was used to identify significant differences between systems. Statistical analysis was performed in MATLAB  and SPSS .Pressure at the patient interface was plotted against applied flow for 0, 3, 5, and 7 cm of submersion for all but the Medijet nCPAP system . For the2O and is shown in Resistance of each system is calculated as the slope of each line from 5–10 L/min at 0 cm HContributions to resistance in the expiratory limb were examined using Poiseuille’s law of resistance. Diameters and lengths of tubing, prongs, and connectors for inspiratory and expiratory limbs of each system were measured. For the first generation Pumani bCPAP, where air flows in parallel to the bottle and patient, only the patient limb of the circuit was included here. Theoretical resistances are plotted in 2O) is shown for the infant, term neonate, and preterm neonate for the Fisher and Paykel, Diamedica and first and second generation Pumani CPAP systems. iWOB for remaining pressure/flow combinations  is shown in Imposed work of breathing (mJ/breath) for three representative flow/pressure combinations . The second generation Pumani bubble CPAP also reduces the iWOB below that of the Medijet nCPAP generator system ,22; it rWe compared our results for imposed WOB to those previously reported for the infant breath profile the first generation Pumani bCPAP, Diamedica, Neopuff, Fisher and Paykel and Medijet systems. Values match those reported in Falk et al. –23; the 2O) and first generation Pumani bCPAP with a submersion depth of 7 cm (11.2±.13 cm H2O).The clinical significance of differences in resistance and iWOB is uncertain . In thisThere are no clinical guidelines for imposed work of breathing, and it varies significantly for commercially available systems, as shown in Although there is scant evidence to demonstrate that system resistance and imposed WOB are clinically significant, laboratory-based performance metrics such as these may help providers select devices appropriate for their clinical setting. This information may also help manufacturers improve device performance by identifying significant contributors to system resistance: for example, tubing diameter is a major contributor to resistance, and also predicts connector size. As resistance varies with the fourth power of diameter, small changes in diameter can result in significant improvements in resistance and iWOB. While tubing length is also an important contributor to overall resistance, small changes in tubing length have only a proportional impact on resistance. In addition, differences in laboratory performance may indicate areas where larger clinical trials are needed.Another area of interest is CPAP provided during or immediately after resuscitation. For term infants in high resource settings, there is no current recommendation for CPAP at birth ,30 and aLaboratory testing under ideal conditions when no leaks are present at the patient interface shows that commercially available CPAP systems differ in resistance and iWOB. There are no available data to suggest that these differences affect clinical outcomes.S1 Fig2O (shown in results); middle: 6 cm H2O; right: 7 cm H2O.Left: 8 cm H(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableFor the Medijet nCPAP generator, pressure cannot be set independently; results for the flow rates of 6, 7, and 8 L/min were measured using ASL 5000 Breathing Simulator.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


To investigate which cardiometabolic factors underlie clustering of osteoarthritis (OA) with cardiovascular disease, and the extent to which these mediate an effect of education.Genome-wide association study (GWAS) of OA was performed in UK Biobank  to obtain genetic association estimates for OA risk. Genetic instruments and association estimates for body mass index (BMI), low-density lipoprotein cholesterol (LDL-C), systolic blood pressure (SBP), smoking and education were obtained from existing GWAS summary data . Two-sample Mendelian randomization (MR) analyses were performed to investigate the effects of exposure traits on OA risk. MR mediation analyses were undertaken to investigate whether the cardiometabolic traits mediate any effect of education on OA risk.MR analyses identified protective effects of higher genetically predicted education  per standard deviation increase 0.59, 95% confidence interval (CI) 0.54–0.64) and LDL-C levels  on OA risk, and unfavourable effects of higher genetically predicted BMI  and smoking . There was no strong evidence of an effect of genetically predicted SBP on OA risk . The proportion of the effect of genetically predicted education mediated through genetically predicted BMI and smoking was 35% (95%CI 13–57%).These findings highlight education, obesity and smoking as common mechanisms underlying OA and cardiovascular disease. These risk factors represent clinical and public health targets for reducing multi-morbidity related to the burden these common conditions. It accounts for 2.4% of all years lived with disability (YLD) and ranks as a leading contributor to global YLDs,Assessing causal effects in observational research is difficult due to environmental confounding or reverse causation. The Mendelian randomization (MR) approach can overcome some of these limitations by using genetic variants related to the exposure of interest as instrumental variables for investigating its effects on an outcomeThe aim of this study was to apply the MR framework to investigate the effects of education and cardiometabolic risk factors on risk of OA. For cardiometabolic risk factors that the MR analyses supported to have a causal effect on OA risk, we aimed to further apply MR mediation analyses to investigate the degree to which these factors might be mediating the effects of educational attainment.,A genome-wide association study (GWAS) for OA was performed in the UK Biobank to obtain genetic association estimates for OA risk. UK Biobank identification of OA was based on hospital-diagnosed OA cases,GWAS for OA was performed in the UK Biobank3. Genetic association estimates for smoking were obtained from a GWAS of 462,690 European-ancestry individuals in the UK BiobankGenetic association estimates for BMI were obtained from the GIANT Consortium GWAS meta-analysis of 806,834 European-ancestry individuals,,,,We obtain genetic association estimates for education, BMI, SBP and smoking from studies that included UK Biobank participantsP < 5 × 10−8) and were independent, i.e., pairwise linkage disequilibrium (LD) r2 < 0.001. To select instruments for multivariable MR (MVMR) in analyses investigating mediators of the effect of genetically predicted education on OA risk, all SNPs related to education or investigated mediators at genome-wide significance were pooled and clumped to pairwise LD r2 < 0.001 based on the lowest P-value for their association with any trait. All clumping was performed using the TwoSampleMR package in RInstruments for each considered exposure in univariable MR analyses were selected as single-nucleotide polymorphisms (SNPs) that associated with the exposure at genome-wide significance  for the risk of OA in MR analysis of each exposure separately, given 80% power, type I error rate = 0.05, exposure GWAS summary statistics sample size and the total variance explained by the genetic instrumentsMultiplicative random-effects inverse-variance weighted (IVW) MR was used as the main analysis for estimating the effects of genetically predicted cardiovascular risk factors and education on OA riskWhen using multiple genetic variants as instrumental variables in MR, a potential source of bias is horizontal pleiotropy, where the genetic variants affect the risk of OA through pathways independent of the considered exposure. To assess the robustness of the findings to the potential bias due to horizontal pleiotropy, we used contamination-mixture method, MR-Egger and weighted median MR as sensitivity analyses2, for LDL-C this was 0.87 mmol/l, for SBP this was 18.68 mmHg and for education this was 3.6 years. For smoking, a one SD increase was equivalent to an individual smoking 20 cigarettes per day for 15 years and stopping 17 years ago, for exampleAll MR estimates were calculated per one standard deviation (SD) unit increase in the exposure under consideration, with SD estimates derived from UK Biobank data. For BMI this was 4.77 kg/m,The genetically predicted cardiovascular risk factors that showed evidence for a detrimental effect on the risk of OA in univariable MR were taken forward for MVMR mediation analysis,,In MVMR, the total effect of each exposure is decomposed to direct and indirect effects. This allows for estimation of potential mediating effects and the proportion of the effect of the main exposure of interest on the outcome that acts via other considered exposuresP value threshold for statistical significance was used. Instead, we interpret the evidence provided by the results by looking at the effect size of interest and the width of its confidence interval (CI), combined with the consistency of the results across the different methods usedNo formal ,,,All data used in this work are publicly available and the studies from which they were obtained had previously obtained relevant ethical approval and participant consentF statistics used in the univariable and MVMR analyses are provided in All genetic association estimates and their P = 0.79), LDL-C: (P = 0.21), and SBP (P = 0.25). There was weak evidence for directional pleiotropy for BMI (P = 0.10) and smoking: (P = 0.09), however in both cases MR-Egger estimate was consistent with the IVW estimate , with consistent findings in sensitivity analyses . There westimate . Given testimate .Fig. 1EfThe proportion of the effect of genetically predicted education mediated through genetically predicted BMI, smoking, and both BMI and smoking together was estimated as 23% (95%CI 1–44%), 25% (95%CI -3%–47%) and 35% (95%CI 13–57%), respectively . The resOur work uses large-scale GWAS data to investigate the effect of genetically predicted education and cardiometabolic risk factors on OA risk within the MR framework, and provides evidence supporting protective effects of education and LDL-C and unfavourable effects of BMI and smoking. These findings add insight into causal mechanisms underlying OA, its clustering with the risk factors of cardiovascular disease, and disparities related to educational attainment.,Our results are consistent with previous MR analyses identifying a protective effect of genetically predicted education and LDL-C, and a detrimental effect of genetically predicted BMI on OA risk,The findings of our study are relevant in both clinical and public health terms. Smoking and obesity have widespread implications on human health that extend far beyond cardiovascular disease. Smoking increases risk of chronic lung disease and many cancers, while obesity is a major contributor towards risk of diabetes,Our results suggest that the protective effect of education on OA risk is mediated through smoking and BMI. However, there was high uncertainty in the estimates, our data being consistent with the mediated proportion being between 13% and 57%. For comparison, approximately half of the protective effect of education on cardiovascular disease has previously been estimated to be mediated together through blood pressure, obesity and smoking,a priori, as mediation and confounding cannot be statistically distinguished,Our study has limitations. Firstly, the MR approach uses the cumulative lifelong effect of genetic variants and should not be extrapolated to presume the effect of a clinical interventionIn conclusion, this study uses genetic data in MR analyses to generate evidence supporting a protective effect of education and detrimental effects of BMI and smoking on OA risk, with evidence that the effect of education is mediated through BMI and smoking. These findings highlight education, obesity and smoking as common mechanisms underlying clustering of OA with risk factors of cardiovascular disease, which may represent clinical and public health targets for reducing multi-morbidity and the burden of these common conditions.DG, and NS designed the study. DG, RM and VK performed statistical analyses. All authors interpreted the results. DG, RM and NS drafted the manuscript. All authors edited the manuscript for intellectual content. All authors take responsibility for the integrity of the study.DG is employed part-time by Novo Nordisk, outside the submitted work. NS has received consultancy fees from Pfizer and Eli Lilly, but has no direct conflicts of interest relating to this project. The remaining authors have no conflicts of interest to declare.10.13039/100004440Wellcome Trust 4i Clinical PhD programme (Grant No. 203928/Z/16/Z) and the 10.13039/501100000274British Heart Foundation Centre of Research Excellence (RE/18/4/34215) at 10.13039/501100000761Imperial College London. NS is supported by a 10.13039/100004440Wellcome Trust Institutional Support Fund (ISSF), Grant No. 204809/Z/16/Z. This project has received funding from the European Union's Horizon 2020 research and innovation programme (666881), SVDs@target , CoSTREAM (to MD); the DFG as part of the Munich Cluster for Systems Neurology , the CRC 1123  and project DI 722/13-1; the Corona Foundation (to MD); the LMUexcellent fond (to MD); the e:Med program  and the FP7/2007–2103 European Union project CVgenes@target . The funding sources were not involved in study design, acquisition of data, analysis, interpretation or manuscript write up.DG is funded by the


Rosmarinus officinalis L.) is a Mediterranean medicinal and aromatic plant widely used due to valuable bioactive compounds (BACs) and aromas. The aim of the study was to evaluate the extraction of intracellular compounds from rosemary combining experimental procedure by means of high voltage electrical discharge (HVED), with a theoretical approach using two computational simulation methods: conductor-like screening model for real solvents and Hansen solubility parameters. The optimal HVED parameters were as follows: frequency 100 Hz, pulse width 400 ns, gap between electrodes 15 mm, liquid to solid ratio 50 mL/g, voltage 15 and 20 kV for argon, and 20 and 25 kV for nitrogen gas. Green solvents were used, water and ethanol (25% and 50%). The comparison was done with modified conventional extraction (CE) extracted by magnetic stirring and physicochemical analyses of obtained extracts were done. Results showed that HVED extracts in average 2.13-times higher total phenol content compared to CE. Furthermore, nitrogen, longer treatment time and higher voltage enhanced higher yields in HVED extraction. HVED was confirmed to have a high potential for extraction of BACs from rosemary. The computational stimulation methods were confirmed by experimental study, ethanol had higher potential of solubility of BACs and aromas from rosemary compared to water.Rosemary ( Rosmarinus officinalis L.) is an autochthonous Mediterranean herb from Lamiaceae family. From ancient times, rosemary has been used as flavoring agent and for medicinal purposes due to its intense aromatic odor and health benefits [Rosemary  techniques are often associated with long extraction time, use of organic solvents in huge amounts and possible thermal degradation of thermosensitive compounds such as antioxidants . For thaA green nonthermal extraction method that has not been investigated for extractions from rosemary is high voltage electrical discharge (HVED)—cold plasma treatment . HVED exMost conventional organic solvents are volatile, flammable, explosive, toxic, and have a negative environmental impact. For that reason, another principle of green extraction is directed towards use of alternative green solvents. In order to define green solvents, Gu and Jérôme (2013) proposed 12 criteria for green solvents that should be fulfilled related to availability, price, recyclability, grade, synthesis, toxicity, biodegradability, performance, stability, flammability, storage, and renewability. The solvent is considered greener when compared to conventional solvents that should be replaced with alternative solvent that fulfils at least some of the mentioned criteria . Among gIt is important to choose an optimal solvent for the desired extraction, and to reduce solvent usage during experimentation. Traditional experimental procedures for solvent selection use high amount of solvents to choose which one is suitable for the extraction of compounds. For that reason, various computational simulations for solvent selection have been developed, such as Hansen solubility parameters (HSPs) and conductor-like screening model for real solvents (COSMO–RS). These models use theoretical predictions for assessing the solubility of targeted compounds in each solvent .Since HVED treatment involves many processes, different reactive compounds are also formed. Beside generation of free radical species, there is also a possibility of electrodes abrasion and release of metals to the sample. For that reason, it is important to monitor preferred substances like phenols, antioxidants and volatile compounds, but also metals and other undesired compounds. Near infrared (NIR) spectroscopy has been utilized widely in the food and agribusiness ventures in the course of the last 20–30 years to decide significant parts in numerous agrarian items and plant materials ,33,34. IThe aim of this work was to understand the green extraction of BACs and volatile compounds from rosemary using computational programs (HSPs and COSMO–RS) and experimental extractions. The experimental solvent extraction was carried out using HVED as a nonthermal technology. The obtained extracts were analyzed for physical and chemical  parameters. Furthermore, analyses of pesticides and metals in dry rosemary leaves and in HVED extracts were also performed.The extraction of BACs and volatile compounds from dried rosemary leaves were assessed by computational simulation methods and experimental analysis using HVED. Computational simulation methods were performed by HSPs and COSMO–RS where various green solvents were assessed in comparison with conventional solvent n-hexane in order to theoretically predict the probability of solution of BACs from rosemary. Experimental extraction method from rosemary was done using water and ethanol solutions (25 and 50%) as green solvents by means of HVED, and it was compared with modified CE under the same extraction conditions. The obtained extracts by CE and HVED were analyzed for physical parameters , non-volatile compounds , antioxidant capacity by 2-diphenyl-2-picrylhydrazyl (DPPH) free radical assay and ferric reducing antioxidant power (FRAP) method, NIR and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS)), volatile compounds by headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS–SPME/GC–MS), and metal content. Additionally, analyzes of pesticides and metals in dry rosemary leaves were performed to assess the safety of raw material for further processing and human consumption. The flowchart of all performed analysis from rosemary is presented in n-hexane (first column). The HSPs for rosemary compounds were assessed at room temperature (20 °C) for different solvents and relative energy difference (RED) values are summarized in The solubility parameters of green solvents for extraction of BACs and aromas from rosemary leaves have been studied by means of the HSP and COSMO–RS theoretical predictions. For both models, solubility results have been presented for various green solvents , dimethyl carbonate, methyltetrahydrofuran (MeTHF), and water) compared to conventional COSMO–RS combines quantum chemical considerations (COSMO) and statistical thermodynamics (RS) to determine and predict thermodynamic properties without experimental data. The computational simulation results derived by COSMO–RS for rosemary compounds at room temperature is presented in Experimental method for assessing the solubility of BACs from rosemary was performed by green extraction method using HVED and green solvents water and ethanol (25 and 50%). For comparison of results, a modified CE by magnetic stirring was performed at same conditions as HVED: at room temperature, extraction times 3 and 9 min, ratio plant:solvent 1 g:50 mL. For all results in experimental procedure, “R” stands for rosemary, “N” for nitrogen, and “A” for argon. For HVED, numbers 1–12 show the order of conducted treatment. For CE treatments, 3 and 9 are referred to extraction time while 0, 25, and 50 stands for concentration of an ethanol solvent (%).Since the aim of the extraction with HVED was to achieve electrical discharge that is responsible for plant cell disruption and consequently the extraction of BACs from intracellular area. During the experiments, it was difficult to achieve discharge using nitrogen under 20 kV, therefore, for nitrogen treatments voltage of 20 and 25 kV were chosen, while lower voltages of 15 and 20 kV were obtained with argon treatments. All results were measured in duplicates and are presented as average ± standard deviation (SD).The experiment design performed in STATGRAPHICS Centurion software is presented in Results of physical parameters of CE and HVED treatment are given The difference between CE and HVED extraction according to results of TPC, antioxidant parameters and extraction yields are presented in The recorded NIR spectra in wavelength range from 904 to 1699 nm were used in the qualitative modelling using principal component analysis (PCA) to identify potential grouping  where th2) and the regression point displacement that is the ratio of the standard error of performance (RPD) and the ratio of the range of reference chemistry values to standard error of prediction (RER). RPD is the ratio of standard deviation of the validation data set (SDv) and the standard error of prediction (SEP). How efficient this quantitative prediction of TPC, FRAP, and DPPH is on the rest of 40 % of the samples is presented in Furthermore, modeling with different wavelength ranges was performed  using thThe colorimetric analyses of rosemary extracts were measured under International Commission on Illumination (CIE)—L*a*b* color system. Results are shown in Data matrix used to identify similarities and/or differences in the data set presenting the samples as well as the physical and chemical properties. The PCAs ability to reduce the dimensionality and increasing the interpretability with minimal information lost was used on the data matrix of experimental data. The matrix included as active variables the physical parameters, total phenolic content and antioxidant capacity, as well as the parameters of color, while the supplementary data set were the experimental conditions . The Extraction type (CE or HVED) were included in the analysis as qualitative variables . To presModeling that has followed included the steps of calibration, validation and prediction. In the modelling were included the NIR spectra what is in detail explained in the UPLC–MS/MS analysis was performed to quantify individual phenolic compounds  from rosemary extracts .Determination of main volatile compounds from rosemary extracts was performed by HS–SPME/GC–MS and analysis included eucalyptol, camphor, borneol and linalool. Results of performed analysis are presented in Results of pesticides and metals measured from dried rosemary leaves are shown in The aim of the present study was to provide the potential of green solvents to extract BACs from rosemary leaves comparing theoretical and experimental methods.n-hexane is one of the most used solvents due to its low polarity, optimal boiling point, easy removal from the product by evaporation and stability. On the other side, n-hexane is a solvent of petrochemical origin, which are nowadays strictly regulated by European Directives. For that reason, industries are forced to replace such solvents with more sustainable alternative solvents [n-hexane. According to RED results, by evaluating compounds with most green color (very good solubility), followed by yellow (medium solubility) and red color (poor solubility), the potential for extractions of BACs from rosemary was in the following order: CPME > limonene > cymene > MeTHF > ethyl oleate > β-myrcene > α-pinene > ethyl acetate > n-hexane > methylacetate > dimethyl carbonate > isopropanol > 1-butanol > methanol > ethanol > water. Results showed that α-pinene, cymene, β-myrcene, CPME, and ethyl oleate had high potential for extraction of volatile compounds such as monoterpenes and sesquiterpenes. For that reason, these solvents should be used for extraction of volatile compounds of essential oil from rosemary. Moreover, water and ethanol showed low potential for extraction of most evaluated compounds from rosemary.For extractions of BACs, solvents . Therefon-hexane > water. It is clear that all green solvents have higher potential for extraction of rosemary compounds compared to conventional n-hexane, except water. According to results, ethyl acetate, methylacetate, ethanol, 1-butanol, isopropanol, methanol, CPME, dimethylcarbonate and MeTHF, showed high probability of solubility for diterpenes, triterpenes and flavonoids, which was not showed with HSPs assessment. Results of COSMO–RS solubility assessment  showed sp ≤ 0.05) only on ethanol content. With higher content of ethanol in the solution, pH increased and conductivity decreased for both extraction types that is expected according to literature data [Results of pH, conductivity, power and temperature during extraction of rosemary BACs using HVED and CE are given in ure data . The power used for the treatment with HVED changed from 7.0 to 27.0 kW. The highest power was noted for sample RN10 (27.0 ± 3.0 kW) which is expected since it was the sample treated for longer time 9 min) and the highest voltage (25 kV). Accordingly, power significantly increased with higher voltage used for the treatment and decreased with higher ethanol content in the sample.p ≤ 0.05). Ethanol content showed different trend for each treatment, with CE, the highest scores were obtained with water, HVED treatment with nitrogen was highest with 50% of ethanol, while treatment with argon had highest yield of TPC with 25% of ethanol. Bellumori et al., (2016) performed extractions from dried rosemary leaves by ultrasound-assisted extraction and microwave-assisted extraction for 10 min. Their results also showed higher amounts of TPC with ethanol as a solvent, compared to water, but their maximum obtained results were slightly higher compared to HVED, 35.0 mg/g for ultrasound and 36.6 mg/g DL for microwave-assisted extraction with ethanol , but different method for calculation was used [The goal of the extraction with HVED was to extract highest yield of phenolic compounds and antioxidants and compare results with same conditions by modified CE (at room temperature) . HVED trwas used . Results of DPPH did not vary notably between extracts. However, the significant correlation was observed for DPPH with treatment time and ethanol content, it was higher with shorter treatment time and higher percentage of ethanol in the solution. Some differences in DPPH results could be due to generation of free radicals that is characteristic for HVED treatment that could influence to the antioxidant activity of extracts and have a possibility to interact with DPPH radical. FRAP results showed similarities with TPC values for HVED extraction, it was higher with longer treatment time, higher voltage used and with treatment using nitrogen, compared to argon. In average, HVED extraction showed 2.39-times higher antioxidant capacity, according to FRAP results, when compared with CE. In total, higher results for phenolic content and antioxidants was noted with ethanol than with water. These results are in line with theoretical predictions with HSPs and COSMO–RS, although pure ethanol was used for calculations, while in experimental procedure 25 and 50% ethanol was used. Additionally, other green solvents were used for experimental assessment for extraction of BACs using HVED including limonene, α-pinene, glycerol, ethyl acetate and dimethyl carbonate. However, no electrical discharge was achieved during the extraction with HVED when mentioned solvents were used. This could be explained by high viscosity and density of these solvents ,42. AlsoThe recorded NIR spectra showed grouping in the range of λ = 904–1699 nm which is the result of different vibrations of molecules as C–H, O–H, and N–H bonds. NIR spectra was specific in two regions: 904–925 nm and 1350–1699 nm. Overlapping of NIR scans is visible with specific differences in the range of 904–925 nm, indicating absorption detecting differences of the third overtone region and detecting different vibration of C–H bonds as well as HOH region at 1400 nm (water λ = 1400–1460 nm) and continues to the end of the recorded spectra indicating differences in the vibrations of the first overtone of C–H3, Ar–CH, C–H, and C–H2 bonds and second overtone of O–H, N–H, Ar–CH, and R–OH ,44.From those findings it was clear that the content of TPC and the antioxidative activity could be predicted. A part of the input spectral data was used for the model training (60%). After the training followed the PLSR model evaluation and its testing on unknown samples. Our aim was to predict those parameters not only qualitative but also quantitatively. In order to gain more accurate models, four different wavelength ranges were used . The first model  which inp ≤ 0.05) on ethanol content—with higher ethanol content, parameters L, ΔC, and ΔE increased, while a*, b*, C, and h decreased. Saturation significantly depended on treatment time and gas used, more saturation was noted with longer treatment time and use of nitrogen during the treatment. The color of the product is an important aspect that specifies the commercial quality of the product and has an effect to the consumer’s final purchase decision . The colIn general, results indicated that HVED extracts had dark greenish-brown color with darker color when compared to CE which is associated also with higher phenolic compounds and antioxidant content. Plasma reactive species induce release of some BACs that are covalently bonded to the plant matrix which accordingly results with increase in TPC and greater antioxidant capacity and consequently with changes in extract colors. The reason is that phenolic compounds result with different color in free and bound forms ,50.Based on the PCA biplot showed in The subjected table to the PCA chart-squared cosines of the variables asserts the HVED treatment time, stirring time, total treatment time and voltage used as experiment condition as values for which the squared cosine is the largest. Biplot in the form where the active variables , and color parameters) are related to the supplementary which are the experimental design parameters, show the correlations between them and for which samples are they dominant. So accordingly, in the fourth quadrant the almost overlap of parameters as the DPPH content and the ethanol content indicating that the antioxidant capacity by DPPH method was higher in those samples where the ethanol content was 25% or 50%  and 31.31% (RA8), respectively. Applying the same rule of data relation analysis in the third quadrant, samples RN12; RA11; and RA12 as well as 3 R25; 3 R50; 9 R25; and 9 R50 will have the highest values of the parameter L*, what confirmed the results presented in Data of UPLC–MS/MS analysis for individual phenolic compounds  showed tStatistical analysis showed that most of the measured compounds significantly depended only on ethanol content  and additionally, apigenin, carnosol, diosmetin, hydroxytyrosol, and luteolin depended on treatment time.HVED is considered to be energy- and cost- saving method for successful extraction of phenolic compounds from rosemary. However, further analysis of energy and environmental impact should be performed. Hirondart et al., (2020) have obtained rosmarinic acid, carnosic acid, and carnosol by PLE in hydroalcoholic solution and conventional Soxhlet extraction. Extract yields of bioactive compounds were similar with both methods, energy consumption was lower for PLE extraction because less solvent had to be heated, and the cost was reduced with a smaller amount of waste generated .When compared with theoretical results, it is clear that a similar trend for solution in water and ethanol was noticed. With higher ethanol content, solubility of most of extracted compounds increased, as well as a sum of all phenolic compounds. According to HSP results, all compounds that were analyzed with UPLC–MS/MS showed poor solubility , compared to camphor (RED = 4.49 for ethanol and 10.45 for water). From COSMO–RS results, it was also predicted that camphor and borneol have low chances for solubility in water, 0.1% and 0.02%, respectively, but different results were given for ethanol: camphor has medium probability of solubility (44.06%), while borneol has high probability (85.11%). Experimental results were opposite and higher concentrations of camphor were found in all extracts, compared to borneol. Moreover, results were closer to COSMO–RS results than HSPs since both compounds were extracted in higher concentrations: 37.83% of camphor was extracted in sample RA10 and 15.99% of borneol in sample RN10. Mena et al., (2016) showed similar results in conventionally performed acetone-based extraction from rosemary, they have extracted more camphor (41.52 ± 6.00 µg/g) than borneol (11.92 ± 2.01 µg/g) in their extracts . SFE is a procedure recently used for the extraction of bioactive compounds and purification of rosemary essential oil. Mouahid et al., (2017) have compared the efficiency of SFE and hydrodistillation of rosemary leaves. The essential oil obtained by SFE had an increase in yield of monoterpenoids for 37%, while yields of individual monoterpenes varied . PereiraAlthough rosemary could be considered as a nutritional supplement, there are still no categories in European Commission (EC) Regulation, herbs or plant tea, therefore the high levels of pesticides and heavy metals that can be found in its dried leaves could possess serious toxicological effects on human health. For that reason, the analysis of pesticides and heavy metals in the rosemary samples were measured and are presented in Residue levels of Lead (Pb) and Cadmium (Cd) in dried rosemary leaves were also below the limit of quantitation of the method, and only the level of Mercury (Hg) was slightly higher than limit of quantitation. However, all these values were quite below MRL according to EC No. 1881/2006. Furthermore, other metals (Chromium (Cr), Nickel (Ni), Manganese (Mn), Iron (Fe), Copper (Cu), and Zinc (Zn)) were measured in selected HVED extracts with high phenolic and antioxidant content  as well and results are presented in Rosemary plant was compliant regarding content of contaminants, pesticide residues and toxic metals. With respect to this, obtained results showed that rosemary samples are safe for use in human dietary. On the other hand, some changes in levels of metals could happen during the treatment and further detailed analyses should be done to assess this issue.The concept of this work is presented in Rosmarinus officinalis L.) were provided by local drugstore . Herbs were stored in polyethylene bags in a dark and dry place until extractions. Dried rosemary leaves were grinded to plant particle size distribution of d(0.1) ≤ 3 9.683 μm; d(0.5) ≤ 224.816 μm; d(0.9) ≤ 425.819 μm measured by the laser particle size analyzer Mastersizer 2000 . For the extraction, 1 g of herb material was weighted into the beaker of 100 mL and mixed with 50 mL of extracting solvent at room temperature (22 °C). Extraction was carried out using distilled water, 25% and 50% aqueous ethanol (v/v) as extraction solvents.Dried rosemary leaves  . For solA potentially good solvent has RED number smaller than 1 (the compound has similar properties and will dissolve), while medium and poor solvents have RED values of from one to three and more than 3, respectively. The chemical structures of the solvents and solutes discussed in this article could be mutually transformed by JChemPaint version 3.3  to their simplified molecular input line entry syntax (SMILES) notations, which were subsequently used to calculate the solubility parameters of the solvents and compounds .10(xsolub)). The logarithm of the best solubility was set to 0 and all other solvents were given relative to the best solvent. Also, the logarithm was transformed into probability of solubility (%). The calculation was performed at room temperature (20 °C) and at boiling temperature for each solvent.The COSMO–RS was developed by Klamt and co-workers as a statistical thermodynamic method for molecular description and solvent screening based on a quantum-chemical approach . COSMO–RHVED was performed with “IMP-SSPG-1200” generator  that generated rectangular pulses using direct current and achieving high voltage. Maximum adjustable current was 30 mA and voltage up to 25 kV. Based on conducted preliminary experiments with different HVED parameters , fixed HVED parameters were chosen as follows: frequency of 100 Hz, pulse width 0.4 microseconds, voltage 15 and 20 kV for argon gas, and 20 and 25 kV for nitrogen gas, the gap between electrodes of 15 mm, treatment duration 3 and 9 min, and ratio mass to solvent 1 g:50 mL (according to pharmacopoeia). Mixture of herb material and solvent was transferred to beaker shaped reactor of 100 mL. This reactor that is opened on both sides was fitted with silicone tops with 1 cm in diameter. Silicone tops were used due to easier mounting of the electrode from the top and needle form the bottom. Gases (argon or nitrogen) were flowed in through the needle with the flow 0.5–1 L/min. Set-up of generator and reactor are shown in For comparison, modified CE (untreated samples) was performed at room temperature as well, by dissolving the dried rosemary material in the solvent with light magnetic stirring during 3 or 9 min. Both extractions, HVED and CE, were performed in duplicates.2CO3 (m/v) was added. After thorough mixing by vortex, the reaction mixtures were incubated at 50 °C for 25 min, followed by absorbance reading at 765 nm against blank . The calibration curve was prepared using 50 to 500 mg/L of gallic acid in ethanol as a standard. The concentration of TPC was expressed in mg of gallic acid equivalents per gram of sample (mg GAE/g of sample).For determination of TPC of rosemary extracts, a Folin–Ciocalteu method was used  with sliThe antioxidant activity of rosemary extracts determined by DPPH method was determined as reported by Shortle et al., (2014)  with sli3 solution in ratio 10:1:1. An aliquot (80 μL) of rosemary extract was mixed with 240 μL of water and 2080 μL of FRAP reagent. Following incubation at 37 °C for 5 min, the absorbance was measured at 595 nm. FRAP values were calculated according to the calibration curve for FeSO4·7H2O and expressed as μmol of Fe2+ equivalents (FE) per g of sample (μmol FE/g of sample).The FRAP assay was conducted according to literature  with modNIR spectroscopy was conducted using the NIR-128-1.7-USB/6.25/50μm  to record sample spectra using the SPEC 32 Control Development software. NIR spectra was recorded in the wavelength range from 904 to 1699 nm. Each sample was recorded in triplicate and the average spectrum was calculated which was used for further processing.Color parameters for all trials was measured by Konica Minolta colorimeter CM 3500d  at CIE Standard Illuminant D65 by 8 mm thick plate. All measurements were conducted in the Specular Component Included (SCI) mode as previously reported . The col2O with 0.1% HCOOH (v/v) and B 100% acetonitrile with 0.1 % HCOOH (v/v) with mobile phase flow 0.40 mL/min. Gradient was set as follows: 1 min 10% B, 2 min 10% B, 15 min 90% B, 25 min 90% B, 27 min 10% B, 30 min 10% B. Determination conditions for MS/MS detector were: ionization -negative ionization mode atmospheric pressure (API)—negative ionization at atmospheric pressure; ionization temperature: 500 °C, i.e., gas temperature combining the mobile phase at the exit from the capillary before ionization. Voltage on the electrode after capillary and next to ionization (Ion Spray Voltage) was −4500 V.UPLC–MS/MS Eksigent Expert Ultra LC 110, SCIEX 4500 QTRAP  method for reference conditions  was condHS–SPME was performed with a manual SPME holder using three fiber covered with DVB/CAR/PDMS obtained from Supelco Co. . For HS–SPME, the finely samples 2 mL were placed separately in 10 mL glass vials and hermetically sealed. The vials were maintained at 60 °C during equilibration (15 min) and extraction (45 min). Thereafter, the SPME fiber was withdrawn and inserted into GC–MS injector (250 °C) for 6 min for thermal desorption. The procedure was similar as in previous paper . n-alkanes (C9–C25), with those reported in the literature [Gas chromatography and mass spectrometry (GC–MS) analyses were done on an Agilent Technologies  gas chromatograph model 7890A equipped with a mass spectrometer (MSD) model 5977E  and HP-5MS capillary column . The GC conditions were the same as reported previously . In brieterature  and thos3 (microwave digestion) with microwave reaction system Multiwave 3000 . Determination of metals were conducted on the Perkin Elmer AAS Analyst 800 and ICP-MS Perkin Elmer NexION 300× , while Hg traces were determined by the Leco AMA254 Hg analyzer .The contents of the pesticides were performed by modified procedures with following national regulations HRN EN ISO 12393-1, 12393-2, and 12393-3: 2013, i.e., extraction with petroleum ether/dichloromethane and determination using the GC-ECD Varian CP-3800 instrument . Metal trace content was determined according to the HRN EN ISO 14084: 2005 procedure, or by wet sample digestion by HNOThe experiment was designed in STATGRAPHICS Centurion  software. Multifactorial design consisting of 12 experimental trials using per gas (argon and nitrogen). The three chosen independent variables for HVED assisted extraction were: treatment time (3 and 9 min), voltage applied and gas type  and concentration of ethanol . For CE, the independent variables included: concentration of ethanol  and treatment time (3 and 9 min). The experimental design is presented in p-values present the statistical significance of each of the factor and it was significant at p ≤ 0.05. Statistics was performed using XLStat (MS Excel 2010) (data not shown).In order to provide information about experimental results, a descriptive statistic was used. Analysis of covariance (ANCOVA) was used for assessment of correlation and all parameters according to dependent variables . The 2 and the regression point displacement that is the ratio of the standard RPD and the ratio of the range of reference chemistry values to RER. RPD is the ratio of SDv and SEP, while RER range, min and max, and minimum and maximum values of the validation set. All data analyses were conducted in MS Excel and its additional statistical tool pack: XLStat.Information investigation of NIR spectra includes preprocessing and calibration modeling  where prIn this study, the potential of high voltage discharges for green solvent extraction of BACs and aromas from rosemary leaves was assessed by computational simulation and experimental method by means of HVED. The experimental results were compared with untreated samples (modified CE) and HVED was presented to yield 2.13-times higher TPC and 2.39-times higher antioxidant capacity. Nitrogen, longer treatment time, and higher voltage yielded higher results of phenolic compounds and antioxidants. Also, NIR spectra and modelling with analytical data were shown as an extremely useful tools that can help in assessing whether there is a “cost-effectiveness” of extracting phenols or antioxidants from specific samples, in a quick and easy way. The results presented that NIR spectroscopy combined with chemometrics approach gave accurate TPC, FRAP, and DPPH content prediction, showing that indicates the potential of the method in estimating the quantitative expected antioxidant potential as well as the content of total phenols. Generally, HVED extracts had a dark greenish-brown color with darker color when compared to CE which is associated also with higher phenolic compounds and antioxidant content. An UPLC–MS/MS showed that main phenolic compounds in rosemary were apigenin, diosmetin, and rosmarinic acid, while the predominant volatile compounds in rosemary extracts was eucalyptol. Altogether, results showed that HVED confirmed high potential for extraction of BACs and food aromas from rosemary with increased yield of individual compounds and total phenolic and antioxidant properties, compared to untreated samples. Furthermore, rosemary was presented as safe raw material for further processing in human nutrition in terms of pesticides and metals. The computational stimulation methods were confirmed by experimental study, ethanol had higher potential of solubility of BACs and aromas from rosemary compared to water. Therefore, these theoretical prediction methods present a new approach for assessment of solubility of individual compounds in selected solvents that could impact to lower solvent usage during experimentation and lower environmental impact.


In this study, we describe the manufacturing and characterization of silk fibroin membranes derived from the silkworm Bombyx mori. To date, the dissolution process used in this study has only been researched to a limited extent, although it entails various potential advantages, such as reduced expenses and the absence of toxic chemicals in comparison to other conventional techniques. Therefore, the aim of this study was to determine the influence of different fibroin concentrations on the process output and resulting membrane properties. Casted membranes were thus characterized with regard to their mechanical, structural and optical assets via tensile testing, SEM, light microscopy and spectrophotometry. Cytotoxicity was evaluated using BrdU, XTT, and LDH assays, followed by live–dead staining. The formic acid (FA) dissolution method was proven to be suitable for the manufacturing of transparent and mechanically stable membranes. The fibroin concentration affects both thickness and transparency of the membranes. The membranes did not exhibit any signs of cytotoxicity. When compared to other current scientific and technical benchmarks, the manufactured membranes displayed promising potential for various biomedical applications. Further research is nevertheless necessary to improve reproducible manufacturing, including a more uniform thickness, less impurity and physiological pH within the membranes. Mulberry silkworm  hold tremendous tensile strengths of up to 740 MPa and are tougher than Kevlar, which itself is predominantly recognized as a benchmark material with regard to high-performance fiber technology [The development of functional materials that also exhibit the capacity for interacting with biological systems poses one of the central challenges in biomedical engineering. Named materials may be derived from nature or may also be synthetically manufactured, such as synthetic polymers, metals or alloys . More rechnology ,6. Furthchnology ,9. The dchnology ,11. In bchnology ,17,18,19chnology ,23,24,252) and then dialyzed for several days before processing [A proper manufacturing process that enables suitable degumming , dissolution and regeneration of silk is a requirement for further biomedical applications in order to properly disintegrate silk fibroin into the desired structural components. Nowadays, different methods are available for degumming, such as enzymatic, soap-assisted or acid-assisted processing, among various others ,27. In tocessing ,29. As oocessing ,30,31. Aocessing ,29,32.2) dissolution. In recent years there have been several works that have established this novel dissolving process using FA/CaCl2 as an integral part of the regeneration process of silk fibroin [The aim of this study was to evaluate the production processes and the resulting properties of casted silk fibroin membranes fabricated via formic acid (FA) and calcium chloride . Apart from negligible contamination by single particulates amounting to a few micrometers, the overall surface exhibited a smooth and homogenous appearance. Isolated cloudy areas revealed a different surface structure. These areas seemed as if “wiped off” and presumably represent residues generated throughout SEM preparation by gold sputtering. Isolated cracks found specifically at the edges of the membrane were thus related to cutting the membrane shape from the casted blanks. Significant differences in morphological appearance in relation to the respective fibroin concentration were not observed. An overall SEM overview and respective close-up images of SF10 and SF12 membranes are exemplarily displayed in Membranes exhibited different thicknesses within the cross-section. The Membranes initially experienced a constant thinning out from both sides under increasing load, with them finally rupturing in exactly these areas. The values displayed in All membranes exhibited fairly small extinction rates between 0 and 0.2 in the visible and infrared range. In the UV range, increasing extinction showed two maxima (first between 230 nm and 240 nm with extinction of 3.5 to 3.8 and second between 265 nm and 275 nm with extinction of 3.45 to 3.95). A higher fibroin concentration was accompanied by higher extinction rates at all wavelengths. 2 could be considered as non-toxic and therefore suitable. Student’s t-test for independent samples was used for all tests in order to detect significant differences between the assessed groups. A p value of p < 0.05 was chosen as the confidence interval threshold where differences were considered to be significant. With reference to the XTT and BrdU tests, the fibroin membranes were compared in a pairwise manner either to the toxic positive control or the non-toxic negative control, respectively. In terms of the LDH assay, the procedure was reversed, with the positive control being used as the presentative basis. Essentially, all cell culture tests provided statistically significant evidence for absent cytotoxicity  of fibroin membranes fabricated via the formic acid and calcium chloride method and both reference materials were examined by fluorescent reflective light microscopy. Representative images were then selected for presentation, as seen in 2, without occurrence of any cytotoxic effects.To confirm the results of the indirect tests, the viability and adhesion of fibroblast cells was examined via direct cultivation on the silk membranes along with their respective controls and subsequent staining of the cultivated cells. Three samples 2+MeHO or LiSCN, among others, the FA/CaCl2 solvent rather showed decreased alterations in molecular weight [In the present study, an alternative approach employing formic acid and calcium chloride as solvents for the production of B. mori derived silk fibroin membranes was used. As previously reported, silk may be efficiently and simply dissolved with a process containing formic acid and CaClsolution ,39,40. Csolution ,42. Tradnofibers ,43. Trannofibers . The disg et al. ,37. Whilng chain ,35,36,37r weight .2 as solvent and simultaneously assessing the most elemental requirements for medical devices, such as biocompatibility, by conducting in vitro assays based on the DIN EN ISO 10993 standard. Thus, we were able to establish an applicable and fast-forward protocol to manufacture silk fibroin devices based on dissolution of silk in FA/CaCl2. The suggested variation of processing sequences for silk fibroin membranes may hold several advantages in comparison to conventional methods, the most pronounced being cost effectiveness and reduced time consumption with regard to the translation towards an industrial manufacturing process. The ability to disintegrate into nanofibrils by applying solvents is another significant advantage of fibroin, thus enabling the formation of hierarchical fibril structures during further processing. Fibroin membranes produced in this study were characterized with regard to their surface, appearance and mechanical properties, as well as their respective transparency.In our study, we mainly focus on an applied research approach, evaluating both, a cost-effective method to manufacture silk fibroin membranes facilitating FA/CaClMechanical properties are an essential characteristic of biomaterials and are fundamental with regard to their reactions and interactions with vital tissue. Results of mechanical tensile testing verified, that an increase in fibroin concentration is accompanied by an increase in tensile strength. Although no definitive statement can be made in terms of significance due to the incremental standard deviations, an increase in fibroin concentration does seem to generate ameliorated mechanical properties (tensile strength) of fibroin membranes. The mechanical properties of the membranes manufactured in this study lie slightly below those described in other studies ,39,40. HIn addition to mechanical properties, optical properties are also of crucial importance for the function of fibroin membranes in biomedical engineering, especially when applied in corneal replacement. Depending on the intended use, here, transparency is either of primary or at least secondary importance. If envisioned as a type of cornea replacement, it is undoubtedly crucial that a non-functionalized membrane is permeable to visible light . However2 solution may also be regarded as advantageous, because the resulting fibers exhibit fibrillary structures of high hierarchical order, higher strength and increased extensibility, especially when compared to conventional manufacturing methods of native silk [The superior physical properties possessed by silk as a biomaterial build upon complex hierarchical structures, which themselves are formed by nanoscale building blocks, namely beta-sheet nanocrystals and nanofibrils . Many ofive silk .To ensure thorough biocompatibility, the physiological pH value (approximately pH 7.4) of surrounding tissues must not be influenced, as cell viability may consequently be impaired ,50. The 2, cytotoxic effects were not detected in any of the test series. Especially LDH, a useful marker in cytotoxicity assessment of materials, was not detectable after incubation with fibroin membrane samples [2 did not have any negative impact on cell viability, cell proliferation and metabolic activity.To further assess biocompatibility, additional in vitro experiments were carried out. For fibroin membranes generated via formic acid and CaCl samples ,52. AlsoIn summary, our test data addressing mechanical and optical properties as well as biocompatibility suggests membranes facilitating 10% casting concentration (SF10) as best case. Since we consider ophthalmological indications, among others, as potential translative applications, we were aiming on one hand on ensuring a high degree of transmission, while on the other hand optimizing mechanical properties, e.g., to prevent membrane damage during insertion or manipulation. In the present study, the SF10 variant represented the sweet spot of the before mentioned characteristics: above 10% fibroin casting concentration, the mechanical properties changed only marginally, but the transmission became noticeably worse.2CO3 solution at 100 °C for 60 min and then washed thoroughly with deionized water for 10 min. Degummed silk fibroin was subsequently dried for 24 h and passed on for dissolution. The solvent was composed of 98% formic acid  supplemented with 4% CaCl2 , which showed preferential (full) dissolution of fibroin in previous experiments [Raw silk was first degummed with 0.02 M Naeriments .The formic acid was added to different amounts of degummed fibroin in order to achieve different concentration levels of fibroin for the respective membranes . Three images were taken for each fibroin concentration, respectively: an overview image with a 30× magnification, a close-up image of the membrane surface with a 2000× magnification and a close-up cross-sectional image. Images were saved and then analyzed with ImageAccess Standard program .The structure of the membranes was analyzed via scanning electron microscopy (SEM) . After undergoing drying for 18 h, two separated pieces were fixed to a holder either in horizontal or upright orientation and subsequently sputtered (Au) in argon medium for 90 s at a vacuum of 3 × 10Membrane thickness was analyzed via light microscopy  with a 100× magnification. Until subjected to the respective measurements, membranes were maintained in a wetted state placed in deionized water supplemented with antibiotics 1% Antibiotic  with 10.00 units/mL Penicillin and 10.00 μg/mL Streptomycin). Membranes were positioned on a vertically oriented microscope slide in order to enable the examination of the membrane cross-section. Respective images were taken at four spots previously marked on the slide, which themselves were equally distributed among the diagonal of the membrane. Images were saved and then analyzed with ImageAccess Standard program .Four membranes per fibroin concentration were analyzed, whereas samples that had ruptured before testing or those that had slipped out of the holding clamps were excluded from further evaluation. Mechanical testing was performed using a zwickiLine materials testing machine Z2.5 . Strips measuring 20 mm in width were cut from the middle area of the wetted membranes, consecutively dabbed off and then fixed in a Vulkollan-coated type 8033 sample holder  with a maximum force of Fmax 200 N. Samples were then pulled at 10 mm/min at a holding length of 50 mm without a respective preload. Force (N) was measured as a function of elongation.® QS cuvette 200–2500 nm . Extinction (E) was measured against a reference cuvette in visible and UV spectral range at wavelengths between 200 nm and 900 nm. Transmittance was then calculated. The transmission degree (T) describes the quotient of the outgoing (I) and incoming light intensity (I0).Five samples per fibroin concentration were examined. Transparency was analyzed by photo spectroscopy . Rectangular strips measuring 25 mm × 8 mm were cut from the middle area of the wetted membranes and subsequently transferred into a quartz glass SuprasilIn spectroscopy measurements, the extinction at a specific wavelength is determined as the negative decadic logarithm of the transmission which can be calculated using following formula.2. Ninety-six-well plates were seeded with 1 × 104 L929 cells per well in 100 µL cell culture medium and then incubated for 24 h under cell culture conditions. The cell culture medium was subsequently discarded and 100 µL of membrane extract was applied to each well. The cells were incubated for another 24 h and consecutively exposed to the BrdU and XTT tests, whereas the respective supernatants were subjected to lactate dehydrogenase (LDH) testing. Similar assays were performed for all extracts, except for the control prepared in order to detect any possible assay interferences, which was formulated without cells. Blank controls  were subtracted from the absorbance values in order to adjust the readout at all times.Novel biomaterials must pass certain toxicity tests based on part 5 of the standard ISO 10993 , Geneva, Switzerland) in order to ensure biocompatibility and safety. Thus, corresponding indirect in vitro assays were performed to check for compatibility of extractive compounds gained from fibroin membranes tinged with formic acid and CaClThe BrdU (colorimetric) test kid  was executed as described in the manufacturer’s instructions. Briefly, cells were labeled with BrdU for 2 h under cell culture conditions and subsequently fixed for 30 min at room temperature with FixDenat reagent.2SO4 for the purpose of reaction termination via scanning multi-well spectrophotometer (ELISA reader) equipped with filters for 450 nm and 690 nm (reference wavelength).Then, the fixed cells were incubated for 1 h with anti-BrdU-peroxidase (POD) antibody and washed 3 times for 5 min in washing buffer. Immune complexes were detected after subsequent substrate reaction with tetramethyl-benzidine (TMB) (20 min at room temperature) followed by the addition of 25 μL 1 M HThe Cell Proliferation Kit II for XTT-assay  was executed as described in the manufacturer´s instructions. Briefly, the electron-coupling reagent was mixed with XTT labeling reagent (1:50 dilution) and 50 μL of the corresponding mixture was then added to the cells. After 4 h of incubation under cell culture conditions, substrate conversion was quantified by measuring the absorbance of 100 μL aliquots in a fresh 96-well plate via scanning multi-well spectrophotometer (ELISA reader) equipped with filters for 450 nm and 650 nm (reference wavelength).The LDH-Cytotoxicity Assay Kit II  was executed as described in the manufacturer´s instructions. Briefly, 10 μL of the respective cell supernatants were incubated with 100 μL LDH reaction mix for 30 min at room temperature. After the addition of stopping solution, absorbance was measured via scanning multi-well spectrophotometer (ELISA reader) equipped with filters for 450 nm and 650 nm (reference wavelength). Paired t-tests were performed to analyze differences between each group for all cytotoxicity tests.For direct cell culture testing, 12 well plates were seeded with 2.4 × 105 cells in 1 mL cell culture medium. Cells were seeded directly onto the surface of the test specimens and incubated under cell culture conditions for 24 h before performing direct assay procedures. To perform live–dead cell staining on the surfaces of the specimens, 60 µL propidium iodide (PI) stock solution, consisting of Phosphate-Buffered Saline (PBS) supplemented with 50 µg/mL PI were added to each well (12 well plate). In addition, 500 µL fresh fluorescein diacetate (FDA) working solution (20 µg/mL in PBS from 5 mg/mL FDA in acetone stock solution) were also added for each well. The wells were subsequently incubated for 3 min at room temperature (RT), followed by rinsing the specimens in preheated PBS and immediately examining them with a fluorescence microscope equipped with a filter for parallel detection of red and green fluorescence.2 solution as an initial solvent followed by a cascaded rinsing series in water and buffer solutions. To avoid the conventional, expensive dialysis process, an alternative washing and neutralization process was investigated. Manufactured membranes displayed favorable physical and mechanical properties, but require improvement concerning pH optimization and prolonged production time. The membranes produced in this study exhibit a compact structure, displaying a proportional correlation of most properties along with increasing fibroin concentration, as, e.g., membrane thickness and transparency. Accordingly, the performance of a fibroin membrane may be predictably controlled by means of fibroin concentration variation. Based on our findings, a concentration level of 10% (SF10) showed the best combination of characteristics, as a further increase of fibroin concentration could not improve the mechanical properties of the membranes, but was detrimental in terms of transmission, which is of utmost importance when for example aiming for ophthalmologic applications. This facilitated and novel process for the manufacturing of fibroin membranes may not only be advantageous in terms of simplicity, cost-effectiveness and efficiency in comparison to conventional methods, but it may also be categorized as less disruptive due to the conservation of the nanofibril structures. Fibroin membranes prepared via formic acid and calcium chloride dissolution displayed thorough biocompatibility in all cell culture tests performed within this study. Nevertheless, further research must be pursued in order to establish silk fibroin as a recognized engineering biomaterial not only in medicine, but to ensure its suitability for a broad variety of other applications.In this study, we introduced a simple and inexpensive method for the production of silk fibroin membranes by using FA/CaCl


Prospective study included 40 cases with acute aSAH. Initial evaluation by Glasgow Coma Scale (GCS) and the severity of aSAH was detected by both the clinical Hunt and Hess and radiological Fisher's grading scales. TCD was done for all patients five times within 10 days measuring the mean flow velocities (MFVs) of cerebral arteries. At the 3-month follow-up, patients were classified into two groups according to Montreal Cognitive Assessment (MoCA) scale: the first group was 31 cases (77.5%) with intact cognitive functions and the other group was 9 cases (22.5%) with impaired cognition. p = 0.03), significantly higher mean Hunt and Hess scale grades (p = 0.04), significantly higher mean diabetes mellitus (DM) (p = 0.03), significantly higher mean systolic blood pressure (SBP) and diastolic blood pressure (DBP) , and significantly higher MFVs measured within the first 10 days. The patients with cognitive impairment were accompanied by a higher incidence of hydrocephalus (p = 0.01) and a higher incidence of delayed cerebral ischemia (DCI) (p < 0.001). Logistic regression analysis detected that MFV ≥ 86  cm/s in the middle cerebral artery (MCA), MFV ≥ 68  cm/s in the anterior cerebral artery (ACA), and MFV ≥ 45  cm/s in the posterior cerebral artery (PCA) were significantly associated with increased risk of cognitive impairment.  Patients with impaired cognitive functions showed significantly lower mean GCS (NCT04329208. Cognitive impairment after the 3-month follow-up phase in aSAH patients was 22.5%. Acute hydrocephalus and DCI are highly associated with poor cognitive function in aSAH. Increased MFV is a strong predictor for poor cognitive function in aSAH. This trial is registered with  Aneurysmal subarachnoid hemorrhage is a form of cerebral hemorrhage that leads to severe injury to the brain with a significant increase in morbidity and mortality –3. It isHütter and colleagues in 1998 determined impaired short and long-term memory, attention, and frontocortical functions in the acute stage following aSAH (average 6 days) .Nordenmark et al. (2019) mention that most studies have depended on the medical variables to predict the cognitive impairment in the chronic stages following aSAH, usually 3 months or 1 year following the onset .The study by Hütter stays the only study of aSAH cases in the acute stage that has associated the medical variables to cognitive impairment and they concluded that the amount of blood (Fisher score), frontal hematoma, intraventricular hemorrhage, and acute hydrocephalus were associated with cognitive impairment in the acute stage .Several researchers found that impaired brain perfusion and cerebral blood flow (CBF) can occur before the onset of the cognitive impairment , 14, sugEarly changes in the cerebral blood vessels wall can be dependably distinguished by ultrasound methods, which permit identifying even small changes . UltrasoThe present study is aimed at predicting the cognitive impairment by TCD and detecting the associated clinical biomarkers of impaired cognition in the aSAH.A single-center, prospective study was conducted on 102 consecutive patients with acute nontraumatic SAH within 24 hours of the onset diagnosed by clinical examination and the initial brain computed tomography (CT). Patients were admitted to the Neuropsychiatry Department, Mataria Teaching Hospital, over a 1-year period initiated on the 1st of January 2018.Adult patients (aged 30-65 years) of both sexes presented by spontaneous aneurysmal SAH, diagnosed by noncontrast brain CT scan at the onset, and confirmed by CT angiography within 1-3 days of onset were included.Patients were excluded from this study in the following cases: marked impaired consciousness, marked mental disorders or previous dementia, marked systemic diseases, marked hepatic or renal impairment, aphasia, or noncooperative patients who could not perform the MoCA. Also, patients with previous stroke, other neuropsychiatric disorders, or drug therapy that impairs cognition were excluded. During the follow-up duration after 3 months, 10 cases died and 5 cases missed . Finallyst-degree relatives. This study was approved by the local Institutional Review Board of Neurology Department, Faculty of Medicine, Mansoura University, Egypt.Written informed consent was provided by all cases or 1A complete history was taken including risk factors that may cause aSAH or may impact the cognition such as age, sex, and history of hypertension, DM, hyperlipidemia, and smoking. General examination was carried out with special consideration of systolic and diastolic blood pressures. Also, a complete neurological examination was carried out for all cases included initial GCS score and Hunt and Hess scale grades.SAH was diagnosed at the onset of admission by noncontrast brain CT scan, and the severity of aSAH was detected by using Fisher's grading scales. Within 1-3 days, the patients were sent for CT angiography and digital subtraction angiography of cerebral arteries to demonstrate the location, number, and size of the ruptured aneurysm . Follow-Initial laboratory investigations were carried out to all cases after admission including full complete blood picture, random blood glucose, prothrombin time and activity, hepatic function tests, urea and serum creatinine, total lipid profile, and serum electrolytes.Clinical evaluation was carried out throughout the day in the intensive care unit (ICU) and the assessment of the cases by GCS score and Hunt and Hess scale grades. Also, continuous observations of any decrease in the level of consciousness or any focal neurological signs were detected.TCD examination was performed using DWL-EZ-Dop machine Compumedics GmbH, Singen, Germany. TCD uses low-frequency pulses (2 MHz) for the measurement of MFV in the cerebral arteries determining systolic and diastolic peaks and MFV. MFV is calculated by (systolic + diastolic)/3 + diastolic  velocities, according to Alexandrov et al. .st, 3rd, 5th, 7th, and 10th days of the onset of SAH. TCD examination protocol for intracranial arteries was performed according to Alexandrov et al. [Initial TCD examination was done after admission serving as a baseline state for cerebral circulation. Follow-up TCD examinations were done at fixed intervals on the 1v et al. .Cerebral vasospasm (CV) is defined as a delayed but reversible stenosis of the cerebral blood vessels mostly involving the proximal arteries of the circle of Willis. Clinical vasospasm is narrowing of cerebral artery causing cerebral ischemia, while angiographic vasospasm is stenosis of arteries as demonstrated on vascular imaging , 24. TCDCognitive function was assessed by using MoCA score after 3 months of the onset of SAH during the follow-up of patients. Impaired cognitive functions were diagnosed if MoCA scores were less than 26 , 33. Accχ2 and Fisher exact tests in categorical variables and the Student t-test for comparison of means in continuous variables.Analyses of data were done by utilizing SPSS version 22. Data are summarized as the mean ± SD and range for continuous variables, and as frequency for categorical variables. Statistical analysis was performed using the p value < 0.05 was considered statistically significant.Spearman correlation analysis was carried out between MFV of cerebral arteries and MoCA. Odds ratio (OR) was estimated, and the 95% confidence interval (CI) was calculated, and to detect the cut-off values for MFV, a receiver operating characteristic (ROC) curve was done. Lastly, a logistic regression analysis was carried out to estimate the independent predictive ability of the detected cut-off values.40 patients with spontaneous SAH were involved in this study with an average age of 51.1 ± 13 years. MoCA scale score after 3 months from the onset of SAH was estimated, and subsequently, cases were classified into two groups: the first included 31 patients with good cognitive functions (77.5%) and the second included 9 patients with poor cognitive functions (22.5%).p < 0.001). Also, there was a significant impairment in the subtests of MoCA especially the domains of memory, executive functions, naming, and attention, while the domains of the visual-spatial ability, language, and orientation were nonsignificantly diminished after SAH with impaired cognition.The total MoCA scores of the cases of SAH with cognitive impairment were significantly less than cases with intact cognition   ( < 0.05) .p < 0.01), with no significant difference regarding the sex between the groups (p = 0.42).The poor cognitive function group was older compared with the good cognitive function group , significantly higher mean SBP , and significantly higher mean DBP , while cholesterol, triglyceride, and smoking were not significantly different between both groups .Cases with impaired cognition in comparison with cases with intact cognitive functions showed significantly higher mean blood glucose level , significantly higher mean modified Fisher scale grades (p = 0.03), and significantly higher mean Hunt and Hess scale grades (p = 0.01).Cases with impaired cognition in comparison with cases with intact cognitive functions showed significantly lower mean GCS .Our results showed that the total cases complicated by hydrocephalus in the acute phase were 7 patients (17.5%) and the cases with impairment of the cognitive functions were accompanied by a higher incidence of hydrocephalus compared with patients with intact cognitive functions .Our study showed that the total cases complicated by DCI in the acute phase were 12 patients (30%) and the cases with impairment of the cognitive functions were accompanied by a higher incidence of DCI compared with patients with intact cognitive functions  was demonstrated in the following cerebral arteries: MCA LT 3, MCA LT 5, MCA LT 7, ACA LT 5, PCA LT 3, PCA LT 5, PCA LT 7, and MCA Rt 5, MCA Rt 10, ACA RT 5, and PCA RT 3 .p < 0.05 for all vessels  were significantly higher than MFVs estimated in cases with intact cognitive functions  and  vessels .p < 0.01), while MFV ≥ 68  cm/s in the ACA is significantly accompanied by a two-fold increased chance of impaired cognition , and MFV ≥ 45  cm/s in the PCA is significantly accompanied by a twofold increased chance of impaired cognition   .In our study, 22.5% of patients had impaired cognitive functions after aSAH in the follow-up after 3 months, and 77.5% of aSAH patients had intact cognition with only mild cognitive dysfunction in memory. Hydrocephalus, CV, DCI, and increased MFV were highly associated with poor cognitive functions.Aneurysmal subarachnoid hemorrhage is a critical cause of short-term or long-term impaired cognition of variable degrees, with a frequency ranged from 7% up to 60% . Researcp < 0.001). Typically similar to past studies on aSAH in the chronic stages, which has demonstrated that memory is the most common and the most frequent cognitive dysfunction [In our study, the patients with impaired cognitive function had a significant reduction of the total cognitive impairment estimated by the total MoCA score compared to patients with intact cognition  showed that the patients with GCS score of ≤7 at the onset, Hunt and Hess grade of ≥3, and Fisher grade of ≥3 were more likely to develop impaired cognition .Patients with impaired cognitive functions were accompanied by a more incidence of CV compared with patients with intact cognitive functions. CV is a serious complication of aSAH, possibly leading to delayed ischemia and focal neurological deficits and associated with cognitive dysfunction –49, and Acute hydrocephalus and recent cerebral ischemia associated with increased intracranial pressure after the bleeding, causing microvascular damage and impairment of the autoregulation leading to impaired cognition in the acute stage of aSAH, were demonstrated in the study of Kreiter and colleagues . This lip < 0.001). Also, Chu and his colleagues found that cases with DCI have more frequent impairment of the cognition. The most common affected domains of cognitive functions in aSAH cases with DCI were memory, language, and skill domains. These affected domains were rare in aSAH cases in the absence of DCI [Our study showed that the patients with impaired cognitive functions were accompanied by a higher incidence of DCI in comparison with cases with intact cognitive functions (77.8% versus 16.1%, e of DCI . Similare of DCI . This ree of DCI .Our study detected a significant correlation linking MFV of cerebral arteries and the cognitive impairment, and the Spearman correlation showed an inverse linear relationship linking the total MoCA and the MFV of cerebral arteries. MFV ≥ 86  cm/s in the MCA is significantly accompanied by a fourfold elevated chance of impaired cognition, MFV ≥ 68  cm/s in the ACA is significantly accompanied by a twofold elevated chance of impaired cognition, and MFV ≥ 45  cm/s in the PCA is significantly accompanied by a twofold elevated chance of impaired cognition.TCD assessment gives a clear physical demonstration of the impairment of brain perfusion and provides a suitable, noninvasive tool to evaluate the effectiveness of the medical treatments on cerebral blood flow or uncover early impairment of the cognition  and asseTCD is able to evaluate the cerebral hemodynamics, the arterial perfusion, and the intracranial small vessel compliance –63 and gThis work represents a small-sized sample of SAH. The intermittent recording TCD may miss episodes of cerebral vasospasm. The techniques of TCD are highly operator dependent and significantly limit its clinical utility , 69. So,22.5% of patients had cognitive impairment after aSAH in the follow-up after 3 months. Acute hydrocephalus and delayed cerebral ischemia are the powerful predictors of impaired cognition after aSAH. TCD detected an inverse linear correlation between the average MFV and the total MoCA. MFV ≥ 86  cm/s in the MCA is significantly accompanied by a fourfold increased risk of impaired cognition, MFV ≥ 68  cm/s in the ACA is significantly accompanied by a twofold increased risk of impaired cognition, and MFV ≥ 45  cm/s in the PCA is significantly accompanied by a twofold increased risk of impaired cognition.