Attention-Deficit Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorder characterized by inattention, impulsivity, and hyperactivity. The neurobiological mechanisms underlying ADHD are still poorly understood, and its diagnosis remains difficult due to its heterogeneity. Metabolomics is a recent strategy for the holistic exploration of metabolism and is well suited for investigating the pathophysiology of diseases and finding molecular biomarkers. A few clinical metabolomic studies have been performed on peripheral samples from ADHD patients but are limited by their access to the brain. Here, we investigated the brain, blood, and urine metabolomes of SHR/NCrl vs WKY/NHsd rats to better understand the neurobiology and to find potential peripheral biomarkers underlying the ADHD-like phenotype of this animal model. We showed that SHR/NCrl rats can be differentiated from controls based on their brain, blood, and urine metabolomes. In the brain, SHR/NCrl rats displayed modifications in metabolic pathways related to energy metabolism and oxidative stress further supporting their importance in the pathophysiology of ADHD bringing news arguments in favor of the Neuroenergetic theory of ADHD. Besides, the peripheral metabolome of SHR/NCrl rats also shared more than half of these differences further supporting the importance of looking at multiple matrices to characterize a pathophysiological condition of an individual. This also stresses out the importance of investigating the peripheral energy and oxidative stress metabolic pathways in the search of biomarkers of ADHD. ADHD symptoms include inattention and hyperactivity/impulsivity, with three symptom presentations described by the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5): predominantly inattentive, predominantly hyperactive/impulsive, and combined profiles, which may vary over time. The diagnosis mainly relies on detailed clinical evaluations of the symptoms and careful assessment of the attention and executive functions4. However, the high heterogeneities of the phenotypic presentations and temporal courses of the disorder, along with the numerous comorbidities, render this diagnosis difficult to establish and often delayed5. Besides, false positives and false negatives are not unusual. Thus, there is a crucial need for biomarkers to facilitate an early and reproducible ADHD diagnosis in order to promote appropriate care and follow-up of the patients. Toward this aim, numerous genetic and neuroimaging studies were conducted in ADHD patients over the past two decades but failed to provide relevant tools for reliable diagnosis on a routine basis4.Attention-deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental disorder affecting 5.3% of school-aged children6. Only a few hypothesis-driven and one nonhypothesis-driven studies using various sample types have been performed in ADHD patients. Therefore, additional studies are needed to identify useful peripheral biomarkers8. Metabolomics aims at providing qualitative and quantitative changes of hundreds of metabolites9 and has been increasingly used to identify molecular fingerprints in neurodevelopmental disorders. In this context, metabolomic studies in animal models are of a great interest as it allows to access peripheral but also central compartments to better evaluate how potential peripheral metabolomic changes can reflect central modifications.To date, metabolomic strategies remain largely unexplored in the search for ADHD biomarkers15. Our specific objectives were (i) to characterize the modifications in the brain, blood, and urine metabolomes in this animal model of ADHD, and (ii) to identify peripheral modifications in metabolites/metabolic pathways related to the reported cerebral changes.The goal of the present study was to explore the cerebral and peripheral metabolomes of SHR/NCrl vs WKY/NHsd rats, which is one of the most extensively studied and characterized animal model of ADHD with good face and construct validities11. Animals were housed in groups of 2 or 3 under humidity—and temperature-controlled conditions and a 12:12 light-dark cycle (light on at 7:00 AM) with ad libitum access to food and water. All animal experiments were conducted in accordance with EU guidelines , and approved by a local Ethics Committee .The subjects were eight male SHR/NCrl rats , which were compared to eight male WKY/NHsd control rats ; all rats were 8 weeks old, i.e., before the onset of hypertension in the SHR/NCrl rats, as described by Sagvolden et al.g and 4 °C for 10 min and the plasma retrieved. Samples were stored at −80 °C. The brains were quickly removed and prepared as described below.Urine samples were collected before sacrifice by handling the animals. Immediately after decapitation, blood samples were collected in vials filled with 1.8 mg of EDTA , centrifuged at 11,500 × 2O (1:1), vortexed for 15 s, homogenized by gentle planar shaking at 4 °C for 30 min and centrifuged at 11,500 × g and 4 °C for 15 min . Supernatants (500 µL) were concentrated under vacuum  at 45 °C for 4 h. The resultant dry residues were reconstituted in 100 µL of MeOH/H2O (1:9).Brain samples were freeze-dried  at −107 °C and 0.2 mbar for 12 h. The lyophilized material (3 mg) was mixed with 750 µL of ACN/HUrine samples were diluted to the tenth with ultrapure water and vortexed for 15 s.2O (1:1). The samples were then vortexed for 15 s, homogenized by gentle planar shaking at 4 °C for 30 min, and centrifugated at 11,500 × g and 4 °C for 10 min to collect 500 µL of supernatant. Supernatants were concentrated under vacuum  at 45 °C for 4 h. The dried residues were reconstituted in 100 µL of MeOH/H2O (1:9). Each prepared sample was then transferred to a 96-well plate for liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis. Quality control samples (QCs) were obtained from a pooled mixture of equal volumes of all samples for each matrix. Fifteen QCs were injected to equilibrate the chromatographic system before each analytic batch and QCs were analyzed every 10 samples.Metabolites were extracted from 100 µL of plasma by adding 800 µL of MeOH/H16. For both modes, heated ESI source parameters consisted in a spray voltage of 3 kV, a capillary temperature of 380 °C, a heater temperature of 350 °C, a sheath gas flow of 40 arbitrary units (AU), an auxiliary gas flow of 20 AU, a spare gas flow of 2 AU, and a tube lens voltage of 50 V. The full-scan acquisition ranged from 58 to 870 m/z and the instrument operated at 70,000 resolution (m/z = 200), with an automatic gain control target of 1 × 106 charges and a maximum injection time of 250 ms. The identity of each sample was processed by experimenters blinded its experimental group.LC-HRMS analysis was performed on a UPLC Ultimate 3000 system  coupled to a Q-Exactive mass spectrometer  and operated in positive and negative electro spray ionization (ESI+ and ESI−). Chromatography was performed as previously described17. Each peak area was normalized to the total of peak areas of interest. Metabolites detected with greater than 30% variance (CV%) in QC samples were not selected for further analysis.A library of 495 standard metabolites  was used for the construction of an in-house metabolite database. Targeted molecules from our database were selected and integrated into Xcalibur 2.2  as previously describedwww.kegg.jp). A Venn diagram was performed to assess the complementarity between the rat’s brain, plasma, and urines samples using the jvenn online tool (http://jvenn.toulouse.inra.fr/app/index.html).The metabolic card was performed using the web-based tool iPath 3.0 to represent all the metabolites detected in the brain on the metabolic pathways based on the latest version of the Kyoto Encyclopedia of Genes and Genomes  and partial least squares discriminant analyses (PLS-DA). PCA were performed on brain, plasma, and urine samples to identify outliers (one rat from each group of the brain samples was excluded), and to perform unsupervised clustering of SHR/NCrl and WKY/NHsd rats. PLS-DA were performed on brain, plasma, and urine samples to build discriminant models between the two strains. Each model was characterized by the cumulative modeled variation in the Y matrix R2Y, and the cross-validated predictive ability Q2 values, providing the discriminant metabolites called variable importance in projection (VIP). Metaboanalyst 4.0 was also used for univariate analyses using volcano plots on brain, plasma, and urine samples to select the metabolites whose levels were statistically significantly different between SHR/NCrl and WKY/NHsd rats , and whose concentrations differed between SHR/NCrl rats and controls by less than 0.75-fold or more than 1.25-fold. Pathway analyses using Metaboanalyst 4.0 were conducted on the VIP metabolites. The pathways were considered when displaying FDR values <0.05 and a non-null impact value. Correlation analyses were performed for metabolites that were significantly altered in the brain and plasma and/or urines samples using Metaboanalyst 4.0 and were considered significant when p < 0.05. Linear regression curves were generated using GraphPad Prism 7 .Multivariate analyses were performed using Metaboanalyst 4.0Starting from a library of 495 compounds, the number of metabolites detected in both rat strains ranged from 125 to 182, representing 25–37% of the whole library Fig. . The num2Y value of 0.988, and a predictive ability (Q2) of 0.804 for the two first components    Fig. . In addi26) Fig. .This study provides the first metabolomic characterization of the ADHD model based on the comparison of SHR/NCrl rats vs WKY/NHsd animals used as controls. Here we extensively studied the differences existing between SHR/NCrl rats vs controls in the brain and in the peripheral matrices. We showed that the brain, blood, and urine metabolomes can be used to discriminate SHR/NCrl rats from controls. The main cerebral differences between the two strains mainly consist in modifications in amino acid, energy, and vitamin metabolisms, that were also observed in blood and urines. In addition, malonate and glucuronate concentrations in the brain and urine compartments of SHR/NCrl rats were significantly correlated.23. Sagvolden and collaborators reported genetic and behavioral differences in existing among the strains of SHR and WKY rats, and showed the importance of using specifically SHR rats from Charles River Germany (SHR/NCrl) compared to WKY rats from Envigo U.K. (WKY/NHsd) to model the ADHD-related inattention, impulsivity, and hyperactivity11. As also highlighted by Sagvolden and collaborators, most of the studies questioning the face and predictive validities of SHR/NCrl vs WKY/NHsd rats as a model of ADHD were confounded by the settings used to assess rat’s behaviors11. In this study, we used the recommended strains of SHR and WKY rats to match with the studies that extensively characterized the ADHD-related behaviors of the animal model.Even though SHR compared to WKY rats are one of the mostly used animal model of ADHD, it has also been criticized for its face and predictive validities, based on studies showing no differences in locomotor activity between SHR rats and controls, and others suggesting a lack of effect of methylphenidate on the ADHD-related phenotypes of this animal model24, and cognitive deficits observed in 11-week-old animals25. A recent metabolomic study performed in urine and plasma samples of 40-weeks-old SHR and WKY rats described a significant correlation between urine and blood variations of several metabolites and hypertension26 . Although we cannot completely rule out that our metabolic data could be related to the development of hypertension in our experimental conditions, we worked on 8-weeks-old animals, and our peripheral data are not in line with the urinary and plasma biomarkers significantly correlated with hypertension in these studies.This animal model of ADHD is also confounded by the development of hypertension from 10 to 12 weeks of age28. Thiamine phosphate is a cofactor of the pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and trans-ketolase29, involved in the glycolysis, pentose phosphate pathway and Krebs cycle. Therefore, significant alterations of the cerebral energy metabolism were expected in this ADHD animal model. This is consistent with studies reporting decreased expression of the dihydrolipoyl dehydrogenase, a part of the pyruvate dehydrogenase complex in the medulla oblongata of SHR/NCrl rats30, along with decreased ATP production capacities in discrete brain regions in this animal model of ADHD31.SHR/NCrl rats showed lower brain levels in thiamine phosphate compared to controls. Interestingly, old reports suggested that vitamin supplementations, including thiamine, could carry beneficial effects on ADHD symptoms33 fitting well with the higher levels of succinate detected. Based on the key role of succinate in the Krebs cycle and in the respiratory chain, these results further support a dysfunctional brain energy metabolism in this animal model of ADHD. In addition, elevated succinate levels and mitochondrial dysfunctions have been associated to an increased oxidative stress, which has been extensively described in the brain of this animal model35, and in ADHD patients39. The higher brain levels of 2-ethylmalonate also observed in SHR/NCrl rats are consistent with an increased oxidative stress as it is a reliable lipid peroxidation biomarker40. Thus, the higher brain levels of glucuronate detected could reflect an elevated activity of the pentose phosphate pathway due to the increased oxidative stress, as the pentose phosphate pathway is used by neurons to regenerate glutathione41. In line with these results and other studies reporting protective effects of methylphenidate on oxidative stress38, Fagundes and collaborators already showed that a methylphenidate treatment increases the succinate dehydrogenase and activates the mitochondrial respiratory chain of young Wistar rats further supporting our hypothesis42.In line with such hypothesis, we observed significantly elevated levels in succinate, malonate and 2-ethylmalonate in SHR/NCrl rats compared to controls. Both malonate and 2-ethylmalonate inhibit several Krebs cycle’s enzymes, including the succinate dehydrogenasevs controls. Carnitine plays a key role in energy metabolism by transporting long-chain fatty acids to the mitochondria for the production of acetyl-CoA via the beta-oxidation, and removes short and medium chain fatty acids formed by metabolic processes to prevent their accumulation in the mitochondria43. Thus, these higher brain carnitine levels could reflect modifications in the lipid metabolism in SHR/NCrl rats. Fatty acids and ketone bodies such as 3-hydroxybutyrate are alternative energy substrates during development or when glucose availability is limited31. In our experimental conditions, the cerebral glucose concentrations in the SHR/NCrl rats were not different from controls. Moreover, 3-hydroxybutyrate is no longer a substitute of glucose for brain energy metabolism after 30 days of life in rats44. Studies reported a switch towards other energy substrates to maintain the Krebs cycle supply with acetyl-CoA in conditions of decreased pyruvate dehydrogenase activity47. Based on the lower thiamine levels we observed in SHR/NCrl rats, and on its role as cofactor of enzymes including the pyruvate dehydrogenase, we cannot exclude that the higher carnitine and 3-hydroxybutyrate levels we observed could reflect such a switch in energy substrates. Another explanation could rely in their antioxidant properties50 in response to the higher oxidative stress detected in this animal model of ADHD35.We also detected higher carnitine and 3-hydroxybutyrate brain levels in SHR/NCrl rats 51. Based on the implication of dopamine in the pathophysiology of ADHD, this support that such abnormalities related to neuroenergetic54 and oxidative stress could be one of the molecular mechanism involved in the pathophysiology of ADHD-related phenotypes in SHR/NCrl vs WKY/NHsd rats. Thus, we expected to see modifications in the cerebral levels of various neurotransmitters in SHR/NCrl rats vs controls, as the implication of multiple neurotransmitters has already been suggested in ADHD57, and dopamine along with noradrenaline modifications have already been described in discrete regions in this animal model13. The fact that we analyzed the whole brain rather than discrete regions could explain the lack of results related to neurotransmitters metabolism in our experiments. Instead, we observed the lower brain levels of pyridoxamine-related compounds and higher adrenaline levels at the whole-brain scale in SHR/NCrl rats. Interestingly, lower peripheral levels of pyridoxamine have been reported in ADHD patients, and pyridoxamine supplementation has been suggested to normalize ADHD symptoms60. Pyridoxamine derivatives are co-factors of enzymes crucial to the biosynthesis of multiple neurotransmitters such as GABA, glutamate and monoamines61 further supporting that the lower pyrixodamine levels observed in our study could be related to modifications in neurotransmitters contents in this animal model of ADHD.Intracerebral injections of malonate have been used as an inducer of energy mitochondrial metabolic dysfunctions, and have been shown to induce modifications in dopamine neurotransmission67. Because the adrenergic neurons of the medulla oblongata are involved in the regulation of blood pressure, these higher levels were associated with the development of hypertension elicited by adult SHR rats. Even though our study was conducted before the appearance of hypertension in this animal model13, we cannot exclude that the higher adrenaline levels we observed could be involved in the hypertension development in these rats. However, adrenergic neurons do not only send axonal projections in areas regulating autonomic functions, but also to the locus coeruleus and ventral tegmental area containing noradrenergic and dopaminergic neurons known to be involved in the pathophysiology of ADHD68. Thus, it cannot be excluded that higher adrenaline levels could be related to modifications in dopamine and noradrenaline contents in specific brain regions in this animal model of ADHD.Concerning adrenaline, higher brain concentrations have already been reported in the medulla oblongata in this animal model, along with greater expression of the phenylethanolamine N-methyltransferase, which is responsible for adrenaline biosynthesis69. Since a large number of metabolites are concentrated in urines, this could result in an enhanced sensitivity for the detection of low metabolic variations. In addition, the easy access to urines in humans makes it a compartment of choice for patients in which blood sampling can be more complex to collect. Thus, this further highlight the complementarity between peripheral compartments, and the interest at looking at multiple peripheral matrices to better reflect a cerebral pathophysiological condition.In this study, we were able to differentiate SHR/NCrl rats from controls using discriminant models based on blood and urine samples with accuracies and predictive abilities superior to what was obtained in the brain. Besides, 61% of the metabolites involved in the discriminant model in the brain were also involved in the peripheral models, and almost all these metabolites were significantly different between SHR/NCrl rats and controls in central and peripheral compartments. This good coverage of the brain by the peripheral metabolomes should enable to identify valuable peripheral biomarkers related to the neurobiology underpinning this animal ADHD model. At the scale of metabolomic pathways, the urine compartment shared almost 80% of the differences detected in the blood. The interest of looking at the urine compartment in the context of neuropsychiatric diseases has already been highlighted, as this compartment is not subjected to the homeostatic changes observed in the blood71. However, studies showed results consistent with our findings: (i) impaired responses to oxidative stress have been observed in blood samples of ADHD patients, along with a relationship between low levels of pyridoxine and ADHD;72 (ii) despite inconsistent results regarding tryptophan pathway changes in the blood of ADHD children62, lower serum levels of tryptophan and related metabolites have been reported in adult patients73, in line with the association between altered tryptophan and the ADHD-like behaviors exhibited by dogs60, and as we observed in SHR/NCrl rats. Taken together, this further support the interest of exploring distinct peripheral matrices and metabolic pathways related to energy metabolism, oxidative stress, vitamins, and amino acids in the search for biomarkers of ADHD.As in the brain, our blood and urine results revealed significantly modified pathways related to amino acids, energetic, and vitamin metabolisms along with oxidative stress in SHR/NCrl rats vs controls. Clinical studies already explored peripheral matrices such as plasma, serum, saliva, and urine in ADHD subjects. Most of these studies have examined levels of growth factors, cortisol, melatonin, catecholamines, fatty acids, amino acids, oxidative stress, and pyridoxine metabolisms in ADHD patients, but no reliable biomarkers of ADHD have been uncovered to dateQuestioning the relevance of potential peripheral metabolites of interest in ADHD, animal models represent an interesting opportunity to have access to both central and peripheral compartments. In addition to common differences observed at the scale of metabolic pathways between the brain and periphery of SHR/NCrl rats, we also detected metabolites that were significantly modified in the brain and periphery between SHR/NCrl rats and controls. These metabolites were related to energy metabolism and oxidative stress reinforcing the interest of investigating these metabolic pathways in peripheral compartments in the search of metabolic signature of ADHD. Significant correlations between the brain and urine variations of malonate and glucuronate were also specifically detected in SHR/NCrl rats. Such a relationship is of a great interest as these results emphasize the relevance to look at the whole individual, using cerebral but also peripheral matrices to better understand the biological modifications underlying pathological behaviors.52. This hypothesis suggested alterations in neuronal transmissions due to deficits in energy supply to explain moment-to-moment fluctuations in task performance, which is a common feature of ADHD52. Our results are consistent with this hypothesis and give new insights into the molecular mechanisms underlying such energetic dysfunction. Based on the limited access to brain metabolites in humans, the use of animal models to better understand the molecular mechanisms underlying ADHD pathogenesis remains an important avenue of research. The presence of such metabolomic modifications both in the brain and in the peripheral compartments, further support the relevance to look at these common metabolites and related metabolic pathways in the search of potential clinical ADHD biomarkers.Fifteen years ago, Russell and colleagues proposed the so-called “Neuroenergetic hypothesis” of ADHDSupplementary legendsTable S1Table S2Table S3


Background and Purpose: Optimal periprocedural management of blood pressure during mechanical thrombectomy (MT) remains controversial. This study aimed to investigate the relationship between the duration of blood pressure drops during general anesthesia and the outcomes in large vessel occlusion (LVO) patients treated with MT.Methods: We retrospectively reviewed our prospectively collected data for LVO patients treated with MT between January 2018 and July 2020. Intraprocedural mean arterial pressure (MAP) was recorded every 5 min throughout the procedure. Baseline MAP minus each MAP value recorded during general anesthesia was defined ΔMAP. Cumulated time (in min) and longest continuous episode (in min) with ΔMAP more than 10, 15, 20, 25, and 30 mmHg were calculated, respectively. Poor outcome was defined as 90-day modified Rankin score (mRS) 3–6. Associations between cumulated time of different ΔMAP thresholds and poor outcome were determined using binary logistic regression models.Results: A total of 131 patients were finally included in the study. After controlling for age, atrial fibrillation, baseline NIHSS, baseline ASPECTS, procedure duration of MT, and times of retrieval attempts, the results indicated that cumulated time of MAP drop more than 10 mmHg  and 15 mmHg  were independently associated with poor outcomes.Conclusion: Prolonged episodes of intraprocedural MAP lowering were more likely to have poor outcomes in LVO patients following MT with general anesthesia, which might be helpful in guiding intraprocedural hemodynamic management of patients under general anesthesia. Reperfusion therapy with mechanical thrombectomy (MT) has been a standard treatment for patients with acute ischemic stroke (AIS) due to large vessel occlusion (LVO) . Neverthpost-hoc analysis of randomized controlled trials found that critical BP thresholds and durations during MT, with MAP <70 mmHg for more than 10 min, are linked with poor outcomes  had CT angiography (CTA)-confirmed LVOs presenting within 6 h of symptom onset, including the internal carotid artery, the middle cerebral artery (M1 or M2), and the basilar artery. Patients presenting at the hospital 6–16 h after symptom onset were included if they met the criteria described in the DEFUSE-3 trial . PatientWe retrieved demographic, clinical, laboratory, and radiological data including age, sex, baseline National Institutes of Health Stroke Scale (NIHSS) score, baseline Alberta Stroke Program Early CT Score (ASPECTS), baseline systolic blood pressure (SBP), and diastolic blood pressure (DBP) parameters, as well as comorbid conditions such as history of smoking, hypertension, diabetes mellitus, atrial fibrillation, and congestive heart failure.MT under general anesthesia is our center's standard procedure. Only in a few cases when the anesthesiologist could not arrive at the angio-suite in time, MT under conscious sedation was selected. For the purpose of reducing the heterogeneity of study subjects, we excluded patients with conscious sedation. All data from the anesthesiology reports were collected from the beginning of anesthetic induction until awakening. The current guidelines from American Heart Association/American Stroke Association guidelines recommend to maintain BP ≤ 180/105 mmHg during and for 24 h after procedure . Baseline MAP minus each MAP value recorded during general anesthesia was defined as ΔMAP. As our main measure of the different ΔMAP duration during general anesthesia, cumulated time (in min), and longest continuous episode (in min) with ΔMAP more than 10 mmHg, while a cutoff value of 15, 20, 25, and 30 mmHg were also calculated, respectively.via a structured telephone interview by two certified neurologists with clinical experience for 8 and 13 years, respectively  scores of 2b or 3; a good outcome was defined as mRS score 0–2, and a poor outcome was defined as mRS score 3–6 at 90 days. At 90 days, an outcome assessment was performed with each patient ectively –18.t-test or a Mann-Whitney U-test was used for the continuous variables, depending on the normality of the distribution. Associations of each ΔMAP duration parameter with clinical outcomes were determined using binary logistic regression models adjusted by baseline characteristics with a P < 0.1 in univariate analyses, respectively. All statistical analyses were performed using SPSS, Version 22.0 . A P < 0.05 was considered statistically significant.The patients were dichotomized according to the functional outcome. Clinical characteristic and imaging profiles were summarized as mean ± SD or median (25th−75th percentile) for quantitative variables depending on the normality of the distribution and as frequency (percentage) for categorical variables. The Fisher exact test was used to compare the dichotomous variables between two groups, whereas an independent sample 2-tailed n = 10), conscious sedation (n = 23), and lost to follow-up (n = 1). Of the patients included, the mean age was 67.2 ± 13.0 years, and 46 (35.1%) were female. The median NIHSS score on admission was 19 , mean time from onset to reperfusion was 460.4 ± 206.1 min and median procedure duration was 124.5 ± 59.2 min.A total of 131 patients were finally enrolled after excluding patients due to TICI 0–2a after the procedure  of patients experienced a drop in MAP during general anesthesia. The mean MAP drop (ΔMAP) was 19.5 ± 14.1 mmHg.P = 0.002), had a higher frequency of atrial fibrillation , higher baseline NIHSS score , smaller baseline ASPECTS , longer procedure duration , and underwent more retrieval attempts  than those with good outcome.As shown in P < 0.001), the cumulated time of MAP dropping more than 15 mmHg , and the cumulated time of MAP dropping more than 20 mmHg  were longer in the poor outcome group. There were no significant differences in other variables including baseline BP. After controlling for age, atrial fibrillation, baseline NIHSS, baseline ASPECTS, procedure duration of MT and times of retrieval attempts, we found the results indicated that cumulated time of MAP drop more than 10 mmHg  and 15 mmHg  were independently associated with poor outcomes (The cumulated time of MAP dropping more than 10 mmHg (110.2 vs. 71.0 min, outcomes . For allIn this study, our findings suggest that prolonged episodes of lower MAP compared with baseline are related to a higher likelihood of poor outcome in LVO patients following MT with general anesthesia.A drop in BP during general anesthesia is a common phenomenon in LVO patients receiving MT, which are thought to occur mainly during the induction phase of general anesthesia. The incidence of this phenomenon reported by recent research is about 87% . SimilarConsidering that the SBP or DBP variables are more susceptible to be influenced by measurement errors and short BP fluctuations, we thus specifically chose the MAP instead of the SBP or DBP variables. Moreover, in the setting of arterial non-compliance or stiffness, SBP and DBP often move in the opposite direction, especially in an increasingly aging population . MAP refInterestingly, we found that only a cutoff value of 10 and 15 mmHg in intraprocedural MAP drop were independently associated with poor outcome. A previous study demonstrated that a fall in MAP of >40%, that is, ~40 mmHg, during endovascular therapy from baseline is an independent predictor of worse functional outcomes in LVO patients under general anesthesia (The major difference between our study and other previous studies is that our observation index is the duration of BP drops, not merely the extent of BP drops. Theoretically, BP elevation may be a natural response of the organism to persistent vessel occlusion in the acute ischemic stroke phase, in order to increase the perfusion of salvageable tissue and to minimize the ischemic damage , 27. MaiLimitations include the study being performed in a single center and with small number of cases. We collected data prospectively, which has the potential of selection bias. Second, we recorded the BP values only every five min during the procedure. It would be more informative to record BP every 2 or 3 min or even every min. Third, the site of LVO occlusion may also have played a role in the bias of the hemodynamic data as BP pathophysiology and optimal approach of management could be largely different between anterior and posterior circulation strokes. Fourth, inclusion criteria provide heterogeneity in baseline profiles of patients. We enrolled LVO patients within 6 h of symptom onset between January 2018 and April 2018. Afterwards, patients presenting at the hospital 6–16 h after symptom onset were also included according to the criteria of DEFUSE-3 and DAWN trial. Fifth, we did not explore the impact of different anesthetic drugs and doses on BP and clinical outcome, which are worthy of further study. Sixth, we had no standardized protocol for general anesthesia or intubation, which may lead to selection bias.Prolonged episodes of intraprocedural MAP lowering were more likely to have poor outcome in LVO patients following MT with general anesthesia. Every 1 min extension in duration of ΔMAP more than 10 and 15 mmHg before recanalization increased the odds of poor outcome by an estimated 1.3 and 1.1%, respectively. Larger prospective studies are warranted to confirm our results and give further insights in periprocedural BP management of AIS patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Ethics Committee of Zhejiang Provincial People's Hospital. The patients/participants provided their written informed consent to participate in this study.CX: substantial contributions to study design, data collection for the whole trial, data analysis, interpretation of data, and drafting and revising the manuscript for intellectual content. GL: substantial contributions to study design, cleaning and analyzing the data, and revising the manuscript for intellectual content. ZZ, TJ, and NL: data acquisition, interpretation of data, and revising the manuscript for intellectual content. HM: intraprocedural data collection for the whole trial and revising the manuscript for intellectual content. SY: image data collection for the whole trial and revising the manuscript for intellectual content. ZY: writing the statistical analysis plan. YG: monitoring data collection for the whole trial and revising the manuscript critically for intellectual content. ZS: developing study concept/design, study supervision, interpretation of data, revising the manuscript critically for intellectual content, and final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study describes a new and safe freehand cervical pedicle screw insertion technique using preoperative computed tomography (CT) morphometric measurements as a guide and a medial pedicle pivot point (MPPP) during the procedure. This study included 271 pedicles at 216 cervical spine levels (mean: 4.75 pedicles per patient). A pedicle diameter (PD) ≥ 3.5 mm was the cut-off for pedicle screw fixation. The presence and grade of perforation were detected using postoperative CT scans, where perforations were graded as follows: 0, no perforation; 1, perforation < 0.875 mm; 2, perforation 0.875–1.75 mm; and 3, perforation > 1.75 mm. The surgical technique involved the use of an MPPP, which was the point at which the lines representing the depth of the lateral mass and total length of the pedicle intersected, deep in the lateral mass. The overall success rate was 96.3% . In total, 54 perforations occurred, among which 44 (81.5%) were Grade 1 and 10 (18.5%) were Grade 2. The most common perforation direction was medial . The freehand technique for cervical pedicle screw fixation using the MPPP may allow for a safe and accurate procedure in patients with a PD ≥3.5 mm. Cervical spine surgery is being performed with increasing frequency . In patiCurrently, with technological advances such as computer assisted navigation (CAN) systems such as Stealth Station Spine Surgery Imaging and Surgical Navigation with O-arm  or Ziehm Vision FD Vario 3-D with NaviPort integration , it has become possible to contribute to accurate posterior fixation ,12. HoweWe therefore developed a new freehand technique for cervical pedicle screw insertion; guided by preoperative computed tomography (CT), our procedure may provide accurate and safe screw insertion, with less exposure to intraoperative radiation. In this study, we aimed to describe this novel freehand cervical pedicle screw insertion technique—using a medial pedicle pivot point (MPPP)—through technical statements. We also examined the accuracy and safety of the technique regarding the presence and grade of pedicle screw perforations.Patient populationThis study was approved by our institutional review board and ethics committee. A standardized protocol was used for all enrolled patients, which included pre- and postoperative CT scans to measure anatomic parameters and the accuracy of pedicle screw placement. All procedures were performed in accordance with the relevant guidelines, regulations of our institute and a waiver regarding the need for informed consent. This prospective study included 57 patients  who underwent surgery with pedicle screws inserted via our novel freehand insertion technique between February 2018 and October 2020; all surgeries were performed by a single orthopedic spine surgeon. A pedicle diameter (PD) of 3.5 mm was the minimum cut-off value for 3.5 mm diameter pedicle screw fixation; if the PD was <3.5 mm, lateral mass screw fixation was utilized instead. In the case of abnormalities—such as a high-rising vertebral artery on preoperative CT—an alternative method was used, such as a lateral mass screw. Additionally, for cases in which a CT scan of an individual pedicle demonstrated sclerotic bony change with superior/inferior articular processes and a lack of cancellous track, lateral mass screws were used for conversion. Therefore, the number of pedicle screws did not always correspond with the number of surgically treated levels.Preoperative radiographic measurementsEach patient underwent preoperative plain cervical CT using a multisided scanner . Both the sagittal and axial views of cross-sectional images (1 mm cuts) were obtained in parallel with the pedicle trajectory. For quality control and CT standardization, technicians in the radiology department received regular instruction by the surgeon performing the operations regarding how to obtain correctly oriented sagittal and matched axial scans of the pedicles.To identify the correct starting point and direction of screw insertion for each pedicle, linear and angular parameters were measured on both the sagittal and axial CT images; this was performed for each patient at every cervical level on both the right and left sidesOn sagittal images, the distance (in mm) was measured from the tip of the inferior articular process of the cephalad cervical vertebra to a point in the lateral mass transected by a line drawn through the center of the pedicle; this was defined as the facet-pedicle distance FPD; A. The saIn the axial view, the starting point and designated MPPP were set first B. The laThe linear and angular radiologic parameters measured via the preoperative CT scan were captured at all cervical levels scheduled for surgical treatment; this was presented on a PowerPoint slide in the form of a screenshot, allowing the images to be viewed on a computer monitor in the operating room.Surgical techniqueAfter inducing general anesthesia, patients were placed in the prone position on a Jackson table , with their head placed on a Mayfield headrest  in a neutral position. Shoulders were pulled caudally and maintained on the sides using heavy bandages. The mainstay procedure for realignment of the cervical spine using a Gardner Wells tong was not performed in this study, as results may differ from the preoperative CT measurement. A lateral view radiograph was obtained to ensure that all necessary cervical levels could be visualized, and cervical lordosis was comparable to that preoperatively noted via CT. If the cervical lordosis adjusted with the Mayfield headrest was different from the lordosis observed on the preoperative CT scan, the headrest device was readjusted to match the preoperative image as much as possible.A posterior midline incision was made, and the posterior elements of the cervical levels undergoing instrumentation were subperiosteally exposed. The dissection was then extended laterally to expose the lateral borders of the lateral masses. PowerPoint slides containing measurements of the intended levels and pedicles, based on the preoperative CT results, were referred to. The screw insertion process is described in detail in The point located at the distal tip of the curved pedicle probe that entered the LMD was designated the MPPP in this study. Based on this point, the lateral aspect of the lateral mass was not decorticated to create a funnel-shaped hole ,15, the The pedicle probe was then inserted to a depth 2 mm less than the pedicle total length (PTL), directed at an angle corresponding with the appropriate horizontal and pedicle insertion angles (PIA). As an optional safety measure, anteroposterior and lateral radiographs could be taken at this point using an image intensifier to confirm the direction of the probe, as well as to guide it to the correct horizontal angle, following the direction of the pedicles on lateral X-ray or sagittal CT scans in complex cases. After forming a track with the curved probe, ball-tip probe palpation was performed to confirm the integrity of the pedicle borders. A 2.5 mm diameter tap was used to make screw insertion easier and lessen the torque required to insert the screw. Finally, a ball-tipped probe was used to confirm the integrity of the pedicle borders after tapping; if the integrity of the pedicle wall was not clearly confirmed using the ball-tipped probe or if bleeding occurred from the hole where the tapping was performed, it was judged that the integrity of the pedicle wall was broken. In this case, coagulation was performed using bone wax in the concerned hole, and a lateral mass screw was used as an alternative. A 3.5 mm pedicle screw of appropriate length  was then inserted following the same horizontal angle and PIA. After screw insertion, cervical spine anteroposterior and lateral radiography were performed to determine whether the inserted screw trajectory was appropriate .Postoperatively, anteroposterior and lateral radiographs , as wellStatistical analysisp < 0.05. All statistical analyses were performed using SPSS software, version 22 .The means and corresponding standard deviations were calculated for all linear and angular pedicle parameters, while the intraclass correlation coefficient (ICC) was analyzed to determine the perforation grade. Forward stepwise multiple linear regression analysis was used to identify potential risk factors for pedicle perforation, including age, sex, vertebral level, PD, LMD, FPD, and PIA; statistical significance was set at p = not significant, t-test). Most patients  underwent three- to five-level operations. The underlying diagnoses were cervical stenosis , ossified posterior longitudinal ligament with cervical myelopathy , and multiple-level herniated cervical disc disease . The mean duration of surgery—including decompressive laminectomy and/or additional foraminotomy at the designated cervical spine levels—was 205.7 ± 23.5 (range: 122–341) min; the mean blood loss was 362.9 ± 54.6 (range: 100–1300) mL. Preoperative radiographic parameters, including PD, PIA, LMD, FPD, and PTL, are shown in This study included 38 (66.6%) males and 19 (33.4%) females, with mean ages of 63.9 ± 12.29 years and 63.94 ± 11.47 years, respectively (p < 0.001). Perforations of Grade 1 or higher occurred with 54 pedicle screws, representing an overall perforation rate of 19.9% (54/271). Most perforations (80.1%) among all cervical levels were classified as Grade 0 (no perforation), followed by Grade 1 (16.2%) and Grade 2 (3.7%); Grade 3 perforations were not observed , indicating that the likelihood of perforation increases with increasing PIA.Forward stepwise multiple linear regression analyses were also used to identify potential risk factors—including age, sex, vertebral level, PD, LMD, FPD, and PIA—for the occurrence of perforation (Grade 1 or higher). PIA was positively associated with the presence of perforation ; the remaining perforations were Grade 2 (3.7%). Since the screw diameter was 3.5 mm, following the strict surgical indication of a PD ≥3.5 mm was important for our technique. Interestingly, most previously reported techniques did not consider PD when planning surgery, which may have resulted in a higher rate of pedicle breach or pedicle fractures when the screws were inserted ,19,22,24As revealed in the stepwise multiple regression analysis for the occurrence of perforation in this study, PIA was a risk factor. Cervical pedicle placement using the MPPP can therefore be established as a safe technique for lateral wall perforation; however, when the PIA is very high, it can be perforated through a relatively thick medial wall. Nevertheless, even if the medial wall is perforated by purchasing a pedicle screw centered on the MPPP while using the lateral mass as a buttress, the pedicle screw would have already progressed to the cortico-cancellous core, resulting in minimal violation. Accordingly, nerve root injury should rarely occur with the use of our technique, since the dorsal and ventral nerve roots are located in the inferior portion of the neural foramen, both at and below the disc level . ContrasOur freehand technique for cervical pedicle screw fixation using the MPPP may be safe and accurate in patients with a PD ≥ 3.5 mm. Linear and angular measurements on preoperative CT images for each cervical pedicle can be used as a simple and accurate guide when the PD is ≥3.5 mm.


The presence of ten metals  was investigated in the final discharge of six facilities, including four wastewater treatment plants, which were continuously discharging treated wastewater to the coastal environment in Gran Canaria Island. A four-day sampling campaign was carried out at each facility in July 2020, in which both the spot samplings technique and the diffusive gradient in thin-film technique (DGT) were carried out to measure total dissolved metals and the in situ labile metal fraction, respectively. After the necessary sample preparation steps, measurements were carried out by ICP-MS for both samplings. Raw data referred to the spot total dissolved and DGT-labile metal concentrations were reported. In general, the average metal concentrations were dispersed in a broad range. As expected, the highest metal contents were found in those facilities with larger industrial contributions. The values of annual average environmental quality standards (AA-EQS) were used to assess the total dissolved metal concentrations for every metal in every final discharge. In only one of the studied facilities, some metals (Ni and Zn) exceeded these EQS within the receiving waterbody, highlighting the need for more efficient treatment targeted towards a specific discharging-water quality. In addition, the total dissolved and labile metal daily fluxes of discharge were calculated to estimate the contribution of every effluent to the receiving water bodies. A significant proportion of metals enter marine water bodies via surface runoff and municipal or industrial wastewater discharges, which are important secondary sources of these substances. Indeed, wastewater treatment plants (WWTP), due to their high continuous flow rates, discharge important amounts of trace metals and other contaminants into the marine environment, which cannot be fully retained in these facilities ,2,3. HeaCoastal seawater is one of the most important water resources in the Canary Islands. Its quality not only impacts tourism, which is the main economic activity, but also the seawater intake of desalination plants that provide water for human consumption to most of the island’s inhabitants, particularly in the case of Gran Canaria. Thus, the deterioration of seawater quality is crucial and critical.The EU (European Union) Policy on Water Protection is based on a combined approach in which the emission limit value (ELV) and the water quality objectives (WQO) are mutually reinforcing. The ELV approach focuses on the maximum allowable quantities of pollutants discharged from a particular source into the aquatic environment.−1, total arsenic [500–1200 µg·L−1], total cadmium [14–400 µg·L−1], total chromium [360–3000 µg·L−1], hexavalent chromium [36–500 µg·L−1], total nickel [720–5000 µg·L−1], total lead [200–500 µg·L−1], total aluminum , total copper [500–3000 µg·L−1], total zinc , total tin , total manganese , iron [2000–3600 µg·L−1], total selenium [50–200 µg·L−1], and total titanium [1000–5000 µg·L−1].Three regions in Spain have developed regional regulatory limits on the metals’ levels in the discharges to the sea: Cantabria, País Vasco, and Andalucía. They have set different limits for 15 metals and metalloids to prevent environmental risks from both industrial wastewater and sewage, including total mercury with an ELV ranging from 2.4 to 100 µg LHowever, the regional regulatory limits on the metals’ levels in the discharges to the sea are not currently defined, even though the competent Canarian regulatory body is working on them. Consequently, the requirements for the discharge of metals (and other substances) discharged from any facility that discharges treated wastewater to the sea must be strictly controlled. These requirements must comply with the coastal WQO, particularly with the Environmental Quality Standards (EQS) for metals, to effectively protect water bodies and ensure their “good chemical status”. The chemical status assessment is used alongside the ecological status assessment to determine the overall quality of a water body . In the Metals can occur in the WWTPs as different physico-chemical “species”. Metals may be found in different forms, including solutions, colloids or suspensions, made up by both simple ions (free ions) or particles and complex organic or inorganic compounds . During Reported data indicate that dissolved metals in WWTPs are less efficiently removed, becoming the main fraction in treated effluents and causing an enrichment on the receiving water bodies ,14,15. TDGT devices have been reported to provide accurate time-weighted average concentrations of dissolved labile metals in wastewaters ,19,20,21Although the use of DGTs, as described above, can be advantageous, the assessment of metals by the direct deployment of DGTs in the wastewater discharge pipeline has not been reported in many studies in the literature ,20. On tIn this study, we aimed to measure the dissolved and labile concentrations of ten metals in the final effluents of six facilities (including four WWTP with different wastewater intakes) that continuously discharged into the coastal receiving water bodies of the Island of Gran Canaria. Based on these results, the total dissolved metal concentrations for every metal and effluent would be assessed considering the values of annual average environmental quality standards (AA-EQS). In addition, the total dissolved and labile metal daily fluxes discharges would be calculated to estimate the contribution of each effluent to the receiving water bodies.Gran Canaria is in the middle of the Canary Islands archipelago in the Atlantic Ocean, southwest of Spain and northwest of Africa. All the facilities under study are located on the east coast of the island, where most of the population and economic activity are concentrated .TP-1: WWTP, which mainly collects household wastewater from the city of Las Palmas de Gran Canaria. This plant was considered as a reference for a typical large Canarian WWTP with domestic inputs.TP-2 and TP-3: medium size WWTP, which collects mixed household and industrial sewage inlets.TP-4: WWTP, which collects only industrial wastewater from two industrial areas of the island.TP-5: a coastal thermal power plant, which produces one of the largest discharges of cooling wastewater in the Canary Islands.TP-6: an indoor seawater aquaculture (fish) farm.Six final discharges from different facilities were studied at the same time in July 2020. Several sources of wastewater and types of installations that continuously discharge into the sea were considered. They were chosen to ensure the representativeness of the existing WWTPs in the islands based on their capacity, their treatment technologies, and the diversity of the collected influents. Those finally selected were:The main characteristics of these plants are summarised in The effluents (outlets) were discharged through underwater outfalls into the marine receiving water.3  acid bath overnight. Once rinsed thoroughly with ultrapure water (type I or better: ≥18 MΩ·cm resistivity), they were stored and sealed in clean plastic bags until being used , Ni [0.111–18.044], Pb [0.009–0.580], Cr [0.172–3.546], Cu [0.015–1.774], Zn [5.160–114.816], Al [0.786–697.056], Fe [0.299–9855.652], Mn [1.211–643.383], and Co [0.007–6.659].We measured the total dissolved concentrations the 10 analysed metals in every sampled effluent, and the mean values of each facility fell among these ranges , Fe (9855.65 ± 4358.438 μg·L−1), and Mn (643.383 ± 233.195 μg·L−1) were high compared to the reported values for entirely or mixed industrial wastewaters in the literature , Ni [0.376–7.610], Pb [0.017–0.047], Cr [0.139–0.465], Cu [0.041–0.791], Zn [1.361–19.943], Fe [3.070–6200.034], Mn [1.306–66.146], and Co [0.024–2.372].Note that concentrations of the DGT-measured labile metals shown in Furthermore, we observed the highest concentrations of most of the labile metals in the TP-4 facility, except for four metals: Cd, Cu, Pb, and Mn. The highest contents in Cd and Cu were measured at TP-6, the marine fish farm. The discharge from TP-1, the largest studied WWTP, registered the highest values in Pb. Finally, the highest levels in Mn were found at TP-2, a mixed domestic and industrial WWTP.As expected, differences were observed when comparing results from the 2 sampling techniques used in this study : the DGTThe dissolved fraction comprises the free metal ions, labile inorganic and organic complexes, as well as inert high molecular organic metal complexes and colloids . DGT resDifferences in the fraction measured in spot sampling  and by DGTs (dissolved labile). As mentioned before, different chemical forms are measured depending on the fraction considered. The concentrations found of total dissolved metals tended to be generally higher than the DGT-labile concentrations, so the percentage of the labile fraction being part of the total dissolved fraction is normally less than 100% . AlthougDifferences in the timescale of the spot-sampling measurements and the DGTs measurements. Results do not represent the same sampling timescale . Total d−1), while, at the other facilities, the dissolved oxygen ranged between 6.19 and 7.31 mg·L−1. In addition, as some of the sampled effluents’ water source was seawater (TP-5 cooling water and TP-6 marine fish farm), the overall recorded conductivity (25 °C) ranged widely, between 1.66 and 55.67 mS·cm−1.Differences in the physicochemical characteristics of the analyzed effluents. The physicochemical conditions may impact the forms’ distribution (speciation) for a given metal. In this study, we determined the physicochemical parameters in the different wastewater effluents at each sampling day . The oveTherefore, direct comparison between the concentration measured by spot samples and the concentration measured by the DGT technique is not possible ,24, and The mobility, bioavailability, and toxicity of heavy metals depend on their speciation rather than on their total concentrations in water. Some of the main physicochemical parameters that determine the bioavailability of metal species in wastewater effluents are the DOC  and SPM (by association with suspended particles), which can markedly reduce the free ion concentration of the metal ,19,41.−1, 458.67 mg L−1, and 435 FNU, respectively. Even though in this study the relationship (correlation) between these parameters and metal speciation has not been addressed due to the limited data availability, the high concentrations of DOC in all the samples of TP-4 facility may explain the low % of the labile fraction per the total dissolved fraction in most of the studied metals. In fact, in the other facilities with a much lower range in DOC, [0.5–15.2] mg·L−1, the % of the labile fraction is higher, especially in the conventional WWTPs  exceeded by two orders of magnitude those determined in the effluents from the other facilities. In TP-4, these parameters reached averaged values of 753.43 mg Lal WWTPs . FurtherEffective reduction of heavy metals loads discharged into receiving waters and mitigation of consequent environmental negative impacts require knowledge about their sources and emissions, as shown by the results of this study. The Environment Agencies usually set numerical limits on the content of pollutants in effluents. Thus, they consider both those parameters that the discharge would probably contain and the defined Environmental Quality Standard (EQS) to assess the chemical status of the water bodies. In addition, EQSs represent a target to reach when addressing risk management measures for specific pollutants and, in particular, concerning the reduction of emissions  . As explHere, we considered the AA-EQS established in the Spanish regulation to assess the total dissolved metal concentrations in the effluents in terms of contribution to the EQS exceedance within the receiving water body .DGT) of Cd, Ni, and Pb in transitional and coastal waters.Even though suitable EQS are not currently established for the labile fraction, we also include the labile metal concentrations in −1) of the six regulated metals included in this study  measured in the dissolved and labile fraction of all the sampled effluents, regarding the established AA-EQS values for other surface waters of each metal. Note that we assume that all Cr is Cr VI, according to the precautionary principle.−1) and Zn (114.815 µg·L−1), both in the effluent of the TP-4 facility, clearly exceeded the AA-EQS established for other surfaces waters . These results show that the TP-4 industrial wastewater treatment was not efficient enough to diminish the concentration of toxic heavy metals like Ni and Zn, resulting in an anthropogenic pressure for the receiving waterbody.As shown in In this case, these high metal concentrations in the effluent will probably be reduced in the receiving coastal water body due to the mixing processes and wastewater plume dilution through the submarine outfall ,45, resu−1) in the effluent of the TP-4 facility, clearly exceeded this range established for discharges to coastal waters [2000–3600 μg·L−1]. None of the concentrations of the other nine metals measured in the effluents of the facilities sampled in this study exceeded the strictest value of the threshold established by these three regulations.We cannot provide a compliance assessment as there are no regional (Canarian) threshold values for the levels of metals in the effluents discharged into coastal waters. However, for reference purposes, comparing the average concentrations of the 10 total dissolved metals shown in th sample measured in this study for each facilityci is the mean concentration  of each metal in the iqi is the daily average flow from each facility, and ti is the time interval (1 day).Furthermore, to address the contributions of the total dissolved and the labile metal fractions discharged from the facilities under study, we estimated the daily specific load for each metal and each facility  using the approach given by Equation (3):The results of this estimation are shown in 3/h) leads to high daily loads of metals. Here, loads greater than 1 kg/day of total dissolved or labile, respectively, were calculated: 3466 and 3225 g/day of Ni; 10,170 and 898 g/day of Zn, and 1042 and 862 g/day of Mn. Total dissolved aluminum was 1897 g/day.The daily fluxes of dissolved and labile metals discharged by the TP-5 and TP-4 facilities calculated in this study  show tha3/h). However, as a consequence of the high values of some of the metals measured in its effluent, high daily loads  were observed for Fe , Al , and Mn .On the contrary, the TP-4 facility, which treats exclusively industrial wastewaters, reported the lowest daily flow of the studied facilities (30 mThe mean concentration of the total dissolved and labile metals found in the studied effluents is dispersed in a broad range and shows high variability. Higher metal contents were found in the discharges of those facilities corresponding to WWTPs in decreasing order as: TP-4 > TP-2 > TP-1 > TP-3.The highest values of mean total dissolved concentration of Zn, Fe, Mn, and Al were recorded at the TP-4 facility, which exclusively receives highly loaded industrial wastewater in which Al and Fe salts are added in the treatment. The same behavior was observed for these metals in the labile fraction recorded with the DGTs except for Mn and Al. For this facility, measures for reducing metal contaminations based on better control of the industrial intakes and also on the improvement in the treatments for heavy metals removal would be necessary. These measurements should be coherent with the pending establishment of regional regulatory limits for the levels of metals in the discharges into the sea.We present the results of a study performed only in the discharged effluents. A complete assessment of the impact of metal discharge to coastal marine environment implies the use of an integrated monitoring approach, and these results should have been supported, at least, by the concentrations of such metals in the water column and sediment, but this information could not be obtained.The limitations of low-frequency spot sampling, such as the lack of representativeness in dynamic systems such as discharges, can be compensated with the inclusion of complementary methodologies, such as DGTs, which integrate the system’s metal fluctuations and measure its labile fraction. The labile fraction is easily related to the ecotoxicological effects, improving the quality of the assessment. Besides, the improvement in the knowledge about the speciation of heavy metals is relevant to designing appropriate removal technologies.The present results confirm the robustness of the DGT technique and its viability to measure dissolved labile metals when directly applied on different wastewater effluents, as DGT behaves predictably over a wide range of physicochemical conditions.


Rosmarinus officinalis L.) is known to be an effective potential source of natural antioxidants which confer benefits to human health. Their bioactive properties are mainly due to phenolic compounds but these molecules are highly vulnerable to oxidants, light, heat, pH, water and enzymatic activities. Therefore, the stability and shelf life of phenolic compounds should be increased by being protected from chemical and physical damage by means of encapsulation prior to application. Encapsulation is becoming increasingly important in the pharmaceutical, food, cosmetics, textile, personal care, chemical, biotechnology, and medicinal industries due to its potential for stabilization and delivery of delicate and precious bioactive compounds. The aim of the present work was to describe the polyphenolic profile of Tunisian Rosemary, collected from two different bioclimatic areas, and further loading in silk fibroin nanoparticles. The loaded nanoparticles were characterized in terms of morphology, size, polydispersity, Z-potential, secondary structure of the protein, encapsulation efficiency, loading content, and antioxidant activity. On one hand, HPLC analysis revealed the presence of 18 polyphenolic compounds of whichcarnosic acid and carnosol were found to be the most abundant compounds (46.3 to 76.4 and 22.4 to 43.5 mg of compound per gram of dry plant weight (mg/g DPW) respectively), Total phenolic content (TPC) ranged from 85.8 to 137.3 mg of gallic acid equivalent (GAE)/g DPW in post-distilled rosemary extracts andantioxidant activity reached the values of 5.9 to 8.3 µmol of ascorbic acid equivalent (AAE)/g DPW). On the other hand loaded nanoparticles were almost spherical and presented nanometric size and negative Z-potential. Although the encapsulation efficiency in silk fibroin nanoparticles and the drug loading content were low in the conditions of the assay, the encapsulated polyphenols retained near 85% of the radical scavenging activity against DPPH· after 24 h. of incubation at 37 °C. The results showed that post-distilled rosemary residues had an effective potential as natural antioxidants due to their significant antioxidant activity and seemed to be useful in both pharmaceutical and food industries with beneficial properties that might confer benefits to human health and these silk fibroin nanoparticles loaded with rosemary extracts are thus a promising combination for several applications in food technology or nanomedicine.Rosemary ( Rosmarinus officinalis L.) is a perennial herb that belongs to the Lamiaceae family, which is cultivated as medicinal plant in different areas of the world, such as the Mediterranean, Asia, and Latin America [Rosemary  is a highly versatile protein utilized for centuries in the textile, and cosmetic industries. Silk is a natural polymer obtained from the cocoon of  systems ,18. Thei systems . Formula systems ,21,22. A systems , quercet systems , or curc systems ,26 among systems , and for systems , to our  systems ,30.Rosmarinus officinalis L.), a by-product of the essential oil industry, characterizing their composition and testing their antioxidant capacity. On the other hand, we are interested in presenting the silk fibroin nanoparticles as a useful biopolymeric carrier in order to increase the stability of phenolic compounds recovered from this by-product in Tunisia. This combination being a natural product is stable, scalable and a simple method to protect the antioxidants of rosemary methanolic extracts (RME). This work constitutes a proof of concept of the revalorizing process giving a second opportunity to this valuable product, which would increase the economic value of this rain fed crop for the rural development in this country.On one hand, our study has been undertaken with the aim to identify and quantify the polyphenolic compounds of the methanolic extracts from post distilled rosemary plants  with respect to those plants harvested in closer areas such as ElKef–Menzel Salem (KMS) and being equal to those prospected at Gafsa–Sened (GS) location, where the climatic conditions correspond to an arid lower zone.In the present study locations belonging to two different bioclimatic regions were prospected, showing, as a general pattern, that the differences in polyphenolic content should be attributed more to the genetic inheritance of the plants, than to the area of prospection. For instance, as observed in p < 0.05), located in an arid low bioclimatic area, where the climatic conditions are the most extreme among the areas under study. This behaviour was also detected for salvianolic acid A, since also for these components high temperatures and low rainfall also increase the presence of this polyphenol in rosemary leaves for these components.However, when the diterpenic fraction is analysed this pattern changes, thus, carnosic acid, major diterpene identified and precursor of the rest of diterpenes (carnosic acid’s oxidation derivative products) quantified in these extracts, was detected at the highest concentration in plants harvested at the Gafsa–Orbata (GO) population  on the effect of bioclimatic area and season on phenolics and antioxidant activities of rosemary growing wild in Tunisia showed that there was a strong correlation between antioxidant activity and phenolic content depending on bioclimatic and seasonal effects . However•. Results, shown in Following the characterization of the rosemary methanolic extracts, the study of the antioxidant activity was carried out by measuring the total phenolic content and the radical scavenging activity against DPPHThe high biological activities exhibited by these components, particularly rosmarinic acid and carnosic acid have led to the publications of several studies related to the potent antioxidant power of these phenolic compounds. In this sense, Chkhikvishvili et al. (2013) published their protective effect from the oxidative stress, induced by hydrogen peroxide, evaluated in Jurkat cells . Later Arosemary methanolic extracts presented a pale green color and after freeze drying that could be easily dispersed in water, forming an homogenous suspension. Although is stable for several weeks at 4 °C without change in color, that presented slow particle aggregation and trend to sedimentation after 24–48 h. Field Emission Scanning Electron Microscopy (FESEM) images of the freeze dried RME–SFNs . The DLS measurements also showed the increase of the size of the nanoparticles when compared with the SFN control formed in the absence of the RME. The size distribution of the aqueous suspension of the RME–SFNs with a nanometric Z−1 which was assigned to the C=O stretching of Amide I region and a second strong peak at 1524 cm–1, corresponding to the N–H bending of Amide II. This peak profile is characteristic of the crystalline and insoluble β-sheet structure [An example of infrared spectrum of the loaded nanoparticles is shown in tructure . The chaThe average values of PLC and the EE% of the selected RME in the SFNs are shown in the After the determination of the composition of the polyphenols loaded in the nanoparticles we tested the radical scavenging activity (RSA) against DPPH· and the stability along the time of incubation at 37 °C of the original RME, the RME loaded into SFNs and the free SFNs as control. Results presented in In conclusion, the DPPH·-scavenging activity assay after 24 h. Of incubation at 37 °C confirmed that the RSA of the RME loaded into the SFNs were better preserved in comparison with the free RME.In the present study 40 individual rosemary shrubs were randomly collected in the spring of 2017 from four wild populations located at two different bioclimatic areas (semi-arid superior and lower arid) at the full bloom phenological stage. Voucher specimens of rosemary from every location are deposited at the Herbarium of Departamento de Desarrollo Rural, Enología y Agricultura Sostenible, Instituto Murciano de Investigación y Desarrollo Agrario y Medioambiental (IMIDA). La Alberca .Details of collection sites are given in Bombyx mori reared in the sericulture facilities of IMIDA  and raised on a diet of fresh natural Morus alba L. leaves. Silk cocoons were processed following previously described procedures [Silk cocoons (SC) were obtained from silkworms ocedures . All reaIn order to avoid interferences from the essential oil components, individual plants were firstly distilled in a Clevenger system, after this, the oil free distilled plant material was dried in a forced-air drier at 35 °C for 48 h (until it reached a constant weight) and then ground to pass through a 2-mm mesh. Dried samples (0.5 g) were extracted using 150 mL of methanol in a Soxhlet extractor (B-811) , for 2 h under a nitrogen atmosphere. Methanolic extracts (ME) were taken to dryness at 35 °C under vacuum conditions in an evaporator system (SyncorePolyvap R-96) . The residue was re-dissolved in methanol and made up to 5 mL [The methanolic extracts of 40 individual samples of wild rosemary shrubs from two different bioclimatic areas of Tunisia were characterized by means of HPLC analysis, total phenolic contents, and radical scavenging activity against the DPPH.2, 5 μm pore size, Agilent Technologies, USA) using a guard column  at ambient temperature. Extracts were passed through a 0.45 μm filter  and 20 μL were injected in an Agilent 1200 (Germany) system equipped with a G1311A binary pump and G1315A photodiode array UV/Vis detector. The mobile phase was acidified water containing 0.05% formic acid (A) and acetonitrile (B). The gradient was as follows: 0 min, 5% B; 10 min, 15% B; 30 min, 25% B; 35 min, 30% B; 50 min, 55% B; 55 min, 90% B; 57 min, 100% B and then held for 10 min before returning to the initial conditions. The flow rate was 1.0 mL/min and the wavelengths of detection were set at 280 and 330 nm. Identification of the phenolic components was made by comparison of retention times and spectra with those of commercially available standard compounds. For quantification, linear regression models were determined using standard dilution techniques. Phenolic compound contents were expressed in milligram per gram of dry plant weight (mg compound/g DPW).For the HPLC analysis, a method adapted from Zheng and Wang , was perTotal phenolic content in the extracts was determined by the Folin-Ciocalteu method . BrieflyRadical Scavenging Activity (RSA) was analysed following the method described by Yen and Duh with some modifications . BrieflyResults were expressed as μmol of ascorbic acid equivalents per gram of dry plant weight (μmol AAE/g DPW).2CO3 0.02N for 120 min in order to remove the soluble sericins and obtain smaller nanoparticles [2/ethanol/H2O (1:2:8 in molar ratio) for 3 h at 65 °C. Once, dissolved the silk solution was dialyzed for 48 h against ultra-pure water using a cellulose membrane (cut-off 3.5 KD) and the resultant solution was centrifuged at 6000× g for 10 min and 8 °C (Eppendorf Centrifuge 5810R equipped with a rotor F-34-6-38) in order to remove protein aggregates or impurities and stored at 4 °C until use.Silk fibroin solutions were prepared according to the method previously described by our group without modifications ,53. Briearticles , composeg for 15 min at 8 °C (Eppendorf Centrifuge 5810R equipped with a rotor F-34-6-38).In order to accomplish this assay, three rosemary extract were selected from the GO population, on the basis of their high polyphenolic content and antioxidant capacity. The loading into the silk fibroin nanoparticles (SFNs) was carried out by a coprecipitation method, following the protocol described by Montalbán et al. with slight modifications . BrieflyThe supernatant was decanted and the pelleted nanoparticles were washed with ultrapure water . After lyophilizing the particles for 72 h at −55 °C and 0.5 mbar (Edwards Modulyo 4K Freeze Dryer), the rosemary methanolic extract loaded nanoparticles (RME-SFNs) were obtained in the form of a dry powder. Empty nanoparticles (SFNs) as controls were prepared by using methanol as solvent instead of the methanolic extracts.−1, a spectral range of 4000–550 cm−1, in 64 scans, using N-B strong apodization and Mertz phase correction. The analysis focused on the 1700–1400 cm−1 range, which provides most information on the FTIR spectra of SF.The characterization of the nanoparticles was performed using common techniques such as Field Emission Scanning Electron Microscopy (FESEM), Dynamic Light Scattering (DLS), and Attenuated Total Reflectance-Fourier Transformed Infrared Spectroscopy (ATR-FTIR). The nanoparticles were observed by FESEM using a MERLINTM VP COMPACT . An aliquot (50 μL) of an aqueous suspension of nanoparticles was dropped onto a clean glass wafer before drying overnight and gold-coated. The morphology was studied in images at different magnifications using an SE2 detector . The size distribution of the particles, including mean diameter (Z-average), Polydispersity (PdI) and Zeta potential (ζ), were measured using a Malvern Zetasizer Nano ZSP . The Z-average diameter and ζ values were calculated with the Zetasizer software V. 7.14 provided by the manufacturer. All measurements were performed in quintuplicate (12 runs/measurement) in purified water at 25 °C and values expressed as mean ± SD. Attenuated Total Reflectance Fourier Transformed Infrared Spectroscopy (ATR–FTIR) analysis was performed in order to detect the possible structural changes of SF after loading with the phenolic compounds. Each spectrum was acquired on a Nicolet iS5 spectrometer, equipped with an iD5 ATR accessory  controlled by OMNIC Software Ver. 6.1.0.0038. Measurements were made in absorbance mode with a resolution of 4 cmg for ten minutes and the supernatant was aspired and used for analysis. The extraction was conducted twice and both supernatants were mixture and made up to 2 mL in a volumetric flask. The methanolic extracts were analysed by HPLC and the phenolic compound contents were expressed in microgram of polyphenol per milligram of dry rosemary methanolic extract loaded nanoparticles (µg/mg RME–SFNs). Preparations were performed in triplicate. The EE% was obtained using the Equation (2).The PLC and EE % of the methanolic extracts in the SFNs were determined by HPLC analysis following the previously described protocol in Radical-scavenging activity was analysed following the method previously described in n = 3) and data were reported as means ± standard deviation (SD). A one-way ANOVA was carried out to assess for significant differences  using the IBM SPSS Statistic Program (v. 25). Next, Fisher’s LSD pairwise comparison was performed on the data.All experiments were performed in triplicate (Overall, the major polyphenolic profile of methanolic extract from post distilled rosemary leaves revealed, as expected, the presence of the diterpenes carnosic acid and its derivatives carnosol and 12-me-carnosic as major components, along with rosmarinic acid and hesperidin. Rosemary residues have proven to be an effective potential source of polyphenols and could be useful in replacing or even decreasing synthetic antioxidants in foods, cosmetics and pharmaceutical products. The positive correlation between the phenolic content and the antioxidant capacity confirmed that the phenolic constituents are responsible for the antioxidant activity of rosemary. This knowledge allowed for the selection of three individual plant extracts for the development of the nanoencapsulation assays. The encapsulation of the polyphenols from the rosemary methanolic extracts in silk fibroin nanoparticles increased their size to ~220 nm and shifted their ζ to -17 mV, still useful for cell penetration. Although the described method of encapsulation presents a low encapsulation efficiency and drug loading content, interestingly the adsorption onto silk protects the polyphenols against the degradation. In reference to the loading polyphenol content determinations, diterpene fraction along with the phenolic acids salvianic and rosmarinic acids were retained as major components in the SFNs and although a deeper study should be developed, an initial approach indicated that a higher concentration of the rosemary extract does not always imply greater retention in the SFNs. Thus, further experiments are needed in order to improve the loading efficiency of the aromatic plants extracts nanoencapsulation.


Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium and Bifidobacterium were the most prevalent genera. Increased alpha diversity was associated with a reduction in neopterin concentration. Microbiota composition was different between fecal samples with low and high composite EED scores; increased abundance of Succinivibrio was associated with reduced composite EED scores.Environmental enteric dysfunction (EED) is a subclinical condition of the gut characterized by changes in morphology and function with underlying chronic inflammatory responses. This study characterized composition and diversity of the gut microbiota in rural Malawian children with and without signs of EED. Fecal samples were collected from children aged 1–59 months. Neopterin, myeloperoxidase and alpha-1 antitrypsin concentrations were quantified by ELISA and combined to form a composite EED score using principal component analysis. DNA was extracted from fecal samples and V4-16S rRNA gene sequencing was used to characterize the gut microbiota. The concentrations of all three biomarkers decreased with increasing age, which is consistent with other studies of children living in similar low-income settings.  •Fecal concentrations of myeloperoxidase, alpha-1 antitrypsin and neopterin decreased with age in Malawian children.•A marginal negative association was found between alpha diversity of the gut microbiota and fecal neopterin concentration.•Succinivibrio abundance was higher in children with reduced composite environmental enteric dysfunction biomarker score.•Modelling found 3 metagenomic-metabolic pathways associated with a reduced risk of high composite biomarker score. Gut microbiota; V4-16S rRNA sequencing; PICRUSt2; Environmental enteric dysfunction. Changes in the gut microbiota composition and function have been associated with a number of conditions including environmental enteric dysfunction (EED) , 2. EED et al. [Confirmation of a diagnosis of EED requires visualization of the intestinal epithelium using endoscopy or biopsy, however, these are invasive tests that would be unethical to perform in asymptomatic children. The urinary lactulose: mannitol (L:M) ratio test has been extensively used in the past as a non-invasive test to diagnose EED, however, the test is inconsistent; procedural details, including fasting prior to ingestion, sugar dosage, time of urine collection and method of detection, vary between studies making comparison of results difficult. Leakage of urine sample from pediatric urine bags and urine contamination by stool are other additional challenges in infants. Potential biomarkers that can easily be quantified in fecal samples and blood using ELISA techniques have been tested as biomarkers of EED , 10, 11.et al.  combined22.1This study utilized baseline samples from a nested cohort study, conducted within the framework of a randomized controlled trial (NCT02047981). The study design and methodology for sample selection of both the nested cohort and the parent trial have been previously described , 15, 16.2.2Fecal samples for participating children were collected by their mothers or guardians, who were provided with sterile fecal collection bottles  and given verbal instruction, by a nurse, on how to collect the sample. Fecal samples were returned to the field team as soon as possible after collection. These samples were put in a cool box with ice packs until the end of the day (not more than 8 h after collection) and were then brought to the laboratory where they were placed at -80 °C for storage.2.3Commercially available ELISA kits for MPO , AAT (Immundiagnostik) and NEO  were used for the quantification of each biomarker. Each assay was performed according to the kit instructions using stool diluted 1/500 for MPO, 1/25,000 for AAT and 1/100 in phosphate buffered saline (PBS) for NEO . Fecal s2.416S rRNA gene sequencing and sequence processing were performed as detailed elsewhere . Brieflyde novo-clustered into operational taxonomic units (OTUs) at ≥ 97% identity. Taxonomy was assigned to the OTUs using a naïve Bayes classifier pre-trained on the SILVA 16S database [Raw sequence data was processed in QIIME 2  as per sdatabase  subsequedatabase  and a fr2.5Shannon (H) and Simpson (D) diversity indices were calculated from the OTUs to indicate alpha diversity using the phyloseq R package . The Shaet al. [Median and interquartile range (IQR) concentration of individual fecal biomarkers of EED were used to determine their distribution. Published cut-off values for each of the 3 biomarkers , 13 were2 fold changes was assessed using the default Wald test with Benjamin-Hochberg p value correction. Cut-off for all significant tests was set at p < 0.01. Zero-inflated negative binomial regression analysis was conducted, adjusting for age and sex, to establish the relationship between the differentially abundant OTUs and the composite EED score.Associations between alpha diversity indices and biomarker concentrations were investigated using regression analyses adjusted for age and sex. PERMANOVA with 1000 permutation tests was run on weighted and unweighted UniFrac distances to compare bacteria community compositional differences between groups; differences in UniFrac clustering by groups were visualized using principal coordinate analysis (PCoA) plots. Differential abundance analysis at the genus level was performed to identify OTUs differentially abundant in EED using the DESeq2 package in R . StatistPICRUSt2  analysis2.6This study was conducted in accordance with the Declaration of Helsinki. It was approved by the London School of Hygiene and Tropical Medicine Ethics Committee (UK) and the College of Medicine Research Ethics Committee . Information and consent forms were translated into local languages (Yao and Chichewa) prior to their approval by the local ethics committee. Consent was first obtained at the community level through discussions with the village chief and community elders who then indicated the willingness, or unwillingness, of the community to participate through verbal consent. Written, informed consent (by thumbprint or signature) was then obtained from the parent or legal guardian of each child for inclusion before they participated in the study. During the consenting process, all parents and guardians were informed of their freedom to withdraw their child from the study at any time without giving reason for doing so.33.1One hundred and two baseline samples were analyzed by 16S rRNA gene sequencing as detailed elsewhere . Of thesThe distribution of fecal biomarker concentrations was calculated in all available samples and compared by age. The concentration of all three biomarkers was skewed . A large3.2Age in months was categorized into 4 groups as follows: 0–12, 13–24, 25–36, and 37–60 and the Kruskal-Wallis test was used to test for the differences in biomarker concentration among the groups. Median fecal concentration for all the 3 biomarkers decreased with age .Table 2M3.3Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium, Streptococcus, Bacteroides, Haemophilus, Subdoligranulum, Bifidobacterium and Escherichia-Shigella were the dominant genera. The mean (SD) Shannon index was 1.72 (0.45) and median (IQR) Simpson index was 0.75 (0.16).Characterization of the microbiota of this sample set has recently been described . BrieflyIn the present study, the effect of age on Shannon or Simpson diversity indices and the composition of the microbiota was explored among the 4 age groups. Shannon and Simpson indices were significantly lower in fecal samples from younger children compared to older children, indicating increasing diversity with age   compared to the older age groups  . Additio3.4Linear regression analysis adjusted for age and sex was used to examine the relationship between Shannon or Simpson diversity and fecal concentrations of NEO, AAT and MPO. There was no relationship between Shannon diversity and AAT or MPO concentrations, or between Simpson diversity and AAT or MPO concentrations . However3.52 = 0.04, F = 2.53, p = 0.011) and weighted UniFrac  (Figures PERMANOVA did not detect any differences in bacterial community composition between normal and elevated biomarker concentration for each of the 3 biomarkers . HoweverSuccinivibrio, Butyrivibrio and Enterococcus, were differentially abundant in fecal samples with high composite EED score compared to fecal samples with low composite EED score (Succinivibrio was associated with composite EED score. An increase in the abundance of Succinivibrio was associated with 0.14 reduced odds of a having a high composite EED score (Differential abundance analysis at the genus level, both unadjusted and adjusted for multiple comparison using Benjamin-Hochberg p value correction, was performed to identify genera differentially abundant between low and high EED scores. Three genera, ED score . An age ED score .Table 5D3.6We then used PICRUSt2  analysis4et al. [et al. [et al. [We estimated the magnitude of intestinal inflammation and permeability in rural Malawian children using fecal biomarkers and explored associations with the fecal microbiota, characterized by 16S rRNA gene sequencing. Our study is one of the few studies that has explored the relationship between the gut microbiota and EED using fecal biomarkers. We also inferred metabolic pathways important in EED using PICRUSt2 analysis. We found elevated fecal levels of NEO in almost two-thirds of the children; MPO was raised in one-third of children whilst AAT was elevated in 10% of children. All biomarker levels decreased with age, consistent with trends shown by previous studies from similar low-income settings. In studies by Naylor  [et al.  and McCo [et al. , more th [et al.  and all Actinobacteria and Bifidobacterium, and a lower abundance of the Bacteroidetes and Faecalibacterium compared to the older age groups. These findings are consistent with trends reported by recent longitudinal studies [We also found an effect of age on the microbiota. Microbiota diversity was higher in older age groups compared to younger children. Younger children were shown to have a higher abundance of  studies , 27. The studies , 29.We did not find a relationship between alpha diversity and fecal levels of AAT or MPO, however a marginal negative association was seen between alpha diversity and NEO, a biomarker of intestinal inflammation. To date, there is no other published data available supporting a relationship between fecal microbiota diversity and intestinal inflammation in EED, however, a reduction in fecal microbiota diversity has been reported previously in inflammatory bowel disease , 31, 32,et al. found differences in intestinal bacterial composition between children with increased versus normal intestinal permeability as measured by the L:M ratio test [Succinivibrio, Klebsiella, and Clostridium_XI were less prevalent in children with increased intestinal permeability compared to children with normal levels. Similarly, we found an association between higher abundance of Succinivibrio and low composite EED score. Succinivibrio is a gram-negative anaerobic commensal of the human colon that has been associated with fiber-degradation [Succinivibrio abundance and the composite EED score, which is consistent with the results of others [Succinivibrio in gut function.There is limited published data indicating associations between intestinal bacterial community structure and EED. Recently, Ordiz tio test ; the genradation , 35. Thef others , indicatUsing the 16SrRNA sequencing data generated, PICRUSt2 analysis predicted 67 metabolic pathways of which 5 were identified by the lasso regression model as important for predicting EED. Among the 5 pathways, the fatty acid biosynthesis, pyrimidine nucleotide degradation and tetrapyrrole biosynthesis pathways were significantly associated with composite EED score in an age-adjusted logistic regression model, which, to an extent, supports the prediction accuracy of the lasso regression model. Fatty acid synthase, an enzyme that catalyzes fatty acid biosynthesis, has been shown to help maintain the intestinal mucus barrier by regulating mucin 2 in murine models . In the Succinivibrio in children with low composite EED scores. This bacterium has been associated with metabolism of nondigestible dietary carbohydrates in humans and animals [Succinivibrio abundance, carbohydrate metabolism and gut function.The present study also found a higher abundance of  animals , 35. AmoSuccinivibrio, which was reported by Ordiz et al. [. This bacterium has been associated with fiber-degradation [Succinivibrio, which is consistent with recent findings that used the L:M ratio test to indicate EED [In conclusion, our data suggest that there is an association between intestinal microbiota diversity and intestinal inflammation as measured by fecal NEO. The genus z et al.  to be reradation  and has radation . Future cate EED  undersco5One of the limitations of this study is the use of EED biomarker values from high income countries to define cut-off limits for normal or elevated biomarker concentration. This limitation is not unique to this study; the lack of reference values for the biomarkers of EED in children living in low income settings has been cited by others , 12. TheDavid Chaima & Harry Pickering: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.John D. Hart, Sarah E. Burr, Kenneth M. Maleta, Khumbo Kalua, Robin L. Bailey & Martin J. Holland: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.10.13039/100000865Bill and Melinda Gates Foundation, under grant numbers OPP1066930 and OP1032340.This work was supported by the https://www.ebi.ac.uk/ena/browser/view/PRJEB41238.Data associated with this study has been deposited at The authors declare no conflict of interest.No additional information is available for this paper.