Fatty acid (FA)‐derived lipid products generated by cytochrome P450 (CYP), lipoxygenase (LOX), and cyclo‐oxygenase (COX) influence cardiovascular function. However, plasma measurements invariably ignore 40% of the blood specimen, namely the erythrocytes. These red blood cells (RBCs) represent a cell mass of about 3 kg. RBCs are a potential reservoir for epoxy fatty acids, which on release could regulate vascular capacity. We tested the hypothesis that maximal physical activity would influence the epoxy fatty acid status in RBCs. We used a standardized maximal treadmill exercise according to Bruce to ensure a robust hemodynamic and metabolic response. Central hemodynamic monitoring was performed using blood pressure and heart rate measurements and maximal workload was assessed in metabolic equivalents (METs). We used tandem mass spectrometry (LC‐MS/MS) to measure epoxides derived from CYP monooxygenase, as well as metabolites derived from LOX, COX, and CYP hydroxylase pathways. Venous blood was obtained for RBC lipidomics. With the incremental exercise test, increases in the levels of various CYP epoxy‐mediators in RBCs, including epoxyoctadecenoic acids , epoxyeicosatrienoic acids , and epoxydocosapentaenoic acids  occurred, as heart rate, systolic blood pressure, and plasma lactate concentrations increased. Maximal (13.5 METs) exercise intensity had no effect on diols and various LOX, COX, and hydroxylase mediators. Our findings suggest that CYP epoxy‐metabolites could contribute to the cardiovascular response to maximal exercise. Red blood cells (RBCs) are a potential reservoir for epoxy fatty acids, which on release could regulate vascular capacity. We tested the hypothesis that maximal physical activity would influence the epoxy fatty acid status in RBCs. In our study, we included all RBC FA lipid components in the circulating blood. Epoxides derived from CYP monooxygenase and metabolites derived from LOX, COX, and CYP hydroxylase pathways were measured in RBCs by LC–MS/MS tandem mass spectrometry. ATP release and nitrite reduction to NO have been shown to be involved in regulating this response , leukotrienes (LTs), epoxides, and hydro(peroxy) fatty acids (or oxylipins) are lipid peroxidation products of 20‐carbon (eicosa‐) polyunsaturated fatty acids (PUFA). These products are generated by three separate enzyme families, namely cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) expoygenases. These pathways catalyse lipid peroxidation in a highly regulated manner generating stereo and regio‐specific products Fig. . Their ecis‐ and trans‐EETs are stored in phospholipids and in RBCs. The greater vasodilator potency of trans‐ vs. cis‐EETs may contribute to the antihypertensive effects of sEH inhibitors in rats  have been implicated to serve as a reservoir for epoxide fatty acids, in particular CYP‐derived EETs, which on release may act in a vasoregulatory capacity  signed informed consent forms which outlined the procedures to be taken and the possible risks involved. The study was approved by the Charité University Medicine institutional review board on the use of humans in research. All subjects were non‐trained and not taking medications. Recruitment was primarily via person‐to‐person interview. Following a routine physical examination at baseline levels each subject underwent a maximal treadmill Bruce test, which is recommended by guidelines for ergometry of the German Society of Cardiology , which comprised an inflatable (Riva‐Rocci) cuff placed around the upper arm. Venous blood was collected from a catheter placed in a contralateral forearm vein  of each subject in the sitting position prior to the exercise test , after termination of the test , and 10 min after the end of the running test  . All samples were analyzed for total and free RBC epoxide lipid mediator status. RBCs were separated from EDTA blood by centrifugation and metabolites were determined by liquid chromatography tandem mass (LC–MS/MS) spectrometry as described in Fischer et al., . Arterial pressure was measured P4) Fig. . An addi et al., . Serum l4) Fig. P. All samP) was chosen. The analysis included Mauchly's test of sphericity followed by applying the test of within‐subjects’ effects with Greenhouse–Geisser correction, which is used for repeated‐measures ANOVA when the assumption of sphericity is violated. When significant differences were found, Tukey’s honestly significant difference post hoc test was used for pairwise comparisons. The nonparametric test (distribution‐free) Friedman’s test was used when the normal distribution was violated. Planned hypotheses (one‐tailed or two‐tailed paired t‐tests as appropriate) were tested to follow up the initial ANOVA findings. All data are presented as mean ± SD. All statistical analyses were performed using SPSS Statistics software  or All‐Therapy statistics beta .Descriptive statistics were calculated and variables were examined for meeting assumptions of normal distribution without skewness and kurtosis. In order to determine statistical significance between the trials at the various time intervals, one‐way repeated measures analysis of variance (ANOVA) was conducted and the 0.05 level of significance (P1), after termination of the test , and 10 min after the end of the running test  are shown in Figure P2) was obtained throughout exercise when the heart rate reached 150 beats per minute. After exercise, heart rate and systolic blood pressure returned to resting levels (P1) in the recovery period (P4)   to 9.5 ± 2.1 mmol/L (P3) .Exercise significantly increased heart rate, systolic, and diastolic blood pressure Table . Data prcis‐metabolites in RBCs at: baseline , the time when heart rate reached 150 beats per min (P2), exhaustion (P3), and recovery (P4) , which are potent vasodilators . The data suggest that exercise leads to an accumulation of various CYP (i.e. cis) epoxy‐metabolites into membrane‐bound compartments of RBCs under exhaustive exercise or in ischemia in healthy individuals, which on release may act in a vasoregulatory capacity.Since the impact of acute exercise on individual RBC epoxide fatty acid status is unknown, we initially used an explorative statistical approach and measured the total austion P, and recaustion P, and recaustion P, and recTo provide possible insights into the nature of this accumulation, we measured free epoxy and hydroxy metabolites in RBCs. We detected significant levels of free 13‐HODE, 9,10‐EpOME, 12,13‐EpOME, 9,10‐DiHOME, 12,13‐DiHOME, 14,15‐DHET, 12‐HETE, 12‐HEPE, 4‐HDHA, 14‐HDHA, and 16‐HDHA in RBCs , although no changes occurred in response to exercise , and dihydroxydocosapentaenoic acids (DiHDPAs) by the sEH, respectively Fig.  Spector. Since ain vivo during and post exercise. Similar results were found for the individual metabolites . Notably, we found that EEQs are preferentially metabolized into their diols  compared to EpOMEs, EETs and EDPs  . In our earlier studies, we observed similar results for plasma diols/epoxides in plasma , 16,17‐EDP, 19,20‐EDP occur from increased production and accumulation, rather than altered sEH activity during acute, maximal exercise. Since inhibitors of sEH are in clinical development, these findings could be important for patients.Trans‐EETs are stored in phospholipids and in RBCs in the circulation, and have been proposed to contribute to the antihypertensive effects of sEH inhibitors in rats  and EpOMEs  in RBCs during maximal exercise. 16,17‐EDP and 19,20‐EDP are potent vasodilators in coronary, pulmonary and mesenteric arteries and can lower blood pressure and exhibit cardioprotection by preservation of mitochondrial function . The reason is unclear but may represent a phenomenon similar to the recovery pulse sum, which becomes larger when the loading intensity is above the long‐term performance limit. We suspect that levels of 5,6‐EET, 11,12‐EET, 14,15‐EET, 16,17‐EDP, 19,20‐EDP, 9,10‐EpOME, and 11,12‐EpOME in RBCs will return to normal levels after a longer resting time interval.The Bruce‐protocol was used to ensure that all runners were able to complete a similar highest intensity workload concomitant with robust and significant increases in hemodynamics without fatiguing. Our clinical data Table  show thaOur treadmill testing was primarily developed to identify ischemic heart disease; fortunately, none of our subjects had this condition. Other exhaustive protocols might have been better. Furthermore, the workload was increased in large increments. Primarily the leg musculature was stressed. We obtained venous blood samples and perhaps a source closer to contracting muscle would have been more elucidative (Giordano et al., Our results suggest that maximal exercise affects the levels of numerous CYP epoxy‐metabolites in RBCs. We suggest that RBCs are reservoir for EETs and possibly other epoxide fatty acids, which on release may act in a vasoregulatory capacity. More research is needed to delineate the contribution of RBC epoxy‐metabolites to cardiac performance and skeletal‐muscle blood flow in health and disease.None.


Neoadjuvant chemotherapy (NeoCTx) is performed for most patients with colorectal cancer liver metastases (CRCLM). However, chemotherapy-associated liver injury (CALI) has been associated with poor postoperative outcome. To date, however, no clinically applicable and noninvasive tool exists to assess CALI before liver resection.n = 161) was included to confirm the findings concerning the prediction of postoperative outcome.Routine blood parameters were assessed in 339 patients before and after completion of NeoCTx and before surgery. The study assessed the prognostic potential of the aspartate aminotransferase (AST)-to-platelet ratio index (APRI), the albumin-bilirubin grade (ALBI), and their combinations. Furthermore, an independent multi-center validation cohort , liver dysfunction (LD) , or mortality , and APRI + ALBI had the highest predictive potential for LD . An increase in APRI + ALBI was observed during NeoCTx (P < 0.001). Patients with longer periods between NeoCTx and surgery showed a greater decrease in APRI + ALBI (P = 0.006) and a trend for decreased CALI at surgery. A cutoff for APRI + ALBI at − 2.46 before surgery was found to identify patients with CALI (P = 0.002) and patients at risk for a prolonged hospital stay (P = 0.001), intensive care (P < 0.001), morbidity (P < 0.001), LD (P < 0.001), and mortality (P = 0.021). Importantly, the study was able to confirm the predictive potential of APRI + ALBI for postoperative LD and mortality in a multicenter validation cohort.Higher ALBI, APRI, and APRI + ALBI were found in patients with postoperative morbidity  contains supplementary material, which is available to authorized users. Among these, the aspartate-to-platelet-ratio index (APRI) and the albumin-bilirubin grade (ALBI) have been shown to allow estimation of liver function, as reflected by the Child–Pugh score and indocyanine green (ICG) clearance.25,29,31,32Although APRI was first introduced as a noninvasive marker for hepatitis C-related liver fibrosis,26,37,38Interestingly, studies have found both APRI and ALBI to be predictors of postoperative outcome for patients undergoing liver surgery.34This study aimed to assess the potential of APRI and ALBI to predict a poor postoperative outcome for a homogeneous cohort of CRCLM patients undergoing LR. In addition, the study aimed to assess the potential predictive benefit of APRI and ALBI combined and to elucidate the relation of both markers to NeoCTx.The study enrolled patients undergoing LR between 2001 and 2014 at the Medical University of Vienna who had CRCLM. Data including routine blood parameters were prospectively collected. Information on NeoCTx was documented, and if applicable, blood parameters were assessed before chemotherapy (preNeoCTx) and after completion of chemotherapy (postNeoCTx). Blood was taken from all patients before LR (preOP). Both APRI and ALBI were calculated as specified in Fig. S1.α = 5%, β = 80%).The current study aimed to assess CALI by a rigorous pathologic examination of 170 patients. In addition, the study included a validation cohort recruited at four different institutions . Based on data from the exploration cohort, a sample size calculation was performed, and 158 patients were found to be suitable for validation, based on the proportion of patients with LD in the defined risk groups .P value lower than 0.05 was considered statistically significant. For more information on statistical methods refer to the supplementary material.Statistical analyses, based on nonparametric tests, were performed using SPSS . A Data from 339 patients with CRCLM were collected. The data were prospectively recorded in the institutional database. Patients were included independently of NeoCTx regimen and grouped as “no NeoCTx,” “low hepatotoxicity” including fluorouracil-based regimen, “oxaliplatin,” “irinotecan,” and “oxaliplatin + irinotecan.” Baseline characteristics are presented in Table S1.P = 0.001; Fig. P = 0.064; Fig. P = 0.009; Fig. P = 0.012; Fig. P = 0.037; Fig. P = 0.045; Fig. First, the study aimed to validate previous reports on the predictive potential of preoperative APRI and ALBI concerning postoperative morbidity, LD, and mortality. Accordingly, patients with postoperative morbidity showed higher preoperative levels of ALBI  and APRI alone . Still, APRI and ALBI combined showed the highest discriminatory potential, with an AUC of 0.695 . Thus, further analysis was focused on APRI + ALBI.To compare the potential of APRI and ALBI alone with that of both variables combined to discriminate between patients with and without LD, receiver operating characteristic (ROC) curve analysis was performed. Notably, comparable results were shown by ALBI  and a high-risk group . For the patients above the cutoff, a significantly higher incidences of prolonged hospitalization (> 10 days)  criteria (grades B and C), as visualized in Fig. S3a. Similarly, severe postoperative complications classified higher than Dindo grade 4 were exclusively observed in high-risk patients according to APRI + ALBI . Ultimately, only patients in the high-risk group died within 90 postoperative days  and preoperative gGT  remained as significant independent variables after stepwise forward selection.To test for the independence of the proposed cutoff from other predictors of LD and to identify potential confounding factors, multivariable analysis was performed (Table S2). However, after the proposed cutoff for APRI + ALBI, the extent of resection, the alkaline phosphatase (AP) level, the gamma-glutamyl transferase (GGT) level, and the retention rate after 15 min for ICG clearance were entered into the multivariable model, only the proposed cutoff . Indeed, the decrease in APRI + ALBI was more pronounced for the patients with a period of 6 to 9 weeks after NeoCTx than for the patients who had a break shorter than 3 weeks before LR .To confirm the obtained data regarding the predictive potential of APRI + ALBI for postoperative outcome, a multi-center validation cohort was investigated. This analysis included 161 patients  with a much higher sensitivity. Whereas severe steatosis was detected with a sensitivity of 90%, the cutoff was able to identify SOS with a sensitivity of 77.8%, and detection of CASH showed sensitivities of 86.7% (NAS ≥ 4) and 92.3% (Brunt = 3). This favors the use of APRI + ALBI as a noninvasive tool for the sensitive identification of CALI before surgery.Interestingly, the patients in the high-risk group had a significantly higher prevalence of severe steatosis and CASH and a tendency toward an increased prevalence of SOS. The was paralleled by a substantially higher incidence of adverse clinical outcomes. Importantly, the gathered data could be confirmed in an independent multi-center cohort of CRCLM patients undergoing LR at four institutions. Accordingly, this leads to the suggestion that APRI + ALBI is able to stratify patients as those with clinically relevant forms of CALI and those with only slightly injured livers after NeoCTx.R = − 0.115; P = 0.474), suggesting that the susceptibility of patients to the development of CALI might be more relevant than the duration of NeoCTx itself.Most importantly, the current study introduced APRI + ALBI as a useful tool for timing LR after NeoCTx. We present solid data on the increase in APRI + ALBI during NeoCTx, suggesting a direct correlation between the marker and induced liver damage. Interestingly, this increase was not affected by the number of NeoCTx cycles administered Below is the link to the electronic supplementary material.


Fabry disease is an X‐linked lysosomal storage disorder with heterogeneous clinical expression in female patients ranging from asymptomatic to severe clinical presentations as in classic males. We assessed clinical profiles and compared natural history data of female patients eventually initiated on enzyme replacement therapy (“ERT‐recipients”) with those remaining untreated (“ERT‐naïve”).We analyzed Fabry Registry data from 93 ERT‐recipients, collected prior to ERT initiation, and 76 ERT‐naïve females with classic or unclassified phenotypes from four Latin American countries and evaluated Fabry symptoms, interventricular septum thickness, left ventricular posterior wall thickness, estimated glomerular filtration rate, and severe clinical events.P < .01) and last follow‐up . Reported Fabry symptom frequencies and abnormal echocardiography findings were higher in ERT‐recipients. Functional renal assessments were normal and similar.For 169 patients with available data, median age of first Fabry symptom manifestation was 12.7 years with peripheral pain as predominant first symptom, and diagnostic delay of 10.3 years from the first reported symptom. Female patients had high symptomatic burden during natural history follow‐up, with 83% reporting peripheral pain, 69%‐79% cold/heat intolerance or abnormal sweating, and 32% gastrointestinal symptoms. ERT‐recipients reported similar age at first symptom as ERT‐naïve patients but they were older at diagnosis (median 39.2 vs 24.4 years, Female patients from Latin America have notable diagnostic delays and high symptomatic burden. ERT was prescribed late in females with advanced age at diagnosis and advanced disease. There remained many female patients who had been diagnosed at younger age, had substantial Fabry manifestations, but did not receive disease‐specific treatment. Pathogenic variants of the 500 is a 22.1www.fabrydisease.org/images/ReferencePDFs/fabry-registry-schedule-of-assessments.pdf), but treating physicians determine the actual frequency of assessments according to a patient's individualized need for medical care and routine follow‐up. The Fabry Registry is registered at www.ClinicalTrials.gov (NCT00196742) and is sponsored by Sanofi Genzyme.The Fabry Registry is a global, longitudinal, observational program designed to track the natural history and treatment outcomes of patients with Fabry disease. It was initiated in 2001 in an effort to help practitioners involved in the diagnosis and/or treatment of Fabry disease to increase their understanding of the disease, facilitate clinical management, and develop Fabry disease treatment monitoring guidelines. Patient and investigator participations are voluntary. Recommended schedules of clinical assessments are available  were included in the analysis. Natural history data from females who were subsequently initiated on ERT (“ERT‐recipients”) were compared with those from females who were not treated with ERT (“ERT‐naïve”).GLA variants categorized in the fabry-database.orgGLA variant database (http://fabry-database.org/mutants/) as being associated with “classic” Fabry disease or variants reported to the Fabry Registry but not entered or classified in this database. We excluded patients with reported GLA variants classified as later‐onset phenotypes and variants previously reported to be of uncertain significance, as well as patients whose genotype had not been reported to the Fabry Registry.The analysis population was restricted to females with 2.3We analyzed demographic and clinical data on Fabry disease presentation and manifestations collected during the natural history period. Echocardiographic assessments included interventricular septum thickness (IVST) and left ventricular posterior wall thickness (LVPWT). Assessments of renal function were estimated glomerular filtration rate , urine albumin‐to‐creatinine ratio (UACR), and urine protein‐to‐creatinine ratio (UPCR). Severe clinical events were defined as: (a) cardiovascular events: significant cardiac procedure , arrhythmia , angina pectoris, myocardial infarction, congestive heart failure, cardiac syncope; (b) renal events: chronic dialysis, kidney transplantation; and (c) cerebrovascular events: stroke, transient ischemic attacks (TIAs).2.4Descriptive statistics were calculated for demographic and clinical data. Numbers of patients who ever reported clinical assessments or events were obtained. The last assessment along with the corresponding age parameters during the natural history period was estimated and the last assessment was defined as the last value of any given parameter within a 2‐year window before the end of natural history follow‐up in ERT‐naïve patients, or a 2‐year window before the treatment initiation in ERT‐recipients. We also reported age at the first clinical assessment or age at the first event. In addition, IVST, LVPWT and eGFR assessments were analyzed cross‐sectionally by grouped age at assessment: 18 to <30 years, 30 to <40 years, 40 to <50 years, 50 to <60 years, and ≥60 years.P < 0.05 was considered to represent statistical significance. Statistical analyses were performed using SAS statistical software V.9.2 .Continuous variables were analyzed using the Wilcoxon signed rank test and categorical variables using a chi‐squared or Fisher's exact test between ERT‐recipients and ERT‐naïve patients. All statistical tests were two‐sided and 33.1GLA variants associated with a classic phenotype were reported for 46.7% of the female patients and 53.3% had reported GLA variants either not listed in the fabry-database.org database or listed without a phenotype classification. The phenotype percentages for ERT‐recipients and ERT‐naïve patients were similar.Patient demographics are summarized in Table P = 0.04). Data on the type of first (any) manifestation of Fabry disease and age at onset were available for approximately half of the patients (Table P = 0.58). Peripheral pain was predominantly reported at onset . The median age of disease diagnosis was 29.9 years, with ERT‐recipients being significantly older at diagnosis . For 92 patients with both age parameters available, the median time from the first Fabry disease manifestation to diagnosis was 10.3 years and, although longer for ERT‐recipients, statistically comparable for the two groups  (Table P = 0.36), but ERT‐recipients had a significantly older age at last natural history follow‐up . In ERT‐recipients, treatment was initiated at a median age of 43.2 years.A total of 86.4% of the patients had a family history of Fabry disease and the ERT‐recipients group had a significantly higher family history compared with the ERT‐naïve group  and abnormal sweating . For all types of Fabry symptoms, the frequency of reports at the last natural history assessment were lower as compared with frequencies observed during the entire follow‐up period (ever reported). The percentages of patients with available data were substantially lower for ERT‐recipients for all Fabry symptoms.Frequencies of Fabry symptoms reported during natural history follow‐up and ages at onset are presented in Table 3.3P = 0.93). Last assessments of IVST were performed at an older median age in ERT‐recipients (46.1 [n = 40] vs 30.8 years [n = 26]). Median IVST values for both groups were similar (10.0 vs 9.0 mm), and the percentages of patients with a last assessment of ≥10.0 mm were 62.5% and 42.3%, respectively.Echocardiographic data of IVST and LVPWT were available for 54 ERT‐recipients (58.1%) and 44 ERT‐naïve females (57.9%) Table . The firP = 0.40). Last assessments of LVPWT were performed at older median age in ERT‐recipients (44.8 [n = 41] vs 31.9 years [n = 25]). The median LVPWT values were similar for both groups (10.0 vs 9.0 mm). The percentages of patients with a last assessment of ≥10.0 mm were 53.7% and 24.0%, respectively.The median ages at first abnormal LVPWT value ≥10.0 mm were sim3.42, indicating chronic kidney disease, were similar for both groups . At the last assessment performed at median ages of 46.8 (n = 42) and 32.1 years (n = 30), respectively, median eGFR values for both groups were similar (99.9 vs 103.8 mL/min/1.73 m2), as were the distributions over eGFR categories.eGFR data were available for 56 ERT‐recipients (60.2%) and 51 ERT‐naïve females (67.1%) Table . The med2 for both ERT‐recipients and ERT‐naïve female patients : cardiovascular events: 4.3%, 2.6% ; cerebrovascular events: 10.8%, 3.9% ; dialysis/kidney transplantation: 1.1%, 5.3% .4Our Fabry Registry study provides demographics and natural history clinical characteristics of Latin American female patients with Fabry disease with a breakdown of patients who did eventually receive ERT (ERT‐recipients) and those who did not (ERT‐naïve). We were particularly interested in comparing these two groups with regard to clinical presentation, timing of diagnosis, and the natural course of Fabry disease.GLA mutation associated with a classic phenotype.Females in both groups often experienced a neurologic symptom as their first Fabry disease manifestation  in the second decade of life. They carried a high symptomatic burden during (early) adulthood, with numerically higher rates of all Fabry‐related symptoms in ERT‐recipients at older ages compared with ERT‐naïve patients, consistent with the progressive nature of the disease. Yet, female patients were at risk of being diagnosed late; ERT‐recipients were diagnosed at a significantly older age (median 39.2 vs 24.4 years). The long diagnostic delays (median 13.1 vs 7.8 years), despite a predominance of females with a family history, suggests that early case ascertainment as a consequence of family screeningWe found higher percentages of ERT‐recipients reporting Fabry clinical symptoms during natural history follow‐up, although fewer females had data available, compared with ERT‐naïve patients. Moreover, ERT‐recipients were characterized by cardiovascular involvement, with more patients in the IVST ≥ 10.0 mm category (62.5% vs 42.3%) and the LVPWT ≥ 10.0 mm category (53.7% vs 24.0%) at last natural history assessment, as compared with ERT‐naïve females. This could be because they were diagnosed at an older age (39.2 vs 24.4 years) and therefore had progressed to a more advanced stage of Fabry disease. It is noteworthy that functional renal assessments (medians) were normal and similar for both groups. It should be noted that, as in male Fabry patients, female patients may develop myocardial fibrosis before the development of left ventricular hypertrophy (LVH), suggesting that LVH may not be the only driver of cardiac disease burden in female patients.More ERT‐recipients experienced a cerebrovascular event (10.8% vs 3.9%) and, overall, renal events were the least common type of severe event. Compared with other Fabry Registry studies that used the same severe event definitions,It is encouraging that for ERT‐recipients, there was only a few years delay between diagnosis age 39.2 years) and initiation of treatment (age 43.2 years), but it is worrisome that there was a large proportion of females reporting Fabry symptoms who never received ERT. For example, around 70% of patients in the ERT‐naïve group reported peripheral pain and abnormal sweating, 66% and 54% intolerance to high and low temperatures, respectively, and around 24% pain crises and GI symptoms. Reluctance to treat symptomatic females has been previously reported in other countries..2 years GLA variants have unclassified phenotypes. To minimize the impact of phenotype heterogeneity on the analyses, we restricted the main analysis population to females with GLA variants associated with “classic” Fabry disease or variants that had been reported to the Fabry Registry but had not been entered or classified in the fabry-database.org database. This inclusion criterion was supported by a comparison of the demographics, baseline clinical characteristics and last assessments of the groups of female patients having these variants showing that both groups had similar demographic and clinical features  monitoring outside these centers. Patient reports to the physician and subsequently to the Fabry Registry may have been limited by patient recall. Incomplete data sets for several critical clinical parameters including left ventricular mass index, residual α‐Gal activity, plasma GL‐3, and lyso‐GL‐3 precluded meaningful analysis. Cardiac fibrosis and X‐chromosome inactivation pattern data were not available for analysis. We were unable to meaningfully analyze changes in symptom reports and intensity and frequency, and progression of organ involvement over time in individual patients given the paucity of data, or determine which patient‐specific clinical considerations led, or could have led, to timely initiation of disease‐specific treatment according to published recommendations. The past decades have witnessed improvements in awareness and understanding of Fabry disease, as well as in diagnostic practices. We have not analyzed changes in diagnostic delays and timing of initiation of ERT over time. Finally, Fabry disease has a remarkable genotypic variability and many features .In conclusion, our study confirms that female patients from Latin America do experience symptoms of Fabry disease—predominantly peripheral pain evident from early adolescence—and experience notable delays in diagnosis. Many female patients carry a high symptomatic burden but may not receive sufficiently early disease‐specific treatment. ERT was prescribed late in female patients with older age at diagnosis and advanced disease. There remained many female patients who had been diagnosed at a younger age, had substantial manifestations of Fabry disease, but did not receive disease‐specific treatment.An increase in awareness of the natural history of Fabry disease in female patients and the consequences of late initiation of treatment, and the encouragement of frequent comprehensive monitoring and family pedigree analysis can help identify female patients at an earlier age. Once the patients are diagnosed, timely initiation of treatment could be enabled by researching and understanding the non‐clinical prompts and barriers to starting disease‐specific treatment in female patients in Latin America who suffer from this progressive disease.A.M.M. is a Fabry Registry Board member and received research funds, travel support, and speaking fees from Sanofi Genzyme, Alexion, and Biomarin. G.C. has consulting arrangements with and received speaking fees from Sanofi Genzyme. He received travel support from Sanofi Genzyme and Takeda. F.M. is a Fabry Registry Board member and received honoraria for lectures and board meetings from Sanofi Genzyme and speaking fees from Takeda and Merck Serono. F.S‐O. is a Fabry Registry Board member and received research funds, travel support, and speaking fees from Sanofi Genzyme. R.A.V. is a Fabry Registry Board member and received travel support and speaking fees from Sanofi Genzyme. C.V. is a Fabry Registry Board member and received honoraria for lectures and board meetings from Sanofi Genzyme and speaking fees from Takeda. M.Y. is an employee of Sanofi Genzyme. J.M.P. received honoraria for presentations and board meetings from Sanofi Genzyme, Takeda, and Amicus Therapeutics.A.M.M., G.C., F.M., F.S‐O., C.V., and J.M.P. contributed to the data acquisition, data analysis/interpretation, drafting the manuscript, and critically revising it. R.A.V. contributed to the data interpretation, drafting the manuscript, and critically revising it. M.Y. contributed to the data analysis/interpretation, drafting the manuscript, and critically revising it.A.M.M. accepts full responsibility for the work and/or the conduct of the study, had access to the data, and controlled the decision to publish.Patients' informed written consent to submit their health information to the Fabry Registry and to disclose this information in further analyses is obtained by each independent site.The Fabry Registry protocol, informed consent form, and any locally required authorization documents are reviewed and approved by the local fully constituted Institutional Review Board or Independent Ethics Committee unless the site provides the Registry with documentation that approval is not required or has been waived.Table S1 First manifestation of Fabry disease present at onset by organ systemTable S2 IVST assessments during natural history follow‐up in the Fabry Registry by age groupsTable S3 LVPWT assessments during natural history follow‐up in the Fabry Registry by age groupsTable S4 eGFR assessments during natural history follow‐up in the Fabry Registry by age groupsTable S5 UACR and UPCR assessments during natural history follow‐up in the Fabry RegistryTable S6 Severe clinical events occurring during natural history follow‐up in the Fabry RegistryTable S7 Demographics and clinical characteristics during natural history follow‐up in the Fabry Registry by phenotypeClick here for additional data file.


Hyperglycemia in pregnancy is a medical condition resulting from either pre-existing diabetes or insulin resistance developed during pregnancy. This study aimed to determine the prevalence of hyperglycemia in pregnancy and influence of body fat percentage and other determinants on developing hyperglycemia in pregnancy among women in Arusha District, Tanzania.A cross–sectional study was conducted between March and December 2018 at selected health facilities in Arusha District involving 468 pregnant women who were not known to have diabetes before pregnancy. Blood glucose was tested by Gluco-Plus™ using the World Health Organization criteria at fasting and 2 h after consuming 75 g of glucose dissolved in 300 ml of water. Body fat was measured using a bioelectric impedance analyzer, mid-upper arm circumference using a regulated tape, weight using SECA™, blood pressure using a GT-868UF Geratherm™ machine, and height using a stadiometer. Demographic and maternal characteristics were collected through face to face interviews using a structured questionnaire.2 (SD ± 5.5). One-third of participants had mid-upper arm circumferences ≥28 cm with 25% being overweight and 22.7% obese before pregnancy. Prevalence of hyperglycemia in pregnancy was 16.2% (n = 76) of which 13% had gestational diabetes and 3.2% diabetes in pregnancy. Hyperglycemia in pregnancy was significantly associated with body fat percentage , family history of Type 2 diabetes mellitus , previous delivery of babies ≥4 kg , mid-upper arm circumference ≥ 28 cm , and Type 2 diabetes mellitus symptoms .The participants’ mean age was 28 years (SD ± 6), mid-upper arm circumference 27 cm (SD ± 3.7), body fat 33.72% (SD ± 7.2) and pre-pregnancy body mass index 25.6 kg/mThe prevalence of hyperglycemia in pregnancy was high, particularly among women with history of delivering ≥4-kg babies, increased body fat, mid-upper arm circumference, symptoms and/or family history of Type 2 diabetes mellitus. These findings identify opportunities to further explore the utility of body fat percentage and other determinants for rapid screening and management of hyperglycemia in pregnancy. Hence, HIP is a result of either pre-existing diabetes or insulin resistance developed during pregnancy, a condition known as gestational diabetes mellitus (GDM) which is defined as impaired glucose tolerance first recognized during pregnancy [Hyperglycemia in pregnancy (HIP) is one of the most common pregnancy-specific health challenges . Hyperglregnancy –4. This regnancy . In mostregnancy .Globally, the prevalence of GDM among women aged 20–49 years was estimated to be 17% affecting 21.4 million live births with more than 90% cases occurring in low- and middle-income countries . EstimatIn addition to sedentary lifestyle, maternal height, dietary factors, cigarette smoking, and extreme pregnancy weight gain accompanied by high body fat accumulation place women at risk of HIP. Body fat percentage can alter body composition leading to pregnancy diabetes and other complications, such as pregnancy induced hypertension and predisposition of the newborn to overweight later in life –14. It cFew studies have assessed the effects of body fat on development of GDM with most extant studies considering Caucasian and Asian populations , 23. ThiIn addition to body fat percentage, MUAC has a strong correlation with BMI  thereforThe current study was conducted in Arusha District, which has a known high prevalence of T2DM (16.2%) especially in urban (22.9%) compared to rural (9.9%) areas , althougA cross-sectional study was conducted in urban areas of Arusha District between March and December of 2018. The study involved pregnant women attending antenatal clinics at Ngarenaro and Kaloleni Health Centers in Arusha District. The two centers were purposively selected due to their central location and large numbers of pregnant women (on average 40–100 per day) accessing Ante-Natal Care (ANC) services from across the District, thereby representing the demographics of this predominantly urban patient population. The study included women with gestational ages between 24 and 36 weeks anticipating delivery at one of the participating centers. Pregnant women with previously diagnosed diabetes and selected conditions, such as sickle-cell anemia or cancer, were excluded from the study. The aim, procedure, benefits, and negative effects of the study were explained to all enrolled women agreeing to participate and signed informed consents. The study was approved by the Tanzania National Institute of Medical Research (NIMR) with a reference number NIMR/HQ/R.8a/Vol.IX/2694.n = 468) completed fasting blood glucose tests and face-to-face interviews using structured questionnaires. In addition, nearly all  of the participating women completed Oral Glucose Tolerance Test (OGTT) procedures.The eligible women were selected with assistance of the nurse in-charge resulting in a total of 468 women being involved in the study. Sample size was determined in accordance with the formula for prevalence studies . Due to Recalled information respecting weight before pregnancy, first generation family history of T2DM, previous history of GDM, symptoms of T2DM, and previous delivery of babies weighing ≥4 kg at birth were collected through face-to-face interviews using a structured questionnaire. Other maternal characteristics, such as age, perinatal and prenatal death, gravidity, education level, occupation, marital status, and weight during the first pre-natal visit, were obtained from the participants’ ANC records.Women were required to fast overnight before capillary blood was taken using a finger prick with a sterile lancet after cleaning the site with an antiseptic alcohol swab. Fasting blood glucose and 2 h OGTT following the consumption of anhydrous glucose of 75 g dissolved in 300 ml of water  were meaMid-upper arm circumference (MUAC) was measured using a non-stretchable tape and women were categorized as normal with a MUAC of < 28 cm and overweight with MUAC ≥28 cm. Mid upper arm circumference was used to supplement BMI due to its relative stability during the course of pregnancy and high correlation with pre-pregnancy BMI, making it a better indicator of pre-pregnancy body fat and nutrition status than the BMI , 36. Weip value ≤0.05. Some factors were potential confounders, hence they were introduced into the model during the analysis, such as age, and gestational age. The variables with missing data  were analyzed by “pairwise” deletion where the statistical procedure used cases that contain some missing data. Hence, the entire case/respondent was not left out of subsequent analyses for elements on which data were complete. Other variables, which were missing due to mistakes in recording, were traced back from the participants as their phone numbers and other contacts were recorded. Other missing data were traced from ANC records available in the health center. Student’s t-test was used to compare the means for HIP and non-HIP groups for continuous variables, such as mean OGTT and fasting glucose values.Data collected were analyzed using the Statistical Package for Social Science™ (SPSS) Version 20 and descriptive statistics, such as frequency, mean and percentage, were obtained. Chi-square test was used for comparing the selected categorical variables between women with and those without HIP. Blood glucose levels were dichotomized, and univariate analysis was done for the variables associated with HIP to obtain crude odd ratios. Multiple logistic regression analysis explored whether different factors had significant association with HIP where the quantitative variables such as body fat percent, BMI and MUAC were treated as continuous variables. Statistical inference was based on 95% confidence intervals (CIs) and significance at n = 459), and over half had attended primary school  and were self-employed  primarily in small businesses earning an average income of < 250,000 Tanzanian Shillings (TSH) per month (approximately 110 American dollars). The mean age of the women was 28 years (SD ± 5.84), of which 65.6% were ≥ 25 years old. The mean gestational age at the start of ANC was 18 weeks (SD ± 5.62) and 28.5 weeks (SD ± 3.82) at the beginning of this study where nearly two-thirds  were beyond 28 weeks gestation at time of entry to the study with 50.4% (n = 236) reported as second or third gravidity  were classified as overweight and 22.7% (n = 54) as obese. The measured mean height was 159 cm (SD ± 6.3), body fat 33.7% (SD ± 7.2), and MUAC 27 cm (SD ± 3.8) in which 36.1% (n = 164) had MUAC ≥28 cm . This weight was used to determine pre-pregnancy BMI of the women in which 25.2% (.5. This n = 468) completed fasting blood glucose tests and 97.8% (n = 446) underwent an OGTT. The prevalence of HIP was 16.2% (95% CI: 13–19.9) of which 3.2% (n = 15) had DIP and 13% (n = 61) GDM using the WHO [n = 51) reported to have symptoms of T2DM, such as extreme tiredness, diaphoresis (excessive sweating), and polydipsia (excessive thirst)  mean compared to the non-HIP group . The overall mean for OGTT was 5.5 mmol/L (SD ± 1.06) and was significantly higher (p < 0.001) among the HIP  compared to the non-HIP group  , with the HIP sub-group having a mean of 6.4 mmol/L (SD ± 1.5), yielding a significantly higher (p < 0.000) among women with a family history of T2DM ; MUAC ≥28 cm ; previous delivery of an infant ≥4 kg ; and symptoms of T2DM . The observed prevalence of HIP did not differ significantly among women with histories of prenatal or perinatal death, high blood pressure, high maternal age, or smoking and alcohol intake habits (p > 0.05) , family history of T2DM  and previous delivery of babies ≥4 kg , and/or having any symptoms of T2DM  in the first model. The second model replaced body fat percentage with MUAC and the association remained consistently significant in all factors of the first model with addition of MUAC   of the women were found to have high cut off points which indicated T2DM. This creates a need to promote pre-pregnancy preparation that include regular screening for diabetes before conception for earlier efforts to prevent the development of HIP. Hyperglycemia in pregnancy has few symptoms and should be diagnosed by screening during pregnancy [Nevertheless, some women with HIP reported having one or more symptoms of T2DM, such as extreme tiredness, diaphoresis (excessive sweating), and polydipsia (excessive thirst) and increased frequent urination. These symptoms were found to have a strong independent association with HIP even after adjusting for MUAC, BMI, gestational age and family history of T2DM. Considering the asymptomatic nature of HIP , these wregnancy . Anotherregnancy . Hyperglregnancy , 46. HenSome data were collected based on participants’ ability to recall. For example, BMI was based on recall data where almost half of the respondents could not report their pre-pregnancy weight leading to missing data; hence, interpretations on the rate of overweight and obesity need to be done carefully. In addition, GDM was tested by one out of two elevated glucose levels instead of one out of three values as recommended .The prevalence of HIP was observed to be high in urban areas of Arusha District and significantly associated with family history of T2DM, increased MUAC, body fat percentage, and having one or more symptoms of T2DM. The reported risk factors can be used to identify women at risk of HIP early enough for earlier interventions to be taken to prevent poor pregnancy outcomes especially in resource poor settings where universal screening is challenging. Modifiable risk factors, like body fat percentages for assessing nutrition status in pregnancy, need to be further explored to identify the proper methods for body fat estimation and develop criteria for classification in pregnancy. Prevention strategies for HIP need to be directed towards the knowledge and control of the risk factors reported in this study and others for promoting self-care before, during, and after pregnancy. This proactive approach can potentially reduce the impacts of non-modifiable risk factors, such as age and genetic predisposition in developing HIP.


To study the development of refractive status from 36 weeks to one year of postmenstrual age and to identify factors that contribute to development of myopia, including gender, birth weight, gestational age, and retinopathy of prematurity (ROP).  Premature infants underwent full cycloplegic retinoscopy at 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 3 months, 6 months, 9 months, and 12 months of postmenstrual age. The infants were grouped by gender, birth weight, gestational age, and the severity of ROP to evaluate the correlation with refractive status at each postmenstrual age. r = 0.42, P < 0.0001). After that, the mean spherical equivalent (SE) gradually declined . Boys had lower hyperopia than girls at nine months  and one year  of postmenstrual age. Premature infants with ROP had a lower average SE at most of the postmenstrual ages; however, this value did not vary significantly (P > 0.05). Premature infants with severe ROP were less hyperopic than those without it at every postmenstrual age, and the average SE differed significantly at one year of postmenstrual age . There was no significant difference between each birth weight and gestational age (P > 0.05).  A total of 942 infants were recruited in this study. A total of 2716 readings were obtained. Refractive state had a hyperopic shift until 46 weeks of postmenstrual age ( The dioptric value of premature infants within one year was generally hyperopic. Different gender, birth weight, gestational age, and ROP did not affect the overall development of refractive status. Females may have higher hyperopia at nine months of postmenstrual age. Birth weight and gestational age had little effect on change of refractive status. Severe ROP was an important contributing factor in myopia progression, which may be related to the treatment required. Further study may be carried out to understand the mechanism behind myopia progression in premature infants, including changes in refractive system parameters and emmetropization process. With the improvement of neonatal care technology, the number of premature infants is increasing. Prematurely born children are at increased risk of visual disability. A number of studies have reported presence of refractive errors to be more common in prematurely born children –4. It haIt remains controversial whether either ROP disease or ROP treatment or both of them are myopia-related factors. The two correlative factors have been analyzed, however, without control groups at the same postmenstrual age. This may lead to inexact results. Studies that analyzed large-sample of patients at the same postmenstrual age are still rare.Our purpose was to study the development of refractive status from 36 weeks to one year of postmenstrual age and to identify factors that contribute to the development of myopia, including gender, birth weight, gestational age, and ROP.A total of 942 premature infants who were screened at the hospital for retinopathy of prematurity (ROP) participated in this study. The exam was performed at the Eye Hospital of Wenzhou Medical University from January 2016 to May 2018. Regional Ethics Committee approval and parental consent were obtained in each case. Infants with poor physical condition leading to intolerance of examination, infectious disease, and refractive interstitial opacity such as congenital cataracts were excluded from this study.The eye examinations were performed respectively at 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 3 months, 6 months, 9 months, and 12 months of postmenstrual age. All methodologies adhered to the tenets of the Declaration of Helsinki.One drop of 5 mg/ml topical tropicamide was instilled three times in each eye at an interval of 10 minutes. Full cycloplegic retinoscopy was performed 30 minutes after the last instillation using a streak retinoscope . Eyelids were held open with a pediatric speculum after topical anesthesia with 1 drop of 0.5% proparacaine hydrochloride. Retinoscopy was performed by the same examiner  at the Eye Hospital of Wenzhou Medical University, Hangzhou, China. An allowance of 2.00 diopters (D) was made for a working distance of 50 centimeters. Refractive error was recorded in the form of spherical equivalent (SE), where the SE = sphere + cylinder/2. The basic information, including gender, birth weight, gestational age, postmenstrual age, and presence of ROP, was recorded at each follow-up. Premature infants were classified by gender, birth weight, gestational age, and severity of ROP to evaluate the correlation between these parameters and refractive status. ROP that required treatment was defined as severe ROP.t-tests were used to evaluate the correlation between these parameters  and refractive status at each postmenstrual age. Significance was defined as P < 0.05.The right eye of each infant was used for further data collection and analysis. Statistical analysis was performed using the Statistical Package for Social Sciences . The results were recorded as the means ± standard deviations (SD). Correlation analysis was used to analyze the relationship between SE and postmenstrual age. Independent samples A total of 942 infants  were recruited in this study. The average gestational age was 30.3 ± 2.3 weeks . The average birth weight was 1,485.8 ± 401.8 g . A total of 2716 readings were obtained.r = 0.42, P < 0.0001). After that, the mean SE gradually declined and reached +(0.64 ± 1.12) D at one year of postmenstrual age  (The mean SE was +(1.12 ± 2.10) D at 36 weeks of postmenstrual age followed by a hyperopic shift until the SE reached +(3.62 ± 1.77) D at 46 weeks of postmenstrual age ( 0.0001) .t = 3.10, P=0.003) and one year  of postmenstrual age.The mean SE of boys was more hyperopic than that of girls during the early period. Beginning from 6 months of postmenstrual age, the SE of boys rapidly decreased with a myopic shift . There w≤ 30 weeks; GA > 30 weeks) and birth weight  to evaluate the correlation between refractive status and the two variables, respectively. At the same postmenstrual age, there was no significant difference between the groups (P > 0.05)  Tables .P > 0.05). Premature infants with severe ROP were more myopic than those without severe ROP at each postmenstrual age. The average SE differed significantly at one year of postmenstrual age   .In our research, the mean refractive index was +1.12 D at 36 weeks of postmenstrual age followed by a hyperopic shift until the refractive index reached +3.62 D at 46 weeks of postmenstrual age. Then, the mean SE gradually declined and reached +0.64 D at one year of postmenstrual age. In the study by Yu et al. , the refPremature infants were reported to be more susceptible to myopia development. Through a 10-year follow-up, Larsson and Holmstrom  found thStudies –7 on theROP is an important factor leading to myopia progression. Previous research , 22 founThe refractive state had a hyperopic shift until 46 weeks of postmenstrual age. After that, the mean dioptric value gradually and continuously declined by one year of postmenstrual age. In general, the dioptric value of premature infants within one year is generally hyperopic. Different gender, birth weight, gestational age, and ROP did not affect the overall development of refractive status. Gender may not have an effect on dioptric values at an early age; however, females may have higher hyperopia at nine months of postmenstrual age. Birth weight and gestational age had little effect on change of refractive status. Severe ROP is an important contributing factor in myopia progression, which may be related to the treatment required.The relationship between dioptric value and refractive system parameters, such as axial lengths and corneal curvature, is expected to be further studied to aid in understanding the mechanism of myopia development. We will also conduct a long-term follow-up study on premature infants to observe the emmetropization process, to discuss the physiological reference value for development of refractive state in Chinese premature infants at different stages and to understand the time point for emmetropization, all of which will be valuable in preventing myopia progression.


Aflatoxin B1 (AFB1) exposure is a crucial factor to initiate hepatocellular carcinoma (HCC). However, comprehensive microRNA (miRNA)‐message RNA (mRNA) regulatory network regarding AFB1‐associated HCC is still lacking. This work was aimed to identify miRNA‐mRNA network in primary human hepatocytes after AFB1 exposure.A miRNA expression dataset GSE71540 obtained from the gene expression omnibus (GEO) was used to identify differentially expressed miRNAs (DEMs) after AFB1 exposure using GEO2R. Target genes of these DEMs were identified using TargetScan V_7.2, miRDB, PITA, miRanda, and miRTarBase. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed at Database for Annotation, Visualization and Integrated Discovery (DAVID). miRNA‐mRNA regulatory network was established by analyzing three enriched KEGG pathways significantly correlated with HCC onset and then visualized at CytoScape.In this work, nine upregulated and nine downregulated DEMs were identified. Functional enrichment analyses showed that these predicted target genes were significantly associated with cancer development. Analysis of three enriched pathways related to the onset of HCC identified 13 and nine target genes for upregulated DEMs and downregulated DEMs, respectively. Subsequently, the miRNA‐mRNA regulatory networks were constructed.In conclusion, miRNA‐mRNA regulatory network was established, which will help to understand the mechanism underlying the AFB1‐induced onset of HCC. This dataset contained data from primary human hepatocytes subjected to 1 µM of AFB1 for 5 days followed by a 3 days wash‐out period or not.GSE71540, obtained from Gene Expression Omnibus (2.2p < .05 and |log2 fold‐change| ≥ 1.5. Volcano plot was established using the plug‐in tool at SangerBox (http://sangerbox.com) to help us identify the overlapping genes. Heatmap was also built by plug‐in tool at SangerBox (http://sangerbox.com) to give a direct presentation of the expression status of genes in different samples.GEO2R, an algorithm based on R language limma package, was employed to analyze differentially expressed miRNAs (DEMs) in this dataset. Cut‐off criteria were set as 2.3http://www.targetscan.org/vert_72/, Agarwal, Bell, Nam, & Bartel, http://www.mirdb.org/miRDB/index.html, Liu & Wang, https://genie.weizmann.ac.il/pubs/mir07/mir07_data.html, Kertesz, Iovino, Unnerstall, Gaul, & Segal, http://www.microrna.org/microrna/home.do, Betel, Wilson, Gabow, Marks, & Sander, http://mirtarbase.mbc.nctu.edu.tw/php/index.php, Chou et al., Targets of these identified DEMs were predicted at five miRNA target prediction websites including TargetScan V_7.2  targeted the largest number of genes in the network. Meanwhile, we found only one downregulated miRNA, hsa‐MIR‐4286 in Figure We were interested to identify miRNA‐mRNA networks after AFB1 exposure. Hence, three KEGG pathways related to cancer initiation were selected after pathway enrichment analyses. Three KEGG pathways including miRNAs in cancer, Pathways in cancer, and PI3K‐Akt signaling pathway for upregulated DEMs were selected and 13 overlapping genes were identified for following analyses Figure a. The ex4Aspergillus flavus and the exposure to AFB1 is reported to account for the initiation of approximately 4%–28% of all HCC cases , hsa‐MIR‐199a‐3p (610719), and hsa‐MIR‐222‐3p (300569) were previously reported to be associated with HCC progression  in HCC has not been reported to date and therefore our study implied that hsa‐MIR‐34b‐5p may be related to HCC onset. For the regulatory network constructed by downregulated DEMs, we have noted that the role of hsa‐MIR‐4286 was not reported to be associated with HCC previously. Therefore, it deserves to be investigated in the future.In this study, a miRNA expression profile dataset that contains data from primary human hepatocytes treated with AFB1 for 5 days and then subjected to 3 days wash‐out period was used to investigate the changed miRNAs after AFB1 withdraw. A total of 18 DEMs including nine upregulated DEMs and nine downregulated DEMs were identified. After targets prediction for these identified DEMs, we analyzed the biological functions using GO annotation and KEGG pathway enrichment methods. We found that the pathways of these genes involved were significantly associated with human cancer progression, which indicated that AFB1 exposure will indeed activate the development of cancers. On the basis of these analyses results, we constructed the miRNA‐mRNA regulatory network to help us understand the mechanisms related to HCC onset after AFB1 exposure. This work established miRNA‐mRNA regulatory networks related to the HCC onset by comprehensive bioinformatic analyses. The limitation in this work was that the predicted miRNA‐mRNA link was not experimentally validated, which need further investigations. Hence, future investigations on in vitro cell lines or in vivo animal models are required to validate the prediction results.In summary, our in‐silico analyses results indicated miRNA‐mRNA connections contributing to the HCC onset after AFB1 exposure. We hope that our results can accelerate the process of tackling the AFB1‐induced onset of HCC.The authors declare that they have no conflict of interest.


Context: Amaranth and canola oils have been used traditionally. Amaranth has been identified as being of interest because of its outstanding nutritive value. Amaranth oil is a rich source of highly unsaturated fats and so could be a valuable dietary alternative for individuals affected with obesity. Reactive oxygen species (ROS) are postulated to be involved in systemic inflammation and oxidative stress. Activated polymorphonuclear neutrophils (PMNs) generate high amounts of reactive oxygen species.Objective: Our study investigates the impact of amaranth and canola oils supplementation on oxidative metabolism in patients with obesity. We hypothesized that, due to its lipid-lowering and antioxidant properties, amaranth and canola oil would protect against oxidative stress.Materials and methods: We tested 19 obese patients . The protocol consisted of two stages: a run-in phase of 2 weeks and an experimental stage – canola or amaranth oil supplementation (20 mL/d) with calorie restriction diet for 3 weeks. The neutrophil oxidative burst was expressed by fluorescence intensity (IF).Results: The oxidative burst had increased significantly at the end of treatment in both groups IF:  p < 0.05. The levels of IF were significantly higher in neutrophils of patients who received canola oil (41.05 ± 25.3) compared to those who received amaranth oil (28.4 ± 11.8) p < 0.05.Conclusions: Canola oil exerts possible effects on oxidative burst activity in neutrophils in vivo conditions. Oxidative stress is the result of an imbalance between the production of reactive oxygen species and an antioxidant mechanism. Reactive oxygen species are hypothesized to be involved in the systemic inflammation and oxidative stress in patients with obesity. Adipose tissue consists of adipocytes, but also contains other cells such as pre-adipocytes, lymphocytes, macrophages, fibroblasts and vascular cells; it is mainly found in subcutaneous and visceral depots  oil contains tocotrienols and squalene compounds (which are known to affect cholesterol synthesis in humans), while canola (Brassica napus L.) oil is rich in both α- and γ-tocopherol, which have antioxidative activities  from individuals who have been receiving amaranth/canola oil supplementation in their diet.2, (mean ± SD)] were studied. The inclusion criteria were based on body mass index (BMI)>30 kg/m2. The exclusion criteria were: nonsmokers, history of cancer, autoimmune diseases, pregnancy, psychiatric disorders, ongoing therapy with antibiotics and/or steroid and alcohol and drug addiction. The study was approved by the Ethics Committee of Poznan Medical University and adhered to basic Principles of the Declaration of Helsinki (NCT03346421 retrospectively registered at 14 November 2017). For all of the patients, the protocol consisted of two stages: a run-in phase of 2 weeks (patients on a standard diet) and an experimental stage – canola oil supplementation (20 mL/d) or amaranth oil supplementation (20 mL/d) with calorie restriction diet for 3 weeks. Testing at an experimental (week 0) and completion stage (week 3) included bioimpedance measurement  and blood sampling. The dose of 18 ml per day of amaranth oil was chosen based on the study by Martirosyan et al. . The oils were given to patients at the same time of day (mid-morning) to avoid circadian influences, and a dietician supervised their consumption. The study protocol is as follows .Blood samples (2.5 mL) were collected in lithium heparin tubes. (1) To measure the intensity of ROS production, dihydrorhodamine  was added to all the samples at a concentration of 10 µg/mL and incubated in the dark (37 °C) for 5 min. (2) A stimulator of the oxidative burst, phorbol 12-myristate 13-acetate , was added to the samples at a concentration of 40 μg/mL. The samples were then incubated in the dark and at the room temperature for 15 min. (3) 450 µL of red blood cell lysis solution was added to the samples.Two aliquots of blood were stained with DHR (an uncharged ROS indicator measurable by flow cytometry when oxidized to cationic rhodamine 123). One of the aliquots was stimulated with PMA . The mean fluorescence intensity of both the stimulated and unstimulated tubes was measured. The results were expressed as the fluorescence intensity (IF) ratio: IF DHR + PMA/IF DHR. ROS production was measured by BD FACSAria III  cytometer and all the results were analyzed using the BD FACSDiva Soft wave .t-test, while for the correlation between different parameters, the Spearman correlation coefficient was used. Significance was established at p < 0.05.Statistical analysis was performed using the Statistica 12.5 . Values were expressed as mean ± SD and were compared using the Wilcoxon test and Student’s p < 0.05.The neutrophil oxidative burst expressed as the fluorescence intensity (IF) before and after oil supplementation and caloric restriction treatments are shown in p < 0.05. The other parameters: waist/hip ratio, body fat mass, fat-free mass and basal metabolic rate changed significantly only in canola oil group p < 0.05.The oxidative burst in the group with canola oil supplementation changed significantly , and thir = −0.23, p = 0.33), BMI , waist/hip ratio , body fat mass , fat free mass , and basal metabolic rate . No relations were observed.Oxidative burst at the baseline and at the endpoint was checked for Spearman correlation with the following covariates:  for weight (p < 0.05) . In the study, neutrophils from obese individuals after treatment had a significantly higher oxygen production compared to those 3 weeks of dietary treatment ( < 0.05) . Suzuki  < 0.05)  suggeste < 0.05)  showed t < 0.05)  and 4. I < 0.05)  and Amar < 0.05)  show thag et al.  and Hungg et al.  show thain vivo conditions. The present result is in agreement with other studies that found enhanced oxidative burst in healthy young men after 8 weeks of fish oil supplementation (Gorjão et al. Our study is the first to document the possible modulation of canola oil on oxidative burst activity in neutrophils As canola plant contains high amounts of natural antioxidants, it may exert protective effects through inhibition of ROS production. The study of canola plant by Vu et al.  showed tBrassica napus on neutrophil function.Our findings suggested a complex and multi-level regulatory system. Further investigations are required to determine the roles of In conclusion, the present study suggests an immuno-stimulating effect on oxidative burst after 3 weeks of canola oil-supplementation in obese patients.


Sleep aids the encoding and consolidation of declarative memories, but many adolescents do not obtain the recommended amount of sleep each night. After a normal night of sleep, there is abundant evidence that a daytime nap enhances the consolidation of material learned before sleep and also improves the encoding of new information upon waking. However, it remains unclear how learning is affected when sleep is split between nocturnal and daytime nap periods during a typical school week of restricted sleep. We compared long-term memory in 58 adolescents who underwent two simulated school weeks of suboptimal continuous  or split sleep . In the first week, participants encoded pictures in the late afternoon on Day 5 and were tested after 2-nights of recovery sleep. On 3 consecutive days in the second week, participants learned about six species of amphibians in the morning, and six different amphibians in the late afternoon. Testing was performed in the evening following a night of recovery sleep. In the first week, the split sleep group recognized more pictures. In the second week, they remembered more facts about species learned in the afternoon. Groups did not differ for species learned in the morning. This suggests that under conditions of sleep restriction, a split sleep schedule benefits learning after a nap opportunity without impairing morning learning, despite less preceding nocturnal sleep. While not replacing adequate nocturnal sleep, a split sleep schedule may be beneficial for chronically sleep restricted learners. In the interim, it remains an open challenge to improve learning given the restricted sleep adolescents currently obtain.Sleep after learning stabilizes and integrates memories for long-term storage,13 and adults.18 Recently these findings have been translated to classroom learning by examining the utility of napping using educationally realistic learning materials. When learning detailed facts about ecology, a mid-afternoon nap enhanced memory to a similar extent as when that time was spent cramming.19 A separate study found that a 2 h nap opportunity after a lecture was associated with better test performance in adolescents when compared to those who attended regular classes,20 while a third study found a similar advantage when weekly lessons were followed by naps of longer than 30 min.21Daytime naps consistently enhance memory in laboratory studies involving children19 As such, nap conditions were associated with more total sleep in the 24 h period prior to cognitive tests compared to wake conditions that did not include a nap. Enhanced post-nap cognition could therefore be a function of total sleep obtained, rather than the nap per se. Determining if naps can benefit learning and memory when total sleep duration is held constant is crucial, as it could inform whether educational institutions should make the considerable practical adjustments that would facilitate napping in schools, or whether alternate solutions that focus on nocturnal sleep would provide similar benefits.These findings suggest that it might be preferable to split available sleep time between a nocturnal bout and a daytime nap. Critically however, prior experimental research exploring the memory benefits of napping has only examined napping as a supplement to a fixed amount of nocturnal sleep.22 or two equivalent periods across 24 h.24 Performance on a battery of cognitive tests was compared with continuous nocturnal sleep of the same overall duration. Measures of subjective alertness, psychomotor vigilance and processing speed were found to be determined by total sleep obtained, rather than how that sleep was distributed across the day.24 It remains to be determined how long-term memory is affected when learning takes place under a similar split sleep schedule.A handful of studies—conducted in adults with a view to optimize shift-work—controlled for total sleep obtained by splitting sleep into nocturnal and afternoon nap periods,26 However, the subsequent nap might compensate by enhancing consolidation of what was learned.18 If memory were purely a function of total sleep obtained, then memory for information learned in the morning or afternoon would not differ between split and continuous sleep schedules. This design permits the evaluation of how splitting sleep affects learning that is similar to the way students study and revise across multiple episodes in school. However, this methodology does not allow a clear attribution of group differences to encoding or consolidation.To address these gaps in our knowledge, we compared long-term memory in adolescents who learned across two simulated weeks of restricted sleep in a naturalistic school setting, where sleep opportunities on each day were either split into 5 h nocturnal sleep and a 1.5 h daytime nap, or 6.5 h of continuous nocturnal sleep Fig. . Partici6 in the late afternoon on the fifth day of sleep restriction. Recognition of those pictures was tested after two nights of recovery sleep  = 5.44 ± 0.84) and extension of sleep on weekends . Participants incidentally encoded pictures of landscapes by making building/no-building judgments and were not informed that memory for images would be tested later. Both groups were highly accurate at judging pictures to contain a building or not  and significantly higher in the split sleep group, t(56) = 2.241, p = 0.029 , all participants indicated that they were unaware they would be tested for their memory of the previous session. Recognition of previously viewed images was tested alongside new images. The signal detection measure A′ was utilized to assess memory as it corrects for response bias. A′ scores were significantly higher in the split sleep group, t(56) = 2.505, p = 0.015, Cohen’s d = 0.66  = 1.395, p = 0.168, specific knowledge, t(56) = 0.579, p = 0.565, or picture identification t(56) = 0.349, p = 0.728. General knowledge about amphibians was relatively low , but significantly above chance (p < 0.001). Specific knowledge (M = 49.66 ± 9.03%) and picture identification (M = 52.88 ± 12.31%) did not differ from chance (p > 0.05).Participants performed a pretest prior to learning to establish their existing knowledge about amphibians. Two-alternative forced choice questions tested general facts about amphibians , specific facts about the species they were about to learn (specific knowledge), and ability to identify species from images (picture identification). Participants were also asked to rate their subjective level of knowledge and disgust  when presented with a picture of each species. There were no significant group differences for general knowledge, t(56) = 1.843, p = 0.071, but ratings in this group were still very low considering a score of 1 represented “no knowledge” (M = 1.42 ± 0.64). On average participants rated all species as moderately disgusting (M = 4.69 ± 2.32), but these ratings did not differ between groups, t(56) = 1.069, p = 0.289.There was a non-significant trend for subjective knowledge to be higher in the continuous sleep group, 1 via two-alternative forced choice questions, followed by confidence ratings . Responses within each rating were corrected for response bias (correct–incorrect). Any group differences were expected to be present for participant’s most confident memories (certain responses),19 which are the least prone to noise introduced by guessing. For certain memories, a mixed ANOVA with group (split sleep/continuous sleep) and time (morning/afternoon) showed a significant main effect of group, F = 4.172, p = 0.046, an interaction, F = 5.646, p = 0.021, but no effect of time, F = 0.117, p = 0.734  = 2.7, p = 0.009, Cohen’s d = 0.71, while groups did not differ significantly for morning learning, t(56) = 1.189, p = 0.239, Cohen’s d = 0.31. Within the split sleep group, the higher performance for afternoon compared to morning learning was significant, t(28) = 2.088, p = 0.046, while the numerically lower performance for afternoon compared to morning learning observed in the continuous sleep group was not, t(28) = 1.34, p = 0.191. This indicates that a split sleep schedule improves afternoon learning without any cost to morning learning.Retrieval was tested at 20:30 on day SR2734 Fig. . IndepenF = 3.825, p = 0.055, but no effect of time, F = 0.363, p = 0.549, and no interaction, F = 1.008, p = 0.32. Planned comparisons showed a similar pattern to certain memory, with a significantly better memory for afternoon learning in the split sleep group, t(56) = 2.177, p = 0.034, Cohen’s d = 0.57, but no group difference for morning learning, t(56) = 1.467, p = 0.148, Cohen’s d = 0.39, and no within group differences between morning and afternoon learning (p > 0.19).Overall memory showed a trend for a main group effect, F = 0.273, p = 0.603, time: F = 0.343, p = 0.561, group * time interaction: F = 1.743, p = 0.192), or guess responses  = 0.15, p = 0.7, time: F = 1.388, p = 0.244, group * time interaction: F = 0.637, p = 0.428).There were no significant main effects or interactions for somewhat certain (group: p > 0.05), but there were significant time * group interactions for focus, F = 7.724, p = 0.007, and ability, F = 5.976, p = 0.018. Consistent with the memory findings, focus on the task was significantly higher in the split sleep group in the afternoon, t(56) = 2.647, p = 0.011, but groups did not differ in the morning session, t(56) = 0.894, p = 0.375. The interaction for ability showed a similar pattern . Subjective alertness showed a significant main effect of time, F = 13.072, p = 0.001, and a group * time interaction F = 25.62. p < 0.001, but no group effect, F = 2.6, p = 0.112. Alertness was significantly higher in the afternoon for the split sleep group, t(56) = 3.34, p = 0.001, but groups did not differ in the morning, t(56) = 0.291, p = 0.772.We explored whether a split sleep schedule impacted on participant’s subjective impressions of their learning capabilities while they were studying the material. We asked four questions that probed subjective motivation, focus on the task, ability to learn, and alertness . Morning and afternoon sessions consisted of two 30 min blocks separated by a 2 min break. Questions were administered during the break and at the end of the block. Ratings were collapsed across all three days to create separate means for morning and afternoon sessions Fig. . ANOVA iern Fig. , but dir27 Significant relationships were only observed for the split sleep group, where memory for morning species was positively correlated with focus  and ability , while afternoon memory correlated significantly with ability .Last, we correlated these subjective measures with certain memory scores within the appropriate time period  within each group separately, making 16 comparisons in total .To summarize, the split sleep schedule led to higher levels of subjective alertness and focus in the afternoon after the nap, although correlations between these subjective measures and afternoon memory were not significant.Psychomotor vigilance was assessed shortly before the learning (10:15 and 16:30) and test sessions for picture encoding (16:30) and the factual knowledge task (20:15). Response times during both tasks were also analyzed as indirect measures of alertness, although note that participants were instructed to respond as accurately as possible for both memory tasks without any mention of the speed of responses.5), the continuous sleep group had significantly more lapses, t(56) = 4.806, p < 0.001. Response times during the encoding task were also significantly slower in the continuous sleep group, t(56) = 2.615, p = 0.011, providing a congruent indication of reduced vigilance/sustained attention during the encoding session. Prior to picture retrieval (16:30 on R12), the continuous sleep group still had significantly more lapses, t(56) = 2.607, p = 0.012, but this time they were faster to respond than the split sleep group during the picture retrieval session at 16:45, t(56) = 4.387, p < 0.001. The higher number of lapses for the continuous sleep group prior to picture retrieval raises the possibility that observed memory impairments are related to lapses of attention during retrieval, rather than a deficit to prior encoding. However, PVT lapses (16:30 on R12) did not significantly correlate with memory (A′) in either experimental group (p > 0.05).Prior to picture encoding (16:30 on SR11 to SR23) and analyzed via a mixed ANOVA that included the factors group and time. This showed a significant main effect of time, F = 9.795, p = 0.003, group, F = 8.465, p = 0.005, and a group * time interaction, F = 25.3, p < 0.001. Groups did not differ in the morning, t(56) = 0.851, p = 0.398, although the split sleep group had significantly fewer lapses than the continuous sleep group in the afternoon t(56) = 4.987, p < 0.001.In the second week of the experiment, lapses for PVT’s occurring prior to the factual knowledge task in the morning (10:15) and afternoon (16:30) were collapsed across the 3 learning days  = 1.269, p = 0.21. Similar to the picture encoding task, response times during retrieval were significantly faster in the continuous sleep group, t(56) = 2.147, p = 0.036.There were no group differences for the PVT performed before the factual knowledge retrieval test at 20:15 on R23) and second week (SR21) (p < 0.01). Second, the continuous sleep group obtained significantly more slow wave sleep (SWS) on recovery days R11 and R21 (p < 0.01), indicating greater accumulation of sleep pressure in participants who did not nap in the afternoon prior to recovery sleep (see Supplementary Table p < 0.05) except for N1 (p > 0.05).When considering total sleep during each 24 h period , the split sleep schedule was associated with consistently reduced TST Fig. . There w week SR2 (p < 0.05). Although groups did not significantly differ in the length of any individual sleep stage (p > 0.05), the split sleep group had significantly lower total sleep time, t(53) = 3.173, p = 0.003, by approximately 15 min . Certain memory did not significantly correlate with any sleep stage duration or TST (p > 0.05).Subsequent analyses focused on sleep stage duration during sleep periods within 24 h of each task. For picture encoding, nocturnal and afternoon nap sleep times were combined (SR11–SR23), we examined averaged sleep characteristics across the two days where PSG was recorded , t(53) = 4.682, p < 0.001. Analysis of macrostructure showed that REM sleep duration was significantly reduced by splitting sleep , t(53) = 2.697, p = 0.009, while other sleep stages did not differ (p > 0.05). Again, certain memory in the morning or afternoon did not correlate significantly with any of these sleep metrics (p > 0.05).Since learning in the factual knowledge task took place over 3 days  and a short daytime nap (1.5 h sleep opportunity) resulted in better recall of learned material compared to a purely nocturnal schedule with the same total sleep (6.5 h), specifically for information learned after the nap in the afternoon. Critically, the split sleep schedule did not impair memory for information learned in the morning, despite less nocturnal sleep and less total sleep across 24 h. These findings expand upon prior observations that a daytime nap benefits long-term memory,28 The afternoon learning improvement we observed for both memory tasks suggests that splitting sleep could be one such strategy to improve learning. The 90 min mid-afternoon sleep opportunity in the current study was chosen to maximize the probability of participants obtaining a full cycle of N2, SWS and REM sleep,29 but is toward the upper limits of nap durations that may be practical to implement in schools. Relatively short naps of 30–60 min have been shown to provide benefits to memory lasting up to a week,21 therefore future studies should examine whether splitting sleep with shorter nap opportunities provides similar benefits for cognition.The optimization of sleep schedules has been proposed as a low cost way to improve educational outcomes.Most prior work has examined the benefit of naps in relation to the consolidation of material learned before sleep, but there are several indications that the nap advantage we observed is the result of enhanced encoding, rather than consolidation. The picture encoding task provides a relatively pure measure of encoding capacity, because the incidental nature of the task restricts the use of mnemonic strategies, while only those trials when participants are paying attention and make the correct building/no-building judgment were analyzed. The improved afternoon encoding we observed for the split sleep group on this task also indirectly suggests that improved afternoon learning for the factual knowledge task was related to enhanced encoding, rather than consolidation. This conclusion also seems likely given that the split sleep group obtained less nocturnal sleep in which to consolidate factual knowledge learned in the afternoon.2 provides a plausible mechanistic account. Synaptic connections are potentiated during wakefulness, and slow-wave activity is proposed to globally downscale net synaptic weight to avoid saturation in memory networks. This is proposed to facilitate new learning. Although the split sleep group obtained significantly less total SWS in the night prior to morning learning, most SWS takes place in the first 4 h of nocturnal sleep. Here, both groups obtained this early period of slow-wave rich sleep, which may be sufficient to restore encoding capacity. This hypothesis is supported by a pair of studies that show encoding to be comparable between those obtaining half a night (~4 h) and a full night of sleep (~8 h).31 It is well established that total32 or chronic sleep deprivation6 impairs encoding, but the relatively small difference between 5 and 6.5 h nocturnal sleep appears not to adversely affect encoding. The afternoon nap however, provides an additional period of downscaling for the split sleep group, and this may account for the difference in afternoon encoding capacity we observed.As splitting sleep resulted in less TST over 24 h, it was surprising that this group had an afternoon memory advantage without any negative impact on information learned in the morning. Although the mechanism underlying this observation cannot be directly explained with the current design—where learning was spread over several days and learning sessions—we posit that the synaptic homeostasis hypothesis34 creative problem solving35, and emotional regulation.36 With regard to the latter, recently published data from the current study protocol showed that the split sleep schedule was associated with improved positive mood,37 despite reductions in total time spent in REM sleep. The split sleep schedule could be optimized to facilitate REM sleep, for example by slightly extending morning sleep, or taking naps earlier in the morning. Further studies are needed to optimize sleep schedules, establishing a balance between the cognitive benefits associated with these different aspects of sleep and the practicality of translating this behavior to schools.It is important to note that while splitting sleep had no adverse effects for declarative memory, several other cognitive faculties linked to REM sleep may have been affected, including procedural memory,38 and perhaps also encoding capacity,17 therefore splitting sleep may have minimized the impairment associated with learning at this relatively disadvantageous time of day. This was hinted at by a non-significant reduction in memory performance across the day in the continuous sleep group.Another factor to consider is the circadian variation in cognitive performance, particularly in relation to the afternoon ‘circadian dip’, which may have impacted on both memory tasks. The circadian dip is associated with reduced afternoon levels of alertness39 and are enhanced by daytime naps,41 therefore these factors may have contributed to the afternoon learning advantage for the split sleep group, particularly for the long and demanding learning blocks of the factual knowledge task. Consistent with this, measures of post-nap psychomotor vigilance, subjective alertness and subjective ability to focus during learning blocks of this task were significantly higher in the split sleep group, therefore it is likely that this contributed to their improved learning capabilities.The encoding of any stimulus relies on a complex interaction of factors that includes focusing and sustaining attention on a stimulus, as well as cognitive control processes that allocate resources to the goal of forming an enduring memory trace. These underlying cognitive processes deteriorate with sleep loss44 even though this level of sleep loss produces a consistent vigilance impairment.39 The group differences we observed in vigilance therefore seem unlikely to account for memory impairments, although we cannot entirely rule out this possibility. For the factual knowledge task, vigilance did not differ between groups prior to retrieval. Moreover, we observed an interaction between memory for information learned in the morning and afternoon: if vigilance during retrieval was an influential factor then memory for all information should be equally affected, regardless of when it was learned.A potential limitation to the picture encoding task is that vigilance in the continuous sleep group prior to the retrieval session remained significantly lower than the split sleep group. Thus, impaired memory of the continuous sleep group may be due to decreased vigilance during retrieval, despite the two nights of intervening recovery sleep intended to equalize vigilance across groups. However, attentional lapses did not significantly correlate with memory performance. Additionally, the few studies that have assessed the effect of sleep loss specifically on retrieval have found no significant impairments after a night of total sleep deprivation,3 In the absence of a control group obtaining the recommended amount of sleep, we cannot determine the level of impairment associated with a 6.5 h TIB sleep schedule relative to the recommended sleep duration, or whether splitting sleep is also advantageous for individuals with habitual sleep length that is closer to the recommended amount. However, this sleep schedule provides an accurate reflection of sleep obtained by adolescents undergoing habitual chronic sleep restriction in East Asia,6 and around the world,45 therefore our findings suggest that facilitation of naps in schools would be beneficial for memory in a range of adolescent populations.A significant limitation to broad generalization of our work may be that the sleep length in our split and continuous groups was below the 8–10 h recommended for adolescents.46 Spaced learning was recently shown to limit the deleterious impact of multi-night sleep restriction on vocabulary learning.47 As factual knowledge learning in both conditions of the current study was spaced, further work will be needed to assess whether the interaction between spacing and sleep contribute to the observed effects.Another factor to consider for the potential translation of these findings to schools is the spacing of learning and how that relates to sleep. Spaced learning results in superior retention of memoranda although the underlying reasons are debated.21 in chronically sleep deprived adolescents.To conclude, while adequate nocturnal sleep should continue to be strongly advised, under conditions of sleep restriction, a split sleep schedule appears to be preferable for harnessing the cognitive functions that underpin learning in the afternoon, without negatively impacting upon morning learning. These findings contribute towards growing experimental evidence that facilitating naps in schools may be an effective measure to improve learning outcomes2. We avoided recruiting habitual short sleepers . Participants must not have traveled across >2 time zones 1 month prior to the study, and not consumed ≥5 caffeinated beverages per day.Sixty adolescents (15–19 years of age) were invited to participate in the study from schools throughout Singapore. Participants were screened for known health conditions, sleep disorders, and had body mass index of ≤30 kg/mn = 29) and continuous sleep (n = 29). The groups did not differ significantly in age, gender, consumption of caffeinated beverages, non-verbal intelligence, morning-eveningness preference, symptoms of chronic sleep reduction, levels of daytime sleepiness, subjective sleep quality, self-reported and actigraphically assessed sleep habits, or levels of anxiety and depression  were matched into 2 groups: split sleep . All participants and legal guardians provided written informed consent.The study was approved by the National University of Singapore Institutional Review Board and was conducted in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments. The study was a registered clinical trial . Two sets were presented during both encoding and retrieval (160 old images), while one set was presented only at retrieval (80 new images). The use of each set during encoding and retrieval was counterbalanced across participants.Stimuli were identical to those used in our prior study,Encoding took place in a single 15 min block. Participants were instructed to look carefully at each image and indicate whether it contained a building or not. They were not informed that their memory would be tested at a later date. Each image was presented for 2500 ms before a response screen was displayed showing “(1) Building, (2) No building”. After participants responded with the corresponding keypress, an inter-trial interval (ITI) of 1000 ms elapsed before the start of the next trial. The 160 images were presented in a randomized order.6 analysis focused on two outcome measures: (1) confidence ratings of 4 (probably saw) and 5 (definitely saw) to old images were classed as “hits”, (2) confidence ratings of 4 and 5 to new images were “false alarms”. To correct for response bias toward old/new responses, the signal detection measure A′ was calculated using hits and false alarms, for which 0.5 represents chance performance.Retrieval tested the recognition of 160 old images randomly intermixed with 80 new images. Images were presented above a five-point confidence scale: “(1) Definitely did not see, (2) Probably did not see, (3) Unsure, (4) Probably saw, (5) Definitely saw”. Participants were asked to indicate whether they remembered each image from the previous session. The trial was terminated following a response, or after a 5000 ms time limit had elapsed, and was followed by a 1000 ms ITI. Images that were incorrectly judged to contain buildings or not were excluded from subsequent retrieval analysis, since these trials suggest that images were not adequately attended to. Consistent with prior work,This was performed prior to learning in order to probe knowledge of the species to be learned. This involved four stages: (1) Picture Identification: participants were shown a single image and identified the animal shown from two options. This was repeated for each species. (2) General knowledge: 20 two-alternative forced choice questions regarding general characteristics of amphibians. (3) Specific knowledge: 20 two-alternative forced choice questions about information that they would encounter during the learning session, similar to questions encountered in the main test after learning. (4) Subjective disgust: participants rated the amount of disgust they felt toward each species , to control for the influence of emotion on memory. (5) Subjective knowledge: participants rated their prior knowledge of each species . All tests were self-paced and trials were presented in a random order.Participants were informed that all information they were about to learn would be tested. They were shown example test questions using a species not featured in the learning and instructed to not discuss or peruse information about frogs/amphibians outside of the specified learning blocks.Participants learned about three frogs , 3 toads , three newts  and 3 salamanders  in separate 30 min blocks. Characteristics  were adapted from their actual biology and behaviors.Each day, participants learned 2 blocks in the morning  separated by a 2 min break and 2 blocks containing different animal types in the afternoon . Participants learned the same species in the morning and afternoon on each of the 3 days of the experiment, but the order that they learned them was switched each day. For example, if they learned frogs and then newts on the first morning of the experiment, they learned newts followed by frogs on the second morning. Animal type was counterbalanced across participants for morning/afternoon sessions.The learning materials for each type of animal  contained roughly 80 slides of factual information in the form of numbered points and images. Participants moved forward and backward through slides at their own pace, but were advised to observe a minimum speed to ensure all slides were seen. A time counter was visible throughout and slides included markers informing how much time should have passed at 5 min intervals.To assist learning, some slides asked participants to write on paper what they could recall. Participants were permitted to make notes which were removed at the end of each block. The final slide of each block instructed participants to use the remaining time to recap the information.When the 30 min of each block had elapsed, participants completed a Karolinska Sleepiness Scale (KSS) and were asked 3 questions to rate on a 7-point scale: “Was your attention focused on the task or something unrelated to the task? ”, “How motivated were you to learn the information? ”, and “How well do you feel you could learn the information? ”. These are referred to as subjective “focus”, “motivation”, and “ability”, respectively. Scores were subsequently inverted for analysis so that higher values represent higher levels for each measure.This involved two-alternative forced choice questions followed by a confidence rating  Fig. . QuestioMemory scores were calculated for “certain”, “somewhat certain”, and “guess” responses separately by subtracting incorrect from correct responses. “Overall memory” consisted of all correct responses, including trials where no certainty response was recorded.Participants performed three test batteries daily that included a 10 min psychomotor vigilance task (PVT) beginning at 10:15, 16:30, and 20:15. A counter appeared on screen at random intervals between 2000–10,000 ms, and participants responded with the space bar as quickly as possible. Failure to respond within 10,000 ms elicited an alerting tone. Lapses (responses >500 ms) were measured. All tasks were presented with E-Prime 2.0 .1–B2) of 9 h nocturnal TIB (23:00–08:00) before a two-cycle simulation of a typical school week in Singapore  followed by two nights of 9 h recovery sleep (R11–R12). The second cycle included three nights of sleep restriction (SR21–SR23) and two recovery nights (R21–R22). During sleep restriction nights in both cycles, the continuous sleep group were afforded 6.5 h nocturnal TIB (00:15–06:45 h), while the split sleep group were permitted 5 h TIB at night (01:00–06:00) and a 1.5 h afternoon nap opportunity (14:00–15:30).The experiment was conducted over 15 days as part of the Need for Sleep 4 study. Participants adhered to a 9 h sleep schedule (23:00–08:00) for one week prior to the study. They were afforded two baseline nights  and returned later for the first afternoon learning block (16:45). This was repeated on SR22 and SR23. The factual knowledge retrieval test took place on R21 at 20:30. Participants also performed 3 PVTs on each day of the experiment .All cognitive tasks were administered to participants via individual laptops in a classroom. Picture encoding took place on SR148 utilizing the Z3 score automated EEG scoring system49 and verified by a trained researcher. Sleep architecture was compared across the whole protocol, while main analyses focused on the duration of each sleep stage and TST averaged across nocturnal and nap episodes in temporal proximity to each task. Thus, for picture encoding this focused on SR15, and for factual knowledge this included the mean of SR21 and SR23.Sleep was recorded using portable SOMNOtouch PSG devices , during selected sleep and nap episodes. EEG was recorded from two main channels (C3 and C4 according to the 10–20 system) referenced to contralateral mastoids. The common ground and reference electrode were placed at Fpz and Cz. Left and right electromyogram and electrooculogram were also attached. Impedance <10 KΩ was verified at each electrode. The sampling rate was 256 Hz. Data was scored according to standard criteriat-tests were used for sleep architecture, picture encoding metrics, and follow-up tests to ANOVA. One sample t-tests compared performance on several measures to chance. Spearman’s Rho correlations explored the relationship between memory and other measures. Effect sizes indicated by Cohen’s d for t-tests of key group comparisons. All statistical tests were two-tailed, significance level p < 0.05. All means are presented in the text ± standard deviation.Separate 2 × 2 mixed ANOVA with the factors group (split sleep/continuous sleep) and time  were performed for each measure of the factual knowledge task , as well as subjective measures during learning  and PVT lapses. Independent and paired Supplementary Material