Nephropathy is a major Fabry disease complication. Kidney biopsies reveal glomerulosclerosis even in pediatric patients. The main manifestations of Fabry nephropathy include reduced glomerular filtration rate and proteinuria. In 2016, an oral pharmacological Chaperone was approved to treat Fabry patients with “amenable” mutations. Because (i) Fabry disease is a rare disorder that frequently causes kidney damage, and (ii) a new therapeutic is currently available, it is necessary to review wich biomarkers are useful for nephropathy follow-up among Fabry “amenable” patients receiving migalastat. The literature search was conducted in MEDLINE, EMBASE, SCOPUS, Cochrane, and Google academic. Prospective studies in which renal biomarkers were the dependent variable or criterion, with at least 6 months of follow-up, were included. Finally, we recorded relevant information in an ad hoc database and summarized the main results. To date, the main useful biomarker for nephropathy monitoring among Fabry “amenable” patients receiving migalastat is glomerular filtration rate estimated by equations that include serum creatinine. Fabry disease (FD) is an X-linked lysosomal storage disorder caused by defects in the glycosphingolipid metabolic pathway due to deficient or absent lysosomal α-galactosidase-A  enzyme activity . This reThe “classic” FD phenotype is expressed in males with absent or severely reduced  α-GalA activity, marked Gb3 accumulation in vascular endothelial cells, cardiomyocytes, smooth muscle cells, and podocytes [Males with “late-onset” FD phenotypes present varying levels of residual α-GalA activity  and late clinical manifestations usually limited to a single organ ,3.In heterozygous female FD patients, the spectrum of disease severity is heterogeneous and it is, in part, dependent on the GLA mutation and the X chromosome inactivation (Lyonization) profile ,3.Nephropathy is a major complication of FD . Kidney Main manifestations of Fabry nephropathy include reduced glomerular filtration rate (eGFR) and proteinuria ,2,4,8; “Some FD patients have missense mutations with a reduction in overall α-GalA enzymatic activity due to reduced stability of the mutated protein, caused by protein misfolding and premature degradation, but the α-GalA catalytic activity remains conserved ,12. FiguMigalastat was initially identified as a competitive inhibitor of the α-GalA enzyme. However, at sub-inhibitory concentrations, a binding to the enzyme’s catalytic center can facilitate proper protein folding in the endoplasmic reticulum and appropriate trafficking to the lysosome. This resulted in an increase of enzymatic activity in Fabry patients .This concept of how a small molecule can help a protein to fold correctly, allowing it to enter physiological processing pathways properly, was enunciated as a “pharmacological chaperone” ,14. Enzyme replacement therapy (ERT) is presently available in the form of agalsidase alfa (given at 0.2 mg/kg body weight every other week by intravenous Infusión) and agalsidase β (administered at 1.0 mg/kg body weight once every 2 weeks as an intravenous infusión) .In 2016, migalastat was approved by the FDA and is available to treat FD patients with “amenable” mutations ,15 Figu.A biomarker is a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes or responses to an exposure or intervention. A biomarker may be used to see how well the body responds to a treatment for a disease or condition. . As i) FThe aim of this work is to carry out a bibliographic search regarding which biomarkers are useful for monitoring FD patients and nephropathy treated with migalastat.- Literature reviewThe literature search was conducted in the following electronic databases: MEDLINE, EMBASE, SCOPUS, Cochrane, and Google academic. The search terms used were “Fabry disease”,“renal disease”, “biomarker”, and “migalastat”- Inclusion criteriaProspective studies in which renal biomarkers were the dependent variable or criterion were included. Studies with at least 6 months of follow-up were included  .- Exclusion criteriaPublications that were “preliminary reports” or “sub-analyses” of other works were excluded to avoid duplication of reported cases.- ProcedureTwo investigators conducted the search independently, and a third investigator corroborated the information overlap. Finally, we recorded relevant information in an ad hoc database to sort the publications and summarize the main results.Four studies met the inclusion criteria ,19,20,21n= 11, post analysis n = 2 [n = 0; SCOPUS = Total n = 0, Cochrane Total n = 0 and Google Academic = Total n = 191, post analysis n = 2 [MEDLINE = Total is n = 2 ,19; 3 stis n = 2 ,24,25; 4is n = 2 ,26,27,28is n = 2 ,30. EMBAis n = 2 ,21; : patients who received migalastat during the 30-month and, Group 2  (n = 15): patients who received ERT during the randomized period (18-month) discontinued ERT and started treatment with migalastat (12-month). In this study, measures of the annualized rate of change in eGFR were assessed using the Chronic Kidney Disease Epidemiology Collaboration equation (eGFRCKD-EPI) at baseline, months 1, 3, 6, 9, 12, 15, 18, 19, 21, 24, and 30 were informed [Feldt-Rasmussen et al. reported the results of ATTRACT  at 30-month to follow-up; 48 patients aged 16 to 74 years, enrolled from 25 centers in 10 countries, classified into two groups: Group 1  . In Group 2 : −2.1 mL/min/1.73 m2  [Authors reported stabilization of renal function determined by the annualized rate of change in eGFR CKD-EPI in both groups of patients. In Group 1 , the mean annualized rate of change from baseline to month 30 was −1.7 mL/min/1.73 m1.73 m2) . Müntze et al. described in 14 patients follow-up during 12-months of treatment with migalastat sub-classified into two groups: (1) the treatment-naive group  and (2) the group that had previously received ERT and then switched to migalastat .2 at baseline and 78 mL/min/1.73 m2 at 12-months; and 76 to 72 (73.5–95.5) mL/min/1.73 m2 in Naive group. The authors performed a sub-analysis of these results and reported that of the total population: (i) women, (ii) the group switch, and (iii) patients with the N215S mutation presented stable eGFR during follow-up ; but that a decreased GFR was present in the rest of patients, although within this last group of patients, patients with mutations likely benign such as N139S were included, which does not cause kidney damage. In these cases, an appropriate genotype–phenotype correlation analysis is necessary so as not to attribute treatment failure to patients who are not going to develop kidney damage due to FD, such as patients with late-onset cardiac phenotypes or benign GLA gene variants carriers [In total population the mean eGFR CKD-EPI was 87 (75.5–102) mL/min/1.73 m2; p < 0.0001) and 24 months , whereas patients receiving other hypertensive drugs were stable after 12 months  and 24 months  [Lenders et al.  reported 0.7025) .In this study, patients with GLA genetic variants causing the “late-onset” FD phenotype  or GLA VUS (variants of unknown significance) (T385A) were included. Because these mutations are not the cause of kidney damage in affected patients at the mean age of the included population (45 years) it is appropriate to analyze causes of nephropathy other than FD influencing the worse renal prognosis and also analyze the population sub-classified in FD phenotypes .Riccio et al. reported follow-up results of 7 men  in whom a switch from ERT to migalastat was indicated . This stThe ATTRACT study design defined renal adverse events such as elevated protein ≥ 300 mg. Authors reported that no significant change from baseline in 24 h urine protein was observed in Group 1  from 0 to 30 months, and in Group 2  during both the initial 18-month period (ERT) and the subsequent 12-month open-label extension period . [In the FAMOUS study, no changes in albuminuria were detected after 12 or 24 months of treatment with migalastat in either females or males . This stp = 0.048) in affected males [The AFFINITY group reported that median proteinuria showed a significant decrease during 12-months of follow-up  and Group 2: , but the study design did not include an analysis of the correlation of these low Lyso-Gb3 levels and kidney function parameters .p = 0.319) in the total patient cohort at the 1-year follow-up (n = 14). A significant reduction was observed in the therapy-naive subgroup, from 10.9 (IQR 7.0–15.7) ng/mL to 6.0 (IQR 4.0–8.1) ng/mL  [Müntze et al.: Lyso-Gb3 values showed a decreasing trend; median values declined from 10.4 (IQR 6.5–13.8) ng/mL to 6.1 (IQR 4.0–14.7) ng/mL  in 14 patients during 1-year follow-up, receiving migalastat, either with or without prior ERT [Müntze et al. reported an α-GalA activity significantly increased from a median of 0.06 to 0.2 nmol/min/mg protein . Their results showed that in the 6-months primary end-point analysis, 13 of 32 patients (41%) who received migalastat had a response (≥50% reduction in number of Gb3 inclusions per KIC) and the median change in KIC Gb3 from baseline was −40.8% with migalastat compared versus −5.6% with placebo. The reduction in KIC Gb3 at 6 months remained stable after an additional 6 months of treatment. In this trial, authors reported results of 23 kidney biopsy samples after 12 months of migalastat treatment: patients showed decreases in Gb3 in glomerular podocytes , endothelial cells , and mesangial cells . None of the samples had increases [Mauer et al. reported the results of 8 male patients with “classic” FD with a mean age of 42 years, receiving treatment with migalastat, with a renal biopsy sample prior to initiation and at 6 months of follow-up. The authors show a beneficial effect on the podocyte cell, with decreased cytoplasmic inclusions of Gb3, decreased podocyte volume and a tendency to correlate these findings with urinary protein excretion without statistical significance. Of note, the authors reported a significant correlation between decreased plasma Lyso-Gb3 and a decline in podocyte volume and decreased cytoplasmic podocytes Gb3 inclusions clearance. The limitation of the study is the short follow-up period, which does not allow definitive conclusions regarding this topic .FD patients treated with phamacological chaperones present stable eGFR, while there are no patients reported who has presented ESRD during follow-up. In this regard, patients with “amenable” mutations and kidney damage due to FD, have a favorable response to migalastat and eGFR is correlated with this favorable evolution, but patients must be adequately characterized prior to starting treatment since, as has been discussed, misinterpretation of response to migalastat may occur when: (i) cardiovascular risk factors or other kidney damage causes are not adequately treated or considered, (ii) patients are receiving drugs that can modify eGFR, (iii) patients who carry GLA mutations or variants that typically do NOT affect kidney function. In these cases, it is not appropriate to attribute kidney damage to FD or poor renal response to migalastat. Renal biopsy should be performed in these selected patients, to clarify the etiology of poor renal evolution.Proteinuria has not been included in trial designs of all currently available studies; although it has remained stable on studies in which it was included; no study found a correlation between proteinuria and renal prognosis. The presence of severe proteinuria (>1 g/24 h) has been for a long time, a risk factor recognized for CKD progression in FD. In patients treated with migalastat, proteinuria/albuminuria is a biomarker of nephropathy added to FD and is a criterion for performing a renal biopsy to diagnose another kidney disease cause, different to FD. Increased α-GalA enzyme activity and decreased Lyso-Gb3 have been described in patients of both genders, both “classic” and “late-onset”; but no study found a correlation between renal evolution and those biomarkers among “amenables” FD patients. The work of Mauer et al. described a relationship between Lyso-Gb3 and Gb3 deposits in podocytes and consequently with proteinuria. However, to date, there is no evidence to support the usefulness of these biomarkers in monitoring the therapeutic response to migalastat in FD nephropathy.Regarding renal biopsy, although it has been reported that there is an improvement in renal histological damage due to migalastat treatment in patients affected by “amenable” mutations, including men with the classic disease phenotype, the routine use of renal biopsy is not adequate, since there are non-invasive biomarkers that correlate with kidney damage evolution, leaving its indication for selected cases, as discussed above.Other biomarkers, for example, podocyturia, micro-RNAs, and urinary mRNA have been described in FD nephropathy, although they are not available in routine practice ,34,35. TTo date, eGFR is the main and only useful biomarker, that has been shown in clinical studies for nephropathy monitoring among Fabry “amenable” patients receiving migalastat. However, albuminuria/proteinuria could be useful to assess the indication for concomitant medication or renal biopsy in selected patients.


A meta-analysis of randomized controlled trials (RCTs) was conducted to systematically evaluate the effects of berberine on the inflammatory markers of metabolic syndrome (MetS) and related disorders.Databases that were searched from inception to October 2020 included PubMed, Web of Science, the Cochrane Library, CNKI, VIP, WanFang Data, and ClinicalTrials.gov. Two reviewers independently selected articles and extracted data. The pooled evaluations were entered and analyzed in Review Manager 5.3.p < 0.05], tumor necrosis factor-α (TNF-α) , and interleukin 6 (IL-6)  among patients with MetS and related disorders. However, it did not affect the level of interleukin 1β (IL-1β) .Of the 7387 publications screened, 52 studies were included, and the related trials involved 4616 patients. Pooled estimates showed that the use of berberine could significantly reduce the concentration level of C-reactive protein (CRP) [standardized mean difference (SMD) = − 1.54, 95% confidence intervals (CI) − 1.86, − 1.22, Overall, the use of berberine in patients with MetS and related disorders appeared to significantly decrease several inflammatory markers. Further multi-center and rigorous investigations with larger patient populations are encouraged to confirm the effect of berberine on MetS and related disorders. Metabolic syndrome (MetS) is a cluster of interconnected physiological and metabolic abnormalities characterized by obesity, insulin resistance, hypertension, and hyperlipidemia  were searched from the date of their inception to October 2020. Medical Subject Headings and text search words included patients [“metabolic syndrome” or “acute coronary syndromes” or “coronary artery disease” or “CVD” or “diabetic” or “T1DM” or “T2DM” or “overweight” or “obese” or “chronic kidney disease” or “end-stage renal disease” or “dialysis” or “heart failure” or “myocardial infarction” or “atherosclerotic” or “hypercholesterolemic” or “hypertension” or “high blood pressure” or “dyslipidemia” or “hyperlipidemia” or “polycystic ovary syndrome” or “stable angina” or “unstable angina” or “diabetic nephropathy” or “obesity” or “stable atherosclerotic plaques” or “atherosclerotic”]  the study population consisted of Chinese patients diagnosed with MetS and related disorders. The MetS-related disorders included acute coronary syndrome, coronary artery disease, cerebrovascular disease, diabetes, obesity, chronic kidney disease, heart failure, myocardial infarction, atherosclerosis, hypercholesterolemia, hypertension, dyslipidemia, hyperlipidemia, polycystic ovary syndrome, angina pectoris, diabetic nephropathy, and stable atherosclerotic plaques; (2) the experimental group was treated with berberine or berberine combined with other treatments, and placebo or non-berberine treatments were used as the control group; (3) RCTs comparing outcomes in CRP, TNF-α, IL-6, and IL-1.Studies with the following criteria were excluded from this meta-analysis: (1) duplicate and non-full-text publications; (2) reviews, non-human studies, and retrospective and observational studies; and (3) published in languages other than Chinese or English.κ = 0.879). Conflicts between the two authors were resolved by the opinion of a third author (YQL). Eligibility screening was performed in two steps: (1) title and abstract screening for relevance to the study objective, and (2) full-text screening for eligibility for meta-analysis. For each eligible study, the following information was extracted (1) basic information ; (2) baseline characteristics of intervention and study population; and (3) relevant outcomes, including CRP, TNF-α, IL-6, and IL-1. Two authors (XWZ and JFH) independently extracted data from each selected RCTs using a standard abstraction excel sheet (κ = 0.962).Studies were independently selected by two authors (XWZ and JFH), and they achieved good agreement (κ = 0.973). The assessment domains of the Cochrane Risk of Bias assessment tool include selection bias, performance bias, detection bias, attrition bias, reporting bias, and other biases  using the Cochrane Risk of Bias assessment tool  model was used for meta-analysis. When significant heterogeneity was determined, its source was further evaluated by sensitivity analyses or subgroup analyses. Sensitivity analyses were conducted to assess the effect of each trial on the validity of the pooled overall SMDs using the leave-one-out method. Subgroup analyses were conducted according to the following variables: dosage of berberine (< 0.9 g daily vs. ≥ 0.9 g daily), type of condition (metabolic syndrome vs. type 2 diabetes vs. diabetic nephropathy vs. cardiovascular disease vs. polycystic ovary syndrome vs. other), duration of study (< 3 months vs. ≥ 3 months vs. unclear), and sample size (< 30 vs. 30–60 vs. > 60). In the absence of clinical and methodological heterogeneity, the random-effects (RE) model was used to analyze the outcomes. The results of the meta-analysis were shown in forest plots. Publication bias was detected by funnel plot symmetry tests and Egger’s regression tests. Egger’s regression test was undertaken in Stata /MP version 16.0 .The meta-analysis was undertaken in Review Manager version 5.3 . Standardized mean differences (SMDs) and 95% confidence intervals (CIs) were used to assess continuous outcomes. A total of 7387 articles were retrieved from the initial search. After screening titles and abstracts, 152 studies were potentially eligible, and these were retrieved for full-text review. After reading the full text, 100 were excluded, because they failed to meet the inclusion criteria. Ultimately, 52 studies that fully satisfied the pre-established inclusion criteria of this meta-analysis were included. The search procedure and reasons for exclusion can be found in the flowchart presented in Fig. The 52 included studies were published between 2008 and 2020 , TNF-α , and IL-6 . Moreover, pooled findings from the random-effects model showed that there was no significant impact of berberine on the level of IL-1β .Forest plots that demonstrate the effects of berberine use on the evaluated inflammatory markers are shown in Fig. I2 = 94%; p < 0.10), TNF-α , IL-6 , and IL-1β , as shown in Fig. The meta-analysis showed statistically significant heterogeneity for the outcomes of CRP  and ≥ 3 months  for TNF-α. Furthermore, there were significant differences between before and after subgroup analyses in the stratum of metabolic syndrome for TNF-α  and the stratum of cardiovascular disease for IL-6 . These results of subgroup analyses suggested that type of condition and duration of study may be the source of heterogeneity in the meta-analysis. Table Following subgroup analyses, heterogeneity was changed among some of the strata of subgroups. The heterogeneity changed significantly in the strata of polycystic ovary syndrome  and IL-6  concentration levels. However, as shown in Fig. p < 0.05; 95% CI 1.27, 2.26) of CRP indicated some publication bias, which probably is attributed to unpublished studies with negative findings.Funnel plots and Egger’s regression test were not evaluated for IL-1β levels due to the relatively small number of studies with this endpoint. These tests showed no significant evidence of publication bias for meta-analyses assessing the effect of berberine on TNF-α  signaling pathway (Jeong et al. As an extract from traditional Chinese herbs, berberine has a long history of clinical application and many efficacy trials on humans in China. The meta-analysis included 52 RCTs involving 4616 Chinese patients with MetS and related disorders, which complemented the evidence of the effects of berberine use on inflammatory markers in humans and in China. The results suggested that berberine could reduce the concentration level of CRP significantly, which was consistent with the results of a previous study (Beba et al. There are a few limitations to this meta-analysis. First, the result of the risk-of-bias assessment presented a large proportion of uncertain risks for insufficient information in trial methods. To a certain extent, the potential differences in the methods of random sequence generation, allocation concealment, and concentration measurement among the included studies have caused the high heterogeneity of the meta-analysis results. Second, the study population of all the included studies was Chinese patients, and the sample size of individual clinical trial was small. The results of this meta-analysis are accordingly hard to extrapolate to other ethnic populations or geographical regions. Therefore, more studies with larger sample size, ideally multi-centers, and rigorous design are needed to confirm the effect of berberine on the inflammatory markers of MetS and related disorders.Despite the limitations of meta-analysis, the robust methodology followed in selecting RCTs for inclusion and in completing the evaluation does facilitate the conclusion that berberine use in patients with MetS and related disorders appears to have significantly decreased inflammatory markers, including CRP, TNF-α, and IL-6. This study provides new and useful evidence for supporting clinical medication decisions for MetS and related disorders and encourages undertaking further RCTs.


Number–color synesthesia is a condition in which synesthetes perceive numbers with concurrent experience of specific, corresponding colors. It has been proposed that synesthetic association exists primarily between representations of Arabic digit graphemes and colors, and a secondary, semantic connection between numerosity and colors is built via repeated co-activation. However, this distinction between the graphemic and semantic pathways of synesthetic number–color connection has not been empirically tested. The current study aims to dissociate graphemic and semantic aspects of color activations in number–color synesthesia by comparing their time courses. We adopted a synesthetic priming paradigm with varied stimuli onset asynchronies (SOAs). A number  was presented in one of three notations: digit, dice, or non-canonical dot pattern, and a color patch (target) appeared with an SOA of 0, 100, 300, 400, or 800 ms. Participants reported the color as quickly as possible. Using the congruency effect  as an index of synesthetic color activation level, we revealed that the effect from the graphemic pathway is quick and relatively persistent, while the effect from the semantic pathway unfolds at a later stage and is more transient. The dissociation between the graphemic and semantic pathways of synesthesia implies further functional distinction within “conceptual synesthesia”, which has been originally discussed as a unitary phenomenon. This distinction has been demonstrated by the differential time courses of synesthetic color activations, and suggested that a presumed, single type of synesthesia could involve multiple mechanisms. Synesthesia is a condition in which individuals (synesthetes) automatically perceive certain stimuli with experiences of additional properties, such as seeing achromatic words with certain colors. The physically present stimulus that elicits synesthesia  is termed the “inducer”, and the additional experience  is termed the “concurrent” . In partcongruency effect). Specifically, in priming paradigms for grapheme– and number–color synesthesia, a grapheme or number prime facilitates or interrupts a synesthete’s response to a color target. For instance, for a synesthete who associates the number 5 with color red, a “5” prime will lead to quicker and more accurate color-naming of a red color patch than a green one. In contrast, naïve non-synesthetes (people who do not experience synesthesia and who have not been trained with inducer–color parings for specific research purposes) show no congruency effect in such tasks.To quantitatively measure synesthesia, common behavioral paradigms have been developed, including the synesthetic priming paradigm ,3. In a Studies on number–color synesthesia have found that numbers automatically elicit color processing when presented in various notations, such as Arabic digits, dot patterns, and finger counts ,6,7, demHowever, most number–color synesthetes report no subjective color experience for numbers in less common or non-graphemic forms  ,7,18. Acwithin conceptual synesthesia. Empirically, number–color synesthesia is consensually considered as conceptual/higher synesthesia, and the dissociation of graphemic and semantic pathways is a distinction of number–color synesthesia mechanisms. Theoretically, grapheme identities are considered as concepts in theorizations of synesthesia [Our present study aims to dissociate the hypothesized graphemic and semantic pathways by comparing the time course of synesthetic activation through them. Different from previous studies which distinguished between perceptual and conceptual synesthesia, we are making this graphemic/semantic dissociation esthesia , and semIn our experiment, we used numbers in different notations  dot patterns) as the prime and manipulated the SOAs between the prime and target. Synesthetes and non-synesthetes reacted to a color patch target as quickly as possible. The SOA at which the congruency effect occurs, which we hereafter refer to as the latency of congruency effect, reflects the speed of synesthetic color activation. The maximum congruency effect size across SOA, which we hereafter refer to as the magnitude of congruency effect, reflects the strength of number–color association. Since graphemic processing precedes semantic processing ,20,21, wM = 20.91, SD = 2.39) and eleven gender-matched non-synesthetes  participated in the experiment. Synesthete participants were then assessed and screened with a customized eight-item questionnaire. With this questionnaire, they reported several key features of synesthetic experience  for five numbers (2–6), the stimulus for a given number × notation was fixed, e.g., NC dot configuration for “4” did not change between trials . A colorThe experiment was programmed with the Psychophysics Toolbox-3 ,23,24 imWe adopted a 3 (number notation) × 5 (SOA) × 2 (congruency) factorial design, yielding 30 experimental conditions. Number notations include Arabic digit, dice pattern, and NC pattern. SOAs were picked by a pilot test and set with five levels: 0, 100, 300, 400, and 800 ms. A trial was defined as “congruent” if the target color matched the corresponding color of the number prime and “incongruent” if they did not match.We arranged the trials in 8 blocks. Only two numbers and their corresponding colors appeared in each block. Thus, a color  always had an equal probability (50%) of being either congruent or incongruent to its preceding number prime. Considering that numerical range  would affect the speed and accuracy of object numerosity processing  and may Participants completed the experiment in two days, with four blocks (taking approximately 90 min) each day. Upon arrival at the laboratory on Day 1, they chose colors for numbers 2–6 from the custom font color palette in Microsoft Word (Microsoft Inc.). Synesthetes chose their synesthetic colors, and non-synesthetes assigned any color they preferred to the given numbers. In this way, though color sets were not physically identical across participant groups, they were functionally identical in that they were chosen by participants themselves.Each block contained a color response practice session and a main experiment session. The practice session familiarized the participant with the correspondence of response keys (left and right “shift” on the keyboard) to colors. Participants were instructed to press the left “shift” key when seeing one color, and the right “shift” key when seeing the other. This response should be as quick as possible. Experimental stimuli were presented against a white background. Each trail started with a 300 ms central fixation cross ), and a color stimulus was presented at screen center until response (maximum waiting time: 2000 ms). Immediately after response, a 500 ms “correct/incorrect” feedback was given. The next trial started after an inter-trial interval (ITI) of 1000 ms. There were 80 randomized trials (40 for each color).The main experiment followed the practice session. Participants were instructed to respond to colors with the same keys as in the practice session, and then report numbers by pressing “Y” and “B” keys . The response to color (hereafter referred to as the color response) should be as quick and accurate as possible, and the response to number (hereafter referred to as the number response) could be at their own pace. In each trial, a central fixation cross ) was presented for 300 ms, and a number stimulus was presented at screen center for 50 ms. After a delay (for SOA > 0 conditions), a color stimulus was presented at screen center until color response . Immediately following color response, a prompt for number response was presented until the response was made (maximum waiting time: 5000 ms). No feedback was given throughout the trial. The next trial started after a random ITI of 2000–2500 ms .Within a block, identical trials  were repeated 3 times. Thus, each block contained 30 (conditions) × 2 (numbers) × 3 (repetition) = 180 trials. These trials were randomized. In total, the experiment contained 8 × 180 = 1440 trials.The color patches for each individual are listed in SDs below average, which was thought to indicate inattentiveness. Trials with incorrect responses for either color or number were excluded from analysis. Within each participant, we removed outlier trials (where RT of color response was more than 3 SDs deviant from the mean RT). In all, 7.66% of trials in the remaining blocks were excluded. To check for the congruency effect, using SPSS 25.0 (IBM Corporation), we ran a repeated measures ANOVA on RT with participant group  as the between-subject factor, and congruency, number notation, and SOA as within-subject factors. Overall RTs of synesthetes (M ± SE: 550 ± 28 ms) and non-synesthetes (593 ± 28 ms) were not significantly different  = 1.212, p = 0.284). The RTs for congruent trials (551 ± 18 ms) were significantly shorter than incongruent trials  = 10.868, p = 0.004, partial η2 = 0.352), i.e., there was a congruency effect. There was an interaction between participant group and congruency  = 6.590, p = 0.018, partial η2 = 0.248). Congruency effects were significant in synesthetes  but not in non-synesthetes . These results indicate a synesthetic congruency effect only in synesthetes, typical in synesthetic priming paradigms  = 6.590, p = 0.018, partial η2 = 0.248). SOA had a main effect  = 4.944, p = 0.001, partial η2 = 0.198) due to a larger congruency effect at 300 ms (60 ± 16 ms) than 800 ms (20 ± 8 ms) SOA . The interaction between SOA and participant group was also significant  = 2.500, p = 0.049, partial η2 = 0.111). Bonferroni-corrected post-hoc comparison indicated that synesthetes’ congruency effects were larger at SOA of 100 ms  and 300 ms  than at 800 ms (37 ± 11 ms), whereas non-synesthetes’ congruency effects did not differ across SOAs. Number notation exhibited no main effect  = 2.095, p = 0.136) but interacted with SOA  = 2.178, p = 0.032, partial η2 = 0.098). At 400 ms SOA, congruency effect for digits (60 ± 17 ms) were larger than for dice , and at 800 ms SOA, congruency effect for NC patterns (38 ± 9 ms) were larger than for dice .For each participant and under each condition, we calculated congruency effects by subtracting the average RT of congruent trials from the average RT of incongruent trials. We ran a repeated measures ANOVA on congruency effect with participant group as the between-subject factor, and number notation and SOA as within-subject factors. We found a significant main effect of participant group (synesthete (73 ± 18 ms) > non-synesthete (9 ± 18 ms), F = 5.450, p = 0.001, partial η2 = 0.353) due to larger congruency effect at 300 ms (73 ± 29 ms) than 800 ms (37 ± 13 ms) . This is in line with our expectation that synesthetic interference would occur at short SOAs and later decline. Number notation had a marginally significant main effect  = 4.332, p = 0.050, partial η2 = 0.302) due to larger congruency effect for digits (87 ± 27 ms) than for dice . SOA × number notation interaction was marginally significant  = 1.960, p = 0.062, partial η2 = 0.164). Bonferroni-corrected simple main effect analysis revealed that at 0 ms SOA, congruency effect for digits (109 ± 26 ms) was larger than for NC patterns ; at 400 ms SOA, congruency effect for digits (93 ± 32 ms) was larger than for dice ; and at 800 ms SOA, congruency effect for NC patterns (56 ± 11 ms) was larger than for dice . Taken together, digits tend to yield larger congruency effects than dot patterns at 0 and 400 ms SOAs, while at SOA levels in between (100 and 300 ms), effect sizes were similar across number notations. The congruency effect elicited by digits was persistent throughout the initial 400 ms following stimulus onset, while the congruency effect elicited by dot patterns increased through 0–300 ms SOAs, but decreased from 300 ms onwards, i.e., exhibited a pattern of “rise-and-fall”. In non-synesthetes  = 0.124, p = 0.884; SOA: F = 1.280, p = 0.294; interaction: F = 0.823, p = 0.0585).To determine the origin of interactions, we ran repeated measures ANOVAs with number notation and SOA as within-subject factors, separately in synesthetes and non-synesthetes. In synesthetes a, SOA haIn order to dissociate graphemic and semantic components in number–color synesthesia, we investigated the time course of color activations elicited by numbers in three notations. We implemented a priming paradigm with varying SOAs and compared number–color congruency effects. Throughout the 0–400 ms time window post-stimulus onset when differences between notations were expected to appear, we found differences between digits and dot patterns but not between types of dot patterns . Specifically, congruency effect elicited by digits had a shorter latency  and was persistent, while that elicited by both dot patterns exhibited a “rise-and-fall” pattern. At 800 ms SOA, NC yielded an unexpected larger effect than dice. The three number notations yielded similar congruency effect sizes . Our findings revealed a distinction between effects of graphemic and semantic pathways of synesthesia on the temporal dimension. In contrast to previous studies distinguishing “perceptual vs conceptual” synesthesia, we highlighted that this distinction is within “conceptual” synesthesia. Moreover, the associated two pathways (as well as multiple working mechanisms) could co-exist in individual synesthetes, rather than a between-individual difference or a single mechanism, which could explain the current results (see more details below).As we have expected, digits did show superiority over dot patterns in eliciting synesthesia, but this superiority was reflected in a quicker and more persistent time course rather than a larger magnitude. Thus, the graphemic and semantic pathways differ in time courses in their effects: effect of the former was quick and persistent, while the latter had actions of a relatively slow and transient manner. Previous studies have measured number–color synesthetic interference using either simultaneous stimuli presentation or a fixed SOA ,6,7,8,9,Our distinction of pathways differs from previous studies in two major aspects. First, models of synesthesia typically distinguish between perceptual and conceptual levels of stimulus processing, and either argue for the occurrence of synesthesia on one of the levels , or ideIn sum, our study did not aim to separate different subtypes of number–color synesthesia but to establish a distinction between the graphemic and semantic aspects of number–color synesthesia on the temporal dimension. We revealed the temporal dynamics of the behavioral outcomes in number–color synesthesia, in which the graphemic pathway was quick and relatively persistent in sub-serving the color activation, while the semantic pathway had a slower onset and is more transient. Moreover, effects from the pathways appeared non-additive, possibly implying a common neuronal representation of the synesthetic concurrent. In contrast to previous studies which discussed the heterogeneity of synesthesia by distinguishing between its perceptual vs. conceptual nature and considered each type of synesthesia as a unitary whole, our distinction between graphemic and semantic pathways is a distinction within conceptual synesthesia. Moreover, the two pathways are co-existing mechanisms within individuals, rather than between-individual differences. For future studies, our study has demonstrated that investigating the time courses of synesthetic activation can be an effective methodology of distinguishing between different mechanisms in synesthesia; theoretically, we call attention to the possibility that a single type of synesthesia may involve multiple mechanisms.


The secondary tar reactions initiate from ≈318 °C (BP), 481 °C (SS), and 376 °C (AD). Moreover, negative apparent activation energies are intrinsic to their kinetics after 313 °C (BP), 448 °C (SS), and 339 °C (AD). The results can support in tailoring and controlling sustainable biochar synthesis from slow pyrolysis of MWB.Synthesizing biochar from mineral- and ash-rich waste biomass (MWB), a by-product of human activities in urban areas, can result in renewable and versatile multi-functional materials, which can also cater to the need of solid waste management. Hybridizing biochar with minerals, silicates, and metals is widely investigated to improve parent functionalities. MWB intrinsically possesses such foreign materials. The pyrolysis of such MWB is kinetically complex and requires detailed investigation. Using TGA-FTIR, this study investigates and compares the kinetics and decomposition mechanism during pyrolysis of three types of MWB: (i) mineral-rich banana peduncle (BP), (ii) ash-rich sewage sludge (SS), and (iii) mineral and ash-rich anaerobic digestate (AD). The results show that the pyrolysis of BP, SS, and AD is exothermic, catalyzed by its mineral content, with heat of pyrolysis 5480, 4066, and 1286 kJ/kg, respectively. The pyrolysis favors char formation kinetics mainly releasing CO Biochar is the non-graphitizing porous carbon formed from the progressive dehydration, decarboxylation, dehydrogenation, demethylation, polycondensation, and graphitization (at high temperatures) of biomass in an inert or oxygen-deficient environment. It has versatile applications in carbon sequestration, soil amelioration, energy storage, and gas- or liquid-phase adsorption. The last decade has seen considerable research on the production and applications of biochar derived from wood and other feedstock with low ash and minerals ,2,3,4,5 On the process side, MWB pyrolysis is a complex thermochemical phenomenon with multiple concurrent and/or sequential reactions . Its modHerein, the authors investigate and compare the pyrolysis mechanism from three different kinds of MWB: (a) rich in AAEM minerals—K-rich banana peduncle; (b) rich in ash —sewage sludge (SS); (c) rich in AAEM and ash—anaerobic digestate (AD). The main purposes of this study are to determine the different pyrolysis stages including secondary tar cracking reactions, their associated gas/volatile emissions and heat flow, kinetic triplets, and reaction thermodynamics using linear and non-linear isoconversional methods. This is to tailor the properties of biochar generated from MWB for its application.3, respectively. Their high H/C molar ratio  and AAEM concentration strengthen the possibility for synergetic interactions during pyrolysis n) overlapping steady decomposition. This is further confirmed from the exothermic DSC and the featureless D-DSC (seen later in 9H12O2), G type (guaiacyl C10H12O3), and/or S type (syringyl C11H14O4) /Si low molar ratio of 0.6) ..α transiα gradually increases with temperature/conversions for all substrates, i.e., with increasing devolatilization of organics, the rate control is shifting to alternate paths within a network of parallel reaction pathways available for the biomass components. Then, the concave transition in Eα to negative values hints at the shift in rate control to a reversible step within the same reaction pathway [1) as in Equation (8). Due to preferential char formation, the net heat release (−ΔH0 + ΔH01) is exothermic.During active pyrolysis, E pathway ,125 thatTar2) desorb out of the pores, and char undergoes an exothermic structural change to a more stabilized form. Here, L is an active site, L(tar2) is the active site filled with heavy tar vapor molecule, ϕ is the fraction of active sites filled with Tar2, (1 − ϕ) is the fraction of empty active sites, and a* is the activity of the metastable char*. Based on the microscopic reversibility principle  r1 = rd = 0 at equilibrium conditions (P = eqP and ϕ = eqϕ) [eqP is given by Equation (15).K1 and dK are thermodynamic equilibrium constants for decomposition and desorption, respectively, and are given by the Van Hoff’s Equations (17) and (18).H1 and ΔdH are standard enthalpy changes, and ΔS1 and ΔdS are standard entropy changes associated with decomposition and desorption, respectively. By pseudo steady-state hypothesis, the reaction intermediates remain constant with time, i.e., r1 = dr. If desorption is the rate-limiting step with da*k, aPk ≪ k1, k2a* then:G* is the positive free energy of formation of the metastable char by release of ΔH*. The reaction rate will then be equal to the rate of desorption, the rate-limiting step, r = dr (Equation (20)).After MPT, int.prod is formed from higher ordered cellulose and more stable organics such as lignin. Then, as in Equation (9), int.prod decomposes to porous metastable amorphous char*. The heavy secondary tar (ϕ = ϕeq) . Then, P2), Equation (22) becomes Equation (23). Here, ϕ ≈ 1 when either (a) RT ≫ (ΔG* + ΔG1), i.e., at higher temperatures after MPT, and (b) ΔG1 ≪ −ΔG*, i.e., when decomposition is spontaneous due to the exothermic char formation reactions as seen in DSC. Thus, when the rate control shifts to reaction rd, we obtain negative activation energies. After this transition to negative values, Eα has a general increasing trend with conversion for all the three investigated substrates. It means that reactions again shift to alternate pathways among a network of available pathways. This point of lowest -Eα from which apparent activation energy starts again increasing marks the start of the increasing influence of HeSTR (resulting in secondary char formation) due to longer contact times with the biochar (Equation (21)). An increase in exothermicity (as seen in DSC) and the presence of non-condensable gases corroborate this observation. Lower bulk density increases HeSTR [When pressure gradients within the experiment are considered negligible and when ϕ ≈ 1 ). After this, -Eα arises from the gasification of char and its associated external diffusion limitations. At these temperatures, further interpretations of observed kinetic parameters would be incomplete as, even at particle sizes below 0.5 mm, these regions would deviate away from the pure kinetic regime depending on the Biot and Pyrolysis  numbers [The temperatures between 650 and 700 °C for SS and those above 800 °C for AD shift toward positive E numbers . Tempera numbers , as seenα  between ln A and activation energy. From the review of the conditions that lead to a pKCE, this conclusion becomes apparent. The pKCE arises from the propagation of systematic and computational errors during kinetic measurements and calculation [α estimation [When the frequency factors are calculated based on pseudo KCE (pKCE), the values of ln A should change proportional to Eα ; the colculation  and doesculation . Hence, timation ,133. Thuα as they are calculated from Eyring equations (α − ΔHα) at any conversion, the more favorable the product of formation [α − ΔHα), in α − ΔHα). The process slows down considerably after ≈745, 735, and 690 °C for BP, SS, and AD beyond, and there is also a large increase in CO and CO2 emissions. These MWBs favor exothermic char formation during active pyrolysis and HeSTR during secondary pyrolysis. It is also in agreement with the findings that biomass pyrolysis can conditionally favor exothermic [The enthalpy variation also follows the same trend as Equations . In manyormation ,140,141 othermic ,143,144 othermic ,145,146 2) emissions if the pyrolysis is performed until the HTT of 745, 735, and 690 °C for BP, SS, and AD. Here, mineral-rich and ash-low BP yields the most HoP, while SS and AD also evolve 7–8% CH4 for energy recovery. GHG emissions of CO2 and CO are the highest for BP . Thus, there is a potential for further optimizing biochar synthesis for energy recovery and GHG emissions through the combined pyrolysis of MWB and warrants further investigations. The HeSTR is prominent from ≈318, 481, and 376 °C for BP, SS, and AD, respectively, while HoSTR is from ≈565 °C. The endothermic gasification becomes rate-limiting from 800 °C.The pyrolysis of conventional lignocellulosic biomass is widely investigated for energetic and value-added product applications. The MWB results from anthropogenic activities such as wastewater treatment and chemical fertilization, and it has limited recyclability owing to the high AAEM, silicate, ash, and/or heavy metal content. Their transformation to biochar presents a feasible route for their sustainable recycling in the field of solid waste management. Three different types of MWB—BP, SS, and AD—are investigated to elucidate their thermolysis mechanism and showcase the potential for biochar synthesis in a TGA scale. The biochar synthesis favors char formation kinetics and is exothermic in the order BP > SS > AD. Exothermicity can be maximized without increasing residence time and GHG (CO and COThe pyrolysis of BP has the highest heat release and most kinetic favorability. Its biochar yield is the smallest due to high cellulose and low silicate content.x precursors follows the order SS > AD > BP.Amongst the various stages, the highest contribution of exothermicity is from secondary pyrolysis and emission of NOBased on isoconversional methods, negative apparent activation energies are intrinsic to explain their kinetics after 313, 448, and 339 °C for BP, SS, and AD, respectively.It can also be concluded that:These results can support modeling and controlling biochar synthesis from the slow pyrolysis of MWB sustainably by maximizing heat uptake and minimizing GHG emissions. Such aspects are presently not addressed in the present biochar literature. Furthermore, the detailed methodology applied in this work can be utilized to critically analyze the pyrolysis schemes of other MWBs and their blends. The investigations of physio-chemical attributes of the MWB-derived biochar and the influence of process scaling can complement these results in tailor-made biochar synthesis for the development of their pyrolysis technology. Hence, future studies in the direction of process scale-up, biochar assay, and life cycle assessment are recommended. In the follow-up part-2 publication, the authors explore the properties of biochar for its application in carbon sequestration, adsorption and soil application.


Exosomes have the ability to carry a wide range of chemicals, convey them to target cells or target regions, and act as “messengers.” For the purpose of investigating embryo attachment, it is helpful to comprehend the range of exosomal mRNAs and miRNAs derived from the uterine flushing fluid before and after embryo attachment. In this study, we recovered exosomes from goat uterine rinsing fluid at 5, 15, and 18 days of gestation and used RNA-Seq to identify the mRNA and miRNA profiles of exosomes obtained from uterine rinsing fluid before and after embryo implantation. In total, 91 differently expressed miRNAs and 27,487 differentially expressed mRNAs were found. The target genes predicted by the differentially expressed miRNAs and the differentially expressed mRNAs were mainly membrane-related organelles with catalytic activity, binding activity, transcriptional regulation activity, and involved in metabolism, biological regulation, development, and other processes. This was revealed by GO analysis. Furthermore, KEGG analysis revealed that they were abundant in signaling pathways associated with embryo implantation, including the “PI3K-Akt signaling pathway,” “Toll-like receptor signaling pathway,” “TGF-beta signaling route,” “Notch signaling pathway,” and others. Moreover, our research has demonstrated, for the first time, that chi-let-7b-5p specifically targets the 3’UTR of CXCL10. Our research offers a fresh viewpoint on the mechanics of embryo attachment. An important step in the beginning of mammalian pregnancy is embryo implantation. It describes the period of time when the fertilized egg transforms into a blastocyst in the uterus, forms a temporary connection with the mother’s uterus, and is stationed there. The embryo and the mother’s endometrium engage in sophisticated communication during the engraftment stage . AccordiExosomes are small vesicles with a characteristic lipid bilayer structure and a diameter ranging from 30 to 100 nm. Exosomes were given their current name in 1987 after being discovered for the first time in sheep reticulum cells in 1983. Endocytosis, which collects DNA, RNA, protein, and other molecules in the cytoplasm into vesicular endosomes, produces exosomes, which are eventually released from the cell when they fuse with the plasma membrane ,5. ExosoNumerous studies have demonstrated that exosomes can encourage intercellular communication in the female reproductive tract , and the10 particles/mL; on the fifteenth, they were 77.29 nm and 1.22 × 1011 particles/mL; and on the eighteenth, they were 75.24 nm and 8.96 × 1010 particles/mL . The main mRNAs that differed in expression were those related to “Metabolic pathways,” “Oxidative phosphorylation,” “Spliceosome,” “Thermogenesis,” “Parkinson disease,” etc.; “Metabolic pathways,” “Huntington disease,” “Carbon metabolism,” “Spliceosome,” “Thermogenesis,” etc.; and “Pathways in cancer,” “Phosphatidylinositol signaling system. “The “FoxO signaling pathway,” the “Apelin signaling pathway,” “Adherens junction,“ “Thyroid hormone signaling system,“ “Pathways in cancer, “ etc. were the key signaling pathways where the target genes of miRNAs that changed between days 15 and 18 of gestation were expressed. The “AGE-RAGE signaling route in diabetic problems,“ “TGF-beta signaling pathway,“ “Axon guidance signaling pathway,“ “Rap1 signaling pathway, “ “Platelet activation, “ “PI3K-Akt signaling pathway,“ and “Toll-like receptor signaling pathway“ were the primary complications between days 5 and 15 of pregnancy. Target genes of differential miRNAs were primarily involved in “Axon guidance,” “Protein digestion and absorption,” “Legionellosis,” “Transcriptional misregulation in cancer,” “Leukocyte transendothelial migration,” “Biosynthesis of unsaturated fatty acids,” “Adherens junction,” “AMPK signaling pathway,” etc. between 5 and 18 days of gestation . These fFTH1, PTGS2, CTNNB1, ACSL4, and GPX4) that were shown to be differentially expressed above were verified using RT-PCR. The results matched the sequencing information  and mRNAs . MUC1 has been described as a target gene for members of the let-7 family [CXCL10 has been described as a crucial cytokine for the implantation of goat embryos. These findings suggest that chi-let-7b-5p inhibits CXCL10, which has an impact on the implantation of goat embryos.Goat endometrial epithelial cells were transfected with chi-let-7b-5p mimic or inhibitor, and the transfection efficiency was greater at concentrations of 100 nM and 150 nM, respectively A. These 7 family , while CCXCL10 (CXCL10 mRNA level in tissues on the 18th day of pregnancy was significantly upregulated (p < 0.01)  B, and th < 0.01) C.We performed the twofold luciferase reporting experiment to confirm that chi-let-7b-5p and CXCL10 have a targeting interaction. A 430 bp fragment with the CXCL10 3’-UTR binding site was amplified and cloned into the psiCHECK2 vector where it was given the name psiCHECK2-CXCL10 WT. The mutant sequence of CXCL10 3’UTR was obtained by fusion PCR, and the created mutation vector was given the name psiCHECK2-CXCL10 MUT A. Sequenp < 0.01), but the chi-let-7b-5p inhibitor would have resulted in the upregulation of CXCL10 expression in gEECs  is recognized to participate in a variety of biological activities. In a previous study, the number of implanted embryos dropped on day 3 of gestation when mice were given a particular inhibitor of TGF-, the most specific signal transduction factor Smads, indicated that TGF- may be involved in the process of embryo implantation . The preCXCL10 and MUC1 expression was greatly reduced by the chi-let-7b-5p mimic, but it was significantly boosted by the chi-let-7b-5p inhibitor. One of the let-7 family’s target genes is MUC1 [CXCL10 is a crucial cytokine for embryo implantation. We discovered a binding site between chi-let-7b-5p and CXCL10 using online target gene prediction software for miRNAs. Furthermore, examination of the double luciferase reporter gene demonstrated that chi-let-7b-5p directly targeted CXCL10. During the implantation stage of a human embryo, the mother and the embryo will secrete chemokines that will act on trophoblast cells and encourage their migration [As the number of days of goat pregnancy increased in our study, the expression of chi-let-7b-5p decreased. Members of the let-7 family have been shown to express differently throughout the implantation stage in rats, and they were especially found in the glandular epithelium, luminal epithelium, and decidual cells . Using i is MUC1 , and CXCigration . In addiigration . Numerouigration ,43 can bigration . In addiigration . Let-7b-n = 2), and were kept in the Yangling Northwest Agriculture and Forestry University’s experimental animal center. The female goat and male goat freely mated after estrus, and this was noted as the 0th day of pregnancy. The pregnancy was determined by looking at the tubular or linear pregnancy in the uterus on the fifth day , the fifteenth day , and the eighteenth day . A midline laparotomy and hysterectomy were performed on the pregnant sheep on the fifth, fifteenth, and eighteenth days. The ewe’s uterus was then quickly collected , and the cervix and horn were bound with ropes to prevent the fluids from pouring out. The ewe’s uterus was placed in a thermos bottle and kept at a temperature of 15–20 °C before being transported in 3–4 h to the lab. To preheat, we placed the sterilized normal saline in a water bath set to 35 °C. Then, we added the necessary dosage of penicillin to the uterus before washing it. We washed the outside wall of the uterus three times with regular saline after eliminating the contaminants from outside the uterus. A segment of infusion tube was cut, inserted into the uterus from the cervix, and connected to a 20 mL syringe, sucked, and repeated eight times. The flushed solution was collected into sterilized centrifuge tube, the membrane was sealed, and the samples were stored at 4 °C for standby.Six sexually mature Guanzhong dairy goats were split into three groups at random, with two goats in each group . Next, the solution underwent 110,000× g ultracentrifugation at 4 °C for 2 h. If the liquid was insufficient, then sterile PBS (PH 7.4) was added, and the ultracentrifugation tube was closed. Then, we the ultracentrifuge tube off using sterilizing scissors, removed the supernatant, and left 2 mL of liquid suspended sediment. We filtered the heavy suspension liquid with a 0.22 μm filter  before transferring it to a 6.0 mL ultracentrifuge tube , where PBS was refilled and sealed. Afterward, the solution underwent 110,000× g ultracentrifugation for 70 min at 4 °C. We threw the supernatant, resuspended the sediment in 1 mL of sterile PBS, then moved the heavy suspension to a 6.0 mL ultracentrifuge tube, filled it with PBS, and closed it. The solution underwent 110,000× g ultracentrifugation for 70 min at 4 °C. The precipitate was subpackaged and stored at −80 °C after being resuspended in the appropriate volume of sterile PBS.Ultracentrifugation , densityFirst, samples were sent for detection after 5 μL of exosomes produced by overspeed centrifugation were removed and diluted to 10 μL. Then, 10 μL of the sample was poured onto the copper mesh, where it precipitated for 1 min. Filter paper was used to get rid of the float. On the copper mesh, 10 μL of phosphotungstic acid was poured and allowed to precipitate for 1 min. In addition, filter paper absorbed the float. The samples were at room temperature for a while, and imaging at 80 kV was performed using an electron microscope .Exosomes obtained by overspeed centrifugation were extracted, diluted to a volume of 30 μL, and sent for detection in samples. Only after the instrument performance test had been qualified with the reference material could the exosome sample be loaded. To prevent a sample from clogging the injection needle, close attention was paid to the gradient dilution. Following testing of the material, the exosome content and particle size could be determined using the NanoFCM equipment .To extract the exocrine protein, we used the complete protein extraction kit  and the BCA protein content kit to assess the protein concentration . We transferred the sample to a polyvinylidene fluoride (PVDF) membrane , sealed it with 5% skimmed milk powder for one hour, and then used the first antibody  for an overnight incubation at 4 degrees and the second antibody  for a one-hour incubation at 1:5000 room temperature. We placed the film in the imager  and exposed it after incubating it with ECL luminous liquid . Then, we saved the image.g at 4 °C. Then, we added 1.5 times the volume of anhydrous ethanol to the upper water phase in a fresh centrifuge tube and mixed. We filled the RNeasy MinElute centrifuge column with the liquid from the previous step, centrifuged at 8000× g at room temperature for 15 s, and then discarded the liquid. Next, we added 700 μL of RWT buffer, 8000× g, centrifuged for 15 s at room temperature, and then removed the liquid. The centrifuge column was placed in a fresh centrifuge tube, which was centrifuged at 12,000× g for 5 min. The liquid and centrifuge tube were discarded. RNA was eluted by centrifuging the centrifuge column for 1 min at 12,000× g at room temperature while adding 14 μL RNase-free water to a fresh centrifuge tube. BGI carried out the quality control, library creation, and total RNA in exosomes sequencing.In this work, total RNA was extracted from the exosomes using the miRNeasySerum/Plasma Kit . The exocrine body was removed from the −80 °C refrigerator and placed in a water bath at 37 °C to melt. Then, the 500 μL of the QIAL Lysis Agent was added, mixed upside-down, and left to incubate for 5 min at room temperature. We combined 100 μL of chloroform and vigorously shake for 15 s. Then, we left the solution to incubate for 3 min at room temperature. The solution underwent 15 min of centrifuging 12,000× The reference genome and transcriptome were compared with the filtered clean reads of the raw data produced from sequencing, respectively. p-values to Q values correctly. We defined genes with more than twice the differential multiple and Q value 0.001 as significantly differentially expressed genes and screened them in order to increase the accuracy of DEGs. Various small RNA analyses were performed. A special molecular tag called UMI was initially added to the cDNA during library preparation in order to measure the small RNA. According to Audio S. et al. method’s for sequencing-based differential gene detection [RSEM  was usedetection , which wp-value was corrected by Bonferroni [The analysis of exosome differentially expressed genes by GO and KEGG was performed. We used RNAhybrid , miRandanferroni . The prinferroni . Based o−ΔΔCt method. All reactions were performed in at least three independent experiments. Following the manufacturer’s instructions, the total RNA was extracted using the Trizol  reagent. Evo M-MLV reverse transcription Kit  was used to reverse-transcribe common mRNA, while the miRNA 1st strand cDNA synthesis Kit was used to reverse-transcribe miRNA . For the mRNA and miRNA analyses, real-time quantitative RT-PCR was carried out using the SYBR Green method , utilizing GAPDH and U6 as the internal references, respectively. Data were analyzed using 22O2 dropwise, incubated for 15 min without light at room temperature to inhibit endogenous peroxidase, and then rinsed with PBS. This process was followed by 1 h of pre-hybridization at 37 °C. Using the probe’s hybridization buffer as the negative control, the hybridization solution containing the chi-let-7b-5p probe was hybridized overnight at 42 °C before being washed with the washing buffer. Then, we added the hybridization solution containing Imaging oligo (DIG) dropwise, incubated at 42 °C for 3 h, and then washed with washing buffer solution. For 30 min, normal rabbit serum was sealed at room temperature, and anti DIG HRP was added after removing the sealing fluid. We incubated the solution at 37 °C for 40 min, then washed sections in PBS four times for 5 min each using FITC Tyramide Reagent. After 5 min of dark, room-temperature reaction, the sections were rinsed with PBS; After washing the film, DAPI was incubated in the dark for 8 min before being sealed with an anti-fluorescence quenching agent. The pictures were captured with a microscope .Initially, chi-let-7b-5p was discovered via in situ hybridization in goat endometrium. The chi-let-7b-5p probe with cy3 label was created and manufactured by Servicebio. Simply put, following dewaxing, rehydrating, and repairing antigens on paraffin sections, protease K (20 μg/mL)  digestion occurred for 20 min, followed by PBS flushing. Then, we added 3% methanol-HThe chi-let-7b-5p mimic and inhibitor, as well as the corresponding NC mimic and inhibitor, were bought from the Guangzhou Ruibo biological company. Immortalized gEECs were obtained as previously described  The cellTM SP Kit . The negative control section was switched out for PBS, and the primary antibody CXCL10  was incubated at a 1:300 dilution. Hematoxylin was counterstained for 25 s while the DAB  color development time was monitored under a microscope. After being exposed to a variety of alcohol and xylene concentrations, the slices were sealed with neutral gum.Using immunohistochemistry (IHC), we evaluated the expression of CXCL10 in goat uterus. Slices were taken out of the refrigerator at 4 °C and baked for 30 min at 60 °C. To remove the paraffin from the slices, they were submerged in xylene and a succession of alcohol solutions of varying concentrations. Slices were placed in PH6.0 citrate buffer, heated to a boil in a microwave, and then cooked for 15 min on low heat. The sample was then washed three times with PBS for five minutes each after cooling to room temperature. Histochemical staining was carried out using the ready-to-use immunohistochemistry hypersensitivity UltraSensitive® Reporter Assay System  was used to detect the firefly luciferase and renilla luciferase activity 36 h after transfection. The ratio of renilla to firefly luciferase was used to present the findings.The goat CXCL10 gene’s 3′UTR (untranslated region) was amplified by PCR and introduced into the psiCHECK2 vector. The partial sequences for the CXCL10 3′UTR WT and MUT were 5′-AGAGAGAAGCTACCTCT-3′ and 5′-AGAGAAGAGCATCAT-3, respectively. We employed 293T cell lines that had been frozen in our lab for later transfection. One day prior to transfection, cells under favorable growth conditions and in the logarithmic growth phase were transferred to 24-well plates, and HEK293T cells were co-transfected with NC-mimic or chi-let-7b-5p mimic. According to the kit’s instructions, dual-Luciferaset-test and one-way ANOVA were utilized. Moreover, p < 0.05 indicates a statistically significant difference, and p < 0.01 indicates an incredibly significant difference. We employed two methods that correct p-values to Q-values for the sequencing data developed by Benjamini, Hochberg, Storey, and Tibshirani. Significantly differentially expressed genes (SDEGs) were classified and screened as genes with a Q-value ≤ 0.001 and a difference greater than two times the control.Data are presented as the mean ± standard error of the mean (SEM). The mapping of the data was performed using Graphpad Prism 5.0. All data were analyzed using SPSS software. To examine the statistical significance of the data differences, the non-parametric In conclusion, we found some exosomes in the uterine rinse fluid both before and after embryo implantation. We provide fresh light on mother-fetal communication during goat embryo implantation by contrasting the differential expression of mRNAs and miRNAs in exosomes recovered from uterine rinsing fluid before and after embryo implantation. Investigating the precise functions of these differently expressed mRNAs and miRNAs during embryo attachment will be the main goal of our future work.


The “Total HRQoL” score was correlated to the “Total Fatigue” score . A low “Sleep/Rest Fatigue” score was associated with low “Physical Functioning”, “School Functioning”, and “Total HRQoL” scores . Parents’ HRQoL score was lower than patients’ score on “Physical Functioning” (p = 0.040) and “School Functioning” subcategories (p = 0.045). Conclusions: Fatigue and disturbed HRQoL are mostly observed in CKD 5D and KTx pediatric patients, and are associated with sleep disorders and school dysfunction. Fatigue affects HRQoL, which is perceived as more deteriorated by the patients’ parents.Background: This study investigates the effect of chronic kidney disease (CKD) stage on fatigue and health-related quality of life (HRQoL) in the pediatric population. Material and Methods: The PedsQL  Multidimensional Fatigue Scale  and HRQoL Generic Core Scales  questionnaires were completed by 30 patients aged from 7 to 18 years old with CKD stage 2–4, CKD stage 5 on dialysis (CKD 5D), and kidney transplantation (KTx), as well as their parents. Results: Both low “Total Fatigue” and “Total HRQoL” scores were reported in 16.7% of patients. “Sleep/Rest Fatigue”, “Emotional Functioning”, and “School functioning” were the lowest scored subcategories. CKD 5D/KTx patients presented lower “Sleep/Rest Fatigue” ( Although chronic kidney disease (CKD) is a rare condition in children, its occurrence has increased during the last decades . Apart fFatigue, defined as the difficulty initiating and sustaining activity and attention tasks, known as physical and mental fatigue, respectively, due to a lack of energy, accompanied by a desire to rest, constitutes a common symptom in chronic diseases, ultimately impairing an individual’s functionality and autonomy . FatigueIn the pediatric CKD population, data regarding fatigue are limited. According to a large-scale cross-sectional study in children with CKD stages 1–4, the “low energy” symptom was observed in 11% of patient reports and in 14% of parent reports; the frequency of this symptom was higher in advanced CKD, and “low energy” was also associated with lower HRQoL scores . The assThis cross-sectional study was conducted from March to October 2020 on children and adolescents aged from 7 to 18 years with CKD stage 2–5 or KTx, followed-up in the Pediatric Nephrology Unit of our Department of Pediatrics. Patients with neurological and musculoskeletal disorders not related to CKD, as well as participants unable to complete the questionnaires due to insufficient Greek language knowledge, were excluded. Demographic data, including age, sex, primary kidney disease, and CKD stage, according to the estimated glomerular filtration rate (eGFR) using Schwartz’s formula, were recorded for each patient. The participants were divided into the following three groups based on CKD status: CKD stage 2–4, CKD 5D, and KTx. Moreover, we collected data regarding the parental education level and the highest education level from two parents, or the education level of a single parent in case of a single-parent family. Parent education level was divided into three subcategories: elementary school, high school, and university degree. Finally, we reported the patient residence place .During an outpatient visit, the physician of the pediatric nephrology unit informed the participants about the study protocol, and demanded written consent for study participation. Then, both the patient and parents were asked to fulfill both questionnaires in calm and safe conditions, with a sufficient time provided for completion. The current study analyzed only the fully completed questionnaires. Fatigue and HRQoL were measured using the Greek version of the “PedsQL–Multidimensional Fatigue Scale” and “PedsQoL-Pediatric Quality of Life Inventory” Generic Score Scales questionnaires, respectively.The “PedsQL-Multidimensional Fatigue scale” questionnaire consists of 3 subcategories, including general, sleep/rest, and cognitive fatigue, each of which comprises 6 items. All times are evaluated on a 5-point Likert scale, from 0–4, where 0 = never a problem, 1 = rarely a problem, 2 = sometimes a problem, 3 = often a problem, and 4 = always a problem. For the purposes of our study, the items were reverse-scored and transformed to a 0–100 scale . The “Total Fatigue” score and the score for each subcategory were measured using the average score of all items and the average score of each subcategory items, respectively. For the purposes of our study, we used the teen report for patients aged from 13 to 18 years old, the child report for patients aged from 8 to 12 years old, and the young child report for patients aged 7 years old. The “PedsQL-Multidimensional Fatigue Scale” has been already used for the evaluation of fatigue in various pediatric populations, including patients with pediatric obesity, and chronic rheumatoid, neuromuscular, and respiratory diseases ,15,16.The “PedsQL- Pediatric Quality of Life Inventory” Generic Score Scales questionnaire consists of 4 subcategories, including physical, emotional, social, and school functioning, each of which comprises 8, 5, 5, and 5 questions, respectively. As for the “PedsQL-Multidimensional Fatigue scale” questionnaire, we used the teen report for patients aged from 13 to 18 years old, the child report for patients aged from 8 to 12 years old, and the young child report for patients aged 7 years old. The “Total QoL” score and the score for each subcategory were measured using the same procedure that was followed for the fatigue questionnaire. The “PedsQL-Pediatric Quality of Life Inventory” Generic Score Scales questionnaire has been already applied for the evaluation of HRQOL in general ,18 and vp < 0.05 was considered statistically significant.Statistical analysis was conducted using SPSS software (version 25.0). Continuous data were presented as median values and ranges, and categorical data as number and percentages. The comparison of the distribution of on-study parameters between the patient groups based on CKD status were performed using the chi-squared test and the multiple chi-squared test for quantitative independent variables, and Fisher’s exact test and the Kruskal–Wallis test for quantitative independent variables. Spearman’s correlation analysis was applied for the correlation test between fatigue and HRQoL scores. The Wilcoxon signed-rank test was used for the comparison of scores between patients and their parents. Box plot analysis was used for the illustration of patient and parent report scores regarding fatigue and HRQoL questionnaires. A value less than 50 was considered as a low score for total fatigue and HRQoL, as well as for their subcategories. A value of In total, 30 children  with a median age of 13 (7–18) years old, participated in this study. Of note, two patients were initially excluded from the study because of severe neurological deficits not related to CKD, and two patients were not recruited because of insufficient Greek language knowledge. None of the eligible patients refused to participate in the study. All the questionnaires were fully completed. The patient place of residence was urban in 17 (56.7%) patients and rural in 13 (43.3%) patients. The parent education level involved elementary school in 7 (23.3%), high school in 12 (40%), and a university degree in 11 (36.7%) patients. A single-parent family was observed in only one case.p = 0.308, p = 0.877, p = 0.312, p = 0.520, and p = 0.212, respectively)  had undergone KTx, with a median post-transplant time of 7.1 (5.7–8.9) years; 10 patients  presented CKD 2–4, consisting of 5 patients with stage 2, 4 with stage 3, and 1 with stage 4; and 13 patients  presented CKD 5D, with a median dialysis duration of 4.3 (1.5–6.2) years. Among CKD 5D patients, 10 were on peritoneal dialysis and 3 were on hemodialysis. Primary kidney disease was congenital abnormalities of the kidney and urinary tract in 16 patients, hemolytic uremic syndrome in 5, ciliopathies in 4, focal segmental glomerulosclerosis (FSGS) in 2, polycystic kidney disease in 2, and congenital abnormalities of uric acid metabolism in 1 patient, respectively. In total, confirmed inherited kidney disease was present in 23 (76.7%) patients. The distribution of patient age, sex, frequency of inherited kidney disease, residence place, and parental education level did not significantly differ among the three patient groups (ctively) .p = 0.043). Specifically, a score ≤50 was observed for “General Fatigue” in 7 (23.3%) patients, for “Sleep/Rest Fatigue” in 11 (36.7%) patients, for “Cognitive Fatigue” in 7 (23.3%) patients, and for “Total Fatigue” in 5 (16.7%) patients , 77.1 (45.8–100), 66.7 (29.2–100), 75 (25–100), and 68.1 (36.1–93.1), respectively. The patients’ scores on the subcategory, “Sleep/Rest Fatigue”, were lower compared to that of the other subcategories (patients .p = 0.032). Moreover, the prevalence of low “Sleep/Rest Fatigue” scores was significantly higher in both CKD 5D (53.8%) and KTx patients (57.1%) compared to CKD 2–4 patients (0%) (p = 0.013). In total, “Sleep/Rest Fatigue” scores were significantly lower in patients under kidney replacement therapy (CKD 5D or KTx) compared to those with CKD 2–4 (p = 0.022) (p = 0.004). The prevalence of low “Cognitive Fatigue” and “Total Fatigue” scores was higher in CKD 5D and in KTx patients compared to CKD 2–4 patients, but the statistical analysis did not reach significance .The scores regarding fatigue in patients with CKD 2–4, CKD 5D, and KTx are presented in detail in = 0.022) . Specifip = 0.246). Specifically, a score ≤ 50 was observed for “Physical Functioning” in six (20%) patients, for “Emotional Functioning” in ten (33.4%) patients, for “Social Functioning” in four (13.3%) patients, for “School Functioning” in eight (26.6%) patients, and for “Total HRQoL” in five (16.7%) patients , 78.1 (28.1–100), 67.5 (20–100), 80 (20–100), and 70 (15–100), respectively. Patient scores on the subcategories, “Emotional Functioning” and “School Functioning”, were lower compared to the other subcategories, but the statistical analysis did not reach significance . The prevalence of “School Functioning” scores tended to be higher among CKD 5D (38.5%) and KTx (42.9%), compared to CKD 2–4 (0%) patients (p = 0.062). In total, “School Functioning” scores were lower, whereas the percentage of patients who scored ≤50 on the “School Functioning” subcategory was significantly higher among patients under kidney replacement therapy (CKD 5D or KTx)  .The scores regarding HRQoL in children with CKD 2–4, CKD 5D, and KTx are presented in ctively) . Finallyp = 0.442), “Sleep/Rest Fatigue” (p = 0.640), “Cognitive Fatigue” (p = 0.414), and “Total Fatigue” (p = 0.955) did not differ from those of the patients (p = 0.029) and for the subcategories, “Physical Functioning” (p = 0.040) and “School Functioning” (p = 0.045). The scores on the subcategories, “Emotional Functioning” and “Social Functioning”, did not differ between patients and their parents .The parental questionnaire was completed by the mother in 22 (73.3%) cases, and by the father in the remaining 8 (26.7%) cases. The parents’ score for “General Fatigue” (patients . On the p < 0.001), and significant correlations were observed between the fatigue and QoL subcategories’ scores ; a low “Sleep/Rest Fatigue” score was associated with low “Physical Functioning”, “School Functioning”, and “Total HRQoL” scores ; a low “Cognitive Fatigue” score was associated with a low “School Functioning” score (p < 0.001); and a low “Total Fatigue” score was associated with low “Physical Functioning”, “Emotional Functioning”, “School Functioning”, and “Total HRQoL” scores . In total, 24 patients presented a score > 50 for “Total HRQoL” and “Total Fatigue”, four patients presented a score ≤ 50 for “Total HRQoL” and “Total Fatigue”, and two patients presented a score of ≤50 only in “Total HRQoL” or “Total Fatigue”.The “Total HRQoL” score was correlated to the “Total Fatigue” score (rs = 0.625, ’ scores . Moreove’ scores . In detaFatigue, both physical and cognitive, is a frequent symptom of chronic diseases. Fatigue is well-described in adult CKD, as a result of physical and mental disorders that accompany the disease ,9,10. MoIn our study, we used the “PedsQL-Multidimensional Fatigue scale” for the assessment of fatigue. To our knowledge, this is the first study which investigated the prevalence of multidimensional fatigue in CKD pediatric patients. According to our results, a low “Total Fatigue” score was observed in 16.7% of patients. As expected, “General Fatigue” was more prevalent in CKD 5D patients, observed in 46.2% of patients, confirming the major role of CKD chronicity and severity in the manifestation of this symptom. Our findings are in accordance with Roumelioti ME et al.’s study, where children with measured GFR 40- < 50, 30- < 40, or <30 presented 2.07, 2.35, and 2.59 higher odds of experiencing the “low energy” symptom than children with GFR ≥ 50 . The “GeSleep disorders have already been reported in the pediatric CKD population, with a higher prevalence in the late stages ,27,28. IIn contrast to fatigue, HRQoL has been intensively evaluated in pediatric CKD, with conflicting results among the reported studies. According to some authors, HRQoL scores were lower in CKD, principally in CKD 5D patients, compared to the healthy population ,31,32,33In our study, a low “Total HRQoL” score was observed in 16.7% of pediatric CKD patients, whereas low “Emotional functioning” and “School functioning” scores were the most frequently observed characteristics of disturbed HRQoL. We also remarked that patients with CKD 5D or KTx presented significantly lower “School Functioning” scores compared to CKD 2–4 patients. In detail, low “School Functioning” scores were observed in 40% of patients with CKD 5D or KTx. According to the data of “School Functioning” questionnaire items, these patients commonly presented attenuated classroom attention and working memory, ultimately burdening their school performance. School absences due to regular consultations or dialysis sessions may also contribute to the learning disabilities. Supplementary in-class and out-class educational activities and the enhancement of the hospital quality of educational activity during dialysis sessions might improve the learning difficulties in this vulnerable population.The interaction between multidimensional fatigue and HRQoL has not been previously investigated in the pediatric CKD population. In the current study, the “Total Fatigue” score was correlated to the “Total HRQoL” score, and significant correlations were also remarked between the fatigue and HRQoL subcategories’ scores. Moreover, a low “Sleep/Rest Fatigue” score, which was, as previously reported, the most frequent fatigue characteristic, was significantly associated with low “Physical Functioning”, “School Functioning”, and “Total HRQoL” scores. In general, adequate sleep contributes to physical and mental child health, whereas sleep disorders may have a devastating effect on a child’s behavior, learning ability, and physical development ,27. Our Prompt medical interventions in cases of fatigue are probably the key in order to ameliorate patient HRQoL and well-being. In adult studies, physical activity, erythropoiesis-stimulating agents, bicarbonate supplementation, and vitamin D administration have been proven beneficial for attenuating fatigue occurrence in CKD 5D patients . SimilarIn the current study, parents’ fatigue scores did not differ from that of patients, emphasizing that this condition is perceived to the same degree by both patients and their relatives. Nevertheless, parents’ scores were significantly lower compared to those of patients for “Total HRQoL”, “Physical functioning”, and “School functioning”. Similarly, more favorable patient reports, compared to parent reports, on HRQoL have been observed in other pediatric CKD studies ,23. ThesOur study has several limitations. Firstly, the limited number of included patients, with a variety of ages, precludes us from making firm conclusions. Moreover, the cross-sectional results of our study need to be confirmed in large longitudinal analyses. Furthermore, our study concerned only Greek pediatric patients; therefore, our results might not be representative of the European pediatric CKD population. Additionally, other factors that may affect QoL, such as patient comorbidities and parent socio-economic status, have not been taken into account in our study.A strength of our study is that we investigated both multidimensional fatigue and HRQoL in our cohort, allowing us to explore the interaction between the two conditions in CKD pediatric patients. Moreover, we included patients with both CKD 2–5D and KTx, which permitted us to compare the prevalence of multidimensional fatigue and altered HRQoL across the full spectrum of CKD. Furthermore, this is the first study which employed a multidimensional fatigue questionnaire validated for pediatric use in CKD pediatric patients, allowing us to explore all domains of fatigue in this vulnerable population.Conclusively, according to our results, general fatigue is observed in children with CKD, with a higher frequency of occurrence in CKD 5D. Sleep disorders represent the main fatigue characteristic, encountered in both CKD 5D and KTx patients. HRQoL deterioration is associated with emotional disorders and learning difficulties, especially in CKD 5D and KTx patients. Fatigue seems to affect the patient HRQoL. Finally, although fatigue is perceived equally by children and their parents, the parents’ perception of their children’s HRQoL is worse than that of the children themselves.


Tandem solar cells based on metal halide perovskites are advancing rapidly during last few years –17. The σΔΤ) and temperature changes (ΔΤ) , is used to evaluate the crystal structure of perovskites [RA, RX, and RB are the radius of ions at A, X, and B positions of perovskites (ABX3). An ideal cubic structure can be formed when 0.8 < t < 1. For certain B and X ions, A cation should have a proper size; otherwise, large strain will be formed in the lattice, resulting in phase transition or instability of perovskite films. For perovskites with APbI3 structure, A position can be MA+  or FA+ ((NH2)2CH+, t = 0.99). However, FA+ has a larger size than MA+, which will lead to a larger size mismatch and local lattice strain. To relax stress in FAPbI3 and stabilize the lattice, some small ions, e.g., MA+, Rb+, or Cs+, are incorporated in A position [Perovskite itself can form local stress/strain in its lattice. Goldschmidt tolerance factor (ovskites , 23, wheposition , 45–47.In short, the stress and strain are issues in developing perovskite/silicon tandem solar cells. The issues become severe when the device area is enlarged, causing current mismatch in the sub-cells. Strategies should be proposed to solve these issues.


To sum up, Phosphodiesterase-1 inhibition has been specifically proposed as a critical therapeutic approach for Alzheimer’s disease. This study provides a comprehensive review on the biological and pharmacological aspects of Phosphodiesterase-1, its role on the Alzheimer’s diseases and its significance as Alzheimer’s disease therapeutic target in drug discovery from natural products. This review will help clinical trials and scientific research exploring new entities for the treatment and prevention of Alzheimer’s disease.Phosphodiesterase-1 (PDE1) is a versatile enzyme that has surprisingly received considerable attention as a possible therapeutic target in Alzheimer’s disease (AD) because it maintains the homeostasis of 3ʹ,5ʹ-cyclic adenosine monophosphate (cAMP) and 3ʹ,5ʹ-cyclic guanosine monophosphate (cGMP) in the brain. 3ʹ,5ʹ-cyclic adenosine monophosphate and 3ʹ,5ʹ-cyclic guanosine monophosphate are the two key second messengers that regulate a broad range of intracellular processes and neurocognitive functions, specifically memory and cognition, associated with Alzheimer’s disease. However, the lack of available selective drugs on the market poses challenges to identifying the beneficial effects of natural products. The present review focuses on Phosphodiesterase-1 and its isoforms, splicing variants, location, distribution, and function; the role of Phosphodiesterase-1 inhibitors in Alzheimer’s disease; and the use of vinpocetine and natural products as specific Phosphodiesterase-1 inhibitors. Moreover, it aims to provide ongoing updates, identify research gaps, and present future perspectives. This review indicates the potential role of Phosphodiesterase-1 inhibitors in the treatment of neurodegenerative disorders, such as Alzheimer’s disease. Certain clinical trials on the alleviation of Alzheimer’s disease in patients are still in progress. Among  PDEs hyal cells .2+, and an endogenous protein factor enhances the sensitivity of PDE1 to Ca2+. At normal concentrations, Ca2+ and calmodulin (CaM) form the Ca2+–CaM complex, thereby stimulating PDE1  and guanylyl cyclase (GC), respectively, into inactive metabolites. Subsequently, these inactive products are expelled from cells under the mediation of multidrug resistance-associated protein-4 . PDE1 acing PDE1 . PDE1 ising PDE1 . Both cying PDE1 . Recentling PDE1 .https://www.alzint.org/about/dementia-facts-figures/). Before the end of 2050, 1 out of 85 persons is anticipated to face AD, which is the most common type of dementia that chiefly affects elderly individuals , Google Scholar (https://scholar.google.com.pk/), and Scopus (www.scopus.com). Only 83 papers were found suitable for this review. For this review, we considered four articles that were published in the 19th and 20th centuries, and the remaining articles were recently published from 2012 to 2022. A systematic scheme was adopted for the review of PDE1 and its isoforms, activation, and inhibition in the brain by taking into account the details of the in vitro and in vivo data available. The following keywords were used to search databases: “phosphodiesterase 1,” “phosphodiesterase 1” AND “cognition,” “pde-1” AND “cognition,” “health benefits of plant,” “PDE1 AND plant extract AND cognition,” “PDE1 and pathophysiology of Alzheimer,” “ca AND pde1,” “PDE1 inhibitors, Alzheimer,” “vinpocetine stability,” “citrus fruits, Alzheimer,” “Caesalpinia sappan,” “Heterophragma adenophyllum Seem,” “Claviceps purpurea,” “Periandra dulcare Mart.,” “Nelumbo nucifera,” and “Vinca rosea,” “PDE1, cognitive enhancer,” “Herbs, phosphodiesterase, Alzheimerʹs disease,” “PDE1 inhibitor, pharmacognosy, Caesalpinia sappan L.,” “PDE1, cognitive enhancer,” “Heterophragma adenophyllum Seem,” “Claviceps purpurea, fungus,” “Claviceps purpurea (ergot), ergot alkaloids,” “Periandra dulcare Mart.,” “Periandra dulcare Mart., PDE,” “Nelumbo nucifera,” “neferine alleviates memory, cognitive dysfunction,” “lotus, horticultural plants,” “Vinca rosea, natural plants, brain,” “citrus fruits, brain,” and “neuroprotective, citrus fruit, Alzheimer.” All structures were drawn by using ChemDraw Ultra 12.0 software.We reviewed more than 200 scientific papers from different literature sources, such as PubMed  of the hippocampus and the fifth to sixth layers of the cortex. Correspondingly, PDE1B has been found to have the highest expression in specific areas of the brain , such asSplicing variants or gene variants enhance functional diversity and extend regulatory activities. Specifically, protein isoforms synthesized by splicing variants can have variations in catalytic abilities, protein-protein interlinkages, or subcellular localization. The splicing variants of PDE1 genes contribute to the regulation of normal physiological functions and pathological processes . Each PD2+/CaM. The lack of meaningful data on the functional complications of these specific isoforms of PDE1 and serious challenges in the selectivity of previous inhibitors have considerably decelerated . An inherited missense variant of the PDE1B gene has been found in ASD and schizophrenia. These results indicate the extreme need to modulate PDE1 gene expression by using pharmacological approaches to assess their influence on cognitive behavior in the population  and synthesizes cAMP. In a similar fashion, GC catalyzes guanylyl triphosphate (GTP) and synthesizes cGMP  stated warning against vinpocetine, reporting that during pregnancy, it can be harmful to the fetus or lead to miscarriage. Vinpocetine induces serious agranulocytosis. Anecdotally, several people have observed that the continuous intake of vinpocetine disturbs immune function. Commission E reported that the decreased immune function during the long-term usage of vinpocetine may lead to apoptosis. The prolonged ingestion of vinpocetine slightly reduces systolic and diastolic blood pressure, as well as serum glucose levels to some degree. Other reported adverse effects included nausea, flushing, dry mouth, dizziness, heartburn, headaches, and transient hypertension and hypotension .Functional recovery is likely the most important therapeutic effect to achieve because it greatly improves the quality of life. Functional recovery in AD implicates cognition enhancement . RolipraClinicalTrials.gov Identifier: NCT0O719953). In this phase, vinpocetine was evaluated as a nutritional supplement for alleviating cognitive impairments in elderly individuals. Although it showed memory-enhancing effects and improved cognition in healthy female participants, it was found to be ineffective in improving memory in patients with AD  is a perennial plant that is mostly found in Southern Asia and tropical countries. It is also native to Madagascar. It is locally known in Malaysia as kemunting cina. This plant has many common names, such as bright eyes, Madagascar periwinkle, graveyard plant, Cape periwinkle, old maid, rose periwinkle myrtle, and pink periwinkle. It is used for ornamental purposes because of its various colors, such as pink, purple, and white. It is highly popular due to its medicinal applications. This plant is used in chemotherapeutic regimes to treat cancer and childhood leukemia, hypertension, diabetes, malaria, nonsmall lung cancer, and Hodgkin’s lymphoma and to improve memory. It has antioxidant, antimicrobial, hypolipidemic, antidiarrheal, and wound-healing activities , is a medicinal plant that is widely used as a natural red dye, herbal drink, and traditional herbal medicinal preparation in Yogyakarta, Indonesia. Numerous reports have shown that C. sappan has antioxidant, antibacterial, antidiarrheal, antiinflammatory, anticancer, hepatoprotector, and antidiabetic activities. C. sappan has been scientifically documented to enhance cognition via PDE1 inhibition  is widely cultivated in North Australia, Asia, Egypt, and the Caspian Sea , represents a rich source of nonessential bioactive compounds, particularly flavanones. Numerous preclinical studies have demonstrated that citrus flavonoids have neuroprotective potential, antioxidative and antiinflammatory activities, and mechanistic actions on BBB function or integrity. Therefore, scientists recommend encouraging the consumption of citrus fruits in the form of whole fruit and 100% juices for their potential neurological benefits . Current effects . Rahimi  effects  (Rahimi  effects .P. dulcis Mart. (accepted name: Periandra mediterranea) is native to the northern and middle parts of Brazil and is used in Brazilian ethnomedicine. It performs numerous biological activities, such as expectorant, diuretic, antiinflammatory, laxative, antifungal, antiplatelet aggregation, cytotoxic, and hemolytic activities; inhibits tumor cell proliferation; and lowers blood cholesterol and triacylglycerol levels (P. dulcis Mart. can inhibit PDE1 (46). Yoshitaka Ikeda et al. (1991) reported that compound 1 from this plant is the most potent known PDE inhibitor and inhibited PDE1 20–40 times more effectively than PDE2 and PDE3 (l levels . The litand PDE3  (R. Iwahand PDE3 .Only a few ongoing investigations on PDE1 inhibitors were found . The proRecent studies on PDE1 clearly show some limitations that affect AD therapy. The major issue is the nonspecificity of vinpocetine for other receptors, showing low target protein selectivity . Few perCesalpinea shuppan and PDE1 inhibition requires further studies (The exploration of PDE1 inhibitors remains crucial for the development of novel substitutes for anti-AD drugs . This re studies . Nimodip studies . A doubl studies . Vinpoce studies . The emp studies . Certain studies . Plants  studies . Nutrace studies . Undoubt studies . In the