title stringlengths 1 1.19k | keywords stringlengths 0 668 | concept stringlengths 0 909 | paragraph stringlengths 0 61.8k | PMID stringlengths 10 11 |
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Outcome analysis | SECONDARY | Continuous variables with normality are presented as mean (standard deviation, SD) and variables without normality are presented as median (interquartile range, IQR). The binary variables are presented as numbers (percentages).For the primary outcome, the correlation between left–right SctOFor the secondary analysis, the correlation of left–right SctOStatistical significance was set at | PMC9906862 | |
Results | PMC9906862 | |||
Correlation of baseline left and right SctO | Baseline left and right SctOCorrelation of left–right SctO | PMC9906862 | ||
Secondary outcomes | PMC9906862 | |||
Correlations between left–right SctO | Correlations between the left and right SctO | PMC9906862 | ||
The trend of SctO | The trend of left and right SctOComparison of left and right SctO | PMC9906862 | ||
Correlation between left and right SctO | The correlation of left–right SctOSamples of correlation between left and right SctO | PMC9906862 | ||
Discussion | REGRESSION, SECONDARY | The present study found that OLV increased the discrepancy in left–right SctOAlthough there are data to support the poor correlation of bilateral SctOOne strength of our study is that we employed a linear regression model to adjust for confounders that might affect SctOBecause the correlation at a single time point could not reflect the trend of change, we provided three methods to illustrate the question. First, we used a linear mixed model to compare the trend of SctOAnother interesting finding was that the maximal difference between the left and right SctOThe discrepancy in the left–right SctOOur results show that bilateral SctOOne limitation of the present study is its secondary analysis design. However, all these data were prospectively collected in a previous study which ensured the quality of the data. Second, the sample size was limited to 124 patients. Based on the results, the sample size yielded a statistical power of 0.99. Third, as discussed above, SjvO | PMC9906862 | |
Conclusions | In the present study, we found that the correlation between left and right SctO | PMC9906862 | ||
Acknowledgements | Not applicable. | PMC9906862 | ||
Authors’ contributions | HQ J, DL M helped in concept and design, and data interpretation. CJ Zh, JH M, F J, XH L helped in data acquisition, data analysis. CJ Zh wrote the manuscript. DL M revised the manuscript. All authors are aware of and responsible for the research data. All authors read and approved the manuscript in its final version. | PMC9906862 | ||
Funding | This study was supported by the National Key R&D Program of China (2018YFC2001800). The sponsors had no role in designing or conducting the study; collecting, managing, analysing, or interpreting the data; or preparing and approving the manuscript. | PMC9906862 | ||
Availability of data and materials | The datasets generated and analysed during the current study are not publicly available due to institutional restrictions, but are available from the corresponding author upon reasonable request. | PMC9906862 | ||
Declarations | PMC9906862 | |||
Ethics approval and consent to participate | SECONDARY | This study was a secondary analysis of a cohort study approved by the Clinical Research Review Board of Peking University First Hospital (#2017–1378). Written informed consent was obtained from all subjects and/or their legal guardians. This study was registered in the Chinese Clinical Trial Registry on 10/09/2017 ( | PMC9906862 | |
Consent for publication | Not applicable. | PMC9906862 | ||
Competing interests | The authors declare no competing interests. | PMC9906862 | ||
References | PMC9906862 | |||
1. Introduction | overactive bladder, Overactive Bladder | OVERACTIVE BLADDER, INCONTINENCE, OVERACTIVE BLADDER, URINARY INCONTINENCE, OVERACTIVE BLADDER | Postmenopausal women are at risk of developing an overactive bladder (OAB). Conventional vaginal estrogen has shown promise for symptom relief. Isoflavones have proven effective as an alternative to estrogen treatment against menopause-related symptoms. However, its effect on OAB symptoms has not been studied. This study investigates if fermented red clover isoflavones reduce OAB symptoms in postmenopausal women. In this randomized, double-blinded, placebo-controlled trial, women were administered red clover extract (RCE) or a placebo twice daily for three months. Women filled out the International Consultation on Incontinence Questionnaire Overactive Bladder (ICIQ-OAB) and Urinary Incontinence Short Form (ICIQ-UI-SF), together with a fluid intake and voiding diary. A total of 33 women (16 in the RCE group and 17 in the placebo group) were included in the analysis. Baseline demographics and OAB characteristics were comparable across groups. Intake of RCE did not lead to significant relief in most urinary bladder symptom measures, although a significant reduction in the bother of urinary urgency (Overactive bladder (OAB) is a common medical condition that may significantly reduce quality of life [Isoflavones, a class of phytoestrogens, are estrogen-like compounds naturally occurring in legumes such as soybeans and red clover. The primary isoflavones in soybeans are daidzein and genistein, while formononetin and biochanin A are the major isoflavones found in red clover [Evidence of the effect of isoflavones or other phytoestrogens on OAB symptoms is sparse [This study aimed to investigate the effect of isoflavones on OAB symptoms in postmenopausal women. Isoflavones were administered using a biofermented red clover extract (RCE) with high concentrations of the aglycone forms of the isoflavones formononetin and biochanin A. | PMC10574253 |
2. Materials and Methods | PMC10574253 | |||
2.1. Study Design | incontinence | SECONDARY, INCONTINENCE, OVERACTIVE BLADDER, INCONTINENCE | This study focusing on OAB symptoms was part of a three-month randomized, double-blinded, placebo-controlled trial (ClinicalTrials.gov NCT05013593) investigating RCE as a treatment for postmenopausal women with bladder symptoms. The primary outcome of the trial was changes in the urinary and vaginal microbiota composition, which will be reported elsewhere. In this current article, secondary outcome measures of OAB symptoms are reported. This includes changes in OAB symptom severity and incontinence assessed by two International Consultation on Incontinence Questionnaire (ICIQ) questionnaires—Overactive Bladder (ICIQ-OAB) [The timeline of the study is illustrated in | PMC10574253 |
Randomization and Double Blinding | The women were randomized to either RCE or placebo with a 1:1 allocation ratio between groups using the data management program Research Electronic Data Capture (REDCap) [ | PMC10574253 | ||
2.2. Study Population | PMC10574253 | |||
2.2.1. Sample Size | We aimed for the inclusion of 50 women in each randomization arm. This was based on a previous study on RCE in the treatment of osteopenic postmenopausal women, where treatment effects were observed in a similar group of women [ | PMC10574253 | ||
2.2.2. Participants | endometrial cancer, infections, cancer of the digestive, hematuria, dysuria, cystitis, tenderness/pain | RECURRENT URINARY TRACT INFECTION, ENDOMETRIAL CANCER, RECURRENT URINARY TRACT INFECTION, CNS INFECTIONS, INFECTIONS, HEMATURIA, CYSTITIS | Participants were recruited through advertisements (posters, social media, and personal electronic mail from the public sector) from the community in Northern Denmark, at the North Denmark Regional Hospital, and Aalborg University Hospital. The first woman was enrolled in September 2019, and the last was enrolled in October 2021. Eligible participants were postmenopausal women with OAB with a minimum of five years since their last menstruation. Exclusion criteria were the use of systemic or local hormonal replacement therapy; intake of RCE or other isoflavone-rich supplements; use of prebiotic and/or probiotic supplements or receiving any antibiotics within three months prior to inclusion; recurrent urinary tract infections (defined as ≥2 infections in the last six months or ≥3 infections during the last year); current urinary tract infection (defined as a positive urine culture together with symptoms of cystitis, including dysuria, suprapubic tenderness/pain, hematuria, frequent urination, and urgency); and current or prior history of breast-, ovary-, or endometrial cancer and/or cancer of the digestive and/or urinary tract. Moreover, women who had used a levonorgestrel-releasing intrauterine device within the last five years and/or had a hysterectomy before the cessation of menstrual periods and were younger than 60 years were also excluded. | PMC10574253 |
2.3. Questionnaires and Data Acquisition | PMC10574253 | |||
2.3.1. Demographic and Clinical Data | At the baseline visit, health-demographic information (such as age, body mass index (BMI), smoking status, treatment, self-reported past medical/disease history, etc.) was collected using a structured questionnaire survey. | PMC10574253 | ||
2.3.2. ICIQ-OAB Questionnaire | OAB symptoms were assessed using a modified Danish translation of the ICIQ-OAB [ | PMC10574253 | ||
2.3.3. ICIQ-UI-SF Questionnaire | INCONTINENCE, URINARY INCONTINENCE | Overall severity and bother of urinary incontinence, together with the type of incontinence, were evaluated using the Danish version of the ICIQ-UI-SF questionnaire [ | PMC10574253 | |
2.3.4. Fluid Intake and Voiding Diary | incontinence | INCONTINENCE, INCONTINENCE | To objectively evaluate the frequency of urination, bladder volume, and incontinence episodes, the women were asked to complete a three-day fluid intake and voiding diary, following the considerations of the International Consultation on Incontinence [ | PMC10574253 |
2.3.5. Diet Intake Registration | The women were also asked to complete a three-day food diary to monitor habitual diets, including the consumption of isoflavone-rich food. Food items were either weighed individually or given in portions. Each food diary was typed into an online diet and nutrition calculation program (VITAKOST ApS, Kolding, Denmark) by a single person from the research team. A second team member performed calculations on a subset of randomly selected samples ( | PMC10574253 | ||
2.4. Standard Urine Culture | STERILE, RECURRENT URINARY TRACT INFECTION | At the baseline and follow-up visits, a urine sample was collected using sterile intermittent catheterization to assess if a current urinary tract infection was present. Ten mL of urine was collected in a Urine Monovette with boric acid (Saarstedt, Germany) and analyzed by standard urine culturing. | PMC10574253 | |
2.5. Red Clover Extract and Placebo Formulations | COLD | The RCE and placebo formulations were produced by Herrens Mark ApS (Odense, Denmark). A heterogeneous culture (proprietary) of bacteria was used to facilitate the cold fermentation of pressed red clover to improve the bioavailability of the isoflavones. The isoflavone content in the RCE was determined as previously described [For the study, the two treatments were prepared using RCE from a single batch or water with the addition of stevia (1.2 g/L), natural sugar-free flavor (1.5 g/L orange and 1 g/L raspberry), and brown food coloring (ammoniated caramel) (10 g/L). The RCE and placebo were packed in identical, sealed cardboard two-liter boxes. Participants consumed 35 mL of RCE or placebo twice a day: self-dosing in the morning and evening with a meal, corresponding to a total daily intake of 58.38 mg isoflavones (58.1 mg aglycones/d) for the RCE group and 0 mg isoflavones for the placebo group. | PMC10574253 | |
2.6. Compliance and Adverse Events | ADVERSE EVENT | Compliance was calculated based on self-reported diaries. The women were instructed to register in the diary every time they consumed the treatment, by which the percentage of missing intakes could be calculated. This was subtracted from 100% to calculate compliance. Adverse events were monitored every month during the trial period by a telephone call from a research team representative and at the final follow-up visit. | PMC10574253 | |
2.7. Statistical Analysis | Data were analyzed using R Statistical Software (v4.1.3; R Core Team 2022, | PMC10574253 | ||
2.8. Ethics | The trial was conducted according to the principles expressed in the Declaration of Helsinki and was executed at the North Denmark Regional Hospital. The study was approved by The North Denmark Region Committee on Health Research Ethics (N-20190028). The categories of personal data collected in the project were registered in the processing activities of research in the North Denmark Region in compliance with EU GDPR Article 30. Oral and written informed consent was obtained from all participants. | PMC10574253 | ||
3. Results | PMC10574253 | |||
3.1. Study Participants | A total of 48 women were enrolled in the study ( | PMC10574253 | ||
3.3. ICIQ-OAB Score | A tendency was seen towards a decrease in the ICIQ-OAB score in the RCE group from baseline to follow-up (with a median score of 6.5 (5.0−8.25) at baseline versus 6.0 (3.75−8.25) at follow-up ( | PMC10574253 | ||
3.4. Individual ICIQ-OAB Items | nocturia | NOCTURIA | When looking at individual items in the ICIQ-OAB questionnaire, we found no differences in urinary frequency (question 3a) or nocturia (question 4a) for either the RCE or placebo group ( | PMC10574253 |
3.5. Patient-Reported Symptom Bother (NRS Values) for Urinary Frequency, Nocturia, Urgency, and Urge Urinary Incontinence from the ICIQ-OAB Questionnaire | NRS | In the RCE group, a reduction was detected in the bother of urinary urgency on an NRS (0–10) from baseline to follow-up ( | PMC10574253 | |
3.6. The ICIQ-UI Score | No significant difference in the ICIQ-UI score was found between baseline and follow-up in either the RCE group or placebo group ( | PMC10574253 | ||
3.8. Compliance and Adverse Events | stomach pain | The median duration of the treatment in the trial was 99.5 (91–107) days, and the median compliance was 96% (90.9–98.5%). The treatments were generally well tolerated. Four women taking RCE had difficulties with the taste, and two women, one in each treatment group, experienced stomach pain after intake; these women all dropped out of the study. | PMC10574253 | |
4. Discussion | OAB symptoms, urinary incontinence | STRESS URINARY INCONTINENCE, SYNDROME, URINARY INCONTINENCE, PATHOPHYSIOLOGY | The present study showed no clear improvements in the majority of OAB symptom parameters in postmenopausal women suffering from OAB following treatment with fermented RCE compared to a placebo. OAB is a complex syndrome, and its pathophysiology is not fully understood [By evaluating the ICIQ-OAB questionnaire, we saw tendencies, though not statistically significant, towards a reduced ICIQ-OAB score (The overall burden and bother of urinary incontinence evaluated by the ICIQ-UI-SF were not improved in either of the groups. A reason for this may be explained by the fact that a large part of the women in our study suffered from stress urinary incontinence (50% in the RCE group and almost 65% in the placebo group), which we do not expect the RCE to be effective against.No improvements in urinary frequency or other voiding parameters from the fluid intake and voiding diary were found in women taking RCE. However, women in the placebo group reported a significantly lower number of micturitions. A similar level of improvement (e.g., reduction of around one void/day) has been observed in the control group in other studies aiming to control OAB symptoms [Overall, we observe an agreement between the responses to the ICIQ-OAB items and parameters reported in the fluid intake and voiding diary. For example, although not statistically significant, a slight increase in urine volume was observed in the RCE group. This change may indicate a tendency toward decreased urgency, as demonstrated in the ICIQ-OAB responses. In contrast, a few observations from the ICIQ-OAB and diary do not agree. In the diary, we find a significant decrease in the numbers of micturitions for the placebo group, but this is not demonstrated in the ICIQ-OAB item 3a. Other clinical studies looking at the effect of isoflavones on symptoms suggestive of OAB are sparse and mainly focus on urinary incontinence or vaginal symptoms. Yet, our results are consistent with previous findings where intake of isoflavones was not associated with improvements in urinary incontinence, frequency, or urgency [There may be multiple reasons why we only detected a limited improvement in bladder symptoms. First, it may be attributed to insufficient ER expression in the bladder. Earlier studies have shown that ER-β is the dominant ER in the bladder [ | PMC10574253 |
Strengths and Limitations | Some limitations should be considered. OAB symptom complexity can be influenced by many factors, such as the type of fluid intake. However, we did not have information about the participants’ consumption of different types of fluids, e.g., caffeine-containing drinks, which are known to affect voiding habits. The sample size in our study was small, making it difficult to obtain statistical conclusions and account for heterogeneity among participants. We formerly aimed for 100 participants, but due to problems finding women meeting study criteria, we enrolled a lower number. Additionally, compliance was not based on measuring the residual volume of the RCE and placebo. The use of a randomized, double-blinded, placebo-controlled study design entails both limitations and strengths for the study. It results in a lower number of women included in each treatment group; however, it also enables the investigation of a placebo effect. Another strength is that we evaluated OAB symptom effects using both ICIQ questionnaires in combination with a fluid intake and voiding diary. | PMC10574253 | ||
5. Conclusions | OAB symptoms | BLADDER DISORDERS | The present study has not been able to demonstrate the effect of systemic treatment with isoflavones on the relief of OAB symptoms in postmenopausal women. Larger studies are needed to unravel the possible role of isoflavones in female bladder disorders. In addition, better in-depth knowledge of the mechanisms that isoflavones may have on ER binding in the bladder is warranted. | PMC10574253 |
Supplementary Materials | The following supporting information can be downloaded at: Click here for additional data file. | PMC10574253 | ||
Author Contributions | Conceptualization, A.B.V., P.B.J., S.S. and L.T.S.A.; methodology, A.B.V., P.B.J., S.S. and L.T.S.A.; validation, P.H.; formal analysis, A.B.V., J.N.H.-J., B.K.P., C.B.-N. and J.B.K.; investigation, A.B.V., J.N.H.-J. and B.K.P.; resources, J.B.K., K.G. and L.P.C.; data curation, A.B.V.; writing—original draft preparation, A.B.V. and S.S.; writing—review and editing, all authors; visualization, A.B.V.; supervision, C.B.-N., P.B.J., S.S. and L.T.S.A.; project administration, A.B.V.; funding acquisition, A.B.V., P.D.C.L. and S.S. All authors have read and agreed to the published version of the manuscript. | PMC10574253 | ||
Institutional Review Board Statement | The study was conducted in accordance with the Declaration of Helsinki and approved by The North Denmark Region Committee on Health Research Ethics (N-20190028). | PMC10574253 | ||
Informed Consent Statement | Informed consent was obtained from all subjects involved in the study. | PMC10574253 | ||
Data Availability Statement | All data generated or analyzed in the study are included in this published article and its | PMC10574253 | ||
Conflicts of Interest | Herrens Mark ApS (Odense, Denmark) produced and delivered the RCE and placebo. They had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. | PMC10574253 | ||
References | incontinence, mellitus, Overactive Bladder, NRS | INCONTINENCE, OVERACTIVE BLADDER, INCONTINENCE, UTI, URINARY INCONTINENCE, URINARY TRACT INFECTION | A schematic representation of the study design. Screening was scheduled approximately two weeks before the baseline visit, at which time, if the women decided to participate, they were asked to answer the ICIQ questionnaires to ensure eligibility. Before baseline and follow-up visits, the women were asked to fill out diaries. On the visit days (day 0 baseline and day 90 follow-up), a catheterized urine sample was collected, and questionnaires were filled in. At the baseline visit, the women were randomized and allocated to receive either the RCE or the placebo formulation. ICIQ: International Consultation on Incontinence Questionnaire; RCE: red clover extract.Consort participant flowchart of participation through the present study. UTI: urinary tract infection; RCE: red clover extract.International Consultation on Incontinence Questionnaire Overactive Bladder (ICIQ-OAB) score at baseline and follow-up for RCE (International Consultation on Incontinence Questionnaire Overactive Bladder (ICIQ-OAB) results from questions 3a (International Consultation on Incontinence Questionnaire Urinary Incontinence (ICIQ-UI) score at baseline and follow-up for RCE (Baseline health-demographic characteristics of the study participants according to randomization group.Never smokedFormer smokerSmokerFormer smokerSmokerWetDryUrgeUrge and stressStress No urinary incontinenceLichen sclerosusProlapsDiabetes mellitus 2HypothyroidismPercentages are calculated based on either the total or within each treatment group. Comparison of patient-reported symptom bother (NRS values) from the ICIQ-OAB questionnaire.NRS values are median (QR1−QR3). The NRS to monitor the bother of the symptom ranges from 0 (not at all) to 10 (a great deal). The comparison from baseline to follow-up was analyzed using the paired samples Wilcoxon test. Fluid intake and urinary voiding information as reported by the women in the study diary in accordance with trial randomization status.Mean ± SD are stated for normally distributed data, whereas median (QR1–QR3) values are stated for not normally distributed data. All volumes are in mL. * One woman did not complete the fluid intake and voiding diary at follow-up and was therefore excluded from the analysis. ** Two women did not report incontinence episodes and were therefore excluded from the analysis of incontinence. The comparison from baseline to follow-up was analyzed using the paired | PMC10574253 |
1. Introduction | stunting, diarrhea, malnutrition | MALNOURISHED, WASTED, MALNUTRITION | These authors contributed equally to this work.Deceased.The profile of the intestinal microbiota is known to be altered in malnourished young children in low- and middle-income countries. However, there are limited studies longitudinally evaluating the intestinal microbiota in malnourished young children in resource-limited settings over the first two years of life. In this longitudinal pilot study, we determined the effect of age, residential location, and intervention on the composition, relative abundance, and diversity of the intestinal microbiota in a representative sample of children under 24 months of age with no diarrhea in the preceding 72 h in the urban and rural areas of Sindh, Pakistan nested within a cluster-randomized trial evaluating the effect of zinc and micronutrients on growth and morbidity (ClinicalTrials.gov Identifier: NCT00705445). The major findings were age-related with significant changes in alpha and beta diversity with increasing age. There was a significant increase in the relative abundance of the Firmicutes and Bacteroidetes phyla and a significant decrease in that of the Actinobacteria and Proteobacteria phyla (Nutritional compromise, especially stunting among children under 5 years of age, remains a global problem, with an estimated 149 million children affected worldwide in 2021 and an additional 45 million children wasted [The gut microbiome has been increasingly implicated in malnutrition in young children in resource-limited settings (reviewed in [We undertook a longitudinal study to determine the effect of age and residential location on the composition and diversity of the intestinal microbiota in children under 24 months of age in the urban and rural areas of Sindh, Pakistan nested within a cluster-randomized trial evaluating the effect of zinc and micronutrients on growth and morbidity (ClinicalTrials.gov Identifier: NCT00705445) [ | PMC10005232 |
2. Materials and Methods | PMC10005232 | |||
2.1. Study Design and Participants | This was a planned prospective pilot sub-study nested within a cluster-randomized trial of two micronutrient powder formulations with or without zinc, in children between the ages of 6 and 18 months in two representative populations to assess impact on growth and morbidity. The parent trial and characteristics of the population have been described in detail elsewhere [ | PMC10005232 | ||
2.2. Fecal Sample Collection, DNA Extraction, Amplicon Generation, and 16S rRNA Sequencing | diarrhea | COLD, INFECTIOUS DISEASE | Fecal samples were routinely collected at baseline (3 months) as well as at 6, 9, 12, 18, and 24 months for enteric pathogens, ensuring the absence of diarrhea in the preceding 72 h. Once the subject was identified by the field teams in their weekly visits, a stool kit (comprising of diaper, spoon, container, and gloves) was provided to the family of the subject to collect a fresh fecal sample. A significant portion of the fecal sample was placed in the plastic screw-capped container using the disposable plastic spoon immediately after collection, ensuring the collection only of specimens not in contact with the diaper lining.The collected fecal samples were transported in a special cold storage box with cold chain maintenance at a temperature of 2–8 °C within 6 h of collection from the field site to the Infectious Disease Research Laboratory (IDRL) at the Department of Pediatrics, Aga Khan University. In the research lab, these stool samples were stored at −80 °C. Ten children representing each of the three groups, from both urban and rural sites, and with fecal samples available at all of the time points (3, 6, 9, 12, 18, and 24 months), were randomly chosen for this sub-study. This provided a total of 360 fecal samples for the assessment of intestinal microbiota.DNA was extracted from stool samples in the IDRL using a QIAamp Stool Mini kit (Qiagen, Inc., Valencia, CA, USA) and shipped to Tufts Medical Center in Boston. Upon receipt at Tufts Medical Center, DNA concentration was measured using a Nanodrop ND-1000 (Thermo Scientific, Wilmington, DE, USA) and normalized to 50 µg/mL in all samples by the addition of UltraClean PCR Water (MoBio, Thermo Fisher Scientific, Waltham, MA, USA). For sequencing runs performed on LR70 plates, (Roche, Indianapolis, IN, USA). PCR primers were designed to amplify the V4 hypervariable region of the genes for the 16S rRNA gene of both Bacteria and Archaea. The forward primer contained the V4 specific sequence AYTGGGYDTAAAGNG, an octamer barcode [PCR reactions were carried out in triplicate in parallel with a barcode-specific negative control; reactions yielding no amplicons or those in which the negative controls amplified were repeated. DNA concentration of amplicons was determined using a Quant-iT assay (Invitrogen, Carlsbad, CA, USA) and they were then pooled in equimolar concentrations. The amplicon pool was purified twice using an AMPure XP kit (Agencourt, Indianapolis, IN, USA) and sequenced on a Roche 454 Genome Sequencer GS FLX using the LR70 platform according to Roche protocols at the Virginia Bioinformatics Institute, and subsequently at the Tufts University Core Facility Genomics Core using a Roche 454 Genome Sequencer GS FLX+ and the Lib-L Sequencing kit according to Roche protocols. Replicates of the same sample were sequenced on both systems and the results showed no significant differences. | PMC10005232 |
2.3. Computational and Bioinformatics Analyses | Computational analyses were performed using the open-source software platform QIIME Version 1.5 ( | PMC10005232 | ||
2.4. Statistical Analysis | diarrheal | Statistical analyses were performed in R (Beta diversity, defined as partitioning of diversity among communities, which is characterized using the number of species shared between communities, was determined by principal coordinates analysis (PCoA) of weighted UniFrac distances in QIIME based on age, residential location, and intervention arm. The ellipse R package, V3.3.2 was used to generate 95% confidence intervals and differences were determined using Adonis with 1000 permutations in the vegan R package V3.3.2. Temporal changes in overall alpha and beta diversity measures and taxon-specific relative abundances of the microbiota of all children in both locations and all three intervention arms were analyzed using a linear mixed-effects model (see below). Relative abundances for selected taxa were modeled as a function, of age adjusting for gender, residential location, and reported diarrheal episode.To estimate the age-specific change in alpha diversity indices and relative abundance of major taxa and the degree of change at a group level, accounting for potential serial autocorrelation in the repeated measurements, the separate linear mixed-effects models were fitted to individual trajectories. Each model described outcomes as a function of time with age in days as a random variable and with gender, residential location, and diarrheal episode as fixed effect covariates using the lmer function of the lme4 R package, v3.3.2. Based on the model results, the 3-monthly rate of change for each outcome along with the lower and upper limits of their 95% confidence intervals (CI) were estimated.The linear discriminant analysis effect size (LEfSe) algorithm was used to identify differentially abundant taxa in children in the first and second years of life combined or at each 3-monthly time point, by residential location and by intervention arms, using default parameters [ | PMC10005232 | |
3. Results | PMC10005232 | |||
3.2. Gut Microbiota | diarrheal, well-nourished | MALNOURISHED, WASTED | Beta Diversity: We first assessed the beta diversity of the gut microbial community composition from all the children at each time point, in both locations (urban and rural) and within each intervention arm using PCoA of weighted UniFrac distances. The UniFrac algorithm measures similarity among microbial communities based on the degree to which their component taxa share branch length on a phylogenetic tree [Differences in weighted Unifrac distances between age groups were statistically significant at 9 (Alpha Diversity: As age appeared to be the factor that most accounted for differences in beta diversity of microbial communities, we focused on the analysis of the effect of age on alpha diversity and relative abundance of major taxa at the highest and lowest taxonomic levels, i.e., phyla and genera. To account for covariates, we used a linear mixed-effects model adjusted for gender, residential location, and reported diarrheal episodes. Although there were no significant differences overall in beta diversity of the gut microbiota of children residing in urban and rural locations, we wanted to determine if there were significant differences in alpha diversity indices between children residing in these locations at each of the 3-monthly time points. While all alpha diversity indices have doubled over the study period, as shown in Relative Abundance: Overall, the most abundant phyla were Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes (Overall, the 10 most abundant genera were Differentially abundant taxa: In order to identify differentially abundant taxa at all taxonomic levels between the microbiota of all of the children in rural and urban locations in all intervention arms combined between the first and second years of life, we used the LEfSe algorithm, which identifies genomic features characterizing the differences between two or more biological conditions [We also used the LEfSe algorithm to identify differentially abundant taxa at all taxonomic levels between the microbiota of children in the urban and rural locations and in the three intervention arms, in each age group. These analyses revealed specific taxa that were enriched in the microbiota of children in urban versus rural locations in all intervention groups combined (There were very few differentially abundant taxa in the microbiota of children who received different interventions (The numbers of malnourished (underweight, wasted, stunted) or well-nourished children at each age, in each intervention arm, and at urban or rural sites were too small to determine if there were significant differences in alpha or beta diversity or differentially abundant taxa among them. | PMC10005232 |
4. Discussion | diarrheal, well-nourished | MALNOURISHED, ZINC DEFICIENCY, WASTED | In this pilot study, we investigated the nutritional status and composition, relative abundance, and diversity of the gut microbiota of children in urban and rural areas of Pakistan from 3 to 24 months of age. The most significant findings were age-related changes with a decrease in HAZ score, increase in alpha and beta diversity, and changes in relative abundance of the major taxa at the level of phylum and genus with increasing age from 3 to 24 months of age. Differentially abundant taxa were identified between all of the children in the first and second years of life, between those residing in rural and urban areas, and those who received different interventions at different ages from 3 to 24 months.Several studies, many of them longitudinal, have investigated the gut microbiota using 16S rRNA sequencing in children under the age of five years in low- and middle-income countries [In the present study, we found that the most abundant phyla were Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, in descending order. This pattern of relative abundance of the major phyla differs by country. For example, in a similar longitudinal study from South India, Firmicutes and Proteobacteria were most abundant, followed by Actinobacteria and Bacteroidetes [Increasing age also had an impact on the relative abundance of the major Phyla, with significant increases in Firmicutes and Bacteroidetes and significant decreases in Actinobacteria and Proteobacteria with increasing age. Similarly, there were changes in the relative abundance of the four most abundant genera, with significant increases in In the present study, we did not investigate eukaryotic components of the gut microbiota. Recently, Popovic et al., analyzed stool samples and data from a subset of 80 children at 12 and 24 months of age from the same clinical trial (ClinicalTrials.gov Identifier: NCT00705445) [The age-related changes in the gut microbiota in the current study were similar to those from a longitudinal pilot study comparing low birth weight stunted children to normal birth weight non-stunted children from birth to 2 years of age in South India [Specific taxa associated with linear growth stunting of children in longitudinal studies vary with geographic location, age, and analytic techniques. In the longitudinal study from India referred to above [Zinc deficiency has been shown to be associated with alteration in the gut microbiota in school-age children [There are a few limitations to our pilot study. The numbers of well-nourished and malnourished children at each age, in each location, and intervention arm were small. We did not account for antibiotic use, diarrheal episodes, or diet, all of which are known to impact the gut microbiota. We used the Roche 454 platform for 16S rRNA sequencing, which was what was available at the time. In future studies, we will use the Illumina MiSeq platform for 16S sequencing and will also perform whole genome shotgun sequencing to characterize the gut microbiome of a percentage of subjects. The study design of the parent study did not include a cohort of healthy children. In future studies, we will also use the “relative microbiota maturity index” and “microbiota-for-age Z score” developed by Subramanian et al. [This study was nested within a larger cluster-randomized trial on the effect of zinc and micronutrients on growth and morbidity. Consonant with the findings of the larger main trial [Further studies of larger numbers of malnourished (including underweight, wasted, and stunted) and well-nourished children using additional analytic tools such as “relative microbiota maturity index” and “microbiota-for-age Z score”, as well as metagenomics and metabolomics, are needed to identify differences in the gut microbiota between them and to design microbiota-directed interventions. | PMC10005232 |
Supplementary Materials | The following supporting information can be downloaded at Click here for additional data file. | PMC10005232 | ||
Author Contributions | Conceptualization, E.N.N., C.A.W., H.D.W. and Z.A.B.; Methodology, E.N.N., C.A.W., H.D.W. and Z.A.B.; Validation, Formal analysis, V.B., D.M.D., I.A. and A.R.; Investigation, V.B., D.M.D., A.V.K., S.S. and J.D.; Resources, A.V.K., H.D.W. and Z.A.B.; Data Curation, V.B., D.M.D., I.A., J.M., A.R. and S.B.; Writing-Original Draft Preparation, E.N.N., H.D.W., A.R., M.C. and Z.A.B.; Writing-Review and Editing V.B., A.V.K., E.N.N., H.D.W., A.R., M.C. and Z.A.B.; Visualization, D.M.D., H.D.W. and A.R.; Supervision, V.B., A.V.K., S.S., C.A.W., H.D.W. and Z.A.B.; Project Administration, C.A.W., H.D.W. and Z.A.B.; Funding acquisition, Z.A.B. All authors have read and agreed to the published version of the manuscript. | PMC10005232 | ||
Institutional Review Board Statement | The study was duly approved by the Ethics Review Committee at the Aga Khan University and the Institutional Review Board at Tufts University, as well as the National Bioethics committee of the Government of Pakistan. | PMC10005232 | ||
Informed Consent Statement | All subjects provided written informed consent for the main study inclusive of the microbiome collection and analysis. | PMC10005232 | ||
Data Availability Statement | The de-identified data for the main study is available through the Ki initiative at the Bill & Melinda Gates Foundation. Additional microbiota data can be requested from the corresponding authors. | PMC10005232 | ||
Conflicts of Interest | The authors declare no conflict of interest. | PMC10005232 | ||
Subject terms | depressive disorder, depression, MDD, dysregulation of mood | TSD | JNJ-54175446 is a selective purine P2X7 receptor (P2X7R) antagonist that attenuates microglial IL-1β/IL-18 release. In healthy volunteers, JNJ-54175446 suppressed peripheral interleukin (IL)-1β release, and attenuated dexamphetamine-induced improvements of mood and (visuo)motor performance in a human dexamphetamine-challenge paradigm. In depression, P2X7R inhibition may dampen immune-related dysregulation of mood. These results suggest that the impact of P2X7R inhibition is most prominent in situations where mood regulation is disrupted. Total sleep deprivation (TSD) results in an acute emotional perturbation, which yields a transient antidepressant effect. In the current study, TSD was applied as a behavioral challenge to investigate whether such effects could be modulated by JNJ-54175446. This was a double-blind, placebo-controlled, randomized study to assess the safety and pharmacokinetics of JNJ-54175446 and explore its effects in patients with single episode and recurrent major depressive disorder (MDD) ( | PMC10363543 |
Introduction | neuroinflammation, unipolar and bipolar mood disorders, mood disorders, ’s mood, MDD, depression | TSD, PATHOLOGY, PATHOPHYSIOLOGY | The purine P2X7 receptor (P2X7R) is one of seven identified subtypes of adenosine triphosphate (ATP)-gated P2X ion channels present on various human cell types. The P2X7R is predominantly expressed in central nervous system (CNS) in microglial cells and peripherally in monocytes [Several lines of evidence implicate neuroimmune mechanisms in the pathophysiology of mood disorders [Because of the well-documented role of the P2X7R in IL-1β release, it is hypothesized that P2X7R antagonists may be beneficial for depression associated with neuroinflammation. Iwata and colleagues were the first to show that chronic stress in rodents results in the activation the P2X7R and NLRP3 inflammasome-related release of IL-1β in hippocampus [Currently, a number of CNS-penetrant high affinity and selective P2X7R antagonists, such as JNJ-54175446 and JNJ-55308942, are in development for the treatment of unipolar and bipolar mood disorders [In healthy human studies, JNJ-54175446 demonstrated blood-brain barrier penetration and a clear dose-receptor occupancy relationship in a range of 5–300 mg using positron emission tomography, and was safe and well tolerated [Since the P2X7R is activated under conditions of elevated neuronal activity and/or pathology, pharmacological antagonism is only expected to elicit an effect when the channels are activated by sufficiently high ATP concentrations [TSD and sleep restriction have been shown to increase ATP levels resulting in an increase of peripheral cytokine levels including IL-1β [Although data from both preclinical and healthy volunteer studies with IL-1β as peripheral pharmacological biomarker for P2X7R antagonism seem encouraging, JNJ-54175446’s mood modulating effects are yet to be established in a target patient population such as MDD. The use of TSD to produce a transient change in mood in patients with MDD conceivably may serve as a probe of the effect of a physiological stressor on mood. In this context JNJ-54175446 is expected to modulate and potentially reduce the effect of TSD on mood and mood-related phenomena. It was hypothesized that TSD-associated acute mood enhancement would be attenuated in MDD patients who had received JNJ-54175446 during the 3 days prior to the TSD challenge, as the acute effects of elevated cytokine levels would be blunted by JNJ-54175446. Conversely, if patients only received JNJ-54175446 The main objective of the current study was to investigate the safety and pharmacokinetics of JNJ-54175446 in MDD, as well as to perform a proof-of-concept exploration of its potential effects on mood-related phenomena by applying TSD in a relevant clinical population. | PMC10363543 |
Material and methods | PMC10363543 | |||
Population | Male and female patients aged 18–64 years with a body mass index (BMI) between 18 and 32 kg/m | PMC10363543 | ||
Dose selection | In an earlier single ascending dose study with JNJ-54175446 in healthy male volunteers, full antagonism of peripheral ex-vivo IL-1β stimulation was demonstrated following single doses of ≥50 mg JNJ-54175446 [ | PMC10363543 | ||
Safety evaluations | Suicidal ideation | ADVERSE EVENTS | During the study, safety and tolerability evaluations were performed by monitoring adverse events, clinical laboratory tests (hematology, serum chemistry urinalysis and serum pregnancy test at screening and urine pregnancy tests during the treatment phase). 12-lead electrocardiograms (ECG), vital signs, and physical and neurological examination were frequently monitored and performed. Suicidal ideation and behavior was assessed using the Columbia Suicide Severity Rating Scale (C-SSRS) [ | PMC10363543 |
Pharmacokinetics (PK) | PK blood samples were collected predose, at 1, 2, 4, 8, and 10 h post dose on day 1 and day 10 and at 24 h and 48 h after the last dose of JNJ-54175446. The maximum plasma concentration (Cmax), minimum observed plasma concentration (Cmin) and the area under the plasma concentration-time curve (AUCτ) was measured during dosing interval (τ). Just prior to the beginning or at the end of a dosing interval of any dose other than the first dose, the observed plasma concentration (Ctrough) was determined. Average plasma concentration (Cavg) was measured at steady state over the dosing interval and time to reach the maximum plasma concentration (tmax) was measured. | PMC10363543 | ||
Exploratory proof-of-concept related assessments | PMC10363543 | |||
Procedure for acute total sleep deprivation (TSD) | TSD | TSD | The TSD procedure consisted of restructuring the individual’s diurnal rhythm and providing activities that prevented sleep and promoted wakefulness. As shown in Table | PMC10363543 |
Hamilton depression rating scale, 17 items (HDRS17) | MDD, HDRS17 | The HDRS17 is a clinician-administered rating scale designed to assess the severity of symptoms in MDD patients. Each of the 17 items was rated by the clinician on either a 3- or a 5-point scale; higher scores indicated a worse outcome. | PMC10363543 | |
Inventory of depressive symptoms, clinician rated 30-items (IDS-C30) | The IDS-C30 [ | PMC10363543 | ||
The self-rated quick inventory of depressive symptoms-10/16 (QIDS-SR10/16) | depressive symptoms | TSD | The QIDS-SR10/16 is a subjective measure to assess the severity of depressive symptoms over a 1-week recall period (QIDS-SR16), or within a 2 h recall period (QIDS-SR10). The QIDS-SR16 was administered at screening, baseline, day 3, day 10 and at follow up and the QIDS-SR10 was administered in the morning and evening during the TSD period on day 4 and 5. | PMC10363543 |
Profile of mood states brief form (POMS) | The POMS [ | PMC10363543 | ||
Snaith-Hamilton pleasure scale (SHAPS) | The SHAPS [ | PMC10363543 | ||
The probabilistic instrumental learning task (PILT) | The PILT [ | PMC10363543 | ||
Interleukin-1-beta (IL-1β) | peripheral white blood cells | TSD | IL-1β in peripheral blood was analyzed on day 1 (predose, 2 h and 8 h post dose), on day 2 (24 h after the initial dose of JNJ-54175446), and on day 4 during TSD (predose, 2 h and 8 h post dose). Venous blood samples were taken (TruCulture) to obtain peripheral white blood cells (WBCs) for ex-vivo stimulation using LPS/BzATP to stimulate IL-1β release. At each sampling, 1 mL blood was collected in each of two TruCulture tubes containing 100 ng/mL LPS (cat#782-001087, Myriad RBM, Austin, Texas,USA). The tubes were incubated at 37 °C for 1 h in dryblock heaters followed by addition of 2′3′-BzATP (NuBlocks, Oceanside, California, USA) to one of the tubes to a final concentration of 1 mM (using 100× stock solution). The contents of both the tubes were mixed by inversion and incubated for an additional 1.5 h. The tubes were then centrifuged (1000 | PMC10363543 |
Analysis | PMC10363543 | |||
Sample size calculation | TSD | This safety and tolerability study involved TSD as an exploratory proof-of-concept intervention to induce acute mood improvement and was preceded and/or followed by JNJ-54175446 and/or placebo. Hence, it was impossible to make any plausible assumptions of the effect sizes and/or the interactions between sleep deprivation, treatment (duration) and mood changes. Therefore, formal statistical calculation of sample size was not appropriate and was not performed. Generally speaking, a sample size of 60 patients or more represents the customary size employed in early development studies designed to allow clinical assessment of safety and tolerability and is sufficient to demonstrate clinical mood elevating effects of sleep deprivation and/or antidepressant medication [ | PMC10363543 | |
Statistics | SE, TSD | TSD | Since the study was not primarily powered to assess the pharmacodynamic effects of JNJ-54175446, descriptive statistics were performed for HDRS17, QIDS-SR POMS, PILT and SHAPS, with mean ± SD values and changes from baseline being summarized by treatment group at the respective scheduled time points per instrument. Data were presented graphically for individual subject values or as mean ± SE values over time.In addition to the descriptive statistics, for the HDRS17 total score a mixed-effects model using repeated measures was performed for change from baseline (day -1) to day 10. The model included baseline score as covariate, and antidepressant treatment status (treatment naïve or SSRI treatment), day, treatment and day-by-treatment interaction as fixed effects, and a random subject effect.In blood, both in vivo and ex-vivo, mean values and mean changes from baseline in IL-1β levels were determined and compared to placebo.Following review and interpretation of the exploratory results, a pattern became apparent for the effects of JNJ-54175446 on hedonic tone during the TSD period: all patients receiving JNJ-54175446 before the recovery night (which were both groups A and B, as group B received their loading dose on the last day of TSD) did not demonstrate changes in PILT ‘total amount lost’, whereas placebo group C demonstrated a clear improvement following recovery sleep. Therefore, specific additional exploration of hedonic tone as assessed with the SHAPS and the PILT total amount lost was warranted. To this end a post-hoc analysis was performed to further assess the effect of JNJ-54175446 compared to placebo on TSD induced decrease of the SHAPS total score and PILT total amount lost, between day 3 and day 5 using an unpaired | PMC10363543 |
Results | Sixty-nine patients received at least one dose of JNJ-54175446, and a total of 64 patients completed the study. The study disposition schedule is shown in Fig. | PMC10363543 | ||
Disposition schedule. | This schedule describes how many patients were assigned to each group and completed the study. | PMC10363543 | ||
Adverse events | nausea, abdominal pain, vomiting, influenza-like illness, headache, dysgeusia, deaths, chills | ADVERSE EVENTS, INFLUENZA-LIKE ILLNESS | JNJ-54175446 was well-tolerated and all adverse events were mild to moderate. There were no deaths, serious AEs, or persistent AEs reported in this study. In group A (JNJ-54175446 early start) two TEAEs, headache and abdominal pain lead to study discontinuation. Both were classified as severe and considered by the investigator as possibly related to JNJ-54175446. In group B (JNJ-54175446 late start) the two TEAEs that lead to study discontinuation were an influenza-like illness starting at day 1 that was considered by the investigator not related to JNJ-54175446 and moderate intensity chills and nausea which were considered to be very likely related to JNJ-54175446. It should however be noted that the patient was suffering from nausea before the administration was started.Adjusting for only those patients who received JNJ-54175446, the following TEAEs were the most reported; headache (25.0%), nausea (11.5%), dysgeusia (5.8%) and vomiting (5.8%). No clinically significant changes were observed in laboratory evaluations. Changes from baseline in vital signs and ECG parameters were small and not considered clinically relevant. | PMC10363543 |
Exploratory proof-of-concept related assessments | Table | PMC10363543 | ||
Mood symptoms | PMC10363543 | |||
Hamilton depression rating scale (HDRS) | TSD | At baseline (day-1), the HDRS17 total score (mean [SD] was 20.0 [4.03] for group A (JNJ-54175446 early start); 18.1 [4.38] for group B (JNJ-54175446 late start); and 18.9 [4.04] for group C (placebo), respectively.Effects of TSD per average HDRS17 total score [SD], measured as change from baseline (day -1) on day 4, were −2.3 (3.82) in group C (placebo), somewhat larger (−3.9 (2.97)) in group A, and slightly smaller (−1.5 (3.33)) in group B. Although on day 4 a numerically larger proportion of patients demonstrated a ≥ 25% improvement on the HDRS17 total score in group A [9/25 (36%)] compared with group C [5/27 (29%)] or group B [5/25 (20%)], these differences were not significant (No significant differences (mean change [SD]) on day 10 compared with baseline were observed in any treatment group versus placebo: Group A −4.8 [4.69] | PMC10363543 | |
The self-rated quick inventory of depressive symptoms (QIDS-SR) | TSD, Depressive | TSD | The average Quick Inventory of Depressive Symptoms (QIDS-SR) total score (mean [SD]) at baseline (day -1) was 13.0 [3.98] for group A (JNJ-54175446 early start); 12.2 [4.63] for group B (placebo pre TSD and JNJ-54175446 late start) and 10.8 [3.52] for group C (placebo) (supplemental Fig. TSD decreased the average QIDS-SR10 total score with a maximal decrease observed in the morning of study day 5 (i.e., after recovery sleep). Mean values [SD] for the QIDS-SR10 total score, assessed on Day 4 and Day 5 (between 2 and 8 h postdose on both study days) decreased from 8.2 to 4.0 [−4.2] for subjects in group C, versus 9.2–6.9 [−2.3] and 10.2–5.8 [−4.4] for subjects in the JNJ-54175446 treated groups A and B, respectively. The effects of TSD were sustained until the last day of treatment (day 10) on which the mean total score (SD) was 8.7 [5.24] for group A; 7.3 [5.24] for group B and 5.9 [3.76] for group C. | PMC10363543 |
Profile of mood scales (POMS) | tension/anxiety, anger/hostility | TSD | The Profile of Mood Scales (POMS) total score (mean [SD]) at baseline (day -1) was 38.9 [16.2] for group A (JNJ-54175446 early start); 34.4 [15.9] for group B (JNJ-54175446 late start) and 32.4 [15.7] for group C (placebo).The average POMS total score (SD) decreased following TSD and recovery sleep from day 4 to day 5 in group A from 33.9 (17.8) to 15.9 (17.7), in group B from 35.6 (16.9) to 13.0 (20.2) and in group C from 31.1 (10.8) to 9.9 (14.8). Reductions were observed for the individual sub-scales: tension/anxiety, depression/dejection fatigue/inertia, anger/hostility and confusion/bewilderment, but an increase in was found for the positive mood descriptor vigor/activity. These changes were consistent across all treatment groups, indicating that there was no clear interaction between TSD effect and JNJ-54175446 treatment. The average POMS total score (SD) on day 10 was 17.8 (17.5) for group A; 17.5 (20.6) for group B and 10.8 (13.0) for group C. | PMC10363543 |
Snaith-Hamilton pleasure scale (SHAPS) | TSD | The Snaith-Hamilton Pleasure Scale (SHAPS) total score (mean [SD]) at baseline (day -1) was 8.6 [2.26] for group A (JNJ-54175446 starting before TSD); 8.15 [3.50] for group B (placebo pre-TSD and JNJ-54175446 starting with loading dose on recovery night) and 8.2 [2.68] for group C (all-placebo) (Fig. | PMC10363543 | |
Snaith-Hamilton Pleasure Scale (SHAPS). | This figure displays the Snaith-Hamilton Pleasure Scale (SHAPS) Total Score Mean [SD] over time for group A (green square), group B (blue triangle) and group C (red circle).The TSD-induced decrease of total SHAPS score, measured as the change from day 3 to day 5, was significantly attenuated in group A (JNJ-54175446 early start) (0.0 [3.64]) compared to group C (placebo) (−2.0 [2.58], | PMC10363543 | ||
The probabilistic instrumental learning task (PILT) | The results of the PILT are shown in Table | PMC10363543 | ||
Probabilistic Instrumental Learning Task (PILT). | This figure displays the Mean [SD] over time for group A (green square), group B (blue triangle) and group C (red circle) for the Probabilistic Instrumental Learning Task (PILT) for total points loss ( | PMC10363543 | ||
Interleukin-1-beta (IL-1β) | TSD | Compared to placebo, no differences were observed in either treatment group for plasma IL-1β at the various assessed time points. TSD however increased levels of ex-vivo stimulated IL-1β release on day 4. These elevations were markedly attenuated compared to baseline (day -1) in the early start JNJ-54175446 (pre-TSD) treatment group A (JNJ-54175446 early start) and remained attenuated through to end of study day 10. Levels of ex-vivo stimulated IL-1β release in group B (JNJ-54175446 late start) were comparable on day 1 and day 4, but attenuated at day 10. A detailed description of the IL-1β and ex-vivo IL-1β stimulation results can be found in the supplemental material (supplemental Figs. | PMC10363543 |
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