Supplementation with micronutrients, including vitamins, iron and zinc, is a key strategy to alleviate child malnutrition. However, association of gastrointestinal disorders with iron has led to ongoing debate over their administration. To better understand their impact on gut microbiota, we analyse the bacterial, protozoal, fungal and helminth communities of stool samples collected from a subset of 80 children at 12 and 24 months of age, previously enrolled into a large cluster randomized controlled trial of micronutrient supplementation in Pakistan . We show that while bacterial diversity is reduced in supplemented children, vitamins and iron  may promote colonization with distinct protozoa and mucormycetes, whereas the addition of zinc appears to ameliorate this effect. We suggest that the risks and benefits of micronutrient interventions may depend on eukaryotic communities, potentially exacerbated by exposure to a rural setting. Larger studies are needed to evaluate the clinical significance of these findings and their impact on health outcomes. et al. find that vitamins and iron increase carriage of protozoa and fungi in the gut, potentially disrupting the bacterial microbiome.Micronutrient supplements are key to global efforts to address child malnutrition. Here, in a cohort of children, previously enrolled into a large cluster randomized controlled trial of micronutrient supplementation in Pakistan, Popovic  With increased vulnerability to infection, undernourished children are at elevated risk of death, not least from diarrheal diseases4. Previous studies have demonstrated the role of gut microbiota in malnutrition, with microbiome immaturity  representing a key factor in disease development6. Beyond bacterial communities, parasites such as hookworm, Cryptosporidium, and Entamoeba have been associated with severe diarrheal disease and intestinal malabsorption8. However, much less is known regarding the role of other, potentially commensal, eukaryotic gut microbes in undernutrition. Of particular interest is their ability to interact with and alter bacterial communities. For example, indole-producing gut bacteria were found to confer protection against Cryptosporidium infection, while deworming treatments targeting helminth endemic communities reduced the abundance of protective Clostridiales10. Mouse studies further showed that helminths and protozoa influence bacterial communities by modulating the host immune system12. While the number of published gut microbiome studies has increased rapidly over the last decade, few have explored the composition of eukaryotic gut communities and their potential interactions with bacteria. Previously, we applied 18S rRNA and internal transcribed spacer (ITS) sequence surveys to systematically characterize eukaryotic microbiota in severely malnourished Malawian children and identified a high prevalence of protozoa including commensals and pathobionts13. We furthermore associated Blastocystis colonization with increased gut bacterial diversity.Malnutrition is a global health crisis with 149 million children stunted and 45 million children wasted under the age of 5 years16. Such supplements are thought to address deficiencies that can impair immune responses to infectious pathogens and impact gut bacterial communities20. While beneficial, supplementation, especially with iron, may also promote unintended growth of pathogens with the potential to disrupt the gut environment, particularly where the host is unable to restrict micronutrient bioavailability21. For example, it has been shown that surplus iron promotes the growth of enteropathogens and induces intestinal inflammation in infants23. Furthermore, while known to reduce the duration of childhood diarrheal episodes, zinc supplementation has been associated with increased duration of Entamoeba histolytica infections25. The impacts of complementary foods introduced between 6 and 24 months of age on gut bacteria, and the potential of tailored formulations, often fortified with micronutrients, to alleviate undernutrition by promoting specific bacterial taxa were recently reviewed26. The success of such strategies may also depend on understanding their effect on eukaryotic microbiota and their potential to modulate the gut environment.Global health programs targeting vulnerable child populations include the use of micronutrient supplements, consisting of vitamins as well as essential minerals zinc and iron, that have been demonstrated to improve growth and reduce morbidity27. We show that diverse protozoa, helminths, and fungi colonize the gut of both undernourished children and those within a healthy weight range. Children receiving MNPs have reduced bacterial diversity, while vitamins and iron are associated with increased carriage of specific protozoa and mucormycetes, an effect mitigated by the addition of zinc. Modeling of microbiota interactions reveals a complex landscape of associations with supplementation, place of residence , age, and nutritional status.In this work, we attempt to understand the impact of micronutrient supplementation on the complex interactions between eukaryotic and bacterial microbiota in the maturing infant gut. We perform 18S rRNA and 16S rRNA amplicon surveys on stool samples obtained at 12 and 24 months of age from 80 children, previously recruited as part of a cluster randomized controlled trial (cRCT) conducted in Pakistan. The trial investigated the impact of micronutrient powders (MNP) containing vitamins and iron with or without zinc on growth and morbidity in 2746 children, and has shown an excess of significant diarrheal and dysenteric episodes among children receiving MNPsNCT00705445)27 for profiling of gut eukaryotic and bacterial communities in this study (control (n = 24), MNP (n = 29), and MNP with zinc (n = 27); Fig. 27, we excluded children with reported diarrhea prior to stool collection for the purpose of microbiome analysis.A subset of 80 children (160 paired stool samples at 12 and 24 months of age) were selected from the parent cRCT . Remaining reads were classified to numerous taxa, including known gut parasites such as Enterocytozoon bieneusi, Pentatrichomonas hominis, and the tapeworm Hymenolepis nana as well as uncharacterized alveolates, Amoebozoa, and Cercozoa  of which 7,167,464 were high-quality reads and clustered into OTUs, and 4,386,494 could be classified as a eukaryotic microbe , species known to cause enteric symptoms .Having generated profiles of eukaryotic communities within each sample, we were interested in identifying any links between these communities and variables captured in the parent study. Applying generalized linear models (GLMs), we tested for associations between protozoan and fungal richness (number of OTUs) and four explanatory variables: micronutrient supplementation, nutritional status, age, and rural versus urban residence. Children residing in the rural study site had increased protozoan richness compared to those from the urban setting . We also observed trends of increased carriage of Cryptosporidium and Cryptosporida (coccidians), represented by 46 OTUs in total, in undernourished children , suggesting a possible inhibitory effect by the metal  and micronutrient supplementation , where protozoan communities in children supplemented with MNPs differed significantly from those in control and MNPs with zinc arms . Cluster 1 in particular was enriched in MNP samples, X2  = 26.80, p < 0.001 , and an OTU assigned to an unknown alveolate found exclusively in clusters 1 and 2 . These organisms were highly prevalent in both age groups, at 42 and 45% (Tritrichomonas) and 20 and 21% .We next compared the phylogenetic compositions of protozoan and fungal populations. The fungal composition was not associated with any of the variables studied here, however, analysis of protozoan compositional differences, based on unweighted UniFrac scores, revealed four distinct clusters of samples Fig. . Differe001 Fig. . Key driGregarina and an uncharacterized alveolate, which contributed to the previously observed differences in beta diversity , with no differences between samples from the control and MNP with zinc groups. Similarly, the carriages of three mucormycete genera  were 1.3, 1.5, and 1.8-fold higher, respectively, in the MNP group compared to the control , with no significant differences between the control and MNP with zinc groups. Toxoplasma was the only genus with significantly reduced carriage in children receiving MNPs with zinc, with 11 and 23-fold reductions compared to CTL and MNP groups ; however, we observed non-significant reductions in other organisms such as Cercomonas and Mucor  suggesting possible species-specific effects. Despite previous reports of the impact of zinc on helminths24, we did not detect significant differences in the carriage of the tapeworm Hymenolepis nana among treatment arms.To better understand this association between protozoan community structure and micronutrient treatment, we systematically compared protozoan, as well as fungal, carriage among supplementation groups. We identified significantly higher carriages of seven phylogenetically distinct protozoa and six fungi in children receiving MNPs without zinc, relative to those that were given zinc  accounting for over 50% of all reads  and evenness  , we identified 1108 bacterial OTUs across all 160 samples , while a significant interaction between nutritional status and place of residence indicated that bacterial evenness was reduced in undernourished children from the urban setting   Fig. . We detep < 0.05), but reduced abundance in the MNP group at 24 months  , with a non-significant reduction in those supplemented without zinc . Supplementation tended to reduce overall bacterial richness with an effect that reached significance in the MNP group   but not Bray–Curtis dissimilarities. Specifically, phylogenetic variance, defined as dispersions from the group centroids, differed among groups , with significantly smaller differences among 12-month-old children receiving MNPs and MNPs with zinc  , with six OTUs reduced at 24 months only. MNPs with zinc were associated with significant changes in an additional 46 taxa, and 29 taxa were altered in MNP samples . These included a seven-fold increase in Escherichia-Shigella abundance in 12-month-old MNP-supplemented children, increases in several Lactobacilli, and a 1.3-fold reduction in one Bifidobacterium OTU  Fig. . Firmicu05) Fig.  and infl01) Fig. . This maOTU Fig. . These d29. The greatest difference, with a 2.5-fold increase (p < 0.001), was noted in the networks of children in the MNP arm, which had the fewest taxon interactions at 12 months , but achieved parity with the control and MNP with zinc groups by 24 months , 6 months after supplementation had ceased. Cross-kingdom interactions between bacteria and eukaryotes represented 20 to 30% of all network interactions at 12 months, falling to between 15 and 24% by 24 months of age, a decrease that was significant in undernourished children , we constructed microbial interaction networks Fig. . Interacp < 0.05; MNP with zinc, p < 0.01), greater reductions were observed in supplemented undernourished children (p < 0.001). Since betweenness provides a measure of the degree of coordination within a network, these findings suggest that micronutrient supplementation, with or without zinc, may result in microbial communities that are less organized at 12 months of age. This is further illustrated by the network visualizations , with possible indirect carryover effects to microbial composition at 24 months  and, these changes, in turn correlated with shifts in eukaryotic composition at 24 months . Place of residence also significantly influenced microbial profiles, with the greatest effects observed on eukaryotic profiles . Children from the rural community had increased levels of several alveolates, including Cryptosporidium, at both ages . These children also had higher abundances of several Clostridium OTUs at both ages and sustained higher levels of Lactobacillus at 24 months. A stratified analysis of reference WLZ and undernourished children did not reveal groups to have significant differences in their correlation structure with variables . However, the stability of path coefficients could not be confirmed in our cohort using bootstrapping, a robust second-step validation procedure that iteratively recalculates path coefficients from a random sample of participants. With our sample size, bootstrapping produced large standard errors likely resulting from unevaluated interactions and/or diverse microorganism carriage  could alter age-related differences in the bacterial microbiome. To explore these complex interrelationships, we generated an integrated model of cross-correlations among microbial profiles and explanatory variables using partial least squares (PLS) path modeling Fig. . The ide30. Here, we have shown that the gut microbiota of both undernourished children and those within a healthy weight range includes a diverse group of protozoa, helminths, and fungi, each with the capacity to impact host health. We have also shown that supplementation with MNPs, a strategy used to improve growth and alleviate micronutrient deficiencies16, may influence the development of the microbiome in these susceptible populations.Malnutrition, both undernutrition and obesity, are associated with altered bacterial compositions, wherein the former, underdeveloped bacterial communities have the capacity to induce weight lossH. nana was identified at significantly higher levels in undernourished children. While H. nana infection is usually asymptomatic, high egg burdens in children have previously been associated with diarrhea, abdominal pain, and weight loss31, with exacerbated morbidity in children <5 years32. We associated rural habitation with significantly more diverse protozoan communities and, in particular, increased prevalence of Cryptosporidium. An important cause of infant mortality and childhood malnutrition, Cryptosporidium infection is attributed to unsafe drinking water and inadequate sanitation often associated with rural settings33. While approximately half of all children enrolled in the parent trial had access to piped drinking water , only 4% of children in the Matiari district had access to underground sewage, compared to 95% in the Bilal Colony27, consistent with a lack of wastewater sanitation resulting in higher parasite carriage. While the Global Enteric Multicenter Study (GEMS), the largest childhood diarrhea study to date, with 22,568 children enrolled across seven developing countries in Asia and Africa, reported Cryptosporidium as a leading cause of death in 12–23-month-old children with moderate to severe diarrhea34, we found a high prevalence of this parasite in the absence of diarrhea . As our detection is based on 18S rRNA amplicon sequencing, we may have detected a broader group of species with variable pathogenic potential compared to the GEMS study, which applied a specific oocyst antigen immunoassay. Alternatively, our findings may indicate a high prevalence of asymptomatic infections, with symptomatic infections resulting from additional unknown factors35. The prevalence of Cryptosporidium in our cohort was also higher than previously reported in non-diarrheal stools, using oocyst antigen testing, in the neighboring Naushero Feroze District (5.1% between 12 and 21 months of age), where Cryptosporidium contributed to 8.8 diarrheal episodes per 100 child-years37. This study associated asymptomatic enteropathogen infection, including Cryptosporidium and Giardia, across eight countries with elevated inflammation and intestinal permeability, factors thought to increase risk of stunting and impact the effectiveness of nutritional interventions in low-resource settings37.Consistent with previous studies, we found that bacterial communities became more complex with age. Eukaryotic communities, however, were not significantly impacted by age but instead were associated with micronutrient supplementation and place of residence. Only the tapeworm Toxoplasma and overall protozoan richness. These findings are consistent with a previous report which suggested that zinc has a parasite-specific protective effect against infection and ensuing diarrhea24. Fungal diversity was not impacted by age, supplementation, place of residence, or nutritional status. However, the predominance of Mucoromycota, particularly in children receiving MNPs without zinc, is of concern, as these organisms are responsible for rare but lethal invasive fungal infections that have previously been reported in low birth weight infants and malnourished children38. Although incidence of infections is rising globally, rates of mucormycoses are particularly high in Asia39. Notably, a recent spike in infections, also termed Black fungus, in thousands of active and recovered Covid-19 patients in India was attributed to treatment with corticosteroids to control inflammation, in conjunction with a high prevalence of diabetes40.A major focus of our study was to estimate the effect of micronutrient supplementation on the gut microbiota. We found that children receiving supplements without zinc were associated with distinct eukaryotic communities, featuring an increased prevalence of multiple protozoa and fungal taxa, including species with known potential to cause symptomatic infections and disrupt the gut microbiome; however, the addition of zinc to these supplements alleviated these increases, while significantly reducing the prevalence of 42. Several studies have shown that iron alone or in combination with other micronutrients worsens existing infections, lengthens the duration and severity of diarrhea and increases mortality rates in children41. Consequently, sequestration of free iron by host proteins such as lactoferrin is a key defense mechanism to limit growth of pathogens, including Mucorales43. Iron deficiency has furthermore been suggested as protective against malaria infection45, and provision of supplements containing iron in endemic regions has been cautioned against due to increased malaria-related hospitalization and mortality of children41. While a deficiency in zinc has been associated with impaired immune function and susceptibility to enteroinfections46, supplementation in the context of enteric pathogens was shown to have parasite-specific outcomes. Provision of zinc alone can increase the incidence of Ascaris lumbricoides and duration of Entamoeba histolytica infections, but it has also been shown to reduce the duration of associated diarrheal episodes as well as lower the prevalence of Giardia lamblia infections24. Interestingly, asymptomatic Giardia infections in children in Tanzania were associated with reduced rates of diarrhea and fever, an effect which was lost in children receiving vitamin and mineral supplements, including both iron and zinc47. Our data suggest that while iron, vitamins, or both, may promote growth and survival of commensal and potentially pathogenic eukaryotes, resulting in a shift in eukaryotic community structure and disrupting the gut environment, the addition of zinc may reduce the ability of at least some eukaryotic microbes to infect and persist. The findings of reduced bacterial diversity in 12-month-old infants receiving micronutrient supplements, together with elevated levels of Escherichia–Shigella and reduced beneficial Bifidobacterium, are also consistent with previous reports, where reductions in beneficial bifidobacteria and lactobacilli and increased enterobacteria in infants receiving iron-containing micronutrients were linked to an elevated risk of inflammation and diarrhea48. The parent cRCT associated Aeromonas infection with increased diarrhea in MNP-supplemented groups27. We did not detect this bacterium in our data, possibly due to the exclusion of diarrheal samples.It has been well established that iron supplementation can promote the virulence of particular fungi and parasitesGiardia lamblia and enteroaggregative Escherichia coli, for example, were shown to have a greater impact on growth in protein-deficient mice during co-infection, an effect which was dependent on the resident gut bacteria49. Taken together, our data showing increased carriage of eukaryotic microbes and increased abundance of Escherichia–Shigella in children supplemented with micronutrients, as well as a potential loss of organization in microbial interactions in supplemented undernourished children, may offer at least a partial explanation for previous reports of increased duration and severity of diarrhea as well as increased intestinal inflammation in children supplemented with micronutrient powders27.The impact of micronutrient supplementation also extended to the structure of the microbial communities. Microbial networks, representing significant correlations in the co-occurrence or mutual exclusion of specific bacteria and eukaryotes, revealed higher network connectivity in the control groups, with the networks generated from the undernourished infants receiving both types of supplements revealing a more fragmented structure. This fragmentation suggests a destabilization of species interactions within the developing gut microbiota in undernourished infants. Possibly contributing to this disruption of community associations is the presence of specific eukaryotic microbes, as evidenced by the increased carriage of a number of protozoan and fungal taxa in children receiving MNPs, and/or the expansion of pathogenic bacteria. These microbes may interfere with the maturation of commensal bacteria through predation, competition for resources, and/or modulation of host immunity. In undernourished infants, the cumulative effect of increases in pathogenic organisms on community structure may be more pronounced than in infants within a healthy weight. Enteropathogens Cryptosporidium burden, for example. Networks constructed from small sample sizes also have a greater propensity for prediction of spurious interactions and a reduced capability to detect true associations50. In absence of sample limitations, network reconstructions from larger balanced groups, with additional timepoints, could improve our ability to discern key microbial linkages impacted by interventions during different stages of growth and weaning. We note that lack of spike-in controls in this study did not allow us to estimate the absolute abundances of microbiota. In future studies, such an approach could confirm whether supplementation, for example, influences the relative representation of eukaryotes and bacterial cells in the gut. The notable absence of Giardia, a parasite typically prevalent in this demographic, is likely due to mismatches to the 18S rRNA sequencing primers13. Parasite diagnostic data from the trial did identify Giardia in 37 infants at 12 months and Cryptosporidium in seven, but noted no significant increases in either of the supplemented groups27. Prevalence was nearly two-fold higher at the rural site, consistent with our findings for Cryptosporidium, emphasizing the need for location-specific investigations of the effects of micronutrient supplements. In addition to potential intraspecies variation, our detection of high sequence diversity in Cryptosporidium OTUs specifically, and eukaryotic taxa in general, may be exaggerated by a high proportion of non-overlapping amplicon reads, a consequence we have attempted to minimize through manual curation. Regardless, we report that eukaryotic microbiota are abundant members of the gut microbiome even in infancy, and given the known role of parasitic pathogens in diarrheal disease and the association of fungi with obesity and inflammatory bowel disease52, their role in malnutrition should be further studied.Due to the relatively small numbers of samples, we were unable to generate separate networks for the three treatment arms for 24-month-old children, or further segregate the networks by place of residence. We note that supplementation ceased at 18 months of age, consequently the acute effects of these supplements may have dissipated prior to 24 months. Moreover, micronutrient interventions may impact undernourished children differently in settings associated with high 54, such a framework is expected to play a key role in identifying scenarios where MNP supplementation may require more cautious thinking.Our integrated model of microbial relationships and influencing external factors recapitulated a number of key earlier findings, including the impact of micronutrients and locality on gut eukaryotes. Furthermore, it supports the notion that complex cross-kingdom interactions may influence the gut bacterial composition and provides a valuable framework to dissect the direct and indirect effects of eukaryotic infections or nutritional interventions on the maturing gut microbiome. The instability observed in the pathway coefficients of the model, when participants were randomly resampled, is likely the result of the heterogeneous and stochastic nature of eukaryotic carriage  and ultimately our small sample size. However, given the current debate concerning the use of MNP supplementation and its role in gastrointestinal disordersThis study demonstrates that micronutrient powders impact the infant microbiota, with potentially disruptive effects driven through the promotion of specific organisms during the early stages of microbiome development. These findings are of relevance to micronutrient supplementation strategies, especially those targeting vulnerable children in low-resource settings.https://clinicaltrials.gov/ct2/show/NCT00705445]) of micronutrient powders (MNPs) with or without zinc that was conducted in urban  and rural  communities in Sindh, Pakistan between November 2008 and December 201127. In brief, a total of 2746 children (control (n = 947), MNP without zinc (n = 910), and MNP with zinc (n = 889)) were enrolled in the parent cRCT at 6 months of age  and training on how to self-collect stool, document the time of defecation, store the sample in a shaded area of the household and notify study staff. Stool samples were picked up at participants’ homes within 1 h of defecation and immediately transported to the field research office in a 2–4 °C cold box. Stool was divided into up to eight aliquots , of which 5–6 were marked for future analyses. Aliquots were then transported to Aga Khan University (AKU) within 4–6 h and frozen without additives at −80 °C. Samples analyzed in this study were shipped from AKU to the Hospital for Sick Children (SickKids) on dry ice, where they were stored at −80 °C until analysis.All stool samples and clinical data analyzed in this study were collected in a cluster randomized controlled trial (cRCT) ; and, (4) no reported diarrhea within 7 days of stool collection. Participants within the reference group were further selected to have the fewest WLZ scores < −1 at other time points to represent as healthy as possible a comparator group. Four participants (three from the reference group and one undernourished) with no detectable 18S amplicons were excluded due to lack of data. A further two participants from the reference group were excluded to meet ethics criteria restricting the analysis to the sequencing of 160 samples. Participant characteristics for the microbiome substudy were summarized as medians with interquartile ranges (IQRs) or means with standard deviations (SD) if continuous variables, and percentages if categorical in Table Eighty children were selected from all three supplementation arms of the parent cRCT for microbiome profiling  chemistry . 18S V4 + V5 amplification was achieved using the iProof DNA polymerase  with primers V4-1 (GCGGTAATTCCAGCTC) and V4-4 (GCCMTTCCGTCAATTCC) as previously described13. Briefly, the cycling conditions used were 94 °C for 3 min, 30 cycles of 94 °C for 45 s, 56 °C for 1 min, and 72 °C for 1 min, followed by a 10 min 72 °C extension. Barcodes were ligated, and libraries were sequenced using MiSeq V3 (300 bp × 2) chemistry . Sequencing was performed at the Centre for the Analysis of Genome Evolution and Function .DNA was extracted from 100–200 mg of stool using the E.Z.N.ATM Stool kit  according to the manufacturer’s protocol. Mechanical disruption of cells was carried out with the MP Bio FastPrep-24 for five cycles of 1 min at 5.5 M/s. 16S variable region 4 (V4) amplifications were carried out using the KAPA2G Robust HotStart ReadyMix (KAPA Biosystems) and barcoded primers 515 F (GTGCCAGCMGCCGCGGTAA) and 806 R (GGACTACHVGGGTWTCTAAT)56 and the UNOISE pipeline in USEARCH v11.0.66758. Filtered sequences were clustered to 99% sequence identity, and the resulting operational taxonomic units (OTUs) were classified with a minimum confidence of 0.8 using the SINTAX59 algorithm and the Ribosomal Database Project version 1660.16S data were quality filtered and processed within a cluster computing environment based on Lenovo SD530 Intel-based servers running Linux (CentOS 7), using VSEARCH v2.10.461 and read pairs with a minimum 200 nucleotide length were merged using VSEARCH or artificially joined using a linker of 50 ambiguous nucleotides (N50) using USEARCH. Resultant amplicon sequences were clustered to 97% sequence identity using the UCLUST57 algorithm and taxonomically classified using SINA v1.2.1162 with a minimum 90% sequence similarity threshold. Unclassified sequences were submitted for classification using SINTAX and the SILVA v132 non-redundant reference database63, and those still unclassified were compared to the NCBI non-redundant nucleotide database64  by BLAST 2.2.2865 using a 90% cutoff for both sequence identity and query coverage. Phylogenetic trees were constructed from 16S and 18S OTUs using FastTree 2.166, and visualized using the Iroki viewer67. Prevalences were shown for taxa with a minimum detection of five reads.18S data were quality filtered using Trimmomatic v0.3668. Rarefaction curves for each were generated at 100 read intervals to a maximum of 5000 or 50,000 for eukaryotes and bacteria, respectively, and sample group means and standard errors were plotted. As the intra-individual correlation of richness and evenness at 12 and 24 months was low, we implemented generalized linear models (GLMs) using the glm function in R to test for group differences, using diversity values averaged from 100 independent rarefactions at read depths of 25,000 (bacteria) or 1000 (protozoa and fungi). Read depths were chosen to ensure sufficient sampling of OTUs while maintaining a representation of samples, a concern for eukaryote data where read depths were highly variable , supplementation, and urban versus rural site.Microbiota richness (number of OTUs) and evenness  were calculated using Phyloseq 1.20.070. OTU counts were normalized using the median of ratios method in DESeq2, and only taxa represented by a minimum of five reads in at least 5% of the samples were used in the analysis. The contribution of age to bacterial composition was calculated using the capscale function, and the remaining variables were tested for significance in age-stratified samples using adonis. The compositional variance within groups, measured as distances to centroids, was evaluated using the betadisper function and tested for significance using two-way ANOVA, with pairwise differences delineated using the Tukey HSD post-hoc test. All adonis and betadisper tests were carried out with 9999 permutations. We applied non-metric dimensional scaling (NMDS) to ordinate samples based on their compositional dissimilarity. The envfit function was used to identify taxa whose abundance significantly correlated with the first two ordination axes (candidate drivers of community differences), projected as vectors in the direction of change in abundance and scaled by the root square of the correlation. Differences in protozoan and fungal compositions were evaluated at 1000 read depth based on unweighted UniFrac distance scores, and significance was tested as above. Sample distances were visualized using NMDS  with candidate drivers of community differences identified using the envfit function and projected as vectors, as for bacterial data. Differential taxon abundance was tested with DESeq2 1.22.271 in samples containing a minimum of 1000 reads.Differences in bacterial composition (beta diversity), based on Bray–Curtis and weighted UniFrac dissimilarity scores, were calculated with Phyloseq and vegan 2.5–772.Two-sided Fisher’s Exact or pairwise test from the rstatix 0.7.0 package was used to evaluate differences in eukaryote carriage among participant groups, using a minimum five read detection threshold per OTU and grouping OTUs to the genus level or the lowest assigned taxonomic level. Benjamini–Hochberg correction was applied for multiple testing50, with the neighbor selection (MB) method, nlambda 100 and lambda.min.ratio 1e-02 parameters, and used to construct graphical networks using igraph 1.2.673. Interaction networks were compared among groups of children using topological properties, including degree centrality (the number of significant associations held by each taxon in a network) and betweenness centrality (the number of paths linking each pair of microbes in a network that pass through a particular taxon). Betweenness centrality can be interpreted as the importance of a particular taxon in mediating overall microbial interactions. Differences in degree and betweenness centralities were tested using the Wilcoxon rank-sum test, and proportions of cross-kingdom interactions among groups were evaluated using the Fisher’s exact test. Benjamini–Hochberg correction was applied for multiple testing72.Microbial interactions were inferred from pairwise associations between taxa in children, grouped by one or more explanatory variables . Briefly, bacterial and eukaryotic datasets were first rarefied to 25,000 and 1000 reads, respectively. As before, thresholds were selected to balance sufficient sampling of OTUs with maximizing sample group sizes  was designed to assess the interrelationship between supplementation, place of residence, and microbiomes at different timepoints, including (1) eukaryotes at 12 months, (2) eukaryotes at 24 months, (3) bacteria at 12 months and (4) bacteria at 24 months. Cross-correlations among factors were calculated using the plspm::plspm function with the following parameters: taxa blocks  were set to the PLS-score mode, scaling for supplementation and place of residence was set to Nominal, all other parameters were at default. PLS is a supervised dimensionality reduction technique that parallels principal component analysis. It reduces each high dimensional OTU matrix into a latent variable (PLS-score) that summarizes the interlinked differences in microbial community patterns that are related to supplementation and place of residence. The strengths of these cross-correlations are indicated by path coefficients, which can conceptually be understood as correlation coefficients. To test the stability of the path coefficients, we additionally implemented a bootstrapping procedure within the plspm package, which iteratively recalculates all path coefficients on randomly resampled participants with replacement. We also applied the plspm.groups function to test for differences in path coefficients between nutritional groups.To explore the complex system of relationships between micronutrient supplementation, place of residence and the bacteria and eukaryotes at 12 and 24 months, we conducted partial least squares (PLS) path analysis using the plspm 0.4.9 package in R75.All microbial data and statistical analyses were carried out with R version 4.0.2NCT00705445)27 that was approved by the Ethics Review Committee (ERC) of AKU (752-Peds/ERC-07). All participants enrolled in the parent cRCT (NCT00705445) provided written informed consent for the routine collection of stool specimens for analysis of their intestinal microbial communities. Ethical approval for the retrospective analysis of clinical data and stored stool samples was granted by the research ethics board (REB) at SickKids, Toronto (REB No. 1000054244), the ERC at AKU, Karachi, Pakistan (4840-Ped-ERC-17), and the National Bioethics Committee (NBC) Pakistan (4-87/NBC-277/17/1191). A waiver of consent for this retrospective analysis was granted by each of the above-mentioned regulatory boards.All clinical data and stool samples used for analysis were collected in a previously conducted cRCT (Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesDataset 1Dataset 2Dataset 3Dataset 4Dataset 5Dataset 6Reporting Summary


The occurrence of occult metastases in oral cavity squamous cell carcinoma (OSCC) to lower levels in the neck (levels IV and V) or development of skip metastases that bypass the upper neck levels (levels I to III) and go directly to level IV or V is common. This challenges the efficacy of conventional neck dissection approaches in the treatment of OSCC. Therefore, the decision to include lower levels cervical nodes during elective neck dissection of OSCC remains controversial.This systematic review was designed to assess the prevalence of level IV and/or V involvement or skip metastases in patients with the clinically negative neck (cN0) or positive (cN+) oral squamous cell carcinoma (OSCC). We searched for studies published between December 2000 and December 2020. Potentially relevant abstracts and full-text articles were screened, and data from the studies were extracted. Quality was rated using the Newcastle Ottawa Scale (NOS) criteria.In total, 802 abstracts and 227 full-text articles were screened, and 32 studies were included in this analysis. The prevalence of metastasis ranged from 1.8% to 66.0%. The incidence for skip metastasis to level IV or V was low, reaching 8.5%. Evidence favored elective neck dissection, including levels I to III, in selected patients with OSCC and patients with cN0 or cN+ neck. The literature was non-conclusive on the recommendation for inclusion of lower levels. Oral squamous cell carcinoma (OSCC), constituted by a broad range of tumors with diverse etiologies, is a life-threatening malignant tumor that ranks as the sixth most common cancer by incidence, with 500,000 new cases reported worldwide annually, accounting for 32%-40% of all head and neck cancers ,2. It caSelective neck dissection (SND), which removes lymph node groups at designated anatomic levels (I-III), is accepted as the standard of care for the management of regional disease in OSCC patients with clinically positive node (cN+) involvement ,8, as weIn view of the controversies surrounding the inclusion of lower levels for dissection, the present study was designed with the objectives of conducting a systematic review of all relevant published literature: (i) to study the prevalence and distribution of metastasis levels and related adverse outcomes in clinically N0 and N+ OSCC; and (ii) to determine the frequency of involvement of levels IV and V, as well as skip metastasis to level IV in patients diagnosed with OCSCC without preoperative evidence of neck involvement. We aimed to summarize the recommendations for routine dissection of lower levels of nodes in patients with OSCC.MethodologySearch StrategyA comprehensive search for all relevant articles published in English between January 2000 and December 2020 was performed using the electronic databases PubMed, Embase, Ovid, Google Scholar, and Science Direct. We included retrospective, prospective, clinical trials, and cross-sectional studies. The key search terms used either alone or in combination were neck dissection, radical neck dissection, cN0 neck, cN+ neck, oral squamous cell carcinoma, skip metastasis, occult metastasis, lymph node management, neck metastasis, oral cavity cancer, and tongue cancer. The references of articles and citations were also searched for additional potentially relevant publications.Study Eligibility CriteriaAll studies that included patients who underwent a neck dissection (ND) of at least levels I through III or I-IV and presented information on clinically node-negative (cN0) and/or clinically node-positive (cN+) necks were eligible for inclusion. The inclusion criteria were as follows: (1) any prospective or retrospective cohort, (2) a study population with the histopathologic diagnosis of OSCC, and (3) full text available in the English language. In addition, studies that reported skip metastasis (metastasis solely at neck level IV or V) were also eligible for inclusion. Exclusion criteria were as follows: (1) studies on patients who underwent treatment other than surgery as primary treatment, such as preoperative radiotherapy and chemotherapy, and (2) studies on recurrent tumors or tumors other than SCC.Data ExtractionInformation regarding patient characteristics, primary tumor site, treatment, sample size, metastasis, authors, publication year, and the country was retrieved from the selected articles. Data were initially extracted and evaluated by two authors . The distributions of the T category, the extent of ND, the subsite of the primary tumor, and nodal metastasis were recorded. A skip metastasis was defined as a positive level IV (or lower) node on final pathology without the involvement of higher levels . A level IV nodal metastasis coexisting with nodes at other neck levels was assessed separately. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for reporting the included observational studies .Quality EvaluationThe quality of literature was evaluated according to the Newcastle Ottawa Scale (NOS) evaluation criteria . By qualResultsThe search and selection process of the articles is presented in Figure Description of the StudiesData of 12,309 patients included in the 32 studies were analyzed. In all studies, cases of level IV or V metastasis and cervical IIb metastasis were confirmed by pathologic examination or other technologies. All studies did not, however, have consistent inclusion criteria and exclusion criteria. Five studies ,28,30,40The prevalence of metastasis ranged from 1.8% to 66.0% . Among 2Six articles ,34,38,48Studies Recommending Dissection of Lower LevelsFive studies ,24,45,48Studies Not Recommending Dissection of Lower LevelsThirteen studies -31,35-37Studies With Inconclusive Results on Dissection of Lower LevelsFew studies ,39,41,47DiscussionThis review revealed that the available literature favored either selective neck dissection, including only the upper levels (I-III), or was inconclusive. Most studies support the view that primary neck dissections should be limited to upper levels only, owing to the low rates of lower level (level IV and beyond) metastasis and the difficulty as well as the damage incurred (thereby introducing complications) due to the inclusion of those levels. Through independent studies, most authors have supported that high efficacy and minor morbidity for selecting pN+ OSCC patients may be achievable using SND (I-III) ,49,50. ISkip metastasis, described by Byers et al. , refers Some arguments may be made in terms of benefits archived in ipsilateral, contralateral, or bilateral node infiltration. Although we did not study the laterality of recurrence, the available literature  suggesteSome studies reported data on oral tongue SCC, which is the most common primary site for OSCC, with most studies suggesting metastasis to level IIb ,56, leadStrengths and limitationsThe present review included studies that reported varied study groups and regions, thereby introducing heterogeneity. The heterogeneity of study groups is considered an important confounder. In our case, it resulted in the lack of appropriate data stratification by T stage, subsites, and involvement of other neck levels that we could not address. The retrospective nature of the included studies also introduced bias, which could not be addressed. However, we exercised caution in including studies with primary neck dissection data only. We excluded all studies with patients with revision NDs and omitted all groups lacking this information to eliminate bias from combining the results of the primary neck surgery with those of revision surgeries for neck recurrences, which may falsely inflate the rate of level IV or lower-level involvement. While most studies presented mixed data for cN0 and cN+ necks, we segregated data wherever possible to report the differences according to nodal status. Lastly, the decision for SND or MRND techniques is widely debated due to the lack of universally accepted guidelines for the anatomic limits for the variety of SND procedures available. The exact anatomic boundaries for an SND are also thought to vary among institutions and even among surgeons within an institution . The anaOSCC is constituted by a broad range of tumors with diverse etiologies. It can metastasize to cervical lymph nodes via lymphatic vessels. SND is considered a standard of care for most subsites, even in early-stage disease. Based on the evidence reviewed in the present study, the frequency of lower-level metastasis (level IV or V), as well as skip metastasis in OSCC, was low. Hence, routine dissection of these levels in cN0 and cN+ necks may be avoided except for tongue cancer. Since dissection of level IV/V is a burden with extra time and might expose patients to more complications, dissection might be selected for specific subsites and extension. It is recommended to dissect level IIb and lower levels for tongue cancers without considering the stage of primary lesions or lymph node status. Most studies recommended sparing lower-level neck dissections, while some were inconclusive.


In recent years, several controversial reports of the correlation between altmetric score and citations have been published (range: -0.2 to 0.8). We conducted a meta-analysis to provide an in-depth statistical analysis of the correlation between altmetric score and number of citations in the field of health sciences. z transformation of correlations. Subgroup analyses based on citation source and sampling methods were performed.  Three online databases  were systematically searched, without language restrictions, from the earliest publication date available through February 29, 2020, using the keywords “altmetric,” “citation,” and “correlation.” Grey literature was also searched via WorldCat, Open Grey, and Google Scholar (first 100 hits only). All studies in the field of health sciences that reported on this correlation were included. Effect sizes were calculated using Fisher's p = 0.055), and trim-and-fill analysis estimated one missing study in this meta-analysis.  From 27 included articles, 8 articles comprise several independent studies. The total sample size was 9,943 articles comprised of 35 studies. The overall pooled effect size was 0.19 . Bivariate partial prediction of interaction between effect size, citation source, and sampling method showed a greater effect size with Web of Science compared with Scopus and Dimensions. Egger's regression showed a marginally nonsignificant publication bias . We emphasize on future examinations to assess changes of correlation pattern between altmetric score and citations over time. Altmetric Institution , Plum Analytics , and ImpactStory (http://impactstory.org/). Several academic publishers, including John Wiley & Sons, Inc., Journal of the American Medical Association (JAMA) Network, Taylor & Francis, Springer Nature, and Elsevier Publishing use one of these resources.The increasing demand for investigators to disseminate their research findings on the internet has given birth to new terms such as “Twitter science stars” and “Kardashian Index” (which measures over/under Twittersphere activity) , 2. A prAltmetric Institution is the most widely used provider. Altmetric database traced more than 27 million research output  and found more than 14 million research output with online attention and 122 million total altmetric mentions. As defined by the Altmetric Institution “altmetric score is a weighted count of all of the mentions Altmetric has tracked for an individual research output, and is designed as an indicator of the amount and reach of the attention an item has received.” . If statistical heterogeneity existed   showed a rapidly increasing number of publications after 2014 . Yet, it should be noted that altmetrics is a relatively new concept among scholars and future rise in awareness and use of social media among researchers may increase this correlation.I2 = 87.99%), which we tried to assess by subgroup analyses based on the source of citation number and sampling method . Further analysis showed that 68% of tweets were carried out by members of the public, since obesity and fitness are popular topics. This situation is not comparable with specialized narrow fields of health sciences such as endodontology. Topics discussed in specialized fields generally receive less online attention and are rarely shared and discussed in social media and news outlets. It could be expected that the medical articles with more popular keywords and topics such as cardiovascular disease or obesity may receive more online attention and higher altmetric scores. This may be an explanation for a high level of heterogeneity in this meta-analysis.This meta-analysis involved a high level of heterogeneity  on citation patterns . In addir value for each group and subgroup and correlation assessment method, (6) criteria for selection of articles such as highly cited article, and (7) type of included articles such as original research.With respect to emerging use of social media among scientists, we recommend future examinations to assess correlation pattern changes over time. To facilitate future meta-analysis, we encourage authors to report the following items clearly: (1) time of data collection, (2) source of citations and altmetric data, (3) number of study groups and subgroups, (4) sample size for each group and subgroup, (5) exact Below is an example to assess correlation between altmetric score and number of citations among articles related to COVID-19 pandemic which was funded by the National Institutes of Health (NIH).r = 0.48, 95% C.I 0.45 to 0.50, p < 0.001) among all articles (n = 3266)Moderate correlation between altmetric score and citations  among top 10% articles with the highest altmetric score (n = 328)Moderate correlation between altmetric score and citations  among top 10% articles with the highest number of citations (n = 335)Moderate correlation between altmetric score and citations  among systematic reviews and meta-analysis (n = 37)High correlation between altmetric score and citations  was used to find altmetric data. Source of citations was the Dimensions database. All data were collected/retrieved on December 13, 2020. A total of 3266 articles were found with altmetric mention (82.4%) from 3961 articles. Statistical analyses showed the following:p = 0.004) .This example indicated degree of correlation may meaningfully depend on the sampling method and characteristics of included articles .


Melia azedarach (Dharek) Azadirachta indica plants to protect the quality of fruits. After evaluating these films, their mechanical, specific respirational, coating crystal elongation, elastic, water vapor transmission rate (WVTR), film thickness, and nanoindentation test properties are applied to apple fruit for several storage periods: 0, 3, 6, 9 days. The fruits were evaluated for postharvest quality by screening several essential phytochemical, physiological responses under film coating and storage conditions. It was observed that prepared films were highly active during storage periods. Coated fruits showed improved quality due to the protection of the film, which lowered the transmission rate and enhanced the diffusion rate, followed by an increase in the shelf life. The coating crystals were higher in Film-5 and lower activity in untreated films. It was observed that the application of films through dipping was a simple technique at a laboratory scale, whereas extrusion and spraying were preferred on a commercial scale. The phytochemicals screening of treated fruits during the storage period showed that a maximum of eight important bioactive compounds were present in fruits after the treatment of films. It was resolved that new active films (1–5) were helpful in the effective maintenance of fruit quality and all essential compounds during storage periods. It was concluded that these films could be helpful for fruits growers and the processing industry to maintain fruit quality during the storage period as a new emerging technology.Today, the most significant challenge encountered by food manufacturers is degradation in the food quality during storage, which is countered by expensive packing, which causes enormous monetary and environmental costs. Edible packaging is a potential alternative for protecting food quality and improving shelf life by delaying microbial growth and providing moisture and gas barrier properties. For the first time, the current article reports the preparation of the new films from Ditriterpenoids and Secomeliacins isolated from  The chemical and physical processes of liquid drying films have a universal history in engineering and protection facilities with appealing requirements and barriers. The active compounds forming thin-film recently gained attention as having advanced chemical, photographic, and electrical properties ,2. It foMalus domestica B), the world’s most popular fruit for its health-related elements and nutritional value, belongs to the Pomoideae subfamily Rosaceae. It is ranked third after banana and citrus for worldwide consumption and uses. It was reported that the losses during postharvest, packaging, transportation, handling, storage, and marketing of fresh apples occur by 20–40% [A.indica (Neem) is a rich source of several bioactive compounds with large chemical diversity, which can be used after isolation [A. indica (Neem) belong to two groups triterpenoids, and diterpenoids secomeliacins, such as salanin, azadirachtin non-isoprenoids, coumarin tannins, and dihydrochalcone, and also aliphatic compounds [Apple  and Azadirachta indica (Neem) plants. After preparing new bioactive films, thickness and nanoindentation tests were also applied for the first time. The films after preparation process and protocol development were checked against mechanical, respirational, coating crystal, elongation, elastic nodule, and water vapor transmission rate (WVTR) of the apple fruits and stored to measure their quality, phytochemical screening, quality enzymes, taste-related compounds, and physiological responses of fruits under storage and film application. It was established that newly developed films might be used to preserve food, pharmaceutical products, and chemical industry as future enactment of products coated at the upper surface, such as in this research where apples were used for testing the prepared films and found effective.This article deliberated the preparation of new bioactive films for the first time through Ditriterpenoids and Secomeliacins isolated from The samples of (Dharek and Neem) leaves of both plants were dried at 72 °C temperature, then ground to get dry leaves powder; after that, different concentrations of liquid solutions were prepared with the help of the filtration process, as shown in . The twow/v)  and Tween 80 at 0.2% (w/v)  were added for making a plasticizer form and as a surfactant, respectively. The pH 5.5 of the films was noted.Five pure films were prepared from organic coated materials made by two significant compounds like Ditriterpenoids and Secomeliacins. The compounds were in liquid form and obtained in (3 mL) with adding of 90% of deacetylation, Aqua Premier Co., Bang Lamung/Chon Buri, Thailand and 1% of lactic acid  in solutions added and stirred for 10 min at room temperature until the compounds of Dtriterpenoids + Secomeliacins were dissolved entirely in solutions. The glycerol at 1% (Melia azedarach (Dharek) and Azadirachta indica (Neem) were standardized. The pure solution of both compounds was used in a concentration of 1–3% based on dryness. The prepared films were cast in Petri plate for the drying process. They were stored at 20°C (controlled by laboratory air conditioning system) and 53% RH 2, under vacuum), until further use as a proper film  were prepared in pure solution. Film making and shaping created a film structure and foaming pattern with conventional scattering or suspension. Mixtures of 10–30 mg of diterpenoids + secomeliacins—blended to make homogeneous concentration solutions and lowered endeavoring of mixtures to obtain complete films.The degassing and defoaming of manufactured films is a significant advancement in creating new films of diterpenoids + secomeliacins to eliminate air microbubbles in solutions left in suspended films, entangled the films, and deformities that were mechanically matched to its structure. The plasticized films of 2 mL of corn syrup were used for vacuum degassing films with the evacuation of 30 m bar films sizes in 4 min.The films were created by seat projecting on the rimmed or Palin plates for the manufacturing process. The film thickness was measured in the rimmed plates, drawdown bars, or plain plates . The staThe thickness of each film was measured using a digital micrometer . For each produced sample, six determinations were made at random positions.The water vapor permeability measurement in active films to its potential and suitability for the food industry was conducted .The method of Bertuzzi et al.  was used2), ∆P is the water vapor pressure differential across the film (Pa), and C is the slope of the weight gain of the dish, to the 0.0001 g, versus time.Water vapor permeability (WVP) was calculated from the following equation:The temperature dependency of WVP of the edible films was evaluated using an Arrhenius relationship (Equation (2)) described by Bertuzzi et al. .WVP = WFrom the slope of the fitted regression line, the apparent activation energy (EP) of films was determined at test temperatures.2). DP is the difference of partial vapor pressure of the atmosphere (2337 Pa at 20 °C) and was the weight loss. Two replicates were obtained for each samples.The gravimetrically ASTM E96-92, 1990 determined the water vapor permeability (WVP) as a reported method of Cerqueira et al. . The WVPTensile strength (TS) and duration during breaks (EB) remaining EAEP films were measured by SUNS 6102 Universal Testing Machine  according to standard ASTM D882-12 . The fil2, then placed on a carbon-coated plate and covered with gold. The sample was then placed on an SEM device for small structure monitoring.The structure of the film was detected by scanning electron microscopy  JEOL Ltd. JSM 5310 LV Peabody, Bostan, MA, USA). The film was cut to 0.5 × 0.5 cmTwo mechanical features, modulus and elasticity, and stiffness were determined by nanoindentation. The elastic modulus (E), commonly referred to as Young’s modulus is a measure of stress (soku) pressure (ε) when the twist is fully stretched. In an expansive region, stress and difficulty are proportional to Hooke’s rule: σ = Eε.Hardness films (H) measure resistance to material in conversion by inclusion. The transformation of plastic is caused by the movement of the separation of the atomic structure of an object.Approximately 50 mg of the film were dried at 104 °C for 24 h (until the equilibrium weight was attained). The weight loss was determined, and MC was calculated as the percentage of water removed from the system. The film solubility in water was determined by the reported method .The samples of delicious golden apples were collected from Haripur, Khyber Pakhtunkhwa, Pakistan. The trials were conducted in the Department of Horticulture laboratory, The University of Haripur, and other chemical processes were analyzed in the Department of Chemistry University of Karachi, Pakistan.The apples (Golden) were washed with sodium hydrochloride (1PPM) solution added in one liter of solution. The apples were dipped in the solution, washed after ten minutes, dried, and graded based on their proper size and shape.The film of proper concentration on each apple was applied for 15 min after washing and grading, simply by dipping in solution to cover the entire apple surface. The apples were placed for 3 min for complete dryness of the bioactive wax thickness, then shifted into a plastic basket and placed at room temperature for 9 days storage with 3-day intervals. In each replication, 5 apples were selected.The apples were treated with specific coating materials, used as a source of treatments, and stored at room temperature, where the initial temperature was 32 °C and humidity was 70%. The storage periods were 3, 6, and 9 days. The days after storage was compared with untreated fruits.−3) was measured by the method [The MDA and the Lipid peroxidation contents were determined by the thiobarbituric acid (TBA-MDA) reaction as reported by Hodges et al. , Ali et e method .The total soluble solids were measured with the help of advanced refractometer 1.2 BX-CV, New York, NY, USA.2CO3. The reaction was performed for 20 min at room temperature in darkness, and the absorption was recorded at 765 nm against the control sample. The results were expressed as mg equivalent of gallic acid (GAE) per gram according to the calibration curve built in the range of 0.02–0.10 mg gallic acid used as a standard.Total phenolic contents (TPC) were measured using the Folin-Ciocalteu assay according to the procedure described by Stintzing et al.  with sliThe DPPH radicals scavenging activity was estimated with the method used by Williams et al.  with somPercentage inhibition of free radical DPPH was calculated based on control reading by the following (Equation (4)):The activity of superoxide dismutase (SOD) was determined according to Das et al.  with sli2O2 and 800 uL of 0.01 M phosphate buffer (pH 7.0), and the absorbance was measured at 240 nm. Time taken for a decrease in the absorbance from 0.45 to 0.4 was noted. The enzyme activity was expressed as a mole of H2O2 consumed/min/mg protein.The catalase activity was determined according to Wheeler et al.’s  method w2O2, 100 µL 20 mM Guaiacol, and 800 µL 0.1 M phosphate buffer pH 7.0. 100 µL enzyme extract was added to 100 µL of the reaction mixture, and the absorbance was recorded at 470 nm.The Peroxidase (POX) activity was determined according to Bergmeyer et al.  with sli3 added, light green coloration, shows the presence of tannin. The steroids were measured by mixing 2 mL of the sample with 1 mL of H2SO4; the red layer showed a presence of steroids in the solution. The terpenoids were measured in the juice of apple by adding 2 mL of each sample with adding of 2 mL of chloroform and 3 mL H2SO4, reddish-brown coloration the presence of terpenoids. The phlorotannins were measured in apple juice with 2 mL of juice sample then 1 mL of HCl was added. The presence of red color indicates the presence of phlorotannins. The cardiac glycoside was measured by adding 1 mL of glacial acetic acid into 2 mL of each sample, and 2–3 drops of FeCl3 show green-blue coloration reflecting the presence of cardiac glycosides. The flavonoids were measured by mixing 2 mL of each sample into 2 mL of NaOH and then adding 1 mL of HCl shows a yellow color, and their disappearance shows flavonoids in apple juice. The quinines were measured in 2 mL of each sample, then added 1 mL of NaOH, which resulted in blue-green or red color indicating the presence of quinines.During the storage period, several essential phytochemicals were measured in the apple juice; the saponins test was measured in 2 mL of each sample where 5 mL of distal water was added and well shaken for 30 s, the stable frothing persistent indicate that saponins are present in it. The tannin test was measured into 2 mL of each sample and heated up to 40 °C, then add 5–6 drops of FeClThe HPLC machine is linked with a detector (DAD) with a frequent wavelength of 500–3000 milli-absorbance units. The HPLC system used a  binary water pump attached to a photodiode array detector. The system attached an auto-injector with a water phase analytical column. The mobile phase was checked through ultrasonic waves before the operation. The ultrasonic machine was attached with a digital timer and temperature-controlled power of 400–900 W with a frequency of 45 Khz.v/v, acetonitrile, and aqueous 0.1% v/v 0.1% formic acid and methanol) were evaluated. Formic acid and methanol 0.1% were best for the mobile phase as all components resolved under this condition. The five compounds were separated by elution of isocratic mode, the eluation mode was followed by (A) formic acid 0.1 v/v and (B) methanol for separation a liner program is followed 10–65% B 0–85 min, and the standard mixture should be baseline separated for each chosen marker.The mobile phase composition was optimized for best resolution results with a short analysis time. The major bioactive compounds were isolated by suppressing the acidic mobile phase. The acidic mobile was best for a longer retention time, and a better peak shape was obtained. Diverse mobile phase compositions, such as  formic acid in water (solvent A) and 40:40:20 (v/v/v) acetonitrile: water:formic acid (solvent B), according to the gradient. The injection volume was 20 μL, and the column temperature was 20 °C, whereas the detection length was 260 nm. Before injection in the HPLC system, samples were filtered through a PTFE 0.45 μm pore-size filter. Quantification was performed based on external standards of known concentrations.HPLC was performed with a Varian-Agilent system, consisting of a ternary pump 9050 series and a UV–Vis detector 9050 series-coupled to STAR 4.5 software for data acquisition and elaboration. A Gemini NX C18 110 À column, 250 × 4.6 mm (Phenomenex), protected by a guard column, was at the stationary phase. The five active compounds and two essential compounds under bioactive films were separated by gradient elution with 12%  least significant difference, which gives us mean lettering of the data, was measured. The graph was prepared by the software Graph Pad Prism 7.02. The HPLC-DAD measured the new taste compounds by Chrom gate v 3.31 Knauer software.Results and DiscussionsComparison of Tensile strength (N), elongation at break (EB), Thickness (mm) Elongation (%), and water vapor transmission rate (WVTR) changes in new filmsSignificant differences were found in tensile strength and films activity. Results showed that both compounds’ higher activity was successfully used in edible films. The analysis of variance  showed tFilms properties with stability, activity, and potential applicationsThe new films of (diterpenoids + secomeliacins) were applied to the apple surface before the storage period and accounted for water vapor permeability (WVP), thermal stability, and film stability. Initial values  were recorded at 0 days, as shown in . HoweverRespirational changes in apples during storage periods under a coating of new films2, ethylene, and respirational rates were  were noticed at 0 days, whereas at the others’ storage, like 3, 6, and 9 days showed lower rates shown in 2, ethylene, and respiration were noted, in the untreated fruits.The comparison of storage period and application of new films on respiration changes of apple fruits were measured and shown in Quality enzymatic changes and storage periods of apples (golden delicious)Statistically significant differences were found regarding apple treatment and storage interactions by applying bioactive films (Ditriterpenoids and Secomeliacins) . Slight Bioactive compounds of apple fruits under films coatingSignificant differences were found in treated fruits of apples stored at ambient temperature. The higher value of TSS was noticed for Film-4 and Film-5 shown in Screening of phytochemicals of apples under new filmsThe screening of phytochemicals in (Golden Delicious) during storage periods of  shown in Taste related essential bioactive compounds of stored apples under film coatingThe five essential bioactive compounds were evaluated in fresh and stored golden delicious apples, as shown in Physiological changes in stored apples under films coatingThe physiological parameters showed significant differences between treatments and storage periods of fruits shown in Apples are a highly nutritional crop globally , and theThe greatest challenge encountered by food manufacturers is the loss of quality of food products during storage, which eventually adds to significant wastage and losses. This study used the preparation of new films for the first time with added active compounds Ditriterpenoids and Secomeliacins. The prepared films were tested for their mechanical, specific respirational, and coating crystal elongation, elastic and water vapor transmission rate (WVTR), film thickness, and nanoindentation. Tests were carried out for new films to assess the impact on apple’s storage periods, quality, and shelf life (Golden Delicious). The five new active films were used in this study with 10–30 mg concentration ranges. The treated fruits of apples with coated films attained maximum fruit quality compared to untreated fruits. Eight proper initial screening tests of phytochemicals were conducted with treated and untreated fruits. Higher values were noted in treated fruits of apples with higher ranges than uncoated fruits. The taste-related and color formation bioactive compounds of  isolated in apple pulp through the advanced and suitable method of HPLC-DAD. All essential compounds were saved under films coating during storage periods, and the physiological response under oxidative stress of films stability parameters was also better in all treated fruits. Based on the results, the films help to prevent diseases and maintain fruit quality during storage. The commercial storage life can be extended by applying films or coated materials for fruits industries.


Methods: MRI testing was used to image mouse cerebellar tissue sections and organotypic cultures. Demyelination was induced by lysolecithin treatment. Myelin-targeting liposomes were synthetized and characterized, and their capacity to quantify myelin was tested by fluorescence imaging. Results: Imaging of freshly excised tissue sections ranging from 300 µm to 1 mm in thickness was achieved with good contrast between white (WM) and gray matter (GM) using T2w MRI. The typical loss of stiffness, WM structures, and thickness of organotypic cultures required the use of diffusion-weighted methods. Designed myelin-targeting liposomes allowed for semiquantitative detection by fluorescence, but the specificity for myelin was not consistent between assays due to the unspecific binding of liposomes. Conclusions: With respect to the sensitivity, imaging of brain tissue sections and organotypic cultures by MRI is feasible, and myelin-targeting nanosystems are a promising solution to quantify myelin ex vivo. With respect to specificity, fine tuning of the probe is required. Lipid-based systems may not be suitable for this goal, due to unspecific binding to tissues.Ex vivo models for the noninvasive study of myelin-related diseases represent an essential tool to understand the mechanisms of diseases and develop therapies against them. Herein, we assessed the potential of multimodal imaging traceable myelin-targeting liposomes to quantify myelin in organotypic cultures.  Myelin is an essential element of the central nervous system (CNS) and is implicated in many neurological diseases. The study of myelin loss (demyelination) and regeneration (remyelination) is of paramount importance for a better understanding of such processes, as well as for the design of therapeutic strategies for myelin-related diseases. The development of suitable in vitro models that allow for reliable myelin quantification in the study of de- and remyelination processes is a very important and not yet fully resolved issue that may help to boost the development of novel therapies for neurological diseases .Indeed, the use of in vitro models may facilitate the obtaining of faster results, more controllable and reproducible experimental conditions, and more statistically powerful results than in vivo models. The possibility of isolating the target subject of study from concomitant confounding effects present in the complexity of in vivo settings  is an additional important feature of in vitro models.The development of an in vitro model is a complex task that requires not only the definition of a suitable system that mimics the relevant biological aspects that are expected to be replicated, but the definition of proper experimental techniques that allow for observing and, ideally, quantifying the phenomena under study.In general, in vitro models may be constructed with increasing degrees of complexity, starting from specific cell-type monocultures and scaling to complex co-cultures , organoids, perfused excised organs or tissues, and 3D bioprinted structures. Despite multiple in vitro models having been successfully reported in the literature for the study of the central nervous system (CNS) , for myeHowever, the quantification of myelin in organotypic cultures—not a trivial issue—has traditionally been tackled by invasive experimental methods such as RT-PCR, Western blot, or immunofluorescence . Each ofIn this context, we have explored the possibility of using functional biomaterials for the quantitation of myelin content in OBCs by magnetic resonance imaging (MRI), which has the great advantage of being noninvasive and therefore enabling longitudinal studies in the same cultures. Despite MRI having been widely used for the quantification of myelin in clinical and preclinical in vivo studies, with several MR imaging biomarkers of myelin, such as T2-weighted MR signal or radial diffusivity , the useWe believe that the use of molecular imaging approaches, including the use of targeting imaging probes that help to increase the sensitivity of detection, may offer an opportunity to overcome such challenges; thus, in this work, we describe our attempts to achieve an experimental protocol for the MRI-based noninvasive imaging of tissue sections and organotypic cultures, and to develop myelin-specific imaging nanoprobes that enable the quantification of myelin content in organotypic cultures from the mouse cerebellum.The development of nanomaterials that act as imaging probes for MRI is an active field of research, and examples can be found in the literature ,10,11. IThe results demonstrate that, although it is feasible to image organotypic brain cultures (OBCs) by MRI, and molecular imaging enables the quantification of myelin in ex vivo models, the design of the functional imaging probe is an especially critical issue that requires further development to ensure the sufficient sensitivity for myelin, avoiding specificity-related issues.Diputación Foral de Guipuzcoa and our institutional ethics committee, with license number CEEA17_002). Studies were conducted on three different types of samples.All experiments involving the use of research animals have been performed according to the appliable legislation  and were approved by our Institutional Animal Care and Use Committee (IACUC) and by the local authorities  were sacrificed at postnatal day 7–12, and their cerebellums were sliced to different thicknesses  and fixed in paraformaldehyde (10%) for 40 min. Next, the brain sections were washed 2× (for around 10 min) with phosphate-buffered saline (PBS) and immersed in 2% low melting point agarose  in a 50 mL Falcon tube for imaging. The possibility of performing multiplanar MR imaging allows for scanning sets of several tissue sections in a single imaging experiment by piling the sections inside the agar gels.C57BL/6JRj mice  were sacrificed at postnatal day 10–12 and their cerebellums immediately extracted and placed in an organotypic culture medium prepared with 24 mL 2-mercaptoethanol (BME) , 24% Hanks’ balanced salt solution (HBSS) , 24% horse serum , 0.125% glutamine , 1% antimycotic and antibiotic , and 3.5% glucose , for a total solution volume of 50 mL.2 . Sections were separated and placed on a Millicell Cell Culture Insert membrane  on a P6 plate and incubated for seven days in an organotypic culture medium (as described above) at 37 °C and 5% CO2 .For imaging studies, cultures were fixed with 4% paraformaldehyde for 40 min and kept in PBS (0.05% sodium azide) at 4 °C until use. Organotypic cultures were embedded in 2% agarose before MRI scanning. For this purpose, the support membrane of the culture insert was gently cropped around the organotypic tissue with a scalpel.Thin organotypic culture samples were prepared as described in the previous section, but with slices of 350 µm thickness, which represents the desired target for our experimental ex vivo assay see .When required, demyelination was induced in these cultures after one week of culturing by exposing OBCs for 15–17 h to a medium containing 0.5 mg/mL lysolecithin .MRI-traceable myelin-targeting liposomes were prepared by the lipid film hydration and extrusion method , using a3+) responsible for the generation of T1-weighted MRI contrast were complexed with the lipid DTPA-BSA(Gd) , used as a constitutive element of the liposome membrane. It was possible to tailor the magnetic properties of liposomes to obtain the best performance from the MRI by modifying the DTPA-BSA(Gd) percentage during the lipid film formation. Four different molar fractions of Gd-BSA lipid were assayed  to achieve the highest T1-induced contrast. In parallel, the fluorescent dye DiOC18(3)  was added (300 μL of a 1 mg/mL solution) to the formulation in the organic phase before a lipid film was formed.Liposomes were designed as multimodal imaging probes detectable by magnetic resonance imaging and fluorescence microscopy. Thus, gadolinium ions  and resuspension of the pellet containing the liposomes in HEPES-buffered saline . Transmission electron cryomicroscopy (CryoTEM), dynamic light scattering (DLS), and inductively coupled plasma mass spectrometry (ICP-MS) were used for the physicochemical characterization. A Bradford Assay  was used to verify that antibodies were attached to the surface of the liposomes. Relaxometry properties were measured in a Bruker Minispec MQ60  contrast agent analyzer at 1.5 T and 37 °C.Liposomes were designed to specifically target myelin. Thus, the maleimide groups present in the surface of liposomes were conjugated with the antimyelin basic protein antibody , substituted by anti-IgG protein antibody in control liposomes . For conjugation, the antibody was preactivated by mixing it in SATA solution (1:80 mol/mol). Afterwards, the SATA–antibody solution was added to the liposome solution in a vial (50 µg of protein per 1 µmol of lipids), and the mix was kept overnight at 4 °C under NCell culture inserts were prepared, each of them with three thin organotypic slices lying on the surface of the membrane . After aFor myelin recognition by liposomes, cultures  were fixed with 4% paraformaldehyde for 40 min and washed with DPBS. Tissue was blocked with a solution composed of DPBS, 0.5% Triton , and 10% goat serum  for 1 h at room temperature. A volume of 350 µL of liposomes was added and samples were incubated overnight at 4 °C. Then, samples were washed with 0.1% Triton in DPBS and stained with Hoechst  10% in DPBS for 10 min.Fluorescence images were acquired using a Nikon Eclipse 80i digital microscope  and processed using NIS elements AR 3.2 software (Nikon).3 to 100 × 100 × 500 µm3. Different T2-weighted imaging sequences were tested  with echo times ranging from 10 to 70 ms and repetition times ranging from 2000 to 7000 ms.MRI studies were conducted at 7 Tesla using a Bruker Biospec 70/30 USR MRI system . Images of brain tissue slices and thin organotypic cultures were acquired with spatial resolutions ranging from 25 × 25 × 25 µm2; gradient pulse durations δ = 6 ms and spacing Δ = 14 ms; TR = 2820 ms; slice thickness = 250 µm.For thin organotypic cultures, diffusion-weighted imaging was also conducted with a spin-echo diffusion-weighted imaging (DWI) sequence, using the following parameters: b-value = 1500 s/mmImaging of thick organotypic slices was performed with a turboRARE sequence with the following parameters: TR = 2000 ms; RARE factor = 10; effective echo time TE = 70 ms; field-of-view (FOV) = 10 mm × 10 mm; image matrix = 400 × 400 points; 24 slices with a thickness of 0.3 mm.tissue/SDbackground). The contrast -to-noise ratio (CNR) between gray and white matter was obtained as the difference of the mean of each tissue divided by the standard deviation of the background (CNR = 0.66 × [meangm − meanwm]/SDbackground).Image analytics were conducted using the NIH software FIJI . For theAs discussed in the introduction, the development of remyelination therapies requires experimental models and techniques that enable the evaluation of the effect of a given therapy. Both in vitro and in vivo models have been proposed in the literature, with growing interest in mixed models such as the use of ex vivo organotypic brain cultures (OBCs), in which the advantages of in vitro systems such as reproducibility, homogeneity, high-throughput capability, etc. can be applied to brain tissue samples with a more complex structure than a culture. Thus, OBCs are being exploited for the study of remyelination therapies. However, a reliable and noninvasive technique thar allows for the longitudinal assessment of myelin content in organotypic cultures is still an unresolved issue ,15 that First, we tested the capability of MRI to image thick and large portions of cerebellar tissue, fixated in formalin briefly after excision from mouse brains. This does not represent a proper model of organotypic cultures per se but allowed us to establish a starting point from manageable samples in the MRI scanner, assess the limits of the achievable sensitivity of detection , and find the working limits of image slice thickness and spatial resolution affordable for our MRI scanner.Thus, several tissue sections of different thicknesses  were prepared, imbibed in agar gels, and scanned with multiple acquisition sequences, in order to image such thick tissue slices, optimizing the balance between imaging time, SNR, CNR, spatial resolution, and image quality .effective = 64 ms); FOV = 1.28 × 1.28 cm2; image matrix = 512 × 512 (in-plane resolution: 25 × 25 µm2), slice thickness = 300 µm; N = 20 averages; total scanning time = 2 h 4 min.The best results were achieved using a RARE  sequence with a RARE factor of 8, an echo time of TE = 16 ms . The minimal imaging slice thickness is determined by hardware constrictions (gradient strengths and SNR of the radiofrequency coils) and the imaging time . Our experimental conditions allowed us to acquire a minimum imaging slice thickness of 300 μm, in practice, and at this condition <1/3 of the voxel was filled with tissue, with the rest being occupied by agar. When ideal conditions were met, it was possible to obtain images from OBCs B,E, but In light of these results, we attempted a different approach for conducting diffusion weighted imaging, whereby the signal arising from water molecules in the agar gel is suppressed by diffusional gradients B. In thiAs we have mentioned, sensing technology uses sensors to detect a physical, chemical, or biological property with an experimental technique, and convert it into a readable signal that allows for quantification. The development of nanomaterials acting as imaging probes for MRI is an active field of research ,10,11. ILiposomes doped with imaging probes (gadolinium for MRI detection and a fluorescence probe for microscopy) and decorated with antimyelin basic protein antibody (anti-MBP) were prepared (as well as liposomes decorated with unspecific anti-IgG protein as a control) and incubated with thin organotypic tissues , as desc9, 40.2 × 109, 61.7 × 109, and 112 × 109 Gd atoms per liposome unit, as measured by inductively coupled plasma mass spectrometry (ICP-MS), and characterized by measuring their longitudinal relaxivity (r1), calculated as the slope of plots of the relaxation rate  of solutions of different concentration of those liposomes versus the concentration of liposomes , resulting in a longitudinal relaxivity of 2.17 mM−1s−1, with respect to the concentration of liposomes. As we anticipated, higher loads of gadolinium resulted in a partial quenching of T1 contrast (An important aspect of the development of imaging sensors is the optimization of the payload of imaging probes (gadolinium content per liposome) since, in the case of MRI, it has been demonstrated that an excessive payload of this lanthanide in the probe could quench the T1 effect and reduce the sensitivity of detection and image contrast . Thus, scontrast B.The decoration of the surface of the naïve liposomes with antimyelin protein or IgG (control) protein attached to polyethylene (PEG) chains has no significant effects on liposomes’ sizes, as determined by dynamic light-scattering studies  and optimization of imaging properties, which is outside of the scope of this work. Work along this line is currently in progress in our laboratories.A limitation of this study is that we do not report the MRI detection of myelin in OBCs through the use of prepared liposomes. Due to the much higher sensitivity of detection of fluorescence imaging versus MRI, we performed tests of specificity with this technique. Since multimodal imaging liposomes were nonspecific for myelin sensing, we decided not to continue MRI studies with them and instead will wait until an optimized nanomaterial is available. We will continue with the search for alternative materials that may be effective for the specific labeling of myelin in organotypic brain cultures.The development of experimental protocols for the noninvasive in vitro characterization of myelin in cultures may represent a breakthrough for the performance of high-throughput studies of treatments, in highly reproducible experimental conditions. In this work, we have shown that the MRI imaging of freshly excised and fixed brain slices is feasible with high SNR and CNR, demonstrating the potential of this technique for the quantification of myelin, but such systems are inadequate as ex vivo models, since the preservation of cells in the interior of thick sections in culture is highly compromised by the limited diffusion of nutrients and oxygen through them.We have also shown that thin organotypic cultures are inherently problematic for imaging and quantification by MRI and that, alternatively, sensing of myelin by molecular recognition with multimodal imaging nanomaterials can be a suitable experimental strategy, providing proof-of-concept for this approach, but we have not yet succeeded in finding a suitable molecular platform to achieve the required level of specificity required for this task, since liposomes have high unspecific affinity for biological tissues in general. Ongoing research in this line will tell us if this is a good approach for obtaining experimental ex vivo tools for the longitudinal study of demyelination and remyelination processes.


Elymus nutans has been widely planted together with other perennial grasses for rebuilding degraded alpine meadow atop the Qinghai‐Tibetan Plateau. However, the rebuilt sown pastures begin to decline a few years after establishing. One of the possible causes for the degradation of sown grassland may come from allelopathy of planted grasses. The purpose of this study was to examine allelopathic potential of Elymus nutans. Three types of aqueous extract from Elymus nutans and its root zone soil were prepared, and 5 highland crops and 5 perennial grasses were used as recipient plants. Elymus nutans exhibited strong allelopathic potential on germination and seedling growth of 5 crops, but different crops or perennial grasses respond to the extract differently. The pieces aqueous extract have stronger inhibition than whole plant extract and root zone soil extract. Hordeum vulgar var. nudum, Avena sativa, and Festuca sinensis were the most affected, while Chenopodium quinoa and Elymus sibiricus were the least affected. Elymus nutans presented less influence on Poa pratensis and Poa crymophylla than on Festuca sinensis. It is recommended that the species combination of mixture for restoration should be considered for allopathic effects on the coseeding to decrease the seeding rate ratio of Elymus nutans. The annual dicot crop seeds of Chenopodium quinoa and Brassica napus can be used as alternative subsequent crop for the seed field of Elymus nutans monoculture. Seeds, including 8 species of monocotyledon and 2 species of dicotyledon, were tested. (Photograph by XLQ) Crop seeds of Hordeum vulgar var. nudum, Avena sativa, Triticum aestivum, Chenopodium quinoa, and Brassica napus, which were harvested in 2019, were acquired from the Academy of Agriculture and Forestry Sciences, Qinghai University. These crops can grow well in the agricultural area of the province. All seeds were stored in the refrigerator at 4℃ before experiment. Plants of Elymus nutans were collected in early August 2019, when the plant growth was in peak period. Robust whole Elymus nutans plants with roots were dug out with a small shovel. The plants were cut into aerial parts and belowground parts on site. The root zone soils were collected by shaking the roots parts. All plant and soil materials were put in plastic bags and kept in a refrigerator and taken back to the laboratory for experiment.Grass seeds of 2.2Two kinds of plant extract were prepared. The whole plant extract was prepared by soaking whole fresh aboveground parts in distilled water . The pieces aqueous extract were prepared by cutting plants into 1–2 cm and soaking them in distilled water . The containers of soaked plant materials were shaken at 500 rpm for 5 min per 12 hr in 72 hr at room temperature. Soil extract was prepared by soaking fresh root zone soil in distilled water  and shaken at 200 rpm for 24 hr in gyratory shaker at room temperature.The resulting mixtures were passed through qualitative filter paper and 0.45‐μm aqueous membrane, respectively. Thus, 0.25 g/ml plant extract and 0.5 g/ml root zone soil extract were obtained. The solutions were stored at 4℃ until use.2.3All seeds were surface‐sterilized by soaking in 0.5% sodium hypochlorite solution for 10 min and rinsed several times with distilled water. Five replicates of 50 seeds were placed in Petri dishes (90 × 15 mm) lined with 2‐layer filter paper. The seeds and filter paper were wetted with 3 ml distilled water or extract solution. To maintain moisture, the petri dishes were put in plastic bags and placed in a growth chamber at temperature of 20/16℃ for 12:12 h (light/dark) period. The light intensity was 3,000 lux. A seed was considered germinated when the root protruded ≥2 mm. Germination was counted at 24‐hr interval over 15 days, and the first count was carried out on the fourth day.2.4To evaluate the effects of different extract on seedling growth, a completely randomized block design with three replications was applied to conduct seedling growth tests on growth plate . The media were a mixture of vermiculite/pearlite . Three pregerminated seeds (5 days old) were transplanted into each cup, and ten cups were used as a replicate. The growth plates were placed in growth chambers with temperature of 20/16℃ for 12:12 h (light/dark) period. The light intensity was 3,000 lux. The moisture content of the media was maintained at a ratio of 1.5:1 solution or water to media on weight basis by spraying different plant extract every three days. After 10 days of growth, the seedling with roots was carefully washed from media. Ten seedlings of a replicate were selected, and the plumule length and radical length of each seedling were measured. The seedlings were oven‐dried to constant weight at 65℃ and weighed to obtain dry weights.2.5N1, N2, N3,…, nN: the number of germinated seeds in the first, second, and final counts, and 1, 2,…, n was the first, second, and final evaluation days.Germination force (GF), germination percentage (GP), and germination index (GI) were calculated according to the following equation , shoot length (SL), and dry weight (DW), respectively. Positive values indicate stimulating effects, while negative ones indicate inhibitory activity of the aqueous extract.The allelopathic effect response index (RI) and comprehensive allelopathic effect response index (CE) were calculated using the equation suggested by Williamson and Richardson :RI=1‐C/TTwo‐way analysis of variance (ANOVA) model was used to test plant species, extract effects, and the species ×extract interaction effects. One‐way ANOVA was used to compare treatment effects of different extract on the same recipient plant. Statistically significant difference was assumed at the probability level of 0.05, and means were separated by using LSD.33.1Elymus nutans, and their interactions (A × B). RL of the highland crops was significantly influenced by crop species and aqueous extract type but not by their interaction  and seedling growth  of the highland crops were significantly affected by crop species (A), aqueous extract type (B) of on Table .Elymus nutans had no significant effect on the GF of Hordeum vulgar var. nudum, Avena sativa, and Brassica napus, but decreased GF of Triticum aestivum and increased that of Chenopodium quinoa. Significant inhibitory effects of pieces aqueous extract on the GF of Hordeum vulgar var. nudum, Avena sativa, Triticum aestivum, and Brassica napus were detected except for Chenopodium quinoa. Root zone soil extract reduced the GF of Hordeum vulgar var. nudum, Avena sativa, Triticum aestivum, and Brassica napus and increased that of Chenopodium quinoa.GF of the tested crop seeds to different extract was dependent on crop varieties and extract types Figure . CompareAvena sativa, Triticum aestivum, and Brassica napus presented similar pattern, and their GP decreased by three kinds of extract compared with control, but there were no differences among the extract treatments. The pieces aqueous extract of Elymus nutans significantly inhibited the germination of Hordeum vulgar var. nudum and Chenopodium quinoa. Hordeum vulgar var. nudum germination was sensitive to pieces aqueous extract.All the three kinds of extract significantly reduced the GP of tested crops as shown in Figure Brassica napus and Chenopodium quinoa, but they had greater impact on that of Hordeum vulgar var. nudum, Avena sativa, and Triticum aestivum .The allelopathic effect response index and comprehensive allelopathic effect response index of 3.2Elymus nutans, and their interactions , aqueous extract type (B) of Elymus sibiricus was greater than 65% and that of other four grasses was less than 50%, suggesting Elymus sibiricus seed has strong vitality and the emergence is uniform. The pieces aqueous extract treatment resulted in significant decrease in germination force of all tested perennial grasses, with a biggest decrease by 58.8% of Festuca sinensis and smallest decrease by 14.6% of Poa pratensis compared with control. The pieces aqueous extract treatment leads to a significant decrease in germination force of Elymus sibiricus, Festuca sinensis, and Poa pratensis compared with those of whole extract treatment. Root zone soil extract had no significant allelopathic effect on germination force of Poa pratensis and Poa crymophila, but significantly lowered the germination force of Elymus sibiricus, Festuca sinensis, and Elymus nutans itself.The effects of the three types of extract on germination force of perennial grasses are shown in Figure Elymus nutans, Festuca sinensis, Poa pratensis, and Poa crymophila except for Elymus sibiricus. The pieces aqueous extract and root zone soil extract showed significant inhibitory on GP of Festuca sinensis and Poa crymophila compared with control and whole extract.The GP of the control of the five perennial grasses was less than 70%. The GP of perennial grasses was decreased by the three kinds of extract Figure . The whoElymus nutans and Poa crymophila. The pieces aqueous extract reduced Festuca sinensis (47.3%), Poa crymophila (40.2%), and Elymus sibiricus (38.1%) significantly. The root zone soil extract increased the GI of Poa pratensis by 24.3%, but decreased that of other four grasses.The germination indexes of all control were less than 40, indicating lower seed vigor of the tested perennial grasses Figure . The redElymus nutans and Elymus sibiricus was significantly inhibited by the three kinds of extract, and the differences among extract treatments were not significant , and the minimum was Poa pratensis (−0.120). The Elymus nutans extract mainly inhibited the germination of fescue seeds, and the root and bud growth of Poa crymophila, Poa pratensis, Elymus sibiricus and Elymus nutans.The comprehensive allelopathic effect index of y Figure . The max4Agropyron repens, Lolium perenne, Festuca rubra, and Poa pratensis, have also been proved to be allelopathic to other plants  and germination rate (>85%) of the control of five crops were relatively higher, reflecting their good germination uniformity. Of the five crops, Hordeum vulgar var. nudum and Avena sativa are susceptible and Chenopodium quinoa is tolerant. Of the five perennial grasses, Elymus sibiricus is the least affected and Festuca sinensis is the most affected. The responses of seed germination and seedling growth of the same crop or grasses vary with the extract from different sources. The pieces aqueous extract has stronger inhibition than others.Elymus nutans has less allelopathic effects on Poa pratensis and Poa crymophila than on Festuca sinensis. It is recommended that the co‐seeding combination for restoration should consider allopathic effects of different species and reduce the seeding rate of Elymus nutans in mixture. The subsequent crop followed seed production of Elymus nutans monoculture should take planting Chenopodium quinoa or Brassica napus into consideration.None declared.Xiaolong Quan: Writing‐original draft (lead). Youming Qiao: Supervision (lead). Mengci Chen: Data curation (supporting). Zhonghua Duan: Data curation (supporting). Huilan Shi: Data curation (supporting).https://doi.org/10.5061/dryad.tht76hdzv.This article has earned an Open Data and Preregistered, for making publicly available the digitally‐shareable data necessary to reproduce the reported results. The data is available at DOI