To validate these findings in a distant vertebrate species, we used single-cell RNA sequencing of lck:GFP cells in zebrafish and obtained the first transcriptome of specific immune cell types in a nonmammalian species. Unsupervised clustering and single-cell TCR locus reconstruction identified three cell populations, T cells, a novel type of NK-like cells, and a smaller population of myeloid-like cells. Differential expression analysis uncovered new immune-cell–specific genes, including novel immunoglobulin-like receptors, and neofunctionalization of recently duplicated paralogs. Evolutionary analyses confirmed the higher gene turnover of trans-membrane proteins in NK cells compared with T cells in fish species, suggesting that this is a general property of immune cell types across all vertebrates.The immune system of vertebrate species consists of many different cell types that have distinct functional roles and are subject to different evolutionary pressures. Here, we first analyzed conservation of genes specific for all major immune cell types in human and mouse. Our results revealed higher gene turnover and faster evolution of  The immune system of vertebrate species has evolved into a highly complex structure, comprising many different types of both innate and adaptive immune cells. Adaptive immune cells are broadly classified into B and T lymphocytes that can directly recognize antigens with great specificity. Innate immune cells include a variety of myeloid cells such as monocytes, neutrophils, basophils, eosinophils, and mast cells. A third major type of lymphocytes, the Natural Killer (NK) cells, has also been historically classified among innate immune cells . TraditiRag-mediated somatic V(D)J (variable diversity joining) rearrangement, and this process is conserved across all jawed vertebrates J variable regions have been recently annotated  has emerged as a promising technology to unravel the landscape of cell types in heterogeneous cell populations without relying on specific antibodies . The simlck promoter –specific genes. Next, we analyzed immune cells in zebrafish, a powerful model in biomedical research . To thispromoter  and perfImmune related genes tend to evolve more rapidly than other genes, and between functionally distinct immune cells, the selective pressures might vary significantly. Here we performed a conservation analysis of the most differentially expressed genes in resting T, B, NK, and myeloid cells in the mouse and human at the genome-wide level (see Methods) .trans-membrane (TM) or secreted protein coding genes, those specifically expressed in NK cells have proportionally fewer orthologs across all vertebrates compared with other immune cells. The difference is most evident between NK and T cells, although these are closer from a functional and ontogenical point of view . Examples of other mouse or human NK TM genes poorly conserved across vertebrates include Fc receptors, granulysin (GNLY), CD160, CD244, and IFITM3. In addition, among conserved protein coding genes, NK-cell–specific genes consistently had lower sequence identity across all vertebrates for TM genes but not for cytoplasmic ones .Our analysis revealed that among  of view A,C. No d of view B,D. As edN/dS ratio) of one-to-one orthologs between human and mouse can provide a good estimation of the evolutionary pressure acting on a gene. Our results indicate that NKs’ TM genes evolve faster  compared with T cells’ TM genes .The ratio between nonsynonymous and synonymous substitutions  cells. This transgenic line expresses GFP under the control of the lymphocyte-specific protein tyrosine kinase (lck) promoter, and it was proposed to be mainly restricted to zebrafish T cells  zebrafish may therefore provide an ideal model to investigate the large difference in conservation between T- and NK-cell–specific genes observed in mammalian species. To simultaneously obtain information about cell morphology and high-quality gene expression profiles, we used high-throughput single-cell RNA sequencing combined with FACS (fluorescent-activated cell sorting) index sorting analysis of two adult zebrafish (3- and 10-mo-old) spleen-derived lck:GFP cells.As reliable antibodies to isolate pure immune cell populations in fish species are not available, we used single-cell transcriptome analysis of zebrafish  T cells . However+ cells isolated from the spleen of two different fish from a different clutch and different age (see Methods). Following quality controls (see Methods) , 15 cells were removed, and gene expression profiles for the remaining 263 cells were generated. Average single-cell profiles showed good correlation with independent bulk samples (Pearson's correlation coefficient [PCC] = 0.82) . Correlations between single-cell gene expression profiles were used to calculate cell-to-cell dissimilarities (see Methods), and these were represented into low-dimensional space using classical multidimensional scaling . Interestingly, a clear cell subpopulation structure emerged   and determine whether absence of expression of specific markers in many cells was mainly due to technical limitations of single-cell RNA-seq technology . Indeed, the vast majority of the cells displayed clear expression of one of the gene signatures . We named the obtained Clusters 1 (T-cells), 2 (NK-like cells), and 3 (myeloid-like cells).We first generated and sequenced libraries from 278 single GFP emerged A showingke cells , and spiMethods) . Next, wgnatures B, suggesTCR locus (see Methods) . Occurrence of V(D)J recombination was associated with Cluster 1 , which provides additional genomic evidence of the T-cell identity. As expected, V(D)J recombined segments were also strongly associated with expression of the T cell receptor beta constant 1   .To further support the hypothesis that most cells in Cluster 1 are bona fide T cells, we adapted a recent method for detection of V(D)J recombination events of the Methods) . With a  < 10−5) . InteresT cells) A, 3 and nitr and dicp genes, the second cluster expressed NK lysins with high specificity (in particular nk-lysin tandem duplicate 2 and 4) (see recombination activating gene 1–deficient (rag1−/−) zebrafish (In addition to d 4) see , which hebrafish . This fucd4, cd8, nitr, dicp, and spi1b) is clearly visible .The clustering structure of our fish immune cells was further validated in a set of more than 300 single cells from a third fish and additional cells from the first fish, where despite much lower coverage due to external RNA contamination of the samples, the separation between cells expressing the different markers (P = 1.4 × 10−5). This is consistent with NK-like cells possessing dense cytoplasmic granules (P = 1.6 × 10−5). The high granularity of cells in Cluster 3 further supports the hypothesis that these cells originate from a subpopulation of lck+ myeloid cells, such as granulocytes  (Since ic mice)  and humaSupplemental Table S2).To identify additional genes specific for each cell population, we performed differential expression analysis of each cluster versus the other two (see Methods)  whose expression is highly correlated with the alpha chain cd8a within Cluster 1 (cd28 (ENSDARG00000069978), and an uncharacterized Ig-like protein (ENSDARG00000098787) related to CD7 antigen  indicates activation of the death-receptor-ligand pathway . Expression of these genes further shows that zebrafish presumably resting NK-like cells transcriptionally resemble effector CD8 T cells, as observed in mammals , the interleukin 2 receptor beta , tnfsf14 , and chemokines of the families ccr38 and ccr34. In addition, we detected differential expression of putative activating NK receptors’ adaptors (ITAMs), Fc receptor gamma subunit (fcer1g), hematopoietic cell signal transducer (hcst), CD247 antigen like (cd247l), and multiple putative transcription factors . Finally, within the top differentially expressed genes of these NK-like cells, we found putative homologs of mammalian granzyme B that is expanded in ray-finned fish genomes , and many uncharacterized putative immunoglobulin-like receptors and cytokines, such as immunoglobulin V-set domain-containing proteins or interleukin-8-like domain-containing chemokines . Other differentially expressed genes included Fc receptor gamma subunit–like (fcer1gl), hck, a member of the Src family of tyrosine kinases mostly expressed by phagocytes in mammals and potentially implicated in signal transduction of Fc receptors and degranulation (zgc:158446), and id2 (a transcription factor interacting with spi1b)  see .P = 0.008; see Methods). Similarly, the comparison of differentially expressed genes in Cluster 2 with human gene expression data confirmed a significant enrichment in NK-specific genes (P = 0.009), thus supporting the conservation of a core transcriptional program between mammalian and zebrafish NK-like cells (see Methods). Finally, differentially expressed genes in Cluster 3 were weakly enriched in human myeloid-specific genes .We next compared differentially expressed genes in each cell population to human transcriptomic data of homogeneous FACS sorted immune cells . For genlck+ cells, compared with 41% of post-speciation duplicated paralogs. As expected, prespeciation duplicated immune genes were more likely (94%) to functionally diverge  compared with the more recent post-speciation paralogs (62%). Ray-finned fish–specific duplicated genes with conserved expression patterns included, for instance, the NK receptors nitr, which although expanded in zebrafish, have kept their cell-type specificity. In contrast, other fish-specific paralogs show distinct expression, suggesting possible neofunctionalization events  provide an interesting example of such recent functional divergence. In our data, nkl.4 was expressed in both myeloid- and NK-like cells. However, nkl.3 was only expressed in myeloid-like cells, while nkl.2 expression was restricted to NK-like cells (Fc receptor gamma subunit (fcer1g), which in mouse (Fcer1g) and human (FCER1G), is highly expressed in myeloid and NK cells  was expressed in myeloid- and NK-like cells, while its paralog Fc receptor gamma subunit–like (fcer1gl) expression was restricted to the myeloid-like cells . Interestingly, genes more recently duplicated (ray-finned fish specific) show lower expression in our data set. For example, 53% of prespeciation duplicated genes showed expression in ents see . Notablyke cells , 4. A seNK cells . In zebrke cells . Other eP < 10−4)  , suggest < 10−4) , as obsenitr and dicp among NK-like specific genes , Ig-like molecules , NK lysins, and the NK receptors see also , as well mammals , 5.Supplemental Fig. S2). Moreover, as in human and mouse, zebrafish TM genes conserved in vertebrates (for which orthologs were detected) were more divergent in NK/NK-like cells than in T cells (P = 0.03) . In contrast, cytoplasmic and nuclear T-cell–specific genes displayed similar sequence identity compared with other genes .When compared at the sequence identity level, the conserved TM genes specifically expressed in either T or NK/NK-like cells from human, mouse, or zebrafish had lower sequence identity than other genes across vertebrates J recombination in 22 cells . Interestingly, a single TCR recombinant was found in each cell , which is consistent with allelic exclusion. Although V(D)J recombination was clearly correlated with T-cell identity, five cells with evidence of V(D)J recombination fall in Cluster 2 and three of them show clear expression NK genes. It is tempting to speculate that these cells could be NKT cells. However, in mammals, the process of TCR rearrangement first initiates in uncommitted hematopoietic progenitors before NK/DC/B/T divergence. Therefore, incomplete rearrangements are also observed in subpopulations of non-T cells, such as NKs , specific transcription factors, and multiple novel putative NK-specific receptors and chemokines  . This suggests that rapid evolution of NK TM genes is key for their function in all vertebrates. As NK genes cannot undergo somatic rearrangement, we propose that this fast evolution reflects, at least partly, a need for NK cells to possess a diverse repertoire of species-specific germline encoded receptors and associated proteins to perform their functions. In particular, both T and NK cells recognize the fast-evolving and highly polymorphic MHC molecules. While T cells do so by rearranging their TCR sequence, NK cells possess an expanded family of receptors. The fast evolution of these receptors may be the result of a need to adapt to MHC rapid evolutionary changes. Our observations also suggest a model of high gene turnover and faster evolution of immune TM/secreted genes but, at the same time, conservation of core cytoplasmic immune genes from zebrafish to mammalian species  zebrafish single cells, see Overall, our work expands the analysis of immune cell types and their evolution to lower vertebrates. To our knowledge, this is the first study to characterize T and NK cells at the whole-transcriptome level in a nonmammalian species and one of the first studies to analyze NK cells’ gene expression at the single-cell level   were ordered based on expression fold-change, and the top 100 genes unique for each cell type were selected as “signature genes” for downstream analysis . Results were robust to different cut-offs for the top N differentially expressed genes . Human and mouse dN/dS ratios  of one-to-one orthologs between these two species were obtained from Ensembl version 82. The two protein groups enriched in (1) TM and secreted proteins and (2) cytoplasmic and nuclear proteins were defined based on the presence of predicted TM domains and/or signal peptide. Statistical significances of differences in sequence identity and dN/dS differences were assessed using Wilcoxon rank-sum tests. Statistical significances of differences in proportion of orthologs were assessed as follows: (1) in a specific species  by comparison against a null-model distribution generated from 10,000 random permutations of gene/cell-type specificity class pairs, and (2) globally across all species  using paired Wilcoxon rank-sum test .Orthologs of mouse and human protein-coding genes and their sequence identities, as well as TM domains and signal-peptide predictions were downloaded from BioMart/Ensembl Genes 82 . For gen3.28.14) . SignifiTg(lck:EGFP) lines were maintained as previously described (Wild type (Tubingen long fin) and transgenic zebrafish escribed , in accoTg(lck:EGFP) adult fish from a different clutch and at different ages (3 and 10 mo) and one adult wild-type fish were dissected and carefully passed through a 40-μm cell strainer using the plunger of a 1-mL syringe, and cells were collected in cold 1× PBS/5% FBS. The nontransgenic line was used to set up the gating and exclude autofluorescent cells. Propidium iodide (PI) staining was used to exclude dead cells. Individual cells were sorted, using a Becton Dickinson influx sorter with 488- and 561-nm lasers . The Smart-seq2 protocol .The spleens from two heterozygote m lasers  and collprotocol  was usedhttp://sourceforge.net/projects/bbmap) with parameters minlen=25 qtrim=rl trimq=10 ktrim=r k=25 mink=11 hdist=1 tbo.Following Illumina HiSeq2000 sequencing (125-bp paired-end reads), single-cell RNA-seq reads were quality trimmed and cleaned from Nextera adaptor contaminant sequences using BBduck . Next, gene expression levels were quantified as Ei,j = log2, where TPMi,j refers to TPM for gene i in sample j, as calculated by RSEM 1.2.19 . On average, 1240 expressed genes per cell were detected . Cells having fewer than 500 detected genes or fewer than 10,000 reads mapped to transcripts were excluded from further analyses.An average of 2.1 million paired-end reads were obtained per single cell  for dimensionality reduction . MDS attempts to preserve distances between points generated from any dissimilarity measure. PCCs between full transcriptional profiles were used to define cell-to-cell similarities, and 1 − PCC was then used as MDS's input dissimilarity measure.In order to visualize cell heterogeneity at the transcriptomic level, we used classical MDS .To correct for batch effects and remove unwanted variation between the first and second fish, we used the ComBat function from R Bioconductor's  package . After thttps://github.com/epierson9/ZIFA in July 2016) that explicitly models gene drop-out events . As input to ZIFA we used ComBat-adjusted log2 (TPMs + 1) having a minimum variance across cells of one (6350 genes passed this filter). As the batch effect adjustment can produce negative expression values and ZIFA requires all values to be positive, we set all negative values to zero. ZIFA was run in the fast “blocks” mode with k = 5.Similar low-dimensionality projection was obtained using zero inflated factor analysis (ZIFA) , NK-like cells , and myeloid cells (spi1b/pu.1). We calculated a score for each set of marker genes [mean log2 (TPMs + 1)] and assigned identities to cells having a score higher than one in only one of the three sets, yielding 91 T cells, 44 NK-like cells, and five myeloid cells . These extended signatures were used to color all cells (hclust using Ward.D2 method) applied on the first four principal coordinates generated by the MDS. The choice of the components was based on the eigenvalue decomposition of the MDS . Eigenvalues decrease very smoothly after the fourth component, i.e., contributing less significantly to the overall variability. The number of clusters  was determined by maximizing the mean silhouette coefficient .To identify different cell populations, we first defined, based on literature, minimal sets of marker genes for candidate T cells  and Rag-dependent V(D)J recombination are found in zebrafish J recombination.All four ebrafish . Howevernnotated . To expl aligner , with lo aligner  using it aligner  using thSCDE R package v1.99 .Estimated gene counts obtained from RSEM were used as input for ge v1.99  that exphttps://CRAN.R-project.org/package=pheatmap), using “correlation” distance with “Ward.D2” criteria to cluster rows, and whole-transcriptome distance ; and (2) 19,499 genes that underwent “early” duplication, where their most recent common ancestor was mapped to bony vertebrates (Euteleostomi) or any of its parent taxa . Many of these genes suffered multiple duplication events both before and after the fish common ancestor. Therefore, to compare differences in expression between these two groups, we did not include the set of overlapped genes and obtained 3235 unique recently duplicated genes and 8609 unique early duplicated genes. From these, 1315 (41%) and 4569 (53%) were detected in our data (genes with >0 TPM in at least 1% of the cells).A list of paralogs in zebrafish was obtained from Ensembl Compara GeneTrees (version 82) . We defiFor the analysis of expression pattern divergence, we searched pairs of paralogs where both genes show some specific expression pattern  according to one of the following criteria: (1) within the top 100 differentially expressed genes in Cluster 1, Cluster 2, or Cluster 3; (2) within the top 100 differentially expressed genes in Cluster 2 and Cluster 3 versus Cluster 1 , Cluster 1 and Cluster 3 versus Cluster 2 , or Cluster 1 and Cluster 2 versus Cluster 3 ; or (3) expressed in all the three clusters (in at least 10% of the cells of each cluster). In the latter case, we only considered pairs of paralogs where only one gene is expressed in the three clusters, and the second is either specifically expressed or depleted from the major Clusters 1 or 2. Pairs of paralogs where both genes are expressed in all three clusters were not considered since most of them are not immune-related genes, while Cluster 3 is too small to accurately assess enrichment/depletion.Supplemental Table S3.Next, we identified cases where both paralogs belong to the same expression pattern group (duplicate genes with conserved expression pattern) and cases where they differ . For recently duplicated genes, we found 23 pairs with distinct expression patterns and 14 pairs with the same expression patterns , while for early duplicated genes, we found 121 pairs with distinct patterns and eight pairs with the same expression patterns , as shown in Z-score >1 and sorting by fold-change , although results were robust to different cut-offs . To assess orthologs’ conservation among nondifferentially expressed genes, we first excluded lowly expressed genes from the analysis . The reason for this is that we observed a bias of higher gene conservation among highly expressed genes compared with lowly expressed genes. After this filter, conservation of differentially expressed genes could be compared with that of nondifferentially expressed  genes as in Supplemental Table S4 shows, for all analyzed genes, the gene sequence identity  shared with orthologs in the vertebrate species analyzed.Orthologous genes of zebrafish in vertebrate species and their sequence identities were downloaded from BioMart/Ensembl Genes 82. For comparisons between differentially expressed genes between Cluster 1 (T cells), Cluster 2 (NK-like cells), and Cluster 3 (myeloid-like cells), we chose the top 100 differentially expressed genes after filtering by https://www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-4617.Raw and processed sequence data sets from this study have been submitted to ArrayExpress (


In the last two decades, numerous powered ankle-foot orthoses have been developed. Despite similar designs and control strategies being shared by some of these devices, their performance in terms of achieving a comparable goal varies. It has been shown that the effect of powered ankle-foot orthoses on healthy users is altered by some factors of the testing protocol. This paper provides an overview of the effect of powered walking on healthy and weakened users. It identifies a set of key factors influencing the performance of powered ankle-foot orthoses, and it presents the effects of these factors on healthy subjects, highlighting the similarities and differences of the results obtained in different works. Furthermore, the outcomes of studies performed on elderly and impaired subjects walking with powered ankle-foot orthoses are compared, to outline the effects of powered walking on these users. This article shows that several factors mutually influence the performance of powered ankle-foot orthoses on their users and, for this reason, the determination of their effects on the user is not straightforward. One of the key factors is the adaptation of users to provided assistance. This factor is very important for the assessment of the effects of powered ankle-foot orthoses on users, however, it is not always reported by studies. Moreover, future works should report, together with the results, the list of influencing factors used in the protocol, to facilitate the comparison of the obtained results. This article also underlines the need for a standardized method to benchmark the actuators of powered ankle-foot orthoses, which would ease the comparison of results between the performed studies. In this paper, the lack of studies on elderly and impaired subjects is highlighted. The insufficiency of these studies makes it difficult to assess the effects of powered ankle-foot orthoses on these users.To summarize, this article provides a detailed overview of the work performed on powered ankle-foot orthoses, presenting and analyzing the results obtained, but also emphasizing topics on which more research is still required. Walking is the most common form of locomotion to move from one place to another. Despite its apparent simplicity, it is a complex movement that requires a precise coordination of multiple body segments and muscles. Although the human gait pattern appears to be energetically optimized , walkingThe capability of the ankle joint to deliver these functionalities can be reduced as a consequence of aging, pathologies, and injuries. It has been shown that the elderly walk slower, take shorter steps, and exhibit a smaller range of motion in the joints of the lower limbs , 7. FurtSubjects with weakened capabilities of the ankle joint due to injuries or diseases, such as strokes, hemiplegia, and incomplete spinal cord injuries, show an altered gait pattern when compared to that of healthy subjects –10. The The crucial role of the ankle joint in human walking, in the last two decades, led to the development of numerous powered ankle-foot orthoses (PAFOs). Their aim is to improve the gait pattern of impaired users or decrease the biological effort of healthy subjects during walking. Some of the developed PAFOs share the same combination of type of actuators and type of controllers , but theThe aim of this paper is to collect the results of studies assessing the assistance provided by PAFOs while walking on healthy and impaired users and compare their outcomes to give an overview of the effects of walking with a PAFO in both groups of subjects. For this purpose, in this paper only articles which analyzed the effects of PAFOs on users during walking experiments were included. On the other hand, articles that reported exclusively on the design of PAFOs, the results of characterization tests which did not involve users, or in which the protocol of the experiments did not involve walking trials, were excluded. Furthermore, articles involving walking experiments in which the discussed results were only about the performance of the actuator of the PAFO and not its effect on the user, were also omitted. Some studies that were performed with a soft exosuit providing ankle assistance on healthy and impaired users were included, due to the relevance of their findings with respect to the aspects discussed by this article. However, it is important to highlight that these devices also provide passive hip assistance along with active ankle assistance –27. The Basic Science PAFOs: PAFOs that have been developed to study human physiology and biomechanics by analyzing the user’s response to external ankle actuation , in which the action of the PAFO is proportional to the activity of a predefined user’s muscle 21] is siGalle et al.  also assFew studies have assessed the effects of the different onset timing on users walking uphill Table . LookingAnother parameter that alters the effect of the PAFO on users is the assistance magnitude. The influence of this parameter has been analyzed in few studies in unloaded , 21 and Galle et al.  assessedThe results obtained in , 21 showProviding positive assistance magnitude to the ankle joint of a user can help in reducing the metabolic cost of loaded walking also, as shown in Table The PMc has the advantage of being better synchronized with the user, resulting in a more physiological controller because the user has direct control over the timing and amplitude of the actuation;The P-Bc has lower complexity and it does not need sensors on the user’s limbs, since they can, in general, all be placed on the device.As previously introduced, the proportional myoelectric controller (PMc) and the phase-based controller (P-Bc) are the main types of controllers used in PAFOs. The benefits and drawbacks of the two controllers have already been discussed in several studies –43 and tThe determination of the specific effects of the two controllers on the user is a very interesting point since it could help define whether one of the two controllers is more suitable for a specific goal. A discussion about this topic is provided in the sequel.A highly debated point is one that refers to the ease of adaptation of the subjects to the two controllers. On one hand, the PMc is considered to be more natural for subjects, making it easier for them to learn how to walk with the device . On the Cain et al.  found noA more systematic comparison of an adaptive gain PMc (Ag-PMc) and a P-Bc was performed by Koller et al. , 42. TheKoller et al. , 42 asseThe bigger reduction of the user’s muscle activation found by Koller et al. , 42 withIn the studies performed by Mooney et al. –49, the p+ and pdis are the average positive and negative mechanical power provided by the exoskeleton, per leg in a stride, i.e. the positive and negative assistance magnitude, η is human muscle-tendon efficiency, mi and βi are the added mass and the location factor related to the ith human segment, respectively. The muscle-tendon efficiency η is found to be equal to 0.41 in [βi are equal to 14.8 W/kg, 5.6 W/kg, 5.6 W/kg and 3.3 W/kg for the foot, shank, thigh and waist, respectively [MetAdvnorm of powered walking was determined in [where  0.41 in , 30, 37;ectively . The relmined in  and giveMetAdvnorm represent a decrease in the metabolic cost of powered walking as compared to normal walking.in which the positive values of MetAdvunpow), as described in Eq. Galle et al.  determinTon) and the positive assistance magnitude summed for the two legs (P+). Furthermore, the weight of the device is not considered, since it affects both the unpowered and powered conditions.In contrast to the formulation by Mooney et al. Eq.  and 2, EThe possible advantages of using a PAFO as a tool to assist or rehabilitate subjects with ankle deficiencies are well illustrated in literature , 70, 72.Contrary to augmentation PAFOs, the main goal of assistive and rehabilitation PAFOs is to improve the altered gait pattern of users with weakened ankle capabilities. Although there is no agreement on what the metrics are for assessing the improvement, Ward et al.  designatThe main goal of assistive and rehabilitation PAFOs is to improve the altered gait pattern of their users, by correcting the ankle RoM and preventing the occurrence of drop foot in subjects with weakened ankle dorsiflexors. Another goal of these PAFOs is to improve the subjects’ walking speed, which is usually reduced by the impairment . Some suIn the following section, the effects of assistive and rehabilitation PAFOs on these parameters are discussed.The results reported in Table Different effects of the PAFO on the gait symmetry of the user have been found by different studies , 53. WheAnother interesting result was reported by Awad et al. . In theiTable Interesting results are reported by Kim et al. , 59, whoOnly a few studies that have been performed with assistive PAFOs assessed the effect of a powered push-off during level walking on muscle effort and the metabolic cost of walking of elderly , 56 and Sawicki et al.  and TakaThe effect on the metabolic cost of a powered push-off in assistive PAFOs was assessed by Galle et al.  and NorrTakahashi et al.  found noIn opposition to this, Awad et al.  and Bae Some common trends of the effects of PAFOs on healthy and weakened users have been identified in the previous sections. In addition to them, some divergences can be noticed in the results presented by different studies. A discussion about these findings is addressed in this section.As previously introduced, the assessment of the adaptation of the user to powered assistance is important to compare the effects of the PAFO in different studies. This is due to the changes in the kinematics, muscle activation, and metabolic cost of walking between the adaptation and the steady-state period , 40, 43.Some differences have been noted in the time to achieve a steady state in works with similar protocols Table . This suThe time needed to achieve a steady state during powered walking has not been assessed in elderly and impaired users. Assessing whether the subjects achieved a steady state is important in assistive and rehabilitation PAFOs to evaluate their effects. For example, assessing the achievement of a steady state at the end of each training session with rehabilitation PAFOs could help distinguish whether the changes in the gait pattern of the subject between sessions are an effect of the robotic training or of the adaptation of the user to the PAFO.As already discussed in the previous sections, it is not easy to define a trend for the influence of the onset timing and the assistance magnitude on the reduction of the soleus activation and the metabolic cost of walking. One of the reasons for this is the fact that the comparison between studies performed on different actuation setups, is not always straightforward.As presented above, two formulae that relate the metabolic advantage of the PAFO to the assistance parameters have been proposed by Mooney et al. –49 Eqs. and 2 anThe results reported in the previous section highlight the advantages of using a PAFO to improve the ankle kinematics and walking speed of its users and to rehabilitate impaired subjects. However, while numerous studies have analyzed the effects of the assistance parameters on the biological effort of healthy young users , 46, 56,Some of the presented studies on PAFOs assess the activity of lower limb muscles that are not directly related to ankle movements, such as the vastus lateralis, vastus medialis, biceps femoris, rectus femoris, medial hamstring, lateral hamstring, and gluteus maximus , 43, 65.Galle et al.  did not Some of the works that reported a reduced activity of more proximal muscles also measured the total  ankle work during powered and unpowered walking. A common finding of these works is that the subjects’ total ankle work during powered walking was higher than it was during unpowered walking. This effect would suggest that in these studies, the enhancement of human capabilities was achieved by the PAFO augmenting the action of the human ankle, instead of replacing part of it. Other works found an increase in the total ankle work during powered walking with a PAFO , 39, 41,From these results, it seems that there are two possible effects of augmentation PAFOs providing powered push-off to the user: the PAFO could replace part of the biological ankle work, or it could augment it and assist not only the ankle joint, but also the hip joint. However, due to the small sample of works that analyzed the effects of the PAFOs on more proximal joints, it is difficult to explain which are the elements that make the PAFO augment rather than replace the biological ankle work.A very important aspect that should be studied in PAFOs is the interaction of the device with the user’s body. This interaction can be divided into two levels: a physical and a neural one. The physical interaction plays a very important role in accomplishing the successful transmission of torques and forces from the device to the user . It is kAn example of the importance of assessing the physical human–robot interaction when analyzing the effects of the PAFO on the user is given by Van Dijk et al. . In theiAnother important aspect that should be considered in the development of PAFOs is how the nervous system will respond to the provided assistance, i.e. the neural interaction. Kao et al.  showed tA similar consideration was made by Kinnaird et al. . In theiPAFOs have been shown to have great potential to enhance the capabilities of healthy users and assist or rehabilitate the ankle joints of weakened ones. However, more research is necessary to improve the understanding of the impact of these devices on the user.From the results presented in this paper, it seems that the adaptation time of healthy users is influenced by the assistance parameters. The determination of a relationship between these variables is complicated by the lack of information regarding the onset timing and assistance magnitude in most of the studies. More research should be performed to determine the influence of the assistance parameters on the adaptation time, both on healthy and weakened users.Future studies should be conducted with more combinations of onset timing and assistance magnitude to assess their interplay in the determination of the metabolic advantage of the PAFO.More studies are needed on elderly and impaired subjects. The small number of studies performed on these subjects makes it difficult to accurately compare the results obtained by different studies. These studies should focus on the influence of the assistance parameters on the effect of the PAFO on the user. Furthermore, the time needed by these subjects to reach a steady state during powered walking should be assessed.An interesting topic to be investigated is also the influence of the type of controller on the response of weakened subjects to the assistance provided by the PAFO. The determination of distinctive effects of the different controllers would define whether a specific controller is more suitable for a certain group of subjects or for a precise objective.Another open question to be addressed is how long the subjects can retain the steady-state walking pattern that has been learned during powered assistance. This would be particularly interesting for rehabilitation PAFOs since it could determine the frequency of robotic rehabilitation sessions.Additionally, the effect of powered walking on more proximal joints should be studied to explain the parameters determining whether a PAFO will augment or replace the biological ankle work.Furthermore, another aspect that should be better studied is the physical interaction of the device with the user, which is of great importance in the understanding of the effect of a PAFO on the user.The performance of PAFOs in terms of healthy and weakened users varies between studies with similar protocols and goals. The effect of powered walking on users is influenced by a set of key factors, which have been identified in this article. It has been shown that these factors mutually impact the performance of PAFOs on users, thus, the influence of each one of them cannot be considered independently from the others. In this paper, it has been highlighted that the comparison of the results of different studies is not always straightforward. This is due to the fact that the behavior of a PAFO is greatly influenced by the dynamics of its specific actuation setup and the comparison of the results obtained from different actuation setups is difficult to make. This comparison would be facilitated with the development of a standard methodology to benchmark actuators, which is, however, still an open issue , 77. FroThe results presented in this paper lead to the conclusion that more experiments need to be performed on elderly and impaired subjects. In the future, studies should specify the parameters used in the protocol  and report, together with the results, whether the subjects had reached a steady state in the experiment. This is particularly relevant for studies performed on elderly and impaired users. Assessing the influence of these parameters on these users would simplify the analysis of the effects of powered walking.


M/T method and T method are adopted to work out the rotational speed. Experiments were conducted on a laboratory-scale test rig to compare the proposed method with the auto-correlation method. The largest relative errors of the auto-correlation method with the sampling rate of 2 ksps, 5 ksps are 3.2% and 1.3%, respectively. The relative errors using digital approaches are both within ±4‰. The linearity of the digital approach combined with the M/T method or T method is also superior to that of the auto-correlation method. The performance of the standard deviations and response speed was also compared and analyzed to show the priority of the digital approach. In industrial production processes, rotational speed is a key parameter for equipment condition monitoring and fault diagnosis. To achieve rotational speed measurement of rotational equipment under a condition of high temperature and heavy dust, this article proposes a digital approach using an electrostatic sensor. The proposed method utilizes a strip of a predetermined material stuck on the rotational shaft which will accumulate a charge because of the relative motion with the air. Then an electrostatic sensor mounted near the strip is employed to obtain the fluctuating signal related to the rotation of the charged strip. Via a signal conversion circuit, a square wave, the frequency of which equals that of the rotation shaft can be obtained. Having the square wave, the  In industrial applications, rotational speed measurement is a crucial part for condition monitoring, speed control, and protective supervision of rotation equipment, such as generators, steam turbines, and gas turbines. Various kinds of tachometers based on different mechanisms, such as optical, electrical, and magnetic induction, have been developed and widely used to measure the rotational speed of target objects. W.H. Yeh presented a high-resolution optical shaft encoder to monitor the rotation behavior of a motor  and J. NIn order to overcome the hash condition, such as a high temperature, heavy dust environment, the electrostatic method has been used to realize the rotational speed measurement. The electrostatic sensor is adaptable for the speed measurement in various industrial conditions for the advantages of contactless measurement, low cost, simple structure, and easy installation and maintenance. Recently, Y. Yan and L.J. Wang utilized electrostatic sensors and a correlation algorithm to calculate the period or elapsed time and successfully obtained the rotational speed of a rotational shaft ,9. The eThe principle of rotational speed measurement using an electrostatic sensor is shown in By now, electrostatic sensors in conjunction with correlation methods, including the cross-correlation method using dual electrostatic sensors and the auto-correlation method using a single electrostatic sensor, have been used to determine rotational speed ,9,13. Fix(t) and y(t) is:In the time domain, the definition of the cross-correlation function between real power signals cv  can be calculated by the sensor angle spacing s):x(t) and y(t) are the same signals obtained by one electrode, Equation (1) turns out to be the auto-correlation function. With respect to the auto-correlation method, only one channel of the electrostatic signal is needed. The time-delay τ between signal x(t) and signal y(t) is the rotational period T(s) and av (rpm) can be obtained as follows:If Obviously, the correlation method needs to locate the coordinate of the first dominant peak in the waveform of the correlation function, which is influenced by the sampling rate to a great extent. At the same time, the waveforms collected by inducing the signal from a cylinder dielectric sleeve contain complex information and a faint sign of the periodical component. Although the correlation calculation of the waveform has good performance and successfully extracts the elapsed time, the computational accuracy of the correlation method is obviously affected by the sampling rate and signal noise .For the sake of improving the performance of rotational speed measurement via the electrostatic method, this paper proposes an approach to generate a square wave from an electrostatic sensor in order to obtain the rotational speed via digital methods, thus eliminating the influence of the sampling rate and signal noise, and also simplifying the system complexity. In the following article, “square wave” refers to the output waveform from the comparison circuit which generates a pulse every rotational period. Implementation of a rotational speed measurement system based on this method is presented. Compared to the rotational speed measurement method using an electrostatic sensor in conjunction with correlation, this designation leaves out the AD converter and simplifies the computation code which is more adaptive for the implementation in a microprocessor system.Inspired by the photoelectric method which fixes a strip of a reflection element, this experiment uses a strip of polytetrafluoroethylene (PTFE) stuck to the rotational shaft. The measurement principle is shown in T method, which calculates the reciprocal of the duration between consecutive pulses to obtain the frequency; (2) pulse counting, commonly termed as the M method, which counts the number of pulses generated within a prescribed period of time; and (3) constant elapsed time, commonly termed as the M/T method, is a combination of pulse counting and measuring elapsed time  are zeros and the waveform of function rf(θ) is the same as in circleq(θ) and the function rf(θ), thus rf(θ) can be regarded as a filter function. The low pass filter property can also be obtained from [rf(θ) is influenced by the rotational speed (rf(θ0 + wt)). The cutoff frequency of rf(θ) increases with the speed . Through the analysis, the electrostatic electrode in this case of application can be regarded as a low pass filter which adaptively adjusts its cut-off frequency. Thus, the waveform of the signal mainly contains a low frequency component if the electro-magnetic interference is well shielded, which helps to explain the signal obtained in the experimental part.As shown in f range −0, 180 ar8 Ω resistors connected in series, which determines the transimpedance gain. In actual application, a feedback capacitor is needed to guarantee the stability of the circuit by inhibiting the high frequency noise.The sensor board shown in The relationship between the output voltage and the input current from the electrode can be calculated according to Equation (17). Thus, the sensitivity of the circuit is 0.2 V/nA. The purpose of the balance resistor and capacitor is to make the impedance of the two inputs equal, thus, the bias current of the amplifier generates no additional offset voltage on the output.When the electric field near the electrode varies with the rotation of the charged strip, a small current signal will be generated and transformed into a voltage signal via the feedback resistance on the amplifier. The voltage output of the sensor board is collected by the condition unit via a shielded cable to avoid electromagnetic interference in the space.The condition unit in our experiment is shown in The amplifying circuit uses the same amplifier chip with the sensor board to meet the performance requirements. The voltage gain of the amplifier can be adjusted by a slide rheostat. Then a third-order Butterworth low pass filter with Sallen-Key topology is used for filtering and inhibiting the noise. The passband frequency is 400 Hz and the stopband frequency is 2.4 kHz. A smooth waveform improves the stability of the square wave. Finally, a hysteresis comparator is utilized to transform the waveform into a square wave which is connected to the DSP board for speed calculation.A laboratory-scale test rig is designed and built for rotational speed measurement. T method and M/T method transformed from the DSP for each point were saved for analysis. A photoelectric reflection digital tachometer with an accuracy of ±0.05% of the reading plus 1 rpm was used to provide a reference speed in our experiment. The ambient temperature was controlled between 20 °C and 24 °C and the relative humidity was kept between 55% and 65%. The square wave was connected to the external interrupt pin to contend with the square wave immediately. The code of realizing the T method and the M/T method were programed and written into the DSP board separately to test the measurement performance. The DSP transmitted the measurement results of the T method and the M/T method to computer via RS232 serial communication. In the experiment, the prescribed time of the M/T method is set to be 1 s.Experiments were conducted on the rig using the same dimension parameters as the simulation. The rotational speed of shaft was adjusted from 300 rpm to 3200 rpm with an increment of 100 rpm via the VFD. To make a comparison between the digital approach and the correlation method, each point was measured five times. Meanwhile, five values of the Seen from the principle of the correlation method, it can be found that the auto-correlation method can be regarded as a particular case of the cross-correlation method, which leaves out the influence of the installation angle error, the distance differences of the two electrodes to the shaft, and the differences between two channels’ circuits. These factors make the accuracy of cross-correlation method not as good as the auto-correlation method. Meanwhile, the cross-correlation method needs two channels of circuits, which is not consistent with the setting in this experiment. Thus, the experiment only makes a comparison between digital approaches and the auto-correlation method.The proposed approach utilizes the electrostatic sensor to induce the charge on the strip of PTFE, which obtains a strong periodic signal. T method and the M/T method are plotted in T method and M/T method are about 0.81‰ and 1.31‰, correspondingly. The measurement results are highly consistent with those of the photoelectric tachometer. Meanwhile, the differences between the T method and the M/T method are hardly discernible by eye.The mean values of the measurement results for the As seen from the principle, the proposed digital method needs no sampling via an analog-digital converter, while the sampling rate is an important factor that determines the accuracy of the method based on the correlation algorithm. In order to make a comparison between these two methods, the analog signals are also collected at different sampling rates. The auto-correlation functions are calculated using the filtered analog signal. T of the rotation can be obtained. It can be observed that the waveform near the first peak after 0 s is very smooth, which benefits confirming the accurate and stable value of the period. However, due to the discretization of the data series, the obtained period T will be a time length away from the actual time of the rotation period with a significant probability despite the auto-correlation method confirming the nearest time point to the ideal time point. Moreover, when the signal contains an obvious level of noise or a weak periodicity, the waveform near the peak of the auto-correlation function will be fluctuant, which impairs the result’s accuracy.The waveform in M/T method and the T method.In order to show the accuracy of this digital method, signals are collected at the sampling rate of 2 ksps and 5 ksps and analyzed using the auto-correlation method. Relative errors of the two methods are plotted and compared in M/T and T methods both have significantly small standard deviations. The standard deviations of the auto-correlation method in In order to research the robustness of the proposed method, the standard deviations of each measurement point are listed in M/T and T methods in M/T method are much smaller than those of the T method. As seen from the principle, the M/T method can be regarded as a mean value of several consecutive T methods. Due to the high response speed of the T method, it is more sensitive to the variation of rotation, which makes its standard deviations greater than those of the M/T method. The minor standard deviations of the M/T and T methods mainly arise from the slight fluctuations of the actual rotation state, which is probably related to the unsteady output rotational speed of the motor and the slippage of the belt on the sheave. With respect to the digital approaches, no matter the M/T method or T method, both have very little spread in the measured speed.The standard deviations of the M/T method, the response time is decided by adding extra time to the prescribed time. Regarding the analog method, usually the sampling length should be predetermined. Thus, the time needed to acquire one measurement result is nearly confirmed. Even if the auto-correlation method self-adaptively adjusts the sampling length according to the nearest obtained rotational speed, the response speed is still not as fast as the T method for the reason that the auto-correlation method needs at least two periods of rotation to achieve the correlation calculation. Moreover, data collection and processing also consume a certain amount of time.The response time of each approach can be determined from their principles and data process procedures. Regarding the T method can output a result every rotational period for the reason that the counter in the DSP can work independently from the code and the DSP only needs to perform an easy computation of the counter number and serial communication. Experiments were conducted to test the capability of the T method to measure the variable speed. The motor was adjusted by the VFD output frequency to work at three stages: acceleration by increasing the frequency from 0 Hz to 20 Hz over 5 s, 4 s of constant speed, and deceleration by decreasing the frequency from 20 Hz to 0 Hz over 3 s. T method, which successfully monitors the acceleration and deceleration processes. The rising and decline curves are not perfectly straight lines because the acceleration of the shaft is not absolutely constant. It can be observed that at a constant frequency, the measurement results are of good stability.By contrast, the M/T method and T method were adopted to calculate the speed in a DSP system. Experiments were conducted to compare the digital approaches with the auto-correlation method. Through experimental analysis, several conclusions can be summarized as follows:The work in this paper dedicates to find a more effective approach to cooperate with electrostatic sensors to improve the performance of rotational speed measurement. The proposed approach utilized the electrostatic sensor to induce a charge on a strip PTFE, which obtained a strong periodical signal. Simulation results also described the expected waveform when a strip of charges rotates near an electrode. By adopting a suitable signal condition unit, a square wave, the frequency of which was equal to that of the rotational speed, has been obtained. Having the square wave proportional to rotational speed, the M/T method and T method both have an obviously higher accuracy. The linearity of the M/T method and T method are about 0.81‰ and 1.31‰, correspondingly, which are much better than those of the auto-correlation method sampled at 2 ksps (3.17%) or 5 ksps (1.33%). Due to the signal discretization, the auto-correlation method can only obtain some discrete values. Improving the sampling rate, calculation quantity, and storage space, the hardware cost will also increase correspondingly.1. Accuracy: Compared with the auto-correlation method, the M/T method and T method obtained particularly small standard deviations among all the measurement points, both within 1 rpm. The M/T method acquired more stable results than the T method due to differences of their respective principles.2. Robustness: The auto-correlation method has a stable performance in some measurement points and also has some obvious standard deviations, which resulted from the signal discretization. However, the T method has the fastest response speed. The correlation method and M/T method have relatively slower response speeds. Experiments also shows that the T method is capable of detecting the variable speed.3. Response speed: The proposed approach combined with the M/T method can be adopted for constant speed measurement or a mean value of rotational speed during a certain time and the T method can be employed for dynamic measurement of variable rotational speed. In actual programming, the M/T method and T method can be written into one piece of a DSP or FPGA simultaneously. An FPGA is more recommended to deal with the square wave for its property of parallel processing and high code execution efficiency.Indeed, having the square wave related to the rotational period, the There are several factors limit the application of this method working at a low speed. The amount of charge on the strip is unstable and the response time is poor at low speed. Further studies can be conducted to deal with these issues by adopting an electret material, adding adaptive numbers of strips and electrodes, and improving circuit properties.


Medication errors are commonly reported in the pediatric population. While evidence supports the use of e-prescribing to prevent certain errors, prescribing with an electronic health record (EHR) system is not devoid of errors. Furthermore, the majority of EHRs are not equipped with functionalities addressing pediatric needs. This study analyzes three unique EHRs in three pediatric clinics. It describes the functionality of each system and identifies errors found in e-prescribed prescriptions. Finally, the study estimates the proportion of e-prescribing errors that could have been avoided if those EHRs had met requirements set by the American Academy of Pediatrics (AAP). The number of prescriptions reviewed for Clinics 1, 2, and 3, respectively, were: 477, 408, and 633 with total error rates of 13.2%, 8.8%, and 6.6%. The clinic EHRs included 21%, 26%, and 47% of the AAP pediatric requirements for safe and effective e-prescribing for children. If all AAP elements had been included in the EHRs, over 83% of errors in the examined e-prescriptions could have been prevented. This study demonstrates that EHR systems used by many pediatric clinic practices do not meet the standard set forth by the AAP. To ensure our most vulnerable population is better protected, it is imperative that medical technology tools adequately consider pediatric needs during development and that this is reflected in selected EHR systems. The Joint Commission  reports that errors linked to medications are believed to be the most common medical related errors . FurtherSince 2008, there has been significant growth in the adoption of EHR and e-prescribing as tools to increase the quality and safety of health care . The US While e-prescribing may reduce pediatric medication errors, pediatric practices have historically been slow adopters of e-prescribing with few of those adopters actually using a pediatric-supportive system . More evIn support of the American Academy of Pediatrics (AAP) recommendation that providers working with children adopt e-prescribing systems with pediatric functionality , this st® 2016 database  for analysis. All results were de-identified with no link to protected health information (PHI) and the study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the University of Oklahoma Health Sciences Center Institutional Review Board (#3463).A cross-sectional, descriptive study design was carried out with three clinics representing three unique EHR systems and practice settings. Including three unique systems provides both depth and breadth as we define systems’ characteristics and capabilities. To be included, clinics must provide care for children, utilize an EHR, and transmit electronic prescriptions to community pharmacies. To estimate a 10% error rate (as observed in prior research ) with 5%Three clinics within the same community were included in the study. Each clinic represented an office within a bigger system with a unique EHR. Clinic 1 is a pediatric clinic within a Federally Qualified Health Center (FQHC) which was the second largest FQHC in the state. Clinic 1 is not a teaching facility and was staffed by three pediatricians. All prescriptions written for a 2-month period were included in the review. Clinics 2 and 3 are both part of university-based health systems representing different state universities, but similar in that they are teaching facilities staffed by trainees/medical residents, nurse practitioners or physician assistants, and faculty physicians. Clinic 2 is a family medicine clinic (only prescriptions for pediatric patients were included in the study) and Clinic 3 represents a pediatric clinic. To obtain the needed pediatric prescriptions to review for Clinic 2, two months of review was required. Whereas, Clinic 3 only required the review of one month of prescription data. EHRs analyzed include Success EHS (Clinic 1), Centricity™ EMR (Clinic 2), and Epic (Clinic 3).To accomplish study aim 1, each of the three clinic EHR systems was assessed for its compliance with the AAP pediatric requirements for safe and effective e-prescribing in the categories of patient information, medication information, cognitive support, pharmacy information and data transmission . The ninFor study aim 2, over 400 new prescriptions from each practice setting were used to identify and classify e-prescribing errors by reviewing the name of medication, patient age, patient weight, medication directions/dose, quantity, and indication. The method for prescription review has been previously described . PrescriFinally, the resulting medication errors were reviewed to determine if they could have been prevented if the EHR system had met AAP recommendations. As with study aim 2, two expert panel members independently completed the review with a third adjudicating any differences.All three clinics provided a list of de-identified, retrospective prescriptions that represented either one or two months of prescription data to meet or exceed the study-determined 400 prescriptions. The number of prescriptions reviewed for Clinics 1, 2, and 3, respectively were: 477, 408, and 633 with total error rates of 13.2%, 8.8%, and 6.6%, respectively . Errors All three EHR systems were assessed for compliance with the AAP requirements for safe and effective e-prescribing . Table 1Compliance with the AAP EHR recommendations could have prevented over 83% (range: 83.3–88.9%) of the medication errors at the clinics . A numbeChildren have unique needs compared to their adult counterparts when it comes to the delivery of safe and effective medical care. Their unique needs must be reflected in the EHR systems used to create and transmit e-prescriptions. This study highlights the problem of pediatric prescribing errors in the outpatient setting found when using an EHR and identifies errors that could have been avoided if an EHR had appropriate pediatric focused functionality. In 2013, AAP published their policy titled “Electronic prescribing in pediatrics: toward safer and more effective medication management” . Using tTo further assist in evaluating an EHR, AAP has created an EHR shopper’s guide for clinicians caring for children . ProvideEven an AAP compliant EHR would have failed to avoid up to 17% of errors identified in this study. A solution to some of these problems could involve EHR customization. The use of indication specific dosing and the inclusion of weight based dosing recommendations and calculations would resolve the majority of dosing errors. The use of a customized pediatric-clinic specific medication list within the EHR could additionally minimize errors with dosage form selection or dosage form specific dosing recommendations. For example, the albuterol dosage form error earlier identified would have been avoided if the prescriber had a customized pediatric list that included the commercially available albuterol 0.083% (2.5 mg/3 mL) vials. A custom medication list would also further promote proper medication selection.On the other hand, some errors would not have been avoided even with full AAP compliance and custom lists. One error, for example, revealed an issue with the dosing calculator for certain ferrous sulfate products. In a 220 mg/5 mL oral solution formulation of ferrous sulfate, 44 mg/5 mL is elemental iron. Dosing should be based on this elemental (44 mg/5 mL) component, however the dose was calculated using 220 mg/5 mL leading to an 80% under dose. It is clear that EHR systems are further along in meeting AAP requirements related to patient information and medication information; while less equipped to meet cognitive support, pharmacy information or data transmission needs.Limitations exist with this novel review of EHR shortcomings related to pediatric use. This study represents a retrospective, descriptive study that identifies and demonstrates a need for EHR systems to address pediatric needs. However, a larger, more vigorous assessment would be beneficial to help develop a more robust, statistically driven plan. In addition, each of the three clinics reviewed utilized different EHR systems so comparing error rates and types with different user groups of the same system may be enlightening and could reveal other issues including education differences with respect to system use. Finally, seasonal variation is not accounted for with the snapshot of prescriptions reviewed at the clinics.With only a small percent of AAP criteria being met by each EHR used in the clinic setting today, there is much work to do in providing technology tools with the pediatric patient in mind. EHR systems should meet a minimum standard for pediatric support without requiring institution specific customization in order to provide appropriate safety checks and balances. Transmitting the patient’s weight to the pharmacy to allow dose checking; and providing mathematical conversion of fluid doses from mg/kg to mg then to milliliters per dose should be standard for all systems.Lastly, more research is needed in the area of pediatric medication safety related to technology in the outpatient environment. A growing volume of data exists looking into EHR safety related to children in the hospital setting, but little data is available examining EHRs in the outpatient pediatric setting. Some error types are consistent between the inpatient and outpatient settings, such as improper dose. However, unique errors in the hospital setting such as wrong rate or monitoring errors are not applicable to our outpatient-focused study . Of noteThis study demonstrates that EHR systems used by many pediatric clinic practices do not meet the standards set forth by the AAP. To ensure our most vulnerable population is better protected, it is imperative that medical technology tools adequately consider pediatric needs during development and be reflected in selected EHR systems.


Administering anti-vascular endothelial growth factor (anti-VEGF) by intraocular injection has been shown to have a safe systemic profile. Nevertheless, incidents of acute kidney injury following anti-VEGF injection have been reported. We assessed the long-term effect of multiple intravitreal anti-VEGF injections on measures of renal function in patients with diabetes including rate of change of estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (ACR).A retrospective review of patients receiving diabetic macular oedema (DMO) treatment was undertaken. Serum creatinine, ACR, number of intravitreal anti-VEGF injections and clinical characteristics were collected from electronic healthcare records (EHR). A co-efficient of eGFR and ACR change with time was calculated over a mean duration of 2.6 years. Regression modelling was used to assess variation in the number of anti-VEGF injections and change in eGFR and ACR.p = 0.22) or ACR change over time  was detected, following adjustment for hypertension, cerebrovascular disease, T2DM, and medications taken.The EHR of 85 patients with DMO  were reviewed. On average, 26.8 intravitreal anti-VEGF injections were given per patient over a mean duration of 31 months. No association between increasing number of anti-VEGF injections and rate of eGFR decline (beta = 0.04, 95% confidence intervals [CI]: − 0.02, 0.09; Our data suggests regular long-term intravitreal VEGF inhibition does not significantly alter the rate of change in eGFR and/or ACR with increasing number of treatment injections. Vascular endothelial growth factor (VEGF) inhibitors have transformed the therapeutic management of several retinal ophthalmic conditions. By improving visual acuity, they have surpassed the ability of laser photocoagulation to limit visual deterioration . DespiteIn addition to its role in the eye, VEGF plays a crucial part in maintaining normal renal function. VEGF released from podocytes interacts with VEGF receptor 2 on glomerular capillaries and promotes integrity of endothelial fenestrations and resultant glomerular barrier function . Loss ofSustained hyperglycaemia secondary to diabetes mellitus (DM) has been shown to activate abnormal metabolic pathways that trigger a complex cascade of inflammatory and vasogenic responses in the eye . VEGF isUse of VEGF inhibition therapy as an ophthalmic therapeutic involves local administration into the vitreous humour by intra-ocular injection with the dosage used approximately 400 times lower than that used in oncology , 11. AflIntravitreal ranibizumab (IVR) exists as a human monoclonal Fab antibody fragment with a molecular weight of 48 kDa and binds to all isoforms of VEGF-A . IntraviA pooled analysis of 751 population-based studies reported a global increase in the number of adults with DM from 108 million in 1980 to 422 million in 2014 . It is pThis was a retrospective, cohort, observational study using electronic healthcare records to access information on patients with DMO receiving intravitreal anti-VEGF treatment in the Belfast Health and Social Care Trust. This study received approval by the Office for Research Ethics Committee Northern Ireland (MREC Reference: 14/NI/1132).Serum creatinine measurements (μmol/L) and ACR (mg/mmol) were collected from the Northern Ireland Electronic Care Record (ECR) system. Each eGFR measurements was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation (CKD-EPI). In this study, participants received aflibercept, ranibizumab or both throughout the course of their treatment. The number of aflibercept, ranibizumab and total intravitreal anti-VEGF injections was recorded for each participant. Changes in eGFR and ACR over time were calculated using multiple eGFR and ACR measurements. These included a measure of renal function before the start of anti-VEGF therapy and after the defined injection period. Data was collected on demographic factors, glycaemic parameters and clinical variables including co-morbidities and medications.> 60 mL/min/1.73 m2. Study participants with an ACR > 3 mg/mmol or an eGFR < 60 mL/min/1.73 m2 were classified as DKD.This study included patients who were administered their first to last recorded anti-VEGF injections between 25th April 2012 and 22nd January 2018. For inclusion, each patient was required to have renal function measurements prior to their first anti-VEGF injection and after their last injection was administered. Patients were excluded on the basis of insufficient number of renal function measurements or if they experienced an acute decline in eGFR or rapid increase in ACR. Patients with diabetic kidney disease (DKD) can be classified depending on their level of kidney function (eGFR) and the amount of protein present in the urine (ACR). This information forms the basis of DKD staging which is useful for planning follow up and management. Individuals were classified as ‘No DKD’ if they had an ACR < 3 mg/mmol and an eGFR P < 0.05 was considered statistically significant.Independent samples T-tests, chi squared or Fishers exact tests were used to compare the distribution of demographic factors, glycaemic parameters and clinical variables between patients with DKD and those without DKD. Covariates significantly associated with a diagnosis of DKD were adjusted for in subsequent linear regression modelling. Simple and multiple linear regression models were used to generate beta estimates (β) and 95% confidence intervals (CI) for the total number of intravitreal anti-VEGF injections against the change in eGFR and ACR over time. In cases where ACRs were not present as absolute values (e.g. < 3 mg/mmol), arbitrary values were used to facilitate slope calculation. A previous study has demonstrated that the median ACR value for patients with an ACR < 3 mg/mmol was 1.06 mg/mmol and we used this as an arbitrary value for ACR values categorised as < 3 mg/mmol on the ECR .Data was collected on 90 patients undergoing regular intravitreal anti-VEGF treatment for DMO in the Belfast Health and Social Care Trust. Although, episodes of acute kidney injury following anti-VEGF injection have been reported previously, in order to evaluate the long-term effect of intravitreal anti-VEGF treatment on renal function and limit potential confounding from co-morbidities, five patients were excluded because an obvious reported co-morbidity led directly to an acute decline in renal function. A total of 42 participants were classified as ‘No DKD’ controls and 43 individuals were classified as ‘DKD cases’. Study cohort characteristics, co-morbidities and glycaemic parameters are summarised Table . The meaThe eGFR data met the assumptions of linear regression including normal distribution, homoscedasticity and absence of multicollinearity. However, ACR data was skewed lacking normal distribution and homoscedasticity. Importantly, absence of multicollinearity remained. Log transformation of ACR data did not improve the distribution curve and as a result, no log transformation was performed.2 to a mean follow-up eGFR of 65.9 mL/min/1.73 m2 with a mean rate of decline of 2.6 mL/min/1.73 m2 /year (Table p = 0.21) and remained non-significant following adjustment for T2DM, cerebrovascular disease (CVD), hypertension and treatment with proton pump inhibitors .Participants demonstrated a decline in eGFR from a mean baseline of 75 mL/min/1.73 m2 compared to 83.8 ± 13.3 mL/min/1.73 m2 /year in patients without DKD (p < 0.01). Additionally, patients with DKD also had significantly lower follow-up eGFR at 57.1 ± 24.6 mL/min/1.73 m2 compared to 75.7 ± 15.9 mL/min/1.73 m2 (p < 0.01). Patients with DKD did not have a greater rate of eGFR decline (− 2.5 ± 3.6 mL/min/1.73 m2 /year) compared to individuals without DKD (− 2.7 ± 3.4 mL/min/1.73 m2 /year).As expected, participants with DKD had a significantly lower mean baseline eGFR of 66.5 ± 24.4 mL/min/1.73 mp = 0.91) and remained non-significant following adjustment for T2DM, CVD, and treatment with beta blockers and proton pump inhibitors .Study participants had increased ACR from a mean baseline value of 17.9 ± 62.1 mg/mmol to a mean follow-up ACR of 18.8 ± 48.5 mg/mmol with a rate of increase of 0.7 ± 12.3 mg/mmol/year. In an unadjusted analysis the rate of change of ACR over time was not significantly associated with the number of intravitreal anti-VEGF injections increased . Additionally, participants with DKD had significantly higher ACR at follow-up 35.4 ± 64.3 mg/mmol compared to 1.8 ± 2.6 mg/mmol (p < 0.01).Participants with DKD had significantly higher mean baseline ACR of 34.4 ± 84.6 mg/mmol compared to 1.0 ± 0.67 mg/mmol in patients without DKD (p = 0.59). The mean number of aflibercept injections received by participants with DKD was 9.0 ± 7.0 injections compared to 11.2 ± 4.5 injections in those without DKD (p = 0.09).Across all participants, the mean number of ranibizumab injections received by those with DKD was 16.0 ± 9.7 injections compared to 17.2 ± 10.5 in those without DKD  and meta-analysis  investigated the systemic safety profile of IVA and IVR respectively, in DMO, neovascular age-related macular degeneration and retinal vein occlusion, by pooling data from existing randomised controlled trials but found no difference in the incidence of adverse systemic events between either intravitreal anti-VEGF treatment and placebo . AdverseIt is important to highlight that the clinical trials investigating IVR and IVA in DMO were not designed or powered to evaluate differences in low frequency systemic events, mainly as a consequence of their small sample sizes. Therefore, a firm conclusion on the systemic safety profile of intravitreal anti-VEGF is limited. Larger prospective studies with a longer follow up period and sufficient power to assess low frequency systemic adverse effects are required. A greater focus on the systemic safety of intravitreal anti-VEGF in high-risk groups is also needed. A population based, nested case-control study including 91,000 participants assessed post-marketing data on intravitreal anti-VEGF injections and found no significant increased risk of stroke, myocardial infarction, venous thromboembolism or congestive heart failure . While tThe Diabetic Retinopathy Clinical Research Network measured baseline and 52-week follow-up urinary ACR in 654 patients receiving ranibizumab, aflibercept or bevacizumab. On average, each patient had 9–10 injections during the treatment period. Across all three treatment groups, over 77% of patients maintained their baseline urinary ACR, while 10–16% of patients experienced a worsening of ACR by the 52-week follow up period, with more than 7% of patients experiencing an improvement in ACR. In the absence of a control group no definitive assessment could be made on the influence of anti-VEGF treatment. However, intravitreal anti-VEGF treatment did not appear to increase the risk of developing or worsening proteinuria .2 increased from 26% at baseline to 39% at follow up, following an average duration of 2.6 years of anti-VEGF treatment. The difference observed for both renal markers highlights the variation in the sensitivity of their measurement outcomes and the importance of monitoring both in diabetic populations.In our study 54, 34 and 12% of patients had a baseline ACR < 3, 3–30 and > 30 mg/mmol, respectively, with no significant change detected over the 2.6 year treatment period. In comparison, the percentage of participants with an eGFR < 60 mL/min/1.73 m2 compared to an eGFR of 78.1 mL/min/1.73 m2 for patients with T1DM. The mean follow-up eGFR was also lower for T2DM patients with an eGFR of 64.9 mL/min/1.73 m2 compared to T1DM with an eGFR of 71.3 mL/min/1.73 m2. The mean rate of eGFR decline was 2.9 mL/min/1.73 m2 /year compared to 1.6 mL/min/1.73 m2 /year for T2DM and T1DM respectively. Eighty four per cent of patients with T2DM had a diagnosis of CKD compared to 16% of patients with T1DM. Our findings reflect those from a large US study which showed a significantly higher prevalence of CKD in T2DM compared to T1DM patients  [In this study, 66 patients with DMO had T2DM and 19 patients had type 1 DM (T1DM). The mean baseline eGFR for patients with T2DM was lower at 74.1 mL/min/1.73 m< 0.001) .There are a number of limitations with our study including the inability to perform a sensitivity analysis to assess the relative contributions of IVR and IVA on change in renal function over time. However, a secondary analyses of a randomised comparative effectiveness trial, known as Protocol T, carried out by the Diabetic Retinopathy Clinical Research Network, showed no significant difference in renal function as assessed by urinary ACR over a 52 week follow-up period between patients who received intravitreal ranibizumab, aflibercept or bevacizumab for the treatment of DMO . In addiDespite these limitations, our study had several strengths. In collecting prospective eGFR/ACR data we were able to assess long-term changes in renal function that would not have been reported as an adverse event. Additionally, we collected data on a wide range of co-morbidities and glycaemic parameters, which allowed for appropriate adjustment of potential confounding factors. We used the CKD-EPI equation rather than the Modification of Diet in Renal Disease equation to calculate estimated glomerular filtration rates. The CKD-EPI equation is generally considered to be a better predictor of renal function, particularly at higher eGFR values .This study supports the previously demonstrated effective renal safety profile of intravitreal anti-VEGF in patients with DMO. Regular long-term intravitreal VEGF inhibition does not significantly alter the rate of change in eGFR and/or ACR with increasing number of treatment injections. The long-term assessment of renal function provides additional evaluation and detection of subtle changes in eGFR and ACR that may not present clinically as adverse events. Larger prospective and post-marketing trials, using renal markers including eGFR, ACR and Cystatin C, as well as assessing incidence of AKI and CKD, are required to strengthen the renal safety of intravitreal anti-VEGF treatment modalities. A greater focus on at-risk groups such as those with CKD is required.


Soil and the human gut contain approximately the same number of active microorganisms, while human gut microbiome diversity is only 10% that of soil biodiversity and has decreased dramatically with the modern lifestyle. We tracked relationships between the soil microbiome and the human intestinal microbiome. We propose a novel environmental microbiome hypothesis, which implies that a close linkage between the soil microbiome and the human intestinal microbiome has evolved during evolution and is still developing. From hunter-gatherers to an urbanized society, the human gut has lost alpha diversity. Interestingly, beta diversity has increased, meaning that people in urban areas have more differentiated individual microbiomes. On top of little contact with soil and feces, hygienic measures, antibiotics and a low fiber diet of processed food have led to a loss of beneficial microbes. At the same time, loss of soil biodiversity is observed in many rural areas. The increasing use of agrochemicals, low plant biodiversity and rigorous soil management practices have a negative effect on the biodiversity of crop epiphytes and endophytes. These developments concur with an increase in lifestyle diseases related to the human intestinal microbiome. We point out the interference with the microbial cycle of urban human environments versus pre-industrial rural environments. In order to correct these interferences, it may be useful to adopt a different perspective and to consider the human intestinal microbiome as well as the soil/root microbiome as ‘superorganisms’ which, by close contact, replenish each other with inoculants, genes and growth-sustaining molecules. The large diversity of microbiota in soil affects its microbial ecology, including its primary productivity and nutrient cycling. In addition, soil is part of the habitat of humans, providing space for living, recreation and food production . From eaMeanwhile, the human microbiome has become a major field of biomedical research, especially the intestinal microbial community, which plays a major role in human health and disease . The intIn view of the functional similarities between the intestinal microbial community and the soil microbial ecosystem, a relationship between both appears possible. Looking at the entire ecological system, the human body and its microbes can be regarded as an extended genome .Therefore, the question ‘To what extent does a relationship between both systems exist, for example by human exposure to different soil microbiological environments?’ arises. As human activities are changing the distribution and abundance of soil microorganisms, e.g., by agricultural land use , the resIn this context, we discuss the soil microbiome and its potential link to the (human) intestinal microbiome and assess the possible interrelation of the human intestinal microbiome and the soil microbiome.Since the start of the Human Microbiome Project in 2007, aiming at sequencing all microbes  inhabiting human body sites, the Human Microbiome Project has developed into a major field of biomedical research focussing mainly on the intestinal microbial community that plays a major role in human health and diseases ,10. The In view of the fact that, phylogenetically, humans developed in close contact with soil as their physical basis for living, providing shelter and water as well as food for daily life, the question arises as to whether the soil microbiome as an exogenous parameter affects the development of the human intestinal microbiome. Soils existed globally a long time before mammals and hominids came into existence and are by far the most extensive natural microbial gene reservoir on earth .Since 2010, the Earth Microbiome Project has focused on this gene reservoir. It is a major collaborative effort to characterize microbial life on this planet by using DNA sequencing and mass spectrometry of crowd-sourced samples to understand patterns in microbial ecology across the microbiomes and habitats of the earth ,16.On a per gram basis, the intestine, specifically the colon, has the highest concentration of cells of all biomes listed in The main factors that presumably determine the human intestinal microbiome are (i) host genetics and metabolism (heritage), (ii) lifestyle (environment) in particular, and (iii) diet and nutritional habits ,20,21.The microbial diversity in the human gut is a coevolution between microbial communities and their hosts . “AncienTo identify the evolutionary history of the biosphere, it is crucial to explore the microbiome of different hosts and habitats. Human-associated intestinal microbial communities are more similar to one another than to those of other mammalian species . When maInterestingly, the gut microbiomes of several mammalian lineages have diverged at roughly the same rate over the past 75 million years ; ContrarThe microbial population of the human gut derived from the ancestors, individually from the mother through vertical transmission during gestation, during birth, and after birth through contact with maternal body sites, with the greatest contribution of the maternal gut . Within Alterations in the foraging behavior and diet of early Homo species also included interaction between family members but in a different way. In the ‘grandmother hypothesis’ , foraginIt is suggested that dietary intake has a stronger influence on gut microbial composition than host genetics . The GI The importance of the environment is also shown by the fact that with increasing age, the variation between individuals decreased . FurtherThe living environment of urban dwellers shows a lower natural biodiversity and exposure to environmental microbes . The losThere is further evidence that soil biodiversity is interrelated with the gut microbiome. In particular, the gut microbial diversity in mice was increased by exposure to soil microbes . Gut micThe health of mammals is largely affected by domestication, which can be related to a less diverse gut microbiome observed in horse species, compared to undomesticated species . This reA recent study of the gut microbiome of terrestrially living baboons showed that soil is the most dominant predictor for shaping the gut microbiota with a 15 times stronger effect than host genetics . While tThe close link between reduced soil biodiversity  and gut microbial richness in baboons is an aspect that deserves particularly critical scrutiny in view of the global megatrend of biodiversity loss, especially for sustaining human health. Rural environments that are rich in microbiota, such as traditional farms, have been shown to have health benefits in humans . In partEven the recycling of human feces in the form of ‘night soil’ that re-entered the agricultural sites depicts the closed cycling of resources in those times . NowadayIncreasing global population and the need for housing and food have intensified agricultural practices and urbanization. Growing industrialization of agriculture results in reduced soil biodiversity . AlreadyOf particular importance in this context is the fact that the richness of gut bacterial species in adults is higher in rural societies as compared to urban communities ,54,55, wMartínez and colleagues  proposedHygienic measures reduce the risk for transmission of pathogens, but also of gut symbionts . This liThus, fecal contamination of water resources poses a significant risk for human health by spreading infectious diseases from fecal pathogens . SanitatBesides the many ecological functions, such as the production of biomass and support of biodiversity, soil has a unique function to provide clean drinking water . This soUrban citizens do not only lose contact with soil but also with feces , e.g., bAs mentioned above, with protection from any form of pathogens, artificial environments such as cities can be seen as habitats that eventually share a lower number of beneficial microbes and may concentrate pathogens. Even in most cases, the exposure to feces has a great potential to perturb an otherwise healthy gut microbiome . The hygAccess to more biodiverse areas in urban environments, such as green spaces and parks, is related to health benefits regardless of socioeconomic status , which cTherefore, we assume that the modern human lifestyle and the loss of direct contact with soil cause interruptions in the microbiological cycle in urban environments in contrast to pre-industrial rural environments. Soil is therefore a key primary source of a healthy intestinal microbiome of humans. However, the exact way how soil and the environment shape the human gut microbiome and how lifestyle changes affect the gut microbiome needs to be further elucidated. It is a dynamic research topic with relevance for preventive medicine.Besides the urban lifestyle and loss of contact with nature, our diet has also changed within the last decennia. In order to preserve food for long transport, storage and distribution, it is often sterilized. In addition to more processed nutrition, the intake of more energy-rich food, abundant in sugars and fat, decreases the biodiversity of the intestine . Often, Medication, in particular the intake of specific drugs, mainly drives the gut microbiome of Western populations and explains the greatest total variance of the fecal microbiome as shown by a large size Danish study . IncreasA large scale study of fecal microbiomes including clinical and questionnaire-based covariates reported that stool consistency had the greatest effect size on the fecal microbiome, while oral medication explained the greatest total variance . Among tThat diet is relevant for shaping the human gut microbiome is further supported by the study of Martinez et al.  which suThe gatherer–hunter community in Tanzania showed a low content or total absence of Bifidobacteria in the gut microbiome , which uThe microbiomes of non-Westernized populations resemble those of vegetarians and vegans ,70. The In this context, it is interesting that, in a Danish study, the dietary habits that were considered as relevant for shaping the gut microbiome identified a set of carbohydrates as relevant, such as fruit, bread and alcohol consumption .Specific types of food result in predictable shifts in intestinal microbiota, and hence the human intestinal microbiome can be directly affected by the diet . Human dThe change in human lifestyle also includes several post-harvesting operations before consumption . These oAs with bitter plant species, the consumption of fresh fruit that are almost un-processed is beneficial for human health specifically as soil biodiversity stimulates the secondary metabolite production . While tIn contrast to traditional smallholder farming, large scale farms, rather common in many industrialized countries, perform intensive farming practices, such as monoculture cropping of few plant species for optimizing yields. This has reduced the variety of food for humans and additionally increased the potential threats through contaminants due to the use of agrochemicals. Therefore, organically grown vegetables show a higher biodiversity of microbial endophytes and epiphytes than those conventionally grown . A recenWe conclude that on top of antibiotic medication, the elimination of microbes from food via processing has direct impacts on the human gut microbiome. Anyhow, the intake of diverse food rich in fibers and secondary plant metabolites, with living microbiota, from a diverse soil environment may positively influence the gut.https://www.globalsoilbiodiversity.org/atlas-introduction/). Thus, it has become possible to compare the geographical data of soil microbiomes with human gut microbiomes [Globally, soils are highly diverse, as are their microbes. There are only a few species that can be found in all soils, while there are numerous rare species that only occur in particular soils or geographical areas . This enrobiomes .Unexpectedly, Tasnim et al.  reportedBeside phylogenetic similarities between the plant rhizosphere and the human gut microbiome, there are many functional similarities : (i) TheIn order to sustain rich rhizosphere biodiversity, we have to understand the major drivers of this functional ecosystem: The rhizosphere microbiome is related to soil type, moisture, age, plant genotype and root lysates and exudates . SimilarAlso biofilms can be found in both the human GI tract/gut ,90 and tThe gut as well as the rhizosphere microbiome can be considered as “superorganisms” contained in/around the host, which are of paramount importance for the health and performance of the host, because (i) the gut microbiota are important for producing essential amino acids and vitamins such as B12 and K, and (ii) the root microbiota for producing hormones that are promoting plant health by improved nutrient acquisition, resistance to abiotic  and biotic  stress, and by sustaining growth .The deficiency of some micronutrients in humans, derived from nutrient-depleted soils, can have substantial effects as co-factors in metabolism, modulating enzyme activities, or functioning as coenzymes . Any redWorldwide urbanization as well as the mechanization of agriculture have dramatically increased during the last century. In combination with the use of agrochemicals such as mineral fertilizers and pesticides, soil biodiversity is reduced. In human medication too, a strong transformation occurred during this time by the use of antibiotics and hormones. The substantial effect of medication on the gut microbiome of the Western population was recently proven .From the above-mentioned structural and functional similarities between the soil rhizosphere and the human gut microbiome, we conclude that both can be considered as functional ecosystems, which interact which each other. This interaction has been decreasing in recent times, potentially reinforcing losses of biodiversity, which have occurred in both systems.Recent research data indicate that the modern lifestyle/environment is the most active driver in shaping the human intestinal microbiome despite the confounding influence of dietary habits, culture, and host genetics. The soil (rhizosphere) microbiota clearly influence the quality and storage of our food apart from the impact of post-harvest processing. In this context, more research is necessary to demonstrate how biodiversity of beneficial microbes in our food can be preserved. Furthermore, specific ways of agricultural practices, especially soil management, may improve the current food quality.Basically, recent findings suggest the investigation of the soil and root microbiota in more detail may identify effects on human health, possibly, among others, by adopting a lifestyle of former generations. This lifestyle, such as the reduced consumption of livestock and dairy products and the intake of a higher diversity of nutritional fibers and bitter substances may have beneficial effects on our health. The intake of mostly unprocessed organically grown regional products is one way towards this goal. Further, wild relatives of the currently used high-yielding crop varieties could increase aboveground and belowground biodiversity, and hence provide benefits to soil and human health, e.g., through reintroducing lost beneficial microbes.A rich soil microbiome would also have several advantages for the terrestrial ecosystem through increased nutrient use efficiency and uptake, which may improve plant yields as well as plant resistance and resilience against global climatic change and biotic stressors. The fact that the gut microbiome of hunter-gatherers has a higher species richness than that of humans that are nourished by Westernized food argues for agricultural practices that promote sustainable soil use and human health. Regarding food security under the aspect of predicted changes in human demographics and environmental change, it is of paramount importance to ensure the biologically sustainable use of land and soil.The soil contributes to the human gut microbiome—it was essential in the evolution of the human gut microbiome and it is a major inoculant and provider of beneficial gut microorganisms. In particular, there are functional similarities between the soil rhizosphere and the human intestine. In recent decades, however, contact with soil has largely been reduced, which together with a modern lifestyle and nutrition has led to the depletion of the gut microbiome with adverse effects to human health. Therefore, we suggest increasing research on the geographical and functional relationships to identify the causes and effects between soils and gut microbiota in order to benefit human health and the environment.