Peripheral nerve injuries are common and can have a devastating effect on physical, psychological, and socioeconomic wellbeing. Peripheral nerve transfers have become the standard of care for many types of peripheral nerve injury due to their superior outcomes relative to conventional techniques. As the indications for, and use of, nerve transfers expand, the importance of pre-operative assessment and post-operative optimization increases. There are two principal advantages of nerve transfers: (1) their ability to shorten the time to reinnervation of muscles undergoing denervation because of peripheral nerve injury; and (2) their specificity in ensuring proximal motor and sensory axons are directed towards appropriate motor and sensory targets. Compared to conventional nerve grafting, nerve transfers offer opportunities to reinnervate muscles affected by cervical spinal cord injury and to augment natural reinnervation potential for very proximal injuries. This article provides a narrative review of the current scientific knowledge and clinical understanding of nerve transfers including peripheral nerve injury assessment and pre- and post-operative electrodiagnostic testing, adjuvant therapies, and post-operative rehabilitation for optimizing nerve transfer outcomes. Nerve injuries are common and can have devastating effects on an individual's physical, psychological, and socioeconomic wellbeing –4. The mNerve transfers involve coapting a healthy expendable donor nerve to the end or side of the injured nerve. This can be done outside the zone of injury and closer to the target end organ to restore the lost sensation or motor function , 13. RecAs our knowledge and understanding of nerve anatomy and regeneration expands, nerve transfer techniques continue to be developed and refined. Alongside this rapid growth in the field of nerve transfers has come the expansion of electrodiagnostic studies for diagnosis and management of these injuries –20. The The assessment of nerve injuries begins with a thorough history and physical examination which can often be enough to diagnose specific deficits. Prognostically, determining the timing and etiology of the nerve injury is critical. Nerve injuries can be broadly classified into open  and closed  injuries. Injury mechanism plays an important role in understanding the potential severity and predicted recovery. For instance, sharp penetrating injuries confer a higher suspicion for complete nerve transection and are typically explored and repaired early. Conversely, gunshot penetrating and closed injury mechanisms are typically followed clinically to monitor for spontaneous improvement and to further characterize the degree and distribution of the injury.Physical examination of motor and sensory deficits characterizes the sequelae of nerve injury. Sensation can be assessed with a variety of techniques including light/sharp touch, the ten test, two-point discrimination, and Semmes Weinstein monofilaments, all of which have advantages and limitations –27. IdenIn some cases, imaging modalities offer further diagnostic and prognostic value to clinicians' pre-operative evaluation. Magnetic resonance (MR) and ultrasound imaging modalities are the most often utilized for their superior soft tissue resolution and have multiple applications in a patient with suspected nerve injury . ConventTogether with a thorough history and physical examination, electrodiagnostic testing (EDX) serves an important role in optimizing outcomes after nerve injury. Although there are indications for proceeding to surgery without EDX, such as in sharp/penetrating nerve injuries which warrant urgent/emergent exploration and repair, most patients undergoing nerve transfer procedures benefit from pre-operative planning that includes electrodiagnostic evaluation . Due to Pre-operative EDX serves three main purposes: first, to characterize the extent of nerve injury including affected and unaffected nerves; second, to characterize the severity of the nerve injury and provide prognostic information to identify targets for nerve transfer; and third, to identify the availability of suitable donor nerves.As a complement to the history and physical examination, EDX using nerve conduction studies (NCS) and needle electromyography (EMG) delineates which muscles and which sensory nerves are affected or spared. Because of normal anatomical variants in peripheral nerve anatomy, and because nerve injuries may spare some nerve fascicles, these electrodiagnostic techniques provide important diagnostic clarification as to the extent of nerve involvement and can be used to establish the approximate localization of injury, if not already known.In addition to establishing the extent of nerve injury, EDX serves to characterize nerve injury severity. Using NCS and EMG after sufficient time has elapsed  can further characterize whether the injury pathology is demyelinating, axonal, mixed, or pre-ganglionic. For instance, in the setting of a suspected lower trunk brachial plexopathy, the presence of reduced motor NCS responses and denervation potentials with reduced or absent motor recruitment for the ulnar and thenar muscles together with normal sensory NCS responses at the dorsal ulnar cutaneous and medial antebrachial cutaneous nerves suggests a pre-ganglionic injury such as a C8/T1 nerve root avulsion or upper motor neuron injury . For posHowever, electrodiagnostic testing does have limitations, such as being unable to distinguish purely post-ganglionic injuries from mixed pre- and post-ganglionic injuries and being unable to distinguish axonotmetic from neurotmetic injuries in a single evaluation. Moreover, sensory NCS may overestimate the severity of post-ganglionic nerve injuries in mixed motor/sensory nerves . As a reBy establishing the severity of injury, the electrodiagnostic evaluation confers prognostic information about likely recovery to guide management. Predominantly demyelinating (neurapraxic) nerve injuries typically recover within weeks to months; nerve transfers are therefore unlikely to offer functional improvements. Conversely, proximal axonal injuries (whether axonotmetic or neurotmetic) are unlikely to undergo spontaneous and complete recovery within 12–18 months if more than 15–20 cm (6–8 inches) from their target organ, as in brachial plexopathies . ConsequIn addition to the insights about extent of injury, approximate localization of injury, and which nerves would benefit from transfer, EDX serves as an invaluable tool to identify suitable donor nerves. Nerve transfer requires a healthy, expendable donor nerve, which typically serves a redundant or less crucial function , 46. NeeIndications for nerve transfers continue to expand and evolve but in general include nerve root avulsions, proximal injuries where the distance to reinnervation is expected to be too long for meaningful recovery, multi-level injuries, large nerve gaps, and situations where it is not safe or feasible to operate in the original zone of injury , 13. GarAnother indication for nerve transfers is in upper limb reconstruction post-spinal cord injury (SCI) as stand-alone procedures or in conjunction with tendon transfers , 54. In Indications for specific nerve transfer techniques are also evolving. SETS transfers remain somewhat controversial. The goal with SETS transfers is to supplement existing nerve function with new axons, “babysit” distal motor endplates, reduce Schwann cell degeneration, and provide additional growth factors, all while avoiding the sacrifice of possible proximal nerve recovery as is necessary with standard ETE transfers , 55–61. For any nerve injury, the timing of surgical intervention is critical: denervated muscles undergo atrophy and after a certain amount of time, motor end plate loss is irreversible and reinnervation does not occur . IdeallyThis section outlines the role of adjuvant therapies, defined as interventions which occur outside the operating room and are designed to improve outcomes in nerve reconstruction. Although they may be considered adjuvant therapies, this portion of the review will not include formal therapy before (“pre-hab”) or after surgery as these topics are covered elsewhere in this paper. This portion of the review also does not review locally applied  substances that have been shown to enhance outcomes in the laboratory but for which efficacy in humans is lacking.The factors which lead to suboptimal outcomes after nerve injury and repair have been elucidated in a series of elegant studies , 66. A nAdjuvant therapies trialed in human subjects include platelet-rich plasma (PRP), tacrolimus (FK506), and electrical stimulation. PRP has gained widespread use in the treatment of various musculoskeletal conditions such as lateral epicondylitis and Achilles tendon rupture . PRP conFK506 (tacrolimus) is a potent inhibitor of T-cell mediated immunity and is widely used in solid organ transplantation as an immunosuppressant. When given systemically, FK506 appears to enhance the rate of nerve regeneration and may act via other mechanisms such as diminishing neural scarring. Because of the potential toxicities of FK506, combined with a narrow therapeutic dosage window, some investigators have worked on delivery of the drug at the site of nerve injury via delivery systems such as microspheres and nerve conduits , 72. ResOf all adjuvant therapies, brief electrical stimulation (ES) has shown the most promise and is closest to widespread clinical availability. Electrical stimulation enhances the rate of axonal growth after nerve injury. A number of methods of applying electrical stimulation have been described in both animal models and humans , 74. In More recently, even greater enhancements in nerve recovery have been demonstrated with pre-operative (“pre- conditioning”) ES. Clinically, this is likely to be more practical and cost-effective than applying ES peri-operatively. Senger et al. have demonstrated improvements in both a nerve graft and nerve transfer rat model which appears to be superior to post-operative ES , 79. WhiIn summary, various systemically given or locally applied pharmacologic substances hold promise to enhance recovery after nerve injury and repair. However, currently, none are available for routine, practical use. Brief electrical stimulation shows real promise as an adjuvant therapy. Larger clinical trials and routine use await easily available and utilized devices. All adjuvant treatments require more robust scientific evidence to determine which are truly effective and how they can be incorporated into nerve reconstruction techniques, including nerve transfers, to optimize patient outcomes.After nerve transfer, rehabilitation plays a crucial role in optimizing recovery and patient outcomes. Although the focus of this review is on addressing motor dysfunction with nerve transfers, addressing sensory deficits is important for patients' outcomes. Patients with sensory deficits often benefit from sensory relearning programs, a field with established techniques . CliniciThe goal of nerve transfer rehabilitation is to restore function and independence through both cortical and peripheral plasticity to perform daily activities that were once lost.In the protective phase, during the first 3 weeks following nerve transfers, avoiding excessive tension on the neurorrhaphy through active or passive motion is the crucial consideration. Although the recipient and donor nerves are loosely coapted, orthotic intervention may be necessary to ensure minimal tension on the neurorrhaphy , 84. In Orthoses that patients may have worn before surgery for positioning and/or increasing ROM can be reintroduced in this phase, provided they do not impose tension on the nerve coaptation.The period between 3 weeks post-operative and the beginning of early reinnervation is known as the “silent phase” due to the lack of active motion , 87. EarAlthough no active motion is observed during this period, cortical reorganization (neuroplasticity) may be stimulated through motor imagery (MI), as new connections need to be established for good motor function of recipient muscles , 88, 89.Retraining begins with early reinnervation (MRC 1) until the patient demonstrates active range of motion in a gravity-eliminated position (MRC 2). This phase is characterized by a greater number of immature polyphasic motor units on needle EMG. The primary therapeutic goal is to facilitate motor relearning and cortical reorganization  to help patients reproduce the newly targeted motion independently. This is initially done by so-called “donor activation” in which the patient activates the original target muscle of the donor nerve to produce motion in the recipient muscle. For instance, in the case of the flexor digitorum superficialis fascicle of the median nerve used as a donor to reinnervate brachialis, elbow flexion may be initiated by concomitant finger flexion , 91, 92.To assist in this process, EMG-biofeedback is a valuable modality that provides patients with visual and auditory information about targeted recipient muscle activity, even before any clinically appreciable muscle function is noticed . When coMirror therapy (MT) is also valuable for stimulating cortical plasticity. MT was initially described by Ramachandran for treating phantom limb pain in amputees , 95. It Exercises in this phase are performed in gravity-eliminated or gravity-lessened positions, with or without the assistance of dynamic-assist orthoses or sling/suspension systems, as illustrated in Patients transition to the strengthening phase when their strength progresses from MRC grade 2–3, at which point patients can overcome gravity and some light resistance. This phase is characterized by a few mature and increasing numbers of polyphasic motor units on needle EMG studies. The main goal of this phase is to increase the strength and endurance of recipient muscles while consolidating the motor control of the newly acquired function .In this phase, EMG-triggered electrical stimulation (EMG-ES) can be a highly effective tool. EMG-ES combines EMG-biofeedback with traditional neuromuscular electrical stimulation (NMES), requiring active patient participation to activate the neuromuscular stimulation . Hence, In denervated muscles, significant wasting and loss of motor units occur, leading to fatigue and potential adverse effects when newly innervated muscle fibers are overpowered. Therefore, lower frequency  parameters are used to stimulate muscle fibers slightly above the threshold to produce tetanic contraction and below the threshold to stimulate anaerobic fibers. While the ideal functional electrical stimulation parameters in human models have not yet been determined, recent research has shown that peri-operative nerve stimulation at 20 Hz can improve recovery after nerve injury and repair –108.As the quality of motion improves and the connection between donor and recipient nerves becomes established, the reliance on donor nerve activation to initiate new motion is expected to decrease. This, in turn, will increase the ability to selectively activate donor and recipient nerves separately , 87, 99.When performing donor de-activation exercises, EMG-biofeedback continues to be crucial in promoting cortical plasticity by providing feedback on muscle function. These exercises involve various techniques, including: (1) EMG-biofeedback of recipient muscle(s) while simultaneously eliciting contraction of the recipient muscle(s) and the antagonist muscle(s) to the donor nerve ; (2) EMGIt is essential to evaluate the muscles that antagonize the recipient muscles during nerve transfer rehabilitation, as weak muscles may be easily overpowered by the newly innervated muscles . For exaAs muscle function and motor control improve during rehabilitation, reliance on dynamic-assist orthoses and support systems, such as suspension slings, gradually declines. However, compensatory movements may persist during exercises and functional use of the limb. To address this issue, orthoses and/or performing exercises in front of a mirror for visual feedback may be excellent adjuncts to reduce compensatory movements , 86, 90.This rehabilitation phase typically starts when patients can demonstrate full range of motion against gravity (MRC 3 and beyond) and can easily activate both donor and recipient nerves simultaneously. EMG studies indicate an increase in new and maturing motor units during this phase. The primary objective of this stage is to build endurance and facilitate the return to as many meaningful functional activities as possible.NMES can be used as a standalone modality with similar parameters as the aforementioned EMG-ES to help increase resistance and endurance. MI is also implemented to help patients mentally replicate various tasks they need or desire to return to. During this phase, exercises are performed against gravity, without the assistance of dynamic orthoses, and with some resistance , 110.It is worth noting that some patients may not have fully mastered the ability to activate donor and recipient nerves' function selectively. Therefore, in addition to building muscular strength and endurance, reducing reliance on donor activation for function should remain a critical therapeutic goal during this phase .Post-operative electrodiagnostic assessments fulfill two key functions after nerve transfer surgery. The first, primary function is to monitor for evidence of recovery; the second is to guide rehabilitation.Post-operative electrodiagnostic assessments allow for monitoring of recovery following nerve transfer. Needle EMG is an effective tool to identify if motor nerve transfer has resulted in reinnervation of new axons to recipient muscles, evidenced by nascent units . Timing The electrodiagnostic reassessment must focus on needle EMG of the most proximal muscle innervated by the recipient nerve, as this will be the first to be reinnervated. In addition, needle EMG must include attempts at activating the muscle through the donor nerve function  and the recipient nerve function  , 111. FoIf sufficient time has passed and reinnervation should be evident on needle EMG and is not, then clinicians should consider re-assessment of surgical or other corrective options, such as tendon transfers or alternative nerve reconstruction procedures , 112. IfIn addition to monitoring for axonal regrowth, the post-operative electrodiagnostic assessment can guide rehabilitation. As outlined in the preceding rehabilitation section, the presence of multiple nascent units on needle EMG after the 3-week protective phase allows for progression from the Silent/Corrective Phase to the Re-education Phase. Beyond the re-education phase, needle EMG can also be used to determine to what extent a patient relies on donor activation. For instance, in the Silent/Corrective Phase, nascent units may only be detected with activation of the donor nerve function. Thereafter, there may be an appreciable difference in the number and recruitment of motor unit action potentials seen on needle EMG with greater motor unit activation and recruitment with donor and recipient co-activation than recipient nerve activation alone. As donor-recipient nerve co-activation improves and cortical reorganization occurs, this difference diminishes, and patients may have full interference patterns observable with recipient nerve activation alone. If this process is delayed or incomplete, and donor activation continues to be critical in the later phases of recovery, identifying this on needle EMG can help redirect therapy to address decreasing reliance on donor nerve activation.The optimal frequency, number, and total duration of post-operative electrodiagnostic assessments is unknown. Those patients whose recovery would benefit from reassessment, whether through detection of reinnervation, confirmation of the need for alternative interventions, or to direct rehabilitation, may have multiple electrodiagnostic assessments in the post-operative periods at appropriate time intervals based on the expected trajectory of axonal regrowth.As noted above, there is a finite window in which denervated muscle may undergo reinnervation. Consequently, particularly for very proximal nerve injuries, the decision to proceed with surgery must occur before the full extent of spontaneous recovery. This concept, as well as the expected timeline for recovery, require careful education for patients. The timeline for recovery after nerve injury, including for patients who undergo nerve transfer, can be lengthy. The aim of nerve transfer is to ensure reinnervation after peripheral nerve injury is achieved within 12–18 months of the injury. The initial time to reinnervation after nerve transfer is proportionate to the distance from the nerve transfer site to the muscle or sensory target, as nerves grow 1 mm per day. Therefore, in the immediate post-operative period, patients who have undergone nerve transfer will not see new motor or sensory functions. Once initial reinnervation is achieved, motor or sensory function may still be unsatisfactory until additional axonal growth and collateral sprouting have taken place. The time for patients to progress through the above phases of motor rehabilitation will vary based on patient factors, injury factors, the distance to reinnervation, and other factors. For some patients, recovery may still be incomplete despite surgical interventions to promote reinnervation.As the indications for, and use of, nerve transfers expand, the importance of pre-operative assessment and post-operative optimization intensifies. Comprehensive pre-operative history, physical examination, and, where appropriate, electrodiagnostic testing improve diagnosis and management of nerve injuries. Nerve transfer surgeries, whether alone or in conjunction with conventional nerve reconstruction/grafting or tendon transfer procedures, can improve functional recovery. Post-operative rehabilitation and electrodiagnostic assessments can optimize outcomes following nerve transfer. The future role of adjuvant treatments, including ES, have yet to be precisely elucidated but show promise for further enhancing recovery from peripheral nerve injury.


To extract insights from these complex datasets, new computational tools have facilitated the integration of information across different data types and the use of machine learning approaches. Here, we summarize current experimental and computational methods for generation and integration of single cell multi-omic datasets. We focus on opportunities for multi-omic single cell sequencing to augment therapeutic development for kidney disease, including applications for biomarkers, disease stratification and target identification.Single cell sequencing technologies have rapidly advanced in the last decade and are increasingly applied to gain unprecedented insights by deconstructing complex biology to its fundamental unit, the individual cell. First developed for measurement of gene expression, single cell sequencing approaches have evolved to allow simultaneous profiling of multiple additional features, including chromatin accessibility within the nucleus and protein expression at the cell surface. These multi-omic approaches can now further be applied to cells  The development of single cell sequencing technology focused initially on profiling the transcriptome ; howevervia this assay, enabling interrogation of gene expression without the need to pre-select a set of target genes. A key benefit of this target-agnostic approach is the ability to explore fundamental aspects of cell biology without the constraints of a priori assumptions. For example, each cell’s “transcriptomic signature” can be used for cell type classification, typically via comparison with reference maps or by clustering and manual inspection of expression of marker genes. With unbiased single-cell resolution, novel cell types may be appreciated that were missed by conventional classification schemes, typically relying on a handful of pre-defined markers (Single cell transcriptome profiling by RNA-sequencing (scRNA-seq) entails the generation of a barcoded complementary DNA (cDNA) library from the total pool of RNA transcripts present in a given cell at a given time. The chemistries commonly employed for high-throughput single cell cDNA generation typically bias the assay towards the 5’ or 3’ end of the most abundant polyadenylated transcripts , while l markers . LikewisSingle cell epigenomic profiling by assay for transposase-accessible chromatin (scATAC) entails fragmentation and tagging of the genome with barcoded adapters for high-throughput sequencing by Tn5 transposase . BecauseSingle cell proteomic profiling by sequencing entails labeling cells with DNA-barcoded antibodies against protein targets, enabling quantitative measurement of their expression. Targets profiled can include those on the cell surface  , as wellThe conversion of transcript expression, protein expression, and chromatin accessibility to a common sequence-based readout makes it possible to simultaneously obtain multiple modalities from individual cells in a single experiment. The first reports of multi-omic single cell profiling involved the combined profiling of transcriptomes and proteins. These early multi-omic assays included Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) , and RNAJoint profiling of transcript expression and chromatin accessibility was described shortly after the introduction of CITE-seq and REAP-seq , and thivice versa. These methods are still in their infancy, and further refinements will be necessary to aid their widespread adoption; however, they offer a glimpse of what promises to be a more holistic era in cell biology.Joint single cell profiling of chromatin accessibility and protein expression has been described more recently . These mKidney is an inherently challenging organ for single cell analysis, owing to the need to first dissociate cells from the matrix in which they are embedded. This typically entails enzymatic digestion and/or mechanical disruption, processes that can result in cell death and/or stress responses. The latter can be particularly confounding for single cell analysis, as transcriptional responses to stress may obscure native cellular phenotypes . Use of Single nucleus RNA sequencing (snRNA-seq) has emerged as a helpful alternative to single cell sequencing in cases where cell dissociation is necessary and poses a challenge . Nuclei The availability of multiple assays, or modalities, for single cell sequencing can provide a much deeper understanding of the biological processes compared to single modalities. On their own, individual modalities can present complementary evidence or serve as independent validation of biological findings. To unlock a greater potential of multi-omic datasets, the modalities need to be explicitly integrated, which can then substantially expand the insights that can be obtained from the individual modalities.A range of integration approaches and tools have been developed for multi-omic single cell datasets. Some of these were developed for specific combinations of modalities, while others can handle a broad range of modality combinations. Here we outline the key distinguishing features of these tools noting that the selection of an optimal tool is largely determined by the nature of the data and the biological insights that are sought.Data from multiple modalities can be from the same cells (paired data) or from different but similar cells (unpaired data) . Paired Another principal distinction between various integration methods is the analysis stage when integration is performed: early stage and late stage . For earIn contrast, late-stage integration does not explicitly combine the different modality datasets, but rather combines analysis results obtained for the individual modalities. For example, a typical step in single cell analysis is the construction of a neighbor-graph that describes the similarities of the cells. The neighbor-graph is then used as input for clustering analysis or reduced dimensionality visualization, such as a uniform manifold approximation and projection (UMAP) or t-distributed stochastic neighbor embedding (t-SNE) map. In a multi-modal context, neighbor-graphs can be constructed for the individual assays, which can then be merged and subsequently used for integrated clustering analysis, visualization, and possibly cell type and state annotation  Hao et .A third example, which can be regarded as late stage as well, is the identification of subpopulations using one modality, which are then extracted and further analyzed using the second modality . This alFinally, methods are either designed to handle specific combinations of modalities (see next paragraphs for examples), or for any combination of modalities. Examples of the latter that have been used extensively include the Weighted Nearest Neighbor (WNN) approach implemented in the widely used Seurat package , MOFA + The availability of commercial kits and protocols has increased the accessibility of combined profiling of the transcriptome and surface proteome. Computational approaches for the integration of paired transcriptome and surface proteome data have been increasingly needed for analyzing these datasets.An example of a late-stage integration approach developed specifically for transcriptomic data and the surface proteome is CiteFuse . Cell-toIntegration of transcriptomic and ATAC data is a special case as it can take advantage of ‘feature correspondence’ . The scAThe combination of scRNA-seq and scATAC-seq modalities has proven useful in various ways, as illustrated in a recent study on mouse kidneys . First, With the advent of single-cell methods came efforts to build cell atlases for tissues or even entire species . More reCommunity efforts such as the Multimodal single cell data integration challenge  can playAdvances in single-cell sequencing technologies have enabled the analysis of individual cells obtained from tissues, such as kidney biopsies, and biofluids, such as urine and blood, at an unprecedented resolution, revealing cellular signatures of inflammation, cellular injury, and fibrosis in various kidney diseases. These signatures enable a deeper understanding of pathophysiology and can also facilitate development of precision therapeutics for these diseases. Single-cell profiling of kidney biopsies, PBMCs, urine samples and skin lesions from patients with lupus nephritis, acute kidney injury, diabetic nephropathy and focal segmental glomerulosclerosis present potential novel approaches for the diagnosis and monitoring of disease activity . These aLN is a form of glomerulonephritis that constitutes one of the most severe organ manifestations of systemic lupus erythematosus (SLE). Despite increased knowledge of disease pathogenesis and improved treatment options, LN remains a substantial cause of morbidity and death among patients with SLE. Conventional markers of disease activity and response to therapy typically consist of measuring auto-antibody levels, markers of complement activity and laboratory parameters like proteinuria and estimated glomerular filtration rate (eGFR).Single cell transcriptomics and urine proteomics were used to identify biomarkers that are upregulated in LN, including IL-16, which is found to be highly expressed at sites of kidney injury . Other sAKI is a major health issue, the outcome of which depends primarily on damage and reparative processes of tubular epithelial cells. According to a recent meta analysis, 1 in 5 adults (21.6%) and 1 in 3 children (33.7%) experienced AKI worldwide . MechaniIn a recent study , scRNA-sα-2 macroglobulin (A2M), elevated glomerular endothelial cell score and loss of canonical podocyte markers in patients with FSGS compared to patients with MCD. These findings may help in differentiating FSGS from MCD and in predicting response to treatment  are common causes of nephrotic syndrome and share many common features, such as diffusely effaced podocytes. However, the response to treatment is variable in FSGS, and there is a higher risk of progression to chronic kidney disease (CKD) in patients with FSGS, compared to patients with MCD, who typically have a benign course. Multiple single cell studies  have idereatment .Fibrosis is a characteristic feature in all forms of CKD. Deposition of pathological matrix in the interstitial space and within the walls of glomerular capillaries as well as the cellular processes resulting in this deposition are increasingly recognized as principal factors resulting in progressive kidney damage. It has been challenging to study kidney fibrosis in patients, since kidney biopsies are usually not performed in patients with established CKD. Use of multi-omic techniques  have helDKD has a high global disease burden and substantially increases the risk of kidney failure and cardiovascular events. Despite treatment, there is substantial residual risk of disease progression with existing therapies. There is an urgent need to better understand the molecular mechanisms driving DKD to help identify new therapies that slow progression and reduce associated risks . snRNA-sSingle cell sequencing provides rich and complex phenotypic portraits of the cellular and molecular circuits involved in disease. These large datasets lead to the identification of an array of cellular and molecular features which are upregulated, downregulated or, in some cases, unique to disease states. Differences observed in disease vs. control samples provide hypotheses of new therapeutic targets, which may have become dysregulated at the cellular or molecular level. Nevertheless, challenges remain in deciphering which observed changes drive pathogenic processes versus those that are passenger effects.Genetic variants and their loci from genome-wide association studies (GWAS) provide complementary evidence to single cell sequencing datasets for the involvement of genes in the development of diseases. A challenge, however, in identifying specific targets from genomic variant studies is that the large majority of disease-associated loci are in non-coding regions, thus complicating their linkage to specific gene products or cell types that could be candidates for therapies. By integrating single cell sequencing datasets with GWAS candidate genes/loci, greater linkages can be established to identify genes, pathways or cell types with causal relationships to disease. In one analytical framework, genes near disease-associated loci from GWAS studies are compared to various cell subsets identified by scRNA-seq to nominate disease-relevant cell subtypes and pathways. This framework led to the identification of cell clusters enriched for expression of TLR7 (nucleic acid sensing), HIP1 (endocytic participating protein implicated in DC regulation), and LBH  in LN patients . When muSince functionally important gene regulatory regions are mostly nucleosome-free, chromatin accessibility data can be used to aid understanding of candidate risk loci in non-coding regions. Additionally, expressed quantitative loci (eQTL) approaches are commonly used to map variants to causal genes. However, a critical limitation in chromatin accessibility and eQTL analyses for mapping causal genes arises from cell type heterogeneity; bulk methods capture aggregated expression or accessibility across multiple cell types, with cell type diversity and proportionality complicating interpretation [reviewed in ]. In a pTo further facilitate target identification and drug discovery, single cell profiling has been combined with gene perturbation methods as a forward genetics screening approach to explore phenotypic impacts of gene modulation. Perturb-seq and related approaches integrate pooled CRISPR screening with single cell profiling, enabling systematic determination of the impact of inhibitory and activating perturbations to large numbers of candidate genes . The pheA high-resolution picture of all cell types from a disease tissue sample provides a comprehensive and deep interpretation of the underlying biology and should be informative to diagnostics and prognostics. Single cell sequencing can provide a comprehensive capturing of all cell types, as well as information regarding their molecular pathways, thereby providing greater detail of disease endotypes and potential for therapy responsiveness. Indeed, studies have used single-cell profiling of peripheral blood immune cells to identify correlates of anti-PD1 responsiveness in cancer patients .For diagnostics and biomarkers, a high-resolution molecular picture of the kidney would be of great value, however, a key challenge in molecular diagnostics, including single cell profiling, for kidney disease is the limited ability to obtain kidney biopsy samples for such analyses. As performing a kidney biopsy carries some risk of complications for patients, they are commonly performed at time of diagnosis and at limited additional timepoints, as needed for patient care. Core biopsies contain a relatively low number of cells (single digit thousands for single cell profiling). Moreover, the tissue must be dissociated to single cell suspension, which can introduce artifacts, such as protease-induced changes in gene expression, and lead to biased loss of cell types. To address these key challenges in diagnostics and biomarkers, emerging studies have begun to analyze surrogate tissue sources of cells from urine for single cell profiling, as well as spatial profiling from limited tissue derived from kidney biopsy cores.Urine sedimentary analysis has been used for decades to inform diagnoses for kidney diseases, however, until the development of single cell profiling approaches, robust and accessible methods to generate an unbiased determination of cell types and their molecular features were lacking. Single cell profiling of urinary cells from DKD patients demonstrated an ability to detect nearly all cell types of the kidney . Emerginin situ hybridization (ISH) and next-generation sequencing (NGS)-based methods. ISH methods can provide subcellular localization information but are limited to a selected (and therefore biased) set of several hundred gene probes. In contrast, NGS-based spatial transcriptomics methods combine scRNA-seq technology with spatial barcodes on a specialized slide, enabling amplification of copied transcripts while retaining localization information. The NGS-method provides unbiased, genome-wide transcriptomics but is limited in spatial resolution due to the spot size on the slide. The current 10x Visium methodology incorporates spot sizes of 55 μm, which falls short of single-cell resolution. To improve the resolution, separate conventional  scRNA-seq data can be integrated with the spatial data, and combined with computational deconvolution methods, used to estimate single cell contributions  tissues, making them more amenable to the limited material of kidney core biopsies from clinical settings that use FFPE as standard of practice. Commercialization of spatial transcriptomic methods has reduced the technical barriers of access to these complex methods, with FFPE-compatible platforms including from 10x Genomics (Visium and Xenium), Nanostring , Vizgen (MERSCOPE), among others. The technologies are rooted in one of two fundamental methods:  reviews . Studies reviews , develop reviews , cell-me reviews , and sma reviews . Multi-o reviews . SpatialMulti-omic single cell sequencing has facilitated the creation of high-resolution cellular and molecular maps in the context of kidney disease, providing new insights into disease mechanisms and opportunities for therapeutic intervention. New experimental methods enable measurement of multiple molecular features simultaneously, including gene expression, surface protein expression, TCR/BCR sequences and chromatin accessibility. Computational tools to integrate these large and diverse datasets have enabled derivation of rich biological insights, from pathogenic mechanisms to new therapeutic targets. And while most studies applying single cell sequencing methods in the context of kidney diseases have utilized single modality datasets , increasingly multi-omic approaches are being pursued.Single cell profiling is increasingly accessible to researchers, due to developments of more user-friendly experimental workflows  and data analysis tools . However, tissue availability from kidney core biopsies has been a major limitation for single cell profiling in the context of kidney disease. Emerging alternative approaches compatible with single cell profiling are being developed, including urinary cell analysis and spatial profiling. With studies in which nearly all kidney cell types can be identified in urine by single cell profiling, the potential for routine, non-invasive monitoring of biological changes of individual diseased patients is an exciting one, with ramifications for more optimal treatment selections . These developments portend a future of collection of these high-resolution datasets in a variety of clinical settings, from observational studies to therapeutic interventional trials. Collectively, these advancements are heralding new opportunities for precision medicine in kidney disease, from diagnostics and patient segmentation to prognostics and new targeted therapies, with the potential to better match the right therapies with the right patients at the right time.


A recent study by Blank, Alink and Büchel, uses multivariate neuroimaging to investigate how the human brain processes the strength of face-related expectations and explores whether these expectations are represented in the same regions that process facial stimuli. In line with predictive coding theories, their study presents compelling evidence that the brain adjusts its processing based on the certainty of expectations. This occurs exclusively within high-level face-sensitive regions, rather than across the entire processing hierarchy. Here we critically discuss these findings and outline potential directions for future research to better understand how the human brain expects, processes, and perceives images. When we navigate our environment, we have preconceived expectations of what or who we will likely encounter. However, these expectations are not all equal; they carry varying degrees of likelihood based on our past experiences, that is, we are likely to expect a colleague’s face at our workplace but a family member’s face at home. A recent study by Their data revealed that only high-level face-sensitive regions, such as the anterior temporal lobe (aTL), coded for the representation of expected faces, both the graded and high response models were significant, while the u-shape model was not. By contrast lower-level face-sensitive regions like occipital face area (OFA) and fusiform face area (FFA) did not code for expected faces. This result confirms that when scene priors that evoke face expectations (but do not contain images of faces themselves) are presented, predictions and their strength are indeed selectively represented in higher-level regions instead of the entire processing hierarchy.Once faces were actually encountered, only the OFA revealed expectation-dependent coding, again both the graded and high response models were significant. This increase in similarity of representation pattern for presented faces which were highly expected was interpreted by the authors as sharpening in perceptual features, consistent with sharpening theory.Finally, univariate analyses for presented faces uncovered a U-shape pattern in FFA, showing increased activity for both highly expected and unexpected faces. This observation was interpreted to suggest the simultaneous occurrence of two processes in lower-level face-sensitive regions: enhanced processing of highly expected faces and heightened processing of prediction errors for unexpected faces.According to PC theory, human brains constantly generate predictions about forthcoming experiences. This PC theory has gained substantial influence in explaining various cognitive processes such as perception, learning, and memory . PreviouWhile acknowledging the limitations of small effect sizes and the use of right-hemisphere-restricted regions of interest, as discussed in the paper, we present several potential avenues for future directions.First, the authors found increased similarity of multivariate patterns in OFA for observed faces that were highly expected, which was interpreted as sharpened facial features. According to sharpening theory, neural activity becomes more selective to preferred features after image repetition, resulting in increased similarity in representations of the repeated image. However, it is worth noting that more similar representations can also be accounted for by PC theory, which explains the activation through two distinct neural populations : (a) neuOne possibility involves separating the similarity matrix for highly and lowly expected faces. According to PC theory, lowly expected faces generate greater prediction errors, leading to greater activity and pattern similarity for lowly expected faces than highly expected faces. Directly testing this prediction can be accomplished by using the data presented in this paper or through future studies. Another way to test prediction error units is to measure laminar responses with high spatial resolution fMRI, for example, at 7 T. According to PC theory, decreased activity and pattern similarity for repeated/expected images are represented by prediction error units which are located at superficial layers.The second consideration, which has also been acknowledged by the authors, is the potential impact of participants engaging in mental imagery of highly expected faces during the 4- to 8-second interval between cues (scenes) and targets (faces). This mental imagery process could significantly influence the representational patterns of scenes, activating mental representations of faces. Consequently, this might affect the similarity between the presented faces in the localiser scan and the scene presentations in the test sessions. Moreover, participants may exhibit varying degrees of engagement in mental imagery, leading to increased variability of activation patterns within their brains. An intriguing avenue for future studies is to investigate whether our brains automatically generate predictions during all stages of perception and if so, when this predictive process occurs. Techniques with high temporal resolution, such as electroencephalography (EEG) and magnetoencephalography (MEG), offer the potential to answer this question.Another promising direction for future research is to explore PC theory in terms of the connectivity between brain regions. Although the authors have demonstrated that higher-level regions encode predictive information, it would be valuable to investigate whether and how this information is propagated from higher-level to lower-level regions. To accomplish this, future studies could employ multivariate connectivity measures such as informational connectivity , dynamicIn summary, the study conducted by


One of the most common cognitive problems that arises during the course of human senescence is a progressive decline in memory. Elders who otherwise live independent and robust lives often complain of forgetting or misplacing items as part of their daily activities. This is not a great revelation for any individual who interacts with an older family member, friend, or acquaintance. On face value, this may be considered one of the vagaries of aging. In fact, age-related memory loss has been the fodder of entertainment and literature for millennia. However, a deeper evaluation reveals many nuances regarding memory loss and potential opportunities to improve peoples' lives with mnemonic preservation.Memory encompasses many modalities, including acquisition, encoding, processing, and recall, among others Squire, . Any oneRigorous and reproducible studies in live humans, postmortem human brains, and relevant animal models of aging and memory, including precise neuroanatomical tracing, neuroimaging, molecular, cellular, physiological, and behavioral studies have identified the hippocampal formation as the principal hub for mnemonic functions  often associated with pathological aging, including AD, in a healthy aging memory clinic cohort of elders employing the Mild Behavioral Impairment Checklist (MBI-C) tool. The authors found the entorhinal cortex was associated with total score MBI-C performance. Moreover, in this nondemented cohort, NPS is associated with medial temporal lobe atrophy, which indicates the MBI-C may be useful for early identification of individuals with NPS who are at a higher risk for dementia.In an original article, Koehl et al., in an original article investigated the impact of Vangl2 on neurogenesis and memory in middle aged mice. Through the study of a Vangl2 mutant model construct, the authors demonstrate normal Vangl2 expression impacts granule cells born in adults and is a key regulator of cognitive flexibility. These provocative preclinical results suggest targeting Vangl2 may be useful in attenuating age-related memory decline.Leveraging recent findings that Vang-Gogh like 2 (Vangl2), a key constituent of the planar cell polarity (PCP) signaling pathway, is localized to the adult murine dentate gyrus, Zhou et al., evaluated five exercise regimens, aerobic training, combined training, high-intensity interval training, resistance training, and aerobic training plus resistance training, on serum levels of the neurotrophin brain-derived neurotrophic factor (BDNF) in healthy aging and diseased cohorts. Bayesian network meta-analysis of 39 randomized controlled trials with 2,031 participants rank ordered resistance training as the optimal exercise approach for both children and elders in terms of sustaining serum BDNF levels to support CNS-related benefits.In a systematic review, Mukli et al., in an original article, tested the hypothesis that gait variability and gait asymmetry are reliable predictors of cognitive decline in elders with cerebral small vessel disease (CSVD). Cognitive performance, as measured by the Cambridge Neuropsychological Test Automated Battery (CANTAB) and gait were evaluated in a cohort of elders with CSVD. Results indicate that step lengths in individuals with CSVD had greater asymmetry than controls. Moreover, gait variability had an inverse association with sustained attention, most notably in individuals with CSVD. These data are supportive of the concept that gait variability and asymmetry are viable predictors of cognitive decline in elders with CSVD.The evidence presented in these articles complements the existing body of knowledge in the literature (Santín-Márquez et al., Frontiers in Aging Neuroscience is to increase awareness and interest in an underserved area with significant public health importance to the aging community writ large. It is our hope this special issue drives greater interest and encourages new investigators from other scientific backgrounds to begin studying aspects of memory dysfunction in aging and AD/ADRD.A general caveat regarding this Research Topic, “Hippocampal mechanisms in aging and clinical memory decline” is that it is not possible to articulate all of the basic, translational, and clinical research components driving memory decline in aging in the context of a circumscribed set of manuscripts on this broad topic. Rather, a stated goal of the Research Topic series in SDG and ST wrote and edited the manuscript and take responsibility data integrity of and the accuracy presented herein. Both authors contributed to the article and approved the submitted version.


Apis mellifera), since both are frequent recipients of chronic pesticide exposure. Clear linkages between pesticide mode of action and host-specific microbiome functionalities are identified in relation to potential antimicrobial risks. For example, inherent differences in nitrogen metabolism of plant- and insect-associated microbiomes may dictate whether neonicotinoid-based insecticides ultimately exert antimicrobial activities or not. Several other context-dependent scenarios are discussed. In addition to direct effects , pesticides may indirectly alter the trajectory of host-microbiome coevolution in honey bees via modulation of social behaviours and the insect gut-brain axis - conceivably with consequences on plant-pollinator symbiosis as well. In summary, current evidence suggests: (1) immediate action is needed by regulatory authorities in amending safety assessments for “non-antimicrobial” pesticides; and (2) that the development of host-free microbiome model systems could be useful for rapidly screening pesticides against functionally distinct microbial catalogues of interest.There is emerging concern regarding the unintentional and often unrecognized antimicrobial properties of “non-antimicrobial” pesticides. This includes insecticides, herbicides, and fungicides commonly used in agriculture that are known to produce broad ranging, off-target effects on beneficial wildlife, even at seemingly non-toxic low dose exposures. Notably, these obscure adverse interactions may be related to host-associated microbiome damage occurring from antimicrobial effects, rather than the presumed toxic effects of pesticides on host tissue. Here, we critically review the literature on this topic as it pertains to the rhizosphere microbiome of crop plants and gut microbiome of pollinator insects (namely managed populations of the western honey bee,  This includes, but is not limited to, the facilitation of marine and atmospheric biogeochemical processes, regulation of soil-plant nutrient cycling, and maintenance of healthy animal communities. Emerging ideologies such as “Planetary Health” and “OneHealth” emphasize these fundamental roles of microbial metabolic processes in supporting macroscopic reality at the systems-level, and further suggest that microorganisms should be viewed as unified constituents rather than as separate entities, as they have been historically regarded,3.Arguably, there is a microbial component inherent to all known systems on Earth with cumulative evidence supporting that niche-adapted microbial communities play unequivocally important roles in total ecosystem functioning which posits that host-microbe co-adaptation has driven functional interdependence between many animal species and their gut microbiomes . Exemplifying this interdependence, animal species frequently rely on their gut microbiomes for nutrition, pathogen exclusion, and immunomodulatory functions. The human gut microbiome has been well characterized in this regard, although there is substantial evidence from insect species too - including the western honey bee (Apis mellifera). In particular, this eusocial insect species relies heavily on the bacterial members of its gut microbiota as a result of depauperate immune and detoxification gene repertoires. Similar relationships exist between plant hosts and the microbe-dense soil zone surrounding plant roots, known as the rhizosphere (or “microbe storehouse”), that plays a multifunctional role in supporting plant growth and is a critical factor influencing crop yields in agriculture. The functional similarities between the gut microbiome of animals and the rhizosphere microbiome of plants have been discussed previously, as has the related theory of “the host microbiome as an ecosystem on a leash”.Consistent with these schemas is the holobiont (or hologenome) theory of evolution), and that many can exert unintentional antimicrobial activities that disrupt host-microbiome homeostasis. These activities include the microbicidal or microbiostatic properties exhibited by various herbicides, insecticides, and fungicides, which together constitute over 95% of all pesticides used worldwide. To note, these effects are often unforeseen  and are not adequately monitored by regulatory agencies since most pesticides are classified as “non-antimicrobial” chemicals. It is thus conceivable that widespread extinction of plant and animal host-adapted microbes may already be occurring, undetected, as a result of chronic sub-lethal pesticide exposures . Nonetheless, it is difficult to ascertain the extent of damage, since baseline host-associated microbiome data is often lacking.Here, we draw attention to the neglected fact that anthropogenic activities (primarily those over the past century relating to farming practices) have introduced an astonishing number of pesticides and other agrochemical xenobiotics into the environment  and excessive use of disinfectants . Importantly, these reductions in microbial diversity are directly associated with altered functionality of the gut microbiome, and are thought to represent a major instigating factor behind the growing global epidemic of chronic, non-communicable, metabolic disease. Such metabolic disorders include irritable bowel syndrome, type-2 diabetes, obesity, atherosclerosis, and several types of cancer. Thus, while the use of antibiotics and disinfectants have undoubtedly revolutionized clinical healthcare and tremendously reduced the spread and lethality of infectious diseases, persistent exposure to antimicrobial agents may pose significant long-term health complications.We can gain some insight into the long-term consequences of microbiome damage from industrialized human societies that have undergone a systematic depletion in host-adapted microbes due to transgenerational antibiotic exposure  on the basis of their proclivity to encounter pesticides, their unsustainable colony loss over the past decade, and their importance to agriculture and global food security.An analogous scenario could be the case for the effects of pesticides, which have revolutionized the agricultural industry  but may pose serious risks to wildlife metabolic health and long-term environmental sustainability. Previous reports have exhaustively described the consequential physiological effects of pesticides on off-target plant and animal tissuecrobiome . Specifi. It is important to note, however, that compliance with regard to ecological assessments extends only to “non-target plants, fish, and wildlife species” without any legislative guidance provided for microorganisms.The Environmental Protection Agency (EPA) of the United States broadly classifies pesticides to be any chemical compound utilized for the purpose of killing crop pests that interfere with agricultural production - most commonly referring to that of herbicides, insecticides, and fungicides. A longstanding issue surrounding the use of pesticides, however, is the off-target deleterious effects they can have on a broad range of species found in terrestrial and aquatic ecosystems. In efforts to make sound regulatory judgements about these risks, authoritative bodies worldwide have attempted to implement minimum data and safety information requirements in relation to a given pesticide product’s potential for causing unreasonable adverse effects. For example, the EPA’s regulation “Data Requirements for Registration”  specifies that risk assessments for the registration of new pesticides must evaluate the Ecological Risks, Human Health Risks, and Environmental Accumulation Risks. In nearly all cases, though, these criteria are nullified by the presence of additional claims , which result in the product  being classified as a “non-antimicrobial pesticide”. Perplexingly, agricultural fungicides are also considered a type of “non-antimicrobial pesticide” despite their registered intent as antimicrobial chemicals targeting fungal species. Legislative loopholes in classification such as these present a major concern as they obscure scientific communication and largely ignore the potential health hazards that common agrochemicals pose on plants and animals through interactions with their host-associated microbial communities.Only recently in 2013 did the EPA promulgate the final rules on data requirements (revised part 158 W) to provide distinct jurisdiction for “antimicrobial” and “non-antimicrobial” pesticides under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). Many pesticides with potential antimicrobial properties are nonetheless still registered as “non-antimicrobial” products  as the result of mutually exclusive categorization schemas and a long list of exemptions. For example, an “antimicrobial pesticide” is defined under section 2 (mm) of FIFRA as any pesticide designed to disinfect, sanitize, reduce, or mitigate the growth of bacteria, viruses, fungi, protozoa, algae, or slime moldIt is foreseeable that any chemical, in great enough quantity, could impede cellular biological function. Thus, the peak concentration and type of exposure , as well as dose-dependent effects, are important considerations when evaluating the off-target antimicrobial effects of pesticides. Discussion in this review will accordingly focus on the antimicrobial mechanisms of insecticides, herbicides, and fungicides at environmentally realistic exposures. A brief in-text summary is provided for each of the relevant mechanisms shown in A site of the gamma-aminobutyric acid (GABA) chloride ionophore complex, which ultimately causes paralysis and/or death via dysregulation of nerve cell membrane polarization. GABA is notably the most common inhibitory neurotransmitter in both vertebrate and invertebrate systems, and is especially crucial to honey bee foraging and grooming. In situ investigations on OCLs demonstrate they can also have strong inhibitory effects on microbial growth and overall metabolic activities at the community level in soil,46. Data from in vitro culture-based studies on hundreds of soil bacterial isolates confirm these effects, showing inhibition of ~74%-100% of the tested Gram-positive strains when exposed to field-realistic concentrations of γ-hexachlorocyclohexane, bandane, chlordane, heptachlor, and other OCLs,47. Dichlorodiphenyltrichloroethane (DDT) is another well-known OCL with potent antibacterial properties, and under field conditions causes a ~60% decrease in active soil bacterial biomass and a ~93% increase in fungal biomass - the latter of which likely represents an indirect response to the former via reduced competition, rather than a stimulatory response to DDT since OCLs are notoriously recalcitrant to breakdown.The bulk of pesticide applications almost invariably reaches the soil, facilitating direct interaction with soil microbes . As a re. That is, the antimicrobial effects of OCLs were thought to be random and independent from their designed functions of inhibiting insect GABAergic signalling. Recent evidence, however, suggests that GABA signalling  plays a major role in cross-kingdom chemical communication and quorum sensing events that actively regulate bacterial-archaeal-fungal community structure. GABA has also been found to represent an essential bacterial nutrient in certain Gram-positive bacteria, such as the recently identified human gut isolate, Candidatus “Evtepia gabavorous”. Taken together, this suggests that the antimicrobial effects of OCLs are not random and may in fact be related to their inhibitory effects on GABAergic signalling. While OCLs are now banned in most countries due to their environmental persistence (facilitated at least in part by inhibition of their own bioremediation) and association with a broad range of other wildlife health concerns, they provide an exemplary account of host-microbiome interconnectedness and how intentional insecticidal properties can directly translate into unintentional microbicidal properties  is the responsible factor involved and can exert differential antimicrobial properties on the basis of EPSPS class I (sensitive) and II (resistant) binding affinities. The honey bee symbiont, Snodgrassella alvi, encoding a class I-type EPSPS has been reported to be consistently lower in abundance during exposure to glyphosate. Together with evidence of S. alvi-mediated immunoregulatory roles, this could potentially strengthen the otherwise somewhat obscure linkages between field-realistic glyphosate exposure and apparent susceptibility of honey bees to viral (Deformed wing virus) and fungal (Nosema ceranae) pathogens.Lastly, glyphosate (commonly known as “RoundUp”) is the most popular herbicide used worldwide for weed control but has been associated with extensive disruption to plant and animal microbiomesFusarium spp. in the plant rhizosphere microbiome by inhibiting plant symbionts which otherwise antagonize the growth of the pathogen. Moreover, the directly antifungal effects of glyphosate can impair mycorrhizal colonization and alter plant-soil nutrient cycling dynamics - an effect that can lead to long-term stunting of plant growth and a gradual reduction of crop yields. While some studies contest the microbiome-mediated stunting effects of glyphosate,73, a meta-analysis on the topic suggests that the phenomenon is dependent on soil pH differences, which may govern the microbial degradation rates of glyphosate to its inactivate metabolite, AMPA. Collectively, the current literature indicates that glyphosate (and many other types of herbicides) can directly exert unintentional antimicrobial effects on plant- and animal-associated microbial communities, and that these changes can consequently impair host developmental processes and disease resistance.Glyphosate can similarly increase the prevalence of root rot-inducing . One such topic of controversy is the administration of neonicotinoids, which are designed as “systemic pesticides” intended for uptake and distribution within plant tissue - the goal being to maximize target pest exposure while minimizing environmental contamination. However, neonicotinoids also accumulate in the plant root exudate and pollen (in the case of angiosperm plants), meaning that the rhizosphere microbiome and the gut microbiome of pollinating insects  are the primary recipients of chronic off-target exposure. Nonetheless, studies on the antimicrobial effects of neonicotinoids have been inconsistent across the literature.Neonicotinoid-based insecticides, which induce neurotoxic effects via selective inhibition of insect nicotinic acetylcholine receptors, are widely popular as a result of their very low toxicity towards humans, but have faced much scrutiny with regard to their controversial association with declining pollinator populationset al. elegantly demonstrated that there were no obvious impacts on genus-level bacterial diversity metrics (measured via 16S rRNA gene sequencing) after five days of in-hive exposure. In the same study, in vitro exposure of 16 honey bee gut-derived bacterial isolates showed essentially no sensitivity (nor degradation ability) of the strains towards imidacloprid in mono- or mixed-culture experiments. Despite these seemingly conclusive findings, a follow-up study testing longer exposure durations  found that imidacloprid (and thiacloprid) exerted a time-dependent decrease in both bacterial and fungal community alpha diversity, with the most significant changes occurring after five weeks. Empirical evidence also suggests that clothianidin, nitenpyram, thiamethoxam, and other types of neonicotinoids can exert bee microbiome-disrupting side effects during longer periods of chronic exposure, although the responsible mechanism remains unclear.In considering the effects of imidacloprid (a common neonicotinoid) on the honey bee microbiome, Raymann ) act to reduce host-mediated selective pressures on microbial communities. This notion is supported by the fact that antimicrobial peptides (AMPs) and other effector molecules produced by the insect innate immune system  possess crucial microbiome-shaping properties via their differential activities against phylogenetically distinct microbial lineages. For example, the honey bee AMP, apidaecin, demonstrates magnitudes lower activity against Gram-negative symbionts  compared with Gram-negative opportunistic pathogens . A noteworthy point to highlight is that host-adapted symbionts possess unique functions, that in return, help shape innate immune signaling. That is, the persistence of host-adapted microbial communities is a bidirectional process, and thus this may functionally explain how immune dysregulation by neonicotinoids could (indirectly) result in a loss of microbial diversity over time under realistic scenarios of chronic exposure. It is also foreseeable how these effects could exacerbate the loss of microbial diversity during concurrent exposure to chemicals that do possess antimicrobial capacities, such is the case for antibiotics and fungicides - the latter of which is consistent with results showing a near doubling of the apparent honey bee mortality risk over a four-month period during neonicotinoid co-exposure. Collectively, the current literature suggests that the microbiome-disrupting effects of pesticides are not always as clear as direct inhibition, and in the case of neonicotinoids in bees, appear to be mediated indirectly via host immune dysregulation [One explanation could be that the immunosuppressive effects of neonicotinoids  could foreseeably have a higher probability of interacting with plant-associated microbial communities. In the case of rice (Oryza sativa) crops, imidacloprid has been found to decrease diversity metrics in the rhizosphere microbiome, with impact severity shown to be both dose- and duration-dependent. Thus, this could potentially imply an indirect plant-defense mediated response  given that imidacloprid (and thiamethoxam) can significantly reduce plant immune-related gene expression. Nonetheless, laboratory studies on sandy soils  instead suggest that neonicotinoids can have a direct inhibitory effect on nitrifying organisms, specifically reducing that of ammonia-oxidizing archaea and bacteria in a species-specific manner. These findings are supported by other soil experiments also showing a dose-dependent decrease in important soil enzymatic activities  alongside a ~37% reduction in culturable fungi after 30 days of imidacloprid exposure. Given that neonicotinoids can persist in soil for > 1000 days in some cases, these effects could foreseeably disrupt long-term microbial homeostasis.The potential direct antimicrobial effects of neonicotinoids cannot be ruled out based on bee microbiome studies alone. Nicotine represents a plant-produced signaling molecule known to interact with soil bacteria and fungiin vitro studies of soil isolates with neonicotinoid-degrading properties. Interestingly, many of these isolates are nitrifying or N2-fixing bacteria such as thiacloprid-degrading Microvirga flocculans, thiamethoxam-degrading Ensifer adhaerens, imidacloprid-degrading Pseudomonas putida. Moreover, in each of these cases, as well in fungal degradation of imidacloprid by Aspergillus terreus, the breakdown is directly coupled with growth impairment of the metabolizing strain - suggesting that neonicotinoid metabolites  are the responsible antimicrobial factors involved, rather than the parent compounds. Other examples exist with at least 40 neonicotinoid-degrading isolates identified so far, mostly from soil or water environments (see review). Notably, the preferential impact of neonicotinoids on plant growth-promoting N2-fixing bacteria appears to have long-term adverse outcomes in chickpea and soybean crop yields,96, although data from corn crops show inconsistencies. While these differences could potentially be attributable to variation in soil parameters  known to influence the behaviour of neonicotinoids,99, there is, overall, a lack of literature on the topic and further studies are needed before any solid conclusions can be drawn.Overall, very little mechanistic work has been done to understand the antimicrobial effects of neonicotinoids, with the bulk of literature focusing on . It is nonetheless interesting to consider how insect-mediated detoxification of neonicotinoids  could have insidious effects on the gut microbiome through activating the antimicrobial effects of these compounds. This has yet to be tested and would be a worthy direction for future studies. To note as well, microbial biotransformation-dependent toxicity of pesticides is not a unique process to neonicotinoids  and 3,5,6-trichloro-2-pyridinol  [-105. Together, these findings provide a basis to speculate on how “biopesticides”  could be favorably utilized in tandem with pesticides to increase their insecticidal properties by in vivo biotransformation to more toxic metabolites - an approach that has been proposed in several emerging microbiome management strategies for agroecosystems.Unlike animals, which rely on their diet for nitrogen via protein consumption, plants absorb nitrogen through their roots, often with the help of their associated microbiomes. This might explain why honey bee-associated microbial communities cannot degrade neonicotinoids and appear to be largely unaffected by their presence in cultureoids see . Symbionrobiome) . FurtherOverall, the discussed material cumulatively highlights three major points: (1) plant- and animal-associated microbiomes intrinsically differ in their sensitivity towards certain pesticides; (2) microbiome-mediated biotransformation of pesticides can produce antimicrobial metabolites from otherwise non-antimicrobial parent compounds; and (3) strategic modulation of host-associated microbiomes in agricultural systems has potential to offset adverse pesticide interactions.A. mellifera) as an example, individual worker bees, which number in the tens of thousands per colony, forage separately for pollen and nectar. However, their industry is not selfish - instead, they return their foraged goods to the hive where it is concentrated into honey and pollen stores and ultimately fed to developing larvae. The social foraging and otherwise colony-oriented behaviour of worker bees thus naturally concentrates any trace environmental contaminants from afar into higher, localized concentrations that can then accumulate and in some cases reach toxic levels. Honey bees and other eusocial insects with this type of centralized foraging are thus vulnerable to bioaccumulation of neonicotinoids and other applied contaminants. The direct effects of pesticides on honey bee physiology have been intensely investigated but less is known about the indirect effects that likely arise from pesticide-mediated microbiome disruption and the downstream effects that this dysbiosis can have on individual and social behaviour.The antimicrobial effect of pesticides on pollinating insects can be amplified to a systems level through indirect effects on foraging and other social behaviours . Using t consisting of 8-10 species clusters within the genera Gilliamella, Snodgrassella, Bombella, Lactobacillus, Apilactobacillus, and Bombilactobacillus. This community is remarkably consistent across environments, suggesting a strongly co-adapted symbiosis is crucial to the maintenance of bee health and immunity. For honey bees and other social insects in which individual behaviour has become integrated into a whole, the composition of symbiotic gut microbes can influence not only the behaviour of individual insects but also the collective behaviour of entire societies in which they live. The brain-gut-microbiome axis is one mechanism by which gut microbiomes can influence the individual performance and social behaviour of workers within the hive. Perturbation of this axis via pesticide-mediated depletion of core microbiome members should therefore affect bee behaviour in predictable and potentially manageable ways. For example, antimicrobial effects on bee gut microbiomes may alter foraging behaviour via individual performance or dietary preference, which when amplified across all individuals and colonies, can impact pollination services.Unique among insects, honey bees (as well as bumble bees) possess a “core” gut microbiome structure. Underpinning this phenomenon could be the substantial fluctuation in gut microbiome structure known to occur between winter and summer seasons. There is ample opportunity for feedback between host and microbiome if, for example, initial changes to microbial communities then bias foraging preference to influence the plant-associated microbes the bees are exposed to. Subtle behavioural changes  could thus hinder natural plant-pollinator microbial exchange processes and influence the long-term maintenance of co-adapted symbionts. Some remediation may be possible through the application of beneficial bacteria that off-set the dysbiosis inadvertently caused by the well-intended application of commercial antibiotics or pesticides to hives.Interestingly, the learning performances of honey bees are differentially affected by imidacloprid according to the season, we expect any significant variation in the gut microbiome to affect the bee’s most fundamental behaviours, including recruitment, hygienic, defensive and appetitive behaviours - all of which are essential to colony’s eusocial structure. For humans, communication along the brain-gut axis is mediated through immune mechanisms, elements of the nervous system and microbial metabolites that relay nutrition and health status from the gut to the brain. For insects and bees in particular, the mechanics of this axis are less well defined, however, because social insects have evolved a particularly strong dependence upon gut symbionts, there is a strong rationale for investigation of this topic in future studies.Given that bee social behaviour is highly coordinated, where the worker caste can specialize into behavioural subcastes that, besides foragers, include nurses, guards, hygienists, undertakers and scouts towards pesticides may consequently impact microbial response to antibiotics through both generalizable and specific mechanisms. This represents a major human health concern in relation to the rise of multidrug-resistant pathogens (or “superbugs”) and hospital-acquired infections that are increasingly difficult to treat.Pesticides, like antibiotics, represent chemical stressors that can exert selective pressures on microbial communities. Mounting evidence suggests that the evolution of tolerance, resistance, and persistence), which are membrane-bound transporters that can export multiple toxic substrates out of the cell. For example, Kurenbach et al. found that pre-exposure of E. coli to two herbicides (glyphosate and dicamba) could significantly increase subsequent tolerance against two broad-spectrum antibiotics (chloramphenicol and kanamycin) via overexpression of the AcrAB efflux pump - an effect that failed to occur in the presence of efflux pump inhibitor Phe-Arg β-naphtylamide. Others have also reported that biocide usage selects for overexpression of efflux pumps. Alternatively, a study on realistic co-exposure of 23 pesticides in E. coli demonstrated that streptomycin-resistance emerged rapidly via selected for mutations in acrR  as well as biofilm, heat shock, oxidative stress defense, and carbon starvation genes.One mechanism of overlapping resistance is through efflux pumps  can confer multidrug resistance by inactivating chloramphenical, monochrotophos, ampicillin, cefotaxime, streptomycin and tetracycline antibiotics. The noteworthy point is that similar or identical OPHs have been found in Pseudomonas, Flavobacterium, Sphingobium, and Agrobacterium spp.-132, with molecular evidence suggesting the response genes have probably evolved within the past 70 years. This coincides directly with the introduction of organophosphate insecticides and the global rise of antibiotic resistance, and thus it is interesting to speculate how these processes have co-impacted the evolutionary trajectory of microbial life. Similar comparisons can be made for several oxidoreductases, transferases, and lyases in terms of conferring overlapping pesticide and antibiotic resistance properties.In the case that pesticides cannot be pumped out of the cell, intrinsic or acquired enzymatic functions can facilitate overlapping resistance to antibiotics. For example, the plasmid-encoded organophosphorus hydrolase (OPH) of insecticide-degrading Several important areas have been highlighted in this review that deserve future scrutiny. These are summarized below:(1) Currently, regulatory oversight of agrochemical usage is inadequate and fails to address potential effects on ecosystem microbiomes which are in turn critical to environmental health. A reassessment of the legislative framework that governs the use of agrochemicals is urgently warranted.vs. benefit of agrochemical use.(2) Agrochemical toxicity is generally defined as a construct of their direct and acute harmfulness towards plant and animal species, whereas their detrimental effects on plant- and animal-associated microbiomes are likely to have more subtle, accumulative consequences. Long-term studies of plant and animal health  following exposure to agricultural compounds are required to allow balanced calculation of risk (3) Microbial biotransformation of agrochemicals is understudied and requires urgent evaluation if we are to fully understand the impact of a given compound on the environment. Relatedly, bioremediation efforts need to account for potential adverse effects of breakdown metabolites on not only plant and animal physiology, but also their host-associated microbial communities.(4) Antimicrobial resistance is a current global threat to health, and it is imperative that the role of agrochemical use in the development of antimicrobial resistance is fully studied and appreciated.(5) The gut-microbiome-brain axis is an emerging field of interest with relevance to pesticide-neuroimmune interactions and merits particular attention in eusocial bee species, such as honey bees, which exhibit strong interdependencies on their gut microbiomes.in vivo observation alone [in vitro can be extremely informative, although not all microbes are culturable and pesticide co-culture experiments with single strains are not adequately representative of the highly complex polymicrobial interactions that may occur under natural conditions .In addressing these issues as they relate to microbiome-mediated pesticide toxicity, proof of causality is an important factor to consider. Multi-omics technologies have massively improved our ability to identify taxonomic- and functional-based correlations of host-associated microbial communities in response to agrochemical exposures. However, demonstrating an association between pesticide exposure and microbiome change is not enough to authenticate an antimicrobial effect. As discussed in this review, it remains challenging (due to confounding host variables) to delineate between direct antimicrobial properties and indirect microbiome disrupting activities based on on alone . Indeed, and SHIME systems) and are actively being used to decipher microbe-drug interactions relevant to human disease treatments. Notably, these systems may be easily adapted for agricultural purposes as well, specifically allowing for high-throughput evaluation of microbe-pesticide interactions pertaining to honey bee health, commercial crop yield, and environmental health as a whole. Future development and testing of such models should be an immediate priority based on the fact that current pesticide risk assessment strategies could also benefit from their use by improving the veracity of new product safety claims prior to their release.Collectively, this indicates a substantial need for model development of host-free rhizosphere and insect gut microbiome systems. Bioreactor models for the human gut microbiome already exist  physiology. This is not easy to do, since most microbial ecosystems are highly complex, and so much of the microbial world remains unstudied. However, tools to study microbiomes and their functions are now increasingly accessible, and should be exploited to study microbial ecosystem modulation by herbicides, insecticides and fungicides across all agricultural sectors as a matter of great urgency.


Spinal cord injury results in multiple, simultaneous sensorimotor deficits. These include, but are not limited to, full or partial paralysis of muscles below the lesion, muscle spasms, spasticity, and neuropathic pain. Bowel, bladder, and sexual dysfunction are also prevalent. Yet, the majority of emerging spinal stimulation-based therapies focus on a single issue: locomotor rehabilitation. Despite the enormous potential of these translational advances to transform the lives of people living with spinal cord injury, meaningful recovery in other domains deemed critical priorities remains lacking. Here, we highlight the importance of considering the diverse patterns of neural transmission that underlie clinically similar presentations when developing spinal stimulation-based therapies. We also motivate advancement of multi-modal rehabilitation paradigms, which leverage the dense interconnectivity of sensorimotor spinal networks and the unique ability of electrical stimulation to modulate these networks to facilitate and guide simultaneous rehabilitation across domains. The sensorimotor consequences of spinal cord injuries (SCI) are wide ranging and intensely individual . UnfortuRecent and emerging technological advances in spinal electrical stimulation systems have positioned the neurorehabilitation field to capitalize on so-called “smart”, or person-specific, neuromodulatory therapies. But the full potential of precision neuromodulation is unlikely to be realized without a dedicated focus on certain key areas of research. Here, we motivate the neurorehabilitation and neural engineering fields to consider some of the basic scientific and translational areas we feel are critical for strategic growth of this exciting new application of spinal stimulation.We begin by revealing the diversity of intraspinal neural transmission in a ubiquitous pre-clinical model of SCI, highlighting the pitfalls of implicitly assuming homogeneity across animals (or people) when developing neuromodulatory therapies to enhance rehabilitation. We then focus on the promise of closed-loop spinal electrical stimulation paradigms, including the importance of therapies that promote beneficial neural plasticity and afford multi-modal therapeutic benefits. And finally, we discuss the need for increasing our understanding of network-level sensorimotor dynamics, both in the neurologically intact spinal cord and following SCI.It is well documented that impaired voluntary motor control is a canonical feature of SCI, owing to reduced neural transmission in motor pathways descending from the brain to regions of the spinal cord below the lesion. In contrast, spinal responses to sensory feedback often become pathologically increased below the lesion, resulting in hyperreflexia. Overactive spinal responses to sensory feedback also contribute to SCI-related neuropathic pain (SCI-NP) in ∼40%–70% of people living with incomplete SCI , 3. PhysBeyond a general understanding that each individual's clinical sequela is somehow related to their unique injury profile, few neural mechanisms have been identified that consistently predict a person's symptom severity, the extent to which they will respond to a given therapy, or their overall recovery potential. As a result, there is often a paucity of actionable mechanistic information with which to guide therapy. This lack of knowledge has far too often led to a one-size-fits-all approach to interventions and a high degree of variability in therapeutic efficacy across individuals.Given the diverse sensorimotor consequences of SCI, it is reasonable to wonder how the research portfolio of the neuromodulation field compares. A recent search of the National Library of Medicine PubMed database returned 820 manuscripts since 2000 describing spinal stimulation-based approaches for enhancing motor recovery after SCI . By compin vivo  differentially modulates neural transmission across these animals. In the face of such profound heterogeneity, and with the knowledge that the clinical efficacy of currently available spinal stimulation paradigms is not broadly generalizable, what strategies can the neurorehabilitation and neural engineering fields employ to address the unmet need for precision neuromodulation? Of the myriad possibilities, the following three interrelated areas seem ripe for consideration.rehabilitate lost function rather than replace lost function.First, the field could more readily focus on closed-loop neuromodulation. In closed-loop paradigms, delivery of spinal electrical stimulation is contingent upon and synchronous with detection of a functionally relevant neural event. The goal of this approach is to enhance the intrinsic ability of the central nervous system to reorganize and repair by restoring and reinforcing natural patterns of neural transmission in spinal networks below the lesion. It is distinguished from open-loop stimulation, in which the delivery of stimuli is not correlated with a specific neural event. For example, ISMS of motor pools associated with forelimb extension can be synchronized with the detection of volitional attempts to recruit those muscles, evinced from motor unit action potentials in the target muscles . This apBiomechanical events can also trigger spinal stimulation in a closed-loop paradigm. Reduced yet detectable movements of the ankle, knee, and hip have been used to trigger epidural stimulation of spinal segments associated with producing those movements for locomotion , 10. TheOptimization of closed-loop spinal stimulation paradigms is not trivial, however. Indeed, even small amounts of electrical current delivered directly to spinal motor pools can lead to widespread modulation of neural transmission over a region far exceeding that which would be predicted from direct current spread alone , 11. ThiSpinal stimulation paradigms currently available clinically, as well as those currently undergoing clinical testing, are parameterized for one consequence of SCI alone. Overwhelmingly, reports of new spinal stimulation paradigms and technologies focus on enhancement of locomotion . Yet, as aforementioned, a multitude of sensorimotor consequences affect people living with SCI. Although locomotion is routinely rated amongst the top rehabilitation priorities of people living with paraplegia following SCI, it is rarely ranked as a greater priority than bowel, bladder, or sexual function .via recruitment of sensorimotor reflex pathways. The potential for such an effect is illustrated in Amelioration of pain is frequently noted as a priority for individuals living with SCI-NP. And although spinal stimulation paradigms for management of non-SCI-related chronic pain are clinically available, they have yet to gain traction for SCI-NP. Interestingly, though, it has recently been demonstrated in pre-clinical models that ISMS parameterized to enhance voluntary motor output concurrently reduces nociceptive neural transmission integral to the development and persistence of SCI-NP . HoweverA key challenge both for pain-related  and multi-modal (broadly) applications is determining how to configure such systems for closed-loop operation. Finding the optimal trigger sources for stimulation will be essential. For systems parameterized only to enhance voluntary motor output, these sources are somewhat straightforward. But for a phenomenon as enigmatic and multi-faceted as pain, considerably more basic scientific discovery is warranted. It is encouraging to see that clinical research in this area is beginning to emerge for non-SCI-related applications , as presAs a final note on the development of multi-modal rehabilitation platforms, we strongly encourage researchers and clinicians already pursuing epidural spinal stimulation paradigms—especially those intended to enhance motor output—to incorporate into their studies assessments of off-target effects including sensation and pain. Because ISMS requires additional time to move through the translational pipeline, determining the extent to which epidural spinal stimulation could also serve as a viable candidate for providing multi-modal therapeutic benefits would be an invaluable resource. Given the wealth of pre-clinical and clinical work already ongoing in the epidural spinal stimulation space and the existence of many efficient, validated instruments for assessing sensation and pain, this would seem to be a straightforward way to meaningfully advance the field.Finally, an essential step in moving towards smart, more efficacious, spinal stimulation paradigms is elucidating how patterns of intraspinal neural transmission relate (or not) to action and perception. As highlighted throughout We suggest that a more detailed characterization of network-level patterns of intraspinal neural transmission associated with movement, pain, or other functions is a tractable yet physiologically rich entry point. There is much to be gained in this area by adapting to the spinal cord the concept of functional connectivity, now well-established in supraspinal networks. For example, it has recently been reported that seemingly spontaneous discharge patterns and associated intraspinal functional connectivity may be non-random and physiologically purposeful  , 6. In FIt is indeed an exciting time in the neuromodulation field, with the pace of technological development and translation seemingly accelerating daily. But along with this rapid pace comes the need to renew our focus both on the diverse, multi-faceted priorities of people living with SCI and on our conceptualization of the spinal cord as a dynamic, integrated system wherein no single function can be modulated in isolation. With these considerations in mind, the promise of precision neuromodulation and multi-modal rehabilitation may become a reality sooner rather than later.