Despite significant improvement in the rates of acute allograft rejection, proportionate improvements in kidney allograft longevity have not been realized, and are a source of intense research efforts. Emerging translational data and natural history studies suggest a role for anti-donor immune mechanisms in a majority of cases of allograft loss without patient death, even when overt evidence of acute rejection is not identified. At the level of the donor and recipient genome, differences in highly polymorphic HLA genes are routinely evaluated between donor and recipient pairs as part of organ allocation process, and utilized for patient-tailored induction and maintenance immunosuppression. However, a growing body of data have characterized specific variants in donor and recipient genes, outside of HLA loci, that induce phenotypic changes in donor organs or the recipient immune system, impacting transplant outcomes. Newer mechanisms for “mismatches” in these non-HLA loci have also been proposed during donor–recipient genome interactions with transplantation. Here, we review important recent data evaluating the role of non-HLA genetic loci and genome-wide donor-recipient mismatches in kidney allograft outcomes. Kidney transplantation is the preferred treatment for patients with end stage kidney disease owing to the survival advantage it confers, compared to maintenance dialysis. Despite success in reducing acute rejection episodes, long term allograft survival has remained an elusive goal and a major research focus for the community , 2. WhilIt is well-known that donor–recipient (D–R) genetic mismatches at the human leukocyte antigen (HLA) region are directly associated with acute renal allograft rejection. For this reason, the organ allocation process has been geared towards precise typing of donor and recipient HLA for organ allocation. In conjunction with patient-tailored induction and maintenance immunosuppression based partly on HLA-matching, this has led to a dramatic improvement in episodes of acute rejection and overall short-term allograft outcomes. However, instances of acute antibody-mediated rejection (AMR) still occur in well-matched and HLA-identical transplants , 7. In fvia the direct pathway (donor major histocompatibility complex [MHC] plus peptide on donor cells), indirect pathway (donor‐derived antigens presented by recipient antigen presenting cells [APC]), or the semidirect pathway (presentation of self‐peptides by donor MHC on recipient APC via membrane transfer). Fundamentally, alloreactivity  is based on specific peptide/MHC differences between the host (recipient) and donor cells giving rise to a classical adaptive immune response. At the level of the genome, the processes that recognize the donor organ as non-self and result in acute organ rejection (AR) are determined by differences in the human leukocyte antigen (HLA) region between the donor– and recipient (D–R) pair or HLA-mismatches. Indeed, AR itself has been repeatedly shown to be associated with decreased allograft survival  pair of genomes contains vast permutations of non-synonymous amino-acid differences that can serve as potential triggers of alloimmune responses even outside of mismatches at the highly polymorphic HLA locus. Several data have now interrogated non‐HLA mismatches in multi-ethnic and heterogenous renal allograft cohorts in a quantitative and genome‐wide basis –19. ThesHowever, novel mechanisms have also been invoked, without predicted donor-recipient (D–R) peptide dissimilarity or polymorphic peptide production , 22. Firvia genome‐wide association studies (GWAS) in D–Rs have thus associated novel genetic loci with graft outcomes  , 33. Recoutcomes –37. Thesoutcomes . Besidesoutcomes . This isoutcomes . Recent outcomes .exonic, intronic, or intergenic. Exonic variations in non-HLA regions may be non-synonymous or missense changes altering the resultant amino-acid, and in turn impacting protein structure/function and potentially conferring unique biologic properties. Here, alternate proteins coded by donor variants could promote anti donor non-self, classical immune responses. However, these loss- or gain-of-function variants could impact donor renal cell structure/function or in recipients, modulate immune responses, without directly altering immunogenicity of the donor organ. Non-coding variants may affect regulation of gene expression without impacting protein sequences (expression quantitative trait loci or eQTL) or alter gene splicing. Indeed, regulatory non-coding variants could themselves also induce loss or gain-of function. Consistently, a summary of GWAS reported that near 80% of GWAS-identified loci for complex traits localize to noncoding regions  have been recognized as an important predictor of chronic kidney disease and end stage kidney disease in individuals of African ancestry . Kidney outcomes . Thus APoutcomes . An ongooutcomes . Since aoutcomes , the APODonor ABCB1 polymorphisms have been associated with long-term kidney function among kidney transplant recipients who received CNI-based regimen . A GWAS via recruitment of rho kinases to facilitate apical myosin contraction (In GWAS efforts to identify susceptibility loci for chronic kidney disease (CKD), loci in SHROOM3 have repeatedly emerged as associated with CKD using either cystatin or creatinine-based equations, especially in Caucasian-predominant cohorts. In a prospective cohort of kidney transplants, we studied the impact of the top ranked Shroom3 locus from CKD-GWAS, rs17319721, on allograft histology and survival. Shroom3 is an actin binding protein with roles in epithelial morphogenesis traction , 52. We traction  associattraction . The rectraction . In thisIntriguingly, the same Shroom3 locus was also associated with reduced albuminuria in CKD populations . A similOther donor Variants: Caveolin-1 (CAV1) is the primary component of caveolae, membrane invaginations that are abundant in endothelial cells, promote cellular transport and regulate signal transduction . An intrSeveral excellent reviews have tabulated associations of donor and recipient SNPs with allograft outcomes , 59, 60.In the first recipient-only GWAS of 326 patients, O’Brien et al. were unable to find any loci of genome-wide significance but did find associations between SNPs on chromosomes 14 and 18 with medium-term serum creatinine and long-term graft survival. However, these findings failed to be validated by a subsequent study .Another recipient-only study  looked a−5. In three large validation cohorts , recipients who were homozygous for the deletion-tagging allele (G/G) had an 84% higher risk of rejection than A/A or A/G. Functionally, the rs893403-G risk allele associated with lower LIMS1-mRNA expression in renal tubulo-interstitium. LIMS1 is a cytoplasmic protein normally expressed in the human distal nephron and endothelium and with a role in cell-adhesion and integrin signaling. Interestingly, LIMS1 expression was further induced by ischemia with appearance of cell-surface LIMS1. The authors then demonstrated the presence of anti-LIMS1 IgG in a subgroup of high-risk D–R pairs with AR.Steers et al. in their investigation of the concept of ‘genomic collision’ identified the LIMS1 locus as associated with allograft rejection in a time-to-event analyses. Genomic collision refers to a loss of function variation in nonessential genes in the recipient that triggers an alloimmune response to the normal variant expressed in the donor leading to poor allograft outcomes. In the innovative approach used in the first phase of their study, they focused on CNV-tagging SNPs that would associate with loss or reduced copy-numbers when homozygously inherited by the recipient . In theiA subsequent study by Caliskan et al. (841 recipients) examined LIMS1 rs893403- risk alleles in a single center study for the outcome of any rejection . Using aAs discussed above, the presence of APOL1 risk variants in the donor and association with outcomes in well-established. However, Lee et al. were the first group to examine the presence of APOL1 risk alleles in African American recipients of kidney transplants and their impact on transplant outcomes. In their cohort of 119 patients, 48% were carriers of two APOL1 risk alleles but no association was found between the presence of any number of risk alleles and allograft loss or death-censored allograft loss . The donEx vivo transcriptome data obtained from peripheral blood mononuclear cells (PBMCs) pre-transplant and from healthy controls, showed a clear signature of immune activation in CD4, CD8, and NK cells in patients with APOL1 G1 or G2 variants vs G0, implicating APOL1 risk alleles with immune activation in these cells. These provocative data will be examined in the ongoing nation-wide APOLLO study and lay the ground for mechanistic work to understand the immunomodulatory role of APOL1.In contrast, in a recent work, we re-evaluated the role of APOL1 variants in the recipient in determining allograft outcomes . Two traAs observed in the data described above, several donor-only or recipient-only GWAS reported either lack of discovery of loci of genome-wide significance or, lack of independent validation of previously identified loci. Several important reasons exist for these predominantly negative findings, namely, the lack of large sample sizes, heterogeneity of phenotypes tested , and insufficient accounting for baseline ancestry-based variation. In addition, transplantation involves an interaction of two independent genomes, and resulting mismatches have complex mechanisms and potentially could have larger effect sizes than conferred by donor- or recipient variants alone. In the next section, we discuss locus-specific mismatches  by the development of unique non-HLA antibodies, and genome-wide mismatch data in kidney transplantation.Major histocompatibility class I-related chain A (MICA) is a surface glycoprotein expressed on endothelial cells, epithelial cells, fibroblasts and other cells, but not expressed on peripheral blood lymphocytes. Following transplantation, exposure to allogeneic MICA can result in formation of antibodies. These antibodies would not be detected by traditional cross-matching techniques, as they are not expressed on peripheral-blood lymphocytes. It was proposed that these antibodies would contribute to allograft rejection as MICA antigens expressed on endothelial cells can be cytotoxic in the presence of serum complements.A study by Zou et al. included 1,910 kidney transplant recipients who received deceased donor kidneys and measured IgG against MICA antigens . Of the de novo development of AVA after transplantation suggesting either auto-immune or allo-immune mechanisms for AVA generation.Antigens expressed on endothelial cells, could be considered to be the “first responders” in the setting of inflammation and ischemia-reperfusion injury. Vimentin is an intermediate-filament protein expressed by endothelial cells and regulates cellular cytoskeletal structure, cell signaling and proliferation. In both heart and kidney transplant recipients, anti-endothelial cell antibodies have been identified, including anti-vimentin antibodies. Anti-vimentin antibodies (AVA) may be produced after tissue injury or infections, situations wherein vimentin is expressed on cell surfaces and “seen” by the immune system . In thesDelville et al. evaluated kidney transplant recipients with acute microvascular rejection (AMVR) within 3 months post-transplantation who did not have anti-HLA DSA identified (on Luminex single antigen bead assay and biopsy) . They mePerlecan is a vascular basement membrane proteoglycan that helps maintain endothelial integrity. Pathogenic antibodies targeting Perlecan were eluted from a Fisher-to-Lewis rat transplantation model . Subsequvia vasoconstriction or vasodilation. Binding of ETAR by antibodies to ETAR (anti-ETAR) results in sustained activation (Endothelin-1 type A receptors (ETAR) are G-protein coupled receptors that are expressed widely in the human body, including renal vascular smooth muscles. Activation of ETAR by endothelin 1 regulates blood flow tivation , 75. Alltivation . In peditivation .Kamburova et al. assessed the presence of 14 non-HLA antibodies with allograft survival using a customized non-HLA antibody assay in a nationwide dataset of 4,770 recipients. In recipients of deceased donor kidneys, they found presence of autoantibodies to Rho GDP-dissociation inhibitor 2 (ARHGDIB) was associated with graft loss. They also found a 13% lower death-censored graft survival at 10-years post deceased donor transplants among recipients with these antibodies, independent of anti-HLA antibodies . The assConsistent across these and mechanistic data  is the nIn 2005, Dragun et al. studied 33 patients with steroid-refractory acute allograft rejection in a landmark study . They evFirst, the association with graft survival of anti-AT1R levels even before transplantation is demonstrable , albeit Next, an extension of anti-AT1R mediated injury to non-kidney organs and a more generalized role for this autoantigen in allo-transplantation is also identifiable , 88. HowRecent data has described the “classical” phenotype of anti-AT1R associated AMR, with a higher prevalence of hypertension, more vascular injury and arterial inflammation, higher levels of endothelial-associated transcripts, and lack of complement deposition in allograft capillaries . Indeed,In the Steers et al. paper, the signal for increased risk of TCMR was enriched when donors with A/A or A/G introduced into G/G recipients were considered vs all other G/G recipients. These findings were validated across 3 cohorts where donor genotype was available. This intriguing finding suggested that while the risk of increased rejection may primarily travel with having G/G genotype in recipients, transplantation of A-allele donor kidneys made this risk manifest for unclear reasons. Furthermore, these data suggested a “directionality” for rs893403 mismatch which associated with worse outcomes when A-allele was introduced into G/G recipients, and not vice versa. Within a subgroup of this cohort, the authors identified the presence of anti-LIMS1 antibodies in rejection cases with high-risk direction mismatch only, suggesting that a classical adaptive immune response is involved in these findings. Since the intronic rs893403 variant does not induce a polymorphic or generate dissimilar peptides, the overall mechanism underlying these exciting findings needs further definition.It must be observed that while most of these studies demonstrate associations of specific non-HLA antibodies with rejection or graft survival, mechanistic data testing causation in animal models are limited. Further, current commercial Luminex or ELISA panels for these targets have been used only in research settings and are not licensed for use in clinical practice. AT1R assays are exceptional in this regard and frequently utilized in clinical practice.via Killer cell immunoglobulin-like receptors (KIRs), which deliver inhibitory signals to NK cells preventing NK-activation and injury  during graft rejection is the histologic hallmark of antibody mediated injury. Using 129 kidney transplant recipients, Koenig et al. demonstrated that histologic evidence of microvascular inflammation was noted independent of the presence of circulating antibodies in half of the cases . They pod injury . KIRs and injury . KIRs ard injury . In suchIn another large study of 924 kidney transplant recipients , missingThe same group investigated whether missing self-amplified DSA-dependent NK cell activation in chronic AMR and explained some of the heterogeneity in AMR outcomes. In 135 kidney biopsies suggestive of complement-independent chronic AMR, those that had MS among D–Rs, had worse overall allograft outcomes (P = 0.02) . ComplemTranscriptomic signatures of renal allograft biopsies have also revealed enrichment of the FCGR3A (CD16) transcripts correlating with DSA and ABMR . In addiThe first ever genome-wide association study (GWAS) that combined the analysis of kidney donors and recipients was undertaken by the Wellcome Trust Case Control Consortium (WTCCC) . They anAlthough a negative study, the first donor–recipient GWAS highlighted the limitations of small study populations at single transplant sites and the complexity of donor and recipient covariates and disease-related phenotypes. As such, the International Genetics & Translational Research in Transplantation Network (iGeneTRAiN) consortium has been established to minimize limitations due to population size . This isMesnard et al. devised an allogenomics mismatch score based on all possible cell surface antigen mismatches between D–R pairs based on amino-acid mismatches in transmembrane proteins . This stvia exome sequencing and outcomes of biopsy-proven rejection versus no rejection on 28 D–R pairs  cohort, we identified a novel correlation between quantitatively estimated recipient ancestry estimated by ADMIXTURE, and post-transplant outcomes. To estimate genome-wide differences, specifically in intra-ancestry donor–recipient pairs, we utilized the proportion of genome-shared identity by descent variable (pIBD) measuring relatedness between any given D–R pairs in this cohort. We observed that this pIBD variable which included coding and non-coding variants significantly associated with long-term death censored graft loss . It mustThe increasing focus towards improving long-term allograft outcomes has spurred investigations to understand the full extent of anti-donor responses that culminate in allograft injury and organ failure after transplantation. These efforts have expanded from an HLA-centric view to a broader examination of genetic variants in the donor and recipient genomes and “mismatches” thereof. These new and emerging data show a clear role for genome-wide mismatches (independent of the HLA region) in rejection and/or long-term renal allograft outcomes. However, there still remain questions regarding the application of these findings to clinical scenarios. Using the TRIPOD checklist benchmarks  for evalPJ and AR performed literature review and wrote the manuscript. LB reviewed and edited the section on non-HLA antibodies. MM was responsible for drafting, editing and organizing the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.MM acknowledges funding from the NIH DK122164, and past pilot funding from the CTOT-19 study  to study non-HLA donor-recipient genetic differences.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite recent advances in skull base reconstructive techniques, including the multilayer technique during the last decade, complete reconstruction of grade 3 intraoperative high-flow cerebrospinal fluid (CSF) leak remains challenging. This study was designed to investigate the role of injectable hydroxyapatite (HXA) used in the multilayer technique on the clinical outcome of skull base reconstruction for intraoperative high-flow CSF leak.This study enrolled 187 patients who experienced intraoperative high-flow CSF leak after endoscopic endonasal surgery for anterior skull base or suprasellar pathologies between January 2014 and July 2021. All skull base defects were reconstructed using the conventional multilayer technique including a vascularized naso-septal flap  and the combined use of HXA with the conventional multilayer technique . We retrospectively evaluated the efficacy of the HXA group by 1:2 propensity score matching analysis.Overall, 17 of 187 patients (9.1%) showed postoperative CSF leaks, resulting in second reconstruction surgery. There were no statistical differences in patient age, sex, body mass index, tumor location, tumor type, and degree of resection, except for the follow-up period between the two groups. The HXA group showed a significantly lower incidence of postoperative CSF leak than the control group . Postoperative lumbar drain (LD) was performed in 8.7% of the HXA group compared to 46.1% of the control group (p < 0.01). CSF leak-related infection rates showed a decreasing tendency in the HXA group compared to the control group . A total of 46 patients in the HXA group were well matched with the control group (92 patients) at a 1:2 ratio. In the propensity score-matched control group, there were higher rates of postoperative CSF leaks than in the HXA group.The use of HXA combined with the conventional multilayer technique completely reduced postoperative CSF leaks in this study. This technique resulted in reduced CSF leakage, even without postoperative LD, and decreased infection rates. Further randomized comparative studies are required to confirm our findings. Endoscopic endonasal surgery is now recognized as an essential surgical tool in the skull base field as its indications have expanded into more complex lesions –3. RegarSince the vascularized naso-septal flap (NSF) method was introduced by Hadad et al., the incidence of CSF leak has been remarkably reduced . HoweverHydroxyapatite (HXA) is a suitable material for covering bone defects owing to its biocompatibility and favorable tensile properties , 17. SinThe inclusion criteria in this study were patients with anterior skull base or suprasellar non-pituitary tumors who underwent endoscopic endonasal surgery with intra-arachnoidal dissection during tumor removal. These patients received a multilayer reconstructive technique, including an NSF with or without LD for intraoperative high-flow grade 3 CSF leak. The degree of intraoperative CSF leak was assessed using the grading scale suggested by Esposito et al. . BetweenDemographic, clinical, and surgical data of the study subjects were collected and reviewed. The degree of each resection was analyzed as “subtotal” (less than 95% of tumor resected), “near-total” (over 95% but less than 100% of tumor resected), and “gross total” (100% of tumor resected) . Clinica®, Integra LifeSciences, New Jersey, USA). Then, an acellular dermal graft with a thickness of 1 mm  was laid over as an on-lay dura graft after being tailored according to the size of the sellar bone defect. The mucosal membrane surrounding the bony defect was completely removed, and the bony surface was in contact with HXA and NSF as much as possible. Before injecting the HXA, it was important to maintain the dry condition of the bony surface. Injectable HXA  was applied over the surrounding bony area to fully cover the bone defect. At this step, we identified no intraoperative CSF leak from the reconstructed graft site 5–10 minutes after injection. An NSF was covered on the above grafts, and as a result, the HXA was not exposed to the nasal cavity  were inserted into the nasal cavity to compress and stabilize the graft. The key points of the reconstruction procedure are demonstrated in Details of endoscopic endonasal surgery have been described previously , 26. Alll cavity 2. FinalIn contrast, for the control group, the buttonhole method using either a fascia lata graft or allodermis, consisting of inlay and on-lay double layers, was used after plugging a collagen matrix into the arachnoid defect. In some cases, a septal bone graft was placed along with the gasket technique for bony defects. Then, the defect site was fully covered using a pre-prepared NSF that was already harvested at the beginning of the operation; it was also verified that the margin of the NSF was completely grafted on the denuded skull base. Pieces of nasal packing material were also inserted to compress and stabilize the graft.After surgery, three antibiotics  were administered intravenously for 3-5 days in all patients. Postoperative follow-up examinations were performed in all patients to monitor for CSF leaks. Rhinological examination was performed on the second and fifth postoperative days. The packing materials, such as Merocel in the nasal cavity, were removed, and then the reconstructed skull base was evaluated with an endoscope to verify the viability of the NSF and to check for CSF leaks. Postoperative imaging was routinely performed within 1 day after surgery to identify the degree of the tumor resection and the integrity of the NSF  was used for all statistical analyses. For categorical variables, data were expressed as frequencies and percentages. The chi-square test and Fisher’s exact test were used to analyze categorical variables using contingency tables. For continuous variables, data were expressed as the mean ± standard deviation. The mean differences between the HXA and control groups were analyzed using Student’s Propensity score matching was undertaken to compensate for group-wise imbalances in baseline characteristics that had the potential to skew the outcomes. Score matching of each patient, which reflected each patient’s age, sex, body mass index (BMI), lesion location, and degree of resection, was conducted using the 1:2 optimal matching method in the Matchit package of R version 4.0.4. The caliper width was set to 0.1 times the standard deviation of the logit of the propensity score to prevent poor matches. The quality of each match was assessed by comparing selected variables in propensity score-matched patients using the standardized mean difference, in which an absolute standardized difference of greater than 10% is suggested to represent a meaningful covariate imbalance.2. Craniopharyngioma was the most common pathology (129 cases), followed by meningioma (49 cases), germinoma (3 cases), epidermoid cyst (2 cases), dermoid cyst (1 case), teratoma (1 case), neurofibroma (1 case), and pleomorphic xanthoastrocytoma (1 case). Regarding the surgical location, 46 lesions (24.6%) were at the anterior skull base, and 141 lesions (75.4%) were in the suprasellar region. Ten patients (5.3%) had a history of endoscopic endonasal surgery. Ninety-six (51.3%) patients underwent gross total resection, 54 (28.9%) had near-total resection, and 37 (19.8%) had subtotal resection of the target lesion. The mean follow-up period was 34.5 ± 26.8 months  underwent a multilayer reconstruction using NSF with or without LD, and 46 patients underwent a multilayer reconstruction with both NSF and HXA . The mean BMI was 24.8 ± 4.5 kg/m8 months Table 1.Postoperative CSF leak occurred in 17 of 187 patients (9.1%). In the 17 patients with CSF leak, dehiscence of non-vascularized constructs such as fascia graft  and detachment or displacement of NSF  were the main reasons for failure. The median time interval to CSF leak was 10 days . Autologous tissue grafts were performed in 119 of 187 patients (63.6%). Postoperative LD was prophylactically performed in 69 patients (36.9%). CSF leak-related infection occurred in 10 patients (5.3%). Bacterial meningitis was confirmed in six patients (3.2%), fungal meningitis in two patients (1.1%), and ventriculitis in two patients (1.1%). One case (0.5%) was a mortality case in which a patient with bacterial meningitis, followed by fungal meningitis, progressed to a septic condition Table 2.p < 0.001) (p = 0.015) and increased need for autologous tissue grafts . Additionally, in contrast to the HXA group, wherein there was no CSF leakage (even among the patients for whom postoperative LD was not performed), there was CSF leakage in 16.7% of patients in the control group without postoperative LD (p = 0.004). The CSF leak-related infection rate was lower in the HXA group than in the control group (0 vs. 4.4%); however, statistical significance was not observed (p = 0.151).Propensity score matching (1:2) created a total of 46 matching sets . After matching, covariates were well balanced between the two groups, with a standardized mean difference of less than 10% for all selected variables (< 0.001) Table 1.To prevent CSF leak after surgery, several methods that combine autologous, allogeneic, and artificial materials have been implemented for skull base reconstructions –29. The However, LDs have several disadvantages that must be balanced against their advantages. The recommended duration of LD maintenance varies between studies, and no consensus has been reached , 13. In ®) can be applied using a syringe and can be used in the skull base , HXA, and an NSF. They reported postoperative meningitis without cultured microorganisms in 13.5% of patients who underwent reconstruction  to confirm the effectiveness of our reconstruction method. We defined a “high-flow CSF leak” as a situation requiring intra-arachnoidal dissection and violation of a ventricle or cistern, which requires a more robust skull base reconstruction. As this was an inclusion criterion, extra-arachnoid lesions, such as pituitary adenoma or Rathke’s cleft cyst, were excluded from patient selection. In addition, skull base defects after operations on posterior fossa tumors may not be entirely covered by the HXA and NSF due to the extent of this type of large bone defect. Therefore, additional reconstruction and CSF diversion are required in these cases, and inconsistent reconstruction methods in a case-by-case manner may affect the accurate comparison of CSF leak results in this study. Thus, non-pituitary tumors of the skull base region requiring intra-arachnoid dissection, except for lesions in the posterior fossa, were selected as the inclusion criteria for our study.Reconstruction after endoscopic endonasal surgery remains a long-standing challenge. Our multilayer technique using HXA and NSF effectively prevented CSF leakage and associated fatal infections. Although the decision for or against LD placement is made by the surgeons, it is worth noting that there was no CSF leakage in 42 of the 46 patients in the HXA group who did not undergo postoperative LD. Compared with the conventional method, our method prevented CSF leakage while reducing the frequency of postoperative LD. In addition, it has the advantage of reducing harvest site morbidity by decreasing the need for autologous tissue grafts. HXA, which can harden immediately within minutes, can advantageously create a barrier that can be relied upon when non-vascularized constructs such as fascia lata or NSF are being organized with surrounding tissues. Possibly due to the inlay and on-lay coverage of HXA, there were no HXA-associated complications, such as delayed infection, graft protrusion, fistula formation, or crust formation , 43, 45.This study has some limitations. This study was designed as a retrospective study rather than a prospective randomized method, which may have led to bias due to the inherent nature of the study design. Also, data were collected from a single institution by a single surgical team. There was a difference in the follow-up period between the control group and the HXA group due to the modifications in the reconstruction method during the investigated period. The mean follow-up period of 7.5 ± 4.1 months in the HXA group might not be sufficient to detect delayed complications. In addition, analysis of skull base defect size could not be performed because of the retrospective nature of the study. Since this factor may be closely related to the CSF leakage rate, further analysis is required. The use of HXA may make revision surgery challenging; however, this aspect could not be analyzed in this study. Multicenter studies with a prospective randomized design and those that include larger patient cohorts and longer follow-up durations will help to verify the clinical utility of the reconstruction method presented here.The multilayer technique using HXA and NSF provided a reliable reconstructive outcome for high-flow CSF leaks after endoscopic endonasal surgery. This technique effectively prevented CSF leakage while reducing the demand for postoperative LDs and lowering the infection rate.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the institutional review board of Samsung Medical Center. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.SHL: Methodology, Data curation, Resources, Validation, Investigation, Formal analysis, Visualization, Writing - original draft. CMH: Data curation, Resources. SDH: Data curation, Resources, Validation. JWC: Supervision. HJS: Supervision. DHN: Supervision. JIL: Supervision. DSK: Conceptualization, Methodology, Data curation, Resources, Validation, Project administration, Writing - review & editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein, the micro interface between two immiscible electrolyte solutions (ITIES) is exploited for the simultaneous electropolymerization of 2,2′:5′,2′′-terthiophene (TT) and reduction of Cu2+ to Cu nanoparticles (NPs) generating a flexible electrocatalytic composite electrode material. TT acts as an electron donor in 1,2-dichloroethane (DCE) through heterogeneous electron transfer across the water|DCE (w|DCE) interface to CuSO4 dissolved in water. The nanocomposite formation process was probed using cyclic voltammetry as well as electrochemical impedance spectroscopy (EIS). CV and EIS data show that the film forms quickly; however, the interfacial reaction is not spontaneous and does not proceed without an applied potential. At high [TT] the heterogeneous electron transfer wave was recorded voltammetrically but not at low [TT]. However, probing the edge of the polarizable potential window was found to be sufficient to initiate electrogeneration/electropolymerization. SEM and TEM were used to image and analyze the final Cu NP/poly-TT composites and it was discovered that there is a concomitant decrease in NP size with increasing [TT]. Preliminary electrocatalysis results at a nanocomposite modified large glassy carbon electrode saw a > 2 × increase in CO2 reduction currents versus an unmodified electrode. These data suggest that this strategy is a promising means of generating electrocatalytic materials for carbon capture. However, films electrosynthesized at a micro and ~ 1 mm ITIES demonstrated poor reusability.Cu nanoparticles (NPs) have been shown to be excellent electrocatalysts, particularly for CO Motivated to reduce production cost, less expensive and involved preparation methods are being sought. For example, many polymerization methods generate relatively high average molecular weight materials that are robust; however, require electrodeposition onto an anode5, often binding the polymer to the electrode surface, or uses specialized bulky methods such as electrospinning6. In the case of the former, this likely eliminates the possibility of obtaining a free-standing film/conductive polymer electrode as the polymer is difficult to liberate from the anode and thus can limit the type of applications.Owing to their flexibility9; particularly copper (Cu) based NPs which are effective in catalyzing CO2 reduction13. Many metal NP preparation methods have emerged; however, the Brust-Schiffrin method, first described in 199415, reproducibly generated low dispersity Au NPs by exploiting the interface between two immiscible electrolyte solutions (ITIES), i.e., the liquid|liquid interface. Indeed, the ITIES has recently come under increasing activity in the electrodeless synthesis of both metal NPs27 and conductive polymer films36. Initially, efforts were focused on the immiscible water|oil (w|o) interface36; however, these have recently expanded to the water|ionic liquid (w|IL)26 and oil|ionic liquid (o|IL)20 ones. In a simple 2-electrode configuration with one electrode immersed in either phase, the Galvani potential difference can be controlled externally via a potentiostat with the potential drop spanning 1–4 nm across the ITIES, ϕw – ϕo = 37.Meanwhile, metal nanoparticles (NPs) form the basis of numerous analytical and electrocatalytic platforms38 were the first to describe an analytical solution for nucleation of metal NPs at the liquid|liquid interface. In their work, they emphasized the absence of defect sites that are common at a solid/solution interface; thus, there is a large thermodynamic barrier to particle formation at ITIES. Nevertheless, they38 and others40 were able to experimentally demonstrate electrochemically controlled metal NP nucleation at w|o interfaces. Interestingly, Nishi’s group has suggested that the molecular structure of the liquid|liquid interface is transcribed onto the NP framework22. They recently demonstrated that the w|IL interface played an important role mechanistically in the formation of nanostructures. Their IL was modified with a ferrocene (Fc) functional group making it redox active, and was exploited in the formation of Pd nanofiber arrays22.Johans et al.36, and more recently by Scanlon’s group4 and ourselves28. In these later reports, large, free-standing polymer films were formed. In the case of Lehane et al.4 who generated PEDOT ) using Ce4+ in aqueous and EDOT in α,α,α-trifluorotoluene (TFT), the films were shown to be highly stable and biocompatible. Our work showcased the simultaneous electropolymerization of 2,2′:5′,2′′-terthiophene (TT) and electrogeneration of Au NPs at a micro-ITIES (25 µm in diameter)26, building on Cunnane’s study at a large ITIES32. We demonstrated that miniaturization of the ITIES could be used to provide another layer of mechanistic and thermodynamic control towards smaller, low dispersity NPs.Meanwhile, electropolymerization at liquid|liquid interfaces was initially investigated by Cunnane’s group2 reduction; however, films electrosynthesized at the 25 µm and 1.16 mm interfaces demonstrated poor stability and surface coverage.Herein, this is expanded to the simultaneous electrogeneration and electropolymerization of Cu nanocluster incorporated poly-TT films. Electrochemical impedance spectroscopy was used to monitor film growth, while SEM and TEM imaging were used to compare film/NP morphology at different TT concentrations between the large and micro-ITIES. Two large ITIES platforms were investigated, including a 1.16 and 10 mm diameter interface. Initial testing of glassy carbon (GC) electrodes modified with the nanocomposite demonstrate excellent electrocatalytic activity towards CO4, > 98%), lithium sulphate , 1,2-dichloroethane , 1-bromooctane (99%), trioctylphosphine (97%), and 2,2′:5′,2′′-terthiophene  were acquired from Sigma-Aldrich. All reagents were used without additional purification. Ultrapure water from a MilliQ filtration system (> 18.2 MΩ cm) was used throughout to generate aqueous solutions. The tetraoctylphosphonium tetrakis(pentafluorophenyl)borate (P8888TB) ionic liquid used as an oil phase supporting electrolyte was prepared as detailed previously41.Copper sulphate  in four-, three-, and two-electrode configurations at the large and micro-ITIES. In the 4-electrode mode a specialized 10 mm inner diameter cell with two Pt wires annealed into the side of the glass cell were connected to the working (WE) and counter electrode (CE) leads of the potentiostat as shown in Fig. 42. Scheme 42– transfer, whose formal ion transfer potential 16.When using the micro-ITIES in two-electrode mode, one Ag wire (Goodfellow Inc.) was integrated into the specialized pipette holder containing the aqueous phase and connected to the WE port of the head-stage, and another Ag wire immersed in the organic phase was connected to the CE/RE port, see Fig. A second large ITIES electrolytic cell  was created by using the modified holder shown in Fig. Electrochemical impedance spectroscopy (EIS) measurements were performed with a frequency range between 10 and 20 kHz, as well as a 20 mV peak-to-peak perturbation. EIS was only measured using Cell 1 at a 25 µm ITIES in a 2-electrode configuration.Before or in-between experiments, electrolytic cells/capillaries were cleaned using the procedure outlined in the Supplementary Information (SI).Electrocatalysis studies were performed using a 3-electrode cell connected to a CH Instruments potentiostat (model# CHI602E) with a glassy carbon  WE (~ 4 mm in diameter) coupled with Ag/AgCl reference (Dek Research) and Pt wire counter electrodes.All transmission electron microscopy (TEM) images were taken using the Tecnai Spirit transmission electron microscope with samples prepared on 200 Mesh Cu ultrathin/lacey carbon or 2 µm holey Au grids (Electron Microscopy Sciences).https://www.nist.gov/services-resources/software/nist-dtsa-ii.SEM imaging was performed using a JEOL JSM 7100 F equipped with energy dispersive x-ray (EDX) which were analyzed via DTSA II software provided by the National Institute of Standards and Technology (NIST) in the US, see 4 in the aqueous phase. In each case, the polarizable potential window (PPW) is limited by the transfer of the supporting electrolyte ions. The large positive current increase at positive potentials is owing to the transfer of Li+/Cu2+ from water to oil (w → o) or TB– from o → w, while the sharp negative increase in current towards negative potentials is owing to the transfer of SO42– from w → o and P8888+ from o → w43.The black, dashed curves shown in Fig. i-V cycle at the micro-ITIES  at roughly 0.52 V. During the reverse scan, towards negative potentials, a sigmoidal wave was observed with a half-wave potential 44. Thus, this signal is consistent with the transfer of negative charge from o → w during the forward scan, such that the process is diffusion limited by a species in the aqueous phase. Therefore, it was hypothesized that this signal is owing to electron transfer from TT in DCE across the ITIES to Cu2+ in water; whereby, Cu2+ is reduced to Cu0 and forms nanoparticles, while TT is oxidized and electropolymerized.The red, solid trace shows the initial IES Fig. A, after i-V curves overlaid in Fig. ip) signal evolves with each consecutive scan. There is only a small change in ip that may be owing to a localized consumption at the interface, or the formation of a nanoparticle incorporated polymer film at the ITIES. The latter would fundamentally alter the effective surface area of the interface as well as its charge transfer characteristics; both would impact the magnitude of ip. Cell 1 was also tested using [CuSO4] = 0 mM and [TT] = 20 mM; however, no peak-shaped signal was recorded (data not shown). Thus, it is likely that Li+ does not interact with TT and is a good electro-inactive supporting electrolyte. However, when [TT] was decreased to 10 and 5 mM in DCE, no electron transfer wave was recorded voltammetrically (data not shown).To investigate this, the system was cycled a total of 25 times with these 4(aq) and TT(org)28; whereby, a thin conductive polymer film, with embedded Au NPs was formed. Lehane et al.4 also recently employed a large w|DCE interface in the electropolymerization of PEDOT with Ce4+ as oxidant/electron acceptor in the aqueous phase. Moreover, electrodeposition of Cu, Au, Pd, and Pt NPs at large45 and micro39 interfaces have been demonstrated previously using metallocenes as electron donors dissolved in oil.In all cases at the micro-ITIES, an aqueous droplet was ejected onto a TEM grid for imaging using the syringe equipped on the back of the modified holder. Even at 5 and 10 mM TT, a film-like deposit was visible on the substrate under an optical microscope. Therefore, despite no observable electron transfer wave, cycling at the PPW edge-of-scan can induce electrogeneration of the nanocomposite film. These results agree well with our recent work at the w|DCE micro-interface using KAuCl0 or anion adsorption waves. Future work will focus on in situ spectroscopic methods to evaluate these two curve features.An ITIES with a diameter of 1.16 mm using a 3-electrode configuration was also tested voltammetrically, see Fig. 4 The two signals at the positive end of the PPW are irreversible electron transfer similar to the result observed at the micro-ITIES in this potential region. With repeated sweeps of the potential, the irreversible electron transfer wave shifts to more negative potentials indicating a reduction in the required overpotential. As mentioned above, the liquid|liquid interface is initially free of nucleation sites which increases the amount of applied driving force necessary to achieve NP nucleation/polymerization38 versus at a solid/electrolyte one. However, once the film begins forming, the population of viable sites increases which is reflected in the concomitant decrease in peak potential of the electron transfer wave. Simultaneously, the signal decreases in current intensity which is likely owing to the consumption of material in the vicinity of the ITIES. The nanocomposite film formed in the large ITIES cell was thick, brittle, and difficult to extract from the cell. However, pieces were deposited onto glass substrates for SEM imaging and onto a GC electrode for electrocatalytic testing (see below).Next, film generation was investigated at a 10 mm diameter ITIES using Cell 2 4–, it is likely a minor contributor and most anion adsorption comes from SO42– on the aqueous side. Moreover, SO42– is molecularly smaller with exposed dense negative charge on the oxygens, doping the film with sulphate is more efficient in stabilizing and neutralizing the film as it forms. However, during the later stages of film growth, diffusion of anions through the film is inhibited making doping/de-doping a slow process and causing voltammetric peak broadening46. Regardless, the film will likely be p-doped.The voltammetric negative peak during the reverse scan in Fig. Both micro and large ITIES experiments were also performed at open circuit potential (OCP), i.e., without an applied external potential. In both cases, no film or NPs were observed. Thus, an applied potential is required to induce nanocomposite film formation.4 paired with 5 or 20 mM of TT in DCE and deposited on a holey-Au TEM grid. Next, the TEM grids were imaged using both TEM and SEM  spectroscopy performed during SEM imaging (data not shown). 24-h shake-flask experiments performed in a 2 mL vial (large ITIES) using the same electrolyte compositions and TT concentrations as Cell 2 revealed no observable thin film or Cu NPs. Therefore, while not strictly observable voltammetrically at [TT] = 5 mM, by probing the positive edge of the PPW one can initiate Cu NP/poly-TT electrodeposition at relatively low [TT]. Moreover, this can be achieved without the use of extreme overpotentials that risk over-oxidizing the film51.Figure 4] = 5 mM and [TT] = 20 mM after 1000 CV cycles. The film is smooth; however, the Cu NPs cannot be resolved with SEM. Fig. 4] = 1 mM and [TT] = 5 mM. The first and every subsequent fifth scan was plotted. The Cu2+/TT reduction/oxidation and Cu re-oxidation waves are visible at roughly 0.85 and 0.55 V, respectively. The Cu NP/poly-TT film was extracted from the cell and deposited on a glass slide, then imaged in the SEM. Fig. Figure 31, Robayo-Molina et al.52, and recently by us28. The early stages of TT electropolymerization via heterogeneous electron transfer and electrodeposition can be described generally by the following,+• is the radical cation, and TT2 is the dimer. This initial stage is likely thermodynamically prohibitive at the liquid|liquid interface since it lacks nucleation sites38. However, once seeded with Cu0 nuclei and the positively doped TT oligomers as capping agents, the thermodynamics likely greatly improve, as mentioned above. It should be emphasized that the glass walls of the micropipette likely behave as nucleation sites; this would also apply to the walls of large glass ITIES electrolytic cells. Using Eqs. .The overall electron transfer potential  for Eq.  can be wVAC) can be described by55,VDC and V0 are the applied DC voltage and AC voltage amplitude (0.020 V peak-to-peak), respectively, while ω (= 2πf) is the angular frequency and t is time. In each case, VDC of roughly 0.7 V was applied  in parallel.Next, electrochemical impedance spectroscopy (EIS) was employed during electrosynthesis at the micro-ITIES to elucidate the underlying physical and electrochemical dynamics of film formation. The sinusoidal applied potential waveform (RCT) and kinetic resistance (RC). As is common, a resistor was added in series to account for the total solution resistance (Rs). EEC1, a Randles-like equivalent circuit, and EEC2 feature the two terminals at either end for the WE and CE/RE employed experimentally. The 2-electrode configuration limits parasitic impedance artifacts from cabling and/or the CE and RE that are often observed at 3- and 4-electrode cells58. Operating at the micro-ITIES has added benefits and is able to resolve the charge transfer resistance over the solution electrolyte resistance; moreover, by repeatedly using the same micropipette, one can greatly enhance reproducibility.Equivalent electric circuits (EECs) used to model the impedance data have been drawn in Fig. Cgeo), recently modeled by von Hauff and Klotz55 in the context of perovskite solar cells, may be considered analogous to the structural parasitic coupling elements proposed and modelled by Trojánek et al.57 for a 4-electrode cell, which they modelled using a 4-terminal EEC. In either case, Cgeo is normally modelled in parallel with all the other circuit elements. If it exceeded the individual capacitive circuit elements, i.e., CPE1 and CPE2 in Fig. 59. Thus, Cgeo/parasitic coupling elements can be ignored, greatly simplifying EEC modelling61. The faradaic impedance (Zf) is described by57,kf is the apparent rate of charge transfer, A is the surface area of the interface, F is Faraday’s constant , R is the gas constant (8.314 J mol–1 K–1), T is the absolute temperature (298.15 K), z is the number of electrons transferred, and ZW is a Warburg impedance, within which, j2 = –1, and σ is defined as57,Di,o and i in the oil (o) phase, while subscript w indicates their values in the water phase. CPEs were used to account for the dynamic nature of the interface and facilitate the evolving properties of the growing polymer-NP network. CPE impedance can be written generally as62,EIS offers avenues to valuable physical insight into charge transfer processes at the liquid|liquid interface. The geometric capacitance (Q (F sn1–) is a constant and when n = 1 the element is a perfect capacitor, while n = 0.5 is in line with a typical Warburg element for semi-infinite diffusion.In which VDC = 0.7 V; whereby, the CV-EIS pulse sequence was performed a total of 11 times. Figure 4] = 5 mM, i.e., a blank spectrum. The pronounced low-frequency tail is likely owing to the relatively high VDC close to the edge of the PPW where supporting electrolyte ions will undergo transfer, e.g., Li+ w → o. This spectrum agrees well with one shown recently by Mareček’s group59 which was associated with simple tetraethylammonium ion transfer at a micro-ITIES.At a freshly cleaned micro-ITIES, a CV was performed followed by impedance measurement using a At low [TT] Fig. B the imp2+(aq) and TT(org) rather than simple ion transfer. Thus, as the film grows the electron transfer properties change (see below).Figure 28. However, as the film develops, and likely due to the build up of local micro-convections in this domain63, it was found that by pushing the impedance measurement to lower and lower frequencies the interface becomes unstable. This either lead to a higher noise level or the interface itself physically broke down, erupting into the organic phase as an electrophoretically induced droplet. Thus, it was not possible to carry out EIS measurements below 10 Hz.Nevertheless, these data indicate a change in the nature of the interface brought about by the development of a liquid|solid|liquid system that occurs immediately upon application of the first CV-EIS. It has been shown that forming a barrier at the interface mainly affects the low-frequency region4(aq) and TT(org), while EEC1 was used in the absence of TT, see the blue, solid curves in Fig. n varies between roughly 0.96 and 0.98, indicative of a conventional capacitor with likely an inhomogeneity in ion distribution at the interface.EEC2 Fig. D was empn value for CPE2, RCT, and RC increase slightly with [TT] = 5 mM and each CV-EIS pulse iteration, while all three parameters show a much larger increase at [TT] = 20 mM. RCT and RC are ½ and 2 × as high, respectively, when [TT] = 20 mM versus 5 mM. This agrees well with impeded electron transfer or ion diffusion through the growing polymer/NP network. The limited diffusion within the nanocomposite structure likely results in limited anion exchange to neutralize individual TT units contributing to its p-doped nature and accumulation of negative charge on the surface of the Cu NP/poly-TT composite. These values agree with those recently reported by us for Au NP/poly-TT nanocomposites electrogenerated at a micro-ITIES28 after multiple CV-EIS pulses were performed. Herein, the interface was monitored in situ using a CCD camera equipped with a 12 × zoom lens assembly with a 10–12 cm working distance; however, unlike the Au NP/poly-TT film grown previously28, the Cu NP/poly-TT nanocomposite film was clear/colourless and, therefore, no observable change was observed optically.The 3(aq) as supporting electrolyte. Figure 2 or CO2 gas for ~ 15 min prior to polarization.Preliminary electrocatalysis results were obtained by modifying the surface of a glassy carbon (GC) electrode with a layer of Cu NP/poly-TT film and using 0.1 M NaHCO2 saturated case, the cathodic peak at roughly – 0.45 V (vs. Ag/AgCl) is likely H+ reduction. However, this cathodic signal experiences a shift in the onset potential to – 0.57 V when purged with CO2 but maintains the same current intensity. The GC electrode modified with the Cu NP/poly-TT film electrosynthesized at a 1.16 mm ITIES shows a greater than 2 × CO2 reduction current at 0.75 V (vs. Ag/AgCl). To modify the GC electrode with a Cu NP/poly-TT composite at a 10 mm ITIES, the GC plug was dipped into the electrolytic cell after the 25 CV cycles were performed. Afterwards, the i-V response shown in Fig. At a bare GC electrode, and in the NFigure Films developed at the two large ITIES were smooth with a relatively even distribution of Cu NPs and no evidence of folding. Therefore, this phenomenon is likely owing to geometric confinement of the growing polymer film within the micropipette tip.After electrocatalysis, the Cu NPs in the films generated at the 25 µm and 1.16 mm interfaces showed large changes in NP morphology which have grown by orders of magnitude; therefore, the thin, polymer network in these cases is insufficient to protect them against aggregation/agglomeration. The film created at the 10 mm ITIES showed little change; however, further experimentation is required.While preliminary, these results are promising. Future work will focus on controlling Cu NP and polymer film morphology while tracking any changes the nanocomposite experiences during electrocatalysis, as well as detailed product analysis.4. Impedance data confirm that the nanocomposite film forms early through large changes in RCT and RC; moreover, increasing [TT] improves film formation while decreasing the median Cu NP size to < 2 nm.The successful application of a micro-ITIES towards electrodeless synthesis of Cu NP/poly-TT has been demonstrated and compared to films generated at a large  ITIES. At [TT] = 20 mM a well resolved electron transfer wave was observed at the micro-ITIES. However, the large interfaces showed a more complex CV profile with an irreversible electron transfer wave at high, positive potentials and a reversible signal towards the negative end. The latter is likely anion adsorption/exchange at the liquid|solid|liquid interfaces and agrees well with recent results by Scanlon’s groupInterestingly, while no electron transfer signal was observed at the micro-ITIES a low [TT], a film was electrogenerated and imaged using SEM. These data show that simply by probing the edge of the PPW one can facilitate electrodeless synthesis of the nanocomposite and avoid overoxidation of the polymer network.2 reduction current versus an unmodified electrode. However, this film underwent large changes in NP morphology. While a tentative first step, these results are indicators that these films are promising alternative electrode materials for carbon capture; however, more optimization of nanocomposite electrosynthesis is necessary.Preliminary voltammetric results at a GC electrode modified with the Cu NP/poly-TT film electrogenerated at a 1.16 mm diameter interface elicited a > 2 × enhancement in the electrocatalytic COSupplementary Information.


Balaji3, Joshua M. Hare4, Ranjith Ramasamy1Jesse OryJ Urol. 2022 Jun;207(6):1295-1301.DOI: 10.1097/JU.0000000000002437 | ACCESS: 35050717The evidence is inconsistent about the association between testosterone therapy (TT) and subsequent risk of cardiovascular events . AccordiIn this study, the authors tried to find the unsafe hematocrit threshold for men receiving TT and determine whether secondary polycythemia among men receiving TT causes an increased risk of major adverse cardiovascular events (MACE) and venous thromboembolic events (VTE). They performed a retrospective cohort study from a database of 74 million people including two groups of men with low testosterone who received TT and subsequently either did or did not develop polycythemia, and compared 5,842 men in each group. Polycythemia was defined as a hematocrit above 52%, in keeping with the AUA guideline definition. The primary outcome was incidence of MACE and VTE in the first year of TT. The authors found that men on TT who developed secondary polycythemia had a higher incidence risk of MACE and VET than men who did not develop polycythemia. Moreover, they reported that hypogonadal men on TT versus those off testosterone had similar rates of MACE/VTE in the absence of polycythemia . Baseline hematocrit was greater in the polycythemia group (47.4%) versus the non-polycythemia group (42.5%). Nevertheless, I congratulate everyone involved in this study, which is the first to establish secondary polycythemia from TT as an independent risk factor for MACE/VTE using a specific hematocrit-based cutoff. This cutoff can guide our clinical practice and we can tell patients undergoing TT that they are at a higher risk of MACE and VTE if their hematocrit reaches or exceeds 52% during the first year of therapy. Despite these findings, further studies are needed to confirm the association between TT dosage and patient adherence to secondary polycythemia and MACE/VTE.


In vivo studies have predominantly utilized transgenic models harbouring amyotrophic lateral sclerosis-associated gene mutations, which have not hitherto elucidated mechanisms underlying motor neuron death or identified therapeutic targets specific to sporadic amyotrophic lateral sclerosis. Here we provide evidence demonstrating pathogenic differences in CSF from patients with sporadic amyotrophic lateral sclerosis and familial amyotrophic lateral sclerosis patients with mutations in SOD1, C9orf72 and TARDBP. Using a novel CSF-mediated animal model, we show that intrathecal delivery of sporadic amyotrophic lateral sclerosis patient-derived CSF into the cervical subarachnoid space in adult wild-type mice induces permanent motor disability which is associated with hallmark pathological features of amyotrophic lateral sclerosis including motor neuron loss, cytoplasmic TDP-43 translocation, reactive astrogliosis and microglial activation. Motor impairments are not induced by SOD1, C9orf72 or TARDBP CSF, although a moderate degree of histopathological change occurs in C9orf72 and TARDBP CSF-injected mice. By conducting a series of CSF filtration studies and global proteomic analysis of CSF, we identified apolipoprotein B-100 in sporadic amyotrophic lateral sclerosis CSF as the putative agent responsible for inducing motor disability, motor neuron degeneration and pathological translocation of TDP-43. Apolipoprotein B-100 alone is sufficient to recapitulate clinical and pathological outcomes in vivo and induce death of human induced pluripotent stem cell-derived motor neurons in vitro. Targeted removal of apolipoprotein B-100 from sporadic amyotrophic lateral sclerosis CSF via filtration or immunodepletion successfully attenuated the neurotoxic capacity of sporadic amyotrophic lateral sclerosis CSF to induce motor disability, motor neuron death, and TDP-43 translocation. This study presents apolipoprotein B-100 as a novel therapeutic target specific for the predominant sporadic form of amyotrophic lateral sclerosis and establishes proof-of-concept to support CSF pheresis as a therapeutic strategy for mitigating neurotoxicity in sporadic amyotrophic lateral sclerosis.Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by motor neuron degeneration. Approximately 90% of cases occur sporadically with no known cause while 10% are familial cases arising from known inherited genetic mutations.  Using a novel CSF-mediated mouse model of sporadic amyotrophic lateral sclerosis, Wong et al. demonstrate that apolipoprotein B-100 induces motor disability and motor neuron degeneration. Targeted depletion of apolipoprotein B-100 in CSF via immunodepletion or CSF filtration attenuates its neurotoxic capacity, thereby preventing induction of disability and motor neuron degeneration.  Although major advancements in the ALS field have occurred with the identification of over 25 ALS-associated genes since the seminal discovery of the superoxide dismutase 1 (SOD1) gene in 1993,4 pathological mechanisms underlying the sporadic form of the disease remain unclear and therapeutic options are lacking. Successful disease-attenuating therapies tested in transgenic models harbouring ALS-associated gene mutations have failed to translate to the clinic, suggesting that genetic models may not necessarily reflect pathophysiological mechanisms underlying sporadic amyotrophic lateral sclerosis (sALS).6 A diverse array of mechanisms including oxidative stress, glutamate excitotoxicity, mitochondrial dysfunction, altered axonal transport, and RNA and protein processing have all been explored as contributors to disease pathogenesis in ALS, but whether these mechanisms have the same degree of importance in different ALS subtypes is unclear.7Amyotrophic lateral sclerosis (ALS) is a disease characterized by upper and lower motor neuron degeneration that is clinically inexorably progressive, with muscle weakness culminating in respiratory failure and death within 3–5 years.8–14In vitro studies have demonstrated induction of neuronal death within 24 h of incubation in ALS CSF,8 whereas various in vivo studies have reported pathological changes induced by intrathecal ALS CSF injections in neonatal rats,12 disruption of motor cortex neuronal activity, and motor impairments following intracerebroventricular infusions in adult rats13 and TDP-43 transgenic mice.14 However, these previous animal studies did not investigate potential differences in neurotoxicity of CSF from patients with sporadic or familial subtypes of ALS and the identities of neurotoxic candidates in CSF responsible for triggering ALS pathology remain unknown.Prior studies have reported neurotoxic properties of ALS CSF and established the feasibility of using ALS CSF to transfer disease pathology to animals.SOD1, C9orf72 and TARDBP using a novel CSF-mediated mouse model which can reliably detect motor impairments and pathological changes in the spinal cord induced by a single 3 µL injection of CSF into the cervical subarachnoid space. By performing CSF filtration studies to systematically eliminate components by molecular weight and unbiased global proteome profiling of sALS CSF pre- and post-filtration, we identified apolipoprotein B-100 (ApoB) as the neurotoxic agent responsible for inducing motor disability, motor neuron death, and other hallmark ALS pathology in the spinal cord. Importantly, we show that CSF filtration and targeted depletion of ApoB from sALS CSF prevents the induction of motor deficits and motor neuron degeneration, thus providing a novel therapeutic target and therapeutic approach to alleviate disease pathophysiology in the predominant sporadic form of ALS.In this study, we directly compared the neurotoxic capacity of CSF obtained from sALS patients and familial amyotrophic lateral sclerosis (fALS) patients with mutations in CSF was obtained from a total of 18 ALS patients, 11 of whom had sALS and seven had fALS . All of CSF samples were collected from patients with ALS, five multiple sclerosis patients as disease controls (DCs), and four healthy control (HC) volunteers following Institutional Review Board approval and informed consent according to the Declaration of Helsinki. None of the multiple sclerosis patients received any kind of immunomodulatory treatment for at least 6 months prior to CSF collection. Samples were collected with sterile techniques either by lumbar puncture or access port aspiration of surgically implanted pumps. CSF samples were centrifuged at 200 x g for 15 min to remove cells, confirmed to be free of red blood cell contamination by microscopy, then stored in aliquots at –80°C.in vivo experiments. All procedures were approved by the Institutional Animal Care and Use Committee at Mispro Biotech Services (New York). Prior to surgery, mice were anaesthetized with a ketamine (110 mg/kg) and xylazine (10 mg/kg) cocktail and received subcutaneous injections of 0.1 mg/kg buprenorphine, 2.5 mg/kg baytril and 1 ml 0.9% saline. Laminectomies at cervical levels 4 (C4) and 5 (C5) were performed to expose the underlying spinal cord. A 32-gauge Hamilton syringe was inserted underneath the dura mater and 3 µL saline, total CSF, or individual human CSF proteins were slowly injected into the subarachnoid space. Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE  and recombinant human CHIT1 . A minimum of three mice were injected per unpooled, individual patient CSF sample or protein sample. Mice were assigned to different treatment groups in a randomized manner.Adult female C57BL/6J mice (aged 8–12 weeks) purchased from The Jackson Laboratory  were used in all Following intrathecal delivery of CSF or proteins, all mice underwent motor testing at 1 day post-injection (DPI). A separate cohort of mice used for time course analysis was tested during six additional sessions until 28 DPI. Forelimb reaching, gripping and tail flaccidity were evaluated on a 3-point scale. Mice were held by their tails above their cage bars and allowed to reach out and grip the bars for five trials. Mice displaying no motor deficits were given a score of 0. Any deficits in either reaching or gripping were each given a score of 1. Specifically, an inaccurate reach was considered to be a reaching deficit, and weakness in grip strength or clenched forepaws were scored as gripping deficits. Tail flaccidity was also given a score of 1. All motor testing was performed blinded with respect to treatment groups.Mice were habituated to the grip strength metre (TSE systems) for 3 days prior to surgery. Each mouse was given 1 min to explore the grip strength metre, then held by their tails and allowed to grip the bar with both forelimbs for five consecutive trials. After a 30 s rest period, the mouse was given another five trials to grip and then returned to their home cage. Baseline grip strength force was measured at 1 day prior to surgery and grip strength was also measured at 1 DPI. A separate cohort of mice used for time course analysis was tested during six additional sessions until 28 DPI. The mean grip strength force was calculated from five trials. Normalized grip strength values were calculated by dividing mean grip strength force on post-injection testing days by mean baseline grip strength force.Mice were sacrificed using an overdose of ketamine (300 mg/kg) and xylazine (30 mg/kg) and were perfused transcardially with phosphate buffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M PBS, pH 7.4. Spinal cords and brains were dissected out, postfixed in 4% paraformaldehyde overnight and then placed in 30% sucrose overnight for cryoprotection.Cervical spinal cords were cut 0.5 cm rostral and 0.5 cm caudal to the injection site, and 1 cm of thoracic spinal cords were also collected. The 1 cm segments were then embedded and frozen in Tissue Tek® . Spinal cords were sectioned sagittally at 20 µm thickness using a cryostat (Leica) and then slide-mounted onto Histobond® slides . The anatomical orientation of tissue sections, as well as the order and position in which they were mounted onto the slides were kept consistent to facilitate unbiased histological comparisons, as described in further detail below. Brains were sectioned coronally at 30 µm and free-floating sections were stored in 0.01% sodium azide in PBS.Immunostaining was performed on series of spinal cord sections at 100 µm intervals throughout the entire cervical spinal cord or thoracic spinal cord. Slides with spinal cord sections or cells, or free-floating brain sections were washed three times in 0.1% triton X-100 in PBS (PBS/T), then incubated in 10% normal goat serum (NGS) or normal donkey serum (NDS) in PBS/T for 1 h at room temperature. Primary antibodies were diluted in 10% NGS or NDS in PBS/T and incubation occurred overnight at 4°C. After incubation, slides or sections were rinsed three times in PBS and incubated in a 1:750 dilution of the appropriate Alexa-Fluor secondary antibodies (Invitrogen) in 10% NGS or NDS in PBS/T for 1.5 h at room temperature. Slides or sections were rinsed three times in PBS and then counterstained with 1:2500 DAPI in PBS (Invitrogen) for 5 min. After two final washes in PBS, free-floating brain sections were mounted onto slides, and slides were mounted using Fluoromount (Sigma).Primary antibodies used include the following: goat anti-ChAT , rabbit anti-activated caspase-3 , mouse anti-GFAP , rabbit anti-Iba1 , mouse anti-SMI-32 , rabbit anti-GLT-1 , rabbit anti-TDP-43 , mouse anti-NeuN , rabbit anti-Olig2 , and rabbit anti-Ki67 .2O Organics). Slides were then stained in 0.1% cresyl violet (Sigma). After rinsing in water, slides were dehydrated in three washes each of 70%, 90% and 100% ethanol for 5 min. Slides were cleared in three washes of xylene for 5 min each and then mounted using cytoseal (Thermo Scientific).Slides were washed in PBS and then water before immersing in 70% ethanol, 80% ethanol, then 90% ethanol twice, for 5 min each. Slides were incubated in 0.1% luxol fast blue in 95% alcohol with acetic acid (Electron Microscopy Sciences) at 60°C for 2.5 h. Excess staining was removed by rinsing slides in water. Staining was then differentiated by immersing slides in 0.05% lithium carbonate , then stored in aliquots at –80°C until use. CSF protein concentrations were measured using a Pierce BCA protein assay according to manufacturer instructions (Thermo Fisher Scientific). Unfiltered and filtered sALS CSF samples were injected intrathecally into mice, as described previously.sALS CSF samples underwent tangential flow filtration using 5 kDa, 100 kDa, 300 kDa, or 750 kDa MWCO hollow-fibre filters . Filters were primed with sterile-filtered distilled H2O. Images of gels were captured using Carestream MI system and software.Unfiltered and filtered CSF samples were run on a NuPAGE™ 4–12% bis-tris (5 and 100 kDa filtrates) or NuPAGE™ 3–8% tris-acetate protein gel (300 and 750 kDa filtrates) (Thermo Fisher Scientific). MagicMark™ XP protein standard was run on the 4–12% gel and HiMark™ protein standard was used for the 3–8% gel (Thermo Fisher Scientific). Gels were placed in 25% isopropanol with 10% acetic acid for 30 min and then stained in 0.006% Coomassie brilliant blue in 10% acetic acid overnight at room temperature. Following destaining with 10% acetic acid, gels were rinsed in Hn = 5), 5 kDa-filtered sALS CSF (n = 2), multiple sclerosis CSF (n = 5) and HC CSF (n = 5) samples were shipped frozen to Biognosys for label-free unbiased global proteome profiling using hyper reaction monitoring mass spectrometry (HRM™/DIA-MS). In total, 100 µL per CSF sample was denatured using Biognosys’ Denature Buffer, reduced/alkylated using Biognosys’ Reduction/Alkylation Solution for 60 min at 37°C in the dark and then digested into peptides using 1 µg of trypsin (Promega) per sample overnight at 37°C.sALS CSF  according to the manufacturer’s instructions and dried down using a SpeedVac system. Peptides were resuspended in 20 µL LC solvent A  and spiked with Biognosys’ iRT kit calibration peptides. Peptide concentrations were determined using a UV/VIS Spectrometer .Peptides from all analyzed CSF samples were used to generate a sample pool. Ammonium hydroxide was added to a pH value > 10. The fractionation was performed using a Dionex UltiMate 3000 RS pump (Thermo Scientific) on an Acquity UPLC CSH C18 1.7 µm, 2.1 × 150 mm column (Waters). The gradient was 1–40% solvent B in 20 min, solvents were A: 20 mM ammonium formate in water, B: acetonitrile. Fractions were taken every 30 s and sequentially pooled to eight fractions. These were dried down and resolved in 12 µL solvent A. Prior to mass spectrometric analyses, they were spiked with Biognosys’ iRT kit calibration peptides. Peptide concentrations were determined using a UV/VIS Spectrometer .For DDA LC-MS/MS measurements, 1 µg of peptides per sample were injected to an in-house packed reversed phase column  on a Thermo Scientific™ EASY-nLC™ 1200 nano-liquid chromatography system connected to a Thermo Scientific™ Q Exactive™ HF mass spectrometer equipped with a Nanospray Flex™ Ion Source. A modified TOP15 method was used. The full-range MS1 scan was followed by 15 data-dependent MS2 scans.HRM™ MS was performed for each individual CSF sample. For DIA LC-MS/MS measurements, 1 µg of peptides per sample were injected to an in-house packed reversed phase column, as described previously. The DIA method consisted of one full-range MS1 scan and 29 DIA segments.Shotgun and HRM™ mass spectrometric data were analyzed using SpectroMine™ software (Biognosys). The false discovery rate on peptide and protein level was set to 1%. Data were searched against a human UniProt protein database . Search results were used to generate a shotgun-based spectral library of HPRP fractions and an HRM™-based spectral library of all samples. SpectroMine™ was then used to combine the separate libraries into a hybrid, which was subsequently used for HRM™ data quantification.t-test. Gene Ontology (GO) enrichment analysis was performed in Spectronaut® software using a human gene associations file obtained from EBI (2018-02-20). Only terms with a minimum of two members were considered.HRM™ mass spectrometric data were analyzed with Spectronaut® Pulsar software (Biognosys). Differentially regulated proteins were identified using the following criteria: q-value < 0.05 and average fold change > 1.5. The false discovery rate on peptide and protein level was set to 1%, and data were filtered using row-based extraction. Data were normalized using local regression normalization. For testing of differential protein abundance, protein intensities were analyzed using a two-sample Student’s ApoB antibody (Invitrogen) was incubated with Dynabeads® Protein A (Thermo Fisher Scientific) for 45 min at room temperature. Antibody-coated Dynabeads® were then incubated with sALS CSF for 2 h at 4°C. IgG depletion was performed with uncoated Dynabeads®. ApoB or IgG-depleted sALS CSF were centrifuged in an ultrafree-MC centrifugal filter tube at 12 000 x g at 4°C for 6 min. Samples were stored at –80°C until use.ApoB protein in sALS, SOD1, C9, TARDBP, HC, multiple sclerosis, and ApoB-depleted sALS CSF was quantified using a human ApoB ELISA kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.2 in either 6-well plates for RNA extraction, or 8-well chamber slides for immunocytochemistry. Astrocytes were cultured in AGM™ astrocyte growth medium (Lonza) containing 3% fetal bovine serum until they reached 80–90% confluence. Astrocytes were treated with either CSF diluted at a ratio of 1:1 in serum-free DMEM, or 0.01 µg/µL or 0.01 ng/µL human ApoB protein (Millipore), human MOG protein (Sigma-Aldrich), or human haptoglobin protein (abcam) in DMEM. After 24 h, astrocytes were either processed for RNA extraction using a RNeasy Plus Micro Kit  according to manufacturer instructions, or fixed with 4% paraformaldehyde for immunocytochemistry. Using ImageJ, Ki67+ and GFAP+ astrocyte numbers were quantified on a minimum of three images per well and then averaged.Human cortical astrocytes were purchased from Lonza and plated at a density of 10 000 cells/cm2 in 8-well chamber slides coated with 80 µg/µL Matrigel in DMEM/F-12. Motor neurons were cultured in Motor Neuron Maintenance Medium (iXCells Biotechnologies) for 8 days. On Day 8, human ApoB (Millipore), human MOG (Sigma-Aldrich), human haptoglobin (abcam), human ApoC-III (Athens Research & Technology), and human ApoE  proteins were applied at 0.01 µg/µL or 0.05 µg/µL in media, or 50% sALS CSF and ApoB-depleted sALS CSF diluted in media were applied. In a separate ApoB titration experiment, lower doses of 0.005 ng/µL or 0.01 ng/µL ApoB were applied. At 24 h pre- and post-treatment, cells were visualized using a 10X objective on an Olympus IX71 microscope and bright field images were captured on a Hamamatsu Orca-Spark camera using Cell Sens Dimension Programme. Motor neurons were fixed with 4% PFA for immunocytochemistry at 24 h post-treatment. Using ImageJ, areas of ChAT+ motor neuron clusters were quantified from three images per well and then averaged.Human induced pluripotent stem cell (iPSC)-derived motor neurons were purchased from iXCells Biotechnologies and plated at a density of ∼86 000 cells/cmQuantitative real-time PCR (q-RT-PCR) was performed using the Applied Biosystems QuantStudio 7 Flex Real-Time PCR system with individual TaqMan probes . The ΔΔCt method was used to calculate fold changes in RNA expression, with the DMEM group as the calibrator and β-actin as the internal control.+ motor neurons, size of motor neuron clusters, and immunostaining intensities were analyzed using a one-way ANOVA, with Bonferroni post hoc analyses. Motor deficit scores and normalized grip strength for the 28 DPI experiment were analyzed using a repeated measures two-way ANOVA, with Bonferroni post hoc analyses. Protein concentrations and ELISA measurements of CSF ApoB levels were analyzed using a paired, two-tailed Student’s t-test. Statistical significance was determined by P < 0.05. Data are presented as mean ± SEM.GraphPad Prism 8 software was used to perform statistical analyses. Motor deficit scores, normalized grip strength, number of motor neurons and astrocytes, percentage of cytoplasmic TDP-43Data reported in this manuscript are available from the corresponding author, upon reasonable request.SOD1, C9orf72 or TARDBP mutations did not exhibit motor deficits and performed similarly to control mice injected with saline or CSF from HC  in degenerating motor neurons.ted mice  and B. T21 we explored whether ALS CSF-induced NF-H upregulation was associated with changes in glutamate transporter-1 (GLT-1) expression. GLT-1 immunostaining in the cervical spinal cord revealed significant upregulation of GLT-1 in the ventral horns of sALS CSF-injected mice compared to saline and SOD1 CSF-injected mice, perhaps as a compensatory response to sALS CSF-induced excitotoxicity  circulating in sALS CSF responsible for triggering and/or exacerbating motor neuron degeneration, sALS CSF was filtered to narrow down putative factor(s) by molecular weight. After tangential flow filtration through a 5 kDa MWCO filter, the filtered sALS CSF no longer induced an increase in motor deficit scores or weaker forelimb grip strength, and neither motor neuron loss nor TDP-43 translocation were observed . FurtherTo further refine the list of putative neurotoxic protein(s) in sALS CSF, we performed additional filtration studies with larger filters until we obtained filtrate which retained neurotoxic protein(s) with the capacity to induce motor deficits and pathology in mice. We found the neurotoxic capacity of both the 100 and 300 kDa filtrates to be attenuated as neither were able to impair motor function or trigger motor neuron degeneration, while the 750 kDa filtrate induced a similar extent of impairment as unfiltered sALS CSF at 1 DPI . Attenua25–27 was significantly upregulated in sALS CSF versus HC CSF, downregulated in 5 kDa-filtered sALS CSF and the sole candidate which met the molecular weight criteria  as a likely candidate neurotoxic protein. ApoB, a 550 kDa protein previously implicated as a risk and prognostic factor associated with ALS,criteria  and B. Tcriteria , consistcriteria .+ motor neuron loss in the cervical spinal cord  into the lateral ventricles of adult rats29 We also observed evidence of activated caspase-3 in motor neurons of sALS CSF-injected mice; however, future studies are needed to determine the exact mechanisms and cell death signalling pathways by which elevated levels of ApoB in CSF trigger motor neuron toxicity and disease progression in sALS. Potential mechanisms of neurotoxicity could involve cell death signalling pathways triggered by ApoB binding to its receptors such as the low-density lipoprotein receptor (LDLR)30 and/or sortilin receptor.31 For instance, it has been reported that inactivation of liver X receptor (LXR) can lead to degeneration of motor neurons in mice,32 and it is possible that increased CSF ApoB levels could modulate the LXR-inducible degrader of the LDLR (IDOL)-LDLR pathway in a detrimental manner.33 Additionally, sortilin activation is known to involve signalling pathways which eventually lead to caspase activation and cell death,34 and it is possible that sALS CSF-induced motor neuron degeneration is mediated by an ApoB/sortilin-mediated cell death signalling pathway.A caveat of our CSF-mediated sALS model is the assumption that neurotoxicity arises from an overabundance rather than a depletion of a CSF component. Our work does not exclude the possibility that depleted levels of a protein could also lead to dysregulation of pertinent cell survival pathways, for instance, or the possibility that other pathological mechanisms contributing to motor neuron degeneration can occur simultaneously with ApoB-induced cell death. Prior studies have demonstrated that lipid-free ApoB has cytotoxic effects on macrophages, CHO cells and HepG2 cells via plasma membrane disruption, increased calcium influx and caspase-3 activation.SOD1, C9orf72 or TARDBP. Our data clearly show that ApoB is sufficient to induce motor disability and motor neuron degeneration, and its removal from sALS CSF via filtration or immunodepletion attenuates neurotoxicity, establishing a novel sALS-specific therapeutic target and proof-of-concept to support targeted reduction of ApoB via CSF pheresis35–37 as a therapeutic strategy for sALS patients.This study provides a novel CSF ApoB-mediated mechanism of motor neuron degeneration which appears to occur in the most common sporadic form of ALS, but not fALS arising due to mutations in fcac207_Supplementary_DataClick here for additional data file.


The estimation of the sEMG–force relationship is an open problem in the scientific literature; current methods show different limitations and can achieve good performance only on limited scenarios, failing to identify a general solution to the optimization of this kind of analysis. In this work, this relationship has been estimated on two different datasets related to isometric force-tracking experiments by calculating the sEMG amplitude using different fixed-time constant moving-window filters, as well as an adaptive time-varying algorithm. Results show how the adaptive methods might be the most appropriate choice for the estimation of the correlation between the sEMG signal and the force time course. Moreover, the comparison between adaptive and standard filters highlights how the time constants exploited in the estimation strategy is not the only influence factor on this kind of analysis; a time-varying approach is able to constantly capture more information with respect to fixed stationary approaches with comparable window lengths. Surface ElecroMyoGraphy (sEMG) has been widely used as a means for measuring muscle activity during force generation, typically relating the amplitude of the sEMG signal to the amount of force exerted during a particular movement ,2,3,4,5.Estimating the amplitude of the sEMG signal is a critical step in most of the well-established clinical and research analyses ,11; thissEMG envelope estimation is performed by low-pass filtering the rectified version of the raw signal, at cutoff frequencies that vary approximately in the range 2–20 Hz for general applications ,15,16,17In this work, different sEMG amplitude-estimation methods are tested on two different datasets to investigate whether choices in the processing schema and particularly in the filters used for sEMG envelope extraction yield results that are characterized by different correlation levels with the force signal, hence being more or less suitable to be used in sEMG–force relationship analyses.triceps brachii lateral head) and lower limb  muscles. Both tasks have been selected to have an experimental condition in which most of the force is generated by the recorded muscle alone, with minimal contribution from muscles belonging to different anatomical groups. Both experimental procedures have been approved by the local ethics committee of Roma Tre University.For this study, data coming from two different isometric force-tracking experiments have been used. In detail, the two experiments are related to isometric contractions of both upper limb , righthanded were enrolled. Subjects were asked to track a force signal composed by the cyclical repetition of 10 s contractions at 20% and 40% of their maximum voluntary contraction (MVC), separated by a 5 s resting period at a very low force level (10% MVC). Visual feedback was realized by showing a colored line that the subjects had to keep within two limits corresponding to the desired value ±5% MVC .During the whole task, sEMG signals are recorded with a wired StepPC system  and synchronized with the force values recorded with a custom-made load cell . The visual stimulation signal is provided and recorded through a custom LabView panel that manages the whole data acquisition. Sampling frequency has been set to 1 kHz for both sEMG and force signals.tibialis experimental protocol . Participants were asked to avoid any kind of fatiguing activity the day before the measurements.Ten healthy subjects were enrolled for the A visual representation of the experimental protocol is given in tibialis anterior muscle have been recorded through a high-density EMG (HD sEMG) sensor ; from the HD sEMG data, a bipolar signal was selected to mimic in the best way SENIAM recommendations for sEMG recordings. Both sEMG and force signals were acquired at a sampling frequency of 2 kHz. Force signals were recorded with a compression load cell with a full-scale range of 220 N .Surface electromyography signals of the The processing strategy for the sEMG signal was standardized for both the experimental protocols. In more detail, as a first step the sEMG signal was pre-processed using standard denoising techniques (3rd-order Butterworth band-pass filter between 25 and 450 Hz and 3rd-order Butterworth notch filter at 50 Hz with a 1 Hz bandwidth), prior to envelope extraction.The sEMG envelope has then been extracted via rectification and a moving RMS filter with different window lengths, namely 500, 200, 100, 80, 66 and 44 ms  between the normalized time course of the force and the sEMG has been computed  as the Rtriceps dataset and one for the tibialis signals. Post hoc analysis was carried out with a Tukey test.The statistical significance of the differences between algorithms in the performance indicators has been tested by means of a one-way ANOVA test, with the algorithm as a factor. ANOVA assumptions were tested by checking the normality of the residuals of the model with a Shapiro–Wilk test. Three different tests were carried out separately—two for both force levels of the tibialis dataset are significantly lower than all the values recorded for the triceps experiment . For the tibialis data, before calculating the average behavior, all the trials have been interpolated over a fixed number of points , considering the variable duration of the experimental trial; this step has been performed only for visualization purposes, not for the calculation of the actual correlation parameters.The average behavior, over all repetitions and all subjects, of the time-varying filter window length is shown in triceps subject and five for the tibialis participants.The experimental protocols described in the Methods section resulted in an average of eight events for each tibialis dataset, the minimum window length is around 60 ms, different to the 70 ms value that is recorded on the triceps signal;For the tibialis window length is higher, with a noise level that is close to the actual range of the triceps dataset;The variability of the triceps dataset, in which also a contraction offset phase is present, the same local minima for the window length can be found;For the triceps dataset, the local minima are more pronounced for higher-level contractions, both for onset and offset phases.In the In both datasets, the time-varying filter time constant shows pronounced minima in correspondence to the onset of the contractions. Moreover, in both scenarios, the filter window stabilizes at a value of 90 ms during static or quasi-static contraction phases. Dataset-related differences that can be identified are:tibialis dataset show lower correlation values and higher variability, while still maintaining the same trend. The adaptive algorithm performs with comparable correlation values  on the evaluation of the sEMG–force relationship. Data from submaximal isometric contractions have been processed and tested in terms of the correlation values between the estimated amplitude envelope of the sEMG signal and the force curve during the onset phases and of the RMSE between the two signals. The set of classical fixed-time approaches presented here represents a subset of the methods that have been applied in the literature; the results coming from this analysis can be reasonably extended to other methods that need to be selected with some optimality criteria before being applied to sEMG–force estimation analyses.In this work, we designed two experiments to have a condition in which all the force is generated by a single muscle; this condition is far from being often encountered in typical experimental scenarios. However, the multi-muscle force-generation condition requires some mathematical models to be built on top of the envelope-estimation procedure, so that these results presented here can be generalized to these wider conditions.The tested methods are divided into standard, fixed-time constant moving-window procedures, and the adaptive procedure introduced in . It has triceps dataset, this is true for both the onset and offset phases; data in the tibialis dataset are characterized by a higher force output (expressed in terms of fractions of MVC), thus justifying the shorter time windows that are recorded at the onset.In addition, the difference between the steady-state value for the time-varying window and the local minima at the onsets are different as a function of the force output that is generated by the contraction. When analyzing data only coming from the tibialis dataset have a higher noise power with respect to triceps; the difference in SNR is clearly visible in the plateau phase of the contraction in tibialis dataset is not to be ascribed to worse performance of the algorithms, but only to a more challenging condition in the dataset itself. It should be noted that this difference in SNR is not only to be ascribed to the recording and experimental settings, but it is indeed coming from the signal-dependent nature of the noise itself [As an additional difference between the two experiments, it can be noted from e itself ,23; whentriceps brachii contractions the values are still very high . However, in terms of correlation with the force, values coming from the fixed-window approach with time dynamics closer to As a general consideration, the slowly varying nature of the force-tracking experiments that have been analyzed here puts the slower filters in a clear advantage with respect to the shorter time windows; moreover, the nature of the quality parameter  is intrinsically higher for time signals that come from very long time windows that are consequently very smooth. Even with this advantage, and even by estimating an envelope that is less smooth, In this paper, the analysis has been focused on simple yet crucial quality parameters for the estimation of the sEMG–force relationship, namely the maximal correlation value and the RMSE, which has already been proven to be effective in characterizing the quality of force estimation from sEMG . These ptibialis dataset, the signals were extracted from an HD sEMG recording, the results presented here can be considered to be valid also in the case in which the sEMG–force relationship is exploited by this recording technology [Considering the fact that, for the chnology ,21,25. EAn accurate estimation of the sEMG–force relationship can yield relevant parameters not only during the onset and offset phases. For example, it has been demonstrated that the force output is characterized by an increasing variability in the presence of neuromuscular fatigue ,27,28, sIn addition to improve fatigue detection, a repeatable, stable and reliable estimation of the sEMG–force relationship is also able to highlight more advanced and detailed information on force-generation mechanisms and force control in general, such as frequency and coherence behavior ,29,30 orAlthough the results presented here are relative to a well-established research question  and have strong physiological models underlying the experimental design , the con