Dear Editors,Advances in neonatal and metabolic medicines have brought up a new profile of paediatric kidney transplant recipients. More children born with congenital anomalies of the kidneys and urinary tract and metabolic conditions are surviving outside of infancy. Some of those children are needing renal replacement therapy since early childhood and therefore are considered as transplant candidates with a high likelihood of needing more than one kidney transplant in their life. Recent meta-analysis states that pre-emptive transplantation has advantages in overall patient and allograft outcomes over transplantation done following a period on dialysis .In the UK there are currently 101 children waiting for a kidney transplant from a deceased donor ; this nuGiven the scarcity of deceased donor organs and increasing waiting times, there has been extensive research into ABO incompatible (ABOi) transplantation from a living donor since the 1980s, as a way of increasing donor pool and as a possible alternative to deceased donation. While the studies initially focused on adult donors and recipients, over recent years more evidence has been produced to support this practice for paediatric recipients. An analysis of the Japanese Kidney Transplant registry was published in 2018 which described the results of 102 children who had undergone ABOi kidney transplants from living donors. The outcomes of these recipients were compared to the outcomes of the children on their registry who had undergone ABO compatible living donor transplants, with no difference found in patient or allograft survival between the two groups . InitialGiven the increasing evidence of positive outcomes following ABOi kidney transplantation in children, and evidence of better allograft survival of kidneys coming from living donors, perhaps it is time to consider ABOi living donor kidney transplantation in children before being listed for deceased donor organs. To date, there is no prospective study comparing the outcomes between those receiving ABOi living donor transplants and those receiving ABO compatible transplants from a deceased donor. However, the evidence that we do have in the literature so far, supports the idea that ABOi transplants should be considered in the paediatric population as a transplant option prior to proceeding with a transplant from a deceased donor, as it has the potential to lead to better patient and allograft outcomes.


Renal cell carcinoma, shorted as RCC is a well-known urological cancer with high level of morbidity and mortality. Although the regulatory role of the spindle microtubule assembly factor (ASPM) in tumor progression has been established, its relationship to the development of RCC remains unclear. To determine the significance of this gene in RCC, we examined its expression in RCC patients in the TCGA database and compared ASPM level between clinical samples of normal tissues and RCC tissues collected at our center. The prognostic relevance of ASPM was assessed by generating Kaplan-Meier survival curves and log-rank functions. Following alteration of ASPM expression using sh-ASPM or oe-ASPM transfection, RCC cell characteristics were evaluated through CCK-8, Transwell, and colony formation assays. Western blot analysis was conducted to measure levels of genes affected by ASPM, and rescue experiments were performed to explore the involvement of Wnt3a signaling in ASPM-mediated malignancy in RCC. Our findings indicate that ASPM is upregulated in RCC samples, and its levels are associated with the long-term survival of RCC patients. ASPM promotes the migration, proliferation, and invasiveness of RCC cells, and the Wnt3a pathway may be implicated in this process. In conclusion, these results indicate that ASPM contributes to the cancer progression of RCC by targeting the Wnt3a signaling pathway. Renal cell carcinoma, shorted as RCC, is a prevalent urologic cancer that has increasing rates of morbidity and mortality. A study conducted in the United States showed that RCC had approximately 65,000 new cases and 15,000 deathsAssembly factor for spindle microtubules (ASPM), initially discovered as a centrosomal protein influencing neurogenesisThis study aimed to investigate the impact of this gene on RCC. We evaluated ASPM expression levels in RCC patients from the TCGA database and compared them to clinical samples of RCC tissue and normal tissue collected at our center. The prognostic relevance of ASPM was assessed through Kaplan-Meier survival curves and log-rank functions. Following ASPM expression alteration using sh-ASPM or oe-ASPM transfection, we analyzed the characteristics of RCC cell by conducting colony formation assays, Transwell, and CCK-8.https://cancergenome.nih.gov/. The edger function was used to analyze differences in expression levels between normal tissues and tumor, while survival function was employed to analyze patient prognosis. Following data processing, we determined the levels of ASPM and the survival rates of patients with RCC.Bioinformatics analysis was conducted to explore ASPM in RCC as well as paraneoplastic tissues and their associated clinicopathological profiles, downloaded in The Cancer Genome Atlas (TCGA) database. The website of TCGA is We collected 12 pairs of RCC and adjacent non-neoplastic tissues in the department of urology at the First Affiliated Hospital of Soochow University between March 2010 and September 2014. TNM staging was based on the system of Fuhrman histological grading. Patients were followed up until January 2019 for the purpose of tracking cancer recurrence, physical condition, and mortality via telephone and outpatient review. None of the patients underwent radiotherapy or chemotherapy before operation, and the samples that were collected were stored in liquid nitrogen. Informed consents were obtained from all participants, which met human subjects' criteria. This study was confirmed by the hospital's research and project ethics committee. The TNM system is a widely accepted staging system for various cancers, including RCC. It classifies tumors based on their Tumor size and invasion (T), involvement of Lymph Nodes (N), and presence of distant Metastasis (M).For cell transfection and culture experiments, RCC and epithelial cell lines  were obtained from ATCC. RPMI-1640  was used as cultures, except for CAKI-1 cells which were cultured in McCoy's 5A. 10% fetal bovine serum  and 1% penicillin/streptomycin (Invitrogen) were used and incubated in a humidified environment with 5% CO2 at 37°C. To overexpress ASPM and Wnt3a, complete cDNA sequences of ASPM and Wnt3a were inserted into the pLVX-puro vector to generate pLVX-Wnt3a and pLVX-ASPM; an empty vector served as the negative control. GeneChem  constructed the pLKO vectors containing ASPM or Wnt3a shRNA sequences. Lentiviral infections were performed on 769P, and CAKI-1 cell lines using lentiviruses containing sh-Wnt3a, phU6-EGFP-shRNA-ASPM, oe-ASPM, sh-NC, oe-Wnt3a, and oe-NC vectors. Stable transconductor pools were formed by puromycin (4 μg/mL) for two weeks.Trizol reagent  was conducted to lyse cells and isolate RNA. The Primescript R Reagent Kit  was used for reverse transcription of the qualified RNA into cDNA, followed by Premix Ex Taq  in qRT-PCR using SYBR Green. GAPDH was utilized as an internal reference. Each tissue was analyzed in triplicate, and relative levels were calculated using 2-ΔCt and normalized to GAPDH. Oligonucleotide primers were created employing Primer 7.1.2a as follows: ASPM: 5′-CCCCGACACCCGATGCCATTTG-3′ (forward) and 5′-TTAACCACCAAGTGAAGCCCTGTTC-3′ (reverse); GAPDH: 5′-CAGGAGGCATTGCTGATGAT-3′ (forward) and 5′-GAAGGCTGGGGCTCATTT-3′ (reverse).To carry out Western blotting, RCC cells and tissues were lysed with RIPA on ice for 20 minutes. The resultant mixture was centrifuged to isolate protein samples whose concentrations were determined by using the BCA method. To ensure uniformity, protein samples were adjusted to the same concentration, denatured, separated using SDS-PAGE, and transferred onto PVDF membranes. The membranes were then cut into strips based on molecular size and blocked in TBST containing 5% skim milk for 1.5 hours. After that, primary (1:500) and secondary (1:2000) antibodies were incubated, followed by ECL exposure and grayscale analysis using ImageJ software. The primary antibodies against ASPM and Wnt3a were sourced from Abcam, while Cell Signaling Technology supplied MMP2, GAPDH, MMP9, anti-rabbit, and anti-mouse secondary antibodies.To perform the CCK-8 assay, 769P and CAKI-1 cells were added into 96-well plates at 3×10^3 cells/well. Optical density at 450 nm was measured using a CCK-8 kit  on days 1, 2, 3, and 4 to generate a viability curve for each sample.To perform the colony formation assay, 1×10^3 cells of 769P and CAKI-1 were seeded into each well of a 6-well plate. The medium was renewed once during the first week and twice during the second week. After two weeks, visible colonies were washed using PBS, fixed in methanol for 25 minutes, and stained with 0.1% crystal violet (purchased from Sigma-Aldrich) for another 25 minutes. Subsequently, the stained colonies were counted and visualized after being washed again in PBS.The Transwell was performed to assess the invasive and migratory ability of cells. The experiment involved adding 300 microliters of suspension containing 2×10^4 cells to the top of Transwell inserts with an 8 μm pore size . In contrast, 500 microliters of medium containing 10% FBS were added to the bottom of the insert. After 48 hours of incubation, methanol treatment for 25 minutes and crystal violet staining for 25 minutes were used to induce and visualize migrating cells at the bottom of the chamber. Five random areas were selected per sample, captured using a microscope, and cell counts were determined. To assess invasive cells, Transwell chambers pre-coated with Matrigel (Invitrogen) were utilized, following the same procedure as mentioned above.In this experiment, a confluent monolayer of cells is scratched via a sterile pipette tip to form a straight and uniform gap. The cells are then washed with medium to remove any detached cells and debris. Subsequently, the cells are incubated under normal culture conditions, and images of the scratch are captured at regular intervals using an inverted microscope equipped with a camera. The rate of cell migration is determined by measuring the degree of scratch closure over time. This assay can be used to evaluate the effect of different treatments or genetic modifications on cell migration in various cell types.The ASPM protein was evaluated for positive expression in RCC tissues using the immunohistochemical technique. Tissue microarrays were first treated with a primary antibody and then incubated 24h at 4°C. This was followed by another day of HRP-conjugated antibody treatment and staining with diaminobenzidine. The IHC staining outcomes were independently appraised by two skilled pathologists. Based on these results, the RCC samples were classified into high-staining and low- clusters for further analysis.The data analysis was performed using the SPSS 23.0 software package to ensure the accuracy of our statistical analysis. To evaluate the overall survival of renal cell carcinoma (RCC) patients, we utilized Kaplan-Meier survival curves. Additionally, we conducted Chi-square analysis to investigate any potential impact of ASPM on the clinical characteristics of RCC. To ensure the reliability of our findings, all experiments were independently conducted in triplicate. By replicating the experiments multiple times, we aimed to minimize any potential variability and increase the robustness of our results. In our statistical analysis, a p-value of less than 0.05 was considered statistically significant. This threshold allowed us to determine whether the observed differences between groups were likely due to chance or if they reflected true associations or effects. By employing these rigorous statistical analyses and experimental procedures, we aimed to enhance the validity and reliability of our study's conclusions regarding the relationship between ASPM and RCC.From the TCGA database, a dataset containing 523 cases of patients with RCC, and 100 healthy controls was obtained. The analysis revealed that ASPM gene was increased in RCC patients, particularly in those with advanced tumors , and β-catenin proteins. Frizzled receptors for the glycoprotein ligand Wnt and co-receptor Low Density Lipoprotein Receptor Related Protein 5/6 are in the cell membraneIn conclusion, knockdown of ASPM resulted in significant downregulation of Wnt3a, β-catenin in RCC cells, while upregulating of ASPM produced the opposite trend. Moreover, Wnt3a was able to reverse the regulatory effect of ASPM on RCC cell phenotype. This suggests that ASPM exacerbates the malignant process of RCC by activating Wnt signaling.


Human prion diseases are remarkable for long incubation times followed typically by rapid clinical decline. Seed amplification assays and neurodegeneration biofluid biomarkers are remarkably useful in the clinical phase, but their potential to predict clinical onset in healthy people remains unclear. This is relevant not only to the design of preventive strategies in those at-risk of prion diseases, but more broadly, because prion-like mechanisms are thought to underpin many neurodegenerative disorders. Here, we report the accrual of a longitudinal biofluid resource in patients, controls and healthy people at risk of prion diseases, to which ultrasensitive techniques such as real-time quaking-induced conversion (RT-QuIC) and single molecule array (Simoa) digital immunoassays were applied for preclinical biomarker discovery.We studied 648 CSF and plasma samples, including 16 people who had samples taken when healthy but later developed inherited prion disease (IPD) . Symptomatic IPD CSF samples were screened by RT-QuIC assay variations, before testing the entire collection of at-risk samples using the most sensitive assay. Glial fibrillary acidic protein (GFAP), neurofilament light (NfL), tau and UCH-L1 levels were measured in plasma and CSF. Second generation (IQ-CSF) RT-QuIC proved 100% sensitive and specific for sporadic Creutzfeldt-Jakob disease (CJD), iatrogenic and familial CJD phenotypes, and subsequently detected seeding activity in four presymptomatic CSF samples from three E200K carriers; one converted in under 2 months while two remain asymptomatic after at least 3 years’ follow-up.A bespoke HuPrP P102L RT-QuIC showed partial sensitivity for P102L disease. No compatible RT-QuIC assay was discovered for classical 6-OPRI, A117V and D178N, and these at-risk samples tested negative with bank vole RT-QuIC. Plasma GFAP and NfL, and CSF NfL levels emerged as proximity markers of neurodegeneration in the typically slow IPDs (e.g. P102L), with significant differences in mean values segregating healthy control from IPD carriers (within 2 years to onset) and symptomatic IPD cohorts; plasma GFAP appears to change before NfL, and before clinical conversion.In conclusion, we show distinct biomarker trajectories in fast and slow IPDs. Specifically, we identify several years of presymptomatic seeding positivity in E200K, a new proximity marker (plasma GFAP) and sequential neurodegenerative marker evolution (plasma GFAP followed by NfL) in slow IPDs. We suggest a new preclinical staging system featuring clinical, seeding and neurodegeneration aspects, for validation with larger prion at-risk cohorts, and with potential application to other neurodegenerative proteopathies. et al. present longitudinal biofluid RT-QuIC and neurodegenerative biomarker analyses of individuals at risk of and symptomatic for prion disease. RT-QuIC can detect seeding activity up to almost 4 years before disease onset, while neurodegenerative marker trajectories differ between fast and slow inherited prion diseases.Mok https://doi.org/10.1093/brain/awad143) for a scientific commentary on this article.See Minikel and Vallabh ( Inherited prion disease (IPD) caused by autosomal dominant highly-penetrant mutations in the prion protein gene (PRNP) comprises 10–15% of the incidence but produces a wide spectrum of clinical syndromes including CJD, fatal familial insomnia (FFI), Gerstmann-Sträussler-Scheinker (GSS) disease, peripheral PrP systemic amyloidosis from truncation mutations, and long-duration dysexecutive-apraxic syndromes seen in octapeptide repeat insertions (OPRIs).4 Acquired prion disease has historically attracted considerable media, political and public health attention, despite being the rarest manifestation. Relevant exposures include bovine spongiform encephalopathy prions in the diet, and use of blood and blood products for variant CJD (vCJD)5; cadaver-sourced human growth hormone,6 neurosurgery, and lyophilized dura mater in iatrogenic CJD (iCJD),7 and at mortuary feasts in the Eastern Highlands Province of Papua New Guinea in kuru.8Prion diseases are transmissible and inevitably fatal neurodegenerative conditions characterized by recruitment of host-encoded cellular prion protein (PrP) into disease-associated polymeric assemblies which propagate by elongation and fission.9 Prions are transmissible to laboratory rodents by inoculation allowing for study of the sequence of prion infection, propagation and toxicity, which forms two mechanistically distinct phases.11 Specifically, following inoculation, infectious prion titres rise exponentially to reach a plateau, which continues for a considerable time until disease onset. Infectivity and toxicity are therefore uncoupled, with the length of the plateau being inversely proportional to PrP expression level. If this two-phase kinetics model is applicable to human disease, the clinically silent incubation phase marked by high prion titres hypothetically offers a window of opportunity for discovery of fluid biomarkers that predict proximity to onset e.g. potential dynamic changes in measures of seeding activity and/or neurodegenerative markers. Moreover, if borne out in humans, the two-phase kinetics model would provide a foundation for targeted prevention strategies in prion disease.13One of the most remarkable aspects of prion biology is the apparent long incubation phase between prion infection/exposure and disease onset, lasting up to five decades in kuru and cadaver-sourced human growth hormone-related iCJD.14 while those currently at risk of IPD were estimated at 1000 in the UK.15 Accrual of longitudinal biofluid sample resources from these studies not only allows repeated examinations for biomarker discovery, but also for ascertainment of rates of change as an even more sensitive predictor of disease onset.16 It is not feasible to adequately power clinical trials for candidate drugs in prion disease prevention for a simple clinical end point,17 but the characterization of presymptomatic biomarkers could inform different strategies, enrichment in and learning from trials.Longitudinal studies of defined populations with high lifetime risk of prion disease undoubtedly afford the best opportunity to elucidate the sequence of biomarker evolution during the presymptomatic phase in humans. For context, cadaver-sourced human growth hormone (c-hGH) was administered to at least 1849 UK individuals between 1958 and 1985, 81 of whom have so far succumbed to iCJD,−18 g) range in the last decade offers the potential to detect presymptomatic CSF PrP-amyloid seeding activity in at-risk individuals.19 The assay exploits the ability of PrP-amyloid in tested samples to convert recombinant PrP (rPrP) monomers within a reaction mixture, and accelerates the process with cyclical bursts shaking and rest to amplify rPrP amyloid fibrils; alteration of thioflavin T (ThT) emission spectrum from amyloid binding within the reaction mixture is then detected by a microplate reader in relative fluorescence units (RFU) over a certain threshold. Indeed, Orru et al.18 used RT-QuIC to demonstrate high levels of seeding activity in brains and CSF of hamsters experimentally inoculated with 263 K prions in the clinically silent incubation period before disease onset, paralleling prion bioassays in the two-phase kinetics model20; interestingly, Vallabh et al.21 identified presymptomatic RT-QuIC seeding activity in an single elderly carrier of the E200K mutation. CSF RT-QuIC assays in human prion disease to date have been honed primarily to detect sCJD and IPD E200K seeds to high sensitivity (>90%) and specificity (∼100%), far outstripping of its utility in other IPD disease syndromes.22–27 Nevertheless, assay developments along the way have identified key factors  and novel seed-substrate compatibilities [truncated hamster (Ha90) and bank vole (BV) rPrPs], which may pave the way for optimizing RT-QuIC for the more fastidious seed species in IPD.28–31The advent of ultrasensitive real-time quaking-induced conversion (RT-QuIC) assays capable of detecting PrP-amyloid seeding down to the attogram  levels between IPD mutation carriers from symptomatic IPD individuals, and more importantly showed rising levels in the 2 years prior to symptom onset in small numbers of converting individuals examined, through use of Simoa assays.33 Further advances in Simoa technology now allows for multiplex arrays measuring up to four candidate biomarkers, limiting depletion of precious biofluid resources.Neurodegenerative biomarkers in prion disease are essentially downstream products of either neuronal injury, astrogliosis and inflammation, or other secondary disease pathologies. While none of them are strongly discriminatory between neurodegenerative diseases, particularly with cross-sectional values, the tracking of biomarker dynamics over time may segregate mutation carriers approaching disease onset from ageing effects in normal controls. The introduction of digital immunoassay platforms revolutionized biomarker detection sensitivity, now down to single molecule resolution , instead of relying solely on overall chemiluminescence intensity.The National Prion Monitoring Cohort (NPMC) study in the UK was well placed to address this unmet need, having recruited at-risk individuals with contemporaneous acquisition of longitudinal clinical, neuroimaging, neuropsychometric and neurophysiological data, along with assembling an expansive blood and CSF biofluid archive since 2008. In this study, we marshalled the combined utility of disease-specific PrP-amyloid seed amplification assay (RT-QuIC) and ultrasensitive multiplexed Simoa digital immunoassay platform to characterize biomarker discovery and evolution in individuals at risk of prion disease.Biofluid samples from all at-risk and symptomatic prion disease used in this study were drawn from individuals enrolled into the NPMC with written consent. Blood samples were routinely drawn at each assessment from 2008 onwards, while acquisition of CSF samples started in 2015 following an amendment to existing ethical approval for the NPMC through the Scotland A Research Ethics Committee (05/MRE00/63).PRNP mutations, and untested blood relatives of those affected by, or known to carry, pathogenic PRNP mutations. The iCJD-AR population in the NPMC is composed of recipients of cadaver-sourced human growth hormone up to 1985. The schedule of assessments, and hence biofluid sampling intervals, were administered according to the stratum in which a participant falls, determined by the projected rate of disease progression,34 and by clinical need. At each assessment, research blood samples were also taken with written informed consent from willing friends or non-blood relatives as controls.The NPMC enrolled eligible individuals from October 2008 onwards, encompassing those symptomatic of all forms of prion disease , asymptomatic individuals at risk of IPD (IPD-AR), iCJD (iCJD-AR) and vCJD, and healthy controls. The IPD-AR population includes confirmed asymptomatic carriers of pathogenic For Simoa biomarker comparison, healthy control CSF samples were sourced from the spouses and non-blood relatives of patients with young-onset Alzheimer’s disease (AD), the British 1946 Birth Cohort (Insight-46), CONFLUID cohorts , and NPMC ; healthy control plasma samples and data were sourced from NPMC internally (friends and non-blood relatives of patients) and from non-mutation carriers within the Genetic Frontotemporal Dementia Initiative cohort (GENFI). For CSF RT-QuIC analyses, control samples were sourced from Institute of Neuroscience and Physiology at University of Gothenburg , NHNN Neuroimmunology Laboratory (CSF referred for non-neurodegenerative indications) and from NPMC .Age at onset in IPD is highly variable (standard deviation ∼10 years even within a family), therefore many people who carry IPD mutations are healthy beyond their parental or average age of onset for each mutation. Consequently, we developed a new method to estimate the age of onset for IPD-AR, whereby each individual has an estimated age of onset in the future. This method approximates a cumulative normal distribution of risk for each mutation based on literature data, and sets estimated age of onset in the future equal to the accrual of 50% of an individual’s outstanding cumulative risk. Further details and an example are provided in the g for 10 min at room temperature (22°C) on arrival at the laboratory. The supernatant (upper plasma phase) was then divided into aliquots of 0.5–2.0 ml in Nunc Cryovials, and then frozen at −80°C.Whole blood samples collected in EDTA or citrate tubes destined for fractionation into plasma were centrifuged at 2000g for 10 min at room temperature, and supernatant separated into aliquots of 0.5–1.0 ml in Nunc Cryovials. Both were then stored in −80°C freezers.CSF samples were collected in two separate polypropylene tubes (Sarstedt 62.610.018), designated as CSF-R (for RT-QuIC) and CSF-N (for neurodegenerative markers). CSF-R was divided into aliquots of 0.5–1 ml in Nunc Cryovials after gentle mixing. CSF-N was centrifuged at 220036 The full-length human P102L rPrP  construct contained His-tags, and as such was purified using a different protocol with some minor modifications.37 Further details are available in the Full-length human  and bank vole rPrP , and truncated hamster  and truncated bank vole rPrP  were purified according to previously established methods.The standard RT-QuIC reaction mix per well was composed of 10 mM buffer , 130–300 mM NaCl or NaI, 0.1 mg/ml rPrP , 10 μM (ThT), 1 mM EDTA, and 0.001 or 0.002% SDS. Reactions were prepared in 96-well optical clear-bottomed plates . In each well, 80 or 85 µl of reaction mix was seeded with 20 or 15 µl of CSF, respectively, bringing the final volume up to 100 µl per well.38 Samples initially resulting in 1 in 4 positive wells were retested, and if 1 in 4 wells remained positive, were classed as ‘equivocal’. Time cut-offs were determined by incubation temperature i.e. 50 h for 42°C, 30 h for 50°C, and 24 h for 55°C.28Thereafter, the loaded plates were sealed (Thermo Scientific Nunc 232702) and incubated in BMG FLUOstar Omega Lite or POLARstar Omega microplate readers between 42°C and 55°C, at double orbital shake/rest cycles of 60 s/60 s at 700 rpm. ThT fluorescence readings  were recorded at intervals of 45 min. Each sample was tested in quadruplicate and classed as positive if the relative fluorescence units (RFU) in ≥2 of 4 wells exceed the 10% baseline-corrected threshold within the corresponding time cut-off points.39 Each sample was serially diluted by one-third using a single non-prion control CSF sample to reconstitute the total seeding volume per well to 20 µl. We define 50% seeding dose (SD50) as a unit of seeding activity or end point sample dilution that yields positive responses in 50% (e.g. 2 of 4) RT-QuIC reaction wells according to the criteria above. The SD50 can be estimated from the results of a dilution series using:CSF seeding doses were determined through end point quantitation of RT-QuIC PrP-amyloid seeding activity using the Spearman-Kärber method originally used in animal bioassay.xp=1 being the highest log10 dilution with 4/4 positive wells; d = log dilution factor; p = proportion positive at a given dose; ∑p = the sum of values of p for xp=1 and all higher dilutions. Adjustments can then be made to report SD50 per unit of neat sample, e.g. undiluted CSF. When a neat CSF sample (20 µl) yielded only 3/4 positive wells, the Spearman-Kärber method was not used, and instead, this sample was calculated to contain 1.5 SD50 (per 20 µl CSF) because, by definition, one SD50 gives 2/4 positive wells.where 32 In brief, samples were thawed and centrifuged at 10 000g for 5 min at room temperature (21°C) to precipitate any debris; subsequently, the samples were transferred to designated wells on the plates, diluted at 1:4 for plasma and 1:40 (or 1:100) for CSF with sample diluent, and bound to paramagnetic beads coated with capture antibodies specific for human GFAP, NfL, tau and UCH-L1. Longitudinal samples from a single patient where available, were analysed on the same plate. The biomarker-bound beads were then incubated with the respective biotinylated detection antibodies, which in turn are conjugated to streptavidin-β-galactosidase complex, which serves as a fluorescent tag. Hydrolysis of the complex of a resorufin β-D-galactopyranoside substrate results in a fluorescent signal proportional to the concentration of the respective biomarkers present. Measurements from each sample were with biomarker concentrations extrapolated from a standard curve, fitted to a four-parameter logistic algorithm. Coefficients of variation (CVs) were determined using four internal quality control samples, and were <20% and <10% for intra-assay and inter-assay comparisons.Plasma and CSF glial fibrillary acidic protein (GFAP), NfL, tau and ubiquitin C-terminal hydrolase L1 (UCH-L1) were measured by Simoa using the N4PB kit on a HD-X Analyser (Quanterix), following the manufacturer’s protocol.Additional previously measured plasma NfL and GFAP values from GENFI non-mutation carriers were also used to supplement healthy control data; five samples from this group were also analysed in our study to validate that the inter-assay CVs were < 15% for these two markers.33 Log10 transformation of GFAP, NfL, tau and UCH-L1 values reduced skewness across our sample cohorts, rendering them approximately normally distributed, allowing group-wise comparison of means using single factor ANOVA followed by pairwise t-tests. To address the known normal ageing effects on GFAP, NfL and Tau levels,40–43 biomarker values for healthy control CSF and IPD-AR CSF greater than 2 years to predicted onset were normalized to age 60 . Single factor ANOVA followed by pairwise t-tests (assuming α = 0.05) were then applied to compare means of age-normalized values grouped by the respective cohorts—healthy controls, IPD at-risk individuals more than 2 years to predicted/actual clinical onset (IPD-AR > 2 years), IPD at-risk individuals less than 2 years to predicted/actual clinical onset (IPD-AR < 2 years), symptomatic IPD individuals (IPD), sCJD/vCJD/iCJD individuals (CJD), and iCJD at risk individuals (iCJD-AR). Individual biomarker slopes were modelled using mixed effects models, with random effects of slopes and intercepts.Similar to previous experience, the N4PB biomarker values including healthy controls were positively skewed.Confidence intervals (95% CI) for sensitivity and specificity of RT-QuIC assays were determined using the exact Clopper-Pearson interval.Statistical analyses were carried out using GraphPad Prism (version 9.2.0) and STATA v15.1.All raw data, including Simoa values and RT-QuIC relative fluorescence units, are available on request. Corresponding clinical data may be requested, but a data transfer agreement is likely to be required, including restrictions that protect confidentiality and consent terms.PRNP mutations in our combined IPD and IPD-AR biofluid cohorts in this study included 5-OPRI, 6-OPRI, P102L, P105S, A117V, Y157X, D178N-129V, D178N-129M, Y163X and E200K. For CJD, this included samples from sCJD, iCJD (cadaver-sourced human growth hormone) and vCJD; the iCJD-AR cohort only included recipients of implicated batches of cadaver-sourced human growth hormone. IPDs are further broadly classified as ‘fast’ or ‘slow’ according to mutation and its associated median/mean survival ; slow IPD > 12 months e.g. OPRIs, P102L, A117V, Y163X, P157X, etc.). We defined clinical conversion as the emergence of characteristic neurological symptoms and signs along with functional decline measurable by MRC Prion Disease Rating Scale scores, supported by the presence of mutation-specific investigation abnormalities e.g. diffusion weighted imaging abnormalities in E200K, neurophysiological abnormalities in P102L, polysomnographic abnormalities in D178N-FFI, etc.The range of n = 61; individuals = 39), followed by IPD , sCJD/iCJD  and c-hGH iCJD-AR , which were tested against non-prion controls . Three pairs of samples exist from E200K, 6-OPRI and P102L converters, each with one sample before and after conversion. Baseline demographic details are summarized in From 2015 to 2021, 161 CSF samples were accrued for RT-QuIC analysis; IPD-AR samples account for the largest proportion  and CSF  samples capturing the interlude spanning clinical conversion ; in two other IPD-AR individuals, a single plasma sample each was collected within 2 years of clinical conversion but none after. Of the 16 converted IPD individuals, eight had plasma NfL and tau levels measured by simplex Simoa platforms and published previously.44 In addition to the IPD-AR cohort, plasma  and CSF  samples from asymptomatic h-GH iCJD-AR individuals were also tested against symptomatic IPD, CJD , and healthy control cohorts, with baseline demographics summarized in We assembled a total of 416 plasma and 135 CSF samples, from 2008 to 2021, for Simoa N4PB measurements. The IPD-AR cohort accounts for the majority of samples for both biofluids with 217 plasma samples from 69 unique individuals, and 67 CSF samples from 40 unique individuals. Crucially, this included longitudinal plasma ] were first screened with IQ-CSF RT-QuIC.n = 59) tested negative for IQ-CSF RT-QuIC with 20 µl seeding volume, as did all the control CSF (n = 47) using 15 µl seeding volume. Overall, the IQ-CSF RT-QuIC sensitivity and specificity for CJD and E200K IPD samples were both 100%.Initial IQ-CSF RT-QuIC survey of the CJD sample set gave 15 positive (≥2/4 wells) and two equivocal (1/4 wells) results, with the equivocal samples becoming positive after adjustment of seeding volume from 20 µl to 15 µl. All four CSF from symptomatic E200K carriers were strongly positive with IQ-CSF RT-QuIC where all wells became positive within 10 h of incubation. CSF from a single 6-OPRI carrier drawn following an unexpected ‘CJD-like’ transformation with corresponding DWI changes on MRI brain indistinguishable from sCJD after several years of classical 6-OPRI disease progression, was also strongly positive . CSF fro45 CSF samples from three symptomatic P102L individuals with purely cognitive phenotypes and two with classical GSS phenotype tested negative with Hu P102L, wild-type Hu, BV, IQ-CSF RT-QuICs and all other exploratory conditions.A new bespoke variation of RT-QuIC using HuPrP P102L  was positive in four of nine symptomatic P102L carriers. Of note, all four positive samples were from those with classical GSS phenotype at onset, though one underwent a ‘CJD-like’ transformation featuring typical DWI MRI brain changes after 2 years.CSF seeding activity (3/4 wells) in a symptomatic P105S carrier with a CJD-like phenotype with cortical ribboning on DWI MRI Brain, was best demonstrated using Hu rPrP in pH 7.4 with 130 mM NaI at 42°C .Optimum RT-QuIC conditions for D178N-129 M, Y163X and classical 6-OPRI were not found despite extensive exploration . In instWe divided the at-risk samples into the following groups, and matched them to the best available RT-QuIC assay determined in the exploratory phase: (i) E200K-AR and iCJD-AR to Ha90 rPrP in pH 7.4 and 300 mM NaCl at 55°C (IQ-CSF RT-QuIC); (ii) P102L-AR to HuPrP P102L in pH 7.4 and 130 mM NaI at 42°C (Hu P102L RT-QuIC) and BV rPrP in pH 7.4 and 300 mM NaCl at 50°C (BV RT-QuIC); and (iii) Other-AR to BV RT-QuIC.n = 22) revealed four positive results. All of these samples recorded 4/4 wells positive apart from one sample (remains asymptomatic at 3.37 years follow-up) in which 3/4 wells were positive  sample from an asymptomatic at-risk untested individual over the age of 60; 1/57 non-prion control also tested positive . Both th29 None of the Other-AR samples (0/12) nor in the P102L-AR subgroup (0/27) tested positive with BV RT-QuIC. All the non-prion control CSF samples were negative (n = 51).The Other-AR samples were tested with the BV RT-QuIC assay on the basis of BV rPrP being a potential ‘universal acceptor’ in experiments seeded by brain homogenates.post hoc groupwise comparisons . Of note, there were no significant differences in the mean values between the healthy control and IPD-AR > 2 years cohorts . The mean values of the N4PB biomarkers according to cohort divisions, and the P-values from the single factor ANOVA analyses are summarized in Log(GFAP) and log(NfL) demonstrated sequentially incremental and statistically significant mean values between IPD-AR > 2 years and IPD-AR < 2 years, IPD and CJD cohorts on single-factor ANOVA with P = 0.329), healthy control/IPD-AR > 2 years versus IPD (P = 0.1), and IPD-AR < 2 years versus IPD (P = 0.849). Mean log(UCH-L1)  was not statistically different between IPD-AR > 2 years versus IPD-AR < 2 years (P = 0.802). As for the iCJD-AR cohorts, relevant statistically significant mean values were only seen with log(GFAP) against the CJD cohort , and with log(Tau) against normal controls ; the latter was driven by a single outlier in the sample obtained from an iCJD-AR individual with contemporaneous destructive pituitary craniopharyngioma.Mean age-normalized log(Tau) was not statistically significant between IPD-AR > 2 years versus IPD-AR < 2 years  who underwent clinical conversion during follow-up over a median of 7.8 years [interquartile range (IQR) 5.2 years] in whom at least one presymptomatic plasma sample was available for analysis. An incline in plasma log(GFAP) and log(NfL) values was observed, but most consistently in P102L, D178N-FFI and E200K converting individuals. The pattern of log(GFAP) and log(NfL) evolution for the clinically fast IPD converters (E200K ± D178N-FFI)33 tend to exhibit relatively flat lines followed by abrupt rises close to or at the time of clinical onset. In comparison, the clinically slow IPD converters (P102L) showed a slower but more consistent upward trajectory in log(GFAP) and log(NfL) values, with 52.6% (10/18) and 44.4% (8/18) of measurements above the 90th percentile of healthy controls (HC90), respectively in the 2 years before clinical onset  of 2.448 years; for plasma log(GFAP) 0.090 pg/ml/year in slow IPD  with a x-intercept of 4.009 years and 0.458 pg/ml/year in fast IPD .We therefore modelled the linear trajectories of presymptomatic NfL and GFAP using mixed effects regression models with random effects for individual slopes and ‘fast IPD’ or ‘slow IPD’ as factor variables, including data prior to conversion  and one time point up to 6 months after conversion. These models estimated a slope for plasma log(NfL) of 0.108 pg/ml/year in slow IPD  and 1.279 pg/ml/year in fast IPD  with an P = 0.03, IPD-AR < 2 years versus healthy controls P = 0.04, and IPD-AR < 2 years versus IPD P = 2.95 × 10−5). No significant differences were shown between disease stages with log(GFAP). Statistically significant differences were seen for log(Tau) and log(UCH-L1) between IPD > 2 years and IPD < 2 years versus IPD, but not between IPD > 2 years versus IPD < 2 years.In the CSF cohort, only log(NfL) successfully demonstrated incremental, and statistically significant segregation of the mean values between the IPD > 2 years, IPD < 2 years and IPD stages  registered a CSF log(NfL) value (3.07 pg/ml) above the HC90 (2.97 pg/ml) in the 2 years prior to clinical onset; all the other N4PB biomarkers in the remaining two converters were below the HC90 values  change explosively around onset with no definable presymptomatic window. Second, in those IPDs for which we have highly sensitive seed amplification assays, particularly E200K, we found evidence of a presymptomatic CSF RT-QuIC seeding stage, considerably longer than the clinical phase of the disease . These distinct aspects of prion pathophysiology are consistent with the two-phase kinetics model of prion propagation.21 In our cohort, no E200K at-risk individual converted without presymptomatic seeding activity. The E200K presymptomatic seeding period (as long as 3.75 years) appears unexpectedly long for an illness with such an explosive onset and short duration. The utility of tracking CSF SD50 values as a proximity marker remains unknown at present given the conflicting trajectories, and will require analyses of greater number E200K-AR follow-up samples and converters to fully elucidate. None of the presymptomatic RT-QuIC positive samples, including one drawn shortly before conversion, recorded abnormal neurodegeneration biomarkers, indicating that the onset of neurodegeneration is likely to be very close to conversion and potentially unrecognizable at current sampling intervals. As such, for those at incipient risk of clinical conversion, we expect asymptomatic positive CSF RT-QuIC (without evidence of neurodegeneration) to herald onset of biomarker evolution towards clinical conversion, though the intervals between seeding, neurodegeneration and clinical onset remain imprecise. Conversely, E200K carriers with negative CSF RT-QuIC and normal neurodegenerative markers may not be at risk of incipient conversion.Our analytical approach was underpinned by the expectation that any RT-QuIC assay ought to be sufficiently sensitive for detecting seeding activity in symptomatic CSF samples in order to be able to do so in presymptomatic samples. The compatibility of IQ-CSF RT-QuIC for CJD and E200K seeds was confirmed in the symptomatic cohort, and then in the at-risk sample set by picking up four positive samples, one of which belonged to a subsequent converter. This argues that the previously reported asymptomatic positive RT-QuIC in an E200K carrier was not an isolated finding.et al.27 is an exception; see the 47 P102L individuals in these papers were classified as GSS with little information provided about clinical phenotype. Given the recognized phenotypic heterogeneity  of P102L disease, and molecular evidence that these may be driven by distinct prion strains and possibly by non-infectious PrP amyloids accumulation, it is difficult to compare the results. It is quite possible that the few RT-QuIC positive samples reported may very well be due to the enrichment of individuals with the CJD-like clinical phenotype within surveillance cohorts.49CSF from P102L affected individuals has historically been tested by variations of PQ-CSF and IQ-CSF RT-QuIC, usually included as very small subsets within large surveys of national CJD surveillance cohorts, with low sensitivities  and log(NfL) trajectories together with a considerable proportion [including CSF log(NfL)] being above HC90 in the 2 years before onset denote a longer pre-conversion phase of escalating toxicity relative to E200K. Slow IPDs may possess an extended pre-conversion seeding window if appropriately sensitive assays can be developed, but more clearly, show an identifiable presymptomatic neurodegeneration window.We developed a bespoke RT-QuIC assay using Hu P102L rPrP and NaI capable of detecting CSF seeding activity in a subset of P102L diseased individuals and a single untested at-risk individual over 60 years of age . Detailed phenotypic profiling suggest that this assay may work best in the P102L-GSS and P102L-CJD subgroups, but not in the P102L-Cognitive subgroup; due to the small numbers tested, it is unknown whether this observation will hold true when applied to larger P102L CSF sample sets. We speculate that the single RT-QuIC positive control sample may have belonged to an undiagnosed P102L patient, based on its highly selective amplification solely by the Hu P102L RT-QuIC (negative with IQ-CSF and BV RT-QuIC), and compelling kinetic curves, unlike the dubious false positives (‘slight amplification’ and ‘slowly rising curve’) reported in the literature.−4 dilutions) of all but D178N-129V cases can be detected using BV RT-QuIC.29 Neither plasma log(NfL) nor log(GFAP) appeared helpful in identifying D178N-129M individuals at risk of incipient conversion. Little conclusion can be drawn as yet from the inconsistent N4PB biomarker trajectories for our small number of 5-OPRI and 6-OPRI converters.The promise of BV rPrP as a ‘universal acceptor’ did not materialize during the RT-QuIC optimization phase, despite efforts to improve sensitivity. No presymptomatic CSF seeding activity was detected in classical 6-OPRI, P102L, A117V, D178N-129M and D178N-129V at-risk samples using BV RT-QuIC despite previous demonstrations that brain homogenates  are being honed for wider application in tissues and CSF.62–65If seeding assays can be more widely developed and applied in neurodegenerative diseases, as seems likely, these findings might provoke exploration of long presymptomatic seeding phases in other disorders. Discoveries in recent years have revealed fundamental aspects of common neurodegenerative diseases similar to prion diseases, particularly in proteopathic seed propagation, transmissibility and strain biology.-only phase without evidence of neurodegeneration, will open even earlier windows of opportunity for intervention. This has particular implications on timing, and study design for therapeutic strategies against neurodegenerative diseases including human prion disease.The extension of the presymptomatic phase to include a proteopathic seedingawad101_Supplementary_DataClick here for additional data file.


Gluteus medius + minimus  and hamstrings  mCSA demonstrated little to no growth with small differences between groups. mCSA changes were greater in SQ for the quadriceps  and adductors . Squat 3RM increases favored SQ  and hip thrust 3RM favored HT . 3RM deadlift  and wall push strength  similarly improved. All measured gluteal sites showed greater mean sEMG amplitudes during the first bout hip thrust versus squat set, but this did not consistently predict gluteal hypertrophy outcomes. Squat and hip thrust training elicited similar gluteal hypertrophy, greater thigh hypertrophy in SQ, strength increases that favored exercise allocation, and similar deadlift and wall push strength increases.We examined how set-volume equated resistance training using either the back squat (SQ) or hip thrust (HT) affected hypertrophy and various strength outcomes. Untrained college-aged participants were randomized into HT (n = 18) or SQ (n = 16) groups. Surface electromyograms (sEMG) from the right gluteus maximus and medius muscles were obtained during the first training session. Participants completed 9 weeks of supervised training (15–17 sessions), before and after which gluteus and leg muscle cross-sectional area (mCSA) was assessed via magnetic resonance imaging. Strength was also assessed prior to and after the training intervention via three-repetition maximum (3RM) testing and an isometric wall push test. Gluteus mCSA increases were similar across both groups. Specifically, estimates [(−) favors HT (+) favors SQ] modestly favored the HT  Resistance training (RT) presents potent mechanical stimuli that produce robust biological responses . HoweverThe reliance on theory and acute measures to guide exercise selection is especially evident in the hip extension exercise literature, an area of particular interest with applications in rehabilitation , aging , performMuscle tension is the primary driver of muscle hypertrophy but is unlikely to be its sole determinant. Recent evidence demonstrates that RT at long muscle lengths and long-duration static stretching can augment hypertrophic outcomes , suggestDirect evidence is presently needed to compare the outcomes of various exercises. Therefore, the purpose of this study was to examine how RT using either the barbell squat or barbell hip thrust on a set-volume equated basis affected gluteus maximus, medius, and minimus muscle hypertrophy (determined by MRI) and various strength outcomes including the back squat, hip thrust, deadlift, and isometric wall push. As a secondary outcome, we sought to determine how these exercises affected gluteus maximus/medius muscle excitation patterns using sEMG and if sEMG amplitudes forecasted hypertrophy.2) of less than 30, have minimal experience with resistance training, averaging less than or equivalent to 1 day per week for the last 5 years; (c) have not been actively participating in any structured endurance training program  for more than 2 days per week over the past 6 months; (d) free of any known overt cardiovascular or metabolic disease; (e) have not consumed supplemental creatine, and/or agents that affect hormones  within the past 2 months, f) free of any medical condition that would contraindicate participation in an exercise program, (g) do not have conditions which preclude performing an MRI scan , h) and free of allergies to lactose or intolerances to milk derived products that would contraindicate ingestion of whey protein. Eligible participants who provided verbal and written consent partook in the testing and training procedures outlined in the following paragraphs.Before commencing study procedures with human participants, this study was approved by the Auburn University Institutional Review Board (protocol #: 22-588). All approved study procedures followed the latest revisions to the Declaration of Helsinki (2013) except for being pre-registered as a clinical trial on an online repository. Inclusion criteria were as follows: (a) between the ages of 18–30 years old with a body mass index  >0.990 for whole body intracellular and extracellular water metrics on 24 participants  for urine specific gravity assessment (USG). Measurements were performed using a handheld refractometer , and USG levels in all participants were ≤1.020, indicating sufficient hydration. Participants’ heights were measured using a stadiometer and body mass was assessed using a calibrated scale  with body mass being collected to the nearest 0.1 kg and height to the nearest 0.5 cm. Body composition was then measured by bioelectrical impedance spectroscopy (BIS) using a 4-lead  SOZO device  according to the methods described by Moon et al. . Our labicipants , and thi2) was used to obtain transverse image sets. 71 slices were obtained with a slice thickness of 4 mm with no gap between slices. Measurements were taken by the same investigator (R.J.B.) for all scans who did not possess knowledge of the training conditions for each participant.MRI testing assessed the muscle cross-sectional area (mCSA) of both glutei maximi. Upon arriving to the Auburn University MRI Research Center, participants were placed onto the patient table of the MRI scanner  in a prone position with a ∼5-min latency period before scanning was implemented. A T1-weighted turbo spin echo pulse sequence , and these images were imported into ImageJ  whereby the polygon function was used to manually trace the borders of muscles of interest to obtain mCSA. For all participants, image standardization was as follows: (a) the middle of the gluteus maximus was standardized at the image revealing the top of the femur, (b) the image that was 10 slices upward from this mark was considered to be the upper gluteus maximus, (c) the image that was 18 slices downward from the top of the femur was considered lower gluteus maximus, (d) gluteus medius and minimus mCSAs were ascertained at the upper gluteus maximus image, and (e) combined quadriceps (vastii and rectus femoris), adductors , and combined hamstrings  mCSAs were ascertained at the first transverse slice distal to the last portion of the lower gluteus maximus. When drawing borders to quantify muscles of interest, care was taken to avoid fat and connective tissue. Certain muscles were grouped  due to inconsistent and poorly delineated muscle borders within participants. All left- and right-side gluteus muscles were summed to provide bilateral mCSA values at each site. Alternatively, thigh musculature mCSA values were yielded from the averages of the left and right legs. This method was performed on the thigh because ∼10% of participants yielded either left or right thigh images that presented visual artifacts from the edge of the MRI receiving coil. In these situations, thigh musculature from only one of the two legs was quantified.Participants reported to the laboratory (non-fasted) having refrained from any exercise other than activities of daily living for at least 48 h before baseline testing. A tri-axial force plate  with an accompanying amplifier (Bertec model # AM6800) sampling at 1,000 Hz was used to measure horizontal force production in newtons N) during a wall push test. The distance from the force plate to the wall was positioned such that when the subjects’ forearms were parallel with the ground, the torso was at a ∼45° angle with the ground and one rear foot was in contact with the force plate. Hand placement was standardized by distance from the ground and foot placement was standardized by distance from the wall. The subject was instructed to push, using the dominant leg, as hard as possible into the wall while keeping the torso at 45° . Two walFollowing wall push testing, dynamic lower body strength was assessed by three-repetition maximum (3RM) testing for the barbell back squat, barbell hip thrust, and barbell deadlift exercises. Notably, our laboratory has extensively performed 3RM dynamic strength testing on numerous occasions in untrained and trained participants . BrieflyResults). Upper and middle gluteus maximus electrodes were placed based on the recommendations of Fujisawa and others , where were placed in parallel to the fibers of the right upper gluteus maximus, mid gluteus maximus, lower gluteus maximus, and gluteus medius  of the 34 participants who finished the intervention. Data were dropped from analyses due to artifacts produced through either electrode slippage or sEMG electrode jarring during the 10RM sets, leading to persistent distortion. In this regard, sample sizes for each muscle site are presented in the results section.Signal processing was performed using software associated with the sEMG system . sEMG signals from the MVICs and 10RM sets of back squat and hip thrust were first rectified. Signals were then processed with a second-order digital low-pass Butterworth filter, with a cutoff frequency of 10 Hz, and further smoothed using a root mean square moving window of 250 m. The average of the middle 3 s of the filtered MVIC time series was then used to normalize the squat and hip thrust data for each site. Data were then visually inspected for fidelity before calculating the mean and peak sEMG values. Partial sequences of sEMG data were removed in the rare event that tempo was irregular or not maintained, or if a brief artifact was introduced. Final EMG data are presented as mean and peak sEMG amplitudes during the hip thrust and back squat 10RM sets. sEMG issues were only evident for a small portion  and were instructed to consume one serving per day . This was done in the hope of diminishing inadequate protein intake as a confounding variable. Other than this guidance, participants were advised to maintain their customary nutritional regimens to avoid other potential dietary confounders.All participants were instructed to provide 4-day food logs  during the first and last weeks of the intervention. A registered dietician (A.D.R.) oversaw food log analyses using The Nutrition Data System for Research  food log entry and analyses . CalorieInvestigators were blinded to group allocation during the MRI scan and its analysis. Participants were not blinded to group allocation as exercise comparisons were not amenable to blinding. Due to logistical constraints investigators were not blinded to group allocation during strength testing and, thus, bias cannot be completely ruled out in this context. Randomization into SQ and HT groups was performed via a random number generator in blocks of 2 or 4 as participants consented.https://www.jamovi.org) and R (version 4.3.0). We performed three different sets of analyses.Data were analyzed in Jamovi v2.3  on each outcome variable was estimated using linear regression, in which post-intervention scores were the response variable, group was dummy-coded 0 for SQ and 1 for HT, and the pre-intervention score was included as a covariate of no interest  was the response variable; sEMG amplitude, group, and their interaction were fixed effects; and we permitted intercepts and slopes for sEMG amplitude to vary across subjects. Since we are interested in generalizable predictions, we calculated prediction intervals for the slopes by calculating a Wald interval using the sum of the parameter variance and random effects variance. iii) Can the differences in growth elicited from different exercises be accounted for by sEMG amplitude? For this, we calculated the so-called “indirect effect” of sEMG amplitude, which represents the extent to which the group effect on hypertrophy can be explained by sEMG amplitudes. This was done the same way a typical “mediation analysis” is done —we bootstrapped the difference between the group effect (SQ vs HT) when sEMG was not in the model and when sEMG was added to the model. If group-based sEMG differences accounted for group-based hypertrophy differences, then the effect of group on growth would shrink towards 0 and sEMG would absorb the variance in growth.Third, we investigated the extent to which sEMG amplitudes from the first session forecasted growth. There are multiple ways this question could be posed, and since claims surrounding sEMG amplitude’s predictive power are ambiguous, we addressed each of the following questions: i) Do individuals with greater sEMG amplitudes grow more than individuals with lower sEMG amplitudes? For this, we calculated a Pearson correlation for each muscle using changes in mCSA and the sEMG amplitudes. ii) Do regions or muscles with greater sEMG amplitudes grow more than regions or muscles with lower sEMG amplitudes? For this, we used a linear mixed-effects model in which ln  were analyzed using two-way repeated measures ANOVAs.https://www.biorender.com), GraphPad Prism v9.2.0 , and ggplot2.Figures were constructed using Microsoft PowerPoint and through paid site licenses for BioRender  two SQ participants reported 3 days only with the first food log, ii) two SQ participants were missing last-week food logs, and iii) one HT participant did not turn in the first food log. Thus, food log data were analyzed for n = 15 SQ and n = 16 HT participants. No group x time (G x T) interactions were evident for any of the assessed variables (data not shown).versus squat set , whereas small differences existed for the lower gluteus maximus or gluteus medius sites ], mid , and upper  gluteal mCSAs; these point estimates were dwarfed by the variance. Left + right mCSA values for the gluteus medius + minimus demonstrated a lesser magnitude of growth, with a point estimate that also modestly favored HT albeit with appreciable variance .The effect of SQ relative to HT for left + right mCSA was negligible across gluteal muscles . Point e2; CI95% , 2; CI95% , 2; CI95% , Compared to HT, SQ produced greater mCSA growth for quadriceps , middle, [r = −0.03 ], upper [r = 0.50 ], and medius + minimus [r = 0.28 ]. We observed similar results for peak sEMG amplitudes from the lower [r = 0.13 ], middle [r = −0.03 ], upper [r = 0.32 ], and medius + minimus [r = 0.24 ].sEMG amplitude’s ability to forecast muscle growth across-subjects was generally poor and variable. Mean sEMG amplitudes produce negligible to moderate correlations for lower [95% ] and HT  groups. The second model, which used peak sEMG amplitudes, produced even more modest results for both the SQ  and HT  groups.We fit two linear mixed-effects models to assess how differences in sEMG amplitudes across muscles can account for regional growth. Since the response variable was change in muscle size on the log scale, the exponentiated coefficients can be interpreted as the increase in muscle relative to baseline for each additional %MVIC; notably, this effect is multiplicative rather than additive. The first model, which used mean sEMG amplitudes, produced small and variable estimates for both SQ , middle , upper , and medius + minimus . We observed similar results for peak sEMG amplitudes for lower , middle , upper , and medius + minimus . These estimates can be compared to the group effects  earlier in the Results.Mean sEMG amplitude’s ability to capture the group effects was inconsistent for lower [indirect effect = −0.55, CITo further our understanding of hip extensor exercises and the validity of relying on theory and acute physiological measures for exercise selection, here we acutely (sEMG) and longitudinally  compared two common hip extension exercises: the back squat and barbell hip thrust. Acutely, HT sEMG amplitudes were generally greater for the HT. However, this did not appear to translate and accurately capture longitudinal adaptations. Across all gluteus muscle hypertrophy outcomes, SQ and HT training yielded modest differences but meaningful growth occurring, except in the gluteus medius and minimus. Thigh hypertrophy outcomes favored SQ in the adductors and quadriceps, with no meaningful growth in either group in the hamstrings. Strength outcomes indicated that hip thrust 3RM changes favored HT, back squat 3RM changes favored SQ, and other strength measures similarly increased in both groups. sEMG amplitudes could not reliably predict hypertrophic outcomes across several analytical approaches. In the following paragraphs, we discuss these results in the context of available evidence and speculate on their potential implications for exercise prescription.The primary finding of interest was that upper, middle, and lower gluteus maximus muscle hypertrophy was similar after 9 weeks of training with either the squat or hip thrust. This may seem to run counter to the notion that muscle tension in lengthened positions augments growth , since tIn addition to motor control governing how the gluteus maximus contributes to and adapts from the squat, there are study-specific considerations. Both exercises may stimulate similar muscle hypertrophy in untrained populations given that RT in general elicits rapid growth early on, potentially creating a ceiling effect on growth rate and thus observed growth. Alternatively stated, skeletal muscle hypertrophy in novice trainees may be less influenced by nuances in exercise selection. Notwithstanding, our results suggest that a 9-week set-equated training program with either the hip thrust, or squat elicits similar gluteal muscle hypertrophy in novice trainees.Finally, our data show that thigh hypertrophy favored the squat, whereas thigh hypertrophy was minimal in the hip thrust. This is perhaps unsurprising and is consistent with previous literature. The adductors, particularly the adductor magnus, have the largest extension moment contribution at the bottom of a squat. Thus, the nervous system may favor its recruitment for this purpose. In line with this finding, adductor magnus mCSA changes favor a greater squat depth . HamstriBoth groups effectively increased strength outcomes for all exercises tested, with magnitudes in accordance with previous strength literature . HoweverOur secondary aim was to evaluate the ability of sEMG to forecast longitudinal adaptations. In agreement with previous work , gluteusFinally, we also verbally asked participants which exercise they “felt more” in the gluteal muscles after testing both exercises. All participants indicated they felt the hip thrust more in the gluteal region. However, these data were not quantified and, despite these anecdotal sensations and sEMG differences indicating more gluteus muscle excitation during HT, hip thrust RT and squat RT elicited similar applied outcomes. These findings highlight the importance of longitudinal investigations.Our study has a few limitations to consider. First, our participants were young untrained men and women; thus, results cannot necessarily be generalized to other populations including adolescents, older individuals, or trained populations. Additionally, like most training studies, this study was limited in duration. It should also be noted that gluteal hypertrophy was the main outcome, and the MRI coil was placed over this region as subjects were lying prone. Thus, compression may have affected the thigh musculature, and distal measures were not obtained for the thigh. Finally, training volume was equated, and frequency was set at two training days per week. Therefore, results can only be generalized to this protocol.Although we did not consider female participants’ menstrual cycle phase or contraceptive usage, we do not view this as a limitation. In this regard, several reports indicate that contraceptives have no meaningful impact on muscle hypertrophy in younger female participants during periods of resistance training . LikewisFuture research should aim to examine a group that performs both exercises on a volume-equated basis to determine if there are synergetic effects. Comparing these exercises with different volumes/frequencies is also warranted as exercises may have differing volume tolerances. From a mechanistic standpoint, future studies should characterize anabolic signaling between different points on the length-tension curve as well as ascertain where a muscle exists on this curve with more clarity.Squat and hip thrust RT elicited similar gluteal hypertrophy, whereas quadriceps and adductors hypertrophy was superior with squat training. Further, although strength increases were specific to exercise allocation, both forms of RT elicited similar strength transfer to the deadlift and wall push. Importantly, these results could not be reliably predicted from acute data (sEMG). These current data provide trainees with valuable insight concerning two widely popular hip-specific exercise modalities, and this information can be leveraged for exercise selection based on specific structural or functional goals.


The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte–macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ond Histiocytic sarcoma (HS), which can occur in a disseminated or localized form, is a malignant tumor in humans and dogs that has a comparable poor prognosis in both species. Due to the higher prevalence of canine HS compared with its human counterpart, dogs are an interesting translational model for this neoplastic disease ,2,3,4,5.Treatment options for canine HS are still very limited ,2,3,4,5.+ and CD8+ T lymphocytes [Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a link between the innate and adaptive immune systems. It is a highly effective cytokine and one of the most commonly inserted genes in oncolytic viruses . GM-CSF phocytes ,21,22,23+ and CD8+ T cells, was observed in a syngeneic colon adenocarcinoma model in immunocompetent mice [The presence of GM-CSF leads to the polarization of tumor-associated macrophages (TAMs) into pro-inflammatory M1 macrophages, which release anti-tumoral cytokines ,24. In aent mice . The latent mice . Althougent mice ,25,26,27ent mice ,21,28. neon-GM-CSF [neon-GM-CSF infects DH82 cells at a high rate, and the infected cells produce GM-CSF [Canine distemper virus , a morbillivirus closely related to MV, has already demonstrated promising anti-tumor effects against canine histiocytic sarcoma in vitro and in a murine xenotransplantation model ,33,34,35n-GM-CSF ,36). CDVe GM-CSF . neon-GM-CSF in vitro.The aim of the present study was to functionally characterize canine GM-CSF secreted by DH82 cells persistently infected with CDV-Ondneon and CDV-Ondneon-GM-CSF were obtained as previously described [2, in a water-saturated atmosphere). The medium was changed every 2–3 days.Non-infected canine histiocytic sarcoma cells (DH82 cells) obtained from the European Collection of Authenticated Cell Cultures (ECACC No. 94062922) were cultured in Minimal Essential Medium (MEM) with Earle’s salts  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin , and 1% non-essential amino acids . DH82 CDV-Ond pi and DH82 cells persistently infected with CDV-Ondescribed . Human cneon) and CDV-Ond with the insertion of mNeonGreen and canine GM-CSF (CDV-Ondneon-GM-CSF), were obtained as described before [neon, and CDV-Ondneon-GM-CSF and those from non-infected controls were harvested 7 days after seeding and centrifuged . Afterward, the pH of the supernatants was lowered with HCl to a pH of 2 for 30 min to inactivate the CDV. Afterward, NaOH was added to increase the pH of all the supernatants to 7. The measurements of pH were performed using pH indicator strips .Supernatants from non-infected DH82 cells and DH82 cells persistently infected with canine distemper virus (CDV) strain Onderstepoort (CDV-Ond), along with modified strains of CDV-Ond with the insertion of mNeonGreen . Ten-fold dilutions of the supernatants were prepared in DMEM  containing 10% fetal bovine serum , 1% penicillin/streptomycin , and phleomycin D1 . The titration was performed in 96-well microtiter plates  containing Vero.DogSLAM cells (1.5 × 104 cells/well). Cells were incubated with the acidified supernatants for 7 days, and the cytopathic effect (CPE) was evaluated daily. The 50% log10 tissue culture infectious dose per milliliter (TCID50/mL) of cell-free virus was calculated according to the Reed and Muench method [Virus titration was performed in triplicate to confirm the inactivation of CDV as previously described . Brieflyh method .In order to verify a CDV infection or confirm the successful inactivation, Vero.DogSLAM cells were immunolabelled after virus titration using an antibody directed against CDV nucleoprotein as previously described . BrieflyTotal RNA was isolated using the RNeasy Mini Kit  with on-column DNA digestion with RNase free DNase  according to the manufacturer’s protocols. The RNA concentration was spectroscopically measured at 260 nm using the GeneQuant pro . RNA to cDNA transcription was performed according to the manufacturer’s protocols with OmniScript , RNAseOut , and Random Hexamers . Reverse transcription was performed using a Biometra Thermocycler T-Gradient ThermoBlock  under the following conditions: 25 °C for 10 min, 37 °C for 1 h, and 93 °C for 5 min.The primers for qualitative and quantitative RT-PCR for the detection of CDV nucleoprotein mRNA transcripts were taken from the literature . The pri® Gel and PCR Clean-up Kit  was used for the isolation of PCR amplicons from the agarose gel according to the manufacturer’s protocol. Standard curves for the estimation of copy numbers were generated using RT-PCR amplicons in a serial dilution from 108 to 102 copies/µL. RT-qPCR was performed with four samples per condition and negative controls, as previously described [The NucleoSpinescribed . Quantitneon, and CDV-Ondneon-GM-CSF was carried out in three independent samples, as previously described [TM BCA Protein Assay Kit . The same amount of protein was separated on 15% (immunoblotting of GM-CSF) or 6% (immunoblotting of CDV) SDS-PAGE gels. The proteins were transferred to a nitrocellulose membrane  and blocked with 5% milk blocking buffer  in 0.1% Tween 20 in phosphate-buffered saline (PBS) for 1 h. Immunoblotting was performed using a primary antibody directed against canine GM-CSF , CD116 , and CDV nucleoprotein (NP) , respectively. Horseradish peroxidase (HRP)-conjugated rabbit anti-goat , goat anti-rabbit , and rabbit anti-mouse  antibodies were used as secondary antibodies for the detection of GM-CSF, GM-CSFR, and CDV nucleoprotein, respectively. Protein bands were visualized using a chemiluminescent substrate  and a ChemiDoc MP Imaging System . Densitometric analysis was performed to quantify the band sizes and intensities using ImageJ version 1.51.0 (https://imagej.nih.gov/ij/). The immunoblotting of supernatants obtained from non-infected DH82 cells or DH82 cells infected with CDV-Ond, CDV-Ondescribed ,29. The neon, and CDV-Ondneon-GM-CSF were used. HeLa cell pellets with a known expression of the GM-CSF receptor (CD116) served as positive controls. Immunohistochemistry was carried out as previously described [Immunohistochemistry for the GM-CSF receptor (CD116) was performed to investigate the expression in DH82 cells. Therefore, formalin-fixed, paraffin-embedded cell pellets obtained from non-infected DH82 cells and DH82 cells persistently infected with CDV-Ond, CDV-Ondescribed . Immunolescribed . Briefly5 cells/well into 96-well microtiter plates  with 200 mL of Minimal Essential Medium (MEM) with Earle’s salts  supplemented with 10% fetal bovine serum , 1% penicillin/streptomycin  and 1% non-essential amino acids . Cells were cultured for 2 days under standard conditions . When cells reached 99% confluence, the cell monolayer was scratched in a straight line with a p100 pipette tip. The medium was discarded, and the cell monolayer was washed with a washing medium  with Earle’s salts ). Acidified supernatants obtained from non-infected DH82 cells or DH82 cells infected with CDV-Ond, CDV-Ondneon, and CDV-Ondneon-GM-CSF, respectively, were added to the scratched wells. Culture supernatant supplemented with 2 ng/mL of canine GM-CSF  was used for additional samples. Pictures were taken at the same position directly after performing the scratch (T0) and after 24 h (T24) using a phase contrast microscope  equipped with an Olympus DP-72 camera and Olympus cellSens standard software version 2.3 . The percentage of cell-free area was calculated with ImageJ 1.52p according to the following formula: 100 − percentage of cell-covered area, as previously described [0 − Area T24|)/Area T0 ∗ 100. The scratch assay was performed as previously described . Brieflyescribed . The cha4 cells in quadruplicate in 24-well plates in MEM medium  with the addition of recombinant human GM-CSF , canine GM-CSF , or acidified supernatants obtained from non-infected DH82 cells and DH82 cells persistently infected with CDV-Ond, CDV-Ondneon, and CDV-Ondneon-GM-CSF, respectively. Commercially available human and canine GM-CSF was supplemented at 5 µg/mL. A culture medium supplemented with caGM-CSF or rhGM-CSF was used in an amount of 500 µL/well. The acidified supernatants obtained from DH82 cells infected with CDV-Ond, CDV-Ondneon, or CDV-Ondneon-GM-CSF were diluted to 2:3 with MEM. Cell numbers were quantified after 6 and 12 h. Therefore, cells were detached from the bottom of the well with 0.05% trypsin/0.02% EDTA . Trypsinized cell suspensions were centrifuged for 10 min at 250× g at 4 °C. The obtained cell pellets were re-suspended in 30 µL MEM medium, and 20 µL of the cell suspension was added to 40 µL of trypan blue to obtain a final 1:2 dilution. Counting was performed using a 0.100 mm Neubauer counting chamber  and a light microscope  [DH82 cells were seeded at a density of 2 × 104 cells in quadruplicate in 24-well plates in DMEM  with the addition of recombinant human GM-CSF , canine GM-CSF , or acidified supernatants obtained from non-infected DH82 cells and DH82 cells persistently infected with CDV-Ond, CDV-Ondneon, and CDV-Ondneon-GM-CSF, respectively. Commercially available human and canine GM-CSF was supplemented at 5 µg/mL. A culture medium supplemented with caGMCSF or rhGM-CSF was used in an amount of 500 µL/well. The acidified supernatants obtained from DH82 cells infected with CDV-Ond, CDV-Ondneon, or CDV-Ondneon-GM-CSF were diluted to 2:3 with DMEM. The cell number was quantified after 6 and 12 h. Thereafter, cells were detached from the bottom of the well with 0.05% trypsin/0.02% EDTA . Trypsinized cell suspensions were centrifuged for 5 min at 300× g at 4 °C. Obtained cell pellets were re-suspended in 30 µL of DMEM, and 20 µL of the cell suspension was added to 40 µL of trypan blue to obtain a final 1:2 dilution. Counting was performed as described above.HeLa cells were seeded at a density of 2 × 10www.sas.com). The level of significance was set at p ≤ 0.05. Graph creation was carried out using GraphPadPrism version 8.0.1 for Windows .For descriptive statistics, the median and range were calculated. For the analysis of data obtained from the virus titration, immunofluorescence, RT-qPCR, and immunoblotting and proliferation assays, the non-parametric Mann–Whitney U and Wilcoxon signed-rank tests were used. Statistical analysis was performed with SAS software version 7.1.5.0  was observed compared with non-acidified supernatants independent of the virus strain . After acidification, the number of CDV nucleoprotein mRNA transcripts was significantly lower in supernatants from all treated cultures compared with corresponding native controls  and CDV-Ondneon-GM-CSF-infected cultures (p = 0.0404) . Immunoblotting with an anti-CDV nucleoprotein antibody displayed protein bands at 58 kDa in supernatants from DH82 cells infected with CDV-Ond, CDV-Ondneon-GM-CSF. The amount of GM-CSF did not differ between native and acidified supernatants obtained from DH82 cells infected with CDV-Ondneon-GM-CSF .Immunoblotting with an anti-GM-CSF antibody revealed protein bands at 14 kDa in native and acidified supernatants obtained from DH82 cell cultures infected with CDV-Ond samples . In natineon-GM-CSF and the confirmation that virus neutralization by acidification did not affect the amount of GM-CSF, the next step was to analyze the effect on DH82 cells and to assess its functionality.After the verification of the presence of GM-CSF in supernatants obtained from DH82 cells persistently infected with CDV-Ondneon, and CDV-Ondneon-GM-CSF. DH82 cells expressed CD116 independently of the infection status, and no significant differences in the CD116 protein amount were observed. (p > 0.05).To assess the potential growth-stimulating effect of GM-CSF on histiocytic sarcoma cells (DH82 cells), the presence of the GM-CSF receptor (CD116) was investigated in non-infected DH82 cells and DH82 cells persistently infected with CDV-Ond, CDV-Ondneon, or CDV-Ondneon-GM-CSF. Commercially available canine GM-CSF (caGM-CSF) was added to the non-conditioned medium as a control. There were no significant differences in the percentage of scratch closure after 24 h between groups . After 6 h, the cell duplication assay did not reveal significant differences between the groups  or from DH82 cells infected with CDV-Ond (p = 0.0152) or CDV-Ondneon (p = 0.0147) or supernatants supplemented with commercial caGM-CSF (p = 0.0303). For the verification of the overall functionality of GM-CSF produced by CDV-Ondneon-GM-CSF did not influence histiocytic sarcoma cell proliferation and migration, but at the same time, it exerted its function by stimulating HeLa cell proliferation in vitro.Overall, the present study demonstrated that GM-CSF in supernatants obtained from DH82 cells persistently infected with CDV-Ondneon-GM-CSF-infected canine histiocytic sarcoma cells (DH82 cells) and explore the effect on non-infected canine histiocytic sarcoma cells in vitro. The treatment of canine histiocytic sarcomas is often of limited success and leads only to partial or transient regressions or a delay in disease progression ,42,43. Tneon-GM-CSF-infected DH82 cells, the first step was to inactivate the infectious virus, which was also present in the supernatant of these cultures, to avoid additional CDV infections, which would hamper the interpretation of observed effects. Morbilliviruses are stable at pH 4.5–9 [neon-GM-CSF were acidified and inactivated at pH 2. Supernatants of all control groups  were treated analogously. Supernatants of non-infected DH82 cell cultures served as an additional control. Successful treatment was confirmed by the absence of cytopathic effects in Vero.DogSLAM cells upon virus titration. Furthermore, a significantly lower number of CDV mRNA transcripts was present in the acidified supernatants of infected cultures, while the protein amount of CDV nucleoprotein remained unchanged. Despite the acidification, the protein amount of GM-CSF did not change significantly compared with untreated supernatants, proving the stability of GM-CSF at low pH values. To allow an investigation of the effect of GM-CSF produced by CDV-OndpH 4.5–9 , whereaspH 4.5–9 ,46. Therneon-GM-CSF-infected DH82 cells or commercially available canine GM-CSF did not result in increased cell proliferation or cell motility. This might be caused by defects in the intracellular signaling pathway since GM-CSF is known to play an important role in myelopoiesis and the function of myeloid cells [The function of GM-CSF is transmitted by the GM-CSF receptor CD116), which is composed of subunits α and β, and leads to activation of the Janus Kinase 2 (JAK2)-dependent signaling pathway , which i. CD116 sneon-GM-CSF displayed increased proliferation after 12 h of stimulation, confirming the functionality of GM-CSF produced by CDV-Ondneon-GM-CSF-infected DH82 cells.To access the overall functionality of GM-CSF, HeLa cells were used. These cells are known to show increased proliferation after stimulation with GM-CSF . HeLa ceneon-GM-CSF-infected DH82 cells secreted increased amounts of functionally active GM-CSF, while the proliferation and migration of histiocytic sarcoma cells was not influenced. Consequently, this genetic modification might result in an increased were capacity of this CDV-Ond strain compared with that of the non-modified parenteral strain. However, the present results only depict in vitro findings and can only partially mimic the complex interactions in the tumor microenvironment. Therefore, the functionality and efficacy of CDV-Ondneon-GM-CSF need to be further detailed in more complex situations, such as three-dimensional co-cultures of neoplastic cells with other components of the tumor microenvironment, and especially in controlled in vivo situations, such as a murine xenotransplantation model.To summarize, CDV-Ond


The quality of the endothelial cell layer is a major criterion for the approval of organ-cultured human donor-corneas for transplantation. We wanted to compare the predictive capacities of initial endothelial density and endothelium cell morphology for the approval of donor corneas for transplantation and for the clinical outcome after transplantation.The endothelial density and endothelium morphology in organ culture were examined by semiautomatic assessment of 1031 donor corneas. We performed a statistical analysis for correlations of donor-data and cultivation parameters regarding their predictive capacities for the final approval of donor corneas for transplantation and the clinical outcome of 202 transplanted patients.Corneal endothelium cell density proved to be the only parameter with a certain predictive capacity with regard to the final decision, whether donor corneas are suitable for transplantation — however, the correlation was low (area under the curve [AUC] = 0.655). Endothelial cell morphology lacked any predictive power (AUC = 0.597). The clinical outcome regarding visual acuity seemed to be largely independent from both corneal endothelial cell density and morphology. Sub-analyses on transplanted patients stratified for their diagnoses vindicated these findings.2), as well as better endothelial morphology do not seem to be critical for transplant-corneal functionality in organ culture and up to 2 years after transplantation. Comparable long-term studies on graft survival are recommended to determine, whether the present endothelial density cut-off levels might be too stringent.Higher endothelial density (above a cut-off level of 2000 cells/mmThe online version contains supplementary material available at 10.1007/s00417-023-06079-0. According to the estimations of the World Health Organization (WHO), corneal opacities are the 3pulation -3.Over a century after being first established as a surgical procedure, allogeneic cornea transplantation still plays an important role as a remedy of last resort for a great variety of corneal conditions. It is the most common type of solid tissue transplantation worldwide: about 150,000 keratoplasties are performed worldwide every year, predominantly in North America and Europe .A high transplant-quality — mainly defined by biomechanical integrity and transparency — is essential for the long-term success of the treatment of corneal diseases by keratoplasty. One of the key factors is the functionality of the corneal endothelium, as it regulates nutrition and osmotic equilibrium of the cornea, thus warranting corneal transparency. The correlation between endothelium cell density (ECD) and functionality is well known and has been studied in detail -6.Several factors have been studied for a possible influence on the ECD in donor corneas such as donor diseases, cause of death, corneal retrieval techniques and proficiency of the staff performing the microsurgery procedures , 8. HoweIn functional corneal endothelium, mainly hexagonal, but also pentagonal cells are prevalent, as their geometry allows a mathematically economic, homogenous cover of the Descemet membrane following the corneal curvature . In caseNew semi-automated methods of microscopic assessment of donor cornea endothelium not only allow the standardized quantification of mean cell density, but also facilitate an easier assessment of the morphology data of endothelial cells. Nonetheless, to our knowledge, no surveys have studied whether the endothelium cell morphology itself may have a cell-density-independent effect on the stability of human donor corneas in culture or on the clinical outcomes after transplantation.The objective of this study is to explore whether endothelial morphology data of donor corneas (obtained after donation and during organ culture) could serve as an independent predictive parameter for the functional stability during in vitro cultivation and the clinical outcome after transplantation; this is done in comparison with data on the endothelial density in donor corneas, the actual main discriminative parameter for transplantation approval of donor corneas.This retrospective case study was conducted at the Hamburg Eye Bank (HEB).For the purpose of this study, we analyzed 1031 donor corneas that were processed in the Hamburg Eye Bank between 2012 and 2016. For this study, endothelial cell counts and endothelial cell morphology of the initial and the final examinations of donor corneas (see below), cultivation data  and donor data  were assessed for all corneas.All corneal tissue was retrieved from deceased donors by the department of forensic medicine of the University Hospital Hamburg. Consent calls with relatives of the donor were performed in accord with the current law. Cornea recovery was performed by the medical forensic staff or by the trained technicians of the HEB. Tissue processing, preparation, and cultivation were performed by trained technicians of the HEB.The retrieval of the donor cornea was performed by removing the corneoscleral disc in situ, without enucleating the eye. Both eyes of the donor were disinfected with PVP–iodine for 5 min and subsequently washed with 500 mL sterile, physiologic sodium chloride solution. After removing the conjunctiva using a sterile pair of Westcott scissors, the corneoscleral discs (CDs) were retrieved from the donor eyes using a 16-mm trephine. The excised CD was transferred to 12 mL sterile primary culture medium.2) for up to 4 weeks. Media were exchanged once a week. All used media were checked for contamination. Only CDs with proven sterility of feeding media, functional endothelium , unsuspicious donor serology and medical history were approved for transplantation. At least 24 h before transplantation, CDs were placed in cell culture medium (s.a.) supplemented with 6% dextran 500 for de-swelling.Organ cultivation was performed according to the methods established by Schroeter and Rieck . Summari2) and stratifies the measured cells according to their form in pentagonal, hexagonal and heteromorphic cells , and after de-swelling for at least 8 h (before transport into the operating theatre). To determine endothelium cell densities (ECDs), the cornea first underwent osmotic preparation in balanced salt solution for 60 s to achieve optimal visibility of the cells, after which the cornea was placed under a binocular inverted phase-contrast microscope  and photographed. The image was then transferred to the semi-automated EAS software . The automatic analysis function was used . In this mode, the software determines the mean endothelial cell density  after transplantation were analyzed. For each time-point group, median visual acuity and objective refraction values respectively were determined. Then, patients were grouped to a more- or less favourable clinical status depending on their individual value being above or below their common time-point mean value.The mean donor age was 66.1 years . There was data on the gender of 513 donors of whom 322 were male and 191 female, yielding a gender ratio of 1.68:1. Further data  are given in Table Clinical data of 202 patients who underwent corneal transplantation from 27.06.2012 until 12.10.2016 using grafts that underwent the automatic endothelial counting and stratification was analyzed. Table mean initial  ECD and ECM values and their stabilities in culture expressed by the relative losses in culture per day (rel.ECD-loss/d and rel.ECM-loss/d respectively) and respective statistical parameters.Overall, the endothelial density (ECD) and morphology (ECM) of 853 corneas could be determined at introduction in organ culture; from these, 556 corneas still displayed measurable ECDs and ECMs at final examination before their transplantation or discard. Table U analyses. P-values > 0.05 were assigned as significant.Statistics and graphs were created by Microsoft-Excel and SPSS. Descriptive statistics comprised means, standard deviations, confidence intervals, medians and inter-quantiles. As the distribution patterns of the endothelial cell density and morphology variables turned out to be non-parametric , correlation analyses were done using Rho-Spearman tests while corresponding group comparisons were tested by nonparametric Mann–Whitney-area under the curve (AUC), whereas a value of 0.5 indicates the complete failure of the tested predictor , while a value of 1 indicates a perfect prediction.ROC-analyses were applied to compare predictive capacities (prospective sensitivity and specificity) of endothelial cell density and morphology on transplantation release (yes or no) or on more- or less favourable clinical status of transplanted patients  at specified time-points after transplantation. A central reference in ROC curves is the The descriptive data Tables It is logical and widely known amongst cornea researchers that endothelial morphology and density correlate with each other; this was vindicated by our own analysis Table .Thus, to prevent an interference of this correlation in our comparisons, we applied ROC analyses to compare the individual predictive capacities of initial ECD, initial ECM and their relative losses in organ culture per day on the selection of corneas for transplantation and their post-transplantation development in the following analyses.As shown in Fig. The examination whether initial ECM and ECD of corneas would be a suitable predictive parameter for the clinical outcome after transplantation is depicted in Fig. To test whether diverging diagnoses  could be used as a supplemental parameter to estimate the prospective quality of donor corneas after organ culture — and thus their suitability for transplantation.A unique feature of our study is the additional analysis of clinical data obtained after the transplantation of the donor corneas. To our knowledge, this has not been done so far.It is obvious that such analyses face several challenges:First, the base collectives  used for the comparisons should be widely homogeneous. Although there was a prevalence of male donors in our study group  — which can be assumed to influence each other; we indeed demonstrated that these parameters are correlated positively: the higher the corneal ECDs, the better the corresponding corneal ECMs  despite their evident correlation: as their individual areas under the curve (AUCs) always include the mutual correlation, differences of the AUCs necessarily are independent from it. A higher AUC of one of these variables (f.e. ECD or ECM) thus could indicate a higher predictive power which is independent from the other variable.The main findings of our study are as follows:Firstly, initial ECD or ECD development in culture displays only a limited predictive power  as well as the ECM development during organ-culture do not provide any additional information to estimate the suitability of donor corneas for transplantation or their visual performances after transplantation — at least when semi-automated measurements were employed.2 (for deep lamellar transplantation even at 2500 cells/mm2). Now, corneal endothelium usually still is functional at ECDs of 600 to 800 cells/mm2 [2 for transplant-cornea approvals could be too high to enable a detection of a putative predictive power of initial ECD for visual performance after transplantation in our study setting (i.e. within 2 years of observation). Noteworthy, in side analyses on sub-collectives from donors older than 80 years , the predictive capacity of initial ECD for transplantation approval proved to be somewhat higher  is that the usual cut-off level of corneal ECDs at their final assessment (at the end of organ culture) for transplantation approval (or against) in our institution lies at minimum 2000 cells/mmIn this study, high numbers of donors, transplant-corneas and (to a lesser extent) transplanted patients were analyzed thoroughly. However, its results and conclusions still need to be tested carefully in future surveys, as our study has some limitations.The first obstacle is a consequence of the nature of semi-automated morphology analyses; it can only be as accurate, as the software performing it. In its current version, the EAS system used in this work mainly quantifies the number of 6- and 5-edged cells compared to pleomorphic cells. Some phenomena as cell agglomeration or degenerative aspects were not considered. A new version of the software will improve accuracy in the future.The second limitation is that we measured the clinical outcome of corneal transplantation by comparing postoperative visual acuity and objective refraction. It is well known that there are a multitude of factors influencing these parameters — such as concurrent (known and unknown) ocular pathologies, subjective collaboration of the patient, size and position of the transplant, etc. While we presumed that — given a sufficient number of outcomes analyzed — these effects could cancel each other out, we admit that only a robust predictive corneal-quality marker could overcome all of these effects. This in mind — and under the premise that the main interfering factor might be the diagnostic background of the eye — we repeated the post-clinical comparative ROC analyses on two diagnostic groups . The results of these sub-analyses support the main observations and indicate that the poor/lacking predictive capacities of initial ECD/ECM (or their stability in organ culture) on the clinical outcomes of transplanted patients might be largely independent from their diagnosis. However, due to low  case numbers, the reliability of these sub-analyses should be viewed critically — more comprehensive analyses are needed to test these observations.n = 178). In contrast, only 24 posterior-lamellar keratoplasties (DMEKs) were performed  than expected. These observations could suggest considerations on less restrictive cut-off levels for the endothelial cell density for transplantation approval; however, further studies are needed.Supplementary file1 (PPTX 108 KB)Supplementary file2 (PPTX 94 KB)Supplementary file3 (PPTX 116 KB)Supplementary file4 (XLS 22 KB)Below is the link to the electronic supplementary material.