TBX5), which is an important regulator in heart development. Knock‐down of lncRNA TBX5‐AS1:2 via promoter hypermethylation reduced TBX5 expression at both the mRNA and protein levels by affecting its mRNA stability through RNA‐RNA interaction. Moreover, lncRNA TBX5‐AS1:2 knock‐down inhibited the proliferation of HEK293T cells. In conclusion, these results indicated that lncRNA TBX5‐AS1:2 may be involved in TOF by affecting cell proliferation by targeting TBX5.Tetralogy of Fallot (TOF) is the most common complex congenital heart disease (CHD) with uncertain cause. Although long non‐coding RNAs (lncRNAs) have been implicated in heart development and several CHDs, their role in TOF is not well understood. This study aimed to investigate how dysregulated lncRNAs contribute to TOF. Using Gene Expression Omnibus data mining, bioinformatics analysis and clinical heart tissue sample detecting, we identified a novel antisense lncRNA TBX5‐AS1:2 with unknown function that was significantly down‐regulated in injured cardiac tissues from TOF patients. LncRNA TBX5‐AS1:2 was mainly located in the nucleus of the human embryonic kidney 293 (HEK293T) cells and formed an RNA‐RNA double‐stranded structure in the overlapping region with its sense mRNA T‐box transcription factor 5 ( We further demonstrated that lncRNA TBX5‐AS1:2 expression was markedly decreased in injured heart tissue from patients with TOF. In vitro, lncRNA TBX5‐AS1:2 down‐regulation, mediated by DNA hypermethylation in the promoter region, significantly suppressed cell proliferation by reducing the expression of TBX5 at both the mRNA and protein levels, the reason of which was that lncRNA TBX5‐AS1:2 affected the stability of TBX5 mRNA through the formation of an RNA‐RNA duplex.In the current study, we characterized the lncRNA and mRNA profiles in human foetal and adult heart tissues by Gene Expression Omnibus (GEO) data mining and bioinformatics analysis, and focused on a previously unreported antisense lncRNA TBX5‐AS1:2 with unknown function. LncRNA TBX5‐AS1:2 was significantly up‐regulated in foetal heart and was predicted to adjust the expression of its sense gene 22.1https://www.ncbi.nlm.nih.gov/geo/) using the keywords lncRNA, human, heart development or CHD. Differentially expressed lncRNAs were analysed using the DESeq package, and WikiPathways database was applied to screen mRNAs related to heart development or CHD with differential expression. A coding and non‐coding co‐expression (CNC) network was established followed by these procedures: (a) data pre‐processing: for same gene, median value of different transcripts for same genes represents gene expression value; (b) data screening: assessing differential expression of lncRNA and mRNA; (c) calculation and removal of subset of data based on Pearson's correlation coefficient (PCC) and calculation of correlation coefficient of PCC between lncRNA coding genes using R values; (d) screening with a standard of PCC ≥0.9 or ≤−0.9 and P ≤ .05 as meaningful subset and constructing CNC network using Cytoscape. Meanwhile, mRNAs adjacent to lncRNAs in the CNC network (≤10 kbp on the genome) were annotated. Target lncRNA was selected based on the following requirements: (a) in the CNC network; (b) adjacent mRNA was one of the differentially expressed in relation to heart development or CHD; and (c) adjacent mRNA co‐expressed with the lncRNA.Online data mining was performed in the GEO database (2.2® RNA Stabilization Solution (Thermo Fisher Scientific) after surgery or autopsy and were stored at −80°C.This study was reviewed and approved by the Institutional Research Ethics Committee of the Children's Hospital of Fudan University (2016‐56). Cardiac tissue samples were obtained from 53 patients with TOF from the Biobank of the Children's Hospital of Fudan University, Shanghai, China. The diagnosis of TOF was based on echocardiography carried out at our hospital. None of the patients included in the study had been diagnosed with extracardiac anomalies or had any common chromosomal anomalies, such as 22q11 microdeletion. Cardiac tissues were removed from the blocked right ventricular outflow tract during surgery. Thirteen normal cardiac tissues samples were acquired from the Department of Forensic Medicine, Fudan University, Shanghai, China. They died as a result of traffic accidents and had no abnormal cardiac structures at autopsy. All tissue samples for RNA extraction were maintained in RNAlater2.3GAPDH) and analysed according to the relative quantification method (2−ΔΔCt). The 2−ΔΔCt was used to calculate the relative expression of RNA. Three repeated measurements were carried out for each sample.Total RNA was extracted from cardiac tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The quantity and quality of the extracted RNA were assayed using a NanoDrop ND‐2000 spectrophotometer (Thermo Fisher Scientific) and agarose gel electrophoresis. A total of 1000 ng of RNA from each sample was used to synthesize cDNA using a Prime Script RT Reagent Kit (Takara), followed by quantitative polymerase chain reaction (qPCR) with SYBR Premix Ex Taq™ (Takara) on a StepOnePlus™ Real‐Time PCR System (Thermo Fisher Scientific). Gene expression was normalized to the housekeeping gene glyceraldehyde 3‐phosphate dehydrogenase  cells were cultured in a mixture of Dulbecco's modified Eagle's medium , 10% foetal bovine serum  and 1% penicillin‐streptomycin  at 37°C in 5% CO2.5https://www.ncbi.nlm.nih.gov/). Empty vector (pcDNA 3.1) was used as a negative control (NC). When the cells plated in 6‐cm dishes reached about 75% confluence, they were transfected with plasmids using Lipofectamine 3000 (Invitrogen). The cells were harvested 48 hours after transfection.Human lncRNA TBX5‐AS1:2‐pcDNA 3.1 and human TBX5‐pcDNA 3.1 were obtained from GeneRay. The plasmids were sequenced and shown to be consistent with the sequence in the National Center for Biotechnology Information database (2.6TBX5) were designed and cloned into the lentiviral vector PGMLV‐SC5‐GFP by Genomeditech. The lentivirus was amplified in HEK293T cells and concentrated using polyethylene glycol (System Biosciences). The interference efficiency was detected by qPCR. HEK293T cells infected by the most interference‐efficient lentivirus were used to establish stable cell lines by selection with puromycin (Sigma‐Aldrich).Three short hairpin RNAs (shRNAs) targeting lncRNA TBX5‐AS1:2  was added to each well followed by incubation for 3 hours at 37°C. Cell viability was used to represent for proliferation and evaluated by the absorbance at 450 nm. All samples were prepared in triplicate and normalized to blank controls. Three time‐points were set at 40, 56 and 72 hours after transfection or incubation. The experiments were repeated three times.Cell proliferation was determined using a Cell Counting Kit‐8  according to the manufacturer's protocol. A total of 1 × 102.86 HEK293T cells and staining using an Annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) Kit (Dojindo). Cells were stained successively with 5 µL FITC reagent and 5 µL PI reagent, and incubated in the dark for 15 minutes at room temperature each time. Flow cytometry analysis was performed using a FACSCalibur (BD Biosciences) and apoptosis was analysed using FlowJo software.Cell apoptosis was detected by harvesting 1 × 102.9Nuclear and cytoplasmic fractions were isolated from HEK293T cells using a PARIS kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Actin was used as a cytoplasmic control, and U1 was used as a nuclear control. RNA levels of lncRNA TBX5‐AS1:2, Actin and U1 in the cytoplasm and nuclear components were measured by qPCR after RNA extraction and reverse transcription.2.10Cells were grown in a 4‐chamber slides for 24 hours, fixed with 4% paraformaldehyde for 20 minutes and dehydrated in an ascending series of ethanol solutions. Cells were hybridized overnight at 42°C with probe. Non‐specific probe was removed using 0.5× saline sodium citrate containing 50% formamide at 37°C. Biotin‐labelled lncRNA TBX5‐AS1:2 was detected using anti‐biotin monoclonal antibody and secondary antibody. Finally, the slides were stained with DAPI  and subjected to fluorescence signal detection under Leica TCS SP8 laser confocal microscope (Leica).The probe of lncRNA TBX5‐AS1:2 used was as follow:CY3‐GGUUUCGAUUAAGAUACACCAUAGGCUCUACACGAUCGGC.2.11Human embryonic kidney 293 cells were collected and lysed with RIPA buffer (Yeasen) containing a protease inhibitor cocktail (Sigma‐Aldrich). Proteins were determined using a BCA Protein Assay Kit (Pierce) and equivalent amounts of proteins were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . The membranes were blocked with 5% milk in Tris‐buffered saline Tween for 1 hour at room temperature and then incubated with specific primary antibodies at 4°C overnight, followed by secondary antibodies at room temperature for 1 hour. The primary specific antibodies used were anti‐TBX5  and anti‐Actin , and the secondary horseradish peroxidase‐conjugated antibody was antimouse IgG . The proteins were then visualized on X‐ray film using Clinx ChemiScope (Clinx Science Instruments).2.12TBX5 employing two sets of distinct primers designed to target the overlapping region of the TBX5 sense and antisense transcripts, and the non‐overlapping region of TBX5, respectively. The primer sequences were as follows:Each RNA sample from HEK293T cells was incubated for 1 hour at 37°C and then treated with RNAse A+T cocktail (Ambion) to digest single‐stranded but not duplex RNAs. After incubation for 30 minutes at 37°C, samples were dealt with proteinase K (Yeasen). A NC without RNAse treatment was operated in the same way. After ribonuclease protection assay (RPA), reverse transcription (RT)‐PCR and gel electrophoresis were used to detect TBX5‐overlapping region‐F: CCCTCATTCCTCCGGAGAAAG,TBX5‐overlapping region‐R: GCCGGGTCTGCGCAGCCACAG;TBX5‐non‐overlapping region‐F: ACATCGTGAAAGCGGATGAA,TBX5‐non‐overlapping region‐R: GTGATCTTGTGGTTCTGGTAGG.2.132, 0.5% NP‐40, RNase Inhibitor 160 U/mL cocktail). The supernatant was mixed with biotinylated lncRNAs and antisense transcripts for 2 hours at 4°C, followed by the addition of Dynabeads MyOne Streptavidin T1 (Invitrogen) and incubation overnight at 4°C. After washing beads four times, RNA was extracted using TRIzol reagent. The lncRNA‐associated RNAs were subjected to cDNA synthesis and qPCR using the following primers:Full‐length biotinylated lncRNAs and antisense transcripts were transcribed using an AmpliScribe™ T7‐Flash™ Biotin‐RNA Transcription Kit (Epicentre Technologies). After purification, HEK293T cells were lysed for 40 minutes with RNA pull‐down buffer  for 4, 6, 8 and 10 hours, respectively. Total RNA was extracted after treatment at each time‐point and subjected to qPCR for TBX5 mRNA quantification.RNA stability was measured by plating HEK293T cells at 2.5 × 102.15https://www.ncbi.nlm.nih.gov/nuccore/NC_000012.12?from=114408195&to=114412832&report=genbank), and CpG islands were predicted using MethPrimer (http://www.urogene.org/cgibin/methprimer/methprimer.cgi). The DNA methylation statuses of the selected CpG islands were analysed by bisulphite sequencing PCR (BSP). Genomic DNA extracted from six TOF and five normal cardiac tissues was subjected to bisulphite conversion using an EpiTect Fast DNA Bisulfite Kit (Qiagen) according to the manufacturer's instructions. LncRNA TBX5‐AS1:2 CpG islands from bisulphite‐modified DNA were then amplified by EpiTaq HS DNA polymerase (Takara), and the PCR products were purified and cloned into the T/A cloning vector pGEM T‐Easy (Promega). Ten positive clones were isolated and sequenced. Methylation analysis was performed using BiQ Analyzer 2.0 and QUMA (http://quma.cdb.riken.jp/). The primers used were as follows: lncRNA TBX5‐AS1:2‐I2‐F1: TTTTAGTAAAATAAAGAGGTAATTAGG,The DNA sequence of the lncRNA TBX5‐AS1:2 regulatory region was obtained from the GenBank database . The recombinant plasmid was treated with CpG methyltransferase (M.SssI) (New England Biolabs) for 2 hours at 37°C and then purified using an AxyPrep DNA Gel Extraction Kit (Axygen) to generate a patch‐methylated construct. Whether the plasmids were methylated was detected by methylation‐sensitive restriction enzymes (MSREs)  (New England Biolabs) digestion and DNA gel electrophoresis assay. Methylation efficiency was evaluated by BSP and the PCR products were used for direct pyrosequencing. The primers used were as follows: lncRNA TBX5‐AS1:2‐I2‐F2‐1: TGTGAATYGATAGTATTAATATAYGTTT,lncRNA TBX5‐AS1:2‐I2‐R2‐1‐tail:aaccttcaacaccccaaccatataTAATTTATATCTTTATTTATTCCCRAAACC;lncRNA TBX5‐AS1:2‐I2‐F2‐2: TTAGTGTAAGTGTAGGTGTTAGAATATT,lncRNA TBX5‐AS1:2‐I2‐R2‐2‐biotin: aaccttcaacaccccaaccatata..2.174 cells per well were seeded in 96‐well plates and incubated at 37°C overnight. The respective methylated and unmethylated reporter plasmids were co‐transfected with pGL3‐Renilla vector into HEK293T cells using Lipofectamine 3000. After transfection for 48 hours, the HEK293T cells were treated with a Dual‐Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. Both firefly and Renilla luciferase activities were measured using an EnSpire plate reader (PerkinElmer). All samples were prepared in triplicate. The firefly luciferase activity normalized to the Renilla luciferase activity represented the transcriptional activity of the lncRNA TBX5‐AS1:2.Human embryonic kidney 293 cells at 1 × 102.18t tests with GraphPad Software. Data were shown as mean ± standard error (SEM), and a P value < .05 was considered statistically significant.All experiments were repeated three times. All statistical analyses were performed using paired two‐tailed Student's 33.1https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68279) was filtered out, which was from an article related to the lncRNA profile in human foetal and adult hearts.TBX5   in HEK293T cells with down‐regulated and up‐regulated lncRNA TBX5‐AS1:2. TBX5 mRNA and protein levels were both significantly down‐regulated after lncRNA TBX5‐AS1:2 knock‐down   Figure , but the) Figure .3.9Hypermethylation of CpG islands can inhibit the transcriptional activity of lncRNAs. We therefore transfected methylated and unmethylated lncRNA TBX5‐AS1:2 reporter plasmids containing CpG island 2 into HEK293T cells. The unmethylated construct was digested by MRSEs whereas the methylated construct was not Figure C. BSP re4CHD is resulted from abnormal cardiogenesis during early embryonic development. Cardiogenesis involves the orderly proliferation, apoptosis, migration and differentiation of a variety of cells, requiring tight and precise regulation by appropriate spatiotemporal gene expression.TBX5. Antisense lncRNAs account for approximately 50%‐70% of all lncRNAs and are transcribed from the opposite strand of their endogenous sense counterparts.TBX5 were down‐regulated in injured heart tissue from TOF patients. We therefore performed in vitro experiments to clarify the molecular mechanism underlying this clinical phenomenon. Given that no human myocardial cell lines are available and lncRNA TBX5‐AS1:2 has no homologous sequence in mice, we used the tool cell line HEK293T, as in previous studies of CHD or human heart development.TBX5 mRNA and protein abundances in vitro. LncRNA TBX5‐AS1:2 and TBX5 formed an RNA duplex by RNA‐RNA interaction, thus preventing TBX5 mRNA degradation and increasing the mRNA stability of TBX5. In addition, lncRNA TBX5‐AS1:2 knock‐down significantly inhibited HEK293T cell proliferation, which could be rescued by overexpression of TBX5. These results showed that the influence of lncRNA TBX5‐AS1:2 on cell proliferation may be mediated by its interaction with TBX5. TBX5 is a member of the T‐box transcription factor family primarily known for its role in cardiac development.TBX5 can increase the risk of CHD, including TOF.TBX5.TBX5 plays a critical regulatory role in cell proliferation during cardiogenesis.Among the differentially expressed lncRNAs, we focused on the novel antisense lncRNA TBX5‐AS1:2, which showed a relatively high fold change in foetal compared with adult heart, and predicted to target its cognate coding mRNA TBX5 mRNA and protein expression via an RNA‐RNA interaction regulatory mechanism to inhibit cell proliferation in TOF. However, further studies are needed to determine the detailed mechanisms by which lncRNA TBX5‐AS1:2 modulates cardiac development and its function. Moreover, if there are ideal human cardiomyocyte lines or cardiac stem cells and the parallel data can be obtained from in vitro experiments with these cell lines, the conclusions will be more credible. In summary, this study identified the novel lncRNA TBX5‐AS1:2 as being down‐regulated in human TOF heart tissue and further explored its part of function in cardiac development by in vitro experiments.We therefore suggest that the previously unknown non‐coding antisense transcript lncRNA TBX5‐AS1:2 may be involved in TOF. Hypermethylation could mediate the down‐regulation of lncRNA TBX5‐AS1:2, leading to decreased The authors declare no conflict of interest.JM, DM and GH were responsible for the idea, project design and concept of the paper. JM performed bioinformatics analysis. WS, WC and XM collected the clinical samples and information. SC, JM, LH and BZ performed the clinical sample detection and in vitro experiments. JM, SC, DM and GH wrote, edited and revised the manuscript. All authors read and approved the manuscript.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.Table S4Click here for additional data file.


Hypercalcemia is a common clinical laboratory abnormality with a majority of cases attributed to malignancy or hyperparathyroidism. Although hypercalcemia is a common manifestation of sarcoidosis, it is rarely the initial presentation. Here we present a case of acute hypercalcemia in a 60-year-old gentleman, which was diagnosed as sarcoidosis following an elaborate workup, including radiological assessment and multiple organ biopsies. This case highlights the diagnostic dilemma of sarcoidosis due to varying clinical presentation that can mimic multiple conditions, including malignancy. Biopsy showing noncaseating granulomas is pathognomic of sarcoidosis. Due to its multisystemic and nonspecific presentation, selecting an appropriate biopsy site is key to diagnosis. Sarcoidosis is an inflammatory multisystem disease, histologically characterized by noncaseating granulomas. Over 90% of cases have lung involvement; however, 30% of patients can have extrapulmonary manifestations . PresentA 60-year-old Caucasian gentleman with a past medical history significant for hypertension and diabetes presented to his primary care physician with abdominal pain for one month. The pain was insidious in onset and associated with poor oral intake, nausea, and vomiting. His diet had been restricted to clear liquids due to unremitting nausea and vomiting, resulting in 32 pounds of weight loss. He denied recent sick contacts, diarrhea, fever, chills, dysuria, or hematuria. Family and social history were insignificant. Examination revealed a blood pressure of 177/94 mmHg, a pulse rate of 87 beats per minute, afebrile, and epigastric tenderness with no other focal systemic findings.Initial laboratory workup revealed hemoglobin of 10 mg/dL, platelet count of 250,000 cell/µL, creatinine of 3.32 mg/dL (baseline creatinine 1.13 mg/dL), calcium of 13.8 mg/dL, and phosphorous of 2.5 mg/dL. Initial evaluation for hypercalcemia revealed low parathyroid hormone (PTH) level of 11.3 pg/mL, parathyroid hormone-related peptide (PTHrP) of 1 pmol/L, 25-hydroxyvitamin D level of 17 ng/mL, and a 1,25-dihydroxyvitamin D level elevated to 68 pg/mL. The constellation of abdominal pain, anemia, acute kidney injury, and hypercalcemia guided further evaluation for multiple myeloma. Serum protein electrophoresis and urine protein electrophoresis revealed no monoclonality. Serum immunofixation revealed imperceptible free lambda light chains and elevation in both kappa and lambda free light chains with a ratio of 1.73. A bone marrow biopsy revealed normal cellularity, no significant increase in plasma cells, and no plasma monoclonality. He was started on intravenous fluids, pamidronate, and calcitonin for hypercalcemia. Due to worsening renal function, a renal biopsy was obtained which revealed acute tubular injury with chronic and subacute thrombotic microangiopathy, but no evidence of multiple myeloma or sarcoidosis. He was briefly started on steroids for possible sarcoidosis with improvement in serum calcium; however, a normal serum angiotensin-converting enzyme (ACE) level and lack of granulomas on biopsy arrested concerns for sarcoidosis. Due to microangiopathy on renal biopsy, workup including autoimmune antibody, rheumatoid factor, anti-double-stranded DNA antibody, anti-Sjogren's syndrome A (anti-SSA) antibody, anti-Sjogren's syndrome B (anti-SSB) antibody, and lactate dehydrogenase were all negative. CT of his chest revealed small bilateral pleural effusions and pulmonary edema  scan was obtained, which revealed multiple tiny fluorodeoxyglucose (FDG)-avid lymph nodes in the chest, abdomen, and pelvis, as well as abnormal FDG labeling in the liver and spleen. Due to concerns for lymphoma, he underwent biopsy of an inguinal lymph node which revealed noncaseating granulomas with giant cells Figure . He was Calcium ions play an important role in cellular function, physiological processes, cardiac contractility, and blood coagulation. Therefore, the extracellular concentration of calcium is tightly regulated through feedback mechanisms that involve PTH and 1,25-dihydroxyvitamin D. Integrating signals from the kidney, parathyroid gland, bone, and intestine orchestrate the regulating mechanisms of calcium.In healthy individuals, vitamin D is stored as a provitamin D in the skin within the epidermis and dermis. Ultraviolet light from the sun converts it into a biologically inactive previtamin D. This previtamin D is enzymatically activated by a two-step hydroxylation reaction in the liver and kidney respectively to 1,25-dihydroxyvitamin D. Vitamin D and its potent metabolites increase extracellular calcium concentration by increasing intestinal epithelial calcium absorption and increased bone resorption through osteoclast production. Though PTH and hypophosphatemia stimulate and enhance renal 1-hydroxylase enzyme; 1, 25 dihydroxyvitamin D in turn inhibits the secretion of PTH, providing a negative feedback regulation.The cause and pathogenesis of hypercalcemia can be understood based on derangements in normal physiological regulation. Broadly based on the inciting mechanism, hypercalcemia is classified into PTH-dependent and independent causes. The most common causes of hypercalcemia include PTH-dependent hyperparathyroidism and malignancy which causes a PTH-independent hypercalcemia through PTHrP. Although rare, chronic granulomatous diseases can cause a PTH-independent hypercalcemia through enhanced conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D. Sarcoidosis alone has been found to be the cause of hypercalcemia in only 1% of patients [Sarcoidosis is a multisystem inflammatory disease histologically characterized by noncaseating granulomas. The disease is seen worldwide with an estimate prevalence of 20 to 60 per 100,000 population . DemograDespite advances in immunopathogenesis, the exact etiology of sarcoidosis remains elusive. The formation of the characteristic sarcoid granuloma involves a complex interplay between immune cells and their mediators. These granulomas can produce 1,25-dihydroxyvitamin D independent of PTH. This has been shown through studies of increased calcitriol in an anephric patient with sarcoidosis, in vitro studies of alveolar macrophages and lymph node tissues which showed increased 1,25-dihydroxyvitamin D, as well as increased 1-hydroxylase RNA in alveolar macrophages from patients with sarcoidosis . In sarcDiagnosis of sarcoidosis can be challenging when hypercalcemia is the presenting symptom. Although hypercalcemia is a well-studied electrolyte abnormality in sarcoidosis, it is seen in 10% of cases, with severe hypercalcemia of more than 14 mg/dL rarely reported . The incTreatment of hypercalcemia in sarcoidosis consists mainly of corticosteroids. Furthermore, hypercalcemia is an indication to start steroid therapy even in the presence of only mild symptoms. However, other causes of granulomatous diseases including histoplasmosis and tuberculosis should be ruled out prior to steroids. Although steroids are the mainstay of treatment in sarcoidosis, they play an important role in managing hypercalcemia by inhibiting macrophage 1-hydroxylase activity . They reThis case shows an unusual presentation of sarcoidosis, as acute hypercalcemia is rarely the initial manifestation. This highlights the importance of including sarcoidosis in the differential diagnosis, as it has a wide and varying presentation. As biopsy is key to diagnosis, locating an appropriate site to biopsy is crucial as sarcoidosis can have a multisystemic involvement. In the setting of high clinical suspicion along with elevated 1,25-dihydroxyvitamin D and corticosteroid response, treatment can be initiated while awaiting histological confirmation.


Finally, we evaluate the therapeutics that may best prevent the onset or spread of pathology in ALS and discuss what lies on the horizon for treating this currently incurable disease.Amyotrophic lateral sclerosis (ALS) is characterized by the progressive degeneration of the motor neurons that innervate muscle, resulting in gradual paralysis and culminating in the inability to breathe or swallow. This neuronal degeneration occurs in a spatiotemporal manner from a point of onset in the central nervous system (CNS), suggesting that there is a molecule that spreads from cell-to-cell. There is strong evidence that the onset and progression of ALS pathology is a consequence of protein misfolding and aggregation. In line with this, a hallmark pathology of ALS is protein deposition and inclusion formation within motor neurons and surrounding glia of the proteins TAR DNA-binding protein 43, superoxide dismutase-1, or fused in sarcoma. Collectively, the observed protein aggregation, in conjunction with the spatiotemporal spread of symptoms, strongly suggests a prion-like propagation of protein aggregation occurs in ALS. In this review, we discuss the role of protein aggregation in ALS concerning protein homeostasis (proteostasis) mechanisms and prion-like propagation. Furthermore, we examine the experimental models used to investigate these processes, including  A major pathological component of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, frontotemporal lobar degeneration (FTLD), and amyotrophic lateral sclerosis (ALS) is the prion-like propagation of misfolded and aggregated proteins in the central nervous system ; however, it can be converted into a β-sheet-rich pathogenic conformation (PrPSc) that can recruit and convert normal PrP to become pathogenic PrPSc through a template-directed manner  containing many chaperones due to the role it plays in protein synthesis. Protein disulfide isomerases (PDI) are a family of molecular chaperone proteins, localized primarily to the ER, that have oxidoreductase activity, which allows them to reduce and oxidize disulfide bonds in the proteins they are chaperoning  is characterized by the progressive degeneration of motor neurons in both the motor cortex, brain stem, and spinal cord  repeat expansions in the C9ORF72 gene, which result in the transcription of large (tens to thousands of repeats) hexanucleotide repeat RNA molecules and subsequent repeat-associated non-AUG (RAN) translation into dipeptide repeat (DPR) proteins , Pro-Gly (PG), Pro-Arg (PR), Gly-Ala (GA), and Pro-Ala (PA) polypeptides. These DPRs are thought to elicit different levels of toxicity and react differently within the cell based on their physicochemical properties , accounting for roughly 90% of cases. The remaining cases are familial (fALS) and, although some of the underlying genetic causes remain unidentified, are typically associated with a family history of the disease . UBQLN2 is an fALS-associated protein responsible for many interactions that facilitate protein degradation in vitro study of purified proteins and cultured cells, as well as in vivo in invertebrate models  and mouse models. For reviews that incorporate zebrafish (Danio rerio) models, see Babin et al.  and the proteins  in an assay, a more fundamental understanding of the processes underlying protein misfolding and prion-like propagation can be achieved.E. coli) or yeast  and electrostatic loop (loop VII), being responsible for the coordination of metals or guidance of superoxide substrate to the enzymatic site, respectively. Furthermore, a conserved intramolecular disulfide, that stabilizes the tertiary structure and promotes dimerization, is formed between residues Cys57 and Cys146  prevents excessive FUS fibril formation in condensation experiments in vitro  is resistant to detergent denaturation and proteolytic degradation would be useful to determine if purified FUS can form pathogenic amyloids. For a visual summary of the assays that can be performed with purified protein, see Indeed, current understanding of ALS as a prion-like disorder is supported strongly by experimentation of both SOD1 and TDP-43, however, experimentation with FUS is in early stages , HeLa cells, SH-SY5Y cells, Neuro-2a, and NSC-34, however, other cell lines are used too. NSC-34 cells are a hybrid cell line generated from a fusion of murine neuroblastoma cells and murine embryonic spinal cord enriched in motor neurons  disease-associated mutant forms of SOD1 are significantly more aggregation-prone than wild-type SOD1  provide unique opportunities to investigate the pathological role of ALS-associated protein aggregates. Here we will discuss models for neurodegeneration using C. elegans has been used for decades in the study of neuronal development  with expression restricted to specific cells or tissues, including the remarkable ability to express transgenes in individual neurons. Also, the transparent nature of C. elegans enables imaging of fluorescence-tagged proteins in specific tissues/cells within a whole organism  C. elegans transgenic lines to investigate the mechanisms through which ALS-associated mutations in SOD1, TDP-43, and FUS contribute to protein aggregation, neurodegeneration, and behavioral defects concerning proteostasis mechanisms and prion-like propagation. Other aspects of ALS (and other neurodegenerative disease) pathology are covered in detail in reviews elsewhere  or mutant SOD1 (hSOD1) tagged with fluorescent proteins to facilitate visualization of protein localization . These studies have shown that expression of disease-associated mutant forms of SOD1  is associated with increased aggregation and behavioral deficits. The formation of aggregates during aging is correlated with defects in the proteostasis network  despite all hSOD1(mutant) lines showing clear aggregate formation  including heat shock factor 1 (HSF1), Hsp110, a DnaJ family member A2 (DNJ-19), an Hsp70 family member (STC-1), E3 ubiquitin ligase components SEL-10 and RBX-1, and proteins involved in SUMOylation [small ubiquitin-like modifiers (SUMOs)] UBC-9 and GEI-17. Several of these hits were validated through genetic crosses  confirmed earlier findings, showing that expression of GFP-tagged hSOD1-G93A is correlated with reduced locomotion and the formation of large aggregates  via Mos1-mediated single copy insertion (MosSCI). Toxic effects were more subtle in MosSCI transgenic lines compared with conventional multi-copy arrays expressing hSOD1 : single-copy insertions of mutant ceSOD1 showed significantly higher hSOD1WT-YFP inclusions compared with ceSOD1WT or the empty vector control. The authors theorize that the hSOD1WT-YFP inclusions were possibly seeded by misfolded mutant C. elegans SOD1 protein in MosSCI models, suggesting a prion-like propagation effect , ALS-associated mutant forms (Q331K and M337V), or the C-terminal 25 kDa fragment  led to severe locomotor defects  receptor DAF-2, which requires HSF1 for pro-longevity effects  and hTDP-43 transgenic lines improved locomotion and reduced hTDP-43-YFP aggregation  in all transgenic lines  lines was able to partially rescue the locomotion defects observed in G290A and M337V transgenic animals  hTDP-43 also showed similar locomotion defects, with some evidence of aggregate formation in the nucleus of C. elegans led to adult-onset, age-dependent paralysis and progressive motor neuron degeneration in hTDP-43(mutant) lines  hTDP-43 to GABAergic motor neurons of C. elegans ortholog of TDP-43 , the authors showed that ceTDP-43 knockout was linked to sensitivity to oxidative and osmotic stress, as well as increased lifespan at moderate (20°C) temperatures. Unlike effects on hTDP-43 transgenic animals, the effects of the IIS pathway on ceTDP-1-mediated stress responses and lifespan are less clear—two distinct DAF-2 insulin receptor mutants have opposing effects, making it more difficult to derive a conclusive mechanistic link. Nonetheless, ceTDP-43 does appear to be required for the stress resistance in DAF-2 mutants as well as age-dependent proteotoxicity . The authors conclude that mutated FUS is likely to be pathogenic through a gain-of-function effect, rather than through titrating WT-FUS from the nucleus. In future work, it would be interesting to assess whether the colocalization of FUS with stress granules can be initiated by stressors other than acute heat stress  in e Olsen, .C. elegans model expressing wild type and S57Δ mutant hFUS in GABAergic motor neurons, hFUS transgenic lines showed adult-onset, age-dependent locomotion deficits  and complex array of behaviors that can be rapidly assessed experimentally, provides a powerful model animal to pursue investigations into the mechanisms of neuronal dysfunction  system , and is a more practical and ethical system than mammalian models to perform large-scale genetic or pharmacological screens . Interestingly, although the hSOD1-G85R line showed the most significant locomotor defect, this did not correlate with a gross reduction in the number of motor neurons in the thoracic ganglia or with the formation of insoluble species in these neurons  in the eyes leads to significant levels of tissue degeneration. This toxicity was completely reverted by the co-expression of EGFP-tagged 12xQ/N. The authors proposed that dTDP-43 may be sequestered in aggregates formed by EGFP-12xQ/N, thereby titrating excess dTDP-43  of TDP-43 in the Drosophila models expressing FLAG-tagged hTDP-43WT, hTDP-43 with a defective NLS  or hTDP-43 with a defective NES . Neuron-specific expression of these transgenes was larval lethal, so an inducible system was used to bypass developmental effects  species, although hTDP-43ΔNES and hTDP-43ΔNLS expression showed more HMW species compared with hTDP-43WT. When probed with an antibody against phospho-TDP-43(S409/410), hTDP-43ΔNES HMW species, but not those of hTDP-43WT or hTDP-43ΔNLS, were strongly labeled by the pTDP-43 antibody. Interestingly, immunostaining did not reveal the presence of inclusions formed by the expression of any of these transgenes, suggesting that toxicity  does not require the formation of inclusions. This study also demonstrated cell-type-specific toxicity resulting from the expression of hTDP-43 transgenes: in adult neurons, both nuclear and cytoplasmic accumulations of TDP-43 result in toxicity, whereas in muscle and glial cells, only the accumulation of cytoplasmic species of TDP-43 was toxic ] as a modulator of hTDP-43 protein levels  in its 3′ UTR  is highly expressed in neurons and functions in synaptic vesicle exo- and endocytosis  or microautophagy functions (Hsc70–43KA). This suggests that hTDP-43 compromises both chaperone and microautophagy activities of Hsc70–4  do not develop an ALS phenotype  of doxycycline-regulatable transgenic mice expressing a human TDP-43Δ NLS in forebrain neurons (under a CAMKIIa promoter). Recipient mice displayed pTDP-43 inclusions within the ipsilateral injection site within 1-month post-injection, with relatively few inclusions observed on the contralateral side. Furthermore, spatiotemporal immunohistochemical analysis of mice injected with TDP-43 seeding material showed a transneuronal-like spreading pattern of TDP-43 pathology in the 1–9 months following injection. Unsurprisingly, a large majority of TDP-43 aggregates were observed within forebrain neuronal cells where the CAMKIIa promoter controls the expression of TDP-43Δ NLS. However, TDP-43 aggregates were also found within the subcortical regions including the nucleus accumbens and caudate-putamen, suggesting regional spreading.Observations from ALS and FTLD post-mortem tissue suggests that TDP-43 also exhibits prion-like propagation and spreading throughout neuroanatomical tracts. Until recently there has been a lack of a et al.  were ablin vivo evidence for the prion-like characteristics of TDP-43, further research is required to identify the specific regions of TDP-43 that are important for seeding. As mentioned previously, full-length TDP-43 and its CTFs have multiple amyloidogenic regions  overexpression mouse models under various promoters. Mitchell et al.  were theFUS mutations are inherited in an autosomal dominant fashion, with FUS-R521C mutations commonly observed in FUS-associated fALS patients. Qiu et al.  developeRecently, Zhang et al.  employedTo distinguish the pathological role of FUS aggregates without the confounding effects on RNA metabolism, Shelkovnikova et al., developed a transgenic mouse expressing a truncated human FUS protein (residues 1–359), which lacked the RNA recognition motif and NLS but still retained an N-terminal QGSY prion-like domain , locomotor deficits measured by rotarod test (140 days vs. 120 days), and mortality , however the mechanism was not described -1-piperazinyl]phenyl]-2-thiophenecarboxamide. In vivo experiments using a SOD1-G93A mouse model showed that the drug was well tolerated, with toxicity estimated to be 75 mg/kg. These mice also showed a significant decrease in SOD1 protein levels, but aggregation was not described  in a randomized double-blind clinical trial in Poland, led to a reduction in ALS severity  and median survival time (212–248 days depending on dose) in SOD1-G37R mice compared to vehicle control . Administration of riluzole in conjunction with CuATSM did not exhibit any additive effect , a continuation of the study ongoing (NCT04313166), and a placebo-controlled study currently recruiting (NCT04082832) at the time of publication.Anti-sense oligonucleotides (ASO) can bind to mRNA and reduce protein expression, representing a potential therapeutic strategy  over 11.5 h in phase I, randomized trial of SOD1 fALS was demonstrated to be well tolerated, however, the therapeutic benefits were not measured   is an important chaperone that prevents protein aggregation  in Huntington’s disease patients (Reilmann et al., Years of intensive study into the disease mechanisms of ALS strongly implicates a pathological role for both declining proteostasis and prion-like propagation. There are intimate links between both of these concepts that explain much of the pathological manifestation of ALS. For example, the observation of proteinaceous inclusions in affected tissues, the apparent spread of pathology through neuroanatomically connected tracts, and the preferential degeneration of motor neurons over other cell types. SOD1 and TDP-43 can misfold and aggregate into conformations that can propagate within and between cells in a prion-like manner, as evidenced by the studies mentioned above ranging from biophysical assays of protein stability to whole-organism studies of aggregate seeding. Indeed, this knowledge of proteostasis dysfunction and protein aggregation has led to the development and examination of multiple therapeutics for ALS. The questions that remain, however, are what the conformation of these spreading strains are, the precise mechanism by which they contribute to pathology, and if targeted therapies can be designed to prevent their formation or spread. Also, a question of significant importance to the field is whether FUS can spread pathology from cell-to-cell in a pathological prion-like manner. This knowledge would unify our understanding of the underlying pathomechanisms in ALS.LM directed the manuscript. All authors wrote and edited the manuscript. NRC and JJY conceptualized the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It appears that shortening the vertical gate distance is a better and safer way to reduce speed in Super-G than increasing the gate offset.In Super-G alpine ski racing mean speed is nearly as high as in Downhill. Hence, the energy dissipated in typical impact accidents is similar. However, unlike Downhill, on Super-G courses no training runs are performed. Accordingly, speed control through course design is a challenging but important task to ensure safety in Super-G. In four male World Cup alpine Super-G races, terrain shape, course setting and the mechanics of a high-level athlete skiing the course were measured with differential global navigation satellite systems (dGNSS). The effects of course setting on skier mechanics were analysed using a linear mixed effects model. To reduce speed by 0.5 m/s throughout a turn, the gate offset needs to be increased by + 51%. This change simultaneously leads to a decrease in minimal turn radius (− 19%), an increase in impulse (+ 27%) and an increase in maximal ground reaction force (+ 6%). In contrast, the same reduction in speed can also be achieved by a − 13% change in  Including overuse injuries and injuries sustained off season, the rates are even higher5. Accordingly, alpine ski racing is recognized as having one of the highest injury risks of all Olympic winter sports8.In each World Cup (WC) alpine ski racing competition season, approximately one third of all athletes suffer an injury10. Since speed is the driving factor for forces that are dissipated during crashes and collisions with the surroundings11, impact injuries are frequent in these two competition disciplines12. Consequently, spill zones and impact protective equipment such as netting along the race courses are commonly used countermeasures13. Additionally a reduction of speed in general, and before potential injury hotspots in particular, may be helpful in preventing impact injuries14. An earlier study of Downhill races assessed whether speed, as a key driver for impact injuries, could be reduced by changes in ski geometry and binding plates. The study, however, revealed that equipment alterations have only small effects on speed and that lower speed does not substantially increase the time athletes have to anticipate and prepare for technically difficult situations in Downhill15. Similar changes in equipment did not have substantial effects on speed in Giant Slalom (GS), but did positively affect the skiers' motion patterns and kinetics17. Hence, studies suggest it is difficult to adequately increase ski–snow friction forces via changes in equipment properties, particularly in the speed disciplines of Super-G and Downhill.In Super-G and Downhill speed is high compared to the other disciplines18, turn entrance speed18 and course setting21 are measures that have an effect on skier speed in technical disciplines. For GS it was found that increasing the gate offset or reducing the vertical gate distance both reduced speed18. However, increasing the gate offset to reduce speed caused an increase in injury risk through increased impulse , reduced minimal turn radius and increased maximal force, while a reduction in the vertical gate distance did not lead to such increases in injury risk18. It is, however, unknown whether speed can be controlled in Super-G in a similar manner to that demonstrated for GS, and it would be beneficial if similar quantitative recommendations could be generated for Super-G. It would also be helpful to determine the negative side effects of course-setting manipulations on other injury risk factors, such as turn radius, impulse and ground reaction forces. Therefore, this study aimed to: (1) assess whether course-setting characteristics, entrance speed and terrain incline influence speed through a turn; and (2) assess whether changes in course-setting aimed at reducing speed have negative consequences on turn radius, impulse and turn forces in Super-G.An alternative approach to reducing speed in Super-G or Downhill is course design. Recent studies have shown that terrain inclineDuring two winter seasons, data was captured at four male Super-G World Cup races in Kitzbühel (AUT), Hinterstoder (AUT), and twice in Crans Montana (SUI). The races were chosen to cover the range of typical World Cup Super-G courses. A forerunner, testing the course directly prior to competition, was tracked using a differential high-end global navigation satellite carried by the skier. For each location a different forerunner was chosen to ski the course. The forerunners were athletes competing at European Cup level. The study was approved by the Ethics Committee of the Department of Sport Science and Kinesiology at the University of Salzburg. Each subject provided written informed consent prior to taking part in the study and the methods were carried out in accordance with the relevant guidelines and regulations.2 were measured. At terrain transitions this number was substantially increased and was reduced in uniform terrain10.Start, turning gate positions, finish line and the terrain were captured prior to the respective race using static dGNSS: Alpha-G3T receivers with GrAnt-G3T antenna  and Leica TPS 1230 + . To reconstruct the snow surface in enough detail, on average 0.3 points per m22. A GPS/GLONASS dual frequency (L1/L2) receiver  collected antenna position at 50 Hz. The antenna  was mounted on the forerunner’s head. To calculate the carrier phase double difference position solutions, two (for redundancy) base stations were located at the start of the course and equipped with GNSS antennas  and Alpha-G3T receivers .Athlete tracking during skiing was conducted using a high-end differential global navigation satellite system (dGNSS) carried by the athlete on the back and helmet10. To calculate skier antenna position data, double difference carrier phase solutions were processed from GPS and GLONASS satellites and frequency L1/L2 using the geodetic software’s  KAR algorithm22. Turns for which the geodetic solution ambiguities failed to be solved were excluded from the study.Static dGNSS measurements on the terrain surface to represent the snow surface, and gate positions to represent the course, were calculated in a post-processing procedure, using the geodetic GNSS software Justin 10. The local incline of the terrain was geometrically derived using the local terrain surface normal vectors. To represent the average slope incline of a turn, the local incline vectors associated with a turn were averaged over the area of each turn10. The average turn incline was named TerrainINCLINE and expressed as the angle to the horizontal10.The shape of the snow surface was surveyed using static dGNSS. A digital terrain model was generated from the point cloud captured by dGNSS by triangulation of the point cloud, using the Delaunay method, and gridded on a rectangular gridGateVERTICAL) and the gate offset (GateOFFSET), also named horizontal gate distance in other studies  to the projection of gate (i) onto the vector between (i − 1) and (i + 1), as shown in Gilgien et al.10. GateOFFSET for gate (i) was calculated as the normal projection of gate (i) on the vector from gate (I − 1) to gate (i + 1). When two consecutive gates formed a delay turn (a long turn marked with two gates) the gate with the larger GateOFFSET was selected as the one to represent the turn10.Courses in alpine skiing are marked with gates, with a turning gate and a gate on the outside of the line skiers take. The setting of the course is characterized by two gate distances; the vertical gate distance  when the CoM turn radius was below 75 m14. The speed at which an athlete entered a turn (SpeedIN) was defined as the instantaneous speed at the point when the turn radius dropped below 75 m prior to the gate passage. Exit speed was the instantaneous speed at the point when the turn radius exceeded 75 m for the first time after the gate passage. The change in speed through a turn (∆Speed) was computed as the difference of SpeedIN and Exit speed. The minimal turn radius through a turn (RadiusMIN) was also considered as an injury proxy and was defined as the smallest radius in the turn20. The maximal ground reaction force (GRFMAX) was defined as the highest instantaneous ground reaction force during a turn24. To measure the effort athletes excerpt to the surrounding over the time of the course impulse was calculated as the time integration of the external forces. Impulse might also serve as an approximation of accumulated fatigue since both duration and the intensity of the physical effort are reflected in this measure. Impulse as a proxy of fatigue was defined as the integral of ground reaction force and air drag (in body weight) through the turn time and expressed in body weight seconds (BWs)25.The position of the dGNSS antenna that was mounted on the helmet of the athlete was filtered using bi-cubic spline functions. To better estimate the center of mass (CoM) position, a mechanical pendulum model was attached to the antenna position with a global position accuracy of 9 ± 12 cmThe data from all four races were pooled together to create a dataset of 192 turns. To characterize the central tendency and variability of the course setting and athlete mechanical parameters, mean and standard deviation (SD) were calculated.SpeedIN, TerrainINCLINE, GateOFFSET and GateVERTICAL. The mixed model was run for each outcome variable: RadiusMIN, ∆Speed, GRFMAX and Impulse. To generate comparable results between the different outcome variables, the fixed effects of the model were adjusted by division by two SD. The results of this intermediate step calculation can be found in the GateVERTICAL is required to change ∆Speed by two standard deviations?".To determine the relationship between course setting and adjustments in course setting and their effect on skier mechanics, a linear mixed effects model was established ). Since snow conditions were not quantified and the forerunners were different in each race location, the model contained the random variables “Skier ID” (3 different athletes), and “Skier ID*Race ID”. As model predictors we included the variables: SpeedIN, TerrainINCLINE, GateOFFSET and GateVERTICAL) to result in a reduction in speed of 0.5 m/s (∆Speed = − 0.5 m/s) from turn start to turn end. The effect that this change in the predictors (to reduce speed by 0.5 m/s) had on the other outcome variables, RadiusMIN, GRFMAX and Impulse, was also determined.The mixed model was re-run on all turns to calculate the adjustment required for each predictor variable (GateOFFSET has much higher variability than GateVERTICAL or linear gate distance.Table SpeedIN have on ∆Speed (reduction in speed induced by the given adjustments through a turn). All model predictors have a significant effect on speed.Table GateOFFSET needs to be increased by 51% of the existing GateOFFSET, or an average of 6.84 m. For the same decrease, GateVERTICAL would need to be shortened by − 6.31 m, which is − 13% of the existing GateVERTICAL.. Further, if course setting and terrain is held constant, but skiers enter the turn at 2.2 m/s or 9% higher entrance speed (SpeedIN), due to the phenomenon of the "velocity barrier" (i.e. a skier’s voluntary control of speed in order to avoid mistakes), they would lose 0.5 m/s speed throughout the turn. And, for a given course setting and SpeedIN, a 3.68° steeper TerrainINCLINE would result in a speed reduction through the turn of 0.5 m/s compared to a turn with the given TerrainINCLINE.To achieve a reduction in speed of 0.5 m/s throughout a turn, (∆Speed) caused by an increase in GateOFFSET by + 6.84 m .The data in Table  m Table  typicallAL Table  leads toGateOFFSET of + 6.84 m (Table GRFMAX of + 0.13 BW (i.e. + 6%). No increase in GRFMAX is found if GateVERTICAL is shortened by − 6.31 m to reduce speed by − 0.5 m/s.Moreover, an increase in  m Table  to reducGateOFFSET is increased by + 6.84 m to reduce speed by − 0.5 m/s. In contrast, impulse is actually decreased by − 0.07 BWs (i.e. − 2%) if GateVERTICAL is shortened by − 6.31 m to reduce speed by − 0.5 m/s.Finally, impulse increases by + 0.87 BWs (i.e. + 27%) if GateOFFSET to reduce speed by − 0.5 m/s throughout a turn  simultaneously leads to a decrease in RadiusMIN (− 19%), an increase in Impulse (+ 27%) and an increase in GRFMAX (+ 6%). To reduce speed by − 0.5 m/s with a change in GateVERTICAL, compared to GateOFFSET, a much smaller percentage change is required . Further, shortening GateVERTICAL to reduce speed by − 0.5 m/s causes a much smaller reduction in RadiusMIN (− 4%) and Impulse (− 2%), and no increase in GRFMAX.The main findings of this study were: increasing GateOFFSET and decreasing GateVERTICAL lead to a reduction of speed; however, for the same speed reduction (− 0.5 m/s) the required percentage changes are almost four times higher for GateOFFSET (+ 51%) than for GateVERTICAl (− 13%), while the absolute changes are similar  in terrain incline reduced speed by 0.5 m/s, which is in line with previous observations in other disciplines28. However, as entrance speed and terrain are only indirectly modifiable by course setting for a specific race venue, they must be considered as important confounders rather than manipulatable prevention approaches.Similar to the findings of earlier studies in slalom and giant-slalom, higher entrance speed led to a higher speed reduction (energy dissipation) throughout the turn. This finding further supports the presence of a so called "velocity barrier"; the idea that skiers need to dissipate energy (control speed by braking) at certain points along the course in order to avoid making mistakesGateOFFSET or decreasing GateVERTICAL. However, the different percentage-wise impact on speed-reduction (larger for decreasing GateVERTICAL) is not the only relevant aspect in the decision of whether to control speed by adjustments in GateOFFSET or GateVERTICAL. As shown in this study, the increase in GateOFFSET to reduce speed by 0.5 m/s resulted in an adverse decrease in minimal turn radius, an increase in maximal ground reaction forces and an increase in impulse  appear much higher than those achieved with modified ski equipment15. To date, it has not been scientifically investigated whether a clothing-induced increase in air drag could adequately reduce speed in the speed disciplines31. Additional padding and use of race suit fabrics with higher air drag coefficient would increase air drag and are likely to reduce speed. Padding might also improve crash impact absorption. Snow properties may also have substantial impact on speed32, but these are only partly in the race organizers’ hands, especially in SG and downhill, where courses are long and environmental factors play an important role in the generation of snow properties.The results of this study show that course setters can control and reduce speed by either increasing se Table . As thes10 and consequently the impact energy in impact accidents is only slightly lower than in Downhill; (2) courses are technically more challenging; and (3) skiers do not have training sessions on the courses prior to competitions as is the case in Downhill. More specifically, Super-G courses are steeper, include more turns with smaller radii and more frequent and more pronounced terrain transitions10. Ground reaction forces are higher and the total physical load (impulse) of a competition is only 13% lower than in downhill25. Super-G courses also include an average of 2.3 jumps (4.2 for downhill), with jump length being only 21% and airtime being only 6% shorter than in Downhill14. Hence, jumps in Super-G can be as challenging as in Downhill, since a given rotation impulse at take-off can cause nearly the same off-balance rotation as in Downhill14.Preventing injuries in Super-G is challenging, since (1) mean speed is only slightly lower (− 2 m/s) than in Downhill33; mostly on easier and shorter slopes than in competition. In this connection, the findings of the current study may help course setters to develop a better understanding of how to set safe courses and adequately control speed in Super-G.In Downhill it is a safety requirement that athletes need to participate in at least one official training run on the course prior to competition to become familiar with the course. In Super-G athletes only have the opportunity to inspect the course some hours prior to competition and need to anticipate their skiing strategy and find a balance between performance and safety. Preventing injuries in Super-G is not only difficult for skiers, but also for organizers and course setters. In Downhill, course setting is kept similar from year to year, which allows organizers to learn and improve courses over the years, while Super-G courses are set differently by different coaches each year and change accordingly. Hence, course setters and the race directors of the International Ski Federation FIS, who carry the ultimate responsibility for course safety, have no opportunity to test the Super-G course for safety prior to competition. Therefore, experience in course setting is important. However, this might take years to build, since WC coaches only set about 40 Super-G training courses per yearThere are some limitations to be aware of when interpreting the study findings:18. This leaves us with the problem that we cannot provide information on how adjustments in course setting depend on terrain incline. Second, the study analysis did not look at the interaction of consecutive turns or entire course sequences and hence, experimental course set testing for a given situation seems indicated. Third, the applied dGNSS methodology did not provide direct data describing the influence of course setting on the mode of ski–snow interaction. Thus, we can only speculate as to why speed is reduced as a function of ski–snow interaction. Future studies should investigate the effect of course setting on skidding and the underlying ski–snow interaction in more detail. For the four races, three different athletes were employed as forerunners. The effect of forerunner however was compensated for in the mixed model.First, the number of turns was not sufficient to split the analysis into terrain incline sub-groups as shown in a similar study on GSSupplementary Information.


This research developed an effective and efficient approach for improving the slow convergence in the scattering analysis of a one-dimensional graphene grating, made of a periodic array of parallel graphene strips, illuminated by a TM-polarized plane wave. Specifically, the electric fields over the graphene strips and slit regions in a unit cell are individually expressed as an expansion of local basis functions inherently satisfying edge conditions. Interestingly, convergence rate is highly improved compared to the customary and modified Fourier modal method. Additionally, with the aid of local basis functions, the Gibbs phenomenon occurring at both edges of graphene strip can be removed. Moreover, graphene-based grating composed of graphene sheet has been a continued research interest14 in both theoretical studies and practical applications in recent years.The plane wave scattering by a one-dimensional (1D) grating made up of dielectric or metallic mediums has been intensively and extensively studied. Some numerical methods such as the rigorous coupled-wave analysis (RCWA)1 is known to be slowly converging for 1D metallic gratings in TM polarization . Fortunately, the inverse rule, by invoking adequate Fourier series of the permittivity and reciprocal permittivity functions of a periodic medium to reformulate the eigenvalue problem, was developed to achieved a highly improved convergence rate for the scattering analysis of a metallic grating in TM polarization18. Additionally, the Floquet modes in a periodic medium with the unit cell composed of a dielectric slab and a metal layer having finite conductivity can be determined by solving the dispersion equation19. However, finding their complex roots is a difficult task. Consequently, use of Fourier series expansion to calculate Floquet modes in a periodic medium, in general, is the most reliable and effective approach in handling a diffraction grating problem.In the RCWA method, both the permittivity function of a periodic medium and electromagnetic fields are expanded into Fourier series and Floquet-Fourier series, respectively. Therefore, the electromagnetic boundary-value problem can be converted into an eigenvalue problem. Such an approach is efficient in handling the grating with an arbitrary profile and a finite stack of multiple gratings, as well. However, the RCWA methodnm); therefore, the electromagnetic fields are merely in the upper and lower homogeneous mediums, as shown in Fig. x-axis, electric and magnetic fields can be presented in the standard form of Floquet-Fourier series (or Rayleigh expansions). Moreover, the electrical property of graphene strips can be modeled with a surface conductivity  throughout this paper.In this paper, we aimed at studying the numerical convergence of plane wave scattering by a graphene grating in TM polarization. Here, the structure under study is a 1D periodic array of graphene strips (ribbons) deposited on a dielectric substrate. The graphene sheet is assumed to be near-zero thickness  that takes into account the effective conductivity due to the displacement current in slit region without graphene. The effective conductivity is non-zero everywhere; therefore, the inverse rule can be successfully applied. Although FMM with ABC convincingly achieves numerical convergence, the convergent value varies in accordance with the enclosed-loop height (h), shown in Fig. h.Unfortunately, the poor convergence occurs in conventional FMM for the scattering analysis of periodic arrays of graphene ribbons reported in the literatureIn this research, the respective local basis functions (LBFs) taking into account the electric-field edge conditions over the graphene-strip and slit regions in a unit cell are developed to replace the global basis functions (Floquet–Fourier series in a homogeneous medium). As will become clear later on, the tangential electric-field expanded by the local basis functions exhibits the fidelity of discontinuous behaviour, which is not seen in the conventional FMM, enabling a fast convergence in the scattering analysis of graphen-strip gratings in TM polarization.This paper is organized as follows. We begin with the mathematical formulation using the conventional FMM that is taken as a general framework for theoretical analysis. Moreover, ABC is employed to reformulate the FMM by invoking the inverse rule. Additionally, the present approach, namely, FMM incorporating LBFs, will be comprehensively elaborated. Finally, convergence behaviour will be examined for the three aforementioned methods. Specifically, electric field distribution on graphene grating surface will be demonstrated for various incident conditions.y-axis and the electromagnetic fields have no variation along that direction. The strip width and period along the x-axis are denoted as Figure 11:e is the electron charge, T is the ambient temperature.Incidentally, graphene conductivity (x-axis can be represented by Rayleigh expansions (or Floquet–Fourier series) in the form5z-axis In a homogeneous medium, the electric and magnetic field components of TM polarization in the presence of periodicity 16 and equaling the same Fourier coefficient corresponding to the same harmonic on both sides, one obtains the system of linear equationsm is running from negative to positive infinity.Owing to the zero thickness approximation of the graphene grating, discontinuity in the tangential component of magnetic fields at the interface (graphene grating surface) between two uniform mediums equals to the conduction current induced on the graphene strips array, yieldingy in Eq.  and condn in Eq. . Using Lm, n) entry Equation  can be en in Eq. ; the \do5. Equation ,,\documenough Eq. . One obtwith Eq. , one obtNotably, for a graphene grating incident by a single plane wave, the wave vector via Eqs.  and 18)\documentz-axis over a grating period is defined asMoreover, the time average Poynting power along the n runs from − N is denoted as truncated order. The total number of space harmonics is then designated as Notably, the mathematical formulation in FMM is rigorous and the result is exact when the space harmonic index 16, the slow convergence in the RCWA for TM polarization is not caused by the Fourier series expansion but the form where the Fourier series of the permittivity and the reciprocal permittivity functions are utilized. The same problem also occurs in graphene gratings with thickness of almost zero. Nevertheless, the inverse rule16 is not applicable in the graphene grating because 20 in slit region. In doing so, the reciprocal function H) on the graphene grating between two uniform mediums equals to the sum of conduction- and displacement-current. At the left-hand side of Fig. x-axis; while it equals to the displacement current filled in the rectangular box at the right-hand side figure. Combining these two results, Eq.  entry of Now, the reciprocal function mentclasspt{minimah is still a core factor affecting the value of convergence, as will be demonstrated in the next section. Moreover, the criterion for determining the appropriate h remains to be studied in detail. So far, we have implemented a computer program based on FMM with ABC for calculating the scattering characteristics of a graphene grating as well as the electric-field  to use only a few basis functions to approach the correct solution, and (2) to have closed forms in the overlap integral between the local basis functions and the space harmonic in Eq. .n is ranging from 1 to More specifically, the 22. They are expressed as follows:n runs from 0 to On the other hand, in the slit region, we have the singular basis functions with singularities at its two edges, which are commonly used to approximate the current parallel to the edges in a micro-strip linely, Eqs.  and 24)22. They In a unit cell on the graphene grating surface (at m is ranging from -N to +N. The notation of a(x) and b(x) in the range of sinc(x) is the unnormalized sinc function defined as n is the index of the local basis function.Due to electromagnetic boundary condition,  of Eqs.  and 25)\documentG]] and [[S]] have the size 19, therefore, we have round[.] rounds a real number towards the nearest integer. In doing so, we have a square matrix Equation x-axis, one obtainsSubstituting of Eqs.  and 25)25) into  and Eq. , the sysAfter performing matrix operations with Eqs.  and 34)34), we oHere, we obtain a totally different admittance matrix while the input-output relation in Eq.  remains _ in Eq.  can be rFurthermore, the power dissipated on the graphene strips array can be directly determined byA free-standing graphene-strip grating is taken as an example to examine the convergence behaviour for the three approaches. The parameters of graphene are N running from 1 to 200. In Fig. h) in FMM with ABC are considered: 20, the conventional FMM (green dotted curve) indeed runs into the serious problem of oscillating convergence behaviour. On the other hand, the FMM with ABC can improve the convergence rate; however, the convergent value changes with h accordingly. Interestingly, the result with We first calculate the absorptance against the truncated order N) are used to examine the performance of numerical convergence. It is obvious to see that FMM with ABC (yellow solid curve) and our approach (purple solid curve) agree very well for the case of Additionally, the absorption versus frequency for both convincing methods including the FMM with ABC and our approach are demonstrated in Fig. x in a unit cell on the graphene grating surface. The red dotted curve is calculated based on FMM with ABC, while the solid curve in blue colour is obtained using FMM incorporating LBFs. The region for x-direction. Therefore, the induced current exhibits the first normal mode (standing wave) pattern. It is very similar to the current induced on a radio-frequency (RF) dipole antenna excited at its first resonant frequency.Figure  in Eqs.  and 24)\documentx-axis was demonstrated for the five cases with different incident angles including 0°, 15°, 30°, 45°, and 60°, respectively. Fig. 13, the resonance effects are in connection with the leaky plasmonic modes existing in individual graphene strip but with weak coupling between strips. Such a normal mode is a source-free solution; therefore, its mode pattern is almost independent of the incident angle. Contrarily, the asymmetric distribution is observed in the slit region for oblique incidence, which maybe caused by unbound waves with continuous spectrum.Since the approach of FMM incorporating LBFs can effectively remove the Gibbs phenomenon, it should be in a good position to observe the effect of incident angles on the electric field  in conventional FMM disappears. Furthermore, the convergence rate of the present approach is superior to the other two methods. Additionally, a new admittance matrix is obtained in the present approach while the whole formulation can still fit into the standard procedure of FMM. Significantly, the inverse rule and ABC for FMM are no longer needed. Such an approach can drastically reduce the required number of space harmonics and is more efficient for scattering analysis of stacked multiple graphene gratings.


Redundancy analysis identified specific conductivity, dissolved oxygen, agricultural land, ammonium, urban land, and water transparency as the dominant environmental factors influencing the bacterioplankton communities in the Pearl River. Variation partitioning analysis indicated that both physicochemical factors and land use had direct effects on the bacterioplankton community, and that land use may also shape bacterioplankton communities through indirect effects of physicochemical factors on riverine ecosystems. This study provides fundamental information on the diversity, spatial patterns, and influencing factors of bacterioplankton communities in the Pearl River, which should enhance our understanding of how such communities change in response to environmental gradients and anthropogenic activities.River ecosystems are critical for human and environmental health, with bacterioplankton playing a vital role in biogeochemical cycles. Unveiling the spatial patterns of bacterioplankton communities in relation to environmental factors is important for understanding the processes of microbial variation and functional maintenance. However, our understanding of the correlations among bacterioplankton communities, physicochemical factors, and land use, especially in large rivers affected by intensive anthropogenic activities, remains relatively poor. Here, we investigated the bacterioplankton communities in July 2018 in three main tributaries of the Pearl River, i.e., Beijiang, Xijiang, and Pearl River Delta, based on 16S rRNA high-throughput sequencing. Results showed that the most dominant phyla, Proteobacteria, Actinobacteria, Cyanobacteria, and Planctomycetes accounted for 33.75%, 22.15%, 11.65%, and 10.48% of the total abundance, respectively. The bacterioplankton communities showed remarkable differences among the three tributaries in terms of composition, structure, diversity, and predictive functional profiles. Mantel and partial Mantel tests revealed that the bacterioplankton communities were affected by physicochemical variables ( The microbial community is a key component of aquatic ecosystems and plays an important role in biogeochemical cycling processes such as ammonia oxidation, nitrification, sulfate reduction, and methane production ,2,3,4. URiver ecosystems are critical for human and ecological health. They are a major source of drinking water and provide irrigation for agriculture, habitat for fish, and space for recreation. Rivers also offer important freshwater habitats for microorganisms. Bacterioplankton communities exhibit complex responses to environmental stresses and disturbances, as manifested by changes at both the population and community levels ,13. Many9 m3, including 7 × 109 and 2 × 109 m3 of domestic and industrial wastewater, respectively [The Pearl River is the largest river in southern China, stretching some 2400 km in length . The rivectively . Such waectively . Althougectively ,23,24,25In this study, we analyzed the bacterioplankton communities in three main tributaries of the Pearl River in July 2018, a typical flood season, using 16S rRNA high-throughput sequencing. We aimed to (1) characterize the composition, diversity, and spatial patterns of bacterioplankton communities in the Pearl River and (2) elucidate the roles of physicochemical factors and land use in shaping these communities. To the best of our knowledge, this is the first study to report on the effects of land use and physicochemical factors on bacterioplankton communities in the Pearl River, which should enhance our understanding of how aquatic ecosystems change and function in response to environmental gradients and anthropogenic activities.A total of 30 water samples were collected in July 2018 from 15 sites ; SK, YD, LF, QY, LB for Beijing River (BJ); and SH, GM, JJ, WH, ZZ for Pearl River Delta (PRD)  along th4-N), nitrate (NO3-N), nitrite (NO2-N), total phosphorus (TP), phosphate (PO4-P), chlorophylla, and suspended solids (SS), were measured in the laboratory following standard protocols [Physicochemical variables, including dissolved oxygen (DO), temperature, salinity, pH, total dissolved solids (TDS), specific conductivity (SpC), and oxidation-reduction potential (ORP), were measured in situ with a YSI multiparameter probe . Water transparency (cm) was measured by a Secchi disk  (20 cm diameter). Ammonium  (2016–2018) were used to determine land use cover. The satellite images were corrected and stitched in ENVI 5.3 , and then they classified into land use types using knowledge-based supervised and maximum likelihood classification methods. Land use was classified into agricultural land, forestland, grassland, urban land, highway, and bare soil. The 1:100,000 scale map of land use was then interpreted. ArcGIS 10.2.2  was applied to extract land-use percentages using a 10 km × 2 km buffer zone upstream of the sampling points.Satellite datasets from the Landstat 8 OLI database . The 16S rDNA V3-V4 hypervariable region was amplified using primers 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT). After being quantified with QuantiFluorTM fluorometer, purified amplicons by PCR were pooled in equimolar amounts and paired ends were sequenced (2 × 250) on an Illumina HiSeq2500 platform according to the standard protocols at Gene Denovo Biological Technology Co. Ltd. . The 16S high-throughput sequencing raw data was uploaded in the NCBI Sequence Read Archive with accession no. PRJNA579235.The raw reads were filtered using FASTP. Paired-end clean reads were then merged using FLASH v. 1.2.11 . Noisy sPhysicochemical factors and land use were analyzed by principal component analysis (PCA) to identify potential spatial patterns in the different tributaries.p < 0.05).Alpha diversity, including Good’s coverage, Abundance-based Coverage Estimator (ACE), Chao1, Simpson, and Shannon indices, were calculated via QIIME. Differences in diversity indices among the different tributaries were examined with Kruskal–Wallis tests for global comparison and Wilcoxon tests for pairwise comparisons (http://huttenhower.sph.harvard.edu/lefse/).Nonmetric multidimensional scaling (NMDS) and analysis of similarity (ANOSIM) were performed to compare bacterioplankton communities based on the Bray–Curtis dissimilarity index using the Vegan package in R . Linear The Mantel test was used to examine the correlation between the environment  and bacterioplankton communities, and a partial Mantel test was used to determine the relationship between a certain type of factor  and bacterioplankton community, controlling for the effects of other factors . The Mantel and partial Mantel tests were conducted with the Vegan package . RedundaFunctional changes in the bacterioplankton communities among the different tributaries were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) . Rarefie4-N. Axis 2 was highly positively correlated with highways and forests, and negatively associated with agricultural land.Spatial variations in 15 physicochemical factors and six land use types were analyzed by PCA to identify differences among tributaries . The fir4N were relatively higher in BJ than in XJ and PRD, whereas DO and ORP were relatively higher in XJ than in BJ and PRD. PRD had relatively higher values for agricultural land, urban land, SS, and NO2N, but lower values related to water transparency, forests and highways.Both XJ and PRD were close to each other, but distant from BJ A. BJ washgcI_clade, CL500-29_marine_group, Planctomyces, Synechococcus, Aquabacterium, Ideonella, Exiguobacterium, CL500-3, Polynucleobacter, and Acinetobacter, which accounted for 10.81%, 6.03%, 3.84%, 3.71%, 3.25%, 1.92%, 1.86%, 1.74%, 1.33%, and 1.23% of total bacterioplankton community, respectively  among the three tributaries, whereas diversity indices (Shannon and Simpson) showed significant spatial variation . The Shap < 0.01). The BJ group was separated from the other groups, whereas XJ and PRD demonstrated partial overlap. These spatial patterns were consistent with environmental factors , and Dechloromonas (genus), were identified as potential biomarkers for PRD , Methylophilaceae (family), Candidatus for PRD . Genera r = 0.638; p = 0.001). Partial Mantel tests revealed that the bacterioplankton communities were affected by both physicochemical variables  and land use . Forward selection and Monte Carlo permutation (499 iterations) of RDA showed that environmental variables SpC , DO , agricultural land , NH4-N , urban land , and water transparency  significantly influenced the Pearl River bacterioplankton communities . The conditional effect of physicochemical variables was 42.6% . The conditional effect of land use was 8.9% . The shared effect of these two groups was 6.1%. In total, 42.4% of variation remained unexplained. The Mantel tests indicated that environmental factors significantly shaped the bacterioplankton communities , multifunctional diversity also increased, but when OTU diversity approached 8, multifunctional diversity reached saturation, indicating functional redundancy within the bacterioplankton communities , and Dechloromonas (genus) were mainly restricted to PRD . NO2-N, TP, and PO4-P are essential nutrients for primary production and microbial growth [Anthropogenic factors such as agricultural and urban land use significantly contributed to the variability in the bacterioplankton communities among the three tributaries. This result may be explained by both direct and indirect anthropogenic influences on bacterioplankton community compositions. Firstly, bacteria may enter riverine waters directly through a variety of point and non-point sources such as urban runoff, wastewater treatment plant outflows, and agricultural areas ,55. The l growth ,61. SuspThis study investigated the bacterioplankton communities in July 2018 in three main tributaries of the Pearl River, i.e., Beijiang, Xijiang, and Pearl River Delta, based on 16S rRNA high-throughput sequencing. We revealed that the three tributaries formed distinct bacterioplankton communities in relation to the environmental gradients. Both physicochemical factors and land use had direct effects on the bacterioplankton communities, and land use may shape bacterioplankton communities through indirect effects of physicochemical factors on the riverine ecosystem. However, physiochemical factors outweighed land use in structuring bacterioplankton communities in this large subtropical river. This study provided fundamental information on the diversity, spatial patterns, and influencing factors of bacterioplankton communities in the Pearl River, which should enhance our understanding of how bacterioplankton change in response to environmental gradients and anthropogenic activities. However, as the current study only covered one season, future studies are needed to provide insight into microbial communities and functions at both the spatial and temporal scale, including biological factors and other physicochemical variables.