Hemiplegic shoulder pain (HSP) is a common complication in patients with stroke. The pathogenesis of HSP is complex, and muscle hypertonia, especially the hypertonia of internal rotation muscles of the shoulder, may be one of the important causes of shoulder pain. However, the relationship between muscle stiffness and HSP has not been well studied. The purpose of this study is to explore the correlations between the stiffness of internal rotation muscles and clinical symptoms in patients with HSP.A total of 20 HSP patients and 20 healthy controls were recruited for this study. The stiffness of internal rotation muscles was quantified using shear wave elastography, and Young's modulus (YM) of the pectoralis major (PM), anterior deltoid (AD), teres major ™, and latissimus dorsi (LD) were measured. Muscle hypertonia and pain intensity were evaluated using the Modified Ashworth Scale (MAS) and Visual Analog Scale (VAS), respectively. The mobility of the shoulder was evaluated using the Neer score. The correlations between muscle stiffness and the clinical scales were analyzed.P < 0.05). YM of internal rotation muscles on the paretic side during passive stretching was significantly higher than that at rest (P < 0.05). YM of PM, TM, and LD during passive stretching were correlated with MAS (P < 0.05). In addition, the YM of TM during passive stretching was positively correlated with VAS and negatively correlated with the Neer score (P < 0.05).YM of internal rotation muscles on the paretic side was higher than that of the control group in the resting and passive stretching positions (Increased stiffness of PM, TM, and LD was observed in patients with HSP. The stiffness of TM was associated with pain intensity of the shoulder and shoulder mobility. Hemiplegic shoulder pain (HSP) is one of the most common complications after stroke, and patients with HSP often have shoulder pain and limited mobility . The preFocal and characteristic muscle dysfunction is often observed in HSP patients . InvestiThe clinical evaluation of hypertonia mainly includes clinical scale evaluation, biomechanical evaluation, and electrophysiological evaluation. The commonly used clinical scale is the Modified Ashworth Scale (MAS), which can only assess the overall condition of the patient and is highly subjective with limited reliability and validity. Moreover, biomechanical evaluation and electrophysiological evaluation are complicated and expensive. Shear wave elastography (SWE), an emerging technique, has the advantages of non-invasive, simple, and timely detection. Most importantly, it has a certain pathological basis for muscle hypertonia assessment, monitoring the structure and viscoelasticity properties of hypertonic muscle. The principle of SWE is to generate shear waves by creating acoustic radio frequency force impulse, stimulating tissue vibration to quantify muscle stiffness . Muscle In this prospective observational study, we hypothesized that the stiffness of internal rotation muscles was related to the clinical symptoms of HSP. Using the SWE technique, we quantitatively measured the YM of internal rotation muscles and analyzed the relationship between muscle stiffness and hypertonia, pain intensity, and shoulder dysfunction.A total of 20 stroke patients with HSP and 20 healthy controls participated in this study. The inclusion criteria for the stroke patients with HSP were as follows: (1) patients with cerebral hemorrhage or cerebral infarction confirmed by computer tomography or magnetic resonance imaging , (2) patThe participants were recruited for this study from October 2021 to April 2022. This study was approved by the Ethics Committee of Weifang Medical College, and all participants provided written informed consent before participation.Before the ultrasonic measurement, all patients were evaluated by experienced physiotherapists using the MAS, VAS, and Neer scores for the evaluation of shoulder muscle hypertonia, pain, and function, respectively. MAS is one of the most commonly used scales to evaluate muscle hypertonia in clinics. The clinician passively moved the patient's upper limb in the 0° to 90° range of external rotation and rated the resistance from 0 (no increase in muscle tone) to 4 (the joint is rigid) , and theUltrasound images were captured using the SWE ultrasound system  with a 2–10 MHz linear transducer array . All subjects were seated in a neutral position with the torso, shoulder abduction 0°, and upper limbs naturally relaxed on the legs. The probe was applied perpendicularly to the skin, paralleling the muscle bundle. First, the measurement position was determined by the B-mode grayscale. Thereafter, SWE was switched to establish the region of interest (ROI), set to an ROI of 1 mm diameter, and a depth of 0–2 cm . YM was The measurement sites were as follows: PM was measured at the midpoint between the greater tubercle of the humerus and sternoclavicular joint; anterior deltoid (AD) was measured at a site 2 to 3 cm below 1/3 lateral to the midpoint of the clavicle. The probe is perpendicular to the line between the inferior angle of the scapula and the acromion to measure TM; LD was measured at the position of the eighth thoracic vertebra parallel to the lower part of the inferior angle of the scapular.t-test was used for the comparisons as appropriate, and the Shapiro–Wilk test was used to check whether the data conformed to normal distribution. The gender distribution between HSP and healthy controls was compared using the chi-square test. The correlations between muscle stiffness and the MAS, VAS, and Neer score were analyzed using Spearman's rank test. The p-values of multiple comparisons and correlation analysis were corrected by controlling the false discovery rate (FDR) at a level of 0.05 .The demographic data are summarized in The results of the clinical evaluation for the HSP group are summarized in P > 0.05) (P < 0.001) (P < 0.001)  . The dom< 0.001) . Then, t< 0.001) .P = 0.727), AD , TM , and LD  at rest. However, YM of PM , TM , and LD  were correlated with MAS during passive stretching (P = 0.901) did not correlate with MAS. YM of TM was positively correlated with VAS  (P = 0.046)  . In addi= 0.046) .Using ultrasonic SWE, we found that the stiffness of shoulder internal rotation muscles was significantly increased on the paretic side of patients with HSP. Moreover, as to paretic side muscles, the muscle stiffness was significantly higher during passive stretching than at rest. The stiffness of PM, TM, and LD during passive stretching was correlated with MAS. We also found that the stiffness of TM was positively correlated with the VAS and negatively correlated with the Neer score. These results suggested that the increased stiffness of the shoulder's internal rotation muscles may be related to the severity of HSP.In this study, SWE was used to evaluate the internal rotation muscles of the shoulder. The results of our study were consistent with the study of Lee et al. . They reIn stroke patients, it has been reported that shoulder pain was related to muscle hypertonia, motor, sensory disturbance, and musculoskeletal system problems . PreviouIn this study, we analyzed the correlation between the stiffness of internal rotation muscles of the shoulder and VAS and Neer scores, finding that only the stiffness of TM was correlated with them. This is probably because TM is the main internal rotation muscle, and when TM hypertonia occurs, it leads to limited external rotation and shoulder abduction and causes pain. The results indicated that HSP patients with higher muscle stiffness may have more severe pain, and treatment to reduce muscle stiffness may relieve pain. This finding was consistent with the results of Itoigawa et al. . They alThere are several limitations to this study. First, SWE cannot fully observe the stiffness of the subscapular due to the special anatomical position, which may affect the judgment of the author. Second, we only assessed the pain intensity of HSP patients using the VAS scale, but not the frequency and duration of pain, and the VAS had a large range, which could have influenced the results. At present, self-reporting is an effective method to measure pain. However, VAS can still be used as a method to evaluate pain due to the special population. Third, the SWE has some limitations. For example, transducer placement, muscle position, and the lack of a uniform measure have limited the development of SWE in the musculoskeletal field. Fourth, although we measured multiple times in order to reduce the error, we did not calculate intraclass correlation coefficients to evaluate the reliability of SWE in the HSP group. In addition, the relationship between MAS grade 4 and muscle stiffness could not yet be determined because patients with MAS grade 4 were unable to cooperate with passive stretching movement. Finally, due to the small sample size, the results of this study need to be validated in a larger cohort.In conclusion, our study found increased stiffness of the internal rotation muscles in patients with HSP. The stiffness of the TM was associated with the pain intensity and function of the shoulder. The stiffness of internal rotation muscles can be used as a quantitative indicator for HSP evaluation.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Weifang Medical College. The patients/participants provided their written informed consent to participate in this study.FJ, X-RZ, L-YK, and X-ZY developed the study concept and design. FJ and X-RZ collected clinical and imaging data and interpreted the data. S-YZ and X-ZY designed and revised this manuscript. J-CF, Z-JZ, and L-ZL analyzed the data. FJ wrote the first draft. All authors critically reviewed this manuscript, contributed to this article, and approved the submitted version.


Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor that is characterized by its high proliferative and migratory potential, leading to a high invasiveness of this tumor type. However, the underlying mechanism of GBM proliferation and migration has not been fully elucidated. In this study, at first, we used RNA-seq together with bioinformatics technology to screen for C-X-C motif ligand 1 (CXCL1) as a proliferation-related gene. And exogenous glial cell line-derived neurotrophic factor (GDNF) induced proliferation and up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we manipulated the CXCL1 expression by using a lentiviral vector (CXCL1-RNAi) approach. By this, the proliferation of C6 cells was decreased, suggesting that CXCL1 plays a key role in proliferation in these cells. We hypothesized that exogenous GDNF promoted NF-κB nuclear translocation and therefore, analyzed the interaction of CXCL1 with NF-κB by Western Blot and immunofluorescence. Additionally, we used BAY 11–7082, a phosphorylation inhibitor of NF-κB, to elucidate NF-κB mediated the effect of GDNF on CXCL1. These results demonstrated that GDNF enhanced the proliferation of rat C6 glioma cells through activating the NF-κB/CXCL1 signaling pathway. In summary, these studies not only revealed the mechanism of action of exogenous GDNF in promoting the proliferation of C6 glioma cells but may also provide a new biological target for the treatment of malignant glioma. Glioblastoma (GBM) is the most common primary malignant brain tumor, characterized by a high invasiveness . Like otGDNF is a member of the transforming growth factor beta (TGF-beta) superfamily, which was first isolated and purified in 1993 . The proCXCL1, also known as GRO-α (growth related oncogene-α), is a proangiogenic and chemotactic factor, which mediates neutrophil recruitment by binding to CXCR2 receptors. Recent studies indicated that the proliferation and migration of cancer cells can be linked to some chemokines of the tumor microenvironment , 17. TheIn this study, we found CXCL1 was a proliferation-related gene by RNA-seq together with bioinformatics technology, and exogenous GDNF up-regulated the level of CXCL1 in rat C6 glioma cells determined by sqPCR and ELISA. Then, we suggested that CXCL1 plays a key role in proliferation in these cells by CXCL1-RNAi approach. Furthermore, we demonstrated exogenous GDNF promoted NF-κB nuclear translocation and the interaction of CXCL1 with NF-κB, and NF-κB mediated the effect of GDNF on CXCL1 by Western Blot and immunofluorescence. This study not only reveals the mechanism underlying the promotion of C6 cell proliferation after exogenous GDNF administration but also provides a new biological target for the treatment of malignant glioma.The rat C6 glioma cell line was obtained from the Chinese Academy of Sciences . The cells were cultured in F12/DMEM medium supplemented with 10% FBS for routine culture and with 0.1% FBS for hunger culture. The C6 cells were grown in 10 cm dishes and subjected to exogenous GDNF. Untreated C6 cells were used as a control. The AST cells were obtained from our laboratory at Xuzhou Medical University.Antibodies were used at the following dilutions: Monoclonal mouse anti-β-catenin , 1:500; polyclonal rabbit anti-CXCL1 , 1:2000; polyclonal rabbit anti-NF-κB , 1:2000; monoclonal mouse anti-β-actin , 1:1000; polyclonal rabbit anti-Histone H3 , 1:500; IRDye 800CW goat  anti-rabbit IgG , 1:15000; IRDye 800CW goat  anti-mouse IgG , 1:15000. BAY 11–7082 .C6 cells were cultured and treated with GDNF for 0, 0.5, 1, and 24 h, respectively and total RNA was extracted from the four sample groups using Trizol reagent. All experiments were run in triplicate. After passing the electrophoretic quality inspection, performed on an Agilent 2100 Bioanalyzer , total RNA was purified with a RNA clean XP kit and RNase free DNase set. Bidirectional RNA-Seq of the four samples were performed with an Illumina hiseq2500 high-throughput sequencer.P was determined by controlling the false discovery rate (FDR). The obtained P value was corrected by a multiple hypothesis test and the corrected P value was designated as Q value. Screening conditions for DEGs: Q value ≤ 0.05 and fold change ≥ 2 [The reads were converted into the number of reads per 1000 bases compared to exons in the reads per 1 million comparisons and the expression amount was standardized to calculate the gene’s fold change (FC). At the same time, the DEGs of different samples were analyzed by edger and the threshold value of ange ≥ 2 .http://bioinfogp.cnb.csic.es/tools/venny/index.html)In this study, we used the heatmap3 package of R language for data visualization . AdditioP < 0.05 was considered as being statistically significantly different [We used the David tool to perform GO and KEGG pathway enrichment analysis on the DEPs information. ifferent .In this study, we used the STRTING tool to construct a PPI network (medium confidence: 0.400) for DEPs, GDNF, and CXCL1 that were both rising in cytoplasm and nucleus, and thereafter used the Cytoscape software for further module analysis .http://gepia.cancer-pku.cn/). GEPIA is an online analysis tool for processing high-throughput RNA sequencing expression data of bulk tumorous and normal samples based on the Cancer Genome Atlas (TCGA)  and the Genotype-Tissue Expression  databases. We analyzed the correlation between CXCL1 expression and the overall survival (OS) of GBM patients and the expression of CXCL1 in GBM tissues.For gene expression profiling and overall survival analysis, we conducted bioinformatics analysis on the GEPIA platform  and gel analysis. Then, the RNA samples were reverse transcribed into cDNA using a Transcriptor First Strand cDNA synthesis kit . After this, semiquantitative real time PCR was performed to analyze the gene expression using the SYBR Green PCR master mix . The 20 μL reaction mixture included 10 μL 2×SYBR® Premix Ex Taq , 0.5 μL each of 10 μM forward and reverse primers, 2 μL cDNA template and 7 μL RNase-free HF:CGGATTTGGCCGTATCGGR:TGAGGTCAATGAAGGGGTCGGAPDH F:GCAGACAGTGGCAGGGATTCR:CGGTTTGGGTGCAGTGGGCXCL1 F:GAGGCAGGCAGTGGGGTTAR:TGGAGGCTCTGGTAGGCAAACx3cl1 F:CTGCCCCGCCTCCTCAR:CGCCACTGTGCCGCTCTSerpine1 F:CACCTCCCAAAGCCCTCGR:TGCTTGAGCCGCTCGTTCTDdit3 F:CCGCTGGCGTCTACGAGTCR:GGCTATGTCGCCCTCATCTTCGadd45g F:CCAACTGCTCCTGCTCCACCR:GCACTTGTCCGAGGCACCTTMt1 F:CCAGCCACAGTGGGAGCTTR:CCCCGGCGTCCGAATCRap1gap F:AAGGAGGTTTGCTCGTTCCGR:GGCAGCTTCTTGGCTGATAGGKlf10 F:TGACCTGGTCTCGCTCCTGTR:GACGCTGGCCTCCAAACACNfkbia For in situ staining, cells were washed with PBS, then fixed with 4% paraformaldehyde (PFA), washed 3× in PBS and placed in 10% normal goat serum with 0.3% Triton X-100 in PBS for 1 h. Afterwards, the cells were incubated in blocking solution with anti- NF-κB antibody overnight at 4°C. After that, the cells were washed 3× in PBS and incubated with goat anti-rabbit antibody in PBS for 1 h, washed 3× in PBS and incubated with DAPI . Finally, the cells were washed 3× with PBS and analyzed under a fluorescence microscope .For total protein extraction, cells of each group were washed with ice-cold phosphate-buffered saline and lysed in RIPA lysis buffer  containing the protease inhibitor PMSF . Cytoplasmic and nuclear protein extraction was subsequently performed. The samples were washed with ice-cold phosphate-buffered saline and treated with a nuclear protein extraction kit  according to the manufacturer’s protocol: The cell pellet was incubated in buffer A containing PMSF and thereafter incubated for 10 min. on ice. Then, buffer B was added and the samples were incubated for another 1 min. After centrifugation at 10,000 g for 5 min at 4°C, the cytoplasmic protein (the supernatant) could be isolated. The nuclei in the pellet were isolated by further centrifugation and resuspended in nuclear extraction buffer containing PMSF for 30 min at 4°C and vortexing every 5 minutes. After centrifugation at 10,000 g for 10 min at 4°C, the supernatant contained the nuclear protein. The protein concentration was determined with the help of a BCA assay kit  and was used according to the manufacturer’s protocol. The isolated proteins were stored at −80°C until use.To detect the expression of specific proteins, 40 μg of total protein was separated on a SDS-polyacrylamide gel  and wet transferred to a nitrocellulose transfer membrane . The membrane was probed with a primary and a secondary antibody. The protein bands were scanned and visualized using an Odyssey® Infrared Imaging System by LI-COR Biosciences (USA).CXCL1, we generated a luciferase expression construct containing the CXCL1 promotor. Firstly, a Luciferase reporter gene vector was constructed by designing primers  to amplify the CXCL1 promoter sequence. A Luciferase reporter gene vector (1 μg/well) and pRL-TK (50 ng/well), expressing Renilla luciferase as an internal control, were co-transfected in C6 cells (1×105) and incubated for various time points. Relative luciferase activity was assessed by the Promega dual-luciferase reporter assay system .To monitor the expression of the GDNF-induced 50 μl of standard, 40 μl of sample diluent and 10 μl of sample were accurately added to the wells to be tested. After gentle shaking and mixing, the plate was closed and incubated at 37°C for 30 min. Then, 50 μL developer was added into each well, gently mixed and incubated for 10 min. at 37°C and protected from light for color development. After this, the wells were washed. Finally, 50 μL termination liquid were added into each well after which eventually the absorbance of each well was measured at 450 nm .TTCTCCGAACGTGCACGTAA. The interference sequence was: AGAACATCCAGTTGAAGGTGAT. The designed sequence was cloned into a pHB-U6-MCS-CMV-ZsGreen-PGK-PURO  to generate the lentiviral vector. Then, the expression vector together with the packaging vector were used to transfect 293T cells with lipofitertm . After 48 h, the supernatants containing HBLV-r-CXCL1 shRNA3-GFP-PURO and HBLV-GFP-PURO NC lentiviruses were collected. After that, the lentiviruses were purified by ultracentrifugation after which the virus titers were detected.The siRNA target was designed according to the transcripts of the r-CXCL1 gene. The siRNA sequence of the control virus vector was: CXCL1 promoter sequence containing putative SRY-related protein-binding sites . Specific ChIP primers used for PCR were as follows: CXCL1-1; F: TTGGGAGTGGAGCAAGGGG; R: TGGAGCTGGTTTAGGATCTGAGTC; CXCL1-2; F: CGTCCTCAGCCCAGAAAAAAC; R: CTCTTCTTTGCTTTTTGAACTCGG; CXCL1-3; F: TGGAGTCCTAGGTGGCGTGG; R: TTTTTGCTTTTTGCCCCAAAGTC.A ChIP Chromatin Immunoprecipitation kit  was employed for the ChIP assay. Briefly, C6 cells were fixed with 1% formaldehyde for 10 min at 25°C and then washed with ice-cold phosphate-buffered saline buffer containing protease inhibitors. The nuclei were collected and resuspended in SDS lysis buffer containing protease inhibitor after which they were sonicated to generate crosslinked DNA fragments with 200–1000 bp length. The soluble supernatant was obtained after brief centrifugation whereupon it was incubated with Protein G Agarose. An aliquot of the supernatant was saved as a DNA input control and the remainder was incubated with anti-NF-κB antibody . A rabbit IgG antibody was used as a negative control. Immunoprecipitation was carried out overnight at 4°C and immune complexes were collected using Protein G Agarose beads. The immune complexes and the input were eluted and protein-DNA complexes were de-crosslinked overnight at 65°C. ChIP DNA was purified and subjected to PCR, which amplified the 3 cells were plated in clear bottom 96-well plates, processed according to the manufacturer’s instructions using a Cell Counting Kit-8  and afterwards quantified on a microplate reader .For cell viability assays, 1×103 cells/well) were cultured for 12 h in 96-well plates before being treated with different concentrations of rapamycin BAY 11–7082 for 24 h. Cell viability was evaluated using an 3--2,5-diphenyltetrazolium bromide (MTT) assay with absorbance measurement at 490 nm.C6 cells . The percentage of tested genes increased with increasing sequencing depth, and the correlation between samples was high . Differentially expressed genes are displayed by a heat map drawn with the help of the R language software package. Compared to the 0 h group, 11, 37, and 87 genes were differentially expressed (DEG) in the 0.5 h, 1 h, and 24 h group, respectively, of which 5, 29, and 37 were up-regulated and 6, 8, and 50 were down-regulated . In order to find the intersection of DEGs, we used Venny’s online software to draw a Venn diagram. There were two common up-regulated DEGs (CXCL1 and AABR07024908.1) and one common down-regulated DEG (LOC100910835) in the 0.5, 1, and 24 h group .It has shown that GDNF can significantly increase the proliferation and migration of glioma cells . In ordeFig 2). Then, the David software was used to enrich DEGs with KEGG. The results showed that 2 of the 5 DEGs were found to be located on the KEGG pathway. However, the enriched KEGG pathway resulted in 0 in the 0.5 h group  ; However, 20 of the 29 DEGs were found to be located on the KEGG pathway and the enriched KEGG pathway resulted in 7 in the 1 h group  ; 16 of the 37 DEGs genes were found to be located on the KEGG pathway with the enriched KEGG pathway result of 1 in the 24 h group  . Finally, STRING and Cytoscape software was used to analyze the protein-protein interaction (PPI) network of DEGs and further module analysis. The results showed that rich protein interactions only existed in the 1 h group and that there were mainly four functional modules: the TNF signaling pathway, the NF kappa B signaling pathway, the I-kappa B/NF-kappa B complex, and the beta interferon production pathway . In order to validate the RNA-seq results, gel electrophoresis was performed to prove the integrity of RNA at this time. Fig 3A showed that the 18S and 28S bands of the target RNA were clear under agarose gel electrophoresis, indicating that the RNA is relatively complete and not degraded. In addition, for some genes a sqPCR analysis was carried out. As a result, the relative expression level of CXCL1 could be shown to be significantly increased in the 0.5, 1, and 24 h group . Because CXCL1 was up-regulated in the DEG analysis and is known to be involved in cellular proliferation in GO gene annotation , CXCL1 turned out to be the most noteworthy target gene.Furthermore, the DEGs were enriched for GO function and KEGG analysis. The number of GO terms mapped resulted in 18, 325, and 461 in the 0.5, 1, and 24 h group, respectively , we firstly analyzed the correlation between CXCL1 expression and the overall survival of GBM patients. We found that CXCL1 was negatively correlated to the OS of GBM patients . In addition, we found that the expression of CXCL1 was up-regulated in GBM tissues compared to normal tissues . To explore the expression and secretion of CXCL1, protein and supernatant from AST and C6 cells were extracted. AST cells were obtained by isolation and purification of rat primary cells with a positive GFAP rate in the cytoplasm of higher than 95% . Western blot and ELISA results showed that the CXCL1 protein expression level and the supernatant content in C6 cells were significantly higher than those in rat primary astrocytes, respectively . These results demonstrated that CXCL1 was up-regulated in C6 cells and GBM.We used RNA-seq combined with bioinformatics technology to screen CXCL1 as a GDNF-induced proliferation-related gene which could not be confirmed by the experiments. Present studies show that CXCL1 acts as a carcinogen in glioma which promotes the proliferation and migration of glioma cells . TherefoFig 5A and 5B). The CCK-8 assay results showed that the OD450 value of the CXCL1-RNAi group was lower than that of RNAi-vector group . The above results suggest that knockdown of CXCL1 inhibits the proliferation of C6 cells.To further validate the effect of CXCL1 on the proliferation of C6 glioma cells, we constructed a lentiviral vector with an interfering RNA (RNAi) targeting the rat CXCL1 gene and which was used to infect C6 cells. Western blot results showed that the expression level of CXCL1 was significantly down-regulated in the CXCL1 knockdown group (CXCL1-RNAi) compared to the control group (RNAi-vector) .In order to detect the effect of exogenous GDNF on CXCL1, we used a double luciferase reporter gene activity assay to detect the effects of GDNF on the CXCL1 promoter region. The results showed that compared to the CXCL1 group, the CXCL1 promoter activity increased significantly after GDNF treatment .To validate the effect of GDNF on CXCL1, exogenous GDNF (40 ng/mL) was added to C6 cells and the CXCL1 mRNA level was analyzed by sqPCR. In addition, cell supernatants were collected and the secretion level of CXCL1 was detected by ELISA. The sqPCR results showed that GDNF could promote the expression of CXCL1 and ELISA results showed that GDNF could significantly promote the secretion of CXCL1 in C6 cells . These results demonstrate that CXCL1 mediates the proliferation of C6 cells after exogenous GDNF administration.In order to prove that CXCL1 is able to mediate the proliferation of C6 cells after GDNF-induction, C6 cells were infected with a CXCL1 knock-out lentiviral vector. After administration of exogenous GDNF (40 ng/mL) to the C6 cells, CCK-8 was used to detect the cell activity of each group. Compared to the control group, the activity of the C6 cells in the CXCL1-RNAi group was decreased. Moreover, compared to the GDNF treatment group, the C6 cell viability was decreased in the CXCL1-RNAi and the exogenous GDNF co-treatment group . In most tumors, NF-κB is abnormally activated and regulates various properties of cancer cells, including proliferation, invasion, inhibition of apoptosis, and angiogenesis [Fig 7B).In order to explore the possible mechanism of the GDNF promoted C6 cell proliferation via promotion of the CXCL1 gene transcription, we used a Qiagen online software to analyze the transcription factor (TF) that initiates the CXCL1 transcription at its corresponding binding sites (BS). The results showed that the most relevant transcription factors were NF-κB, NF-κB1, STAT1, STAT1 α, STAT1 β, C/EBP α, C/EBP β and P53, in which the TF-BS positions of NF kappa B on the CXCL1 gene promoter were NC_74723660, NC_74735035, and NC_74735036, respectively . These results suggest that GDNF promotes NF-κB nuclear translocation and identified an interaction of CXCL1 with NF-κB.To clarify the effect of GDNF on nuclear translocation of NF-κB in C6 cells, we used a Western blot analysis. The results showed that the expression level of NF-κB in the GDNF group was higher than that in the control group. Additionally, we performed immunofluorescence stainings and found that exogenous GDNF promoted the translocation of NF-κB to the nucleus . Western blot results showed that the expression level of CXCL1 protein in the BAY 11–7082 treated group was significantly lower than that of the control group. Compared to the GDNF group, the expression level of CXCL1 protein was decreased in the BAY 11–7082 and the exogenous GDNF co-treatment group . These results suggest that NF-κB is able to mediate the effect of exogenous GDNF on CXCL1.In order to further study the role of NF-κB in the proliferation of C6 cells induced by GDNF, we used BAY 11–7082, a NF-κB specific inhibitor that decreases the phosphorylation level of IκB-α. Here, it was observed that C6 cells may tolerate up to 10 μM of BAY 11–7082 in the MTT assay, and the expression of NF-κB in nucleus was suppressed in the BAY 11-7082-treated cells . These results demonstrate that the GDNF enhanced CXCL1 promotor activity can be inhibited by BAY 11–7082.Previous experimental results showed that exogenous administration of GDNF enhanced the activity of the CXCL1 promoter. In order to further clarify the role of NF-κB in this process, we used a double luciferase reporter gene assay. The results indicated that compared to the CXCL1 group, the CXCL1 promoter activity was significantly decreased after treatment with BAY 11–7082, suggesting that BAY 11–7082 is able to inhibit CXCL1 promoter activity. Compared to the GDNF group, the CXCL1 promoter activity was also significantly down-regulated in the BAY 11–7082 and the exogenous GDNF co-treatment group . These results indicate that NF-κB mediates the exogenous GDNF-induced proliferation of C6 cells.Furthermore, we investigated the effect of GDNF on the proliferation in rat C6 glioma cells. CCK-8 results showed that the proliferation ability of C6 cells in the BAY 11–7082 group was lower than that of the control group. Compared to the GDNF group, the proliferation ability of C6 cells in the BAY 11–7082 and GDNF co-treatment group was inhibited  may be a membrane receptor that mediates the proliferation of C6 glioma cells after exogenous GDNF administration . This prIn order to clarify the GDNF gene target responsible for promoting C6 glioma cell proliferation, we employed RNA-Seq analysis. Comparing the distribution of DEGs in the 0.5, 1, and 24 h group with the 0 h group, the results suggested two common up-regulated DEGs: AABR07024908.1 and CXCL1. Since AABR07024908.1 has not been annotated in the existing gene annotation system and CXCL1 was related to cell proliferation (GO: 0008283) in GO gene annotation, we hypothesized that CXCL1 may be the most noteworthy target gene.Fig 4). To further verify the effect of CXCL1 on the proliferation of C6 glioma cells, we constructed an interfering RNA (RNAi) lentiviral vector targeting the rat CXCL1 gene. The CCK-8 kit experiments showed that knockdown of CXCL1 inhibited the proliferation of C6 cells . In addition, by sqPCR and ELISA we demonstrated that exogenous GDNF was able to increase the expression and secretion of CXCL1 in C6 cells . Compared to the GDNF-treated group, the viability of C6 cells was decreased in the co-treatment group of exogenous GDNF and CXCL1-RNAi . Therefore, CXCL1 indeed mediated the effect of exogenous GDNF on C6 cell proliferation.Studies have shown that the stimulation of GDNF-RET in immune cells was able to cause the up-regulation of the chemokine CXCL1 . In ordeFig 7B) and that exogenous GDNF promoted the translocation of NF-κB into the nucleus .How does GDNF mediate the transcriptional regulation of CXCL1? By using the Qiagen online software tool, we tried to shed some light on this by selecting the transcription factor NF-κB as a target. We could show that NF-κB existed in almost all animal cell types and was involved in the response of cells to stimuli such as stress, cytokines, free radicals, heavy metals, ultraviolet radiation, oxidative LDL, and bacterial or viral antigens . It has Fig 8A and 8B). Next, we studied the role of NF-κB in the proliferation of C6 cells promoted by GDNF. The results showed that the enhancement of CXCL1 promoter activity by GDNF could be inhibited by BAY 11–7082 . Finally, CCK-8 experiments revealed that compared to the GDNF treatment group, the proliferation of C6 cells was reduced in the BAY 11–7082 and GDNF co-treatment group . It is suggested that NF-κB mediates the proliferation of C6 cells after exogenous GDNF administration. Thus, we hypothesize that NF-κB mediates the proliferation of C6 cells by exogenous GDNF administration via interaction with the promoter region of the CXCL1 gene, thereby promoting its transcriptional activity.To further study the role and mechanism of NF-κB in GDNF-induced CXCL1-mediated C6 cell proliferation, we used BAY 11–7082, a phosphorylation inhibitor of NF-κB. Compared to the GDNF treated group, the expression level of CXCL1 protein was lower than that in the BAY 11–7082 and the GDNF co-treated group  The sequenced Q-value box diagram. The X-axis was the base position in reads, and the Y-axis represented the mass value of all bases. The green, yellow and red backgrounds represented the high, reasonable and low quality of the numerical area respectively. The blue line represented the average value of the quality value. (B) Base distribution map of sequencing. The X-axis was the position of each base in reads, and the Y-axis was the proportion of each base. Red: T; Blue: C; Green: A; Black: G. (C) Saturation analysis diagram of four samples groups. X-axis was the sequencing depth (unit: thousands of reads); The Y-axis was the percentage of genes covered. NO0_1: GDNF 0 h group; No1_1: GDNF 0.5 h group; No2_1: GDNF 1 h group; No3_1: GDNF 24 h group. (D) Heat map of gene expression correlation of four samples groups. The color depth represented the correlation, and the value was the correlation coefficient.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(A) KEGG function note table. (B) DEGs enriched KEGG table.(TIF)Click here for additional data file.S4 Fig(A) KEGG function note table. (B) DEGs enriched KEGG table.(TIF)Click here for additional data file.S5 Fig(A) The group of exogenous GDNF for 0.5 h. (B) The group of exogenous GDNF for 1 h. (C) The group of exogenous GDNF for 24 h.(TIF)Click here for additional data file.S6 FigP<0.05, ***P<0.001).(A) Cytotoxicity of BAY 11–7082 to C6 cells. (B) Western blot showed that the expression level of CXCL1 in the BAY 11–7082 treated group and the control group. (C) The statistical results of (A). (*(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Starch’s crystalline structure and gelatinization temperature might facilitate or hinder its use. Ball milling has frequently been mentioned in the literature as a method for reducing starch size and as a more environmentally friendly way to change starch, such as by increasing surface area and reactivity, which has an impact on other starch properties. In this study, starch samples were milled for varying durations  and at different starch-to-ball mass ratios (1:6 and 1:20). Microscopy and XRD revealed that prolonged milling resulted in effective fragmentation and a decrease in crystallinity of the starch granules. Increasing milling times resulted in an increase in amylose content. Rheology and thermal studies revealed that gelatinization temperatures dropped with milling duration and that viscosity and thixotropy were directly influenced. The samples milled for 10, 20, and 30 h at a ratio of 1:20 were the most fragmented and upon drying formed a transparent film at ambient temperature, because of the lower gelatinization temperature. Starch ball milling could lead to the use of this material in thermosensitive systems. Starch, a biopolymer and one of the main polysaccharides on the planet, is a natural and abundant molecule extracted from various plants, such as but not limited to cassava, corn, and potatoes . AnhydroStarch’s main components (amylose and amylopectin) have entirely different properties. Amylose, a macromolecule formed by glucose units joined by α-1,4 glycosidic bonds, gives rise to a primarily linear chain with a few long ramifications. Amylopectin, formed by glucose units joined by α-1,4 and α-1,6 glycosidic bonds, creates a smaller, highly branched structure that is organized in a double helix conformation. As a result, amylose tends to retrograde, producing tough gels and strong films, while amylopectin dispersed in water forms soft gels and fragile films ,6. TheseThe scope of starch application in its natural form is enhanced and limited due to its unique structure. For example, when in water and subjected to heating 60–90 °C), it forms a viscous paste that becomes a brittle gel after cooling, which can be desirable or not, depending on the final application. Another example of its unique structure influencing the application is its semicrystalline organization. The compact molecules in the crystalline region inhibit water and other chemicals from accessing and reacting with the molecules in this region, increasing the gelatinization temperature and decreasing the starch chemical reactivity . Several0 °C, it A greener route to modify starch would be the milling process, as it is a process that generates no residues that could harm the environment, unlike hydrolysis, for example, which uses acids and/or organic solvents. Starch milling is widely reported in the literature as a technique for reducing the size of starch granules and ensures higher safety, simplicity, and cost-efficiency compared to other treatments. Ball milling performs the partial breakdown of the starch structure through friction, impact, and shear, caused by the collision of the balls with the starch and the starch with the mill wall, increasing the surface area and reactivity and reducing the granule size of the starch . HoweverAs a result, this research aims to study the thermal and rheological characteristics of these starches by using ball milling processing, which affects the granule surface area, as a greener way to alter starch properties, particularly its gelatinization temperature, and determine the structural changes affected by milling time . Further considerations on possible applications for the obtained starches are discussed.This work used commercially available food-grade corn starch  as feedstock, with a humidity content of 11%.A ceramic ball mill (20 cm diameter) with ceramic balls (1.5 cm) was used with two different starch/ball mass ratios, 1:6 and 1:20 ,12. OperFor scanning electron microscopy (SEM), starch samples were mounted in stubs, covered with a thin layer of gold in a metallic covering device , and visualized under a voltage acceleration of 15 kV in different increments on a Quanta 450-FEG, FEI.Light microscopy analysis was performed to observe the Maltese cross in the starch granules using an optical microscope with polarized light. Samples of starch (0.01 g) were added to 1 mL of a glycerol solution (50% m/v), and 0.5 mL of this suspension was transferred to a microscope slide and covered with a coverslip for visualization under polarized light.® Spectrum Two spectrometer  with a resolution of 4 cm−1, wavelength range from 4000 to 600 cm−1, and 32 scans. The starch spectra were obtained via the attenuated total reflection (ATR) method.The spectra of the starch samples were obtained using a PerkinElmer−1 are sensitive to changes in the starch structure, especially those at approximately 995 and 1044 cm−1, where the first is commonly related to the amorphous structure of the starch and the latter to the ordered structure of the starch [−1 intensity ratio was used to indicate the degree of order in the starch structure.The effect of ball milling on the ordered structure of the starch was also evaluated using FTIR spectra. It has been reported that bands between 950 and 1100 cme starch ,16. TherThe amylose content was determined based on the methodology proposed by Chrastil et al. and Hu et al. ,18, with® TGA 6000 instrument  with temperatures ranging from 50 to 600 °C, a heating rate of 10 °C/min, under a nitrogen atmosphere with a flow of 40 mL/min.Thermogravimetric measurements were performed using starch samples (~10 mg) in a PerkinElmerDSC analysis was performed in a TA Instruments DSC  with a hermetic aluminum pan. The pans were hermetically sealed after being filled with approximately 1.5 mg of the starch sample and 5 µL of distilled water. The temperature ranged from 30 to 100 °C, with a heating ramp of 10 °C/min, and the experiment was carried out in a nitrogen atmosphere.For the polycrystalline samples, the DRX diffraction graphs were produced using a Co tube at 40 kV and 40 mA with a diffractometer, model XpertPro MPD—Panalytical . The sweep speed was 0.5 °/min, and the 2-theta angle ranged from 5 to 40°. Diffractogram deconvolution was performed, and the crystallinity index (CrI), which measures the proportion of crystalline peak regions to all deconvoluted peaks, was determined using the equation below Equation (1).SP and S% were calculated using a modified version of the approach described by Fu et al. and Mandala et al. [W0) of the starch samples. After adding distilled water to achieve a final starch concentration of 2.0% (w/w), the tubes were vortexed at room temperature (25 °C). Finally, the tubes were centrifuged for 15 min at 1006 g. The supernatant was carefully drained off in a petri dish, and the residue weight was noted (pW). The tubes with the residue and the petri dishes with the supernatant were dried in an air oven (105 °C) until constant weight . The swelling power (SP) and the solubility (S%) of the samples were calculated using the equations below Equations (2) and (3):a et al. ,20. A ceRheological studies were conducted in the linear viscoelastic zone of the samples using a Thermo Scientific Rheometer . The samples for gelatinization temperature were prepared by making a 5% (w/w) starch suspension and using a gelatinized starch solution (5% starch in water heated at 90 °C for 10 min), as a 5% gelatinized starch solution is more viscous than water, the starch solution was employed in place of water to reduce the effects of starch precipitation. The samples for viscosity (oscillatory rheology experiment) and thixotropy analysis were prepared by heating to 90 °C for 10 min in a 5% suspension of the starch samples in distilled water.−1 sheer rate and a heating rate of 7.2 °C/min from 30 to 75 °C. The gelatinization temperature was defined as the temperature at which the sample began to undergo a sudden change in viscosity.An oscillatory rheology experiment was performed for 2 min at 25 °C and 1% deformation using a cone plate geometry with a gap of 0.05 mm (C60/1-TiL) to estimate the dynamic viscosity, storage module (G′), and loss module (G″) of the samples. The same measuring geometry was used for gelatinization temperature measurements in a rotational experiment with a 1 s−1 in 120 s and then held constant at 10 s−1 for 30 s before decreasing (linearly) to 0.1 s−1 in 120 s. The areas of thixotropy (defined as work performed for the demolition and reconstruction of a sample inner structure) were computed by adding the areas of certain trapeziums between the “up” and “down” curves.The thixotropy measurements were performed in a rotational experiment utilizing the previously described geometry. The shear rate was increased linearly from 0.1 to 10 sFor the film-forming ability assays, milled starch aqueous suspensions  were prepared at room temperature, subsequently poured into Petri dishes, and then dried for 16 h in a vacuum oven at 30 °C. The resulting films were registered with pictures to observe the effect of milling on the starch film forming capacity at low temperatures.p < 0.05 was considered statistically significant.The statistical analysis was performed using the analysis of variance produced with the software Statistica10 . Tukey’s test was applied to detect significant differences; The microscopic images obtained via SEM showed that the starch granule was fractured throughout the ball-milling process . The effThe most fractured and heterogeneous group of granules was shown by the starch milled using the 1:20 starch/ball ratio. However, the particle size does not always decrease with milling time due to the agglomeration of the damaged starch granules. This agglomeration can be undone by suspending the starch in some solvent if the application of the material calls for it ,14. ThisThe light microscopy analysis of the control starch clearly showed birefringence , which i−1, the C–H stretch band at 2910 cm−1, and the anhydroglucose stretching vibration band at approximately 950 cm−1, all characteristic of starch. No uncharacteristic band appeared, which indicates that the energy involved in the milling process did not cause the addition or removal of any chemical groups [−1 bands was used as an indicator of the ordered structure of the starch [The distinctive starch bands visible in all the observed infrared spectra include the O–H stretch band at 3500–3000 cml groups ,24,25. Te starch . These r−1 is sensitive to the short-range double helix structural alteration [−1 band diminishes as the amorphous stage increases, the 995/1044 cm−1 ratio grew as milling time increased by a total of 9%, indicating a drop in the starch structure order [−1 ratio, indicating a more amorphous structure than the other samples, corroborating the light microscopy analysis (−1 band of the 30 h milled starch (both ratios) in contrast to the control starch, which indicates the increase in the 995/1044 cm−1 ratio and the resulting decrease in the structural order of the starch granules.Changes within this range can be utilized as a disturbance sign of the ordered structure of the starch granule because the spectral area between 1050 and 950 cmteration . Since tre order ,28. The analysis  and the analysis . Figure The amylose content peaked at 20 h of milling for both ratios and then reached a plateau . An incrAs milling time and ratio rise, there is a more visible change in enthalpy, not only does the gelatinization process begin at a lower temperature but less energy is required to gelatinize the milled starch . The peaThe diffractograms of the examined samples are shown in For both ball/mass ratios, the crystalline peaks showed reduced intensity as milling time increased, indicating a decline in structural organization and resulting in a lower crystalline index (CrI), as shown in The starch crystalline structure is broken when it is heated in excess water due to the breakdown of intramolecular and intermolecular hydrogen bonds. Water molecules then establish hydrogen bonds with the exposed hydroxyl groups of amylose and amylopectin, causing granule swelling to rise. The amount of interaction between starch chains within the amorphous and crystalline domains has a major impact on swelling power ,39. MoreDuring temperature and frequency sweep testing of starch suspensions, the dynamic rheometer enables continuous assessment of dynamic moduli and viscosity. The storage modulus (G′) is a measure of the energy stored and recovered from the material every cycle, whereas the loss modulus (G″) is a measure of the energy dissipated or lost per cycle of sinusoidal deformation . When coThixotropy is a property associated with starch gel and paste rheoinstability. Simply put, thixotropy measures how well or poorly a gel or paste may return to its initial conformation after being subjected to a shear rate fluctuation. The greater the thixotropy, the greater the gap between the initial and end states and the higher the rheoinstability of the gel or paste. The thixotropy of the samples decreased as the milling time and ratio increased, reaching low numbers (<2.2) for the 10, 20, and 30 h (1:20 ratio) samples. This behavior occurs because the more-milled samples have smaller granules, and their structure is formed by weaker bonds that can be easily restored once the sheer rate decreases, whereas the less-milled and control samples have larger granules and thus more complex bonds that would take more time to completely restore, if ever ,43,44. AAccording to Goiana et al. and Oliveira et al. ,25, mostA more fractured granule has a larger surface area and is more susceptible to the effects of water. Water can enter the grain quickly, swelling it and causing gelatinization of the starch. This behavior displayed by the milled starch is related to the fracture caused by the milling process. Therefore, milling is an efficient way to reduce the starch gelatinization temperature ,10,11.Ball milling altered the starch granule physical makeup by shattering it. These fractures made it easier for water to access the granule, boosting the granule’s reactivity and decreasing its gelatinization temperature. The 10 h milled starch at a 1:20 starch/ball mass ratio underwent enough physical and gelatinization temperature changes, with the shortest milling time possible, to be used as a thickening agent in thermally sensitive applications, such as the pharmaceutical industry and some food products. However, samples milled at different times can have different applications, such as the 1 h milled starch at a 1:6 mass ratio forming a more viscous gel than the control starch, making it a more efficient thickening agent than the non-milled starch. In addition, this research made a discovery: a process as simple as milling starch can drastically change the gelatinization temperature of starch leading to effects on its rheological properties.


Evidence-based practice (EBP) is the integration of best research evidence with clinical expertise and patients’ values and preferences. Little is known about knowledge, attitudes, and application of EBP among chiropractic students and trainees. Our aims were to (1) examine the feasibility of implementing a new journal club format within a Swiss university chiropractic healthcare education setting, and (2) assess the associations between the new journal club implementation and EBP characteristics among chiropractic students.A before-and-after study was conducted through a newly implemented journal club with 5th and 6th year chiropractic students and postgraduate trainees between 1 February 2021 and 31 July 2021. The journal club was developed based on the “community of practice” and “team-based learning” conceptual frameworks. EBP knowledge, attitudes, personal application, and future use, were assessed with a validated questionnaire. We summarised participant characteristics using descriptive statistics, estimated before-and-after EBP total and subscale scores , and conducted an exploratory subgroup analysis based on journal club attendance .Among 32 eligible students and trainees, 29 participants  were enrolled: 25 (78%) responded to the pre- and 29 (91%) to the post-assessment surveys. Most (80%) were chiropractic students and 20% were postgraduate trainees. Group A consisted of 12 (41%) and Group B of 17 (59%) participants, respectively. We found reasonable feasibility for the new journal club format and our findings were compatible with no difference in before-and-after EBP scores . Exploratory subgroup analyses based on journal club attendance were consistent with our overall findings.Our study suggests that the newly implemented journal club and embedding chiropractic educational research within the journal club were feasible and acceptable. Small before-and-after differences in the EBP subscale scores for knowledge, attitudes, personal application, and future use were observed in chiropractic students and postgraduate trainees. The small study size and short timeframe during a single semester limit potential inferences.The online version contains supplementary material available at 10.1186/s12998-023-00494-0. Journal clubs have been used as a teaching format within academic medicine for almost 150 years . EBP hasWithin the department of chiropractic medicine at Balgrist University Hospital, journal clubs had been used to facilitate exchange between researchers, clinicians, and students. While it was routine for experienced researchers to lead discussions and critically appraise the literature, chiropractic students with clinical interests and less research experience often found this format challenging. Common student feedback, for example, suggested that some students felt isolated and not sufficiently supported during the journal club preparation phase and found the journal presentations intimidating . Similar experiences have been reported in other academic health institutions, with reports of medical journal club content lacking clinical relevance and containing an unnecessarily large focus on biostatistics , 10. GivTo examine the implementation of this new journal club format, we followed Kirkpatrick’s four-level model — a widely recognized method of evaluation of education programs . The objThe department of chiropractic medicine is integrated within Balgrist University Hospital, a leading musculoskeletal (MSK) specialized hospital affiliated with the University of Zurich, in Switzerland. Chiropractic medicine students at the University of Zurich complete a six-year curriculum. To fulfil the academic requirements to become a chiropractor, students must first complete a three-year bachelor’s program in human medicine and a subsequent three-year master’s program in chiropractic medicine. During the last year, students spend 6 months in the outpatient chiropractic polyclinic  and 6 moWe carried out a before-and-after study with 5th and 6th year chiropractic students and postgraduate residents during the Spring 2021 semester, between 1 and 2021, and 31 July 2021. All 5th year chiropractic students of the chiropractic masters study program, and 6th year chiropractic students , as well as postgraduate residents and PhD students, from the chiropractic polyclinic and research, respectively, were eligible and invited to participate.Our study was reported according to the SQUIRE-EDU  guideline  , two PhD candidates with chiropractic clinical experience , a chiropractor and masters of medical education candidate (MH), and a chiropractic postgraduate resident (LH). MH, LH, and LN had first-hand experience of the previous journal club format as former students in the chiropractic medicine program.We implemented the new chiropractic journal club format in February 2021 to bring together chiropractic students, clinicians, and researchers to critically appraise and discuss diverse and relevant clinical research topics. The chiropractic journal club is mandatory for all 6th year students within the chiropractic polyclinic rotation and postgraduate residents. To increase earlier engagement of students, the 5th year students were also invited to join, if interested and able. One month prior to the first session, an electronic needs assessment survey was conducted where potential participants were asked about their various research and clinical interests. Over one study semester , five journal club sessions took place. All journal club sessions took place online via the ZOOM web application due to the ongoing COVID-19 pandemic public health restrictions. During the first session, participants were given a presentation about the definition and goals of EBP, and how to critically appraise a study based on an example using a risk of bias (RoB) tool . FurtherFor the following four sessions, we used ‘community of practice’ and ‘team-based learning’ as conceptual frameworks for the journal club sessions . UndergrKirkpatrick’s model assesses the effectiveness of training programs at four levels: (1) reaction of the participant to the training program, (2) learning of professional knowledge or skills, (3) change of behaviour or performance, and (4) results on an organizational level .Level 1 (reaction) was measured after the last journal club using two questions — “Which aspects of the chiropractic journal club were helpful?” “Which aspects of the chiropractic journal club do you think need improvement?” — requesting participant feedback to qualitatively gauge feasibility, acceptability, and areas for improvement of the journal club. Participants had the opportunity to raise three points that were helpful and three points that could be improved.Levels 2 (learning) and 3 (behaviour) were assessed using an EBP questionnaire that has been previously validated in various healthcare teaching and learning contexts in different health professional students –25. The Level 4  was not evaluated in this preliminary feasibility study. Immediately after each session participation was recorded through a short questionnaire and confirmed with the participant list extracted from the ZOOM video conferencing application. All data were captured via an automatically secured online data collection system  using electronic questionnaires.Descriptive statistics were used to describe participants’ characteristics. We summarised continuous data using means and standard deviations, or medians and interquartile ranges (IQR), as appropriate. Total scores and EBP subscale scores  of the EBP questionnaire were indicated as raw scores and calculated as percentages and presented as medians and IQRs. This was performed to provide a better understanding of the differences between the four subscales in our study population before and after the implementation of the new journal club format.A prespecified exploratory subgroup analysis was conducted based on actual participant journal club attendance during the semester . Additionally, we reported item-, subscale-, and full scale-level findings of the EBP questionnaire in our study population, including psychometric properties , 27 suchResponses to Level 1  questions were grouped and presented thematically. Answers that were provided in German were translated into English for thematic analysis by native German and English speakers. Data were extracted from REDCap into R  for processing and analysis .Between 4 and 2021 and 12 February 2021, 32 students and trainees  were identified as eligible for our healthcare education feasibility study and were invited to participate. 25 (78%) students completed the ‘before’ survey, and 29 (91%) the ‘after’ survey. Most of the study participants (79%) were female, with a mean age of 26 ± 4 years. 35% (n = 10) were in the 5th year of the chiropractic study program, 45% (n = 13) in 6th year, and 21% (n = 6) were postgraduate trainees. Group A (3–5 sessions attended) consisted of 12 participants, and 17 participants made up group B (≤ 2 sessions attended). Table Our findings were compatible with no difference in before- and after- overall EBP scores following journal club implementation . Small before-and-after within group differences were found for the EBP subscale scores of knowledge, attitudes, personal application, and future use , with example representative participant responses, were:Working in a team: e.g., “Grouping with a more advanced fellow”; “Discussion with a fellow”.Using risk of bias assessment tools: e.g., “Working with appraisal tools”; “Working with paper-checklists”.Discussing clinically relevant topics: e.g., “Talking about practice”; “Interactive discussion”.The three most common “room for improvement” themes  were:More time for discussion: e.g., “More time to discuss”; “Lack of time”.Introduce risk of bias tools earlier in the program of chiropractic studies: e.g., “In 5th year already using quality assessment tools”.Preference for in-person journal club rather than online: e.g., “It should be in person not online”; “Active discussion was hard online”.Our preliminary before-and-after study provides an initial understanding of the associations between a newly implemented journal club and EBP knowledge, attitudes, and behaviours among chiropractic students and trainees over one academic semester. Overall, the new journal club format was feasible to implement and acceptable in a chiropractic educational setting and our findings were consistent with small before-and-after differences in the EBP factor scores for knowledge, attitudes, personal application, and future use. In addition, our study demonstrated the feasibility and acceptability of embedding chiropractic educational research within a newly implemented journal club.This study achieved an acceptable participation proportion, with 78% (before) and 91% (after) of eligible students completing the surveys. Although the journal club was feasible to implement, all participating 5th year students were present at 2 or fewer journal club sessions. This poor participation of the 5th year students is likely the result of scheduling conflicts and the optional nature of the journal club for this group students. In contrast, 6th year students within the chiropractic department, for whom the journal club was mandatory attended 3–5 times (Group B). Our more novel journal club methods, such as the involvement of students in the design and study selection phase, were meant to promote participation and engagement. However, these methods alone may not be enough to entice student participation. Educational institutions which offer journal clubs may benefit in incentivising students for participation and providing time necessary for attendance.Our study found an increase in the EBP domains of knowledge and future use over the course of a semester for participants who attended 3 to 5 sessions (group A) when compared to participants who attended 2 or fewer journal club sessions (group B). A similar increase in the EBP knowledge score was found in the original validation study of the Johnston questionnaire . This inThe subscore of EBP attitudes in our full study sample remained unchanged after the completion of the chiropractic journal club. These findings are similar to Cheng and colleagues , who comIn an international sample of clinicians, Shi et al. showed that the knowledge subscore can trend towards a celling effect  in a diverse group of physicians, nurses, occupational therapists, physical therapists, and psychologists. We found similar results in this sample of chiropractic students, with 4 out of 5 knowledge subscale items demonstrating a ceiling effect. As discussed by Shi et al., the ceiling effects may be a result of the high priority clinical education settings now place on EBP teaching . In addiOverall, the small differences observed between the before and after measurements generates some testable hypotheses. First, participants’ attendance (or lack thereof) in journal club sessions may have played a role. Notably, participants in Group A (more attendance) showed higher scores in both the knowledge and future use subscales, whereas Group B (less attendance) had no change in these two subscales. Second, we acknowledge that explicit teaching about EBP concepts and values occurred only during the first journal club session. It is plausible that journal club sessions that integrate critical appraisal of research reports with more content on EBP concepts, behaviours, and values could potentially yield more meaningful improvement in the knowledge, attitudes, personal application, and future use of EBP.Our course evaluation according to Kirkpatrick’s model showed that the team-based learning approach, introduced risk of bias assessment tools, and interactive clinically relevant discussions were highly valued by the students and trainees. The most frequently mentioned point for improvement was that the course should be held on-site and in-person rather than online. The journal club was initially planned to be fully in-person sessions throughout the Spring semester, but later changed to an online format due to rapidly changing COVID-19 public health guidance. This feedback was implemented the following semester, as the chiropractic journal club moved to a hybrid format.In summary, an undergraduate student-led, team-based journal club format was shown to be feasible and highly valued by chiropractic students. Based on student feedback, the journal club will be repeated with slight modifications, but always with the long-term goal to promote EBP and inspire lifelong learning in chiropractic students. To improve behaviours of EBP personal application, future journal club sessions may select studies not only through student interest, but also in the context of common patient cases seen at Balgrist University Hospital. It may also be prudent for follow-up EBP questionnaires to be provided to participants earlier and not coinciding with student examinations.A strength of our study is the methodology’s grounding in established conceptual frameworks, including ‘community of practice’ and ‘team-based learning’ for the journal club intervention and ‘Kirkpatrick’s model’ for the measurement of EBP. This study is one of only a few investigating EBP in chiropractic education and helps to fill an important knowledge gap.Our study has important limitations. We acknowledge that our study is a small, exploratory feasibility study that used an uncontrolled before-and-after design, which limits inferences that can be made about the effects of the new journal club. It is therefore uncertain whether our findings are a result of the journal club intervention, regression to the mean, or other factors that may have influenced EBP knowledge, attitudes, and behaviours over the course of the study semester. Our results on journal club implementation and EBP outcomes should be considered preliminary evidence and interpreted cautiously.Given the paucity of research on the use of journal clubs in chiropractic education, future studies should focus on developing larger student cohorts with lengthier follow-up periods to better assess outcomes related to EBP. In addition, it would be important to consider outcomes at Level 4 of Kirkpatrick’s model. This level would assess the impact of the new journal club as a healthcare education improvement intervention at the level of the organization. However, the ability to determine which specific outcomes and organizational impact occurred due to student participation in a new journal club format make measuring results at the organizational level quite challenging. Conceivable metrics on the educational program level might be, for example, the proportion of students that get involved in research projects, the proportion of students that choose to write a nonmandatory clinical research dissertation , or student performance in EBP-related topics of the final federal exam for full licensure in Switzerland. By including Level 4 evaluation in a future study, it would be possible to provide a more comprehensive understanding of the impact and effects of the assessed healthcare education improvement intervention. Furthermore, there is a need for randomized controlled trials in the larger medical education space to assess the effectiveness of journal clubs on the beliefs towards EBP in attending participants.Our study suggests that embedding chiropractic educational research within a newly implemented journal club is feasible and acceptable. Small before-and-after differences in the EBP factor scores for knowledge, attitudes, personal application, and future use were observed in chiropractic students and postgraduate trainees. Causal inferences about the effect of the new journal club on EBP knowledge, attitudes, personal application, and future use should be avoided.Below is the link to the electronic supplementary material.Additional file 1: SQUIRE-EDU checklist; Additional file 2: Information about each journal club session; Additional file 3: The Chiro Journal Club (Chiro JC): 7 simple steps to success; Additional file 4: Modified Johnston EBP questionnaire; Additional file 5: Item-level and subscale score analysis


Ocular microtremor (OMT) is the smallest of three involuntary fixational micro eye movements, which has led to it being under researched in comparison. The link between OMT and brain function generates a strong rationale for further study as there is potential for its use as a biomarker in populations with neurological injury and disease. This structured review focused on populations previously studied, instrumentation used for measurement, commonly reported OMT outcomes, and recommendations concerning protocol design and future studies. Current methods of quantifying OMT will be reviewed to analyze their efficacy and efficiency and guide potential development and understanding of novel techniques. Electronic databases were systematically searched and compared with predetermined inclusion criteria. 216 articles were identified in the search and screened by two reviewers. 16 articles were included for review. Findings showed that piezoelectric probe is the most common method of measuring OMT, with fewer studies involving non-invasive approaches, such as contact lenses and laser imaging. OMT frequency was seen to be reduced during general anesthesia at loss of consciousness and in neurologically impaired participants when compared to healthy adults. We identified the need for a non-invasive technique for measuring OMT and highlight its potential in clinical applications as an objective biomarker for neurological assessments. We highlight the need for further research on the clinical validation of OMT to establish its potential to identify or predict a meaningful clinical or functional state, specifically, regarding accuracy, precision, and reliability of OMT. Eye-tracking can provide insights into underlying cognitive mechanisms, such as attentional processing  may provide an objective measure for clinical use. First described in 1934, OMT is one of three involuntary eye movements that are present even when the eye appears still  to modern non-invasive technological assessment with eye-tracking devices  the populations previously studied with OMT; (2) instrumentation used to measure OMT; (3) commonly reported OMT outcomes; and (4) recommendations concerning protocol design and future studies.Four electronic databases were searched: PubMed, SCOPUS, Science Direct, and Web of Science. The key search terms in this review were “ocular microtremor” and “ocular micro tremor”. The search was limited to papers published between 1990 and April 2023 full journal articles only and articles written in English language to eliminate the potential for translational errors creating confusion due to complex language used. Studies were considered relevant if they incorporated terminology which contained the search term “ocular microtremor” in the title, abstract, or keywords. Findings were then screened, and any duplicates were removed. The remaining articles were then reviewed with consideration of the predetermined inclusion and exclusion criteria. These studies were then included in the review shown in Table Table Studies that met the inclusion and exclusion criteria were reviewed, and data pertaining to OMT were scrutinized by the reviewer (LG). Article titles and abstracts were also reviewed by a second reviewer (JD), and any discrepancies were resolved by an additional reviewer (RM). Data were extracted and synthesized into table format which were again confirmed by the second reviewer (JD). Key information was extracted from the articles, including demographics, sample size, control samples, and methods used. Study inclusion and exclusion criteria were also analyzed.Figure n = 16) investigated healthy individuals (n = 8)   (Brown n = 1)   . The participants included in the reviewed studies were male dominant,  with participants ranging from 17 to 88 years old.The reviewed articles 2) Brown , individ Brown , n = 10). When they were included, history of neurological trauma or ocular disease was frequently identified as a source for exclusion (n = 6), as was presence of medication which might affect brain stem function (n = 2). Age was also identified as a limiting criteria (n = 4) with two restricting the research to 18 + years old     (Brown n = 1)  Brown , and lasn = 11). These were all piezoelectric studies apart from one which used contact lens-mounted accelerometers . Eye retractors were also reported (n = 1). In some studies, head movement was anticipated as a source of noise and so to eliminate this, a headrest or a bite bar was used (n = 5). Recordings were taken over various time periods with some studies taking serial readings and some taking continuous recordings for up to 1 min. Where reported, OMT measurement was collected monocularly. If both eyes were tested, this was done independently.Table In all studies, the only reported outcome measure was OMT frequency. In healthy participants, the reported frequency ranged from 78.27 Hz (± 3.9 Hz)  the populations previously studied with OMT; (2) instrumentation used to measure OMT; (3) commonly reported OMT outcomes; and (4) recommendations concerning protocol design and future studies. Current methods of quantifying OMT were reviewed to analyze the efficiency and reliability of OMT as a measure. This review found that OMT frequency differs between different populations and possesses major clinical potential. However, the current methodologies are highly invasive and limit the clinical applicability of OMT measurement.n = 8–214). Reviewed studies with larger samples involved healthy participants, those under general anesthesia or brainstem death. Smaller sample sizes were seen for neurological cohorts, such as PD and MS, which will impact reported data and reduce results generalizability. PD and MS are heterogeneous conditions and therefore larger sample sizes are required to reflect the populations adequately. For example, some participants experienced blepharospasm (a painful spasm of the eyelids) when the eyes were held open with tape or instruments . Overall, regardless of group the OMT frequencies range between 70 and 150 Hz, in line with previous reports (Bolger et al. This is the first review of OMT measurement that has shown that protocols vary between studies and that very few studies have examined clinical cohorts. OMT is a quantifiable involuntary eye tremor that can be measured with various technologies, with more recent devices being non-invasive. OMT is generally reduced in those under anesthesia, or with neurological conditions, and is absent in those with brain stem death. Further quantification of OMT is needed to determine the effect of specific clinical conditions on its frequency, and aid in the development of further OMT outcome measures. Moreover, further research on the clinical validation of OMT is required to establish its potential to acceptably identify or predict a meaningful clinical or functional state, specifically regarding accuracy, precision, reliability, and validity of OMT.


Hepatitis E Virus (HEV) is a major cause of acute and chronic hepatitis. The severity of HEV infection increases manyfold in pregnant women and immunocompromised patients. Despite the extensive research on HEV in the last few decades, there is no widely available vaccine yet. In the current study, immunoinformatic analyses were applied to predict a multi-epitope vaccine candidate against HEV. From the ORF2 region, 41 conserved and immunogenic epitopes were prioritized. These epitopes were further analyzed for their probable antigenic and non-allergenic combinations with several linkers. The stability of the vaccine construct was confirmed by molecular dynamic simulations. The vaccine construct is potentially antigenic and docking analysis revealed stable interactions with TLR3. These results suggest that the proposed vaccine can efficiently stimulate both cellular and humoral immune responses. However, further studies are needed to determine the immunogenicity of the vaccine construct. Hepatitis E virus (HEV) is a common cause of acute hepatitis and is mainly transmitted through contaminated food and drinking water. It has been estimated that almost 20 million HEV cases, 3.4 million symptomatic infections, and 70,000 deaths occur annually in the world .Endemic Although scientists are constantly working on developing an HEV vaccine during the last several decades, an effective HEV vaccine, either inactivated or live-attenuated, has not been produced due to lack of effective cell culture systems for robust HEV replication . FortunaImmunoinformatic analysis has played an important role in determining the immune response of various antigenic regions by using computational techniques and resources. This allows the selection of epitopes from the viral and bacterial genomes. The ideal epitopes can be taken as potential vaccine candidates to trigger effective immune responses in the hosts. The immunoinformatic-based multi-epitope vaccine candidate can stimulate both cellular and humoral immunity . Targete+ T cell response in mice and human cells. Flunisyn™ or FP-01.1 is a chemically synthesized multi-epitope vaccine that also produces CD4+ and CD8+ T cell responses in mice, when compared with native peptides [Previously, numerous immunoinformatic methods have been used to develop vaccines against various pathogens. The vaccine produced by conventional methods can increase the potential for allergic reactions. However, the epitope-based vaccine might be able to overcome these limitations. In addition, this method is rapid, easy, and cost-effective when compared with other vaccine production methods. Multimeric-001 (M-001) is an example of an epitope-based vaccine that induced humoral and cellular immune responses and is under clinical trials  FLU-v ispeptides . These s+, CD8+) and B cell responses. The HEV whole genome is composed of three open reading frames (ORFs). Among them, ORF2 encodes the major structural protein and has the highest sequence identity. Therefore, HEV capsid protein (ORF2) is the main target for neutralizing antibodies, and most of the experimental vaccines against hepatitis E are based on this antigen [An ideal HEV multi-epitope vaccine should stimulate effective T cell ;Screening of experimentally-validated antigenic regions of conserved HEV ORF2 regions through data mining;Prediction of T cell, IFN-γ and B cell epitopes;Screening of overlapping T cell, IFN-γ and B cell, epitopes and experimentally-validated ORF2 antigenic regions;The fusion of immunogenic, antigenic epitopes with appropriate linkers and adjuvant;Validation of the proposed vaccine construct (Vu) structure and interactions.The present study was divided into six key parts SelectioWe retrieved 53 ORF2 sequences of HEV genotype 1, 133 of genotype 3, and 105 ORF2 sequences from genotype 4 from the GeneBank. All leading genotypes of the HEV  were included in the study. The sequences of the HEV ORF2 region were aligned using BioEdit and CLC Workbench 7  to produAn extensive literature search was performed to screen out all experimentally validated epitopes or antigenic regions in HEV ORF2. Studies evaluating the role of different antigenic regions of ORF2 were considered, and each antigenic region was detailed for further evaluation of selected T cell epitopes. http://tools.iedb.org/bcell/ (last accessed on 22 July 2022)), was utilized. We applied highly restrictive thresholds and screen out epitope sequences which overlapped with experimentally validated epitopes in the IEDB. Furthermore, for the prediction of IFN-γ epitopes, the IFNepitope (http://crdd.osdd.net/raghava/ifnepitope/ (last accessed on 22 July 2022)) server was used. This server predicts the results of the amino acid composition of experimentally validated IFN-gamma-inducing epitopes.MHC I, and MHC II-based T cell epitopes, B cell epitopes, and IFN-γ inducing epitopes were predicted by using the consensus sequence of selected genotypes. HLApred  was usedAll T cell epitopes overlapping with the prophesied epitopes of IFN-γ and B cell were prioritized. The selection was extended to prioritize overlapping epitopes lying within conserved regions and experimentally validated antigenic regions. Screened epitopes were then again checked for their antigenicity using VaxiJen  with a cBLASTp  was usedIn this study, we adopted strict criteria for the prediction of a multi-epitope vaccine construct against HEV. Linkers, such as AAY, GPGPG, and GGGGGS were added between prioritized epitopes to achieve better expression levels, solubility, and the perfect folding of multi-epitope structures. Each linker was either added individually adjacent to each epitope or in combination with other linkers. Their antigenicity and allergenicity were further verified with different combinations by VaxiJen  and Allehttp://mordred.bioc.cam.ac.uk/~rapper/rampage.php (accessed on 15 August 2022).PSIPRED  software+ and Cl− ions using GROMACS tool Genion [Molecular dynamic simulations were run against predicted vaccine constructs using GROMACS at 10 ns for 50,000 steps. This program provides a realistic cellular environment to the given biological model. In a dodecahedron box, the vaccine construct was enclosed, and an Optimized Potential for Liquid Simulation force-field (OPLSA) was employed. Spc216.gro water was used as a solvent to simulate all proteins. The overall charge on proteins was calculated and neutralized by addition of Nal Genion . For docl Genion . Furtherl Genion .Conserved regions of virus genomes are considered one of the potential regions for vaccine design. Therefore, we used a conserved region HEV genome to design a HEV universal vaccine construct (Vu) against common genotypes  and performed conservation analysis. CLC workbench  and BioEThe consensus sequence of selected genotypes  was used to predict 9-mer T cell (MHC class I and class II) epitopes. In this study, the T cells with binding affinity to the maximum number of human alleles were prioritized. A total of 71 MHCI epitopes and 32 MHCII unique epitopes against all major alleles were identified. IFNepitopes and IEDB servers predicted 100 IFN-y epitopes and 20 mer B cell epitopes. Furthermore, 48 MHC-I and II epitopes overlapped with the IFN-y and B cell epitopes, and conserved regions were prioritized. BLASTp  indicateIn this study, all possible antigenic regions and epitopes of HEV ORF2 regions were explored and validated with experiments. Different words were used for exploring antigenic regions of HEV . By usinAll prioritized 41 epitopes were linked by flexible linkers . Each linker was added between the epitopes, their allergenicity and antigenicity were further checked with VaxiJen and AllergenPro. We also checked different combinations of linkers between MHCI and MHCII epitopes for their antigenicity and allergenicity . We founPSIPRED was used to predict the 2D structure of the Vu . It showThe vaccine construct showed peculiar characteristics. The charge on the protein was +10. Therefore, ten chloride ions were added by substituting water molecules at atoms 23212, 49258, 40630, 141907, 144796, 31747, 57016, 14941, 38455, and 135937. The energy minimization was achieved at 50,000 steps. The potential energy was −2.714397e + 6, and the average potential energy of the system was −2.04264e + 06, with a drift of −255,072 KJ/mol. The temperature of Vu was maintained at around 300K. RMSD of the protein is shown in HEV is a fecal-orally transmitted foodborne pathogen that causes acute hepatitis in humans and emerged as an important public health concern in the world . HEV has+ cytotoxic T lymphocytes will be helpful in developing vaccines with a more specific immune response. The selected 41 epitopes were revealed to be non-toxic, non-allergen, and impotent of initiating autoimmune responses, which represents their potential as effective vaccine candidates. The screened epitopes were further connected with different linkers GPGPG, AAY, GGGGGS, and the best combination with high antigenicity was selected as a vaccine candidate. The EAAAK linker, a helical protein, was also added between β-defensin and the combined epitopes. The vaccine candidate was predicted by I-TASSER and was refined by GalaxyRefine and ModRefine. Moreover, the structural validation highlights the high structural integrity of the vaccine construct by showing the maximum percentage (89.3%) of residues in the favorable zone. The Vu was also shown to be a stable entity by predicted RMSD. The effective vaccines of HEV should be proficient in activating both cellular and humoral immune responses in humans. This study proposes a set of immunogenic epitopes that could produce both T cell and B cell immune responses. This could help to develop an epitope-based vaccine against HEV infection. Our proposed methodology in finding immunogenic epitopes involves the identification of overlapping regions of T cell, IFN-y, and B cell epitopes from ORF2 of HEV, mainly at those spots where these epitopes are present in reported HEV antigenic regions. Based on the results, we have shortlisted 37 epitopes of the consensus sequence. These epitopes have T cell epitopes overlapping B cell. IFNy epitopes thus can stimulate T cell, B cell, and IFNy responses, simultaneously. IFN-gamma is the signature cytokine of adaptive and innate adaptive immune systems, consequently epitopes having the ability to induce interferon gamma could increase the immunogenicity of any vaccine to combat the pathogen . IFNγ isIt has been revealed by previous studies that a highly glycosylated vaccine has low antigenicity and affords significantly less protection than a low glycosylated protein . DiffereEpitope-based vaccines can only produce a steady immune response if it is effectively recognized by the target immune cell receptors . Toll-liWith the help of various immunoinformatic approaches, we have designed a conserved universal epitope vaccine against major HEV genotypes. This vaccine may have the ability to stimulate both humoral and cellular immune responses and can tackle the emerging RNA mutations of HEV. The potential immune association between TLR3 and vaccine construct was also observed, presenting it as an ideal candidate against the HEV vaccine. However, further experimental validations are still required to confirm these findings.


Biodegradable interference screws (IFS) can be manufactured from different biomaterials. Magnesium was previously shown to possess osteoinductive properties, making it a promising material to promote graft-bone healing in anterior cruciate ligament reconstruction (ACLR). The purpose of this study was to compare IFS made from magnesium to a contemporary biocomposite IFS.In a porcine model of ACL reconstruction, deep porcine flexor tendons were trimmed to a diameter of 8 mm, sutured in Krackow technique, and fixed with either 8 × 30 mm biocomposite IFS (Bc-IFS) or 8 × 30 mm magnesium IFS (Mg-IFS) in an 8 mm diameter bone tunnel in porcine tibiae. Cyclic loading for 1000 cycles from 0 to 250 N was applied, followed by load to failure testing. Elongation, load to failure and stiffness of the tested constructs was determined.After 1000 cycles at 250 N, elongation was 4.8 mm ± 1.5 in the Bc-IFS group, and 4.9 mm ± 1.5 in the Mg-IFS group. Load to failure was 649.5 N ± 174.3 in the Bc-IFS group, and 683.8 N ± 116.5 in the Mg-IFS group. Stiffness was 125.3 N/mm ± 21.9 in the Bc-IFS group, and 122.5 N/mm ± 20.3 in the Mg-IFS group. No significant differences regarding elongation, load to failure and stiffness between Bc-IFS and Mg-IFS were observed.Magnesium IFS show comparable biomechanical primary stability in comparison to biocomposite IFS and may therefore be an alternative to contemporary biodegradable IFS. Interference screws (IFS) are the most frequently used implants for tunnel aperture fixation of soft tissue anterior cruciate ligament (ACL) grafts . While oA possible alternative material for biodegradable IFS may be magnesium (Mg). It closely resembles the stiffness of human bone and was shown to elicit osteoinductive effects, both in vitro and in vivo, which may facilitate a quicker graft healing in ACL reconstruction , 47, 49.Thus, the purpose of this study was to investigate the biomechanical primary stability of ACL graft fixation with a novel Mg-IFS, and to compare it to a biocomposite IFS (Bc-IFS). We hypothesized that Mg-IFS show equivalent primary stability for ACL graft fixation.Frozen (-20 °C) Porcine knees and lower legs were obtained from a local butcher, who confirmed adequate health and comparable age of all used animals. Magnesium IFS were kindly provided by Medical Magnesium GmbH . All other implants and materials were commercially purchased. Ethical approval was waived by the institutional review board of our institute.FastThread™ biocomposite  IFS  and 8 × 30 mm mm.IF WE43MEO  magnesium IFS  were used (each n = 10). Porcine knees were defrosted at 7 degrees Celsius for 24 h (no repeated freezing and thawing), muscles and soft tissue were removed, and the tibia embedded into an aluminum mount using synthetic resin . In case of visible osteoarthritis, or fracture of the specimen, it was excluded. Deep porcine flexor tendons were prepared from porcine hind feet and trimmed to a diameter of 8 mm and a length of 100 mm, to resemble the thickness of a quadriceps ACL graft typically used in the clinical setting [The following implants were used and compared in this study Fig. : For int setting . The diaFor biomechanical testing, a servo-hydraulic uniaxial testing machine , equipped with a 0 to 20 kN sensor, was used. The mount containing the embedded porcine tibia was fixed to the base of the machine with two clamps. The proximal end of the graft was fixed 20 mm above the tibial joint line to the testing machine using a cryoclamp, simulating the intra-articular part of the graft. Before starting the experiment, the graft was manually pretensioned to 20 N . All conp-value ≤ 0.05 was defined as a significant difference.An a priori power analysis was performed using G*Power 3.1  to calculate the sample size needed for this study . To deteBoth IFS were able to withstand the torques during insertion without breakage. None of the IFS were damaged during insertion into the bone tunnel.After 1000 cycles at 250 N, elongation was 4.8 ± 1.5 mm in the Bc-IFS group, and 4.9 ± 1.5 mm in the Mg-IFS group (Table Mode of failure was tendon pullout in 8 specimens of the Bc-IFS group, and in 7 of the Mg-IFS group. In the rest of the cases, the constructs failed by elongation of the graft, followed by a rupture at the proximal tunnel aperture. However, elongation and load to failure between the different failure modes did not differ. There was no visual slippage at the cryoclamp and no damage to the cortex, or fracture of the bone in any specimen, after load to failure testing Fig. .Fig. 3BiThe most important finding of this study was that IFS made from magnesium have similar biomechanical primary stability, compared to a contemporary IFS made from a biocomposite material. The loads acting on the ACL during a gait cycle and other everyday activities like sitting and squatting were reported to range from 0 to 454 N , 27, 37.Primary stability of magnesium IFS was previously compared to other materials in few biomechanical studies. In a robotic biomechanical study, utilizing cadaveric knee specimen, IFS made from pure magnesium were compared to polylactic acid IFS for ACL graft fixation, in a human cadaveric knee model of ACL reconstruction . No signThe integration of a tendon graft into the bone tunnel is known to undergo different phases during which the graft remodels and attaches to the tendon-bone interface , 36, 45.This biomechanical study has several limitations to mention. Previous studies showed that the porcine knee anatomy and ligament biomechanics are adequately similar to humans, making it a frequently used model to assess biomechanical primary stability of orthopedic implants , 17, 31.Magnesium IFS show comparable biomechanical primary stability in comparison to biocomposite IFS and may therefore be an alternative to contemporary biodegradable IFS.