In vitro dissolution experiment, the Res release of the loaded sample (CMR) showed slowly release behavior and reached about 87% at 36 h. In vitro at the cellular level and in vivo at the animal level experiments demonstrated that CMR could alleviate significantly oxidative stress by reducing level of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and increasing glutathione peroxidase (GSH) level. Additionally, immunofluorescence  and western blot  were used to detect the expression of related factors, which verified that CMR could also reduce inflammation and neuronal apoptosis. These results indicated that CM, as a potential central nervous system drug delivery material, was suitable for SCI treatment.Spinal cord injury (SCI) is a serious central nervous system disease, and secondary injury, including oxidative stress, the inflammatory response and accompanying neuronal apoptosis, will aggravate the condition. Due to the existence of the blood–spinal cord barrier (BSCB), the existing drugs for SCI treatment are difficulty to reach the injury site and thus their efficacy is limited. In this study, we designed chitosan-modified hollow manganese dioxide nanoparticles (CM) for the delivery of resveratrol to help it pass through the BSCB. Resveratrol (Res), a poorly soluble drug, was adsorbed into CM with a particle size of approximately 130 nm via the adsorption method, and the drug loading reached 21.39 ± 2.53%.  Dimethyl sulfoxide (DMSO), phloretin, carboxymethylcellulose sodium, and 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Aladdin Chemical Reagent Co. ROS assay kit, malondialdehyde (MDA) content assay kit, superoxide dismutase (SOD) content assay kit, total glutathione (GSH-Px) content assay kit, and Hematoxylin-Eosin/HE staining kit were bought from Beyotime Biotechnology Co. Anti-HIF-1α, Anti-iNOS, anti-cox-2, anti-IL-1β, anti-IL-10, anti-bax, anti-bcl-2, anti-Cl caspase-3, anti-β-Tubulin, and anti-actin were purchased from Abcam. PC12 cells and BV2 cells were obtained from the cell bank of Chinese Academy of Sciences.Tetraethyl orthosilicate (TEOS), potassium permanganate (KMnO2.2.2) were prepared by the sol–gel method and used as templates  under stirring, ultrasonicated for 30 min, and stirred at room temperature. The reaction lasted for 6 h. After centrifugation, the precipitate was obtained. The prepared MnO2-coated SiO2 was dispersed in 2 M Na2CO3 aqueous solution at 60 °C for 24 h to remove the SiO2 template. After centrifugation, the obtained product was defined as HM. HM (500 mg) was dispersed into 100 ml of chitosan solution (1 mg/ml), and the system was stirred for 4 h. After centrifugation, the obtained product was defined as CM.First, solid silica nanoparticles . The zeta potential and particle size of the prepared HM and CM were measured with Zetasizer Nano ZS . The current state of the drug was observed with differential scanning calorimeter ). The crystal structure was measured by X-ray diffraction .2.4.HMR and CMR were prepared by the adsorption method. Res (100 mg) was dissolved in acetone (1 ml). HM or CM (100 mg) was weighed and dispersed into an acetone solution of Res, stirred at room temperature for 6 h, centrifuged, and dried to obtain HMR and CMR. Then, the drug content was determined by UV–Vis spectrophotometry  at 306 nm, and the drug loading was calculated as follows:2.5.Drug release was examined using a shaker . Phosphate buffered saline  was used as a drug release medium. The samples  were added to the dissolution medium (100 ml) and then shaken at 50 r/min in water bath with 37 °C. Next, the release solution (3 ml) was collected at a predetermined time interval for filtration. At the same time, the corresponding volume of PBS was supplemented to maintain a constant release medium volume. Finally, the concentration of Res was measured using UV  at a wavelength of 306 nm. This experiment was performed three times in parallel.2.6.the in vivo safety of HM and CM. PBS was used to wash fresh mouse blood until the supernatant was not red to obtain red blood cells. Red blood cells were then diluted to 2% (V: V) using PBS. After mixing different concentrations of HM and CM  with 2% red blood cells, the mixtures were allowed to stand at room temperature for 3 h and centrifuged at 10,000 r/min for 3 min. Finally, the supernatant (100 μl) was taken into a 96-well plate and detected at 570 nm using a microplate reader . Water and PBS were used as positive and negative controls, respectively. Each sample was analyzed three times in parallel. The hemolysis percentage was calculated by the following formula:Hemolysis tests were used to evaluate A hemolysis rate of more than 5% is regarded as hemolysis.2.7.2.7.1.2 atmosphere and digested with 0.25% trypsin during cell subculture. In addition, FBS supplemented with 10% DMSO was used to preserve cells at −80 °C.PC12 cells and BV2 cells were taken from the Cell Bank of the Chinese Academy of Sciences. The culture medium was DMEM  containing fetal bovine serum (10%), penicillin (100 units/ml), and streptomycin (100 μg/ml) . All cells were cultured at 37 °C in a humidified 5% CO2.7.2.First, to simulate the environment of hypoxia and lack of sugar, the oxygen concentration in the three-gas incubator was adjusted to 1%, and then the medium was changed to Earl’s equilibrium salt solution without glucose. Afterward, PC12 cells and BV2 cells were treated with hypoxia and glucose deficiency for 3 h and then returned to normal culture for 24 h to use for further determination.2.7.3.An MTT assay was conducted to evaluate the cytotoxicity of HM and CM on PC12 cells and BV2 cells. PC12 cells and BV2 cells were seeded into 96-well plates at a density of 5000 cells/well and allowed to adhere overnight. Then, different concentrations of HM and CM  were added to the 96-well plates and incubated for 48 h at 37 °C. Next, MTT aqueous solution (5 mg/ml) was added to 96-well plates and cultured for 4 h in the dark. After that, the supernatant was removed from the 96-well plates, and DMSO (150 μl) was added to each well. Finally, the absorbance values (OD) were measured at 492 nm using a microplate reader  after 10 min of shock at room temperature. The cell survival rate was calculated with the following formula:ODt represents the absorbance of the treated cells, and ODc represents the absorbance of the control cells.2.7.4.CM and HM were labeled with fluorescein isothiocyanate (FITC). First, HM (500 mg) was added to an ethanol solution containing 4% APTES. Then, the suspension was placed in a round-bottomed flask and stirred in reflux at 77 °C for 10 h. After vacuum drying, the obtained product was defined as NHM. Subsequently, NHM (50 mg) was dispersed in 1 mg/mL FITC anhydrous ethanol solution and stirred for 3 h. After centrifugation, the precipitate was vacuum-dried to obtain FITC-labeled HM. Finally, chitosan was coated to obtain FITC-labeled CM.FITC-labeled HM and FITC-labeled CM were used to observe cellular uptake. First, PC12 cells and BV2 cells were cultured in a confocal culture dish until reaching 80% confluence, and then incubated in DMEM containing HM-FITC and CM-FITC (40 μg/ml) for 3 h at 37 °C. After that, the cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton for 15 min, and then incubated with goat serum for 2 h to block nonspecific staining. Next, the primary antibody (anti-β-Tubulin) was incubated overnight at 4 °C. The cells were washed with PBS three times and incubated with secondary antibody  for 2 h. Then, after the cells were washed with PBS, the nuclei were stained with DAPI. Finally, the cellular uptake was observed and imaged by confocal laser scanning microscopy .2.8.2.8.1.All animal protocols were approved by the Experimental Animal Ethics Committee of Jinzhou Medical College for Scientific Research. Ten to twelve-week-old C57BL/6J  mice were used for the experiment and raised in the SPF experimental Animal Center of Jinzhou Medical College. Allen’s method was utilized to establish the model of SCI. In brief, after mice were anesthetized with pentobarbital sodium, the back hair was removed, and povidone iodine was used to disinfect the back. Then, a T9/T10 laminectomy was performed to expose the thoracic spinal cord. Next, a modified impactor  was used to strike the exposed dorsal surface of the spinal cord. Mice in the sham group were subjected to laminectomy without contusion. Mice with SCI were then randomly divided into five groups including Sham group, SCI group, CM group, Res group, and CMR group. CM, Res, and CMR were dispersed in saline solution (5 mg/kg) and injected by the caudal vein (injection volume 100 μl). Among them, the mice of CM group, Res group, and CMR group were given after waking up 1 h. The frequency of administration was once a day and lasted for 7 days. The sham group and SCI group were injected with physiological saline. Penicillin was administered daily to prevent infection, and manual urination was performed twice daily until bladder function was restored.2.8.2.in vivo imaging system  at excitation wavelengths of 518 and 494 nm.FITC-labeled CM and HM were injected into SCI mice via the tail vein. The heart, liver, spleen, lung, kidney, brain, and spinal cord were removed at different time points, and the fluorescence signals were detected using an 2.8.3.Proteins were extracted from the spinal cord tissue of each group (1 cm around the injury site) by RIPA lysis buffer. The protein concentration of each group was quantified with a BCA protein kit. After equal amounts of samples in each group were added to a sodium dodecyl sulfate polyacrylamide gel, SDS-PAGE was utilized to separate the proteins. Then, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% skim milk powder for 2 h and incubated with primary antibodies at 4 °C overnight. The primary antibodies and dilution ratios were as follows: anti-bcl-2 (1:1000), anti-bax (1:1000), anti-Cl caspase-3 (1:1000), anti-IL-6 (1:1000), anti-IL-1β (1:1000), and anti-cox-2 (1:1000). The next day, the secondary antibody was incubated at room temperature for 2 h. Finally, the signal was displayed by the Tanon 5500 gel imaging system . ImageJ software was used to analyze the signal intensity.2.8.4.n = 3). Res, HMR, and CMR  were injected into the rats through the tail vein. Blood was collected at different time points , and plasma was obtained by centrifugation at 3000 rpm. Phloretin solution  as an internal standard was added to rat plasma (200 μl), and then methanol solution (400 μl) was added to precipitate the protein. After swirling for 5 min, the samples were centrifuged for 10 min at 10,000 rpm. The obtained supernatant (20 μl) was analyzed by high-performance liquid chromatography (HPLC) (Shimadzu LC-2030). Acetonitrile and water (70:30) were used as the mobile phase, and the detection wavelength was 306 nm. The pharmacokinetic parameters were calculated using Pksolver version 2.0.Pharmacokinetic (PK) studies were performed using Sprague–Dawley rats. The SD rats (200 ± 20 g) were purchased from the SPF Experimental Animal Center of Jinzhou Medical College. Twelve SD rats were randomly divided into three groups .2.9.5 per well) were seeded into 24-well plates and allowed to adhere overnight. After PC12 cells were treated with OGD for 3 h, CM, Res, and CMR were added to each well and cocultured for 24 h. Then, a ROS fluorescent probe (DCFH-DA) was added to the cells and incubated for 30 min in the incubator. Finally, the production of ROS by cells was immediately observed with a fluorescence microscope.PC12 cells .Similarly, the protein of the spinal cord in different groups was extracted to detect SOD activity and content of GSH and MDA in tissues.2.10.The spinal cords were placed in 10% sucrose solution, 20% sucrose solution, and 30% sucrose solution to remove water and cut into slices after OTC imbedding. Frozen sections of spinal cord were dried at room temperature and then washed three times with PBS. Next, 0.3% Triton X-100 was used to drill for 15 min. After washing three times with PBS, goat serum was employed to block for 2 h. Subsequently, the primary antibody  was incubated with the sections overnight at 4 °C. The next day, the appropriate secondary antibody was added and incubated for 2 h at room temperature. Finally, the sections were stained with DAPI for 15 min and filmed with a FV10I confocal microscope. The fluorescence intensity of each group was analyzed with ImageJ software.Cell samples were fixed with 4% PFA for 30 min, and the rest of the experimental steps were carried out as described above. The used the primary antibodies were anti-β-tubulin, anti-iNOS, and anti-Cl caspase-3. Finally, the cells were imaged with a confocal laser scanning microscopy .2.11.The Basso Mouse Scale (BMS) system and footprint analysis were used to assess the recovery of mice hind limb motor function after SCI. On Days 3, 7, 14, and 28 after SCI, the mice were placed in an open space and observed by two examiners blinded to the experimental design. After 5 min of observation, the motor scores were evaluated, and footprints were collected. The BMS score ranges from 0 to 9 points. Among them, 0 points indicates complete paralysis, and 9 points indicates normal exercise. In addition, the weight of the mice was recorded immediately after the behavioral assessment to preliminarily assess the recovery of the mice in the different treatment groups.2.12.P value was less than 0.05, it was considered to be statistically significant.All data were presented as the mean ± standard deviation (SD), and statistical analysis was performed using GraphPad Prism (version 8.0). The data were analyzed by one-way analysis of variance (ANOVA) to compare the mean difference between groups. When the variance was not equal, the Kruskal–Wallis test was used. When the 3.3.1.The main preparation process of CMR and its mechanism for treating SCI are shown in −1 . This meant that CM could effectively regulate the release rate and release site (rapid release under acidity at the injury site) of Res, which contributed to increased bioavailability. The results of the pharmacokinetic experiment supported this point of view. As shown in t1/2 values of free Res, HMR, and CMR were 0.991 ± 0.981 h, 2.19 ± 0.613 h, and 5.01 ± 0.698 h, respectively. The AUC0-t of Res, HMR, and CMR were 14.4 ± 2.66 μg/ml*h, 4.36 ± 1.99 μg/ml*h, and 0.857 ± 0.172 μg/ml*h, respectively. The AUC0-t of CMR was eightfold greater than that of Res and threefold greater than that of HMR. This indicated that CMR significantly improved the bioavailability of Res. The cytotoxicity test and hemolysis test were used to study the biological safety of CM. MTT results showed that compared with HM, the survival rate of PC12 cells and BV2 cells treated with CM were higher, both reaching more than 95%, indicating that CM can reduce cytotoxicity and has good biocompatibility . The results of the cytotoxicity test hemolysis test and H&E staining showed that CM had good biosafety. Based on these conclusions, CM has potential as a central nervous system drug delivery vehicle.As shown in −1 . Due to −1 . Studies−1 . Based otibility . In addi3.2.In vitro cell and in vivo imaging experiments were conducted to test the CMR’s cellular uptake and the crossing the BSCB ability. The BSCB, as one of the unique barrier structures of the central nervous system, is mainly responsible for restricting and regulating the entry of extramedullary molecules into the spinal cord and maintaining spinal homeostasis  was the strongest and that of the CM group (101.6 ± 3.726 a.u.) and Res group (86.7 ± 2.214 a.u.) decreased successively, among which the fluorescence signal of the CMR group (36.4 ± 3.327 a.u.) was the lowest, indicating that CMR had the best effect on alleviating oxidative stress  was the strongest after OGD induction, indicating that OGD caused BV2 cells to be in an inflammatory state. The relative fluorescence intensity of iNOS in the CM group (0.783 ± 0.0164 a.u.) and Res group (0.581 ± 0.0167 a.u.) decreased sequentially, and the iNOS relative fluorescence intensity of the CMR group (0.481 ± 0.0291 a.u.) was the lowest except for the Sham group, which was only 0.465 times that of the OGD group (in vivo experiments were carried out using immunofluorescence and western blot. The results showed that compared with the SCI group (1.03 ± 0.0540 a.u.), IL-1β expression in the CM group (0.783 ± 0.0471 a.u.), and Res group (0.581 ± 0.0171 a.u.) was decreased, while the expression of IL-1β in the CMR group (0.481 ± 0.0302 a.u.) was the most significantly decreased, which was 0.456 times that of the SCI group (Inflammation plays a major role in the pathological process of secondary injury, as does oxidative stress (Fan et al., GD group . This inCI group . FurtherCI group . Taken t3.5.in vitro immunofluorescence assay was used to verify the anti-apoptotic effect of CMR on neurons. In in vivo immunofluorescence experiments were carried out. The anterior horns of spinal cord were co-stained with Neun/Cl caspase-3. As shown in Both inflammation and oxidative stress induce neuronal apoptosis after SCI, which can affect the recovery of motor function in SCI sufferers (Wang et al., 3.6.To further determine the effect of CMR treatment on the recovery of neurological function after SCI, behavioral tests, body weight analysis, and Nissl staining were performed. The BMS scores from 1 d to 28 d after SCI were shown in 4.In vitro cell and in vivo animal experiments showed that CM could effectively transport Res to the injury site, inhibit oxidative stress, the inflammatory response, and neuronal apoptosis, and promote motor function recovery in SCI mice. Therefore, CMR can provide a new therapeutic strategy for the clinical treatment research of SCI.In summary, this study successfully synthesized a nanoplatform of chitosan-modified hollow manganese dioxide nanoparticles to deliver Res for the treatment of SCI. As the carrier of Res, CM had high drug loading and presented sustained release. Click here for additional data file.


A. baumannii is a common clinical pathogen that often causes pneumonia and bloodstream infections in ICU patients. Sequence types (ST) are used to investigate the distribution and spread of A. baumannii. Biological characteristics such as virulence and resistance may play a role in A. baumannii becoming a specific dominant ST strain. To characterize the biological, genetic, and transcriptomic differences between the DST and non-dominant ST strains in A. baumannii, we performed several biological experiments and genetic, and transcriptomic analyses. The DST group displayed more resistance ability to desiccation, oxidation, multiple antibiotics, and complement killing than the NST group. However, the latter had higher biofilm formation ability than the former. The genomic analysis showed the DST group exhibited more capsule-related and aminoglycoside-resistant genes. Besides, GO analysis indicated that functions involved in lipid biosynthetic, transport, and the metabolic process were up-regulated in the DST group, while KEGG analysis manifested that the two-component system related to potassium ion transport and pili were down-regulated. In short, resistance to desiccation, oxidation, multiple antibiotics, and serum complement killing are important reasons for the formation of DST. Genes related to capsule synthesis and lipid biosynthesis and metabolism play an important role at the molecular level in the formation of DST. Moreoveaumannii .A. baumannii in the hospital setting  guidelines from 2020. Resistance breakpoints determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) were used for polymyxin B.All 2.2Growth curves were performed as described previously with slight modifications  Briefly,2.3Desiccation resistance assays were completed as previously described  Log-phas2.4A. baumannii in the log-phase of growth were adjusted to OD600 = 0.5 and spread evenly on an MH agar plate. A sterile filter paper disk with a diameter of 6 mm was placed in the middle of the inoculated agar, and 10 μl 20% (v/v) hydrogen peroxide was pipetted onto the filter paper. The plates were incubated at 37°C for 18 h, after which the growth inhibition zone around the disk was measured using Vernier calipers.2.5A. baumannii in the log-phase of growth were adjusted to 2 × 106 CFU/ml and mixed with normal or inactivated serum at a ratio of 1:9, respectively. After incubation for 1 h at 37°C, samples were serially diluted and spread on MH agar plates. After overnight incubation at 37°C, bacteria colonies were counted, and the bacterial survival rate was calculated by the following formula: Bacterial survival rate =  × 100%.Blood collected from healthy volunteers was centrifuged and filtered with a 0.22 μm pore size syringe filter to obtain sterilized serum. Half of the serum was inactivated by heating in a 56°C water bath for 30 min. 2.6A. baumannii in the log-phase of growth were adjusted to 0.5 absorbance units (OD600), before 200 μl aliquots were pipetted into the wells of a 96-well plate. Bacteria were cultivated in an incubator for 24 h at 37°C and 5% CO2, to allow biofilm formation, before nonattached bacteria were removed from the wells by aspiration. Biofilms were gently washed with ddH2O, after which the 96-well plate was inverted on absorbent paper for 15 min at room temperature. Biofilm cells were fixed by adding 4% (v/v) paraformaldehyde to each well for 20 min and were then stained with 1% (w/v) crystal violet for 15 min. Biofilms were washed thoroughly with ddH2O, before the bound crystal violet was eluted with 200 μl 95% ethanol, and were then incubated at 37°C for 30 min. The crystal violet eluted from each biofilm was measured colorimetrically at OD570.2.7A. baumannii in the log-phase were adjusted to 1× 107 CFU/ml. G. mellonella larvae weighing ~300 mg was divided randomly into groups, with each group containing 15 larvae. Each larva was infected with 10 μl of the adjusted bacterial suspension, incubated at 37°C, and observed once every 12 h for 3 days. Larval survival status was assessed using the acupuncture method, and no response was considered as death.2.8A. baumannii genomes were extracted using QIAamp DNA Mini Kit , whole genome sequencing was performed by Illumina HiSeq 2500 , and the raw sequencing results were quality-controlled using FastQC v.0.11.5. Trimmomatic v.0.40 was used to trim splice regions. The trimmed reads were assembled and annotated by SPAdes(http://cab.spbu.ru/software/spades/) v.3.6 and RAST(https://rast.nmpdr.org/), respectively. Antibiotic resistance genes and virulence genes were identified using the ResFinder . The raw data were first processed by a Perl script to remove reads with adapters, those containing poly-N and low-quality reads. The sequencing error rate for a single base position should be less than 1% and no more than 6% (2.10A. baumannii MDR-ZJ06 (accession No. CP001937.2) using Bowtie2-2.2.3. Counting the reads numbers mapped to each gene was achieved using HTSeq v0.6.1. Differential expression analysis for the DST and NST groups was performed using the DESeq R package (1.18.0), and p-values were corrected using the Benjamini–Hochberg method to control for false discovery rate  enrichment analysis of DEGs was performed using the GOseq R package. GO terms with 2.12A. baumannii RNA was first stabilized with RNA protection solution  and then extracted and purified according to the instructions of the RNeasy Mini Kit . One microliter RNA was added to nine microliter reaction buffer from the PrimeScript™ RT reagent Kit  in the appropriate ratio, and then reverse transcribed to generate cDNA as follows: 37°C for 15 min, 85°C for 5 sec, and 4°C. cDNA was added to the reaction system of the TB Green® Premix Ex Taq™ , and PCR was carried out as follows: 95°C, 30 s for one cycle (stage 1); 95°C, 5 s, 60°C, 34 s for 40 cycles (stage 2); and 95°C, 15 s, 60°C,1 min, 95°C 15 s (stage 3). The genes and primers used are shown in recA was selected as the internal reference. The relative expression of different genes was calculated by the 2-ΔΔCt method.For analysis of expression of specific genes, 2.13p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.The biological experiments were repeated at least three times, except for the growth curve experiment and anti-desiccation assay, where two biological replicates were done, and the biofilm experiment, where four biological replicates were done.Statistics were performed using prism9. Two groups were compared using the Mann-Whitney t-test, and the five groups were compared using a one-way ANOVA, with horizontal lines representing means ± standard error of means; *33.1p = 0.42).We first compared their growth and there was no difference in the growth rates of strains in the two groups , indicating that the DST strains were more resistant to oxidative stress than the NST strains , than the NST group , middle (36 h), and late (72 h) stages of infection, however, all this difference was not statistically significant  and two genes related to iron acquisition.Twenty-four virulence genes associated with CPS were found only in strains of the DST group. These include glycosyltransferase-related genes A. baumannii, were unique to members of each of the two groups, consistent with the high variability previously reported for the middle region at the K locus -Ia_1 and armA_1 (aminoglycoside resistance), blaTEM-1D_1 (β-lactam resistance), mph(E)_1 (macrolide resistance), and sul1_5  ≥ 2, 128 genes were identified as being significantly upregulated in the DST group, including nine CPS synthesis-related genes, one efflux pump gene, and three T6SS secretion system-related genes. Loci corresponding to gene_ids for all these genes were identified in the genomes of the DST strains being analyzed (2 FoldChange (DST/NST) ≤-2, 53 genes were identified as being significantly downregulated in the DST group, including three genes related to CPS biosynthesis and six genes related to iron uptake , with 492 genes upregulated and 128 genes downregulated, in DST compared with NST, accounting for approximately 15% of all protein-coding genes Figure 4wecB, capD, and galE genes involved in CPS synthesis, as well as the fabB, fabF, and psd genes involved in lipid syntheses. Other functional groups enriched in the DST upregulated genes group included “lipid metabolism process” and “carbohydrate transmembrane transport activity” , which contains three genes, nthesis” Figure 73.5Differences in DEGs associated with virulence and antibiotic resistance were next investigated since these two phenotypes are closely linked to the clinical significance of bacteria and to their survival in the environment.wza/wzb/wzc, etc.), iron uptake , type VI secretion systems , and biofilm formation . In addition, the toxin-antitoxin cdiA/cdiB, RNA chaperone hfq, sensor protein pilS, and the redox-sensitive transcriptional activator soxR were also highly expressed in strains of the DST group  and the pilus regulatory protein gene (PilR). In addition, genes downregulated in the DST group contained genes related to iron uptake, CPS synthesis, potassium transport, chemotactic and competition functions, type II secretion system, and phospholipase synthesis  ≥2) were CPS biosynthesis and T6SS component-associated genes, whereas the downregulated virulence factors genes (log2Foldchange(DST/NST) ≤-2) were iron uptake related. This may be due to the fact that the capsule promotes the survival of A. baumannii both in the hospital setting and in patients. T6SS also contributes to the dominance of A. baumannii owing to its competition with other bacteria  technique was used to identify 50 genes required for survival, in human serum, of a strain of myelitis . SeveralfabB and fabF involved in this pathway exhibit a prolonged effect on fatty acid synthesis, and the knockdown of fabB in E. coli leads to unsaturated fatty acid nutrient deficiency and failure to grow properly  that may influence the evolution of the strains.A. baumannii strains, while CPS biosynthesis, T6SS, and especially lipid synthesis pathway-related genes also play an indispensable role in this process. Our study further revealed the relationship between antibiotic resistance, virulence, and specific dominant ST of A. baumannii, laying a foundation for better prevention, control, and treatment of infections by this organism in the future.In conclusion, tolerance to desiccation, oxidation, and complement killing all play an important role in the evolution of DST https://www.ncbi.nlm.nih.gov/,PRJNA906176.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: XK completed most of the experiments and wrote the first draft of the manuscript. TC and PL contributed in toxicity testing of bacteria analysis. LG and YZ performed the bioinformatics analysis. YX contributed to conception and design of the study and revised the manuscript. All authors contributed to the article and approved the submitted version.


To explore the diagnostic value for chronic kidney disease (CKD) between 640-slice computed tomography (CT) kidney scan and conventional CT scan. n = 60) and the control group (n = 60). Patients in the control group received the conventional CT plain scan and enhanced scan, and for patients in the experimental group, CT plain scan was performed first, the range of 640-slice CT dynamic volume scan was determined, and after bolus injection of contrast agent, dynamic volume scan was performed for scanning in the cortical phase, myeloid phase, and secretory phase. The imaging quality and effective scanning dose were compared between the two modalities, and the relationship between CT values obtained from 640-slice CT scan and conventional CT scan and the renal impairment was analyzed.  A total of 120 CKD patients who received kidney plain scan plus enhanced examination in the CT room of the Medical Imaging Department of our hospital from June 2019 to September 2019 were selected and randomly divided into the experimental group (P < 0.05); the effective radiation doses of the experimental group and the control group were, respectively, (1.89 ± 0.32) mSv and (3.26 ± 0.47) mSv, indicating that the dose was significantly lower in the experimental group than in the control group , and the correlation analysis showed that the relationship between the sum of CT values in the cortical phase of both kidneys and kidney injury in the experimental group was r = 0.835, P < 0.001.  Compared with the control group, the image quality of 640-slice CT scan conducted in the experimental group was significantly better ( Both 640-slice CT kidney scan and conventional CT scan can be used in the diagnosis of CKD. 640-slice CT has a lower radiation dose, better image quality, and higher application value. Chronic kidney disease (CKD) is a collective term for kidney diseases with impaired renal function, and patients mostly present with hallmarks of kidney injury, such as albuminuria and abnormal urinary sediment . In receThis retrospective study was conducted in our hospital from June 2019 to September 2019 to explore the diagnostic value of 640-slice CT kidney scan and conventional CT scan for CKD. It was a double-blind study, meaning that neither the subjects nor researchers understood the trial grouping, and the study designer was responsible for arranging and controlling the full trial.n = 60) and the control group (n = 60). In the experimental group, there were 38 males and 22 females, the patients' mean age was (40.28 ± 5.19) years, mean body mass was (64.98 ± 2.65) kg, and BMI was (22.65 ± 2.14) kg/m2, and in the control group, there were 35 males and 25 females, the patients' mean age was (40.65 ± 5.13) years, mean body mass was (65.05 ± 2.74) kg, and BMI was (22.74 ± 2.23) kg/m2. No significant differences in the baseline data between the two groups were observed (P > 0.05), so these patients could be selected as the subjects.A total of 120 CKD patients who received kidney plain scan plus enhanced examination in the CT room of the Medical Imaging Department of our hospital from June 2019 to September 2019 were selected and randomly divided into the experimental group (2), meaning mild impairment; for CKD stages 2 and 3, GFR = 15–29.9 ml/(min × 1.73 m2), meaning moderate and severe impairment. Patients at stages 4 and 5 of CKD had a serious renal impairment and were not suitable for using the contrast agent, and thus they were excluded from the study. In addition, those who suffered from hearing disorder, language barrier, unconsciousness, mental illness, and other factors, who quit the treatment in the middle of the study, who had other severe organic diseases [All patients met the diagnosis criteria for CKD , had no diseases , who werThe study met the principles of the World Medical Association Declaration of Helsinki (2013)  and was The patients were told to empty the stomach and hold the urine. They were in the supine position and kept smooth breathing, foot first was selected, and the parameters of Toshiba 64-slice CT scanner (NMPA (I) 20063300657) were set as follows: tube voltage of 120 kV, tube current of 310 mA, noise index of 8.63, speed of 0.6 s/circle, slice thickness of 5 mm, pitch of 0.984, and FOV of 36 cm × 36 cm. Low-concentration iso-osmotic non-ionic contrast medium, iodixanol , was administered via intravenous injection through the right median cubital vein by a single-tube pressure syringe, with a total dose of 65 ml and a rate of 3.5 ml/s. After injection, enhanced scans in the cortical phase, myeloid phase, and secretory phase were performed, and the CT values were measured by the workstation.Patients received CT plain scan first; after the range of 640-slice CT dynamic volume scan was determined, Toshiba 320/640 volume CT (NMPA (I) 20143303426) was used to determine the scan position of both kidneys according to the scout view, the detector effective width was ensured to an extent of containing bilateral whole kidneys, and the dynamic volume enhanced perfusion scan was performed with the following parameters: tube voltage of 100 kV, tube current of 50 mAs, rotation time of 0.5 s, collimation of 0.5 mm × 230, and slice thickness of 0.5 mm. Low-concentration iso-osmotic non-ionic contrast medium, iodixanol, was administered via intravenous injection through the right median cubital vein by a double-tube pressure syringe, with a total dose of 65 ml and a rate of 3.5 ml/s. After injection, enhanced scans in the cortical phase, myeloid phase, and secretory phase were performed, and the CT values were measured by the workstation.K value, the dose-length product was generated automatically by the computer, and the K value referred to the European Guidelines on Quality Criteria for Computed Tomography [After finishing the experiment, the experimental data and test data were collected to perform subjective scoring of the images, the effective radiation dose (dose-length product × mography ), signalX2 test and t-test. The differences were considered statistically significant at P < 0.05.In this study, the data processing software was SPSS18.0, and the picture drawing software was GraphPad Prism 7 . The data included enumeration data and measurement data, tested by P < 0.05)  see .P < 0.001).The SNR was significantly higher in the experimental group than in the control group .The CNR was significantly higher in the experimental group than in the control group .The effective radiation doses of the experimental group and the control group were, respectively, (1.89 ± 0.32) mSv and (3.26 ± 0.47) mSv, indicating that the dose was significantly lower in the experimental group than in the control group  HU, (125.65 ± 9.55) HU, and (87.66 ± 9.65) HU, and in the control group, the CT values of patients with mild, moderate, and severe renal impairment in the cortical phase were, respectively, (145.98 ± 9.55) HU, (140.65 ± 9.57) HU, and (102.98 ± 9.57) HU.In the experimental group, the sums of CT values of patients with mild, moderate, and severe renal impairment in the cortical phase were, respectively, (288.98 ± 9.65) HU, (245.98 ± 9.65) HU, and (170.98 ± 9.50) HU, and in the control group, the sums of CT values of patients with mild, moderate, and severe renal impairment in the cortical phase were, respectively, (310.98 ± 9.54) HU, (279.65 ± 9.10) HU, and (200.98 ± 9.55) HU.P < 0.05). Noise and CNR are the important criteria for evaluating the image quality, with higher CNR indicating better image quality. Also, the experimental group obtained a higher subjective score, proving that the image quality of 640-slice CT is more stable. In addition, the effective radiation doses obtained by the experimental group and the control group were, respectively, (1.89 ± 0.32) mSv and (3.26 ± 0.47) mSv, demonstrating that 640-slice CT can reduce the scanning radiation dose while obtaining images with better quality, which is conducive to reducing the adverse risks of ionizing radiation to the patients and presents exact application value.There are many kinds of kidney diseases, and the common ones include calculus, inflammation, tuberculosis, tumors, and congenital dysplasia , 15. Ther = 0.835, P < 0.001, proving significant correlation of the sum of CT values in the cortical phase of both kidneys and kidney injury in the experimental group. Scholars Hori et al. reported that Pearson analysis of CT contrast-enhanced scan correlation measures can similarly yield a correlation between the sum of CT values in the cortical phase of both kidneys and renal injury [Scholars You et al. showed that the blood flow of renal cortex obtained by 640-slice CT after scanning with reduced radiation dose was not significantly different from that of the conventional dose group, indicating that further reducing radiation dose still did not affect the accuracy of 640-slice CT, so this examination modality can effectively reflect the situation of the renal cortex . Renal cl injury , but witIn conclusion, both 640-slice CT kidney scan and conventional CT scan can be used in the diagnosis of CKD. The 640-slice CT has a lower radiation dose, better image quality, and higher application value. Therefore, the study of 640-slice CT kidney scan can be deepened to provide more references for the clinic and increase the application frequency of 640-slice CT kidney scan.


Thus, a new and sensitive RRS-ET method for the detection of SO32– (0.160–5.00 μmol/L) was developed accordingly using AuMOF as nanoprobes, with a detection limit of 0.0800 μmol/L. This new RRS method was applied to determination of SO32– in food and SO2 in air samples. The recoveries of food and air samples were 97.1–106% and 92.9–106%, and the relative standard deviation (RSD) was 2.10–4.80% and 2.10–4.50%, respectively.A stable Au metal organic frameworks (AuMOF) nanosol was prepared. It was characterized by electron microscopy and molecular spectral techniques. In pH 6.8 PBS buffer solution, AuMOF nanoprobes exhibit a strong resonance Rayleigh scattering (RRS) peak at 330 nm. After basic fuchsin (BF) adsorbing on the surface of AuMOF, the RRS energy of the nanoprobe donor can be transferred to BF receptor, resulting in a decrease in the RRS intensity at 330 nm. Both sulfite and BF taken place an addition reaction to form a colorless product (SBF) that exhibit weak RRS energy transfer (RRS-ET) between AuMOF and SBF, resulting in the enhancement of the RRS peak. As the concentration of SO Due to its characteristics of good stability, adjustable pores, large specific surface area, and polymetallic sites, it has been found that MOFs can be used for sensing some analytes such as Htal ions , 2. Hu etal ions  synthesital ions  synthesi32–) and phosphate (PO43–) can react with ammonium molybdate to form silicomolybdate heteropoly acid (SiMo) and phosphomolybdate heteropoly acid (PMo), PMo decomposed by the addition of citric acid, and SiMo/PMo combined with N/Au co-doped carbon dots (CDN/Au) show good RRS analytical properties. With the increase of SiO32–, SiMo/PMo reacted with CDN/Au probe to form large particles, resulting in enhanced RRS intensity at 555 nm. Based on this, Li et al. established a new RRS-ET method to detect SiO32– and PO43– continuously  is a double-edged sword. A large number of studies have proved that excessive intake of SO32– will not only induce emergency and various respiratory diseases in some individuals, but also induce cardiovascular diseases and neurological disease, such as abdominal pain, urticaria, asthma, respiratory lung cancer, myocardial ischemia and brain cancer. In food processing, the oxidation of sulfur dioxide and sulfites can effectively inhibit the non-enzymatic browning; with the reducing and bleaching properties of tetravalent S, it can also be used as a preservative to inhibit the growth of mold and bacteria. Therefore, in the production and processing of food, sulfur dioxide and sulfites are often added to make the food fade and free from browning, improve the appearance quality and extend the shelf life. Once these additives are used in excess and without follow-up sulfur dioxide removal technology, it will inevitably lead to excessive sulfur dioxide residues. This will not only damage the quality of the food, but also seriously affect the health of consumers. Because of its potential toxicity, many countries have strictly limited the amount of sulfites in food. The Joint Expert Committee on Food Additives (JECFA) of Food and Agriculture Oganization (FAO)/World Health Organization (WHO) has assessed the risk of sulfur dioxide as a food additive as follows: the Acceptable Daily Intake (ADI) of sulfur dioxide is 0–0.700 mg/kg (as SO2)  . In addia et al.  developeo et al.  reported methods . For exa methods  used mel methods  prepared°, a model of FEI Talos F200S field emission transmission electron microscope , a model of F-7000 fluorescence spectrophotometer , a model of TU-1901 dual-beam ultraviolet-visible spectrophotometer , a model of C-MAG HS7 heating magnetic stirrer , a model of constant temperature magnetic stirrer , a model of KC-6120 atmospheric integrated sampler  were used.A model of H-800 transmission electron microscope , with a dot pitch of 0.45 nm, an acceleration voltage of 200 KV, and a tilt angle of ± 25–3mol/L or 1.00 × 10–4mol/L when used. A 1.00 × 10–3mol/L basic fuchsin (BF) solution was prepared as follows, weigh 8.10 mg BF and dissolve in 25.0 mL volumetric flask, then fix the volume and shake well. Dissolve with ultrasound and dilute to 1.00 × 10–5mol/L when used. A 100 mmol/L pH 7.50 4-hydroxyethyl piperazine sodium ethanesulfonate/hydrochloric acid buffer solution (HEPES-HCl) was prepared as follows, 0.260 g HEPES was dissolved in water, then 370 μL of 1.00 mol/L HCl was added and the volume was fixed to 10.0 mL. 0.0100 mol/L silver nitrate solution, 30.0% H2O2 solution, 0.100 mol/L sodium borohydride solution (ready to use), 1.00% chloroauric acid solution , and 1.00% trisodium citrate solution were used. Anhydrous ethanol , 1,2,4-benzenetricarboxylic acid , N,N-dimethylformamide  and triethylamine  were also used in this experiment. The reagents used were all analytically pure, and the water used for the experiments was secondary distilled water.A 0.0126 g of sodium sulfite was dissolved in 10.0 mL water to obtain 0.0100 mol/L sodium sulfite stock solution, and store away from light before being diluted to 1.00 × 104 was separately dissolved in 20 mL DMF/C2H5OH/H2O (1:1:1 v/v) before the two solutions were mixed with stirring. Then 0.100 mL of 7.00 mol/L TEA was added, and the synthesis mixture was placed in a Teflon jar, sealed and irradiated in a microwave oven at 110 °C for 4.00 h. After the reaction, it was cooled to room temperature, and the solid product was obtained by centrifugation. The solid product was washed with DMF/H2O to remove unreacted BTA, and dried at 100 °C for 12.0 h. The above synthesis method for preparing AuMOF is the original method, and it has not been reported to use this method to synthesize AuMOF by this method.A 1 mmol BTA and a 1 mmol HAuCl4 was added under stirring. The solution was continued to boil for 10.0 min and removed, stirred and cooled to room temperature under no heating conditions. The volume was fixed to 100 mL and stored in a sterile seal. The particle size of the colloidal gold was about 10.0 nm. The concentration of the gold nanoparticles was 58.0 μg/mL (0.290 mmol/L).AuNP: 100 mL water was placed in a clean conical flask and boiled, then 7.00 mL of 1.00% aqueous sodium citrate was quickly added, and 1.00 mL of 1.00% HAuCl–2 mol/L AgNO3. After mixing thoroughly, 4.00 mL of 0.500 mg/mL sodium borohydride was added slowly drop by drop, and the solution changes from light yellow to dark yellow. Keep stirring for 10.0 min before the solution was fixed to 50.0 mL and stored at 4.00 °C. The concentration of the silver nanoparticles was 0.180 mmol/L.AgNP: 40.0 mL water was added into a 50.0 mL conical flask, and stirred with a magnetic stirrer while adding 3.50 mL of 10.0 g/L trisodium citrate and 385 μL of 2.40 × 10–5 mol/L BF, and a certain volume of 1.00 × 10–4 mol/L SO32– was successively added into a 5.00 mL stoppered graduated tube, and shake well. After standing for 2 min, 200 μL of 1.00 μg/mL AuMOF was added, then mixed to 2.00 mL and shake well. The RRS spectra were obtained by simultaneous scanning with a fluorescence spectrophotometer under the conditions of volt = 450 V, excited slit = emission slit = 5.00 nm, emission filter = 1.00% T attenuator, λex–λem = Δλ = 0. The RRS intensity of the solution at 330 nm was measured as Inm330 , and the intensity of reagent blank without SO32– was recorded as (Inm330 )0. The value of ΔInm330  = Inm330 -(Inm330 )0 was calculated.A 100 μL of PBS buffer solution at pH 6.80, 150 μL of 1.00 × 1032– in the solution, the addition reaction of BF does not occur, and strong RRS-ET occurs between BF and AuMOF, so the RRS signal at 330 nm was weak. When SO32– was added, which can make BF undergo an addition reaction to form a colorless BF product (SBF)  . The weantration . Accordi32–-BF-AuNP system, it can be seen from the TEM results that the aggregation degree of nanogold becomes smaller and smaller as the SO32– concentration increases  of the nanogold system was obtained by transmission electron microscopy (voltage: 200 kV) according to the experimental method (32–-BF-AuNP system was shown (The transmission electron microscopy (TEM) of the system and AuMOF were obtained according to the experimental method . For AuMncreases . That isl method , and theas shown . The EDSThe RRS and UV-Vis absorption spectra of AuMOF nanosol were studied. The results showed that the RRS and Abs intensities linearly increased with the concentration of AuMOF . In addinm330 ) did not fluctuate greatly within 7 days, indicating that the prepared AuMOF had good stability. A 21.6 μmol/L AuNP and 13.5 μmol/L AgNP were placed in the same environment for 7 days, and the signal changes of Inm370  at different times were recorded. The results show that the Inm370  of AuNP/AgNP increased with time in 7 days, indicating that the prepared AuNP/AgNP continued to aggregate with time. In addition, the electrolyte resistance of AuMOF/AuNP/AgNP were also investigated. A 400 μL of 0.100 g/L AuMOF, 150 μL of 0.290 mmol/L AuNP, and 150 μL of 0.180 mmol/L AgNP solution were added to 3 groups of glass tubes, respectively, with 11 glass tubes in each group. A 0–500 μL of 0.100 mol/L NaCl solution was added to each group to make up to 2.00 mL. The results show that the RRS of AuMOF remained stable when the NaCl concentration was as high as 25.0 mmol/L, while the Im370n of AuNP/AgNP increased with the increase of NaCl, indicating that NaCl would induce the aggregation of AuNP/AgNP. Therefore, AuMOF has good electrolyte resistance compared to AuNP/AgNP. So, the AuMOF nanosol was chosen for the analytical experiment.In this experiment, the stability of AuMOF/AuNP/AgNP was tested. The AuMOF was configured as a homogeneous suspension with a concentration of 20.0 μg/mL, placed at room temperature (25°C) for 7 days, and the RRS signal of changes at different times were recorded . The res32– reacted with BF to form colorless addition reaction product SBF. Due to the RRS energy transfer between AuMOF/AuNP and BF, as the concentration of SO32– increases, the BF in the system decreases, and the energy transferred from AuMOF/AuNP decreases. However, the RRS-ET between AuMOF/AuNP and BF was weak. The peak intensity at 330 nm increased linearly with the increase of SO32– concentration. So, the peak at 330 nm for AuMOF system and 370 nm for AuNP system were chosen to determine SO32– in this experiment. For the AuMOF system, it is a decreasing trend for the absorption peak at 540 nm produced by BF . Fo. Fo32– rt 540 nm . However32– and an experimental group with a certain amount of SO32– were set up, the RRS signal intensities I0 and I were measured, respectively, and the difference ΔI was calculated. By changing the conditions to be optimized and comparing ΔI, the optimal analysis conditions can be screened. As can be seen from 32– with BF was investigated. Two min later, the reaction was basically complete, so AuMOF/AuNP was added after the addition of SO32– and left for 2 min.The analysis conditions were studied by the univariate method, and the optimal analysis conditions were selected. In the experiment, a blank control group without SO32–-BF-AuMOF, the slope of working curve was largest and the detection limit (DL) was lowest, with a linear range (LR) of 0.160–5.00 μmol/L. What’s more, the AuMOF nanoprobe stability was better than the AuNP and AgNP. Thus, it was chosen for the determination of sulfite. Compared with the reported methods for the determination of sulfite  in different kinds of beer and fruit and vegetable juices samples that bought from the market were analyzed according to the procedures. The results show that the sulfite residues in commercially available samples of draft beer and fruit and vegetable juices exceeded the standard, while the sulfite residues in pineapple beer and apple cider vinegar were within the standard  contents in the air samples of inside the laboratory  and outside the laboratory (Sample 4) were examined according to the experimental method, respectively. Sampling procedure was as follows: 5.00 mL of 5.00 mmol/L absorbing solution was added into the U-shaped absorbing tube before it was connected to the atmospheric sampler, and sampled at a flow rate of 0.500 L/min for 20 min. Then the sampled solution was transferred to a 5.00 mL test tube, and a blank control was used for SO2 concentration detection. According to China National Standard GB/T17097-1997 for sulfur dioxide in indoor air, the daily average maximum permissible concentration of sulfur dioxide in indoor air should not exceed 0.150 mg/m3. As shown in The sulfur dioxide  will be developed to study RRS-ET to achieve the detection of target substance, to develop high-sensitivity methods and multimodal methods.A new, stable and strong RRS AuMOF nanoprobe was fabricated and characterized by electron microscopy and molecular spectral techniques. Based on the principle of addition reaction between sulfite and BF to form colorless SBF, strong RRS-ET between AuMOF donor and BF receptor, and weak RRS-ET between AuMOF and SBF, a new RRS-ET method was established for detecting SOof SO32– , 32–34, The original contributions presented in this study are included in the article/XL: formal analysis, data curation, and writing—original draft preparation. YL and SZ: data curation and writing—original draft preparation. MW: writing—original draft preparation. GW and ZJ: conceptualization, formal analysis, supervision, project administration, funding acquisition, and writing—review and editing. All authors contributed to the article and approved the submitted version.


Valid epidemiological data about distal fibular fractures and their treatment strategies are missing. Innovative osteosynthesis techniques were introduced and improved during the past 15 years. The aim of this study was to investigate the epidemiologic development and the implementation of new treatment strategies in a nationwide register in Germany over a period of 15 years.Data of the German Federal Statistical Office from 2005 until 2019 were screened. Adults with a fracture of the distal fibula were included. Data were separated for gender, age and treatment strategy.p = 0.436). 60.1% ± 0.6% of all fractures occurred in females. The annual incidence for male was nearly constant over the different age groups, whereas for female, there was a clear increase in incidence above the age of 40. Whereas 66% of fractures in between 20 and 30 years of age occurred in male, approximately 70% of fractures above the age of 60 occurred in females. The relative quantity of locking plates increased from 2% in 2005 to 34% in 2019. In 2019, only 1.02% of the patients were operated with an intramedullary nail.During the past 15 years, there was a steady annual incidence of distal fibula fractures of 74 ± 32 per 100,000 people without any significant changes (Operatively treated distal fibular fractures revealed an age dependent increase in incidence in postmenopausal women compared to younger females. Regarding the treatment strategy, there was an increase in application of locking plates. The data implicate a typical fragility fracture related age and gender distribution for distal fibula fractures.The online version contains supplementary material available at 10.1007/s00402-021-04232-0. Ankle fractures are among the most common fractures in adult people . There i. [Reported incidence rates range from 42 to 187/100,000 people per year . To our .  publishe. [Thur et al.  did also.  and intr.  for dist. –19. HoweThe aim of the present study was, therefore, to evaluate the incidence and treatment strategies for operatively treated ankle fractures with a fracture to the lateral malleolus in the most populous country in Europe over a 15-year period based on national registry data.The herein presented study is a longitudinal national registry study based on anonymized data sets. Therefore, the study needed no approval of the local ethics committee.In Germany, any in-house medical procedure must be reported to the German Federal Statistical Office to receive payment for the reported case . TherefoThe aim was to identify all procedures related to distal fibula fractures in adult patients (≥ 20 years). Therefore, the data set was screened for the following OPS codes: 5–790.3r, 5–790.4r, 5–790.4r, 5–793.3r, 5–793.ar, 5–793.br, 5–793.cr, 5–793.kr, 5–794.2,, 5–794.ar, 5–794.br, 5–794.cr, 5–794.gr, 5–794.kr (Supp. 1).The resulting 15 individual data sets were merged, and the age groups recalculated to 10-year increments . The annual incidence was calculated as the sum of all procedures per year  divided by the year- and age-matched German population. Annual population data were also retrieved from the German Federal Statistical Office The second aim of this study was to evaluate the treatment trends over the past two decades for anatomical locking plates and intramedullary nails. Therefore, the OPS codes and incidences were grouped according to these treatment strategies: Conventional plates , angular stable plates , fibular nails . Based on these data sets, the treatment trends over the 15-year period as well as a possible influence of age or gender was investigated.p < 0.05.Next to descriptive statistics, a linear regression analysis was performed to analyze the incidence and age distribution. A Chi-squared-test was conducted for analysis of treatment strategies. Values are presented as mean ± standard deviation if not stated differently. All statistics were performed with SPSS Statistics . In all cases, significance was set at p = 0.436).Between 2005 and 2019 a total of 745,823 ankle fractures with an involvement of the lateral malleolus were treated surgically in Germany. On average, 49,721 ± 5,464  fractures were treated surgically per year . This age–gender dependency is further highlighted in Fig. Figure p < 0.0001; Fig. R2 = 0.937, p < 0.001; Fig. p = 0.712; Fig. Figure R2 = 0.658, p < 0.001; Fig. R2 = 0.664, p = 0.014, see Fig. 2 = 0.976, p < 0.001; Fig. Figure The analysis of national registries do not only allow to calculate precise epidemiological data, but also give an unbiased inside into the treatment reality and trends. The herein analyzed OPS data sets of the German Federal Statistical Office revealed a rather constant incidence for operatively treated ankle fractures involving the lateral malleolus over the past 15 years. Incidence rates varied considerably per age and sex. Whereas 66% of all fractures occurred in male patients in the age group 20–30 years, approximately 70% of all fractures occurred in female patients in the age group 60 years and above. Over the observed time period the proportion of locking plates for the distal fibula increased 17-fold and for fibular nails tenfold.. described a massive increase of the incidence of ankle fractures in elderly patients from 57/100,000 people per year in 1970 to 150/100,000 people per year in 2000 [As outlined in the introduction, published incidence rates range considerably from 42 to 187/100,000 people per year . In cont in 2000 . However. [. [. [. observed an almost four times higher incidence for female ankle fracture than for mens’ [Similar to previous studies, the herein presented study revealed an effect of gender and age to the incidence rates. Scheer et al.  analyzed. . In line. . Juto et. [.  analyzed. [. . Again, . [. [.  conducte. [. [. . In a pror mens’ . Consequor mens’ , 26–28. or mens’  and a rior mens’ –32.This age-related impaired bone quality , 28, 33 . demonstrated a prevalence of intramedullary fixation for ankle fractures up to 20% in a recent british long-term register analysis [Intramedullary nails for distal fibular fractures bear the advantage of reducing the invasiveness of the procedure at the cost of the quality of reduction. However, previous studies demonstrated not only fewer complications of intramedullary nails compared to standard plate osteosynthesis, but also an equal functional outcome , 40. Desanalysis . [There are certainly limitations to this study. The OPS data are a nationwide comprehensive data set, but as they provide data only on operatively treated patients, one cannot conclude the true incidence of these injuries, but rather the incidence of the performed surgeries. However, in Germany, there is a traditional low threshold for a surgical indication, especially in younger patients . Therefo. , who sho. , 41, 42.In conclusion, the herein presented study is the first to present the incidence and treatment strategies for operatively treated distal fibula fractures between 2005 and 2019. Based on national registry data, a constant incidence rate was found with a gender and age dependent distribution. The treatment strategy over the observed 15-year period showed a steep increase for locking plates and intramedullary nails.Supplementary file1 (DOCX 19 kb)Below is the link to the electronic supplementary material.


A large number of studies have studied the influence of emotional experience on an individual’s estimation performance, but the influence of implicit emotion regulation is still unknown. Participants were asked to complete the following tasks in order: idiom matching task, multiplication computational estimation task (MCE task), gender judgment task (GJ task), and emotional experience intensity assessment task. The words matching task was adopted to achieve the purpose of implicit emotion regulation . Behavioral results showed that implicit reappraisal and implicit suppression equally contributed to improving an individual’s estimation speed (but not ACC (accuracy)). The MCE task related ERP  results showed that the influence of implicit emotion regulation on estimation consisted of two phases. In the first phase (encoding phase), implicit reappraisal both enhanced (larger P1 amplitudes) and weakened  an individual’s encoding sensitivity, while implicit suppression enhanced an individual’s encoding sensitivity (larger P1 amplitudes). In the second phase , implicit reappraisal (but not implicit suppression) cost more attention resources (larger LPC2 and LPC3 amplitudes). The present study suggested that both implicit reappraisal and implicit suppression contributed to improving an individual’s estimation performance, and the regulation effect of implicit suppression  was better. Previous studies showed that an individual’s estimation performances might be affected by emotion experience ,2,3, ageCombined with the emotion priming paradigm, Liu et al.  asked paHowever, the result of Liu et al.  was baseA recent study has explored the influence of emotion regulation on an individual’s estimation performance . In thisThe dual-process framework theory proposed that both explicit and implicit emotion regulation were important for us, though researchers paid more attention to explicit emotion regulation in previous studies . ExpliciTo our knowledge, only a few studies have studied the relationship between emotion regulation and an individual’s arithmetic performance ,31; therReferring to a previous study , a totalThe experimental materials consisted of four-character words, facial images, and two-digit multiplication problems.(1)Four-character wordsFour hundred and fifty four-character words were divided into three categories according to their meanings, by 32 participants, i.e., view, reappraisal, and suppression words. In order to distinguish neutral facial expressions from neutral words, neutral words were named “view” (consistent with the “freely view” strategy in the field of emotion regulation). For a certain idiom, only if not less than 75% (24 participants) of the total number of participants classify it into one of the three groups mentioned above, the idiom can be used in the material of the formal experiment. Additionally, twenty-five college students were recruited to evaluate the familiarity of each idiom using a 9-point scale, where “1” means “very unfamiliar” and “9” means “very familiar”. Another twenty-five participants were asked to use a 9-point scale to evaluate the relevance of the meaning of each idiom to “describe natural scenes” (view), “none of my business” , and “hide one’s own emotional experience” (suppression), where “1” means “not relevant”, and “9” means “highly relevant”. Referring to previous studies ,35, the p = 0.253. The familiarity towards the three groups of words was as follows: view, M ± SD, 7.70 ± 0.96; reappraisal, 8.06 ± 0.41; expression, 7.98 ± 0.65, indicating that the subjects were equally familiar with the three groups of words. (2) The relatedness between the words and “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”: A. There was a significant difference in the relatedness between the three types of words and “describe natural scenes”, F = 2354.29, p < 0.001, the relatedness between the view words and “describe natural scenes” were significantly higher than (ps < 0.001) the reappraisal words and suppression words; there was no significant difference between the latter two (p > 0.05). B. There was a significant difference in the relatedness between the three types of words and “none of my business”, F = 1083.09, p < 0.001, the relatedness between the reappraisal words and “none of my business” were significantly higher than (ps < 0.001) the view and suppression words, and the suppression words were higher than the view words (p < 0.001). C. There was a significant difference in the relatedness between the three types of words and “hide one’s own emotional experience”, F = 1120.45, p < 0.001, the relatedness between the suppression words and “hide one’s own emotional experience” were significantly higher (ps < 0.001) than the view and reappraisal words, and the reappraisal words were higher than the view words (p < 0.001). The above results showed that there were significant differences among the three types of words in terms of the relatedness to any of the meanings “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”. (3) The relatedness between a certain idiom group and “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”: A. There was a significant difference between the relatedness of view words and “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”, F = 4255.54, p < 0.001; the relatedness between view words and “describe natural scenes” were significantly higher (ps < 0.001) than the relatedness to “none of my business”, and “hide one’s own emotional experience”, and the difference between the relatedness of the latter two was not significant (p > 0.05). B. There was a significant differences between the relatedness of reappraisal words and “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”, F = 962.27, p < 0.001; the relatedness between reappraisal words and “none of my business” were significantly higher (ps < 0.001) than with “describe natural scenes” and “hide one’s own emotional experience”, and the relatedness to “hide one’s own emotional experience” was higher than to “describe natural scenes” (p < 0.001). C. There was a significant difference between the relatedness between suppression words and “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”, F = 561.38, p < 0.001; the relatedness between suppression words and “hide one’s own emotional experience” were significantly higher (ps < 0.001) than “describe natural scenes” and “none of my business”, and the difference between the relatedness to the latter two was not significant (p > 0.05). The above results showed that, for a specific group of words, there were significant differences between the relatedness to “describe natural scenes”, “none of my business”, and “hide one’s own emotional experience”. (4) There was no significant difference in the emotional experience elicited by the three types of words, F = 0.89, p = 0.416, and the emotional experience intensity elicited by the three types of words were as follows: view, M ± SD, 5.16 ± 0.52; reappraisal, 5.15 ± 0.56; suppression, 5.35 ± 0.34.Referring to previous study , the fam(2)Facial imagesp < 0.001, η2 = 0.630. The valences of happy  images were significantly higher than those of fear  and neutral  images, and the valence of neutral images was significantly higher (p < 0.001) higher than for fear. The main effect of arousal was not significant, F = 0.49, p = 0.617, η2 = 0.028. The arousals of happy, fearful, and neutral pictures were M ± SD, 5.62 ± 0.49, 5.70 ± 0.45 and 5.57 ± 0.50. All facial images were presented with the same visual angle (4.52° × 6.75°).The facial images used in the present study were selected from the NimStim Database . Eightee(3)Two-digit multiplication problemsA total of 54 two-digit multiplication estimation questions suitable for DU strategy were used. The principles for selecting the arithmetic problems were in accordance with previous studies ,2,12. ThThe experimental procedure was programmed with E-prime 2.0 . The formal experiment consisted of 9 blocks, a single block sequentially containing the idiom matching task, MCE task, gender judgment task (GJ task), and emotional experience intensity assessment task . The speThe present study was conducted in a quiet room. It was ensured that participants fully understood the experimental procedure; the eighteen practice trials idiom matching task, nine practice trials MCE task, nine practice trials GJ task, and one practice trials emotional experience intensity assessment task were provided before the formal test. Feedback was provided in the practice trials, while it was not provided in the formal experiment. The experimental materials  used in the practice trials were not used in the formal experiment. To relieve fatigue, all participants were required to rest for 2 min after each block.The electrophysiological signals elicited by completing the task were recorded using Vision Recorder 2.0  with 64 scalp sites (10–20 system), while adapting FCz as the reference electrode. Both the horizontal and vertical electrooculogram (EOG) were recorded. The horizontal EOG was recorded from the electrode site at the outer canthi of the right eye, while the vertical EOG was recorded from the electrode placed 1 cm below the right eye. The electrode impedance was kept below 5 kΩ. The electrophysiological and EOG signals were amplified with a band pass of 0.01–100 Hz, and sampled at a rate of 500 Hz. Offline signal processing had been adopted to obtain a global average reference, by using Vision Analyzer 2.0 . The data were filtered with a 0.1 Hz high-pass cutoff and a 35 Hz low-pass cutoff (24 dB/octave), respectively. Trials with EOG artifacts (mean EOG voltage exceeding ±80 μV) were excluded from averaging. The EEG data were corrected for eye movements with ICA ocular correction. Noisy EEG sensors were identified through visual inspection, and the bad electrode was interpolated by using spline interpolation procedure. The TTL synchronization with stimuli presentation was adopted, which was based on a synchronization signal sent through a cable connection between the computer that was presenting the experimental materials, and the other computer that was recording the experimental data. Meanwhile, the TCP/IP Synchronization had been adopted. Using the TCP/IP connections between E-prime 2.0 and Vision Recorder 2.0  allowed us to send events from E-prime 2.0 to Vision Recorder 2.0. In the present study, different experimental conditions were defined by E-prime 2.0 before the experiment; thus, we could configure E-prime 2.0 to send an event at every onset of a stimulus. Additionally, event marking was adopted for post-processing synchronization.The average epoch for ERP was 3000 ms, including a 200 ms baseline. According to previous studies ,2,37, thp values, while Bonferroni’s correction was applied to post hoc tests. Additionally, partial eta-squared  and type of emotion regulation strategies . Additionally, three-factor repeated-measures ANOVA was performed on the ERP data, and the independent variables were implicit priming emotion type, type of emotion regulation strategies, and hemisphere (left hemisphere vs. right hemisphere). Dependent variables were the amplitudes of the ERP component mentioned above. SPSS 16.0 was used for statistical analysis, the Greenhouse–Geisser correction was applied to the (1)The ACC and RT of completing the idiom matching taskp = 0.196. The ACC of completing the idiom matching task under view, implicit reappraisal, and implicit suppression conditions were as follows: M ± SD, 0.997 ± 0.054, 0.993 ± 0.085, and 0.999 ± 0.038, respectively. Additionally, the independent sample t-test results showed that the ACC of completing the idiom matching task under the above three conditions was significantly higher than (ps < 0.001) the random level (0.5).ACC: The results of one-way ANOVA showed that the main effect of implicit emotion regulation strategy type was not significant, F = 1.631, p < 0.001. The RT of completing the idiom matching task under implicit reappraisal  and implicit suppression (1389.248 ± 507.836) conditions were shorter (ps < 0.001) than under neutral (1624.857 ± 671.522) condition, while the RTs under the implicit reappraisal and implicit suppression conditions showed no significant difference (p > 0.05).RT: The results of one-way ANOVA showed that the main effect of implicit emotion regulation strategy type was significant, F = 33.812, (2)The intensity of the emotional experiencep = 0.002. Participants’ emotional experience intensity under view  conditions were higher than under implicit reappraisal  and implicit suppression  conditions, while their emotional experience intensity under the latter two conditions showed no significant difference (p > 0.05).The results of one-way ANOVA showed that the main effect of emotional experience intensity was significant, F = 6.333, (3)Individual’s ACC and RT of completing the MCE taskp = 0.188, p = 0.611, p = 0.203, ACC: Two-way repeated-measure ANOVA showed that the main effect of implicit emotion regulation strategy type was not significant, F = 1.727, p < 0.001, ps < 0.001) than under implicit reappraisal and implicit suppression conditions, while their RT under the latter two conditions showed no significant difference (p > 0.05). The main effect of implicit priming emotion type was not significant, F = 3.016, p = 0.080, p = 0.138, RT: Two-way repeated-measure ANOVA showed that the main effect of implicit emotion regulation strategy type was significant, F = 16.427, (1)ERPs elicited by processing facial expressionp < 0.001, p < 0.001) than in the left hemisphere (−2.871 ± 5.172 μV)  Repeated measurement ANOVA revealed a significant main effect of hemisphere, F = 12.719, .172 μV) . The mai(2)ERPs elicited by completing the MCE taskp = 0.001, p = 0.001) and implicit suppression  conditions were larger  than in the right hemisphere (0.259 ± 4.089 μV)  Repeated measurement ANOVA revealed a significant main effect of implicit emotion regulation strategy type, F = 9.758, e larger . The mai.089 μV) . The maip < 0.001, p < 0.001) than in the right hemisphere (−3.568 ± 4.694 μV). The interaction effect between implicit priming emotion type and implicit emotion regulation strategy type was significant, F = 3.234, p = 0.049, p = 0.027) and implicit suppression  conditions were larger (p = 0.047) and implicit suppression  conditions, the T-N170 amplitudes under implicit reappraisal (−3.988 ± 4.947 μV) condition were larger (ps > 0.05). The main effect of implicit emotion regulation strategy type was not significant, F= 0.828, p = 0.443, p = 0.795, p = 0.259, p = 0.386, p = 0.191, T-N170 (1340–1380 ms) Repeated measurement ANOVA revealed a significant main effect of hemisphere, F = 19.004, e larger ; B. undee larger ; C. undep < 0.001, p < 0.001) than in the right hemisphere (−1.729 ± 4.818 μV). The main effect of implicit priming emotion type was not significant, F = 0.172, p = 0.747, p = 0.338, p = 0.114, p = 0.682, p = 0.602, p = 0.107, T-LPC1 (1340–1380 ms) Repeated measurement ANOVA revealed a significant main effect of hemisphere, F = 17.973,  = 0.094 .p = 0.001, p = 0.001) than in the right hemisphere (−1.171 ± 4.740 μV). The interaction effect between implicit priming emotion type and implicit emotion regulation strategy type was significant, F = 3.261, p = 0.034, p = 0.016) and implicit suppression  conditions were smaller (ps > 0.05). The main effect of implicit emotion regulation strategy type was not significant, F = 1.812, p = 0.182, p = 0.708, p = 0.635, p = 0.427, p = 0.138, T-LPC2 (2200–2600 ms) Repeated measurement ANOVA revealed a significant main effect of hemisphere, F = 12.808,  smaller ; B. undep = 0.012, p = 0.001) than in the right hemisphere (−0.958 ± 4.529 μV). The interaction effect between implicit priming emotion type and implicit emotion regulation strategy type was significant, F = 3.127, p = 0.032, p = 0.014) and implicit suppression  conditions were smaller (ps > 0.05). The main effect of implicit emotion regulation strategy type was not significant, F = 0.655, p = 0.524, p = 0.452, p = 0.734, p = 0.271, p = 0.131,T-LPC3 (2600–3000 ms) Repeated measurement ANOVA revealed a significant main effect of hemisphere, F = 7.288,  smaller ; B. undeIn order to enhance readability, the comparisons of ERP amplitudes induced by different experimental conditions were summarized , participants were asked to complete the idiom matching task (an implicit emotion regulation method). Meanwhile, they were asked to complete the MCE task with the DU strategy. The present study explored the relationship between implicit emotion regulation and an individual’s estimation performance. The behavioral results showed that participants completed the idiom matching task carefully, and that implicit reappraisal and implicit suppression equally reduced an individual’s emotional experience intensity. Both implicit reappraisal and implicit suppression contributed to reducing an individual’s RT (but not ACC) of completing the MCE task. In addition, the ERP results showed that both the encoding of the estimation question (T-P1 and T-N170) and the retrieval of arithmetic facts (T-LPC) were influenced by implicit emotion regulation.A large number of previous studies have demonstrated that the idiom matching task was an effective way of implicit emotion regulation ,26,41,42The emotional experience intensity score-related results showed that, compared with the baseline level (view condition), implicit reappraisal and implicit suppression effectively reduced participants’ emotional experience intensity after completing the MCE task, and the regulation effect of implicit reappraisal and implicit suppression were equivalent. A previous study showed that through implicit emotion regulation, an individual’s anger experience can be reduced , and theConsistent with previous study , the preAdditionally, the present study found that a participant’s reaction speed of completing the MCE task was quicker under implicit reappraisal and implicit expression conditions, than under view conditions, and their reaction speed under implicit reappraisal and implicit expression conditions showed no significant difference; these results seemed to indicate that implicit emotion regulation could provide more efficient processing for arithmetic tasks, through modulating the cognitive and emotional control capacities, thus leading to a faster response speed under implicit reappraisal and implicit suppression conditions which was consistent with a previous study . TotallyA large number of previous studies ,46,47 inThe present study showed that the C-N170 amplitudes elicited by processing different facial expression images  showed no significant difference. Previous studies showed that N170 was an effective indicator of facial expression processing ,48, and The present study showed that when participants complete the MCE task with the DU strategy under different implicit emotion regulation conditions, the T-P1, T-N170, and T-LPC amplitudes are elicited, indicating that the different stages of individuals completing the MCE task are affected by implicit emotion regulation. The present study showed that the T-P1 amplitudes elicited by completing the MCE task were larger in the left (vs. right) hemisphere, suggesting that the left hemisphere was more sensitive to completing digit processing tasks, which was consistent with previous studies ,54. In tThe present study showed that the T-N170 amplitudes were influenced by the interaction effect of implicit priming emotion type and implicit emotion regulation strategy type. To be more specific, compared with the baseline (view) and implicit suppression, implicit reappraisal contributed to estimation question encoding  under the implicit neutral priming condition. However, inverse results were found under the implicit fear priming condition, while an individual’s encoding performances were not influenced by implicit emotion regulation under the implicit happy priming condition. Totally speaking, both T-P1 and T-N170 belong to the ERP components induced in the encoding stage of the MCE task; T-N170 (but not T-P1) amplitudes were influenced by the interaction effect of implicit priming emotion type and implicit emotion regulation strategy type, which clearly reveal that the dynamic process of encoding are modulated by implicit emotion regulation. These results indicate the superiority of analyzing several small time windows (T-P1 and T-N170) separately at the encoding stage, instead of adapting a general analysis of the ERP amplitude changes over a longer period of time window, such as 0–200 ms ,56, 0–25LPC is a positive component that appears in the visual cortex during the late stages of processing the target stimuli, and it is often regarded as an effective indicator of resource allocation in response to attention ,59,60. TTo sum up, the behavioral results of this study showed that participants successfully carried out implicit emotion regulation by completing the idiom matching task, and participant’s RT (but not ACC) of completing the MCE task with the DU strategy was influenced by implicit emotion regulation. Implicit reappraisal and implicit suppression equally contributed to improving a participant’s estimation performance under different implicit priming conditions. The ERP results revealed the dynamic time course of implicit emotion regulation affecting estimation performance. Firstly, in the encoding stage, the influence of implicit reappraisal on encoding sensitivity was increased first (larger T-P1 amplitudes) and then decreased , while implicit suppression contributed to improving a participant’s encoding sensitivity (larger T-P1 amplitudes). Secondly, in the retrieval stage, implicit reappraisal (vs. view) cost more attention resources (larger T-LPC2 and T-LPC3 amplitudes) when completing the MCE task under the implicit fear priming condition, which reflected the advantage effect of implicit expression .The present study investigated the influence of implicit emotion regulation on an individual’s estimation performance under implicit emotional priming condition for the first time. The behavioral results showed that an individual’s estimation performances were equally improved (shorter RTs) by implicit reappraisal and implicit suppression, while the ERP results further expanded our understanding of the relationship between implicit emotion regulation and an individual’s estimation performance. In detail, both implicit suppression and implicit reappraisal contributed to improving an individual’s estimation performance, and the regulation effect of implicit suppression was better than implicit reappraisal, especially under the implicit fear priming condition. However, it should be noted that the ACCs around 0.99 suggested that the difficulty of the estimation task may have been too low in order to allow variations, generated by the emotion regulation strategy, to be induced. The behavioral results showed that an individual’s ACCs of completing the MCE task were not influenced by the implicit emotion prime and implicit emotion regulation; this may be related to the fact that only one estimation strategy has been adopted in the present study, and future studies can verify this speculation by adopting harder estimation strategies .The present study revealed that an individual’s estimation performance was modulated by implicit emotion regulation. Compared with view, both implicit reappraisal and implicit suppression contributed to reducing an individual’s RT of completing the MCE task. Implicit reappraisal and suppression were equivalent in reducing an individual’s emotional experience intensity when they completed the MCE task. Compared with view, implicit reappraisal first enhanced (larger T-P1 amplitudes) and then weakened  an individual’s encoding sensitivity, while implicit suppression only enhanced an individual’s encoding sensitivity (larger T-P1 amplitudes). Additionally, under implicit fear priming condition, compared with view, there was implicit reappraisal (but not implicit suppression) along with more attention resource cost (larger T-LPC2 and T-LPC3 amplitudes). Totally speaking, both implicit reappraisal and implicit suppression contributed to improving an individual’s estimation performance, and the regulation effect of implicit suppression  was better.