Centrioles are non-membrane-bound organelles that participate in fundamental cellular processes through their ability to form physical contacts with other structures. During interphase, two mature centrioles can associate to form a single centrosome—a phenomenon known as centrosome cohesion. Centrosome cohesion is important for processes such as cell migration, and yet how it is maintained is unclear. Current models indicate that pericentriolar fibres termed rootlets, also known as the centrosome linker, entangle to maintain centriole proximity. Here, I uncover a centriole–centriole contact site and mechanism of centrosome cohesion based on coalescence of the proximal centriole component cNap1. Using live-cell imaging of endogenously tagged cNap1, I show that proximal centrioles form dynamic contacts in response to physical force from the cytoskeleton. Expansion microscopy reveals that cNap1 bridges between these contact sites, physically linking proximal centrioles on the nanoscale. Fluorescence correlation spectroscopy (FCS)-calibrated imaging shows that cNap1 accumulates at nearly micromolar concentrations on proximal centrioles, corresponding to a few hundred protein copy numbers. When ectopically tethered to organelles such as lysosomes, cNap1 forms viscous and cohesive assemblies that promote organelle spatial proximity. These results suggest a mechanism of centrosome cohesion by cNap1 at the proximal centriole and illustrate how a non-membrane-bound organelle forms organelle contact sites. During interphase, two mature centrioles can associate to form a single centrosome; this "centrosome cohesion" is important for processes such as cell migration, but how is it maintained? This study combines live cell quantitative imaging, expansion microscopy and ectopic tethering to provide insights into the mechanisms by which centrioles maintain spatial proximity inside human cells. Organelle contact sites are critical to diverse cellular functions. Membrane-bound organelles associate via dedicated molecular complexes that perform functions such as membrane tethering . How nonCentrosomes are microtubule-organising centres that mediate fundamental cellular processes including cell division, polarity, and motility. Centrosomes exist in the cellular interior without a bounding membrane, dynamically interacting with structures such as the cell membrane and mitotic spindle . During CROCC) is a key component of rootlets [Rootlets, also known as the centrosome linker, are pericentriolar fibres found at centrioles ,9,10. Acrootlets ,11–14. Rrootlets ,15,16. Orootlets ,9,17–19.rootlets –24.cNap1  is a rootletin paralog found at the proximal centriole, spatially adjacent to rootlet fibres ,19,25,26Here, by studying the biophysical properties and nanoscale architecture of cNap1 at centriole–centriole contact sites, I discover that it directly maintains centrosome cohesion. Live-cell imaging and expansion microscopy of endogenous cNap1 shows that it bridges between dynamic centriole–centriole interfaces. cNap1 accumulates at micromolar concentrations, forming supramolecular assemblies with viscous material properties that cohesively maintain organelle spatial proximity. I propose a model of centrosome cohesion explaining how organelle solidity is balanced against organelle plasticity using dynamic cNap1 assemblies.S1A–S1C Fig and Materials and methods). Clones were screened to identify cells with all cNap1 alleles homozygously modified. cNap1-mScarlet-I and rootletin-meGFP functionality was ensured by measuring centrosome cohesion, which was indistinguishable in genome-edited and wild-type cells . These considerations confirmed that cNap1 and rootletin were tagged at functional, endogenous levels.To simultaneously track the spatiotemporal behaviour of both proximal centrioles and rootlets in living cells, genome editing was used to create U2OS cells expressing endogenously tagged fluorescent cNap1 and rootletin (cNap1-mScarlet-I and rootletin-meGFP). Cell lines were carefully validated; precise genomic tagging was ensured by a combination of overlapping genomic PCR and imaging , as expected from prior electron microscopy [Fig 1B and S1F and S1G and S1 Movie). Proximal centrioles thus transiently formed contacts as they collided together during continuous movement. Rootlet fibres were also mobile during centriolar movements. Rootlets from both centrioles could either form contacts, or alternatively move independently, apparently not in contact . Throughout these dynamic movements, cNap1-mScarlet-I foci were always present at the centrosome-proximal termini of rootletin-meGFP fibres . Thus, simultaneous imaging of endogenously tagged cNap1 and rootletin reveals that both proximal centrioles and rootlets form dynamic contacts during centrosome cohesion .I reasoned that if centrosome cohesion is mediated by direct links between centrioles, these contact points might be visible with imaging. cNap1-mScarlet-I enriched strongly at centrosomes relative the surrounding cytosolic pool, forming either 1 or 2 foci corresponding to the proximal centrioles  . U-ExM iFig 1), centriole pairs occupied variable orientations relative to each other in populations of U2OS cells -validated antibody showed that it accumulates at the proximal centriole  [Fig 2C). A montage of approximately 60 different cells is presented in S2B Fig to document this variability in cNap1 orientation and shape. cNap1 bridging between centrioles was also observed in a different cell type, hTERT-HPNE , both in ciliated and non-ciliated cells .Consistent with the live-cell imaging data (and S2A) . StrikinFigs 2E and S2D). With centrioles adjacent, rootlets could either be oriented radially into the cytoplasm without overlap, or formed contact points with the rootlets from the adjacent centriole . Together, these results demonstrate that cNap1 can either form separate assemblies on each proximal centriole or can bridge centriole pairs as a contiguous structure.U-ExM of rootletin showed that it too was present in multiple orientations, also consistent with the live-cell imaging data . Centrosomal cNap1 concentrations were high compared to the cytosolic pool, with a mean centriolar concentration of approximately 862 ±59 nm, compared to approximately 10 ±1 nm in the cytosol  . Using a measure of total cNap1 centriolar size, this concentration corresponds to a mean of approximately 160 cNap1 proteins per centrosome. This is in broad agreement with a previous proteomics-based estimate [I investigated further the biophysical properties of cNap1 that allow it to bridge across centriole pairs. Fluorescence correlation spectroscopy (FCS) calibrated imaging was used to measure endogenous cNap1-mScarlet-I protein copy number in living cells, where FCS measurements on freely diffusing components are used to calibrate fluorescence confocal images into absolute concentration  [S3C Fig). U-ExM of cDNA-expressed mScarlet-I-cNap1 showed similar morphologies to endogenous protein, differing primarily in size, being larger at higher levels . Time-lapse imaging on a timescale of minutes to hours showed that like endogenous cNap1, overexpressed cytoplasmic and centrosomal mScarlet-I-cNap1 had the capability to coalesce, through the formation of intermediate bridged shapes prior to one contiguous structure . mScarlet-I-cNap1 patches also showed shape changes over time independent of coalescence, suggestive of cohesive properties within each patch  ,34,35. CFig 3E). Bead movement within cNap1 was slowed relative to the cytosol, as seen in a lower relative mean squared displacement (MSD) over time . In a log–log plot of MSD, diffusion within a viscous fluid results in a diffusive exponent α of 1 (MSD = 4DTα) [S3E Fig and Materials and methods). Bead diffusion coefficient within cNap1 was 0.00017 μm2/s (25th to 75th percentile 0.0001 to 0.0002), indicating a viscosity of approximately 15.6 Pa-s (25th to 75th percentile 9.8 to 25.2) according to the Stokes–Einstein relation . This is approximately 15,000 times more viscous than water at 21°C. Consistently, fluorescence recovery after photobleaching (FRAP) indicated that cNap1 within condensates had limited turnover over short timescales of up to approximately 30 s  ,39. Micry 30 s . Taken Fig 4A and S7 Movie). Thus, approximately 63% of eGFP-rootletin fibres were coincident with mScarlet-I-cNap1 patches, approximately 18 hours after mScarlet-I-cNap1 transfection in a population of cells. cNap1 predominantly bound to the ends of cytoplasmic rootletin fibres, attaching to either one or both ends . mScarlet-I-cNap1 was not just temporarily colocalised with rootletin fibre termini, but stably attached, since they could translocate together over minutes .cNap1 has previously been shown to bind rootletin by yeast 2-hybrid and coimmunoprecipitation ,9. I theFig 4C; respectively named here; NT, CT, M1, M2, or M1/2). An R188 mScarlet-I-cNap1 truncation was also created, since it is the site of a truncating mutation (R188) identified in a consanguineous family with retinal impairment [To investigate whether individual cNap1 protein domains are sufficient for condensate formation and rootletin binding, cNap1 was divided into a series of separate fragments . These ppairment ,28 and cpairment .Fig 4D). Instead, dependent on the domain, 0% to 70% of cells formed cytoplasmic condensates, relative to approximately 80% in wild type. I investigated whether condensate formation influenced cNap1 rootlet-end binding. Rootletin fibre end-binding correlated with condensate formation capability, when analysing only cells that formed condensates. Wild type thus had the highest rootletin fibre end-binding and other truncations had lower levels . Thus, in agreement with previous work [Fig 4G). These results together suggest that condensate formation promotes cNap1-rootletin fibre end-binding but is not essential for centriolar localisation.No single cNap1 protein domain was fully sufficient for condensate formation at wild-type levels . FRAP of NT, CT, and R188 also revealed increased exchange rates at centrosomes relative to wild-type protein .Previous loss of function studies have shown that cNap1 is required for centrosome cohesion ,15,29,30Figs 3), suggesting that cNap1 itself possesses cohesive properties that promote organelle cohesion. Such a model theorises that separate cNap1 pools coalesce to maintain spatial proximity . Since rootlets are already known to promote centrosome cohesion [Materials and methods for details). Note that cNap1 has not been reported to localise to these organelles. Lyso-cNap1, Golgi-cNap1, and mito-cNap1 had different shapes related to the structure and dynamics of the targeted organelles. Lyso-cNap1 formed spherical shells surrounding lysosomes marked by LysoTracker . Mito-cNap1 and Golgi-cNap1 formed adjacent to or coincident with either mitochondria or the Golgi, respectively , demonstrating that the shape of cNap1 assemblies changes when targeted to different organelles, forming an external coating in each case.mScarlet-I-cNap1 forms supramolecular assemblies that coalesce Figs , suggestFig 5E and 5H). Time-lapse imaging revealed that lyso-cNap1 coated lysosomes had reduced movements relative to controls, over both minutes . To test whether cNap1 condensates are also sufficient to promote centrosome cohesion, I expressed mScarlet-I-cNap1 in U2OS cells, which maintain high levels of centrosome cohesion [Fig 5I). Together, these results show that cNap1 promotes centrosome cohesion in the absence of rootlets.Given that these organelles do not contain rootlets, this suggests that cNap1 itself can mediate organelle cohesion, and indeed rootlet fibres were not detected after co-staining for rootletin . Proximal centrioles form dynamic contact sites that are directly bridged on the nanoscale by cNap1 . cNap1 accumulates at micromolar concentrations on centriole termini and forms viscous supramolecular assemblies that promote organelle cohesion, even in the absence of rootlets . This model may explain previous observations that proteins with no known role in rootlet formation are required for centrosome cohesion [The precise mechanisms of centrosome cohesion are unclear, despite the identification of key molecular players ,26. Currcohesion .Previous electron microscopy data in bovine thymocytes described an “amorphous density” or “hinge” between proximal centriole pairs and partially inside the centriolar lumen ,43, suppFigs 1 and 4). The data here therefore suggest a dual model of cNap1 function, both anchoring rootletin fibres as 2 foci, and directly promoting centrosome cohesion through coalescence of the foci . One interpretation is that cNap1 condensates create a phase-separated environment that promotes rootlet fibre anchoring or nucleation at the proximal centriole. This model is reminiscent of others proposed for the pericentriolar material—a different centriole protein coat that has also been suggested to phase separate to nucleate microtubules [S3K Fig.) [cNap1 anchors or nucleates rootlets at centrioles, since cNap1 disruption prevents rootlet formation at centrosomes ,9,26,29.otubules ,46. cNap3K Fig.) , supportOverall charge has been suggested to regulate cNap1 oligomerisation, through multisite phosphorylation from the kinase Nek2 . Since mIn contrast to membranous organelles, less is known about how non-membrane-bound organelle fusion and fission is maintained. cNap1 intra-organelle assemblies at centriole contacts have a number of the characteristics that are used to define membrane–membrane contact sites , includiFigs 4 and 5), illustrating how the effects of other mutations may be rationalised in future work. However, it should be noted that unidentified protein–protein interactions may also be the primary source of disruption in truncated cNap1 mutants.The data here provide a framework to understand the effects of cNap1 disease-causing mutations in the future. One possibility is that disease-causing mutations alter the material properties of cNap1 molecular assemblies. The cNap1 R188 truncation disrupts centrosome cohesion as a dominant negative and forms cytoplasmic condensates with reduced viscosity and altered molecular mobility  and DMEM, with 5% fetal calf serum, 2 mM glutamine, 10 ng/ml EGF, and 750 ng/ml puromycin. Cilia were induced in hTERT-HPNE by 24 h growth in complete medium with 0.2% fetal calf serum. Cell lines were confirmed as mycoplasma free. All tissue culture reagents were purchased from Sigma-Aldrich unless otherwise stated. DNA transfection was with lipofectamine 3000 (Invitrogen) or jetPRIME (Polyplus) according to the manufacturer’s instructions.Human U2OS cells were obtained directly from the American Type Culture Collection (ATCC HTB-96). U2OS and HeLa Kyoto cell lines were grown in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum, Glutamax, and 100 μg/ml penicillin/streptomycin and maintained at 37°C with 5% COcNap1 or CROCC genomic reference sequence. These arms were inserted into plasmids such that they flank mScarlet-I or meGFP coding sequence. The cNap1 donor plasmid was purchased from Thermo Fisher GeneArt. The CROCC donor plasmid was constructed by In-fusion cloning (see molecular cloning for In-fusion methods).Endogenously tagged cNap1-mScarlet-I and rootletin-meGFP (CROCC-meGFP) U2OS cells were produced with the methods described in . Donor pcNap1 were: TCCAGGTAGCAGCCACAGCC (Strand 1), CTGTGGCTGCTACCTGGAGG (Strand -1), TCCTGGCTGTGGCTGCTACC (Strand -1). Guide RNA sequences (5′—3′) for CROCC against the +ve strand were as follows (5′—3′): CCAGCAGGAGCTCATTTCTC, CCAGAGAAATGAGCTCCTGC, and CAGGAGCTCATTTCTCTGGG.sgRNA sequences were designed in Benchling software, selecting optimal on and off target activity as close to the target site as possible. Guide RNAs did not target the donor plasmid. Guide RNA sequences (5′—3′) for Guide RNA sequences were cloned into pSpCas9(BB)-2A-Puro (PX459) (Addgene plasmid 48139) for expression. Guide RNA and the donor plasmid were co-transfected. After a week, single cells were sorted for mScarlet-I or meGFP positivity and grown as clones for PCR screening. Genomic insertion of fluorescent proteins was screened by overlapping genomic PCR. PCR primers were designed either side of the insertion site in clone manager suite, ensuring no false priming. PCR primer annealing temperature was optimised across a temperature gradient before screening clones. For CROCC-meGFP, primer sequences were as follows 5′-3′: GGCTTGGATCTAAGGAGG and GGCTGGCCTTACCTTCCCTT. For cNap1-mScarlet-I primer sequences were as follows 5′-3′: GATTCGTGTATGTGGTAGAG and CTATCACAGTGCATGGTGTA. Tag insertion was detected based on PCR product size, approximately 700 bps larger with fluorescent protein insertion. PCR for screening was with DREAMtaq (Invitrogen) according to the manufacturer’s instructions, run with Hyperladder 1 DNA marker (Bioline). Selected clones were confirmed to have centrosomal localisation as expected by Airyscan confocal imaging, concurring with previously reported antibody staining. Cell lines were also validated by removing fluorescent signal using siRNA-mediated knockdown of either cNap1 or rootletin, using the methods described in the siRNA methods section.S3A Fig were acquired on a Carl Zeiss LSM 880 Airyscan confocal laser scanning microscope, controlled by Zen Black software. Lenses used were 100× NA 1.46 oil, 63× NA 1.4 oil, 40× NA 1.3 oil, and 40× NA 1.2 water immersion objectives, with optimised cover glass correction where possible. Airyscan images were acquired in SR mode and processing was performed with automatic settings in Zen Black. Pixel size and Z-slice size were optimised, depending on the scan area of each experiment (which was variable), using the optimal function in Zen. Other imaging parameters including scan speed and image averaging were variable for each experiment, but did not change between comparative samples. Laser power was adjusted to minimise bleaching and cellular toxicity in live-cell experiments. Detector gain was adjusted to ensure pixel intensities were never saturated or clipped. The median (± median absolute deviation) lateral and axial resolution of the system was measured at 198 ± 7.5 nm and 913 ± 50 nm , respectively, through imaging of a sub resolution fluorescent bead. Brightness and contrast were adjusted on images for display purposes, but never unequally between comparative samples.All images except those in 2-independent medium at 37°C. Cell health in the conditions used was optimal since cell growth continued. For live-cell time-lapse, image size and frequency of acquisition varied depending on the timescale of events observed and scan area. For high-resolution imaging of centrosomes, a 100× NA 1.46 oil lens was used. Autofocus was at every time point using the Zeiss definite focus autofocus system. A stage top piezo was used for high-speed z-stack imaging. FRAP was performed essentially as described previously [Cells were imaged in L15 COeviously . Selecte2O) at 95°C for 90 min [2O prior to and post antibody incubation. Primary antibody labelling was either overnight at 4°C or 3 h at 37°C in 2% BSA-PBS at 1:250 dilution. Secondary antibody labelling was at 37°C in 2% BSA-PBS at 1:500 dilution for 2.5 h. Gels were washed with PBS 0.1% triton-X after both antibody staining steps. Nuclei were labelled with Hoechst 33342 dye in the final wash step. Gels were mounted in Ibidi μ-slide 2-well glass bottom #1.5 dishes (80286), which were pre-treated in poly-L-lysine or poly-D-lysine and imaged using Airyscan imaging. Primary antibodies used were mouse anti-acetylated tubulin , rabbit anti-rootletin , rabbit anti-cNap1 , mouse anti-γtubulin . For anti-γtubulin only, cells were fixed in 100% ice-cold methanol for 5 min prior to formaldehyde fixation. Secondary antibodies were Alexa 488, Alexa 568, or Atto 565 conjugates. The expansion factor was calculated from measurements of the gel diameter pre and post expansion, and from centriole size in final images when stained with anti-acetylated tubulin, giving values around 4.2 to 4.6. Calculation of the percentage of cells with cNap1 bridges between centrioles was done from 3 independent experiments, measuring a total of approximately 80 cells.U-ExM was as described in ,32,53. CS2A Fig). In standard immunofluorescence, anti-cNap1 staining closely matched cNap1-mScarlet-I fluorescent protein.cNap1 antibody was validated specifically in U-ExM by confirming that signal was removed by siRNA targeting cNap1 in comparison to non-targeting control siRNA  into the vector pcDNA 3.1, according to the manufacturer’s instructions. Briefly, primers containing complementary 15 bp extensions were designed in the TaKaRa Bio In-fusion online design tool. Both the vector and inserts were amplified by PCR with CloneAmp DNA polymerase. Amplified DNA length was verified by agarose gel electrophoresis. In-Fusion ligation was performed on gel extracted DNA using In-Fusion HD enzyme premix in a total volume of 5 μl at 50°C for 15 min. Clones were screened by Sanger DNA sequencing, restriction digest and microscopy after transfection into mammalian cells. The original cNap1 cDNA template was provided by Andrew Fry .LAMP-1) [Mito-cNap1 consists of an N-terminal fusion of a pair of mitochondria targeting sequences from cytochrome c oxidase subunit VIII (COX8) , separatLAMP-1) , to creaLAMP-1) , consistCROCC) was as previously described by [plus pools from Horizon Discovery . siRNA transfection was with RNAiMAX (Thermo Fisher Scientific), following the manufacturer’s instructions. Briefly, for 96-well transfections, cells were transfection with 25 nm of siRNA, and 0.25 μl lipofectamine per well. Cells were analysed either 48 or 72 h after transfection. The efficacy of both cNap1 and rootletin siRNA knockdown was confirmed by loss of fluorescence in cNap1-mScarlet-I and rootletin-meGFP cells.siRNA targeting rootletin/CROCC (gene name ribed by  and is aribed by  and was 4Cl in PBS (pH 7.4). Cells were permeabilised in 0.1% Triton in PBS and blocked in 3% bovine serum albumen (Thermo Fisher Scientific) in PBS. Antibodies used were: mouse anti-gamma tubulin GTU-88 , rabbit anti-GM130 , rabbit anti-PCNT , and rabbit anti-CEP135 . MitoTracker deep red (Thermo Fisher Scientific) was incubated at culture conditions at 100 nm for 30 s before replacing with fresh medium for imaging. LysoTracker was used at 75 nm, added directly prior to imaging, and kept in the imaging medium.Cells were fixed in either 4% paraformaldehyde in PBS pH 7.4 for 15 min or ice-cold 100% methanol for 5 min. Fixatives were freshly prepared. Paraformaldehyde was quenched in 0.1 M NHFCS-calibrated imaging was performed as described in ,57, to mApproximately 100 nm Alexa 568 dye in water was used to measure the size of the confocal volume , approximately 20 μm above the coverslip. Cellular FCS measurements were taken with four 10-s readings in the same position, in the cytoplasm away from any discernible structure. Wild-type U2OS cells without any fluorescent fusion protein were measured to determine the background fluorescence count rate in FCS. To make a calibration line for FCS-calibrated imaging, 12 cells overexpressing mScarlet-I-cNap1 were measured in the cytoplasm, taking care to avoid photobleaching. Cells were measured with both FCS and imaging (as below). Subsequent imaging of further transiently transfected mScarlet-I-cNap1 samples or endogenously tagged samples were with identical imaging settings.Imaging was in standard confocal mode (not Airyscan), using the same water immersion lens, laser power and pinhole diameter settings used for the FCS. Since cytoplasmic concentration was of primary interest in overexpressing cells, imaging settings were optimised for the cytoplasm (rather than for cNap1 cytoplasmic condensates), with pixel size and dwell time optimised for the dimmest sample. Image x, y, and z pixel size was 100 and 400 nm, respectively. In calibration cells, images were taken before FCS measurements to minimise the effect of photobleaching from FCS.I is the intensity and <> time averaging. The “base frequency” was set to 1,000,000 in Fluctuation Analyzer. Dye calibration measurements were fit to a 1-component model of diffusion with triplet-like blinking: k. N is the apparent number of molecules in the confocal volume, ɵT is the fraction of molecules in a nonfluorescent state, and τT their lifetime. The width of the confocal volume was calculated using: dye is the diffusion coefficient of the dye, and 521.46 μm2/s was used. This method gave a value of approximately 225 nm for the confocal volume lateral waist size. The confocal volume was calculated with: A is the Avogadro constant.FCS data analysis was performed in Fluctuation Analyzer software , to calcData was discarded if it showed significant bleaching or cell movements, visible in the count rate. The “base frequency” was set to 100,000 in Fluctuation Analyzer to calculate the autocorrelation function, and the offset was determined from measurements in wild-type cells not expressing fluorescent protein. To obtain the number of particles in the effective confocal volume (N), autocorrelation functions were fitted with a 1-component model of anomalous diffusion with fluorescent protein blinking: An experiment-specific conversion factor was used to convert relative fluorescence intensity measurements into absolute concentrations, by using images and FCS measurements taken in the same cells. The mean intensity of a 6 × 6 voxel area was taken centred on the FCS measurement position. Plotting image intensity versus absolute concentration in the cytoplasm from a population of cells gave a straight line from which the conversion factor was obtained, indicating use of the microscope in the linear range when correlating FCS and imaging measurements.A is the Avogadro constant, and Δp is pixel volume in x, y, and z. The number of cNap1 proteins per centrosome was calculated as the product of this N and cNap1 centriolar size. Centriolar cNap1 size was calculated from the number of manually segmented cNap1 positive pixels in each single confocal image. For each cell, the mean of both centrioles was calculated. This measure of centriolar size agreed well with an estimate from the expansion microscopy shown in Fig 2. Intensities at centrosomes were taken by manual segmentation to acquire the mean pixel intensity.Concentrations were converted into number of molecules per pixel using: 6/ml. After 18 h, this resulted in a population of cells in which beads were encapsulated inside cNap1 condensates. Medium was removed, washed twice with warm media, and then replaced with fresh, CO2-independent Leibovitz’s L-15 medium imaging medium. Cells without fluorescent bead encapsulation were excluded from the analysis. Time-lapse imaging of bead movement was with a Zeiss LSM 880 confocal laser scanning microscope operating in standard confocal mode with a 100× NA 1.46 oil immersion objective. The time interval was 10 s between frames, acquiring images with xy scaling of 0.082 μm and 0.393 μm z-scaling. A single z-slice was acquired. Two colour images were acquired, with filters 493 to 594 and 599 to 696 nm, to acquire images of both microspheres and mScarlet-I, respectively. Shorter time interval imaging  was also tested and resulted in a similar diffusion coefficient. The CTD truncation formed very few condensates which did not encapsulate beads, and hence was not measured. Tracks were acquired in 3 independent experiments for wild-type cells and 2 independent experiments for NTD and R188 mutants. Cytoplasmic measurements were obtained in untransfected cells. These samples occasionally showed beads with rapid directional movement rather than diffusion, and these beads were not included in the analysis.Cells were seeded overnight in μ-slide 8-well dishes . The next day, they were transfected with mScarlet-I-cNap1 cDNA using lipofectamine 3000, and simultaneously incubated with green fluorescent microspheres (PS Speck P7220 Thermo Fisher Scientific), at a bead concentration of 2.25 × 10α, where D is the particle diffusion coefficient and α is the diffusive exponent. α and D were obtained from fits in MSD Analyzer, and tracks were corrected for drift as described [2/s, calculated from immobile beads dried to the bottom of a well.Single-particle tracking was performed with the FiJi Trackmate plugin , using tescribed ,59. D waIntrinsically disordered regions in cNap1 were predicted using the Predictor of Naturally Disordered Regions (PONDR) algorithm .Statistical analyses and graphical representations were in GraphPad Prism 5 software. Statistical tests are listed in the figure legends.S1 FigA) Junction PCR screening of genomic DNA for insertion of mScarlet-I at the C-terminus of cNap1. Clone 7 was selected since it is homozygous for cNap1-mScarlet-I. The selected clone is indicated by *. The DNA ladder is Hyperladder 1 from Bioline. (B) Junction PCR of genomic DNA, screening for insertion of mScarlet-I at the C-terminus of cNap1. This shows a comparison of clone 7 with a heterozygous pool. Lanes are loaded in triplicate to exclude the possibility of lane-to-lane variability. (C) Junction PCR of genomic DNA, screening for insertion of meGFP at the C-terminus of CROCC. This shows a heterozygous clone. (D) Centrosome cohesion in wild type and cNap1-mScarlet-I/rootletin-meGFP cells, assessed by immunofluorescent staining of centrosomes with anti-PCNT antibody in a population of cells. Centrosomes were classed as split if 2 PCNT positive foci were present and separated by more than 1.6 μm, measuring in 240 and 329 wild-type and genome-edited cells, respectively. The images show maximum intensity projections of confocal Airyscan z-stacks. Scale: 10 μm. (E) Endogenously tagged cNap1-mScarlet-I co-stained with anti-CEP135. Scale: 1 μm. (F) Distance between the centroids of cNap1-mScarlet-I foci of the cell shown in Fig 1B during time-lapse imaging. (G) Distance between the centroids of cNap1-mScarlet-I foci during time-lapse imaging. Each colour plots a different cell. Data underlying this figure can be found in ((PDF)Click here for additional data file.S2 Fig(A) Validation of anti-cNap1 U-ExM staining with siRNA. U2OS cells were treated with either siRNA targeting cNap1 (left panel), or non-targeting siRNA (right panel), and then processed identically for U-ExM. (B) U-ExM expanded U2OS cells stained with anti-cNap1 (grey) and anti-acetylated tubulin (magenta). Each image is a different cell. Maximum intensity z-projections are shown. (C) U-ExM expanded hTERT-HPNE cell stained with anti-cNap1 (grey) and anti-acetylated tubulin (magenta). (D) U-ExM expanded U2OS cells stained with anti-rootletin (green) and anti-acetylated tubulin (red). Each image is a different cell. Scale: 200 nm throughout.(PDF)Click here for additional data file.S3 FigA) cDNA-based cNap1-mScarlet-I expression results in either centrosomal or cytosolic patches (red). Centrosomes are co-stained with gamma-tubulin (green), and centrosome position is indicated with arrows. White lines in the bottom panels denote nuclei. Scale bar: 4 μm. The histogram shows the number of mScarlet-I-cNap1 patches per cell in a population of 388 cells, acquired with automated imaging and analysis as detailed in Materials and methods. The xy graph plots mScarlet-I-cNap1 area against aspect ratio (long axis/short axis) in approximately 4,000 patches, where a circle has an aspect ratio of 1. (B) mScarlet-I does not form condensates when overexpressed (top panel—red), but cNap1 does (bottom panel—green). (C) FCS-calibrated imaging of cDNA-expressed cNap1-mScarlet-I. The dot plot shows the cytoplasmic concentration in cells either with or without cytoplasmic condensates. Each dot is a single cell. The dashed line indicates a concentration of 110 nm. The example image is coloured relative to concentration. (D) Live-cell time-lapse imaging of a single cytoplasmic mScarlet-I-cNap1 patch over minutes, showing viscous liquid-like shape changes over time. Scale: 1.5 μm. (E) Log–log plot of fluorescent microsphere movement inside cNap1 condensates. The graph plots the mean from 51 tracks. The diffusive exponent alpha has a value of 0.93 + 0.04 (goodness of fit adjusted R2 = 0.98), obtained using the MATLAB class MSD Analyzer [F) Half-bleaching FRAP of an mScarlet-I-cNap1 patch in the cytosol shows limited exchange over approximately 30 s. The bleached region is located at the bottom and images show successive indicated time points. Scale bar: 2 μm. (G) Mean squared displacement of fluorescent microspheres diffusing in cNap1 NT, R188, or wild type. The lines show weighted means (±SEM) from N = 51 (wild type), N = 40 (NT), and N = 20 (R188) tracks. (H) Right panel: dual colour time-lapse imaging of lyso-cNap1 (red) and LysoTracker (green). Scale: 5 μm. Left panel: dual colour time-lapse imaging of Lamp1-mScarlet-I  and LysoTracker (green). Time-lapse was performed over minutes as indicated. Scale: 5 μm. (I) Top panel: immunofluorescent imaging of mito-cNap1 (red), DNA , and MitoTracker (yellow). Bottom panel: immunofluorescent imaging of Golgi-cNap1 (red), DNA (blue), and anti-GM130 (green). The arrows in both panels denote a cell expressing either mito-cNap1 or Golgi-cNap1, respectively, and the stars denote untransfected control cells in the same image. Scale bars: 7 μm and 10 μm in the top and bottom panels, respectively. (J) Lyso-cNap1, Golgi-cNap1, and mito-cNap1 do not induce de novo rootlet formation, as detected by anti-CROCC staining. Scale: 5 μm. (K) Predicted cNap1 disorder in each residue according to PONDR VL-XT score [(XT score . A score(PDF)Click here for additional data file.S1 Raw Images(A) S1A (B) S1B (C) S1C.(PDF)Click here for additional data file.S1 Raw Data(XLSX)Click here for additional data file.S1 MovieA single slice of a z-stack is shown. Scale: 1 μm.(AVI)Click here for additional data file.S2 MovieA sum projection of a z-stack is shown.(AVI)Click here for additional data file.S3 MovieA sum projection of a z-stack is shown.(AVI)Click here for additional data file.S4 MovieMaximum intensity projections at 30 min time intervals in U2OS cells. Scale: 10 μm.(AVI)Click here for additional data file.S5 MovieFig 3D. Scale: 2 μm.Time frames are taken at 3-min intervals, related to (AVI)Click here for additional data file.S6 Movie(AVI)Click here for additional data file.S7 MovieMaximum intensity projections are shown. Scale: 10 μm.(AVI)Click here for additional data file.S8 MovieScale: 2 μm.(AVI)Click here for additional data file.S9 MovieA single z-slice is shown. Scale: 3 μm.(AVI)Click here for additional data file.S10 MovieA single z-slice is shown. Scale: 3 μm.(AVI)Click here for additional data file.S11 MovieA single z-slice is shown. Scale: 3 μm.(AVI)Click here for additional data file.S12 MovieA single z-slice is shown. Scale: 3 μm.(AVI)Click here for additional data file.


Staphylococcus aureus showed a consistently decreasing trend, while that with 3GC- and fluoroquinolone-resistant Escherichia coli significantly increased. Unlike the national rate, the rate of colonization with antibiotic resistant-Klebsiella pneumoniae did not increase and that of 3GC- and fluoroquinolone-resistant Pseudomonas aeruginosa significantly decreased. Stepwise implementation of core ASP elements was effective in reducing antibiotic use despite a lack of sufficient manpower. Long-term multidisciplinary teamwork is necessary for successful and sustainable ASP implementation.To optimize antibiotic use, the US CDC has outlined core elements of antimicrobial stewardship programs (ASP). However, they are difficult to implement in limited-resource settings. We report on the successful implementation of a series of ASP with insufficient number of infectious diseases specialists. We retrospectively collected data regarding antibiotic administration and culture results of all patients admitted to a tertiary care teaching hospital, Seoul National University Bundang Hospital (SNUBH), from January 2010 to December 2019. Trends of antibiotic use and antibiotic resistance rates were compared with those from Korean national data. Trend analyses were performed using nonparametric, two-sided, correlated seasonal Mann–Kendall tests. Total antibiotic agent usage has significantly decreased with ASP implementation at SNUBH since 2010. National claim data from tertiary care hospitals have revealed an increase in the use of all broad-spectrum antibiotics except for third-generation cephalosporins (3GC). In contrast, at SNUBH, glycopeptide and fluoroquinolone use gradually decreased, and 3GC and carbapenem use did not significantly change. Furthermore, the rate of colonization with methicillin-resistant IMPORTANCE Antimicrobial stewardship programs aimed to optimize antibiotic use are difficult to implement in limited-resource settings. Our study indicates that stepwise implementation of core antimicrobial stewardship program elements was effective in reducing antibiotic use in a tertiary care hospital despite the lack of sufficient manpower. Antimicrobial resistance (AMR) has been a global issue for decades. An increase in antibiotic use in the past has exerted selective pressure on susceptible bacteria, leading to the rise of AMR . PreviouClostridioides difficile infection  encouraged all hospitals in the United States to implement ASP and outlined the core elements of hospital-based ASP . Some honfection , 8. Howenfection .Here, we summarize the findings from a tertiary care hospital with limited resources where an ASP was successfully implemented for over a decade and analyze the effects of ASP on antibiotic use and AMR rates.P < 0.01; Table S1 in the supplemental material). Glycopeptide (P < 0.01) and fluoroquinolone  use gradually decreased, while third-generation cephalosporin , fourth-generation cephalosporin (P = 0.29), and beta-lactam/beta-lactamase inhibitor (P = 0.21) use did not significantly change. There was no significant change in carbapenem use, although use of the group 1 carbapenem ertapenem increased (P = 0.02). Total antibiotic use was lower in SNUBH data than in Korean National Health Insurance Service (NHIS) data. In addition, the use of every broad-spectrum antibiotic except 3GC continuously increased at other Korean tertiary care hospitals. Although glycopeptide and carbapenem use were higher in SNUBH data than in NHIS data, all types of broad-spectrum antibiotic prescriptions except for group 1 carbapenem showed no increase, and at least maintained a stable level, at SNUBH.Staphylococcus aureus significantly decreased . The rates of 3GC and FQ resistance increased in Escherichiacoli (P < 0.01), did not significantly change in Klebsiellapneumoniae (P = 1.00), and decreased in Pseudomonasaeruginosa (P = 0.01). The proportion of carbapenem-resistant Acinetobacter baumannii (CRAB) seemed to increase, but the trend was not statistically significant (P = 0.07).S.aureus (MRSA) colonization was consistently lower, and that of 3GC- and FQ-resistant E. coli colonization was higher, in the SNUBH data than in the Korean Antimicrobial Resistance Monitoring System (KARMS) data. National data revealed increasing trends for FQ-resistant K. pneumoniae and carbapenem-resistant P. aeruginosa (CRPA), but SNUBH data showed no significant changes in the resistance rates for these organisms. Although the carbapenem-resistance rate of A. baumannii in the SNUBH data was 40% lower than that in the KARMS data in 2010, the difference between the two decreased over time.The trend of methicillin-resistant E. coli increased like that in the nationwide data, the AMR rates of other important pathogens did not increase at SNUBH.The ASP team at SNUBH not only introduced new activities one by one but also ensured that they were continued. With the implementation of strong ASP at SNUBH, not only total antibiotics use but also broad-spectrum antibiotic prescriptions except for group 1 carbapenem showed no increase compared to nationwide trends. Although AMR rates of There are a few studies on the application and outcomes of ASP implementation. Although most of them were single-center studies, they showed that the use of antibiotics  and the rates of CRPA and CRAB decreased after ASP implementation , 11. HowFor implementing new ASP activities, interdepartmental teamwork involving pharmacists, laboratory medicine physicians, and infection control nurses, as well as support from the senior leadership, are vital. To increase clinician compliance, we gradually expanded ASP activities based on small successes. For example, the duration of routine perioperative antibiotic prophylaxis was changed to less than 5 days, and this was reflected in clinical decision support systems, as shown in a previous study . After cThe designation of an ID pharmacist as a co-leader was important in maintaining the expanded ASP. The addition of new activities alongside the continuation of existing ones led to a steady decrease in total antibiotic use. Also, in the case of ASP activities in which the ID pharmacist was the lead, physician adherence was maintained over 79% through continuous monitoring of the ASP performance , 19. BecIn this study, the proportion of MRSA decreased, which might have been related to decreased FQ use, as mentioned in a previous study . AlthougBeta-lactam/beta-lactamase inhibitors and 3GC were used to treat most of the common infections, such as hepatobiliary and gastrointestinal tract infection, skin and soft tissue infection, urinary tract infection, and pneumonia. Although these antibiotics were not restricted or monitored if used for less than 2 weeks, their use did not significantly increase at SNUBH, possibly because education about the optimal empirical antibiotic therapy for common infections was periodically provided to physicians. Since the ASP team cannot monitor the prescription of each antibiotic, ASP activities with a low work burden, such as education, should be consistently provided.E. coli). Replacing group 2 carbapenems with ertapenem for empirical antibiotic therapy in critically ill patients might help reduce CRPA and CRAB colonization; however, previous studies on this strategy have obtained conflicting results , and the requirement for obtaining written informed consent was waived due to the retrospective nature of the study.The ASP implemented at SNUBH are summarized in The first action performed as part of the ASP was the preauthorization of restricted antimicrobial agents. Beginning in August 2011, as the number of ID physicians increased, we implemented a system in which ID physicians were automatically consulted before restricted antibiotics were prescribed. At the same time, when blood culture results indicated bacteremia, ID physicians were automatically consulted and the ASP team recommended the type and duration of antibiotics to be administered .After the establishment of these activities, more aggressive ASP activities were gradually added with the consent of the clinicians. For example, the two ID pharmacists (one full-time and one part-time basis), as ASP co-leaders, performed additional activities to reduce the use of redundant anti-anaerobic antibiotics and intravenous FQ , 19. NotIn recent years, with the aim of long-term reduction of antibiotic prescription, ID specialists regularly provide lectures to residents and clinicians about antibiotic use for common infections. Additionally, a booklet on antibiotic dosage according to renal function was created and distributed to clinicians.S.aureus, E.coli, K.pneumoniae, P.aeruginosa, and A.baumannii, were reviewed using the WHONET program, which collects the types of clinical samples and bacteria, and the AMR information of all culture results (S. aureus (oxacillin), E. coli and K. pneumoniae (3GC [cefotaxime or ceftazidime] and FQ [ciprofloxacin]), and P. aeruginosa and A. baumannii .Data regarding antibiotic use were extracted from a clinical data warehouse , particu results . To avoi results . The proTo compare antibiotics use at SNUBH with that at the national level, we reviewed the NHIS database. The NHIS is the sole health insurance provider in Korea and is responsible for all hospitalization-related costs. We randomly extracted data regarding 40% of all antibiotic-related claims in patients admitted to Korean tertiary care hospitals from 2010 to 2019. Tertiary care hospitals specialize in high-quality medical treatment for severe diseases, as designated by the Ministry of Health and Welfare, with at least 20 departments and 500 beds . AntibioP < 0.05 indicating a statistically significant increase or decrease. The magnitude of change per year was estimated using Sen’s method (The trends of antibiotic use and AMR rates were analyzed using nonparametric two-sided correlated seasonal Mann–Kendall tests, with s method . Statist


Infection is a major threatening side effect after total hip arthroplasty (THA) that its management is so difficult and is accompanied by different complications. The aim of this study was to determine the outcomes of patients underwent 2-staged THA after a course of antibiotic therapy. It was an observational prospective study performed during 2009 and 2019. We managed 51 patients with infected THA using a method in which antibiotic prophylaxis was performed after THA. We followed the same protocol for treatment of patients included 2-staged revision: in first stage, removal of infected instruments were performed and insertion of a hand-made antibiotic-cement spacer was done until erythrocyte sedimentation rate and CRP were normalized. In second stage, an un-cemented prosthesis was re-implanted in femoral side and post-operative IV antibiotic were administered for a week. Patients were monitored for about 15 months. Data were analyzed. There were 3 patients developed recurrent infection required girdlestone due to the aging. One of them needed to remove implant and 2 other with 3 times of re-infection were treated by antibiotic therapy. Other 10 cases were treated first by re-changing the cement. The rate of successful treatment was 78.4% (40 of 51) after the primary surgery and antibiotic therapy. This rose to 92.1% (47 of 51) following more debridement and antibiotic therapy. The merging of staged surgical debridement, using spacer of cement-antibiotic and re-implant beside 1-week intravenous antibiotic therapy, leaded to appropriate early outcomes in this series. Profound infection followed by total hip arthroplasty (THA) is a destructive side effect, which encountered orthopedics in a challenge for better treatment planning.–4 There are different approaches for treating infections consisted of 1-stage revision, long-term antibiotic suppression treatment, multiple debridement with implant conservation and 2-step revision.–6 The most used technique for management of infections is staged re-implantation of prosthesis after systemic antibiotic therapy. Despite of lower rate of infection after primary THA (<1%),,5,6,8–11 the rate of infection after 2-step THA is reported between 3.2% and 13%.–15 Recent guidelines have mentioned the improvement in diagnostic approaches of pre-operative infections.–19Using antibiotics has been changed in different aspects such as kind and family of antibiotics, sensitivity, and resistance pattern to various antibiotics. Despite of success in 1-step revision and debridement in selected cases,,20 the standard preferred surgical technique in management of prosthetic hip infections (PHIs) is staged arthroplasty included removing all components beside debridement of infected tissue and antibiotic therapy followed by re-insertion of new prosthesis at final stage. The use of an antibiotic-loaded cement spacer (ALCS) has been changed to a frequent technique. Using ALCS can used for a temporary hip joint implant and also a technique for direct antibiotic delivery. The role of ALCSs in the treatment of PJI is 2-fold: mechanical and pharmaceutical. The ALCS fills the gap, which occurs after the removal of the infected prosthesis and surgical debridement. Therefore, it prevents extensive scarring, preserves the quality of the bone and the length of the limb and facilitates final prosthesis implantation.There is no consensus for necessity of post-operative antibiotic therapy, currently.In this study, we determined the outcomes of patients underwent 2-staged THA after a course of antibiotic therapy.This was a prospective observational investigation. We evaluated patients who were underwent 2-stage revision of a THA because of peri-prosthetic infection. Moreover, we assessed patients with revision surgery due to aseptic loosening. All patients had at least 15 months’ follow-up. We included patients by census-manner sampling and all eligible patients with sufficient data were enrolled.During the study period, 51 revision THA procedures were performed by the senior author of the article (orthopedic surgeon) in Erfan hospital. From patients enrolled, 12 revisions were due to aseptic etiologies, and the left for a periprosthetic hip infection (loosening). Two stage revision surgery was performed for infected joints. Therefore, subjects were first underwent removal surgery of components and temporary antibiotic spacer were placed and simultaneously intravenous antibiotic were administered for at least 6 weeks. Then, reimplant was done if signs of infection were not resumed. Infected re-implantation THA were performed at the authors’ center. In all subjects, antibiotic treatment during surgery and 24 hours after it was prescribed. Primary demographic data and history of diseases are listed in Table PHI was determined by the existence of at least 1 of the next clinical criteria:A purulent sinus correlated with the joint;Perioperative evidence of suppuration; or We defined “Persistent infection” as the existence of PHI after first-stage operation. It was the primary outcome. “Re-infection” was defined as PHI which happened after the finishing of SEA and antibiotic treatment. It was the secondary outcome in this study. Re-infection of the 2-stage revision THA was determined as profound infection of the hip prosthesis after first operation which needs re-operation to be treated. If seromas or superficial infection were increased by the deeper layers or to the hip prosthesis, it was considered as failure.Laboratory or pathologic findings of infection or isolation of pathogens from 2 exclusive samples of articular aspiration or other samples.After finishing intravenous antibiotic therapy within time of cement spacer placement, all subjects were underwent re-implantation. Infectious disease consult was done for giving longer period of antibiotic treatment after re-implant operation. At the centers of this study, there was no indications for prescription of oral antibiotic post-THA operation. If in any cases, oral antibiotic was started postoperatively, it was resumed for at least 14 days empirically and based on probable positive culture.After re-implant operation, we followed up subjects in 4 time points: 1 week, 3 weeks, 3 and 6 months and then every year for at least 2 years. In each visits, they were evaluated for history and physical examination for finding signs of erythema, pain or discharge and dehiscence and others. Moreover, AP and lateral radiographs were taken in each visits for component placement or radiolucency assessment. If there was any evidence of re-infection, complete sepsis workup panel were performed.All cases were asked to fill written informed consent for participation in the study. All steps of the study was in line with ethical committee of hospital and Helsinki declaration.t test for comparing before and after operation factors. Linear regression test was applied in finding predictors and Kaplan–Meier test used to estimate survival rate at 5 years. SPSS  software was used in all analyses.Descriptive statistical analyses was used to show mean and standard deviation of quantitative variables. We used paired sample Fifty-one patients was included in the study. Most of them were female (68.6%) and the mean age of them was 69.23 ± 15.79 years old. The mean of BMI was 29.89 ± 4.56. Other baseline data is listed in Table After 2-staged revision operations, 40 cases had no problem until follow-ups. Eleven cases had persistent infection (27.5%) by severe hip pain, high CRP levels and poor wound improvement which needed to change their cement. Two cases (5%) needed 3 times for changing the prosthesis. Re-infection rate was 3 of 51 cases. Of these cases, one needed to removal of implant and 2 others managed by antibiotic therapy. List of infecting microorganisms is shown in Table The success rate of infection treatment, which is determined as the elimination of infection, was 78.4% (40 of 51) after the first operation and antibiotic treatment. This increases to 92.1% (47 of 51) after more debridement and antibiotic therapy.None of other subjects received debridement, revision operation or antibiotic therapy for PHI within the follow-up period. Therefore, 47 patients had no infection at the final follow-up. The mean preoperative hip score was 8.5 ± 2.1 (6.2–9.1) which was improved at last follow up to 15.2 ± 1.7 (13.3–16.4). There were no complications except infection as mentioned earlier until last follow up.,5,6,8 remarkable percent of patients could lead to re-infection after 2-staged re-implant surgery. Eradication of such infections is so hard and have a high cost about 100 dollars per each revision. So, finding easy, cost-effective and available approaches to increase outcomes of patients to decrease re-infection rate after 2 stage revision THA, is necessary. In our investigation, the success rate was nearly 92%, which was better or equal to other studies.,25–31In this investigation, we demonstrated that short-term results of 2-staged revision of infected hip prosthesis is a safe method with low complications and high rate of success. Despite of lowing the rate of infection risk after primary THA to about 1%,,33 The important question, regarding a spacer’s efficacy in the treatment of PJI is, whether a spacer can achieve a certain antibiotic concentration in vivo or not. Downes et al reports negligible antibiotic levels beyond the fifth postoperative day in an in vivo study. In a systematic review, the in vivo antibiotic concentrations were measured during the first week after spacer implantation (7 studies), on the day of spacer removal or both. It was demonstrated a large increase in the antibiotic concentration on the first day after implantation, and then a rapid decrease during the next days. Furthermore, the antibiotic concentration at 6 weeks to 4 months after spacer removal, although lower than during the first week, was always higher than the MIC for the causative bacteria. In another in vivo study, it was shown that antibiotic washout from ALCSs was at least 4 months by loading 3.6 g of tobramycin and 1 g of vancomycin per 40 g package of bone cement. The efficacy of a spacer as a local carrier of antibiotics is based on its ability to reach high concentrations of the antibiotic at the site of infection for a suitable period of time.Previous investigations have shown that the antibiotic release from ALCS is great and constant.In another study, McDonald and his colleagues evaluated 82 patient with PHI after SEA and showed that re-infection happened in 3 of 7 subjects with retained cement. They also reported only 8 re-infection within the other patients (75 subjects) with cement removal.,18Using antibiotic perioperatively in 2-stage revision arthroplasty has been negotiated for its duration, special agents, and their impact on special organisms (such as methicillin-resistant Staphylococcus aureus). The suitable duration of parental antibiotic treatment is a controversial topic. This success rate can be achieved by radical debridement. In a study of 31 consecutive infected THAs, McKenna et al explained that there was no recurrence in infection. They indicated that shortening postoperative antibiotic therapy leaded in infection treatment. Bassetti et al evaluated the duration of parenteral versus oral antibiotic (linezolid). They showed that both of them were effective in orthopedic procedures. Moreover, Oussedik et al demonstrated that linezolid is effective for the oral treatment of infected joint arthroplasties.Hsieh and his colleagues showed that antibiotic washout from ALCSs, was abrupt and great and the antibiotics (vancomycin and aztreonam) retained their therapeutic ability for several months.,27 It is sometimes preferred to monitor the CRP level as an index of the response to treatment. This way seems to be more practical method than continue the fixed interval. Despite of not being specific to infection the CRP measurement, it is sensitive, accessible and most valuable when serially monitored. The use of a shortened antibiotic therapy between stages is not new.,39 Stockley et al reported a success rate of 87.7% (100 of 114) in infection control with depot antibiotic treatment in the absence of long-term antibiotic therapy in PHI. Nelson et al, in a study of 28 patients who had an infection at the site of a hip or knee arthroplasty, reported that patients who were managed with the implantation of antibiotic cement beads in conjunction with <5 days of parenteral antibiotic therapy, and those who were managed with 6 weeks of intravenous antibiotic therapy, had comparable results.The optimal time of the re-implant operation is yet controversial. Prior investigations have suggested different intervals between steps, ranged from 1 month to 1 year.Data from our prospective study showed that short-term antibiotic therapy in SEA could increase the rate of treatment failure in patients with PHI. Using effective staged surgical debridement, spacer of cement-antibiotic usage with monitoring erythrocyte sedimentation rate and CRP levels and performing re-implant operation with shortened IV antibiotic treatment, has led to better outcomes in this series of revised chronic hip joint prosthetic infection. Our results suggested that the routine usage of protracted antibiotic treatment courses might be beneficial. Performing large-scale, prospective, longer follow-up and randomized clinical trial to approve the findings is recommended.Conceptualization: Afshin Taheriazam.Data curation: Amin Saeidinia.Investigation: Afshin Taheriazam, Amin Saeidinia.Project administration: Afshin Taheriazam.Writing – original draft: Afshin Taheriazam, Amin Saeidinia.Writing – review & editing: Afshin Taheriazam, Amin Saeidinia.


As it is known by everyone, CAPES governs graduate programs in Brazil. It certifies and assesses the programs, classifying them in relation to their excellence at an international level and as far as readership is concerned. The main goal of strictu sensu graduate programs (academic master's degree and PhD) is to train researchers and to create knowledge which can impact science. For that, the first assessment parameter, besides the flow of student titles, is the impact of the publications resulting from the programs' dissertations and theses.At a first moment, this criterion caused much discomfort among advisors and students, and also many questions: would it be more important to create a publication in a national circulation journal, in Portuguese, which would reach most otorhinolaryngologists in Brazil, or publish in international journals, in English, where but a few Brazilians would read? We believe that both publications are important, for otorhinolaryngologists who work as physicians and who also do research. We have two publications which travel on this road: the Brazilian Journal of Otorhinolaryngology, indexed at Medline and at the JCR (awaiting the impact index), and the International Archives of Otorhinolaryngology - indexed at Scielo.There are no doubts that this CAPES pressure brought about results, and deserves praise. Brazil improved significantly in the quantity and quality of its publications, today holding the 13th position concerning publications in the world, with a growth curve much higher than that of other countries. Behind this leap in publications are the graduate programs, governed and stimulated by CAPES. Otorhinolaryngology also increased, very much, the quantity and quality of publications, having seen the high and growing number of papers presented in meetings and published in journals, many of them stemming from dissertations and theses, gaining the well-deserved respect and acknowledgement of the world otorhinolaryngology.On the other hand, otorhinolaryngology has been much harmed by the system utilized to classify the publications. CAPES adopted the so called Qualis system, which classifies them according to their index of impact  within the many fields of knowledge. Otorhinolaryngology belongs to Medicine III - a surgery field, in which Qualis is currently classified into: A1: impact > 3.30, A2 > 2.63, B1 > 1.51, B2 > 0.90, B3 > 0.01, B4: indexed at MedLine, Scielo, LILACS, B5 and C. Although it is a very transparent and honest system, it does not involve the particularities of the surgical field, which is very heterogeneous. Otorhinolaryngology journals have a mean impact of 1.20, which is the lowest in Medicine III .Only one ENT journal is Qualis A2 . Our best and best-known journals, where the most renowned international ENT researchers publish, are B1 and B2. Since they are considered of second line by CAPES, it harms the concept of our graduate programs. However, we should not be compared to researchers from ophthalmology or urology, but rather with our own ENT peers. We have no doubts that we publish in the main journals of our specialty, but one single Qualis to the entire Medicine III is not sensitive to this fact. It is a method which does not fulfill its goal. It compares the otorhinolaryngology researcher with all of those in surgery, but not our peers. The sample is heterogeneous and the conclusions are wrong.The persistence and idealism pushes us to continue publishing and growing in our specialty, but it is important to state the limitations of the graduate programs' coordinators vis-à-vis the criteria adopted by CAPES.


Chronic sclerosing sialadenitis is commonly regarded as a manifestation of IgG4-related disease. We previously found that a high IgG4 expression or IgG4-related disease could accompany nonspecific sialadenitis, whereas chronic sclerosing sialadenitis was not directly associated with IgG4-related disease. Our previous findings lead us to hypothesize that these inflammatory conditions of the submandibular gland signify a continuous progression of disease rather than different disease entities. We, therefore, aimed to determine the presence of IgG4-positivity and genuine IgG4-related disease in a cohort of 165 submandibular gland specimens from patients who underwent surgery due to chronic nonspecific sialadenitis or sialolithiasis. To do so, we re-evaluated histopathological features and divided samples into three groups: (A) nonspecific sialadenitis without known sialolithiasis, (B) sialadenitis with sialolithiasis, and (C) sialolithiasis without sialadenitis. We performed immunohistochemical staining for IgG4, IgG, and CD31, and assessed the Boston consensus statement criteria for IgG4-related disease in IgG4-positive samples. We also reviewed patient records and supplemented follow-up data with a questionnaire among patients with IgG4-positive samples. IgG4-positive plasma cells (range 1–344) were found in 131 samples. Among these, 19 samples were classified as IgG4-positive (≥70 IgG4-positive plasma cells/high-power field). Two IgG4-positive samples were histologically highly suggestive of IgG4-related disease, but only one had a clinically confirmed diagnosis of IgG4-related disease. Our results indicate that patients with sialadenitis and sialolithiasis often present with IgG4-positive lymphoplasmacytic infiltrates, but exceedingly rarely present with genuine IgG4-related disease. In sialolithiasis without sialadenitis, IgG4-positive plasma cells are often absent or appear in small numbers. These results support our hypothesis of a continuum of disease, and indicate that progressive inflammation of the submandibular gland leads to the development of more specific pathological features over time. In the last decade, a specific form of submandibular sialadenitis, known as chronic sclerosing sialadenitis or a Küttner tumor, has often been regarded as a presentation of IgG4-related disease of the submandibular gland3. However, few previous patient series exist to support this conclusion, specifically among Western countries4.Immunoglobulin G4 (IgG4) related disease often manifests at sites in the head and neck frequently affecting the submandibular gland5. Another significant finding was the presence of elevated numbers of IgG4-positive plasma cells in specimens associated with sialolithiasis and nonspecific forms of sialadenitis. This phenomenon had not been described previously7.Contrary to current consensus, our previous results from a Finnish patient cohort revealed that chronic sclerosing sialadenitis is rarely associated with IgG4-positive plasma cell infiltration. Among 51 patients diagnosed with chronic sclerosing sialadenitis, only two presented with genuine IgG4-related disease. Furthermore, these two specimens did not entirely fulfill the histopathological criteria for chronic sclerosing sialadenitis, but were instead classified as nonspecific sialadenitisThe finding that IgG4-positive plasma cell infiltrates were not strictly found in chronic sclerosing sialadenitis alone raised the question of whether these inflammatory conditions of the submandibular gland could form part of a disease continuum. Specifically, it remains unclear if nonspecific forms of sialadenitis develop features characteristic of chronic sclerosing sialadenitis or even IgG4-related disease over time. Another area of interest speculates that IgG4 plays a role in this proposed continuum of disease.In this study, we examined the occurrence of IgG4-positive plasma cell infiltrates and other features characteristic of IgG4-related disease in a comprehensive cohort of submandibular gland samples from patients diagnosed with nonspecific sialadenitis, sialolithiasis, or both.We retrospectively identified patients diagnosed with nonspecific sialadenitis and sialolithiasis from among all patients who underwent submandibular gland surgery between 1 January 2000 and 31 December 2012 in the Helsinki University Hospital catchment area, comprising 1.6 million inhabitants. To do so, we searched the Q-pati database used in the Department of Pathology (HUSLAB) for a diagnosis of sialadenitis or sialolithiasis, and included all patients who underwent submandibulectomy during the study period for histopathologically confirmed nonspecific sialadenitis, sialolithiasis, or both. Patients with diagnoses of Sjögren’s syndrome, sarcoidosis, lymphoepithelial sialadenitis and granulomatous sialadenitis were not included in this study. Tissue samples and hematoxylin and eosin (H&E) stained slides for each sample were acquired from Helsinki Biobank. The local research ethics committee approved the study design, and we received institutional permission to conduct this study (HUS/967/2017).Clinical patient data including sex, age at the time of surgery, and the preoperative diagnosis were collected from the Helsinki University Hospital patient records and pathology reports. Specimens were divided into three groups based on the preoperative diagnosis and the final histopathological diagnosis: (A) nonspecific sialadenitis without known sialolithiasis, (B) sialadenitis with sialolithiasis, and (C) sialolithiasis without sialadenitis. A mild, periductally located inflammatory infiltrate was included in group C. The presence of a sialolith was determined based on either the clinical history or the finding from the histological specimen.9. The degree of fibrosis and density of the inflammatory infiltrates were scored, and presence of acinar atrophy was assessed. All researchers evaluated the samples independently, and if the scores differed between researchers the assessment was made in tandem.Three researchers including two experienced pathologists , evaluated all histological H&E-stained submandibular gland samples and assessed the histopathological criteria for IgG4-related disease as follows: lymphoplasmacytic infiltrates, presence of lymphoid follicles, storiform fibrosis, obliterative phlebitis, and eosinophiliaThe formalin-fixed submandibular gland samples were cut into 4-µm-thick sections and stained for IgG4 using a mouse monoclonal anti-human IgG4 antibody . IgG4-positive specimens with plasma cell infiltrates with ≥70 IgG4-positive plasma cells/high-power field  were further stained for IgG using a rabbit polyclonal anti-human IgG antibody  and for CD31 using a mouse monoclonal (Clone JC70A) anti-human CD31 antibody . Deparaffinization and antigen retrieval were performed in a Pre-Treatment module  using a pH 9 retrieval solution  for 15 min at 98 °C. The sections were then stained in an Autostainer 480S (LabVision) using the EnVision Flex Detection System . Samples were treated with EnVision Flex peroxidase-blocking reagent (SM801) for 5 min. All slides were incubated with mouse monoclonal IgG4 antibody (1:100) for 1 h and the IgG4-positive specimens (≥70 IgG4-positive plasma cells/HPF) were incubated with rabbit polyclonal IgG antibody  and mouse monoclonal CD31 antibody (1:50) for 1 h. Appropriate dilution and incubation time was optimized for each antibody, Dako REAL Antibody diluent was used for antibody dilution. Subsequently all slides underwent 30-min incubation with peroxidase-conjugated EnVision Flex/HRP (SM802) rabbit/mouse (ENV) reagent. Slides were visualized using DAB chromogen  for 10 min. Mayers hematoxylin  was used for counterstaining.5 and were included for further study and assessment of genuine IgG4-related disease. To do so, all IgG4-positive specimens were additionally immunostained for IgG, whereupon the IgG4/IgG ratio was calculated after determining the number of IgG-positive plasma cells at three HPFs. For the IgG4-positive samples where obliterative phlebitis could not be excluded based on histological evaluation, CD31 staining was performed. CD31 staining was used instead of elastic stain to assess obliteration of venules as well.Based on the IgG4 staining, samples were classified as either IgG4-positive or IgG4-negative by counting the plasma cells immunopositive for IgG4 at three different HPFs (x40) in each specimen. We used the same cut-off point for IgG4-positivity as in our previous study: specimens with ≥70 IgG4-positive plasma cells/HPF were categorized as IgG4-positive8.IgG4-postitive specimens were assessed for the probability of genuine IgG4-related disease according to the Boston consensus statement criteria. The Boston consensus statement criteria for the diagnosis of IgG4-related disease in the salivary glands consist of ≥100 IgG4-positive plasma cells/HPF, an IgG4/IgG plasma cell ratio ≥40%, and at least two of three histopathological features. These features are a strong lymphoplasmacytic infiltrate with lymphoid follicle formation; fibrosis arranged, at least focally, in a storiform pattern; and obliterative phlebitis, which, however, is sometimes absent in the salivary glandsFollow-up data for patients with IgG4-positive samples were gathered from patient records and supplemented with a questionnaire. The questionnaire was sent to Finnish-speaking patients with IgG4-positive tissue specimens to determine if any further symptoms related to the salivary glands or other conditions developed following a submandibulectomy. Patient records were reviewed, and the questionnaires were sent to patients in October 2021. The follow-up period was calculated from the time of surgery until the date the questionnaire was returned or the last date when patient records were reviewed. For patients who died, the follow-up period was calculated from the time of surgery until the time of death.5.During the study period, a total of 395 submandibulectomies were performed in the Helsinki University Hospital catchment area. Among all patients undergoing submandibulectomy during this period, 169 patients (43%) received a histopathological diagnosis of nonspecific sialadenitis, sialolithiasis, or both. From these, we included a total of 165 patients whose samples were available for further study  were assigned to group A , 56 patients (34%) were assigned to group B , and 50 patients (30%) were assigned to group C . From all patients, 80 (48%) were male and 85 (52%) were female. The median age of patients was 47 years (range 8–86) at the time of surgery.The evaluation of H&E-stained samples revealed some degree of lymphoplasmacytic infiltrates in all 165 samples, ranging from a localized, mild inflammatory infiltrate to strong lymphoplasmacytic infiltrates affecting the entire gland. In 22 samples (13%), the inflammatory infiltrate could be described as strong. Lymphoid follicles were observed in 57 samples (35%) and acinar atrophy in 47 (28%). Some degree of fibrosis was observed in 107 samples (65%), and in 25 (23%) of these the fibrosis could be described at least focally as storiform. Obliterative phlebitis was not conspicuous upon H&E staining in any of the specimens. Table We observed IgG4-positive plasma cells in 131 specimens (79%) with a median of 14 IgG4-positive plasma cells/HPF (range 1–344 IgG4-positive plasma cells/HPF). Table The histological and immunological evaluation revealed two patients whose tissue samples were histologically highly indicative of IgG4-related disease Table . In the At the time of the submandibulectomy, the patient presented with swelling of both submandibular and parotid glands, and was preoperatively diagnosed with chronic sialadenitis before undergoing excision of the left submandibular gland. A diagnosis of IgG4-related disease was established 2 years later when the patient presented with pancreatic symptoms. The patient received prednisolone, but persistently showed elevated serum IgG4 levels despite treatment. Eleven years following the IgG4-related disease diagnosis, the patient presented with a suspected IgG4-related pseudotumor of the retroperitoneum. The abdominal mass did not respond to prednisolone treatment and was surgically removed. The lesion was postoperatively identified as a liposarcoma of the retroperitoneum.This patient experienced prolonged swelling of both the submandibular and parotid glands, and upon sialendoscopy sialoliths were observed in all of the major salivary glands. The patient received several prescriptions for antibiotics and corticosteroids, but ultimately the right submandibular gland was excised due to persistent symptoms. Following the submandibulectomy, the patient no longer received corticosteroid treatment. Furthermore, the patient records made no mention of IgG4-related disease in any other organ or data on the serum IgG4 levels. Clinically and histologically, the patient received a diagnosis of sialolithiasis. Nine years after the submandibulectomy, a nasal polyp was removed from the patient at our clinic, shortly after which the patient died. During the 9-year period following the submandibulectomy through the patient’s last hospital visit, no follow-up visits regarding the salivary glands were registered.According to the patient records, none of the 17 remaining patients with IgG4-positive tissue samples showed signs or symptoms indicative of IgG4-related disease.The questionnaire regarding additional salivary gland symptoms was mailed to 12 of the 19 patients with IgG4-positive specimens. Among the remaining patients, five died during follow-up and two were not Finnish-speaking. In total, 10 of 12 patients (83%) responded to the questionnaire.The questionnaires revealed symptoms in other salivary glands in three patients. Symptoms included mild intermittent pain and transient swelling during mealtimes, but none of these patients sought treatment for the symptoms reported. The median follow-up time among the 19 patients with IgG4-positivity was 15 years (range 2–19 years).n = 131) of samples. Quite often (19 of 131), the amount of IgG4-positive plasma cells reached the cut-off point for IgG4-positivity (≥70/HPF). Furthermore, two IgG4-positive samples showed histological and immunological features highly indicative of IgG4-related disease8. Of these two patients, one received a diagnosis of IgG4-related disease confirmed following submandibular gland surgery. IgG4-positivity was most frequently found in samples with sialadenitis, both with and without associated sialoliths. In contrast, the absence or only small amounts of IgG4-positive plasma cells were most frequently registered in tissue samples accompanying sialolithiasis without sialadenitis (group C). These results support our previously hypothesized continuum of disease5. That is, as opposed to the early stage of disease, more severely affected salivary glands frequently showed more specific pathologic features associated with chronic sclerosing sialadenitis or even IgG4-related disease, such as IgG4-positive plasma cell infiltrates, storiform fibrosis, glandular atrophy, and lymphoid follicles.In our cohort of 165 patients diagnosed with sialadenitis, sialolithiasis, or both, IgG4-positive plasma cells were observed in the majority  based on the presence of inflammatory infiltrates and sialoliths. IgG4-positive plasma cells ranging from 11 to 327/HPF were most frequently identified in specimens from group B, where 70% of the samples showed ≥11 IgG4-positive plasma cells/HPF. In the two other groups (A and C), IgG4-positive plasma cells ranging from 11 to 344/HPF were found in 39% and 24% of the samples, respectively. Previous studies rarely identified IgG4-positive plasma cell infiltrates in nonspecific sialadenitis or sialadenitis with sialolithiasis. Thus, this study is among the first to report the presence of IgG4-positive plasma cells in a large cohort of patients diagnosed with nonspecific sialadenitis or sialolithiasis. Previously, Harrison et al. reported a series of 129 cases of chronic sialadenitis, where >50 IgG4-positive plasma cells/HPF were observed in only three samples. In addition, Harrison et al. identified IgG4-positive plasma cells locally around dilated ducts7.In our cohort, the presence and quantity of IgG4-positive plasma cells correlated with the degree of inflammatory infiltrates. All IgG4-positive samples except those belonging to group C showed either strong or moderate lymphoplasmacytic infiltrates Table . Accordi5. The incidence of IgG4-related disease in this patient series agrees with findings from other retrospective studies. Specifically, Harrison et al. found no cases of genuine IgG4-related disease in their cohort of 129 patients with submandibular sialadenitis, while Gunasekara et al. identified three cases of actual IgG4-related disease in their cohort of 137 patients originally diagnosed with chronic sialadenitis19.In our cohort, one of 165 patients had a confirmed IgG4-related disease manifesting in the salivary glands and the pancreas. In another patient with sialolithiasis, the histology revealed findings highly suggestive of IgG4-related disease. But, the patient record revealed no disease manifestation in other organs or any clinical signs of IgG4-related disease during follow-up. IgG4-related disease can thus exist in nonspecific sialadenitis and sialolithiasis, although such a finding is exceedingly rare. In our previous study, we examined the relationship between chronic sclerosing sialadenitis and IgG4-related disease in a cohort of 51 patients, identifying only two patients with genuine IgG4-related disease. Furthermore, roughly one-third of patients originally diagnosed with chronic sclerosing sialadenitis appeared histologically more compatible with a diagnosis of nonsclerosing chronic sialadenitis. This also held for both patients with confirmed IgG4-related diseaseWhile IgG4-positive plasma cells were identified in 131 samples, a significant number of these samples exhibited only small or negligible amounts of IgG4-positive plasma cells (range 1–10 IgG4-positive plasma cells/HPF) Table . Further22. Currently, therefore, it would be difficult to collect a similar patient series. In addition, in our study, we achieved a long follow-up period for IgG4-positive patients, since follow-up from both patient records and questionnaires continued until October 2021. One limitation regarding the follow-up data is that patients who underwent submandibulectomy due to nonspecific sialadenitis and sialolithiasis did not receive regular follow-up care. Thus, our follow-up relies on a questionnaire and a review of patient records. However, we must note that we had access to all hospital records within the entire catchment area. A second limitation lies in the lack of data on serum IgG4 levels in patients whose tissue samples presented high numbers of IgG4-positive plasma cells, since the serum IgG4 measurement was not a routine practice among patients with sialadenitis. Currently, we recommend serum IgG4 measurement for those patients whose tissue samples revealed high levels of IgG4-positive plasma cells.The samples for this study were collected over a 12-year period from 2000 through 2012. One strength of our study is that we could gather a large number of samples with sialolithiasis and mild inflammatory lesions, since the standard treatment for sialolithiasis at our institution consisted of submandibulectomy during the early phase of the study period. Over time, the treatment of sialolithiasis shifted toward sialendoscopy with minimally invasive procedures, enabling gland preservationIn conclusion, lymphoplasmacytic infiltrates in the submandibular glands affected by sialadenitis and sialolithiasis can exhibit elevated levels of IgG4-positive plasma cells, although IgG4-related disease in this patient population remains exceedingly rare. The existence of IgG4-positive inflammatory infiltrates appears to represent a part of a continuous inflammatory process in the submandibular gland, most likely a phenomenon distinct from genuine IgG4-related disease.


Prestressed concrete sleepers are an important track component that is widely used in railway ballast track. Prestressed concrete sleepers have high strength, strong stability, and good durability; thus, their operation and use in railways are beneficial. However, in different countries and regions, certain damage to sleepers typically appears. Existing research on concrete sleepers focuses primarily on the structural design method, the application of new materials, theoretical analysis, and bearing strength test research, while ignoring sleeper damage. There are a few sleeper damage studies, but they look at only one type of damage; thus, there is no comprehensive study of prestressed concrete sleeper damage. The damage forms of prestressed concrete sleeper damage are thus summarized in this study, and the theory of the causes of prestressed concrete sleepers is analyzed based on the limit state method for the first time. The findings indicate that sleeper structure design is the primary cause of its operation and use status, and that special measures should be considered based on sleeper use conditions. In addition to meeting design requirements, materials, curing systems, product inspection, and other factors must be considered during manufacturing to improve the sleepers’ long-term performance. Keeping the track in good condition, including but not limited to the state of fasteners, ballast bed, and track geometry is also an important aspect of preventing sleeper damage. The outcomes of this study provide better insights into the influences of damage to railway prestressed concrete sleepers and can be used to improve track maintenance and inspection criteria. Railways are one of the most important modes of transportation for both passengers and freight worldwide. Railway traffic is increasing due to the widespread use of high-speed lines, and new railway lines are being built in many countries. Parallel to these developments, the production and the use of railway sleepers are increasing. Railway sleepers are key components in railway tracks that carry the loads transferred from vehicles to the rails, support the rails, protect the gauge, and withstand horizontal and vertical rail movement ,2,3,4. SThe use of prestressed concrete sleepers has grown in recent years due to their high quality. Furthermore, prestressed concrete sleepers are more environmentally friendly than creosote-treated wooden sleepers ,8,10. CuMost prestressed concrete sleepers are in good operating condition, but some are damaged in different ways ,13. ManyBerntsson and Chandra investigated calcium chloride damage in concrete sleepers and concluded that calcium chloride is harmful to concrete . Pawluk Transverse cracks are perpendicular to the long axis of the concrete sleeper. Because of the change in the ballasted bed support state and the randomness of the trainload, the concrete sleeper may experience a load-bending moment that exceeds its strength during service, resulting in transverse cracks. According to the appearance position, the transverse crack of the concrete sleeper can be classified into two types: a transverse crack of the rail seat section and a transverse crack of the central section.The transverse crack of the rail seat section can be divided into the lower part crack and upper part crack, as shown in A transverse crack of the central section can be divided into a lower part crack and upper part crack, as shown in Cracks along the long axis of the sleeper are collectively referred to as longitudinal cracks. For the prestressed concrete sleeper, stress was applied to the concrete in the design, manufacturing, construction and maintenance links of improper treatment, which may lead to the occurrence and development of longitudinal cracks. A longitudinal crack in a sleeper is generally categorized as an end surface crack, a longitudinal crack on the upper surface, a horizontal longitudinal crack on the side, a longitudinal crack at the embedded parts and a longitudinal crack through the sleeper  refers to the concrete hole solution by cement or alkali admixture, mineral admixture, and the environment released Naenomenon . Figure The aggregate in the concrete has alkali activity;There is a certain amount of soluble alkali in concrete;Sufficient water or wet environmental conditions are present.There are three conditions for AAR occurrence:The alkali-aggregate reaction can primarily be divided into two types of reaction types: alkali-silicic acid reaction (ASR) and alkali-carbonate reaction (ACR).2 in the aggregate to form the alkali silicate gel. The gel volume is greater than the SiO2 volume before the reaction (as shown in Equation (2)). The gel water absorption causes concrete expansion and cracking, and the internal reaction mechanism is shown as follows. The ASR generally has a large swelling area that accounts for cracking within 10–20 years after the construction of the concrete structure and can further develop damage to the entire structure:ASR is the chemical reaction between the alkali in concrete and the active SiOACR is the reaction of the alkali in the concrete with the dolomite crystals contained in the active carbonate aggregate, producing concrete expansion and cracking, as shown in Equations (2) and (3). Studies have shown that LiOH can be used to distinguish ASR from ACR. The ACR reaction development speed is fast, and the general concrete project is built in 2~3 years of expansion and cracking, and cannot be repaired and reinforced:The Delayed Ettringite Formation (DEF) is a form of sulfate erosion in cement concrete with sulfate ions from the inside of cement concrete. Therefore, the definition of delayed alum rock can be considered as follows: in hardened cement concrete, not from the process of sulfate outside the cement concrete, the harm caused by delayed alum rock often appears only months or years later. The delay of slurry expansion caused by ettringite formation will cause cracks in the interface between the cement slurry and aggregate-cement slurry , which lTypically, delaying the formation of ettringite occurs when the cement concrete experiences a temperature of 70 °C and causes serious expansion cracking in an environment of high relative humidity. Between 70 °C and 100 °C, the higher the temperature is, the more severely inflated;The expansion and development of cement concrete in wet air are slower than those in water, but its macrocracks are larger;The expansion caused by delayed ettringite begins from the outside of the cement concrete and gradually expands to the inside;The expansion caused by delayed calcium ettringite is affected by the nature and size of the aggregate used;When the concrete structure has been damaged, delayed calcium damage is typically accompanied by an AAR.Results show that the primary characteristics of delayed calcium generation are as follows:There are still some disputes about the mechanism of DEF caused by concrete structure damage, which primarily includes the following aspects. First, a real concrete structure engineering environment is diverse, and the causes of concrete damage are complex. For example, AAR consumes the alkali in the liquid phase, which causes the solubility of calcium in the liquid phase to decline and precipitate out. Therefore, when concrete experiences cracking failure, it is often considered the joint action of AAR and DEF, and the reaction of AAR occurs before DEF. In addition, other factors can also affect DEF, which cannot show whether the primary cause of concrete destruction is DEF. Second, calcium ettringite is widely used as an indispensable component of concrete shrinkage compensation and cement early strong and fast hard, even if the discovery of calcium ettringite cannot prove its real impact on the performance of concrete.. Third, laboratory small size test blocks can be observed in the water after several days of the new formation of ettringite, marginally larger mortar test blocks can see the delayed formation of ettringite, and the real engineering of large size concrete to several years to see this phenomenon. Therefore, the action mechanism and destruction process must be investigated in more detail.The load effect under accidental conditions must be taken into account in the sleeper design in addition to bearing the operating load of the design. In rare circumstances, these unintended load effects can harm the sleeper. When a train is derailed , the sleFor sleeper damage with an accidental load effect, the proportion of damaged sleepers is not large, and the characteristics are important. Combined with the situation on the site, it is easier to judge the cause of the damage.Sleepers are a track component in the ballast track structure when the track structure is in poor condition  will also lead to sleeper damage or even failure, as shown in Sleeper damage and poor railway conditions have an interaction. Inadequate railroad conditions can cause sleeper damage, and in the other direction, poor track conditions can be made worse by sleeper damage . ExampleWhen prestressed concrete sleepers are used in a track design, if the strength is insufficient, damage will occur during its service life. For one type of sleeper, the same damage form usually occurs in different lines, different manufacturers and under similar operating conditions. The design strength of the sleeper structure is insufficient; typically, the reasons for this phenomenon are a small concrete sleeper section, an unreasonable prestressed steel wire configuration, insufficient configuration or no stirrups.Typical sleeper damage caused by insufficient structural design strength is a transverse crack at the rail seat section, as shown in (1)Raw material qualityIn addition to the structural design leading to the insufficient structural resistance of prestressed concrete sleepers, the production quality also has an important impact on the structural resistance of sleepers, and the general production quality of sleeper damage will appear in different external load superposition forms. In the production and manufacturing process, the resistance of sleepers is primarily affected by the material quality and curing process.Select the inactive aggregate;2O+0.658K2O), which should not exceed 0.6%;Control the alkali content of cement Curing processUnder the action of the sleeper mentioned above, the AAR will lead to damage, which is closely related to the raw material quality of the sleeper. An important indicator of high-quality materials is not to produce or cause AAR damage to concrete. Steps to prevent AAR are typically required to eliminate the reaction:Determine the relationship between the temperature of the sleeper concrete and the surrounding air temperature throughout the process of the sleeper life cycle, and avoid a large temperature difference between the temperature of the sleeper concrete and the surrounding air temperature;The temperature change of the sleeper core should not exceed 15 °C/h, and the cooling rate should not exceed 15 °C/h;The difference between the temperature of the sleeper surface and the outside environment when leaving the curing pool should not exceed 15 °C.A prestressed concrete sleeper is typically made using steam curing, as shown in The external load effect and the structure’s resistance are used to analyze the primary causes of prestressed concrete sleeper damage. However, the causes of sleeper damage during operation are frequently caused by multiple factors rather than a single factor. For example, the quality control of sleeper manufacturing lax alkali activity of materials exceeds the standard. This sleeper is used in a humid environment and certain acidic medium, and it is likely to exhibit sleeper surface cracking damage caused by AAR, which is the comprehensive factor of manufacturing quality and environmental effect.In addition, the comprehensive sleeper damage mentioned above can also be caused by the comprehensive superposition of multiple factors.(1)Structural design guarantee of prestressed concrete sleeperThis paper summarizes the damage forms of concrete sleepers and analyzes the damage mechanism of prestressed concrete sleepers from the three aspects of the external load effect, structural self-resistance and comprehensive factors. Based on the results of this paper, prestressed concrete sleepers can prevent damage and improve the quality level from the following aspects:Sleeper structure design is the fundamental cause of its operation and use status. An excellent structural design can avoid damage to a large extent and should consider the appearance size, reinforcement design, manufacturing, operation, maintenance systems and environmental conditions of the sleeper. Therefore, the prevention of concrete sleeper damage starts with improving the design quality of the sleeper structure.(2)Manufacturing quality controlIn addition to structural design, the production quality is also an important factor to prevent concrete sleeper damage. During manufacturing, in addition to meeting design requirements, materials, curing systems, product inspection and other aspects must be considered to improve the long-term sleeper performance.(3)Use of environmental condition adaptabilityTemperature, humidity and chemical erosion in the use environment are also important factors that can lead to sleeper damage. Therefore, in the sleeper design process, special measures should be considered based on these use conditions. For example, when used in cold areas, sleeper design must consider the ability to resist freezing and thawing; in acid rain or sea regions, sleeper design must consider resistance to chemicals.(4)Track status improvementA concrete sleeper is a portion of the railway track, and the deterioration of the overall state of the track will inevitably lead to a sleeper bearing a greater load effect, which can lead to damage. Therefore, an important aspect of preventing sleeper damage is to keep the track in good condition, including but not limited to the state of fasteners, the ballast bed and track geometry.