The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3–5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4–5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF  to evaluate the effect on the proteomic profile. Even with a 0.5 ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on whether ‘matches between runs’ was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5 ml of canine CSF. Recently, other extracellular nanoparticles, including small (< 50 nm) non-membranous exomeres and supermeres have also been discovered5. Alternative EV subgroups have been classified based on the size and density of the particles since these are properties that can be used to fractionate EVs6.Extracellular vesicle (EV) is an umbrella term for various kinds of cell-released membranous particles. Traditional EV subtypes include exosomes, that are secreted from endosomal pathway, microvesicles from plasma membrane budding, and apoptotic bodies released mainly during cell death9. An important role of EVs has been demonstrated for the central nervous system, such as regulation of synaptic communication, synaptic strength, and nerve regeneration11. Thus, analysis of EVs secreted into CSF can offer a way to study changes in brain tissue. So far changes in CSF EV cargo have been detected in patients with Alzheimer's disease and multiple sclerosis15, thus providing proof of concept that CSF derived EVs can be used as a source of biomarkers.EVs are a promising tool for biomarker discovery, since EV cargo reflects that of the cells the EVs are secreted from, and it is possible to isolate them from different biofluids. Cerebrospinal fluid (CSF) is a liquid located close to the central nervous system and is secreted mostly by cells in the choroid plexus16. The selection of isolation method is a balance between EV yield and purity, which poses challenges for EV biomarker discovery; optimising purity is likely to enhance test specificity, whilst optimising EV yield is necessary to maximise test sensitivity. Ultrafiltration combined with size-exclusion chromatography (UF-SEC) outperforms the differential centrifugation method and/or commercial precipitation-based kits for EV isolation from cell culture supernatant, and urine in terms of purity18. For CSF EVs, UF-SEC has been reported to increase both yield and purity19. Density gradient ultracentrifugation produces highly pure EV preparations but is time consuming and requires expensive ultracentrifugation facilities that might not be available in all clinical settings20. SEC, on the other hand, is relatively fast and cost-effective, and requires no specialised laboratory equipment16. However, it is not capable of separating EVs from similarly sized lipoprotein particles, the latter of which are especially abundant in plasma22.There are various methods available for isolation of EVs, including differential centrifugation, precipitation and size-exclusion chromatography (SEC). Different isolation methods have been described in more detail in a review by Sidhom et al.23. Also, lipoprotein contamination is a problem with CSF samples. ApoA1 and ApoA2 that are present in CSF are suggested to originate from plasma, and ApoE and ApoJ are suggested to be made within the blood–brain-barrier24. In addition, small quantities of plasma proteins, such as albumin, are present in CSF7 and can cause contamination.CSF derived EVs have not been widely studied to date, likely because of the invasive collection method and limited volume that can be obtained from individual patients, and because it is difficult to acquire samples from healthy control patients. In a survey performed by International society for Extracellular Vesicles (ISEV) Rigor and Standardization Subcommittee in 2019, only 10% of the respondents had used CSF in their EV studies, which was only a few percentages more compared to the previous survey performed in 201525. Hence, more information about the minimum CSF starting volume for isolation of a sufficient amount of EVs is needed. Compared to humans, it is easier to collect high volumes of fresh CSF from deceased animals, which enables methodological studies.Canine spontaneous diseases are utilised in translational research and therefore studying canine CSF can be beneficial also for human studies. In dogs, limited amounts of CSF can be collected (recommended 1 ml maximum per 5 kg body weight), especially from small individualsOur overall objective was to validate the UF-SEC method for isolation of CSF derived EVs and to determine the minimum CSF volume required for EV proteomic analysis. We first performed a literature review to investigate the current state of CSF EV research, which revealed a need for basic characterisation of EVs. Secondly, we used the UF-SEC method for EV isolation from canine CSF and used a variety of techniques to confirm the presence of EVs and characterise CSF derived EVs. Finally, we compared the EV proteomes obtained from different volumes of CSF in order to find the minimum volume needed for successful proteomic analysis from CSF EVs. Our results indicate that the UF-SEC protocol can be used to isolate purified EVs from CSF, and that a sufficient amount of EVs can be isolated from 0.5 ml of CSF to permit EV proteomic analysis. To our knowledge, this is the first article describing EV isolation from canine CSF.At first, we performed a literature review to investigate the present state of EV analysis from CSF samples. A total of 123 articles were included in the review  isolated EVs from 1 to 6 ml of CSF, whereas 31 articles (25%) used starting volumes under 1 ml. Unfortunately, a large number of studies  did not report the starting volume . Five separate CSF pools were used in the study, two for the method set-up and three for the proteomics analysis.The EV characterisation was done with EVs derived from two separate 7 ml pools of CSF. The highest number of particles (determined by NTA) was detected in fraction 3, as expected Fig. a. ProteiThe size distribution of the particles was measured with NTA and by nano-flow cytometry (NanoFCM). In general, NanoFCM showed a smaller particle diameter with a narrower particle size distribution compared to NTA. The particles in fraction 3 had a modal size of around 100 nm when measured with NTA and around 70 nm with NanoFCM. The measured particle size slightly decreased in fractions 4 and 5 with both analysis methods as the main peak moved towards smaller particles Fig. c. Raw da26  and lowest number from whole CSF samples   Fig.  and S3.FFor the further comparisons, protein identification results using MBR were used. For each group , a list of core proteins found in all three replicates was generated  was used to probe the presence of EV and non-vesicular nanoparticle-related proteins in each fraction. In general, ranked lists for GSEA from all the samples identified ApoA1, ApoE and ApoJ (Clusterin) among the 6 highest ranked proteins, in other words they had high intensities. In addition, albumin was among the top 4 proteins in other groups apart from fraction 4, where it was ranked as the 11th most abundant protein. Using Vesiclepedia top 100 proteins as a protein list, the greatest positive enrichment for EV associated proteins was detected within fraction 3 with a nominal 29. All apolipoproteins had highest intensities in fractions 4 and 5, indicating a presence of lipoprotein particles. To further explore EV related proteins, we used a general EV marker listing prepared by Crescitelli et al.(Table 1 in their article)30. Apart from individual proteins , the highest intensity was consistently detected in fraction 3. Lastly, albumin and L1-CAM were also added to the list as they are considered relevant regarding CSF EVs32, both of which had highest intensity in fraction 5, likely reflecting the presence of non-vesicular proteins.As a final analysis, we compared the mean normalised Intensity Based Absolute Quantification (iBAQ) values from selected individual proteins from SEC fractions 3–5 Fig. d. First In addition to fraction comparison from 6 ml of pooled CSF, UF-SEC isolation was performed from 3 ml, 1 ml and 0.5 ml of each CSF pool. For those samples, proteomic analysis of fraction 3 was performed to compare the effect of starting volume on EV yield and number of proteins identified in the EV enriched fraction.9 ± 3.4 × 109 particles) and the lowest in the 0.5 ml starting volume (4.7 × 108 ± 1.7 × 108 particles)  and the lowest number of proteins identified in the 0.5 ml starting volume (743 ± 77 or 345 ± 88 proteins)  were found in fraction 3, and enrichment for supermere proteins was only identified in fraction 3. Also, the exomere listing was not enriched in fractions 3–5. Fractions 4 and 5 also had the highest relative intensity of apolipoproteins, supporting the premise that the particles identified in fractions 4 and 5 with TEM were mainly lipoproteins.Interestingly, TEM imaging also revealed distinct particle populations in fractions 4 and 5, with fraction 5 morphology resembling that of HDL38.Despite separate protein profiles, the most abundant proteins were similar in all the SEC fractions and in whole CSF samples, and the number of shared proteins was high. Fraction 4 was enriched with Vesiclepedia top 100 proteins, but with lower abundance compared to fraction 3, whereas fraction 5 contained more non-vesicular proteins than the earlier SEC fractions. These data indicate that i) fraction 3 includes the majority of EVs, but also small quantities of lipoprotein particles, and ii) a small amount of EVs is present also in the other fractions. Although SEC did not fully fractionate EVs from other components of the CSF, fraction 3 did include the highest number of individual proteins compared to whole CSF, likely representing a reduction in the abundance of contaminant proteins  that might mask the presence of other proteins of lower abundance. Together with the Western blot analysis for ApoA1 and albumin, our proteomics data shows that our UF-SEC protocol was successful in reducing the quantity of non-vesicular proteins and lipoprotein contaminants from the EV fraction, which is in line with previous articles showing successful SEC-based EV isolation from CSF31, although it was recently suggested that L1-CAM or its isoforms are not carried by EVs in either human plasma or CSF32. In our study, L1-CAM was identified from the unpurified CSF and from the SEC fractions 4 and 5, and was not consistently identified within fraction 3. These data indicate that L1-CAM might not be strongly associated with EVs in canine CSF, supporting the data from the aforementioned study32.L1-CAM has traditionally been considered as a marker for neuronal EVs39. This could be a useful option for further clinical proteomics studies on CSF EVs if it is possible to include one sample with larger starting volume to boost the overall number of identifications. In addition, 180 common proteins were identified in all the replicates of all the four volumes showing reproducibility. In the future, these proteins could further be evaluated with larger study sets.The previous proteomics studies from CSF EVs have used variable starting volumes and methods for EV isolation, and most used pooled CSF. Pooled samples are useful in methodological comparisons, but in the clinical setting analysis of individual patient samples is usually required, although the limited CSF volume that can be obtained from patients ante-mortem can be a limiting factor. As expected, both absolute EV yield and the number of proteins identified correlated with the starting volume used. The number of particles varied between the pools showing biological variability. Promisingly, sufficient amount of EVs could be isolated from 0.5 ml of CSF to permit proteomic analysis, with 293–447 proteins identified from such samples without the MBR setting active, thus providing proof of concept that proteomic analysis of EVs could be performed on individual canine patient samples. In addition, when the MaxQuant analysis was performed using the MBR setting, we were able to identify 696–832 proteins from the 0.5 ml samples. This is in line with recent findings of Hirschberg et al., also successfully isolating vesicles from 0.5 ml of CSF and being able to identify a sufficient amount of proteins in the vesicle fractionDespite the challenges, CSF EVs hold high potential for biomarker studies of brain related diseases. There is an ever-growing interest in EV research, but CSF EVs are yet to be studied on a larger scale. Our data suggest that the UF-SEC method effectively enriches EVs and reduces the amount of contaminant proteins present in EV enriched fractions. The NTA and proteomic analysis showed that sufficient EVs can be isolated from 0.5 ml of CSF to permit proteomic analysis, although a volume of ≥ 3 ml of CSF is likely to allow more complete proteomic analysis.For the literature review, we searched articles about CSF EVs from the following online databases: Web of Science, PubMed, EV Track, Wiley Online Library and Google Scholar using the search term cerebrospinal fluid combined with the terms extracellular vesicles, exosomes, microvesicles, ectosomes and apoptotic bodies. An asterisk was used when necessary to broaden the search results. We only included original research articles published in 2015 or later in the study and the searches were done between April 2021 and August 2022. We reviewed the articles for the starting volume of CSF, for the isolation and characterisation methods, and the number of different characterisation methods used per study. The objectives of the studies were also noted. Articles analysed for the literature review can be found in the Supplementary file Cerebrospinal fluid was collected from dogs euthanised at the Veterinary Teaching Hospital of the University of Helsinki, Finland. The study was approved by the Viikki Campus Research Ethics Committee at the University of Helsinki, Finland and carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. The dogs were euthanised for various reasons unrelated to our study and all the carcasses were donated to teaching and research purposes with written informed consent from the owner. CSF was collected from a total of 21 dogs.CSF was collected via a cisternal tap with a sterile puncture within one hour of euthanasia. As much CSF as possible was drained from each dog into 2 ml plain tubes. Samples were visually assessed to be clear of any visible red blood cell contamination. Within 30 min of collection, total nucleated cell count (TNCC) and total protein concentration were determined. The TNCC and red blood cell count were determined by directly placing undiluted and well-mixed CSF into a Bürker counting chamber . One side of the chamber was filled, and all cells were counted using a Nikon ECLIPSE Ci-L microscope  at 40X magnification. Pleocytosis was defined as TNCCs > 5 cells/μl. The remainder of the sample was centrifuged at 4000 × g for 10 min (with brake) at room temperature to pellet any cellular material . The supernatant was transferred into a new tube, from where it was divided into Protein LoBind tubes , snap-frozen in liquid nitrogen and stored at − 80 °C until further use.g for 20 min at 4 °C to remove any remaining debris. For method set-up, two 7 ml pools of CSF were used. For proteomic analysis, three separate pools of 12 ml were divided into four volumes: 6 ml, 3 ml, 1 ml, and 0.5 ml, which were then processed individually. The individual pools used for the experiments were created by combining CSF from 2 to 8 dogs.Prior to EV isolation, CSF aliquots were thawed on ice, pooled, and 1/100 volume of protease inhibitor  was added to eliminate any proteases that could destroy the proteins in the samples. The pool was centrifuged at 4000 × g at 21 °C using Amicon® Ultra-4 Centrifugal Filter Unit with 100 kDa molecular weight cutoff . After sample concentration, the final volume of each sample was adjusted to 130 μl with PBS. SEC was performed with qEV single/70 nm Legacy columns , according to manufacturer's protocol. Briefly, the column was rinsed with a minimum of 10 ml of PBS and 130 μl of the concentrated sample was loaded into the column. PBS was added as needed and nine 500 μl fractions were collected.For the UF-SEC isolation of EVs, the samples were first concentrated by centrifugation at 4000 × Before the proteomics comparisons, an aliquot of 50 μl was taken from each fraction for NTA and BCA analysis and the rest of the fraction was stored at − 80 °C prior to proteomic analysis.The protein concentration of each fraction was measured using the Pierce BCA Protein Assay Kit  according to the manufacturer's instructions. Briefly, two sets of standards and 25 μl of each sample were mixed with 200 μl of working reagent on a well plate. The plate was incubated at 37 °C for 30 min and read at a wavelength of 562 nm with the Multiskan GO instrument and the Thermo Scientific SkanIt software . The BCA assay was done once per sample.Western blot analysis was performed for SEC fractions 2–9. The samples were combined with a self-prepared 10X sample buffer (sodium dodecyl sulfate (4%), tris hydrochloride (0.125 M), glycerol (20%), bromophenol blue (0.006%), 2-mercaptoethanol (10%)) and boiled for 10 min. Of each fraction, 10 µl  or 20 µl (for the ApoA1 gel) were loaded into 4–20% Mini-PROTEAN® TGX™ Precast Protein gels  and run at 200 V for 35 min. The proteins were transferred to nitrocellulose membranes using Power Blotter Select Transfer Stacks  and semi dry transfer with Power Blotter Station . Membranes were blocked for 1 h in Tris Buffered Saline with Tween  5% milk , and incubated overnight with primary antibodies against albumin  or ApoA1  in the blocking buffer. Membranes were washed three times and incubated with secondary antibodies: Donkey anti Sheep/Goat IgG , Goat Anti-Rabbit Immunoglobulins/HRP  for 1 h in room temperature, after which the washing steps were repeated. SuperSignal™ West Atto Ultimate Sensitivity Substrate  was applied on the membranes and the chemiluminescence was detected using FujiFilm LAS-3000 imager.The samples were diluted in ultrapure water and the numbers of particles were measured using ZetaView® PMX120 . Measurements were done once at all 11 positions and the video quality was set to medium. Lower detection limit was provided by the manufacturer. The dilution factor varied between 50 and 500 depending on the sample. The measured volume was 1 ml and the measurement time 10 s. The chamber temperature was set to 22 °C, and the sensitivity of the camera to 85. Data were analysed using the ZetaView® analysis software version 8.05.12 with a minimum area of 10, a maximum area of 1000 and a minimum brightness of 30.A NanoAnalyzer U30 instrument  with a 488 nm laser and single-photon counting avalanche photodiode detectors (SPCM APDs) was used for determination of size and concentration of individual particles using side scattered light through a bandpass filter of 488/10 nm. The gravity-fed sheath fluid system consisted of HPLC grade water, focusing the sample core stream diameter to ~ 1.4 µm. Measurements were taken over 1-min durations at a sampling pressure of 1.0 kPa. Particle concentrations were determined against a standard of 250 nm silica nanoparticles of known concentration. For particle sizing, a standard consisting of a 4-modal silica nanosphere cocktail  with diameters of 68, 91, 113 and 155 nm was used. Lower detection limit was provided by the manufacturer. Each sample was measured once for size and concentration. Between 4000 and 7000 events were recorded for each sample. Flow rate was 50.71nL/min. Triggering was done via side scatter channel (SS-H). Thresholding is performed by the analysis software to be set at three standard deviations and the mean of the background, with only events above this threshold being plotted as true particle events.For assay controls, the Tris–EDTA buffer used as the diluent was analysed separately. This was then used as a blank, whereby the number of events in the blank and their size profile was removed from final sample results. The instrument was calibrated using 250 nm SiNP QC beads  that fluorescence in both FITC and APC range. The intensity of this fluorescence as well as the side scatter intensity of the 250 nm particles were used to align the lasers and detectors to optimum detection resolution and sensitivity within the software. These beads were of a known concentration (2.37E10 particles/ml), thus providing the concentration standard for the samples to be compared against. For size calibration four silica nanospheres of known sizes  in a cocktail are analysed and the 4 distinct peaks mapped by the software to create a standard curve relating intensity of side scatter to the size in nm of the particles, providing the basis for sizing of the samples.A standard curve was generated based on the side scattering intensity of the four different silica particle sub-populations using the NanoAnalyzer software. The laser was set to 10mW and 10% side scatter decay. Data processing and analysis was performed using the NanoFCM Professional Suite v2.0 software.The grids used were 200 square Mesh copper 3.05 mm . The film was made by hand in 2% biofoform in chloroform solution . Carbon coating was done with Bal-Tec CED030 carbon thread evaporator  and Emitech K100X Glow discharge unit  was used as the glow discharge system. Grids were then placed on a 10 μl droplet of sample on parafilm for 2 min. Buffer salts were removed by transferring the grids twice to a fresh drop of distilled water and incubated for 5 s each. The excess fluid was removed with filter paper and the grids were transferred to one drop of uranyl acetate (1.5% in distilled water) and incubated for 1 min. Excess fluid was removed with filter paper and the grids were air dried prior to imaging. The samples were imaged using a Jeol JEM-1400 transmission electron microscope with 80,000 V and magnifications of 6000X and 20,000X.40. First, acetonitrile (ACN) was added to EV samples so that the final ACN concentration was 70%, proteins were precipitated using MagReSynAmine beads (ReSyn Biosciences), captured with magnetic rack and washed with 100% ACN and 70% ethanol. Then 50 mM NH4HCO3 was added to the beads, and proteins were reduced with DTT, alkylated with iodoacetic acid and on-beads digested with trypsin (Promega) over night. In addition to fraction 3  and SEC fractions 4–5 (obtained from 6 ml of CSF), also intact and depleted CSF samples were analysed with liquid chromatography with tandem mass spectrometry (LC–MS/MS). Intact CSF samples were prepared using the same protocol used with EVs. For depletion of the most abundant proteins from CSF ProteoSpin Abundant Serum Protein depletion kit (Norgen Biotek) was used according to manufacturer’s instructions, and proteins were in-solution digested with trypsin after depletion.EV samples (~ 450 µl) were lysed, proteins precipitated and digested into peptides with trypsin using a previously published protocol for protein aggregation captureThe resulting tryptic peptides were purified using home-made C18 Stage tips-columns followed by nanoLC-MS/MS analysis with nanoElute coupled to timsTOF fleX (Bruker). Peptide separation was done using 60-min linear separation gradient with 0–35% ACN using 25 cm Aurora C18 column (Ion Optics). The timsTOF fleX was operated in PASEF mode. Mass spectra for MS and MS/MS scans were recorded between m/z 100 and 1700. Ion mobility resolution was set to 0.60–1.60 V·s/cm over a ramp time of 100 ms. Data-dependent acquisition was performed using 10 PASEF MS/MS scans per cycle with a near 100% duty cycle. A polygon filter was applied in the m/z and ion mobility space to exclude low m/z, singly charged ions from PASEF precursor selection. An active exclusion time of 0.4 min was applied to precursors that reached 20 000 intensity units. Collisional energy was ramped stepwise as a function of ion mobility.For protein identification the data from LC–MS/MS was analysed by MaxQuant ver 2.0.1.0 against dog (Canis lupus) database downloaded from Uniprot in November 2021. MaxQuant searches were done with and without ‘match between runs’ active. The searches were done using MaxQuant’s default parameters with a false discovery rate of 1% on protein and peptide level.https://bioinformatics.psb.ugent.be/webtools/Venn/). Pathway analysis was done with FunRich version 3.1.4, with the level of significance set on p ≤ 0.05.In the proteomics data analysis, two different methods of identifying proteins were used: identification by LC–MS and identification by matching to a library created from all the analysed samples. For Venn diagrams, an online tool from Bioinformatics Institute Ghent was used (p-value < 0.05 were selected4. Out of the 103 proteins, 58 were identified from the proteomics data and were used as the protein list. Supermere protein list was generated by combining the top 20 most abundant proteins in supermeres derived from three cell lines presented in Supplementary Table 43. Out of the 34 proteins, 28 identified from the proteomics data were used as the protein list.GSEA was performed with GSEA 4.1.0 program using ‘GSEApreranked’ option. A rank list was generated for each sample by giving the lowest rank number for protein with highest total intensity. After this an average of the ranks of three replicate samples was calculated and the final group rank list was generated based on that. Protein list for Vesiclepedia top 100 proteins was taken from its website on June 14th 2022. Protein list for exomeres was generated from differential expression data between small EVs and distinct nanoparticles from Zhang et al. 2019, proteins with positive logFC and adjusted Proteomic and NTA data are presented as mean ± standard deviation unless otherwise stated.Supplementary Figures.Supplementary Tables.Supplementary Information.


This is achieved by the action of adrenaline on β1 receptors of heart muscle cells leading to enhanced contractility, faster heart rate and faster relaxation (lusitropy) via activation of the cyclic AMP-dependent protein kinase, PKA. PKA phosphorylates serines 22 and 23 in the N-terminal peptide of cardiac troponin I. As a consequence the rate of Ca2+release from troponin is increased. This is the key determinant of lusitropy. The molecular mechanism of this process has remained unknown long after the mechanism of the troponin Ca2+ switch itself was defined. Investigation of this subtle process at the atomic level poses a challenge, since the change in Ca2+-sensitivity is only about twofold and key parts of the troponin modulation and regulation system are disordered and cannot be fully resolved by conventional structural approaches. We will review recent studies using molecular dynamics simulations together with functional, cryo-em and NMR techniques that have started to give us a precise picture of how phosphorylation of troponin I modulates the dynamics of troponin to produce the lusitropic effect.Ca This, 2+ release from troponin C in diastole, is increased 2–threefold and recent studies indicate that at physiological temperatures Ca2+-dissociation can be rate-limiting for relaxation . Presum2+ sensitivity  but not affecting Ca2+ affinity, thus the concept of a conformational relay between the TnI phosphorylation site and the switch peptide was established.To understand the mechanism of regulation by troponin I phosphorylation and the effects of mutations, methods that can describe the dynamics of troponin such as NMR techniques  have become the method of choice for understanding the phosphorylation-dependent modulation the cardiac troponin.  of the simulation  and cTnC lysine 39 tend to form a cluster of ionic bonds with the phosphate groups of phosphoserines 22 and 23. .Consequential upon this phosphorylation-induced change, the pulling of Lys 39 upwards impinges upon the helix B and consequently its orientation relative to Helix A and the hydrophobic patch that the switch peptide binds to, manifested as the change in mean helix A/B interhelical angle. Longer range allosteric consequences of the helix B rearrangement include a repositioning of the switch peptide and changes in the peptides crossing the interdomain interface that can account for the observed changes in mean interdomain hinge angle.2+-sensitivity change was studied (Biesiadecki et al., As a reality check, the G159D mutation in troponin C that causes dilated cardiomyopathy and also uncouples phosphorylation from the Ca2+-sensitivity and release rate to the atomic level. As a further test of our molecular dynamics analysis we Simulated the effects of small molecules that can restore coupling to mutations (See Fig. Overall, these changes in dynamics are compatible with the NMR studies, described above, and extend our understanding of the linking of phosphorylation to CaSee Fig.  (SheehanSee Fig. ; in factSee Fig. .2+ (Yamada et al., Finally, it should be understood that, in the muscle thin filament, troponin is associated with actin and tropomyosin, therefore studies based on the troponin core only are incomplete. Cryo-EM studies of the thin filament have precisely located troponin in relation to tropomyosin and actin in the absence and presence of bound CaThese simulations did not include the other disordered parts of troponin such as CcTnT, so further studies may uncover different interactions between troponin, tropomyosin and actin involved in the response to NcTnI phosphorylation at serines 22 and 23.In conclusion, molecular dynamics simulations together with functional, cryo-em and NMR techniques have started to give us a better picture of how phosphorylation of troponin I modulates the dynamics of troponin to produce the lusitropic effect. Remarkably, as summarised in Fig. There is still much more to be found out about this system using these new tools.


Murine studies indicate that, after allogeneic haematopoietic stem cell transplantation (aHSCT), donor-derived macrophages replace damaged microglia and alloreactive T-cells invade the central nervous system (CNS). The clinical relevance of this is unknown. We assessed CNS immune surveillance and metabolic activity involved in neuronal survival, in relation to fatigue and cognitive dysfunction in 25 long-term survivors after aHSCT. Patients with cognitive dysfunction exhibited increased proportions of activated T-cells and CD16 + NK-cells in the cerebrospinal fluid (CSF). Immune cell activation was paralleled with reduced levels of anti-inflammatory factors involved in T-cell suppression , NK-cell regulation , and macrophage and microglia activation . Additionally, the CSF mRNA expression pattern was associated with neuroinflammation and oxidative stress. Furthermore, proteomic, and transcriptomic studies demonstrated decreased levels of neuroprotective factors, and an upregulation of apoptosis pathway genes. The kynurenine pathway of tryptophan metabolism was activated in the CNS of all aHSCT patients, resulting in accumulation of neurotoxic and pro-inflammatory metabolites. Cognitive decline and fatigue are overlooked but frequent complications of aHSCT. This study links post-transplant CNS inflammation and neurotoxicity to our previously reported hypoactivation in the prefrontal cortex during cognitive testing, suggesting novel treatment targets. Allogeneic haematopoietic stem cell transplantation (aHSCT) remains the only curative option for high-risk haematological malignancies. While significant improvements have been made to decrease short-term morbidity and mortality, long-term complications still hamper treatment efficacy , 2. FatiCompelling evidence suggests that the aHSCT procedure can affect brain structure and function , 6: ReduCentral nervous system (CNS) dysfunction after aHSCT may have multiple causes, including neurotoxic chemotherapy and infections. Recently, several lines of evidence that transplantation causes persistent neuroinflammation have emerged. Activated microglia and T cell infiltrates were reported in the brains of aHSCT treated mice and in post mortem brain tissue from acute graft-versus-host disease (GVHD) patients . Sailor We previously reported a descriptive cross-sectional study on patients 1–5 years after aHSCT for haematological disease . A selecIn the present study, we hypothesized that dysfunctional neuronal activity in patients with cognitive dysfunction and fatigue after aHSCT associate with impaired CNS immune surveillance, altered metabolism, and/or a failure in neuroprotective processes. Compared to a brain biopsy, cerebrospinal fluid (CSF) can be obtained relatively non-invasively. Since CSF is drained from the brain interstitial fluid, it comprises mediators that reflect ongoing inflammation and homeostasis in the parenchyma . For exaBy combining analyses of immune cell subsets, protein and mRNA expression, and activation of metabolic pathways regulating inflammation and neuroprotection in the CSF, this study attempted to decipher the pathophysiology of post-transplant neurocognitive sequelae.The CSF samples were obtained from a biobank at Karolinska University Hospital (KUH), Sweden of patients sampled 1–6 years post aHSCT for haematological disease . In our Inclusion and exclusion criteria from the original study are summarized in supplemental Table n = 26) with available stored CSF samples. The participants were divided into groups based on the MFS and CANTAB results. Participants with an MFS score of ≥14 were labelled as fatigued (n = 13). The non-fatigued group contained all other patients (n = 13). Cognitive dysfunction was defined as a global deficit score (GDS) ≥0.5 (n = 7) and no cognitive dysfunction as GDS < 0.5 (n = 19) [The present study included all participants ((n = 19) , 13. Cogg for 10 min at 4 °C and the supernatant was frozen in aliquots to −80 °C. The cell pellets were resuspended in pooled human AB plasma supplemented with 10% (v/v) dimethyl sulfoxide  and stored in liquid nitrogen.On average, 5.3 ml of CSF (previously suggested as adequate for flow cytometry ) was colA total of 274 proteins were measured in the CSF using the Olink® Target 96 Metabolism, Neurology and Inflammation panels , employing a Proximity Extension Assay (PEA) technology  particles and debris. Subsequently, RNA was extracted using TRI reagent  and precipitated according to Hagey et al., 2021 [CSF supernatants were centrifuged at 2000 x l., 2021 . cDNA wal., 2021  and 50 bAs CSF cells are scarce, we opted to analyse the full samples, without prior counting. CSF cells were thawed and washed twice with RPMI media supplemented with 10% (v/v) fetal calf serum . Cell pellets were first stained with 1ul of anti-CCR7 for 30 min at 37 °C. Subsequently, they were incubated with the additional antibodies for another 20 min at 4 °C. Supplemental table Tryptophan, kynurenine, kynurenic acid (KYNA), quinolinic acid (QUIN), picolinic acid (PIC), 3-hydroxykynurenic acid (3-HK), nicotinamide (NAM) and nicotinic acid were quantified in CSF and plasma by UPLC-MS/MS system using a Xevo TQ-XS triple-quadrupole mass spectrometer  . The samp < 0.05) were compared using ANOVA with post-hoc Tukey’s honest significant difference test, or student’s T-test. Non-normal populations were compared using Kruskal–Wallis’ test with post-hoc Dunn’s test, or Wilcoxon rank sum test. Statistical analyses were performed using R version 4.1 [Unless otherwise specified, normality was determined using the Shapiro-Wilk test. Normally distributed populations (sion 4.1 . Principn = 55) were excluded from further analysis. NPX values from the proteomic analysis were then compared using t-tests. The analysis was performed using the OlinkAnalyze package for R version 4.1 [Potential outliers were examined by plotting interquartile range vs sample median. No outliers were found. Proteins that were below the limit of detection in ≥50% of the samples , with all genes passing this threshold used as a control group. Gene ontology term fold enrichment for up- or downregulated genes were then divided by the fold enrichment in the control group to produce the relative term fold enrichment.Differential expression was assessed using the Deseq2 package to compare duplicate samples in R . Up- andCSF immune cell subsets and kynurenine pathway metabolites were correlated to protein levels using simple linear regression. One model was constructed for each correlation with either a kynurenine metabolite or a cell subset as dependent variable, and a protein as the independent variable.The proteomic, mRNA expression and correlation analyses were adjusted for multiple comparisons using the Benjamini-Hochberg method. As this was an exploratory, hypothesis-generating study, further adjustments were not made.Demographic and clinical characteristics of the study cohort are summarized in Table +CD14low T-cells was detected in both patients with fatigue and cognitive dysfunction, compared to patients without fatigue or cognitive dysfunction , CD6 and C-X-C Motif Chemokine Receptor-3 (CXCR3), compared to T-cells in the FSClowSSClow gate  and/or tryptophan 2,3-dioxygenase  or neuroprotective , and therefore of relevance in psychiatric disorders , 45, 61.By comparing to non-transplanted controls, our study revealed increased activity of the kynurenine pathway in all aHSCT patients, both in the periphery and the CNS, without direct association to cognitive impairments or fatigue. Since the resulting increased levels of QUIN in the CSF were not counterbalanced by PIC or KYNA (demonstrated by the decreased PIC/QUIN and KYNA/QUIN ratios), the activation likely contributed to a neurotoxic environment. In plasma, the PIC/QUIN and KYNA/QUIN ratios were not different between transplanted patients and controls (Supplemental Fig. At onset of both acute and chronic GvHD, peripheral kynurenine pathway activity is stimulated by CXCL9, and correlates with disease severity –65. Our Neurobiological aberrations underlying the previously demonstrated reduced prefrontal cortex activity in these patients , may ultLarger longitudinal studies are needed to prove the cause-and-effect of an unbalanced immune activation on neurocognitive sequelae after aHSCT. Such mechanistic understanding will pave the way for targeted treatments and prevention strategies, directed at enhancing neuroprotection, synaptic plasticity, and immune regulation. Limiting the entry of kynurenine pathway metabolites to the CNS after aHSCT could also prove efficient in this regard. Importantly, this study demonstrates that CSF analyses can quantify immune dysregulation and neuronal homeostasis in the brain of aHSCT recipients. Therefore, future clinical trials of treatments for cognitive complications after aHSCT can be readily monitored using similar analyses.Supplemental Material


In Brazil, donor anonymity is mandatory; however, the tendency of Brazilians towards the practice is unknown. In this study, an attempt was made to investigate whether couples undergoing assisted reproductive technology (ART) have a different perception of anonymous versus identity-release gamete donation than a target population in Brazil.This cross-sectional study was performed from September 1, 2020 to December 15, 2020. For that purpose, surveys through online platforms were conducted, including either patients undergoing ART  or subjects interested in the theme  randomized by age at a 1:4 ratio. The survey collected information on the participants’ attitudes towards anonymity of gamete donors, and answers were compared between the groups.Most participants stated that the relationship between children and their parents would be affected by the child’s knowledge of the origin of its conception. Most participants in the ART-group believed that the gamete donor’s identity should not be revealed to the child, while only half of the interested-group stated the same. Most of the participants stated that “the donor’s identity should be revealed if the child questions its biological origin”. “From birth” was the second most common response, while “when the child turns 18 years old” and “sometime during teenage years” were less common answers.The attitudes of ART patients about anonymity are conservative, with most participants believing that family relationships may be affected if the child is aware of the origin of his/her conception. These patients also believe that the identity of the gamete donor should not be revealed to the child. Gamete donation is a well-established treatment in ART . Sperm dOver the past years, fertility treatments using donated oocytes have widely increased, and not only are a large number of children born following donor conception , 6, but Donor conception has traditionally been performed with anonymous donors ; howeverCurrently, legislation concerning whether donor conception should be anonymous ranges from mandating donor anonymity in some countries, such as Brazil, to prohibition of anonymous gamete donation in others. In some countries, such as Japan, there is no statutory law regulating ART or third-party reproduction . In one Legislation on identity-release gamete donation was first introduced in 1985 in Sweden , and curIn Brazil, the last resolution of the National Health Surveillance Agency (ANVISA) published in December 2022, determined that confidentiality of all information relating to the sample to be used must be respected. The assisted reproduction center is responsible to preserve the confidentiality of documents and records involving gamete donors .Since the 1990s, a growing debate about whether donor-conceived people should have a legal right to access information about their donor has been observed; yet a shift in attitudes towards a more open view has emerged which argues that donor-conceived people should be told about the way they were conceived and they must have access to information about their gamete donor –18.It has been argued that identity-release gamete donation protects the offspring’s interest in knowing their genetic heritage, securing accurate information about potential health problems, and making future medical decisions. In contrast, those who defend anonymous gamete donation state that donor anonymity protects offspring from potential negative consequences for family relationships if the truth is revealed .There are few publications comparing disclosing and nondisclosing families, and apart from some conflicts observed between mother and children among these families , parent-Although there is a rich body of literature on the discussion about anonymous or identified gamete donation, there is limited knowledge of patients’ attitudes towards disclosure issues, especially in countries where anonymous donation is required by law. It is also unknown whether the assisted reproduction treatment, and its physical and emotional implications could influence the opinion of individuals regarding donor anonymity.Therefore, the goal of the present study was to investigate whether couples undergoing infertility treatments have a different perception of anonymous versus identity-release gamete donation than a target population in a country (Brazil) where anonymous donation is required by law.This cross-sectional study was performed from September 1, 2020 to December 1, 2020, in which surveys were conducted through online platforms, including either patients undergoing ART  or those interested in the subject who accessed the website of a university-affiliated IVF center . Participants were randomized by age, in a 1:4 ratio, into one of the two groups. Those in the ART group were invited via e-mail, with a cover letter outlining the survey and a link to access it, and participants in the interested-group obtained the questionnaire via a website. The survey collected information on demographic characteristics and participant attitudes towards anonymity of gamete donors, and answers were compared between the groups.This study was approved by the Faculdade de Medicina de Jundiaí, Institutional Review Board, and participants provided the con-sent for publication of questionnaire data.i) In the case of children conceived through ART, do you believe that revealing the method of conception may affect the relationship between children and their parents?Response options: “Yes, it may affect the relationship with their parents” or “No, it wouldn’t affect the relationship with their parents”.ii) In the case of oocyte donation, if the method of conception is revealed, do you believe that the child has the right to know the oocyte donor? Response options: “Yes, the child has the right to know his/her origin” or “No, the child should not be told”.iii) If you had to reveal the identity of the gamete donor to your child, when do you think that the identity of the oocyte donor should be disclosed? Response options: “the oocyte donor’s identity should be revealed if the child questions its biological origin”, “the oocyte donor’s identity should be revealed since birth”, “the oocyte donor’s identity should be revealed when the child turns 18 years old”, or “the oocyte donor’s identity should be revealed sometime during teenage years”.iv) In the case of sperm donation, if the method of conception is revealed, do you believe that the child has the right to know the sperm donor? Response options: “Yes, the child has the right to know his/her origin” or “No, the child should not be told”.v) When do you think that the identity of the sperm donor should be disclosed?Response options: “the sperm donor’s identity should be revealed if the child questions its biological origin”, “the sperm donor’s identity should be revealed since birth”, “the sperm donor’s identity should be revealed when the child turns 18 years old”, or “the sperm donor’s identity should be revealed sometime during teenage years”.The questionnaire contained questions regarding demographic data. Participants were asked to provide information about age (open response format), professional activity (open response format), and marital status . There were three more questions regarding their perceptions concerning the anonymity of gamete donors:vs. 21  statistical program. Variables were tested for normality distribution and group homogeneity using the Shapiro Wilk and Levene’s tests, respectively. Age was compared between the groups using Student’s t-test, while other variables were compared using the Chi-square test. Age was described as the mean±standard deviation, and the other variables were described as the percentage±standard deviation. The considered significance level α was 5%.Data were analyzed using the SPSS Statistics vs. 36.9±6.3, p=0.076, for ART-group and interested-group, respectively, vs. 82.7%, for ART-group and interested-group, respectively, p=0.868, There was no difference in age of two groups .When asked if the sperm donor should be identifiable, most participants in the ART-group answered that the sperm donor identity should not be revealed to the child, while only half of the interested-group stated the same . “From birth” was the second most common response , while “when the child turns 18 years old” , and “sometime during teenage years”  were less common answers  argues for the right of the child to be informed about the origin of his/her conception and about the donation . In factWhile donor anonymity was the rule in most countries in 1990s, identity-release donation is nowadays mandatory in at least 14 countries . At the In our study, among participants who stated that the children should be informed about their genetic origin and that the gamete donor should be identifiable, most were unsure about suitable timing for disclosure. One-third of them answered that the donor’s identity should be revealed if the child questions its biological origin. The second most common response was “since birth”, while “when the child turns 18 years old” and “sometime during teenage years” were less common answers.When children who were conceived through gametes from identity-release donors grow up, parents need to deal with the question of disclosure to the child. In fact, in a previous report from Sweden, it was observed that as children grow older, reaching an agreement about what to tell the child concerning donor conception might be a source of stress in parenthood . It has It has been reported that a common age for disclosure to donor-conceived children is when they are approximately 5 years old , 33, 41.The perception that the child is too young and needs to reach sufficient maturity to understand donor conception is a commonly reported reason to postpone disclosure , 47. In Lack of adequate opportunities to conduct face-to-face interviews and lack of knowledge of the real condition of the website participants concerning infertility or being involved in ART may have biased these results. While there is a movement in favor of a more open approach regarding disclosure of donor identity which is inspired by the United Nations Convention on the Rights of the Child  in many Despite the global tendency towards identity-release gamete donation programs, donor anonymity is still mandatory in Brazil, and attitudes among ART patients seem to be more conservative, with most participants believing that family relationships may be affected if the child is aware of the origin of his/her conception. In addition, these patients believe that the identity of the gamete donor should not be revealed to the child. Participants interested in ART seem to have an open-minded view; however, if facing the problem, future parents’ intentions to disclose may not be borne out in practice. Finally, despite the children’s right to know their biological origin, disclosure of the donor conception to offspring remains a challenge for many of parents.


Chitinase -3-like 1-protein (CHI3L1) is a glycoside secreted by monocytes, microglia, and activated astrocytes. Its distribution in inflammatory lesions denotes its role in astrocytic response to modulate CNS inflammation. In multiple sclerosis (MS), CHI3L1 levels have been found to be influenced by disease severity, activity, and progression. We aimed to measure CSF level of CHI3L1 in patients with MS and correlate its level with disability measures for a possible role as a biomarker for disease progression.Fifty-two MS patients (30 relapsing-remitting MS and 22 progressive MS) and thirty-five age and sex-matched healthy controls were included. They all underwent full clinical assessment , radiological assessment, and CSF level of CHI3L1.Patients with MS had higher CSF level of CHI3L1 than controls. Patients with progressive forms had higher levels than relapsing forms. There were positive correlations between disease duration, number of attacks, total EDSS, and CSF level of CHI3L1. Patients who had higher level of CSF CHI3L1 showed worse performance in MMSE and BICAMS and more lesions in T2 MRI brain. A cut off value of 154 ng/mL was found between patients with RRMS and PMS patients.CHI3L1 can be considered as a biomarker of disease progression. CHI3L1 level increases in progressive MS more than RRMS. Also, high CSF level of CHI3L1 was associated with more disability including motor, cognitive, and radiological aspects. Multiple sclerosis (MS) is a complex autoimmune disease that affects the central nervous system (CNS), which leads to chronic inflammatory demyelination and neurodegeneration. Despite advances in diagnostic and therapeutic approaches, MS continues to pose significant challenges due to its heterogeneous nature in terms of clinical presentation, radiological and histopathological changes, and therapy response. Lesion load in the central nervous system, as detected by magnetic resonance imaging (MRI), and clinical characteristics such as relapse rate and disability progression, has long been important factors in the diagnosis and prognosis of multiple sclerosis. However, recent research has highlighted the importance of atrophy and cortical lesions, instead of focusing on the amount of lesions in white matter and relapse rates, in predicting progression and cognitive impairment in MS . UnfortuRecent studies have highlighted the potential of chitinase-3-like-1 protein (CHI3L1), also known as YKL-40, to serve as a promising biomarker for MS progression. CHI3L1 is a glycoprotein secreted by monocytes, microglia, and activated astrocytes and has been implicated in various inflammatory diseases, including MS, where it is believed to play a significant role in modulating CNS inflammation. CHI3L1 can be detected in both cerebrospinal fluid (CSF) and serum, making it a potentially accessible biomarker for MS patients. CHI3L1 is also produced by a broad spectrum of cells including macrophages, neutrophils, chondrocytes, synovial cells, osteoblasts, and vascular smooth muscle cells. Its expression is upregulated in response to inflammatory stimuli, and it has been implicated in various chronic inflammatory diseases . StudiesNevertheless, additional research is required to thoroughly comprehend the role of CHI3L1 in MS pathogenesis, and its potential as a biomarker. Specifically, longitudinal studies that measure CHI3L1 levels over time in MS patients are necessary to evaluate if alterations in CHI3L1 levels correlate with disease progression or treatment response. Additionally, further studies are needed to investigate the underlying mechanisms by which CHI3L1 contributes to MS pathogenesis, which could lead to the development of novel therapies for this debilitating disease.In this context, the aim of our study is to measure CSF level of CHI3L1 in MS patients and correlate its level with different aspects of disability including EDSS, cognition, and radiological aspects and if it could be used as an objective biomarker for disease progression. We hope to gain insights into the underlying mechanisms of MS progression and identify novel targets for therapeutic intervention.This was a case-control study conducted on 87 Egyptian individuals divided into two groups. The patient group (52 MS patients) who were diagnosed according to revised McDonald Criteria 2017 , and conThe study included patients aged between 18 and 60 years of both sexes who were relapse free at least 1 month before CSF sampling. Also, patients were off steroids at least 1 month before CSF sampling. We excluded patients and controls whose criteria are mentioned in Table All patients underwent clinical assessment in the form of full history taking, general medical examination and neurological examination, evaluation of disability using the expanded disability status scale (EDSS)/Functional System Score (FSS) , neuropsAlso, they underwent laboratory assessment of CSF level of Chitinase-3-like-1 protein (ng/ml) using ELISA technique  test was done. Exact test was used instead when the expected frequency is less than 5 [p-value) [p-values less than 0.05 were considered statistically significant, and all statistical tests were two-tailed.Our data were analyzed using the statistical package for the Social Sciences (SPSS) version 26 . The data was summarized using mean, standard deviation, median, minimum, and maximum in quantitative data and using frequency (count) and relative frequency (percentage) for categorical data. Comparisons between quantitative variables were done using the non-parametric Kruskal-Wallis and Mann-Whitney tests . For coms than 5 . Correlap-value) . ROC curn=52) had a mean age of 34.85 years (SD = 8.9), with 23.1% male and 76.9% female participants. The control group consisted of 30 individuals with a mean age of 37.91 years (SD = 11.1), of whom 50% was male and 50% was female. Also, educational level (in years) was obtained.Table Table z-score for each.Table Lesion load varied between patients, with a mean lesion number of 10.8 (SD = 3.2). Table Table Table Comparison between RRMS and PMS in CSF CHI3L1 levelsComparison between PPMS and SPMS in CSF CHI3L1 levelsCut-off value in CSF level of CSF CHI3L1 between RRMS and PMS by using ROC curve = 154 ng/ml; Fig. 2Correlations were measured between all studied parameters and CSF CHI3L1 level, but only significant correlations were reported here.p > 0.001, r = 0.608).There was a highly statistically significant good positive correlation between duration of the disease and CSF level of CHI3L1 .Also, there was a statistically significant good positive correlation between number of attacks and CSF level of CHI3L1 .In addition, there was a highly statistically significant good positive correlation between EDSS and CSF level of CHI3L1 . Episodic memory was the most correlated cognitive domain with CSF CHI3L1 level.The severity of cognitive impairment (either MMSE or BICAMS) showed significant good positive correlation with CSF CHI3L1 level .There was a highly statistically significant good positive correlation between T2 lesion number and CSF level of CHI3L1 .Also, there was a highly statistically significant good positive correlation between presence of MRI brain atrophy and CSF level of CHI3L1 .In addition, there was a highly statistically significant good positive correlation between presence of MRI brain black holes and CSF level of CHI3L1 .In MRI CS, there was statistically significant positive correlation with presence of demyelinating lesions, cord atrophy, and CSF level of CHI3L1 level (r = 0.92), but SIMOA was more sensitive than ELISA, detecting CHI3L1 in samples that were negative by ELISA [r > 0.8), but SIMOA was the most sensitive, followed by Luminex and ELISA [r = 0.85) [Within CNS, CHI3L1 is mainly secreted by reactive protoplasmic astrocytes, and primed microglial cells in lesions with low inflammatory activity. CHI3L1 could be measured in serum and CSF, where increase throughout MS stages seems to be derived from the extension of diffuse brain inflammation associated with neurologic impairment rather than acute inflammation during relapses and the detrimental role of astrocyte activation in disease pathogenesis [by ELISA . Anothernd ELISA . A study = 0.85) . In our  = 0.85) . Rucsand = 0.85) , 19. In  = 0.85) . Regardi = 0.85) . While a = 0.85) , 23. Fur = 0.85) . This ca = 0.85) . Our res = 0.85) . Also, S = 0.85) , 28. Als = 0.85) . Our stu = 0.85) . Our stu = 0.85) . Interes = 0.85) , 32. Als = 0.85) . Cogniti = 0.85) . One pot = 0.85) . CHI3L1, = 0.85) . Our stu = 0.85) , 38. A s = 0.85) . This go = 0.85) . Also, t = 0.85) . Moreove = 0.85) . Overall = 0.85) . There w = 0.85) , 38. Als = 0.85) . A possiBased on the available evidence, it can be concluded that CHI3L1 is a promising biomarker of disease progression in multiple sclerosis (MS). Studies have shown that the levels of CHI3L1 in the cerebrospinal fluid (CSF) are elevated in patients with progressive MS compared to those with relapsing-remitting MS (RRMS). Moreover, higher CSF levels of CHI3L1 are associated with greater disability, including motor, cognitive, and radiological aspects of the disease. These findings suggest that CHI3L1 could be a useful tool for monitoring disease progression and predicting outcomes in patients with MS. However, further research is needed to fully establish the clinical utility of CHI3L1 as a biomarker in MS.


Rana dybowskii were investigated during post-reproductive dormancy and post-dormant activity. The results showed that R. dybowskii live individually under leaves, soil, stones or tree roots. Furthermore, although the dormant sites of frogs are significantly different, the distribution of male and female frogs in these sites is similar. Shading and shelter significantly affected the exposed body percentage, burrow depth and body-soil contact percentage of frogs compared with soil. In the stone group, soil and stone form the frog's refuge/burrow, whereas in the soil group, the refuge/burrow is composed entirely of soil. Even though the soil group has a deeper burrow and a larger area of soil contact with the body, it still has a higher exposure rate than the stone group. Frog activity frequency was affected by shelter and shade; the interaction of shelter and time and the interaction of shading and time were significant. The soil group had a higher activity frequency than the stone group, and the no-shade group had a higher activity frequency than the shade group. Shelter and shading differences do not significantly affect frog survival; however, the death rate during post-reproductive dormancy is lower than that during the active period.Amphibians are facing population declines and extinctions, and protecting and supplementing refuges can help species survive. However, the microhabitat requirements of most species are unknown, and artificial shelters or burrows have not been well tested for amphibians. Some amphibians exhibit complex behaviour during the transition from post-reproductive dormancy to activity. However, little is known about the ecology, post-reproductive dormancy, and terrestrial activity of amphibians. Here, habitat site selection in experimental enclosures and the effects of shelters  and shade (with and without shade netting) on the activity, exposed body percentage, burrow depth, body-soil contact percentage, and survival of The online version contains supplementary material available at 10.1186/s40850-022-00163-4. Amphibians are experiencing catastrophic population decreases and extinction due to infectious illnesses, habitat loss, climate change, invasive species, and chemical contaminants, underscoring the critical need for conservation . Many amThere are many causes of amphibian declines, and in recent years, a growing body of research has focused on how amphibians are being affected by global climate change , 6. In aRana dybowskii) require high-quality aquatic and terrestrial habitat [R. dybowskii go from entering post-breeding dormancy to becoming active again sometime later, and R. dybowskii exhibits very complex behaviour, influenced by complex environmental factors [Many amphibian species require a variety of habitat types to complete their life cycle . Failing habitat . After m factors . Consequ factors . HoweverIn place of natural refuges, artificial refuges are man-made buildings designed to provide secure areas for animals to use during breeding, dormancy, hibernation, or when seeking shelter , 13. AroR. dybowskii) is mainly distributed in northeastern China, where it is widely hunted for its medicinal and commercial value and where the degradation of its habitat has drastically decreased its number [Rana dybowskii has a significant fatality rate during the post-reproductive phase, which could be due to harsh weather in early spring and poor post-reproductive dormancy habitat conditions [R. dybowskii during the post-reproductive period is a serious challenge to establishing captive breeding and reintroduction programs [R. dybowskii exposed to harsh spring circumstances may depend on their ability to identify abundant climate refuges locally [R. dybowskii that frequently inhabit these microhabitats [The brown frog   and 25.11 ± 6.19 g   and 17.36 ± 4.57 g  and 29.65 ± 5.32 g  . All frogs were weighed before spawning. All experimental enclosures were located outdoors near spawning ponds and in the natural habitat.R. dybowskii in post-reproductive dormancy habitats, we chose 120 spawning frogs  and placed them in the experimental enclosures to observe their dormant sites, activity and death.To understand the specific dormant position of 2 in zone and had spawning ponds. The depressions (spawning ponds) were spawning ponds for amphibian reproduction. At the beginning, the water at the centre of the spawning pond was 85.3 cm in depth and ~ 130 m2 in zone. The vegetation in the enclosure was well preserved and consisted of weeds and a small number of dwarf trees (59 trees), with approximately 340 m2 of the enclosure covered by fallen leaves. To prevent the frogs from escaping, we sealed the four site edges each with a plastic cloth fence (30.0 × 30.0 m2) supported by wooden sticks at an interval of 1–2 m. The bottom of each plastic cloth was embedded in soils, and the fence floor was approximately 1.0 m high.The enclosure was 900 mR. dybowskii, took the frogs out of the enclosure, and calculated their survival rates .Experiments were conducted in April and May 2018. On 10 April, vigorous wild frogs were placed in a spawning pond to lay eggs. After approximately 2 days, the frogs had finished laying eggs , and the four dormancy microhabitats  are described below. The total zone of the four experimental enclosures was 48 mI2) surrounded by a fence  was divided into three dormancy subzones (each 2.0 × 2.0 m2). Each of the subzones was regarded as one replicate.Stone shelter without shading group: A dormancy zone . The cobblestones were placed in ridges with a gap of 20 cm between them and the fence. A transparent plastic cloth wider than the dormancy zone was installed in the upper part (1.6 m) of the zone to protect it from rainfall. To prevent strong wind and animal escape, we placed another fence (the outer fence) outside the dormancy zone  of The frogs were randomly and periodically sampled to examine their resting status, exposed body percentage, burrow depth and body-soil contact percentage. The exposed body percentage was computed as the proportions of the abdomen and back that were exposed . The body-soil contact percentage was computed as the proportion of the body in contact with soil and was estimated in the same way as the exposed body percentage. Burrow depth was defined as the distance between the top of the highest surrounding soil and the abdomen of the frog.R. dybowskii was investigated on 6 May and 20 May separately.The occurrence frequency (activity frequency) at which frogs appeared above ground was observed daily from 10 April to 25 May. “Appearance above ground” was defined as a frog exposing more than 75% of its back. If this condition was met, the corresponding frog was recorded as “appearing on the ground” Fig. S. We recoAll results are expressed as the mean ± standard deviation (x ± SD). Fisher's exact test was used to compare habitat distributions and survival rates in males and females. The Scheirer Ray Hare test (a nonparametric test) was used to analyse a two-way factorial design for the effect of shelters and shading on the body exposure rate, body-soil contact rate, and burrow depth, and the Nemenyi test was used for post hoc analysis.Generalized estimating equations (GEE) were used to analyse the repeated-measures design for the effect of shelters, shading and time by selecting the frequency of activity at four time points: the beginning of post-reproductive dormancy (11 April), during post-reproductive dormancy (25 April and 10 May), and at the end of post-reproductive dormancy (25 May). The same method was used by selecting the surviving number at two time points: day 1 (6 May) and day 2 (20 May). Least-squares means estimated from GEE were used for pairwise post hoc multiple comparisons while applying the Bonferroni method to calculate the adjusted p values.P < 0.05 indicated a significant difference.All statistical analyses were performed utilizing R software (version 3.5.3), the Scheirer Ray Hare test was conducted via the rcompanion package , and theRana dybowskii tended to rest in dark and wet conditions at low temperatures (> 0 °C) in cool zones with small day-night temperature differences. The majority of the frogs entered dormancy alone, with all four limbs bowed. The ventral surfaces of the forelimbs were positioned behind the anterior borders of the eyes; the frogs' eyes were tightly closed, and they breathed slowly.During the experimental period, from 8 April to 30 May, the overall rise of the land at the experimental site was slow but slightly fluctuating Fig. S. Rana dyF = 115.090, df = 4, P < 0.01). The majority of the frogs rested beneath leaves , beneath the soil , and beneath stones ; nevertheless, a minor number rested beneath water  or beneath roots   Fig. .Fig. 2SiP = 0.943, P = 0.937, and P = 0.824, respectively; Fig. Male and female frogs did not differ in their habitat selection. The occurrence distribution of male and female frogs in these dormant sites did not differ significantly on 25 April, 1 May, or 5 May .There was no difference between male and female frogs in terms of survival rates. On 25 May, a total of 112 frogs were found, with 37 females and 39 males surviving and 18 females and 18 males dying. The difference between female and male survival (67.27% (37/55) vs. 68.42% (39/57)) was not significant  , 30.21 ± 7.21% (soil with shading group), 7.35 ± 6.29% (stone without shading group), and 7.28 ± 6.76% (stone with shading group) , 67.26 ± 10.52% (soil with shading group), 61.50 ± 7.28% (stone without shading group), and 61.43 ± 3.98% (stone with shading group) , 3.42 ± 0.41 cm (soil shelter with shading group), 2.33 ± 0.28 cm (stone shelter without shading group), and 2.09 ± 0.35 cm (stone shelter with shading group) , was not different during post-reproductive dormancy (25 April and 10 May)  and was different at the end of post-reproductive dormancy (25 May)  (Table SAt some points in time (11 April and 25 May), frogs appeared more frequently in the shelter soil than in the shelter stone. In the comparison between shelter stone and soil, behaviour towards frogs was different at the beginning of post-reproductive dormancy (11 April) ; there was no difference during post-reproductive dormancy (25 April)  and during post-reproductive dormancy (10 May) ; and at the end of post-reproductive dormancy (25 May), soil without shade was significantly different from soil with shade , and stone without shade was not significantly different from stone with shade  , frogs appeared more frequently in the nonshade group than in the shade group. Comparisons between shade and nonshade on frog behaviour were different at the start of post-reproductive dormancy (11 April)  Table S.P > 0.05; Table SGEE was used to investigate the influence of shelter, shading and time on the survival rate of activity at two time points Table S. The surP < 0.05), with the number of surviving frogs being smaller on day 2  and day 2 (20 May) was statistically significant ; low temperature inhibits frog activity [R. dybowskii behaviourally regulates body temperature by altering the daily or seasonal use of shaded or sunny microhabitats [During dormancy (between 25 April and 10 May), there was no significant effect of shading on the activity. Shade has no discernible impact on  dormant . Rana dyactivity , 38. To habitats , 39Amphibians use refuges or shelters for a variety of reasons, including ambush feeding, buffering, reducing osmoregulatory and thermal stresses, and predator avoidance . Often tR. dybowskii in our study used shelters (rocks and loose soil holes) during the dormant period; some frogs even dug holes to make shelters during dormancy . Thus, the fate of R. dybowskii exposed to harsh springtime conditions will depend on whether they can find locally abundant refuges that buffer against life-threatening conditions [R. dybowskii repose, allowing them to evade predators and providing a safe environment for the frogs during dormancy [Almost all ncy Fig. . The typnditions , 12. Altnditions . Previounditions . By provnditions –44. Duridormancy .In this study, the majority of frogs found refuge or shelter at the experimental site. Frogs in soil shelters were more active than those in stone shelters, but there was no difference in their survival rates. Previous research has demonstrated that individuals without shelter spend more time digging and that all frogs spend a considerable amount of time beneath shelter . SignifiThe two strategies used by frogs to dig burrows to avoid desiccation appear to be different, and the type of soil has an effect on both strategies , 46. In Xenopus, shelter cover appears to confer behavioural benefits but has no impact on the growth or body conditions of this type of frog, whereas for nonmodel taxa, shelter cover might exert positive effects on growth as well as behaviour [There are currently few meaningful management actions that will have a tangible effect on amphibians' vulnerability to climate change . Numerouehaviour , 40.R. dybowskii in terrestrial habitats. It is necessary to consider a type of shelter conducive to a particular frog's dormancy. Previous research suggested that PVC pipes and cover boards could provide suitable shelter for some amphibians [R. dybowskii, does not irritate their skin, is easy to clean and disinfect, and is slow to conduct heat in direct sunlight. However, in the dormant state, many R. dybowskii individuals are crowded together, pressing against each other; if some individuals move, the remainder may be easily disturbed [In this study, stones were used as a shelter during the dormant period for phibians , 13. Thephibians . We recoisturbed . Therefoisturbed .R. dybowskii lives individually under leaves, soil, stones or tree roots. Furthermore, although the dormant sites of frogs were significantly different, the occurrence distributions of male and female frogs at these sites were similar. Shading and shelter significantly affected the exposed body percentage, burrow depth and body-soil contact percentage of frogs compared with soil. Frog activity frequency was affected by shelter and shade, and there are interaction effects between shelter + shade and time. Shelter and shading differences did not significantly affect frog survival; however, the death rate during post-reproductive dormancy was lower than that during the active period. These findings advance our understanding of amphibian behaviour ecology by demonstrating how the interaction of external factors and individual behaviour results in observed patterns of movement and habitat use.The results showed that Additional file 1: Figure S1. Phenological time of R. dybowskii emerging from hibernation, post-reproduction dormancy, activity, and feeding. Figure S2. Diagrams of microhabitat II described in Section 2.2.2. Figure S3. The occurrence frequency of frogs was measured by their presence on the ground. Figure S4. The temperatures in the experimental enclosures. Additional file 2: Table S1. Scheirer Ray Hare test for two-factor design with shelter and shading demonstrated a statistically significant interaction for body exposure rate, body-soil contact rate, and ground hole depth. Table S2. Comparing the body exposure percentage, body-soil contact percentage and borrows depth among all 4 groups by Nemenyi test. Table S3. Used GEE to investigate the influence of shelter, shading and time by selecting the frequency of activity at four time points. Table S4. The results indicated that interaction of shelter and time, interaction of shading and time had significant. Table S5. Comparing frog behavioural differences in refuge stones and soil at four time points. Table S6. Comparisons between shade and non-shade on frog behaviour at four time points. Table S7. Used GEE to investigate the influence of shelter, shading and time by survival rate of activity at two time points. Table S8. The survival rate of frog was reduced in the post-dormancy activity period.


Conclusions: Elevated levels of CXCL10 in the CSF of MS cases are associated with increased T cells but appear to be independent of peripheral CXCR3 expression. These results support the importance of elevated CXCL10 in MS and suggest the presence of an alternative mechanism of CXCL10 outside of solely influencing immune cell trafficking.Background: Cerebrospinal fluid (CSF) is an important sampling site for putative biomarkers and contains immune cells. CXCL10 is a multiple sclerosis (MS)-relevant chemokine that is present in the injured central nervous system and recruits CXCR3+ immune cells toward injured tissues. Objective: Perform a comprehensive evaluation to determine a potential relationship between CXCL10 and various immune cell subsets in the CNS of MS and control cases. Methods: In MS and control cases, CXCL10 was measured in the CSF and plasma by ELISA. Immune cells within both the CSF and peripheral blood were quantified by flow cytometry. Results: Compared to non-inflammatory neurological disease (NIND) cases, MS cases had significantly higher CXCL10 in CSF ( Multiple sclerosis (MS) is a chronic disease characterized by an inflammatory insult to the central nervous system (CNS) that results in demyelination and neurodegeneration. The cerebrospinal fluid (CSF) plays an immunologically important role within the CNS . PeripheCytokines and chemokines are critical for the induction, maintenance, and resolution of inflammatory processes throughout the body. In individuals with inflammatory conditions, like MS, cytokine and chemokine profiles are often reflected by an increased pro-inflammatory and decreased anti-inflammatory molecular profile. CSF has been previously investigated to identify the repertoire of cytokines and chemokines present in the CNS in MS. Profiling studies have revealed significant increases in several cytokines in MS, many of which were related to Th1-mediated immune responses ,8,9. HowCytokine and chemokine profiles in CSF are likely rich sources of putative biomarkers. However, exactly which marker (or combination of markers) is the most valuable remains elusive and depends on the type of biomarker being considered. CXCL10 is a chemokine produced in response to pro-inflammatory stimulation and is involved in the chemoattraction of macrophages, monocytes, and activated T and NK cells via binding to the CXCR3 receptor . In MS, While CXCL10 has been recognized as being dysregulated in CNS inflammation and MS, a comprehensive evaluation of its associative levels and effects on cellular infiltration into the CNS compartment have yet to be investigated. Herein, we demonstrate an increase in CSF CXCL10 in MS compared to controls and that CXCL10 is associated with increased T cells within the CSF compartment but not with the expression of its receptor, CXCR3, in the periphery.All experiments involving living human participants and human samples were approved by the Newfoundland Health Research Ethics Board, which follows the declaration of Helsinki. Written informed consent was obtained from all participants prior to study initiation.CSF and blood samples were obtained from patients recruited from the Health Sciences Center Neurology Clinic  or Horizon Health . For CSF and cell analyses, all patients were DMT-naïve; 39/84 patients were treated with steroids at the time of clinical relapse (1–166 months prior to LP). For plasma analyses, RRMS patients were recruited through the Health Research Innovation Team in Multiple Sclerosis (HITMS), an MS patient registry and biorepository at Memorial University of Newfoundland, St. John’s NL, Canada. All clinical parameters were determined based on assessment by an MS neurologist. Clinical and demographic information for the various cohorts is presented in g) to remove cells. Cell-free CSF was aliquoted, frozen at −80 °C, and transferred to liquid nitrogen. Cells not used for flow cytometry were lysed in QIAzol®  and stored at −80 °C for RNA isolation and qPCR analysis.CSF was drawn by lumbar puncture (LP), and 2.5 mL was centrifuged to collect cells for flow cytometry. The remaining sample was also centrifuged (300× g). Peripheral blood mononuclear cells (PBMCs) were isolated as described previously [®). PBMCs were isolated from whole blood using Ficoll density gradient centrifugation  and SepMate™ Tubes . Once isolated, PBMCs were cryopreserved and stored in liquid nitrogen.Plasma was obtained by centrifugation of whole blood samples .Assays were performed according to the manufacturer’s instructions. CSF levels of 27 cytokines, including CXCL10, were measured by Bio-Plex Pro™ Human Cytokine 27-plex Assay and analyzed on a Bio-Plexg), resuspended in 100 uL flow buffer , applied to a DURAclone IM Phenotyping BASIC tube , mixed, and incubated at 4 °C for 30 min. The cells were washed in flow buffer and fixed with 100 µL 2% paraformaldehyde. Data were acquired using the CytoFLEX , and all cells in the sample were quantified.CSF cells were quantified by flow cytometry in a subset of patients . Cells wPeripheral PBMCs were quantified from 10 healthy controls and 20 RRMS cases . Of the ® Micro Kit with DNAse treatment  and quantified by NanoDropSM 1000 spectrophotometer . A total of 200 ng RNA was reverse transcribed using M-MLV reverse transcriptase kit . Gene expression assays were performed using TaqMan Fast Universal PCR Master Mix  and TaqMan probes and primers for cxcr3 .Total RNA was isolated using the RNeasyp ≤ 0.05 was considered significant. In all analyses, the data were assessed for normality by the Shapiro–Wilk test, and nonparametric tests were performed if the data were not normally distributed.Statistical analyses were conducted using Prism 9 . Flow cytometry data were analyzed using FlowJo™ v10.0 software . Data are presented as mean ± SEM unless otherwise indicated, and t-tests (sex), simple linear regression, Pearson’s correlation test  or Spearman’s correlation (EDSS), and one way ANOVA (DMT use).Bio-Plex and ELISA data were analyzed by t-tests comparing RRMS and NIND cases and included a correction for multiple comparisons using the Holm–Sidak method when necessary. For Bio-Plex, values measured using extrapolation below the lowest standard were included in the analyses. The percentage of extrapolated values for each cytokine is documented in Flow cytometry data were gated independently for each sample. For CSF flow cytometry, cell counts were correlated with CXF CXCL10 concentration using Spearman’s correlation test. CXCR3 expression in PBMC populations was analyzed between HC and RRMS groups using one-way ANOVA and was correlated to CSF CXCL10 using Spearman’s correlation test. qPCR for CXCR3 in cells isolated from CSF was displayed as 1/CT and correlated to CXCL10 levels by Spearman’s correlation test.p = 0.049, Initially, using Bioplex analysis, we wished to obtain an unbiased immunological profile of cytokines and growth factor expression within the CSF from RRMS patients and non-inflammatory neurological disease controls NINDs; . In totap < 0.0001; p = 0.012, Moving forward, all subsequent measurements of CXCL10 within CSF of RRMS and NIND cases were performed using an ELISA. A validation of the CXC10 results was therefore performed in a larger cohort  using an ELISA . Comparep = 0.515, p = 0.012, p = 0.922, p = 0.478, p = 0.995, p = 0.951, p = 0.380, To determine whether the increase in CXCL10 levels within the CSF of RRMS could also be detected in the more easily accessible blood plasma, we performed a correlational analysis of CSF CXCL10 levels vs. plasma CXCL10 levels within the same subjects. Interestingly, CSF and plasma CXCL10 were not correlated within the same subjects A, thus sp = 0.013, p = 0.017, p = 0.026, p = 0.341, p = 0.121, p = 0.071, Following centrifugation, using immune cells isolated from the CSF of documented RRMS cases, flow cytometry was used to phenotype the cells within the CSF; the gating strategy used is displayed in p = 0.805). Additionally, 0.402 ± 0.112% of PBMCs were double-positive for CD14 and CXCR3 in the HC group compared to 0.582 ± 0.133% (high CSF CXCR3) and 0.591 ± 0.109% (low CSF CXCR3) in the RRMS groups (p = 0.128). Finally, 0.863 ± 0.138% were double-positive for CD19 and CXCR3, compared to 1.18 ± 0.257% and 0.971 ± 0.249% in the RRMS groups .Following analysis of CSF cells, flow cytometry was conducted on previously cryopreserved PBMCs from both healthy controls and RRMS cases. These experiments were conducted in order to measure levels of CXCR3+ (the cognate receptor for CXCL10) immune cells within circulation and to determine whether these cells were impacted by CSF levels of CXCL10. Expression of CXCR3 was assessed based on the gating strategy outlined in p = 0.629). In addition, 13.9 ± 1.61% were double-positive for CD8 and CXCR3 in the HC group, whereas 15.4 ± 2.14% (high CSF CXCR3) and 17.0 ± 2.40% (low CSF CXCR3) were observed double-positive for CD8 and CXCR3 in the RRMS groups (p = 0.668). qPCR for CXCR3 was performed on cells collected from MS CSF. We observed that the expression of cxcr3 mRNA in CSF cells was not correlated with CSF CXCL10  in relation to CXCL10 levels. This could explain why our results demonstrate a significant correlation between CSF CXCL10 and T cells in the CSF, while others did not. As the CSF flow cytometry performed in this study was a routine protocol performed as part of the general immunophenotyping of patient samples enrolled in our biorepository, flow cytometry for CXCR3 on CSF cells was not included, which is a limitation of our current study. We did perform qPCR for CXCR3 on pooled cells in the CSF samples and found that CXCR3 expression was not correlated with CSF CXCL10 , suggestF CXCL10 . These dAlong with CXCL10, the CXCR3 receptor also binds CXCL9 and CXCL11 . Like CXNevertheless, in summary, the present study provides evidence that CSF CXCL10 is elevated in RRMS cases compared to NIND and that it is not significantly associated with clinical or demographic variables. We also show that plasma CXCL10 is significantly correlated with disease duration but with no other clinical or demographic variables investigated. We provide evidence that CSF CXCL10 is significantly correlated with the number of T cells in the CSF, but this is unrelated to the expression of CXCR3 in the CSF or in the periphery. Taken together, these results support the importance of elevated CXCL10 in MS and other neurological disorders and support an alternative mechanism of CXCL10 outside of immune cell trafficking into the CNS.


Pectobacterium spp. infect many horticultural crops worldwide and lead to serious crop losses. Zinc-uptake-regulator (Zur) proteins are present widely in prokaryotes and play an important role in pathogenicity. To uncover the role of Zur in P. odoriferum, we constructed mutant (ΔZur) and overexpression [Po (Zur)] strains of a Zur, and a virulence assay showed that the Po (Zur) was of significantly lower virulence, while the ΔZur displayed significantly increased virulence on Chinese cabbage compared to their respective control strains, wild-type P. odoriferum (Po WT) and P. odoriferum harboring an empty vector (Po (EV)) (p < 0.05). The growth curves of the ΔZur and Po (Zur) showed no obvious differences from those of the control strains. Comparative transcriptome analysis showed that Zur overexpression in P. odoriferum induced differentially expressed genes (DEGs) related to flagellum and cell motility, while mutating Zur resulted in DEGs mainly corresponding to divalent-metal-ion transport and membrane transport. Phenotypic experiments on the Po (Zur) showed that flagellum numbers and cell motility were reduced in comparison with the control, while those of the ΔZur did not change. Collectively, these results show that the Zur negatively regulates the virulence of P. odoriferum and might function via a dual mechanism dependent on dose. Zinc (Zn) is the second most abundant transition-metal element among all the essential trace elements in living organisms after iron . It is aznuABC, a high-affinity zinc-transport system and the low-affinity ZIP family Zn transporter ZupT , which were cultured in standard LB growth medium, were sequenced and two comparisons were performed—Po (Zur) vs. Po (EV) and ΔZur vs. Po WT. Each strain included three biological replicates, and a total of 30.09 Gb raw data and 29.89 Gb clean data were produced. Each sample contained 2.21–2.85 Gb clean data, with a Q20 quality score of 97.67–98.39% and a Q30 of 93.22–94.83%  < 0.05 and fold change (FC) ≥ 2 were identified as differentially expressed genes (DEGs). A total of 79 DEGs (22 upregulated and 57 downregulated) were identified in the comparison between Po (Zur) and Po (EV), while for ΔZur vs. Po WT, there were 72 DEGs in total, including 56 upregulated and 16 downregulated ones  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the obtained DEGs identified in Po (Zur) vs. Po (EV). In the GO enrichment analysis, DEGs in Po (Zur) vs. Po (EV) were associated with 15 functional groups belonging to two main categories: cellular components (CC) and biological processes (BP). In the CC category, DEGs were enriched in GO terms such as bacterial-type flagellum, cell projection, and bacterial-type flagellum part, and in the BP category, DEGs were enriched in eight functional groups, all of which were related to motility functions, including bacterial-type flagellum-dependent swarming motility, locomotion, and cilium- or flagellum-dependent cell motility , Po (EV), ΔZur, and Po WT, showed that the Po (Zur) exhibited significantly less flagellum (none) in comparison with the control strain Po (EV) (1–2 flagella per bacterium cell) (p < 0.05), while the ΔZur had no obvious difference in the number of flagella in comparison with the control strain Po WT, both having 1–2 flagella per bacterium cell (p > 0.05)  a,b. ThesZur) had a remarkably reduced halo compared with its control strain, the Po (EV), which nearly swam to the edge of the medium plate , BCS7_RS09455 (encoding zinc ABC transporter ATP-binding protein ZnuC), and BCS7_RS09450 (encoding zinc ABC transporter permease subunit ZnuB). These three genes were all upregulated in the ΔZur, suggesting a negative regulating role of the Zur on the znuABC system in P. odoriferum. This result is unsurprising as the Zur has been reported to regulate zinc homeostasis via the znuABC zinc-uptake system in bacteria  compared to their respective control strains, Po WT and Po (EV), was determined in vitro by macerating the petioles of Chinese cabbage. For the inoculation, we largely followed the method described by Li et al. , were cultured in LB with Rif from a 1% dilution of their seed cultures (OD600 = 1.0) overnight (16 h). One milliliter of the cultures was centrifuge-pelleted at 8000 rpm for 2 min and washed once with RNA-free water before total RNA extraction using TRIzol. The obtained total RNA was used to prepare sequencing libraries using the NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®  following the manufacturer’s recommendations, and the sequencing was performed on an Illumina NovaSeq 6000 platform. The raw reads of each sample were filtered to produce clean data by means of removing reads containing adapters or poly-N and low-quality reads. We mapped the clean data to the P. odoriferum BCS7 reference genome (CP009678.1) using STAR software (2.7.10b) [p-value (padj) < 0.05 and an absolute value of log2(fold change) ≥ 1 were considered as DEGs. The DEGs were annotated using the GO and KEGG databases. GO enrichment was performed using clusterProfiler software (v3.18.0) with a p-value threshold of 0.05 in Fisher’s exact test [For our transcriptome sequencing, three replicates were included for each strain. Cells of the 2.7.10b)  and obta2.7.10b) . Only geact test . The KEGZur) vs. Po (EV). The cDNA was prepared from the remaining total RNA after transcriptome sequencing by EasyScript One-step gDNA removal and cDNA synthesis supermix  and subjected to qPCR using TB Green® Premix Ex Taq™ II (Tli RNaseH Plus)  as previously described [Zur) compared with Po (EV) was analyzed using the 2−ΔΔCt method [Twenty DEGs related to cell motility were selected for validation by means of qPCR from the transcriptome data of Po (escribed . RecA wat method . All qPCZur), Po (EV), ΔZur, and Po WT were cultured overnight in LB medium with Rif for 12–16 h, and the cultures were adjusted to OD600 = 1.0 using the original medium. Subsequently, cell suspensions were diluted with 20 mL LB medium containing Rif at a ratio of 1:100 for growth at 200 rpm, and the OD600 value was monitored after incubation for 0, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 h with an Eppendorf BioPhotometer Plus . Flagella staining and observation: bacterial staining was conducted using the silver staining method as previously described [Zur), Po (EV), ΔZur, and Po WT was cultured in the LB medium with Rif for 12–16 h to OD600 = 1.0. The cell motility of each strain was visualized based on swimming haloes on an LA plate containing 0.3% agar 16 h after 3 μL of the cell suspension was patched onto it and incubated at 28 °C. The experiment was repeated three times with at least three replicates.Bacterial growth curves: cells of Po (escribed , and flat-test or one-way ANOVA in IBM SPSS Statistics 20.A statistical analysis of data differences was performed using an independent