Clostridioides difficile infection. Moreover, new evidence also supports a role for FMT in other conditions, such as inflammatory bowel disease, functional gastrointestinal disorders, or metabolic disorders. Recently, some studies have identified specific microbial characteristics associated with clinical improvement after FMT, in different disorders, paving the way for a microbiota-based precision medicine approach. Moreover, donor screening has become increasingly more complex over years, along with standardization of FMT and the increasing number of stool banks. In this narrative review, we discuss most recent evidence on the screening and selection of the stool donor, with reference to recent studies that have identified specific microbiological features for clinical conditions such as Clostridioides difficile infection, irritable bowel syndrome, inflammatory bowel disease, and metabolic disorders.In the past decade, fecal microbiota transplantation (FMT) has rapidly spread worldwide in clinical practice as a highly effective treatment option against recurrent  In the last decade, multiple studies have expanded knowledge in the field of gut microbiota, including pathogenesis, diagnosis, and therapeutics . To dateC. difficile infection (CDI) [Fecal material has been used in medicine since almost two thousand years. The first description of the use of fecal material for medical purposes dates back to about 1700 years ago; traditional Chinese medicine in particular had perceived the potential role of this biological material and used it for several clinical indications such as gastrointestinal, nervous system, skin, and gynecological diseases . In WestThe first randomized controlled trial that investigated the role of FMT for recurrent CDI was published by van Nood et al. in 2013. They reported that a single infusion of fecal material by nasoduodenal route was superior to standard therapy with vancomycin . In furtOver the years, further issues have emerged, which are still not clarified to date. In particular, in view of the growing number of patients who could benefit from FMT, it is necessary to identify innovative ways to storing fecal material to be used if necessary. Indeed, in the early experiences, FMT was performed only with fresh material from occasional healthy donors, but this approach is not feasible for large-scale use of FMT. To solve this problem, the possibility to create structures to bank the feces after manipulation was suggested, and this approach is supported by the evidence of the effectiveness of FMT performed with frozen material . In consTo date, the only recommendation for FMT in clinical practice is the treatment of recurrent CDI, although a large number of emerging indications are being experienced in several studies .Enterobacteriaceae, Veillonellaceae, and Lactobacillaceae, and lower relative abundance of Ruminococcaceae, Bacteroidaceae, and Lachnospiraceae [CDI is a burdensome clinical issue and represent the most relevant cause of antibiotic-associated diarrhea; its incidence has evolved in recent years and the risk of recurrence after standard antibiotic therapy has widely increased ,23. The piraceae . Furtherpiraceae .To date, several systematic review and meta-analyses have shown an overall cure rate of FMT of up to 90% in preventing further CDI recurrence ,32. MoreC. difficile over infection. A recent clinical trial, including patients affected by UC or Crohn disease with recurrent CDI, reported that FMT has a curative effect on the recurrence of CDI, but has no apparent beneficial effect on the IBD course [Alongside well-established indications such as CDI, several studies have found emerging clinical conditions for which FMT may represent a promising alternative to standard therapies. Most evidence comes from inflammatory bowel disease (IBD) studies. Several alterations of gut microbiota has been proposed as factors contributing to the development of the aberrant immunological response in IBD , but it D course .Gut microbiota disturbance was also involved in other gastrointestinal diseases such as irritable bowel syndrome (IBS). A systematic reviews with meta-analysis showed that FMT may be beneficial in IBS , but thiFurthermore, metabolic and hepatic diseases are also considered emerging indications for FMT. There is great interest towards the modulation of the gut microbiota in metabolic syndrome, as two studies reported promising results in improving peripheral insulin sensitivity ,46. UnfoFMT was also proposed in the treatment of several other clinical conditions, but evidence is limited and results were reported by small studies; thus, the application is limited to clinical studies and selected cases. For instance, FMT was reported as effective in the decolonization of patients carrier of multi-drug resistant organism , in reduDonor selection represents a fundamental challenge in view of the implementation of FMT programs worldwide. To date, there is a broad debate regarding the preference of donor selection, whether the stool donor should be known to the patient or whether it is preferable to use feces from unrelated donor. Moreover, in the case of non-related donor, fecal material could be banked at dedicated structures that provide support to the hospital that will perform FMT .In particular, the ideal stool donor should be a healthy volunteer, without risk factors for infectious or other chronic diseases, and who is willing to “donate” frequently if needed. Unfortunately, although the conditions do not seem too selective, it is not always easy to identify an adequate number of donors to meet the needs of the FMT program. Indeed, data from large stool bank suggest high rates of donor drop out due to high commitment required ; furtherThe screening of potential donors consist in two key landmarks, the preliminary interview and the laboratory testing . A preliThe optimal donor correspond at young individual . Furthermore, blood tests can be considered in case of anomalies of the first round of laboratory tests, endemic spread of some pathogens, emergence of new pathogens or selected cases of recipients . In particular, there is debate about the usefulness of serology for EBV and CMV, as the high prevalence of prior exposure among adult individuals weakens the diagnostic power of this approach, limiting the clinical utility to IgM CMV in donors dedicated to immunosuppressed recipients. Of course, it is not appropriate to exclude subjects with prior exposure to EBV or CMV from the donation because of the unlikely risk of transmission, unless clinical or laboratory suspicion of reactivation. Finally, the candidates could be considered for testing the serology for nematodes, based on social and geographical features and tests availability .Clostridium difficile, fecal parasites, and Helicobacter pylori antigen (this last exam only for upper route of FMT delivery). Enteric pathogens, which must also be investigated in asymptomatic subjects, should be detected with conventional methods  and/or with molecular diagnosis (PCR-based panels) that have shown a high specificity and sensitivity compared to conventional methods in rapid detection of pathogens [Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), extended-spectrum β-lactamase-producing Enterobacteriaceae, and carbapenem-resistant Enterobacteriaceae/carbapenemase-producing Enterobacteriaceae), considering the burden of the gastrointestinal carriage in asymptomatic subjects [Stool testing should include common enteric pathogens, athogens . Furthersubjects ,62 and tsubjects . Nowadaysubjects .Finally, if all blood and fecal tests are negative, the candidate is accepted to become a stool donor. Especially in the fecal bank program, the donor should be available to donate on many occasions over time. For this reason, it is advisable to repeat the screening tests every 8–12 weeks and administer a short questionnaire on the same day of the donation to check for any recent-onset harmful events.In this paragraph we have reported the general rules to select and to screen potential donor for FMT, mainly to treat CDI that is cured by the restorative effect of fecal transfer on gut microbiota. However, for other clinical indications, which find their rationale in the modification of metabolic and inflammatory pathways mediated by gut microbiota, it would be appropriate to identify a specific donor for each case. This issue will be discussed later.The correct recruitment of healthy donors is essential for a standardized and safe FMT procedure ,13. FMT Caudovirales, together with a decreased Caudovirales diversity, richness, and evenness, have been reported in patients with CDI. Moreover, CD eradication was associated with the colonization of a higher abundance of donor-derived Caudovirales contigs detected during follow up. These findings could possibly explain why bacterial fecal filtrate infusion resulted in effective treatment of CDI [C. difficile colitis [The research of the ideal donor in recurrent CDI is obviously a widely debated topic of study. For example, one study identified the optimal donor among nine healthy vegetarian or vegan candidates, selecting the candidate who had a balanced Bacteroidetes/Firmicutes ratio, the highest alpha diversity among screened individuals, and high butyrate concentration. After 10 weeks from a single or multiple FMT, none of the 10 patients experienced CDI recurrence . Of intet of CDI , and shi colitis ,74. HoweFusobacterium spp and Sutterella spp in recipients’ fecal microbiota after the FMT [Akkermansia muciniphila, unclassified Ruminococcaceae, and Ruminococcus spp. was more likely to induce remission compared to antibiotics alone [Many studies analyzed the microbial profile of donors and tried to relate it with clinical and laboratory outcomes in patients with IBD. Clinical outcomes and immunological changes after FMT in patients with IBD were significantly related to the variations of several specific strains in recipients of fecal microbiota . For ins the FMT . Furthercs alone . As alsocs alone ,77,78, ics alone . Furthercs alone .Faecalibacterium, Bifidobacterium longum, Collinsella aerofaciens, and Enterococcus faecium) when compared to mice that received “non responder-like” microbiome  [Several preclinical and clinical studies supported the rationale for donor selection based on gut microbial profile in other disorders associated to gut dysbiosis. Indeed, in the field of anti-cancer treatment, it has been reported that microbiota can influence chemotherapy response . Preclinoidales) ,83. Neveoidales) .Streptococcus, Dorea, Lactobacillus, and Ruminococcaceae spp in the donor microbiota was associated with efficacy in relieving IBS symptoms [Bifidobacterium, Prevotella, and Desulfovibrio). Conversely, Prevotella, and Desulfovibrio were more represented in recipients after FMT than in the donor samples, suggesting that unknown factors changed the intestinal ecosystem, making it more hospitable to these strains [Recently, a randomized placebo-controlled trial of FMT in IBS reported that the abundance of symptoms . Interes strains .Within the context of metabolic diseases, the effect of allogenic FMT post-Roux-en-Y gastric bypass donors was compared with metabolic syndrome donors on glucose metabolism and other parameters in treatment-naïve patients with metabolic syndrome. The authors assessed a decrease of insulin sensitivity in recipients who received FMT from donors with metabolic syndrome compared with using post-surgical donors. Moreover, they identified several microbial OTUs possibly predictive of metabolic response, suggesting a microbiota-related transmissible mechanism of insulin resistance . SimilarTo date, these results appear promising but partially controversial; thus, findings need to be confirmed with stronger evidence and by standardized clinical trial. Further research is needed to identify the favorable microbial signature of donor or other ideal features in disease-specific settings.In this review, the stool donor screening process has been described, and recent evidence has been reported that try to identify the optimal donor for each clinical condition .To date, the clinical characteristics of the donor are well defined; in particular, they are recommended to be a healthy volunteer with a balanced lifestyle, without chronic diseases or family history of metabolic diseases or cancer, and defined laboratory exams must certify the current absence of disease. However, identification of the ideal donor through the microbiological typing of the stool is currently not suitable. First of all, understanding the role of the intestinal microbiota in each chronic disease is an indispensable condition before hypothesizing a personalized approach through FMT. In fact, while the restorative mechanism of FMT in recurrent CDI is now understood, many aspects still need to be understood regarding the treatment of other chronic conditions. Interesting evidence has been reported regarding dysbiosis in IBD or in other chronic conditions, but the contrasting results reported in clinical trials of FMT could be justified by the choice of unsuitable donors. The identification of the microbiological characteristics of the ideal donor for each disease appears to be an achievable goal but still far from being accomplished due to the lack of clinical studies. The current evidence is still limited and insufficient for explaining and resolving the complexity of the interaction between the intestinal barrier and its role in gut-related chronic diseases. However, further studies need to be designed to confirm the encouraging results that have been reported in recent years. In particular, it will be necessary to type the fecal microbiota of the donor and the recipient, and to understand how environmental factors, such as diet, or individual features may benefit (or not) the clinical response to FMT. Understanding the microbial characteristics of the optimal donor, in particular if they are modifiable through lifestyle changes or pharmacological measures, could increase the therapeutic potential of FMT.


Pneumococcal meningitis (PM) remains a global public health concern and affects all age groups. If acquired during infancy or childhood, permanent neurofunctional deficits including cognitive impairment, cerebral palsy, and secondary epilepsy are typical sequelae of neuronal injury. Determination of patients at risk for the development of brain injury and subsequent neurofunctional sequelae could help to identify patients for focused management. Neurofilament light chain (NfL) is an axonal cytoskeletal protein released upon neuronal injury into the cerebrospinal fluid (CSF) and blood. As little is known about the course of neurofilament release in the course of PM, we measured CSF and serum NfL levels longitudinally in experimental PM (ePM).Streptococcus pneumoniae and treated with ceftriaxone. At 18 and 42 h post-infection (hpi), the blood and CSF were sampled for NfL measurements by a single molecule array technology. Inflammatory cytokines and MMP-9 in CSF were quantified by magnetic bead multiplex assay (Luminex®) and by gel zymography, respectively.Eleven-day-old infant Wistar rats were infected intracisternally with p < 0.0001). CSF and serum NfL correlated at 18 hpi  and 42 hpi . Both CSF and serum NfL at 42 hpi strongly correlated with CSF levels of IL-1β, TNF-α, and IL-6 and of MMP-9 depending on their individual kinetics.In ePM, CSF and serum NfL levels started to increase at 18 hpi and were 26- and 3.5-fold increased, respectively, compared to mock-infected animals at 42 hpi (Current results demonstrate that during the peak inflammatory phase of ePM, NfL levels in CSF and serum are the highest among CNS disease models studied so far. Given the strong correlation of CSF versus serum NfL, and its CNS-specific signal character, longitudinal measurements to monitor the course of PM could be performed based on blood sample tests, i.e., without the need of repetitive spinal taps. We conclude that NfL in the serum should be evaluated as a biomarker in PM. Streptococcus pneumoniae and Neisseria meningitidis are the most prevalent causative pathogens for childhood bacterial meningitis (BM) [tis (BM)  with fattis (BM) . Pneumoctis (BM) , 4.In reaction to the bacterial infection, the host’s immune response causes an excessive inflammation that leads to injury to the central nervous system (CNS) , 5. The Neurofilaments (NFs) are a major component of the axonal cytoskeleton and consist of three subunits: NF light chain (NfL), medium chain, and heavy chain. Upon axonal damage, NFs are released into the extracellular space and eventually into the CSF and blood. Their increased levels can be used as a biomarker of neuronal damage , indicatWith the advent of the single-molecule array (Simoa) technology, the sensitivity for quantitation of NfL has been increased to a degree that levels in serum or plasma can be accurately quantified . This teOnly one study has evaluated NFs as a biomarker for neuronal injury in BM across an array of infectious agents. CSF concentrations of NF heavy chain were increased by 85% (22/26) children with BM; peak levels in patients with neurological sequelae were significantly higher than in those without .Present results are the first to investigate the course of NfL in ePM. We hypothesized that serum NfL is a useful biomarker to monitor disease activity in BM, as its kinetics accurately correlated with those in CSF, as well as with typical inflammatory mediators upregulated in ePM.Streptococcus pneumoniae (serotype 3) was cultured overnight in brain heart infusion (BHI) medium, diluted tenfold in fresh, prewarmed BHI medium, and grown for 5 h to reach the logarithmic phase. The bacteria were centrifuged for 10 min at 3100 × g at 4 °C, washed twice, and resuspended in saline (NaCl 0.85%). The resuspended bacterial solution was then further diluted in saline to the desired optical density (OD570nm). The inoculum concentration was determined by serial dilution and culturing on Columbia sheep blood agar (CSBA) plates.A clinical isolate of 5 CFU/ml of living S. pneumoniae serotype 3. Control animals received an equivalent volume of saline. PM was confirmed by the quantitative analysis of bacterial titers in the cerebrospinal fluid (CSF) at 18 hpi. Five microliters of CSF was gained by puncture of the cisterna magna, followed by serial dilution and cultivation on CSBA plates. To confirm PM in animals which were not punctured at 18 hpi, the blood was cultivated on CSBA plates. The numbers of animals per independent experiments was determined by a maximum litter size of 14 pups. Given the probability of premature abortion of experiments due to reaching humane endpoints or spontaneous death in infected animals, or unsuccessful infection, experiments were designed with 4 mock-infected and 10 infected animals per experiment. Four independent experiments were performed, for a total of 56 animals. Animals without confirmed PM were excluded (n = 9). All animals received ceftriaxone  at 18 and 24 hpi. With the dosing regimen applied in the present study, therapeutic concentrations are maintained until the end of the experiment, so there is no resurgence of bacterial infection at later time points. Animals were weighed and clinically scored according to the following scoring scheme  at 0, 18, 24, and before sacrificing at 42 hpi. Spontaneous mortality was documented. Blood sampling in all animals was performed at 18 hpi by puncturing the facial vein using a 20-gauge needle. Blood  was collected in Microvette® 200 Z (clotting activator/serum) (Sarstedt), kept at room temperature for at least 30 min, and then centrifuged for 10 min at 13,000 × g at 4 °C. The supernatant (serum) was collected and kept at − 80 °C for later analysis. Following blood sampling, CSF samples of half of the animals (n = 28) were obtained by puncture of the cisterna magna using a 30-gauge needle. CSF samples were only collected from half of the animals to assess if this additional puncture causes an artefactual increase in NfL level. CSF samples were then centrifuged for 10 min at 13,000 × g at 4 °C, and supernatants were stored at − 80 °C for later use. Animals were treated with ceftriaxone after the collection of specimens. At 42 hpi, the blood and then CSF were sampled again from all animals, followed by sacrificing with pentobarbital . Of note, throughout the experiment, each animal was only punctured once to avoid a possible artificial increase of NfL in CSF and serum by multiple puncturing. Accordingly, if an animal was not punctured successfully or died spontaneously, no CSF could be collected which then resulted in lower datapoints.All animal studies were approved by the Animal Care and Experimentation Committee of the Canton of Bern, Switzerland (license no. BE 01/18). A well-established infant rat model of PM was used , 27. EleCytokines known to be upregulated in PM  were assessed using a magnetic multiplex assay  on a Bio-Plex 200 station (Bio-Rad Laboratories) as earlier reported . Five miNfL in CSF and serum were analyzed using a SIMOA® immunoassay  as previously described ; the ant2O) to a loading volume of 20 μl and electrophoresed under non-reducing conditions in 10% SDS-polyacrylamide gels containing type A gelatin from porcine skin  as proteinase substrate. After electrophoresis for 2 h at 100 V, gels were incubated for 2 × 30 min in SDS-removing buffer (1% Triton X-100), washed 5× with dH2O, followed by incubating in incubation buffer  for 18 h at 37 °C on a shaker. Gels were then stained with Coomassie blue  to visualize the substrate lysis zones of MMP-9 (92 kDa) and MMP-2 (72 kDa). The gelatinolytic activities of MMP-9 and MMP-2 were analyzed using ImageJ software. The amount of expressed MMP-9 was determined as a percentage of that of the constitutively expressed MMP-2 for each sample [The amount of MMP-9 in CSF was assessed by gel zymography as reported earlier . Brieflyh sample .Statistical analyses were performed with GraphPad Prism . Data were first tested for normal distribution with the Kolmogorov-Smirnov test.p value of 0.05 was considered as statistically significant, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.To compare the differences between the non-normally distributed data, a non-parametric Mann-Whitney test was used. Correlations were assessed by using the Spearman correlation coefficient. The results are presented as median with interquartile range. A S. pneumoniae or saline in a first step and to collect CSF samples later. To determine whether this mechanical trauma leads to an artifactual neuronal damage, we measured serum NfL levels of animals before any intervention and compared them with animals at 18 hpi which have been inoculated with saline at baseline.In the present ePM model, intracisternal punctures are performed to inoculate p = 0.18)  at 42 hpi increased 1.6-fold compared to those having been punctured only at baseline (299.3 ± 137.2 pg/ml) (p = 0.33). Accordingly, CSF levels of NfL showed a 1.8-fold increase in animals having had a second intracisternal puncture (789 ± 303.9 pg/ml) compared to those without (436.7 ± 163.2 pg/ml) (p = 0.01)  were not different from those of mock-infected animals at 18 hpi (293.7 ± 168.3 pg/ml) (p = 0.06) and at 42 hpi, they showed a 26-fold higher level  compared to mock-infected controls (590.1 ± 296.4 pg/ml) (p < 0.0001) , while at 18 hpi the levels between the two groups were similar  and in serum (p < 0.0001), whereas those of mock-infected animals remained stable in both fluid compartments  trended to be higher compared to mock-infected animals (590.1 ± 186.9 pg/ml)  with an average CSF concentration (1908.6 ± 2877 pg/ml) being 6.9-fold higher than that in serum (276.9 ± 144.8 pg/ml)  with average CSF levels (9848.6 ± 29768 pg/ml) being 9.8-fold higher than in serum (1007.3 ± 1029 pg/ml)  Fig. .p = 0.02)  and 42 hpi (p < 0.0001)  Fig. c. The re01) Fig. d.Fig. 3p < 0.0001; Suppl. Fig. p = 0.04, r = 0.5126) and a trend at 42 hpi   have been found in patients  and in aIn contrast, the strong correlation of serum and CSF measures of NfL makes it an ideal marker to monitor CNS-based pathology. Present results demonstrate that NfL is upregulated in CSF and serum during ePM and confirm the close relationship between the two fluid compartments. Further, we showed that the increase of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in CSF during an early stage of the disease correlated with the later degree of NfL release. Accordingly, CSF levels of MMP-9, an effector molecule of tissue damage that is released in the function of cytokine expression , 36, werBecause our kinetic CSF study required cisterna magna punctures at two different time points in the same animal, we had to evaluate whether this procedure does lead to artifactual neuronal damage interfering with disease-related levels of NfL. The baseline intracisternal puncture with saline did not cause an increase in serum NfL concentration as compared to animals without any intervention. As our primary interest is to demonstrate the value of NfL as a serum-based marker for ePM and to limit the use of animals, we therefore refrained from performing referring CSF measurements.At 42 hpi, mock-infected animals punctured for a second time at 18 hpi for CSF collection showed a small increase of CSF NfL concentrations compared to animals punctured only at baseline, while serum levels were unaffected. We conclude that this intervention, likely in combination with a local inflammation due to the first puncture, caused a minor neuronal damage. Because the amount of artifactually induced NfL release was less than 2% of that found in ePM at 42 hpi, we conclude that the NfL levels measured are almost exclusively reflective of the infection-related pathology.NfL can reach the systemic circulation by the bulk flow through the ventricular system and then exiting into the subarachnoid space where it is then reabsorbed into the blood stream . In addiIn ePM, TNF-α, IL-6, and IL-1β appear early, prior to neutrophil recruitment, indicating that they are released by brain-resident cells , 41. TNFDuring BM, microglia are activated by several stimuli including pathogen-associated and damage-associated molecular patterns (PAMPs & DAMPs) and pro-inflammatory mediators, leading to the production of cytokines and chemokines. Nevertheless, excessive microglial activation can result in neuronal damage . In the This study has several limitations. We used an experimental model where bacteria are directly injected into the CSF which therefore does not reflect the natural course of the disease. Nonetheless, this method showed a higher success rate compared to methods of hematogenous infection, where successful meningitis is only developed in around 50% , 48. In In the present study, the degree of cortical damage was limited and gross cortical necrosis was not observed (data not shown) and NfL release resulted mainly from brain-diffuse damage. The correlation of NfL release with defined morphological features of a neuronal damage is the next step underway in our laboratory.Nevertheless, this study indicates that NfL is a very sensitive marker able to quantitate neuronal damage before it manifests with the most severe stage of typical PM pathology.During the acute phase of BM, the upregulation of inflammatory mediators causes neuronal damage. Although inflammatory mediators could be used as predictors, they are not a direct indicator of neuronal injury. Furthermore, a clinically useful biomarker for BM needs to be blood-based to avoid the invasive procedure of lumbar puncture. Serum NfL is a sensitive biomarker which reflects neuronal damage with high specificity. Given the strong correlation between CSF and serum, current results provide the conceptual evidence for the use of NfL as a promising biomarker for the evaluation of neuroprotective agents in BM.Additional file 1: Figure S1. At 18 hpi, inflammatory cytokines – TNF-α, IL-6 and IL-1β – correlated with each other (n=17).Additional file 2: Figure S2. IL-1β significantly correlated with MMP-9 at 18 hpi  (A), but showed only a trend at 42 hpi  (B).


Atherosclerosis, calcific aortic valve disease (CAVD), and bioprosthetic heart valve degeneration  represent three diseases affecting distinct components of the circulatory system and their substitutes, yet sharing multiple risk factors and commonly leading to the extraskeletal calcification. Whereas the histopathology of the mentioned disorders is well-described, their ultrastructural pathology is largely obscure due to the lack of appropriate investigation techniques. Employing an original method for sample preparation and the electron microscopy visualisation of calcified cardiovascular tissues, here we revisited the ultrastructural features of lipid retention, macrophage infiltration, intraplaque/intraleaflet haemorrhage, and calcification which are common or unique for the indicated types of cardiovascular disease. Atherosclerotic plaques were notable for the massive accumulation of lipids in the extracellular matrix (ECM), abundant macrophage content, and pronounced neovascularisation associated with blood leakage and calcium deposition. In contrast, CAVD and SVD generally did not require vasculo- or angiogenesis to occur, instead relying on fatigue-induced ECM degradation and the concurrent migration of immune cells. Unlike native tissues, bioprosthetic heart valves contained numerous specialised macrophages and were not capable of the regeneration that underscores ECM integrity as a pivotal factor for SVD prevention. While atherosclerosis, CAVD, and SVD show similar pathogenesis patterns, these disorders demonstrate considerable ultrastructural differences. Atherosclerosis and calcific aortic valve disease (CAVD) represent chronic inflammatory disorders which are characterised by endothelial dysfunction, lipid deposition, macrophage infiltration, and the maladaptive remodelling of the extracellular matrix (ECM) in the arterial wall and valve leaflets ,2,3,4,5.Bioprosthetic heart valves (BHVs) are fabricated from bovine/porcine pericardium or porcine heart valves and undergo structural deterioration (SVD) over time due to the lack of a regenerative capability upon chemical fixation ,7,8. In In agreement with the common mechanisms of development, atherosclerosis, CAVD, and SVD share a number of risk factors, including smoking, arterial hypertension, dyslipidaemia, diabetes mellitus, and chronic kidney disease ,14,15,16With the aim to solve this issue, we have previously developed an original protocol for the staining, embedding, and backscattered scanning electron microscopy visualisation of the mineralised cardiovascular specimens ,18,19 whHere, we employed the above-mentioned approach for performing an ultrastructural investigation of atherosclerotic plaques, calcified native aortic valves (AVs), and degenerated BHVs to compare the pathological phenomena characteristic of the inflamed cardiovascular tissues. Among them were lipid retention, macrophage infiltration and specialisation, neovascularisation, intraplaque/intraleaflet haemorrhage, and mineralisation. Lipid retention, haemorrhages, and calcium deposits were frequently detected across plaques and affected valves. However, intraplaque haemorrhages were associated with leaky neovessels, whereas intraleaflet haemorrhages were provoked by the tissue delamination. Abundant neovascularisation was specific for plaques, while macrophage diversity was a feature of SVD. We first performed a semi-quantitative analysis of all the samples . As underlying health conditions could impact on the development of histopathological features, we have also analysed the prevalence of the patient comorbidities, but did not find any statistically significant differences between the groups .Each of the disorders has been characterised by a specific histopathological pattern . Foam ceWe further carried out a detailed analysis of the indicated ultrastructural features. As atherosclerosis, CAVD, and SVD all involve macrophage infiltration and are largely driven by lipid accumulation, we primarily focused on the formation of foam cells. Depending on the dehydration efficiency, they appeared as large round, oval, or irregularly shaped cells full of black or white globules A. Among We next investigated the histopathological diversity of the macrophages in the affected vascular and valvular tissues. The majority of the macrophages had elliptic or round large nuclei and did not have any specific inclusions in the cytosol, suggesting their moderate activity in the ECM remodelling A. HoweveMonocyte infiltration followed by a macrophage-driven ECM disintegration represents a multi-step process. Electron microscopy snapshots enabled to illustrate its events, including rolling A, adhesiLeukocyte rolling and attachment are induced by endothelial activation and the loss of endothelial integrity, although endothelial dysfunction does not necessarily mean endothelial injury, and re-endothelialisation is a common regenerative phenomenon. Atherosclerotic plaques and native AVs were covered by a layer of endothelial cells elongated along the direction of flow, while BHVs had single endothelial-like cells attached to the prosthetic surface A. MesencWe found intraplaque haemorrhage as a frequent event in the atherosclerotic plaques because of leaky neovessels D. In conMineral deposits were characterised by a remarkable heterogeneity. Both plaques and native/bioprosthetic valves contained macrocalcifications between the inflow and outflow surfaces A, which The concept that the development of atherosclerosis and heart valve disease is interrelated was a matter of debate during the last decade ,25,26,27Despite lipid deposition being prominent regardless of the tissue type, it was most pronounced in atherosclerotic plaques where the foam cells formed multiple conglomerates in contrast to native and prosthetic valves. However, the macrophage diversity in plaques  was limited, whereas BHVs contained a number of macrophage subtypes . The blood vessels in plaques and native AVs had distinct structural properties. Plaque neovessels frequently leaked and were responsible for intraplaque haemorrhages. While native AVs also relied on the microvasculature to meet the nutritional needs, it generally did not leak and intraleaflet haemorrhages have been associated with stress-driven ECM degradation and delamination. Substantial delamination and ECM disintegration were characteristic of BHVs which were incapable of regeneration. The calcification patterns in the arteries and heart valves were similar and included both large mineral deposits and microcalcifications. The schematic representation of the study findings is depicted in Macrophage infiltration is evident in both atherosclerotic and valvular disease, being responsible for the neointimal and dystrophic remodeling, respectively ,35,36,37Recent single-cell sequencing studies have indicated the leading role of non-lipid-laden macrophages in promoting the neointimal inflammation ,44,45. I(Neo)vascularisation provides a route for the migration of macrophages and other immune cells to the degraded ECM and altered microenvironment, thereby promoting inflammation and calcification in plaques and AVs ,48,49,50The ultimate outcome of inflammation, mechanical stress, and intraplaque or intraleaflet haemorrhage is calcification ,57,58, wDifferences in the prevalence and localisation of different ultrastructural features in distinct cardiovascular tissues may be linked to the site- or stage-specific alterations. Seemingly, the deposition of lipids in the ECM and their internalisation by macrophages play a key role in both the initiation and progression of atherosclerosis, as foam cells are abundant across the entire neointima. However, the localisation of lipid-laden macrophages in dysfunctional native or bioprosthetic valves is typically restricted to their inflow or outflow surfaces, suggesting that, in these settings, lipid retention and foam cells are responsible mainly for the initiation and do not have a pivotal significance further.The main shortcoming of this study is that is has been limited to the observational approach and did not include any interventions. Nevertheless, here we focused on drawing parallels between the main inflammation-, remodeling-, and calcification-related disorders of the circulatory system components that did not require interventional study. To conclude, atherosclerosis, CAVD, and BHV failure share general pathogenetic mechanisms, while almost every of them has specific nuances and ultrastructural features which deserve further investigation.n = 36) were excised during carotid endarterectomy conducted because of chronic brain ischemia. Calcified native AVs (n = 12) and BHVs  were obtained from the patients who underwent primary heart valve replacement because of CAVD or repeated heart valve replacement due to SVD, respectively. In-stent restenosis was the exclusion criterion for the patients with carotid atherosclerosis, whereas bicuspid AV, rheumatic heart disease, and infective endocarditis were exclusion criteria for those with CAVD. Median of BHV functioning was 5.75 (interquartile range 4.69–11.37) years. All surgical interventions were performed in the Research Institute for Complex Issues of Cardiovascular Diseases  in 2019–2020. The investigation was carried out in accordance with the Good Clinical Practice and the Declaration of Helsinki. The study protocol was approved by the Local Ethical Committee of Research Institute for Complex Issues of Cardiovascular Diseases . All the patients provided a written informed consent after receiving a full explanation of the study.Atherosclerotic plaques  for 24 h, stained in 2% osmium tetroxide for 48 h, dehydrated in ascending ethanol series , stained in 2% alcoholic uranyl acetate  for 5 h, dehydrated in isopropanol  for 5 h and acetone (1 h), impregnated with acetone: epoxy resin  mixture (1:1) for 6 h and with epoxy resin for 24 h, and were finally embedded into the fresh epoxy resin at 60 °C. The samples were then grinded, polished , and counterstained with Reynolds’s lead citrate  for 7 min. After a brief washing in double distilled water, samples were sputter coated (10 nm thickness) with carbon  and visualised by means of backscattered scanning electron microscopy at 10 or 15 kV voltage .Excised plaques as well as degenerated and calcified fragments of all available valve leaflets were fixed in two changes of 10% neutral phosphate buffered formalin  for 24 h at 4 °C, postfixed in 1% osmium tetroxide  and cut on a cryostat (Microm HM. Sections (5 μm thickness) were then fixed in 4% paraformaldehyde  for 10 min, washed thrice in phosphate-buffered saline , incubated in isopropanol for 5 min, stained with Oil Red O  for 15 min, briefly washed in 60% isopropanol, counterstained with modified Mayer’s hematoxylin , washed in tap water for 5 min and in double distilled water briefly, and finally mounted . Visualisation was performed by light microscopy .p values ≤ 0.05 were regarded as statistically significant.Statistical analysis was performed using GraphPad Prism 7 . Data were represented by the proportions or by the median and interquartile range. The proportions were compared by Pearson’s chi-squared test or Fisher’s exact test.


Rapid and accurate diagnosis of central nervous system (CNS) infections is important, and laboratory tests help diagnose CNS infections. Even when the patient has symptoms, laboratory tests often do not reveal any specific findings. The potential of vitamin D-binding protein (VDBP) to be used as a biomarker for viral and bacterial CNS infections was studied.GC gene encoding VDBP was also performed. VDBP levels were analyzed and compared by CNS infection, pathogen, CSF opening pressure, and GC genotype.A total of 302 subjects with suspected CNS infection who underwent lumbar puncture were included. Clinical and laboratory data were collected retrospectively. VDBP levels were measured in the cerebrospinal fluid (CSF) samples. Genotyping for the n = 90) and a non-CNS infection group (n = 212) were studied. In terms of its receiver operating characteristic, CSF VDBP showed an area under the curve of 0.726 for the diagnosis of CNS infection. CSF VDBP levels were significantly different between the CNS infection and non-infection groups. The CNS infection group with enterovirus showed a statistically lower distribution of CSF VDBP levels than the other virus groups. The group with CSF opening pressure > 25 cmH2O showed higher CSF VDBP levels than the other groups. There was no significant difference in GC gene allele distribution between the CNS infection and non-infection groups.A CNS infection group (CSF VDBP levels were increased in patients with CNS infection. The CSF VDBP showed potential as a new biomarker for viral and bacterial CNS infections. The central nervous system (CNS) can be infected with numerous infectious agents, which results in significant morbidity and mortality, despite the effectiveness of available antimicrobials –3. GlobaThe gold standard for the diagnosis of CNS infections is microbiological culture. However, this method shows low sensitivity, and the diagnosis may be delayed . MoreoveIn clinical situations, the initial diagnosis of CNS infection, including meningitis, usually relies on the cytological and biochemical characteristics of cerebrospinal fluid (CSF), culture results, neuroimaging, and clinical manifestations. A lumbar puncture, also referred to as spinal tap, is the commonly performed procedure to obtain CSF to diagnose meningitis , 8. The GC) has a high rate of polymorphism. Two single-nucleotide polymorphisms (SNPs), rs7041 and rs4588, have given rise to three major polymorphic isoforms of VDBP . Their frequencies also differ among ethnic populations [Vitamin D-binding protein (VDBP) is a 58-kDa multifunctional protein known to be an acute phase reactant whose level can change depending on various conditions –15. Althulations . The levVDBP is known to potentially modulate the inflammatory and immune responses in the blood and body fluids . In a prThis prospective study enrolled 302 patients who underwent lumbar puncture for the purpose of diagnosis from September 2017 to May 2020. We collected clinical and laboratory data including age, sex, cellular differential counts of CSF, chemical analysis of CSF (including glucose and protein levels), and the final diagnosis from electronic medical records (EMR). Culture and molecular laboratory tests, including multiplex PCR  and FilmArray  Meningitis/Encephalitis ME panel were utilized at the clinicians’ discretion for the identification of pathogens. The remaining CSF samples were stored at − 80 °C until the CSF VDBP measurements.These enrolled patients were classified into two groups (CNS infection and non-CNS infection) depending on the results of CSF analysis and the final diagnosis. We classified patients as having CNS infections if the results of CSF analysis, imaging studies, or clinical symptoms strongly suggested meningitis, meningoencephalitis, or encephalitis. The differential diagnosis for meningitis, encephalitis, and meningoencephalitis is mainly made by clinical symptoms, and the criteria are as follows . Meningi3 in differential counts for CSF, it was regarded as traumatic tapping and excluded from this study. With reference to a previous study [2O and ≤ 25 cmH2O). The distributions of VDBP in the two groups were compared.When the number of red blood cells exceeded 1000 per mmus study , 25 cmH2CSF VDBP levels were measured using an enzyme-linked immunosorbent assay kit  according to the manufacturer’s protocol.GC gene genotyping for rs7041 and rs4588 was performed using a TaqMan SNP Genotyping Assay  and an ABI ViiA 7 Real-Time PCR System  according to the manufacturer’s instructions. GC gene alleles were determined as follows: Gc1f , Gc1s , and Gc2 . In addition, the distribution of CSF VDBP levels according to GC allele was also compared in the CNS infection group and non-CNS infection group, respectively. For simplicity of comparison, only CSF VDBP levels from subjects with homozygous GC alleles were used.Genomic DNA was isolated from peripheral blood leukocytes using a DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. GC genotype and pathogen. All statistical analyses were performed using MedCalc Statistical software, version 17.2 . P values < 0.05 were considered statistically significant.T-tests or Mann-Whitney U tests were used to compare variables depending on the normality of CSF VDBP distribution. Pearson’s Chi-square test was used to compare the proportion of categorical variables. Box-and-whisker plots were used to demonstrate the VDBP distribution. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic value of CSF VDBP for CNS infection. The Kruskal-Wallis test was used to test for differences in the VDBP distribution by A total of 302 patients were enrolled in this study. The median age of the study subjects was 31, and the number of patients under 18 years old was 129. A total of 166 male subjects were included, and they comprised 55.0% of the subjects. Among them, 90 (29.8%) and 212 (70.2%) patients were classified in the CNS infection group and non-CNS infection group, respectively, based on the final diagnosis in the electronic medical records. There were no significant differences in median age, the number of patients under 18 years old, or the number of males between the two groups. In the CSF analysis, the CNS infection group showed higher total protein levels, WBC counts, and VDBP levels than the non-CNS infection group. The general characteristics of the study subjects and the results of the CSF analysis are shown in Table n = 29), Epstein-Barr virus (EBV) (n = 3), varicella zoster virus (VZV)(n = 4), herpes simplex virus type 2 (HSV2) (n = 1), human herpesvirus 6 (HHV6)(n = 1), and EBV and VZV detected simultaneously (n = 1), whereas Neisseria meningitidis (n = 2) was detected by culture. The remaining 39 patients were clinically diagnosed based on an imaging study, CSF cell counts, and clinical findings checked by neurology physicians. Among the eight encephalitis patients, one patient had a pathogen detected in a molecular test , and no pathogen was detected in the other seven patients. Among the two meningoencephalitis patients, one patient had a pathogen detected in a molecular test , and no pathogen was detected in the other patient. The etiologies of the CNS infections are summarized in Table Among the 90 patients with CNS infections, 80 were diagnosed with meningitis, 8 with encephalitis, and 2 with meningoencephalitis. Of the 80 patients with meningitis, 41 patients had pathogens detected in laboratory tests, including molecular tests. These pathogens included Enterovirus (n = 90) and non-CNS infection group (n = 212) was 3.20 (1.70–5.10) μg/mL and 1.10 (0.80–1.80) μg/mL, respectively. CSF VDBP levels in the CNS infection group were significantly higher than those in the non-CNS infection group (P < 0.001)  and the pathogen-non-identified group (n = 47) was 2.34 μg/mL (1.46–4.53) and 3.76 μg/mL (2.25–5.21), respectively, and the difference between the groups was not significant (P = 0.075)  and CNS infection group with pleocytosis in the CSF cell count . The difference in CSF VDBP levels between the two groups was statistically significant (P = 0.035).CSF VDBP levels in the study subjects are summarized in Table oup n = 22 was 3.2oup n = 22 was 3.2P < 0.001) of 0.726 , the enterovirus group  showed a significantly lower distribution of CSF VDBP levels than the EBV  and VZV  groups (P = 0.022) . Neisseria meningitidis was identified in two cases, and the CSF VDBP levels in those two cases were 10.53 μg/mL and 14.74 μg/mL, respectively. The CSF VDBP levels of other pathogens identified in only one case are as follows: HSV2 (18.69 μg/mL), HHV6 (0.76 μg/mL), and simultaneous detection of EBV and VZV (6.67 μg/mL).We compared the CSF VDBP levels in the CNS infection group to determine whether CSF VDBP levels were different depending on the pathogens of CNS infection. In the comparison of the distribution of CSF VDBP levels among infections caused by pathogens that were identified at least three times   Fig. .Fig. 4Tn = 82, 27.15%) was the most common genotype, followed by Gc2-1f type  and Gc1f-1f type  among all study patients. The three most frequent genotypes were in the order Gc1f-1f > Gc1s-1f > Gc2-1f in the CNS infection group, and Gc1s-1f > Gc2-1f > Gc1f-1f in the non-CNS infection group. There was no significant difference in genotype distribution between the two groups  was the most common allele, followed by Gc2  and Gc1s  among all study patients. The most frequent alleles were in the order of Gc1f > Gc2 > Gc1s in the CNS infection group, and Gc1f > Gc1s > Gc2 in the non-CNS infection group. There was no significant difference in allele distribution between the two groups  or the non-CNS infection group (P = 0.135).Gc1s-1f , the potential for CSF VDBP to be used as a single diagnostic marker of CNS infection in the group with normal CSF cell counts could not be determined from our data.Our research team had observed that the CSF VDBP level was significantly increased in patients with meningitis and has reported that CSF VDBP concentration can potentially be considered a new biomarker for the diagnosis of meningitis, in a previous study . HoweverVDBP is a protein that has many functions, including the transport of vitamin D metabolites, actin sequestration, and regulation of immune responses . VDBP inn = 5) and VZV (n = 4), were not sufficient to statistically compare with the enterovirus cases; thus, it may be difficult to determine clinical significance and draw conclusions from the current results. Further studies are necessary to elucidate this observation.In the comparison of CSF VDBP levels among cases with identified pathogens , the enterovirus group showed a lower distribution than the other two groups. Enterovirus is the most common causative virus of meningitis and cases in which aseptic meningitis is caused by either enterovirus, EBV, or VZV are usually self-limited with good prognoses . The imm2O) than in patients with CNS infections with low opening pressure (≤25 cmH2O). Therefore, this result suggests that CSF VDBP level might be useful not only for the diagnosis of viral and bacterial CNS infections but also for the assessment of meningitis severity. CSF opening pressure has been reported to increase in bacterial meningitis and approach the reference range in viral meningitis and also to increase due to posture and patient anxiety [2O, and CSF VDBP in this group showed a higher distribution than in the group with opening pressures ≤25 cmH2O. Therefore, it will be worth further research to determine whether CSF VDBP could be an objective biomarker that specifically increases in bacterial CNS infections.The opening pressure during CSF tapping reflects the intracranial pressure (ICP). Meningitis causes increased intracranial pressure (IICP), which leads to clinical symptoms such as headache, nausea, vomiting, and cranial nerve palsy , 31. It  anxiety , 36. In GC genotype and allele frequencies are known to vary among ethnicities [GC allele frequencies than African American and Caucasian individuals [GC genotype and allele frequency did not show any statistical correlation with the presence or absence of CNS infections. The GC genotype exhibits differences in affinity for vitamin D metabolites with the hierarchy of affinity binding being Gc1f > Gc1s > Gc2 [GC genotype can influence serum VDBP levels [GC alleles (P = 0.561). This might originate from the difference between CSF and blood, and further studies are needed to evaluate the difference in function and concentration of CSF VDBP among GC genotypes.The major nicities . The majnicities . It has ividuals . A previividuals , which wividuals , the GC 1s > Gc2 . In addiP levels , 40. FurP levels . HoweverOur study has several limitations. First, our study did not differentiate between children and adults. The common CNS infection pathogens and the concentration of CSF VDBP may differ by age group, so further research for examining different age groups is needed. Second, in the CNS infection group, not all patients were found to have laboratory-confirmed pathogens. In addition, cases of infection in the non-CNS infection group cannot be ruled out because PCR and culture tests were not performed for all potential pathogens.In summary, we reconfirmed that CSF VDBP levels were increased in patients with viral and bacterial CNS infections. We also observed that the CSF VDBP level has the potential to reflect the severity of meningitis. Thus, through this study, we suggest that CSF VDBP has clinical utility as a new biomarker for the diagnosis of viral and bacterial CNS infections.


Introduction: The diagnosis of Parkinson’s disease (PD) mainly relies on clinical manifestation, but may be difficult to make in very early stages of the disease, especially in pre-motor PD. Thus, there is great interest in finding a biomarker for PD. Among diagnostic biomarkers, the most promising molecules are those which reflect the pathophysiological mechanisms of the disease. Until now, only α-synuclein, a classical CSF Alzheimer’s disease biomarker, and neurofilament light (NFL) chains have turned out to be helpful in differential diagnosis between PD and healthy control subjects.Aim: To assess whether CSF molecules related to some pathological processes present in PD might be of interest in the diagnosis of PD and whether they correlate with disease severity.Methods: CSF levels of S100B and neuron-specific enolase (NSE) were measured in 58 PD patients and in 28 healthy control subjects. Correlations were determined between the levels of these CSF molecules and measures of disease severity , as well as disease duration and levodopa dose.Results: CSF S100B and CSF NSE were both significantly increased in PD subjects vs. healthy controls . CSF S100B was significantly positively correlated with measures of disease severity (H-Y score and UPDRS part III), as well as disease duration (p < 0.05). No correlation was found between CSF NSE levels and disease severity or disease duration (p > 0.05). CSF S100B levels alone provided a relatively high discrimination (AUC 0.77) between PD and healthy controls, with 60.7% sensitivity and 88.5% specificity (p < 0.001) at a cut-off value of 123.22 pg/ml. Similarly, CSF NSE levels alone provided a relatively high discrimination (AUC 0.775) between PD and healthy controls, with 78.6% sensitivity and 74.1% specificity at a cut-off value of 51.56 ng/ml (p < 0.001).Conclusions: Our results show that both CSF S100B and CSF NSE seem to be promising markers of the axonal and glial degeneration present in PD. Additionally CSF S100B may be a promising marker of PD progression. Parkinsonian syndromes involve a large group of different disorders, with most frequent Parkinson’s disease (PD), but also rarer atypical parkinsonisms. Differential diagnosis of these disorders may be sometimes challenging, especially in very early stages of the disease. Presently, the diagnosis of PD is based on the clinical manifestation and the exclusion of atypical features suggestive of atypical parkinsonism.That is why the search for blood and CSF biomarkers which could be helpful in PD diagnosis is so crucial. Other currently available biomarkers for PD are either expensive or not easily available. Additionally, there is no specific CSF biomarker for PD, to date. This is partially due to the mixed pathology of PD, with different underlying pathological processes  as a marker of neuronal damage.S100B is a calcium binding protein mostly produced and released by astrocytes in the central nervous system . S100B levels were presented in picograms per milliliter (pg/ml).CSF NSE levels were measured using human Elisa kit (Demeditec). NSE levels were expressed in nanograms per milliliter (ng/ml). All samples were analyzed in duplicates.Additionally, we collected CSF samples from 28 healthy controls matched for age and gender. Control subjects were also patients admitted to the Department of Neurology, who required lumbar puncture for different reasons  and had pathological processes in the CNS excluded.The study was approved by the local ethics committee of Medical University of Lublin, Poland (consent ID KE-0254/292/2017), and all study participants gave written informed consent for participation in the study.t-test for NSE, and a non-parametric Mann Whitney test for S100B. Correlations are presented with Pearson’s coefficient, for NSE with age, disease duration, disease severity  and levodopa dose as well as levodopa equivalent; and with nonparametric correlation coefficient rho Spearman’s for all S100B correlations. P < 0.05 was considered statistically significant.Comparison between groups was made by using a parametric To establish the optimal cut-off point the Youden index was used, which maximizes the sum of sensitivity and specificity.p) were tested by the GraphPad Prism, version 7.0 (GraphPad Software Inc.).Descriptive statistics were calculated for every group. Significances .The PD and healthy controls group were matched for gender and age, no statistically significant differences between investigated groups were found in terms of age or gender  vs. 84.74 (50.75–110.23) ng/L; R = 0.5917, p = 0.01).Among PD patients a significant positive correlation was found between CSF S100B levels and disease severity assessed in H-Y scale .A positive correlation was also found between CSF S100B levels and disease duration (p = 0.07). No correlation was also found between CSF S100B levels and levodopa dose (p = 0.34) or levodopa equivalent  between PD and healthy controls, with 60.7% sensitivity and 88.5% specificity , overall levodopa dose (p = 0.84) or levodopa equivalent (p = 0.95).No significant correlations among PD subjects were found between CSF NSE levels and the patients’ age  between PD and healthy controls, with 78.6% sensitivity and 74.1% specificity .No correlations were found between NSE and S100B neither in PD subjects group nor in healthy control subjects  or disease duration. The reason for such results may also be a short disease duration of PD patients involved in our study. At earlier stages of the disease the neurodegenerative process is mainly limited to the midbrain, and is not widespread to the whole brain. It is possible that involvement of PD in later stages of the disease would change the results of the correlation analysis.Interestingly, we found no correlations between CSF NSE and CSF S100B either in PD subjects’ group or in healthy control subjects. This could potentially be explained by pathological specificity of neurodegenerative processes present in PD. It is known that the loss of dopaminergic neurons observed in brains of PD patients is typically accompanied by a glial reaction (Teismann et al., Since NSE seems to be a non-specific marker of neuronal damage in different neurological disorders, it cannot be treated as a single CSF biomarker to differentiate PD patients from controls. Nevertheless, the results of our study do show that increased CSF NSE probably reflect neuronal damage present in PD. The same applies to increased CSF S100B level in PD, which should be treated as non specific marker of glial damage in PD. Since increased CSF NSE and S100B levels showed a relatively high discriminatory value for differentiating between PD and control subjects, so their levels may be helpful in differential diagnosis, especially when used with other CSF biomarkers for PD (Parnetti et al., The limitations of our study need to be mentioned. The first limitation is limited number of participants. This is mainly due to the fact that lumbar puncture requires hospitalization, is not widely accepted by all patients and their families, and its performance does not directly influence the diagnostic process. Secondly, the diagnosis of PD subjects involved in the study was based on clinical symptoms, which is however a typical diagnostic approach in neurodegenerative departments.Presently, there is no single CSF biomarker which allows to differentiate between PD and healthy control subjects. This is partially due to different underlying pathological processes present in PD, but also due to spatially restricted neurodegenerative process present in PD, limited mainly to the midbrain, especially in the early stage of the disease. Nevertheless there is sufficient evidence for some CSF biomarkers, including α-synuclein, NFL and classical AD biomarkers, to be helpful in differential diagnosis of PD. But still the highest performance accuracy can be achieved while assessing a combination of biomarkers. Since no single CSF brain derived protein can alone differentiate PD from healthy subjects, that is why different other particles have to be considered and evaluated to find the proper biomarkers for PD.Our results require confirmation on larger group of PD patient, in different disease stages, nevertheless both CSF S100B and CSF NSE seem to be promising markers of axonal and glial degeneration, respectively, as parallel processes present in PD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Medical University of Lublin, Poland. The patients/participants provided their written informed consent to participate in this study.EP: conceptualization, data collection, formal analysis, investigation, and writing—original draft. EP and KR: methodology, software, and writing—review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The mDurance® system is an innovative digital tool that combines wearable surface electromyography (sEMG), mobile computing and cloud analysis to streamline and automatize the assessment of muscle activity. The tool is particularly devised to support clinicians and sport professionals in their daily routines, as an assessment tool in the prevention, monitoring rehabilitation and training field. This study aimed at determining the validity of the mDurance system for measuring muscle activity by comparing sEMG output with a reference sEMG system, the Delsys® system. Fifteen participants were tested during isokinetic knee extensions at three different speeds , for two muscles  and two different electrodes locations . The maximum voluntary isometric contraction was carried out for the normalization of the signal, followed by dynamic isokinetic knee extensions for each speed. The sEMG output for both systems was obtained from the raw sEMG signal following mDurance's processing and filtering. Mean, median, first quartile, third quartile and 90th percentile was calculated from the sEMG amplitude signals for each system. The results show an almost perfect ICC relationship for the VL (ICC > 0.81) and substantial to almost perfect for the RF (ICC > 0.762) for all variables and speeds. The Bland-Altman plots revealed heteroscedasticity of error for mean, quartile 3 and 90th percentile (60 and 300 deg/s) for RF and at mean and 90th percentile for VL (300 deg/s). In conclusion, the results indicate that the mDurance® sEMG system is a valid tool to measure muscle activity during dynamic contractions over a range of speeds. This innovative system provides more time for clinicians  and sport trainers , thanks to automatic processing and filtering of the raw sEMG signal and generation of muscle activity reports in real-time. The application of surface electromyography (sEMG) has been widely used to measure muscle activity signals, facilitating access to electrophysiological processes that cause the muscle to generate force and produce movement De Luca, . In mediThe electromyographs present valued benefits providing depth knowledge as an assessment tool in the prevention, monitoring rehabilitation, and training field. The most advanced and complex electromyographs in the field of research have incorporated numerous sEMG channels and synchronization with other technologies such as accelerometry, force platforms and motion capture systems  and software (proprietary android app and cloud service), has not been validated yet, so a joint validation (software and hardware) is found fundamental. Hence, the aim of the current study is to evaluate the concurrent validity of the mDurance system, both hardware and software, for measuring muscle activity by comparing sEMG output with a reference sEMG system .Fifteen participants  were recruited for this study. All participants met the inclusion criteria: (1) older than 18 years old; (2) be able to extend and flex the knee with a range of motion >90° (between 90 and 180°); and (3) free of any neurological disorders or musculoskeletal injuries within the last 6 months. After receiving detailed information on the objectives and procedures of the study, each participant signed an informed consent form in order to participate, which complied with the ethical standards of the World Medical Association's Declaration of Helsinki (2013). It was made clear that the participants were free to leave the study at their convenience. The study was approved by the Universidad de La Frontera (Chile).Participants were refrained from severe physical activity for, at least, 48 h before the tests. All tests were conducted, at least, 3 h after eating. Prior to all testing, height and body mass were measured using a precision stadiometer and balance  for descriptive purposes. For the measurements, the vastus lateralis (VL) and rectus femoris (RF) were collected from both legs and with both sEMG systems simultaneously  is a portable and low-cost sEMG system that consists of three parts. First, a Shimmer3 EMG unit , that is a bipolar sEMG sensor for the acquisition of superficial muscle activity. Each Shimmer sensor is composed of two sEMG channels, with a sampling rate at 1,024 Hz. Shimmer applies a bandwidth 8.4 kHz, the EMG signal resolution is 24 bits and overall amplification of 100–10,000 V/V. The electrodes used were pre-gelled Ag/AgCl with a diameter of 10 mm and an interelectrode distance of 20 mm. Second, the mDurance (Android) mobile application is responsible for receiving data from the Shimmer unit and sending it to a cloud service. And, third, the mDurance cloud service where the sEMG signals are stored, filtered, and analyzed, generating the reports.TrignoTM Wireless System , henceforth Delsys, was the reference system used to compare with the novel electromyograph. It is a widespread commercial sEMG system with 16 channels and a sampling rate at 2,000 Hz. Besides, Delsys applies a built-in filter of 20–450 Hz bandwidth, the EMG signal resolution is 16 bits, and the inter-sensor latency <500 us. The acquisition was carried out via the EMGworks® Software .Both sEMG systems were compared through their natural hardware configurations despite their differences , and it was taken into account for the processing and filtering of the signal.A HUMAC Norm  isokinetic dynamometer was used to control and measure angular displacement during knee extension sets. The dynamometer was used to reduce variability in the performance of the movement by controlling motion speed and position.For the validation testing, participants were instructed to cycle for 10-min on a stationary bike at a self-selected pace followed by mobility exercises  for the warm up . Then, wFor the processing and filtering of the raw signals of the two independent systems into the sEMG output signal, we implemented in Python the mDurance methodology used in its cloud service. (1) First, both the isometric and dynamic tests were filtered using a fourth order Butterworth bandpass filter with a cut-off frequency at 20–450 Hz, only for mDurance raw signal since Delsys already includes this filter by the hardware. (2) Second, the signal was smoothed using a window size of 0.025 s root mean square (RMS) and a overlapping of 0.0125 s between windows for both systems separately. (3) And third, the MVIC value was calculated using the average of the three maximum peaks of the RMS signal during the 5 s isometric test, and being the MVIC used for normalized the RMS signal of each system. The use of the average (following mDurance mythology) instead of the peak for the MVIC calculation will not affect the magnitude of the sEMG outputs inter-systems Burden, . Python To determine the onset and offset of each muscle contraction, a threshold of 10% was applied to the normalized RMS signal from the MVIC  De Luca. Subsequt-test) was conducted between systems, and the Cohen's d effect size (ES) was used to interpret the magnitude of the differences . Descriptive statistics are represented as mean and standard deviation (SD). In order to examine the concurrent validity between systems , the level of agreement between sEMG data from both devices was examined. To that end, a comparison of means  between systems for measuring the sEMG activity of the RF at different speeds (r > 0.7) between data from both systems for all parameters at 60, 180, and 300 deg/s. The ICCs reported almost perfect relationships (ICC > 0.81) for every variable at any speed condition .Despite the pairwise comparisons showed some significant differences , and substantial to almost perfect ICCs (ICC > 0.762) for all parameters at every speed.Regarding the other muscle , the means comparison revealed the lack of differences at any speed . The corIn order to ensure the lack of effect of electrode position , a between systems comparison  was performed for every parameter at every speed for both right and left leg. Almost perfect ICCs were obtained, with ICCs > 0.88, where the confidence interval spans between moderate and almost perfect (0.411–0.980) in all cases.The Bland-Altman plots revealed heteroscedasticity of error for some sEMG-related parameters of the RF at 60 deg/s , at 180 deg/s , and 300 deg/s  .For the VL , the BlaThis study aimed at determining the validity of the mDurance system for measuring muscle activity during isokinetic knee extensions at three different speeds , for two muscles  and two different electrodes locations , by comparing sEMG output with a reference sEMG system, the Delsys. The results obtained suggest that the mDurance system provides valid sEMG output data for VL and RF during knee extensions at 60, 180, and 300 deg/s speed, showing a strong concurrent validity as compared to Delsys sEMG system, based on Pearson's coefficients and ICCs referring to a relative validity. However, Bland-Altman plots suggest an acceptable absolute validity, due to the random errors, going from ±8% for the mean to ±15% for the Perc90.r > 0.7, p < 0.001) reported by the Pearson correlation analysis for the RF, and significant for the VL , for all speeds and all variables. In comparison with our results, Burns et al.  and sports  applications. Thirdly, the lack of standardized methods and analysis for sEMG validations studies may make difficult to compare with similar validations. Finally, an important consideration is that validation data were obtained from an analysis based on within-participant variation (CV) rather than on different days  so, our current reliability statistics might not generalize to runs performed several days apart.The results indicate that the mDurance® sEMG system is a valid tool to measure muscle activity during isokinetic contractions over a range of speeds, tested in laboratory conditions. Our results revealed an excellent relative validity for all variables, and an acceptable to good-enough absolute validity, with a systematic bias of 1.62 ± 8% random error for the mean. From a practical standpoint, mDurance as a system made up of Shimmer sensor and proprietary software, is a valid sEMG system, as compared to Delsys. This novel system includes a user-friendly software, a light hardware and a reasonable price, which make it more affordable and accessible for clinicians and sport trainers. In addition, it provides more time for interpretation and for addressing patients' pathologies or making recommendations to athletes instantly, thanks to the generation of real-time reports. Further research is required to establish reliability (test-re-test) over several days and to check the validity of mDurance measurements in more muscles from other areas and sizes, in more exercises modalities  and at different submaximal intensities.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Universidad de La Frontera, Temuco, Chile. The patients/participants provided their written informed consent to participate in this study.AM-M, FC-P, MD, and OB defined the experimental design. AM-M and ER-M collected the data. FC-P carried out in Python the mDurance's cloud computing simulation for signal processing and filtering. FG-P carried out the statistical analysis. AM-M conceptualized the approach and together with FG-P wrote the paper. LR-S, MD, OB, ER-M and FG-P reviewed the manuscript for scientific content. All authors have read and agreed to the published version of the manuscript.MD and OB were affiliated with the company mDurance Solutions S.L. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Osteoporosis is a metabolic bone disorder that leads to low bone mass and microstructural deterioration of bone tissue and increases bone fractures. Resveratrol, a natural polyphenol compound, has pleiotropic effects including anti-oxidative, anti-aging, and anti-cancer effects. Resveratrol also has roles in increasing osteogenesis and in upregulating mitochondrial biogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, it is still unclear that resveratrol can enhance osteogenic differentiation or mitochondrial biogenesis of periosteum-derived MSCs (PO-MSCs), which play key roles in bone tissue maintenance and fracture healing. Thus, in order to test a possible preventive or therapeutic effect of resveratrol on osteoporosis, this study investigated the effects of resveratrol treatments on osteogenic differentiation and mitochondrial biogenesis of PO-MSCs.The optimal doses of resveratrol treatment on PO-MSCs were determined by cell proliferation and viability assays. Osteogenic differentiation of PO-MSCs under resveratrol treatment was assessed by alkaline phosphatase activities  as well as by extracellular calcium deposit levels (a late biomarker). Mitochondrial biogenesis during osteogenic differentiation of PO-MSCs was measured by quantifying both mitochondrial mass and mitochondrial DNA (mtDNA) contents.Resveratrol treatments above 10 μM seem to have negative effects on cell proliferation and viability of PO-MSCs. Resveratrol treatment (at 5 μM) on PO-MSCs during osteogenic differentiation increased both ALP activities and calcium deposits compared to untreated control groups, demonstrating an enhancing effect of resveratrol on osteogenesis. In addition, resveratrol treatment (at 5 μM) during osteogenic differentiation of PO-MSCs increased both mitochondrial mass and mtDNA copy numbers, indicating that resveratrol can bolster mitochondrial biogenesis in the process of PO-MSC osteogenic differentiation.Taken together, the findings of this study describe the roles of resveratrol in promoting osteogenesis and mitochondrial biogenesis of human PO-MSCs suggesting a possible application of resveratrol as a supplement for osteoporosis and/or osteoporotic fractures. Elderly people occupy the fastest-growing segment of populations throughout the world. Accordingly, age-associated bone diseases such as osteoporosis have been increasing. Due to low mineral density and concomitant change of microarchitecture of the bone tissue, osteoporosis manifests as an increased risk of bone fracture . The lifAdult mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into various cell types including adipocyte, chondrocyte, and osteocyte . In addiRecently, the metabolic shift from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) is taking on increased importance in stem cell differentiation , 17. GlyResveratrol is a natural polyphenol found in red grapes, peanuts, berries, pomegranates, etc. , 21. Res2 in 100-mm culture dishes containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Resulting adherent cells were passaged by gentle trypsinization and reseeding in a fresh medium. The cell culture medium was changed every 3 days during the isolation period. For osteogenic differentiation, PO-MSCs were cultured using osteogenesis induction medium (OM) which is composed of DMEM, supplemented with 50 μg/mL l-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate [All chemicals used in this study were purchased from Sigma-Aldrich . Cell culture media and fetal bovine serum were purchased from Invitrogen . Human periosteal tissues were obtained from patients who granted informed consent for the collection of the tissues, as required by the Ethics Committee of Gyeongsang National University Hospital (GNUH 2014-05-012). PO-MSCs were then isolated as described previously . Brieflyhosphate .4 cells were seeded in 24-well plates and were cultured in OM medium. Resveratrol treatment was performed by treating cells with vehicle (ethanol) or a range of resveratrol concentrations from 500 nM up to 20 μM. Five-day and 10-day cultures were trypsinized, and resulting detached cells were counted with a hemocytometer. The viability of PO-MSCs treated with resveratrol was determined by MTT assays. Shortly, 2 × 104 cells were seeded in 24-well plates and cultured in OM medium with resveratrol treatments (500 nM~20 μM). Five-day and 10-day cultures were subjected to a colorimetric MTT assay.The proliferation of PO-MSCs was measured by cell counting. Briefly, 2 × 10Alkaline phosphatase (ALP) activities were determined colorimetrically using an ALP Assay Kit  according to the manufacturer’s instructions. In brief, whole-cell lysates were prepared using a NP-40 lysis buffer . Cell lysates were then incubated with p-nitrophenylphosphate, a colorless substrate for ALP, in a Tris-HCl buffer (pH 9.5) at 37 °C for 15 min. Upon splitting off the phosphate group of p-nitrophenylphosphate by ALP, p-nitrophenol is released and detected spectrophotometrically . These ALP activities then were normalized to cellular protein contents determined by the Bradford method.Mineralization of PO-MSC cultures with resveratrol treatments (500 nM and 5 μM) in the OM medium was assessed by von Kossa staining . Calcium5 cells were stained in PBS with MitoTracker Green FM dye  for 30 min, washed, and resuspended in 200 μL PBS with 1% fetal bovine serum. Cellular fluorescence signals were then analyzed using a FACSCalibur . Resulting flow cytometry data were analyzed using the FlowJo software version 8.7.3 . For fluorescent microscopy, PO-MSCs cultured on chamber slides were stained with MitoTracker Green FM dye. Cellular mitochondria were visualized under a fluorescent microscope , and images were analyzed using the ImageJ software .Mitochondrial mass of cultured PO-MSCs was quantified by flow cytometry. Briefly, 1 × 10For mtDNA copy number analysis, total cellular DNA was extracted from cultured PO-MSCs. A quantitative real-time PCR method was used to determine the relative abundance of mtDNA versus nuclear 18S rDNA using corresponding mitochondrial and nuclear PCR primer sets in two parallel PCR reactions as described previously . Relativt test was used for the determination of the statistical significance between the groups, and a p value of < 0.05 was considered statistically significant.All experiments were performed using at least three independent cell cultures. Error bars in all figures represent the mean ± SEM, and statistical analyses were computed using the GraphPad Prism 7 software . Student’s two-tailed In order to see the effects of resveratrol on cell proliferation and viability during osteogenesis, PO-MSCs were cultured in the OM medium for 5 and 10 days. For the same periods of cultures, these PO-MSCs were also treated with various concentrations of resveratrol from 500 nM up to 20 μM. At day 5 and day 10 cultures, cells were harvested and counted with a hemocytometer for cell proliferation or were subjected to an MTT assay for cell viability. As shown in Fig. An increase of ALP activity is known as an early biomarker for osteogenic differentiation or osteoblast activity. To test an effect of resveratrol on osteogenic differentiation of PO-MSCs, ALP activities were assessed by a colorimetric assay described in the “Methods” section. Figure Mineralization by calcium deposits is known as a late biomarker for osteogenesis. In order to test if resveratrol can bolster mineralization during osteogenic differentiation, PO-MSCs were cultured in OM conditions for 2 and 3 weeks with or without resveratrol in the culture medium. From von Kossa staining images which mark mineralization shown in Fig. Recently, the upregulation of mitochondrial biogenesis has been reported as a characteristic of differentiated MSCs. Moreover, it is well known that resveratrol has a beneficial effect on mitochondrial biogenesis in various cell types. Thus, to test whether mitochondrial biogenesis is upregulated by resveratrol during osteogenic differentiation of PO-MSCs, mitochondrial mass and mtDNA contents were measured. PO-MSCs were cultured and induced to osteogenic differentiation by OM with or without resveratrol treatments. The resulting differentiated PO-MSCs were stained with a fluorescent dye, Mitotracker Green FM (Invitrogen), which is localized in the mitochondria in the cell. The stained cells were then assessed for mitochondrial mass by flow cytometry. In Fig. In this study, we demonstrated that resveratrol treatments (5 μM) upregulate both ALP activities and calcium deposits during osteogenic differentiation of PO-MSCs compared to the untreated controls. These results indicate that resveratrol can enhance osteogenic differentiation of PO-MSCs. In other studies, resveratrol was reported to promote osteogenic differentiation of bone marrow- or adipose tissue-derived MSCs at higher concentrations –32. HoweIn addition, this study showed that resveratrol treatments increase both mitochondrial mass and mtDNA contents during osteogenic differentiation in PO-MSCs. These findings indicate that resveratrol can upregulate mitochondrial biogenesis during osteogenic differentiation of PO-MSCs and are consistent with other reports regarding mitochondrial biogenesis by resveratrol in various cell types , 38–42. Currently, the most widely used therapeutic agents for osteoporosis are anti-resorptive drugs such as bisphosphonate and selective estrogen receptor modulators (SERMs) . HoweverFracture healing in the osteoporotic bone is poor compared to fractures occurring at a young age. For example, the fixation failure or nonunion rates of osteoporotic fractures were reported to be approximately 15~24.9% depending on specific fractures , 59. CelIn conclusion, this study demonstrates that resveratrol promotes osteogenic differentiation and mitochondrial biogenesis of PO-MSCs. Therefore, enhancing osteogenesis of PO-MSCs by small molecules such as resveratrol may be clinically valuable for the treatment of osteoporosis and accompanying osteoporotic fracture.


Brain metastases explain the majority of mortality associated with lung cancer, which is the leading cause of cancer death. Cytology analysis of the cerebrospinal fluid (CSF) remains the diagnostic gold standard, however, the circulating tumor cells (CTCs) in CSF (CSF‐CTCs) are not well defined at the molecular and transcriptome levels.We established an effective CSF‐CTCs collection procedure and isolated individual CSF cells from five lung adenocarcinoma leptomeningeal metastases (LUAD‐LM) patients and three controls. Three thousand seven hundred ninety‐two single‐cell transcriptomes were sequenced, and single‐cell RNA sequencing (scRNA‐seq) gene expression analysis was used to perform a comprehensive characterization of CSF cells.Through clustering and expression analysis, we defined CSF‐CTCs at the transcriptome level based on epithelial markers, proliferation markers, and genes with lung origin. The metastatic‐CTC signature genes are enriched for metabolic pathway and cell adhesion molecule categories, which are crucial for the survival and metastases of tumor cells. We discovered substantial heterogeneity in patient CSF‐CTCs. We quantified the degree of heterogeneity and found significantly greater among‐patient heterogeneity compared to among‐cell heterogeneity within a patient. This observation could be explained by spatial heterogeneity of metastatic sites, cell‐cycle gene, and cancer‐testis antigen (CTA) expression profiles as well as the proportion of CTCs displaying mesenchymal and cancer stem cell properties. In addition, our CSF‐CTCs transcriptome profiling allowed us to determine the biomarkers during the progression of an LM patient with cancer of unknown primary site (CUP).Our results will provide candidate genes for an RNA‐based digital detection of CSF‐CTCs from LUAD‐LM and CUP‐LM cases, and shed light on the therapy and mechanism of LUAD‐LM. Our study firstly investigated CSF‐CTCs characteristics of five LUAD‐LM patients and one CUP‐LM patient.CSF‐CTCs enriched metabolism and cell‐adhesion pathways.CSF‐CTCs exhibited heterogeneity manifested by cell cycle, mesenchymal and cancer stem cell, and cancer‐testis antigen properties BMsbrain metastasesCNScentral nervous systemCSCcancer stem cellCSFcerebrospinal fluidCTAcancer‐testis antigenCTCscirculating tumor cellsCUPcancer of unknown primary siteDEGsdifferentially expressed genesECMextracellular matrixEMTepithelial‐to‐mesenchymal transitionGSEAgene set enrichment analysisLMsleptomeningeal metastasesLUADlung adenocarcinomaMRImagnetic resonance imagingNSCLCnon‐small cell lung cancerPDXpatient‐derived xenograftscRNA‐seqsingle‐cell RNA sequencingt‐SNEt‐distribution stochastic neighbor embedding1Lung cancer is the second common cancer type in both men and women.The diagnosis and monitoring of NSCLC‐LMs are mainly based on medical history, clinical symptoms, imaging and CSF examinations. Gadolinium‐enhanced magnetic resonance imaging (MRI) of the brain and spine is the best imaging technique for LMs detection in solid tumors, with sensitivity of 70‐85% and specificity of 75‐90%.CSF‐CTCs are relatively rare in patient CSF samples, and ≥1 CSF‐CTC/mL was defined as a cutoff for diagnosis.22.1All human sample materials used in this research were collected at Huashan Hospital, Fudan University. The consent forms and the proposed studies were approved by Institutional Review Board of Huashan Hospital . Patients diagnosed as LUAD‐LM  or CUP‐LM (P8) without any other cancers were included in the study. Three CSF samples (N1‐N3) were collected from patients who had pulmonary cryptococcal infection without CNS symptoms, and screened for potential CNS infections. The examination results showed the three CSF samples (N1‐N3) were normal without cryptococcal infection. Clinical information of patients is listed in Table S1.2.2Antibodies  and labeling dye for live cells  were used per manufacturer recommendations. Pathological CSF samples were diagnosed by cytology, and CSF‐CTCs in 3 mL leftover CSF per patient sample (Table S1) with a larger cell diameter than lymphocytes were selected by fluorescence‐activated cell sorting  by STAR,Genes expressed in less than 10 cells were filtered out from the gene expression matrix of CSF samples. Individual cells with fewer than 600 covered genes and over 20% mitochondrial reads were filtered out, and 1986 single cells remained (401 immune cells and 1585 CTCs) for subsequent analysis using the Seurat 3.0 software packageWhen we analyzed the transcriptome characteristics of CTCs, we selected tumor cells with more than or equal to1000 covered genes, and 1360 CTCs retained  for analysis. The mean number of genes detected per CSF‐CTCs was 2070.2.5Cells were clustered by non‐supervised t‐distribution stochastic neighbor embedding (t‐SNE) dimensionality reductionCD2, CD3D, CD3E, and CD3G), B cells , monocytes , lung cells , epithelial cells , and proliferation cells (CCND1 and TOP2A). The ImmuneScore was computed based on Estimation of STromal and Immune cells in MAlignant Tumors using Expression data (ESTIMATE) R package.To infer the cell type identity, Seurat 3.0 was used to generate expression heatmaps of selected gene markers of known cell types, including T cells  was used for functional enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways.Significantly differentially expressed genes (DEGs) between samples were detected by DESeq22.7Cell cycle assignment was performed in R version 3.6.0 using the CellCycleScoring function included in Seurat 3.0 package. Cycling cells are defined as cells with either G1/S.Score or G2/M.Score greater than 0.2. The criterion of intermediate cells is 0 < G1/S.Score or G2/M.Score ≤ 0.2. The rest cells are defined as non‐cycling cells.2.8P values except for DEGs analysis were obtained using Wilcoxon Rank‐Sum test, and P < .05 was considered significant.All other 33.1To characterize the single‐cell transcriptomes and the composition of CSF cells under healthy conditions, we sequenced the single‐cell transcriptomes of 624 cells from three normal CSF samples (N1‐N3) using Smart‐seq2 single‐cell RNA‐sEquation (scRNA‐seq) technology Figure . On aver3.2We developed an effective and highly reproducible protocol for cell isolation from the CSF samples of LUAD‐LM patients, and six LM patients were enrolled in the scRNA‐seq study Table S1). In total, 1776 candidate CTCs from five LUAD‐LM patients  were FACS sorted , SFTPB,NAPSACCND1 and TOP2A  gene superfamily mainly found in bronchial epithelial cells. It is a growth factor during fetal lung development with anti‐inflammatory function in the lung.10P‐adj = 24.08, log2FC = 1.84; Figure CEACAM6, SCGB3A2, and C3 have great potentials in a developing CSF immunoassay for LUAD‐BM diagnosis.Two hundred ninety genes were significantly upregulated in patient CSF‐CTCs compared to normal CSF cells , indicating greater heterogeneity in clinical CSF samples ,CD44,ALDH1A1,ALDH1A3,ALDH3A1ABCG2ALDH1A. 27.7% (268/967) of CSF‐CTCs across five LUAD‐LM patients had CD44 expression, a stem cell marker for CTC aggregation and polyclonal metastases.PROM1 or ABCG2 positive were extremely rare. Fifty‐seven CTCs had both CD44 and ALDH1A1 expression.Cancer stem cells (CSCs) exhibit the properties of asymmetrical division, differentiation, and self‐renewal, as well as increased intrinsic resistance to therapy.FN1, VIM, and CD44), 113 CSF‐CTCs in P1 (33.2%) and 54 CSF‐CTCs in P4 (42.5%) had that both epithelial and mesenchymal/CSC markers scores were greater than 0.5 , or the EMT regulator TGFβ.EMT has been suggested as a driver of epithelial tumor spreading. Figures  and 4D, LAMA3, LAMA5, LAMB2, LAMC1), integrinsITGA3, ITGB4), and CD47.We also examined the ECM‐related markers, which is another class of EMT features. Compared to normal CSF cells, the ECM receptor interaction pathway was significantly enriched in CSF‐CTCs  of CTCs across five patients at high level  whose expression is typically restricted to normal male germ cells, providing unprecedented opportunities for clinical development of cancer diagnosis and immunotherapy.7 Figure . LY6K wa6 Figure . SPAG9 wl Figure , with th3.8CK7, CK8, CK18, CK19, and MUC1) and a prolactin‐induced protein PIP/GCDFP15  in 2017. CUP is a well‐recognized clinical disorder accounting for 3‐5% of all malignant epithelial tumors; metastatic adenocarcinoma is the most common CUP histopathology (80%).CD45 expression  were not detected in P8, except for CEACAM6, which was shared across P8 and all LUAD‐LM patients  expression, which was different from LUAD CSF‐CTCs (20/967). All P8 CTCs had high expression of epithelial genes . 73.8% (290/393) of P8 CTCs were in stem‐like phenotype (PROM1+CD44), and 9.2% (36/393) of P8 CTCs had detectable expression of mesenchymal genes (FN1+VIM) .The proliferation ability of P8 CTCs was similar to LUAD‐LM CSF‐CTCs, with three of 131 (P8‐1) and eleven of 262 (P8‐2) cells in high‐cycling state . Interestingly, a large proportion of (118/393) P8 cells had 4CTCs and cell‐free DNA (cfDNA) in CSF samples can reflect the real‐time status of leptomeningeal disease and have promising potential for characterization and monitoring of LM development. However, false negatives in CTC capturing4.14.1.1One major advantage of single‐cell RNA‐seq approach is the ability to characterize the expression variation among individual cells. Our study found significant among‐patient heterogeneity and among‐cell heterogeneity within a given patient, which could be explained by spatial heterogeneity of metastatic sites, cell‐cycle gene, and CTA expression profiles, as well as the proportion of CTCs displaying mesenchymal and CSC properties. In addition, the temporal difference during disease progression and patient‐specific mutations also contributed to the heterogeneity among CTCs, suggesting the need of personalized diagnosis and expression profiling.The scRNA‐seq approach could detect potential temporal heterogeneity during tumor progression. We obtained CSF samples from two different time points for both patient P1 (P1‐1 vs P1‐2) and P8 (P8‐1 vs P8‐2). Collected within a 2‐month time interval, P1‐1 and P1‐2 CSF‐CTCs from two datasets formed a single homogeneous cluster Figure , indicatEGFR  gene and ALK (anaplastic lymphoma kinase) rearrangements are keys in the development of BMs.EGFR (19del) and ALK (Arg1192Trp) mutations, along with TP53 (Trp53Ter) mutation and low frequency of KRAS mutation were detected in P1 CSF cfDNA (Table S4). EGFR (Leu858Arg) mutation and TP53 (Arg248Gln) mutation were found in P4 CSF cfDNA, and P7 CSF cfDNA only exhibited TP53 (Asp281Tyr) mutation (Table S4). We have not performed CSF cfDNA mutation detection by NGS in P2 or P6, but the EGFR mutation in P6 tumor was detected by other method in 2012. These mutational profiles guided different targeted therapy strategies, especially for EGFR. In summary, the patient clinical characteristics (Table S1) including age, sex, duration of disease, primary LUAD site, metastatic sites in LMs  have been detected by next‐generation sequencing (Table S4). The discovery of common driver mutations and the development of targeted therapies have dramatically improved the treatment efficacy of intracranial tumors and prolonged survival. Activating mutations of the s Figure  or other4.1.2VIM and FN1) within single CSF‐CTC , suggesting that stem cell markers and EMT markers were not intrinsically linked in CSF‐CTCs. Similar results have also been observed in pancreatic blood‐CTCs.EMT is a process related to tumor invasion and metastases. It has been reported that some NSCLC blood‐CTCs have a dual epithelial‐mesenchymal phenotype.Notably, we also identified the unexpected abundant expression of ECM genes in CSF‐CTCs Figures  and 4D, 4.1.3MMP7  is another adhesion‐related gene, which was greatly upregulated in CSF‐CTCs. Claudins are solely involved in tight junctions and critical for cell‐to‐cell adhesion in epithelial cells, CLDN2 and CLDN7 have been shown to facilitate the adhesion of cancer cells to the ECM, which is important for cancer metastasis.CLDN7 in LUAD‐LM are still unknown. Whether CLDN7‐mediated adhesion to the BBB is advantageous for LUAD‐LM warrants further study.Endothelial cells are the main component of the blood‐brain barrier (BBB), and the disruption of tight junctions between endothelial cells by disease or drugs can compromise the leptomeningeal.ICAM1; Table S7; log2FC = 2.12) was also significantly upregulated in most LUAD‐LM CSF‐CTCs. ICAM1, a member of an immunoglobulin‐like superfamily of adhesion molecules, is involved in various processes in lung cancer development and signal transduction across leukocyte‐epithelial cell interactions.VCAM1, mediates the interaction between breast‐CTCs and neutrophils.ICAM1 contributes to the formation of CSF‐CTC‐WBC clusters in LUAD‐LM patients. MMP7, CLDN7, and ICAM1 have great potential as therapy targets to decrease the metastatic ability of CSF‐CTCs.Intercellular adhesion molecule‐1 , lung origin markers , blood‐CSF barrier‐associated genes (MMP7 and C3), and cell‐cycle genes CCND1  and adhesion‐related genes (CLDN7 and ICAM1) also correspond to the characteristics of LUAD‐LM CSF‐CTCs , seven CTCs from P8 had high expression levels  under accession number PRJNA602172. All data generated or analyzed during this study are included in this published article. All the data are available from the corresponding author upon reasonable request.SUPPORTING INFORMATIONClick here for additional data file.