In multiple sclerosis (MS), activated T and B lymphocytes and microglial cells release various proinflammatory cytokines, promoting neuroinflammation and negatively affecting the course of the disease. The immune response homeostasis is crucially regulated by the activity of the enzyme adenosine deaminase (ADA), as evidenced in patients with genetic ADA deficiency and in those treated with cladribine tablets. We investigated in a group of patients with MS the associations of a single nucleotide polymorphism (SNP) of ADA gene with disease characteristics and cerebrospinal fluid (CSF) inflammation. The SNP rs244072 of the ADA gene was determined in 561 patients with MS. Disease characteristics were assessed at the time of diagnosis; furthermore, in 258 patients, proinflammatory and anti-inflammatory molecules were measured in the CSF. We found a significant association between rs244072 and both clinical characteristics and central inflammation. In C-carriers, significantly enhanced disability and increased CSF levels of TNF, IL-5 and RANTES was observed. In addition, lower CSF levels of the anti-inflammatory cytokine IL-10 were found. Finally, the presence of the C allele was associated with a tendency of increased lymphocyte count. In MS patients, ADA SNP rs244072 is associated with CSF inflammation and disability. The selective targeting of the ADA pathway through cladribine tablet therapy could be effective in MS by acting on a pathogenically relevant biological mechanism. Autoreactive T and B lymphocytes play a crucial role in disease pathogenesis ,2. T cel lesions  and have lesions . Additio lesions . Accordi lesions , and con lesions .ADA) is associated with the toxic accumulation of triphosphorylated deoxyadenosine and lymphocyte depletion, as shown in ADA genetic deficiency and in patients treated with cladribine (2-chloro-2′-deoxyadenosine) tablets, a purine deoxynucleoside analogue resistant to the action of ADA, approved for the treatment of highly active relapsing–remitting (RR)MS [ADA have been reported to be altered, respectively, in peripheral and central compartments of patients with MS compared to healthy subjects [Targeting T and B lymphocytes constitutes a promising therapeutic approach in MS. Lymphocytes are sensitive to impaired nucleotide metabolism because it affects their entire life, from DNA synthesis and integrity to the inflammatory response. In particular, the altered activity of the enzyme adenosine deaminase (g (RR)MS ,9. Accumg (RR)MS , but fewsubjects ,12.ADA gene and cerebrospinal fluid (CSF) inflammation as well as clinical characteristics. We hypothesize that, between the complete absence of ADA and its full expression and function, there could be intermediate conditions related to individual genetic variability which, although not altering the normal immune responses, could influence the pathophysiology of autoimmune diseases and particularly of MS.In this study, we investigated, in a large cohort of patients with MS, the association of a single nucleotide polymorphism (SNP) in A group of 561 consecutive MS patients were enrolled in the study. Patients were admitted to the neurological clinic of the University Hospital Tor Vergata in Rome and Neuromed Research Institute in Pozzilli, Italy, between 2010 and 2017, and diagnosed with MS on the basis of clinical, laboratory and MRI parameters . The EthADA gene with potential regulatory functions of mRNA expression were identified by using the UCSC Genome Browser [SNPs in non-coding regions of  Browser  .Thus, genotyping for In 258, MS patients’ CSF concentrations of inflammatory cytokines were analyzed. CSF was collected at the time of diagnosis, during hospitalization, by lumbar puncture (LP). No corticosteroids were administered before LP. Disease modifying therapies were initiated after the confirmed diagnosis when indicated. CSF was stored at −80 °C and then analyzed using a Bio-Plex multiplex cytokine assay . CSF cytokines levels were determined according to a standard curve generated for the specific target and expressed as picograms/milliliter (pg/mL). Samples were analyzed in triplicate. The CSF cytokines analyzed included interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, tumor necrosis factor (TNF) and RANTES.ADA SNP rs244072 and peripheral white blood cells .In an independent cohort of 230 MS patients, we explored the association between ®, Becton Dickinson, Milan, Italy). For the blood exam we used a hematology analyzer ADVIA 2120i system  whose technology utilizes peroxidase staining that permits the cytochemical differentiation of myeloid and lymphoid cells. We calculated the neutrophil/lymphocyte ratio (NLR) by dividing the neutrophil number by the lymphocyte number [Peripheral blood samples were collected by standard venipuncture  false discovery rate controlling procedure was applied for multiple testing. A p value ≤ 0.05 was considered statistically significant. To show statistically significant differences in CSF cytokines levels and EDSS scores between two categories, a box plot was used. To better show the cytokines levels’ distribution, the values were depicted on logarithmic scale. All analyses were performed using IBM SPSS Statistics for Windows .Shapiro–Wilk test was used to evaluate normality distribution of continuous variables. Data were shown as mean  or median . Categorical variables were presented as absolute (n) and relative frequency (%). Chi-square or, when necessary, Fisher exact test, were employed to explore the association between categorical variables. Difference in continuous variables between the ADA gene, we identify the SNP rs244072 as a good candidate to test our hypothesis. This SNP is placed in the second intron of the ADA gene (IVS2-1496T>C) inner putative binding sites for five different modulators of mRNA processing, as emerged by RBPmap prediction  and SRSF3 (Serine and arginine Rich Splicing Factor 3), which are fine regulators of lymphocyte activation [By in silico analysis of non-coding regulatory regions of ediction A. In partivation ,19,20. Sp = 0.238; n = 469; 83.60%), TC , CC . For further analyses, patients were grouped as: CT + CC  or TT.The genetic screening of a population of 561 patients with MS showed no significant departure from Hardy–Weinberg equilibrium in the study population   . No otheADA gene could influence central inflammation in MS, we studied the possible association between rs244072 alleles and the CSF levels of proinflammatory and anti-inflammatory molecules in 258 patients. As shown in p < 0.001), IL-5  and RANTES . In addition, patients with the C allele showed lower levels of IL-10  in the CSF. These results indicate that the different genetic variants of ADA SNP rs244072 could participate in determining the CNS inflammation in MS patients.To explore whether individual genetic variability in ADA group (TT vs. CT/CC) was associated to WBC count, number of Neutrophils, Lymphocytes, and NLR, in an independent cohort of MS patients ; MS Phenotype RR/CIS/RIS = 194 (84.3%); SP/PP = 36 (15.7%); Age, mean (SD) = 39.13 (12.52), EDSS, median (IQR) = 2 (1–3)). No significant differences emerged in the demographic and clinical characteristics of the two groups of MS patients.We explored whether the ADA enzyme, no statistically significant differences were found between the two groups : “TT” = 6.77 (5.8–8.28); “CT/CC” = 7.125 (6.07–8.49); p = 0.298. Neutrophils, median (IQR): “TT” = 4.15 (3.4–5.4); “CT/CC” = 4.25 (3.77–5.2); p = 0.654. Lymphocytes, median (IQR): “TT” = 1.8 (1.5–2.2); “CT/CC” = 2 (1.57–2.54); p = 0.108. NLR, median (IQR): “TT” = 2.28 (1.72–3.09); “CT/CC” = 2.17 (1.71–2.85); p = 0.412).Although WBC and particularly lymphocyte were slightly reduced in the TT group , suggestADA activity is critically involved in immune response homeostasis, the influence of ADA individual variability on MS course and CNS inflammation has not been investigated. In the present study, we found that ADA gene SNP rs244072 is associated with disability and with different CSF levels of specific proinflammatory and anti-inflammatory molecules at the time of diagnosis. Particularly, patients bearing at least one C allele showed both exacerbated CSF inflammation and higher EDSS compared to TT patients.Although Different studies have demonstrated that CSF inflammatory milieu critically influences the clinical course of MS. Increased CSF levels of specific proinflammatory molecules at the time of diagnosis were found to be associated with enhanced prospective disease activity, disability and neurodegeneration . ConversADA gene and a proinflammatory CSF milieu may negatively influence the disease course of MS. In particular, the presence of a C allele of ADA gene SNP rs244072 is associated to enhanced expression of TNF, RANTES and IL-5, and reduced concentrations of anti-inflammatory molecules, such as IL-10. The role of these cytokines in the pathogenesis of MS has been clearly demonstrated both in preclinical and clinical studies.Our results suggest that the association between individual variability of TNF is a major proinflammatory cytokine with a crucial pathogenic role in MS ,4,24, exADA SNP and IL-5 levels, the role of this cytokine in MS pathogenesis is uncertain. IL-5 is released by Th2 lymphocytes and is mainly involved in eosinophil differentiation, and its production was found to be increased by glatiramer acetate [Although we found an association between  acetate .+ T cell proliferation, mediated by IL-10, was shown to be less effective [Our observation that C-carriers in rs244072 SNP present higher EDSS and lower CSF levels of the anti-inflammatory cytokine IL-10, is coherent with previous studies showing that IL-10 exerts several beneficial effects in EAE and MS. IL-10 induces regulatory T cells resulting in reduced microglia activation, less neuroinflammation and neurodegeneration . Conversffective .The effect of different disease-modifying therapies (DMTs) may be mediated by the modulation of the inflammatory milieu. Treatment with IFN-B in MS is associated with reduced production of TNF compared to untreated patients , decreasADA activity could beneficially influence CSF cytokine milieu in MS, possibly reducing disability progression and neurodegeneration. Indeed, despite the fact that the functional consequences of the different rs244072 alleles are poorly understood, the SNP is an intronic variant with possible regulatory implications because it is included in the RNA binding site for important regulators of lymphocyte activation, such as PTBP1 and SRSF3. In combination with the results of the in silico analysis, the small decrease in lymphocyte count in TT patients may be explained by a reduction in ADA activity, possibly due to less efficient gene expression, justifying, at least in part, reduced central inflammation and disability in these patients compared to C-carriers. Additionally, its relevance for MS may be indirectly inferred by recent evidence that genes that are disrupted in Mendelian disorders  are dysregulated by noncoding variants in complex traits [ADA affect the enzyme’s activity  in MS patients and further studies exploring different SNPs of ADA gene are required to better characterize the relationship between ADA genetic variability and MS pathogenesis. It is also important to prospectively investigate MRI measures of neurodegeneration, and the association with CSF biomarkers of neuronal damage, including tau protein, neurofilaments, and β amyloid.Our result suggests that the modulation of x traits . Furtherx traits ,40. ThesThe present investigation highlights, for the first time, the potential role of genetic individual variability of ADA gene in regulating central inflammation and clinical manifestations in MS. These results deserve further investigation to better comprehend the possible mechanisms of action of cladribine tablet treatment in MS.


E. colihns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the  E. coli RNAP and two binding sites for this alarmone have been localized on the core enzyme -labeled hns probes obtained from pPLc11 -ATP [The 221 bp DNA fragment carrying the 32P]-ATP . ReactioThe E. coli RNA polymerase was purified according to Gross et al. . The amohns expression was initially investigated by means of antibiotics as chloramphenicol (CAM) and kanamycin (Kan), which were known to affect the intracellular level of this unusual nucleotide. In fact, these two drugs, by blocking protein synthesis, have been shown to cause a dramatic reduction in (p)ppGpp [hns expression, the level of hns mRNA was monitored in a time course experiment after treating the cell with high doses of CAM and Kan. As seen in hns transcript compared to the untreated cells as control. Steady-state level of hns mRNA was further investigated using a strain defective for RelA synthetase, the enzyme catalyzing (p)ppGpp production along with double mutant relA/spoT that, in addition to relA, carried an inactivated bifunctional (p)ppGpp synthetase/hydrolase SpoT. Xiao et al. [relA and from relA to relA/spoT backgrounds. Accordingly, northern analysis demonstrates that basal level of hns transcript ppGpp. Thus, this stimulation decreases from wt (fivefold) to relA/spoT (1.4-fold) through relA (twofold) cells; given that the intracellular level of (p)ppGpp is already very low in relA/spoT strain, CAM effects are almost completely lost in this double mutant  and this difference gradually disappears as the cells approach the stationary phase (A600 > 1.5) (hns expression is inversely correlated with the intracellular concentration of (p)ppGpp.The role of (p)ppGpp on (p)ppGpp . Thus, to et al.  found thanscript A, time 0hns mRNA A,C. Indee mutant C. To rei0 > 1.5) B,D. Takehns transcript in cells carrying mutations in relA and spotT genes might simply reflect a stabilization of mRNA rather than an activation of hns promoter, the decay rate of hns mRNA was analyzed, comparing wt and relA/spotT double mutant after blocking the transcription by rifampicin (½) of hns message, expressed as the time required for degrading 50% of the existing mRNA molecules, slightly changes in the two different genetic backgrounds, being t½ = 2.0 min and t½ = 1.2 min for wt and relA/spoT strains, respectively. Notably, the chemical stability of hns mRNA is higher in wt than in double mutant, ruling out that RNA t½ can account for the elevated levels of hns transcript observed in relA/spoT cells. Thus, these findings indicate that the hns promoter is responsive to (p)ppGpp, and the low level of this unusual nucleotide, as a consequence of the inactivation of relA and spoT genes, induces de novo transcription of hns.Furthermore, to explore whether the higher amounts of fampicin E. The hahns. To this aim, the intracellular (p)ppGpp levels were artificially increased by overexpressing the RelA alpha fragment in plasmid pKT31, carrying the (p)ppGpp catalytic domain independent of ribosome binding. The isogenic plasmid pKT40, because of the insertion of a stop codon in relA gene, encoded a truncated protein and was used as control. The production of functional RelA and its inactivated variant from pKT plasmids was achieved by adding IPTG to the cell cultures [hns transcription ppGpp on hns transcription through an in vitro purified system as a function of increasing concentrations of this alarmone. As shown in hns transcription of ~10% and hns::cat mRNA becomes undetectable when this regulatory nucleotide is present at 1 mM, value that corresponds to the concentration estimated in vivo after amino acids starvation [These results suggest that (p)ppGpp exerts a targeted repressive action on arvation . Importahns. The yield of DNA-RNAP complexes was investigated by electrophoretic mobility shift assay (EMSA) performed as a function of increasing periods of time without and with a fixed concentration of (p)ppGpp . Summarizing, EMSA results suggest that (p)ppGpp negatively affects the extent of complexes trapped in transcription that are therefore considered to be productive, hence accounting for transcriptional inhibition of hns promoter by (p)ppGpp as observed both in vivo and in vitro as well ppGpp A. To conomplexes . As show as well .Bacterial cells, lacking homeostatic mechanisms, withstand changes in environmental conditions, deeply reshaping transcriptional activity. Hence, prompt adaptation to stresses, in particular to amino acid starvation, is coupled with an intracellular accumulation of the second messenger (p)ppGpp, also known as “magic spot”. This signaling molecule modulates the stringent response by affecting several fundamental cellular processes, including transcription.hns gene, coding for the DNA-binding protein H-NS, is under control of (p)ppGpp. Specifically, treatments with antibiotics capable of arresting the protein synthesis and null mutations in relA and spoT genes responsible for (p)ppGpp production reduce the alarmone level and as consequence elicit an activation of hns promoter in vivo ppGpp itself. In fact, H-NS, besides its role in DNA-packaging and nucleoid organization, shows high pleiotropic effects, and is currently considered as a global repressor of transcription in bacteria. The majority of genes belonging to H-NS regulon are involved in counteracting adverse growth conditions, and in particular H-NS is able to sense shifts of temperature, osmolarity, and pH [hns expression limited to the exponential phase of growth [hns expression patterns of wt and relA/spoT strains that become similar as the cell density increases ppGpp: (i) by directly impacting the intrinsic activity of hns promoter; (ii) by depleting the hns gene of its natural activator FIS.In this study, we demonstrate that the expression of  in vivo . Converspromoter . Accordi, and pH . Thus, ppGpp, investigated through an in vitro transcription system (hns transcription declines as much as 5–10% for higher (p)ppGpp concentrations. These amounts of (p)ppGpp are very close to those used in other in vitro studies [Importantly, an adaptative stress-response also takes place when pathogenic bacteria face up transitions from free-living to host-associated states. In this contest, H-NS has been found in important human pathogens such as rminants ,49,50,51n system , undenia studies ,52,53 an studies ,54.hns as a (p)ppGpp-responsive gene using transcriptomics techniques as RNA-seq [hns mRNA level at 5 and 10 min after the production of (p)ppGpp by an inducible relA plasmid system. Using an analogous experimental approach . Under our experimental conditions ppGpp-mediated regulation of hns might have been overlooked in previous studies [hns transcription rate in a (p)ppGpp-independent manner probably because, at this time, other factors  may contribute mostly to the regulation of this gene. Remarkably, H-NS is also able to bind and repress transcription from its own promoter [hns mRNA in both wt and relA/spoT strains, as observed at the onset of stationary phase  whose in vivo expression was affected at least twofold by the modified nucleotide [hns promoter was superimposed to the histograms of base distributions for promoters inhibited by (p)ppGpp as determined by Sanchez-Vazquez et al. [hns shows an adenine at position −8 that was found to interact with RNAP [hns sequence, three cytosines occupy positions −4, −3 and −1, respectively, thus contributing to constitute a robust discriminator ppGpp as validated through in vitro experiments ppGpp directly regulated genes has been obtained thanks to an extensive in vitro transcription analysis carried out on more than 100 promoters ppGpp concentration in the presence of DksA as previously described for rRNA genes [hns transcription rate in response to small changes of intracellular levels of p(p)ppGpp as observed in vivo using drugs addition, relA/spoT mutants and RelA over-expressing systems (In addition to (p)ppGpp, a second effector, playing a crucial role during the stringent response, is the transcription factor DksA. This small protein 17 kDa) binds RNAP through the secondary channel  and larg kDa bindNA genes . Ultimat systems . The mol


Gallus gallus) and evolutionarily derived parrots: budgerigar (Melopsittacus undulatus) and cockatiel (Nymphicus hollandicus). Because cockatiel lacks a complete genome information, we compared the cross-species protein identifications using the reference proteomes of three model avian species: chicken, budgerigar and zebra finch (Taeniopygia guttata) and found the highest identification rates when mapping against the phylogenetically closest species, the budgerigar. In total, we identified 483, 641 and 458 unique proteins consistently represented in the CSF and plasma of all chicken, budgerigar and cockatiel conspecifics, respectively. Comparative pathways analyses of CSF and blood plasma then indicated clusters of proteins involved in neurogenesis, neural development and neural differentiation overrepresented in CSF in each species. This study provides the first insight into the proteomics of adult avian CSF and plasma and brings novel evidence supporting the adult neurogenesis in birds.Cerebrospinal fluid (CSF) proteins regulate neurogenesis, brain homeostasis and participate in signalling during neuroinflammation. Even though birds represent valuable models for constitutive adult neurogenesis, current proteomic studies of the avian CSF are limited to chicken embryos. Here we use liquid chromatography–tandem mass spectrometry (nLC-MS/MS) to explore the proteomic composition of CSF and plasma in adult chickens ( Being from ~ 80% plasma (PL)-derived and ~ 20% brain-cell-derived2, CSF dynamically reflects both peripheral and CNS physiological processes. Despite the paramount importance of the relationship between blood and CSF, little comparative effort has been made in non-mammalian vertebrates to characterise the two vital fluids integrating processes in and out of the CNS.The central nervous system (CNS) function is regulated by a vivid communication with other tissues. This is also achieved through the cerebrospinal fluid (CSF) that supports the brain cell functions3. In addition, several in vitro and in vivo studies have provided evidence that, CSF can promote neurogenesis even in the adult brain9. In particular, spatial gradients of CSF proteins induced the migration of neuroblasts from the subventricular zone to the olfactory bulb in the adult mouse brain8. Also, when rat CSF was applied as a culture medium to neural stem cells and cortical explants, the cells survived without additional supplements, particularly for age-matched CSF samples and tissues9. Therefore, increased interest has been raised to study and describe the molecular composition of CSF, especially in humans16.There is widespread evidence that the birth and growth of neurons also occurs in adult invertebrate and vertebrate CNS and that these cells add onto existing neuronal networks or replace apoptotic neurons12, species differ in physiological requirements of their tissues and different species show different neurogenesis rates as well as anatomical features17. Birds have been proposed as suitable models to investigate the molecular basis of the regulation of adult constitutive neurogenesis18. Nonetheless, the available avian CSF proteomic analyses have addressed only embryonic chicken CSF. Based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometry (MS) or tandem mass spectrometry (MS/MS) methods, the chicken embryonic CSF (eCSF) consists of hundreds of proteins of the extracellular matrix, regulators of osmotic pressure, ion carriers, hormone-binding proteins, regulators of lipid metabolism, and various enzymes and their regulators11. However, the advent of methods such as gel-free-LC/MS for comparative protein analysis has resulted in several advantages over the label-based methods used by the aforementioned studies, including the greater coverage of the proteome19.Although CSF proteomic composition shares many similarities across species such as rat, human and chickenGallus gallus, GG, order Galliformes) serves as a basic avian model organism in developmental biology and neuroscience21, current evidence shows that its neural densities are much lower than in some other, evolutionarily derived birds23. Importantly, recently, parrots (order Psittaciformes) have been proposed as models for neurobiological research24. Similarly to humans, parrots have large brains relative to their body size25, high density of neurons in the forebrain22, exhibit outstanding cognitive skills such as object permanence27 and tool use28, cooperative problem solving29, complex social organization30, learning of vocalizations through cultural transmission32, and some species are on par with great apes in number of cognitive domains35. Furthermore, despite their high metabolism, they show extended developmental periods36 and longevity37.While the domestic chicken  and the cockatiel , representing two distinct parrot clades . Since the cockatiel genome is not presently available, a condition that may affect the peptide/protein identification, we first test the consistency of the proteomic annotation by comparing the MS/MS identification success rates after mapping all spectra against the reference proteomes of three model avian species: chicken, budgerigar and zebra finch (Taeniopygia guttata). Second, using the quality-filtered and consistent data, we describe the proteomic profiles of PL and CSF in the three bird species, searching for the pathways common to differentiate the avian PL and CSF proteomes.In the present study, we focus for the first time on avian adult CSF and PL proteomes, comparing the domestic chicken to two parrot species: the budgerigar  were common to all species  and all sample types (PL or CSF) Fig. a, while We ranked the shared identified proteins according to their normalized logarithmic abundance means. There was a considerable inter-individual variability in the expression of the lowly abundant proteins in both PL and CSF. In contrast, among the highly abundant proteins, the protein composition appears relatively consistent in both fluids between the three species, parrot species being more similar Fig. . Among tIn general, the proteins commonly represented in CSF and blood PL were involved in metabolic and signalling pathways and most of their functions are common to CSF and plasma Fig. . As expep value < 0.05 and fold change cut-off ≥ 2. Similarly, 332 proteins were significantly differentially represented in MU CSF compared to PL proteome, including 99 under-represented and 224 over-represented proteins  and, for NH, the analysis revealed 289 differentially represented proteins, 46 under-represented and 206 over-represented in CSF compared to PL . The fold change differences in the common proteins across species are shown in volcano plots at Fig. Raw and normalised data were inspected by their log2 distributions and correlation of their principal component 1 of the shared genes. After data normalization, the distributions showed almost no variation between the samples and their first principal component positively correlated with the first principal component of raw data  are under-represented in the CSF of parrots but over-represented in chicken. We did not identify any proteins over-represented in the CSF of chicken but under-represented in parrots. STRING analysis of the 72 commonly over-represented proteins in CSF of all species indicates a network of protein interactions, containing 72 nodes and 199 edges  based on GO Biological functions classification and pathway enrichment in CSF compared to plasma that revealed differential representation in proteins involved in the neural function. Enriched pathways in plasma were mostly related to immune functions. Supplementary Table CSF connects the bloodstream with the CNS and reflects the physiology of the brain. Here we have described the adult CSF and PL protein composition in three avian species  using gel-free nLC-MS/MS. First, we have confirmed that the phylogenetically closest avian reference proteome yields the highest efficacy in protein identification success rates, although a proportion of proteins  was mapped only using other references. In total, following the data filtering, we identified in the CSF and PL 483 proteins in the chicken, 641 proteins in the budgerigar and 458 proteins in the cockatiel. GO pathway analyses of the core proteomes revealed components of several signalling and metabolic processes such as regulation of peptidases, synapse organisation, regulation of IGF transport and uptake by IGFBPs in CSF and blood coagulation, complement activation and glycolysis in PL. Comparative analysis of the proteomes derived from the two biological fluids showed that the CSF and PL are clearly distinguishable in terms of their protein content. In CSF, the pathways involved in neurogenesis, and neural development and differentiation are over-represented when compared to blood plasma.Xenopus laevis microtubule-associated proteome40, but this is the first study reporting a paradigm of cross-species quantitative proteomics in diverse avian taxa.Current research increasingly adopts investigation in diversified taxa lacking complete and annotated genomes and proteomes. In this study, we investigated also the cockatiel, a species currently lacking a genome sequence. We found that by cross-species protein identification using the sequence database from its evolutionarily closest relative, the budgerigar, we could identify 458 proteins represented in the CSF and PL. This is more than if other reference genomes were used . This trend was consistent with our findings in other species included in this study. Interestingly, however, some proteins were identified only when mapped against the other mapping combinations. Quantitative proteomics of unsequenced organisms is still a challenge and, when de novo sequencing of the unidentified proteins is unavailable, the reference proteome of a close-related phylogenetically species is utilised to identify the proteome in question. An analogous approach has been used to characterise the proteome of unsequenced micro-organisms and 42. In a cohort of more than 100 individuals, Dayon et al. identified 790 and 422 proteins in parallel CSF and PL samples respectively with an overlap of 255 proteins shared by both fluids41. In a previous study, a maximal protein set of 3,081 and 1,050 CSF and PL proteins was identified respectively with 877 proteins common in both fluids from a group of 21 healthy individuals42. The reasons for the differences in the number of protein identifications between these two studies could be attributed in different sample preparation approaches and detection techniques. Thus, although the protein concentration is likely higher in PL compared to CSF43, the variety of proteins is greater in the CSF compared to PL, being enriched from ca. 20% with proteins expressed in the choroid plexus and brain44. Blood and serum comprise receptor ligands, immunoglobulins, tissue leakage products, aberrant secretions and proteins that are extraneous to the animal. Many blood proteins are synthesized in liver45. Although quality of CSF collection can influence the protein content observed, the CSF concentration of CNS-derived proteins remains unaffected following contamination of the fluid with blood46. In the present dataset, we found the presence of haemoglobin subunits and carbonic anhydrase that can be attributed to some minor blood contamination47. Yet, like in Smith et al.13, although haemoglobin subunits were found amongst the most CSF abundant proteins, our clustering analysis clearly differentiated CSF and plasma samples in all studied individuals and 58.5% of the shared proteins across species, such as gamma-enolase, neuronal growth regulator 1 or neural cell adhesion molecule 1, could be identified selectively in CSF.We found more proteins in CSF compared to plasma, a finding which was consistently found in all avian species in this study and similar to what has been previously reported for humans in paired samples48. Human eCSF contained 188 proteins, 44% of those also present in rat eCSF. Functional analysis revealed protease inhibitors, extracellular matrix proteins, and transport proteins as well as signalling and intracellular proteins12. Comparing the data available for human eCSF with our results in birds, 39%, 50% and 47% of the proteins overlap with adult CSF from chicken, budgerigar and cockatiel, respectively. In chicken embryos investigated at the developmental stage HH24, using 2D-PAGE, 26 proteins were identified, related to the extracellular matrix, regulation of the osmotic pressure and metal transport, cell survival, transport of retinol and vitamin D as well as mitogen-activated protein kinase activators, antioxidant and antimicrobial proteins, intracellular proteins and some unknown proteins. We found that 50% of these proteins were included also in our data. Many of these proteins regulate the development in systems other than CNS, while others are altered during neurodegeneration11.Using gel-based techniques and mass spectrometry, the proteomes of eCSF from rat, chicken and human have been previously described to elucidate the molecular composition of CSF and its biological role during embryonic neurogenesis15. However, expectedly, interindividual variation is accountable for identification of many of the rare proteins. For example, out of 2,615 CSF proteins, only 20% of the identified proteins were shared by all six healthy subjects16. In a combined dataset from three studies the full human CSF proteome of 4814 proteins was revealed, but only about 40% of these proteins were common. We found that about a half of the proteins identified in either plasma or CSF in each species showed a high degree of inter-individual variability, while the other half was relatively consistently represented across all individuals. In our study we have shown that 71.22 and 55.27% (GG), 41.97 and 45.31% (MU), and 56.92 and 38.87% (NH) proteins identified for CSF and PL respectively were common to all individuals within species and only 193 proteins were common across all three species. Thus, the number of proteins that are individually specific is high. The reasons for this variation, however, remain unclear50. The molecular variations in CSF composition can be possibly increased by post-translational modifications, age, blood contamination47 and circadian rhythm51.LC-based methods allowed the identification of a dramatically increased number of proteins in CSF. Most recently, using LC–MS/MS after abundant protein depletion, Macron et al. identified 3,174 proteins in a commercial pool of “normal” human CSF samples54. It has been suggested that these proteins are waste products eliminated from the brain extracellular space by the CSF. A small proportion could also indicate cellular contamination of the samples54. Post-mortem samples have been previously reported to contain also cytoskeletal proteins, glycolysis and antioxidant enzymes released into the CSF following cell necrosis55.We also found proteins of intracellular origin in the CSF, unlike in PL. These were consistently observed in human, rat and chick CSF/eCSF and classified mainly as “binding proteins”NEGR1) and reelin (RELN), 72 were shared across all the three avian species. A PPI network constructed based on these proteins, as well as the enrichment analysis of biological functions revealed namely representation of pathways associated with cell adhesion, nervous system development and neurogenesis. In addition, using GSEA, we indicated in CSF enrichment of pathways involved in neural functions across the studied species. In addition to those revealed by differential analysis, the CSF samples shared several gene products related to neurogenesis for example neogenin 1 (NEO1), neurofascin (NFASC), neuromodulin (GAP43), or pigment epithelium-derived factor (SERPINF1). Our results suggest that adult neurogenesis occurs in all the avian species studied. Previous reports have stressed the importance of various growth factors present in CSF which bind to receptors located on the apical membrane of cortical progenitor cells and promote the development and growth of neural stem cells and cortical explants56. In this study, we identified proteins such as 14–3-3 proteins  and fibroblast growth factor receptor 1 (FGFR1-4) participating in the fibroblast growth factor pathway57, molecules interacting with the Wtn9 pathway such as the Wtn inhibitor Dickkopf-related protein 3 (DKK3) and cadherins , amyloid beta A4 protein precursor (APP)12, insulin-like growth factor II (IGF2) and insulin-like growth factor-binding proteins 58. However, how these proteins are regulated in the context of systems level, their lifetime in CSF, as well as the contribution of interindividual variability requires further investigation.Our comparative analysis shows that CSF and plasma were distinguishable based on their proteome compositions. From the proteins over-represented in CSF compared to plasma, such as homologs of neuronal growth regulator 1 . The functional role of adult neurogenesis in birds remains unknown. It has been proposed to be associated with continuous learning. In adult mammals, the proliferative hot spots may disappear altogether62, but comparative approaches between birds and mammals have elucidated similarities on the origin of neurons, the phenotype and proliferative mechanisms of stem cells and the migration and differentiation of neurons as well as differences such as the spatial distribution of adult-born neurons, their mode of migration and phenotypic diversity72. We found that chicken and parrots share common proteins and pathways associated with the modulation of neuronal cell proliferation and plasticity in CSF. Being based on a single methodological approach, our identification of these gene sets and pathways in birds serves for an initial insight and subsequent experimental verification is required. Nevertheless, our study forms an essential reference for further molecular analysis.In vitro studies of adult human CSF treatment in neural stem cells showed that adult human CSF promoted gliogenesis, but not neurogenesisThe advent of the proteomic techniques has allowed new insights into molecular processes driving the CNS function, with research in CSF forming its cornerstone. For the first time, we concurrently explored in birds the proteomes of two main biological fluids, PL and CSF. Importantly, we focused on multiple species, the chicken and two parrot species, the budgerigar and cockatiel, investigating adult individuals. The proteins over-represented in CSF were mainly classified into functional pathways involved in neural function, providing a novel molecular evidence for adult neurogenesis in birds. Consistently with histological evidence for adult neurogenesis in birds, understanding the molecular components involved in this process is essential for designing future experimental work in this system.2 and mounted on a stereotaxic instrument . After incision in the dorsal neck region and exposure of the skull occipital region, a needle of a 0.5 ml sterile insulin syringe was inserted into cisterna magna73. Approximately 3–10 µl of clear, colourless CSF was collected per animal and stored at −80 °C until analysis preparation. Blood was extracted from the carotid artery and PL obtained by centrifugation  was stored at −80 °C, until further use. Experimental design involving animals was performed by accredited researchers . The research was approved by the Ethical Committee of Charles University, Faculty of Science (permits MSMT-1373/2016-4 and MSMT-30397/2019-5) and was carried out in accordance with the current laws of the Czech Republic and European Union and in compliance with the ARRIVE guidelines.CSF and PL samples were collected from a total of seven chicken, five budgerigars and five cockatiels. The animals were originated from legal imports for the pet trade  who confirmed their status as healthy, adult birds and were kept in our animal facility (accreditation No. 37428/2019-MZE-18134). The animals were euthanised by COThe ProteoSpin detergent-free total protein isolation kit  was used for isolation and purification of the total protein content from CSF and PL according to the manufacturer's instructions and then frozen at −20 °C. Protein precipitation was performed using acetone  at −20 °C for 1 h. All precipitated samples were centrifuged at 14,000 g and 4 °C for 15 min. After centrifugation, the supernatant was discarded, the samples dried for 30 min at 37 °C and were cleaved with trypsin  at 37 °C overnight before mass spectrometry analysis.74. The eluting peptide cations were converted to gas-phase ions by electrospray ionization and analysed on a Thermo Orbitrap Fusion mass spectrometer . Survey scans of peptide precursors from 350 to 1400 m/z were performed at 120 K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS/MS was performed by isolation at 1.5 Th with the quadrupole, high-energy collision dissociation fragmentation with a normalized collision energy of 30 and rapid scan MS analysis in the ion trap. The MS/MS ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with a charge state of 2–6 were sampled for MS/MS. The dynamic exclusion duration was set to 45 s with a 10-ppm tolerance around the selected pre-cursor and its isotopes. Monoisotopic precursor selection was turned on and the instrument was run at top speed with 2 s cycles.Nano reversed-phase columns were used to elute peptide cations using a previously described method75. False discovery rate (FDR) was set to 1% for identification of all peptides and proteins. We set a minimum peptide length of seven amino acids. The Andromeda search engine was used for the MS/MS spectra search against the chicken Gallus gallus UniProt reference proteome , budgerigar Melopsittacus undulatus UniProt reference proteome  and zebra finch Taeniopygia guttata UniProt reference proteome , with all duplicates removed. Enzyme specificity was set as C-terminal to Arg and Lys, also allowing cleavage at proline bonds76 and a maximum of two missed cleavages. Dithiomethylation of cysteine was selected as a fixed modification and N-terminal protein acetylation and methionine oxidation as variable modifications. Quantifications were performed with the label-free quantification algorithms using a combination of unique and razor peptides75. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE77 partner repository with the dataset identifier PXD021633.All data were collected and quantified using MaxQuant software version 1.6.10.4378 and the budgerigar79, but not for the cockatiel. Thus, mapping cockatiel reads on its own reference proteome was not possible. To estimate the effect of this condition, we compared the MS/MS identification success rates of chicken, budgerigar and cockatiel proteomes, each mapped against three applicable avian reference proteomes, the chicken, budgerigar and zebra finch. We performed the following data filtering to eliminate low-scoring spectra: from further analysis we excluded all the unlabelled peptides, peptides only identified by site or reverse, all contaminants, proteins identified by < 2 peptides and all proteins occurring in less than 1/3 of the samples for each group. For the purpose of comparison, chicken ortholog gene codes were assigned to all identified proteins using the database OrthoDB (v10.1)80. When the gene code was not possible to retrieve, the UniProt protein ID was used.Annotated reference proteomes are presently available for the chicken81. To find overrepresented Gene Ontologies (GOs)82 in the core proteomes of CSF and PL, we launched the g:profiler against the chicken reference list  and the annotation data sets of the Gene Ontology and biological pathways 85. The Benjamini–Hochberg FDR threshold was set to 0.05 of all identified proteins in PL and CSF of chicken, budgerigar and cockatiel.For protein classification, we used the online Protein Analysis Through Evolutionary Relationships (PANTHER) library86, Microsoft Excel and Perseus software platform . The Venn diagrams were prepared using Biovenn87 and Jvenn88. In order to remove minor technical variation between samples, the abundances of the identified proteins were previously normalised using the Variance stabilization normalization method90 and DEP 1.12.0 R package. Missing data were imputed using random draws from a manually defined left-shifted Gaussian distribution applying the default settings of Perseus software. PCA was performed to examine the variation between the PL and CSF fluids in these species using Perseus and R. Significantly differentially represented proteins were identified by a Student’s t-test corrected by the Permutation Based FDR . In order to visualise biological protein groups clustered together in either CSF or PL, hierarchical cluster analysis was performed in Perseus using default parameters . To indicate the proteins significantly differentially represented in CSF and PL in the three studied species, a Volcano Plot analysis was performed in Perseus  and R. In the volcano plot, protein intensity fold-change between the fluids is represented as log2. The Retrieval of Interacting Genes 91 web-tool was used to analyse and construct the PPI network of the common group of proteins across the three species that were over-represented in CSF compared to PL  < 0.05, identified by the t-test analysis described above) . The interaction sources included experimental data and significant PPIs were considered those with a combined score > 0.4 and annotations from human (Homo sapiens). For the network visualization, also using annotations from Homo sapiens, we used ShinyGO (v0.61)92 online tool to determine the GO terms of biological functions which are significantly overrepresented in CSF compared to blood PL in the same set of common gene products over-represented in the CSF compared to PL. GSEA was also performed. All proteins were ranked based on the association between their abundance and the class distinction (CSF or PL), using annotations from Homo sapiens (Human Gene Symbol with Remapping MSigDB.v7.2). We used the GOs analysis of biological processes, nominal p value < 0.05 and adjusted p value < 0.25, as previously suggested. An FDR of < 25% is recommended for discovering candidate gene sets to be further validated as a result of future research93.The statistical and bioinformatic analysis conducted in the R softwareSupplementary InformationSupplementary Table S1. nLC-MS/MS output from different mapping approaches.Supplementary Table S2. Core proteomes of CSF and PL.Supplementary Table S3. GO Classifications and Pathway analyses of CSF and PL.Supplementary Table S4. Differentially represented proteins in CSF vs plasma.Supplementary Table S5. GSEA results.


This study was carried out to investigate the irritation of the prodigiosin isolated from Zooshikella rubidus on the skin and eyes in New Zealand white rabbits. The tests were performed on the basis of Korea Food and Drug Administration (KFDA) guidelines. Prodigiosin induced severe eye irritation at high concentration (0.5 g/site/ml) but there was no eye irritation at low concentration (0.3 mg/site/ ml). The primary irritation index was calculated from higher concentration (0.5 g/site/ml) to lower concentration (0.3 mg/site/ml). There were found non-irritation or induced mild irritation at lower concentration of prodigiosin application. On the basis of this study, it could be concluded that the prodigiosin may be non-irritant to mild irritant of usual application at lower concentration (0.3 mg/site) resulting it is safe and useful in dyeing technology of fabrics.


ALB) and TMAO (QTMAO) quotient and evaluating QTMAO values in individuals with and without blood-CSF barrier dysfunction. A total of 290 patients, who underwent diagnostic lumbar puncture with QALB and QTMAO determination, were evaluated. Serum and CSF TMAO measurements were performed on a tandem mass spectrometry SCIEX QTRAP 4500  coupled with an Agilent 1260 Infinity HPLC system . Serum and CSF albumin were measured on the Atellica® NEPH 630 system . CSF TMAO levels were positively correlated with serum TMAO levels . The QALB was significantly associated with the QTMAO . A total of 117 patients with blood-CSF barrier dysfunction had significantly higher median (Q1 - Q3) QTMAO values (4.7 (2.8 - 7.5) vs. 3.8 (2.5 - 5.7) x 10-1, p = 0.002) compared to 173 individuals with normal blood-CSF barrier function. CSF and serum TMAO concentrations were significantly associated in 290 CSF/serum pairs from lumbar punctures of clinical routine. QALB showed a relevant influence on QTMAO. Present results indicate that TMAO may cross the blood-CSF barrier.Recently, the microbiome-derived trimethylamine-N-oxide (TMAO) was shown to be present in human cerebrospinal fluid (CSF). However, data on the potential of TMAO crossing the blood-CSF barrier are still lacking. This retrospective study aimed at investigating possible associations between the CSF/serum albumin (Q The small organic molecule trimethylamine-N-oxide (TMAO) is generated in a gut microbiota-dependent way. The gut microbiome metabolizes L-carnitine and phosphatidylcholine containing nutrients  to trimethylamine (TMA), which is absorbed into the bloodstream and oxidized by the hepatic flavin mono-oxygenases (FMOs)  has been widely accepted as a reliable indicator of the blood-CSF barrier function . The CSter, 2001). Since ALB) and the CSF/serum TMAO (QTMAO) quotient and investigated the QTMAO in individuals sub-grouped by the presence or absence of blood-CSF barrier dysfunction.The aim of this retrospective study was to evaluate the albumin and TMAO concentrations in 290 consecutive CSF/serum pairs of lumbar punctures. We assessed possible associations between the CSF/serum albumin  in a one year period (2019), were retrospectively examined. Patients < 18 years of age were excluded. Informed consent was obtained from all patients. The ethical approval of this study was provided by the Ethical Committee of the Medical University Graz  and carried out with the current version of the declaration of Helsinki.In total, the medical records of 290 consecutive patients, who received diagnostic lumbar puncture with Q®  Z No Additive and 5 mL VACUETTE®  Z Serum Clot activator tubes . Samples were kept at 4 °C and batch analyzed within one week. The serum (reference range: 3.5 - 5.2 g/dL) and CSF (reference range: ≤ 0.35 g/L) concentrations of albumin were determined by nephelometric method on the Atellica® NEPH 630 system . The intra- and inter-day coefficients of variation (CVs) ranged between 2.7 - 3.1 and 1.7 - 3.5 %. The QALB was calculated to assess the blood-CSF barrier function .CSF and venous blood were taken simultaneously and collected in sterile 2 mL VACUETTEal., 2001). Accordal., 2001; BrettscSerum and CSF TMAO were measured using a stable-isotope-dilution assay and high-performance liquid chromatography (HPLC) with electrospray ionization tandem mass spectrometry on a SCIEX QTRAP 4500 triple quadrupole instrument  equipped with an Agilent 1260 Infinity HPLC system . The intra- and inter-day CVs ranged between 2.2 - 5.5 and 7.6 - 9.9 %. The serum TMAO reference range (0.98 - 15.5 µmol/L) was calculated according to the literature .The Kolmogorov-Smirnov test was performed to calculate data distribution. As all analyzed continuous variables were not normally distributed, they were expressed as medians (Q1 - Q3). To assess potential correlation between two continuous variables the Spearman's rank correlation coefficient  was used. Linear regression models were performed to assess the association between variables. The exact Mann-Whitney U test was used for subgroup comparisons. A p-value < 0.05 was considered statistically significant. For statistical analysis, the Analyse-it® software version 4.92  was used.The baseline characteristics of the study population are shown in Table 1ALB was 6.8 (5.0 - 9.9) x 10-3. All in all, 117 and 173 individuals were identified with and without blood-CSF barrier dysfunction. The median TMAO CSF and serum concentrations (Q1 - Q3) were 0.9 (0.5 - 1.4) and 2.1 (1.3 - 3.4) µmol/L.The median (Q1 - Q3) QCSF TMAO levels were positively correlated with serum TMAO levels . The univariate regression model for CSF TMAO is presented in Figure 1TMAO and the QALB is illustrated in Figure 3ALB showed a statistically relevant influence on the QTMAO . As shown in Figure 4TMAO values (4.7 (2.8 - 7.5) vs. 3.8 (2.5 - 5.7) x 10-1, p = 0.002) compared to 173 individuals with normal blood-CSF barrier function. Raw data are provided in Supplementary Table 1.The univariate regression model between the Qin vivo. The CSF TMAO levels were positively correlated with serum TMAO levels  and in the linear regression model serum TMAO showed a strong influence on CSF TMAO .In the present study, possible CSF and serum TMAO associations were assessed at the blood-CSF barrier in 290 liquor/serum pairs obtained from clinical routine. It was hypothesized that TMAO could cross the blood-CSF barrier. This aspect has never been demonstrated In comparison, Del Rio et al. measured for the first-time CSF TMAO in 58 subjects with diagnostic lumbar punctures . UnfortALB and the QTMAO . These results indicate, that the small molecule TMAO may cross the blood-CSF barrier via passive diffusion. Small molecules have been shown to penetrate the blood-CSF barrier into a much larger number compared to large molecules  or facilitated by active transporters  . Hereinuer, 1974). Since uer, 1974), an addALB . In a pber, 2001). The blber, 2001).TMAO values compared to 173 individuals with normal blood-CSF barrier function (p = 0.002). Many neurological diseases have been shown to be accompanied by an altered blood-CSF barrier permeability . This cber, 1994).The major advance of the present study is the high number of diagnostic lumbar punctures obtained from neurological patients. Nevertheless, the major limitation of this retrospective analysis is that it is not possible to draw conclusions about possible links between CSF TMAO concentrations and different neurological conditions. Conducting prospective studies may contribute to this gap of knowledge in the future.ALB showed a relevant influence on QTMAO. Patients with blood-CSF barrier dysfunction had significantly higher CSF TMAO concentrations compared to individuals with normal blood-CSF function. Significant association of CSF and serum TMAO concentrations was observed in 290 diagnostic CSF/serum pairs of clinical routine. QThe authors declare that there is no conflict of interest.


The prolongation of the classic swine fever (CSF) outbreak in Japan in 2018 was highly associated with the persistence and widespread of the CSF virus (CSFV) in the wild boar population. To investigate the dynamics of the CSF outbreak in wild boar, spatiotemporal analyses were performed. The positive rate of CSFV in wild boar fluctuated dramatically from March to June 2019, but finally stabilized at approximately 10%. The Euclidean distance from the initial CSF notified farm to the farthest infected wild boar of the day constantly increased over time since the initial outbreak except in the cases reported from Gunma and Saitama prefectures. The two-month-period prevalence, estimated using integrated nested Laplace approximation, reached >80% in half of the infected areas in March–April 2019. The area affected continued to expand despite the period prevalence decreasing up to October 2019. A large difference in the shapes of standard deviational ellipses and in the location of their centroids when including or excluding cases in Gunma and Saitama prefectures indicates that infections there were unlikely to have been caused simply by wild boar activities, and anthropogenic factors were likely involved. The emergence of concurrent space–time clusters in these areas after July 2019 indicated that CSF outbreaks were scattered by this point in time. The results of this epidemiological analysis help explain the dynamics of the spread of CSF and will aid in the implementation of control measures, including bait vaccination. Pestivirus and the family Flaviviridae. CSFV exhibits a variety of disease modes in host animals with infections that may be acute, subacute, chronic, late-onset, or asymptomatic. It is known that disease severity depends on the virulence of the CSFV, age and species of a host animal, and status of individual or herd immunity. CSFVs with moderate virulence have recently been isolated in Mongolia and China [Classical swine fever (CSF) is a highly contagious disease causing a multisystemic infection in domestic and wild pigs. CSF is distributed worldwide and causes enormous economic losses in husbandry due to its high virulence in domestic pigs . The caund China ,3.Japan once achieved the elimination of CSF through the application of the attenuated CSFV vaccine . Since 1One year after the initial CSF notification, the lack of success in controlling the outbreak is concerning. To provide another perspective that could be of assistance, we investigated the dynamics of CSF spread in wild boar by analyzing the transmission pattern of CSFV in wild boar temporally and spatially. The identification of CSF cases which were unlikely to have been transmitted via wild boar would suggest important opportunities for biosecurity measures in farms and a disease containment strategy.From September 2018 to the middle of November 2019, a total of 6,594 wild boars, including 826 dead and 5768 captured animals were tested for CSF infection . After tIn general, the direct distance between the locations of the initial CSF notification and cases in wild boar increased proportionally with time . HoweverFor each case of CSF notified in wild boar, the distance from the initial CSF case and time since the initial case was plotted.The two-month-period prevalence showed the first peak around the area of the initial farm case in November–December 2018 A. The meThe intensity of the red color indicates the estimated two-month-prevalence of CSF in wild boar at the municipality level, with an intensity of 1.0 indicating a prevalence of 100%. A: November–December 2018, B: January–February 2019, C: March–April 2019, D: May–June 2019, E: July–August 2019, F: September–October 2019.Standard deviational ellipses (SDEs) for the three phases were overlaid on a map with CSF-positive notifications to illustrate the directional trends and dispersion of CSF notifications . The posStandard deviational ellipses for three time periods . Ellipses and their centroids (green and pink plus signs) were overlaid with CSF notifications distinguishing domestic pig (white square) and wild boar (dot) cases. Black dot: CSF notification of wild boar in September 2018–March 2019, green dot: April–June 2019, blue dot: July–September 2019, red dot: October–November 2019. For the last two periods, standard deviational ellipses that exclude the notification data in Gunma and Saitama prefectures are shown.A total of 13 significant space–time clusters were identified from the CSF notification dataset by space–time permutation analysis based on the 26-km upper limit on cluster size set in the software . ClusterDuring the study period, from September 2018 to November 2019, 13 significant clusters were observed in or around the disease notification area. Detailed information for each cluster is given in At the time of writing, November 2019, more than one year has passed since the initial CSF notification in September 2018, and the outbreak has still not been terminated. In this period, 46 CSF outbreaks in pig farms were reported with approximately 120,000 killed animals, but the wild boar population was considered to play a critical role in the spread of the disease. Disease notifications were concentrated at locations near the initial cases in the early phase of the current outbreaks and then became more widespread over time. The results from the present study indicate that the disease could have spread via the movement of wild boar to nearby contiguous areas was confirmed from spring 2019 onward through spatial changes in period prevalence. The risk of CSFV infection at a farm located at a 5-km distance from a CSFV-positive wild boar within 28 days was estimated at more than 5% in Hayama et al. [Since the 1990s, wild boar has been recognized as an important reservoir of CSFV due to a change in pathogenicity from high to moderate virulence in wild boar as well as domestic pigs. Transmission routes of CSFV are comparable in wild boar and domestic pigs, and occur either through direct contact between diseased animals or indirectly via feces, food, and carcasses . During Sus scrofa leucomystax) breed piglet in April-June, especially in May, it would have been necessary to complete bait vaccination in the affected areas no later than May 2019 when the CSFV spread further by the movement of the wild boar [Bait vaccination with the commercial vaccine  for wild boar was utilized twice between March and May 2019 in selected areas in Aichi and Gifu prefectures where CSF positive cases were found . Thirty ild boar ,22. In aCSFV infection in domestic pigs was continuously notified in Japan since September 2018 and spread more widely mainly through wild boar movement. The implementation of effective control measures in wildlife, such as bait vaccination under a well-planned strategy and the involvement of a surveillance program using hunting or a capture scheme, is essential for successful containment. Though biosecurity measures were strengthened at pig farms to prevent CSF introduction, unexpected outbreaks occurred in pig farms in areas where the wild boar were unlikely to have been infected with CSFV. The current control measures both for domestic pigs and wild boar should be intensively reviewed.−, is performed in Livestock Hygiene Service Centers under the direction of the National Institute of Animal Hygiene as one of the diagnostics of CSFV detection [Epidemiological data of CSF notification and reverse transcription polymerase chain reaction (RT-PCR) test results of CSFV detection in domestic pigs and wild boar between September 9, 2018, and November 15, 2019, were collected from the websites of 15 prefectures. In Japan, the RT-PCR based on the Vilcek et al., using a positive control of the attenuated CSFV strain GPEetection ,24. The etection . A totaletection , as wellThe dates and locations of CSFV detection from both dead-found and captured wild boars were used to investigate the relationship between the time elapsed and distance from the location of the initial CSF notification in the domestic pig farm to each of the CSF cases in wild boar. The dates and locations of wild boars tested for CSFV, including those produced negative results, were used for the calculation of weekly positive rates of CSFV among both dead-found and captured animals, and among only captured animals, respectively, to describe the temporal trend of CSF positive rates in wild boar in expanding infected areas.Two-month-period wild boar diagnostic positive and negative results based on PCR tests were aggregated at the municipality level for the period between September 2018 and October 2019, and the period prevalence in each administrative unit was estimated using an integrated nested Laplace approximation (INLA) with zero-inflated binomial errors using the package R-INLA in the statistics software R version 3.6.1  . IntrinsSDE analysis was performed to describe the trend and spatial characteristics of CSF notifications in the study area using ArcGIS v10.6.1 software . This provided the orientation and shape of a distribution, and dispersion of the diseases in domestic pigs and wild boar, following an approach similar to those in previous studies ,26,27. TK function. Detailed information on the method for calculating the maximum distance of relationships, which yielded the highest Diff K value, was described in a previous study [p < 0.05). Analyses were conducted in SaTScan software v9.6  [A multi-distance spatial cluster analysis tool in ArcGIS v10.6.1 was used to identify the maximum distance of the relationships between CSF notifications by applying the common transformation of Ripley’s us study . A spaceus study . A MonteMA, USA) . The habMA, USA) .


In single-space combined spinal-epidural anesthesia (CSEA), it is important to correctly determine if the fluid coming out of the spinal needle is cerebrospinal fluid (CSF) or the liquid used in the loss of resistance (LOR) technique. In this study, we used mepivacaine for LOR and measured the pH values of CSF and mepivacaine to determine whether the pH test is a reliable method to confirm CSF when performing single-space CSEA.This clinical trial included 47 full-term pregnant women who underwent cesarean section. Single-space CSEA was administered at the lumbar intervertebral space using a small amount of mepivacaine for LOR. The pH values of CSF and mepivacaine were determined by the color of the test strip immediately after dropping. The area under the curve (AUC) for the pH values was calculated to determine the cutoff value for distinguishing between CSF and mepivacaine.The median pH values were 7.7 (7.1–8.0) and 6.2 (5.9–6.8) for CSF and mepivacaine, respectively. When the cutoff value of pH for distinguishing CSF from mepivacaine was 7.1 or greater, the AUC was 1.0 (100% sensitivity and specificity). Our result demonstrated that CSF can be correctly distinguished from mepivacaine in patients undergoing cesarean section under single-space CSEA using a cutoff value of pH 7.1.The pH test is a simple and reliable method to confirm CSF when performing single-space CSEA with mepivacaine for LOR.UMIN000036454. Registered 1 May 2019Accuracy of pH test paper for cerebrospinal fluid during spinal anesthesia: prospective study in healthy pregnant women under scheduled caesarean section; University Hospital Medical Information Network,  Combined spinal-epidural anesthesia (CSEA) is widely used for cesarean sections due to its advantage of long-lasting postoperative analgesia. When single-space CSEA is performed, the epidural space is identified by the loss of resistance (LOR) technique using saline. Subsequently, the subarachnoid space is punctured using a spinal needle inserted through the epidural needle, and the local anesthetic is administered intrathecally. Since the saline used in the LOR technique may come out of the spinal needle, care should be taken to ensure that the liquid is cerebrospinal fluid (CSF) for successful anesthesia.The qualitative sugar test is a convenient technique for confirming CSF that provides quick results in 30–60 s. However, small traces of blood in the CSF can deem the sugar test positive. Once a vascular puncture occurs during needle insertion, it is difficult to confirm CSF in a sugar test. Therefore, this test may not be suitable for confirming CSF in pregnant women with a dilated epidural venous plexus.The pH test is another simple technique for confirming CSF. Previous studies showed the pH test to be as useful as the sugar test , 2. SincIn this study, we used mepivacaine for LOR and measured the pH values of CSF and mepivacaine to determine whether the pH test is a reliable method for confirming CSF when performing single-space CSEA. We also determined a cutoff value of pH to distinguish between CSF and mepivacaine.The study was approved by the Institutional Ethics Committee (#3040) on April 16, 2019, and was registered at the University Hospital Medical Information Network (ID UMIN000036454) on May 1, 2019. Written informed consent was obtained from all participants. This study was conducted at the Seirei Hamamatsu General Hospital in Shizuoka, Japan, between May 2019 and July 2019.We included pregnant women aged > 20 years who were scheduled for elective cesarean section with single-space CSEA. Parturients with an American Society of Anesthesiologists physical status ≥ 3 and those whose native language was not Japanese were excluded. Demographic data, including the patient’s age, gestational week, and the indication for cesarean section were obtained from the medical records.In the operating room, single-space CSEA was administered at the L2/3, L3/4, or L4/5 interspace with the parturient in the lateral decubitus position. Oxygen saturation, non-invasive blood pressure, and electrocardiogram were continuously monitored for all patients. After local infiltration with 1% mepivacaine , single-space CSEA was performed using a combined spinal-epidural needle . The epidural space was identified using the LOR technique with a small amount of 1% mepivacaine. After confirming that there was no dural puncture, the spinal needle was passed through the epidural needle and advanced to the subarachnoid space. When a clear liquid came out of the spinal needle, it was dropped onto a pH paper  placed on a white gauze. The pH values were determined under fluorescent light by two members of the medical staff in the operating room, where they indicated the most complementary color to that of the pH test paper , post-dural puncture headache (PDPH), and nerve injury, were also assessed.κ coefficient takes a value of 0–1, with values greater than 0.6 indicating substantial agreement.The sample size was calculated based on a two-sided test with the area under the curve (AUC) of 0.75, a power of 0.9, and a significance level of 5%. The determined required sample size was 25. Statistical analysis was performed using computer software R . The AUC for the pH values was calculated, and the cutoff value was decided by the intersection of the diagonal line and the receiver operating curve. The AUC takes a value of 0.5–1.0, with values 0.9–1.0 indicating a high predictive ability. The reliability of the pH test for identification of both CSF and mepivacaine was assessed using Cohen’s weighted kappa coefficient. The n = 26), breech presentation (n = 8), twins (n = 4), placental abnormalities (n = 4), and others (n = 5).Of the 51 eligible participants, three were excluded because their native language was not Japanese, and one patient did not provide consent. Thus, 47 pregnant women with a mean age of 34.7 ± 5.0 years and a mean gestational age of 37.9 ± 0.5 weeks were included in this study. The indications for cesarean section were past cesarean section  and 6.2  for CSF and mepivacaine, respectively  does not overlap with that of CSF . Future The limitation of this study was that the fluid obtained from the spinal needle was CSF in all cases. We did not measure the pH value of mepivacaine administered into the epidural space by the LOR. The cutoff value of pH to distinguish between CSF and mepivacaine in non-pregnant patients may differ from our findings.The pH test is a simple and reliable method to distinguish between CSF and mepivacaine when performing single-space CSEA, and the cutoff value of pH was determined to be 7.1.


We aimed to investigate whether troponin clearance calculated according to this formula had an effect on short-term mortality.  In patients with CKD, cTn concentrations may be elevated in the absence of AMI, which is a predicted finding caused by chronic structural heart disease rather than acute injury. The increase in troponin level observed in noncardiac conditions provides conflicting results when predicting mortality. Low lactate clearance was associated with increased mortality. Lactate clearance is calculated as follows: (early lactate − late lactate/early lactate) t-test was used for the parametric data, and the Chi-squared test for the nonparametric data.  The study included 300 patients with chronic renal failure who had a sepsis-related organ failure assessment (SOFA) score ≥3. By taking the baseline troponin at the time of emergency presentation as reference and comparing them with the fourth-hour troponin values, troponin clearance was investigated in the evaluation of mortality among hospitalized patients with CKD within the first month after discharge. The data obtained were analyzed using the SPSS data analysis software version 20.0. Student's P=0.0000083).  Of the 300 patients evaluated, 189 patients survived (mean age 66.20 ± 14.597 years), and 111 died (mean age 74.81 ± 12.916 years). Troponin clearance was detected in 40 of the 111 patients in the mortality group and 119 of the 189 patients in the survival group. Troponin clearance was significantly more frequent in surviving patients ( Troponin clearance can be considered as a valuable leading indicator of survival, but higher levels of troponin clearance did not lead to higher survival rates. Cardiac troponin I (cTnI) and cardiac troponin T (cTnT), which are sensitive biological markers of myocardial injury, are used to diagnose acute myocardial infarction (AMI) –4. AlthoIncreased cTn has been observed in the absence of acute coronary syndrome (ACS) in both cardiac and noncardiac patients. There are additional questions concerning the causes of elevated cTn and the reasons for the observed differences between the increases in cTnT and cTnI. To answer these questions, it may be helpful to examine the synthesis, susceptibility, destruction, and/or clearance process of cTn .The association between elevated cTnT and low glomerular filtration rate (eGFR) is stronger compared to that of cTnI to eGFR , 11, sug∗ 100 [Some emergency departments and intensive care units use serum lactate concentration as a qualitative indicator of resuscitation in patients with sepsis. Bolvardi et al. revealed that low lactate clearance was associated with increased mortality; however, direct lactate elevation yielded contradictory results as a predictor of mortality . Similar∗ 100 . In the Patients who presented to the Emergency Department of Haydarpasa Numune Training and Research Hospital with CKD between January 2013 and December 2015 and scored ≥3 in the sequential (sepsis-related) organ failure assessment (SOFA) were retrospectively analyzed using the hospital data screening system. Of the 6,000 patients with CKD, a total of 300  were included in the study.By taking the baseline troponin at the time of emergency presentation as reference and comparing them with fourth-hour troponin values, troponin clearance was investigated in the evaluation of mortality among hospitalized patients with CKD within the first month after discharge. Serum cTnT levels were measured using the dimension analyzer  through application of the one-step sandwich principle immunoassay.th hour troponin were deemed measurable. Conversely, patients with a 4th hour troponin level higher than baseline levels were deemed to have no measurable troponin clearance. In patients with measurable troponin clearance, the mean value of their respective groups' clearances was analyzed as well. The parameters analyzed from the records were age, gender, blood urea nitrogen (BUN), creatinine, baseline and fourth-hour troponin values, and mortality within one month of discharge from hospital.The patients were analyzed in two ways; firstly, they were analyzed in terms of presence or absence of measurable troponin clearance. Patients with a baseline level of troponin higher than their subsequently measured 4Patients were divided into two groups based on their mortality or survival. Patients who died within a month of admission were assigned to the mortality group, while surviving patients were assigned to the survivor group. The inclusion criteria were as follows: presence of stages 4 and 5 CKD, an increase in troponin due to non-ACS causes, being above 18 years of age, a SOFA score of ≥3, and availability of data regarding first-month mortality. The patients with missing files, unknown troponin levels or ACS, those who died in the emergency department before examination, those with additional diseases that might cause elevated troponin, and those who were referred to the hospital from another health center were excluded.t-test was used for the parametric data and the Chi-squared test for the nonparametric data. P < 0.05 was considered statistically significant.The data obtained were analyzed using the SPSS data analysis software version 20.0. Student's P < 0.05). Of the survivors, 84 were female, and 105 were male; while there were 50 female patients and 61 male patients in the mortality group. There was no statistically significant relationship between gender and mortality (P=0.507).Of the 300 patients evaluated, 189 patients survived (mean age: 66.20 ± 14.597 years), and 111 died (mean age: 74.81 ± 12.916 years). There was a statistically significant difference between the two groups in terms of mean age   .P=0.174) . Of the 159 patients with troponin clearance, 119 survivors had a mean troponin clearance of 31.32 ± 42.90, while the 40 who died had mean troponin clearance of 24.38 ± 21.11. There was no significant difference between the mortality and survival groups in terms of the mean troponin clearance values (P=0.156)  , i.e., aA large number of studies have shown that in patients with end-stage renal disease (ESRD), both cTnI and cTnT are strong prognostic indicators for death of all causes and although cTnI increases less than cTnT, it is associated with a higher risk of mortality –19.μg/l, 53% were diagnosed with ACS and 41% of these cases were associated with nonthrombotic causes . For the remaining 6% of patients, the reason for the elevated cTnT could not be determined. The results of that study show that although a significant proportion of the patients admitted to hospital had high cTn levels, this was not due to coronary artery disease [In a study by Alcalai et al., the cTnT levels were measured in all patients who presented to the hospital for various reasons within a 10-month period. Of the 635 cases with a cTnT value of >0.1  disease .Since coronary ischemia is frequently present in patients with renal failure, it is important to ascertain whether the cTn elevation is caused by ischemia or chronic progression of impaired renal function. Although there is no specific method to appropriately make this distinction, increased cTn levels in follow-up are mostly interpreted in favor of acute ischemia . In patiIncreased cTn, in the absence of ACS, is observed in patients with both cardiac and noncardiac conditions. This raises additional questions concerning the causes of elevated cTn and the observed differences between the increases in cTnT and cTnI. To answer these questions, it may be helpful to examine the synthesis, susceptibility, destruction, and/or clearance process of cTn . To the Life-threatening complications of renal failure, such as pulmonary edema associated with fluid overload, hyperkalemia, other electrolyte and acid-base disorders, increased acute coronary frequency, dyslipidemias, and chronic volume overload, also increase the cardiovascular burden, leading to an imbalance in myocardial oxygen consumption, coronary vasoconstriction, and myocardial injury . ContinuIt is important to acknowledge the potential limitations of the current study. As a single-center retrospective study, the effects of unrecorded factors that may have led to confusion cannot be denied. Furthermore, the exact etiology of CKD may not have been documented by the treating clinical team in all cases.In conclusion, in this study, measurable troponin clearance was shown to be associated with increased probability of survival in critical cases, but a higher mean value of troponin clearance did not lead to a higher survival rate. According to the overall results, presence of measurable troponin clearance may be valuable as a leading indicator of survival; however, for troponin clearance to be used as an independent predictor of survival, our results should also be confirmed in patient groups with conditions other than CKD. For this purpose, multicentered studies involving larger case series are needed.


It remains unclear how introduction of high-sensitivity troponin T testing, as opposed to conventional troponin testing, has affected the diagnosis of acute myocardial infarction (AMI) and resource utilization in unselected hospitalized patients. In this retrospective analysis, we include all consecutive cases from our center during two corresponding time frames (10/2016–04/2017 and 10/2017–04/2018) for which different troponin tests were performed: conventional troponin I (cTnI) and high-sensitivity troponin T (hs-TnT) assays. Testing was performed in 18,025 cases. The incidence of troponin levels above the 99th percentile was significantly higher in cases tested using hs-TnT. This was not associated with increased utilization of echocardiography, coronary angiography, or percutaneous coronary intervention. Although there were no changes in local standard operating procedures, study site personnel, or national coding guidelines, the number of coded AMI significantly decreased after introduction of hs-TnT. In this single-center retrospective study comprising 18,025 mixed medical and surgical cases with troponin testing, the introduction of hs-TnT was not associated with changes in resource utilization among the general cohort, but instead, led to a decrease in the international classification of diseases (ICD)-10 coded diagnosis of AMI. Since their implementation into clinical practice, conventional troponin I (cTnI) assays have enabled physicians to diagnose myocardial infarction more accurately than any other biomarker assay. They have significantly facilitated treatment selection and patient-risk stratification and have become cornerstones of routine clinical practice ,2,3,4,5.The present work is a retrospective single-center study conducted at a 1300-bed German university hospital. The study was approved by the local ethics committee.® Cardiac Troponin I Assay, Dimension Vista® 1500, Siemens Healthineers, Eschborn, Germany-and 10/2017–04/2018 for hs-TnT–Elecsys® Troponin T hs, Cobas® e801, Roche Diagnostics, Mannheim, Germany). By definition, two cases could potentially represent the same individual being evaluated in both consecutive time frames, but analysis was restricted to the first individual presentation per assay to avoid double counting. If the patient underwent coronary angiography, the recorded troponin values were restricted to timepoints before the procedure, to exclude elevated values as a result of the intervention. Three hundred sixteen patients with ST-elevation myocardial infarction (STEMI), as well as 595 patients under the age of 18 were excluded. We restricted our analysis to patients of all departments except the cardiac surgery department (906 patients excluded), due to potential problems in data acquisition of external diagnostics . The data collected consisted of age, gender, in-hospital mortality and procedural data, hs-TnT or cTnI values, as well as international classification of diseases (ICD)-10 codes and codes of the German procedure classification (OPS). The following tokens for Germany (ICD-10-GM and OPS) were used for the analysis: German OPS Code 1-275 for coronary angiography (CAG), 8-837 for percutaneous coronary intervention (PCI), 8-98 for admission to the critical care unit (CCU), 8-77 for cardiopulmonary resuscitation (CPR), I21.4 and I21.9 for acute myocardial infarction (AMI) other than STEMI and lastly I25.1 for coronary heart disease (CHD).We used the hospital’s patient data management system and laboratory database to include in the analysis every case where at least one troponin measurement was obtained during the two corresponding time frames of October to April in an all-comers fashion, regardless of ambulatory or hospitalized status  remained the same throughout the study period.A 99th percentile value for cTnI was considered 0.05 ng/mL and for hs-TnT 14 ng/L, as recommended by the manufacturers.Subsequently, we analyzed different subgroups: patients hospitalized in the cardiology department (Subgroup 1), patients hospitalized in other departments (Subgroup 2), and lastly, to investigate the findings in a cohort of patients with high a priori probability for the diagnosis of acute myocardial infarction (Subgroup 3), we applied clinically relevant laboratory criteria to all inpatients of the cardiology department in a separate analysis: with high-sensitivity troponin testing based on the ESC hs-TnT 0/1h algorithm ,8,24,25 The primary endpoint was defined as diagnosis of myocardial infarction based on ICD-10 coding. Secondary outcome measures were: total number of troponin values above the 99th percentile, total number of performed echocardiograms, coronary angiographies, percutaneous coronary interventions, admissions to the critical care unit, cardiopulmonary resuscitation, and in-hospital mortality.p < 0.05 was considered significant; p-values are two-sided where appropriate. Statistical calculations were performed using IBM SPSS Statistics 25 .Baseline characteristics were described as frequencies and percentages for categorical variables, mean and minimum/maximum, and standard deviation for continuous variables. Categorical variables were analyzed using Pearson’s chi-square or Fisher’s exact tests, and continuous variables with the T-test. A value of In total we included 18,025 cases, representing 12,863 individuals with at least one troponin measurement. Of these cases, 9065 (50%) had been tested using the cTnI assay (10/2016–04/2017) and 8960 (50%) cases with the hs-TnT (10/2017–4/2018). Subsequently, we analyzed different subgroups: patients hospitalized in the cardiology department (Subgroup 1), patients hospitalized in other departments (Subgroup 2) and lastly, patients with high a priori probability for the diagnosis of acute myocardial infarction (Subgroup 3).p < 0.001) and below the limit of detection (LoD)  as well as the number of coded acute myocardial infarction (AMI) . The rate of outpatient treatment increased significantly from 28.2% to 30.1% (p < 0.01). The extent of consecutive testing increased after introduction of hs-TnT from 34.7% to 39.1% (p < 0.001).The baseline characteristics of both cohorts were clinically comparable, with the mean age being 61 years in both groups and a slight predominance of male gender  between the two time frames. There was no significant change in the number of CCU admissions, CPR-rate, or in-hospital mortality. The percentage of patients examined with echocardiography (81.6% vs. 83.2%) or CAG (38.3% vs. 37.1%) as well as the rate of PCI (44.7% vs. 46.6% of patients undergoing CAG) remained stable. The number of coded AMI decreased upon the introduction of hs-TnT .The mean age of hospitalized cardiology patients was 70 (cTnI) and 71 years (hs-TnT), with a significant increase in male patients since the introduction of hs-TnT  as well as a decrease in diagnosed AMI  after introduction of hs-TnT (p < 0.05).Similar to the previously detailed groups, we noticed a significant increase in troponin results above the 99th percentile . The significant drop in diagnosed AMI was even more pronounced in comparison to the beforementioned groups . No significant changes were noticed for echocardiograms, coded CHD or in-hospital mortality.Hospitalized patients in the cardiology department with elevated troponin represent the collective with highest probability for the diagnosis of myocardial infarction. In consideration of existing guidelines on non-ST elevation myocardial infarction and to represent daily clinical decision-making, we selected patients with markedly elevated levels of cTnI  or a 50% change between two measurements of cTnI or when testing hs-TnT a single value above 52 ng/L or a Delta 0/1 h > 5 ng/L for analysis in this high probability group. In our collective, in both groups 39% of hospitalized patients met these criteria. The frequency of CAG decreased significantly after introduction of high-sensitivity testing  with a high-sensitivity troponin assay . From our data, we cannot determine if the decrease in CAG had an impact on patient treatment and outcome.Several studies have investigated resource utilization in different settings, e.g., the ED or chest-pain unit, before and after introduction of high-sensitivity troponin testing, giving rather ambiguous results: some of the studies showed increased use ,35,36,37In a recent analysis of the SWEDEHEART registry, Odqvist et al. showed that the introduction of hs-TnT resulted in an increased diagnosis of myocardial infarction, which was combined with an increased use of coronary angiography and revascularization . InteresThe decreased rate of diagnosis of AMI documented in the present study is concerning. Even if the analysis is restricted to cases in the cardiology department with troponin values above the 99th percentile receiving PCI, ICD-coded diagnosis of AMI is significantly lower after introduction of high-sensitivity testing. This observation could have profound impact on in-hospital treatment and future care: misidentification of AMI may lead to detention of oral antiplatelet therapy and other measures of secondary prevention, reduced awareness with ambulatory follow-up visits and ultimately a higher rate of potentially preventable MACE. These findings highlight the urgent need for future prospective analyses on this topic.We noticed no significant change in the rate of admission to the critical care unit or cardiopulmonary resuscitation in all the different subgroups. Receiver operating characteristic curve analysis demonstrated a correlation with hs-TnT and in-hospital mortality with an area under the curve (AUC) of 0.81 . The in-The present investigation contains data of every inpatient case receiving troponin testing at a large tertiary care hospital. The unselected mixed cohort of patients from different surgical and medical departments reduces the risk of selection bias. To our knowledge, this is the first study analyzing the introduction of hs-TnT in a non-ED, non-chest-pain unit setting with both surgical and medical patients included.Due to the retrospective approach of the presented study, we used the existing ICD-coding of patient data without further verification or secondary adjudication of diagnoses. The indication for troponin testing was not fully traceable and, to some extent, may be attributable to preoperative risk-stratification directives in the absence of symptoms. The use of coronary angiography and revascularization was not standardized and left to the treating physician, therefore, the true prevalence of significant coronary artery disease in both cohorts still requires further clarification.In the present single-center retrospective study comprising 18,025 mixed medical and surgical cases with troponin testing not restricted to the emergency department or ACS setting, the introduction of hs-TnT led to a decrease in the diagnosis of acute myocardial infarction. We noticed significantly reduced use of invasive imaging, restricted to a subgroup of patients with high probability for myocardial infarction, but no change in resource utilization or in-hospital mortality in the overall cohort.


Chemotherapy-induced neutropenia (CIN) increases the risk of infections and mortality in cancer patients. G-CSF therapies are approved for the treatment of CIN, but non-G-CSF therapies are needed to increase efficacy and minimize side effects. Plinabulin is an inhibitor of tubulin polymerization that ameliorates CIN caused in patients by the microtubule stabilizer docetaxel. The present study evaluates the potential of plinabulin to reduce neutropenia induced by chemotherapies of different classes in a manner not dependent on increasing G-CSF.The anti-CIN benefits of plinabulin were tested in rodents co-treated with docetaxel, cyclophosphamide or doxorubicin. Effects on G-CSF levels were evaluated in tissues by immunoassay. Flow cytometry was utilized to test treatment effects on femur bone marrow cell counts from immunocompetent mice-bearing orthotopic 4T1 breast cancer tumors.−/Sca1+/c-Kit+ (LSK) hematopoietic stem/progenitor cells (HSPC) in murine bone marrow collected 2 days after treatment was not affected by docetaxel monotherapy despite increased plasma G-CSF in this group. LSK cell number was, however, increased when plinabulin was combined with docetaxel, without affecting G-CSF.Plinabulin alleviated neutropenia induced by microtubule stabilizing, DNA cross-linking and DNA intercalating chemotherapies, yet did not affect bone marrow or blood G-CSF levels. The number of lineageResults support the clinical testing of plinabulin as a non-G-CSF-based treatment for CIN associated with chemotherapies of different mechanisms. Results also support HSPC as a focal point for future mechanism-of-action work aimed at understanding the ability of plinabulin to reduce this serious side effect of cytotoxic therapy in cancer patients.The online version of this article (10.1007/s00280-019-03998-w) contains supplementary material, which is available to authorized users. Myelosuppression is the primary toxicity of many chemotherapy regimens, and neutropenia in particular is a frequent and potentially life-threatening complication . Chemoth® (pegfilgrastim)  in rat bone marrow samples flushed from the femur with PBS, and total protein was evaluated by bicinchoninic acid assay. G-CSF was measured in EDTA plasma isolated from blood collected by cardiac puncture in anesthetized non-tumor-bearing and 4T1 tumor-bearing animals described above, with the bead-based LEGENDplex™ immunoassay (BioLegend).p < 0.05 statistically significant. ANC time course data were analyzed by two-way ANOVA with Treatment and Time as factors. G-CSF concentration, single time point ANC data and flow cytometry data were analyzed by one-way ANOVA. If the ANOVA reached p < 0.05, Tukey’s or Sidak’s multiple comparison test was utilized to compare groups.Data were plotted and analyzed with Prism software (GraphPad) using two-sided tests and considering p < 0.0001). A more detailed evaluation of the time course for ANC, utilizing a higher dose of cyclophosphamide and IV dosing, found a more dramatic rise in ANC from 7 to 12 days when plinabulin was added to cyclophosphamide  and again demonstrated a late rise in ANC from 5 to 11 days after treatment. On the other hand, SC pegfilgrastim at a lower dose level than previously utilized caused a dramatic increase in ANC 2–3 days after chemotherapy dosing, with little effect from 5 to 11 days. ANC effect profiles for pegfilgrastim alone , cyclophosphamide (DNA cross-linking) or doxorubicin . With IP docetaxel, a transient lowering of ANC was detected 2 days after dosing, relative to untreated rats . Plinabulin alone or in combination with chemotherapy had no effect on bone marrow G-CSF levels. To incorporate the potential effect of a tumor on G-CSF levels , with a mechanism thought to involve increased production of neutrophils in the bone marrow, possibly by relieving a docetaxel-induced accumulation of murine HSPC , despite changes in G-CSF  and vehicle-treated tumor-bearing mice (p = 0.048). The trend for an increase compared to docetaxel-treated mice did not reach statistical significance (p = 0.15). In contrast to LSK cells, more differentiated common myeloid progenitor cells  and their levels tended to be further reduced 2 days after docetaxel monotherapy. Adding plinabulin to docetaxel had no significant effect on total bone marrow CLP or CMP cell numbers (p > 0.96) at 2 days after treatment, nor other cells relevant to the myeloid lineage (Online Resource 2).Neither the presence of tumors nor treatment with docetaxel significantly affected the total number of LSK cells :2068], a marker for hematopoietic stem and progenitor cells in human that is reportedly low or negative on murine hematopoietic stem LSK cells [The consistent increase in ANC found in plinabulin-treated rats, beginning approximately 7 days after therapy, may be related to the 1–2 weeks necessary to form mature neutrophils from HSPCs , 4. MoreSK cells .In summary, plinabulin has beneficial effects on chemotherapy-induced neutropenia induced by chemotherapies of different classes, with a mechanism distinct from G-CSF-based therapies. Results reported here support the continued development of plinabulin as an alternative and/or combinatorial approach to G-CSF therapy for the treatment of CIN.p value indicated is for the effect of treatment by one-way ANOVA (PDF 119 kb)Online Resource 1 Dose-dependent effects of plinabulin on doxorubicin-induced neutropenia. Blood absolute neutrophil count (ANC) 2 days after intraperitoneal treatment with plinabulin (7.5 mg/kg), or intravenous treatment with doxorubicin (3 mg/kg), followed 1 h later by intraperitoneal plinabulin  or plinabulin vehicle (n = 6 rats/group). Data are presented as the mean ± SEM. Statistical a Gating strategy example for flow cytometry analyses of bone marrow collected from both femurs of untreated mice or 4T1 tumor-bearing mice, 2 days after a 15 min intravenous infusion of docetaxel  or docetaxel vehicle (7.5% ethanol/7.5% Tween-80), followed 15 min later by IP injection of plinabulin (7.5 mg/kg) or plinabulin vehicle (Veh) twice, 3 h apart. Control tumor-bearing animals received both vehicles (gating strategy example shown). Total number of, b CD45 + Lineage-multipotent progenitors , c common myeloid progenitors , d granulocyte/macrophage progenitors , e neutrophils (CD115-CD11b + Ly6Ghi) and f monocytes (c-kit-CD115+) collected from both femurs. Data are presented as the mean ± SEM for n = 5 mice per group (PDF 304 kb)Online Resource 2 Effects of treatment on bone marrow cells involved in myeloid lineage hematopoiesis. Below is the link to the electronic supplementary material.