Sepsis is a complex syndrome characterized by simultaneous activation of pro- and anti-inflammatory processes. After an inflammatory phase, patients present signs of immunosuppression and possibly persistent inflammation. Hematopoietic growth factors (HGFs) are glycoproteins that cause immune cells to mature and/or proliferate. HGFs also have a profound effect on cell functions and behavior. HGFs play crucial role in sepsis pathophysiology and were tested in several clinical trials without success to date. This review summarizes the role played by HGFs during sepsis and their potential therapeutic role in the Management of sepsis-related immune disturbances. Infectious diseases are a major cause of death and morbidity worldwide and especially in intensive care units (ICUs) , 2. DecaSepsis is a complex double-face syndrome. Once having crossed the defensive barriers of the body , pathogens will activate the innate immune system and induce inflammation. As seen in most of biological processes, inflammation is well balanced by a counter-inflammation process driven by cellular reprogramming and anti-inflammatory cytokines , 7. The Indeed, after the initial phase of cell activation, the immune system appears to be “blunted” by the assault and patients face an immunosuppressive state. Poor response to infection, lymphopenia, and decreased reparative properties of immune cells are observed. Some authors termed this phase a persistent inflammation, immunosuppression, and catabolism syndrome (PICS) , 9. TherThis whole sequence is compartmentalized in space and time. Immune disturbances and time variations are observed in the whole body, the hematopoietic organs , the blood and the tissues.During sepsis, immune cells undergo profound phenotypic modifications in their activation state, response to stimuli, localization, and numbers. These phenomena are finely regulated by various cytokines and hematopoietic growth factors (HGFs).An HGF is defined as a relatively stable, secreted, or membrane-bound glycoprotein that causes immune cells to mature and/or proliferate. They also have profound effects on cell functions and behaviors.Hematopoietic growth factors are deeply involved in sepsis pathophysiology both in the initial phase and the late phase. They were naturally identified as potential therapeutic targets to treat septic patients. However, until now, there is no evidence of clinical benefit for the use of HGFs during sepsis.In this review, we will detail how the most studied HGFs are involved in sepsis, explore the findings from clinical trials, and discuss the perspectives for HGF-mediated immunotherapy of sepsis.α) and a subunit chain that is common to interleukin (IL)-5-and IL-3-receptors . GM-CSF-R signal is mediated via Extracellular signal-regulated kinase (ERK) 1/2, phosphoinositide-3-kinase (PI3K), mitogen-activated protein (MAP) kinase, and Janus kinase (JAK) – signal transducer and activator of transcription (STAT) pathways. Forty years after its discovery, it appears that this protein action is far more complex than just a proliferative agent; it has pleiotropic effects ranging from cell activation, survival, differentiation, chemotaxis  was discovered after observing that a factor present in lung-conditioned medium was able to induce the formation of granulocytes and macrophages , 16. GM-emotaxis , and proemotaxis . GM-CSF Until recently, most of literature regarding the role of GM-CSF during sepsis was focused on the early inflammatory phase. In murine/rat models and human cells culture, GM-CSF modulation through antibody mediated blockade, genetic invalidation, or protein supplementation helped to understand its functions in the host response to infections.−/− mice have reduced abilities to phagocyte and kill pathogens, have reduced Fcγ receptors (FcγR) expression, and have lower membrane expression of TLRs and subsequent lipopolysaccharide (LPS) or peptidoglycan-induced tumor necrosis factor alpha (TNFα) release. GM-CSF-deficient alveolar macrophages have markedly reduced reactive oxygen species (ROS) production and adenovirus-elicited Interferon (IFN)γ, IL-18, and IL-12 production. GM-CSF also increases the expression of scavenger receptors such as macrophage receptor with collagenous structure (MARCO) and other class A scavenger receptors (SR-As)  , 24. The (SR-As) . Most ofIn caecal ligation and puncture (CLP) model, recombinant murine (rm)GM-CSF treatment improves survival and reduces bacterial translocation .Interestingly, in some studies, injection of GM-CSF or genetic invalidation did not impact mice survival after CLP whether due to a timing of administration or dose issues. Inflammatory cytokines levels are higher when GM-CSF is present or enhanced , 28. In Regarding the late phase, proliferative capacities of monocytes during sepsis in response to GM-CSF are reduced in a time-dependent manner. Early myeloid-derived suppressor cells (MDSCs) obtained 3 days after CLP procedures produced more macrophages and DCs after GM-CSF stimulation than late MDSCs obtained 12 days after CLP .H)1 response. During sepsis, GM-CSF and IFNγ treatment can restore IL-12 production by splenic DCs. Mayuzumi et al.  . GM-CSF i et al.  showed tGranulocyte macrophage – colony stimulating factor has also pro-angiogenic effects and promotes endothelial cells proliferation –37; thusThe GM-CSF receptor is downregulated in human monocytes during sepsis  and in hin situ GM-CSF production demonstrates the importance of the spatial and temporal organization of this growth factor secretion.The source of GM-CSF during sepsis was unknown and thought to be mainly due to macrophages activation. In 2012, a study conducted in Swirski Lab in Boston  tackled this question. It appears that the main source of GM-CSF following abdominal sepsis is a new cell originating from a B1-type B cells . This neIt appears that a lack of GM-CSF is responsible for an immunosuppressed status which in turn could be associated with a worst outcome. Treatment with GM-CSF does not appear suitable for the initial cytokine storm-associated phase. However, immunosuppression features can be reversed by GM-CSF making this protein a potential candidate as an immune therapy for SIS.in vitro, monocytes functions in septic patients. GM-CSF treatment increases respiratory burst activity, integrin, and CD14 expressions. Same results are observed when AIDS patients with Mycobacterium Avium Complex bacteremia are treated with GM-CSF -DR in a common feature of SIS, GM-CSF was shown to be effective to increase monocyte (m)HLA-DR expression. Injection of GM-CSF is more efficient than G-CSF to restore HLA-DR levels in septic neonates . IntrapeA clinical trial conducted by Presneill et al.  showed tThe most famous trial regarding the use of GM-CSF in sepsis was conducted by Meisel et al. . Thirty-n = 147 patients). Mortality was consistently found to be unchanged.Bo et al.  conducteA multicenter prospective randomized controlled trial is now ongoing in France (NCT02361528).Use of GM-CSF in sepsis studies is characterized by a small number of patients and high heterogeneity of diagnosis and clinical presentation.To date, results of clinical trials show no benefit of GM-CSF treatment during sepsis. At least, there are no major adverse events observed after nearly 20 years of clinical use. It is insufficient to conclude in a lack of safety threat but still there is no big signal of risk and there are potential benefits. There is no definitive answer to the appropriateness of GM-CSF as an immunotherapy for certain subgroups of septic patients. The timing and the phenotype of the patients that could benefit from such treatment is to be elucidated. Due to the finely regulated GM-CSF response to sepsis in time and space, intravenous or subcutaneous injections could have reached negative results because of mixed benefic and adverse effects that could be ameliorated by adding GM-CSF in specific tissues or organs and/or timepoints.Granulocyte – colony stimulating factor, also known as CSF-3, is a 25-kDa glycoprotein coded by the Csf3 gene that is located on chromosome 17 in humans. G-CSF levels are low in steady state and rise after inflammatory stimuli . TNFα, IGranulocyte – colony stimulating factor is now widely used in routine to treat or prevent chemotherapy-induced neutropenia. G-CSF treatment is recommended by experts in various clinical scenarios –61.Granulocyte – colony stimulating factor exerts proliferative effects but also enhances mobilization of neutrophils in a direct and indirect chemotactic effect. Intravenous or subcutaneous injection of G-CSF is rapidly followed by a marked neutrophilia together with a release of progenitors and immature cells. G-CSF has also an impact of the generation of regulatory DCs and indirectly on T-cell populations.P. aeruginosa infection, G-CSF-deficient mice have decreased survival and augmented neutrophils apoptosis while local production of cytokine remains unchanged   . The tren = 480) .n = 83) or placebo (n = 83). Mortality does not differ between groups but the rate of liver dysfunction and elevation of troponin raises in the G-CSF group. Thus, G-CSF appears not only to be non-beneficial but detrimental in septic patients. These results point out the risk of increasing inflammation without precise guidance.Another trial focused on patient having nosocomial pneumonia showed no clinical benefit of G-CSF treatment but was associated with a trend lower apparition of sepsis features compared with placebo . A studyIn the meta-analysis of the impact of G-CSF during sepsis conducted by Bo et al. , G-CSF aThe limits of the described trials are nearly the same for G-CSF than for GM-CSF. The main difference is that GM-CSF is a better candidate to the reversal of various features of SIS, while G-CSF is only effective on neutrophils.Macrophage – colony stimulation factor, also termed CSF-1, is an 85-kDa glycoprotein in its homodimeric (secreted) form. M-CSF also exists as a membrane bound protein. The M-CSF receptor (M-CSF-R) is coded by the c-fms protooncogene. M-CSF-R has a ligand-inducible tyrosine kinase activity. Binding of M-CSF to M-CSF-R induces a dimerization, auto-phosphorylation, and activation of the kinase activity. M-CSF is also essential during pregnancy for the development and biology of the placenta.Macrophage – colony stimulation factor increases monocyte production of G-CSF, GM-CSF, IL-6, and TNFα after LPS stimulation . TNFα anMacrophage – colony stimulation factor plays a fundamental role in bone homeostasis; mice lacking M-CSF are osteoporotic. Using CLP model, Ogiku et al. found that the deficit of M-CSF is associated with a decreased survival, reduced phagocytosis, and increased HMGB1 levels .Several studies showed that M-CSF contributes to monocytes- and macrophages-mediated immune response and bacterial clearance in response to various pathogens –81.K. pneumoniae pneumonia, M-CSF increases locally in the lung, promotes monocytes and macrophages survival in the lung and the liver, and enhances bacterial killing (In a model of  killing .Macrophage – colony stimulation factor selectively expands CD16 + monocytes in human and primates .Macrophage – colony stimulation factor added to culture of murine macrophages enhances the macrophages response to TLR4 agonists while lowering the response to TLR9 agonists (CpG) .Macrophage – colony stimulation factor-elevated levels during sepsis are associated with the occurrence of hemophagocytosis and thrombopenia .Macrophage – colony stimulation factor can also control DC production . RecentlTo date, there was no clinical trial testing the impact of M-CSF treatment during sepsis.Interleukin-3, also named multicolony-stimulating factor (MSF), contributes to leukocyte production, proliferation, and survival . IL-3 stIn steady state, the main sources of IL-3 are activated T-helper cells. IL-3 has important effects on macrophages-DC and mastocytes in synergy with IFNγ .We recently published that published that IL-3 has a crucial role in the pathogenesis of the early phase of sepsis . We showhigh) monocytes and neutrophils in blood and increases inflammation. Surprisingly, the sources of IL-3 during sepsis are the IRA B cell making these cells a producer of two crucial HGFs during sepsis (GM-CSF and IL-3)  .Importantly, we showed that during human sepsis, high levels of plasma IL-3 at admission were correlated with a better survival at 28 days after sepsis in two independent cohorts of patients. IL-3 levels are associated with responsiveness to corticosteroid therapy during septic shock .The study of the role of IL-3 during sepsis is at its very beginning. The roles of IL-3 during the reparative phase of sepsis in mice and human are to be elucidated. As a potent DC function regulating cytokine , IL-3 coInterleukin-7is a 25-kDa glycoprotein (152 amino acids in humans) mainly produced by stroma epithelial cells of the thymus and the bone marrow. IL-7 gene is located on chromosome 8.Interleukin-7 receptor (IL-7R) is composed of two subunits: IL-7Rα (CD127) and the common gamma chain (γc) (CD132). IL-7R is expressed on the lymphoid lineage. IL-7 effects are mediated through JAK3 activation and STAT1,2,3,5, and PI3K pathways.The main effects of IL-7 aim to maintain lymphocytes survival. It has been recognized as a potential treatment of an HIV-related lymphopenia in a phase-II trial .Sepsis is associated with a lymphopenia. All types of lymphocytes, except for the regulatory T cells, see their numbers reduced in blood and tissues. The remaining lymphocytes, essentially the T cells, present signs of immunosuppression . Markers of apoptosis are elevated, while the ability to proliferate is reduced together with reduced cytokines productions.Unsinger et al.  used CLPRecently, Terashima et al. identified osteoblasts as a major source of IL-7 during sepsis . DepletiA human clinical study shows that IL-7 gene expression is reduced during sepsis but remains surprisingly normal during bacteremia; however, IL-7 level is unchanged . AnotherErythropoietin is a 30-KDa glycoprotein mainly secreted by the peritubular interstitial fibroblasts in the kidneys. EPO gene is located on chromosome 7. EPO binding to its receptor  activates JAK2 signaling and increase erythropoiesis. EPO is well known for its impact on acute and chronic anemia, especially during chronic kidney disease, hematologic disease, or after chemotherapy. EPO was originally seen as potential treatment of sepsis-associated anemia. EPO levels are usually low in critically ill patients  but wereIt appears that EPO effects on the vascular tone and its anti-apoptotic properties could also be beneficial.Several studies have shown anti-apoptotic effects of EPO during inflammation.In CLP model, EPO reduces renal and pulmonary damages in mice . EPO is During endotoxemia, EPO reduces AKI through decreased apoptosis  and actiErythropoietin impedes lymphoid cell apoptosis after CLP without major effect on mortality in rats .The effects of EPO treatment on critical illness-associated anemia and especially sepsis-associated anemia are debated. Two major clinical studies show conflicting results , 119 andOne clinical trial is ongoing to test the effect of EPO on microcirculatory alterations NCT1087450) during sepsis (Table 087450 duFigures The understanding of sepsis is still limited. Tremendous efforts have been made in order to decipher its complex pathophysiology. HGFs play a crucial in both early and late phases of sepsis but there is to date no positive clinical trial regarding their use. The various pathways involved and the wide range of clinical presentation may explain partly the negatives results of clinical trials. As emphasized earlier, included patients in clinical trials are highly heterogeneous. Therefore, a potential benefit of some HGFs could have not been seen because of its futility in some clusters of patients. In addition, HGF effects are compartmentalized in space and time. Therefore, the timing of administration and the route of administration are crucial and require more developments. Most of the efforts regarding the use of HGFs during sepsis are now concentrated on the immunosuppressed patients. One important underlying question is that of the appropriateness of reintroducing inflammation in these patients. The boosting of the immune system could be exaggerated and at the end detrimental. The undergoing trial of GM-CSF and IL-7 during sepsis will help to have a better idea of their utility in this indication. EPO and G-CSF treatments seem to be deleterious during sepsis.Anyway, there is a crucial need to be able to identify the “endotype” of sepsis patients. The biological effects of HGFs are incompletely understood and require further investigations. Next generation of sepsis trials will use this advanced knowledge and will be biomarker guided trials as recommended by experts . FundameBoth authors contributed to the literature search and the writing of the review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It has been reported that donor age affects patient outcomes after liver transplantation, and that telomere length is associated with age. However, to our knowledge, the impact of donor age and donor liver telomere length in liver transplantation has not been well investigated. This study aimed to clarify the influence of the length of telomere and G-tail from donor livers on the outcomes of living donors and recipients after living donor liver transplantation. The length of telomere and G-tail derived from blood samples and liver tissues of 55 living donors, measured using the hybridization protection assay. The length of telomeres from blood samples was inversely correlated with ages, whereas G-tail length from blood samples and telomere and G-tail lengths from liver tissues were not correlated with ages. Age, telomere, and G-tail length from blood did not affect postoperative liver failure and early liver regeneration of donors. On the other hand, the longer the liver telomere, the poorer the liver regeneration tended to be, especially with significant difference in donor who underwent right hemihepatectomy. We found that the survival rate of recipients who received liver graft with longer telomeres was inferior to that of those who received liver graft with shorter ones. An elderly donor, longer liver telomere, and higher Model for End-Stage Liver Disease score were identified as independent risk factors for recipient survival after transplantation. In conclusion, telomere shortening in healthy liver does not correlate with age, whereas longer liver telomeres negatively influence donor liver regeneration and recipient survival after living donor liver transplantation. These results can direct future studies and investigations on telomere shortening in the clinical and experimental transplant setting. Liver transplantation (LT) is a standard treatment for end-stage liver disease and liver malignancies. In a globally aging society, a declining pool for living donor liver transplantation (LDLT) and cadaver LT has become a critical issue. The possibility and safety of donations from marginal donors should be considered, particularly those of elderly and obese donors. It remains controversial whether donor age impairs recipient outcomes after LDLT . HoweverFor a long time, the liver was recognized as an organ that could regenerate; yet, the mechanism of liver regeneration is remains unclear. Eukaryotic organisms senesce as they get older, and organ function and regeneration ability decline. It has been reported that liver regeneration in elderly people and rats after hepatectomy slows down . The res5'-TTAGGG-3' at the ends of chromosomes, appear to be deeply involved in tissue regeneration, lifespan, and cell division [Telomeres, double-stranded DNA containing repeat sequences of division . It has division . Accordidivision . HoweverWe investigated the influence of telomere and G-tail length from donor blood and liver tissues on donor liver regeneration and recipient outcome after LDLT.Overall, 223 patients underwent LDLTs at Hiroshima University Hospital between 1991 and 2015. Blood and liver samples from 55 donors were collected at LDLT between 2010 and 2015. Written informed consent was obtained from all participants before surgery, in accordance with the Declaration of Helsinki. This study was approved by the Hiroshima University Institutional Review Board (HiM129-28). The procedure to protect the identity of the patients was subject to approval by the institutional review committee and met the guidelines of the responsible governmental authority.LDLTs were performed at Hiroshima University Hospital, following the Japanese Liver Transplant Society guidelines. Donors were healthy adults who voluntarily applied to donate their liver. The size of the graft had to be more than 0.8% of the recipient's body weight . Our donPerioperative volumetric measurements of hepatic morphology were performed as previously described , 9. ReseDonor outcomes included morbidity, post-hepatectomy-liver-failure (PHLF), and ERI. Recipient outcomes included morbidity and survival. The definition of PHLF was based on the International-Study-Group-of-Liver-Surgery (ISGLS), included the following: decrease in liver synthesis excretion, detoxification function, or symptoms of elevation of high bilirubin value; and international normalized ratio of prothrombin (PT-INR) value on day 5 (PT-INR-5), or later compared with the previous one if there was a high preoperative value, or a normal value that required “artificial supplements from the outside to maintain” , 12. TheQuantification of relative telomere length (RTL) and relative G-tail length (RGL), double-stranded and single-stranded, was performed using the telomere hybridization protection assay (HPA) method, as described previously , 14. TheAll statistical analyses were conducted using JMP 13 . The univariate analysis for continuous variables with normal distribution was compared using the Student's t-test. Continuous variables without a normal distribution were compared with Mann-Whitney's U test. A descriptive comparison was performed with the chi-squared test. Correlations between the presence of PHLF and continuous variables were expressed using Pearson's correlation coefficient. The area under the curve was calculated using the receiver operating characteristic (ROC) curve for the sensitivity and specificity of the value of the limit of telomere length. The difference between the two sides was considered statistically significant when the p-value was 0.05 or less. All relevant data are shown within Supporting Information files .Clinical characteristics of donors and recipients are shown in Next, we investigated the correlation between B-RTL and L-RTL, as well as that between B-RGL and L-RGL. L-RTL was not associated with B-RTL , and B-RWe investigated the correlation between telomere and G-tail length from blood and liver tissues and donor age. As shown in We reviewed preoperative characteristics of LDLT donors. Among 55 donors, 5 donors had PHLF, according to the ISGLS criteria. We were unable to identify any significant preoperative factors which affected PHLF . NietherWe assessed the factors which may affect postoperative liver regeneration. To investigate the differences between the regeneration rates after left and right hemihepatectomy, we examined ERI separately for left and right hemihepatectomy. Donor age was not correlated with ERI in any donor, regardless of the procedure they underwent . ERI wasWe reviewed the postoperative outcomes of recipients after LDLT . While tThe impact of donor age on LT has been analyzed in several studies , 15. SevLiver regeneration rate after hepatectomy has been shown to be inversely correlated with age , 23, 24.5'-TTAGGG-3' at the ends of chromosomes. Those repeat sequences (TTAGGG) are single strands of approximately 50 to 300 bases at the furthest 3' ends (G-overhangs), called G-tails. These G-tails are normally protected by forming a loop, except when telomerase, a telomere extension enzyme, interacts with the telomere, for instance, during DNA replication [Telomeres appear to be deeply involved in tissue regeneration, lifespan, and cell division . They arlication . Telomerlication .In the present study, B-RTL was inversely correlated with age, consistent with previous studies. However, B-RGL, L-RTL, and L-RGL were not correlated with donor age, and L-RTL was not correlated with B-RTL. Telomere shortening in liver tissues during aging has been reported in some studies –32: for This is the first study investigating the length of liver telomere/G-tail and liver regeneration after hepatectomy in LDLT. Several reports have investigated telomere length and telomerase activity in patients with liver disease. Aikata et al. reported that telomere repeats were shorter in the liver of patients with chronic diseases than in normal age-matched livers , and HarCellular senescence is considered to be a stress-response limiting the proliferation of damaged cells and leading to permanent cell-cycle arrest . The telTo our knowledge, this is the first study investigating the association between telomere length of donor tissues and recipient outcome in LDLT. In organ transplantation, it remains unclear whether donor tissue telomere length is associated with recipient survival. A previous study showed that longer donor telomere length is associated with improved recipient survival among hematopoietic cell transplant recipients with aplastic anemia . In contThis study has several limitations. First, although this is the first study to investigate specifically on telomere and G-tail length from healthy donor liver tissue, we had a relatively small cohort with a limited follow-up period, and the age of donors included was limited to less than 65 years only. Second, a cell cluster of whole liver tissue used in our study included not only hepatocytes, but also other cells including cholangiocytes, and sinusoidal cells. Third, we did not assess telomerase activity or the telomere/G-tail in transplant recipients. Fourth, we assessed telomere length by HPA assay, as a flow-based assay. Telomere length is measured as fluorescence intensity relative to internal control, not as an absolute length .Telomere shortening in healthy liver tissue was not correlated with age, whereas longer liver telomeres negatively impact donor liver regeneration and recipient survival after LDLT. These results can direct future studies and investigations on telomere shortening in the clinical and experimental transplant setting.S1 Table(XLSX)Click here for additional data file.


Elevated plasma concentration of retinol binding protein 4 (RBP4) has recently emerged as a potential risk factor as a component of developing metabolic syndrome (MS). Therefore, this study aimed to analyse the relationship between components of MS and concentrations of plasma RBP4 in a population of subjects 65 years and older.The study sample consisted of 3038  participants of the PolSenior study, aged 65 years and older. Serum lipid profile, concentrations of RBP4, glucose, insulin, C-reactive protein, IL-6, and activity of aminotransferases were measured. Nutritional status (BMI/waist circumference) and treatment with statins and fibrates were evaluated. Glomerular filtration rate (eGFR), de Ritis ratio, and fatty liver index (FLI), as well as HOMA-IR were calculated.p < 0.001).Our study revealed a strong relationship between components of MS and RBP4 in both sexes: plasma RBP4 levels were increased in men by at least 3×, and in women by at least 4×. Hypertriglyceridemia was most strongly associated with elevated plasma RBP4 levels. Multivariate, sex-adjusted regression analysis demonstrated that chronic kidney disease [OR 1.86 (95% CI 1.78–1.94)], hypertriglyceridemia [OR 1.52 (1.24–1.87)], hypertension [OR 1.15 (1.12–1.19)], low serum HDL cholesterol [OR 0.94 (0.92–0.97)], and age > 80 years [OR 0.86 (0.81–0.90)] were each independently associated with RBP4 concentration (all In Caucasians 65 years and older, RBP4 serum levels are associated with a number of components of MS, independent of sex and kidney function. Hypertriglyceridemia as a component of MS is most significantly related to RBP4 concentration. Metabolic syndrome (MS) is a set of abnormalities which include visceral obesity, carbohydrate metabolism disturbances or type 2 diabetes, hypertriglyceridemia, decreased HDL cholesterol levels, and elevated blood pressure, –5. NumerInsulin resistance related to visceral obesity is the major pathway in the pathogenesis of MS –5. IncreIt has been shown that retinol binding protein 4 (RBP4) is one of the adipokines that participates in the development of insulin resistance by impairing insulin signalling at both the receptor and post-receptor levels, as well as by stimulation of liver gluconeogenesis . The priPlasma RBP4 level correlates with abdominal fat volume , waist cThe association between plasma RBP4 levels and BMI, HOMA-IR, or other components of MS in obese patients is questioned by some researchers , while oSo far, no studies have assessed the relationship between RBP4 levels and components of MS in older adults. Hence, the aim of this study was to evaluate if there is a relationship between circulating RBP4 levels and components of MS in a large (65+), population-based Polish cohort.The PolSenior study, performed in the years 2007–2012, recruited six, similarly sized, age cohorts . During three visits performed by specially trained nurses, a questionnaire survey, comprehensive geriatric assessment, and measurements of body mass, height, waist circumference, and blood pressure were performed . The curHeight and body mass were measured with the subjects wearing light clothes and no shoes  with an accuracy of 0.5 cm and 0.1 kg, respectively. The body mass index (BMI) was calculated according to the standard formula.Waist circumference was measured midway between the last rib and the iliac crest in a standing position with the anterior axillary line guiding the tape-measure near the umbilicus with an accuracy of 0.5 cm.Blood pressure (BP) measurement was performed on the right upper arm, three times, in the sitting position after a 5-min rest using a fully automatic oscillometric BP measuring device accurate to 1 mm Hg , with the cuff selected according to the arm circumference. Thirty min before BP readings, the subjects did not smoke cigarettes, drink coffee, or engage in physical exercise . Mean vaPlasma RBP4 concentrations were measured by ELISA  with a 0.9-pg/mL limit of sensitivity and with mean intra- and inter-assay coefficients of < 5.0 and < 9.8%, respectively. The standard range in adults reported by the manufacturer is 20–75 µg/mL.Serum insulin concentration was assessed by electrochemiluminescence immunoassay (ECLIA) using commercially available kits on a Cobas E411 analyser  with an inter-assay coefficient of variability of < 3.8%. Plasma interleukin-6 (IL-6) was measured by ELISA  with a sensitivity of 0.04 pg/mL and mean intra-assay and inter-assay coefficient of variance < 7.8 and < 7.2%, respectively.Serum total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides, glucose, albumin, creatinine, and C-reactive protein concentrations, and activity of alanine (ALT), aspartate (AST) transaminases, and gamma-glutamyl transpeptidase (GGT) were previously assessed by an automated system  in a single certified laboratory with inter-assay coefficients of variability below 1.7, 1.2, 1.3, 1.8, 1.7, 1.7, 2.3, 5.7, 4.4, 3.2, and 1.4%, respectively.Components of MS were diagnosed according to the IDF modified criteria . Any thrInsulin resistance was assessed by HOMA-IR calculated with a standard formula. The de Ritis ratio (AST/ALT) was calculated and values greater than 1 were considered to be a surrogate marker of liver damage. Fatty Liver Index (FLI) was calculated according to the equation derived from the population of the Dionysos Nutrition & Liver Study ; a scorep value below 0.05. All tests were two-tailed. Nominal and ordinal data were expressed as percentages, while interval data were expressed as the mean value ± standard deviation in the case of a normal distribution or as median (lower quartile–upper quartile) in the case of data with skewed or non-normal distribution. Distribution of variables was evaluated by the Shapiro–Wilk test and quantile–quantile (QQ) plots. The Levene test assessed homogeneity of variances. To evaluate the relationship between variables and RBP4 serum levels in three groups according to tertiles, the one-way ANOVA with Dunnett’s post hoc test and two-way ANOVA (metabolic syndrome components and sex) with contrast analysis were used. The stepwise backwards multivariate ordinal logistic regression was used to assess factors influencing RPB4 serum levels. Comparison of data in ordinal and nominal scale was made either with the χ2 test or χ2 trend test.Statistical analysis was performed using STATISTICA 10.0 PL  and StataSE 12.0 . Statistical significance was set at a p = 0.96). Therefore, lower and upper tertiles were calculated jointly for men and women after logarithmic conversion because of heavy-skewed RBP4 distribution . Regardless of similar RBP4 values, but due to the differentiated distribution of components of MS between men and women, univariate analyses were done separately for men and women.Similar plasma RBP4 levels were found in men and women  and men (p < 0.001). Hypertriglyceridemia (p < 0.001 for both sexes), occurrence of type 2 diabetes or fasting plasma glucose over 100 mg/dL  were substantially related to RBP4 levels . Simple main effects analysis showed a significant association of the number of components of MS (p < 0.001) with the level of RBP4, but there were no differences between men and women (p = 0.39) , RBP4 serum levels were higher in groups with at least three components for men and with at least four components for women Fig. .Our large, population-based, cohort study shows that increased RBP4 serum levels are strongly associated with the presence and the number of components of MS in an 65 + Caucasian population. These findings correspond with previous studies performed in multi-ethnic and over 65-year-old populations –39. The In older adults the association between RBP4 levels and components of MS, especially carbohydrate disturbances, is weaker and affected by impaired renal function. The strong associations between RBP4, insulin resistance, and the percent of trunk fat in young subjects were shown to disappear in older adults . HoweverA significant effect on RBP4 metabolism is impaired kidney function, which is frequently found in older adults. This supposition is supported by previously published data showing normalisation of RBP4 levels after kidney transplant –44 and bThe most significant findings of our study are the associations between the levels of RBP4 and hypertriglyceridemia, as well as low HDL cholesterol levels . This suggests that RBP4 might play an important role in the disturbance of lipid synthesis in the liver, related to insulin resistance, and perhaps is a surrogate marker of liver steatosis. It was shown that RBP4 expression was aberrantly elevated in non-alcoholic fatty liver disease (NAFLD) in middle-age human and animal models, as well as positively associated with hepatic mitochondrial dysfunction combined with increased hepatic triglyceride accumulation , 46. In A strong association between circulating levels of RBP4 and triglycerides seems to reflect the increased lipogenesis in the study subjects. This supposition is strengthened by Rocha et al., who demonstrated that on middle-aged, morbidly obese patients, RBP4 independently correlates with triglycerides, HDL, LDL, VLDL (very-low-density lipoprotein), and small-HDL subfractions of cholesterol; all particles enriched in triglycerides . There aThis study has some limitations, such as the lack of validation of the utilised ELISA kit by a quantitative western blot analysis , liver sIn older adult Caucasians, RBP4 serum levels are associated with a number of components of MS, independent of sex and kidney function. Hypertriglyceridemia as a component of MS is most significantly related to RBP4 concentration.


Non-inflamed CSF samples from patients with Bell’s palsy and normal pressure hydrocephalus (n = 19) were used as controls. Analysis of 91 metabolites revealed considerable metabolic reprogramming in the meningitis samples. It identified phosphatidylcholine PC.ae.C36.3, asparagine, and glycine as an accurate  combined classifier for enterovirus meningitis overall, and kynurenine as a perfect biomarker for enteroviral meningitis with an increased CSF cell count . Remarkably, PC.ae.C36.3 alone emerged as a single accurate  biomarker for enteroviral meningitis with normal cell count, and a combined classifier comprising PC.ae.C36.3, PC.ae.C36.5, and PC.ae.C38.5 achieved nearly perfect classification . Taken together, this analysis reveals the potential of CSF metabolites as additional diagnostic tools for enteroviral meningitis, and likely other central nervous system (CNS) infections.Enteroviruses are among the most common causes of viral meningitis. Enteroviral meningitis continues to represent diagnostic challenges, as cerebrospinal fluid (CSF) cell numbers  may be normal in up to 15% of patients. We aimed to identify potential CSF biomarkers for enteroviral meningitis, particularly for cases with normal CSF cell count. Using targeted liquid chromatography-mass spectrometry, we determined metabolite profiles from patients with enteroviral meningitis ( Typical symptoms of meningitis are headache, fever, or neck stiffness. Altered mental status or neurological deficits appear additionally when meningitis evolves into meningoencephalitis, in which brain tissue is also affected [A variety of processes can lead to meningeal inflammation. Viruses are the most common cause of infectious meningitis, followed by bacteria. Fungal or parasite infections are found rarely. There is also a variety of causes of non-infectious meningeal inflammation, such as carcinomatosis, adverse drug effects, or autoimmune diseases. Pathogens can reach the meningeal structures via the blood stream and infiltrate the membranes that surround the brain and spinal cord. Another way of infection is by infiltration affected ,2.Of all neurotropic viruses that can cause inflammation of the meninges, enterovirus infections are the most common, comprising 40–77% of all cases. Infections with herpes simplex virus (HSV) or varicella zoster virus (VZV) are less common . EnterovSociodemographic and routine laboratory parameters are summarized in Of the 188 potentially detectable metabolites, 89 passed quality assessment because of detection >LOD in ≥75% samples . Overalln = 18) and decreased (n = 5) concentrations were detected  curve analysis to assess the degree of concentration changes in the 91 metabolites and to screen the resulting biomarker potential . A markedetected A. As expdetected C, but evdetected B. When cdetected D. This mp values of the ROC curves, we then determined the optimal single biomarker for each comparison. Their concentration ranges are shown in Cross-validation by the leave-one-out method was then used to identify the most robust biomarkers. For each comparison, the 10 most frequently selected markers and their parameters from ROC analysis are summarized in Combining biomarkers into complex classifiers may improve diagnostic accuracy, particularly when intraclass correlations are negative . Random Enteroviral meningitis continues to present diagnostic challenges, as up to 15% of patients with this infection may present with normal CSF cell counts and turn-around of PCR diagnostics may be insufficient (see Introduction). We therefore performed a targeted metabolomics screen of CSF from patients with enteroviral meningitis and identified accurate biomarkers, not only for enteroviral meningitis with the expected elevated CSF leukocyte count, but, notably, also for a subgroup of patients with meningitis and CSF cell counts in the normal range.Our results showed an elevation of several metabolites in both groups of enteroviral meningitis patients, those with and without pleocytosis. The lack of correlation with an increased cell count in CSF suggests that the elevation of those metabolites is based not only on the inflammatory reaction and modulation of the immune system, but also on specific pathological processes within the meninges. Compared to the standard diagnostic CSF parameters such as leukocyte count, protein, or lactate concentrations, the most robust metabolite biomarkers were more sensitive and specific in diagnosing enteroviral meningitis. Furthermore, selecting complex classifiers comprising three metabolites improved classification accuracy further. These results solidify our previous findings that “metabolite signatures” constitute highly accurate diagnostic biomarkers for CNS infections that may be superior to routine CSF diagnostics . Phosphatidylcholines constituted two of the metabolites that were best validated as differentially upregulated in enterovirus meningitis. Of these, PC.ae.C36.2 was most closely associated with enteroviral meningitis independent of pleocytosis, whereas the nearly identical metabolite PC.ae.C36.3 was a highly accurate biomarker for the clinically challenging subgroup of meningitis without pleocytosis. N-methyltransferase choline requiring protein 2 (Cho2p), an enzyme which catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholines. This interaction induces activity of this enzyme and leads to phosphatidylcholine increase in the infected cell [Phosphatidylcholines are important constituents of the cell membranes in eukaryotes. They make up about 50% of all phospholipids. Increased phosphatidylcholine levels have been described in vitro in the context of brome mosaic virus replication as well as after infections with dengue virus, polio virus, or hepatitis C virus . Brome mted cell ,16. Thisted cell . A specited cell . It is tMeasurements of PC.ae.C36.2 concentrations in septic shock, breast cancer, or Huntington’s disease in peripheral blood samples did not reveal any significant concentration changes ,19,20. THaving shown that PC.ae.36.3 is a highly accurate biomarker for enteroviral meningitis with normal CSF cell count, the question arises whether it is feasible to translate this finding into clinical applications, for instance as a rapid diagnostic tool. Fluorometric assays can be used to quantify total phosphatidylcholine levels and are offered in routine laboratories. However, analyzing a single sample is expensive and time-consuming, so samples are often collected over a period of time and then analyzed in batch. However, this is not feasible for an acute situation like meningitis. The development of simple point-of-care kits, such as a lateral flow assay for CSF ,22, woulAs opposed to the subgroup with normal cell count, regulation of amino acid and biogenic amines metabolism was apparent in the pleocytosis subgroup, and an amino acid “signature” persisted in the combined group also containing the non-pleocytosis sample, suggesting that alterations in the associated pathways  also occur in the absence of CSF pleocytosis. Asparagine plays an important role in cell proliferation. Inside cells it controls uptake of certain amino acids whose levels are critical for activity of the mTOR complex, a promotor of proliferation . Glycine is an inhibitory neurotransmitter that regulates several neurological functions ,25. FurtOur study is limited by the small number of patients, especially in the enterovirus subgroups. In order to gain robust data even with low numbers of participants, we performed an internal validation. Thus, it was still possible to identify several potential biomarkers. These require validation in a larger group of patients, including calculation of sensitivity and specificity, which was not possible with our data. Furthermore, the control group was not formed by healthy patients, and CSF findings in this group may therefore not entirely reflect findings expected in healthy individuals from the general population. However, lumbar puncture in healthy individuals is ethically disputable. We thus used CSF obtained during routine diagnostic evaluations from patients where, in the final diagnosis, inflammation of the nervous system was excluded. Finally, our mass-spectrometric assay allowed detection of only a portion of all metabolites that are found in the CSF, and it is therefore possible that we did not capture the full spectrum of metabolite biomarkers.Taken together, our results suggest: (1) that metabolite profiles in CSF of patients with enteroviral meningitis differ considerably from patients without infectious CNS disease, and (2) that this high degree of metabolite “reprogramming” reveals accurate CSF biomarkers that can aid in the diagnosis of enteroviral meningitis, even in the clinically challenging subgroup of patients with a clinical suspicion of meningitis but normal CSF cell count. n = 10, abbreviated as EntM ) were clinical suspicion of meningitis and detection of enteroviruses in CSF by polymerase chain reaction (PCR). This group could be subdivided into 50% (n = 5) with normal CSF cell count ) and 50% (n = 5) with elevated cell count ). The non-inflamed control group (n = 19) consisted of patients with normal CSF cell count (0–4 cells/μL) and a clinical diagnosis of (1) idiopathic facial paresis without evidence of an infectious etiology  and (2) normal pressure hydrocephalus , defined as normal CSF pressure, cranial computed tomography, or magnetic resonance tomography indicative of NPH in combination with at least one symptom of the Hakim triad [CSF samples were obtained during routine lumbar puncture and collected prospectively as part of establishment of a CSF biobank encompassing a variety of neurological disorders. This study was approved by the Ethics Committee of Hannover Medical School  and was conducted according to the Helsinki Declaration. Inclusion criteria for enteroviral meningitis  and MetIDQ™ software (Biocrates) for peak integration and calculation of metabolite concentrations. A two-step quality screen was applied to select the subgroup of detectable analytes to be included in the present analysis. First, all analytes that were detected >limit of detection (LOD) in ≥75% of all samples were selected. Values <LOD were then replaced by k-nearest-neighbor imputation, as described in the R package “Biocomb” [CSF metabolite concentrations were measured with the AbsoluteIDQ™-p180 kit , using a combination of liquid chromatography tandem mass spectrometry (LC-MS/MS) and direct flow injection analysis MS/MS, as described by Kuhn et al., 2018 . With thBiocomb” . Second,p <0.05 unless stated otherwise. Discriminatory ability of the metabolite biomarkers was assessed by receiver operating characteristic (ROC) curve analysis, defining significant areas under the curve (AUC) by asymptotic p values of <0.05 and lower bound confidence intervals (CI) of >0.5. The group with the higher CSF cell count constituted the enumerator for calculating ratios of mean concentrations (“fold change”), and the positive state in ROC analysis. Due to the small sample size and lack of an external validation cohort, an internal cross validation was performed via the leave-one-out (jackknife) procedure. In this method, the cohort is divided into a training subset  and a validation subset (n = 1). The best classifier on the training subset is computed and tested on the validation subset in an iterative process, where each sample is used as the validation subset. The classifiers are then ranked in order of selection frequency. To select optimal biomarker combinations, we constructed a random forest classifier based on the biomarker candidates with the highest AUC values and tested the classifier on the single sample that was left out during the corresponding iteration of the jackknife method. The most frequently selected classifiers were then re-evaluated by the jackknife method once again. To reduce selection bias, we repeated the procedure with 1000 bootstrap samples in order to compute a confidence interval for the AUC of the final classifier.Significance of between- and across-group comparisons was determined with the Mann-Whitney U and Kruskall-Wallis tests, respectively, defining significance as Our results demonstrate: (1) that despite its relatively mild clinical manifestations, there is a considerable degree of CSF metabolite reprogramming in enteroviral meningitis, and (2) that CSF metabolite analysis can be a helpful tool in establishing a differential diagnosis in clinically suspected enterovirus meningitis. Further validation of PC.a.e.36.3. as a potential biomarker and establishing easy and quick detection assays might enable its use as a point-of-care test (for instance in the emergency room or at the bedside) to trigger further investigations, including PCR for enteroviruses.


V˙O2), cytosolic metabolite  concentrations, and pH during whole‐body constant‐power exercise (CPE) (6 min), step‐incremental exercise (SIE) (30 W/3 min), and slow (10 W/min), medium (30 W/min), and fast (50 W/min) ramp‐incremental exercise (RIE). Different ESA (each‐step activation) of oxidative phosphorylation (OXPHOS) intensity‐ATP usage activity relationships, representing different muscle fibers recruitment patterns, gave best agreement with experimental data for CPE, and for SIE and RIE. It was assumed that the muscle V˙O2‐power output (PO) nonlinearity is related to a time‐ and PO‐dependent increase in the additional ATP usage underlying the slow component of the V˙O2 on‐kinetics minus the increase in ATP supply by anaerobic glycolysis leading to a decrease in V˙O2. The muscle V˙O2‐PO relationship deviated upward (+) or downward (−) from linearity above critical power (CP), and the nonlinearity equaled +16% (CPE),+12% (SIE), +8% (slow RIE), +1% (moderate RIE), and −2% (fast RIE) at the end of exercise, in agreement with experimental data. During SIE and RIE, changes in PCr and Pi accelerated moderately above CP, while changes in ADP and pH accelerated significantly with time and PO. It is postulated that the intensity of the additional ATP usage minus ATP supply by anaerobic glycolysis determines the size of the muscle V˙O2‐PO nonlinearity. It is proposed that the extent of the additional ATP usage is proportional to the time integral of PO ‐ CP above CP.A computer model of the skeletal muscle bioenergetic system was used to simulate time courses of muscle oxygen consumption ( Step‐incremental exercise (SIE) and ramp‐incremental exercise (RIE)  can increase with different speed with time. For instance, PO can be elevated step‐wise by 30 W every 3 min  or increase continuously with time at speed of 10 W per min , 30 W per min , or 50 W per min .V˙O2 kinetics or blood lactate and blood pH, were measured or estimated in these kinds of exercise. On the other hand, such variables concerning directly the skeletal muscle bioenergetic system kinetic properties during exercise as muscle V˙O2, PCr, and cytosolic ADP, Pi, ADP, and pH that can be measured for constant‐power exercise (CPE) in contracting calf  (see below). This nonlinearity in SIE was related to the slow component of the V˙O2 on‐kinetics . In ramp‐incremental exercise (RIE) the nonlinearity of the pulmonary V˙O2‐PO relationship tends to be highest in slow RIE, medium in medium RIE, and lowest  in fast RIE  component of the V˙O2 on‐kinetics present in intensive exercise. The slow component appears just after exceeding the lactate threshold (LT), that is PO above which the blood lactate is elevated  and glycolysis are directly activated by some cytosolic mechanism during the rest‐to‐work or low‐to‐high work transition in skeletal and heart muscle cells. While ESA is essentially the only mechanism acting in intact heart in situ, where metabolite concentrations are approximately constant during work transitions, in skeletal muscle it cooperates with the negative feedback through the increase in ADP and Pi , during work transitions is each‐step activation (ESA) (Korzeniewski AOX) depends in a saturating manner on the ATP usage activity (AUT) during voluntary CPE in humans  , step‐incremental exercise (SIE) (30 W/3 min), as well as slow (10 W/min), moderate (30 W/min), and fast (50 W/min) ramp‐incremental exercise (RIE). The second aim is to decide whether the regulation of OXPHOS is the same in CPE, on the one hand, and in SIE and RIE, on the other hand. Third, this study is intended to predict the changes over time of muscle V˙O2, ADP, PCr, Pi, and pH in all considered exercise modes. These variables were not measured experimentally in SIE and RIE, mostly because of technical difficulties.The first aim of this theoretical study is to propose the mechanism(s) underlying the AOX‐AUT relationship is present in CPE, then the kinetic properties of the skeletal muscle bioenergetic system in SIE and RIE are better explained by the declining‐type AOX‐AUT relationship. It was assumed that the muscle V˙O2‐PO nonlinearity and its magnitude is related to the increase in V˙O2 due to the time‐ and PO‐dependent increase in the additional ATP usage underlying the slow component of the V˙O2 on‐kinetics minus the decrease in V˙O2 due to an increase in ATP supply by anaerobic glycolysis.It was assumed, in order to reproduce appropriately experimental data, that while the saturating‐type V˙O2‐PO nonlinearity will be greatest and positive (upward deviation) in CPE , smaller in SIE (30 W/3 min), even smaller in slow RIE (10 W/min), in medium RIE (30 W/min), and smallest  in fast RIE (50 W/min). It is hypothesized that the AOX‐AUT relationship is different in CPE, on the one hand, and in SIE and RIE, on the other hand, due to different muscle fibers recruitment patterns. It is expected that (end‐step) PCr will decrease and (end‐step) Pi will increase linearly with time and PO in SIE and in RIEs of different steepness below CP, while these changes will accelerate moderately above CP. It is expected that the increase in ADP and decrease in pH will significantly accelerate with PO and time at all PO values.It is hypothesized that for the assumptions made within the model the muscle the V˙O2‐PO nonlinearity in various exercise modes is proportional to the intensity of the additional ATP usage minus the intensity of ATP supply by anaerobic glycolysis. It is proposed that the extent of the additional ATP usage is proportional to the time integral of the PO ‐ CP difference above CP. It is argued that the regulation of OXPHOS is different in CPE, on the one hand, and in SIE and RIE, on the other hand.Generally, it is postulated that the size of the muscle AOX, ESA intensity (relative OXPHOS activity); AUT, relative ATP usage activity; CP, critical power; CPE, constant‐power exercise; ESA, each‐step activation; OXPHOS, oxidative phosphorylation; PO, power output; SIE, step‐incremental exercise; RIE, ramp‐incremental exercise; τp, characteristic transition time of the primary phase of the V˙O2 on‐kinetics; and t0.63, time to reach 63% of the V˙O2 amplitude, analogous to τp. Other abbreviations are used only in equations within this The following abbreviations are used throughout the text: This is a purely theoretical study that did not involve any experiments on humans or animals.β‐oxidation, MAS, etc.), particular OXPHOS complexes , proton leak through the inner mitochondrial membrane, glycolysis (aerobic and anaerobic), ATP usage, creatine kinase (CK), and proton efflux/influx to/from blood. The complete model description is located on the website: http://awe.mol.uj.edu.pl/~benio/.The computer model of OXPHOS and the entire bioenergetic system in intact skeletal muscle (Korzeniewski and Zoladz AUT (relative increase in its rate constant kUT in relation to rest) between 1 (rest) and 105 (maximum AUT) was used in computer simulations.The relative activity of ATP usage AOX on AUT is most probably different in various modes of muscle work and depends on the pattern of muscle fiber recruitment by motor units  is the relative OXPHOS (+ NADH supply) activity (activation in relation to rest), AUT is the relative ATP usage activity (activation in relation to rest), and the power coefficient pOX is a measure of ESA strength  dependence of AOX‐AUT relationship:AOX  is the relative OXPHOS (+ NADH supply) activity (direct activation in relation to rest), AUT is the relative ATP usage activity (activation in relation to rest), AOXmax = 5 is the maximum AOX−1 , and KAUT = 5 is the half‐saturating AUT value for the increase in AOX.In CPE during voluntary exercise  a mixture of different fiber types is recruited at different work intensities, with the fraction of glycolytic type IIx and IIb fibers with low ESA intensity increasing with work intensity (power output). Thus, only or mostly oxidative type I and IIa fibers with high ESA intensity are recruited at the onset of exercise at low work intensities, while a mixture of oxidative type I and IIa fibers as well as glycolytic type IIx and IIb fibers with low ESA intensity is recruited at the onset of intense CPE in cortically stimulated muscle. Nevertheless, even at highest work intensities a mixture of oxidative and glycolytic fibers is recruited at the onset of exercise. It was shown  AOXmax and AUT0 = 206 is the “reference” ATP usage activity. The values of these parameters were adjusted in order to represent appropriately experimental data, for instance, the dependence of τp on AUT (power output) in SIE and the size of the V˙O2‐PO nonlinearity in SIE and RIE, as well as reasonable values and their changes with time and PO of muscle V˙O2, ADP, PCr, Pi, and pH in SIE and RIE.Finally, in SIE and RIE most probably only or mostly oxidative, fatigue‐resistant type I (and IIa) muscle fibers with high ESA intensity are recruited in the initial period of exercise at low power outputs below CP  and 105 (maximum AUT) was used in computer simulations. One AUT unit is equivalent to ~3 W of power output (PO) for whole‐body exercise  ; AUT = 5 (unloaded exercise) corresponds to total PO = 12 W, unloaded PO = 12 W, and loaded PO = 0 W; AUT = 15 (first step in SIE) corresponds to total PO = 42 W, unloaded PO = 12 W, and loaded PO = 30 W; AUT = 105 (maximum work) corresponds to total PO = 312 W, unloaded PO = 12 W, and loaded PO = 300 W.It was postulated that the additional ATP usage  (Korzeniewski and Rossiter vvUTadd) of the increase in the absolute additional ATP usage flux (rate in mmol/L min−1) vUTadd is described in this study by the following equation:vvUTadd (mmol/L min−2) is the rate of the increase in the absolute additional ATP usage flux (in mmo/L min−1) (vUTadd), kUTadd = 0.0005 min−1 is the “rate constant” of the increase in the absolute additional ATP usage flux in time, AUT is the relative normal  ATP usage activity (activation in relation to rest) (unitless) and AUTcrit = 60 (unitless) is the critical relative ATP usage activity . This is the kinetic description of the activity of the additional ATP usage used in Korzeniewski (AUT (and therefore PO) with time in SIE and RIE. This equation implies that the additional ATP usage flux vUTadd increases both with time and with power output (ATP usage activity). In other words, the current additional ATP usage is proportional to the time integral of AUT ‐ AUTcrit, or, what is equivalent, to the time integral of power output over critical power (the PO ‐ CP difference) above CP. The parameter values, especially kUTadd, were chosen in order to give realistic values of the V˙O2‐PO nonlinearity.The additional ATP usage, postulated to underlie in CPE and SIE the slow component of the eniewski  for wholvUTtot (in mmol/L min−1) is equal to the sum of the normal and additional absolute ATP usage flux:The total absolute ATP usage flux AUT = 1) to maximal work with the relative ATP usage activity AUT = 105  was simulated in CPE. This is demonstrated in Figure kUT was increased AUT times in relation to rest. At the same time the activity of OXPHOS and NADH supply was elevated AOX times  in relation to rest. Glycolysis activity was increased AGL times (the rate constant of glycolysis kGL was increased AGL times) in relation to rest. AOX = 5.77 was calculated from Equation AGL equaled AUT0.55=12.93 (Korzeniewski The transition from rest (AUT‐fold increase in kUT) during on‐transient was applied in computer simulations for voluntary CPE. The additional ATP usage activity increased with time according to Equation V˙O2 related to both ESA and ADP and Pi increase). The time‐dependent activation after the onset of elevated work in CPE was described by the following equation (Korzeniewski and Rossiter X is OX (oxidative phosphorylation + NADH supply) or GL (glycolysis), mX is the current (at time t) relative activity of X  of its rate constant(s)), t(ON)X is the characteristic activation time of X, and t is the time after the onset of exercise (rest‐to‐work transition), t(ON)OX = 3 sec and t(ON)GL = 6 sec , a computer simulation without the additional ATP usage was carried out.An instantaneous increase in the activity of ATP usage  to 105 . The simulated exercise lasted 6 min.Also a series of computer simulations for CPE, assuming the saturating‐type AUT = 5 . AUT was increased instantaneously by 10 AUT units (30 W) after every 3 min. The activity of OXPHOS (and NADH supply) was increased in subsequent steps AOX times in relation to rest, according to Equations AOX‐AUT relationship). The relative activity of glycolysis AGL equaled AUT0.55. The time‐dependent activation of OXPHOS and glycolysis after the onset of elevated work in subsequent steps was described by the following equation:X is OX (oxidative phosphorylation + NADH supply) or GL (glycolysis), mX is the current (at time t) relative activity of X  of its rate constant(s)), AXn is the X activity in a given step n, AXn−1 is the X activity in the previous step n−1, t(ON)X is the characteristic activation time of X, and t is the time after the onset of elevated work in step n, t(ON)OX = 3 sec and t(ON)GL = 6 sec, as for CPE. The additional ATP usage activity increased above the critical ATP usage activity  according to Equation t0.63 for each step, a computer simulation without the additional ATP usage (and thus the slow component of the V˙O2 on‐kinetics) was made. However, this procedure gave only approximate estimations of t0.63 values for particular steps in the presence of the additional ATP usage shown in Figure Step‐incremental exercise Fig.  was starUT units 0 W afterAOX‐AUT relationships: power type  representing unloaded work. In three different protocols  AUT increased linearly with time by 3.33, 10, and 16.67 AUT units  per min, respectively  increased in time according to Equation Ramp‐incremental exercise was started from the baseline ly Figs. . The relIt must be stressed that the same set of parameter values was used in all computer simulations concerning constant‐power exercise, step‐incremental exercise, and ramp‐incremental exercise. Therefore, in real situation they could concern one particular human subject. For this reason, the results of simulations for different exercise modes can be directly compared.2 = 30 μmol/L was assumed in all simulations.The oxygen concentration OAUT = 105) used in this study was simulated in order to establish a reference frame for further simulations concerning step‐incremental exercise and ramp‐incremental exercise. The theoretical results are presented in Figure V˙O2 (due to ESA and rise in ADP and Pi), a clear slow component of the muscle V˙O2 on‐kinetics can be observed  and thus allowed to match the continuously increasing ATP usage . PCr and pH slowly decreased with time after the initial significant fall  , and finally started to be significantly inhibited by accumulating protons  would appear. For the assumed high work intensity  and the size of the additional ATP usage .The simulated additional sage Eq.  the consV˙O2 on‐kinetics t0.63 for transition from unloaded work (12 W), and thus zero loaded work (0 W), to 300 W of loaded work in CPE equaled ~ 24 sec for the parameter values used in this simulation.The simulated characteristic transition time of the principal phase of the muscle V˙O2 on‐kinetics appeared in the steps with PO above CP. This is demonstrated in Figure i increased linearly with time and PO below CP. These changes accelerated moderately above CP  the slow component of the muscle V˙O2 on‐kinetics and accelerated changes in PCr and Pi above CP. Generally, OXPHOS was the main ATP supplier in this type of exercise. CK supplied some ATP at the beginning of each step, while the contribution of anaerobic glycolysis to ATP production was generally rather small, although its absolute value increased with PO   extracted from Figure The slow component of the muscle t0.63 of the V˙O2 on‐kinetics on PO is strongly nonlinear in SIE for the declining‐type AOX‐AUT relationship. This is demonstrated in Figure t0.63 increases from 18 to 48 sec (2.7‐fold) between 30 and 300 W of loaded work (power output). However, these values are only approximate, as they were obtained in a computer simulation for SIE without the additional ATP usage, and thus the slow component of the V˙O2 on‐kinetics. On the other hand, when the saturating‐type AOX‐AUT relationship, postulated to be present in voluntary constant‐power exercise (Korzeniewski t0.63 was little dependent on power output (PO): a slight decrease in its value with PO from about 20 to 15 sec can be observed. Finally, when the power‐type AOX‐AUT relationship, postulated to be present in electrically stimulated muscle , with a certain contribution of ATP synthesis by anaerobic glycolysis, especially at higher POs, and negligible contribution of ATP production by CK  RIE the increase in muscle  CP Fig. B. On the CP Fig. A. The AT CP Fig. C. This w CK Fig. C.V˙O2, PCr, cytosolic Pi, ADP, and pH  and end‐exercise (300 W) loaded PO was used for both kinds of RIE, the exercise duration was threefold shorter for medium RIE (10 min), than for slow RIE (30 min), as the increase in PO in time was threefold faster in the former than in the latter. Within the model, it was assumed that the size of the additional ATP usage is not only PO‐dependent but also time‐dependent (compare Eq. V˙O2‐PO relationship would be greater in the absence of ATP supply by anaerobic glycolysis). This, in turn, led to a much smaller positive nonlinearity of the muscle V˙O2‐PO relationship in medium RIE (+ 1%) than in slow RIE (+ 8%)  RIE was qualitatively similar to slow RIE (10 W/min), although differed from it quantitatively. This is demonstrated in Figure  pH Fig. A and B wV˙O2, ATP supply by anaerobic glycolysis diminished V˙O2. As a result, the nonlinearity of the muscle V˙O2‐PO relationship in fast RIE was negative (deviated downwards) (−2%), as the decrease in muscle V˙O2 due to the increase in ATP supply by anaerobic glycolysis prevailed at high POs over the increase in muscle V˙O2 due to the additional ATP usage. For the same reason, also metabolites and pH changed less for the same PO values than in slow and medium RIE  RIE differed quantitatively  from slow RIE even more than medium RIE. This is demonstrated in Figure RIE Fig. .V˙O2 on loaded work (power output) was different for the three kinds of RIE, for SIE and CPE. This is demonstrated in Fig. V˙O2 for a given PO was highest in CPE (6 min), lower in SIE (30 W/3 min), slow RIE (10 W/min), medium RIE (30 W/min), and lowest in fast RIE (50 W/min). This was caused by the highest end‐exercise additional ATP usage in CPE and lowest end‐exercise additional ATP usage in fast RIE. On the other hand, the relative contribution of anaerobic glycolysis (and creatine kinase) to ATP supply that lowers V˙O2 and thus diminishes the V˙O2‐PO nonlinearity was comparable in all discussed exercise modes  for CPE, was used (not shown).The dependence of muscle r in SIE 0 W/3 mint in CPE  min, lowAOX ‐AUT (ATP usage activity) relationship in skeletal muscle in different exercise modes; 2. The mechanisms underlying the nonlinearity of the muscle V˙O2‐PO relationship and the differences in its magnitude in various exercise modes. The following exercise modes were considered: whole‐body constant‐power exercise (CPE) (6 min), step‐incremental exercise (SIE) (30 W/min), as well as slow (10 W/min), medium (30 W/min), and fast (50 W/min) ramp‐incremental exercise (RIE), all with the same maximum loaded PO = 300 W; and 3. Time courses of muscle V˙O2, metabolites , and cytosolic pH in CPE, SIE, and three modes of RIE.The present theoretical study dealt with three main problems: 1. The form of the V˙O2‐PO nonlinearity constitutes a manifestation of the additional ATP usage magnitude minus ATP supply by anaerobic glycolysis. The additional ATP usage increased linearly with time and PO. Its magnitude was a derivative of the time integral of the PO ‐ CP difference above CP. Computer simulations demonstrated that various exercise modes considered in this study can be ordered in the following descending sequence taking into account the size of the muscle V˙O2‐PO nonlinearity: CPE (+16%) > SIE (+12%) > slow RIE (+8%) > medium RIE (+1%) > fast RIE (−2%). This theoretical study shows that the time‐ and PO‐dependent increase in the additional ATP usage can, at least potentially, explain the muscle V˙O2‐PO nonlinearity and account for the differences in its magnitude between various exercise modes encountered in experimental studies (see below). The V˙O2‐PO nonlinearity was lessened, especially at higher POs, by an increase in ATP supply by anaerobic glycolysis.Within the computer model used it was assumed that the muscle AOX‐AUT relationship applied for SIE and RIE leads to a significant increase in t0.63 with PO in SIE. On the other hand, the saturating‐type AOX‐AUT relationship, showed previously to describe well the system properties in CPE during voluntary exercise, results in t0.63 that depends little on PO . Additionally, the declining‐type AOX‐AUT relationship leads to greater changes in metabolites and pH, greater contribution of anaerobic glycolysis to ATP supply, and lower (or more negative) V˙O2‐PO nonlinearity in relation to the saturating‐type AOX‐AUT relationship. These predictions agree well with experimental data (see below). Therefore, it is postulated that the phenomenological (averaged over the entire muscle) regulation of OXPHOS is different in CPE, on the one hand, and in SIE and RIE, on the other hand. This difference is postulated to result from different muscle fibers recruitment pattern.It was demonstrated that the declining‐type V˙O2, ADP, and Pi rose, whereas PCr and pH fell quickly and significantly after the onset of exercise , and then changed with a moderate pace. This prediction agrees well with numerous experimental data . Computer simulations show that, for the assumptions made, (end step) PCr decreases, while (end step) Pi increases linearly with time and PO below CP in SIE and RIE, and that these changes accelerate moderately above CP. On the other hand, the increase in (end step) ADP and decrease in (end step) pH accelerate significantly with time and PO at all PO values in SIE and RIE. These variables have not been measured in experimental studies in SIE and RIE yet.In CPE above CP, the simulated AOX‐AUT relationships: power‐type relationship in electrically stimulated muscle, saturating type during voluntary CPE, and declining type during SIE and RIE.The muscle fiber recruitment pattern is most probably different in CPE in electrically stimulated muscle, in CPE during voluntary exercise as well as in SIE and RIE during voluntary exercise, as described in V˙O2, following a large and quick initial rise, increased continuously with a slower pace after the onset of CPE of high intensity (loaded PO = 300 W). This is the so‐called slow component of the V˙O2 on‐kinetics  was assumed Eq. , say by V˙O2 on‐kinetics appears above PO value (corresponding to CP), at which the additional ATP usage appears. This is demonstrated in Figure V˙O2‐PO relationship. Figure V˙O2‐PO relationship extracted from Figure V˙O2‐PO relationship was extracted in Korzeniewski , especially at highest power outputs.In the computer model used, the discussed nonlinearity in the muscle V˙O2 on‐kinetics significantly slowed down with increasing PO in subsequent steps of SIE (30 W/3 min) for the declining type of the AOX‐AUT relationship. This is demonstrated in Figure t0.63 extracted from simulations concerning SIE without any additional ATP usage (and therefore slow component of the muscle V˙O2 on‐kinetics) for three types of the AOX‐AUT relationship. For the declining type of this relationship, postulated to be relevant for SIE (and RIE), the muscle t0.63 increased 2.7‐fold from 18 sec at 30 W to 48 sec at 300 W. A noticeably greater relative lengthening of the transition time (τp) of the primary phase of the pulmonary V˙O2 on‐kinetics from 21 to 98 sec (4.7‐fold increase) was encountered in Keir et al. (t0.63 (or τp)‐PO dependence was similar both in computer simulations and experimental studies. On the other hand, t0.63 (τp) slightly decreased with PO (from 20 to 15 sec) when the saturating‐type AOX‐AUT relationship, postulated to be present in voluntary CPE, was used in the simulation concerning SIE  was encountered both in computer simulations . t0.63 decreased significantly with PO, when the power‐type AOX‐AUT relationship, postulated to be present in electrically stimulated muscle, was applied  can be of course the fact that the model used is (over)simplified, at least in the discussed aspect. This is a one‐compartment model  that deals with variable (fluxes and metabolite concentrations) values averaged over entire muscle and all its fiber types. It has been postulated  , and oxygen consumption by auxiliary tissues can contribute to the significant slowing down of the pulmonary V˙O2 on‐kinetics between low and high power output in SIE.However, the difference between the relative increases in pulmonary and muscle Rossiter . Additioτp (t0.63) of the muscle and pulmonary V˙O2 on‐kinetics in subsequent steps in SIE.Taking into account all these reservations, a good, at least semiquantitative agreement can be observed between computer simulations Fig.  and expet0.63 equaled ~ 24 sec for CPE for 0 → 300 W loaded work transition, assuming the saturating type of the AOX‐AUT relationship. Therefore, t0.63 for transition to 300 W in CPE was decidedly lower, than t0.63 for transition to 300 W in step‐incremental exercise (SIE) for the declining type of the AOX‐AUT relationship (48 sec). This finding further supports the conclusion that, unlike in CPE, the declining‐type, and not saturating‐type AOX‐AUT relationship better accounts for the kinetic system properties in SIE.Simulated V˙O2‐PO dependence, the initial nonlinearity at the beginning of exercise is ignored. In other words, the problem concerns the “time‐shifted” V˙O2‐PO relationship  (but lower than in SIE) in slow RIE (8%) . As the latter effect prevailed over the former effect in fast RIE, a negative nonlinearity in the muscle V˙O2‐PO relationship was predicted in this exercise mode.The nonlinearity in the simulated muscle 8%) Fig. , close t8%) Fig. , and sliV˙O2‐PO relationship was observed in different RIE modes. Keir et al. (V˙O2‐PO relationship in different subjects for medium RIE (30 W/min). Takaishi et al. (V˙O2‐PO nonlinearity for slow (10 W/min) RIE, approximately zero (0%) nonlinearity for medium‐slow (20 W/min) RIE, negative (−5%) nonlinearity for medium (30 W/min) RIE, and negative (−6%) nonlinearity for medium‐fast (40 W/min) RIE. Even higher positive (+ 11%) pulmonary V˙O2‐PO nonlinearity was encountered by Keir et al. . Scheuermann et al. (V˙O2‐PO nonlinearity for very slow (8 W/min) RIE and a negative (−5%) pulmonary V˙O2‐PO nonlinearity for very fast RIE (64 W/min). Davies et al. (V˙O2‐PO nonlinearity for medium‐slow (20 W/min) RIE and medium (30 W/min) RIE, and a small negative nonlinearity for fast (50 W/min) RIE  RIE. Generally, the simulated muscle V˙O2‐PO relationships seem to agree rather well with experimental pulmonary V˙O2‐PO relationships. The experimental pulmonary V˙O2‐PO relationship in RIE tends to be positively nonlinear (upward deviation from linearity) in slow RIE, approximately linear in medium RIE and negatively nonlinear (downward deviation from linearity) in fast RIE. Perhaps the main slight difference between computer simulations and experimental data is that the negative nonlinearity in fast RIE seems frequently to be somewhat greater for experimental pulmonary V˙O2‐PO relationship than for simulated muscle V˙O2‐PO relationship  were assumed (not shown). The discussed phenomenon was due to the highest additional ATP usage at a given PO in CPE and lowest in fast RIE, especially at higher POs.CPE, SIE, and particular modes of RIE are characterized by different muscle V˙O2‐PO relationships for different RIE modes were generated using a simple model by Keir et al. , medium‐slow (20 W/min), medium (30 W/min), and medium‐fast (40 W/min) RIE are rather small in experimental studies by Takaishi et al. , when the saturating‐type AOX‐AUT dependence, instead of the declining‐type AOX‐AUT dependence, is used in computer simulations (not shown). The nonlinearity in this case is also slightly positive for fast RIE. As such kinetic behavior of the system differs more from the experimental pulmonary V˙O2‐PO relationship than the behavior generated using the declining‐type AOX‐AUT dependence used in this study, it seems likely that also in RIE (like in SIE) the muscle V˙O2‐PO relationship is better accounted for by the declining‐type AOX‐AUT dependence, than by saturating‐type AOX‐AUT dependence. Therefore, the regulation of OXPHOS seems different in RIE and SIE, than in CPE, as discussed above.It should be stressed that the muscle V˙O2max) and the power output achieved at it (POpeak), related to muscle fatigue, in CPE, SIE, and three modes of RIE. While the pulmonary V˙O2max seems to be identical in various exercise modes , then POpeak can differ very significantly, being highest in medium ramp‐incremental exercise (RIE) and lowest in constant‐power exercise (CPE) (8 min) for the same individual , lower CP, and/or faster increase in the additional ATP usage with time and PO were used (not shown).This study does not deal explicitly with the maximum oxygen consumption , the simulated changes in metabolites and pH in SIE and RIE cannot be compared with experimental data, as these variables were not measured so far in SIE and RIE. Therefore, these model predictions will have to be verified by future experimental studies.While the simulated time courses of ADP, PCr, PV˙O2 increase after the onset of exercise is ADP independent, and therefore V˙O2 must be quickly elevated by some other mechanism. This mechanism was also confirmed by Wüst and co‐workers on the basis of the V˙O2‐ADP relationship during on‐transient in skeletal muscle , NADH supply block, and glycolysis/glycogenolysis are directly activated by some cytosolic mechanism, together with the stimulation of muscle contraction (actomyosin‐ATPase and Ca2+‐ATPase) by Ca2+ ions. The ESA mechanism is able to account for various, apparently not related to each other kinetic properties of the skeletal muscle and heart bioenergetic system, in particular OXPHOS (see Korzeniewski The each‐step activation (ESA) mechanism, being a special case of the parallel‐activation mechanism, was proposed in relation to OXPHOS in skeletal muscle by Korzeniewski . Accordi2+ of three irreversible TCA (tricarboxylic acid) cycle dehydrogenases    and metabolite concentrations averaged over entire muscle and different muscle fiber types  with different patterns of recruitment in various modes of exercise . On the other hand, the model was tested mostly for one‐compartment experimental data, like measured muscle A two‐compartment model of skeletal muscle bioenergetics was developed  were made for the same parameter set and this is a great advantage that allows a direct comparison of the V˙O2‐PO nonlinearity and the differences in its magnitude in various exercise modes, namely, the linear increase in the additional ATP usage with time and PO, is correct. Nevertheless, the model can demonstrate that this mechanism offers a plausible explanation that works at least semiquantitatively.Of course, a computer model cannot prove that the postulated mechanism underlying the V˙O2‐PO relationship. Additionally, different extents of the additional ATP usage are proposed to be responsible for different magnitudes of this nonlinearity in different exercise modes. The size of the additional ATP usage was proportional to the time integral of the PO ‐ CP difference above CP. The extent of the muscle V˙O2‐PO nonlinearity was diminished by an increase in ATP supply by anaerobic glycolysis. Using the same parameter values, computer simulations predicted that the muscle V˙O2‐PO nonlinearity is largest in constant‐power exercise  (+16%), lower in step‐incremental exercise (30 W/3 min) (+12%), in slow ramp‐incremental exercise (10 W/min) (+8%), in medium ramp‐incremental exercise (30 W/min) (+1%), and lowest  in fast ramp‐incremental exercise (50 W/min) (−2%). The last theoretical result was caused by the fact that the increase in V˙O2 due to the increase in the additional ATP usage was smaller than the decrease in V˙O2 related to elevated ATP supply by anaerobic glycolysis. These theoretical predictions agree well, at least semiquantitatively, with experimental data concerning the pulmonary V˙O2‐PO nonlinearity in different exercise modes.It was postulated in this theoretical study that the assumed linear increase in the additional ATP usage with time and power output (PO) above critical power (CP) is the mechanism responsible for the nonlinearity of the muscle AOX ‐AUT (relative ATP usage activity) relationship, then the declining‐type AOX‐AUT relationship seems to predict correctly the kinetic behavior of the system in step‐incremental exercise and ramp‐incremental exercise. For instance, the declining‐type AOX‐AUT relationship is able to account for the significant increase in t0.63  in step‐incremental exercise and the negative V˙O2‐PO nonlinearity in fast ramp‐incremental exercise (through stimulation of ATP supply by anaerobic glycolysis). Both phenomena were encountered in experimental studies. Therefore, it was proposed that the phenomenological (averaged over whole muscle) regulation of OXPHOS is different in constant‐power exercise, on the one hand, and in step‐ and ramp‐incremental exercise, on the other hand.It was also postulated that while the kinetic properties of the system in voluntary constant‐power exercise can be satisfactorily accounted for by the saturating‐type None declared.


Circulation of HFRS-associated orthohantaviruses, such as Seoul, Gou, Amur, Dobrava and Hantaan, are supposed to be restricted to Eurasian countries even though their hosts can be a worldwide distribution. Few confirmed HFRS orthohantavirus infections in humans have been reported in American countries, but due to lower medical awareness of the symptoms of this zoonosis, it could be associated to viral underreporting or to misdiagnosis with several tropical hemorrhagic diseases. Serological evidence of orthohantavirus infections, using enzyme-linked immunosorbent assay for the presence of immunoglobulin M and G against recombinant nucleoprotein protein, remains as an essential assay for viral surveillance. In this study, we aimed to identify In silico analysis were performed using Seoul orthohantavirus nucleoprotein (SHNP) sequence as a model. Linear B-cell-epitopes on SHNP and its immunogenicity were predicted by BepiPred-2.0 and Vaxijen algorithms, respectively. The conservancy of predicted epitopes was compared with the most clinically relevant HFRS or HCPS-associated orthohantavirus, aiming to identify specific sequences from HFRS-orthohantavirus. Peptide validation was carried out by ELISA using Balb/c mice sera immunized with purified recombinant rSHNP. Peptides cross-reactivity against HCPS orthohantavirus were evaluated using immunized sera from mice injected with recombinant Juquitiba orthohantavirus nucleoprotein (rJHNP).In silico analysis revealed nine potential immunogenic linear B-cell epitopes from SHNP; among them, SHNP(G72-D110) and SHNP(P251-D264) showed a high degree of sequence conservation among HFRS-related orthohantavirus and were experimentally validated against rSHNP-IMS and negatively validated against rJHNP-IMS. Taken together, we identified and validated two potential antigenic B-cell epitopes on SHNP, which were conserved among HFRS-associated orthohantavirus and could be applied to the development of novel immunodiagnostic tools for orthohantavirus surveillance. Seoul orthohantavirus, associated with HFRS, was isolated in America and its natural host (Rattus norvegicus) are widely distributed around the world, it raised the question if the viral underreporting is associated to lower medical awareness of the symptoms or if it is associated to misdiagnosis with other tropical hemorrhagic diseases . In this context, considering that the HFRS are clinically indistinguishable from order hemorrhagic diseases, and that serological tests are predominantly based on serology tests against nucleoprotein, a highly conserved protein among different orthohantavirus, we hypothesize that current available tests do not detect all HFRS-associated orthohantavirus. In this sense; we aimed to identify B-cell linear epitopes exclusively conserved on HFRS-associated orthohantavirus nucleoprotein, using a combination of in silico and experimental approaches, to identify targets that could be applied in the development of novel immunodiagnostic tools able to identify different HFRS orthohantavirus species.Orthohantaviruses are the etiological agents of serious rodent-borne neglected human diseases named as hemorrhagic fever with renal syndrome (HFRS) and orthohantavirus cardiopulmonary syndrome (HCPS). These distinct clinical manifestations of disease are related to specific orthohantavirus species and it is believed that HFRS-associated orthohantavirus mainly circulate into Old World (Asia and Europe) whereas HCPS-associated orthohantaviruses are predominant into New World countries (Americas). However, since  Orthohantavirus, of the family Hantaviridae, which comprises a diverse group of infectious negative-sense single-stranded RNA viruses. They are zoonotic in nature and transmitted to humans through the inhalation of contaminated aerosols present in the excreta, saliva, and urine of infected rodent animals [2]. T016  and . (2016) , were us. (2016) . The low. (2016) , 30 and . (2016) . The ene. (2016) .Table 1, nine sequences (SHNP(K30-E48); SHNP(G72-D110); SHNP(G145-K173); SHNP(P182-G196); SHNP(D231-G141); SHNP(P251-D264); SHNP(T347-F361); SHNP(Q363-I380); SHNP(D394-E405)) presented BepiPred and Vaxijen Scores above threshold values  were predicted as antigenic linear B-cell epitopes in SHNP. Among sequences predicted as B-cell epitope by BepiPred 2.0, only SHNP(E5-V29) and SHNP(D231-G141) were predicted as non-antigenic epitopes by Vaxijen and were excluded of further analyzes.To predict linear B-cell epitopes with potential antigenicity, the full sequence of SHNP (NCBI ID: AAT45728.1) was analyzed by the BepiPred 2.0 and Vaxijen algorithms. As showed in Nucleoprotein is a conserved protein among orthohantavirus, presenting similarity level ranging from 60% to 99%. However, when compared to orthohantavirus related to HCPS , the nucleoprotein from orthohantavirus related to HFRS  were only intermediary conserved, ranging from 62% to 74% of similarity .Based on this variable degree of conservation of nucleoprotein and aiming to identify immunodominants B-cell epitopes specific to orthohantaviruses related to HFRS, we compare the similarity level of SHNP predicted epitopes among nucleoproteins of HFRS Hantavirus and HCPS Hantavirus. The levels of similarity were classified as very low ; low (25%>X>50%), intermediary (50%<X<75%) and high (75%<X<100%).(G145-K173), SHNP(P182-G196), SHNP(T347-F361), SHNP(Q363-I380), SHNP(D394-E405) presented an conservation degree ranged from 67% to 100%, being the sequence SHNP(Q363-I380) the most conserved epitope, with an similarity level of 100% when compared to the most of orthohantavirus associated to HFRS and 94% when compared to virus or isolates associated with HCPS ((K30-E48) and SHNP(G72-D110) presented high conservation degrees  in comparison with to the most of orthohantaviruses associated to HFRS and intermediary degree  when compared to sequences of virus or isolates associated to HCPS. Remarkably, the predicted epitopes SHNP(P182-G196) and SHNP(P251-D264) were considered specific to Seoul orthohantavirus, once both presenting 100% of similarity with Gou specie and low level of conservancy  in comparison with orthohantavirus associated HCPS.The similarity level observed among the epitopes of each orthohantavirus compared was shown in ith HCPS . Besidesin silico analyzes, sequences of epitopes SHNP(G72-D110), SHNP(P251-D264) and SHNP(I266-A283) were synthetized as linear peptides and tested by ELISA against sera sample from BALB/c mice immunized with rSHNP. This step aimed to validate experimentally epitopes with low (SHNP(P251-D264), SHNP(I266-A283)) and intermediary (SHNP(G72-D110)) conservation degree among orthohantavirus associated to HCPS. Firstly, we confirmed that rSHNP is immunogenic in animal models because immunized mice presented specific antibodies against it at 28 days after the first immunization (IgG titer: 336829 ± 92407) and presented an increase of 2.5-fold in the IgG serum title against rSHNP after the third immunization (IgG titer: 855671 ± 118257). In relation to the specific humoral response against predicted peptides, mice immunized with rSHNP elicited specific antibodies against peptides SHNP(G72-D110) and SHNP(P251-D264). Both peptides, were specifically recognized, presenting similar levels of antibodies level in the first (anti-SHNP(G72-D110) IgG titer: 5925 ± 2747; anti-SHNP(P251-D264) IgG titer: 1604 ± 1212) and second bleeding (anti-SHNP(G72-D110) IgG titer: 7550 ± 1474 and anti-SHNP(P251-D264) IgG titer: 1911 ± 1119). On the other hands, we did not observe any humoral response against SHNP(I266-A283) peptide (After  peptide .(G72-D110), SHNP(P251-D264) and SHNP(I266-A283) were tested by ELISA against samples from mice immunized with both recombinant proteins, rSHNP and rJHNP and with non-immunized mice, at dilution of 1:100.In order to verify the specificity of predicted B-cell epitopes, the reactivity against rSHNP, rJHNP, SHNP(G72-D110) and SHNP(P251-D264) were highly specific to SHNP  once that non-immunized mice  and mice immunized with rJHNP  ((G72-D110) and SHNP(P251-D264) between non-immunized mice and rJHNP immunized mice.Initially, the immunization with rSHNP and rJHNP elicited a high cross reactivity, once that the immunizations results in similar recognize of recombinant proteins, rSHNP and rJHNP, by both immunized groups, immunized with rSHNP and immunized with rJHNP . Despitectively) . BesidesTable 3.Search of homologous SHNP structures (GenBank: AAT45728.1) were carried out using the SWISS-MODEL server, using information from Protein Data bank (PDB), and resulted in three protein structures which were used for homology modeling: 5FSG, 4FI5 and 5E04 with sequence identity of 82.91%, 84.62% and 66.08%, respectively. The complete template description is shown in From the alignment of the target sequence with the template sequences, 200 models were built and the lowest energy model was selected. The Molprobity server and WinCoot software was used to evaluate the stereochemical quality of the selected model from Ramachandran plots. After energy minimization, it was possible to reach 97.19% (415/427) of residues in preferred regions, 2.58% (11/427) in allowed regions and only 0.23% (1/427)– 114 GLY—outliers in Ramachandran graph. The 3D structure of the model presented a score of 75.52% on Verify 3D, which demonstrates the quality of the model obtained.(G72-D110) ((P251-D264) ((I266-A283) ((G72-D110) ((P251-D264) ((I266-A283)  , SHNP(P251-D264)  and SHNP66-A283) —were dem72-D110)  is at a 51-D264)  and SHNP66-A283)  peptidesin silico identification and experimental confirmation of two B-cell linear epitopes on SHNP. These data could be helpful to improve the development of new diagnostic tools, resulting in a better and more specific HFRS-associated orthohantavirus diagnose, especially in the New World.Orthohaantavirus species associated with hemorrhagic fevers are worldwide spread; however, they could be misdiagnose as viral or bacterial hemorrhagic diseases, like dengue, yellow fever or leptospirosis, especially in countries where these diseases are endemic. In this context, the development of novel, specific and broader diagnostic tools, able to recognized HFRS-associated orthohantavirus species, have become an urgent demand to public health. Immunodiagnostic tools are vital in the study of orthohantavirus infections due to the limited success with molecular detection as the viremia will be short-lived. Based on this, our study allowed the in silico identified as B-cell linear epitopes may be used for development of geographic region-specific immunoassays [Seoul orthohantavirus, using a combination of in silico approaches to predict antigenic B-cell linear epitopes, non-conserved among hantaviruses associated to HCPS, and to validate its immunogenicity by the reactivity of serum samples from mice immunized with the recombinant protein. Initially, we predicted nine sequences as immunogenic epitopes, in which eight (88.9%) were, entirely or partially, inserted on sequences described in Kalaiselvan’s study [Recently, Kalaiselvan and collaborators suggested that 20-mers peptides noassays , however’s study . In our (K30-E48), SHNP(G72-D110), and SHNP(I266-A283) were highly conserved just among species associated to HFRS, presenting a conservation level ranging from moderate to low, when compared to HCPS orthohantavirus species. Besides, the epitope SHNP(P251-D264) was considered specific epitope of Seoul orthohantavirus, presenting a very high similarity when compared with Gou (100%) and low conservancies (<36%) in comparison with species associated to HCPS or HFRS. Remarkably, all epitopes considered non-conserved among HCPS orthohantavirus (SHNP(K30-E48), SHNP(G72-D110), SHNP(P251-D264) and SHNP(I266-A283)) presented low to moderate conservation degrees, ranging from 17% to 59%, when compared with Puumala nucleoprotein sequence. Despite of the association of this orthohantavirus specie with HFRS [Puumala orthohantavirus was also associated with HCPS [in silico predicted with prevalence in C-terminal region of the nucleoprotein.Aiming to identify epitopes non-conserved among HCPS orthohantavirus, we compared the conservation of each predicted sequence among orthohantaviruses associated to HFRS and HCPS. The epitopes SHNPith HFRS –35, Puumvancies <% in compith HCPS , a mild ith HCPS , 17, in in silico approaches to predict epitopes against several diseases, the experimental validation of predicted sequences remains scarce, despite being a critical step to the development of novel vaccines and diagnostic tools. In this context, this was the first study reporting the experimental validation of specific B-cell linear epitopes against orthohantavirus, resulting in the recognition of SHNP(G72-D110) and SHNP(P251-D264) epitopes as promising targets to further development of monoclonal antibodies and/or highly-specific tools to HFRS orthohantavirus diagnosis. Moreover, about the non-reactivity of predicted epitope SHNP(I266-A283) and considering the use of in silico strategies to select B-cell epitopes, the oligomerization mode of selected target seems to be an important aspect to epitope selection. Here, we initially predicted three specifics B-cell epitopes in HFRS orthohantavirus and evaluated their localization in tertiary monomeric structure of SHNP, considering all epitopes exposed in protein surface. Controversially, considering that nucleoprotein oligomerizes in dimeric or hexameric forms [(I266-A283) could be explained by its localization in quaternary structure.Despite the increasing use of PI266-A28 and consic forms , we beliFig 2). Based on this cross reaction and considering that Juquitiba genotype nucleoprotein is highly conserved among ANDV and SNV , both orthohantavirus associated to HCPS in Americas, we believe that Juquitiba genotype could be used as HCPS model. Remarkably, despite the cross reaction among JHNP and SHNP, we identify epitopes specific to Seoul orthohantavirus. SHNP(G72-D110) and SHNP(P251-D264) epitopes were only recognized by sera from SHNP immunized mice, suggesting that these epitopes are non-conserved among HCPS-associated orthohantavirus and corroborating their potential as HFRS target. This strategy, based on experimental immunization assays using recombinant proteins, have been used to confirm in silico findings [Seoul orthohantavirus in Americas and the necessity of differential diagnosis of HFRS in New world, experiments using samples of naturally infected hosts (human and/or rodents) should be conducted in order to fully support the present data and to confirm the real potential of these epitopes as differential diagnostic tools.Currently, orthohantavirus commercial serological kits are based on nucleoprotein detection however the cross-reactivity among hosts infected by HCPS and/or HFRS-associated orthohantavirus could not be discarded. In our study, we experimentally immunized mice with recombinant nucleoprotein from Juquitiba genotype, a HCPS-associated orthohantavirus that mainly circulates in Brazil , 40, elifindings –44, howein silico identified and experimentally validated the sequences (72)GKNIGQDRDPTGVEPGDHLKERSALSYGNTLDLNSLDID(110) and (251)PIAGSLSGNPVNRD(264) as linear B-cell epitopes on SHNP, which could be helpful to improve the development of novel immunodiagnostic tools against HFRS-associated orthohantavirus species. These epitopes, as synthetic peptides, could be anchored on solid surface or membranes for enzyme immunoassays; or used in the development of monoclonal antibodies, applicable to tools like sandwich ELISA, immunohistochemistry and immunofluorescence tests, which may be relevant to better and more specific HFRS-associated orthohantavirus diagnose, especially in the New World.In summary, we S1 TableSeoul orthohantavirus nucleoprotein (SHNP) and its predicted epitopes among orthohantaviruses associated to HFRS–Gou (GOUV), Hantaan (HTNV), Amur (AMRV), Dobrava-Belgrade (DOBV) and Puumala (PUUV)—and related to HCPS–Juquitiba genotype (JUQV), Andes (ANDV), Laguna-Negra (LANV) and Sin Nombre virus (SNV). The levels of similarity were classified as intermediary  and high .Values express the percentage of similarity of (DOCX)Click here for additional data file.