We show that crystallized, but not soluble, UA induces production of pro-inflammatory mediators in human whole blood. Soluble UA is internalized in blood cells but does not potentiate or reduce LPS-induced release of cytokines.Several epidemiological studies have pointed at serum uric acid (SUA) as an independent risk factor for mortality, diabetes, hypertension, cardiovascular and kidney disease; however, no clear pathogenic pathway is established. Uric acid (UA) crystals show pro-inflammatory properties and can thus create or contribute to the state of chronic low-grade inflammation, a widely accepted pathogenic mechanism in several of the above-mentioned pathologies. On the other hand, soluble uric acid possesses antioxidant properties that might attenuate inflammatory responses. We aimed to explore the net effects of experimentally rising SUA in human whole blood cultures on several mediators of inflammation. Production of TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8 was assessed upon addition of 200 µM UA, 500 µM UA or monosodium urate (MSU) crystals in the presence or absence of 5 ng/ml lipopolysaccharide (LPS). RT-qPCR and multiplex bead based immunoassay were used to measure mRNA expression and cytokine release at 2 and 4 h of culture, respectively.  Hyperuricemia is also a common problem after renal, liver and cardiac transplantation13.Hyperuricemia is a modifiable condition that can lead to gout, and may be an independent risk factor for mortality14, but others did not18.Epidemiological studies are conflicting whether elevated serum uric acid (SUA) is an independent risk factor for cardiovascular disease. Some studies have found SUA to be an independent cardiovascular risk factor19. Deposition of MSU crystals can also occur in other tissues and organs in humans, although it is reported seldom22. Formation of MSU crystals may also occur in the blood from hyperuricemic patients22.In humans, the concentration of uric acid (UA) can reach its theoretical supersaturation threshold in extracellular fluids. This is due to multiple missense mutations in the uricase enzyme responsible for degrading UA to a more soluble allantoin in most other species. Precipitation of uric acid into monosodium urate (MSU) crystals in joints elicits acute aseptic inflammation, the gouty arthritis, prevalence of which increases with increasing SUA levels25. However, the loss of functional uricase and increase in SUA might represent an evolutionary benefit for humans. UA is recognized to be a major antioxidant in human plasma26 and some studies have suggested a neuroprotective effect29. UA´s potential physiological role is further supported by the high rate of reabsorption of filtered UA in the kidneys30. Soluble UA has been shown to have both antioxidative and pro-oxidative properties in different animal and in vitro experimental models31. Anti-inflammatory effects have also been reported for other antioxidants added to lipopolysaccharide (LPS)-stimulated whole blood cultures33. One doctoral thesis reported that UA attenuated inflammatory responses to LPS indirectly by inhibiting the release of pro-inflammatory cytokines TNF-α and IL-1ß in a human monocyte culture, this effect, however, could not be attributed to antioxidant properties of UA34. Little is known with respect to the direct effect of elevated SUA on inflammatory markers in blood. Therefore, we aimed to investigate whether short-term exposure of blood cells to high concentration of UA elicit a pro-inflammatory response or compromise cell viability, either directly or through precipitation into MSU crystals. Alternatively, UA might elicit protective effects in blood cells exposed to inflammatory stimuli. We also examined to what extent UA is internalized by blood cells.MSU crystals have recently been suggested as a damage associated molecular pattern (DAMP) which stimulates both the innate and adaptive immune system9/L, 4.3 ± 1.0 109/L, 4.3 ± 0.9 109/L vs 4.4 ± 1.0 109/L respectively, n = 10) , LPS (4.1 ± 0.6 109/L) or LPS + UA (4.2 ± 0.6 109/L) for 4 h compared to vehicle . When the baseline vehicle sample was compared against vehicle incubated for 4 h, there was a significant decrease in WBC´s .Measurements made in blood cultures at 15 minutes (baseline) and 1 h 45 min of incubation showed that pH did not significantly change during incubation . At the start of incubation, the mean pH in the whole blood cultures was at the upper limit of the reference range of physiological pH for arterial blood (7.35–7.45). As expected, glucose concentration was significantly lower, while lactate concentration was significantly higher at the end of the incubation at 1 h 45 minutes Fig. . There w10) Fig. . As showNo significant changes in protein concentration of TNF-α, Il-1ß, MCP-1, IL-8 and Il-1RA were observed following exposure of blood cultures to increasing concentrations of soluble UA for 4 h Fig. . At the de-novo production, expression of TNF-α, IL-1ß, MCP-1, IL-8 and Il-1RA mRNA was assessed with qPCR. As illustrated in Fig. In order to differentiate between the release of pre-formed cytokines from their in vitro. After 4 h of incubation, LPS caused significant release of all the studied cytokines but neither 200 µM or 500 µM UA modulated LPS-responses  of ely Fig. . WhereasHumans lack the UA-degrading enzyme uricase. However, UA can still be degraded by non-specific oxidation. We did not detect any sign of UA degradation in the blood culture supernatants during 4 hours of incubation (data not shown). The uptake of UA was unaffected by the presence of LPS (5 ng/ml) in the blood cultures, either when added simultaneously or following 1 h of pre-incubation with UA (data now shown). Addition of transporter inhibitors probenecid and tranilast to blood cultures failed to decrease the level of cell-associated radioactivity or the rate of internalization of radiolabeled UA.ex vivo to whole blood.Addition of soluble UA to whole blood cultures from healthy male volunteers did not illicit an acute pro-inflammatory response or modulate LPS induced stimulation as judged by unchanged plasma concentration of TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8. This also applied to UA added in supranormal concentrations. Similarly, the gene expression analysis showed no effect of soluble UA on TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8 mRNA expression in the cultures. Results from the UA uptake experiment show that UA is internalized by blood cells when added 35. With regards to the pro-inflammatory properties of soluble uric acid in other species, the reports in the literature are somewhat different. Although not being pro-inflammatory per se, uric acid was convincingly reported to have adjuvant properties as it stimulated expression of co-stimulatory molecules CD86 and CD80 on dendritic cells but did not affect their rate of phagocytosis of particulate antigens. Of note, the concentrations of uric acid that up-regulated co-stimulatory molecules were very close to concentrations at which crystal formation occurs, at least in humans23. Another study found that soluble uric acid was able to induce IL-1ß release followed by production of mitochondrial ROS and caspase 1 activation36. Both studies used bone-marrow derived in vitro maturated macrophages from rodents. Rodents have considerably lower uric acid levels in vivo due to presence of functional uricase gene, and the rise in uric acid that macrophages became exposed to in vitro (from an assumed baseline of 13 μΜ37 to 900 μΜ) was relatively higher than concentration delta in our model (from an assumed baseline of 300 μΜ to 500–800 μΜ). Moreover, in the study by Braga et al.36 the dose-dependent increase in IL-1ß production in response to soluble uric acid was only observed in the presence of LPS in cultures of rodent macrophages, and no signs of activation were detected in human cells. These results therefore were completely in line with our data.Our results are in line with a similar study which showed no pro- or anti-inflammatory effects of supraphysiological concentrations of soluble UA on human whole bloodex vivo have shown that the main chemotactic factors for monocytes and neutrophils, MCP-1 and IL-8 respectively, are regulated by reactive oxygen species (ROS). Adding the exogenous oxygen radical scavengers - N-acetyl cysteine and dimetyl sulfoxide - to LPS-stimulated whole blood, was reported to reduce the release of MCP-1 and IL-833. However, a study in monocytes found UA to attenuate the LPS-induced release of cytokines TNF-α and IL-1ß without affecting intracellular ROS, which suggests that the effect might have been mediated by other than antioxidant properties of UA34. Due to the natural levels of UA in whole blood from healthy males, isolated monocytes in culture might be a more suitable model to study the potential effect of UA as a molecule with immune-regulatory properties. If UA has immune-regulatory properties, normal SUA levels of healthy individuals might be sufficient for this to occur and there is no additional gain of further increasing UA ex vivo.Addition of soluble UA to LPS-stimulated blood did not have any attenuating effect on the elicited pro-inflammatory response as could be expected from antioxidant properties of UA. Earlier studies in whole blood Nevertheless, addition of crystallized UA to our whole blood cultures caused significant upregulation of mRNA and protein release of several pro-inflammatory cytokines, particularly chemokine IL-8 and IL-1RA. These results contrast a lack of stimulatory effect of soluble UA.12. It is now well established that uric acid crystals are recognized as endogenous danger-associated molecular patterns39. MSU-crystal represents a repetitive purine structure that is likely to be sensed as foreign polynucleotide-like molecule by the cells of immune system. The ability of MSU-crystals to activate pro-inflammatory pathways in phagocytes is linked to engagement of specific receptors44, membrane rearrangements45 and complement activation46. Accordingly, lack of sufficient expression or engagement of such receptors as CD14, TLR-2 and −4 and Fc-receptors, for example due to immaturity of phagocytes, insufficient opsonization or blockage of binding sites on MSU-crystals would all prevent an inflammatory response from full activation.Uric acid is an end product of purine degradation pathway, and an acute rise of serum uric acid in the circulation takes place in situations with massive cell death and break-down of nucleic acids. It is logical that such a sign would be perceived by the immune system as a danger signal. Indeed, high serum uric acid is a predictor of adverse events as it is longitudinally associated with increased mortality and morbidity53. Indeed, a surprisingly small proportion of people with hyperuricemia experience MSU crystal deposition disease, suggesting either subclinical disease, or that additional factors are needed for MSU crystal formation in human tissue. The conditions in our model with relatively good cell viability  suspended in plasma and not with adherent maturated macrophages. It has been shown that silica aluminum salt crystals did not induce IL-1β release in human PBMCs unless cells were primed by LPS56, a well-known modulator of macrophage differentiation57. While studies of mature phagocytes in serum-free environment is relevant as a model for inflammatory response in tissue/joints, whole blood suspension cultures used by us, suit better to study the effects of uric acid in the circulation. As NLPR3 has intracellular localization58, its activation with subsequent IL-1ß production requires phagocytic uptake of MSU crystals56. Indeed, addition of Cytochalasin D blocks MSU-crystal phagocytosis and abolish activation of NLPR3 inflammasome and IL-1β release56. The weak IL-1ß upregulation in our experiments could be attributed to differences in the efficiency of phagocytosis and/or/ density of cell surface receptors between enriched cultures of mature phagocytes and peripheral blood cells. Another possible explanation is the presence of plasma in our cultures which is known to contain elements that can non-specifically antagonize pro-inflammatory responses to crystallized uric acid, such as CD-4459 and apolipoprotein B60. At the same time, MSU-crystals in our model induced significant increase in the production of other pro-inflammatory cytokines, such as TNF-a and IL-8, confirming that activation of pro-inflammatory pathways has taken place. A more feasible explanation is therefore sub-optimal timing for IL-1ß assessment.NLRP3 induced IL-1ß production has been previously implicated as a key element in crystal-elicited pro-inflammatory response61. The results from our UA uptake experiment indicated that UA is indeed internalized into blood cells. However, the type of cells responsible for the uptake remains to be determined. Rough separation of blood suspension 4 h after incubation with 14C-UA into erythrocytes and buffy coat fractions showed that the majority of cell-associated radioactivity was present in the erythrocyte fraction (data not shown). Whether white blood cells contribute to 14C-UA internalization remains to be determined.UA has been shown to reduce ROS intracellularly in cultured fibroblast by uptake through GLUT9 (SLC2A)62. Therefore, the most probable mechanism for UA uptake in blood cells is via transmembrane transporters. The possible candidates of UA transporters are GLUT9, URAT1, OAT10 and OAT4 among others31. However, addition of inhibitors probenecid and tranilast did not affect uptake of UA (data not shown). Probenecid and tranilast are considered by many to be inhibitors for all the UA transporters mentioned above63. However, some authors define probenicid solely as URAT1 inhibitor. Nevertheless, our data indicate that neither GLUT9, URAT1, OAT10 nor OAT4 are involved in UA uptake by blood cells, while it has been previously reported that probenecid retards the uptake of UA, at least in human erythrocytes64. It should be noted that with renal tubular epithelial cells as exception, urate uptake mechanisms are not well described. Since only overall UA uptake but not rate of uptake were higher in male group the observed difference in UA uptake between sexes may be due to typically higher erythrocyte numbers in male compared to female subjects.The cell-associated radioactivity detected at the first time point  indicates a very rapid initial uptake of UA. When the uptake experiments were conducted on ice almost no cell-associated radioactivity was detected (1.3%) and no changes in dynamics observed during 4 h (data not shown). This suggests that the uptake of UA by blood cells is not free diffusion across cell membrane the process that would not be significantly hindered by low temperature. Furthermore, the rate of UA uptake suggests that fluid phase pinocytosis is unlikely to be the mechanism behind. Receptor-mediated endocytosis is also unlikely to be involved since no endocytic receptor for UA has been described in humans so far. However, receptor-mediated endocytosis of UA was demonstrated in yeast65. Based on earlier kinetic experiments performed in similar whole blood models with LPS as stimulant we chose to incubate our cultures for four hours prior to cytokine analysis66. However, it might be that longer incubation is needed when using MSU crystals as stimulant. No measurements of oxidation products or oxidative burst were conducted in our experiments, and consequently, it is not possible to exclude that UA may have an effect on ROS-production that we did not detect. The timing for sample collection might not be optimal for all the cytokines we wanted to study. Because earlier kinetic experiments performed in similar whole blood models indicated that two hour incubation was probably too short to observe the full effect on MCP-1 and IL-8 release66, we extended the study with sampling also at four hour time point. Another limitation with the whole blood model for studying chemokines is the presence of Duffy-receptor on red blood cells, which is known to scavenge MCP-1 and IL-8 from plasma67 and mask the effect of UA on production of these chemokines. At the same time scavenging would doubtfully mask changes in cytokine gene expression, which in our experiments were consistent with ELISA results. In addition, the relatively low number of study participants, the use of young, healthy and mainly male donors are all important limitations.This study has several limitations. We were not able to measure UA in the cultures because we used anti-coagulated blood and the final concentration of UA in cultures is therefore unknown. The use of only male donors in the experiments studying inflammatory responses weakens the external validity of the study. The number of cytokines and chemokines we measured was limited due to financial restrains. The response to MSU crystals in our study was also somewhat weaker than described by others. This may be due to differences in experimental design, such as longer MSU crystals incubation times or the production of smaller or otherwise qualitatively different crystals in other studiesex vivo inflammation model. Exposure of whole blood to high concentrations of UA allows studying the uptake and responses to this metabolite in a setting that is more similar to the situation in the circulation than what can be achieved by using cultures of purified blood cell types or human cell lines. Results from the hematological differential counting and assessment of metabolic parameters indicate that cell viability and metabolism were not considerably affected by incubation of blood for up to four hours ex vivo. Humans have higher SUA levels and different purine metabolism than most other mammals, including the typical laboratory animals - rats and mice30. The external validity of the study is therefore also superior compared to studies in animal models.The strength of this study is the use of freshly drawn human whole blood in an Crystallized, but not soluble, UA elicits production of pro-inflammatory cytokines TNF-α and IL-1ß, chemokines MCP-1 and Il-8, and IL-RA in short-term cultures of whole blood from healthy young men. Soluble UA was taken up by blood cells but did not seem to potentiate or reduce LPS-induced release of cytokines. The subgroup of blood cell(s) responsible for UA internalization and the mechanism behind need further investigation.The study has been approved by the Regional Committee for Medical and Health Research Ethics (2014/73). All the study participants signed the informed consent form prior to participation. The biological material in the study was used without keeping any linked information on the identity or health status of the donors. All methods were performed in accordance with the relevant guidelines and regulations.E. coli 026:B6 (Sigma-Aldrich), EDTA 1% (w/v) in PBS without Ca2+ and Mg2+ , RPMI-1640 cell culture medium (Sigma-Aldrich), Tissue solubilizer “Solvable” (PerkinElmer), “Ultima Gold” scintillation cocktail (PerkinElmer), 14C labelled uric acid (50 mCi/mmol) . Probenecid and tranilast were purchased from Sigma-Aldrich.Uric acid  (Sigma-Aldrich), Lipopolysaccharide (LPS) from A 4 mM UA stock solution was prepared by dissolving UA powder in 4.06 mM NaOH and pH was adjusted to 8.5 to obtain long-term stable stock solution. The 4 mM UA stock solution was further diluted with 1.8% NaCl to produce 2 mM UA/0.9% NaCl stock solution. All solutions were filtered through a 0.2 µm syringe filter  before use. MSU crystals were prepared by addition of NaCl to 4 mM UA stock solution to 0.9%, followed by cooking at 100 °C for 6 h and allowing to precipitate at room temperature under sterile conditions for 5–7 days. The crystal formation was checked microscopically. The crystals were typically 5–25 µm long. LPS was reconstituted in 0.9% NaCl to 500 ng/ml working solution.68. Healthy male study participants, aged 18–40 years, were recruited from the university, hospital staff members and students. Only males were invited to avoid introducing heterogeneity in the donors from cyclical changes of estrogens during menstrual cycle known to affect immune response and cytokines production71. Exclusion criteria were self-reported concurrent use of medications or any chronic or acute illness. Blood samples were slowly aspirated from the antecubital vein into a 20 ml polypropylene  syringe, with a 19-gauge needle and anticoagulated with Dalteparin sodium  10 IE/ml blood. Aliquots of blood were immediately transferred to 50 ml polypropylene tubes to establish 3 ml blood cultures. UA stock solutions were added to achieve final concentrations of 200 µM or 500 µM on top of the donors’ level to represent moderate and high level of SUA72. MSU crystals were added to one of the cultures to a final concentration of 0.78 mg/ml. The volumes of uric acid in solution and crystal suspension added to whole blood cultures, were the same and comprised 1% of total culture volume. The tubes were capped, inverted gently and incubated in a rotary shaker incubator at 37 °C for 1 h, followed by addition of 30 µl of LPS working solution to a final concentration of 5 ng/ml or vehicle. At 2 h the tubes were gently inverted to mix and an aliquot of 2 ml was transferred to a Tempus tube (Applied Biosystems) mixed and kept frozen at −20 °C. The remaining 1 ml of blood cultures continued incubation in a rotary shaker incubator at 37 °C for additional 2 h, followed by centrifugation at 2000g for 10 min. Plasma was then collected and kept frozen at −70 °C until further analyses.A human whole blood suspension model was used as earlier described by othersTotal RNA was extracted using the PerfectPure RNA Blood Kit (5′Prime) or RNeasy Mini Kit (Qiagen) according to the manufactures instructions. Isolated RNA was quantified by NanoDrop 1000 Spectrophotometer (Thermo Scientific). Isolated RNA samples were diluted to final concentration 25 ng/µl and cDNA synthesis was conducted with High CapacitycDNA kit (Applied Biosystems) using 500 ng mRNA in a total reaction volume of 20 µL. cDNA synthesis were performed with and without RT enzyme to generate cDNA and RT-free negative controls. Quantitative PCR (qPCR) was performed using gene-specific primers (Table t) with adjustment for efficiency of amplification73.The PCR efficiency for all primer sets was determined by performing a serial dilution of pooled cDNA from all individuals. Melting curves were assessed to control for non-specific amplification. RPL-13A was used as a reference gene for normalization of cytokine gene expression  was used for simultaneous measurement of IL-1ß, IL-1RA, TNF-α, IL-8, MCP-1 and ICAM-1 (negative control). The plasma samples were diluted with PBS prior to analysis and analyzed in duplicates according to the manufactures instructions. The chemiluminescense was read on a VERSAmax Absorbance Microplate Reader (Molecular Devices). Final plasma concentrations were calculated using the Bioplex software supplied by the manufacturer. The standard curve was constructed using a 4-parameter logistic function.2, pCO2, Na+, K+, Cl− and Ca2+ concentrations were analyzed with an ABL 800 blood gas analyzer (Bergmann diagnostika).Aliquots of blood were pipetted into 15 ml polypropylene centrifugation tubes  containing either vehicle (0.9% NaCl), uric acid (200 µM and 500 µM), MSU crystals (0.78 mg/ml) or LPS (5 ng/ml) to a total volume of 2 ml. The tubes were inverted ten times and incubated with closed caps at 37 °C in a shaker incubator. At baseline and following two or four hour incubation, the experiments were stopped by placing the tubes on ice. A 1000 µl of blood was pipetted to standard EDTA tubes for hematological differential cell counting performed at Clinical Chemistry Department, University Hospital of North Norway. Lactate, glucose, pH, pO14C-UA and 1000 µM non-labelled UA. A total volume of 1790 µl of the mixture was pipetted into six well cell culture plates  and incubated at 37 °C 5% CO2 on a shaker board. Incubation was terminated at 0, 0.5, 1, 2 and 4 h by transferring the cell suspensions to 2 ml microcentrifuge tubes (Eppendorf) and centrifuged at 2000 G for 10 min at 5 °C. Supernatants and cellular fractions were separated and hydrolyzed by incubation with tissue solubilizer “Solvable” at 55 °C for 3 hours according to manufacturers’ instructions. To the hydrolyzed cellular fractions, 50 µl of 0.2 M EDTA and 500 µl of 30% hydrogen peroxide were added and the mixtures were incubated at room temperature for 30 min, and then at 55 °C for 60 min in order to decolorize the samples. After cooling down, both fraction types were mixed with 17.7 ml “Ultima Gold” scintillation cocktail. Radioactivity (d.p.m.) was measured in a Packard Liquid Scintillation Counter. The extent of UA degradation was determined by counting radioactivity after addition to cell culture supernatants of ZnCl2 that precipitates only non-degraded UA.Blood from six healthy volunteers  was collected as described above. Blood was mixed 1:1 with RPMI-1640 cell culture medium containing 1 µM 74 and endocytic uptake and facilitated transport (membrane transporters) where effect of temperature lowering is significant75. Addition of probenecid  and tranilast  to blood culture was used to analyze whether GLUT9, URAT1, OAT10 and OAT4 transporters were responsible for uric acid uptake63.Incubation on ice was performed in order to differentiate between free diffusion across cell membrane where the dependence on temperature is negligible14C-UA-associated radioactivity was analyzed using non-linear regression. The model that best described the radioactivity uptake data, (one phase association model), was selected using the Akaike’s Information Criterion (AIC)76. The differences between sex groups were determined by comparing best-fit values of parameters using F-test.Prism 6 and 7 (Graph Pad software inc.) were used to make figures and conduct statistical analyses. A paired t-test was used when two groups were compared with each other. A repeated measures ANOVA with a Geissner-Greenhouse correction for not assuming sphericity was used to compute P values for the main effects in the cytokine experiments. Provided significant differences were observed on an alpha level of ≤ 0.05 in the repeated measures ANOVA, Dunnett’s multiple comparison test was conducted. P-values were adjusted for multiple comparisons. For UA uptake studies, the dynamics of blood cells’


Unstable angina pectoris (UA) is one of the most dangerous clinical symptoms of acute coronary syndrome due to the risk of myocardial ischemia, which can lead to high morbidity and mortality worldwide. Though there are many advantages in understanding the pathophysiology of UA, the identification of biomarkers for the diagnosis, prognosis, and treatment of UA remains a challenge in the clinic. A global metabolomics research based on ultra-performance liquid chromatography (UPLC) combined with Q-TOF/MS was performed to discover the metabolic profile of health controls, UA patients, and UA patients with diabetes mellitus (DM), and screen for potential biomarkers. Twenty-seven potential biomarkers were determined using pattern recognition. These biomarkers, which include free fatty acids, amino acids, lysoPE and lysoPC species, and organic acids, can benefit the clinical diagnosis of UA. Pathway analysis indicated that arginine and proline metabolism, glycerophospholipid metabolism, and purine metabolism were affected in the UA patients, uniquely. Additionally, alterations in the metabolic signatures between UA and UA-complicated DM were also explored. As a result, six differential metabolites with an area under the curve (AUC) of more than 0.85 were identified as biomarkers for the diagnosis of UA and UA complicated with DM. Pathway analysis implied tryptophan metabolism was a key metabolic pathway in UA patients with DM, which provides new insights into the pathological study and drug discovery of UA. Unstable angina pectoris (UA), is a clinical manifestation of acute coronary syndrome (ACS), which presents as ST-segment elevation myocardial infarction (STEMI) or non-STEMI (NSTEMI). Resulting from platelet adhesion due to the rupture of fibrous plaques on the surface of the atheroma, UA occurs secondary to acute exacerbation of coronary occlusion. Compared with stable angina, UA is more painful with longer episodes, occurs spontaneously at rest (onset angina) and more frequently, which is also a progressively worsening disorder ,2. HowevMetabolomics has its advantages in high throughput metabolite analysis and is feasible to apply in disease diagnosis and mechanism research. Integrating bioinformatics and biostatistics, metabolomics enables the quantification and identification of biological molecules to explore the response in an organism to a disease . PreviouDiabetes mellitus (DM), which can aggravate the deterioration of cardiac function, is one of the main complications of UA. Distinguishing the features of UA from the features of UA complicated with DM will have a benefit on the precision treatment of UA and slowdown the progression of UA. However, little attention has been given to the identification of biomarkers and pathological metabolisms of UA complicated with DM. Therefore, in the present study, the biomarkers of UA and UA complicated with DM were identified based on global metabolomics. Furthermore, the metabolic differences between UA and UA complicated with DM were also investigated systematically. The results of the present study will assist in the diagnosis of UA and UA complicated with DM in the clinic, and provide further understanding for the pathophysiologic process of UA from the initial occurrence through progression.The protocol of the present study was approved by The Ethical Committee of Beijing University of Chinese Medicine (No. 81302914), and informed consent of the protocol was obtained from all participants. Patients (n = 40) were recruited from the Beijing Anzhen Hospital and were diagnosed with UA between January 2016 and September 2016. The diagnostic criteria of UA included: (1) severe and newly developed angina; (2) angina pectoris occurs at rest or with minimal exertion; and/or (3) angina with crescendo pattern. Healthy controls (n = 39) were recruited from the medical center of an affiliated hospital of the Beijing University of Chinese Medicine. The exclusion criteria included liver disease, vascular disease, cancer, thyroid disease, renal disease, and acute/chronic inflammatory disease. The patient and control participants in the present study were well-matched, and the detailed information is shown in HPLC-grade methanol and acetonitrile were purchased from Burdick & Jackson (USA). Formic acid (98%) was purchased from Sigma-Aldrich (USA). Ultrapure water was prepared from the Milli-Q system .The blood samples from the participants were collected on the same day of inclusion. Patient information, including gender, age, electrolyte, ejection fraction, creatinine, BNP, hemoglobin, urea, fasting blood glucose, platelet count, high-sensitivity C-reactive protein (hs-CRP), triglyceride, total cholesterol, and medication, was recorded at inclusion. Associated diabetes, dyslipidemia, or hypertension were also added to the patient record. The venous blood of the participants was collected with EDTA as an anticoagulation and centrifuged at 3500 rpm for 15 min. Plasma samples were divided into equal aliquots and stored at −80°C. The plasma samples went through no more than two freeze-thaw cycles before metabolomics analysis.The metabolites extraction was performed following a previous protocol with some modifications . An aliqPlasma metabolites were acquired using Agilent 1290 ultra-performance liquid chromatography (UPLC) equipped with 6540 Q-TOF/MS. Zorbax Eclipse plus C18 chromatography column  was adapted for chromatography separation. Acetonitrile (solvent B) and water (solvent A) with 0.1% formic acid were used as the mobile phase with a gradient program as follows: 0–2 min, 5% B; 2–4 min, 5% B to 40% B; 4–5 min, 40 to 50% B; 5–10 min, 50% B; 10–15 min, 50 to 70% B; 15–19 min, 70 to 80% B. The flow rate was 0.8 ml/min, and the injection volume was 5 μl. The column temperature was 40°C. MS was performed in ESI and positive ion mode. The MS parameters were as follows: the mass range was from 100 to 1700; voltage was 3.5 kV; dry gas flow rate was 5 l/min; nebulizer gas was set at 40 psig; capillary temperature was 350°C; internal reference was used for the real-time correction to ensure the stability and accuracy of instrument. Quality control (QC) samples were obtained by pooling 20 µl aliquots of all plasma samples for assessing the quality of the metabolomics workflow. All of the samples were analyzed randomly.https://xcmsonline.scripps.edu) was applied for data preprocessing. First, the raw data from the MS were converted to mzData files and then analyzed using XCMS. The important XCMS parameters are shown below: polarity, positive mode; retention time format, 60 min; ppm, 30; minimum peak width and maximum peak width, 10 and 60, respectively; signal/noise threshold, 6; mzdiff, 0.01; prefilter intensity, 500; and profStep, 0.5. Metabolomics data were preprocessed through peak discrimination, filtering, and alignment, and normalized using probabilistic quotient normalization (PQN), log transformation, and Pareto scaling based on MetaboAnalyst 3.0 [http://www.metaboanalyst.ca/MetaboAnalyst/). Pattern recognition was performed using orthogonal partial least squares discriminant analysis (OPLS-DA) with SIMCA-P 13.0 software . Significance analysis was performed on SPSS Statistics17.0 software. Heatmap of specific differential metabolites was visualized using MeV software, and the classification performance (specificity and sensitivity with the highest accuracy) was assessed by the receiver operating characteristic (ROC) curve based on the SPSS 17.0 statistics software.XCMS online   and Human Metabolome Database (HMDB), were applied for related metabolic pathways analysis. Metscape, a plug-in for Cytoscape , was app2XR = 0.72, 2YR = 0.997, 2Q = 0.977), which verifies the validity of the original mode. A response permutation test with 200 iterations was adopted to avert over fitting of the OPLS-DA model  more than 30% in each group were removed from further statistical analysis. For detecting subtle metabolomics differences between UA patients and the healthy controls, PCA-DA and OPLS-DA were performed as pattern recognition methods to discriminate between the above two groups A,B. The DA model C.U test (P< 0.05)  . Heat maMetabolic pathway analysis has practical significance for systematically evaluating the metabolic status of disease and revealing the related metabolic functions. This analysis indicated that three pathways were found to be obviously affected in UA patients, including glycerophospholipid metabolism, arginine and proline metabolism, and purine metabolism. The detailed results of this pathway analysis are shown in 2XR = 0.268, 2YR = 0.981, 2Q = 0.817). The characteristic biomarkers of UA complicated with DM are listed in DM, which aggravates the deterioration of cardiac function, is one of the most common complications in CAD and also a risk factor for UA. UA complicated with DM is more likely to progress to a myocardial infarction; therefore, prevention and treatment are particularly important. In the present study, the differential metabolism between UA and UA complicated with DM has been explored, providing valuable references for clinical diagnosis and precise treatment. Similar to UA, the potential metabolic biomarkers between UA and UA complicated with DM were identified through OPLS-DA pattern recognition A. StatisA metabolic pathway analysis was performed using the above 22 biomarkers, and three distinctive pathways were discovered for UA complicated with DM, including arginine and proline metabolism, purine metabolism, and tryptophan metabolism. Compared with the three pathways of UA, arginine and proline metabolism, purine metabolism were indicated as significant in UA and UA complicated with DM. Of the pathways, glycerophospholipid metabolism has a greater impact on UA patients and tryptophan metabolism has a greater impact on UA patients with DM. This is the first study identifying tryptophan metabolism as a critical pathway in UA complicated with DM, which will initiate further research revealing the related mechanisms.Up to now, the clinical diagnosis of UA has been a challenge for the prevention and treatment of CAD. Meanwhile, advanced understanding of the metabolic alteration that occurs in CAD patients with complications is quite urgent. For example, DM is known to increase the severity of heart function. In the present study, a UPLC-QTOF/MS-based global metabolomics approach was performed on the plasma of patients with UA and UA complicated with DM to screen for potential biomarkers and reveal the pathways associated with those diseases. Additionally, the present study filled a gap in understanding UA complicated with DM and provided new insights into the biological mechanisms surrounding this disorder.Plasma metabolomics data demonstrated that specific metabolites exhibited various patterns between UA and UA complicated with DM. Rather than evaluating the diagnostic performance of the metabolomics results, the critical metabolites discriminating patients with UA, UA complicated with DM, and healthy controls were identified. Ultimately, 27 biomarkers in UA patients and 22 biomarkers of UA complicated with DM were determined, which has great advantages in visualizing the metabolic differences between UA and UA with complications, and improving the diagnostic performance of our model. Six common biomarkers, including creatinine, 12-methyl-tridecanoic acid, acetylcarnitine, myristicic acid, uric acid, and cycloleucine, were identified between the two groups. Based on our analysis in the present study, it is reasonable to believe that the expression changes of these six metabolites have a vital impact on the onset and development of UA. Noteworthy, creatinine is phosphorylated to phosphocreatine in muscles, which is the most common indicator of renal function ,14. AddiIn addition, metabolic pathways are modified by a series of enzymatic reactions, which participate in specific biological processes. Many diseases are proven to be network phenomena, and a metabolic network could help to describe the complex relationship among various kinds of metabolites, contributing to drug discovery. Here, MetPA and Metscape were used to reveal the pathways related to UA and UA complicated with DM, and construct metabolic networks separately. The pathway analysis indicated that arginine and proline metabolism, purine metabolism, and tryptophan metabolism were the distinctive pathways of UA complicated with DM. It is worth noting that tryptophan metabolism is a typical metabolic pathway of UA complicated with DM. Therefore, the key metabolites and enzymes in the tryptophan metabolism pathway were investigated systematically; the detail information is shown in The identification of biomarkers is one of the main applications of metabolomics. In the present study, a global metabolomics strategy based on UPLC combined with Q-TOF/MS was used to screen for biomarkers of UA and UA complicated with DM. There were 27 and 22 plasma biomarkers identified in patients with UA and UA complicated with DM, respectively. The identified metabolites associated with UA were key participants in energy metabolism, lipid complexes, and amino acids, which provided a reference for clinical diagnosis. Pathway and network analysis based on the biomarkers revealed that tryptophan metabolism was a key metabolic pathway in UA patients with DM, which provided a new understanding in the biological metabolic mechanism of UA complicated with DM. It is believed that systematically exploring potential biomarkers and distinguishing the metabolic mechanism of UA from its complications will promote personalized medicine and drug discovery.


Afghanistan has been a country blighted by war over the past five decades and limited research is available on its demography. This study seeks to assess the suitability of recent survey data for Afghanistan and the 2015 Afghanistan Demographic and Health Survey (ADHS)) for estimating levels and trends in fertility. As several fertility measures rely on the quality of age data, we first apply demographic tools for the identification of age misreporting, finding evidence that it is severe. We then explore the consistency of fertility reporting across the two surveys, finding that the 2015 ADHS reports higher fertility among older women than the 2010 AMS although the seasonal pattern of fertility is consistent across the two surveys. We then estimate total fertility rates in 2008–2010 and 2012–2015 and measures of Bongaarts’s key proximate determinants of fertility for Afghanistan and its provinces for urban and rural areas separately. The results show that fertility is similar in urban and rural Afghanistan. Although most of the provincial data on the proximate determinants is reasonably consistent with the fertility rates, there are anomalies in some provinces which indicate the possible under-reporting of births. Overall, we conclude that the fertility data in the two surveys can be used with care to give an indication of broad regional fertility patterns and trends in the country. Many aspects of the demography of the Islamic Republic of Afghanistan remain under-researched. The exact population of the country is not known due to the lack of a recent census. According to the Central Statistics Organization of Afghanistan (CSOA) the total population of the country is estimated to be 32.2 million people . The urbThe fertility transition in Afghanistan has attracted limited attention in demographic research . AlthougNeighboring countries of the South Asian region have shown a noticeable decline in fertility. For instance, Iran’s total fertility rate is 2.0 and that of Pakistan is 3.1 ,5.In this paper we examine the quality of the data on fertility collected in the only two recent surveys that did include birth histories: the Afghanistan Mortality Survey carried out between April and December 2010 (hereafter 2010 AMS) and the Afghanistan Demographic and Health Survey conducted between June 2015 and February 2016 (hereafter 2015 ADHS).There exists an enormous literature on the types of error  affecting estimates of fertility ,7. In thThe 2010 AMS used a sampling frame provided by the CSOA. It aimed to provide estimates of demographic variables which were representative of the whole country, and of urban and rural areas within three domains . The domThe 2015 ADHS used an updated version of a sampling frame provided by the CSOA. The sampling frame used information about 34 provinces, control areas, districts and urban or rural residence. In the 2015 ADHS a two-stage stratified sample design was used . The firThe 2015 ADHS only interviewed ever-married women aged 15–49 years. For comparative purposes, we compare these women with the 26,730 ever-married women in the same age range in the 2010 AMS. The distribution of these women in both surveys by province and urban-rural residence is presented in This paper examines the quality of the birth history data in the two surveys using the following approaches. First, we measure age misreporting using two well-known methods: Whipple’s Index and Myers’s Blended Index. Whipple’s Index measures age heaping on ages with digits ending in 0 and 5 in the adult age range . Its norBecause we only have data on women aged 15–49 years, we calculate Whipple’s Index using the age range 18–47 years.Modifications and extensions to Whipple’s Index have been suggested in the literature ,12. HereSecond, we look at the consistency of the estimates of fertility produced by the birth histories in the two surveys. We do this in two ways, first by looking at the seasonal pattern of births and, second, by computing age-specific fertility rates (ASFRs) and the total fertility rate for four-year periods from 1984–1987 to 2012–2015 using data from the two surveys. Because both surveys are designed to be representative of the majority of the population, ASFRs for the whole country estimated for the same four-year period using weighted data should be similar for both surveys. We compute the ASFRS using weighted data to maximise comparability. The ASFRs are computed using the exact exposure method. The numerators are the total numbers of births reported by the women in each survey in each four-year period when they were in each five-year age group at the time of giving birth. The denominators are obtained by working out the exact exposure (in years and fractions of a year) for each woman in each age group in each four-year period and summing these over all women.Third, we use the ASFRs in the most recent period for each survey to estimate total fertility rates for the urban and rural populations in each province. To maximise comparability, we have amalgamated strata with fewer than about 200 women in either survey to produce a set of sub-samples defined on the basis of province and urban-rural residence which are comparable across the two surveys and which include approximately 200 women or more. We estimate the total fertility rate for each of these sub-samples for the period 2008–2010 from the 2010 AMS, and for the period 2012–2015 from the 2015 ADHS.We then examine some key determinants of fertility in the same set of sub-samples. John Bongaarts, in his classic work, built on the ideas of Kingsley Davis and Judith Blake, and identified the four most important proximate determinants of the fertility outcome in any population: the prevalence of marriage, the use of contraception, the use of abortion and the impact of breastfeeding ,5,14. HeThe pattern revealed by Myers’s Blended Index is similar to that for Whipple’s Index; in particular, in no province is the value of Myers’s Blended Index below 10, indicating substantial levels of preference for certain terminal digits in all areas of the country.An important feature of age reporting in Afghanistan is that it is not obviously better in urban areas than in rural areas in either 2010 or 2015 . It is, We turn now to the comparison of fertility patterns revealed by the birth history data in the two surveys. within each the age groups arising from the fact that we are estimating period fertility using data from a cohort aged 15–49 years at the date of interview.The total fertility rate for the period 2008–2011 based on the 2010 AMS was 6.46 and that for the same period based on the 2015 ADHS was 7.05. This also represents a substantial difference. The total fertility rate for the period 2012–2015 based on the 2015 ADHS is 6.61, still higher than that reported for the earlier period by the 2010 AMS, but marking a decline in fertility since 2008–2011.The ASFRs reported in Finally, Tables A comparison of the reported total fertility rates by province among urban areas suggests either that fertility has increased between 2008–2010 and 2012–2015 in several areas , or that fertility was under-reported in these areas 2010 AMS relative to the 2015 ADHS. Regional patterns are hard to discern, save perhaps especially high fertility in urban areas in Eastern province. The low fertility in the city of Herat is consistent with a very high (by Afghan standards) prevalence of modern contraception.In rural areas the figures suggest fertility increases between 2008–2010 and 2012–2015 in several provinces . However, some of the reported total fertility rates are suspiciously low. In 2008–2010 those in Kunduz, Daykundi and Nimroz provinces seem unreasonably low when compared with the contraceptive prevalence rates among ever-married women, which in Kunduz and Daykundi provinces are also some of the lowest in the country. In 2012–2015 the total fertility rate of 3.5 in Ghazni province seems unreasonably low when the contraceptive prevalence rate of 12.1 per cent is taken into account.There are also some dramatic changes in the total fertility rate between 2008–2010 and 2012–2015 in provinces such as Logar . In parBreastfeeding for between one and two years is common in all areas of Afghanistan. In all provinces, and in urban and rural areas, at least half the children aged 12–23 months at the time of the 2015 ASHS were being breast fed Tables . It seemWe focus this discussion eventually on the quality of the birth history data but, first, let us make some remarks about recent fertility trends in Afghanistan. Our results confirm the observations of Thomas Spoorenberg based only on the 2010 AMS that Afghanistan entered its fertility transition around the turn of the century . The ferThe fertility data in both the 2010 AMS and the 2015 ADHS have their shortcomings. In common with other neighbouring south Asian countries, age heaping on ages ending in the digits 0 and 5 is very pronounced. This has the potential to affect reported fertility rates but the precise effects will depend on the nature of the misreporting . The age heaping is a feature of both urban and rural populations, but is slightly less prevalent among women with some education than those with no education. Trends in age-specific fertility revealed by the two surveys are broadly consistent, especially at ages 15–19 and 20–24 years. At older ages, the 2015 ADHS reports higher fertility than the 2010 AMS, and there are probably several factors contributing to this, all tending in the same direction.Regional patterns of fertility are difficult to discern in either survey, and in some provinces there is reason to believe that the birth history data in one or both surveys are deficient. A few provinces have very low reported fertility, and in others there are dramatic increases or decreases in the current and recent fertility levels reported by the 2010 AMS and the 2015 ADHS. Nevertheless, some regional patterns seem robust. High fertility seems characteristic of Eastern region and rural areas of Urozgan province. The city of Herat has low fertility and a high contraceptive prevalence rate.Afghanistan has only conducted two nationally representative surveys which collected birth history data: the 2010 Afghanistan Mortality Survey (AMS) and the 2015 Afghanistan Demographic and Health Survey (ADHS). In this paper we have examined the accuracy of age reporting in the two surveys, the consistency between the estimates of the trends in age-specific fertility they generate, and the plausibility of the provincial fertility estimates when set alongside estimates of the proximate determinants of fertility.Our conclusion is that both surveys have weaknesses, notably in the reporting of women’s ages. The 2010 AMS suffers from the omission of female births in the south of the country, and perhaps among older women, but this was rectified to some extent in the 2015 ADHS. Some provinces have unusually low fertility rates given the reported rates of use of modern contraception. Despite this, the two surveys both reveal the onset of the fertility transition in Afghanistan around the turn of the century, and there is a broad consistency in the reported trends in age-specific fertility. Overall, we conclude that the fertility data in the two surveys can be used with care to give an indication of broad regional fertility patterns and trends in the country.


In tropical regions, as in temperate regions where seasonality of forage production occurs, well-preserved forage is necessary for animal production during periods of forage shortage. However, the unique climate conditions (hot and humid) and forage characteristics (high moisture content and low soluble carbohydrate) in the tropics make forage preservation more difficult. The current study used natural ensiling of tropical forage as a model to evaluate silage characteristics under different temperatures (28°C and 40°C).Four tropical forages  were ensiled under different temperatures (28°C and 40°C). After ensiling for 30 and 60 days, samples were collected to examine the fermentation quality, chemical composition and microbial community.Cyanobacteria originally dominated the bacterial community for these four tropical forages and was replaced by Lactobacillus and Enterobacter after ensiling.High concentrations of acetic acid (ranging from 7.8 to 38.5 g/kg dry matter [DM]) were detected in silages of king grass, paspalum and stylo with relatively low DM  content, acetic acid production was promoted with increased temperature and prolonged ensiling. Small concentrations of organic acid (ranging from 0.3 to 3.1 g/kg DM) were detected in silage of white popinac with high DM content (50.8% FM). The microbial diversity analysis indicated that Lactobacillus and Enterobacter were the most probable genera responsible for tropical silage fermentation.The results suggested that forage silages under tropical climate conditions showed enhanced acetate fermentation, while high DM materials showed limited fermentation.  With the rapid economic development in tropical areas, the demand for animal products is increasing greatly. It is well known that tropical forage is sufficient or even available in excess for animal feed during the fast growing season, whereas there can be limited supply in winter when the forage grows slowly. This has become the main factor limiting the improvement of animal product quantity and quality in these regions. Therefore, a suitable storage technique for green forage is necessary to cover these shortages in the tropics .Ensiling has been regarded as a common way for preserving green forage in temperate areas, because of its long shelf life, good palatability and high nutrition . HoweverTemperature is one of the crucial factors impacting silage quality. Different temperature conditions can affect the activity of microorganisms in silages, which can also influence the fermentation process . In genePennisetum purpureum×P. americana), paspalum , white popinac , and stylo (Stylosanthes guianensis) are widely cultivated forage species with high yield and good palatability for livestock in this region. Increasing efforts are devoted to improving their preservation for animal production maintenance. This study was designed to evaluate the natural fermentation characteristics of these tropical forage silages under different temperatures (28°C and 40°C).Hainan is the southernmost province of China with the typical tropical climate of warm to hot and moist throughout the year. King grass  were harvested at the vegetative stage in July, 2016.The experiment was conducted at the experimental base of the Tropical Genetic Resource Institute of the Chinese Academy of Tropical Agriculture Science, Dan Zhou, Hainan, China . Four forages , and the air was removed using a vacuum sealer. The silos were stored at low temperature (28°C in incubator) and high temperature (40°C in incubator) for 30 and 60 days. Each treatment of the four forages was conducted in triplicate.−1 to 10−5 in sterilized water. Bacterial counts were estimated for lactic acid bacteria (LAB) using de Man, Rogosa and Sharpe agar . Yeasts and molds were enumerated on spread plates of potato dextrose agar. The plate cultures were incubated at 30°C for 48 h . Microbial counts were converted to log10 and presented on a fresh material (FM) basis.Silage samples (10 g) were blended with 90 mL of sterilized water and serially diluted from 103-N) and organic acid contents were determined from water extracts. The pH was measured using a glass electrode pH meter . The NH3-N content was analyzed using the method described by Broderick and Kang [Silage pH, ammonia nitrogen (NHand Kang . Lactic Dry matter (DM) content was determined by drying the material in an oven at 65°C for 48 h. Crude protein (CP) was analyzed according to the guidelines provided by Latimer . NeutralThe four pre-ensiled forages and silage samples were added to a 20×volume of sterilized phosphate-buffered saline (pH 7.4), and DNA extraction was performed as described by Ni et al . The V3–http://drive5.com/uparse/), and chimeric sequences were identified and removed using UCHIME. The taxonomy of each 16S rRNA gene sequence was analyzed using the ribosomal database project (RDP) Classifier (http://rdp.cme.msu.edu/) against the SILVA (SSU115) 16S rRNA database with a confidence threshold of 70%. The alpha-diversities of the samples, mainly the Shannon index, Chao richness estimator, and Good’s coverage, were calculated using Mothur . Taxonomic classification at the genus level was performed using the RDP algorithm to classify the representative sequences of each OTU [Operational taxonomic units (OTUs) were clustered with a similarity cutoff of 97% using UPARSE  factorial treatment structure. The means were then compared for significance using the Duncan’s multiple range method. All statistical analysis was performed using the general linear model procedure in SAS 9.0 . Significance was declared at p<0.05 unless otherwise noted.The chemical composition and microbial community composition of the four tropical forages prior to ensiling are shown in 3-N content increased with prolonged ensiling time (p<0.001) and ranged from 10.1 to 17.5 g/kg DM for king grass silages. There was no significant effect of storage temperature on NH3-N for these tropical silages. Similar to the results of organic acid contents, white popinac silage had the lowest NH3-N content .Fermentation characteristics of the four tropical silages are shown in Chemical composition of the silages is shown in The interaction of ensiling days and storage temperature had significant effects on the population of LAB by enumeration using culture-based approaches p<0.001; . After 3High-throughput sequencing of 16S rRNA gene amplicons was conducted to comprehensively characterize bacterial communities in four tropical forage silages. A total of 766,028 bacteria sequences  were obtained. After bioinformatics analysis, a total of 552,860 sequences were classified. As listed in The dynamic variance of bacterial community structure of four tropical forage silages was demonstrated by principal component analysis PCA; , where pCyanobacteria accounted for 71.7%, 38.9%, 93.2%, and 71.4% in pre-ensiled king grass, paspalum, white popinac and stylo, respectively; its proportion decreased to <1% after 30 days ensiling and increased to 33.4% after 60 days ensiling at 40°C in king grass silages. The opposite trend was found in white popinac, where the proportion of Cyanobacteria decreased gradually with prolonged ensiling and increased temperature; the proportion of Cyanobacteria was only 0.4% after 60 days ensiling at 40°C. For paspalum and stylo, Cyanobacteria decreased to marginal levels after ensiling. Lactobacillus and Enterobacter became the two predominant genera after ensiling, accounting for 11.6% and 33.7% of the total sequences, respectively. These two high abundance genera corresponded to the bacterial genus analysis in Lactobacillus ranged from 8.2% to 25.8% in king grass, 0.2% to 7.4% in paspalum, 14.2% to 44.4% in white popinac, and 2.9% to 7.2% in stylo; while the proportion of Enterobacter ranged from 9.2% to 60.7% in king grass, 27.5% to 64.4% in paspalum, 0.9% to 26.7% in white popinac and 63.3% to 74.6% in stylo. Many other LAB genera, i.e., Enterococcus, Pediococcus, Weissella, and Lactococcus, were also detected after ensiling  was mainly detected at 40°C after 30 days ensiling. Bifidobacterium was also detected in these silage samples, mainly in king grass silages, and it increased gradually with increased temperature and prolonged ensiling (0.4% to 26.5%).Differences in bacterial community among the pre-ensiled forages and silages at the genus level with the threshold at >2.0% of the total, are shown in ensiling . The proAlthough ensiling technique is quite mature and has been widely applied in temperate regions, it is not common in tropical areas, despite the high yield of tropical forages. Feeding silage is expected to improve the productivity of livestock in the tropics and increasing attempts are being carried out to develop a suitable ensilage process ,16. The It is well known that pH and organic acid are important indicators reflecting microbial activity and silage fermentation. Previous studies reported that high temperatures (around 40°C) were unfavorable for LAB fermentation, leading to decreased organic acid concentration, increased pH and having a detrimental effect on the fermentation process for many temperate forage silages  ,6. HowevIt is well documented that lactate type fermentation is dominant in temperate forage silages. In the present study, acetic acid content was much higher than lactic acid in king grass, paspalum and stylo silages, and its content increased as the temperature increased and the ensiling was prolonged. Similar results were found in other studies that acetic acid was considered as the main acid responsible for preservation in tropical grass silages ,3,4. NisEnterobacter and hetero-fermentative LAB [The acetic acid production was higher at high storage temperature (40°C) when compared with that at low temperature (28°C); a similar trend was also demonstrated with guinea grass, maize and total mixed ration silages in temperate areas ,18,19. Ttive LAB . This mi3-N was measured to follow potential protein breakdown that might have resulted from elevated temperature and prolonged ensiling time. Previous studies on temperate forages reported that high temperatures (around 40°C) usually led to detrimental effects on the fermentation process by facilitating the growth of proteolytic bacteria, and enhancing proteolysis, thereby exacerbating the NH3-N production. Our results were inconsistent with this trend, as high temperature exhibited no significant effects on NH3-N in the present study. A possible explanation for such differences might be that the high acetic acid content at high temperature inhibited some proteolytic microorganisms [NHrganisms .Lactobacillus was present at higher proportions when stored at 40°C than 28°C after 30 days ensiling in these four silages, which could account for the increases in the lactic acid content, and decreases in pH observed at high temperature. The dominance of Lactobacillus is desirable during silage fermentation as it is associated with increasing lactic acid concentration while reducing the pH [Lactobacillus proportion declined with increases in organic acid and the gradual decline in pH in silages at 40°C after 60 days ensiling. This might because the microflora evaluation was only performed at the beginning of the process and at 30 and 60 days of fermentation, whereas the fermentation products were accumulated throughout the fermentation process. Another possible explanation is that, in addition to Lactobacillus, some other bacterial genera could regulate the fermentation of tropical forages over long periods of ensiling.Microorganisms play an important role in silage fermentation; however, investigations on the microflora associated with tropical fermentation are rare. In this study, the microbial diversity of the four tropical forage silages was systematically analyzed by both culture-based and culture independent methods. The results indicated that numbers of yeasts and molds decreased during the ensiling process, especially for the silages stored at high temperature, where yeasts and molds were not detected after ensiling. The LAB counts were higher in silages stored at 40°C than at 28°C after 30 days ensiling, and this was consistent with the results of the high-throughput sequencing of metagenomic DNA, g the pH ,21,22. HEnterobacter and high acetic acid content in tropical guinea grass silages by denaturing gradient gel electrophoresis (DGGE) analysis, however, the abundance of this genus was unclear, as DGGE analysis is not quantitative. In the present study, the bacterial community analyzed by high-throughput sequencing confirmed this suggestion. Enterobacter was the predominant genus for the silages of king grass, paspalum and stylo with high acetic acid concentration. Enterobacter is a common genus found during silage fermentation and, various species of the Enterobacter genus were found in forage silages like corn, alfalfa, guinea grass and other forages in the tropics [Enterobacter might account for the rapid decrease in undesirable microorganisms during the ensiling process and might be responsible for the findings of Ostling and Lindgren [Enterobacter exhibited a positive effect on silages, with an unexpected increase in storage stability.Nishino et al  suggeste tropics ,23,24. T tropics . The mai tropics ,27. Rega tropics , the relLindgren  who repoBifidobacterium is well known as an important intestinal probiotic for humans and animals. This genus possesses a unique fructose-6-phosphate phosphoketolase pathway employed to ferment carbohydrates to acetic acid and lactic acid [Bifidobacterium was identified in this study, mostly in king grass silages, and the abundance at high temperature was higher than that in low temperature silages; this might partly be related to the high contents of lactic acid (7.1 g/kg DM) and acetic acid (38.5 g/kg DM) that were found in king grass after 60 days ensiling at high temperature. Similar results were also reported for natural fermented alfalfa silage [Bifidobacterium abundance increased with high acetic acid content throughout the ensiling period.tic acid . Bifidoba silage , that BiEnterococcus, Weissella, Pediococcus, and Lactococcus were found at considerable proportions in these silages; similar results were also found in other temperate forages, i.e., oat, soybean and grass silages [Some other LAB genera i.e.,  silages –33. Ni eLactobacillus. The low content of organic acid and high pH of white popinac silage in this study might imply that the DM content affected the mechanism of the forage fermentation and that the quantity of LAB did not guarantee efficient tropical silage fermentation.In this study, similar fermentation characteristics were observed for the tropical silages tested, except for white popinac, which produced few fermentation products. The chemical composition of white popinac showed that it had the highest DM content before ensiling with the highest residual WSC after ensiling, which might imply that the extent of fermentation was restricted. McDonald et al  reportedCyanobacteria, as the predominant microorganism before ensiling, was mainly replaced by Lactobacillus and Enterobacter after ensiling. These two genera were probably most responsible for the unique fermentation pattern in these four tropical forages.The fermentation characteristics, chemical composition and microbial community of the four tropical forages  under different temperatures (28°C and 40°C) of ensiling were studied. The results indicated that the four tropical forage silages are prone to enhanced acetic acid production with increased temperature and prolonged ensiling. The fermentation of white popinac with high DM was slow with low concentrations of fermentation products.


The bacterial count decrease seemed greater with the EBHS compared with the detergent/disinfectant, but the difference was not significant. Therefore, within the limits of a single application, the EBHS was an efficacious means of BP cuff disinfection. However, the repeated exposure to emollients contained in EBHS may require further studies before validating these results.Blood pressure cuffs (BP cuffs) have been implicated in some nosocomial outbreaks. We compared the efficacy of an ethanol-based hand sanitizer (EBHS) with a detergent/disinfectant for the disinfection of BP cuffs. The inner sides of 30 BP cuffs were sampled for bacterial culture. Then, the same area was divided into halves. One half was disinfected by a detergent/disinfectant and the other was disinfected by an EBHS. The bacterial count decreased significantly with both disinfectants ( As with many other patients with cardiovascular risk, patients with chronic kidney disease check their blood pressure very often, to reduce the complications of high blood pressure. Regrettably, blood pressure cuffs (BP cuffs) are implicated in nosocomial infections, with potentially high clinical and economic costs ,2,3,4,5.Most guidelines recommend BP cuff disinfection between each patient with a detergent/disinfectant , but theStaphylococcus aureus (MRSA), vancomycin-resistant enterococci, Acinetobacter baumanii and Clostridium difficile-associated diarrhea [Staphylococcus aureus [Improving environmental disinfection can reduce health-care associated infections, notably those due to methicillin-resistant diarrhea . In a Jadiarrhea . Similars aureus .Ethanol based hand sanitizers (EBHS) include an emollient to protect the skin. They are well tolerated and easily available in hospital wards, but their efficacy in disinfecting BP cuffs has not been evaluated. The aim of this study is to compare the efficacy of an EBHS and of a detergent/disinfectant for the disinfection of BP cuffs.Thirty nylon BP cuffs, used daily, from 14 medical units of a general hospital  were analyzed. The 14 units were medicine wards . In the hospitalization wards, each nurse made a “blood pressure tour” per day, for 11 patients on average. As there are three nurse shifts per day in each ward, each BP cuff was used on average 30 times per day. The recommended practice is to disinfect the cuff with a detergent/disinfectant after each measure, but the common practice is to disinfect the BP cuff after it has been used on a carrier of multiresistant bacteria, on immunosuppressed patients, and at the end of the “blood pressure tour”. The nurses disinfect the BP cuffs with the detergent/disinfectant, they wear gloves, pour the detergent/disinfectant on a cloth, and rub the inner surface of the BP cuff.TM, Copan) an area of 10 cm × 10 cm, in the inner side of each BP cuff. Then, this area was divided into halves. One half was disinfected with 2 mL of detergent/disinfectant  and the other half was disinfected with 2 mL of an EBHS NPCR, Anios France). Once fully dry (10 min), each half area was sampled. Samples were cultured using tryptone soy agar with sheep blood plates (BioMérieux®) and chromogenic media for detection of Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL)-producing enterobacteria, and vancomycin-resistant enterococci (VRE). After 48 h incubation at 37 °C, bacteria colony forming units (CFU) were quantified and the two dominant species were identified by mass spectrometry with a matrix-assisted laser desorption ionization time-of-flight .For the study, we sampled with a swab  were made by a non-parametric test (Wilcoxon for paired samples), taking into account the Bonferroni correction, considering that we performed three tests.Each BP cuff was disinfected three times a day in 57% of the units, and once a day in the remaining units (43%).Staphylococci 77%, including Staphylococcus lugdunensis 1.7%; MRSA 3.3%), bacteria derived from the mucosae , and from the environment (Acinetobacter johnsonii and lwoffii 5%). Non-pathogenic bacteria (8.3%) included Micrococcus luteus (5%) and Bacillus sp. (3.3%) (Most of the bacteria found (55/60 = 91.7%) were potential pathogens. These included cutaneous bacteria (coagulase-negative . (3.3%) .2. After disinfection with the detergent/disinfectant and the EBHS, the average bacteria CFU was 16 and 10 CFU/100 cm2, respectively (p < 0.0001 for each). The bacterial count decrease seemed greater with the EBHS compared with the detergent/disinfectant, but the difference was not significant (p = 0.338). After disinfection, the virulent bacteria MRSA, Staphylococcus lugdunensis, and Enterococcus faecalis were no longer isolated. Before disinfection, the average bacteria CFU was 82 CFU/100 cmectively . The Wil2 (range 0 to 2.3).The number of bacteria colony forming units is presented in log10 per 100 cmp < 0.0001) decrease of bacteria counts with the detergent/disinfectant and the ethanol hand sanitizer (Wilcoxon non-parametric test). The difference between the detergent/disinfectant and the hand sanitizer is not significant (p = 0.338).Statistical analyses show a significant , and Staphylococcus lugdunensis ; other authors found this predominance too, with 8% to 45% of BP cuffs contaminated ,12. Sixtis 3.3%, . Other a% to 33% ), and vaStaphylococcus lugdunensis, Enterococcus faecalis), pneumonia (Pseudomonas aeruginosa), severe diarrhea (Clostridium difficile), and device-related infections . This may be of particular importance in immunocompromised patients, such as many patients with chronic kidney diseases, and in patients with medical devices like prostheses, cardiac devices, and catheters, as is the case for many dialysis patients. Most of these bacteria may cause life threatening infections like bacteremia, endocarditis (MRSA, The downside of the EBHS is that it contains emollients. The routine use of such solutions would lead to the build-up of a sticky residue on the surface of the cuffs, necessitating periodic washing. Furthermore, it is not known if the accumulation of the emollient could interfere with EBHS efficacy. Further research may be addressed to test the disinfecting efficacy of the EBHS after repeated applications on BP cuffs.Within the limits of a reduced sample size, and of testing only one application of the EBHS or of detergent/disinfectant, our study has the merit of driving attention on a neglected aspect of daily care, BP cuff disinfection, and, in a broader sense, of making us reflect on the importance of simple maneuvers in infection control in medical wards. Our study, within the limits of a small sample size and a single application, suggests that EBHS may be an efficacious means for BP cuff disinfection. In hospitalization wards, hand sanitizer is usually more easily available and is better tolerated than detergents/disinfectants. However, the repeated exposure to emollients contained in EBHS may require further study before validating these results.


Viruses will explore the impact of VSGs on the outcome of virus infection and the role of these genes within the infected cells and organism. We have focused on the most recent discoveries in VSG research, including the molecular biology of related virus–host interactions. Topics include studies on various steps of gene induction, innate immune responses to virus infection, and the mechanisms of virus immune evasion of related host defense pathways. The clinical presentation of VSG-driven pathology and strategies to use VSGs to cure chronic viral infections are also discussed. In this special Issue, we endeavored to assemble a collection of research papers and reviews that, together, will offer a comprehensive view on new areas of research pertaining to VSGs. Over the last few decades, accumulating evidence has demonstrated that numerous Interferon (IFN) stimulate genes (ISGs) can be directly upregulated following virus infection independent of IFN signaling. Indeed, whereas typical ISGs are driven by JAK-STAT signaling, other virally-stimulated genes are upregulated through the IRF3 and NF-κB pathways . These gMajzoub et al.  contribuDespite the recent progress in our understanding of these host defense responses, much more is left to be unraveled with respect to the regulation of antiviral responses triggered by distinct viral species. Clearly, IFN is a major component of host defense responses, but there are many ISGs whose antiviral activities remain to be clarified that can be regulated independently of IFN. These new genes would likely be ideal candidates to be targeted through therapeutic intervention as novel antiviral strategies.We hope this special issue provides a wealth of basic scientific data and introduces new concepts pertaining to ISGs that are regulated independently of IFN signaling.


Crosstalk between the BMP and TGF-β signaling pathways regulates many complex developmental processes from the earliest stages of embryogenesis throughout adult life. In many situations, the two signaling pathways act reciprocally. For example, TGF-β signaling is generally pro-fibrotic, whereas BMP signaling is anti-fibrotic and pro-calcific. Sex-specific differences occur in many diseases including cardiovascular pathologies. Differing ratios of fibrosis and calcification in stenotic valves suggests that BMP/TGF-β signaling may vary in men and women. In this review, we focus on the current understanding of the interplay between sex and BMP/TGF-β signaling and pose several unanswered questions. The distinct developmental mechanisms that bring about the dramatic differences in male and female characteristics are well studied. However, understanding of the impact of sex-associated signaling on the bone morphogenetic protein (BMP), transforming growth factor (TGF)-β and other pathways in developing animals and during adult life is incomplete. Cardiac valvulogenesis is just one of the many complex developmental processes where both BMP and TGF-β signals, along with WNT, fibroblast growth factor (FGF), NOTCH, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) signals, orchestrate differentiation and morphology ,2,3. As Bmp15 (GDF9B) maps to the X chromosome in humans, rodents and sheep  were identified in a screen for X-chromosome inactivation modulators . ChromatUnanswered questions: Human X-chromosome inactivation and reactivation have profound consequences on cellular reprogramming and disease . LikewisGenetic Imprinting is another influential cellular process controlled by sex, in this case, that of the parents. Imprinted genes are expressed on either the maternal or paternal allele, but not both. This monoallelic expression causes a variant or mutated allele to produce a different phenotype based on the parental origin of that imprinted gene. Two hundred and fifty human and 150 mouse imprinted genes were surveyed . BMPs acription . An excecription ,36,37,38cription . Althougcription ,40. Unanswered questions: A daunting web of BMP/TGF-β additive, synergistic and antagonistic actions among members of the ligand superfamily and signal mediators occurs in reproductive tissues. Each interaction within this panoply “may” occur in other tissues. The challenge is to identify which interactions also occur in other tissues., phosphorylated SMAD1/5/8(9), are potent pro-osteogenic signals strongly implicated in pathological calcification [In just the context of cardiovascular biology, sex profoundly influences heart and vascular health, with estrogen generally playing a protective role ,52. Thatfication ,68,69,70fication ,71,72. Ufication ,73.Unanswered questions: Clear sex-specific differences in the relative levels of calcification and fibrosis in aortic valves occur. Although BMP/TGF-β signaling strongly influences these processes, few studies have addressed the regulation of these pathways in each sex. What are the relative levels of each BMP/TGF-β ligand in healthy and diseased valves in men and woman? How do sex and hormonal status influence the relative activities of signaling mediators and extracellular and intracellular antagonists of signaling? Most importantly, what therapeutic strategies may modulate the balance of BMP/TGF-β signaling optimally for each sex?Many factors lead to sex-specific differences in disease incidences and manifestations and in therapeutic efficacy . These i


Pearson analysis showed that there was a statistically significant correlation between UA levels with mean corpuscular hemoglobin, mean corpuscular hemoglobin concentrations, mean corpuscular volumes, RBC counts, and hemoglobin (p < 0.05). Multiple linear regression analysis suggested that there were statistically significant positive correlations between UA levels and RBC counts , as well as UA levels and hemoglobin concentrations . Furthermore, similar results were observed in both the male and female subgroups. The serum UA levels may be independently associated with RBC parameters, regardless of sex, and UA may protect RBCs owing to its antioxidant effect.Uric acid (UA) is a major antioxidant molecule in the human blood, and it has been linked with cell longevity. However, it is unclear whether serum UA levels are associated with red blood cell (RBC) indexes. This cross-sectional study included 10,759 Chinese subjects, recruited from the Shanghai Xuhui Central Hospital from January 2014 to December 2017. The participants were categorized into gender groups and then further divided into three different subgroups according to their UA reference range as follows: low , female (UA < 0.143 mmol/l)), normal , female (0.339 mmol/l > UA ≥ 0.143 mmol/l)), and high , female (UA ≥ 0.339 mmol/l)). The associations of UA levels with RBC parameters were analyzed using 1-way ANOVA, Pearson correlations, and multivariate linear regression. The levels of mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, RBCs, and hemoglobin were lowest in the low UA group, followed by the normal UA group and high UA group ( This loss of UA oxidase may have evolutionary advantages [p < 0.05) [Uric acid (UA) oxidase, an enzyme that converts UA to 5-hydroxy isourate and Hvantages , 2, as tvantages . UA, a nvantages –5. The l < 0.05) –9.There has been increasing experimental and clinical evidence suggesting that higher plasma UA levels may protect humans from cancer, multiple sclerosis, central nervous system diseases, glaucoma, and other life-shortening disorders –16. For RBCs play an important role in the transport of oxygen from the lungs to other tissues. There is growing evidence to suggest that oxidative stress plays a significant role in damaging the RBC membrane and impairing its deformability –20. DuriAlthough UA has been shown to play a role in RBC storage in vitro as an endogenous antioxidant, to our knowledge, the relationship between UA levels and RBCs in human health has not been studied previously. It can be hypothesized that low levels of UA are associated with increased oxidative stress and inflammation, and UA may provide protection for RBCs through its antioxidant properties. Thus, we performed this large-sample cross-sectional study to assess the association between serum UA levels and RBC indexes.The study was approved by the Ethics Committee of the Shanghai Xuhui Central Hospital (2018025), Shanghai, China, and was conducted according to the Declaration of Helsinki. Subjects were recruited from participants in yearly health screenings of the Shanghai Xuhui Central Hospital from January 2014 to December 2017 according to the inclusion criteria listed below.Medical examinations were performed by respective physicians for all subjects at the Shanghai Xuhui Central Hospital. Those on treatment with agents affecting laboratory parameters, including patients with hematological disorders and chemotherapy treatment, were excluded. Subjects had no diabetes, hypertension, anemia, hematological system diseases, active infections, or major systemic diseases (autoimmune diseases and cancer) that could affect the levels of erythrocyte parameters. Children (age < 14) were also excluded. Based on these criteria, 7791 subjects were excluded from the study. In addition, 488 subjects were excluded because of missing information. As part of the standard of care at the Shanghai Xuhui Central Hospital, peripheral blood samples were routinely collected and tested for UA levels and complete blood counts. Clinical and demographic information were obtained from the medical data platform of the hospital.Complete blood counts that included mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), red blood cell distribution width (RDW), RBCs, and hemoglobin were measured with the Mindray BC-5500  automatic blood counting system. The blood samples (2 ml) were collected in ethylenediaminetetraacetic acid (EDTA) tubes. RBC parameters were measured within 0.5 hours after blood collection. Then, another 4 ml blood sample, which was obtained in the morning after the subjects had fasted for 8 hours, was used for UA and creatinine (Cr) measurements. Serum levels of UA and Cr were measured within 3 hours after blood collection by enzymatic colorimetry using a commercially available kit . Internal controls were analyzed daily over the 3-year period, with typical monthly CVs of 2%–7% and no significant changes in measured values.The reference range of UA was 0.2023-0.4165 mmol/l for males and 0.1428-0.3392 mmol/l for females. Therefore, the subjects were categorized into gender subgroups. According to the level of serum UA, the male subjects were divided into 3 subgroups (low UA group (UA < 0.202 mmol/l), normal UA group (0.417 mmol/l ≥ UA ≥ 0.202 mmol/l), and high UA group (UA > 0.417 mmol/l)). The female subjects were also divided into 3 subgroups (low UA group (UA < 0.143 mmol/l), normal UA group (0.339 mmol/l > UA ≥ 0.143 mmol/l), and high UA group (UA > 0.339 mmol/l)).p < 0.05 was considered statistically significant.The data were analyzed using SPSS13.0  and GraphPad Prism 6 software . The results are presented as the mean ± standard deviation (SD). The UA/Cr ratio is reported as UA∗1000/Cr. Normality was assessed with the Kolmogorov-Smirnov test. The chi-square tests were used to compare the categorical variables among the three groups. The one-way ANOVA test was used to compare the continuous variables among the three groups. The associations between UA levels and RBC parameters were analyzed using Pearson correlation analysis. After simple linear correlation analysis, multivariate linear regression analysis was performed to confirm RBC parameters that were associated with UA levels and UA/Cr ratios. A two-sided A total of 10,759 subjects  were studied according to the inclusion criteria. Subjects' average age was 50.17 ± 16.22  years. The general and clinical characteristics of the study participants are presented in p < 0.001). Similar results were also observed in the male subgroup (p = 0.815) (p < 0.001); however, no correlation was found between RBC levels and MCH or MCHC levels  . HoweverC levels .p < 0.05). Furthermore, similar results were also found between the UA/Cr ratio and RBC parameters. The specific analysis results are shown in Pearson analyses were performed to identify the associations between serum UA levels and the UA/Cr ratio with RBC parameters. UA levels significantly correlated with MCH, MCHC, MCV, RBC, and hemoglobin levels p < 0.0. FurtherB = 0.245, p < 0.001, 95% CI = 0.003 to 0.092), as well as UA levels and hemoglobin levels , the UA/Cr ratio and RBC counts , and the UA/Cr ratio and hemoglobin levels . Furthermore, similar results were also observed in both the male and female subgroups  in either the outer or inner monolayer of erythrocyte membranes . UA was p values, the r values varied from small to moderate power in some cases. The reason may be that the diurnal variation of UA levels and day-to-day in-person variation of red blood cell indicator variables were not taken into account. Moreover, several subjects with UA levels as low as 0.05 mmol/l may influence the results of simple linear correlation. Therefore, further research was necessary in order to confirm our results.Strengths of our analysis include the relatively large sample size, the subgroup analysis including all subjects, and an analysis further adjusted for an extensive set of possible confounding variables. However, our study also has some limitations that should be considered. First, as this is a cross-sectional study, our ability to explore the exact mechanisms underlying the associations between serum UA concentration and RBC remained limited, and we did not measure endogenous and exogenous uric acid separately. Second, although children and other patients with conditions that can affect UA concentrations, including diabetes, hypertension, and anemia, were excluded from this study, several subjects with UA levels as low as 0.05 mmol/l were identified. The reason may be that it is more difficult to detect hereditary hypouricemia and xanthurenic aciduria through medical screenings; thus, such patients were not excluded from this study. Moreover, we did not consider how differences in exercise habits, smoking habits, drinking habits, and dietary preferences might affect serum UA concentrations. Third, despite the significantly low In summary, our study suggested that there was a significant positive correlation between UA levels with RBC counts and total hemoglobin concentrations. To the best of our knowledge, this is the first study to report that serum UA levels may be independently associated with RBC parameters. Our findings support the hypothesis that UA plays a beneficial role in RBC longevity.


Development of organ dysfunction discriminates sepsis from uncomplicated infection. The paradigm shift implicated by the new sepsis-3 definition holds that initial impairment of any organ can pave the way for multiple organ dysfunction and death. Moreover, the role of the systemic inflammatory response, central element in previous sepsis definitions, has been questioned. Most strikingly, a so far largely underestimated defense mechanism of the host, i.e., “disease tolerance,” which aims at maintaining host vitality without reducing pathogen load, has gained increasing attention. Here, we summarize evidence that a dysregulation of critical cellular signaling events, also in non-immune cells, might provide a conceptual framework for sepsis-induced dysfunction of parenchymal organs in the absence of significant cell death. We suggest that key signaling mediators, such as phosphoinositide 3-kinase, mechanistic target of rapamycin, and AMP-activated protein kinase, control the balance of damage and repair processes and thus determine the fate of affected organs and ultimately the host. Therapeutic targeting of these multifunctional signaling mediators requires cell-, tissue-, or organ-specific approaches. These novel strategies might allow stopping the domino-like damage to further organ systems and offer alternatives beyond the currently available strictly supportive therapeutic options. In this “perspective,” we summarize evidence that substantial changes in these signaling pathways during sepsis occur not only in the immune system but also in parenchymal cells. To target  cellular metabolic and signaling dysfunctions will open new avenues for therapeutic interventions.Severe metabolic dysregulation and circulatory failure are central to the definition of septic shock , 3 and sA paradigmatic example of sepsis-related organ failure is excretory dysfunction of the liver. Sepsis accounts for approximately 20% of admissions for jaundice, a rate that is only surpassed by malignant compression of the bile duct . WhereasLiver dysfunction also strongly challenges overall metabolic and immunological homeostasis in the critically ill and frequently promotes progression to multi-organ failure. Prolonged hepatic dysfunction interferes with the adaptive immune system . Accordivia two distinct strategies: resistance and disease tolerance  effects. Hence, before signaling mediators can be used therapeutically, it will be necessary to overcome the significant hurdles posed by these multifunctional signaling molecules. Nanoparticle-based targeted drug-delivery , 22, 59 The manipulation of key signaling pathways can be expected to significantly add to armamentarium of sepsis therapy, in particular, in the light of increasing antibiotic resistance.MB, SC, ML, SW, BL, and RW wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.MB declares that he is co-founder and share-holder of a company aiming to develop PI3K inhibitors for target-specific treatment of organ dysfunction. RW holds patents related to the therapeutic use of PI3K inhibitors. All other 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.