Low transferrin saturation (TSAT), calculated by serum iron divided by total iron-binding capacity (TIBC), indicates iron deficiency. Because malnutrition and inflammation are associated with low TIBC in chronic kidney disease (CKD), TSAT might not reflect iron status or risk for anemia. We examined whether low serum iron was a risk factor for anemia in CKD patients with normal TSAT. Thus we compare the risk for anemia in 2500 CKD stage 1–4 patients divided by TSAT (cutoff: 20%) and serum iron . Our results confirmed low TIBC (< 200 μg/dL) was associated with hypoalbuminemia and high C-reactive protein. In fully-adjusted logistic regression, both “normal TSAT low iron” and “low TSAT low iron” groups were associated with baseline anemia (hemoglobin < 11 g/dL) (odds ratios (OR) 1.56; 95% confidence interval (CI) 1.13–2.16 and OR 2.36; 95% CI 1.76–3.18, respectively) compared with the reference group . Sensitivity tests with different cutoffs for TSAT and iron also showed similar results. In patients without anemia, both groups were associated with anemia after 1 year . In conclusion, CKD stage 1–4 patients with normal TSAT but low serum iron are still at risk for anemia. It is defined as hemoglobin (Hb) concentration < 13 g/dL in men and < 12 g/dL in women according to World Health Organization (WHO) criteria2. The prevalence of anemia defined by WHO criteria varies geographically from 15.4%, 45.0 to 51.5% in the United States, South Korea and China respectively5. The severity of anemia is associated with cardiovascular risk, hospitalization, all-cause mortality, and decreased health-related quality of life9. Underproduction of endogenous erythropoietin by atrophic renal cortex is one of the major causes of anemia in CKD patients, and treatment with erythropoiesis-stimulating agents (ESAs) increases Hb effectively1. However, the use of high dose ESAs is associated with adverse events, such as vascular access thromboembolism, stroke, and tumor progression11.Anemia is a common comorbidity in patients with chronic kidney disease (CKD)1. There are two forms of iron deficiency recognized. One is absolute iron deficiency, and the other is functional iron deficiency. Absolute iron deficiency is defined by severely decreased or absent iron storage in bone marrow, whereas functional iron deficiency is characterized by adequate or increased total body iron stores that are not available to provide sufficient iron to erythroid precursors for erythropoiesis12. Iron therapy might be useful for correction of anemia in CKD patients with absolute or functional iron deficiency. In addition, it could lessen the dosage of ESAs in CKD patients13.Iron deficiency is also common in CKD and is another substantial mechanism of anemia in CKD. It is caused by impaired dietary iron absorption, uremic bleeding, and insufficient iron replenishment for Hb synthesis15. The Kidney Disease Outcomes Quality Initiative (K/DOQI) 2006 guidelines recommend iron therapy to maintain ferritin > 100 ng/mL and TSAT > 20% in non-dialysis chronic kidney disease (ND-CKD) patients16, while the Kidney Disease Improving Global Outcomes (KDIGO) 2012 guidelines recommend iron therapy in ND-CKD patients with TSAT < 30% and serum ferritin < 500 ng/mL if an increase in Hb levels is desired17.The definition of iron deficiency in CKD is not clear. The transferrin saturation (TSAT) and serum ferritin levels have limited sensitivity and specificity in patients with CKD for prediction of bone marrow iron stores and the response to iron therapy18, is not uncommon in CKD patients owing to malnutrition and inflammation, and proteinuria21. A gold standard for iron deficiency, defined as TSAT < 15% and Ferritin < 15 ng/mL in general population22, is difficult to be applied to CKD patients with routine laboratory measurements. Moreover, iron deficiency is but one contributor to anemia in this population. Nonetheless, serum iron levels may be useful in reflecting iron availability and utilization in hemoglobin production. A recent review suggested that serum iron may provide more information to guide iron therapy than TSAT23. We thus compared the risk for anemia in CKD stage 1–4 patients with low TSAT or with low serum iron. The aim of our study was to investigate additional indications of iron therapy in CKD patients who could benefit from iron therapy aside from current guidelines.However, TSAT, calculated by serum iron divided by total iron-binding capacity (TIBC), could overestimate iron availability in the circulation if TIBC is low. Low transferrin, which is statistically correlated with TIBC24, and the estimated glomerular filtration rate (eGFR) was calculated using the equation of the 4-variable Modification of Diet in Renal Disease (MDRD) study. We excluded patients suffering from CKD stage 5 or those who were lost to follow-up in less than three months; therefore, the final study population was composed of 2500 CKD stage 1–4 patients. All participants in our study provided informed consent to participate. The protocol in the study was approved the Institutional Review Board of Kaohsiung Medical University Hospital. We performed the methods according to the approved guidelines.This prospective, observational study was conducted in two affiliated hospitals  of Kaohsiung Medical University in southern Taiwan. Totally, 3749 patients who joined the Integrated CKD Care Program Kaohsiung for Delaying Dialysis from 11 November, 2002 to 31 May, 2009 were included. CKD was staged according to the definition of K/DOQI guidelinesTo study the impact of TSAT and iron on anemia at baseline, we divided these 2500 CKD patients into four groups—“normal TSAT normal iron”, “normal TSAT low iron”, “low TSAT normal iron”, and “low TSAT low iron”. The cutoff value of TSAT was 20%, while the cutoff values of serum iron were 70 μg/dL in men and 60 μg/dL in women. To study the impact of TSAT and iron level on anemia after 1 year of follow-up, we retrospectively selected 1621 patients without anemia (Hb ≧ 11 g/dL) at baseline, after excluding patients with dialysis, mortality, loss of follow-up, and < 3 hemoglobin measurements in 1 year.25.Baseline variables such as demographic features (age and sex), medical history , clinical parameters , and laboratory data  were collected. The demographic features, medical history and clinical parameters were obtained from medical records and interviews with patients at enrollment. The medical history was obtained by chart review by 2–3 nephrologists. DM and hypertension were defined by clinical diagnosis. Hyperuricemia was defined as uric acid > 7.2 mg/dL in men, > 6.5 mg/dL in women, or under urate-lowering therapy. CVD was defined as clinical diagnosis of ischemic heart disease, congestive heart disease, and cerebrovascular disease. The definition of metabolic syndrome was the presence of three or more of the following five criteria proposed by the Health Promotion Administration, Ministry of Health and Welfare of Taiwan at 2007 (1) fasting blood glucose ≥ 100 mg/dL or diabetes mellitus; (2) systolic blood pressure ≥ 130 mmHg or diastolic blood pressure ≥ 85 mmHg or hypertension; (3) HDL cholesterol > 40 mg/dL in men or > 50 mg/dL in women; (4) triglycerides ≥ 150 mg/dL; and (5) waist circumference ≥ 90 cm in men or ≥ 80 cm in womenThe MAP was calculated by using the formula one-third averaged systolic blood pressure plus two-thirds averaged diastolic blood pressure measured 3 months before and after the enrollment. BMI was calculated as the baseline enrolled measurement by using the formula weight in kilograms divided by height in meters squared. High sensitivity CRP was measured in serum using near the near-infrared particle immunoassay rate method by Beckman Coulter UniCel-DxC 800 (Beckman Coulter); ferritin was measured in serum using two-site immunoenzymatic assay by Beckman Coulter UniCel-DxI 800 (Beckman Coulter); while iron was measured in serum using Fe reagent via timed-endpoint method by Beckman Coulter UniCel-DxC 800 (Beckman Coulter). Transferrin was measured in serum using transferrin reagent via the turbidimetric method by Beckman Coulter UniCel-DxC 800 (Beckman Coulter). And the TIBC value was gained by multiplying transferrin (mg/dL) by a coefficient of 1.4. Laboratory data were averaged and analyzed three months before and after enrollment of the CKD care system.26. MIS, modified for CKD patients, has 10 components including body weight change, dietary intake, gastrointestinal symptoms, functional capacity, comorbidity, fat stores, muscle wasting, BMI, albumin, and total iron-binding capacity. Each score component is classified according to 4 levels of severity, from 0  to 3 . Measurements were based on Subjective Global Assessment. Malnutrition-inflammation was defined by MIS > 4, based on ROC curve for outcome prediction27.Malnutrition-inflammation score (MIS) was first proposed by Professor Kalantar-Zadeh for dialysis patients28. We further defined more severe anemia as Hb < 10 g/dL since ESAs therapy was usually started below this level by current clinical practice29. Hemoglobin at 1 year was determined by simple linear regression with all hemoglobin measurements in this 1 year.The main study outcome was anemia at baseline and after 1 year. Anemia was defined as Hb < 11 g/dL as the definition in the study of Trial to Reduce Cardiovascular Events With Aranesp Therapy (TREAT)Statistical results of baseline characteristics of the subjects in the four groups were expressed as mean ± SD for continuous variables with normal distribution, median  for continuous variables with skewed distribution, and percentages for categorical data.The significance of differences for normally-distributed continuous variables among groups was tested using one-way ANOVA analysis with post hoc analysis by Fisher’s least significant difference test. And the difference for non-normally-distributed continuous variables was tested using Kruskall–Wallis analysis with post hoc analysis by Dunn–Bonferroni method. The difference in the distribution of categorical variables among groups was tested using the Chi-square test with post hoc analysis by Bonferroni method. Multivariable logistic regression models were used to identify the factors associated with anemia or low TIBC. The adjusted hierarchical covariates were as follows: model (1) demographic factors; model (2) plus comorbidities factors; and model (3) plus other covariates such as albumin, CRP log, ferritin log, and phosphorus. Statistical analyses were conducted using SPSS 21.0 for Windows . Statistical significance was set at a two-sided p-value of < 0.05.To assess the robustness of our findings, we performed a series of sensitivity analyses, including of: (1) redefining the result of low iron as < 65 μg/dL in men and 50 μg/dL in women, according to the cutoff in our hospital; (2) redefining TSAT < 30%; (3) separating the two “low iron” groups into four subgroups according to serum ferritin levels .2 (interquartile range [IQR] 24.5–48.7 mL/min/1.73 m2). There were 1230 patients (49.2%) suffering from DM, 1513 (60.5%) had hypertension, and 553 (22.1%) had CVD. The mean Hb of patients was 12.2 ± 2.2 g/dL. The subjects in the group of “normal TSAT low iron” were more likely to be older and male, and they also tended to have cerebrovascular disease, lower BMI, higher MIS, lower eGFR and higher UPCR. The post hoc analysis shown in Table The baseline demography, medical history, clinical and laboratory parameters of study participants are shown in Table The iron status of participants is shown in Table In Table As the result of the multivariable logistic regression analysis depicts in Table As compared with the subjects in the “normal TSAT normal iron” group, the subjects in both “low iron” groups had significantly higher OR for Hb < 11 g/dL  and for Hb < 10 g/dL  Table  and TSATWe followed 1695 patients with hemoglobin ≧ 11 g/dL at baseline and 22/1091, 9/220, 0/50 and 9/334 patients encountered dialysis or mortality in “normal TSAT normal iron”, “normal TSAT low iron”, “low TSAT normal iron” and “low TSAT low iron” groups, respectively, in the first year of follow-up. After further excluding those loss of follow-up, and < 3 hemoglobin measurements, 1621 patients without anemia entered the analysis. 164 of them had Hb < 11 g/dL after 1 year. The subjects in both “low iron” groups had significantly higher OR for Hb < 11 g/dL after 1 year , after adjustment for baseline hemoglobin and other variables in Tables In our observational study, CKD stage 1–4 patients with low serum iron still had the risk of anemia no matter what the TSAT levels. Notably, the subjects of the “normal TSAT low iron” group had the highest percentage of anemia, malnutrition, and inflammation. Furthermore, we also discovered that low TIBC was significantly associated with malnutrition and inflammation. The possible explanation of these 4 groups was presented in Supplementary Table 22, is quite different in CKD groups since the majority of CKD patients suffer from functional iron deficiency rather than absolute iron deficiency30. Although current guidelines use TSAT and serum ferritin as the standard tools for stratifying CKD patients who will/will not respond to iron therapy, there are still some limitations. For example, serum ferritin concentration may increase in inflammation, malignancy, and liver disease31. Therefore, other iron indices such as reticulocyte Hb content (CHr), percentage of hypochromic red blood cell (PHRC), soluble transferrin receptor test (sTfR), and hepcidin have all been utilized to evaluate iron metabolism in CKD patients36. Hepcidin, which regulates the dietary iron absorption from gut and the release of iron from macrophages, can help us to clarify the capacity of iron availability of the patients37. sTfR can reflects erythropoietic activity and is not affected by inflammation37. However, it cannot reflect iron availability completely38. Instead, the ratio of sTfR to log10ferritin is superior to sTfR alone in differentiating iron deficiency anemia from anemia of chronic disease39. CHr and PHRC reflect iron availability to the bone marrow and iron-restricted erythropoiesis37. All of these biomarkers can help us to evaluate the response to iron therapy especially for intravenous iron36. However, the cost of techniques and the availability of extensive testing must be concerned, whereas in contrast, serum iron is inexpensive and used widely. Our study suggests that low serum iron value is independently associated with anemia in CKD group. To our knowledge, this is the first paper to utilize serum iron to predict anemia in CKD subjects.The first important finding of our study was identification of low iron value as an independent risk factor of anemia in CKD stage 1–4 patients regardless of TSAT levels. Iron deficiency anemia, defined as TSAT < 15% and Ferritin < 15 ng/mL in the general population19, has a high statistical correlation with TIBC18. Kalantar-Zadeh et al. indicated that low serum TIBC is related to protein-energy wasting, inflammation, poor quality of life, and mortality19. They found that TIBC is positively associated with nutritional biomarkers such as serum albumin and negatively associated with several inflammatory markers such as log CRP. Our data shown in Table 40, who found that serum iron is low while serum ferritin is high in poorly nourished groups. Indeed, they also recommended not to use TSAT as a diagnostic tool of iron deficiency if serum TIBC is less than 200 mg/dL. They believed that TIBC, as the denominator of iron saturation ratio, might mistakenly lead TSAT ratio to normal or increased ranges especially in malnourished patients of whom the TIBC value is decreased40. Since the highest percentage of hypoalbuminemia and low TIBC value in the group of “normal TSAT low iron” was shown by our findings, it is inferred that the TSAT in this group is erroneously high due to low TIBC levels caused by malnutrition. By comparison, from this group, it appears that low serum iron is more associated with anemia in CKD patients. Our study cannot determine if iron replacement or supplementation in patients with low serum iron and normal transferrin saturations will be beneficial. This combination of findings should at the very least prompt further scrutiny of nutritional and other factors that may affect the TIBC in CKD patients.The second important finding of our study is that the subjects of the “normal TSAT low iron” group had the highest percentage of anemia, malnutrition, and inflammation. We further interpret the characteristics of the patients in this group as “anemia of chronic inflammation”. Among them, low TIBC is an important associated factor for malnutrition and inflammation. Transferrin, a 90-kDa beta-globulin, primarily synthesized by the liverSimilarly, the concept of low TIBC caused by malnutrition and inflammation could be utilized to explain the subjects in “low TSAT low iron” group, who was interpreted as “iron deficiency combined with inflammation”. Unlike the patients with absolute iron deficiency anemia, the subjects in “low TSAT low iron” group have higher malnutrition-inflammation, ferritin and CRP levels and lower TIBC values. These findings might be partially explained by inflammation.41. Our new analysis of malnutrition-inflammation score may explain the obesity paradox in CKD population27. In addition, none of patients with “low TSAT normal iron” and hemoglobin ≧ 11 g/dL encountered dialysis or mortality in the 1st year, which showed the best survival, compared with the other groups. We hypothesized that the stability of serum iron levels was driven by reductions in hepcidin levels. The low hepcidin levels would improve the iron absorption from gut and the iron release from macrophage, which induced low ferritin levels. Besides, an increase in TIBC also improved iron delivery to the bone marrow. Thus, even if TSAT was low, the increased iron bioavailability allowed adequate erythropoiesis. According to the above characteristics, we interpreted the subjects in “low TSAT normal iron” group as “iron deficiency with greater iron availability ”.“Low TSAT normal iron” group had lower percentage of malnutrition-inflammation, although higher percentage of metabolic syndrome. Our previous study had shown an obesity paradox in CKD population that high BMI was not associated with all-cause mortality, especially in female10ferritin, PHRC, and CHr, which help to determine iron availability or erythropoiesis activity, and also to predict the response to iron therapy. Especially for hepcidin, it might prove that serum iron is better than TSAT to define anemia in CKD patients with inflammation. Further study is needed to clarify the state of iron deficiency and the capacity of iron availability by using these new iron indices.There are several limitations in this study. First, the subjects was Taiwanese without iron and EPO treatment, more data in other populations and patients treated with EPO are needed before broader application. Second, we did not include patients of CKD stage 5 without dialysis. In these patients, EPO deficiency and ESAs treatment could be more important. Third, we only got the data of serum iron and transferrin in the beginning of enrollment. This might cause variability and affect clinical decision making of iron therapy. Fourth, only a few patients (0.8%) in our study received iron treatment. Neither causality nor the temporal relationship between serum iron and anemia can be determined with this observational study. Fifth, the longitudinal study about the impact of TSAT and iron on anemia after 1 year was retrospective and did not evaluate many confounders. Finally, we did not examine novel markers of iron homeostasis such as hepcidin, sTfR/logIn conclusion, our study suggests that CKD stage 1–4 patients with normal TSAT but low serum iron are still at risk for anemia. “Normal TSAT low iron” group is associated with inflammation and malnutrition, and could be at risk for “anemia of chronic inflammation”. Low serum iron might be a considerable risk factor of anemia in CKD stage 1–4 subjects regardless of TSAT levels. Further research is needed to confirm the effect of iron treatment in CKD stage 1–4 patients with low serum iron value.Supplementary Information.


Tetanus is a fatal but vaccine‐preventable disease. The currently available tetanus vaccines are tetanus toxoid (TT)‐based. Although these vaccines are generally effective, challenges in vaccine development and access remain. A randomized, double‐blind, dose escalation, placebo‐ and positive‐controlled, phase 1/2 trial (ChiCTR1800015865) is performed to evaluate the safety and immunogenicity of an alternative recombinant tetanus vaccine based on the Hc domain of tetanus neurotoxin (TeNT‐Hc) in healthy adult volunteers. The primary outcome is the safety profile of the recombinant tetanus vaccine, and immunogenicity is the secondary outcome. 150 eligible participants were enrolled and randomly assigned to receive one of the three doses of recombinant tetanus vaccine (TeNT‐Hc 10/20/30 µg), TT vaccine, or placebo. The recombinant tetanus vaccine shows a good safety profile. The frequency of any solicited and unsolicited adverse events after each vaccination does not differ across the vaccine and placebo recipients. No serious treatment‐related adverse events occur. The recombinant tetanus vaccine shows strong immune responses , which are roughly comparable to those of the TT vaccine. In conclusion, the findings from this study support that recombinant tetanus vaccine is safe and immunogenic; thereby, it represents a novel vaccine candidate against tetanus. This study is the first randomized, phase 1/2 trial comparing dose escalation recombinant tetanus vaccine (TeNT‐Hc) with TT vaccine and placebo in healthy adults. All three doses of recombinant tetanus vaccine show a good safety profile, and strong immune responses, which are roughly comparable with TT vaccine. Thus, recombinant tetanus vaccine might be a novel vaccine candidate against tetanus. In developed countries, tetanus cases are occasionally reported, most often among the older population (aged ≥60 years) or injection drug users. However, in low‐ and middle‐income countries, tetanus mortality rates remain high. According to the Global Burden of Disease Study, an estimated 56 000 deaths annually were reported from tetanus worldwide in 2015. Treatment of tetanus involves providing supportive care, neutralizing tetanus toxoid (TT) antibodies, and eradicating bacteria at the wound site. In the absence of medical intervention, the case fatality rate of tetanus remains extremely high (near 100%), even in resource‐abundant regions. Fortunately, tetanus is a vaccine‐preventable disease. The TT vaccine was first developed in 1924 and since then has become part of the recommended immunization schedule for children owing to its effectiveness in protecting against tetanus. By 2018, nearly 85% of infants worldwide (116.3 million infants) received three doses of diphtheria‐tetanus‐pertussis (DTP3) vaccine. However, DTP3 immunity is short‐lived in many individuals. Global tetanus cases and deaths have dramatically declined within the past two decades due to the scale up of immunization programs. However, to maintain an adequate concentration of protection, additional booster doses should be administered every 10 years, especially in high‐risk populations, including women of reproductive age, soldiers, the older population, or injection drug users. Barriers to access of the tetanus vaccine, especially monovalent tetanus vaccine, is one of many challenges in achieving universal coverage of the tetanus vaccine. Major impediments remain regarding the production of the current TT vaccine, including environmental pollution caused by during the formaldehyde detoxification process and low‐profit earnings, leading to low yields. Moreover, the quality of TT and how it is stored are important. Evidence has shown that 15 TT lots, in use from eight manufacturers in seven countries, had potency values below World Health Organization (WHO) requirements. These challenges, among others, demonstrate that ease and standardization of tetanus vaccine preparation are needed.Tetanus is a serious, often fatal, bacterial infection caused by tetanus neurotoxin (TeNT) produced by the anaerobic bacterium m tetani.1 In d With the advent of recombinant technologies, recombinant vaccines are being seen as promising solutions by the scientific community because of their relatively low production cost and innate stability for storage and transportation purposes. The recombinant tetanus vaccine mainly consists of the Hc domain of TeNT (TeNT‐Hc), which possesses high immunogenicity and full binding affinity to neuronal cells via gangliosides in lipid rafts. This first‐in‐humans trial aims to assess the safety and immunogenicity of recombinant tetanus vaccine in a dose range of 10–30 µg. The primary focus of this study is to prevent tetanus; further, the investigational recombinant tetanus vaccine is a recombinant TeNT‐Hc‐based candidate vaccine that may be developed into a novel vaccine against tetanus.Advancements in vaccine development have been ongoing since the beginning of the genetic engineering revolution.15 Wit22.1The recombinant tetanus vaccine is composed of the Hc domain of TeNT—the recombinant protein TeNT‐Hc based on a patented technology (Chinese invention patent No: ZL2009101359720). All three doses of the recombinant tetanus vaccine and placebo were qualified by the China Institutes for Food and Drug Control. The inspection items included appearance, volume, identification test, osmotic pressure molar concentration, protein concentration, aluminum hydroxide concentration, sterility test, abnormal toxicity test, endotoxin test, and vaccine efficacy test. The placebo contains all the recombinant tetanus vaccine excipients but without any TeNT‐Hc. The recombinant tetanus vaccine and placebo were produced by the Beijing Institute of Biotechnology and Sichuan Zihao Times Pharmaceutical Co., Ltd. The TT vaccine used in the study was a monovalent adsorbed TT vaccine made by Olymvax Biopharmaceuticals .2.2n = 30) or withdrawal of consent (n = 3). A total of 150 participants received either a low‐dose recombinant tetanus vaccine, medium‐dose recombinant tetanus vaccine, high‐dose recombinant tetanus vaccine, TT vaccine, or placebo as prime immunization on day 0, with 30 participants in each treatment group. A total of 149 participants received booster immunizations. In this trial, 8 (5.3%) participants discontinued or were excluded from the per‐protocol population—three from the low‐dose group, two from the high‐dose group, two from the TT vaccine group, and one from the placebo group. A total of 150 participants were included in the safety analysis, and 142 (94.7%) participants in the immunogenicity analysis . Among the 90 participants who received the recombinant tetanus vaccine, 32 (35.6%) reported at least one solicited adverse reaction within the first 7 days after the initial vaccination, and 42 (47.2%) reported after booster vaccination. The injection‐site reactions were frequent after both doses, with redness  most commonly reported, followed by injection‐site pain without touching , induration , itching , and rash . Systemic adverse reactions are typically rare, and none have been reported. Redness was the only injection‐site adverse reaction reported more frequently in the medium‐dose (p = 0.02) and high‐dose (p = 0.01) recombinant tetanus vaccine groups than in the TT vaccine groups. However, increased rates of injection‐site reactions were not associated with increases in TeNT‐Hc dose, and increases in the severity of local reactions were not linked to booster vaccinations. At the end of the study, all local reactions were completely resolved. None of the injection‐site reactions required any medical treatment.Our results suggest that all three doses of recombinant tetanus vaccine were highly safe for the healthy adult participants in China. Some of the subjects experienced solicited local and general reactions of mild or moderate intensity Table . The occp = 0.26, p = 0.79) on day 28 after each vaccination did not differ across the vaccine and placebo recipients. In the recombinant tetanus vaccine group, mild (46 [51.0%]) and moderate (21 [23.0%]) unsolicited adverse events were reported most frequently; however, no significant differences were found between the recombinant tetanus vaccine and placebo groups .The occurrence of any unsolicited adverse events (n = 7 [58.3%]), followed by high creatine kinase (n = 3 [25.0%]) and hypertransaminemia  at day 28 and in the low‐dose  and medium‐dose groups  at day 208 than in the TT vaccine group  antibodies reached 81.2% (95% CI: 0.727–0.897)/87.1% (95% CI: 0.798–0.943) after the first immunization (day 28), increased to 100% (95% CI: 1.00–1.00)/96.5% (95% CI: 0.925–1.00) after the second immunization (day 56), and remained at a high level (97.6%/92.9%) for 6 months after the second immunization (day 208). Participants who received the medium‐dose recombinant tetanus vaccine reported a lower seroconversion rate of anti‐TT antibody 28 days after prime vaccination than those who received the TT vaccine  after the first vaccination, from 358.7 (95% CI: 304.8–422.2) to 3988.1 (95% CI: 3014.5–5276.3) and 38‐fold to 13551.9 (95% CI: 11297.3–16256.4) after the second vaccination (Table p < 0.001) after the first vaccination, from 493.0 (95% CI: 395.6–614.3) to 6194.6 (95% CI: 4902.1–7828.0) and 28‐fold to 13887.5 (95% CI: 11696.2–16489.4) after the second vaccination  of anti‐TT and anti‐TeNT‐Hc IgG antibodies increased rapidly in both the recombinant tetanus vaccine and TT vaccine groups Figures3A and Bp < 0.05, Figure p < 0.05, Figure Compared to the TT vaccine group, the GMT of anti‐TT IgG antibodies was lower in the low‐dose group at 14, 28, 35, and 42 days and in the medium‐dose group at 14, 28, and 35 days after the first vaccination. However, the high‐dose group showed higher GMT of anti‐TeNT‐Hc IgG antibodies than the TT vaccine group at 7, 14, and 28 days after the first vaccination (2.5γ (IFN‐γ) showed moderate increases after prime vaccination, with 115.0/20.0 spot‐forming cells (IQR 65.0–218.0/3.0–35.0) in the low‐dose group, 80.5/11.5 spot‐forming cells (30.0–245.0/0–35.0) in the medium‐dose group, and 70.0/10.0 spot‐forming cells (40.0–141.5/1.5–30.0) in the high‐dose group; these were significantly higher than those in the TT group  and interferon‐γ by T‐cells than the TT vaccine group. IL‐2 responses in the low‐dose and high‐dose groups were higher than those in the TT vaccine group at 14 and 35 days after the first vaccination. All three doses of recombinant tetanus vaccine showed a higher response for IL‐2 than the TT vaccine group at days 28, 42, 56, and 208 after the first vaccination. The responses for IFN‐γ were higher in the low‐dose group at days 35 and 42 and in the high‐dose group at days 7 and 42 than those in the TT vaccine group. In addition, all three vaccine doses elicited greater IFN‐γ expression by T‐cells than the TT vaccine group at day 28 as well as 6 months after the second vaccination  value of 0.1–0.2 IU/mL is the level of adequate protection for humans,20 all The heavy chain contains an N‐terminal translocation domain (HN) and a C‐terminal receptor binding domain (Hc). In this study, TT induced the production of antibodies against both TeNT‐Hc and TeNT‐HN as well as against the light chain. However, the antibodies induced by TT against TeNT‐Hc are the primary, protective neutralizing antibodies. The recombinant tetanus vaccine is mainly composed of the TeNT‐Hc protein, which is expressed as a soluble protein with appropriate folding and high yields. The bioengineering characteristics of TeNT‐Hc, including its ability to retain full binding affinity to neurons and induce a protective immunological response against the full‐length holotoxin, are described elsewhere. Another paper reported that TeNT‐Hc can induce equine tetanus immunoglobulin similar to TT as an immunogen in horses. Moreover, TeNT‐Hc can be used as a carrier protein for polysaccharide vaccines like TT to induce immune memory. TeNT‐Hc has previously been cloned and expressed with several other vaccine platforms. Several studies succeeded in producing a high expression of TeNT‐Hc; however, they failed in reaching productization due to poor efficacy compared with the commercial TT vaccine. The recombinant tetanus vaccine in this trial was based on studies on the soluble protein TeNT‐Hc and our previous patent, which was the first report on the purification of a non‐tag TeNT‐Hc isoform from E. coli in large quantities for potential human application.TeNT is a protein of about 150 kDa and consists of a 50 kDa light chain and a 100 kDa heavy chain.21 The In this study, we found that the recombinant tetanus vaccine induced significantly more specific CD4+ T‐cells than the TT vaccine, especially in the IFN‐γ T‐cell response. Altogether, our data further indicate that recombinant tetanus vaccine may be a suitable vaccine candidate, given the differences in special cell‐mediated immunity between the recombinant tetanus vaccine and TT vaccine.Vaccine efficacy is based on whether the host immune response against an antigen can elicit a memory T‐cell response over time. Once infection occurs, T‐cells transform into a group of effector T‐cells that can quickly acquire and expand the effector function after antigen re‐exposure, ultimately surviving long‐term and providing continual immunity.33 In  In our study, the recombinant tetanus vaccine showed equal safety and immunizing potency as the TT vaccine. Conventional vaccines (inactivated vaccine and attenuated vaccine) require a mass culture of associated or related organisms and long incubation periods; further, special requirements during storage and transportation add to the cost of vaccine preparation. With advances in technology, safe, cheap, and effective vaccine candidates have been produced, and the field began to focus on the development of recombinantly expressed antigens known as subunit vaccines. The recombinant vaccine is a subunit vaccine that uses recombinant proteins from pathogens. This implies that once the proteins having the capacity to vaccinate have been identified, they can be produced in sufficient quantity at a low cost. For these reasons, recombinant tetanus vaccine can be used as a candidate vaccine against tetanus.Previous papers have reported that TT vaccine is effective in protecting against tetanus, with a failure rate of 4/100 million immunocompetent people. Further, reactions to the TT vaccine are estimated to be 1 in 50 000 injections, and most reactions are not severe; local tenderness, edema, flu‐like illness, and low‐grade fever are the most often encountered reactions.16 In Although the data presented here suggest good safety and immunogenicity of the recombinant tetanus vaccine, results from this phase 1/2 trial in a small proportion of healthy adults from a single region in China could be limited in generalizability. A phase 3 study of the recombinant tetanus vaccine should be conducted in a large population across China. Meanwhile, the sample size of a phase 1/2 study limits the detection of all rare vaccine‐related adverse events. Extended trials will have to confirm the safety of recombinant tetanus vaccine and the persistence of functional antibodies in a larger, more heterogeneous population.4In summary, the present preliminary clinical trial results showed that the recombinant tetanus vaccine induced adequate protective neutralizing antibodies (anti‐TT and anti‐TeNT‐Hc antibodies) and specific T‐cell responses in healthy adults. No significant differences in adverse reactions were found between the recombinant tetanus vaccine and placebo groups, and no serious treatment‐related adverse events occurred. This trial revealed that recombinant tetanus vaccine might have the potential to serve as a safe and effective vaccine against tetanus.5The authors conducted this randomized, double‐blind, dose escalation, placebo‐ and positive‐controlled, phase 1/2 trial in Hangzhou City, Zhejiang Province, China, from May 10, 2018, to December 31, 2018. A total of 150 healthy adults of both sexes, aged 18–60 years, meeting all eligibility criteria were enrolled. Two doses each of the recombinant tetanus vaccine, TT vaccine, and placebo were administered as intramuscular injections in the deltoid muscle at an interval of 28 days. The primary exclusion criterion was a previous history of TT or DTP vaccination within the past 5 years. Women who were pregnant, breastfeeding, or planning to become pregnant within the next 3 months were excluded. A complete list of the inclusion and exclusion criteria is provided in the Supporting Information. All participants provided written informed consent prior to the initiation of the study. The study protocol and its amendments were reviewed and approved by the Ethics Committee of the First Affiliated Hospital, Zhejiang University School of Medicine. The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The trial was registered with Chictr.org under the identifier ChiCTR1800015865.n = 30 per dose group), TT vaccine , or placebo . As the recombinant tetanus vaccine, TT vaccine, and placebo were clearly labeled, three unmasked study nurses were assigned to prepare all vaccines and placebo preparations. The study investigators, participants, and other team members were masked to the vaccination allocation throughout the study. An emergency unmasked process could be performed only on special requests from the study investigators or ethical committee if ethical and emergency clinical concerns were raised.The eligible participants were sequentially enrolled in ascending dose groups of three stages, and participants in each stage were randomly assigned at a 3:1:1 ratio based on computer‐generated random numbers to receive one of the three doses of recombinant tetanus vaccine —among the first 50 participants, 30 were randomized to low‐dose recombinant tetanus vaccine, and 10 each to TT vaccine or placebo after a blinded review showed no evidence of dose‐related reactogenicity or toxic effects within 7 days; the next 50 participants were randomized in the same ratio, but with 30 to the medium‐dose, and when this was similarly seen to be safe, the last 50 participants were randomized in the same ratio, now with 30 to the high‐dose  and vital sign checks  were performed at baseline and days after each vaccination for safety analysis. Participants were required to keep a diary for 56 days after the first vaccination to record any abnormalities  or general reactions and their severity . Diaries were reviewed by a physician during each visit to verify the accuracy of the reported symptoms. Further, participants were interviewed for adverse events, and their responses were recorded by clinical research coordinators at each visit. The severity of clinical adverse events was graded on a three‐point scale from mild to moderate to severe, indicating the subjective intensity of the symptoms. The severity grading of adverse events was based on the standard guidelines issued by the China Food and Drug Administration.γ and IL‐2 enzyme‐linked immunosorbent spot (ELISPOT) assays with peripheral blood mononuclear cells stimulated with recombinant TeNT‐Hc protein, and the duration of specific immune responses was assessed up to 6 months after the second vaccination.Participant serological biomarkers, including TT and TeNT‐Hc indicators, and cellular immune assays comprised immunological specimens. We obtained specimens at baseline and at 7, 14, and 28 days after each vaccination and 6 months after the second vaccination for immunogenicity analysis. Antibody titers against TeNT‐Hc and TT were determined by IgG enzyme‐linked immunosorbent assay (ELISA) at baseline. An immune response was defined as seroconversion to a TeNT‐Hc or TT antibody titer >1:200 (baseline titer <1:50) or a fourfold titer increase (baseline titer ≥ 1:50) after one or two vaccinations. The antigen‐specific CD4+/CD8+ T‐cell responses were assessed using IFN‐The primary outcome was the safety profile of two doses of recombinant tetanus vaccines in healthy adults aged 18–60 years, including occurrence of solicited adverse reactions within 7 days after each vaccination, solicited and unsolicited adverse events within 28 days after each vaccination, abnormalities in laboratory measures within 0–7 days after each vaccination, and any serious adverse events throughout the follow‐up period. The secondary outcomes were the immune responses measures by the percentage of seroconversion of anti‐TeNT‐Hc or anti‐TT antibody 28 days after each vaccination, the GMT of antibodies against TT and TeNT‐Hc, and the magnitude of antigen‐specific CD4+/CD8+ T‐cell responses.χ2 tests  for categorical variables and analysis of variance (ANOVA)  for continuous variables. Multiple comparisons were performed using the Bonferroni procedure when there was a significant difference across groups. Statistical analysis was performed using SAS software, version 9.4 . Statistical significance was defined by p < 0.05 (two‐tailed). The safety endpoints were summarized in an intention‐to‐treat cohort that included all participants who received an injection. The immunological analysis was based on a per‐protocol cohort that included all participants who adhered to the study protocol and had no major protocol deviations.Data transformation, normalization, and evaluation of outliers were not used in this study. Continuous variables presented as mean (± standard deviation) or median (interquartile range). Considering a 5% dropout rate, the authors estimated that a total of 150 participants were needed for this non‐inferiority trial. They considered the seroconversion rate of anti‐TT IgG for the traditional TT vaccine to be 90%. They assumed that it is adopted if the recombinant tetanus vaccine has a seroconversion rate of at least 80%. The actual difference in the seroconversion rate of anti‐TT IgG between the traditional TT vaccine and recombinant tetanus vaccine was set to ±5%. The randomization allocation ratio was set to 3:1:1 for the recombinant tetanus vaccine, traditional TT vaccine, and placebo groups. Comparisons between different groups were performed using The authors declare no conflict of interest.X.X., R.Y., L.X., J.W., and M.Y. contributed equally. L.L. and X.X. are the principal investigators of this study. L.L., W.C., and R.Y. conceived the idea and designed the study. X.X., R.Y., and L.X. contributed to the study design, data interpretation, drafting of the manuscript, and revision of the report. J.X., Y.T., K.H., X.O., S.W., and X.M. contributed to the laboratory analyses, data interpretation, and revision of the report. J.W., M.Y., X.X., S.B., X.W., J.J., L.Y., and H.Z. participated in the site work, including recruitment, follow‐up, and data collection. R.J. contributed to vaccine management. R.Y., Q.W., and J.S. supervised the entire study process and took responsibility for all data from both sites and laboratories. R.Y. and L.X. contributed to the statistical analyses. All authors have reviewed and approved the final version of the manuscript.Supporting InformationClick here for additional data file.


Mechanical power (MP), defined as the amount of energy produced by mechanical ventilation and released into the respiratory system, was reportedly a determining factor in the pathogenesis of ventilator-induced lung injury. However, previous studies suggest that the effects of MP were proportional to their involvement in the total lung function size. Therefore, MP normalized to the predicted body weight (norMP) should outperform the absolute MP value. The objective of this research is to determine the connection between norMP and mortality in critically ill patients who have been on invasive ventilation for at least 48 h.This is a study of data stored in the databases of the MIMIC–III, which contains data of critically ill patients for over 50,000. The study involved critically ill patients who had been on invasive ventilation for at least 48 h. norMP was the relevant exposure. The major endpoint was ICU mortality, the secondary endpoints were 30-day, 90-day mortality; ICU length of stay, the number of ventilator-free days at day 28.p <  0.001]. Similarly, norMP was correlated with ventilator-free days at day 28, ICU length of stay. In the subgroup analysis, high norMP was associated with ICU mortality whether low or high Vt . But high norMP was associated with ICU mortality only in low PIP .The study involved a total of 1301 critically ill patients. This study revealed that norMP was correlated with ICU mortality [OR per quartile increase 1.33 (95% CI 1.16–1.52), Our findings indicate that higher norMP is independently linked with elevated ICU mortality and various other clinical findings in critically ill patients with a minimum of 48 h of invasive ventilation.The online version contains supplementary material available at 10.1186/s12871-021-01497-1. When performing surgery or in critically ill patients, mechanical ventilation is a vital component of supportive treatment since it preserves respiratory function and minimizes respiratory effort –3. HowevThe severity of VILI is determined by the ventilator settings . VariablIncreases in MP measured on the second day after intensive care unit (ICU) admission was correlated with increased hospital mortality in a recent study on 8207 critically ill mechanically ventilated patients . In addiThe objective of this study was to explore the prognostic role of mechanical power normalized to the predicted body weight in the clinical outcomes of intensive care patients.The Massachusetts Institute of Technology’s Laboratory for Computational Physiology maintains the Multiparameter Intelligent Monitoring in Intensive Care III  database, which includes data on over 50,000 patients admitted to the intensive care unit at Beth Israel Deaconess Medical Center between 2001 and 2012 . We atteThe establishment of the database was approved by the institutional review boards of the Massachusetts Institute of Technology  and Beth Israel Deaconess Medical Center . The author Jiang extracted the data for this study after passing the National Institutes of Health’s online training course (certification number: 9322422).In total, 58,976 intensive care unit (ICU) patients were recorded in the MIMICIII database, of these, we included in our study patients who were older than 16 at the time of their initial admission and who underwent invasive ventilation for a minimum of 48 consecutive hours. Patients were excluded if they met the criteria: had incomplete ventilatory variables to calculate MP and norMP, received pressure support ventilation, had > 1% missing data, were extubated, or had died during the first 48 h. We used only data from the patient’s initial ICU admission or initial hospitalization.2). The following equation was used to calculate mechanical power [The structured query language (SQL) was used to extract data from the database, and included tidal volume (Vt), positive end–expiratory pressure (PEEP), peak inspiratory pressure (PIP), RR, and the inspired fraction of oxygen  = 0.098 × Vt × RR × (PIP – ΔP × 0.5), where the driving pressure (ΔP) = PIP – PEEP .− 3 J/min/kg) = MP/PBW [norMP  were collected: age, gender, ethnicity , height, weight, comorbidities, and disease severity scores  , 19. VitTo gather information about ICU patients’ status, the follow-up followed from ICU admission and ended at death. The major endpoint was ICU mortality, the secondary endpoints included 30-day, 90-day mortality; ICU length of stay (ICU_LOS), the number of ventilator-free days at day 28 .Continuous variables are presented in the tables as the median with interquartile ranges. The required Mann-Whitney U test, or Kruskal– Wallis test, was applied. Chisquared test or Fisher’s exact test was used for categorical variables, which are presented as a percentage. Patients were categorized into groups according to ICU mortality.2, temperature, pH, PaO2 / FiO2, PaCO2. These variables were selected due to their clinical relevance. The final models were built using a stepwise backward elimination method with a significance level of 0.05. Additionally, subgroup analyses were conducted to determine the relationship between norMP and the primary outcome according to the Vt and PIP levels. According to the concepts of protective ventilation [2O.The median and interquartile range of norMP was used to classify all patients. For all outcomes, univariate and multivariate regression were used to account for potential confounding variables. Relevant covariates known to predict outcome were entered into the model including age, sex, ethnicity, BMI, admission type, comorbidities, APACHE, heart rate, MAP, SpOtilation  and a prtilation , and thep <  0.05. SPSS software was used for all statistical analysis .Statistical significance was described as a two-sided − 3 J/min/kg) than non-survivors (245.4(183.8–333.4) × 10− 3 J/min/kg) (p <  0.001), but the MP has no significant difference across the entire cohort.Finally, 1301 patients fulfilled the requirements for the study ; from 166.5 × 10− 3 J/min/kg to 226.4 × 10− 3 J/min/kg, quartile 2 (n = 325); from 226.5 × 10− 3 J/min/kg to 300.9 × 10− 3 J/min/kg, quartile 3, (n = 325); greater than 301.0 × 10− 3 J/min/kg, quartile 4 (n = 326). The clinical outcomes of patients in various groups were summarized in Table p <  0.05, all). However, there was no evidence that 30-day mortality and 90-day mortality between the groups was statistically different (p > 0.05).Across the entire cohort, norMP had a median of 226.5 × 10p <  0.001). In addition, norMP in the second 24 h still had a strong correlation with increased ICU mortality even after adjustment for covariates .Figure Figure p = 0.004; OR = 1.32, 95% CI 1.08–1.62, p = 0.008, respectively). The analysis revealed . Our results also (eTable p = 0.004) than for those with high PIP levels .In the subgroup analysis Fig. , regardlThe essential findings of this research can be summarized as follows: (a) norMP during the second 24 h of ventilation was independently correlated with increased ICU mortality of critically ill patients, who received invasive ventilation for more than 48 h; (b) increased norMP was independently correlated with a longer ICU stay, a lower number of ventilator-free days and alive at day 28; and (c) high norMP was associated with ICU mortality regardless of Vt, but high norMP was associated with ICU mortality in patients with low PIP only.In invasive ventilation, a lung-protection ventilation strategy that provides adequate gas exchange while minimizing VILI should be used , 23. VILSince the two important factors of ventilator parameters are tidal volume and airway pressure, we evaluated the effect of norMP on the prognosis of patients with different tidal volumes and airway pressure levels. In line with our hypothesis, we discovered that high MP was correlated with ICU mortality, even when Vt was low. This suggested that norMP added more information aside from the volume. Our research also demonstrated that a high norMP was associated with ICU mortality in patients with low PIP only. We propose a possible explanation for this finding. Our results showed that PEEP levels were higher in the group of patients with high PIP levels than in their counterpart. Patients with high PIP levels may be more severely ill, as sicker patients may be default be receiving higher PEEP levels. This may explain why low norMP was not associated with decreased ICU mortality in patients with high PIP. The majority of mechanical power in patients with higher levels of PEEP may be secondary to the applied PEEP. This further emphasizes the need to consider the PEEP component in the analysis. We failed to do a simple sensitivity analysis taking out the PEEP (focused on patients without PEEP), because the sample became very small in this situation. However, we have performed an analysis to determine the relationship between norMP and ICU mortality according to the different level of PEEP. Considering this analysis complicated the results so much, we decided not to report. Therefore, further investigations  are necessary to explore the relationship between norMP and mortality in patients without applied PEEP.These findings suggested that norMP might be a useful marker to predict clinical outcomes because it combines the effects of different ventilator parameters. Modifying a single parameter has little effect on the amount of energy transmitted to the lung tissue, and it does not always protect the lungs . AccordiIn addition to not considering the PEEP component in the analysis, our current analysis had some limitations. First, in order to determine patients with more serious illnesses and ample exposure time, only patients who underwent invasive ventilation for at least 48 h were selected. However, the current findings cannot be generalized for patients who were extubated or died within the first 48 h. Second, norMP was calculated only once and not during the ICU stay. Therefore, it did not accurately represent the temporal changes in norMP administered to the patient. Third, since the datasets used in this study were from publicly available data, the airway pressure may not have been collected under consistent standard conditions. Such is the case in patients without spontaneous breathing efforts. Finally, it was difficult to quantify functional lung size. In the present study, we indirectly described the functional lung size through PBW. However, other conditions leading to decreased functional lung size, such as ARF and ARDS, were not considered. In the future, further studies investigating normalizing MP to respiratory system compliance or lung volume determined using CT  are neceHigh norMP is independently correlated with increased ICU mortality and many other clinical outcomes in critically ill patients who undergo invasive ventilation for at least 48 h. Due to the ease with which norMP can be determined using ventilator parameters, monitoring norMP can help predict the early outcome of ICU patients undergoing invasive ventilation.Additional file 1: eTable 1. Comparisons of PEEP between different PIP level.


Microalgae is one of the most important components in the aquatic ecosystem, and they are increasingly used in food and medicine production for human consumption due to their rapid growth cycle and survival ability in the harsh environment. Now, the exploration of microalgae has been gradually deepening, mainly focused on the field of nutrition, medicine, and cosmetics. A great deal of studies has shown that microalgae have a variety of functions in regulating the body health and preventing disease, such as nitrogen fixation, antitumor, antivirus, antioxidation, anti‐inflammatory, and antithrombotic. Furthermore, microalgae can synthesize various high‐valued bioactive substances, such as proteins, lipids, polysaccharides, and pigments. In this paper, we have briefly reviewed the research progress of main bioactive components in microalgae, proteins, lipids, polysaccharides, pigments, and other nutrients included, as well as their present application situation. This paper can provide the guidance for research and development of industrial production of microalgae. The manuscript has reviewed the research progress of main bioactive components in microalgae, proteins, lipids, polysaccharides, pigments, and other nutrients, as well as their potential application in industrial production. Simultaneously, this review has briefly demonstrated that microalgae have huge advantages in regulating the body health and preventing disease, such as nitrogen fixation, antitumor, antivirus, anti‐oxidation, anti‐inflammatory, and anti‐thrombotic. Microalgae represent the microscopic photosynthetic group that consists of eukaryotic organisms and prokaryotic cyanobacteria , they have applications in many industries, food, health products, animal husbandry, aquatic feed, and cosmetics. Furthermore, there is growing interest in biomacromolecules as a potential drug, marine organisms being a great source. Hence, microalgae could be included in screening programs for promising bioactive substances.Microalgae have much higher photosynthetic efficiency and are not sensitive to the seasons compared with higher plants, which allows for high yields of valuable molecules such as proteins, lipids, carbohydrates, and pigments, combined with the diversity of microalgae  in microalgae can differ greatly. Marine microalgae are high in protein content, that is, 6%–70% of its dry weight (DW), and a majority having about 50%, as shown in Table 2.1Cyanobacteria as well as some algae. The complex is formed based on apoprotein and covalently bound phycobilins serving as the chromophores. Phycobiliproteins are classified into four groups: phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC), and phycoerythrocyanin (PEC). PC was isolated from Spirulina and mainly exists in Cyanophyta, Rhodophyta, and Cryptophyta [80]. It is usually divided into C‐phycocyanin (C‐PC) and R‐phycocyanin (R‐PC), where the former is found in Cyanophyta, the latter in Rhodophyta, and both in Cryptophyta. PC is a protein–pigment complex that comprises of protein and nonprotein components, which participate in various biological effects, such as antioxidant, anti‐free radical, and antitumor activities  results showed the expression of TrpA on trichome surface, with no typical localization pattern  fixation microorganisms were studied. The N2 fixation microorganisms were less diverse based on nitrogenase gene (nifH) diversity. Most nifH sequences retrieved can be divided into two categories, including one constituted by Trichodesmium sequences and other by the α‐proteobacterial group. At present, Trichodesmium is the highest abundant diazotroph, with as high as 6 × 105 nifH gene copies/L , isoelectric point (4.8), Mo (2 mol/mol), S2− (20 mol/mol), and Fe (20 mol/mol) contents, as well as amino acid (AA) composition similar to components that contained Mo and Fe, was subjected to homogeneous purification and separated based on additional bacteria , which had antimicrobial activity against the Gram‐negative bacteria, such as E. coli, P. aeruginosa and S. typhimurium, as well as against the Gram‐positive bacterial strains, for example, B. cereus, M. luteus, L. monocytogenes, and methicillin‐resistant S. aureus. Several studies have also demonstrated that peptides from microalgae had antitumoral and antihypertensive activity (Table Bioactive peptides (BP) as a specific protein fragment play a vital role in the physiological activity of most living organisms. BP was reported to have several therapeutic activities, including antioxidant, anti‐inflammatory, antitumoral, antiproliferative, antihypertensive, and antimicrobial properties, as shown in Table d Cherng  also atti et al.  evaluaten et al.  reportedty Table .3Microalgal oil has attracted much attention due to its short breeding cycle, its ability to capture carbon dioxide and accumulate a large amount of lipids, and its rich omega‐3 long‐chain polyunsaturated fatty acids. As shown in Table Phaeodactylum tricornutum, remarkably increased EPA and PUFAs yields. It indicated that P. tricornutum could be utilized as an aquacultural food organism as well as the candidate EPA source. Ryckebosch et al.  levels . Several animals and in vitro experiments supported the microalgal oil effect against inflammation. Nauroth et al.  suggestei et al.  were the4Spirulina provides biological activities beneficial for tissue engineering. It could produce nanofibers and also contains various biomass contents to treat spinal cord damages or serve as the matrix in stem cell cultures  PS obtained from P. cruentum did not exhibit the antioxidation effect, whereas the low‐molecular‐weight (LMW) fragments, following degradation, showed inhibition against OS , showed an anticancer effect on Graffi myeloid tumor in hamsters in vitro and in vivo. After treatment with the sulfated polysaccharide, delayed tumor growth was observed with an extended median survival time by 10–16 days, thus suggesting that the Porphyridium‐derived EPS may serve as a potential candidate antitumor drug. Furthermore, Akao et al. found that PS isolated from Spirulina platensis could enhance the activity of natural killer cells in mice, thus play an essential role in antitumor  rats fed with PS from 5Natural pigments play a vital role during algae photosynthetic metabolism and pigmentation. Microalgae account for the primary photosynthesizers that generate several essential pigments, including chlorophyll, β‐carotene, phycobiliproteins, astaxanthin, and xanthophylls with applications in food, nutraceutical, pharmaceutical, aquaculture, and cosmetic industries Table . ChloropMost pigments are biologically active, with antiobesity, anti‐inflammatory, anticancer effects, mainly due to their potent antioxidant activity employed to protect the body from OS , hydroxyl radical (HO•), hypochlorous acid (HOCl), and anion peroxynitrite (ONOO⁻) . Vitamin B12 has also been found in some macro‐ and microalgae. Edelmann et al.  with applications in some medical and industrial fields. These bioactive substances from marine microalgae provide new and vast sources for the development of human health care and industrial production.Although there are many advantages in using microalgae to produce high‐value products, its economic cost is too high due to a series of factors, such as low content of microalgae, low biomass, and much difficulty in harvesting, which greatly limited the utilization of microalgae resources. With the rapid development of synthetic biology, the use of this technology in microalgae artificially builds high‐value biosynthetic pathways of natural compounds to realize the goal of synthetic products ; Data curation ; Formal analysis ; Investigation (lead); Methodology ; Writing‐original draft (lead). Xinzhe Gu: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing‐original draft (lead); Writing‐review & editing (lead). Danlu Yang: Data curation ; Formal analysis ; Investigation . Shannan Xu: Formal analysis ; Project administration (lead); Resources ; Supervision (lead). Shaoyun Wang: Investigation ; Methodology ; Project administration ; Supervision . Xu Chen: Data curation ; Formal analysis ; Project administration . Zhengwu Wang: Investigation ; Methodology ; Project administration ; Resources ; Supervision .


Although the prognostic role of neutrophil‐to‐lymphocyte ratio (NLR) has been assessed in patients with metastatic castration‐resistant prostate cancer, data on its impact on oncological outcomes of patients with metastatic castration‐sensitive prostate cancer (mCSPC) are scarce.This study aims to examine the influence of elevated pretreatment NLR on time to prostatic‐specific antigen (PSA) progression and overall survival (OS) of patients with mCSPC.We retrospectively reviewed patients presenting between June 2007 and June 2019 with mCSPC. Survival was estimated by the Kaplan‐Meier method and compared by the log‐rank test. Multivariate analyses were used to assess the factors influencing time to PSA progression and OS.n = 37) compared to patients with NLR < 4 (n = 146); 11.3 and 18.3 months, respectively, P = .015. Patients with NLR ≥4 also had inferior median OS . On multivariate analysis, NLR ≥4 was not an independent factor for time to PSA progression. However, NLR ≥4 was an independent factor of inferior OS . Other independent factors predicting inferior OS included Eastern Cooperative Oncology Group Performance Status ≥1, high‐volume status, and Hb < 12.A total of 189 patients were included; median age = 69 years, median PSA = 155 ng/mL, 41(22%) had visceral metastasis. Median time to PSA progression was shorter for patients with NLR ≥4 (Elevated pretreatment NLR independently predicts inferior OS in newly diagnosed patients with mCSPC. However, NLR was not a predictor of time to PSA progression. On the contrary, NLR was not associated with oncological outcomes in patients with localized disease.Inflammation is a hallmark of cancer development and progression.Although the impact of NLR on OS has been well studied in metastatic castration‐resistant disease, it is not explored in newly diagnosed patients with mCSPC.The CHAARTED and LATITUDE trials highlighted the importance of prognostic stratification on initial therapy selection.22.1This study aims to examine the prognostic role of NLR on the oncological outcomes of patients with newly diagnosed mCSPC. Study primary endpoints are to assess the impact of high NLR on time to prostatic‐specific antigen (PSA) progression and OS.2.1.1After Institutional Review Board approval, we identified all patients with metastatic prostate cancer who were referred to our center between June 2007 and June 2019. All patients were required to have pathologically confirmed prostatic adenocarcinoma and to have a metastatic disease detected on conventional imaging studies (computed tomography (CT) scan, magnetic resonance imaging, and bone scan). Patients with metastasis detected only on prostate‐specific membrane antigen scan without being detected on conventional imaging studies were excluded. Patients who develop a metastatic disease while being treated with ADT for biochemical relapse following definitive primary radiotherapy or radical prostatectomy or those presenting with castration‐resistant metastatic disease were also excluded.Variables: age at presentation, date of diagnosis of metastatic disease, sites of metastasis, number of metastatic bone lesions, GS, ECOG PS, and date of initiation of ADT, defined as the date of the first injection in patients treated with medical castration (GnRH agonists) or the date of orchidectomy for patients treated with surgical castration were abstracted. Concurrent administration of docetaxel and abiraterone was noted. Finally, pre‐treatment laboratory values, including blood counts, serum PSA, ALP, and serum calcium, were extracted. The pretreatment neutrophil‐to‐lymphocyte ratio was calculated for every patient by dividing the absolute neutrophil over the absolute lymphocyte count from the CBC before commencing ADT treatment.Also, we extracted data on the time of first PSA progression after starting ADT. PSA progression was defined according to the prostate cancer working group criteria (PCWG3).Metastatic burden was stratified into high volume and low volume disease based on the definition from CHAARTED trial. Accordingly, high volume disease was defined as the presence of more than three metastatic bone lesions with at least one lesion outside the spine and pelvis or the presence of visceral metastasis,2.1.2Descriptive statistics were interpreted as means, medians, SDs, and proportions. Time to PSA progression and OS were assessed by the Kaplan‐Meier method. Survival comparisons were carried out by the Log‐rank test.P < .05), utilizing the backward stepwise cox‐regression method.As the cutoff value for high vs low NLR is not well defined in literature, we opted to assess the correlation of NLR  on the median time to PSA progression. In addition, we utilized cutoffs of 3 and 4 utilized and compared in the literature.P < .05 in the univariate analysis were forced into a multivariate analysis for OS utilizing the backward stepwise cox‐regression method. All P‐values <.05 were considered statistically significant. All statistical analyses were performed using SPSS version 21 .We priori planned to utilize the cutoff value of NLR significantly associated with time to PSA progression in OS analysis. Univariate analysis to assess the possible impact of factors other than the NLR on OS, including: GS, age, serum PSA, visceral vs non‐visceral metastasis, ECOG PS, serum alkaline phosphatase, serum calcium, serum Hb, volume status, and risk status was conducted. All factors with 33.1We identified a total of 280 patients with metastatic prostate cancer. Forty‐three 15%) were excluded because they presented as new cases of mCRPC. Seventeen (6%) were excluded because they came for a second opinion and continued treatment at other centers or lost follow up, 9 (3%) because the pathology did not confirm prostatic adenocarcinoma, 4 (1%) because of diagnosis of a concurrent second malignancy, and 4 (1%) because the metastasis was not detected on conventional imaging studies. In addition, 14 (5%) were excluded for missing data on complete blood counts prior to starting ADT. Thus, a total of 189 patients were included in the final analysis. Patients' characteristics are summarized in Table 5% were eMore than 90% (174/189) of patients had bone metastasis. Of 174 patients with bone metastasis, 37 (21%) had fewer than three bone lesions, and 135 (78%) had ≥3 bone lesions, whereas the number of lesions was not documented in 2 (1%). Approximately three‐quarters of patients had CHAARTED‐trial defined, and LATITUDE‐trial defined high‐volume and high‐risk status. Initial therapy administered was ADT alone for 134 patients (72%), ADT plus docetaxel  for 48 (24%), and ADT plus abiraterone and prednisone for 7 (4%).The median NLR was 2.7, mean NLR 3.2 . A total of 39 patients (21%) had NLR ≥4.3.1.1P = .015) . There was no impact of NLR ≥ median vs < median on time to PSA progression .The median time to PSA progression for the entire cohort was 15.6 months. Patients with NLR ≥4 had a shorter time to PSA progression compared to patients with NLR < 4; 11.3 and.18.3 months, respectively .At a median follow‐up of 26 months, the median OS for the entire cohort was 47.1 months. Median OS was inferior in patients with NLR ≥4 compared to patients with NLR < 4 [23.9 vs 49.5 months, P‐values were not statistically significant).Other factors that predicted OS in the univariate analysis included ECOG PS, volume status, risk status, serum ALP level, Hemoglobin level, and initial therapy administered Table . Age, GSNLR≥4 remains an independent factor for inferior OS on multivariate analysis and was associated with three‐fold increased risk of death Table .4The importance of identifying pre‐treatment factors in patients with mCSPC that correlate with poor oncological outcomes stems from the fact that these patients might benefit from early treatment intensification. In this study, we assessed the impact of pre‐treatment NLR on oncological outcomes of patients with mCSPC and found that an elevated ratio was associated with inferior OS. Notably, after controlling for other patients and disease factors, elevated NLR remains an independent factor predicting inferior OS.The role of several inflammatory markers such as platelet‐to‐lymphocyte ratio (PLR), CRP, albumin, and NLR have been extensively studied in the context of various malignancies, as these markers might reflect the interaction between the immune system and tumor microenvironment.P   <  .001) and progression‐free survival  of patients with mCRPC. On the contrary, NLR was not associated with oncological outcomes in patients with localized disease.P = .002).Previous reports have assessed the prognostic value of NLR in the castration‐resistant stage of metastatic prostate cancer or those with non‐metastatic disease. In the meta‐analysis conducted by Yin et al, elevated NLR predicted poor OS .


Chronic migraine (CM) and episodic migraine (EM) are associated with substantial headache-related disability, poor quality of life and global societal burden. In this subgroup analysis from the CONQUER study, we report efficacy outcomes from a pre-specified analysis of galcanezumab versus placebo in patients with CM or EM and 3–4 prior preventive medication category failures due to inadequate efficacy (after at least 2 months at maximum tolerated dose), or safety or tolerability reasons. The patient population is of particular interest due to evidence of decreased quality of life and increased economic burden among patients with migraine that is inadequately managed and is of interest to decision-makers globally.Key outcomes included overall mean change from baseline in monthly migraine headache days and proportions of patients achieving ≥30% (CM), ≥50%, and ≥ 75% reduction (response rates) in monthly migraine headache days across Months 1–3. Patient functioning and disability were evaluated at Month 3.P ≤ .001) larger overall mean reduction in monthly migraine headache days . Similarly, the ≥50% response rate was significantly (P ≤ .001) higher with galcanezumab versus placebo . In the CM group, the ≥30% response rate was significantly higher in the galcanezumab group than the placebo group  as was the ≥75% response rate . Galcanezumab also resulted in significant (P < .0001) improvements in patient functioning and reductions in disability.Of the 462 randomized patients, 186 (40.3%) had a history of 3–4 preventive category failures. Galcanezumab versus placebo resulted in significantly (Galcanezumab was effective in a difficult-to-treat population of patients with CM or EM who had failed 3–4 prior preventive medication categories.NCT03559257.CONQUER. Clinicaltrials.gov identifier:  P < .001) higher in patients who switched to their fourth ($5004) class of drugs within 1 year compared with patients who switched to either their third ($2997) or those who never switched ($2420) , P: 4.8 ; P ≤ .0001, Fig. P ≤ 0.010).In the total population, the mean percentage of patients with ≥50% reduction from baseline in monthly migraine headache days was significantly (P ≤ .0001). The ≥50% responders were significantly higher in the galcanezumab group compared with placebo . The same was also true for the ≥75% responders who were 13.3% in the galcanezumab group and 2.6% in the placebo group .In the subgroup of patients with CM, galcanezumab 120 mg versus placebo resulted in significantly larger ≥30% reduction in monthly migraine headache days .In the subgroup of patients with EM, the percentage of ≥50% responders was significantly higher in the galcanezumab group versus placebo , the overall rate could not be estimated with the planned analyses. Similarly, the 100% response rate could not be estimated in patients with CM or EM due to the low number of patients achieving this response rate.P < .0001), in the MSQ-RFR domain score , the MSQ-RFP domain score , MSQ-EF domain scores  and MIDAS total score . This result was similar in patients with CM or EM ; P < .0001; EM: − 3.5 versus − 0.7; mean change difference from placebo, − 2.8 (0.8); P = .0008, Table Galcanezumab-treated patients in the total population had a significantly greater reduction in the number of monthly migraine headache days with acute medication use compared with placebo ; Of note, galcanezumab 120 mg reduced the overall proportion of patients who satisfied the ICHD-3 criteria for acute medication overuse from 60.6% to 11.7 , or safety or tolerability reasons in the past 10 years demonstrate that galcanezumab 120 mg versus placebo is efficacious across multiple endpoints. In patients with CM, ≥30% reduction in monthly migraine headache days and in patients with EM, ≥50% reduction in monthly migraine headache days, is considered clinically meaningful –33. In tFurthermore, the significantly larger mean reduction from baseline in monthly migraine headache days with galcanezumab 120 mg versus placebo observed in this subgroup analysis is consistent with earlier published subgroup findings , 22, 28.This is a pre-specified subgroup analysis of the larger population enrolled in the CONQUER study and, as such, included a smaller number of patients. These analyses were pre-specified, with the same models defined for the analyses on the full CONQUER population. Due to limited sample sizes and zero event count in the placebo group for the 75% responder definition, the planned model did not converge and therefore the difference between placebo and galcanezumab for the EM population could not be estimated. Analyses were not multiplicity adjusted.The current pre-specified analysis demonstrated the efficacy of galcanezumab versus placebo in reducing monthly migraine headache days in patients with CM or EM, who had documented treatment failure of 3–4 standard-of-care migraine preventive medication categories in the past 10 years owing to inadequate efficacy (after at least 2 months at maximum tolerated dose), or safety or tolerability reasons, or both. This population is of particular interest due to the evidence of decreased quality of life and increased economic burden among patients with migraine that is inadequately managed  and is o


Musical training is thought to be related to improved language skills, for example, understanding speech in background noise. Although studies have found that musicians and nonmusicians differed in morphology of bilateral arcuate fasciculus (AF), none has associated such white matter features with speech-in-noise (SIN) perception. Here, we tested both SIN and the diffusivity of bilateral AF segments in musicians and nonmusicians using diffusion tensor imaging. Compared with nonmusicians, musicians had higher fractional anisotropy (FA) in the right direct AF and lower radial diffusivity in the left anterior AF, which correlated with SIN performance. The FA-based laterality index showed stronger right lateralization of the direct AF and stronger left lateralization of the posterior AF in musicians than nonmusicians, with the posterior AF laterality predicting SIN accuracy. Furthermore, hemodynamic activity in right superior temporal gyrus obtained during a SIN task played a full mediation role in explaining the contribution of the right direct AF diffusivity on SIN performance, which therefore links training-related white matter plasticity, brain hemodynamics, and speech perception ability. Our findings provide direct evidence that differential microstructural plasticity of bilateral AF segments may serve as a neural foundation of the cross-domain transfer effect of musical experience to speech perception amid competing noise. There is a great deal of evidence that musical training experience has a pervasive positive effect on auditory cognitive functions  and results in wide-spread structural and functional changes in the human brain . HoweverSIN perception, or the “cocktail party phenomenon,” requires skilled perceptual and cognitive processes  in order to pick up meaningful units . The musThe AF/SLF has been proposed as the anatomical basis of the auditory “dorsal stream” for mapping speech phonological information onto articulatory motor representations . Note thBased on lesion studies and brain electrical stimulation, the direct AF has been implicated in syntactic and phonological processing, the anterior AF in articulation, and the posterior AF in phonological processing . Using dHowever, the white matter substrates supporting SIN perception in musicians have not been studied. In contrast to the relevance of speech and language function with AF microstructure in the left hemisphere, the right AF has been implicated in music processing. For instance, the structure of the right AF predicted melody and rhythm learning speed  and pitcIn the present DTI study, we aimed at investigating whether and how long-term musical training modulates the white matter diffusivity of bilateral AF segments and its contribution to SIN perception ability. The corticospinal tract (CST), which is part of the sensorimotor system and connects cortical motor regions with brainstem, is often modulated by musical experience , but shot26 = −0.72, P = 0.48), postsecondary education years , pure-tone average hearing level , auditory working memory , and nonverbal IQ . Musicians had started training before age 7 (mean = 5.21 ± 1.62), had at least 10 years of musical training (mean = 14.64 ± 3.46), reported practicing consistently (≥3 times per week) over the past 3 years but varied in the type of training . No significant correlation was found between SIN accuracy and musical training years in musicians . The lack of correlation may be due to a narrow range of training duration (11 ~ 22 years) without recruiting amateur or life-long musicians, as well as the small sample size.This task was carried out during a previous fMRI scanning  = 2300 ms, echo time (TE) = 2.98 ms, flip angle = 9°, field of view (FOV) = 256 × 256 mm2, slice numbers = 192 slices, voxel size = 1 × 1 × 1 mm3. DTI data were acquired with the following parameters: diffusion-weighted gradient directions = 99, b-value = 1000 s/mm2, b0 nonweighted images = 10, TR = 9340 ms, TE = 88 ms, flip angle = 90°, FOV = 256 × 256 mm2, EPI factor = 128, slice numbers = 72, voxel size = 2 × 2 × 2 mm3. See Imaging data were collected using a 3.0 T MRI system (Simens Magnetom Trio) with a 32-channel head coil. http://www.nitrc.org/projects/panda/), was employed to process the raw DTI data. The core commands for preprocessing procedure were embedded in the Functional MRI of the Brain (FMRIB) software library . Firstly, the Brain Extraction Toolbox was executed on the diffusion tensor images to obtain skull stripped images. Then the diffusion images were registered to the b0 images with an affine transformation for correcting the eddy current distortion. After that, DTIFIT was applied to build tensor models, and 3 eigenvalues, λ1, λ2, and λ3, were determined through tensor fitting. The λ1 and the averaged λ2 and λ3 were used to profile water diffusion parallel to the axonal direction  and in the perpendicular direction  that differentiate axon and myelin changes, respectively  based on FACT tracking algorithm . The fiber trajectories of the 3 segments of bilateral AF in a typical individual are shown in Before formal fiber tracking, all termination regions in MNI space were mapped into individual native space. Fiber tracking initiated from 10 randomly selected seeds within each voxel of the termination regions in the native diffusion space and terminated if the angle between 2 consecutive orientations exceeded 45° or if the FA value was lower than 0.2. It is suggested in the literature that it was impossible to reconstruct a continuous trajectory of the right direct AF in nearly a half of participants using deterministic tracking ; for ins3). This approach balances the need to minimize extraneous fibers, register subtle differences of AF morphology between groups and hemispheres, and enable the correlational analysis with the behavioral variable across all subjects. Many studies on AF/SLF and other language or music-related tracts used a similar threshold as we did  and cluster size >240 voxels  / (leftFA + rightFA).Lastly, for each individual, the mean diffusivity values  of each tract were extracted from the diffusivity maps that were overlaid with the reconstructed tract templates of each group in MNI space. The FA-based LI was then calculated for characterizing the hemispherical asymmetry of each AF segment and the CST using the following algorithm: LI = (leftt-test for 2 groups). To obtain robust estimations on small samples without making any assumptions about the sampling distribution, nonparametric permutation tests were conducted on tract diffusivity or LI. Firstly, the diffusivity  and LI values of each tract were compared between groups by 2-sample t tests to verify the musical experience-dependent plasticity on white matter microstructure. The significance of laterality of each AF segment was additionally tested by a 1-sample t-test in each group separately. Next, for specifying white matter tracts involved in SIN perception, partial correlations were conducted between the diffusivity or LI values of each tract and the SIN performance, with hearing level, working memory (digit span) and nonverbal IQ as covariates. Additionally, Pearson’s correlations were performed to assess the relationships between musical training time and diffusivity or LI values. Following each above mentioned analysis, a null distribution of test statistics  was generated by randomizing the labels of diffusivity or LI values 10 000 times. The permutation test P value was calculated by ranking of a true statistical value in the shuffled distribution  or 28 measurements .The group differences of age, education years, pure-tone hearing level, auditory digit span, nonverbal IQ, and SIN perception accuracy were examined using parametric tests (independent samples 3) that was significantly higher in musicians than nonmusicians during the SIN task (family-wise error corrected P < 0.001 by main effect of group) and predicted SIN performance in musicians  in our previous fMRI study , normed-fit index (NFI), and comparative fit index (CFI). P of χ2 > .05, RMSEA < 0.07, NFI > 0.95, and CFI > 0.95 indicate an acceptable fit of the model  using maximum likelihood estimation to assess the direct and indirect relationships of the following variates:1) the FA value of the right direct AF that showed significant group difference and correlation with SIN performance; 2) the BOLD activity in the right STG cluster  he model . It is whe model .P = 0.004, FDR-corrected, Cohen’s d = 1.20) and higher AD  in the right direct AF, as well as lower RD  and a tendency of higher FA  in the left anterior AF. The increment of FA with an increment of AD and a stable RD suggests axonal property changes in the right direct AF, whereas the increment of FA accompanied by a decrement of RD and a stable AD are compatible with an enhanced degree of myelination of the left anterior AF in musicians (P > 0.05). With regard to the control tract, no group difference in diffusivity was identified  and the RD of the left anterior AF negatively correlated with SIN performance  in all participants. In addition, the RD values of the left direct AF, the right anterior AF and the left posterior AF as well as the AD values of bilateral posterior AF negatively correlated with SIN perception across the subjects , although the group difference was not significant. All the correlation estimates are listed in P = 0.001, FDR-corrected, P > 0.05 for all, To identify the white matter correlates supporting musician benefit on SIN perception in a robust way, we report only tract diffusivity values showing both a significant group difference, as well as a significant partial correlation with SIN performance after controlling for hearing level, auditory working memory and nonverbal IQ, and passing FDR correction. As shown in FA − rightFA) / (leftFA + rightFA)] was further calculated using FA values for characterizing each tract’s hemispheric asymmetry and its relationship with SIN performance. As shown in P < 0.001, FDR-corrected, Cohen’s d = 1.77; nonmusicians: LI = 0.033 ± 0.012, P < 0.001, FDR-corrected, Cohen’s d = 2.73) and the posterior AF  were left lateralized. The direct AF exhibited a right lateralization in musicians  but no bilateral asymmetry in nonmusicians . Moreover, compared with nonmusicians, musicians showed a stronger right lateralization in the direct AF  and a stronger left lateralization in the posterior AF . Among the 3 AF segments, only the LI of the posterior AF had a positive correlation with SIN performance in all participants  and the BOLD activity in the right STG  all correlated with SIN accuracy across the entire sample of subjects, mediation analysis was performed to understand their relationships. As shown in P = 0.37; RMSEA = 0.56; NFI = 0.71; CFI = 0.96). Model A could explain 46.8% of variance in SIN perception accuracy. Specifically, the FA of the right direct AF significantly predicted the BOLD activity in the right STG  which in turn significantly contributed to SIN performance . Thus, the indirect effect of FA of the right direct AF on SIN performance with the right STG BOLD activity as a mediator was significant , whereas the direct effect of FA of the right direct AF on SIN accuracy was insignificant . Additionally, to test the possibility that structural changes may mediate the effect of brain function on behavioral performance, Model B with the FA of the right direct AF as a mediator in explaining the effect of the right STG BOLD activity on SIN performance . These results suggest that the structural alterations of the right direct AF drove enhanced SIN perception in musicians by influencing the hemodynamic activity in the right STG.As the FA of the right direct AF ; the Strategic Priority Research Program of Chinese Academy of Sciences (grant XDB32010300); the Canadian Institutes of Health Research (Foundation Grant); an infrastructure grant from the Canada Fund for Innovation.Conflict of Interest: None declared.We thank Lei Zhang, Baishen Liang, Xiaoqian Zhou, and Yiyang Wu for their help on data analysis and results examination. Figure_S1_bhab063Click here for additional data file.Li_Zatorre_Du_Supplementary_information_bhab063Click here for additional data file.