Adenomyosis is a benign gynecological condition in which endometrial tissue or endometrial-like tissue develops within the uterine myometrium. Few cases of disseminated intravascular coagulation has been reported in the patients with adenomyosis. Although hysterectomy is indicated for refractory massive uterine bleeding in the patients with advanced uterine adenomyosis, conservative treatment is often desired in women in the late reproductive age. Recently such cases are increasing due to the social trend of late marriage.A 37-year-old woman with huge adenomyosis, gravida 2 para 0, was referred to our hospital to terminate her pregnancy. Acute, non-septic, disseminated intravascular coagulation (DIC) developed after early pregnancy was terminated in a woman with huge adenomyosis. Massive bleeding and DIC occurred 3 days after the dilatation and curettage. There was no evidence of infection as the cause of the DIC, because neither bacteria nor endotoxin could be detected in her blood, and antithrombin 3 (AT3), which would be expected to decrease in septic patients, was not decreased. Hemorrhage in the adenomyotic tissue after the termination presumably developed inflammation, with numerous microthrombi and necrosis in the adenomyotic tissue, which subsequently promoted coagulation and fibrinolysis, leading to the onset of massive uterine bleeding and DIC. Although severe hyperfibrinolysis is observed in peripheral blood, the fibrinolysis state in the uterine myometrium is considered to be even more severe. The newly formed clots for hemostasis under the uterine mucosa could be removed due to the excessive activation of fibrinolytic system happened in the adjacent myometrium, leading to the onset of massive uterine bleeding. Massive bleeding and DIC resolved quickly after the patient was treated with nafamostat mesilate, which is effective for both excessive coagulation and fibrinolysis.Adenomyosis could cause massive bleeding and DIC when pregnancy is terminated. Massive bleeding was considered to occur because the excessive fibrinolysis system inside adenomyosis affected the adjacent endometrium. Before considering hysterectomy to control refractory uterine bleeding, nafamostat mesilate should be considered as one option, thinking the pathophysiology of the massive bleeding due to uterine adenomyosis. Disseminated intravascular coagulation (DIC) is a coagulopathy that can occur in a large number of clinical conditions, including malignant tumors and obstetrical events –5. HowevA 37-year-old woman, gravida 2, para 0, was referred to our hospital for termination of early pregnancy. She suffered from huge adenomyosis and had been treated with a GnRH agonist for 15 months in total. She also had a history of transfusion due to severe uterine hemorrhage when a miscarriage occurred at the age of 33 years.3 (3.3 × 103–9.0 × 103/μl), hemoglobin (Hb) 8.8 (11.5–15.0 g/dl), platelets 24 × 104 (12 × 104 – 40 × 104 /μl), C-reactive protein (CRP) 0.9 mg/dl (< 0.5 mg/dl), anti-thrombin 3 (AT3) 108 (80–130%), thrombin antithrombin III complex (TAT) 10.9 (< 3.0 ng/ml), D-dimer 6.1 (< 1.0 μg/ml), plasminogen activator inhibitor-1 (PAI-1) 91.4 (< 50 ng/ml), and plasmin α2-plasmin inhibitor complex (PIC) 0.8 (0.4–0.8 μg/ml) , AT3 87%, TAT 36.0 ng/ml, D dimer 111.1 μg/ml, PAI-1 27.3 ng/ml, and PIC 13.7 μg/ml  for 60 h, which has effects against both excessive coagulation and fibrinolysis, and cefmetazole (2 g/day) for 2 days. A decrease in bleeding was observed in a few hours. There was rapid improvement of her symptoms of abdominal pain and uterine bleeding and the laboratory data  and is thus an inhibitor of fibrinolysis. As it inhibited activation of blood fibrinolysis, DIC might not occur at that time. Five days after the procedure, the Hb level and platelet count were decreased, and PAI-1 was decreased to 27.3 ng/ml in normal range. However, TAT was increased to 36.0 ng/ml, and D dimer and PIC were increased to 111.1 μg/ml and 13.7 μg/ml, respectively, although AT3 was still in the normal range. Based on these data, the patient could be diagnosed as developing DIC with both excess coagulation and fibrinolysis. We thought that a large number of thrombi had already formed 3 days after the procedure. Considering the changes of PIC and D dimer, fibrinolysis was excessive with increased coagulation after 3 days of the procedure. Therefore, we thought that a large number of microthrombi had formed inside the adenomyotic tissue around 3 days after the procedure, which subsequently led to the rapid onset of DIC with excessive fibrinolysis. Since severe fibrinolysis dysfunction was confirmed in the peripheral blood, it was presumed that the fibrinolysis dysfunction inside the uterus was further enhanced. For this reason, massive uterine bleeding occurred, and it is thought that a rapid decrease in the Hb level and a decrease in the platelet count were observed.Four cases of cerebral infarcts associated with adenomyosis have been reported . InteresIn the present case, nafamostat mesilate was administered because the coagulation function was still activated, and nafamostat mesilate has effects against both excessive coagulation and fibrinolysis to prevent the development of infarcts , 20. AdmShe was very grateful that she saved her uterus as she was unmarried and desired to get a child in future.To the best of our knowledge, this is the first report of successful conservative treatment of acute non septic DIC after termination of early pregnancy in a woman with adenomyosis. The clinical course strongly suggested that bleeding inside the adenomyotic tissue promoted excessive coagulation and fibrinolysis, which was then related to the development of DIC. Although severe hyperfibrinolysis is observed in peripheral blood, the fibrinolysis state in the myometrium is considered to be even worse. The newly formed clot for hemostasis under the uterine mucosa could be removed due to the excess fibrinolytic system in the adjacent myometrium and could cause massive bleeding. This case serves as a warning that adenomyosis might induce DIC with excessive fibrinolysis after termination of pregnancy as a trigger and the usefulness of nafamostat mesilate to treat it in patients with huge adenomyosis who want to preserve their uterus. Before considering hysterectomy to control refractory uterine bleeding, nafamostat mesilate should be considered as one option, thinking the pathophysiology of the massive bleeding due to uterine adenomyosis. Conservative treatment can be considered in the case of “women who wants to conserve her fertility”.


Objectives. To elucidate the mutation in the novel compound heterozygous CFTR causing CF in Chinese family. Materials and Methods. Clinical samples were obtained from a Chinese family, the brother and sister with recurrent airway infections, hypoxemia and obstructive ventilatory impairment, sinusitis, clubbed fingers, salty sweat, and nasal polyposis. We performed whole-exome sequencing on the family and validated all potential variants by Sanger sequencing. Results. Next-generation sequencing showed a novel compound heterozygous CFTR mutation (c.400 A > G p.Arg134Gly and c.3484 C > T p.Arg1162∗) which resulted in CF in the family. Conclusions. As this mutation is consistent with the observed clinical manifestations of CF and no other mutations were detected after scanning the gene sequence, we suggest that their CF phenotypes are caused by the compound heterozygous mutation, c.400 A > G p.Arg134Gly and c.3484 C > T p.Arg1162∗. As c.400 A > G is not currently listed in the Cystic Fibrosis Mutation Database, this information, regarding the CF-causing mutations in two Chinese patients, is of interest.Cystic fibrosis (CF) is one of the most common autosomal recessive disorders among Caucasians of Northern European descent but is uncommon in the Chinese population.  It is characterized by nasal polyposis, chronic obstructive pulmonary disease, and congenital bilateral absence of the vas deferens (CBAVD). Cystic fibrosis is diagnosed by clinical symptoms, laboratory tests including immunoreactive trypsinogen (IRT) testing, and sweat chlorometry . Of courte to CF .Approximately 50% of Caucasian CF patients are homozygous for the ΔF508 mutation, which results in a complete loss of CFTR function and the classic, severe manifestations of the disease. About 40% of CF patients have ΔF508 on one chromosome and another less common mutation on the other chromosome. The remaining ∼10% of patients have two rare mutations , 4.∗) in CFTR, with typical clinical characteristics, such as recurrent airway infections, hypoxemia and obstructive ventilatory impairment, sinusitis, clubbed fingers, salty sweat, and nasal polyposis.In European and American countries, CF is inherited as recessive mutations, and both are homozygous. However, Professor Xilun Tian's team at Peking Union Medical College Hospital reported that the CFTR mutations associated with CF in Chinese population are very different from those in western patients; the common CFTR mutation group in Europe and America was used . TherefoIn this report, we elucidated the causative role of this novel compound heterozygous mutation in the pathogenesis of a severe form of CF along with a review of previously reported cases.This study was approved by the Research Ethics Committee of Haihe Hospital, Tianjin University, and all experiments were performed in accordance with approved guidelines.9/L, with 89.5% N. Blood gas analysis showed an oxygen partial pressure of 58.1 mmHg, indicating respiratory failure. Chest CT . The male patient was found to have blocked vasa deferentia but normal sperm activity, and via IVF (in vitro fertilization), he fathered two test-tube babies who were healthy. The female was unmarried. Their parents are not consanguineous marriage. Neither of them had a history of chronic respiratory disease besides their mother having breast cancer.Genomic DNA was extracted from the peripheral blood of both patients according to the manufacturer's protocol .Whole-exome sequencing was performed for both patients, and all exons were captured by the Agilent SureSelect Human All Exon V5 kit on an Illumina Cluster system with SBS enrichment. The average sequencing depth of the target region was≥90×, and the sequencing depth for 95% of the target sequence was greater than 20×. Base recognition was performed on all sequenced fragments. This test was established and verified by Mingma Biotechnology . Sentieon software was used in the secondary analysis to analyze the sequencing data. Sentieon BWA was compared with the UCSC hg19 reference genome. The WuXi Next CODE development process was used to annotate variants. In addition, the sequencing depth and variation prediction for each base were obtained from the genomic sequencing data. Variant effect predictor was used for annotation. Three major databases containing known or suspected pathogenic variants, including ClinVar, OMIM, and HGMD, were used to screen for known pathogenic variants, and multiple tools were used to predict the function of missense variants and annotate noncoding regulatory sequences.With the elder brother as the proband, we conducted genetic testing on his father, mother, sister, son, and daughter. The results are shown in Figures 2The genomic DNA sequencing indicated that the brother and sister had the same mutations in the CFTR gene. Sanger sequencing confirmed that they carried the same compound heterozygous mutation (c.400 A > G and c.3484 C > T) in CFTR. Sequencing of the parental DNA showed that the c.400 A > G mutation was inherited from the father, and the c.3484 C > T mutation was inherited from the mother.CF is an autosomal recessive disorder resulting from mutations in the CFTR gene that resulted in deficient or dysfunctional CFTR protein. Mutations in CFTR cause a multisystem disease, and mean survival of patients was about 40 years . CFTR en∗). One mutation (c.400 A > G p.Arg134Gly) was inherited from the father, and the other (c.3484 C > T p.Arg1162∗) was inherited from the mother. The third generation is a set of fraternal twins who have one heterozygous CFTR gene mutation (c.400 A > G p.Arg134Gly) and are disease-free.Generally, it was believed that patients must be homozygous for a CFTR mutation to develop CF, because carriers with a single CFTR mutation have approximately 50% of the normal CFTR function, which is enough to maintain their health . HoweverThe CFTR gene is located in chromosomal region 7q31.2 and is composed of 27 exons , and enc∗, was previously reported. This mutation is located in exon 22 and is a C ⟶ T substitution. It is a nonsense mutation that changes the CGA codon at position 1162, which encodes arginine to a UGA stop codon (R1162∗) [We screened all exons of CFTR and detected two mutations in the family, and one mutation, c.3484 C > T p.Arg1162∗) detected in our study resulted in CF when combined with a previously unreported CFTR mutation (c.3119 T > A p.L1040H). The R1162X mutation carrier in our study is healthy, without any respiratory issues or other CF manifestations. The same is true for the p.Arg134Gly carriers. This suggests that this novel, compound heterozygous mutation causes CF and results in CBAVD.CF caused by compound heterozygous mutations were reported. Terlizzi et al.  investigCF is common among Caucasians but is rare in Chinese populations. Until now, only dozens of CF patients have been reported in Chinese populations. However, the numbers are increasing because of genetic testing , 21–23. ∗ heterozygous carrier or c.400 A > G p.Arg134Gly heterozygous carrier, cannot induce CF. The effect of the c.400 A > G p.Arg134Gly variation is unknown. Although these gene loci appear to be far away from one another, functional supplementation may occur. The study results suggest that the p.Arg134Gly variation may supplement the function of the R1162X heterozygous mutation to promote the development of CF. The variant spectrum in the Chinese population is different from that of the Western population; thus, a CFTR mutation database for the Asian population is greatly needed.In conclusion, we found a novel heterozygous mutation in a Chinese family. One of the detected CFTR mutations alone, either c.3484 C > T p.Arg1162


Aortic thrombosis (ATh) is an uncommon condition in dogs, with limited understanding of risks factors, outcomes, and treatments.To describe potential risk factors, outcome, and treatments in dogs with ATh.Client‐owned dogs with a diagnosis of ATh based on ultrasonographic or gross necropsy examination.Multicentric retrospective study from 2 academic institutions.P = .04). Sixty‐five dogs were hospitalized with 41 subsequently discharged. Sixteen were treated as outpatient and 19 euthanized at admission. In‐hospital treatments varied, but included thrombolytics (n = 12), alone or in combination with thrombectomy (n = 9). Fifty‐seven dogs survived to discharge. Sixteen were alive at 180 days. Using regression analysis, ambulation status at the time of presentation was significantly correlated with survival‐to‐discharge (P < .001).One hundred dogs were identified. Anti‐thrombin diagnosis, 35/100 dogs were nonambulatory. The dogs were classified as acute (n = 27), chronic (n = 72), or unknown (n = 1). Fifty‐four dogs had at least one comorbidity thought to predispose to ATh, and 23 others had multiple comorbidities. The remaining 23 dogs with no obvious comorbidities were classified as cryptogenic. Concurrent illnesses potentially related to the development of ATh included protein‐losing nephropathy (PLN) (n = 32), neoplasia (n = 22), exogenous corticosteroid administration (n = 16), endocrine disease (n = 13), and infection (n = 9). Dogs with PLN had lower antithrombin activity than those without PLN  (Dogs with ATh have a poor prognosis, with nonambulatory dogs at the time of presentation having worse outcome. Although the presence of comorbid conditions associated with hypercoagulability is common, an underlying cause for ATh was not always identified. ATEaortic thromboembolismAThaortic thrombosisDVTdeep venous thrombosisTPA, tissue plasminogen activator; PLNprotein losing nephropathy1Aortic thrombosis (ATh) is an uncommon condition in dogs, thought to be associated with a regional prothrombotic environment in the distal aorta.Consensus guidelines on the optimal strategy for the prevention and treatment of thrombosis in animals have been established, but never studied prospectively.Available literature on ATh in dogs is limited to case reports, descriptive studies on small numbers of dogs, and a few larger studies without information on outcome or treatments.2The electronic medical records at Cummings School of Veterinary Medicine at Tufts University (TCSVM) and The Ohio State University College of Veterinary Medicine (OSUCVM) were searched for dogs diagnosed with ATh. Cases were collected between 1997 and 2014 at TCSVM and 2005 to 2011 at OSUCVM. Dogs were included if a diagnosis of a distal ATh was determined by either visualization of a thrombus  by abdominal ultrasound or by direct visualization at necropsy. Dog characteristics, including sex, breed, age, and weight, were recorded. Presenting complaint, physical examination findings, locomotor ability, results of diagnostic tests performed, and initial clinicopathological data were also documented. All diagnostic imaging was performed or reviewed by a board‐certified diagnostic imaging specialist. All laboratory diagnostics were performed at the clinical pathology laboratories supervised by board‐certified clinical pathologists. Assessment of acute and chronic ATh was determined based on chronicity of signs. Dogs showing signs for at least 48 hours were considered chronic, and dogs showing signs for less than 48 hours acute. Underlying conditions possibly contributing to ATh in individual dogs were recorded after review of the medical records. Dogs were classified as cryptogenic in the absence of a concurrent diagnosis of a medical condition known to predispose to a hypercoagulable state. In‐hospital and outpatient treatment, overall duration of hospitalization, survival to discharge, and 180‐day survival were recorded when available.t test for normally distributed data, and a Wilcoxon rank sum for nonparametric data. Categorical data were tested with a χ2 test or Fisher's Exact test (if more than 2 cell counts were less than 5). The following candidate clinical variables were entered into a backward regression analysis: ambulatory status, BUN, sex, corticosteroid administration, hospitalization, pelvic limb deficits, bilateral clinical signs, and neoplasia. Prediction of outcome was tested with the Wald test with respect to variables' association with survival‐to‐discharge. Significance was set at a P < .05. All statistics were performed using commercially available software .Descriptive statistics were used to describe dog characteristics and treatment administered. Normality was assessed by the Shapiro‐Wilk test and parametric data are reported as mean (SD) and nonparametric data as median (range). Comparisons between groups  was tested using a Student's 3From the medical records at TCSVM and OSU‐CVM, a total of 100 dogs with ATh were included . Aortic thrombosis was diagnosed by ultrasonography in 91 cases and by direct visualization at necropsy in 9 cases. Dogs diagnosed with ATh during necropsy did not have an ultrasound performed but exhibited clinical signs compatible with ATh.3.1There were 47 spayed female dogs, 40 castrated males, and 13 intact male dogs. The median age was 10 years (2‐17 years). Median body weight was 24.7 kg (3.2‐45.1 kg). Thirty‐six breeds were represented including: Labrador retriever (n = 16), Greyhound (n = 14), Shetland sheepdog (n = 7), mixed breed dog (n = 7), Border collie (n = 5), Standard Poodle (n = 3), Beagle (n = 3), Rottweiler (n = 3), Golden Retriever (n = 3), Pug (n = 3), Weimaraner (n = 3), Maltese (n = 2), Staffordshire Bull Terrier (n = 2), Pit bull (n = 2), Cavalier King Charles Spaniel (n = 2), Chihuahua (n = 2), Dalmatian (n = 2), and Cocker Spaniel (n = 2).3.2For all cases, the median rectal temperature was 101.2°F (96.8‐105.1°F), median heart rate was 120 beats/min (50‐220), median respiratory rate was 32 breaths/min (12‐70), and the median systolic blood pressure was 162 mm Hg (80‐240). Sixty‐two dogs had a Doppler blood pressure > 160 mm Hg. Pelvic limb physical examination abnormalities were identified in 85/100 dogs; 35/85 were nonambulatory at the time of presentation, 32/85 were considered painful, 62/85 had bilateral locomotor abnormalities, 56/85 had absent femoral pulses bilaterally, and 30/85 were noted to have cold pelvic limbs.3.3P < .001), have pelvic limb neurologic deficits (P = .007), have bilateral signs (P = .01), and be painful (P = .03) compared to dogs with chronic ATh (Table P = .04). However, the percentage of euthanasia‐at‐admission was similar for dogs with acute signs compared to dogs with chronic signs .The median duration of clinical signs before presentation was 10.5 days (0‐912). Twenty‐seven dogs were presented with acute signs, 72 with chronic signs, and 1 dog with an unknown duration of clinical signs. Dogs with acute signs were more likely to be nonambulatory , ALP n = 42), creatine kinase (n = 36), BUN (n = 38), creatinine (n = 21), bilirubin (n = 20), and alanine transaminase (n = 41) above the reference interval (RI) for the laboratory; and albumin (n = 31), hematocrit (n = 21), and protein (n = 20) below the RI . Activated partial thromboplastin time was measured in 47 dogs (27 survived to discharge and 20 nonsurvivors) and was above the upper end of the RI in 9 dogs. Thromboelastrography (TEG) was performed in 21 dogs; 11 dogs were considered hypercoagulable, 7 dogs normocoagulable, and 3 dogs hypocoagulable. We defined a hypercoagulable TEG tracing in our study as having a clot strength G above the breed‐specific reference ranges for each institution, as previously described in dogs.P = .04). The thrombus was located at the terminal aorta in 99 dogs , with concurrent aorto‐iliac thrombosis being noted in 54 dogs. An assessment of the occlusive characteristics of the thrombus was recorded in 90/100 dogs, with complete occlusion recorded in 10/90 dogs, partial occlusion in 64/90 dogs, and 16/90 were nonocclusive. Echocardiography was performed or supervised by a board‐certified cardiologist in 35/100 dogs. The main findings were suspected infective endocarditis (n = 4), a visible thrombus within the left ventricle (n = 3), mitral insufficiency (n = 3), left ventricular hypertrophy (n = 3), cardiac mass (n = 3), main pulmonary artery thrombus (n = 1), and aortic insufficiency (n = 1). Dogs whose echocardiograms identified thrombi in the left ventricle were not diagnosed with concurrent structural heart disease based on echocardiography findings. None of those dogs were considered to have a cardiogenic cause of ATh, as dogs with endocarditis and cardiac masses were classified as infectious and neoplastic, respectively.Prothrombin time (PT) was measured in 43 dogs (28 survived to discharge and 15 nonsurvivors). Prothrombin time was above the upper end of the RI for the specific machine used in 11 dogs. Out of the 11 dogs with prolonged PT, 2 survived to discharge and 9 were nonsurvivors (3.5The most commonly diagnosed systemic condition known to be associated with hypercoagulability in our study population was PLN n = 32), either alone (n = 22) or associated with 1 (n = 8) or 2 other conditions . Cryptogenic dogs survived to discharge at a rate of 60.1% compared to 55.8% from noncryptogenic dogs (P = .81).Twenty‐three dogs were classified as cryptogenic despite diagnostic testing including an abdominal ultrasound (n = 16), a complete blood count (n = 15), a chemistry profile (n = 15), a urinalysis (n = 15), an echocardiogram (n = 10), PT/pro‐thrombin time (n = 9), and/or a TEG (n = 4). Only 6/23 did not have any diagnostics performed and were euthanized upon admission. The mean age of cryptogenic dogs was 10.7 ± 2.8 years. The median body was 28.7 kg (5.9‐44.0) and 56 were males. Twelve different breeds were represented, and the most common was Greyhounds (n = 6). There was no statistically significant between the percentage of Greyhounds in the cryptogenic group (26%) vs the noncryptogenic groups .A total of 57 dogs survived to hospital discharge including the 16 dogs treated as outpatients and 41 of the hospitalized dogs , anticoagulant drug monotherapy (n = 5), combined anticoagulants and antiplatelet medications (n = 12), or combined anticoagulant drugs  dogs .Our study has several limitations. As a retrospective study, dogs were not randomized to treatment, and the treatments used varied greatly in the medications, dosing, and outpatient protocols. Some data points were missing, including more thorough TEG evaluation, and some information such as adverse effects of treatment or clot resolution was not collected. Case selection was based on ultrasound and necropsy findings, and it is possible that some cases of ATh in dogs were not included because of the use of another diagnostic modality. Dogs with ATh might have been underdiagnosed in dogs with mild locomotor or neurological abnormalities because of the lack of recognition and clinical suspicion. As all cases were collected from specialty centers, it is possible that our study population reflects the most severely affected ATh dogs. The cases collected from each of the centers span different timeframes within the period of our study and might reflect an evolving understanding of thrombosis over the 17‐year span of our study. In addition, our logistic regression was determined using several candidate variables chosen specifically by the authors. The variables chosen could be derived from medical records and assessed in isolation. Consequently, some potentially important disease characteristics  might have been overlooked in our regression modeling.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.Authors declare no IACUC or other approval was needed.Authors declare human ethics approval was not needed for this study.


P > 0.05). Our meta‐analysis revealed that the incidence of ligament release in the MA‐TKA group was higher than that in the KA‐TKA group. This meta‐analysis shows that the KA‐TKA group had better clinical outcomes and knee range of flexion than the MA‐TKA group at short‐term follow‐up.The aim of this study was to estimate whether kinematic alignment (KA) improves knee function or clinical outcomes compared with mechanical alignment (MA) in the short term after total knee arthroplasty (TKA). We searched the literature for randomized controlled trials published before January 2020 from PubMed, EMBASE, Google, Web of Science, Cochrane Library, and other databases. The observation markers included “The Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index,” “Knee Society Score (KSS),” “Oxford Knee Score (OKS),” “combined Knee Society Score (KSS),” “Knee injury and Osteoarthritis Outcome Score (KOOS),” “European Quality of Life Measure‐5 Domain‐5‐Level (EQ‐5D‐5L),” range of motion (ROM), lower limb alignment, ligament release, and complications. A total of 11 randomized controlled trial studies were included in the study. During the follow‐up of 6–24 months, the KA‐TKA group was superior to the MA‐TKA group in terms of WOMAC scores, combined KSS, KSS, knee function scores, and knee range of flexion, but there was no significant difference in EQ‐5D‐5L, KOOS, KOOS , complications, knee range of extension, hip‐knee‐ankle (HKA) angle, tibial component slope angle, lateral distal femoral angle (LDFA) or medial proximal tibial angle (MPTA) angle between the MA‐TKA group and the MA‐TKA group ( Although the survival rate of MA‐TKA has improved, approximately 20%–25% of patients remain unsatisfied with the outcome.Therefore, this article to evaluate whether the clinical outcome of KA‐TKA is better than that of MA‐TKA. Osteoarthritis (OA) is the most widespread joint disease in the elderly, and knee osteoarthritis (KOA) is more frequent than OA of the hip or ankleet al.et al. et al.Traditional mechanical alignment (MA) has been used in TKA for more than 30 years, and it is still common worldwide. It is generally believed that a hip–knee–ankle (HKA) angle within less than 3° of the neutral mechanical axis is essential for postoperative limb recovery after TKAet al.While relevant meta‐analyses have been published in recent years, these studies included randomized controlled trials, case reports, and systematic reviewsThis meta‐analysis method was based on the Cochrane Collaboration standard. We searched the literature database for randomized controlled trials (RCTs) published before January 2020. The databases that were searched included PubMed, EMBASE, Google, Web of Science, and Cochrane Library. The retrieval strategy was performed by the method of free words combined with the Medical Subject Headings (MeSH). The literature search was conducted using the keywords “Total Knee Arthroplasty,” “Kinematic Alignment,” “Kinematic,” “Mechanical Alignment,” “Mechanical,” and “biomarker” using Boolean operators (AND), (OR), and (NOT). Literature was retrieved without restricting the language.Criteria for inclusion: (i) randomized controlled trials; (ii) comparisons of clinical results between KA‐TKA and MA‐TKA in total knee arthroplasty; (iii) primary knee replacement surgery; (iv) observation indexes include “The Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index,”, “Knee Society Score (KSS),”“Oxford Knee Score (OKS),” “combined Knee Society Score (KSS),” “Knee injury and Osteoarthritis Outcome Score (KOOS),” ”EQ‐5D‐5L,” range of motion (ROM), lower limb alignment, ligament release, and complications; and v) studies published in English.Criteria for exclusion: (i) basic research or cadaver study; and (ii) inaccessible data or full‐text.Data from the studies from all selected articles were extracted independently by two of the authors using a data extraction template, which was designed before the database searches. From each study, first author, years, type of study and surgery, sample size, follow‐up time, and clinical outcome were extracted from the literature. Any disagreement was resolved through discussion and consensus or consultation with other authors in cases of disagreement. If the data from a study was missing, insufficient, or vague, we contacted the author or corresponding authors by email or telephone to retrieve further information.Literature quality was evaluated independently by two of the authors with the Cochrane Collaboration Network risk evaluation tool. The risk of bias for each indicator was divided into three levels: “low,” “high,” and “unclear.” If we obtained more than 10 articles, a funnel chart or Eggers regression test was used to assess publication bias.The WOMAC is a validated questionnaire to evaluate lower extremity osteoarthritis and joint replacement. The WOMAC questionnaire produces three subscale scores  and a total score. Patients are asked to answer each question about the severity of pain, stiffness, or behavioral difficulties experienced in the previous 48 hours. There are five response options ranging from “none” to “extreme” to choose. A response of “none” was scored as 0,”mild” as 1, “moderate” as 2, “severe” as 3, and “extreme” as 4. The scores of the questions in each subscale were summed together to get scores for pain, stiffness, and physical function. A lower subscale score indicates less pain, less stiffness, or better physical function. A total score of < 70 is considered a severe score, 21–48 is moderate, <21 is mild.The KSS is a condition‐specific validated questionnaire widely used to evaluate the functional capabilities of the knee joint before and after total knee arthroplasty. The scoring system consists of two parts. One part is the knee score. The assessment includes pain (maximum 50 points), stability (maximum 25 points), total range of flexion (maximum 25 points), and other items . The other part is the function score. The assessment includes walking distance (maximum 50 points), ability to climb stairs (maximum 50 points), and the use of walking aids. The highest score for each part is 100 points, and a higher score means better knee function. The evaluation result score is rated as four levels: 80–100 points, 70–79 points, 60–69 points, <60 points.P < 0.05 considered statistically significant. Heterogeneity between studies was evaluated by calculating the I2. If the I2 was greater than 50%, it was considered high heterogeneity, and a random‐effects model was chosen to analyze the data; otherwise, the fixed‐effects model was applied. Enumerated data were presented as the risk ratio (RR) or odds ratio (OR) and 95%CI, while continuous data were presented as the weighted mean difference (WMD) or standardized mean difference (SMD) and 95%CI as a statistical measure of the curative effect. We attempted to use a funnel plot to evaluate the publication bias; a symmetrical funnel plot may indicate a low publication bias, while an asymmetric funnel plot may indicate possible publication bias.Statistical analysis of all extracted data was carried out using Review Manager 5.3 , with We retrieved 11,075 papers from the databases; 6754 duplicates were removed by EndNote X9.1 software Fig. . This leThe 11 papers included were evaluated for risk of bias according to the seven aspects in Fig. I2= 93%). We found that one of these studiesI2= 71%), so the random‐effects model was used for further analysis. The results showed that the WOMAC score of the MA‐TKA group was higher than that of the KA‐TKA group , KOOS symptoms , KOOS pain , KOOS ADL , KOOS sports , and KOOS QoL . Three trials with a total of 291 patients evaluated EQ‐5D‐5L. The meta‐analysis result showed low heterogeneity (I2 = 0%), and hence, a fixed‐effects model was used for further analysis. The results showed that the two groups had similar mean scores , tibial component slope angle , LDFA angle , and MPTA angle . The results of two trials with 181 patients showed that the femoral component internal rotation to sulcus line angles of the two groups were different  but the difference in flexion range angle did . Furthermore, the KA‐TKA group had a higher mean flexion range angle than the MA‐TKA group (P = 0.0005).Our review found a total of seven relevant studies, including four studiesI2 = 0%). The incidence of ligament release was lower in the KA‐TKA group than in the MA‐TKA group  and minor complications .Of the 11 studies, five studiesThe 11 studies (RCTs) that fulfilled the inclusion criteria included 1103 participants: 553 patients in the KA‐TKA group and 550 patients in the MA‐TKA group. Furthermore, follow‐up ranged from 6 months–8 years. Most of the literature reported that the patients were followed up for 6–24 months. The results of the KA‐TKA group were better than those of the MA‐TKA group in terms of the WOMAC score, combined KSS, KSS, knee function score, and knee range of flexion, while the EQ‐5D‐5L, KOOS, KOOS , complications, knee range of extension, HKA angle, tibial component slope angle, LDFA, and MPTA angle in the KA‐TKA group were not significantly different from those in the MA‐TKA group. The incidence of ligament release in the KA‐TKA (29/144) group was lower than that in the MA‐TKA (48/144) group, and the difference was statistically significant.et al.vs MA in TKA, and KA‐TKA reduced the incidence of ligament releaseP = 0.0008). Consistent with findings in previous studiesAlthough the survival rate and the clinical and functional outcomes of TKA are very good overall, approximately 20%–25% of patients remain unsatisfied with the outcomeet al.P = 0.0005). However, Ishikawa et al.P = 0.70). Howell et al.et al.et al.Although the goal of KA‐TKA is to restore normal knee kinematics or prearthritic kinematics and restore the patient to previous functional levels, the concern for increased risk of patellofemoral instability and polyethylene wear was raisedHowever, some limitations still existed in this study. First, high heterogeneity existed in some comparisons. Although we used several subgroups, heterogeneity was still present in some results, and we failed to thoroughly explain the heterogeneity. Second, although the short‐term clinical effect after KA‐TKA has been evaluated, there is a lack of long‐term follow‐up results for the survival rate of the prosthesis. Finally, the chief limitation of this study was the small sample size; thus, a large sampler size, more rigorous RCTs, and longer follow‐up supporting these results will be needed in the future.This meta‐analysis shows that the KA‐TKA had better outcomes than the MA‐TKA on WOMAC score, Combined KSS, KSS (Society and Function) score, and knee range of flexion at short‐term follow‐up.The authors declare that we have no conflict of interest.


This study aimed to clarify the relationship between physical performance and oral function in older women participating in community‐based physical and oral exercise programs.We included 163 older women (mean age: 77.4 ± 8.6 years) who participated in weekly health programs in Takehara City, Hiroshima Prefecture, Japan, from August to December 2018. Physical fitness was assessed using a handgrip strength test, a timed up and go test, a one‐leg standing time with eyes open test, and a 30‐s chair stand test (CS‐30). Tongue pressure, oral diadochokinesis, and dysphagia risk assessment for the community‐dwelling elderly were used to assess oral function.r = .234, p = .009; /ta/: r = .299, p = .001; and /ka/: r = .283, p = .002) in the older group. Multivariable analysis revealed a significant positive relationship between CS‐30 and /ta/ repetition in the older group (p = .016).Participants were divided into women aged 65–74 years (younger group) and those aged ≥75 years (older group). There was no significant difference in oral function measures between the groups. A significant correlation was found between CS‐30 and oral diadochokinesis for /pa/, /ta/, and /ka/  may be associated with physical performance  in women aged ≥75 years. Further study is necessary to clarify sex differences in oral function deterioration. The Japanese Ministry of Health, Labor, and Welfare has implemented a community‐based integrated care system, which can provide community healthcare resources for community‐dwelling older people . We calculated the sample size required for the correlation coefficient using G*Power  with a statistical power of 90%, a significance level of 5%, and an effect size of 0.3 to be 112 subjects. Of 189 women aged ≥65 years, seven older women who had been certified as requiring long‐term care and 19 participants who had been certified as requiring help were excluded. Finally, we enrolled 163 women (mean age: 77.4 ± 8.6 years). The study design was approved by the Ethical Committee of Hiroshima University (No. E‐2573), and all participants signed an informed consent agreement. This program mainly consisted of 40 min of physical exercise and 10 min of oral exercise as preventive care . The mean peak handgrip strength of the right and left hands was used for analysis. The timed up and go test is a functional test to assess mobility, falling risk, and walking ability. It requires the participants to stand up from a chair, walk to a line on the floor 3 m away, turn, walk back, and sit down again, and measures the overall time to complete the series of movements , skeletal muscle mass, the number of remaining teeth, and oral function measures with a p value of <.05 through univariate analysis were used as explanatory variables. Statistical analysis was performed using JMP® Pro 12 software .Spearman's rank correlation coefficient was used for statistical analysis. The Mann–Whitney 44.1The participants' characteristics are summarized in Table 4.2r = .2–.4 were considered weakly correlated; correlation coefficients of r = .4–.7 were considered moderately correlated. There was a significant weak negative correlation between handgrip strength and DRACE score . A significant moderate negative correlation between the timed up and go test time and repetition of the monosyllable /ta/ was found . Additionally, a significant weak negative correlation was found between the one‐leg standing time with eyes open test and the DRACE score . A significant positive correlation was found between CS‐30 score and ODK .The association between physical function measures  and oral function measures  was examined in the younger group Table . Correla4.3p value of <.05 in Table p = .036). There was a significant association between the timed up and go test time and age (p = .02). However, no significant association was found between the one‐leg standing time with eyes open test and clinical factors or between the one‐leg standing time with eyes open test and oral function measures. There was also no significant association between CS‐30 and clinical factors or between CS‐30 and oral function measures.Multivariable analysis for each measure of physical function was performed using physical function measures as the dependent variables and oral function measures showing a 4.4r = .277, p = .002) and between handgrip strength and ODK . Additionally, there was a significant weak positive correlation between the one‐leg standing time with eyes open test and ODK . Furthermore, a significant weak correlation was found between CS‐30 score and ODK /pa/, /ta/, and /ka/ .There were significant weak positive correlations between handgrip strength and tongue pressure . A significant negative association was found between one‐leg standing time with eyes open and age (p < .001). There was a significant relationship between CS‐30 score and repetition of /ta/ (p = .016).Significant independent variables in the multivariable analysis are summarized in Table 5In this study, older participants exhibited significantly lower skeletal muscle mass than younger participants. Muscle strength  was also lower in older participants than in younger participants. Our results suggest that the decline in skeletal muscle volume  and decreased muscle strength may be associated with aging.It was speculated that older participants would show greater deterioration in oral function than younger participants. However, there was only a small difference in the decline in tongue pressure, ODK, and DRACE score in the older group when compared with the younger group. This result suggests that not age but other clinical factors may be involved in the maintenance of oral function in older women. Wakasugi, Tohara, Machida, Nakane, and Minakuchi  reportedWhen assessing clinical factors related to physical function, we found a significant association between handgrip strength and skeletal muscle mass by multivariable analysis in both groups. The findings of this study are consistent with previous research showing a close relationship between skeletal muscle mass and handgrip strength in middle‐aged and older women , low activity in daily living, and poor nutrition .Our previous study revealed that regular physical and oral exercise may be helpful for older women who have not been certified as requiring long‐term care to prevent the impairment of oral function  was importantly associated with physical performance  in women aged ≥75 years. Multivariate analysis of the small number of participants in the younger group may have resulted in a possible bias. Therefore, further additional study is necessary to clarify the correlation between physical and oral function in people aged 65–74 years. Moreover, independent older women may exhibit a slow decline in oral motor function. Further additional study will be necessary to clarify the sex differences in the deterioration of oral function in community‐dwelling older adults.The authors declare that they have no conflict of interest.


Chromochloris zofingiensis. We demonstrate that elevated carbon availability and active path rate of precursors are determinants for product biosynthesis, and the former exhibits a superior potential. As protein content reaches a threshold value to confer survival advantages, carbon availability becomes the major limiting factor for product biosynthesis and cell reproduction. Based on integrated interaction, regulating the C/N balance by feeding carbon source under excess light increases content of high-value products without inhibiting cell growth. Our findings provide a new orientation to achieve great productivity improvements in microalgal production.Accumulation of high-value products in microalgae is not conducive with rapid cell growth, which is the potential conflict in microalgal production. Overcoming such conflict faces numerous challenges in comprehensively understanding cell behavior and metabolism. Here, we show a fully integrated interaction between cell behavior, carbon partitioning, carbon availability and path rate of central carbon metabolism, and have practically overcome the production conflict of  Sun et al. study the effects of light intensities and C/N ratios on microalgal growth and biomass production. They use chemical and transcriptomic assays to demonstrate that feeding carbon source under excess light increases high-value products without inhibiting cell growth. Microalgae, photosynthetic cell factories with high photosynthetic efficiency, are essential for capturing and recycling the global carbon dioxide, and potentially convert it into biomass over time and distance. In addition, microalgae can be exploited in wastewater treatment and biofuel production due to their advantages of nitrogen and phosphorus loading, no competition with food production, high oil content, and rapid growth rate5. Alternatively, microalgae are also considered as the promising cell factories producing profitable natural pigments and lipids in food fields, and offsetting the cost in various treatments during cultivation and harvesting6. Consequently, environmental issues and food revenues make microalgal production a matter of overall consideration. One of the most obvious examples is the conversion of process-compatible products always negatively affects cell growth. Recently, optimized culture strategies and genetic engineering approaches have been developed for alleviating the potential conflict between biomass concentration and its valuable products7. However, the obstacle still remains as productivity of biomass and associated lipids and pigments is controlled by a set of complex but poorly understood environmental and genetic factors1. Furthermore, considering the challenges of applicability and validity under Algae Raceway Integrated Design, microalgal production is needed to coordinate environmental conditions in agricultural and industrial processes9, and thus commercialization are very different. To efficiently address environmental issues with considerable economic benefits, understanding of underlying relationship between the conditions and cell behavior is essential.Bio-based economy is urgently being driven by the changing global climate and the increasing demand of renewable biofuels and oleochemicals12. Among the condition factors, light and nutrient have been reported to take charge in the important physiological and morphological responses13. Though light is essential for photosynthesis, the solar-to-biomass conversion efficiency is <6% for most photosynthetic organisms14. Up till now, light intensity, light quality, and light cycle have been tested in order to confirm their best implementation in cell growth, reproduction and accumulation of high-value products15. On the other hand, nutrient is involved in carbon and nitrogen metabolism, where modes of repletion, starvation, recovery and recycle are considered as valid strategies for increasing the productivity of biomass and high-value products16. Furthermore, coordinating advantages of light and nutrient brings out various powerful strategies for microalgal cultivation such as light-increment culture, flashing-light culture, mixing-light culture, fed-batch culture, gradient-fed culture, semi-continuous culture, and nutrient-starvation stress culture20. However, since most reported cultivations are carried out in a black-box model through a single chemical reaction of nutrient into biomass, precision and adaptability of the strategies proposed above are limited in the realistic cultivation. In this case, global understanding between cell behavior and cell metabolism responding to a certain environmental factor is needed for achieving a higher biomass value.Microalgae excel in adapting to environment or saving energy for survival under various environmental conditions18. Variations in light and nutrient would lead algal cells to grow at a steady-growth state or redistribute carbon sinks for survival. For instance, previous study stated sufficient nutrient preferably increased the biomass, while excess light and nitrogen starvation favored in the accumulation of lipid and carotenoid by elevating pyruvate availability18. Consider excess light has a great potential to promote accumulation of carotenoid and polyunsaturated fatty acids (PUFAs). It concludes that elevated intracellular carbon availability is beneficial for biosynthesis of lipid and secondary metabolite21. However, given the fact that elevated carbon availability under stress condition is at the expense of cell growth18, it is essential to systematically investigate the relationship between carbon efficiency and path rate of the products, since efficient exogenous carbon ensures the cell reproduction and sufficient intracellular carbon availability helps to gain revenues from lipid and secondary metabolites. At the molecular level, tricarboxylic acid (TCA) cycle and synthetic pathways of lipid and carotenoid share pyruvate and acetyl-CoA as carbon precursors, which can be consumed at a fast rate once the synthetic genes are upregulated4. However, the path rates under different environmental factors are seldom studied, limiting the metabolic regulation to suppress or remove the pathways with high-energy consumption. Furthermore, no report has focused on the significant influence of exogenous carbon on cell behaviors and metabolism. Exogenous carbon molecule is the key factor to link cell growth and product accumulation through its complex influence on carbon availability and path rate of carbon metabolism4. When tolerating stress conditions, algal cells commonly pave ways for lipid accumulation through protein degradation, which provides carbon skeletons, energy molecules , and reducing power 4. In addition, as cellular protein stoichiometry is largely dependent on nitrogen availability22, the supplied carbon is then linked to the carbon/nitrogen (C/N) stoichiometric ratio, which is reported to range from 6:1 to 15:1 as a result of acclimatization and subsistence24. Therefore, a comprehensive understanding of the relations among nutrient, light, and C/N balance at the molecular level will no doubt deliver great productivity improvements, and then blaze a new frontier in microalgal production for the highest revenue.In a cell factory, dynamics of biomass component and metabolite analysis have revealed that light and nutrient cause compositional shifts through carbon partitioning and central carbon metabolism13C tracer-based metabolic flux analysis (13C-MFA) are applied to explain the carbon behaviors in central carbon metabolism. In combination with light, exponential fed-batch cultures providing different C/N ratios are designed to explore complex interactions of carbon source, carbon availability and path rates of central carbon metabolism. Such interactions are finally described through metabolic flux analysis and targeted metabolites of precursors for lipid and carotenoid biosynthesis. The results of this study will potentially overcome the conflicts between biomass concentration and high-value product yield, and make it possible to accurately design cultivation processes of microalgae for the highest revenue.The objective of the current study is to comprehensively understand the influence of light and nutrient on microalgal production in carbon partitioning at the molecular level. First, a kinetic model is established to explore the changes of carbon partitioning under different light intensities, followed by a determination of photosynthetic characteristics. Then, transcriptomics and 15. As shown in Fig. −2 s−1, and subsequently reduced at 300 µE m−2 s−1. In addition, intensity at 50 µE m−2 s−1, as the most suitable light intensity, induced the highest growth rate with the maximal biomass at 72 h. The previous study suggested excess light could restrain cell growth by changing carbon partitioning and limiting photosynthetic efficiency18. As shown in Fig. −2 s−1. The decreased protein would lead to growth retardation or even cell death4. Interestingly, although protein degradation at heterotrophic culture was slower than those at 150 and 300 µE m−2 s−1, its lipid accumulation rate was the highest among the cultures. The changes of carbon partitioning under dark and light conditions followed different rules.As light is an essential source for photosynthetic activity, its change in intensity potentially influences the biomass concentrationDP, αC and YCX,max were all the highest at 300 µE m−2 s−1, which suggested excess light induced carbon flux from protein to carbohydrate and then to lipid. However, DP in dark was close to that at 50 µE m−2 s−1, whereas αC and YCX,max were lower than those at 50 µE m−2 s−1, which revealed carbon flux was oriented from protein into lipid directly as the time extended under heterotrophic culture. Therefore, elevated carbon availability for lipid biosynthesis was mainly due to the degraded protein molecules both under excess light and darkness. Moreover, YPX,max and μglucose were at a higher level under heterotrophic culture, which suggested that direct connection of protein and lipid might be beneficial for the conversion of glucose into protein. Therefore, numerous researches suggested that heterotrophic cultivation was an efficient and sustainable approach for some microalgae to produce lipids6.Therefore, a kinetic model was designed to explore the dynamic changes of carbon partitioning. As shown in Table 2 uptake rate and biomass concentration4. As shown in Fig. Fv/Fm and ETR under darkness and excess light were both lower than those under suitable light intensity. As Fv/Fm is a stressing indicator and ETR represents electron transfer, their decreases suggested darkness and excess light inhibited photosynthesis18. NPQ under darkness (p = 0.041) and excess light (p = 0.005) was significantly higher that revealed more energy was quenched as thermal dissipation. Accordingly, reported researches revealed that excess light damaged the photosystem of Chlamydomonas reinhardtii and heterotrophic culture limited the photosynthesis of C. zofingiensis25. In addition, as 0.03% CO2 (V/V) in air was not sufficient for the rapid growth under mixotrophic culture, the photosynthetic efficiency could not achieve the maximum due to the tough limitation by CO226. Besides, the exogenous glucose was also revealed to trigger chloroplasts degradation and decrease photosynthesis of C. zofingiensis27. By comparing photosynthetic characteristics under darkness and excess light, light intensity at 300 µE m−2 s−1 created a more severe stress condition that largely limited photosynthetic efficiency, as its Fv/Fm was lower (p = 0.001) and NPQ was higher (p = 0.035) than those at 0 µE m−2 s−1. In addition, Rubisco-bisphosphate carboxylase/oxygenase (Rubisco) is the first enzyme involved in Calvin cycle, which largely determines the photosynthetic carbon assimilation. As shown in Fig. −2 s−1 (p = 0.002) and 300 µE m−2 s−1 (p < 0.001) was reduced significantly compared with that at 50 µE m−2 s−1, suggesting the photosynthetic carbon assimilation was limited. Furthermore, the limited CO2 potentially reduced the assimilative capacity under the mixotrophic culture26. Therefore, the speediest cell growth under 50 µE m−2 s−1 could be attributed to the higher μglucose and the highest level of Rubisco activity. On the other hand, μglucose and Rubisco activity were the lowest at 300 µE m−2 s−1, which revealed that elevated carbon availability for lipid biosynthesis was mainly due to the conversion of carbon partitioning under mixotrophic culture with excess light.Photosynthesis begins with the activation of assimilative pigment centralized by light harvesting antenna complex. Its activity ensures a higher CO4. Pigment content and composition influence photosynthetic efficiency and cell growth. As shown in Fig. a and chlorophyll b are major light harvesting pigments, their decreases in pigment composition could also cause a limited photosynthetic efficiency28. In addition, energy for cell metabolism under heterotrophic culture is from respiratory action29. However, O2 could be converted into reactive oxygen species (ROS) by electron transfer of NADPH oxidase30. The intracellular ROS at 0 μE m−2 s−1 (p = 0.001) was significantly higher than that at 50 μE m−2 s−1 with active NADPH oxidase. Therefore, heterotrophic culture had a great potential to produce large amounts of NADPH and achieve high ROS levels, which were beneficial for lipid biosynthesis. Furthermore, the highest ROS level was achieved at 300 μE m−2 s−1. In addition to the active NADPH oxidase, the limited CO2 and sufficient O2 under excess light were both prone to induce photorespiration that delivered electrons to O2 and finally generated ROS. Consequently, mixotrophic culture with excess light was the worst condition for cells, which was in accordance with the results of Fv/Fm, ETR and NPQ.Light harvesting antenna complex in microalgae is commonly comprised of about 80% chlorophyll and 20% the rest pigments, which account for around 5% of biomassCz08g30210, and the performance of assimilatory nitrate reduction was enhanced by potentially accelerating amino acid metabolism. Moreover, Cz13g11170 and Cz04g15230 regulating fructose and mannose metabolism from fructose-6-phosphate were downregulated. Since results of carbon partitioning revealed C. zofingiensis under heterotrophic culture tended to guide carbon flux from protein into lipid directly, the changing genes might then be in the charge of this carbon behavior. However, heterotrophic culture had less influence on the photosynthetic capacity  was then applied to summarize the functional genes. Comparing with light intensity at 50 µE m−2 s−1, darkness and excess light both regulated genes related to cell death and apoptotic process, suggesting they influenced the cell cycle progression. The previous study stated that the programmed cell death involved H2O2 in various ways and excess of H2O2 led to changes in gene expression and cell death31. Therefore, excess light exhibited more significant impacts to downregulate the relative genes, which was mainly ascribed to the highest level of ROS. In addition, regulation of Wnt signaling pathway was beneficial for cell division and survival, and its downregulation in Cz13g05130, Cz17g13110, and Cz07g27090 at 0 µE m−2 s−1 revealed that heterotrophic culture induced cell growth, whereas limited its division for biomass accumulation. As phospholipids could be transferred to diacylglycerol for triacylglycerol synthesis, the downregulation of Cz03g23260, Cz03g23240, and Cz03g23250 at both 0 and 300 µE m−2 s−1 suggested the triacylglycerol synthetic metabolism was suppressed to some extent.The algal cells displayed different carbon behaviors and photosynthetic properties as light developed to excess, which meant light acted as a key factor to control the cell metabolism. Nine high-quality transcript profiles were generated with high reproducibility among the three biological replicates  pathway was then decreased, whereas its use for lipid biosynthesis was increased  and FADH2 (p = 0.005), it might then result in the highest ROS level , PP pathway, and TCA cycleght Fig. . As clocp = 1.000), which might be due to the low requirement for protein biosynthesis. At 50 μE m−2 s−1, NRFC significantly heightened biomass concentration more than NSFC (p < 0.001). Our previous study revealed that nitrogen starvation limited the photosynthetic efficiency and nutrient absorption, leading to a lower cell growth rate than those under nitrogen repletion, and thus the complete nutrients were profitable for the green growth18. Interestingly, at 300 μE m−2 s−1, NSFC largely improved biomass concentration with one-fold increase than NRFC at 300 μE m−2 s−1, which was close to that obtained by NRFC at 50 μE m−2 s−1 (p = 0.211). The enhanced biomass accumulation might be ascribed to the active metabolic networks under nitrogen starvation, which reversely induced more carbon molecules absorbed into cells. It could be concluded that nitrogen repletion worked against rapid cell growth under the stress conditions, which then broke the routine that setting suitable environment was the best choice for the highest cell activity and biomass in microalgal production.Although carbon availability can be increased under stress conditions, the strategy to ensure its rapid consumption may be of great significance for the continuous cell progression. Therefore, nitrogen-repletion fed-batch culture (NRFC) and nitrogen-starvation fed-batch culture (NSFC) to regulate cellular C/N balance were applied to create two physiological steady-growth states of algal cells, one for green growth stage and the other for lipid biosynthesis. As shown in Fig. −2 s−1 increased the content and productivity of high-value products . However, NSFC at 300 μE m−2 s−1 resulted in a higher carotenoid content than NRFC (p = 0.044), which was close to that under a suitable condition (p = 0.175). The fed-batch cultures also influenced fatty acid profiles. Considering the stability and caloricity of oil quality, contents of C16:0 and C18:1 were the important indicators for biodiesel34. As shown in Fig. p = 0.004) under NRFC, while showed no effect (p = 0.109) under NSFC. In addition, NRFC significantly increased astaxanthin content (p < 0.001) and decreased lutein content (p < 0.001) at 300 μE m−2 s−1 . However, it limited carbohydrate accumulation that provided precursors and energy for lipid biosynthesis. Therefore, the residual protein, source of carbon molecules, then performed the critical role for subsistence and would not degrade in spite of the fact biosynthetic capacity of lipid and astaxanthin had yet reached the highest. Although transcriptional level and path rate of fatty acid biosynthesis revealed that excess light accelerated lipid accumulation preferably, algal cells inherently constrained carbon molecules as residual protein substances to guarantee their survival advantages. The improved carbon availability induced by NSFC under excess light was guided into lipid directly, not through the transfer station of carbohydrate, which was more efficient for glucose utilization. Therefore, feeding carbon source under stress conditions could emphatically provide a rapid direction for converting nutrients into high-value products, which then had less influences on biomass accumulation.The outstanding advantage of NSFC was that it could achieve a comparable biomass with that under suitable environment and the highest productivity of the most valuable products. This interesting phenomenon might be related to the accessible carbon and nitrogen molecules. As shown in Fig. 35. However, its content under NSFC was rarely detected, which might indicate a fast carbon-conversion rate in carotenoid biosynthesis. As α-carotene was the precursor for biosynthesis of lutein, whose content was the highest at 50 μE m−2 s−1, its decrease under darkness and excess light suggested these conditions were beneficial for biosynthesis of secondary metabolites35. In addition, NSFC significantly decreased lutein content at 0 μE m−2 s−1 (p = 0.009) and 50 μE m−2 s−1 (p = 0.021), while it had no significant influence under excess light (p = 0.058). However, unlike the lutein biosynthesis, the lower precursor β-carotene resulted in a higher astaxanthin content, which revealed that biosynthesis of lutein was in a carbon-storage manner and astaxanthin as a carbon-usage form.Another benefit of NSFC was it constructed a stressing environment through nitrogen starvation, which made it possible for the rapid biosynthesis of lipid and astaxanthin, as well as for continuous cell cycle progression. As shown in Fig. 36. Harnessing cultivation condition and environment is considered as a useful approach to produce the products purposefully. However, by assessment of biomass component in a black box, accuracy and validity of the approach are challenged for the lack of comprehensive understanding of carbon behaviors37. The same treatments will then exhibit different influences on biomass and product accumulation.It is now universally acknowledged that valuable product accumulation in microalgae cannot go without efficient carbon conversion among the main carbon sink−2 s−1, ascribed to the insufficient biosynthesis of protein or its degradation38. Then, results from kinetic model, transcriptome analysis and 13C-MFA suggested there was in dire need of the reservation of carbohydrate and lipid to confirm the survival advantages, and elevated carbon molecules were guided into these carbon sinks. However, darkness downregulated Cz16g00050, Cz02g14160, Cz13g05150, Cz04g09090, UNPLg00257, and Cz01g09160 that participated into lipid synthesis, which demonstrated that the increased lipid content was attributed to the elevated carbon availability  guarantee a suitable condition for rapid cell growth; (2) construct a stressing environment for product biosynthesis, and (3) provide enough carbon availability and high ROS level for both rapid cell growth and product biosynthesis. Regulating cellular C/N ratio was critical in microalgal production as it could realize a higher lipid and astaxanthin contents without affecting cell growth. This study then overcame the biggest conflict between biomass concentration and accumulation of high-value products in microalgal production.Normally, stress-based strategies were capable of enhancing the biomass value by upregulating path rates of lipid and several carotenoids, whereas largely reacting against cell growth or even inducing cell death. Since the strategies elevated available carbon molecules through conversion of the main carbon sinksChromochloris zofingiensis (ATCC 30412) was obtained from the American Type Culture Collection . The strain was cultured in BG-11 media. The cultures were incubated in 250-mL Erlenmeyer flasks containing 100 mL Kuhl media at 25 °C for 4 days and illuminated with a continuous light intensity of 30 μE m−2 s−1. C. zofingiensis grows well in Kuhl medium, which consists of (per L): KNO3 2.02 g, NaH2PO4·2H2O 0.7 g, Na2HPO4·12H2O 0.181 g, MgSO4·7H2O 0.247 g, CaCl2·2H2O 14.7 mg, FeSO4·7H2O 6.95 mg, H3BO3 0.061 mg, MnSO4·H2O 0.169 mg, ZnSO4·7H2O 0.287 mg, CuSO4·5H2O 0.0025 mg, (NH4)6MO7O24·4H2O 0.01235 mg. The pH value was adjusted to 6.5 prior to autoclaving.−1 glucose as the carbon source. The cells were cultivated in 250-mL Erlenmeyer flasks containing 100 mL of growth media at 23 °C under continuous illumination of 0, 30, 50, 150, and 300 μE m−2 s−1. During fed-batch culture, the feeding media and conditions were set according to our previous study40. For metabolic flux analysis of the labeled group, 20 and 100% U-13C glucoses were set as the carbon source and 1 mL seed cells were washed and inoculated in 100 mL labeled media for cell growth. The other conditions were same as the unlabeled.The seed cells were grown in Kuhl with 5 g LQP, QC, and QL were the content of protein, carbohydrate and lipid respectively (g L−1), YPX and YCX represented the protein and carbohydrate yields on biomass concentration respectively (g g−1), YCL and YPL represented carbohydrate and protein yields on lipid yield respectively (g g−1), t was the cultivation time (h), S was the substance concentration (g L−1), DP and αC were the conversion rate of protein and carbohydrate (h−1) respectively. Then, the above equations could be converted into:μ was the specific growth rate of (h−1), μP, μC, and μL were the specific generating rate of protein, carbohydrate, and lipid (h−1) respectively, X0 was initial cell concentration (g L−1). The rates were influenced by protein content and could be described as:The model was constructed to predict storage carbon behaviors based on the following assumptions: (1) the cultivation conditions were constant, (2) the extracellular metabolites during the exponential growth phase were seldom and had no inhibitory effect on microalgae, (3) nitrogen was the only limited factor for carbon partitioning, and (4) carbohydrate and lipid concentrations were proportional to the biomass concentration. The behaviors of the carbon sinks  were initially designed based on the general physiological change of microalgae. The nutrients in the media were absorbed and assimilated into protein and carbohydrate firstly. During the growth stage, a part of protein was degraded and converted into carbohydrate, lipid and other functional compartment. Partial carbohydrate was reallocated into lipid, especially as the environmental conditions got worse. Therefore, the global carbon flux can then be simplified into two specific fluxes, which lead to the final production of lipid. The model was established as the following equations:YPX and YCX were also influenced by the protein content.Therefore, indexes of C. zofingiensis, exponential fed-batch culture was used to supply sufficient nutrients during the exponential growth phase. The feeding nutrients during cultivation were fixed at the initial concentrations, respectively. Therefore, extracellular products were seldom produced and consumption of substance and energy was low for cell maintenance during the phase. The fed-batch culture containing nitrogen was named as nitrogen-repletion fed-batch culture (NRFC), whereas without nitrogen was defined as nitrogen-starvation fed-batch culture (NSFC). The model was expressed as the following equation:FS was the feeding concentration of substrate and YXS was biomass yield on substrate. When the nutrient concentration was maintained constantly, equation was described as:To increase biomass of VFS was the feeding rate (mL h−1), CFS was the nutrient concentration in feeding media (g L−1) and V0 was the initial cultivation volume (mL).Then, the volume was used as the feeding index, Eq.  could be18. The lipid was extracted and determined by measuring the weight. The carbohydrate was extracted and determined by phenol sulfuric acid method. The protein was extracted and collected to measure the protein concentration by protein assay kit .The lipid, carbohydrate, and protein were determined as previously described−1 perchloric acid and measured by the acetyl-CoA assay kit . For determination of pyruvate content, pyruvate was extracted and measured by the pyruvate assay kit .For determination of acetyl-CoA content, acetyl-CoA was extracted by 1 mol L41. The cells were collected by centrifugation at 13,000 × g for 5 min. The pellet was then redissolved with 99.9% methanol and incubated at 45 °C for 24 h in dark. The extract was centrifuged at 13,000 rpm for 5 min and the supernatant was used to measure the absorbance at 470, 652.4 and 665.2 nm. The pigment concentrations were calculated according to the previous study41.The pigment contents were determined photometrically42.The carotenoids were extracted by acetone and analyzed by HPLC  that was equipped with a Shiseido CAPCELL PAK C18 5 μm column (4.6 × 250 mm) and a 2998 photodiode array detector . The operational parameters were set according to the pervious studyFv/Fm) of the photosystem II and electron transport rate (ETR) were measured by a pulse-amplitude-modulated fluorometer  according to the previous study18. The ROS levels were determined with the ROS assay kit  as previously described18. The fluorescence was immediately measured by a fluorescence microplate reader .The maximum quantum yield . For determination of NADPH oxidase activity, the samples were extracted and measured according to kit’s instruction .https://www.thermofisher.com/). The RNA quality and concentration were examined using Agilent 2100 Bioanalyzer (Agilent Technologies) and NanoDrop 2000C (Thermo Scientific). Then mRNA purification was carried out using Sera-mag Magnetic Oligo(dT) Beads (Thermo Scientific). The transcriptome libraries were prepared using the TruseqTM RNA sample pre Kit  and sequenced for 2 × 150-bp runs (paired-end) using a Illumina HiSeq 4000 sequencing system  by Majorbio (Shanghai) Co., Ltd, China. Reads were aligned to the C. zofingiensis genome43  with HISAT2 (https://ccb.jhu.edu/software/hisat2/index.shtml). Reads mapping to more than one location were excluded.Total RNA was extracted using TRIzol reagent . Genes were considered to be significantly differentially expressed if their expression values showed at least a two-fold change with an FDR adjusted P-value < 0.05 between control and experimental conditions. KEGG and Go enrichment analysis were carried out using KOBAS (https://kobas.cbi.pku.edu.cn/nome.do) and Goatools (https://github.com/tanghaibao/GOatools) respectively.Gene expression was measured as the numbers of aligned reads to annotated genes using RSEM software and normalized to FPKM values. The DEGs were identified using DESeq2 software . The glucose uptake was set as 100 to compare the different influences of cultures on central carbon metabolism. The abundance of labeled amino acid was modified by analyzing 100% U-13C glucose, although the contribution of CO2 to biomass was low  was used to detect the significant differences from the respective control groups for each experimental test condition.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The purpose of this study was to examine the association of lean body mass (LBM) to the development of vasomotor symptoms (VMS) as women transition through menopause.This study is a secondary use of data available for public use from follow up visits six through 10 for participants in the Study of Women’s Health Across the Nation. The study examined 2533 women, between the ages 42–52 years, each year over a10-year period. Data was modeled for associations of lean body mass and VMS. Changes in LBM since prior visit and since baseline were also modeled along with differences in means using binary logistic regression, adjusting for covariates.p = .036), percent change in LBM since prior visit (p = .003), percent change since baseline (p < .001), and overall means associations (p = .023). LBM was not significant for VMS at individual visit measures. In mixed regression modeling, time was significant (p < .0001) at all visits. The estimated probability of developing VMS decreases significantly as LBM increases.LBM was significantly associated to concurrent VMS (Lean body mass is negatively associated with incident VMS. Our data suggests that maintaining higher levels of LBM during the menopausal transition may be protective against the development of VMS. Every woman will experience menopause in her life and the ability to potentially prevent the onset of specific symptoms through basic interventions, such as resistance training to increase lean body mass, may positively impact this large population. Menopause is a significant event in many women’s lives as it marks the end of the natural reproductive life. For most women, menopause will occur between the ages of 40 and 58 years with the average being 51 years . SymptomAs they transition through menopause, women will simultaneously experience a decrease in basal metabolic rate and a loss of lean muscle tissue which increases the risk of weight gain and obesity . Sarcopeα) or interleukine 6 (IL-6) [1) - Lower concurrent LBM will be associated with greater concurrent incident reporting of VMS; Hypothesis 2 (H2) - In longitudinal analyses, lower LBM over time, since baseline, will be associated with greater incident reporting of VMS; Hypothesis 3 (H3) - In longitudinal analyses, lower LBM over time, since last annual visit, will be associated with greater incident reporting of VMS.For postmenopausal women, chronic systemic inflammation, oxidative stress, abdominal visceral adipose tissue, dyslipidemia, sarcopenia, and a sedentary lifestyle are all risk factors for metabolic syndrome . A syste6 (IL-6) . A recen6 (IL-6)  suggesti6 (IL-6) . Decreas6 (IL-6) . MaintaiThis study was a secondary analysis of existing data utilizing the public dataset from the SWAN study –19. Deta2)). For the purposes of this study, LBM refers to the level of skeletal muscle mass and will be represented by the variable SMI. Fat free mass, total body water, and percent body fat were all provided by RJL Systems and validated using NHANES III data [2) and fat free mass index (FFMI = kg/m2). Height (m) and weight (kg) were measured in light clothing, without shoes, using a standard protocol with a stadiometer for height and a balance beam scale for weight. Hip circumference (cm) was measured at the iliac crest and waist circumference (cm) was measured at the level of the natural waist or the narrowest part of the torso from the anterior aspect [Analysis was limited to the use of body composition data collected using BIA and included 2533 women. BIA is based on measurement of the transmission speed of a one-quarter volt electrical pulse between electrodes attached at the feet and electrodes attached across the knuckles of the hand. Because fat-free mass is comprised of water, proteins, and electrolytes, conductivity is greater in fat-free mass than in fat mass . Resistar aspect , 27.Hot flashes and night sweats were assessed via questionnaire at each SWAN visit. Women responded to two questions that separately asked them to record how often hot flashes and night sweats were experienced in the 2 weeks prior to the annual visit . Accuracy of recall for VMS among the SWAN participants was previously verified .Covariates were selected on the basis of previously documented associations with VMS  and bodyx2) for categorical variables, and Kruskal-Wallis test for continuous variables. A scatter plot matrix was used to examine linear correlations among variables. For the purposes of modelling, LBM is represented by the SMI variable. Additionally, to account for the nonindependence of longitudinal observations derived from the same woman and data in which the number of observations may differ across women, longitudinal modeling using SAS PROC MIXED incorporated a random intercept term to account for the correlated errors among repeated measures of the same woman. Missing values of time-varying variables were interpolated based on prior and subsequent values for gaps of one to two visits as in previous SWAN analyses [1, incident VMS was modeled as a function of concurrent LBM using logistic regression analysis. To address H2 regarding long term change in LBM, the model was expanded to add within-woman percent change in LBM since baseline and to address H3, regarding recent change in LBM, the model was expanded to add within-woman percent change in LBM since prior visit (approximately 1 year earlier). The overall association between LBM and VMS was estimated in binary logistic regression models. Statistical analyses were one-tailed with an alpha level of 0.05 and conducted using SAS University Edition .Characteristics of the sample were described by means (standard deviation) and frequency (%). At baseline, two VMS groups – any or none – were compared for group differences in, and associations among, demographics , quality of life (SF-36 score), and clinical characteristics , and VMS was estimated using chi square test  were included in longitudinal analyses for VMS with LBM. At visit 10 there were 800 women remaining in the study.At baseline (visit 6) there were 2533 participants remaining in the SWAN study who were on average 52 years old and the differences in the group characteristics are shown in Table 0 (864) = 0.931, p < .0001) and symptoms = any (r1 (1143) = 0.933, p < .0001), and a moderate positive correlation to FMI (r0 (864) = 0.567, p < .0001) (r1 (1143) = 0.579, p < .0001). FMI showed a strong positive correlation to FFMI at baseline for both groups (r0 (864) = 0.820, p < .0001; r1 (1143) = 0.826, p < .0001). Pearson correlation of mean SMI to mean FMI  was moderately strong for ‘none’ (r0 (405) = 0.648, p < .0001) and relatively weak for ‘any’ with (r1 (611) = 0.559, p < .0001). Mean FFMI was strongly correlated to mean FMI in both groups (r0 (405) = 0.860, p < .0001; r1 (611) = 0.829, p < .0001). Mean SMI was very strongly correlated to both symptom groups for mean FFMI (r0 (405) = 0.942, p < .0001; r1 (611) = 0.923, p < .0001). SMI continued to be very strong as expected and the relationship of FFMI to FMI remained consistent over time.At baseline, SMI showed a strong positive correlation to FFMI both for symptoms = none (r1 (VMS associations with concurrent LBM) results are summarized in Model 1 where LBM is significant in both the unadjusted (p < .0001) and adjusted (p = .036) analyses. For H2 (VMS associations with LBM over time since baseline), within woman percentage change since baseline was added to Model 1 and results are presented as Model 2. Again, LBM showed significance in both unadjusted (p = .001) and adjusted (p < .001) modelling. For H3, (VMS associations with LBM over time since prior visit) within women percentage change since prior visit was added to Model 1 and the results are summarized as Model 3. LBM remained significant in unadjusted (p = .009) and adjusted (p = .003) models respectively.Models were developed to address the three hypotheses and are presented as odds ratios and 95% confidence intervals for incident VMS in Table p = .0003), 2 (p < .0001), 3 (p < .0001), and 4 (p = .0073). Race/ethnicity was significant in Models 1 (p < .0001) and 2 (p = .0139) but failed to reach significance for Model 3 or 4. Menopausal status was significant (p = .0052) in Model 4 only. Age was not significant in any model. In mixed regression models of VMS and LBM repeated for time (symptoms as random effect), results were significant (p < .0001) for least squares means at visit (time) 6, 7, 8, 9, and 10. Additionally, significance (p < .0001) was found for the difference of least square means (Tukey-Kramer method) at visit 6 , and for time effect visit 8–10 (p = .007) (data not shown). Further, estimated predicted probabilities were examined to determine the likelihood of LBM to predict the development of VMS. The probabilities estimated for given levels of SMI at each of the four visits following baseline, including the mean over time, for women reporting no VMS at visit 6 (n = 1179) shows significant negative correlation to levels of SMI  at visit 6 to 72% (n = 733) at visit 10, our study was able to document the movement through menopausal transition for the majority of the cohort.This study is among the first to examine the relationship of lean body mass to vasomotor symptoms longitudinally. Using the data for this large, multiethnic sample of mid-age women from the SWAN study, we found that participants with higher relative levels of LBM were less likely to develop VMS as they transitioned through menopause. This effect was found to be independent of sociodemographic factors and levels of fat mass. Additionally, as the average age of the participants in our study increased from 52.0 to 56.6 years and the number of women who were postmenopausal increased from 40% (Considerable recent research has focused on the association of body mass index (BMI) and percent body fat with VMS and has suggested a positive correlation between increasing BMI and the presence of VMS , 32–36. Evidence is emerging on the role of oxidative stress in menopause and its relationship to muscle mass. Lipoperoxide (LPO) levels are considered a measure of oxidative stress and in postmenopausal women are found to be significantly higher than in premenopausal women suggesting that the depletion of estrogen is a risk factor for oxidative stress . A recenCertain limitations need consideration when interpreting results of the current study. The use of BIA measurement involves several assumptions, and while skeletal muscle mass was calculated using a validated equation by Janssen et al.  and fat This study was among the first to examine the longitudinal association of LBM to VMS, providing new evidence that lean mass may provide protection against the development of VMS as women transition through menopause. Using the longitudinal SWAN database that encompasses a large, multiethnic sample of women from across the United States, these findings are particularly relevant and transferable to general populations in North America. Given the continued resistance to hormone replacement therapy as a means to mitigate symptoms, these results provide for the possibility of symptom prevention through resistance training programs. Importantly, the results of our study suggest that the greatest contributing factor to mitigating symptoms was maintaining LBM throughout the menopausal transition.


Cardiorespiratory fitness (CRF) is a powerful predictor of mortality. This study evaluated the predictive value of CRF for mortality in Chilean subjects without atherosclerotic disease compared with the Framingham, European Systematic Coronary Risk Evaluation (SCORE), and 2013 ACC/AHA risk scores and determined the incremental predictive value of CRF when added to these scores.CRF improves prediction of all‐cause and cardiovascular disease (CVD)‐related mortality of the standard international risk scores.Cross‐sectional study, which evaluated 4064 subjects between 2002 and 2016. Cardiovascular (CV) risk factors, anthropometric and biochemical parameters, and blood pressure were measured. CRF was determined by metabolic equivalents during maximum stress test. The Framingham, SCORE, and ACC/AHA risk scores were calculated for all subjects. After a median follow‐up of 9 years, all‐cause and CVD‐related mortality were assessed. Receiver operating curves were built to determine mortality prediction for CRF, the risk scores, and CRF added to the scores.As of August 2016, 99 deaths were reported, 33 of which were CVD‐related. All risk scores and CRF predicted CVD‐related mortality, with CRF identified as the best predictor: CRF: C = 0.88 (95% CI: 0.82‐0.93) vs Framingham: C = 0.68 (95% CI: 0.60‐0.76), SCORE: C = 0.76 (95% CI: 0.70‐0.83), and ACC/AHA: C = 0.79 (95% CI: 0.73‐0.85). Predictive power of the three scores improved when CRF was added to the model, but this was only significant for the Framingham score.CRF is a good predictor of both, all‐cause and CV mortality and a better predictor of CVD‐related deaths than standard risk scores in this population. This program only receives subjects in primary prevention, older than 18 years old, and without known ischemic heart or cerebral disease, heart failure (HF), peripheral and carotid ischemic vascular disease, and any cardiac surgery, congenital heart disease, and pregnant women.2.1.1Upon enrollment, all subjects participated in an interview with the program nurse, during which demographic information and medical history were collected. Also, the following variables were measured: weight, height, body mass index (BMI), waist/hip circumference, and systolic and diastolic blood pressure (SBP). In all patients, fasting venous blood samples were collected to measure glycemia, lipids, plasma creatinine levels, and thyroid‐stimulating hormone. Blood pressure was measured seated three times after 5 minutes resting at 2‐minute intervals, using a device with a brachial cuff with automatic inflation (Omron HEM 742). Subjects who performed leisure‐time physical activity <1 time/week of <30 minutes were considered sedentary.2 and ≥ 30 kg/m2, respectively; and (e) smoking: if the subject had smoked one cigarette or more during the last month.Cardiovascular RFs were defined according to the following criteria: (a) hypertension: previous medical diagnosis of hypertension, with or without pharmacologic treatment, and subjects with two or more BP determinations ≥140/90 mm Hg on alternate days; (b) dyslipidemia: previous medical diagnosis of hypercholesterolemia, with or without pharmacologic treatment, and subjects with total cholesterol ≥200 mg/dL and/or high‐density lipoprotein cholesterol (HDL‐C) < 40 mg/dL in men and < 50 mg/dL in women, respectively; (c) diabetes: was defined as fasting glucose ≥126 mg/dL, or nonfasting glucose ≥200 mg/dL, or reported diagnosis of diabetes coupled with the use of glucose‐lowering medication; (d) overweight and obesity were defined as the World Health Organization (WHO) criteria: ≥25 kg/m2.1.2In those few patients whom the nurse and the cardiologist thought they would not be able to walk to maximum, we used the modified Bruce protocol.CRF was recorded as the maximal aerobic capacity expressed in METS achieved in a symptom‐limited exercise stress test, walking on a treadmill using the Bruce protocol, according to recommendations of the AHA.Framingham, SCORE, and ACC/AHA risk scores were calculated for all subjects according to published equations.All subjects provided a written informed consent approved by the Ethics Committee of the Pontificia Universidad Católica de Chile to analyze their data in academic investigations.2.1.3Samples for glycemia and lipid profile were obtained by venous puncture following 12 hours of fasting. Samples for glucose measurement were collected in gray cap tubes with sodium fluoride and potassium oxalate as inhibitors of Becton Dickinson brand glycolytics: BD Vacutainer tubes. Samples for the lipid profile were collected in tubes with separating gel of the brand Becton Dickinson: BD Vacutainer SST. Glycemia and lipids were analyzed using Cobas from Roche 8000 modular analyzer series . Low‐density lipoprotein cholesterol (LDL‐C) was calculated using the Friedewald formula when triglycerides were < 400 mg/dL.2.2t‐test for different variances with Welch correction for continuous variables and Chi‐square test for discrete variables were used to compare differences between groups. Comparisons between living and deceased and between CRF groups were adjusted by age and sex .Student Discrimination models were built for the prediction of mortality risk using CRF adjusted for age, sex, and the Framingham, SCORE, and ACC/AHA risk scores. The following models were recorded: CRF; Framingham + CRF; SCORE + CRF; ACC/AHA + CRF; Framingham; SCORE; and ACC/AHA. The discriminatory capacity of these models was evaluated by the construction of receiver operating characteristic curves (ROC) with areas under the corresponding curves and 95% confidence intervals (CI) using mortality (CVD‐related) vs nonmortality as a hard endpoint. The ROC curves were based on logistic regression models, and the CIs for the area under the curve (C) were estimated using Bootstrap.We also calculated net reclassification index (NRI) to determine the improvement in prediction of CVD‐related mortality when adding CRF to each risk score.3P < .0001). Framingham risk score was significantly higher in men vs women. No significant differences were observed between men and women for total and CVD‐related mortality rates.Table P < .01).Table P < .001) and CVD‐related mortality (P = .03).Subjects with lower CRF (<10.1 METs) had significantly higher all‐cause mortality .A total of 99 deaths were reported from all causes, 33 of which were CVD‐related, with no significant differences between sexes. Individuals whose deaths were CVD‐related were significantly older, had higher blood glucose, and lower CRF than living subjects  and CVD‐related mortality  and it improved the predictive value of the three risk scores but only was statistically significant for the Framingham risk score .e > 0 = 0.80 ; NRIne > 0 = 0.03 NRIa) Framingham + CRF vs Framingham:e > 0 = 0.64 ; NRIne > 0 = 0.03 NRIb) SCORE + CRF vs SCORE:e > 0 = 0.50 ; NRIne > 0 = 0.05 NRIc) AHA/ACC + CRF vs AHA/ACC:e is the net proportion of events assigned a higher risk or risk category (CVD‐related deaths) and NRIne is the net proportion of non‐events assigned a lower risk or risk category (non‐dead).Where: NRI4In this study of a middle‐aged population, CRF proved to be the best predictor of all‐cause and CVD‐related mortality followed by the ACC/AHA, SCORE, and Framingham risk scores. These results highlight the value of CRF as a significant predictor of prognosis in CVD death in middle‐aged and intermediate‐risk populations. Moreover, NRI confirmed a significant improvement in the risk classification of SCORE and Framingham when adding CRF to the models in this population.20 demonstrated in a large cohort with no prior CVD, that the presence of low fitness was associated with an increased 30 year risk for CVD‐related deaths across all ages, sex, and risk factor strata. Recently, Mansager et alSeveral studies have shown an inverse, graded, and consistent relationship between CRF levels and mortality risk.Recently, Ekblom et al.Aerobic exercise has several cardioprotective effects besides reducing total and CVD‐related mortality: it increases exercise tolerance, decreases cardiac symptoms, has positive effects on lipids, and improves psychosocial factors, such as anxiety, anger, and stress, among others.Despite the previous beneficial effects of CRF on CVD‐related mortality and risk factors, neither American nor European cardiology associations have included CRF to current guidelines to determine CV risk better. The exclusion has been partially explained because an essential mechanism through which exercise influences its cardioprotective effects is the modification of traditional and novel risk factors. Nevertheless, Mora et al2. Our population was followed in an ambulatory setting in a primary prevention program. VO2 testing only is performed when asked by the cardiologist in special populations, that is, HF or NYHA class III entering Cardiac Rehabilitation or pre heart transplant.Although Framingham, SCORE, and ACC/AHA risk scores are widely used in observational studies to predict CVD risk, the highest proportion of CVD events, including CVD‐related mortality, occurs in intermediate‐risk populations, not in individuals defined as high risk by these scores.To our knowledge, there has been only one study showing a relationship between altered stress test and CVD risk prediction. However, in that study, they did not add CRF to SCORE risk score to define improvement in risk prediction by CRF, but only demonstrating that both predicted CVD‐related and all‐cause mortality.Our results indicate that CRF was helpful in both males and females with a low/intermediate ASCVD risk score. In these individuals, someone who had a CRF < 9 METS had a higher risk of death in 9 years. Having this information could spare the realization of CAC scoring. Also, due to the enormous prevalence of free‐time leisure sedentarism in the population, a low CRF could help to encourage regular aerobic physical activity which is lacking in most Latin American countries.Finally, our results demonstrated that SCORE and ACC/AHA risk scores statistically significantly determined CVD‐related mortality. This is important as these are currently the most used risk scores throughout the world. We demonstrated that the addition of CRF to these CV risk prediction models improved more their ability. Therefore, having good discrimination of CVD‐related death underlines that the “old and simple” CRF, determined by a stress test, still has a space in determining risk.4.1Our study has limitations. Given that the subjects included in this study are those who attend a primary CV prevention unit looking for better health, there is a potential for sample bias. Second, we only report on mortality  since there is no reliable registry of CVD events . Moreover, we did not separately report in men and women, given that in the last, only a few deaths occurred. Lastly, CRF was estimated by METS, which is an approximation of an individual's actual maximum oxygen consumption.5In conclusion, this study demonstrates that CRF is a good and independent predictor of CVD‐related mortality, superior to accepted CVD risk scores, and when added to standard risk scores it significantly increased their predictive power in our Chilean Latino population. It would be desirable that current American and European guidelines make it clear that CRF is still a valuable tool in the cardiologic armamentarium.The authors declare no potential conflicts of interest.Mónica Acevedo and Giovanna Valentino were involved in the conceptual design, analysis, and interpretation of the data. Carlos Navarrete was involved in statistical analysis and interpretation. Ramón Corbalán was involved in the conceptual design and interpretation of the data. Lorena Orellana, Fernando Baraona, Mónica Acevedo, and María José Bustamante were involved in the acquisition and interpretation of the data. Mónica Acevedo and Giovanna Valentino drafted the manuscript. All authors revised the manuscript critically for important intellectual content, and approved the final version for publication.