The C0 to C2 region is the keystone for range of motion in the upper cervical spine. Posterior procedures usually include a fusion of at least one segment. Atlantoaxial fusion (AAF) only inhibits any motion in the C1/C2 segment whereas occipitocervical fusion (OCF) additionally interferes with the C0/C1 segment.The purpose of our study was to investigate clinical outcome of patients that underwent OCF or AAF for upper cervical spine injuries.Over a 5-year period (2010–2015), consecutive patients with upper cervical spine disorders were retrospectively identified as having been treated with OCF or AAF. The Numeric Pain Rating Scale (NPRS) and the Neck Disability Index (NDI) were used to evaluate postoperative neck pain and health restrictions. Demographics, follow-up, and clinical outcome parameters were evaluated. Infection, hematoma, screw malpositioning, and deaths were used as complication variables. Follow-up was at least 6 months postoperatively.p = 0.01). Follow-up was shorter in the OCF group compared to the AAF group . No differences were found related to infection  and revision rate . Regarding bother and disability, no differences were discovered utilizing the NDI score . A reduction of disability measured by the NDI was observed with greater follow-up for all patients (p = 0.01).Ninety-six patients  underwent stabilization of the upper cervical spine. OCF was performed in 44 patients (45.8%), and 52 patients (54.2%) were treated with AAF. Patients with OCF were diagnosed with more comorbidities (Theoretically, AAF provides greater range of motion by preserving the C0/C1 motion segment resulting in less disability. The current study did not show any significant differences regarding clinical outcome measured by the NDI compared to OCF. No differences were found regarding complication and infection rates in both groups. Both techniques provide a stable treatment with comparable clinical outcome. The craniocervical junction represents a complex anatomical region consisting of two essential joints, the atlanto-occipital joint and the atlantoaxial joint . MobilitInjuries to the upper cervical spine represent a serious entity including a wide spectrum of pathology ranging from benign to life threatening . EspeciaFor posterior fixation of the upper cervical spine, two different categories of treatment options are available. Atlantoaxial fusion (AAF) procedures only interfere with the atlantoaxial motion segment (C1/C2). The AAF is the more demanding procedure compared to the occipitocervical fusion (OCF) but provides greater range of motion by preserving the C0/C1 motion segment. OCF leads to further and considerable limitation of movement compared to atlantoaxial fusion alone. After OCF, there is virtually no extension, flexion, and rotation in the upper cervical spine.According to our literature research, no comparison of clinical outcome comparing occipitocervical fusion and C1/C2 fusion has been performed previously. Therefore, the aim of this study was to compare clinical outcomes of patients with a totally impaired range of motion at the upper spine (OCF) to patients with a preserved motion of the C0/C1 joint. According to our null hypothesis, OCF patients should have greater impairment regarding pain and everyday activities.This study was an Institutional Review Board-approved retrospective and prospective cohort study of patients who underwent surgical treatment for fractures of the upper cervical spine between January 2010 and August 2015 in one referral trauma center. Surgeries were performed by four fellowship trained spine surgeons. The involved patients were retrospectively identified from the clinics database based on a computer query of Current Procedural Terminology (CPT) codes for fractures of the upper cervical spine. Informed consent was obtained, and patients were prospectively evaluated.Inclusion criteria were age greater or equal to 18 years, injuries of the upper cervical spine (C0–C2), surgical treatment by AAF or OCF, no preoperative paraplegia or tetraplegia, signed patient information, and follow-up greater than 6 months. Exclusion criteria were additional anterior stabilization, metastatic disease or pre-existing infection, and insufficient medical record or radiographic data.For AAF, the Magerl-Gallie technique was performed. Figure Occipitocervical fusion (OCF) was performed in a prone position with the patients’ head fixed with a Mayfield clamp similar to the atlantoaxial fusion under general anesthesia. For occipitocervical fixation, two different cervical spine system were utilized . Screws were inserted as lateral mass screws. Additional bone grafting was performed at the surgeon’s discretion.For clinical outcome measurement, the Neck Disability Index (NDI)  and NumeT test was performed. Fisher’s exact test was utilized to compare nominally scaled data. Significance was defined as p < 0.05. p values were rounded to one decimal place.For statistical analysis, Excel  and SPSS version 23.0  were utilized. To compare continuous variables , a n = 47 (44.3%), female: n = 59 (55.7%)), patients were treated for upper cervical spine injuries. Thereof, 10 (9.4%) patients with preoperative tetraplegia (n = 8), paraplegia (n = 1), or brain damage (n = 1) were excluded. A total of 96 patients , female: n = 54 (56.2%)) met the inclusion criteria. Of our patient population, 44 underwent occipitocervical fusion (OCF group) and 52 underwent transarticular atlantoaxial fusion in the Magerl-Gallie technique (AAF group) based on the surgeon preference. Patient age at the time of operation averaged 79 years .Between 2010 and 2015 106 ). Of these 88 patients, 60 had isolated dens fracture, 7 had odontoid fracture combined with C2/C3 spondylolisthesis, and 21 had odontoid fracture with atlas fracture. Three patients sustained an isolated atlas fracture (Gehweiler type 3). The types of fracture are shown in Table .All patients had a recent history of trauma. In all, the most common fracture was the odontoid process fracture (n = 88 9.7%). Of p = 0.01). There was no significant difference between the two groups in terms of sex, age, types of fractures, time of hospital stay, and body mass index (BMI). Significantly, more OCF patients suffered from hypertension and coronary heart disease compared to the AAF group (p = 0.02). Baseline demographic and clinical data are shown in Table Follow-up averaged 10.4 months (6 months to 5.9 years). Follow-up of the OCF group was shorter compared to that of the AAF group .Surgery was successfully performed in 96 patients. The average operation time (defined as the time from incision to skin closure) was 136.3 ± 52.3 min in the OCF group versus 139.8 ± 31.9 min in the AAF group (p = 0.01). Figure Operative time was related to BMI for both surgical techniques . Postoperative computed tomography (CT) showed malpositioning of screws/rods in one patient with OCF and 5 patients with AAF. Two patients of the OCF group had a persistent instability of the upper cervical spine postoperatively and underwent a reversion surgery. One patient sustained a cardiopulmonary arrest intraoperatively due to suspected primary cardiac event. Therefore, he underwent surgery for OCF a few days later. The postoperative revision rate was 13.6% in the OCF group versus 17.3% in the AAF group (p > 0.05). No differences were found related to hematoma and/or infection rate .Postoperatively, there was no case of nerve damage or cerebrospinal fluid leakage. No soft tissue irritation or wound infections were observed at the donor sites of the autologous iliac bone graft. The most common postoperative complications encountered in our series included hematoma and/or infection in the affected surgical region  died during hospital stay: 4 patients died due to postoperative multiple organ failure, 1 patient died to a heart attack, and finally, 1 death happened due to pacemaker dissociation. There was no significant difference between the death rate of the OCF group compared to the AAF group (14 (31.8%) and 13 (25%), respectively).Among the 96 patients, 27 patients died during the follow-up. There was no intraoperative death. Six patients . Moreover, NDI scores increased with increasing age for both operations (p = 0.01). Figure No significant differences were discovered in both groups related to postoperative neck pain: pain reduction was rated sufficient and satisfying for all patients according to the NPRS. Patients who underwent OCF indicated average pain levels of 2.9. The mean NPRS score for the AAF group was 2.0. Regarding bother and disability in daily living, no differences were discovered utilizing the NDI score. The average NDI score of the OCF group was 37.4% compared to 21.4% in the AAF group. A significant reduction of disability in daily routine measured by the NDI was observed with greater follow-up for all patients  , 22–26. There are multiple OCF techniques currently available, and they have all proved high fusion rate and reduced pain levels , 28. CurDespite distinct impairment of range of motion, OCF often is the first choice for craniocervical instability in the elderly , 39. SevCappuccio et al.  recommenp = 0.0085). All OCF patients (n = 20) complained of severe restriction of cervical spine flexion, extension, and rotation, and only 14 patients were satisfied with their outcome. Both groups had a restricted rotation of the neck, yet the additional OCFs’ restriction of the extension and flexion led to significant self-reported disability. The average age of the OCF group was 53 years (35–78) [In this study, comparisons were made between demographic data, clinical outcomes, and complications after OCF and AAF based on the data of patients with at least 6-month follow-up. Regarding clinical outcomes, no statistically significant differences, such as NPRS or NDI score, were found between OCF and AAF. Moreover, additional fusion of the C0/C1 segment, with lack of flexion, extension, and rotation of the neck (OCF group), did not lead to an increase in pain or disability in daily life. Postoperatively, both study groups presented with nearly the same pain level . There were no statistical differences of NDI scores between both groups, but patients who underwent OCF had a slightly higher NDI score (moderate pain level) compared to patients after AAF (mild pain level). Hu et al.  compared (35–78) , and thep < 0.01). They used the Japanese Orthopaedic Association (JOA) score to assess the severity of clinical symptoms in their patients. According to our results, both groups presented with mild symptoms after fixation of their upper cervical spine. In summary, and according to other studies [A significant improvement in neck pain was also documented by Hu et al. . The ave studies , 47, 48,Our study shows a negative correlation between follow-up time and NDI score. The longer the follow-up time, the better the NDI score. Similar results were found by Yuan et al. . This imRegarding postoperative adverse events, no statistically significant differences, such as infection or revision rate, were found between our study groups. The postoperative revision rate was 13.6–17.3%, and the hematoma and/or infection rate was 4.5–7.7%. In a systematic review, Winegar et al.  reportedWe acknowledge the limitations of our study. The major limitation of this study was its retrospective design and relatively small sample sizes. Due to the advanced age of the collective, many patients were deceased at time of follow-up. The lack of long-term follow-up data is another limitation of this study. Additionally, we did not have data on preoperative scores (NDI and NRPS) or range of motion. Therefore, further data and studies are warranted.Instability of the upper cervical spine endangers patients and neurologic integrity. Stable fixation techniques are essential tools in the treatment of these entities. Occipitocervical fusion and atlantoaxial fusion are both effective and safe treatment options for upper cervical spine disorders. In the hand of an experienced surgeon, both procedures provide safe stabilization for the upper cervical spine. Despite an additional fixation of the C0/C1 segment (OCF group) with a consecutively increased impairment of motion compared to the AAF group, both surgical methods lead to comparable clinical outcome in our elderly study population.


SIRT1 single nucleotide polymorphisms (SNPs), SIRT1, and eNOS  protein expressions, total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) in young patients with premature ST-elevation myocardial infarction (STEMI).  Premature myocardial infarction (PMI) is an uncommon disease, and its incidence varies between 2% and 10%, rising, depending on genetic susceptibility under the influence of lifestyle. The purpose of this study was to investigate the association between SIRT1 gene was performed in 108 consecutive patients (87.0% were men with a mean age of 40.74 ± 3.82 years) suffering from ST-elevation myocardial infarction at the age of ≤45 and 91 control subjects.  Genotyping of the three single-nucleotide polymorphisms  in  The risk for myocardial infarction was increased by 2.31 times in carriers of CC or CG genotypes. SIRT1 protein levels were enhanced and endothelial nitric oxide synthase levels were diminished in ST-elevation myocardial infarction patients regardless of the underlying gene variant. There was no correlation between SIRT1 expression and the amount of endothelial nitric oxide synthase, total antioxidant status, total oxidant status, and oxidative stress index levels in patients and in the control group either. SIRT1 single-nucleotide polymorphisms were associated with premature myocardial infarction, which affected the SIRT1 and endothelial nitric oxide synthase protein expression, irrespective of the underlying SIRT1 genotype. Myocardial infarction (MI) is an atypical disease in young individuals with a varying prevalence between 2% and 10% .An increasing prevalence of risk factors for coronary artery disease (CAD), such as impaired glucose tolerance and obesity in adolescence, is counteracting for the protection featured by young age . Thus, iPathogenesis of myocardial infarction in young people is likely to be different from those in an older population . PatientSIRT1 single-nucleotide polymorphisms (SNPs) are associated with obesity, disturbed glucose tolerance and diabetes, arterial hypertension, dyslipidemia, and coronary artery sclerosis, which may shape the cardiovascular phenotype [SIRT1 gene  were investigated related to the pathogenesis of coronary artery disease through modifying SIRT1 protein expression [It has been shown before that some of the henotype –18. In tpression .SIRT1 gene is located on chromosome 10q21.3  [The er 2014) . The reser 2014) , whereaser 2014) , latter er 2014) .SIRT1 single-nucleotide polymorphisms  and the levels of SIRT1 and endothelial nitric oxide synthase expression, total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI) as well as common cardiovascular risk factors and major adverse cardiac events (MACEs) in young patients suffering from premature ST-elevation myocardial infarction (STEMI).The purpose of this study was to investigate the association between p < 0.001). The difference of SIRT1 protein between both groups (STEMI vs control) was at least 0.7 ng/ml for any subgenotype with a standard deviation of 1.7. Corresponding to the power analysis, a minimum of 94 patients in the STEMI group were necessary to calculate a significant difference.Patients included in this study presented with a history of premature myocardial infarction (PMI) to the Departments of Cardiology of the Bezmialem Vakif University Hospital and Mehmet Akif Ersoy Heart Hospital between January 2012 and May 2015. Patients with malignancies, major trauma or surgery in the previous six months, and acute or chronic infectious disease were excluded. The randomly selected healthy control subjects were selected from people, who came to Bezmialem Vakif University Hospital for a routine examination without known cardiovascular diseases. According to the power analysis , the study groups consisted of 108 patients, who had suffered from a premature ST-elevation myocardial infarction under the age of 45  and 91 control subjects .After 12 hours of fasting, whole blood samples were taken and transferred into plain tubes . After centrifugation for 5 min at 4,500 rpm at +4°C and removal of serum and plasma, samples were stored at −20°C. Parameters like fasting glucose, total cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and very low-density lipoprotein (VLDL) were determined in all patient samples. Body mass index (BMI) was calculated by dividing weight by height square (kg/mEnzyme-linked immunosorbent assay (ELISA) kits from USCN Life Science  were used to determine levels of SIRT1 and endothelial nitric oxide synthase proteins.After incubation of the samples with antibody-coated 96-well plates, enzyme-linked antibodies for the proteins were added. The intensity of the color was measured in a microplate reader  at 450 nm.The measurements of the total antioxidant status (TAS) and the total oxidant status (TOS) were conducted by a microplate reader  as described previously  using anThe genomic DNA was isolated from peripheral blood leukocytes with the DNA isolation kit . Thereafter, DNA samples were stored at +4°C and PCR analysis was performed .SIRT1 rs7895833 A > G in the promoter region, rs7069102 C > G in intron 4, and rs2273773 C > T in exon 5 [The PCR-CTPP technique was used to analyze the gene polymorphisms of n exon 5 , 30. Then exon 5 .Three genotypes for each polymorphism were defined as the following patterns : for rs7Major adverse cardiac events (MACEs) included recurrent nonfatal myocardial infarction, hospitalization due to decompensated heart failure, and target vessel revascularization (TVR). In addition, the synergy between PCI with Taxus and cardiac surgery (SYNTAX) score was applied to all coronary lesions with a diameter stenosis greater than 50% in vessels with a diameter larger than 1.5 mm as described before .The electrocardiogram-guided echocardiographic examination, using a transthoracic approach, was performed by an experienced sonographer who was blinded to the study in terms of infarction type, date, and genetic variation, with the patient in the left lateral decubitus position within 48 hours after admission and 1 year after MI, by using the Philips envisor C HD  echocardiography devices and 2–4 MHz phase transducers. All measurements were obtained as per the criteria recommended by the current guidelines . Left veSIRT1 gene between experimental and control groups.All continuous variables were tested for normality using the Kolmogorov–Smirnov test. Data are presented as percentages, mean ± standard deviation (SD), or median (interquartile range). Chi-squared analysis was used for comparing categorical variables between groups like the distribution differences of the genotypes or alleles in t-test or Whitney U-test for parametric and nonmetric variables, respectively. Statistical analysis was performed using SPSS version 2.0 . Relative risk at 95% confidence intervals (CI) was given as the odds ratio (OR). Pearson's correlation test was also used to determine the relation between parameters of interest. All tests were two sided, and p < 0.05 was regarded as significant.Differences in continuous variables were tested with the Student's The clinical characteristics of the study groups are given in p=0.001), with a higher rate of smokers , and had dyslipidemia  compared to the control group. The prevalence of diabetes mellitus  and hypertension  were significantly higher in the ST-elevation myocardial infarction group. The concentrations of serum triglycerides (p=0.003), very low-density lipoprotein (VLDL) (p=0.026), fasting blood glucose (p < 0.001), and uric acid (p < 0.001) were significantly enhanced in ST-elevation myocardial infarction patients, while high-density lipoprotein (HDL) levels (p < 0.001) were decreased  was more apparent than other myocardial infarction types (51.9%). Seventeen patients underwent percutaneous coronary intervention (PCI) at follow-up (FU), among them 12 with recurrent myocardial infarction, whereas 3 patients were hospitalized due to decompensated heart failure. After myocardial infarction, left ventricular ejection fraction (LVEF) was slightly decreased with an average of 51.9 ± 9.2%, while twenty-five patients had a left ventricular ejection fraction less than 45% .p < 0.001). To investigate the role of SIRT1 protein in premature myocardial infarction, other associated parameters such as levels of endothelial nitric oxide synthase and oxidative stress markers and their relation with SIRT1 protein were analyzed.SIRT1 protein levels were significantly increased in patients as compared to control subjects (p=0.001)  .p=0.026) in patients but not in controls (p > 0.05) (A positive correlation was detected between SIRT1 expression and age (controls . In cont > 0.05) .SIRT1 gene in both groups are given in The frequencies of genotypes and alleles of p > 0.05).For rs7895833 A > G in the promoter region, only the wild-type AA genotype and heterozygote mutant AG genotype were detected, indicating no difference in distribution of genotypes and alleles between patients and control group (p=0.027). The C allele was predominantly found in the patients group, nearly significant (p=0.06)  .χ2: 7.046, p=0.008, OR: 2.31, 95% Cl: 1.24–4.31).The risk for myocardial infarction was increased by 2.31 times in carriers of CC or CG genotypes , while there was no difference in the frequency of gene variations between the patient and control groups (p > 0.05)  and heterozygote mutant (AG) genotype as compared to that od=f the control group ( > 0.05) .p=0.004), while there was no statistically difference in endothelial nitric oxide synthase expression between the patient and control groups (p > 0.05). For the CG and GG genotypes, SIRT1 levels in the patients' group were significantly higher, while endothelial nitric oxide synthase levels were markedly decreased compared to those of the control group   (p=0.014) . Parallep=0.014) .If the cardiac risk factors were analyzed separately in each genotype, there were some significant differences between the groups . High-dep=0.012). In contrast, patients with the heterozygote CG and the homozygote mutant GG genotypes were characterized by enhanced body mass index , elevated levels of triglycerids , very low-density lipoprotein (VLDL) , and triglycerids/high-density lipoprotein . Moreover, levels of uric acid and fasting blood glucose were increased in patients carrying the heterozygote genotype compared to the control collective .Among patients carrying the rs7069102 C > G CC genotype, only high-density lipoprotein levels were decreased without significant differences in the distribution of the other risk factors compared to the control group , while the high-density lipoprotein levels were decreased (p < 0.001). In the patients with AG genotype, very low-density lipoprotein levels were statistically not relevant (p=0.183), but the fasting blood glucose was higher compared to that of the control group (p=0.001). The other parameters were equally distributed as in the AA group , and endothelial nitric oxide synthase levels were similarly decreased  in diabetic and nondiabetic patients compared to the control group. There was no difference in rs7069102 C > G genotype distribution between diabetic and nondiabetic patients (p=0.084).Baseline fasting blood glucose concentration was significantly higher in the ST-elevation myocardial infarction group compared to that of the control group, suggesting that a higher proportion of patients had undetected diabetics or prediabetes. SIRT1 protein levels were similarly increased  without differences in SIRT1 and endothelial nitric oxide synthase expression between both groups. Patients carrying the rs7895833 AA genotype possessed higher SYNTAX score than patients carrying the AG genotype (p=0.04). There was no difference in SYNTAX score values between the three genotypes of the rs7069102 single-nucleotide polymorphism (p=0.548). All patients with recurrent myocardial infarction had their first coronary event under the age of 40. These patients had higher levels of SIRT1 protein (p=0.026) compared to patients without an adverse event, while endothelial nitric oxide synthase levels were similar. There was no difference in SNP distribution. We detected a negative correlation between recurrent myocardial infarction and left ventricular ejection fraction,  as well as between SIRT1 values and left ventricular ejection fraction .Patients with the heterozygote rs7069102 CG genotype tended to undergo anterior myocardial infarction (71.4%) more frequently than other myocardial infarction types (61.5%)  and development of premature ST-elevation myocardial infarction ≤45 years. Levels of SIRT1 and endothelial nitric oxide synthase proteins, total antioxidant status, total oxidant status, and oxidative stress index were measured to investigate the relationship between genetic variation and phenotype; further, we investigated the association between protein expression and cardiovascular risk factor constellation.The purpose of this study was to investigate the association between SIRT1 protein levels were markedly increased in myocardial infarction patients compared to that of the control group . This reHereby, the increase in SIRT1 levels was explained as a compensatory mechanism to induce the production of antioxidants against oxidative stress in patients with cardiovascular disease. Kaneda et al.  had showEndothelial nitric oxide synthase levels were determined in order to investigate the relationship between SIRT1 and endothelial function. In young adults, cardiovascular disease risk factors promote endothelial dysfunction, while in older individuals advancing age per se is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction is a result of decreased nitric oxide synthesis, paralleled by increased endothelial oxidative stress and inflammation, which can be also augmented by cardiovascular disease risk factors in older adults. Moreover, lack of endothelial nitric oxide synthase is associated with different disease constellations like hypertension, ventricular hypertrophy, and diet-induced atherosclerosis –39.In our study, the level of endothelial nitric oxide synthase protein was considerably low in ST-elevation myocardial infarction patients compared to those of the control group , whereasThus, the decreased levels of eNOS and increased levels of SIRT 1 might be disease specific but regulated via different mechanisms.When we analyze the MI susceptibility of the genotype variants, our results revealed that carrying the CC or CG genotypes, additionally the wild-type C allele of the rs7069102 single-nucleotide polymorphism, was associated with an increased myocardial infarction risk. SIRT1 levels were markedly elevated in the ST-elevation myocardial infarction group, irrespective of the underlying genotype . On the p < 0.001), pointing out the protective role of C allele for both single-nucleotide polymorphisms against cardiovascular disease [However, in the previous study, the risk for cardiovascular disease was increased by 2.4 times for rs7069102 C > G and 1.9 times for rs2273773 C > T in carriers of mutant allele compared to carriers of wild-type allele , smoking history (87.1% vs 63.8% p=0.727), and dislipidemia (73.1% vs 12.1% p < 0.001). Uric acid (UA) levels were elevated in the myocardial infarction group . Many experimental and epidemiological data have suggested a possible role for hyperuricemia in inducing endothelial dysfunction and particularly impaired nitric oxide (NO) bioavailability, showing a causal role for UA in the development of coronary heart disease or death from cardiovascular causes [Interestingly, our patients had a high cardiovascular risk profile, including diabetes  were carrying the CG genotype, while only a few patients (n=12) were carrying CC genotype. Thus, we might assume that the risk factor constellation would be equalized between the groups, if more patients would be included in the CC group.This finding seemed to be controversial at the first view, as the C allele was defined as the allele increasing the susceptibility for premature myocardial infarction. Thereby, it should be considered that most of our patients (p=0.068). There was no difference in SIRT1 and endothelial nitric oxide synthase expression between patients with anterior myocardial infarction and other myocardial infarction types . There was no statistically difference in SYNTAX score values between the genotype variants of the rs102 SNP (p=0.548), while SYNTAX score was higher in patients carrying the rs AA genotype compared to that of the AG genotype (p=0.04).Besides the fact that anterior myocardial infarction was more common compared with other myocardial infarction types. Most of these patients were carrying the rs7069102 CG genotype (p=0.026) compared to those of patients without an adverse event, while endothelial nitric oxide synthase levels were similar. Further these results suggest that the SYNTAX score is not related with an increased risk for recurrent myocardial infarction in patients suffering from premature myocardial infarction.All patients with recurrent myocardial infarction had their first coronary event under the age of 40. They had higher levels of SIRT1 protein (There was no difference in the distribution of common cardiovascular risk factors between patients with and without a recurrent event. The moderate increase of the SIRT1 protein may suggest a compensatory mechanism to protect the people from the detrimental effects of reinfarction. The potential cardioprotective role of SIRT1 was demonstrated in two other recent studies.In the first study, conducted by our group, SIRT1 protein was increased in cardiac tissue of patients suffering from postoperative atrial fibrillation, likely to compensate the deleterious effects of tissue hypoxia during/after surgery .In one study, SIRT 1 protected the aging heart from contractile dysfunction, mediated through the inhibition of endoplasmic reticulum stress-mediated apoptosis in cardiac-specific sirtuin 1 knockout mouse model . These tSIRT1 genotype.In conclusion, carrying the CC or CG genotypes, additionally the wild-type C allele of the rs7069102 single-nucleotide polymorphism, was associated with an increased risk of premature myocardial infarction, latter affecting SIRT1 and endothelial nitric oxide synthase protein expressions, irrespective of the underlying The patient number has to be enhanced to determine a clear association between disease phenotype, outcome, and SIRT1 gene variants.


To evaluate the impact of gender on the clinical and metabolic parameters in prepubertal growth hormone deficiency (GHD) children at diagnosis and during GH treatment (GHT).The data of 105 prepubertal children  affected by idiopathic GHD were retrospectively evaluated.Body height, BMI, waist circumference (WC), IGF-I, HbA1c, lipid profile, fasting and after-OGTT glucose and insulin levels, insulin sensitivity and secretion indices were evaluated at baseline and after 24 months of GHT.P < 0.001), BMI (both P < 0.001), WC , IGF-I (both P < 0.001), fasting glucose , fasting insulin (both P < 0.001) and Homa-IR (both P < 0.001), with a concomitant significant decrease in insulin sensitivity index (ISI) (both P < 0.001) and oral disposition index (DIo) . At 24 months of GHT, females showed significantly higher BMI (P = 0.027), lower ISI (P < 0.001) and DIo (P < 0.001), in concomitance with a significant greater change from baseline to 24 months of BMI (P = 0.013), WC (P < 0.001), ISI (P = 0.002) and DIo (P = 0.072), although the latter does not reach statistical significance.At baseline, no significant difference was found in all clinical, hormonal and metabolic parameters between males and females. After 24 months of GHT, both males and females showed a significant increase in height (both Twenty-four months of GHT in prepubertal children leads to different metabolic outcomes according to gender, with a greater reduction in insulin sensitivity in females, regardless of auxological and hormonal parameters. Therefore, prepubertal GHD females should probably need a more proper monitoring in clinical practice. The sexual dimorphism in the mechanisms involved in the regulation of somatotroph axis is well documented, and it might account for some of the sex differences in growth rate and body composition . Indeed,In healthy subjects, GH and IGF-I levels decline significantly with age in both sexes, while men show lower baseline and mean GH levels than women mainly due to the action of estradiol on the neuroendocrine regulation of pulsatile GH release .This complex interplay between sex hormones and the GH/IGF-I axis has a clinically relevant impact in patients with hypopituitarism and the gender-dimorphic GH secretion pattern must be taken into account both in diagnosis of GH deficiency (GHD) and in detection of the appropriate and effective dose of GH replacement treatment (GHT) to be used .Indeed, in hypopituitary patients, GHT produces a greater increase in plasma IGF-I in men, and women require a higher GH dose due to lower responsiveness to standard GH doses , 13.The sexual dimorphism of GH secretion also results in a different expression of some genes involved in glucose and lipid metabolism. There is strong evidence that estrogens can influence the GH-regulated endocrine and metabolic functions in the human liver, and testosterone maximizes the metabolic benefits of GH , 14.Height and weight gain or body composition become sexually dimorphic around the time of puberty, when males more rapidly gain muscle and bone and lose fat than females  and the Conversely, the relationship between GH and gonadal steroid levels in children is difficult to establish because steroid levels are very low prior to puberty. Therefore, although the clinical features related to sexual dimorphism of the somatotroph axis have been well demonstrated in GHD adults, to date, very few studies about gender difference are available in prepubertal GHD children.The aim of this study was to evaluate the impact of gender on the clinical and metabolic parameters in a large series of prepubertal GHD children at diagnosis and during GHT.We retrospectively evaluated the data of 105 children  affected by idiopathic GHD who were consecutively admitted to the Section of Endocrinology of the University of Palermo from January 2013 to December 2017 and underwent GHT for at least 24 consecutive months. Fifty-one healthy subjects, matched for sex , age , stature and pubertal status, were recruited as a control group at baseline among children referred for short stature. In this group, screening for short stature did not reveal endocrine disease and GHD was excluded. We excluded children affected by organic GHD or multiple pituitary hormone deficiency or receiving other hormonal replacement treatment to avoid interference with the metabolic parameters evaluated. All children, including the older ones, were in the first stage of sexual development according to the criteria of Marshall and Tanner  to avoid any interference of puberty with the auxological and metabolic parameters analyzed, and they maintained their prepubertal clinical and hormonal status for the entire follow-up . Similarly, no signs of clinical or biochemical adrenarche were detected during the entire follow-up . GHD was diagnosed according to the criteria of the GH Research Society .s.d.) below the mean and growth velocity more than 1 s.d. below the mean for age, or, without severe short stature, a growth velocity more than 2 s.d. below the mean over 1 year. As radiological criteria, we considered a bone age delay, estimated from an X-ray of the left wrist and hand and evaluated according to the methods of Greulich and Pyle, of at least 1 year with respect to the chronological age  and the insulin tolerance test (ITT), with GH peaks below 8 µg/L according to the Italian criteria of appropriateness of use and reimbursement of GHT in children .Neuroimaging, with magnetic resonance of the hypothalamic–pituitary region, was performed in all GHD children with more severe GHD (GH peak ≤3 μg/L) and did not show significant pituitary abnormalities.All GHD children received GH once daily at bedtime with a pen injection system. IGF-I levels and growth velocity allowed us to determine the GH dose. On average, in all patients, the initial daily dose of GH was 0.025 mg/kg, and it was gradually increased to 0.027–0.028 mg/kg from month 6 to 12, to 0.029–0.030 mg/kg from month 12 to 18 and to 0.030–0.032 mg/kg from month 18 to 24 in order to achieve the above-mentioned targets.s.d.), BMI s.d. and waist circumference (WC). GH secretion was evaluated by GH levels during GST and ITT, performed on two different days. During GST, blood samples were collected at 0, 30, 60, 90, 120, 150, 180 and 240 min after the injection of 30 μg/kg (up to 1000 μg) intramuscularly of glucagon , for measurements of GH. Blood samples for GH were also measured during ITT (0.1 U/kg of body weight of human Humulin R insulin) at baseline and at 30, 60, 90 and 120 min after insulin administration and results were accepted with blood glucose lower than 2.2 nmol/L (40 mg/dL).In all children at baseline, we measured body height , IGF-I concentrations and lipid profile, including total and high-density lipoprotein (HDL) cholesterol and triglycerides. Low-density lipoprotein (LDL) cholesterol levels were evaluated by the following formula: total cholesterol–(HDL cholesterol–triglycerides/5). This sample also served as the baseline sample for an oral glucose tolerance test (OGTT). Blood samples were collected every 30 min for 2 h for glucose and insulin measurements.As surrogate estimates of insulin sensitivity we considered the homeostasis model assessment estimate of insulin resistance (Homa-IR)  and the After the diagnosis of GHD was made, in GHD children in addition to auxological parameters and IGF-I measurement we performed OGTT (for glucose and insulin) after 12 and 24 months of GHT. In the control group, these evaluations were only performed at baseline.For the analyses, we also considered the change (delta) in clinical and metabolic parameters from baseline to 24 months of GHT. The Institutional Ethics Committee of the University of Palermo approved this study. At the time of hospitalization, an informed consent for the scientific use of the data was obtained from both the participants and their parents.Glucose was measured in the centralized accredited laboratories of the University of Palermo with the standard methods. HbA1c levels were determined by HPLC with an ion-exchange resin . Serum insulin was measured by electrochemiluminescence . The sensitivity of the method was 0.4 µU/mL. The normal range (µU/mL) was 2.6–24.9. Serum GH levels were measured by Immunoassay in electrochemiluminescence . The lower limit of detection of the assay was 0.030 µg/L. The intra- and inter-assay coefficients of variation (CV) were 0.6–5.0 and 3.8–5.0%, respectively. We reported GH concentrations in µg/L of IS 98/574. Serum IGF-I levels were measured by means of a chemiluminescent immunometric assay  using murine monoclonal anti-IGF-I antibodies. The standards were calibrated against the World Health Organization second IS 87/518. The sensitivity was 1.9 µg/L. The intra- and inter-assay CVs were 2.3–3.9% and 3.7–8.1%, respectively.s.d. for continuous variables ; rates and proportions were calculated for categorical data. The differences between groups were evaluated with the t-test. A P value <0.05 was considered statistically significant.The Statistical Packages for Social Sciences SPSS, version 19 was used for data analysis. Baseline characteristics were presented as mean ± The clinical, hormonal and metabolic parameters of control subjects and GHD children in full at diagnosis and after 24 months of GHT are shown in P < 0.001), IGF-I (P < 0.001) and GH peak after GST (P < 0.001) and ITT (P < 0.001) than control subjects. No significant difference in BMI and WC between GHD children and controls was found. Similarly, no difference was found in all metabolic parameters analyzed , BMI (P < 0.001), WC (P < 0.001) and IGF-I (P < 0.001).After 24 months of GHT, GHD children showed a significant increase in height (P < 0.001), fasting insulin (P < 0.001) and Homa-IR (P < 0.001), with a concomitant significant decrease in ISI (P < 0.001) and DIo (P < 0.001). No significant change was found in Hba1c levels and lipid profile , BMI (both P < 0.001), WC  and IGF-I (both P < 0.001).After 24 months of GHT, both males and females showed a significant increase in height , fasting insulin (both P < 0.001) and Homa-IR (both P < 0.001), with a concomitant significant decrease in both sexes in ISI (both P < 0.001) and DIo . No significant change was found in Hba1c levels and lipid profile in either males or females , lower ISI (P < 0.001) and DIo (P < 0.001) and higher values of WC (P = 0.059), although not statistically significant , WC  and ISI  and greater delta of DIo  in females, although the latter does not reach statistical significance (In addition, the comparison of delta of all parameters from baseline to 24 months showed significantly greater delta of BMI (0.4 ± 0.3 vs 0.2 ± 0.4 ificance , withoutIn this study, we found that prepubertal GHD females show a worse metabolic profile than males after 24 months of GHT, regardless of the auxological and hormonal data, indicating that gender may impact on the metabolic outcomes during GHT also in children. Indeed, although all children showed a significant improvement in auxological data and IGF-I levels in concomitance with a slight deterioration in glucose metabolism, insulin sensitivity and β-cell response after GHT, females demonstrated a greater reduction in insulin sensitivity and pancreatic function, probably related to higher increase in fat mass.et al. showed similar sensitivity to GHT for body composition changes, lipolysis, lipid and glucose concentrations in males and females during puberty and just a higher IGF-I response in males, which does not translate into differences in linear growth (The sexual dimorphism of the somatroph axis has been well documented in healthy subjects and in adult patients affected by both GH excess and deficiency , 23, 24.r growth . The effr growth . Therefoet al. in a retrospective study evaluated whether this was a sex steroid effect during the first 2 years of GHT in 147 prepubertal children, finding no evidence of gender difference in growth response (Rose response .et al. (et al. in a randomized prospective dose-ranging study demonstrated a clear gender impact on both auxological and biochemical parameters in a larger cohort of GHD children during GHT, concluding that there is a gender difference in GH sensitivity. Indeed, the authors showed that males had a linear GH dose response, whereas females had an apparent plateau of both growth and IGF-I levels, and these data were also confirmed when only prebubertal children were analyzed, indicating that the gender difference observed is not related to the occurrence of puberty (et al. also reported a significant gender difference in 2-year growth response to GHT in a large cohort of prepubertal GHD children, although no difference was observed in the change in IGF-I levels (Similarly, no significant gender differences in metabolic parameters were observed both at baseline and during GHT by Salerno et al. . Convers puberty . SavendaI levels .et al. who demonstrated similar auxological benefits but partial gender-specific metabolic effects of GHT in 62 children. Indeed, the authors showed a significant change in percent body fat both in boys and girls during the first 6 months of treatment, although body fat remained constant in boys but started to increase in girls from the 18th month of treatment (In our study, both GHD children taken together and grouped according to gender showed a significant and similar auxological improvement and normalization of IGF-I levels during GHT. However, if apparently all children had increased BMI and WC, the direct comparison between males and females after 24 months of GHT showed a greater increase in fat mass in females, as confirmed by the calculated delta of BMI and WC. These data are in agreement with a previous study by Kuromaru reatment .Few studies have evaluated the gender differences of metabolic features in children with GHD. GHT is known to impact on glucose metabolism, by increasing insulin secretion and decreasing insulin sensitivity, with a discordant impact on glucose levels , 34. In After 24 months of GHT, regardless of gender, all GHD children showed a significant increase in fasting glucose and insulin levels with a concomitant impairment in insulin sensitivity and β-cell function. Indeed, although fasting glucose and HbA1c levels were always maintained within the normal range, a significant decrease in insulin sensitivity, demonstrated by an increase in Homa-IR and a decrease in ISI, was found in all children after GHT. These data are quite consistent with the majority of existing studies , 42, 43.Therefore, the gender difference in the metabolic outcomes of GHT observed in our patients were unlikely due to being mediated by the IGF-I levels, which were always within the normal range during the observational period and similar in males and females. However, to minimize the impact of the different age of patients on IGF-I, we considered the delta in IGF-I during GHT, rather than absolute IGF-I levels, and it too proved comparable in the two groups of children. Similarly, a different GH dose in males and females can be excluded as the cause of the different metabolic outcomes in this study, since the dose was similar in both groups of patients during the entire follow-up. This finding suggests that there are other factors than IGF-I change or different GH dose that may mediate the gender difference in metabolic outcomes during GHT.Notably, in our study, females showed a greater increase in BMI and WC than males, as demonstrated by the comparison of these parameters at 24 months and as confirmed by the significant difference in delta of BMI and WC between the two groups. Therefore, the greater change in BMI and WC found in females suggests that adiposity might be a mediator of the different metabolic effects of GHT.Indeed, a different gender response to GHT secondary to body composition changes has been widely demonstrated in GHD adults , 47, altThis study has undoubtedly some limitations represented by the relative small size of the population and by the lack of data for the control group during the 24 months of follow-up. Indeed, the insulin sensitivity seems to decrease with age, probably due to the physiological effect of steroids production or the modifications of body composition during the peripubertal period, also in normal children , 50. ForIn conclusion, this study highlights that 24 months of GHT in children leads to different metabolic outcomes according to gender, regardless of auxological and hormonal parameters. Therefore, the follow-up of prepubertal GHD children during GHT should probably also be modulated and optimized not only according to auxological and biochemical response, but also considering the patient’s gender. Specifically, prepubertal GHD females should probably need a more proper monitoring in clinical practice aimed at correct lifestyle during GHT to prevent metabolic alterations, although studies with longer follow-up and a larger cohort of patients are required to confirm these preliminary results and to establish with greater certainty whether GHD females need more surveillance during GHT or, alternatively, different GHT doses.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.This research did not receive any specific grant from any funding agency in the public, commercial or not-for-profit sector.


We used the Surveillance, Epidemiology, and End Results (SEER) database to develop and validate deep survival neural network machine learning (ML) algorithms to predict survival following a spino-pelvic chondrosarcoma diagnosis.The SEER 18 registries were used to apply the Risk Estimate Distance Survival Neural Network (RED_SNN) in the model. Our model was evaluated at each time window with receiver operating characteristic curves and areas under the curves (AUCs), as was the concordance index (c-index).n = 1088) were separated into training  and test sets . The training data were randomly sorted into training and validation sets using 5-fold cross validation. The median c-index of the five validation sets was 0.84 . The median AUC of the five validation subsets was 0.84. This model was evaluated with the previously separated test set. The c-index was 0.82 and the mean AUC of the 30 different time windows was 0.85 (standard deviation 0.02). According to the estimated survival probability (by 62 months), we divided the test group into five subgroups. The survival curves of the subgroups showed statistically significant separation (p < 0.001).The subjects (This study is the first to analyze population-level data using artificial neural network ML algorithms for the role and outcomes of surgical resection and radiation therapy in spino-pelvic chondrosarcoma. The Surveillance, Epidemiology, and End Results (SEER) database has been queried in a series of reports to analyze all primary malignant tumors of the osseous spine, including chondrosarcoma –4. The SMachine learning (ML) provides the opportunity to analyze heterogeneous and complex data due to the greater capability to identify unintuitive patterns in large patient datasets . SeveralIn the present study, we hypothesized that applying ML techniques may be equally valuable in other clinical areas, such as in identifying the prognostic factors in spinal and pelvic chondrosarcoma. We seek to develop and validate recurrent neural network ML algorithms to precisely predict survival following a diagnosis of chondrosarcoma using a national database.http://seer.cancer.gov/). Serial registry data are de-identified and submitted to the United States National Cancer Institute on a biannual basis to make available to researchers. The primary data in the SEER database includes the following variables: age at diagnosis, gender, race, primary site and size of tumor, histology, grade of tumor, stage of tumor, surgical treatment, radiation and chemotherapy, and overall survival (OS) in months. The original data set has 193 variables. (https://seer.cancer.gov/data-software/documentation/seerstat/nov2016/).The SEER database is a longitudinal database that collects information from 17 population-based cancer registries , codes 9220–9243. The sites of presentation were compiled according to ICD-O-3 topography codes and were grouped into vertebral and pelvic sites in a fashion similar to that in previous studies , 8. The /malignant tumors for the patient. The patient’s age at diagnosis was grouped into 3 categorical variables with intervals of 30 years. The tumor size was divided into two groups (> 8 or ≤ 8 cm) according to the T stage classification for bone tumors determined by the American Joint Committee on Cancer. The age, grade, tumor size, and number of tumors were used as discrete ordinal values. The other variables were considered to be categorical. All subjects (n = 1088) were separated into a training set  and a test set . The training data were randomly sorted into the training set and validation set using 5-fold cross validation , age at diagnosis, marital status, primary site (spine and pelvis), tumor size, histologic type, grade, laterality, SEER historic stage, surgery, radical resection, radiation, chemotherapy, and total number of in situects n = 088 were The key point of this model is that the event and time should be located in different dimensions, and the neural network learns these two targets at the same time using a multimodal algorithm  as follows .The parameter of the model (θ) should be inferred using the cosine proximity loss function  = g , {1, 2, …i} ∈ nY_hat  and some will be lost (censored), thus the observed time and event will change in the next time window. The algorithm is trained serially with the different target value at each time window, and the model parameter (θ) will be gradually adjusted to the time serial targets.  at each time window because our algorithm is designed to estimate the prognosis with the first status of the patients.) The algorithm was trained using backpropagation and was optimized using RMSProp at every two months (time window (m) = 2) with a single epoch and risk score (α) = 10.The present study was developed and written according to transparent reporting of a multivariable-prediction model for individual prognosis or diagnosis model development guidelines. Patient outcomes were measured based on OS, the period (in months) between diagnosis and death or loss of follow-up from any cause, as reported in the SEER database. The performance of our prediction model was evaluated at each time window with the receiver operating characteristic (ROC) curves and areas under the curves (AUCs). Using the last follow-up information, the concordance index (c-index) of the model was also evaluated. The AUCs and c-indexes were compared between the groups using the Mann-Whitney test. The calibration curves were estimated by comparing the mean probability of the predicted survival with the actual survival proportion using Kaplan-Meier estimates after subgrouping of the test group into seven equally numerous subsets.p < 0.05. We used the Keras and Theano libraries to obtain the deep learning framework. Data preprocessing was performed using the scikit-learn library. Statistical analysis was performed with SPSS version 24.0 .In the test group, the SEER historic stage and the subgroups classified by the predicted survival probability were compared using Kaplan-Meier curves. Each curve was compared to the neighboring curves using the log-rank test. Comparisons between groups were considered to be statistically significant at The search identified 1089 patients with primary chondrosarcoma of the osseous spine and pelvis between 1973 and 2014. Only one case was excluded owing to an unknown survival period. Among the remaining 1088 patients, 62.0% were men and 86.2% were white . In contrast, radiologic and clinical confirmation were performed in 27 patients (2.5%). Among the 1061 patients who received histologic confirmation, most were diagnosed with chondrosarcoma, not otherwise specified (87.1%). Among other histologic types, myxoid (5.5%), dedifferentiated (4.0%), and mesenchymal (2.1%) were common histologic variants. Regarding grade, 64.1% of cases were of low grade (grade I and II), 16.9% were high grade (grade III and VI), and 19.0% were of unknown tumor grade. The mean and median tumor size at the time of diagnosis were 96.6 mm (standard deviation 61.2 mm) and 88.0 mm, excluding 394 patients with unknown tumor size. The extent of the disease was known in 92.6% cases, with the majority presenting as regionally invasive disease (41.4%). The index site was the first malignant tumor; 868 patients had only the index tumor, while 220 patients had two or more tumors.After diagnosis, 12.3% of patients received both surgery and radiation, 57.9% underwent surgery alone, and 9.0% underwent radiation alone, while 16.2% of patients received neither, and 4.5% had an unknown treatment regimen , with time-dependent target values within a single epoch. The same learning process is repeated with different batches in the second epoch. The median c-index of the five validation sets was 0.84 . The median AUC of the five validation subsets was 0.84  was finally trained with the total training set, and the final RED_SNN specified for spinal and pelvic chondrosarcoma was developed. All of fourteen input variables selected initially were applied in final model. This model was evaluated with a previously separated test set. The c-index was 0.82, and the mean AUC of the 30 different time windows was 0.85 (standard deviation 0.02). The calibration curve analysis showed that the predicted survival probability represented the actual survival proportion within a 10% margin of error Fig. a and b.p < 0.001). According to the estimated survival probability (by 62 months), we divided the test group into five subgroups. The first subgroup included patients with estimated survival probability greater than 96% (n = 19), and the second subgroup included patients with estimated survival probability between 78 and 95% (n = 64). Patients with estimated survival probability between 53 and 77% were in the third subgroup (n = 58), and patients with an estimated probability between 32 and 52% were in the fourth subgroup (n = 44). Patients with a survival probability less than 31% were in the fifth subgroup (n = 33). Kaplan-Meier curves were generated for each subgroup .With the test data set, Kaplan-Meier curves were generated according to the SEER historic stages Fig. a. A cleaAlthough chondrosarcoma is the third most common primary malignant bone tumor, the spine is a relatively rare site that presents a challenge for surgery due to the potential for local recurrence –18. MoreSince cancer is associated with multidimensional factors, conventional linear statistical models have not shown reliable accuracy in predicting prognosis. To build non-linear statistical models, various ML techniques, including Bayesian networks (BNs), semi-supervised learning (SSL), support vector machines (SVMs), decision trees (DTs) and artificial neural networks (ANNs) have been widely applied to develop predictive models, resulting in accurate and powerful decision-making in cancer research. Zhou and Jiang  used DTsDue to the rarity of chondrosarcoma, only a few small case series of treatment outcomes have been reported over several decades at individual institutions , 22–24. ANNs, in particular, may allow accurate performance in the presence of unreliability, including incomplete data or measurement errors. Moreover, ANN could detect and recognize complex non-linear relationships between the variables . In thisIn this study, we developed a novel algorithm specific to survival analysis, and the results indicate it offers better performance compared to those in other studies using both a conventional statistical model and an ML model. Our RED model showed mean AUC of 0.85. In the calibration curve Fig. b, our moYet, the development of model base on ANNs is empirical, and some methodological issues remain to be unresolved. However, implementing statistics based on artificial intelligence could produce valuable information and clinical relevance including disease staging, patient’s prognosis, survival prediction and treatment decision making for physicians in clinical practice and should deserve further attention.However, the present study has limitations. One of limitations is that variations in treatment strategies could not be accounted for, including radiation and chemotherapy and surgical strategies. Previous studies have shown that surgical techniques might have an impact on survival period, as chemotherapy regimens , 31–34. Secondly, artificial neural network has the ability to detect all possible interactions between variables. However it may act as a ‘black box’ and have limited ability to identify variables (or coefficient weights) used to create the models and possible causal relationships.Lastly, the development and validation of the machines learning algorithms described in this study have not been externally validated in an independent physician-collected dataset or other registry such as the national Cancer Database (NCDB). As the model performance would be similar or much lower if an external dataset was used, the generalizability of the algorithm predictions remains to be determined, and future studies should seek to build on the findings presented here by examining prospectively collected registries.RED_SNN is a valid method to predict survival for spinal and pelvic chondrosarcoma, and it appears to be comparable to other methods. RED_SNN may offer the advantage of increased sensitivity for predicting longer or shorter OS.Additional file 1: Figure S1. Experiments Networks (A) Final Network consists of Embedding Layer, LSTM Layer, 4 Fully Connected Layers. (B) Dropout0.3 Network adds Dropout Layer(0.3) between FC Layer and LSTM Layer on origin network. (C) Dropout0.5 Network adds Dropout Layer(0.5) between FC Layer and LSTM Layer on origin network. (D) Dropout0.7 Network adds Dropout Layer(0.7) between FC Layer and LSTM Layer on origin network. (E) Bignode network has randomly increased nodes in some Layers on the origin network.Additional file 2: Table S1. 5-fold valid test accuracy about various networks.


Transient elastography (TE) using FibroScan with M probe has been widely used in adults for controlled attenuation parameter (CAP) and liver stiffness measurement (LSM). In this study, we aimed to assess the feasibility of this approach and reference values of CAP and LSM in healthy preschool children aged 5 years.FibroScan-502 with M probe  and bioelectrical impedance analysis  were prospectively conducted in healthy children aged 5 years from the Shanghai Prenatal Cohort Study. Linear regression models and piece-wise linear regression models were used to explore the factors associated with CAP and LSM.The success rate of a valid TE measurement was 96.5% in 452 healthy preschool children aged 5 years, and 436 children with 236 boys were included for further study. The median, inter quartile range (IQR) and the 5th–95th percentiles of CAP values were 171.50, 162.07–188.13 and 154.21–214.53 dB/m, respectively. The median, mean ± standard deviation and the 5th–95th percentiles of LSM were 3.20, 3.28 ± 0.86 and 2.00–4.78 kPa, respectively. In multivariate linear regression analyses, the CAP but not the LSM value was significantly positively correlated with such anthropometric index as body weight, body mass index, waist circumference, body fat content and body fat percentage.FibroScan-502 with M-probe can be used to measure CAP and LSM in preschool children aged 5 years. The 95th percentiles of CAP values and LSM were 214.53 dB/m and 4.78 kPa, respectively. Further study should be performed to explore the cut-off values of CAP and LSM for diagnosis of hepatic steatosis and fibrosis in children. Liver biopsy is traditionally considered the gold standard for diagnosis of hepatic steatosis and fibrosis, but its use as a screening tool in apparently healthy individuals is limited because of its costly and invasive nature, sample errors, and intraobserver and interobserver variability –3. RecenFibroScan-502 with M-probe has been widely used in adults with chronic liver diseases and during health examinations, and the CAP and LSM values were found to be closely related to liver biopsy-proven steatosis and fibrosis, respectively –9. HowevThe individuals included in the study were 5-year-old (58–62 months) children from the Shanghai Prenatal Cohort Study, which was an ongoing perspective birth cohort that enrolled 1043 Han maternal-child pairs between January 2012 and December 2013 at Xinhua Hospital and International Peace Maternity and Child Hospital in Shanghai. All children in the cohort received annual follow-ups with some medical examinations. The exclusion criteria for the study population were as follows: (i) the diagnosis of any type of liver disease; (ii) extraliver diseases and/or therapeutic drugs that might affect liver fat and liver function tests; (iii) body weight over two standard deviations (SD) of the average weight for healthy children of the same sex, age or height; (iiii) measurement failure of FibroScan-502 with M probe. Ethics approval was obtained by the Ethics Committees of both Xinhua Hospital affiliated with the Shanghai Jiao Tong University School of Medicine and the International Maternal and Children Care Hospital. The parents of all the participating children were required to give informed consent to the study and sign the written documents.All participating children underwent annual medical examination at the health examination center in Xinhua Hospital. Stadiometers  were used to measure height to the nearest 0.1 cm. Digital scales  were used to measure body weight to the nearest 0.1 kg. Fat mass and the percent of body fat were obtained through bioelectrical impedance analysis . Participant characteristics and anthropometric indices, including age, sex, body weight, height, waist circumference, body mass index (BMI), fat mass and the percent of body fat were obtained. Blood samples were collected in the morning after at least 12 h of fasting, and serum levels of alanine amino transferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (AKP), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured with a Hitachi 7600 series automatic analyzer .Following a fast of at least 6 h, all participants underwent FibroScan-502 with M-probe (3.5 MHz) examination  by the same physician (JZ). The device estimates liver stiffness in kilopascal (kPa) and liver steatosis in decibel/meter (dB/m). CAP in dB/m and LSM in kPa were obtained simultaneously by each examination. A TE examination was considered successful when 10 valid measurements with a success rate of at least 60% were conducted and the interquartile range (IQR) was less than 30% of the median LSM value . Subject2 test were used to test intergroup differences between different genders for continuous and categorical variables, respectively. Correlations between the CAP and LSM values and clinical parameters were tested using Spearman’s correlation analysis. The potential influencing factors associated with CAP and LSM values were analyzed using a univariate linear regression model. Multivariate linear regression models and piecewise linear regression models were applied to examine the nonlinear relationships between CAP values and covariates. Statistical analyses were performed using R (http://www.R-project.org). Statistical significance was indicated by two-sided P values < 0.05.Continuous variables were expressed as means ± SD for a normal distribution and median ± IQR for a skewed distribution. The t-test and χP < 0.05).From the Shanghai Prenatal Cohort Study, 746 maternal-child pairs prospectively received a 5-year (58–62 months) follow-up. Of these individuals, 452 participants were enrolled into the TE study, and the success rate of valid FibroScan measurements was 96.5% (436/452) with 16 children excluded . Finally, 436 children aged 5 years old (58–62 months) were included in the analysis . Fig. , Table 4This study is by far the largest survey using FibroScan-502 with M probe in preschool children who underwent healthy checkups each year from birth. The mean values of LSM, median values of CAP and the ranges of LSM and CAP values in a total of 436 healthy children at 5 years old were obtained in the study.p = 0.001), with 3.4 (2.3–4.6 kPa) in children aged between 1 and 5 years, 3.8 (2.5–6.1) kPa in children aged between 6 and 11 years and 4.1 (3.3–7.9) kPa in children aged between 12 and 18 years, respectively, and the mean CAP value was 183 (112–242) dB/m which did not change with age [After the development of TE for more than ten years, FibroScan has been applied in many studies to evaluate adult liver disease –9. Currewith age . They thwith age . Howeverwith age , 23. As The FibroScan probe types include S type (S1 and S2), M type and XL type according to the different detection populations. Previous studies and the manufacturer have suggested that the S type probe should be used in preschool and primary school children, whose thoracic circumferences are less than 75 cm , 24. HowIn our study, the potential influence of gender on LSM and CAP values using FibroScan was also investigated. The results indicated that the LSM and CAP values did not differ according to gender, which is the same result as reported in other similar studies , 26, 27.There are some limitations in this prospective study. First, this was a single-center study that included only a sample of preschool children aged 5 years in apparently good health. Further multi-center large study with large sample preschool children are needed, which will validate the normal ranges of LSM and CAP values and consider some other factors. Second, our study was focused on apparently healthy children who did not undergo liver ultrasound and liver biopsies, although liver ultrasound was not recommended for the diagnosis of fatty liver in children considering its lack of sensitivity and specificity, and liver biopsies are also not appropriate for healthy children. Third, this study only focused on an age level. We should continue to discover the normal range at other age levels in future studies based on this ongoing perspective birth cohort.In conclusion, these promising results supported FibroScan epuipped with M probe can be used to measure LSM/CAP values in the preschool children aged 5 years. The success rate was similar to that in adult. This study also determined the normal ranges of LSM and CAP values in Chinese 5-year-old children which are different from these in adults. CAP values may become a useful tool to access hepatic fat content in children. Further follow-up with this Shanghai Prenatal Cohort and further study with other large sample sizes are needed to validate the normal ranges of LSM and CAP values at different age levels and whether LSM and CAP can identify those children who are at a higher risk of subsequently developing NAFLD.


Prognostic biomarkers in asthma are needed. The biomarker soluble urokinase plasminogen activator receptor (suPAR) has been associated with asthma control and with prognosis in acutely admitted medical patients. We investigated if suPAR and blood eosinophil counts at the time of admission for asthma are associated with readmission and mortality.Our cohort comprised 1341 patients  acutely admitted with a diagnosis of asthma to Hvidovre Hospital, Denmark (November 2013 to March 2017). Patients had suPAR and blood eosinophils measured at admission. Outcomes were 365-day readmission and all-cause mortality. Logistic regression analysis adjusted for age, sex, C-reactive protein, and Charlson comorbidity score was used to assess the association of the two biomarkers with readmission and all-cause mortality.n = 452, 42.3%) or died  had significantly higher suPAR concentrations (p < 0.0001) and lower eosinophil counts (p = 0.0031) at admission. The highest odds of readmission or mortality were observed for patients in either the 4th suPAR quartile (p < 0.0001) or with eosinophil counts < 150 cells/μL at admission. Increasing levels of suPAR were associated with 365-day readmission  and mortality . Eosinophil count > 300 cells/μL was significantly associated with lower odds of readmission  and lower mortality .Compared to event-free patients, patients who were either readmitted (In patients acutely admitted with asthma, elevated suPAR concentrations together with blood eosinophil count < 150 cells/μL at the time of hospital admission were associated with both 365-day all-cause readmission and mortality. Poorly controlled asthma is associated with impaired quality of life, accelerated decline in lung function, and, not least in more severe cases, repeated hospital admissions and increased mortality. The Global Initiative for Asthma estimates that half of the global asthma population has uncontrolled or only partially controlled disease , a risk Soluble urokinase plasminogen activator receptor (suPAR) is the soluble form of the urokinase plasminogen activator receptor (uPAR), a three-domain membrane receptor that is expressed on a plethora of cells ranging from mono- and lymphocytes to endothelial and smooth muscle cells. uPAR plays a central role in the plasmin/plasminogen pathway and is important for chemotaxis, immune system activation, and tissue remodeling . suPAR iIn asthma, sputum, biopsy, and serum suPAR has previously been shown to be elevated in stable asthma patients in comparison to controls –8. Plasm2 asthma, is the most prevalent asthma phenotype comprising up to 50% of asthma patients [2 asthma is seen over a wide disease spectrum from mild disease to difficult-to-control or severe asthma with high symptom burden and frequent exacerbations [2 asthma is characterized by both neutrophilic and paucigranulocytic forms, driven by neutrophils and/or mixed leukocyte populations [2 inflammation. We thus hypothesize that the two biomarkers could be used in conjunction to improve risk stratification in acutely admitted patients with asthma.Another, more intensely studied, asthma biomarker is blood eosinophil count. Eosinophilic asthma, often referred to as Tpatients . T2 asthrbations . Eosinoprbations . Howeverrbations . On the ulations . Interesulations , 15, comBased on a large, unselected cohort of acutely admitted medical patients, we identified those admitted with a diagnosis of asthma, and we tested the hypothesis that suPAR and blood eosinophil count are independent biomarkers associated with both one-year all-cause readmission and all-cause mortality.This study is a registry-based cohort comprising 29,088 medical patients acutely admitted between November 18th 2013 and March 17th 2017, including 1341 with a prevalent diagnosis of asthma. The study was carried out in the Emergency Department (ED), Copenhagen University Hospital Amager and Hvidovre, Hvidovre, Denmark . The ED The index admission was defined as the first admission where a patient had suPAR measured within the study period. Biochemistry data was obtained from the electronic hospital database LABKA via the Department of Clinical Biochemistry. Information on hospital admissions, length of stay, readmissions, and diagnoses were obtained from the Danish National Patient Registry. Vital status of patients was obtained from the Danish Civil Registration System . PatientBlood samples were routinely analyzed at the Department of Clinical Biochemistry. Plasma suPAR (D1D2D3 & D2D3 molecules) levels were measured using the suPARnostic AUTO Flex ELISA  on an automated Siemens BEP2000 platform according to the manufacturer’s instructions, as described in detail previously . Plasma The outcomes of interest in this study were acute all-cause readmission and all-cause mortality within 30-, 90-, and 365 days.n = 282), Q2: 1.9–2.6 ng/mL (n = 266), Q3: 2.61–3.5 ng/mL (n = 237), Q4: > 3.5 ng/mL (n = 283).Population-specific quartiles for suPAR were calculated. The suPAR quartile cut-offs were: Q1: < 1.9 ng/mL (n = 452), medium eosinophils , and high eosinophils  [For blood eosinophil counts, cut-offs were arbitrarily defined as low eosinophils  , 18, 19.Continuous variables are presented as median  and categorical variables as number (percentage).Unadjusted and multivariable adjusted logistic regression analyses were used to estimate the association between suPAR or blood eosinophil count and readmission or mortality. suPAR and blood eosinophil count were used as continuous (log2-transformed) or categorized variables (suPAR quartiles and low/medium/high blood eosinophil count groups). Results are presented as odds ratios (ORs) with 95% confidence intervals (CIs). The multivariable logistic regression analyses were adjusted for sex, age, Charlson comorbidity score, and CRP. Additionally, suPAR analyses were adjusted for blood eosinophil count and vice versa. The Charlson comorbidity score was calculated using a SAS macro as previously described , with weIn the analyses of readmission, death before readmission was considered a competing endpoint. Therefore, all patients who died before the end of follow-up were excluded from this analysis regardless of readmission or not.Cumulative probability for survival and readmission stratified by suPAR quartiles and blood eosinophil count are presented to illustrate the outcome distribution of patients over time. The discriminative ability of suPAR and blood eosinophil count to predict readmission and mortality was analyzed using Area under the Curve (AUC) for Receiver Operator Characteristics (ROC) curves.p-value < 0.05 was considered to be statistically significant.SAS Enterprise Guide 7.11 (SAS Institute) and R 3.2.3  were used for statistical analysis. R 3.2.3 was used to create the Figs. A The study was approved by the Danish Health and Medicines Authority (ref. 3–3013-1061/2) and the Danish Data Protection Agency .The datasets used and/or analyzed during the current study are available from the authors upon reasonable request, but analysis may require approvals from the Danish Patient Safety Authority and the Danish Data Protection Agency.Of 29,088 medical patients acutely admitted to the ED at Hvidovre Hospital during the study period, 1341 patients had a prevalent asthma diagnosis and were included in present study cohort  patients within 30 days, 289 (21.6%) within 90 days, and 452 (42.3%) within 365 days. The corresponding numbers for all-cause mortality were 21 (1.6%) within 30 days, 38 (2.8%) within 90 days, and 57 (5.3%) within 365 days, respectively (Table p = 0.037) between suPAR and blood eosinophil counts was found at 30 days, but not at 90- nor 365 days (data not shown).The median suPAR level was significantly elevated at the time of the index admission in patients who were readmitted within 365 days compared to patients without readmissions. Higher readmission rates were observed especially in the 4th suPAR quartile Table . A weak p = 0.044) after 365 days in the 4th quartile when compared to the 1st suPAR quartile using the multivariable model adjusted for sex, age, Charlson comorbidity score, CRP, and blood eosinophil count.   vs 2.4 ng/mL (IQR 1.8–3.1); p = 0.0002). Although 73.7% of all asthma patients who died belonged to the 4th suPAR quartile at 365 days follow-up  for suPAR in readmission prediction and 0.84 (95% CI 0.79–0.89) in mortality prediction at 365 days, see Additional file Patients with low blood eosinophil count at admission had significantly higher readmission rates at 365 days (Table p = 0.005), when compared to patients with blood eosinophil counts below 150 cells/μL in multivariable, adjusted regression analyses  (Table 50) (Tablp = 0.0007). The lowest odds of death at 365 days was seen in patients belonging to the patient group with high blood eosinophil count with OR 0.25   Table .ROC curve analyses showed an AUC of 0.53 (95% CI 0.50–0.56) for blood eosinophil count in readmission prediction and 0.72 (95% CI 0.65–0.79) in mortality prediction after 365 days, see Figs. E1 and E2 in the online data supplement.When event rates are stratified across both suPAR quartiles and groups of low/medium/high blood eosinophil count, an even distribution of patients was seen in terms of readmission Table . HoweverIn patients with an active diagnosis of asthma acutely admitted to a hospital, this study shows that the inflammatory biomarkers suPAR and blood eosinophil count are associated with hospital all-cause readmission and all-cause mortality. The highest odds of both readmission and mortality were seen in patients with suPAR concentrations > 3.5 ng/mL and in patients with low blood eosinophil counts (< 150 cells/μL).1/T17 T-cells are thought to be part of the pathogenesis of the non-T2 asthma endotypes [2 asthma [While the plasmin/plasminogen-pathway has been extensively investigated in basic research studies, little is known about the mechanistic association between asthma and suPAR. Both in vitro and in vivo studies of acute and chronic disease suggest that neutrophils are an important source of suPAR, as uPAR is shed from the surface of neutrophils, T-cells, and a subset of monocytes upon activation , 22, 23.ndotypes  which arndotypes , 25, andPrevious studies have shown that suPAR is associated with disease progression and severity, i.e., suPAR levels increase with higher disease stages and increasing severity. We have previously shown in unselected acute medical patients that suPAR levels increase with number and severity of comorbidities (Charlson score)  and withWe hypothesized suPAR concentrations to be elevated in hospitalized asthma patients due to the systemic inflammation associated with asthma, especially in uncontrolled disease . In the Mortality rates at 365 days were slightly higher than previous studies on all-cause mortality in asthma patients in Denmark , perhapsThe usefulness of blood eosinophil count as a biomarker for acute eosinophilic inflammation is still debated, and while eosinophils are extensively investigated in mechanistic studies, blood eosinophil count is commonly used as a surrogate marker for eosinophilic airway inflammation. The usage of blood eosinophil count as a surrogate marker is not without controversy, as some studies have found weak correlations between blood eosinophils and the gold standard sputum eosinophils , while oThe relationship between blood eosinophil counts and hospitalization rate has been thoroughly investigated in recent years, with some studies , 33 show2 asthma is relatively treatment refractory, resulting in frequent readmissions and death, perhaps due to a less efficient response to the initial in-hospital treatment in comparison with patients with normal or elevated blood eosinophil counts [Regarding mortality, our study showed that the median blood eosinophil count of patients who died was 30 cells/μL and 75.9% of all patients who died had a blood eosinophil count of < 150 cells/μL. The present study thus adds to the growing body of evidence that non-Tl counts –40.2 inflammation activity, while blood eosinophil counts represent T2 inflammation, but further studies regarding the different inflammatory mechanisms and their association to adverse outcomes in asthma are needed.Our study showed that in patients with low blood eosinophil counts, 31.9% of readmissions and 75.6% of mortalities belonged to the 4th suPAR quartile, suggesting that combined measurement of the two biomarkers upon admission potentially could improve risk assessment, especially in terms of all-cause mortality. With regards to inflammatory profiles, suPAR might reflect current non-TA limitation to the present study is the lack of information on patient smoking status, as suPAR is known to be affected by smoking . SmokingIn conclusion, elevated suPAR concentration and low blood eosinophil count associated with poor 365-day prognosis in this cohort of acutely admitted asthma patients. Usage of the two biomarkers at the time of acute hospitalization could allow for more precise identification of patients with a high risk of adverse events and potentially also patients with low disease control. However, further research is needed, especially effect studies of treatment on suPAR concentrations in asthma patients.Additional file 1: Fig. S1. AUC-graph for suPAR and blood eosinophil count-based 365-day Readmission Prediction Fig. S2. AUC-graph for suPAR and blood eosinophil count-based 365-day Mortality Prediction.