Acinetobacter baumannii as representative for our analysis, since it is one of the most important opportunistic human pathogens, exhibits a significant drug resistance and it has the ability to survive in hospital environments. The E.coli DnaK was also included in the analysis as reference structure due to its wide diffusion. Unfortunately, bacterial DnaK and human Hsp70 have an elevated sequence similarity. Therefore, we performed a differential analysis of DnaK and Hsp70 residues to identify hot spots in bacterial proteins that are not present in the human homolog, with the aim of characterizing the key pharmacological features necessary to design selective inhibitors for DnaK. Different conformations of DnaK and Hsp70 bound to known inhibitor-peptides for DnaK, and ineffective for Hsp70, have been analysed by molecular dynamics simulations to identify residues displaying stable and selective interactions with these peptides. Results achieved in this work show that there are some residues that can be used to build selective inhibitors for DnaK, which should be ineffective for the human Hsp70.DnaK, the bacterial homolog of human Hsp70, plays an important role in pathogens survival under stress conditions, like antibiotic therapies. This chaperone sequesters protein aggregates accumulated in bacteria during antibiotic treatment reducing the effect of the cure. Although different classes of DnaK inhibitors have been already designed, they present low specificity. DnaK is highly conserved in prokaryotes (identity 50–70%), which encourages the development of a unique inhibitor for many different bacterial strains. We used the DnaK of  Heat Shock Proteins (Hsp) are essential for the survival cells and their expression levels rely on cellular conditions. In particular, proteins belonging to the Hsp70 family are involved, under stress conditions, in signal transduction, cell cycle regulation, and programmed cell death. Other conditions that involve these proteins are principally native protein folding, refolding and prevention of protein aggregation . Their eAcinetobacter baumannii [A baumannii [DnaK has been characterized in several pathogenic bacteria and seems to have important functions in stress resistance and pathogenicity in multiple-drug-resistant bacteria, such as aumannii –4, whichaumannii . In detaaumannii –8. DnaK aumannii –8, sinceaumannii  and assiaumannii .Like all other Hsp70 proteins, DnaK is composed of about 650 residues, arranged in two domains: the nucleotide binding domain (NBD) and the substrate binding domain (SBD), these are connected by a highly flexible linker involved in the allosteric communication between the two domains. When the NBD domain hydrolyses an ATP molecule, the SBD domain assumes a closed conformation, which binds a short extended hydrophobic polypeptide sequence . Therefo1,2 and L3,4 in combination with strands 1 and 2 conform the deep peptide binding cavity. αSBD consists of five helices, one of which, indicated as HB, forms a lid over the polypeptide binding site [SDB consists of two sub-domains, βSBD and αSBD. The former is a β-sandwich of two antiparallel β–sheets composed of four strands each one and connected by seven loops. Among these loops, Ling site . The bining site .Short hydrophobic peptides (~7 residues) were identified by experimentally screening libraries of compounds able to bind SBD . The hydRecently some peptides with antibacterial characteristics have been isolated in insects. The optimized forms of these peptides able to bind DnaK are relatively stable to protease digestion, due to the high proline content, are nontoxic to human cell lines, and are predominantly active against Gram-negative bacteria , 16. In A.baumannii DnaK, representative for the opportunistic human pathogens, E.coli DnaK, included as reference and control structure, and human Hsp70-2 in closed conformation, were simulated in complex with Api88, drosocin, heliocin, oncocin, and pyrrhocoricin bound to the SBD. Results achieved for the bacterial complexes were compared to data obtained for the human model. Our analysis resulted in a set of key residues of the DnaK binding site that are involved in stable interactions with the ligands. Moreover, we developed a pharmacophore model, which can be employed in a further virtual screening study to identify ligands with high specificity and selectivity.In this study, we identify SBD hot spot residues that can be exploited for the differential inhibition of the bacterial DnaK in comparison to the human Hsp70. The key residues for the interaction with the inhibitor-peptides were recognized, selecting residues conserved in bacteria and not-conserved in the human Hsp70 family. The study is based on sequence analysis, evaluation of the binding site through molecular dynamics (MD) and interaction analysis. MD is a good approximation for studying protein-peptide binding with atomistic detail, and to take into account the dynamic behaviour of the whole system , 19. In Protein sequences were downloaded from Uniprot . BacteriE.coli DnaK in complex with ADP-Mg2+ (pdb ID: 2KHO) [A.baumannii DnaK and human Hsp70-2 were obtained relying on E.coli DnaK in closed conformation, using the Swiss-model server [The closed conformation of D: 2KHO)  was obtaD: 2KHO) . Structul server .E.coli SBD bound to Api88 (pdb ID: 4E81) [E.coli and A.baumannii DnaK and human Hsp70 to obtain the complexes to simulate. Peptides are composed of 18–22 amino acids, but for each inhibitor only the 8 core-residues were co-crystallized and therefore included in our complexes.Coordinates of the inhibitors were extracted, respectively, from the X-ray structure of D: 4E81) , DrosociD: 4E81) , HeliociD: 4E81) , OncocinD: 4E81)  and pyrrD: 4E81) . These p+ ions were added to complexes for neutralizing the net charge of the system. All complexes were energy relaxed with 1000 steps of “steepest-descendent” energy minimization. MD simulations were performed using the LINCS algorithm [MD simulations were performed with GROMACS 4.0 , by empllgorithm  to constlgorithm . Van derg_cluster module of GROMACS to obtain representative conformations of protein complexes. Clustering of the trajectories was achieved fitting Cα atoms with the gromos method [Analyses of MD simulations were performed using different software from the GROMACS package. System stability was verified analysing the energy components and the RMSD of the trajectory conformations compared to the starting structure. The cluster analysis was performed using the s method , with a s method .g_mdmat tool of GROMACS, which identifies the minimum distance between residues by calculating the smallest distance between any pair of their atoms. A truncation distance of 0.5 nm was employed to obtain the contact map. This analysis was useful for identifying residues in the neighbourhood of inhibitor-peptides during simulations.Contact maps were generated using the g_hbond module of GROMACS, applied to MD simulations, while in-house developed scripts were employed to identify key residues for the interaction with inhibitor-peptides [plot_hbmap.pl [The interaction analysis was performed with the peptides . In parthbmap.pl . Residuehbmap.pl .Computational alanine-scanning was accomplished by employing the Robetta server –38, whicStructure-based pharmacophore models were generated with LigandScout . Interacg_energy. The interaction energy of the three proteins and of residue Met404, Val425, Ser427, Ala429 and Ala435 for the ligands, were analyzed.A previously developed approach was applied to estimate the enthalpy variation caused by the ligand binding  to the pE.coli and A.baumannii as representative for all the possible bacterial proteins. Moreover, this analysis was useful for verifying residue conservation in positions identified as hot spots at the end of the study. The Hsp70-2 was chosen as a representative for human Hsp70s since it is stress-induced, it localizes in the cytoplasm, and it is one of the most diffused in cells [A preliminary analysis of the available bacterial DnaK sequences was performed to evaluate the reliability of employing the DnaK of in cells .E.coli DnaK in closed conformation, with the SBD lid closed over the peptide binding cavity, and the homology model of A.baumannii DnaK compared to the homology model of the human Hsp70 bound to Api88, drosocin, heliocin, oncocin and pyrrhocoricin. For each of the five inhibitors, closed conformations of both DnaK and Hsp70 in complex with ADP were simulated for 100 ns. Overall, we obtained about 1,3 μs of equilibrated simulation. The equilibrated parts of MD trajectories were evaluated by interaction analysis, conformation clustering, and were employed for the identification of residues belonging to the peptide binding cavity. Computational alanine scanning and per residue binding energy decomposition were carried out to evaluate the role of residues in protein binding. Moreover, multiple alignments were employed to assess conservation of residues, with particular attention to the SBD. At last, identified residues allowed the design of a pharmacophore model, which can be employed in virtual screening studies for the identification of selective and specific DnaK inhibitors.A comparative analysis was carried out by simulating complexes of the X-ray structures of A.baumannii were aligned  and the DnaK of  aligned  to ident aligned . DnaK isBoth identities and similarities between sequences analysed in this study have been evaluated. As shown in In order to identify stable DnaK residues anchoring the inhibitor-peptides, the temporal evolution of the interactions was evaluated. The presence of hydrogen bonds and contacts was analysed along the whole MD trajectories. Residues involved in hydrogen bonds with the inhibitor-peptide for at least 25% of the simulation (50% for contacts) were taken into account. Among these residues, only those conserved in DnaK and not in Hsp70 were further considered for identifying selective amino acids for the bacterial protein . Four reFrom the peptide point of view, amino acids involved in interactions with the binding site were analysed. In particular, Met 404 frequently interacts with both side-chain and backbone of residues in the position 4 of peptides, while Ala429 and Gln433 interact with the backbone of residues in positions 5 and 6, respectively. Moreover, Ser427 and Val436 establish contacts with the backbone of residues in positions 2–5 and with the core residue side-chains of the inhibitor-peptides (5 and 6). Concerning residues conservation in peptides, only Proline and Arginine enrichment can be reported.1—L1,2 - β2, β3—L3,4 - β4, L5,6 - β6 and HB . Representative conformations were obtained by cluster analysis, see Experimental section for details. A binding energy threshold (ΔΔGbind) of 1 kcal/mol for residues replaced with alanine was applied to identify residues responsible of protein-protein binding as specified in Kortemme et al. . ResidueWe evaluated the energetic contribution made by the sum of Lennard-Jones and Coulomb terms to the binding enthalpy for each residue of the protein binding cavity displaying differences between DnaK and Hsp70 . For eac1—L1,2 – β2) are highly conserved in bacteria, while in human proteins only positions 405, 409 and 411 are well conserved and 402, 403 are quite conserved. Considering residues of β3—L3,4 – β4, positions 426–429, 431–433, 436, 438 and 440 are highly conserved in bacterial DnaK, while considering human Hsp70 only positions 426, 431 and 436 are conserved. Among residues belonging to L5,6 – β6 of DnaK, only positions 458 and 469 are not conserved, while Arg468 and Ile474 are quite conserved also in Hsp70 sequences. Concerning residues of HB, only Glu531, Asn537 and Asp540 (respectively Asn540 and Glu543 for Hsp70) are highly conserved.At last, the conservation of residues identified with the previous analysis was evaluated. In particular, residue conservation among the 65 DnaK bacterial sequences  and amonThe structure-based pharmacophore model comprises the most important pharmacological features for inhibitors binding. Relying on the identified residues, a pharmacophore was obtained for each DnaK-peptide complex . The finConcerning protein residues, these principally correspond to amino acids identified in previous analysis. In particular, Thr403 and Phe426 are involved only in hydrophobic contacts; Ser427 and Asp/Thr437 are donors or acceptors for hydrogen bonds, while Gln433 results only as an acceptor of hydrogen bond. Moreover, Met404, Thr409 and Ala429 are occupied both in contacts and hydrogen bonds as acceptors or donors.DnaK is the bacterial homolog of the human Hsp70 and it contributes to bacterial multi-drug resistance, which makes this protein a very interesting drug target. For this reason we propose a detailed study of DnaK binding cavity and its interaction capability in comparison to five recently discovered peptide inhibitors  in comparison to the human Hsp70, though structure analysis and molecular dynamics simulations. These methods allow the analysis of complexes in static and dynamic situations. The analysis of known complexes returns residues stably involved in the interaction with inhibitor-peptides, conserved in bacterial DnaK proteins, but not in the human homolog, while more general analyses on the binding cavity residues allow to identify amino acids potentially useful for the design of high affinity and selective ligands.3,4 and L1,2 cavity-forming loops, was experimentally confirmed in contacts with the bound peptide [Overall, residues of βSBD are quite conserved, while residues belonging to helices HA and HB are poorly conserved in bacterial DnaK sequences. Considering only the binding cavity residues, several non-conserved amino acids between bacterial DnaK and human Hsp70 were identified: Met404, Val425, Ser427, Ala429, Ala435, Ile438 and Gly468. These residues are non-conserved in human Hsp70 or are not included in its binding cavity. Our prediction concerning residues, Met404, Ser427, Ala429, Ala435 and Ile438, included in L peptide , while t peptide . None of3) and Ala429  are highly conserved in bacterial DnaK proteins, while in the human Hsp70 they are substituted by non-conserved residues. On the other hand, Met404 is quite conserved in the bacterial DnaK family and no conserved in the human family. Ala429 forms with Met404 the hydrophobic arch over the peptide backbone [Among the identified residues, Met404, Ser427 and Ala429  are invobackbone  and is cbackbone . Experimbackbone –45. MoreThe binding cavity analysis returned four other residues not involved in the binding of known peptides, which could furthermore be exploited for drug design. Two of these residues, Ile438 and Gly468 are highly conserved both in bacterial DnaK proteins and in the human Hsp70. However Val425 and Ala435 are quite conserved in bacterial, but not in the human protein family, although position 425 presents a conserved Thr residue in human proteins. These two differential conserved residues can be useful anchoring points for the design of selective peptides .The SBD domain is partially involved in co-chaperones binding, mainly with proteins carrying the J-domain . In partOur analysis allowed to identify three residues, Met404, Ser427 and Ala429 previously exploited by known inhibitors, and two new anchoring points, Val425 and Ala435, belonging to the binding cavity, probably involved in the inhibition of DnaK binding and selective for bacterial proteins compared to the human Hsp70. In particular, the three known residues localized in the binding cavity anchor the central part of the ligand, while the two new identified residues are on the binding cavity border and can be employed to recognize the external part of the ligand. Moreover, from the binding energy analysis, residues Val425 and Ala435 can be better exploited as anchoring points.Rational drug design of compounds capable of binding these residues may increase the chance of delivering selective inhibitors. On the other hand, a virtual screening strategy, followed by the selection of ligands that bind critical residues, could also be applied for identifying inhibitors capable of selectively impairing the bacterial DnaK with high affinity.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.


The association between body mass index (BMI) and mortality is not conclusive, especially in East Asian populations. Furthermore, the association has been neither supported by recent data, nor assessed after controlling for weight changes.We evaluated the relationship between BMI and all-cause or cause-specific mortality, using prospective cohort data by the National Health Insurance Service in Korea, which consisted of more than one million subjects. A total of 153,484 Korean adults over 30 years of age without pre-existing cardiovascular disease or cancer at baseline were followed-up until 2010 (mean follow-up period = 7.91 ± 0.59 years). Study subjects repeatedly measured body weight 3.99 times, on average.2 and ≥ 30 kg/m2 had higher risks of all-cause and cause-specific mortality compared with the reference group (BMI 23–24.9 kg/m2). The lowest risk of all-cause mortality was observed in subjects with a BMI of 25–26.4 kg/m2 . In subgroup analyses, including the elderly and those with chronic diseases , subjects with a BMI of 25–29.9 kg/m2 (moderate obesity) had a lower risk of mortality compared with the reference. However, this association has been attenuated in younger individuals, in those with higher socioeconomic status, and those without chronic diseases.During follow-up, 3,937 total deaths occurred; 557 deaths from cardiovascular disease, and 1,224 from cancer. In multiple-adjusted analyses, U-shaped associations were found between BMI and mortality from any cause, cardiovascular disease, and cancer after adjustment for age, sex, smoking status, alcohol consumption, physical activity, socioeconomic status, and weight change. Subjects with a BMI < 23 kg/mModerate obesity was associated more strongly with a lower risk of mortality than with normal, underweight, and overweight groups in the general population of South Korea. This obesity paradox was prominent in not only the elderly but also individuals with chronic disease. Obesity, generally defined by high body mass index (BMI), is closely associated with incident chronic disease including hypertension, type 2 diabetes and atherosclerotic cardiovascular diseases (CVDs) ,2. PreviPossible explanations for this paradox, mainly focusing on a study bias and confounding factors, include selection or survivorship bias, unintentional weight loss following chronic disease, treatment bias, and effects of major confounders including smoking status and age . HoweverIt has also been observed that the association between BMI and mortality is largely dependent on race. Asians generally have a lower BMI value, but the risk of obesity-related disorders and CVD is quite comparable to that of Europeans, even for those belonging to lower BMI categories . TherefoWe also note that most studies of East Asians were based on baseline BMI measures from the early 1990s. Since obesity prevalence, disease status, and cause-specific mortality rates are rapidly changing, especially in East Asia ,18, an aA national health insurance system in Korea has been initiated since 1963 according to the National Health Insurance Act, and it became compulsory for all citizens in South Korea to participate. Currently, the National Health Insurance Service (NHIS) maintains and manages all databases of Korea’s health service utilization. The NHIS is also in charge of the national health examination programs, which include a general health examination for all insured employees or self-employed persons over the age of 40, as well as their dependents; it is recommended that this examination be undertaken at least biennially. Moreover, a life-transition examination (for those reaching milestones of 40 and 66 years of age), a cancer examination, and a pediatric examination (for those between 4 and 71 months post-birth) are also managed by the NHIS.The NHIS released a National Sample Cohort (2002–2010) database (The NHIS-NSC [2002–2010]) in July 2014, consisting of 1,025,340 Koreans as an initial 2002 cohort, and followed the subjects for 9 years up to 2010. The cohort data represents about 2.2% of the source population in 2002 , sampled systematically to represent an individual’s total annual medical expenses within each of 1,476 strata defined by age, sex, eligibility status (employed or self-employed), and income level  combinations. This is a semi-dynamically constructed cohort database; namely, the cohort has been followed up to either the time of the participant’s disqualification of health services due to death or emigration, or the end of the study period, while samples of newborn infants are included annually. The database contains eligibility and demographic information regarding health insurance and medical aid beneficiaries, medical bill details, medical treatment and disease histories, as well as prescriptions; such data are constructed after converting insurance claim information to the first day of medical treatment.In the cohort, the subject’s mortality information along with cause of death was included; the latter was classified according to the 10th revision of the International Classification of Diseases (ICD-10) codes, obtained from the Korean National Statistical Office. Furthermore, the laboratory and survey questionnaire data of general and life-transition health examinations for all cohort members was merged.From this cohort, we selected subjects who had received health examinations at least once between 2003 and 2004, noting that citizens are recommended to take the examination at least once biennially. We then selected men and women over 30 years of age who measured their own body weight. We did not use the 2002 cohort data in order to clearly identify the subject’s past medical history as well as to avoid possible medical history inaccuracies in the first year. To minimize reverse causality and any effects of pre-existing disease on mortality, we also excluded subjects who had reported CVD or cancer from 2002 to 2004, and further excluded deaths within less than 2 years after the baseline measurement of body weight. As a result, a total of 153,484 subjects were included in our follow-up study.This study were based on data from NHIS, informed consent was not specifically obtained individually. Data were fully anonymized and de-identified for the analysis. This study was approved by the Institutional Review Board of Korea University Hospital. (IRB number: ED14188)2), as measured at regular medical check-up programs. Most participants measured their body weight and height at 2–5 year intervals, and the mean (SD) number of measurements taken per subject was 3.99 (2.01).BMI was calculated as the subject’s weight (kg) divided by height  were obtained via questionnaire. We conducted statistical analyses using the simplified status classification of smoking , alcohol (drinker or non-drinker), and physical activity . The subjects’ socioeconomic status (SES) was categorized into 3 groups  based on 20 strata of income levels provided in the NHIS-NSC (2002–2010) database.The participants’ medical history was identified by a combination of the following: clinic and pharmacy codes of ICD-10, lists of prescribed medicine, and previous medical histories. To identify diabetes mellitus (DM) and hypertension (HTN) status, laboratory data were also used in addition to medical records and health examination survey results based on the criteria of those with fasting serum glucose ≥126 mg/dL for DM, and those with systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg for HTN.2) to highest (≥32.5 kg/m2). The remaining groups were 18.5–19.9, 20–21.4, 21.5–22.9, 23–24.9, 25–26.4, 26.5–27.9, 28–29.9 and 30–32.4 kg/m2. These groupings were based on the WHO obesity classification for Asian populations and the previous Korean study (KCPS) [2.An association between BMI level and mortality risk was analyzed with Cox’s proportional hazard regression model. The BMI level at baseline was categorized into 8 groups from lowest (<18.5 kg/my (KCPS) , 12. The2) were estimated, after adjusting for the subjects’ age, sex, and BMI change relative to his/her initial baseline value. Adjusting for changes in BMI would eliminate the effect on mortality of intentional weight control in healthy subjects or unintentional weight change in those with disease, providing us with a pure BMI effect on mortality. The same analysis was conducted by additionally adjusting for the subjects’ smoking status , alcohol drinking habits , physical activities , and 20 SES groups. The HRs of BMI on all-cause, cardiovascular, and cancer mortalities were also estimated. Furthermore, in order to evaluate the robustness of the association between BMI and mortality, stratified analyses according to the subjects’ sex, smoking status , age group  and three SES group  were performed.The hazard ratio (HR) and 95% confidence interval (CI) for each groups, relative to the reference , CVD (yes or no), and cancer (yes or no). The interactions between BMI and different stratifications were tested in order to examine differences in HRs across strata. Tukey’s method was used for multiple mean comparisons of different groups or strata. All statistical analyses were performed using SAS version 9.4 . All p-values were two-tailed, and values less than 0.05 were considered statistically significant.2), while the proportion of the upper 30% was relatively high between 23 and 30 kg/m2 BMI categories. However, among women, the smallest proportion of the upper 30% was in the highest BMI group, and the proportion decreased as the BMI group increased from 21.5–22.9 kg/m2 to the highest BMI group (≥32.5 kg/m2). Meanwhile, systolic blood pressure, fasting serum glucose, and total serum cholesterol increased as the BMI increased. By extension, the risk of incident DM, HTN, and CVD was directly proportional to increasing BMI, as expected  resulting from CVD and 1,224 (31.1%) from cancer. The mean age of all participants at baseline was 47.1 ± 11.8 years, 61.1% of whom were male. Baseline demographic characteristics stratified by BMI categories are presented in expected .2 . The highest risk of mortality was observed in the lowest BMI group (<18.5 kg/m2). This association was maintained even after additionally adjusting for subjects’ smoking, alcohol intake, physical activity status, and SES. Further analyses stratified by smoking status and sex also did not alter the associations. There was a trend of increasing risk of mortality in subjects with BMI lower than the reference group. Subjects with a BMI equal to or more than 30 kg/m2 (severe obesity group) showed high mortality risk; however, those with a BMI of 25–29.9 kg/m2 (moderate obesity group) had lower HRs than normal, underweight, overweight, and severe obesity groups.Associations between BMI and all-cause mortality for all participants, stratified by their demographic factors, are presented in We note that a stratification of study population by age and SES showed different associations between BMI and all-cause mortality across subgroups . In subj2. An elevated risk of mortality from CVD or cancer was also consistent in subjects with lower BMI groups than the reference group as for that of all-cause mortality. A positive association between severe obesity and mortality risk was prominent for cases of CVD mortality.This U-shaped association between BMI and CVD and cancer mortalities has also been observed  Tables. We assessed an association between BMI and mortality risk within subgroups stratified by chronic medical disease status, which included DM, HTN, and chronic kidney disease (CKD) . There wIn this recent population-based, prospective cohort study, we performed comprehensive analyses of the associations between BMI and all-cause mortality as well as cause-specific mortalities. In general, there were U-shaped associations between BMI and all-cause, CVD-, and cancer-associated mortalities in the total subjects. An increased risk of mortality in subjects with BMI of less than 23 and more than 30, compared with the reference range  was also found, with the lowest mortality in the moderate obesity group , more specifically in the BMI range of 25–26.4. Further subgroup analyses stratified by sex and smoking status did not change the associations, while they were varied across age and SES subgroups.Previous large epidemiological studies including East Asians showed similar U-shaped associations between BMI and all-cause or cause-specific mortality, but the BMI group with the lowest mortality risk has widely ranged from 18.5 to 27.5 according to study populations and cause of death –16. AmonA previous study in the United States encompassing the first to third rounds of the National Health and Nutrition Examination Survey (NHANES) provided an important clue about this phenomenon . They reIt was also of interest to note that associations between BMI, and CVD and cancer mortalities were, to a certain degree, different each other; the HR of cancer mortality more rapidly decreased in the moderate obesity group than that of CVD mortality. This indicated that high BMI may represent an optimal physical and nutritional state for protecting against catabolic diseases such as cancer. Growing evidence suggests that low muscle mass is a poor prognostic indicator of mortality, and BMI is positively correlated with muscle mass or lean body mass, especially in the elderly ,24. ThisAnother important finding in our study was that SES was revealed as a major determinant of the association between BMI and mortality. First of all, we would like to mention that the identification of study subjects’ SES was almost complete because the Korean national health care system covers all citizens, and an individual`s medical insurance premium is directly proportional to their income. We found that the effect of SES on BMI differed by sex. Although there were inverse U-shaped associations between BMI and SES in both sexes, the main difference among them was that men in the lowest social class were prone to being underweight, while women in a low social class were likely to have a high BMI. It is well known that SES influences not only accessibility to a medical care system but also life expectancy; in a study of mortality, it needs to be taken into account as a major confounder, as our study reflects. Alter et al. showed that those in higher income groups were associated with an increased rate of access to coronary angiography and revascularization for acute myocardial infarction, as well as decreased mortality, compared with those in lower income groups . AnotherWe also found that the association between BMI and mortality varied according to the state of chronic diseases, including DM, HTN, and CKD; the obesity paradox was more profound in patients with chronic diseases than those without. Previous large amounts of data have demonstrated the paradox in patients with chronic diseases. As stated above, some authors have explained the obesity paradox by focusing on a study bias, or confounding factors. For example, most epidemiologic studies that investigated the effect of BMI on mortality excluded subjects with pre-existing CVD, and thus obese CVD patients who were at high risk of death were not included in the analysis, obscuring the actual relationship between obesity and mortality . Another2) had a lower HR of mortality than the reference group, suggesting a protective role for obesity in patients with various morbidities  cohort consists of representative samples of about one million Koreans who were prospectively followed during the study period. We were able to obtain accurate information with regard to critical variables to measure an effect of BMI on mortality risk at baseline as well as during follow-up; BMI was calculated by directly measured body weight and height, and the subjects’ disease status was identified by using health examination survey questionnaires, disease codes, and laboratory or anthropometric data. We were also able to collect detailed histories of smoking, alcohol consumption, physical activity, and SES. Most of all, by considering a major criticism of previous studies regarding the obesity paradox, we incorporated BMI changes with repeated measured body weight in the analyses in order to overcome a reversal causality between unintentional or intentional weight loss and mortality.A limitation of this study includes a potential selection bias. The NHIS recommends, but does not obligate all health insurance subscribers to undergo at least once biennial health examination. Therefore, this cohort probably includes healthier individuals, or those who are more concerned about their health. Another limitation is the lack of confounders such as marital and educational status, and body composition, as discussed previously.2). This suggests that the change of age, combined with social structure, could alter the associations between obesity and health status. Interestingly, the association observed in this study resembles that of developed countries [2. This was largely based on the relationship between BMI and the incidence of obesity-related disorders. However, the “optimal” BMI should take into account mortality as well as morbidity. Furthermore, changes in the epidemiologic characteristics of Asian populations over the last decade, including an increasing prevalence of obesity, should also be considered. The findings of our study likewise suggest a higher optimal BMI for Asians. Moreover, our data suggests that the obesity paradox needs to be interpreted separately, according to individuals’ demographic and health status; this was apparent for older persons and patients with chronic diseases. Health professionals and providers may need to take a different approach to patients to maintain optimal BMI, by understanding and taking into consideration a variety of health status factors.In this study, we identified several novel findings regarding the association between BMI and mortality. The BMI level with the lowest risk of mortality was changed; that is, a shift to the right compared with the Korean data from 10 years ago Click here for additional data file.S1 STROBE Checklist(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.


Previous reports of chromosomal aberrations in different forms of congenital diaphragmatic hernia have been described as comprising aneuploidies , microdeletions, and duplications .de novo partial tetrasomy 4q35.2 in a father with left-sided, isolated renal agenesis and left-sided, isolated congenital diaphragmatic hernia in his son, who inherited the chromosomal aberration from his father.We describe the first association of a de novo in the father and was transmitted to his son, with both presenting with unilateral left-sided developmental field defects, we suggest a gene dosage effect of the tetrasomic region to be involved in the phenotype of our two patients. Furthermore, we suggest performing a genetic workup in multiplex families with congenital malformations.Given that the aberration occurred  The diaphragmatic defect allows an upward displacement of the abdominal organs into the thorax with a consecutive compression of the intrathoracic compartment, leading to hypoplasia of the lung, secondary pulmonary hypertension, and intestinal malrotation . CDH occThe index patient is the first child of his nonconsanguineous parents. Due to reduced fertility of the father, assisted reproduction in the form of intracytoplasmic sperm injection had been performed to achieve pregnancy in the couple. Besides his reduced fertility, the father was born with unilateral agenesis of the left kidney and a poorly formed hypoplastic helix of his left ear. He was a university graduate and was working in a management position.The achieved pregnancy was uneventful until the second trimester, when prenatal ultrasound of the fetus revealed isolated left-sided CDH. The mother gave birth to a boy at 27 + 2 weeks of gestation by cesarean section due to preterm rupture of the membranes and contractions despite intravenous tocolysis. The boy’s birth weight was 950 g (25th percentile), length was 36.6 cm (50th percentile), and head circumference was 25 cm (30th percentile). His Apgar scores were 6 at 1 minute, 8 at 5 minutes, and 8 at 10 minutes. He was intubated immediately after delivery and ventilated. A chest X-ray confirmed left-sided CDH Fig. . After hin situ hybridization (FISH) analysis was performed, which revealed an increased signal at 4qter only. Multiple ligation-dependent probe amplification  of the specific region confirmed a triplication of chromosomal region 4q35.2 spanning approximately 3.73–4.43 Mb  in one of the two chromosomes 4 of the fetus. To test whether the chromosomal aberration occurred de novo, and because the father presented with reduced fertility and unilateral renal and helix anomaly, consecutive karyotyping of the parents was performed, which revealed the same aberration in the father ] and associated CDH, unilateral renal agenesis, and ear anomaly in the carriers. Previous reports of chromosomal aberrations in different forms of CDH described aneuploidies , microdeletions and duplications  , 9. ApprFOG2 , GATA4  presenting with, among other anomalies, unilateral renal agenesis, low-set ears, and ventricular septal defect. Furthermore, a male patient [accession number 278863] presented with, among other anomalies, CDH and posteriorly rotated ears. The phenotypic variation between the father and the son described in the present report might be due to different genetic mutations in the genes downstream of the triplicated genes or that other genes not involved in the triplication may compensate for or inhibit the pathogenesis of the triplication to different degrees [FAT1) lead to CDH and renal agenesis. FAT1 (NM_005245) encodes a tumor suppressor essential for controlling cell proliferation during Drosophila development. According to the Mouse Genome Informatics database (http://www.informatics.jax.org/), homozygous, targeted null mutations exhibit holoprosencephaly, anophthalmia, kidney defects, and perinatal lethality.Regarding the aberrant region on 4q35.2 that we describe in this report, we conducted a medical database search using PubMed ( defects , 16. How [et al.  describe degrees . Hence, Overall, our observations suggest that duplication or triplication of chromosomal region 4q35.2 carries susceptibility for the development of renal, ear, and diaphragmatic anomalies. Furthermore, we suggest performing a genetic workup of multiplex families with congenital malformations.Written informed consent was obtained from the patient’s legal guardian for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Postprandial metabolic response depends on the meals' components and can be different in normal weight and obese people. However, there are some discrepancies between various reports. The aim of this study was to determine the metabolic response after intake of standardised meals with various fat and carbohydrate contents and to determine the differences among normal weight and overweight/obese individuals. The study group comprised 46 healthy men. The participants were divided into two groups and study was carried out using a crossover method. Group I received high- and normal-carbohydrate meals, whereas group II received high-carbohydrate and high-fat meals. Glucose, insulin, triglyceride, and free fatty acids levels were measured at fasting state and at 30, 60, 120, 180, and 240 minutes after meal intake. Despite the lack of differences in glucose levels, insulin levels were higher among overweight/obese individuals after each meal. TG and FFA levels were higher after normal-carbohydrate and high-fat meals. Moreover, in overweight/obese young men after high-fat meal intake postprandial hypertriglyceridemia was observed, even if meals contained predominantly unsaturated fatty acids, and fasting triglycerides levels were in normal range. The conducted study showed that postprandial metabolic response depends not only on the meal macronutrient content but also on the current body mass index (BMI). In the last 30 years, a dramatic increase in number of people suffering from obesity has been observed. The prognosis for the future is alarming, as it is believed that, with current trends, over a billion of people will have been obese by the year 2030, and another 2 billion will have been overweight . The maial state . On the al state . Moreoveal state , 13, 14.The aim of this study was to evaluate the metabolic changes after intake of meals with various fat and carbohydrate contents, using a crossover method, and to analyse the differences between postprandial metabolic responses among normal weight and overweight/obese healthy men. It was expected to discover early postprandial metabolic disturbances in young overweight/obese men in postprandial state, even if the baseline results were in normal range.46 men participated in the study: 23 with normal weight (NW) and 23 were overweight/obese (OO) in the age range of 21–58 years. The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving patients were approved by the local Ethics Committee of the Medical University of Bialystok (Poland) and a written informed consent was obtained from all participants. The participants of the study did not suffer from any glucose metabolism disorders, endocrine disorders, renal or liver failure, and digestive system diseases, did not undergo any gastroenterological and bariatric surgeries or procedures, and did not suffer from any other diseases which could influence the results. People who had received pharmacological treatment or had used any other products with undocumented or unknown influence on metabolism were excluded from the study. Taking into consideration the fact that the levels of some factors can be characterised by sexual dimorphism and its analysis was not the aim of the study, only male participants were selected for the study group, in order to obtain reliable results.2) and men that were overweight/obese . Subsequently, the participants were randomly divided (randomisation using sampling without replacement) into two experimental groups  received a high-carbohydrate (HC) meal , 89% of energy coming from carbohydrates, 11% coming from protein, and 0% coming from fat , and, then, after a period of 1-2 weeks received a meal with normal-carbohydrate (NC) content , 45% of energy coming from carbohydrates, 30% coming from protein, and 25% coming from fat: 12,4% SFA, 59,0% monounsaturated fatty acids (MUFA), 28.7% polyunsaturated fatty acids (PUFA), and n-6/n3 ratio 5,11 .Group II (12 NW men and 11 OO men) received a HC (fat-free) meal , 89% of its energy coming from carbohydrates, 11% coming from protein, and 0% coming from fat , and, then, after a period of 1-2 weeks received a high-fat (HF) meal , 4% of energy coming from carbohydrates, 0% coming from protein, and 96% coming from fat: 10.7% SFA, 60.7% monounsaturated fatty acids (MUFA), 28.6% polyunsaturated fatty acids (PUFA), and n-6/n3 ratio 5,02 .The participants were asked not to change their diet and daily physical activities during the study.The participants were arriving at the lab at 08:00–08:30 in the morning, in the fasting state, with at least 12 hours since their last meal. The following procedures were carried out: height and weight measurements and body fat content measurement . A peripheral venous catheter was placed in the elbow crook and venous blood was drawn in order to determine fasting blood glucose and insulin and free fatty acids (FFAs) and triglyceride (TG) levels. The participants then received a randomly selected meal (at room temperature) and were advised to consume it within 10 minutes. 30, 60, 120, 180, and 240 minutes after meal intake the venous blood was drawn in order to measure the above-mentioned factors levels again.μQuant, BioTek Instruments Inc., Winooski, Vermont, USA).The specimen was drawn and prepared for testing in accordance with the recommendations provided by the laboratory kit producers. The assay of particular levels was carried out immediately after drawing the specimen, while the remaining ones were determined after the specimen was obtained for the whole period of tests. The specimen was stored in accordance with the recommendations of the producers until it was tested, at temperature of −20°C/−80°C. The particular factors levels were determined using the following methods: glucose-hexokinase enzymatic colorimetric assay , insulin-immunoradiometric assay , TG-enzymatic colorimetric assay , FFAs-enzymatic assay  were calculated using the trapezoidal method and underwent the same analysis schema as the rest of the features.In order to obtain the general characteristics of the data, arithmetic mean and standard error of the mean were calculated for all numerical features, which were treated as dependent variables in subsequent steps of the analysis. The aim of the study was to check whether there is a statistically significant postprandial metabolic response depending on meals' components. There were two main null hypotheses stated: (1) different types of meals  have no influence on postprandial metabolic response in normal weight and overweight/obese patients ; (2) there is no difference in postprandial metabolic response to a particular meal in normal weight and overweight/obese patients. The first hypothesis (1) was verified for two pairs of meals: HC versus NC and HC versus HF for normal weight and overweight/obese subjects. Since both meals were given to the same patients, tests for dependent variables were used: either one-way ANOVA or Wilcoxon signed-rank test (both for paired samples)—depending on fulfilling of the condition of the normality of the variables' distribution. The second hypothesis (2) was verified for each type of meal: HC, NC, and HF in order to check whether there are significant differences in postprandial metabolic response between normal weight and overweight/obese subjects. One-way ANOVA or Wilcoxon rank-sum test (both for unpaired samples)—depending on fulfilling of the condition of the normality of the variables' distribution—and the homogeneity of variances were used to test the stated hypothesis. To address the issue of multiple hypothesis testing, false discovery rate P < 0.01) and in OO men .The blood glucose levels were significantly higher after the HC meal intake than after NC meal at 30 and 60 minutes in OO men and also at 120 minutes in NW individuals . SimilarP < 0.0001, and 29648 ± 1378 versus 21327 ± 331, P < 0.0001, for OO men). 240 minutes after HC meal intake the blood glucose levels were significantly lower in comparison to the levels after the remaining meals and glycaemia ≤65 mg/dL was observed in 29% of the participants .The blood glucose levels were significantly higher after HC meal in comparison to HF meal as well, both in NW and in OO men . The AUCP > 0.05) and after NC meal (P > 0.05) in group I and after HC meal (P > 0.05) and HF meal (P > 0.05) in group II.At fasting and postprandial state Figures  blood glP < 0.03) and in OO men .The insulin levels were significantly higher after HC meal in comparison to NC meal, both in NW and in OO men . The insP < 0.0001, and 33075 ± 11070 versus 4533 ± 973, resp., P < 0.01, for OO men).The insulin levels in NW and OO men were also significantly higher after HC meal intake, in comparison to HF meal . In NW mP < 0.01; and after NC meal 12845 ± 1712 versus 5694 ± 716, resp., P < 0.01). In group II, both at fasting state and after HC meal intake, no differences between insulin levels in NW and OO men were observed (P < 0.01).While comparing the insulin levels between NW and OO men, the OO men from group I had significantly higher insulin levels both at fasting state and after HC and NC meals intake . Furtherobserved , which wP > 0.05). In the OO men the AUCs for TG levels were significantly higher after NC meal than after HC meal and amounted to 38888 ± 4640 versus 27057 ± 1879, respectively (P < 0.02).In the OO men the TG levels were significantly higher after NC meal at 120, 180, and 240 minutes, in comparison to the levels after HC meal  which waP < 0.03). In OO men a tendency for higher values of the AUCs for TG levels after HF meal intake, in comparison to HC meal, was noted .When comparing the TG levels after HC meal with the TG levels after HF meal intake, from 120 to 240 minutes in NW and from 180 to 240 minutes in OO men the TG levels were significantly higher after HF meal intake . The AUCNo differences between NW and OO men Figures  were obsP < 0.03) and OO men . The differences were more distinct after HF meal intake, when for 30 minutes of testing the FFAs levels were higher in comparison to FFAs levels after HC meal intake, among NW and OO men (P < 0.000001) and OO individuals .Both, in NW and in OO men, the FFAs levels were significantly higher from 120 to 240 minutes after NC meal intake, in comparison to the levels after HC meal . Moreoved OO men . The AUCP < 0.01).The FFAs levels were similar among NW and OO men, both at fasting and after HC and NC meals intake Figures . HoweverThe study confirmed that metabolic response depends on the components of a meal and on the current body energy balance. Moreover, after HF meal intake postprandial hypertriglyceridemia was noted in OO men, even if fasting TG levels were within the normal range and the meal contained predominantly unsaturated fatty acids. It is worth noticing that the investigated study groups comprised healthy young men, and, in spite of this, we observed very early disturbances in postprandial metabolic response, which in long-term can lead to development or progress of metabolic disorders such as obesity, hypertriglyceridemia, metabolic syndrome, and type 2 diabetes.The highest glucose levels after HC meal, observed in NW men and in OO men, were not surprising and are consistent with the results obtained by other researchers , 16. HowThe study showed that glucose levels in general were not different for NW and OO people, both at fasting and after each of the meals. Other researchers also did not observe any significant differences in glucose levels between people with normal weight and those with obesity after high-carbohydrate and high-fat meals intake , 17. HowThe insulin levels, both in NW men and in OO men, were significantly higher after HC meal intake than after NC or HF meal. The increased secretion of insulin was undoubtedly a result of higher glucose levels after high-carbohydrate meal, which was also confirmed in studies carried out by other authors , 16, 19.Despite the lack of significant differences in glucose levels, the insulin levels were significantly different both in fasting and in postprandial states. OO men had significantly higher fasting insulin levels after HC, NC, and HF meals intake (in comparison to NW men). Other researchers also observed higher insulin levels, at fasting state and after a meal intake in people with obesity, also with a lack of significant differences in glucose levels , 20, 21.When comparing meals with different composition, we have noticed that in OO men, after NC meal intake, the TG levels were significantly higher in comparison to the levels after HC meal intake. Also among OO men the AUC for TG levels was significantly higher after NC meal, whereas among NW men the AUCs for TG levels after HC and NC meal did not differ significantly. The TG levels were also significantly higher after HF meal in comparison to HC meal, both in NW and in OO men. Other researchers also proved that the high-fat meal intake is related to the increase of TG levels, where the maximum values were obtained 4 hours after ingesting the meal . The higAlso the high FFAs levels are responsible for elevated endothelial activation markers, vascular endothelial cell inflammation, and increased prothrombotic activity markers, which can cause vascular and atherosclerotic anomalies resulting in circulatory system diseases . As a paThe conducted analyses did not show any significant differences in fasting FFAs levels between NW and OO people, and the AUCs for FFAs levels after HC and NC meals were the same for NW and OO people as well. However, 60 minutes after HF meal intake higher FFAs levels were observed among OO people in comparison to NW men and remained significantly higher until 180 minutes of the test. Also the AUC of FFAs levels in postprandial state after a high-fat meal intake was significantly higher in OO than in NW men. Imbeault et al.  also obsThe differences in postprandial metabolic effects, especially in lipids response, probably can be explained by the lower numbers of lipoprotein particles of hepatic origin that compete with chylomicrons for lipolysis , which rMoreover, the postprandial response can be dependent also on the other, for example, genetic factors, which we have observed in one of our previous studies .In summary, the conducted study confirmed that postprandial metabolic response depends on the components of a meal and moreover it is different among NW and OO people.The highest blood glucose levels were observed after HC meal intake; however the levels were the same for NW and OO people. Insulin levels were also the highest after HC meal intake, and, despite the lack of significant differences in blood glucose levels, higher insulin levels were observed in OO people, in comparison to the NW group. Triglyceride and FFAs levels were higher after NC and HF meals intake. In OO people, postprandial hypertriglyceridemia was observed after HF meal intake, even if it contained mostly unsaturated fatty acids. Further investigations on the effect of MUFA on the CHD risk are undoubtedly warranted. Nevertheless, our findings showed that metabolic response in OO people to high-fat meals, even if rich in unsaturated fatty acids, caused postprandial hypertriglyceridemia and should be avoided in diet. Even if the study sample size was quite small and our results need to be confirmed, for example, by increasing the sizes of study groups, the obtained results have crucial significance in a period of obesity plague and of the omnipresent trends of diets with modified amount of macronutrients, including high-fat diets.


Lung cancer is an important and common cancer that constitutes a major public health problem, but early detection of small cell lung cancer can significantly improve the survival rate of cancer patients. A number of serum biomarkers have been used in the diagnosis of lung cancers; however, they exhibit low sensitivity and specificity.+, Cl-, carcino-embryonic antigen (CEA), and neuron specific enolase (NSE) in 145 small cell lung cancer (SCLC) patients and 155 non-small cell lung cancer and 155 normal controls. A gene expression programming (GEP) model and Receiver Operating Characteristic (ROC) curves incorporating these biomarkers was developed for the auxiliary diagnosis of SCLC.We used biochemical methods to measure blood levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), NaAfter appropriate modification of the parameters, the GEP model was initially set up based on a training set of 115 SCLC patients and 125 normal controls for GEP model generation. Then the GEP was applied to the remaining 60 subjects (the test set) for model validation. GEP successfully discriminated 281 out of 300 cases, showing a correct classification rate for lung cancer patients of 93.75% (225/240) and 93.33% (56/60) for the training and test sets, respectively. Another GEP model incorporating four biomarkers, including CEA, NSE, LDH, and CRP, exhibited slightly lower detection sensitivity than the GEP model, including six biomarkers. We repeat the models on artificial neural network (ANN), and our results showed that the accuracy of GEP models were higher than that in ANN. GEP model incorporating six serum biomarkers performed by NSCLC patients and normal controls showed low accuracy than SCLC patients and was enough to prove that the GEP model is suitable for the SCLC patients.We have developed a GEP model with high sensitivity and specificity for the auxiliary diagnosis of SCLC. This GEP model has the potential for the wide use for detection of SCLC in less developed regions. Lung cancer is a major cause of cancer death worldwide, representing about 12.7% (1.6 million cases) of all new cancer cases each year and 18.2% (1.4 million deaths) of all cancer deaths. Small c+, Cl-, which can be obtained by common biochemical detection method and don’t need exorbitant agentias or facilities. In a rural and impoverished area, using the approach, a fundamental serum test could warn people who are at higher risk of suffering from cancer and to do an indepth health examination such as CT, PET-CT and so on.Many different techniques have been used in the detection of lung cancers, including Chest Radiograph (x-ray), Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Sputum Cytology, and bronchoscopy. In receTherefore, new technology is urgently needed to find the association information between a large set of biomarkers and for the early detection of lung cancer. In recent years, with the development of science and technology, computer-aided design has become an auxiliary tool for the diagnosis of human cancers. Nowadays, machine learning methods, such as artificial neural networks (ANNs), decision trees, the naive bayesian (NB) algorithm, and support vector machines (SVM) have been utilized in the diagnosis and prognosis prediction of cancers. For insIn this study, we developed a prediction model using the GEP method to improve the diagnostic efficacy of SCLC. A number of biomarkers have previously been demonstrated to be useful for lung cancer diagnosis. Our GEP model suggested a novel multi-analysis of serum biomarkers for the early detection of SCLC.In total 430 cases, including 145 SCLC patients, 130 non-small cell lung cancer (NSCLC) patients and 155 non-cancer controls, were enrolled from the Affiliated Hospital of Qingdao University between July of 2006 and May of 2013. The diagnosis of 145 SCLC patients was based on biopsy and histopathology, and they were proven to be untreated primary lung cancers , the 130The SCLC group included 94 male and 51 female patients, aged between 33 and 78 years old. The control group was composed of 155 non-cancer cases, which underwent examinations proving their health . The NSCLC patients  were included in the negative control to show the difference from SCLC, we selected 130 cases from 155 non-cancer cases as the healthy control. Research approval was obtained from the corresponding ethics committee and written informed consent was obtained from all participants. Samples and health information were labeled using unique identifiers to protect subject confidentiality Tables .We selected six biomarkers that are closely related to lung cancers, especially to SCLC, and that have been widely used in the screening of SCLC. The indexes we chose have been incorporated into the GEP model. Based on previous clinical examination, the serum levels of LDH and CRP in SCLC patients are significantly higher than in healthy controls, but the serum level of sodium and chloride are significantly lower than that in normal controls. The serum level of LDH, which is commonly elevated in neoplastic disorders, has been suggested as a powerful tumor marker for many years. Therefore, these markers have significant meaning in SCLC. For example, lung cancer patients, especially SCLC patients, the Syndrome of Inappropriate Anti Diuretic Hormone secretion (SIADH) is considered to be the leading cause for hyponatraemia and hypochloraemia and can be induced by comorbidity such as lung cancer. Also, the major osmotic active substances that in the extracellular fluid principal contain serum sodium and its accompanying anions chloride31]. The. The31].+, and Cl- were measured by polyacrylamide gel electrophoresis (PAGE), immunoturbidimetry (ITM), and ion selective electrode methods, respectively, using the Hitachi 7600–020 automatic biochemical analyzer . Results were presented as mean values of duplicates after the subtraction of background values. The normal critical values of LDH (99–245 u/l), CRP (0-3mg/l), Na+ , CEA (0–3.4 ng/ml), and NSE (0-17ng/ml) were used as standards.Blood (10 ml) was collected in serum separator tubes, processed immediately, and separated by centrifugation at 3,000 rpm at room temperature for 10 minutes. The separated serum was then aliquoted and stored at -80°C for the measurement of the six biomarkers mentioned above. CEA and NSE were determined by electro-chemiluminescence immunoassay (ECLIA), using the Roche E601 chemical luminescence immunity analyzer with the auxiliary reagent kit . LDH, CRP, NaGEP is an evolutionary algorithm introduced by Ferreira in 2001. It can The algorithm begins with the random creation of the chromosomes in the initial population. Then the chromosomes are expressed and the fitness of each individual is evaluated. According to fitness, reproduction with modification is made, the individuals are then selected and the results lead to new traits. Additionally, the individuals of this new generation are subjected to the same developmental process: expression of the genomes, confrontation of the selection environment, and reproduction with modification. It is repeated for a certain number of generations until a satisfying solution has been found. It is important that the individuals are selected and copied into the next generation according to the fitness by roulette wheel sampling with elitism. This guarantees the survival and cloning of the best individual to the next generation. Each GEP gene contains a list of symbols with a fixed length that can be any element from a function set :The optimum fitness is:Artificial Neural Networks (ANNs) that has the ability of classification is a mathematical model, which original designed to imitate human neural system. Multiple neurons interconnect to each other and arranged in to a wiring layer. ANNs use complicated layers  to deal input and output, the input where each neuron represents an independent variable. ANNs contain a series of different architectures including Multilayer Perceptron (MLP) and Radial Basis Function (RBF) 39]. MLP. MLP39].P values < 0.05 were considered to be statistically significant.Statistical analyses were performed using SPSS 16.0. Differences between groups were calculated by means of a nonparametric Wilcoxon test (Mann–Whitney U test), independent-Samples T Test and chi-square test. The Receiver Operating Characteristic (ROC) curves were used to describe sensitivity of biomarkers, alone and combined, which were graphed by “R programming project 2.15–1”. Using ANNs to compare the detection capability, we can ascertain the optimal algorithm.Research approval was obtained from the Ethics Committee of Qingdao University Medical College and written informed consent was obtained from all participants. The study were followed by the STARD (Standards for Reporting of Diagnostic Accuracy) checklist to improve the accuracy and completeness of reporting of studies of diagnostic accuracy.p <0.001), whereas the concentrations of Na+ and Cl- were significantly lower than in normal controls (p <0.001)  . There a <0.001) . The cor <0.001) .+ and Cl- are meaningful in the detection of lung cancer patients and controls. Model 1 has united all the six biomarkers and model 2 has conjoined four biomarkers that remove serum sodium and serum chloride. The striking difference of the performance in model 1 and model 2 was graphed in The ROC curves to discover the sensitivity/specificity in each biomarker were determined by comparison with the area under the curve, we found the result in serum sodium and serum chloride were lower than any other biomarkers , then buA software known as “Automatic Problem Solver 3.0” was used to run the algorithm. The GEP modeling randomly selected four of five partitions as a training set (240 subjects) for model generation, including 115 SCLC patients and 125 normal controls. Next, the GEP parameters were modified to test the remaining 60 subjects for model validation. The concentration of six biomarkers was input into the GEP model to calculate its detection sensitivity and specificity for the discrimination of SCLC and normal controls. GEP model 1 used all six biomarkers as inputs and the algorithm was:0, x1, x2, x3, x4, and x5 represented the biomarkers LDH, CRP, Na+, Cl-, CEA, and NSE, respectively.If the calculated value of “y” equal to or greater than the rounding threshold, then the record is classified as "1", "0" otherwise. The variables x+, Cl-, CEA, and NSE were used as inputs in model 1. The general experiment setup was summarized in Patients suffered from lung cancer were marked as class “1”, while the healthy subjects were marked as class “0”. The serum concentrations of LDH, CRP, NaWhile the performance of model 1 with 6 biomarkers was good, we wanted to ascertain whether the numbers of biomarkers could be decreased to only four, which could significantly reduce the cost and time for SCLC screening. In model 2, we only chose the markers that were widely used in the detection of lung cancer, including LDH, CRP, CEA, and NSE, with the same function set described above.The algorithm of GEP model 2 was:0, x1, x2, and x3 were biomarkers LDH, CRP, CEA, and NSE, respectively.If the calculated value of “y” equal to or greater than the rounding threshold, then the record is classified as "1", "0" otherwise. In this model, variables xThe accuracy of GEP model 2 was 91.66% and the sensitivity was 86.67% in the test set, which was lower than that in model 1 . All traIn order to compare the classification power between GEP and ANN, IBM SPSS Statistics 18.0 was applied to build ANNs (MLP and RBF models) prediction models. The model1 and model2 were as same to GEP. SCLC patients and controls (0 or 1) were input as a dependent variable as GEP models. Using model 1, MLP indicated accuracy of 85.4%, 80.0% and in RBF acquired an accuracy of 80.0%, 78.3% for training and test phase, respectively. In addition, in model 2 the correct classification rate for MLP represented the identification of 83.3% and 83.3% and for RBF was for 84.2%, 83.3% among training and testing stages, respectively. The software have been ran three times and covariant was different arrange to select the best  Fig 5)..Fig 5).Compared to ANNs, the GEP algorithm proves the supreme predictive rate which has significant strengths. The ROC curve and GEP model showed that the model 1 is the adequate combination to distinguish lung cancer patients from high-risk people.The optimal GEP model 1 was used to make a comparison between early and late SCLC (74 limited stage and 71 extensive stage). We selected 74 cases from the 155 non-cancer cases as the healthy control. Firstly, in order to explore the early SCLC, as the above method GEP model randomly selected four of five partitions as a training set (118 subjects) for model generation, including 59 early SCLC patients and 59 normal controls. Remaining 30 cases  were for model validation. It can be observed that the early SCLC acquired the accuracy of 92.37% (109/118) and 90% (27/30) for training and test set, respectively. Secondly, for late SCLC, 116 subjects  for model generation and 29 cases for model validation, it represented the accuracy of 96.52% (112/116), 91.30% (27/29) for training and test set, respectively. The results showed that the accuracy of late SCLC in GEP model 1 was performed better than early SCLC and total 145 SCLC, but the early SCLC accuracy was close to the result of 145 SCLC, it was still a good performance  S4 Data.To confirm the GEP model 1 test, NSCLC patients have been included in the negative control with healthy subjects. As the above method, GEP randomly selected 208 subjects  for model generation, 52 subjects  for model validation respectively. It indicated that the accuracy of 87.5% (182/208), 86.5% (45/52) for training and test set, respectively. Meanwhile, the results were significantly worse than SCLC patients and were enough to prove that the GEP model is suitable for the SCLC patients  S2 Data.SCLC accounts for approximately 13–18% of all lung cancers, with diverse incidences in different countries. WithoutVarious methods have been used for the detection of SCLC, such as thoracic radiography, sputum cytology, and CT. The efficacy of these tools has been evaluated in clinical trials and it turns out that thoracic radiography and sputum cytology have low sensitivity for early-stage detection of SCLC ,47. Alth+, Cl- and other two tumor biomarkers were selected based on previous studies to establish the GEP model for the detection of SCLC. These biomarkers can be easily tested, even in developing regions, using two kits. For example, LDH and CRP, two important inflammation markers, are routinely tested in most hospitals in China, let alone electrolyte solution Na+, Cl-.Biological markers can be easily detected in biological fluids using minimally invasive procedures, which can significantly enhance the detection rate of a number of human cancers. Several tumor markers, such as ɑ-fetoprotein (AFP), prostate specific antigen (PSA), and cancer antigen125 (CA125), have been proven to be highly sensitive and effective for the screening of liver, prostate, and ovarian cancers . Each biet al.[et al.[+ and Cl- is lower than others and the six biomarkers emerged the best. Then in GEP model 1, incorporating six biomarkers, successfully distinguished 281 of 300 tested samples with an accuracy of 93.75% (225/240) and 93.33% (56/60) for the training and test sets, respectively. Model 2, including four biomarkers, had slightly lower accuracies of 93.75% and 91.67% for the training and test sets, respectively. To confirm the excellent result in GEP, we repeated the models on ANN, Our results exhibited that the accuracy of GEP models performed higher than that in ANNs. The six biomarkers combined in MLP indicated a standout result that seem as to in GEP. Therefore, when compare the detection capability of ROC curve, GEP and ANN, GEP was proved to be the best algorithm which depend on model 1, otherwise, GEP model 2 with four biomarkers may be more suitable for screening SCLC in regions with extremely low incomes.A previous study conducted by Flores, et al.,53,15 inl.[et al. reached l.[et al.. Accordil.[et al.. NeverthTo confirm the GEP model 1 test, NSCLC patients have been included in the negative control with healthy subjects, the results were significantly worse than SCLC patients, also, there are significant differences in the concentrations of LDH, Na, Cl and NSE between SCLC and NSCLC. The results were enough to prove that the GEP model is suitable for the SCLC patients. Furthermore, The optimal GEP model 1 was used to make a comparison between early and late SCLC patients, the early SCLC model 1 acquired the accuracy of 92.37% (109/118) and 90% (27/30) for training and test set. It was close to total data accuracy. Meanwhile, the late SCLC model 1 performed well than early stage which represented the accuracy of 96.52% (112/116), 91.30% (27/29) for training and test set, respectively. The particular poor physical condition of the advanced patients may explain it. Generally, the accuracy of early stage was still good and GEP model 1 can be the optimal test for SCLC early detection.In clinical examination, the serum CRP and LDH levels in SCLC patients are significantly higher than healthy people, but the serum sodium is much lower. The clinical significance of serum level of LDH has been proven to be a strong and independent predictive factor of median survival, both in limited and extensive disease stages of SCLC. In addi+ and Cl-, as well as LDH, CRP, CEA, and NSE, had a slightly better performance for the detection of SCLC than model 2. GEP model 2 did not affect the detection accuracy by much, perhaps because paraneoplastic syndrome caused by SCLC is not common in clinical settings. However, GEP model 2 might be improved by adding more samples, new subjects, or keeping the normalizing criteria to train the model. In addition, clinical information, such as other biomarkers, nodules, and hemoptysis, etcetera, could also be included to improve the GEP performance.It has been reported that the syndrome of inappropriate antidiuretic hormone (SIADH) is the leading cause of hyponatremia and hypochloremia in hospitalized patients. Malignancy pulmonary diseases, particularly SCLC, usually lead to SIADH. TherefoIn summary, we developed an effective GEP model incorporating six biomarkers to screen SCLC patients. This model and measurements of six biomarkers are convenient, economical, and can be widely used in less developed area. However, this model should be further tested and improved with more SCLC patients in different hospitals and regions. With the emergence of new predictive tumor markers, we are no longer going to select several determinate tumor markers joint detection, but going to obtain larger sample size, higher amount of information and larger scale on gene and protein levels. Moreover, due to the intricate parameter selection, the parameters in GEP algorithm may be optimized in the later research. Also, those serology tests couldn't replace lung biopsy to confirm a diagnosis, rather they serve as a good screening tool to auxiliary diagnosis. In addition, the economic cost of this GEP model needs to be comprehensively evaluated before it is widely applied in the clinical screening for SCLC.S1 Dataset(XLS)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.


Low birth weight and unhealthy lifestyles in adulthood have been independently associated with an elevated risk of hypertension. However, no study has examined the joint effects of these factors on incidence of hypertension.We followed 52,114 women from the Nurses’ Health Study II without hypercholesterolemia, diabetes, cardiovascular disease, cancer, prehypertension, and hypertension at baseline (1991–2011). Women born preterm, of a multiple pregnancy, or who were missing birth weight data were excluded. Unhealthy adulthood lifestyle was defined by compiling status scores of body mass index, physical activity, alcohol consumption, the Dietary Approaches to Stop Hypertension diet, and the use of non-narcotic analgesics.Pinteraction <0.001). The proportions of the association attributable to lower term birth weight alone, unhealthy lifestyle alone, and their joint effect were 23.9 % , 63.7 % , and 12.5 % , respectively. The population-attributable-risk for the combined adulthood unhealthy lifestyle and low birth weight at term was 66.3 % .We documented 12,588 incident cases of hypertension during 20 years of follow-up. The risk of hypertension associated with a combination of low birth weight at term and unhealthy lifestyle factors  was more than the addition of the risk associated with each individual factor, indicating a significant interaction on an additive scale (The majority of cases of hypertension could be prevented by the adoption of a healthier lifestyle, though some cases may depend on simultaneous improvement of both prenatal and postnatal factors. Hypertension affects one third of American adults  and is tHowever, no previous study has examined the joint associations of birth weight and adulthood lifestyle with hypertension risk, or evaluated their potential interactions. In this study, we prospectively assessed the joint association of birth weight at term – a marker of fetal growth restriction – and established lifestyle risk factors in adulthood with incident hypertension in the Nurses’ Health Study (NHS) II .In 1989, 116,430 female registered nurses aged 25 to 42 years enrolled in NHS II by completing and returning an initial questionnaire that provided detailed information on medical history, lifestyle, and medications. The dietary survey was initiated using a food frequency questionnaire (FFQ) in 1991, which served as the baseline of the present analysis. Detailed information on lifestyle habits and medical history was updated biennially; FFQ was updated every 4 years. The follow-up for the cohort exceeded 90 % of eligible person-time.2) as those with relative information.In 1991, 100,090 participants returned the questionnaire including a FFQ. For the current analysis, we excluded participants (1) who reported diagnosis of hypertension, ever use of antihypertensive medication, or who reported median systolic blood pressure in the prehypertensive range, greater than 120 mmHg or diastolic blood pressure greater than 80 mm Hg, at or before baseline of the current analysis ; (2) who reported physician diagnosis of other chronic conditions, including hypercholesterolemia, diabetes, cardiovascular disease, and cancer at baseline ; (3) who had missing data on dietary, physical activity, alcohol consumption, use of non-narcotic analgesics, or body weight at baseline ; and (4) who were born preterm , did not provide birth weight data , or who were born of a multiple pregnancy (n = 687). After these exclusions, 52,114 women were included in the analysis. Participants who did not report birth weight or missed lifestyle factors had similar age (mean 36.3 ± 4.7 vs 36.0 ± 4.7 years) and body mass index  , unhealthy lifestyle alone as [(RRg0e1 – 1)/ (RRg1e1 – 1)], and to their interaction using the equations as [RERI/(RRg1e1 – 1)], the detail information on the equation has been previously published [We further proceeded with the decomposition of the joint effect, e.g. the proportions attributable to a lower term birth weight alone as [ using the standard equation  as,\docuThe estimated PAR% was the percentage of incident hypertension in the study population that theoretically would not have occurred if all people had been in the low-risk group, combining a healthy birth weight at term and a healthy lifestyle, assuming a causal relation between the risk factors and hypertension.P <0.05.Data were analyzed using a commercially available software program , and statistical significance was set at a two-tailed Table We documented 12,588 new cases of hypertension during the 20 years of follow-up. We observed a consistent, graded inverse association between birth weight at term and risk of hypertension . The graded decreasing risk of hypertension with increasing of birth weight at term appeared consistent across all levels of unhealthy lifestyle factors  per 1-kg lower birth weight at term and 1.61  per 1-point higher unhealthy lifestyle score. In addition, we observed that the risk of hypertension associated with a combination of low birth weight at term and unhealthy lifestyle factors  was more than addition of the risk associated with each individual factor, indicating a significant interaction on an additive scale  for risk of hypertension. The PAR% for not being in this group was 66.3 %, indicating 66 % of the new cases of hypertension in our cohort could have potentially been prevented if they had all five healthy lifestyle factors combined with a birth weight at term 2.5–4.49 kg (Table P for additive interaction <0.0001). When we applied the expanded low-risk lifestyle score, the RRs of hypertension were 1.28  per 1-kg lower birth weight at term, 1.46  per 1-point higher unhealthy lifestyle score (5- expanded low-risk lifestyle score), and 1.84  for their joint effect, with a RERI of 0.10 . We also performed analysis of the main research question based on the cross-sectional data at baseline including all participants without missing data of birth weight and lifestyle factors. The odds ratios of hypertension were 1.09  per 1-kg lower birth weight at term, 1.62  per 1-point higher unhealthy lifestyle score, and 1.78  for their joint effect, with a RERI of 0.07 .We performed several sensitivity analyses. In order to examine potential confounding of socioeconomic status (SES), we added self-ranking of their standing in US society, including their money, education, and jobs, as a surrogate of the SES to the multivariable-adjusted model. The SES adjusted RRs of hypertension were 1.22  per 1-kg lower birth weight at term, 1.59  per 1-point higher unhealthy lifestyle score, and 1.93  for their joint effect, with a RERI of 0.12 (95 % CI, 0.09–0.14; After 20 years of follow-up of a large population of initially hypertension-free young women, we observed that the risk of hypertension associated with a combination of low birth weight at term and unhealthy lifestyle factors was more than the addition of the risk associated with each of these factors, indicating a significant interaction on an additive scale. Our data indicate that combination of a healthy birth weight and a healthy adulthood lifestyle could prevent 66 % of the cases of hypertension in this population.It is hypothesized that restricted fetal growth reflects maternal vascular stress inherited by her children, and which may also interact with adulthood lifestyle , 29. EveIt is widely accepted that hypertension can be prevented by lifestyle modifications. The National High Blood Pressure Education Program Coordinating Committee published its first statement on the primary prevention of hypertension in 1993  and updaOur study also indicates that 12 % of hypertension cases may only occur if both unhealthy birth weight and unhealthy lifestyle were present, not if only one or the other is present . This fiA major strength of the present study is decomposition of the joint effect of birth weight and adulthood lifestyle factors. Our study, for the first time, quantitatively estimated the joint effects of prenatal and adulthood risk factors on risk of hypertension. Other strengths of the present study include the large number of incident hypertension cases, long-term follow-up, and repeated measurement of lifestyle factors during the 20 years of follow-up.Our study has several limitations. First, our cohorts included mostly Caucasian women and the PAR was population-specific, which limited the generalizability to men or other ethnic groups of women. However, the relative homogeneity of the study populations in educational attainment and SES enhances the internal validity. The prevalence of low-risk factors in the NHS II women is much higher than that among Black and Hispanic women , while tIn conclusion, our findings suggest that the effects of fetal growth restriction and unhealthy lifestyle on the risk of hypertension are greater than additive. Though some cases of hypertension may only be prevented by simultaneous improvement of both prenatal and postnatal factors, the majority of cases of hypertension could be prevented by the adoption of a healthier lifestyle.


Mondini dysplasia, also known as Mondini malformation, is a developmental abnormality of the inner and middle ears that can cause hearing loss, cerebrospinal fluid (CSF) leakage, and recurrent bacterial meningitis (RBM), which is defined as two or more episodes of meningitis separated by a period of convalescence and the complete resolution of all signs and symptoms. An accurate diagnosis of the underlying pathology is crucial to prevent further episodes from occurring. Herein, we present a three-year-old boy with RBM and unilateral sensorineural hearing loss. During the evaluation to determine the etiology of the RBM, cystic malformation in the cochlea and vestibular dilatation consistent with Mondini dysplasia were detected via computerized tomography (CT) of the temporal bone. Recurrent bacterial meningitis (RBM) is defined as two or more episodes of meningitis that are separated by a period of convalescence and the complete resolution of all signs, symptoms, and laboratory findings –3. The mMondini dysplasia, one of the more common congenital inner ear malformations, is thought to result from a developmental arrest of the interscalar septum in which the cochlea characteristically consists of one and a half turns instead of two and a half , 6, 7. I3, an absolute neutrophil count of 48.000/mm3, a platelet count of 413.000/mm3, and a C-reactive protein (CRP) level of 214 mg/L . Furthermore, the results of the CSF examination showed 200 polymorphonuclear leukocytes/mm3, an elevated protein concentration of 214 mg/dL, and a low glucose level of 1 mg/dL along with a simultaneous blood glucose level of 126 mg/dL. However, the CSF pyogenic culture and blood cultures remained sterile, and his cranial magnetic resonance imaging (MRI) was also normal. Ceftriaxone and vancomycin were administered for 14 days because his meningitis had only been partially treated with antibiotics before his admission. He was then discharged after full clinical and laboratory improvement.A three-year-old boy presented to the emergency room with fever and decreased level of consciousness. Two days earlier, he had been admitted to another hospital with a presumed diagnosis of enteritis, and ceftriaxone therapy had been administered. A physical examination revealed fever, weakness, and nuchal rigidity. His medical history showed that he had been vaccinated four times with 13-valent pneumococcal conjugate vaccine (PCV-13) and that his father had been treated for meningitis when he was seven years old. A laboratory investigation revealed the following: a peripheric blood leukocyte count of 52.000/mm3, an elevated protein level of 209 mg/dL, and a low glucose level of 0 mg/dL. Additionally, his white blood cell (WBC) count was 27.200/mm3, and he had an absolute neutrophil count of 23.500/mm3 along with a CRP level of 91 mg/L. Moreover, the CSF polymerase chain reaction (PCR) test for M. tuberculosis was negative. Treatment with ceftriaxone and vancomycin was then restarted. The CSF culture revealed Streptococcus pneumoniae (serotype 19F) which was susceptible to both ceftriaxone and vancomycin, and a repeated lumbar puncture showed a cell count of 10 leukocytes/mm3, a CSF protein concentration of 41 mg/dL, a CSF glucose level of 35 mg/dL, and a simultaneous blood glucose level of 85 mg/dL, but the CSF pyogenic culture was sterile. Hence, the antibiotic therapy was given to the patient for four weeks.Ten days after being discharged, the patient was readmitted with fever and neck stiffness, and he underwent another CSF examination that revealed 10 leukocytes/mmCranial and temporal bone imagings were performed to determine the predisposing conditions for RBM, and temporal bone CT showed decreased aeration in the right middle ear and mastoid cells, a dysmorphic structure of the semicircular canals, vestibular dilatation, and the cystic appearance of the cochlea, all of which are consistent with Mondini dysplasia Figures  and 2. FSurgical repairment of the dysplastic inner ear was planned, but it could not be performed because of parental refusal.Bacterial meningitis is a life-threatening disease with high mortality and morbidity. Recurrent bacterial meningitis is rare, occurring in just 1% of all bacterial meningitis cases, and it poses a diagnostic challenge . Early dAnatomical abnormalities such as spinal defects or abnormalities in the anterior fossa or temporal bone represent the most common cause of recurrent meningitis , 8. Mond S. Pneumoniae [ S. Pneumoniae, which is the infectious agent in more than 50% of patients with RBM [ Neisseria meningitidis is the second most common agent, but it is associated more with immunological abnormalities [Communication between the middle ear and CSF spaces may result in bacterial translocation, with the most common infecting organism beingeumoniae –8, 10. Imalities .In RBM, imaging studies should include CT of the cranium, paranasal sinuses, and temporal bones, and multiple coronal views must be obtained because small defects can be missed in axial views , 11, 12.Treatment options in these cases include the surgical repair of the defect to prevent recurrent meningitis, amplification aids for those with residual hearing, and cochlear implants , 14. WheIn conclusion, we recommend CT of the temporal bones in patients with recurrent meningitis and hearing loss to assess whether or not Mondini dysplasia is present.


N. meningitidis, identified according to the capsular polysaccharide's antigenic structure, only 5  are clinically relevant and responsible for 90% cases of meningococcal invasive disease. In Italy, serogroup B is the principal cause of invasive meningococcal disease [Between 13 different serogroups of There are two types of tetravalent vaccines for serogroups A, C, W-135 and Y, represented by polysaccharide and conjugate vaccines. However, only recently it has been possible to develop a vaccine against meningitis B with the “Reverse vaccinology” technique: after identifying more than 600 proteins as immunological potential targets, 91 have been selected (expressed on the outer capsule). Only 28 of them could induce a bactericidal activity and, among these, 3 have been selected: fHbp, NHBA and NadA, which were able to stimulate an antibody protection, in addition to proteins OMV (4CMenB)[In November 2012, the multicomponent vaccine 4CMenB was approved from the European Medicines Agency (EMA). In January 2013, the European Commission authorized the marketing of the new vaccine 4CMenB (Bexsero®), addressed to immunization from two months of age [In Italy this authorization is implemented by AIFA determines of May 27, 2013.Moreover, in 2014 in the US, a new anti-meningococcal bivalent vaccine was approved: Trumenba® (Pfizer). Currently, 4CMenB has been authorized in 12 countries worldwide, the largest of which are Australia and Canada. Already 151,800 subjects have received at least one dose of vaccine in 18 countries worldwide.In Italy, Basilicata has been the first region that implemented 4CMenB in its vaccination schedule, with resolution in 24 February 2014. A few months later also Puglia, Liguria, Tuscany, Veneto, Sicily, Friuli, Bolzano and Calabria have introduced the meningococcal B vaccine, and other regions are ready to follow them.In the new Italian vaccination calendar, proposed by SLTL, FIMP and SIP, 4CMenB has been prepared in a 4-doses schedule, in consideration of the higher incidence of the infection among the first 4-6 months of life  [


The suprachiasmatic nuclei (SCN) are pointed to as the mammals central circadian pacemaker. Aged animals show internal time disruption possibly caused by morphological and neurochemical changes in SCN components. Some studies reported changes of neuronal cells and neuroglia in the SCN of rats and nonhuman primates during aging. The effects of senescence on morphological aspects in SCN are important for understanding some alterations in biological rhythms expression. Therefore, our aim was to perform a comparative study of the morphological aspects of SCN in adult and aged female marmoset. Morphometric analysis of SCN was performed using Nissl staining, NeuN-IR, GFAP-IR, and CB-IR. A significant decrease in the SCN cells staining with Nissl, NeuN, and CB were observed in aged female marmosets compared to adults, while a significant increase in glial cells was found in aged marmosets, thus suggesting compensatory process due to neuronal loss evoked by aging. The suprachiasmatic nuclei (SCN) are a pair of neurons located in the anterior hypothalamus above the optic chiasma, , 2 and iIn humans, many factors attempt to explain the aging process; among them, mitochondrial dysfunction  and oxidDuring aging, changes in morphological, neurochemical, and circadian rhythms can be observed . The eff post mortem studies in human beings showed that neurodegenerative processes occur in the SCN during senescence, suggesting progressive deterioration of the circadian pacemaker [In mammals, changes in basic parameters of circadian rhythms have been showed, such as alterations in length period, besides reduction of amplitude and phase duration , 23. Theacemaker , 28. Callithrix jacchus a new world primate, the number of neurons in the SCN in aged female. To this aim, a morphometric analysis using Nissl staining and the immunoreactivity of SCN cells to NeuN, CB, and glial fibrillary acidic protein  has been widely used as a neuronal marker, while, in marmosets, CB is used as an important cell marker of the SCN , 29, in trocytes ) were peFour young adult female marmosets and four aged female marmosets , pH 7.4, containing 500 IU heparin , followed by 700 mL 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were removed from the skull, postfixed in the same fixative solution for 2–4 h, and transferred to a solution containing 30% sucrose in 0.1 M PBS, pH 7.4. Each brain was serially cut in the coronal plane into 30 mm thick sections with a freezing sliding microtome. The sections were placed sequentially in six compartments (one section per compartment), with the distance between one section and the next in the same compartment being approximately 180 ails see ).2O2 in 0.1 M phosphate buffer, pH 7.4. The sections were washed  with 0.1 M phosphate buffer, pH 7.4, between each step and at the end of the procedure. The sections were then dried, dehydrated in a graded alcohol series, cleared in xylene, and coverslipped with neutral mounting medium . Sections in which the primary antibodies were omitted and replaced with normal serum from the same species served as controls. Under these conditions, staining was completely abolished. All the immunostainings, for specific substance, were performed concomitantly, minimizing possible differences in background between the animals.For immunostaining, free-floating sections of the one compartment were washed in phosphate buffer and incubated with the primary antibodies to NeuN , CB , and GFAP  for 18–24 h at room temperature, containing 2% normal donkey serum in 0.3% Triton X-100 and 0.1 M phosphate buffer, pH 7.4. Next, the sections were incubated with the biotinylated secondary antibody , for 90 min, followed by incubation with the avidin-biotin peroxidase solution  for 90 min. The reaction was developed by the addition of diaminobenzidine tetrahydrochloride  and 0.01% HThe sections were examined under bright field illumination on a light microscope (Olympus BX-41) and images were captured using a CCD camera (Nikon DXM-1200).In order to estimate the number of SCN cells, six images (objective 40x) of each animal, aged and adult, were selected: one at the rostral level, three at medium level, and two at caudal level, representative of the rostrocaudal extension of area of interest. For all solutions used as cell markers, we use only the right side of each hemisphere to the cell count. A rectangle measuring 125 um × 100 um, corresponding to 52% of the total area of the SCN, was extracted of every image and the number of cells found in this area was counted. All images (adults and aged) were randomly renamed so that the counter did not know which animal belonged to the image, featuring a blind count. Thus, it minimized the suggestive effects of knowing the sample. Some histological characteristics presented by neurons in the thionine dye were used as selection criteria in the cell counting, for example, rounded shape, strong staining, and evident nucleolus.Additionally, GFAP levels in the SCN were assessed by analyzing relative optical densitometry (ROD) of images using the software Image J . Six representative sections of the rostrocaudal extension of SCN were chosen. In each section, fields bypassing throughout the SCN were analyzed. The medium pixels in the target area were subtracted from de medium values of a control region (areas that should not have specific GFAP staining) of the same tissue (optic chiasma). Finally, all values were normalized considering the control group, in order to evaluate proportional alterations.r). The level of significance was set at P < 0.05.To confirm whether there are differences between the animal groups, the nonparametric Mann-Whitney test was applied. Correlations between age of animals and morphometric values were performed using the Spearman test ; aged marmoset: 36.4 (43.68–30.25); data are represented as median (interquartile ranges q3–q1); P = 0.0209; r = 0.857; P = 0.010; Considering GFAP immunoreactivity in the SCN, Mann-Whitney test revealed a significant increase in GFAP expression in aged marmosets compared with adult animals (adult marmoset: 2745.8 (4737.7–1339.1); aged marmoset: 12880.1  CB is shown as an excellent marker of SCN cells in marmosets , 11. CB ffering” . In our ffering”  and primffering” . Moreoveffering” , 52. Arvicanthis [Microcebus murinus), a nonhuman primate also, there is found a CB marking mainly concentrated in the central region of the nucleus [The labeling pattern of CB in the SCN cells of adult marmosets is characterized by nucleus almost completely stained , thus reicanthis . However nucleus . In aged2+ seems to confer protection against agents that promote cell death [2+ levels [The ability of CB binds to the Call death ; for exa+ levels . TherefoWu et al.  in a stuLesions of CB-IR cells in the SCN lead to a failure in the circadian rhythm regulation of body temperature, heart rate, melatonin, and cortisol, suggesting that CB is required to support circadian rhythm functioning . In a st2+, favoring neuronal death.We suggest that the loss associated with the CB-IR neurons in SCN of aged marmosets may increase the vulnerability of these neurons to degenerative processes that occur in aging, such as those involving increased intracellular CaROD analysis showed a significative increase of GFAP expression in SCN of aged marmosets compared to adults. The increase of glial expression in response to neuronal decrease is a recognized pathophysiological reaction, which is mainly seen in neurodegenerative diseases, such as Alzheimer's disease and other neurological disorders .Measurements of gliogenesis rate have been used to estimate the regional overproduction of glia in response to regional decrease of neurons in a variety of disease conditions, as well as during normal aging , 62. It A morphometric analysis of SCN in aged female marmosets compared with adult animals showed a significant decrease in the number of neurons stained with Nissl and labeled immunohistochemically for NeuN, a specific marker for neurons. Other two protein markers, CB which is used as an excellent marker for SCN cells in marmoset, and GFAP were observed. This neuronal decline in aged animals may be responsible for changes seen in the pattern of expression of biological rhythms in animals during the aging process.