Background: Computational modeling of the absorption, distribution, metabolism, and excretion of chemicals is now theoretically able to describe metabolic interactions in realistic mixtures of tens to hundreds of substances. That framework awaits validation.a) evaluate the conditions of application of such a framework, b) confront the predictions of a physiologically integrated model of benzene, toluene, ethylbenzene, and m-xylene (BTEX) interactions with observed kinetics data on these substances in mixtures and, c) assess whether improving the mechanistic description has the potential to lead to better predictions of interactions.Objectives: Our objectives were to Methods: We developed three joint models of BTEX toxicokinetics and metabolism and calibrated them using Markov chain Monte Carlo simulations and single-substance exposure data. We then checked their predictive capabilities for metabolic interactions by comparison with mixture kinetic data.Results: The simplest joint model (BTEX interacting competitively for cytochrome P450 2E1 access) gives qualitatively correct and quantitatively acceptable predictions (with at most 50% deviations from the data). More complex models with two pathways or back-competition with metabolites have the potential to further improve predictions for BTEX mixtures.Conclusions: A systems biology approach to large-scale prediction of metabolic interactions is advantageous on several counts and technically feasible. However, ways to obtain the required parameters need to be further explored. For higher-order interactions, between triples, quadruples, and so forth, the number of assays grows with the cube, the quartic, and so on, of n. To avoid the curse of dimensionality, one can stay within the limits of four to five substances in the mixtures considered. The published modeling literature does not venture much beyond those bounds , for which we have good data. We built and calibrated a predictive interaction model using single-chemical exposure data to obtain enzymatic microconstant values for benzene, toluene, ethylbenzene, and m-xylene individually. We then used the model to predict metabolic interactions in BTEX mixtures. We compared the resulting predictions with experimental mixture data and we explored the sensitivity of the predictions with respect to model structure.To tackle mechanistically the question of realistic mixtures, we recently proposed a systems biology approach that, in its simplest form, addresses metabolic interactions , 2009c. PBPK model.iQ of a substance in liver, fat, and poorly perfused or well perfused tissues were of the formiF is blood flow through tissue i , iV is its volume, iP is the corresponding tissue/blood partition coefficient, and Cart is the arterial blood concentration. Concentration dynamics in venous blood (Cven), arterial blood (Cart), and exhaled air (Cexh) were described by algebraic equations:where Falv is alveolar ventilation rate, Cinh is the inhaled substance concentration in air, and Part is the blood/air partition coefficient. Baseline PBPK model parameters values are reported in where SBML models of metabolic reactions. For each chemical, the basic steps of its cytochrome P450 2E1 (CYP2E1)-catalyzed oxidation were coded in an SBML model  and the CYP2E1 concentration in the above equations.where m-xylene can be metabolized by a second, unspecified enzyme, CYPX. The corresponding differential equations have additional terms, similar to those in X enzymes were assumed to be constant over time.Two sets of alternative SBML models (type II and type III models) were also coded to explore the effect of more complex metabolism. In type II models , benzeneAutomatic code generation for global model. A C language file coding the complete set of differential equations describing the joint transport and metabolism of the four substances  was automatically generated from the individual SBML metabolic pathway files and compiled by GNU MCSim . The transport terms, based on the PBPK template presented above, were generated for each chemical species placed outside the liver cell compartment in SBML. Species placed in the liver  were not transported. Interactions implied by the competition for the same enzyme(s) are automatically taken into account; for example, in type I models, benzene, toluene, ethylbenzene, and m-xylene all bind competitively to CYP2E1  and the Michaelis constant (Km)  , 2001, wionships :Vmax and Km leaves one of the microconstants unspecified. Therefore, we obtained values for k1, k2, and k3 by Bayesian calibration  ] indicated that, besides k3, the volume of fat, alveolar ventilation rate, cardiac output, and blood/air partition coefficient were sensitive. These parameters were therefore treated as random variables and sampled. Given the similarity of the kinetics of benzene, toluene, ethylbenzene, and m-xylene, we fitted the same parameters for the other three BTEX components as well.but knowledge of ibration  of the gthe air) . We firsthe air)  to checkthe air) , 2. Parak1 and k2 were sampled from wide uniform distributions, with informative bounds given by k3 we used a narrower range because we had reasonable prior values for benzene, toluene, ethylbenzene, and m-xylene Vmax  simulations. The volume of fat, alveolar ventilation rate, cardiac output, and blood/air partition coefficient were sampled according to informative prior distributions . Parametene Vmax  and for x  computed from the arithmetic SD given for each data point by R diagnostic.To obtain samples from the joint posterior distribution of the model parameters, five Markov chains were run independently for each chemical. Their convergence was assessed using m-xylene administered together in various mixture combinations. We simulated the mixture dosing conditions of As a cross-validation, the last step was to perform posterior predictive simulations of the kinetics of benzene, toluene, ethylbenzene, and Type II and III model predictions. Those models are more complex and each require 12 additional reaction microconstants. We had no hope of identifying them with the data available and therefore set them to plausible values. For the parameters common to all three models , we used the posterior values of global model I  as for CYP2E1, but CYPX k1´ was set to 1/10 of CYP2E1 k1. CYPX therefore behaves as a lower affinity, lower capacity pathway than CYP2E1. The total quantity of CYPX in rat liver was set at 1.16 × 10–6 mmol, similar to that of CYP2B1 .Model calibration using individual benzene, toluene, ethylbenzene, or m-xylene exposures. Global model type I (see R diagnostic < 1.021 for any parameter). The following results were obtained using 10,000 joint posterior parameter samples. k1 and k2 rate constants (about 20–50% coefficient of variation). We used Km values corresponding to the sampled k1, k2, and k3 values and compared them with previously published values . pe I see , 2, 5 wape I see . Venous Model predictions and cross-validation for BTEX mixtures.m-xylene. On average, the data means and prediction medians differ by about 25%. We also performed predictions for binary 100 ppm toluene and 200 ppm m-xylene coexposures or ternary toluene, ethylbenzene, and m-xylene coexposures (100 ppm each) (http://dx.doi.org/10.1289/ehp.1103510)].pm each) . The resExploration of modeling alternatives. In order to check whether the model predictions would be altered significantly by the addition of a minor metabolic pathway or by the possibility of competition with the metabolites formed, we studied type II and type III global models (see http://dx.doi.org/10.1289/ehp.1103510)] and model and input files that can be used to perform new simulations with GNU MCSim are available at http://www.gnu.org/software/mcsim/supplement_EHP_2011.tar.gz.} Neither model gave a consistent improvement in predictions, but with adequate parameter values they could potentially lead to a refined prediction of interactions.dels see , 4. UsinWe used a modeling framework theoretically able to predict complex metabolic interactions among an unlimited number of drugs, environmental and workplace contaminants, and food-borne natural chemicals , 2009c. ik) for each pair of chemicals considered. That simplifies modeling considerably: to model any mixture, it is enough to draw the individual metabolic schemes of its constituents and write their joint equation system. The same metabolic pathways and parameters values can be reused for different mixtures. Finally, our approach is in the long run more parsimonious in terms of parameters. The Michaelis-Menten formulation requires two parameters per chemical and per reaction (Vmax and Km) and usually two ik for each pair of interacting substances. The number of ik parameters therefore grows with the square of the number n of substances in the mixture. Our approach needs only three microconstants  per chemical and per reaction, whatever the complexity of the mixture, and the number of parameters grows only linearly with it. With four substances, 20 parameters are classically “needed,” whereas our approach requires only 12. The corresponding figures for a mixture of 50 substances are 2,550 versus 150. A little used implication of the Michaelian competitive inhibition model is that the parameters ik are equal to the Km of each chemical . The Michaelis-Menten approximation also gives reasonable results in many cases when studying one or two substances. It is interesting to note that the question of interactions has been reduced in the drug industry to that of “drug–drug” interactions, even if that is a severe reduction of the whole question. Finally, experimentally determined ik do not need to describe a precise mechanism and can approximate a variety of them. In contrast, our approach requires a precise definition of the hypothesized interaction mechanisms.Yet, the use of fundamental constants has drawbacks. Although values for k1, k2, and k3 from whole-body kinetic data ].To demonstrate our approach, we confronted its predictions with well-studied data. The BTEX case, even if on a limited number of chemicals, is important and has been well studied , 1997. Idata see , Table 3ffusions . Their rffusions . In addiOverall, our results show that a microconstant-based model (model I) gives a correct qualitative picture of the interactions observed in BTEX mixtures. Quantitatively, about 50% of the interaction effects are predicted. We did not have sufficient information to precisely calibrate improved models of BTEX metabolism. We know, however, that such models surely exist. The initial steps of benzene metabolism involve CYP-dependent oxidation. CYP2E1 and to some extent CYP2B1 are likely to be the main enzymes for benzene metabolism at high levels of exposure . A seconA first-principles, systems biology, or mechanism-based approach for large-scale prediction of metabolic interactions is technically feasible and worth exploring. Generic PBPK models are an essential ingredient of it. The predictions made here for the BTEX case were basically correct, if not perfect. Better experimental or modeling ways to obtain kinetic microconstants should be explored. This agenda applies to systems biology as a whole, and tools are becoming available to calibrate cell-level models . The maj(315 KB) PDFClick here for additional data file.


Oxytrigona. Individuals of the species Oxytrigona cf. flaveola, members of a colony from Tangará da Serra, Mato Grosso State, Brazil, were studied. The chromosome number was 2n = 34, distributed among four chromosomal morphologies, with the karyotype formula 8m+8sm+16st+2t. Size heteromorphism in the first metacentric pair, subsequently confirmed by sequential staining with fluorochrome (DA/DAPI/CMA3 ), was apparent in all the examined individuals The nucleolar organizing regions (NORs) are possibly located in this metacentric chromosome pair. These data will contribute towards a better understanding of the genus Oxytrigona. Given that species in this group are threatened, the importance of their preservation and conservation can be shown in a sensible, concise fashion through studies such as this. The aim was to broaden knowledge on the cytogenetics of the subtribe Meliponina, by furnishing cytogenetic data as a contribution to the characterization of bees from the genus  The number of stingless bee species (subtribe Meliponina) found in the Neotropics is extremely high, with approximately 400 known to date . In BrazOxytrigona, have specific characteristics. Bees of this genus are commonly known as “cospe-fogo” (fire spitting), due to the peculiar characteristic of secreting a caustic liquid (formic acid) from the mandibular glands, thereby giving rise to severe burns in both animals and humans, while leaving permanent spots on the skin. Besides being highly aggressive, they are also cleptobiotic, as colony robbers of other meliponine species  was placed in evidence by the crushing technique, thereby revealing four morphological types of chromosomes, classified in decreasing order based on size , the cytogenetic study of bees as a whole, is rare , Mato Grosso State, Brazil, to be used for cytogenetic analysis. Voucher specimens are deposited in the Biology Laboratory of the Universidade do Estado de Mato Grosso, Tangará da Serra campus. The material used to obtain metaphase chromosomes was extracted from the cerebral ganglia of 20 post-defecating O. cf. flaveola larvae, according to the methodology developed by A colony of 3 - CMA3) (3 (CMA3) is an antibiotic with affinity for GC base pairs .Conventional staining was carried out with a solution of 1 mL of Giemsa, in 30 mL of Sörensen buffer 0.06 M (pH = 6.8) for 25 min at room temperature, followed by sequential staining with fluorochromes (4'-6-diamidino-2-phenylindole - DAPI and chromomycin A - CMA3) . 4'-6-dise pairs . Further® Plus, version 3.1, Media Cybernetics, 1998).Metaphase cells revealed by Giemsa and fluorochrome staining were captured by a CCD camera  connected to an Olympus TM BX60 microscope equipped with epifluorescence, with a WB filter (λ = 450-480 nm) and immersion objectives at 100x magnification. Graphs and karyograms were constructed using an image analysis program ; and (2) the large chromosome would be the ancestral condition, with the smaller originating from deletion of a portion of the former. Thus, cytogenetic analysis of other O. cf. flaveola colonies might provide relevant data to prove either of the two hypotheses.There are two hypotheses for explaining heteromorphism in pair one: (1) the small chromosome could be an ancestral condition, with the larger originating through 3 in pair 1 of O. cf. flaveola, thereby implying that this chromosome may contain sites of ribosomal DNA sequences, since there is generally an association between the presence of nucleolar organizer regions (NORs) with CMA3 labeling in the same chromosome region (GC bases were prevalent in the region marked by CMAe region .3 and NORs has been reported in several species of the subtribe Meliponina, as Partamona mulata and Partamona vicina (Partamona peckolti (Partamona helleri and Partamona seridoensis (Trigona fulviventris (Trigona (A positive correlation between CMAOxytrigona, underlines the importance of further cytogenetic knowledge of this group, as a whole. This could be useful in orientating taxonomy and conservation methods. Cytogenetics directly affects progress in taxonomy studies, by ensuring biological data with the elimination of subjectivity in systematic classification, especially in the Meliponina, through numerical taxonomy (The scarcity of biological information on bees from the subtribe Meliponina, especially the genus The information obtained in this work, besides being of use in future cytotaxonomic studies, will be of assistance in comparative analyses, as a means of clarifying both taxonomic problems and those phenomena involved in karyotype evolution in this group.


The incidence of tuberculosis (TB) and its risk factors in China remains unclear. This study examined TB incidence and relative risk factors in rural areas of China. Participants  were recruited in Xiangtan County  and in Danyang County (in the eastern area of China) in 2009 and a followed-up study was conducted for one year. The incidence density of pulmonary TB and smear-positive TB were 91.6  per 100,000 person-year and 36.7  per 100,000 person-year respectively in Xiangtan, and 47.3  per 100,000 person-year and 22.7  per 100,000 person-year in Danyang. The medical history of TB was associated with TB, with the relative risk (RR) of 7.00  in Xiangtan and that of 31.08  in Danyang. The association between TB and per capita living space over median was found in Xiangtan, with the RR of 1.86 . No association was found between TB and the insurance status, the contact history with TB, the history of diabetes, smoking, or per capita annual income. The host genetic susceptibility, and social factors such as education and income could be considered in future studies. In 2010 an estimated 8.8 million  new tuberculosis (TB) cases occurred in the world, equivalent to 128 cases per 100,000 population TB incidence–one of the most important surveillance indicators–reflects the impact of TB on public health. Since 1979, China has conducted five national TB prevalence surveys. The infection and prevalence of TB were investigated; however, little data on TB incidence were reported. While a majority of researchers have paid close attention to prevalence, only a few have focused on TB incidence in China Besides incidence, the following risk factors relative to TB occurrence are also unclear in rural populations in China: being male, poor living conditions, smoking, history of TB, and contact with TB patients We could not speculate about TB incidence using the new TB notifying rate directly in most developing countries, because of patients’ incomplete registration forms, so we did not have access to fully accurate TB incidence data or were unable to identify the influencing factors. We conducted a population-based cohort study in two counties of Eastern and Middle China to get TB incidence rates in rural areas and to analyze the association between risk factors and TB.Our study included a baseline study, a one-year follow-up, and an endpoint survey. In our baseline survey, trained staff administered a standard questionnaire to obtain baseline information which included demographic data, medical history of TB symptoms or related chronic diseases, and lifestyle risk factors. We conducted household interviews regarding TB symptoms of all the residents in the study sites. Individuals who had suspect TB symptoms and/or history of TB were encouraged to obtain chest X-rays and collect three sputum specimens for smear and culture tests. According to the TB diagnosis standard , people who did not have TB were eligible for the follow-up cohort observation.The cohort was followed up for one year and TB incidence information was collected. During the observation period, new TB cases were detected through the following procedures during the follow-up period: (1) Routine detection: we carried out a media campaign and health education regarding TB to enhance public awareness of TB prevention and then actively mobilized the follow-up population who had suspect TB symptoms to designated TB health facilities; (2) by strengthening follow-up screening for high-risk groups such as those who had close contacts with smear-positive patients; (3) by detecting new TB cases through household clue investigations, community recommendations, and health examinations.At the endpoint of the follow-up, all existing populations received symptom interviews with the same investigation method as the baseline survey.We used a convenience sampling method to choose selected provinces; economic conditions were important factors for choosing study fields. We selected Danyang County of Jiangsu Province in Eastern China and Xiangtan County of Hunan Province in Middle China as representatives of good and poor financial status in the rural areas, respectively.2. This county ranks 18th of the 100 richest counties in China. During 2006–2008, the average notification rate of active pulmonary TB was 59.74/100,000; that of new sputum smear-positive TB was 33.03/100,000. Another study site was Xiangtan County, in the mid-eastern part of Hunan Province, with a population of 1.09 million and an area of 2,372 km2. The economic status of Hunan is not as good as that in Jiangsu. The average net income of rural inhabitants in Xiangtan County was 6,458 RMB  in 2009, compared with 10,058 RMB in Danyang in the same period. During 2006–2008, the average notification rate of active pulmonary TB in Xiangtan County was 101.10/100,000; that of new sputum smear-positive TB was 63.52/100,000.Danyang is a county in south Jiangsu Province, with a population of 0.81 million and an area of 1,047 kmThe sampling process was based on the average notification rate of new smear-positive TB during 2006–2008. Townships in the two counties were stratified into three levels  based on their TB notification rate. One township was randomly selected from each level. We selected two towns from Danyang County and three from Xiangtan County.All local residents (excluding people absent for more than six months during that year) and permanent residents immigrating from other areas/provinces for more than three months were included in the survey.Gender, age, marital status, occupation, and educational level were considered as background factors. These factors were demographic characteristics which would not change by public health intervention. Insurance status, history of TB, history of contact with TB patients, history of diabetes, smoking (former or current), per capita annual income, and per capita living space were classified as potential risk factors.If a person had access to any type of medical insurance, we defined the insurance status as exposed. A history of TB was determined by medical records as diagnosed and registered by TB dispensaries. History of contact with TB patients was based on self-reported information from the questionnaire. A history of diabetes was identified by a positive response from participants to the question, “Has a doctor ever told you that you have diabetes?” Cigarette smoking was defined as having smoked at least 100 cigarettes in one's lifetime. For analysis, per capita annual income and per capita living space were divided into “below median” and “over median.”mycobacterium tuberculosis (M. tuberculosis). Sputum smear-negative and culture-negative TB were defined as a clinical illness, also chest X-rays consistent with active TB which did not respond to broad-spectrum antibiotics but did respond to anti-TB treatment. Participants were classified as lost-to-follow-up if they did not attend for review (scheduled or unscheduled) during the study period and could not be traced; their records were deleted from analysis.Laboratory-confirmed TB cases were defined as sputum smear-positive (including scanty positive) or sputum culture-positive for We performed all statistical analyses using SPSS for Windows (version 12.0). Continuous variables such as age, per capita annual income, and per capita living space were divided into categorical variables. We used the following calculation of incidence density. The all detected new TB cases during follow-up and endpoint periods was the numerator; person-time based on length of the person’s exposed/unexposed time was the denominator. To compare the incidence density between Xiangtan County and Danyang County, the incidence was standardized by age and gender. A Poisson regression model was fitted to identify risk factors associated with new TB cases. Insurance status, history of TB, history of diabetes, smoking (former or current), per capita annual income, and per capita living space were included as factors in the model.P value less than 0.05 was considered statistically significant, with a 95% confidence interval (95% CI) to determine adjusted relative risk.Each model was then repeated with an adjustment approach for the following potential confounders: gender, age, marital status, education, and occupation. The study protocol was approved by the institutional review boards of the Tongji Medical College, Huazhong University of Science and Technology. We obtained informed written consent from all participants or the next of kin, carers or guardians on the behalf of the minors/children participants after the study protocol had been explained to them.The eligible population in our cohort study was 192,166; after the one-year follow-up, 177,529(92.4%) completed the study: 84,526 participants in Xiangtan County and 93,003 in Danyang County, including 90,574 men and 86,955 women . Over onIn the period under observation, 117 new TB cases occurred: 72 cases in Xiangtan County, 45 in Danyang County. The sex ratio was 4.1∶1 in Xiangtan and 2.5∶1 in Jiangsu. None of the patients were under 15 years of age, in both fields. The factory worker, rural laborer, peasant group had the most patients in the three occupational groups. Primary school and junior school education levels accounted for 85% of the patients.P<0.001).We observed 72 new TB cases in Xiangtan County during the follow-up period, including 29 sputum smear-positive cases. The crude incidence density of active TB was 85.3  per 100,000 person-year in total, and that of sputum smear-positive TB was 34.3  per 100,000 person-year . When itWe detected 45 active cases in Danyang County, including 22 sputum smear-positive TB cases. The overall crude incidence density of active TB was 50.8  per 100,000 person-year in total; that of sputum smear-positive TB was 24.8  per 100,000 person-year. The number dropped to 47.3  per 100,000 person-year and to 22.7  per 100,000 person-year when standardized.The highest incidence density appeared in the population with a medical history of TB in the two study sites . In XianIn Xiangtan County, except for the group of per capita living space over median, people with characteristics such as access to insurance, or a history of diabetes had an incidence below 100 per 100,000 person-year. All the patients in Danyang County had insurance; the incidence changed from 4.8 to 71.5 per 100,000 person-year under different exposure conditions.In univariate analysis, people who had a history of TB or TB contact history were more likely to progress to TB. In addition, smoking and per capita living space over median were significant risk factors in the Xiangtan population, while diabetes was significant in the Danyang population .The Poisson model  also shoIn our cohort study, the incidence density of active TB ranged from 26.3 to 144.3 per 100,000 population; that of sputum smear-positive TB changed between 12.3 to 55.0 per 100,000 population in different gender and study sites. Xiangtan County suffered a more serious TB epidemic situation than Danyang County. Different economic levels may account for this. In the literature review, there were no related data about rural areas of China; however, in rural areas of Africa China has the largest agricultural population in the world: ∼674 million rural inhabitants. We estimate that if there are no effective interventions China will have at least 318,802 new TB cases in rural areas every year. China’s 2010 National TB Prevalence Survey also showed that the prevalence of active TB in rural areas was 569 per 100,000 population. Therefore, effective intervention strategies must be implemented to control TB in rural areas of China.In our study, the TB incidence among men in Xiangtan and Danyang Counties was more than twice that of women. The distinction between public health risks of infection and different lifestyles may account for this gender difference. A recent study showed that concealment of a disease status such as TB happened more among females because of fear of losing social status, marital problems, or harmful reactions from the community M. tuberculosis infection has occurred, the risk for development of TB is influenced by several factors: duration of the infection; intrinsic characteristics such as age, body mass index, Bacillus Calmette-Guerin(BCG)immunization history, HIV infection, host genetic susceptibility, etc. So a higher incidence of TB may be caused by increased risk of exposure to M. tuberculosis.Unlike other studies M. tuberculosis may result in incidence diversity of TB between male and female subjects. A study from a rural district in Malawi, Africa, suggested that identifiable recent contacts with known smear-positive cases accounted for 12.5% of the TB burden M. tuberculosis, may have a substantial effect on controlling TB epidemics M. tuberculosis infection in their households and communities.Moreover, different contact types with intensity and duration, and differences in experiences of exposure to M. tuberculosis, it persists for a long time in a process called latent TB infection (LTBI). LTBI has traditionally been considered to involve the TB bacilli remaining in a non-replicating state (dormant) in old lesions–but still retaining their ability to induce reactivation and to cause active TB when a disruption of the immune response occurs Another risk factor associated with TB is a history of TB, which has been documented by other research The prevalence of diabetes mellitus is increasing rapidly worldwide and has been reported to be significantly associated with increased risk of TB Smoking and alcohol use–two risk factors for TB mentioned in many studies TB research in other rural sites in China showed similar results of negative correlation of TB with socioeconomic status Our study had some limitations. First, as a longitudinal study, one-year follow-up is too short to obtain more useful effects. Second, our study did not include some interesting key variables that have been studied in other settings such as HIV infection, effect of drug resistant patterns and use of mass public transportation due to restriction of conditions, so there might be selection bias. These variables will be included in the further research of our study to examine relative risk factors of TB.TB incidence in rural populations is very high in China. Considering the large agricultural population and characteristics of this disease, TB is still a severe public health challenge in China and may need effective strategies to decrease the TB burden in rural areas.


Vibrio genomes, and, in addition, also calculated the pan- and core-genomes from a data set of more than 250 draft Vibrio genome sequences. These genomes come from 9 known species and 2 unknown species. Within the finished chromosomes, we find a core set of 1269 encoded protein families for chromosome 1, and a core of 252 encoded protein families for chromosome 2. Many of these core proteins are also found in the draft genomes  Of the chromosome specific core protein families, 1169 and 153 are uniquely found in chromosomes 1 and 2, respectively. Gene ontology (GO) terms for each of the protein families were determined, and the different sets for each chromosome were compared. A total of 363 different “Molecular Function” GO categories were found for chromosome 1 specific protein families, and these include several broad activities: pyridoxine 5' phosphate synthetase, glucosylceramidase, heme transport, DNA ligase, amino acid binding, and ribosomal components; in contrast, chromosome 2 specific protein families have only 66 Molecular Function GO terms and include many membrane-associated activities, such as ion channels, transmembrane transporters, and electron transport chain proteins. Thus, it appears that whilst there are many “housekeeping systems” encoded in chromosome 1, there are far fewer core functions found in chromosome 2. However, the presence of many membrane-associated encoded proteins in chromosome 2 is surprising.We have compared chromosome-specific genes in a set of 18 finished  Vibrio genus represents a large subgroup of Gamma subdivision of Proteobacteria, which are abundant, fast growers that can be highly variable. These bacteria have the ability to form biofilm on biotic and abiotic surfaces and are ubiquitous in marine and estuarine environments at notably high densities in fish, corals, shrimps, plankton, and mammals . The initial set included 368 genomes, 18 of them were complete and 350 were retrieved as Illumina raw reads from the NCBI Sequence Read Archive (SRA). Of these, 188 genomes were sequenced using a HiSeq 2000 sequencer and the remaining 162 were sequenced with an Illumina Genome Analyzer II.A set of all publically available Vibrio genomes using RNAmmer  and is more stable, carrying many essential protein coding genes, whereas chromosome 2 is smaller (about 1 Mbp), contains a large genomic island (the “superintegron”), is more variable, and has fewer essential genes. A pairwise comparison of set of 18 genomes for both chromosomes is shown in Figure V. cholerae species, a high similarity within chromosomes can be found with confidence, although on average only 10% of the proteins are shared between chromosomes 1 and 2.The The core-genome of complete strains contains 1269 conserved protein families shared within chromosome 1, and 252 core families shared within chromosome 2; only 104 functional profiles are shared between the two chromosomes. When additional draft genomes were included, the numbers for both chromosome 1 and 2 dropped to 673 core-genomes and 140 protein families, followed by a decrease of shared functional profiles for a total number of 96. The core- and pan-genome summary results are shown in Table V. cholerae strain M66-2), whilst the pan-genome for chromosome 2 (~3740 gene families) is more than three times the size found encoded in a single copy of chromosome 2 . Many of these additional gene families are likely to be found in the super-integron, which is a known variable region of chromosome 2.The pan genome for chromosome 1 (~5500 gene families) is about twice the number of genes encoded in a single copy of chromosome 1 (e.g., 2650 genes in A closer look at the distribution of functions within the core-genomes of two chromosomes showed that all of the shared proteins are found in the PfamA database Figure  and mostIn order to explore the overlap between the core genes in chromosomes 1 and 2, we extracted the core proteins for each chromosome and then examined the overlap with the core of the other chromosome Figures , 5. A toVibrio is comprised of a diverse group of bacteria, which can be either pathogenic or symbiotic to mammals and organisms of marine environments. Species-specific genomes may contain proteins responsible for pathogenicity or they may be crucial for survival in a given environment. To demonstrate the level of specificity between species of the same chromosome, 9 strains representing 7 known and 2 unknown species, a pairwise comparison of specific-genomes, was performed. Within chromosome 1, the fraction of unique proteomes varies from 18 to 33% (Figure The genus % Figure , whereas% Figure .Vibrio cholerae spp. are known pathogens in humans and were chosen to examine for genome specific differences in gene content. Representative strains of V. cholerae species were compared to other strains, as shown in Figure V. cholerae is one of the most important, highly documented, and most sequenced species of Vibrios. Our dataset included 279 V. cholerae strains, 8 completely sequenced and 271 draft genomes. For the draft genomes, chromosome specific genes could not be calculated. However, starting with the known core genomes from the finished genomes, it is possible to look for the presence of the known chromosome core genes across the draft genomes. Thus, core-genome analysis of 279 V. cholerae strains yielded in 776, 250, and 182 protein families, in large, small, and both of the chromosomes, respectively. Further, we examined the pan-genomes of both chromosomes within a set of 18 genomes. The distribution of the total number of 8325 functional profiles is as follows: 2333, 341 and 73 families assigned to PfamA, Superfamily, and TIGRFAM databases, respectively (Figure V. cholerae strains brings at least 2000 possible profile combinations to the pool of previously known functions that represent more than 70 different GO functional categories (Figure Vibrio pan-genome can be quite large, with more than 17,000 gene families, although, any one Vibrio genome will contain only about 3500 genes, or about one-fifth of the size of the pan-genome. There is considerably more variability in chromosome 2 than in chromosome 1.In conclusion, the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several viruses have been identified in recent years in the intestinal contents of chickens and turkeys with enteric problems, which have been observed in commercial farms worldwide, including Brazil. Molecular detection of these viruses in Brazil can transform to a big threat for poultry production due to risk for intestinal integrity. This disease is characterized by severely delayed growth, low uniformity, lethargy, watery diarrhea, delayed feed consumption, and a decreased conversion rate. Chicken astrovirus (CAstV), rotavirus, reovirus, chicken parvovirus (ChPV), fowl adenovirus of subgroup I (FAdV-1), and avian nephritis virus (ANV) were investigated using the conventional polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR). In addition, the infectious bronchitis virus (IBV), which may play a role in enteric disease, was included. The viruses most frequently detected, either alone or in concomitance with other viruses, were IBV, ANV, rotavirus, and CAstV followed by parvovirus, reovirus, and adenovirus. This study demonstrates the diversity of viruses in Brazilian chicken flocks presenting enteric problems characterized by diarrhea, growth retard, loss weight, and mortality, which reflects the multicausal etiology of this disease. The economic impact of enteric virus infections on the poultry industry has been evaluated and ranges from insignificant economic effects to those that are severe and cause devastating losses. Enteric diseases tend to predominantly affect young birds; however, the disease may occur in all age groups, which increases susceptibility to other diseases, decreases feed conversion efficiency, and prolongs the time to market , 2. At pEnteric disease was induced by experimental infection in one-day-old broiler chicks with intestinal content from a broiler flock which has presented enteric problems such as diarrhea, poor performance, and mortality , 4 or wiIn the past, enteric disease has been called the pale bird syndrome and helicopter wing disease and was characterized by poor growth and retarded feather development. These symptoms are observed consistently along with the other less frequent clinical signs including diarrhea, increased mortality, and pancreatic and lymphoid atrophy , 15. EntIn this study, we screened seven related viruses as potential agents of enteric disease in chickens to investigate the highest number of agents and their emergence in the Brazilian poultry production.Two hundred and eighty (280) intestinal contents samples were collected from commercial chicken from nine Brazilian states, as follows: Rio Grande do Sul, Minas Gerais, São Paulo, Paraná, Pará, Rio de Janeiro, Santa Catarina, Goiás, and Ceará. The samples were collected between 2008 and 2010. Two hundred and twelve of the samples were collected from chickens with clinical signs of enteric problems that were described as diarrhea, poor weight gain, malabsorption syndrome, culling, and mortality, and 68 samples were collected from chickens without clinical histories of enteric problems in the last three reared previously flocks. Each sample was composed of a pool of intestinal contents obtained from five chickens of whole intestine, from duodenum to the end of ileum. The age of the chickens varied from three days old to 106 weeks old broilers, broiler breeders, pullet, and layer hen flocks. Samples were preserved at −20°C until shipment to the Laboratory of Avian Diseases  and were kept at −20°C until the processing.2 1.5 mM, pH 7.3) [A 1 : 5 suspension was prepared with Tris-Calcium buffer  . After 3ChPV, adenovirus, CAstV, and ANV strains that had been isolated from Brazilian flocks and whose identity had been confirmed by sequencing were used as positive controls in the molecular assays. Additionally, a Massachusetts vaccine strain (H120), Nebraska calf diarrhea virus, and S1133 strains were used as controls for IBV, rotavirus, and reovirus, respectively.Previously published primer sets were utilized for the reverse-transcriptase polymerase chain reaction (RT-PCR) and for PCR to detect the seven viruses screened in this study. The sequences and references are showed in μL) was used as a template for PCR in a 25 μL reaction mix that contained 10 pmol of each of the forward and reverse primers  mix, 1.25 mM MgCl2, and 1 U of Platinum DNA Polymerase (Invitrogen). The negative control included sterile water. Amplifications were performed in a Biometra DNA thermocycler . After a 5 min incubation at 95°C, 30 cycles of amplification  were performed to detect chicken parvovirus and 34 cycles of amplification  to detect fowl adenovirus subgroup I. The PCRs ended with a final extension step of 10 min at 72°C. The PCR products were visualized after separation by electrophoresis in an agarose gel (1.5%) using Blue Green Dye  to stain the DNA. The size of the amplified product was estimated using the 100 base pair DNA Ladder molecular size marker (Invitrogen).Conventional PCR was conducted to detect chicken parvovirus and fowl adenovirus of subgroup 1, DNA, as previously described , 18. Briin vitro transcribed and amplified using the One-Step RT-PCR Kit . Primer sets that have been previously described were used to detect CAstV, reovirus, rotavirus, and ANV as described in μL reaction mix contained 1 X Qiagen One Step RT-PCR kit reaction buffer, 320 μM of each dNTP, 10 pmol of each forward and reverse primer, 1 μL of Qiagen RT-PCR enzyme blend, and 2.5 μL of extracted RNA. To detect IBV, the primers and reaction conditions described by Cavanagh et al. [RNA isolated from field samples was h et al.  were useAt least one virus was detected in 226 (80.7%) of the 280 examined samples. Of the 212 samples collected from chickens with clinical signs such as diarrhea, lethargy, poor performance, poor weight gain, malabsorption, and mortality, 183 (65.4%) were positive for one or more of the screened viruses and in 43 (15.4%) samples, at least, one enteric-diseases-related virus was detected in samples from healthy chickens . All of The majority of positive samples were detected in broiler chickens 193 (85.4%) during the first seven weeks of age, being more frequent at the first four weeks, following for broiler breeders with 22 (9.7%) of positive samples, and the lowest detection of enteric viruses was present in pullet and layer hens with 4.9%, both types of birds with a homogeneous distribution among the weeks . The minAstroviridae (Chicken Astrovirus-CAstV and Avian Nephritis Virus-ANV) [Bacteria and parasites have been considered the primary etiological agents of gastroenteritis in commercial poultry. However, many viral infections have been associated with enteric diseases of chickens and turkeys, including coronavirus, reoviruses, rotaviruses, adenoviruses, enteroviruses, and the members of Family rus-ANV) –27. Inferus-ANV) , 28, 29 rus-ANV) . Recentlrus-ANV) . IBV canrus-ANV) , and somrus-ANV) .Different denominations or terms have been used to describe the enteric disease in poultry because the clinical signs are infrequent or occur independently of previous conditions, such as the presence of primary or secondary etiological agents, the immune and nutritional status of the host, and environmental conditions , 15, 33.The results obtained in this study demonstrate a high level of infection with one or more of the seven viruses investigated in the chickens with clinical symptoms (65.4%), as shown in Regardless of the symptoms, most of the samples (226 samples) representing 80.7% were positive for one or more of the viruses, which demonstrates the high prevalence of these viruses in Brazilian chicken flocks . In almoThe combinations were scattered proportionally according to the prevalence of each virus and did not present a pattern or a constant frequency. None particular combination of the viruses was observed with significant repeatability. Rarely, a single agent is the sole contributing factor to enteric disease; moreover, the presence of different combinations of viruses could result in varied disease presentations .IBV was the most prevalent virus detected in the samples that contained a single virus  and in aAstroviridae in 2000 [Of the viruses investigated, ANV was the second most prevalent by absolute numbers and was detected in 83 samples (29.6%), as shown in  in 2000  and has  in 2000 . A recen in 2000 . The cha in 2000 , 39.Chicken astroviruses (CAstV) were the third and rotaviruses were the fourth most frequent agents detected in this study, with 21.1% and 17.1%, respectively . PreviouParvoviruses are known to cause gastrointestinal disease in mammalian species and have been implicated as a cause of malabsorption syndrome in chickens and enteritis in turkeys , 44. HowFowl Adenovirus subgroup I had a frequency 9.6% positive samples among the viruses investigated in this study . The subEscherichia coli [Avian reoviruses are frequently detected in the intestinal tracts of poultry with enteric disease and are widely distributed in poultry . Avian rhia coli , and infhia coli . Howeverhia coli , 50.The association between viruses and age showed that the viruses could be detected at different stages in the broilers, breeders, and pullet/layer hens; for example, virus was detected from the first week of age to the last week, especially in broilers. An important finding was the detection of CAstV in one-day-old breeder chicks, which may constitute an indicator of vertical transmission. According to McNulty et al. , in a loIn conclusion, the primary viruses detected in this study were the IBV, ANV, CAstV, and rotavirus, followed by chicken parvovirus, adenovirus, and reovirus. However, the astroviruses (ANV and CAstV) should be regarded as the most important pathogens because coronavirus (IBV), although present in a higher percentage of samples, has not been demonstrated to cause pathogenicity in the enteric tract in our previous experiments (data not published). Detection of virus was high among chickens with and without clinical signs of disease, which demonstrates the high prevalence of these viruses in asymptomatic carriers and indicates that these carries may represent a potential source of infection. Moreover, the presence of CAstV in one-day-old breeder chicks that was detected in this study may suggest a vertical transmission. In addition, the injuries that these viruses cause can interfere with the absorption capacity of the intestinal tract during the first weeks of life of these birds, which enhances the negative impact on productivity for the rest of the production cycle. This is the first study to demonstrate the presence of these viruses in association with enteric disease in Brazil. Experimental challenges should be conducted to establish the role of these viruses and to analyze their impact on commercial chicken productivity.


Brain metabolite concentrations change dynamically throughout development, especially during early childhood. The purpose of this study was to investigate the brain metabolite concentrations of neonates : 30 to 43 weeks) using single-voxel magnetic resonance spectroscopy (MRS) and to discuss the relationships between the changes in the concentrations of such metabolites and brain development during the neonatal period. A total of 83 neonatal subjects were included using the following criteria: the neonates had to be free of radiological abnormalities, organic illness, and neurological symptoms; the MR spectra had to have signal-to-noise ratios ≥ 4; and the estimated metabolite concentrations had to display Cramér-Rao lower bounds of ≤ 30%. MRS data  were acquired from the basal ganglia (BG), centrum semiovale (CS), and the cerebellum. The concentrations of five metabolites were measured: creatine, choline, N-acetylaspartate, myo-inositol, and glutamate/glutamine complex (Glx). One hundred and eighty-four MR spectra were obtained . Creatine, N-acetylaspartate, and Glx displayed increases in their concentrations with PCA. Choline was not correlated with PCA in any region. As for myo-inositol, its concentration decreased with PCA in the BG, whereas it increased with PCA in the cerebellum. Quantitative brain metabolite concentrations and their changes during the neonatal period were assessed. Although the observed changes were partly similar to those detected in previous reports, our results are with more subjects (n = 83), and higher magnetic field (3T). The metabolite concentrations examined in this study and their changes are clinically useful indices of neonatal brain development. In vivo magnetic resonance spectroscopy (MRS) of the brain can provide information about the concentrations of particular metabolites and changes in their levels, which can aid the early detection of abnormalities, e.g., in cases of acute neonatal brain injury in which diffusion imaging and conventional MR imaging (MRI) are negative [negative . The chanegative -13. In cAt our children’s medical center, single-voxel MRS is usually included in routine clinical MR examinations of the brain and provides additional information for radiological diagnosis. Also in the neonates, hundreds of the MRS data are obtained per year. In this article, we focus on neonatal brain metabolite concentrations and their changes by the development. Neonatal subjects : 30 to 43 weeks) with no radiological abnormalities, organic illnesses, or neurological symptoms were selected for this study. 　Note, the quality of the MRS data was also considered during the selection process. Then, the changes in their brain metabolite concentrations were investigated during the neonatal period. This retrospective study was approved by the institutional ethical review board of the Kanagawa Children’s Medical Center, where all of the clinical data in this study was acquired. Written informed consent was waived by the review board.From October 2009 – November 2012, MRS examinations were performed on a total of 235 neonates, who ranged in PCA from 30 – 43 weeks, due to neurological symptoms such as hypoxic ischemic encephalopathy, a suspicion of organic disease, or as routine evaluations before discharge. The inclusion criteria were as follows: the subjects had to be free of radiological abnormalities, organic illness, and neurological symptoms at their latest radiological and clinical examinations. The MR images were interpreted by an experienced pediatric neuroradiologist and clinical and neurological conditions were evaluated by board-certificated neonatologists at our medical center. As a result, 79 neonates , including 60 preterm infants : 23 – 36 weeks; studied at PCA of 30 – 43 weeks; postnatal age (PNA): 39.9 ± 28.3 d) and 19 term infants  were included. Four of the 79 neonates were examined twice in different weeks, which resulted in a total of 83 sets of neonate MR spectra .All proton MRI and MRS scans were performed on a clinical 3T MR system  using a standard 12-element head coil (diameter: 25 cm) in the quadrature detection mode. During the examinations, the neonates were wrapped in vacuum-type immobilization bags  to prevent excessive head movement and protect their hearing. Sedation with thiopental  was employed where necessary (27 out of 83 neonates). During the examinations, heart rate and transcutaneous oxygen saturation were monitored continuously with a pulse oximeter . For single-voxel MRS, the volume of interests (VOI) were placed in three regions: 1) the basal ganglia (BG) as a representative of the subcortical gray matter, 2) the centrum semiovale (CS) as a representative of the white matter, and 3) the cerebellum . T1-weigThe MRS data processing including the signal quantification was performed using LCModel  ,16 on a t-test was used for group comparisons of metabolite concentrations between preterm infants at term (PT) and full term infants at term (FT). For the comparisons of the metabolites between the sexes, the analysis of covariance (ANCOVA) with covariate of PCA was used. P-values of less than 0.05 were considered to be significant. In this study, the concentrations of five metabolites were assessed: tCr, the total concentration of Cr and PCr; tCho, the total concentration of GPC (including other choline-containing compounds ) and PChIn the neonatal MR spectra, the five target metabolites  were clearly observed, and Lac doublet peaks were also frequently detected . There wIn total, 184 MR spectra  were obtained from the 83 neonates. The numbers of each metabolite to meet the quality criteria were as follows: tCr ; tCho ; tNAA ; mIns ; Glx .tCr, tNAA, and Glx displayed increases in their concentrations with PCA . Among tThere were no significant differences in the levels of any metabolite between the PT and FT t-test, . HoweverIn this study, we quantitatively assessed neonatal brain metabolite concentrations  in the BG, CS, and cerebellum using short TE (30 ms) MRS, which was performed with a clinical 3T scanner. To the best of our knowledge, this is the first such study to include a reasonably large number of neonates (n = 83) without radiological or short-term clinical abnormality, with reasonably high MRS data quality . Furthermore, we acquired MR spectra not only from the cerebrum but also from the small neonatal cerebellum, and our all MRS data were obtained on 3T MR system with higher SNR and greater spatial resolution than 1.5T; therefore, our data would provide novel and/or more precious information about the metabolite concentrations and their developmental changes in the neonatal brain.tNAA (NAA + NAAG) showed marked increases in its concentration during the neonatal period, as was reported previously ,4-7,9-13tCr acts as a “battery” for replenishing ATP levels ,5,12,22.tCho is involved in membrane synthesis in the brain and acts as a precursor for the neurotransmitter acetylcholine, which has many functions in the nervous system ,12,22,23The peak observed at 3.56 ppm, which was considered to represent mIns, is also known to contain glycine , and it p < 0.005) with more sample size (n = 23) and higher magnetic field (3T) than previous studies: Hüppi et al. [A novel finding of the present study was the increase in the concentration of mIns observed in the cerebellum  [Regarding the cerebellum, only 24 out of the 83 MR spectra were included in the analysis because the rest had low SNR and/or high %S.D. values, mainly due to the small volume of the cerebellum (about 26.9 cmll term) . HoweverNone of the metabolites displayed concentration differences between the PT and FT , which aLimitations); however, Lac peaks were frequently observed , the subjects have reached corrected ages of 6 months to 3 years. If a subject displayed any abnormalities, such as genetic abnormalities or a reduced developmental quotient, during the follow-up examinations, then their MRS data were excluded from the study.The relaxation times and concentration of water change during development, even during the neonatal period . HoweverDue to our selection criteria, only five metabolites were evaluable. Data for metabolites with low concentrations often display large %S.D. values; i.e., a large degree of uncertainty regarding the existence of the peak. When a metabolite displays a concentration of 0, its %S.D. value is infinite. We often observed Lac peaks; however, quite a few Lac peaks showed large %S.D. values; i.e., there was a greater possibility that the peaks were actually noise rather than Lac peaks, and so had to be excluded from the analysis. Therefore, it is often difficult to obtain publishable results for such metabolites, even if the changes in their concentrations seem to be important. Some advanced methods such as spectral editing and two-dimensional proton techniques may provide accurate information about the low SNR metabolites ,37.Quantitative brain metabolite concentrations and their changes during the neonatal period were assessed. Although our findings were partly similar to those of previous studies, such as concentration increasing trends with development in some metabolites, our results are more quantitative with more subjects (n = 83) and higher magnetic field (3T). Data regarding the ‘absolute’ concentrations of brain metabolites are also valuable because the concentration of each metabolite changes in a different manner, and it would be difficult to determine the changes in the levels of a particular metabolite from the changes in its concentration relative to those of other metabolites. The metabolite concentrations examined in this study and their changes are clinically useful indices of neonatal brain development. Further studies will increase our understanding about such brain metabolites including their specific functions during the neonatal period.


Tumor tissue-associated KLKs  are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer-directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only.Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic drugs to interfere with tumor progression and metastasis. Serine proteases, e.g. plasmin, thrombin, urokinase (uPA), and the KLKs  regulate diverse biological processes such as general protein turnover, embryogenesis and pregnancy, blood coagulation, complement activation, and wound healing.The human genome encompasses close to 600 different proteases, with about 180 serine proteases  are sufficiently specific to predict the course of the disease or response to systemic adjuvant therapy.20Prognosis of tumors of the ovary is poor, owing to late diagnosis and often inefficient primary debulking surgery of this rare malignancy, but because of rapidly developing chemoresistance as well. In general, the term “ovarian cancer” describes epithelial-surface-type tumors of the ovary, accounting for more than 80% of all solid ovarian tumors. Others, such as sex cord-stromal tumors, germ cell tumors, and metastases from for example gastrointestinal tumors are less common. Two-third of the ovarian cancer patients will develop chemoresistance and disease recurrence within the first 5 years after primary surgery. The therapy of choice is paclitaxel plus carboplatin polychemotherapy. Neither vaginal ultrasonography nor analysis of the tumor-associated antigen CA125 in serum, nor other protein or gene expression analyses of the blood or tumor tissue  at the time of diagnosis of the disease represents the major traditional prognostic factor. The 5-year survival of early FIGO stage I patients is more than 90%, while survival of patients with FIGO stage III and IV is only 25%. Other important traditional prognostic factors are size of residual tumor mass after cytoreductive surgery histology of the tumor tissue, tumor grade, and presence of ascitic fluid.KLK expression at the mRNA level is highest for KLK6–8 and 10, whereas low to moderate expression was noted for KLK1, 9, 11, 13 and 14 with no expression for KLK2–5, 12, and 15.  are released into the blood, six of these KLKs are also released into peritoneal ascitic fluid  of ovarian cancer patients.41–48 KLK proteins released into the blood or ascitic fluid may also predict the course of early and/or late stage ovarian cancer. KLK8 protein present in blood (serum) indicates a favorable prognosis for the ovarian cancer patient while elevated protein levels of KLK5, 6, 10 and 11 are markers of a poor clinical outcome.49Owing to well-accepted screening programs and successful therapy of pre-malignant lesions and early stages of cervical cancer, this malignant disease has become a rare disease in the industrialized world, although, malignant tumors of the cervix uteri are still one of the leading causes of death of young women in other countries. Cervical cancer develops stepwise from infection with the human papilloma virus (HPV) and subsequent inefficient immune response to eliminate the virus followed by cervical dysplasia (CIN I–III), subsequently turning into an invasive type of cervical carcinoma.50One of the most important factors to predict the clinical outcome of cervical cancer is clinical stage at the time of diagnosis, thus management of cervical cancer is stage-dependent. Early invasive cervical cancers are subject to surgery, whereby total radical hysterectomy including dissection of the parametries and pelvic lymph nodes, and resection of the vaginal cuff is achieved. In advanced stages of cervical cancer, primary radio-chemotherapy is the therapy of choice52KLK1–3, 12 and 14, high ones for KLK4–11 and 13; KLK15 mRNA is not expressed.i.e. from cervicitis to low-grade cervical intraepithelial neoplasia, high-grade cervical intraepithelial neoplasia, squamous cervical carcinomas, and even cervical adenocarcinomas.57For normal cervix tissue , low to Endometrial cancer, which is a malignancy of the elderly female, derives from the inner glandular layer of the uterus; luckily it is often diagnosed in an early stage of the disease, which leads to expect a favorable clinical outcome. The therapy of choice for endometrial cancer is hysterectomy with bilateral salpingo-oophorectomy, frequently associated with pelvic and paraaortal lymphadenectomy and/or followed by adjuvant radiotherapy. Systemic chemotherapy or endocrine therapy is predominately administered in advanced stages of endometrial cancer, which are rare.58KLK1, 3, 10–12 and 14) , seven were not . At the  and 14) .KLK1–3, 6, 8 and 10) were found to be expressed, for the other nine KLKs no mRNA expression data have been reported. Assessment by immunohistochemical staining demonstrated protein expression of twelve KLKs , no data are available regarding protein expression in the normal endometrium of the other three KLKs.KLK1 was found to be down-regulated whereas KLK6, 8 and 10 are up-regulated. KLK4 and 8 proteins are up-regulated; no data are available for this malignancy regarding protein expression of the other thirteen KLKs.Informative data are available for KLK expression in the normal endometrium, at the mRNA and protein level . Six KLKe.g. oncogenes, tumor suppressor genes, hormones, growth factors, and proteases. Serum/plasma-based bio-markers would be helpful for the early diagnosis of breast cancer, for assessment of the course of the disease, prediction of response or resistance to cancer therapeutics, or monitoring of efficacy of therapy.Even though treatment options such as surgery, radiotherapy, chemotherapy/ endocrine therapy, and immunotherapy are currently available, breast cancer remains the second leading cause of cancer-related deaths among women after lung cancer.In fact, several serum-based biomarkers have been described in the literature and are in clinical application, such as CA 15-3, BR 27.29 (CA27.29), carcinoembryonic antigen (CEA), tissue polypeptide antigen, tissue polypeptide specific antigen, or p105HER2 (the shed extracellular domain of HER2).62KLK15 mRNA is not expressed by normal breast tissue, low to moderate mRNA levels are detected for KLK2–5, 9, 12 and 13, high ones for KLK1, 6–8, 10, 11 and 14. No expression of KLK10 and 12 protein was determined but low to moderate levels for KLK1, 4–8 and 13–15 and high levels of KLK9 and 11. Interestingly, KLK3 is not prostate-specific but expressed in a wide variety of other tissues as well, including the breast of about one third of the women.e.g. milk of lactating women, breast cyst fluid, and nipple aspirate fluid.69Extracellular proteases such as uPA, plasmin, matrix metalloproteases, cathepsins, and the KLKs mediate many of the changes in the tumor micro-environment during tumor progression in disrupting the tumor nest-surrounding the basement membrane and the adjacent extracellular matrix (tumor stroma). With the recent discovery of all of the fifteen members of the KLK family, increasing evidence has indicated that KLKs may play pivotal roles in breast cancer progression and metastasis ,2.6,63–666KLK3, 8 and 11 mRNA expression is not changed in malignant breast tissue compared to normal breast tissue; KLK1, 2, and 5–12 mRNA expression is decreased; KLK4 and 15 are increased. KLK13 mRNA is expressed in breast cancer tissue but comparison with normal breast tissue has not been made available. For KLK6 and 14 both increases and decreases in mRNA expression have been reported. At the protein expression level, only KLK4 is elevated compared to normal breast tissue; KLK6 and 14 protein levels were reported either to be lowered or elevated, depending on the study. KLK3 is decreased or absent in breast cancer tumor tissue. Limited data are available for KLK1, 5 and 10 protein expression since expression levels were not compared to expression levels of those proteins in the normal breast tissue. Several other KLKs  have not been assessed for protein expression in breast cancer tumor tissue yet.KLKs are not only involved in breast tissue development but also in various stages of breast cancer development and progression, indicating a regulating role of KLKs in tumor growth and metastasis. In this cancer, most of the KLKs, except KLK4 and KLK15, show reduced mRNA and/or protein expression levels compared to expression of the KLKs in normal breast tissue.71Nine of the fifteen members of the KLK family are considered potential prognostic and/or predictive cancer biomarkers in breast cancer. Five KLKs predict favorable prognosis , four indicate unfavorable, poor prognosis .NKX3.1, PTEN, ETS, MYC, TP53, AR, RB1, and APC plus miRNAs as potential prognostic biomarkers.82Following lung cancer, prostate cancer is the second most common cancer and cause of cancer-related deaths in men worldwide.KLK4–9, 12 and 13, high levels for KLK1–3, 10, 11, 14 and 15. Low KLK protein expression is reported for KLK4, 5 and 13–15, high ones for KLK1–3, 9 and 11. KLK6, 7, 10 and 12 are expressed as well but expression levels were not scored. In prostate cancer, elevated levels of KLK2, 4 and 13–15 mRNA and/or protein have been reported; KLK3, 5, 7, 10 and 11 are decreased compared to nonmalignant tissue counterparts  is associated with poor prognosis; KLK4 is a marker of a favorable prognosis. Decreased mRNA or protein levels of KLK2, 3, 5–7, 10, 11, 13 and 15 have been reported of which KLK3 and 15 are markers of a poor prognosis and KLK5 and 11 markers of a favorable prognosis.84In normal prostate tissue, all of the KLKs are expressed at the mRNA level and, except for KLK8, at the protein level as well .23,54 Loterparts . mRNA exKLK2 and 3 possess steroid hormone binding sites while KLK1 and 4 possess putative steroid binding elements regulating KLK expression in prostate cancer91Testicular cancer, which is affecting men between age 15 and 35 is relatively uncommon in Asia and Africa, but common among Caucasians; the incidence of this cancer increased during the last century for unknown reasons. Testicular cancer is treatable by surgery, radiotherapy, or chemotherapy with a cure rate of ∼95%.98KLK5, 10, 11, 13 and 14, which are all decreased compared to normal tissue expression.In the normal testis, all of the fifteen KLKs are expressed at the mRNA level, this is also true for KLK protein expression, except for KLK15 which is not expressed.23,54 Soe.g. in cancer of the lung, brain, head and neck, the kidney, urinary bladder the endometrium, cervix uteri, and the testes. For several of these malignancies, the tumor tissue-associated KLKs may serve as novel cancer biomarkers in allowing tumor sub classification, diagnosis and prognosis of the cancer disease or prediction of response/failure to cancer-directed drugs. Since, regarding their clinical utility, for most of the KLKs only single reports have been published, validation of KLK gene and protein expression data in independent patient sets on the basis of standard-operating-procedures is a prerequisite before recommendation which of the fifteen KLKs, and for which cancer disease, should be considered for clinical management to support individualized cancer care and treatment. Likewise, in this context, harmonization of methodologies, tools, reagents, and statistics to assess KLK expression in tumors and bodily fluids  have to be pursued.KLKs are not only known for their strong biomarker value in prostate, ovarian, breast, and gastrointestinal cancers, regarding prediction of the course of the disease and response to cancer therapy, several KLKs appear to be of clinical value in other malignancies as well, At first glance, the KLK peptidases are characterized by high sequence similarities, yet, they show significant differences in their substrate specificities, which will facilitate development of targeted KLK inhibitors. We envision that selective inhibitors to certain KLKs will be developed for future therapeutic application, that aim at blocking their enzymatic activity, in order to interfere with KLK-mediated degradation or activation of other proteins. Nonetheless, one has to bear in mind that KLKs may exist in different enzymatic active and inactive molecular forms. Since reports about the enzymatic state of the various KLKs in different healthy and malignant tissues are scarce at present, the clinical utility of such new synthetic or biological therapeutics is not yet apparent.


Microalbuminuria is associated with increased risk of renal disease and cardiovascular diseases even in non-diabetic subjects. High incidence rates of microalbuminuria have been found in a number of population-based studies. However, the prevalence and risk factors associated with microalbuminuria in the general population in Korea are unclear.The present study was performed to estimate the prevalence of microalbuminuria and investigate the associated risk factors in the general adult population using the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-2) data from 2011.A total of 5,202 participants  were included in the analysis. Microalbuminuria was evaluated in participants of KNHANES V-2 based on the urine albumin–creatinine ratio. Estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease study equation.The weighted prevalence of microalbuminuria was 5.2%  in the general population. The prevalence of albuminuria is increased with age. After adjustment for age and sex, the presence of albuminuria was associated with increased waist circumference, systolic and diastolic blood pressure, aspartate aminotransferase, triglyceride, fasting plasma glucose, and the presence of hypertension and diabetes. In logistic regression analyses, older age, female sex, diabetes, hypertension, and serum aspartate aminotransferase were independently associated with the presence of albuminuria.The prevalence of microalbuminuria was found to be 5.2%, and conventional risk factors for cardiovascular diseases are closely related to the presence of microalbuminuria in Korea. Microalbuminuria may be a useful marker to identify individuals with increased risk of cardiovascular disease. In 2009, the overall prevalence of end-stage renal disease (ESRD) in Korea was reported to be 1,113.6 per million subjects, and the prevalence rate increased by about 12% per year during 2000–2009. The high prevalence of diabetes and the associated renal complications have been described for these years in Korea Microalbuminuria is an important marker of progression to ESRD as well as increased risk of cardiovascular disease (CVD) and mortality across all levels of glomerular filtration rate (GFR) This study used data from KNHANES V-2, a national probability survey conducted by the Korean Center for Disease Control for Health Statistics at 192 survey locations to determine the health and nutritional status of the civilian, non-institutionalized Korean population since 1998. The Korean Center for Disease Control conducted a series of KNHANES, in 1998, 2001, 2005, 2007–2009, and 2010–2012. In KNHANES V-2, an annual total of 3,840 households were selected, and urine albumin was first measured starting in 2011. A standardized interview was conducted in the homes of the participants to collect information on demographic variables, family history, medical history, medications used, and a variety of other health-related variables. Participants were chosen using proportional allocation-systemic sampling with multistage stratification . This study was approved by the institutional review board of Ilsan Paik Hospital, Republic of Korea (IB-1308-031). After approval of the study proposal, the KNHANES dataset was made available at the request of the investigator. As the dataset did not include any personal information and participants' consent had already been given for the KNHANES, our study was exempt from participant consent.The Health Interview included well-established questions to determine the demographic and socioeconomic characteristics of the subjects including questions on age, education level, occupation, income, marital status, smoking habit, alcohol consumption, exercise, previous and current diseases, and family disease history. Smoking status was divided into three categories: current smoker, ex-smoker, and nonsmoker. Subjects were questioned about whether they exercised with an intensity that left them with slight difficulty in breathing and sweating. Subjects who exercised regularly at a moderate intensity were asked about the frequency at which they exercised per week and the length of time per exercise session. Regular exercise was defined exercising as five or more times per week. Alcohol consumption was assessed by questioning the subjects about their drinking behavior during the month before the interview. Heavy alcohol drinking was categorized as drinking four or more times per week.2). Systolic and diastolic blood pressure (BP) was measured by standard methods using a sphygmomanometer with the patient in the sitting position. Three measurements were made for all subjects at 5-min intervals; the average of the second and third measurements was used in the analysis. Blood samples were collected in the morning after fasting for at least 8 h and single-spot urine specimen collected in the first morning void. Analysis of fasting plasma glucose and serum total cholesterol, TG, LDL-C and high-density lipoprotein cholesterol levels were performed by Hitachi Automatic Analyzer 7600 . Serum gamma-glutamyl transpeptidase (GGT) was measured using an enzymatic colorimetric method and aspartate aminotransferase (AST) and alanine transaminase (ALT) were measured using the standardized kinetic method . Obesity was defined as BMI ≥25 kg/m2Height and weight were obtained using standardized techniques and equipment. Height was measured to the nearest 0.1 cm using a portable standiometer . Weight was measured to the nearest 0.1 kg using a Giant-150N calibrated balance-beam scale . Body mass index (BMI) was calculated by dividing weight by the square of height (kg/m2) = 175×−1.154×Age−0.203×0.742  2) into four groups: group 1, eGFR ≥90 (n = 1988); group 2, eGFR 60–89 (n = 2981); group 3, eGFR 30–59 (n = 215); group 4, eGFR <30 ml/min/1.73 m2 (n = 8) 2 in this study.Urine albumin and creatinine concentrations were measured in the same laboratory during all surveys. Serum and urinary concentrations of creatinine were measured using a colorimetric method . The inter-assay coefficient of variation for serum creatinine was <1.4%. Urinary albumin was measured in random urine samples using a turbidimetric immunoassay . Laboratory control measures in KNHANES V-2 indicated that these assays are highly reliable with consistently low coefficients of variation (3.1%). The ratio of urinary albumin to urinary creatinine was reported as albumin-creatinine ratio (ACR) in milligrams per gram of creatinine. Subjects were divided according to albuminuria status, i.e., normoalbuminuria (ACR <20 mg/g Cr), microalbuminuria (ACR = 30–299 mg/g Cr), or macroalbuminuria (ACR≥300 mg/g Cr) P<0.05 was deemed to indicate statistical significance. Statistical analyses were performed using SPSS software .Data are presented as mean and 95% confidence interval (CI). Participants in Korean NHANES were not sampled randomly. Korean NHANES was designed as a complex, stratified, multistage probability-sampling model. Therefore each participant does not have the same power for representation of whole Korean population. If we try to present prevalence in whole Korean population form the dataset, we should consider how much power each participant for representation (sample weight) for whole Korean population. After approval of investigator's proposal by the Korean Center for Disease Control, the Korean Center for Disease Control provide survey dataset including information about survey location, strata by age, sex, and other factors, and sample weight of each participants to investigator. Survey sample weights, calculated taking the sampling rate, response rate, and age/sex proportion of the reference population  into consideration, were used in all analyses to produce estimates representative of the non-institutionalized Korean civilian population. Age and age-adjusted comparisons of clinical characteristics according to the presence of microalbuminuria or macroalbuminuria were performed using analysis of covariance (ANCOVA). To determine which parameters were associated with the presence of microalbuminuria or macroalbuminuria, logistic regression analysis was conducted with age, sex, waist circumference, presence of hypertension, serum AST, serum TG, impaired fasting glucose, and diabetes as variables. Linear regression analysis for log ACR was performed with age, sex, waist circumference, systolic BP, fasting plasma glucose, serum TG, and serum AST as a continuous variable. Two-tailed analyses were conducted, and A total of 10,589 people participated in the KNHANES V-2, 2011. The participation rate for the health examination (including laboratory tests) was 76.1%. Of these, the 5,202 participants aged over 19 years who completed the measurement of ACR were included in the analysis. Sample data were weighted appropriately to account for the complex design and unequal probability of selection of sample subjects to compute prevalence estimates of abnormal albumin excretion levels for the Korean population.2 , serum creatinine level (SCr) was 79.6 µmol/l , and median ACR was 2.34 mg/g Cr . And, 33% of subjects were obese and 9% had diabetes. Twenty-seven percent of subjects was hypertension: systolic BP≥140 or diastolic BP≥90 mmHg without antihypertensive medication , or use of antihypertensive medications irrespective of BP .P<0.001). Generally, the prevalence of microalbuminuria or macroalbuminuria was not different according to sex. In subjects <65 years old, the prevalence was higher in men. The reverse pattern was observed in subjects ≥65 years old, but the difference was not statistically significant (nificant .2) was 2.8%   and 7.1% (4.2–11.8), respectively. The rates in cases with eGFR <30 ml/min/1.73 m2 were 27.8%  and 38.3% (12.9–72.2), respectively (2)  . When suectively . On the vely (2) .Multiple logistic regression analysis showed that age, female sex, AST, hypertension, and diabetes were independently associated with the presence of any albuminuria including micro- and macro-albuminuria . Age, hyThese survey data from a representative sample of the Korean population indicated a microalbuminuria prevalence rate of 5.2% . Older age, female sex, hypertension, serum TG, and diabetes are independently associated with the presence of albuminuria in the Korean adult population. Microalbuminuria has been shown to be an early sign of progressive renal failure and increased risk for CVD, not only in diabetic or hypertensive patients but also in non-diabetic and non-hypertensive subjects The prevalence of microalbuminuria in this study was slightly lower than those in Australia (6.0%) P<0.001). These findings were in agreement with the observation that aging is associated with senescence and interstitial fibrosis and with renal ischemia due to intrarenal arteriosclerosis and cholesterol emboli involvement P = 0.002). This association was also found in NHANES and AusDiab studies Our results were consistent with previous reports in that some studies have demonstrated an association of microalbuminuria with CVD risk factors, including hypertension, hypertriglyceridemia, impaired fasting glucose, and diabetes P<0.001). Insulin resistance was suggested to play a role in the development of microalbuminuria by raising glomerular hydrostatic pressure, increasing renal vascular permeability, aggregating glomerular hyperfiltration, and enhancing renal sodium reabsorption Although the association between hypertriglyceridemia and albuminuria, including microalbuminuria, was not statistically significant in multiple logistic regression analysis, it appeared to be a significant independent predictor of macroalbuminuria in this study. With adjustment for age and sex, the significant positive trend was observed between across categories of albuminuria and all components of metabolic syndrome  had microalbuminuria  Although microalbuminuria has been considered a marker of glomerular injury and an early indicator of progression to renal insufficiency, 64% of the subjects with decreased eGFR had normal albuminuria in the present study. These findings were consistent with the results of the Framingham Heart Study, in which about only one-quarter of subjects with decreased eGFR (<60 ml/min/1.73 mThese results of the present study clearly indicated the prevalence of microalbuminuria and its associated factors in Korea. However, this study had some limitations. The first is that a single urine albumin creatinine ratio result was used in this analysis. A single measurement of urine albumin excretion and serum creatinine for calculating ACR and eGFR could result in misleading classifications of albuminuria and CKD stages. However, repeated measurement of these parameters or direct measurement of eGFR is both time consuming and costly and is therefore not feasible for nationwide health examination studies. Additionally, single-point measurement of ACR has been used to predict increased CVD risk The results of this population-based study indicated the prevalence of microalbuminuria was slightly lower than those in Western population but comparable to those in Asian population. These may suggest ethnic difference although we do not have the data of direct comparison between each ethnic group. However, the presence of microalbuminuria is a matter of concern because this condition is predictive of progressive deterioration of kidney function and is associated with increased risk of CVD events and death. Therefore, identification and management of relevant risk factors in individual with microalbuminuria is an important step to develop targeted preventive efforts in practice because a growing body of evidence suggests that albuminuria is strongly associated with the risk of adverse outcomes even in general populations.