The largest ex situ olive germplasm collection consists of approximately 500 Italian olive varieties and corresponding to 85% of the total Italian olive germplasm is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia (CRA-OLI), in Italy. In this work, eleven preselected nuclear microsatellite markers were used to assess genetic diversity, population structure, and gene flows with the aim of assembling a core collection. The dendrogram obtained utilizing the unweighted pair group method highlights the presence of homonymy and synonymy in olive tree datasets analyzed in this study. 439 different unique genotype profiles were obtained with this combination of 11 loci nSSR, representing 89.8% of the varieties analyzed. The remaining 10.2% comprises different variety pairs in which both accessions are genetically indistinguishable. Clustering analysis performed using BAPS software detected seven groups in Italian olive germplasm and gene flows were determined among identified clusters. We proposed an Italian core collection of 23 olive varieties capturing all detected alleles at microsatellites. The information collected in this study regarding the CRA-OLI ex situ collection can be used for breeding programs, for germplasm conservation, and for optimizing a strategy for the management of olive gene pools.The olive is an important fruit species cultivated for oil and table olives in Italy and the Mediterranean basin. The conservation of cultivated plants in  Olea europaea L. subsp. europaea var. europaea) is an important fruit species cultivated for oil and canned fruit in Italy and the Mediterranean basin. The existing ex situ collections of olive tree germplasm may valuably provide either raw material for plant breeding or plants which are directly valid for a sustainable production. With respect to the latter, we refer to those local varieties that evolved for a very long period in a location, that developed adaptative traits which are well integrated with the environmental, agronomic, cultural, and traditional features of the site, and that have been relatively recently replaced with new varieties [ex situ olive germplasm collections, including Cordoba (Spain), Marrakech (Morocco), Porquerolles (France), and Cosenza , which hosts the majority of olive varieties. Currently, on the basis of estimates from the FAO Olive Germplasm Plant Production and Protection Division, the world olive germplasm contains more than 2.600 different varieties [http://ec.europa.eu/agriculture). The European Union, with about 32%, is also the major producer of world's table olives. Even, in this case, the largest producing European countries are Spain, Greece, and Italy. Italy has about 600 olive cultivars and holds the world record for the number of cultivated varieties, representing 25% of the known world olive germplasm [ex situ olive germplasm collection consisting of approximately 500 Italian olive varieties, and corresponding to 85% of the total Italian olive germplasm and to more than 18% of the total world olive germplasm, is maintained at the Consiglio per la Ricerca e sperimentazione per l'Agricoltura, Centro di Ricerca per l'Olivicoltura e l'Industria Olearia  in Italy [http://www.certolio.org/data-base-molecolare/).The olive  and synonymy  , 10. It The identification of cultivars and accessions using molecular markers is a crucial aim of modern horticulture, with applications in breeding programs and in germplasm collection management. Traditionally olives, like other tree species, were characterized by morphological traits . Howeverex situ olive collections.In recent years, many studies about molecular characterization of germplasm in olive trees have been performed, but generally on a small number of Italian olive cultivars and without taking into account the presence of a core collection , 12–15. The purpose of this study was to investigate the genetic structure of the entire Italian olive germplasm CRA-OLI collection, including all 489 accessions, using eleven nuclear microsatellite (SSRs) markers. The genetic structure of Italian olive germplasm was investigated using a model-based Bayesian clustering method to assign individuals into defined gene pools. This work is the first that takes into account such a large number of Italian olive cultivars (489 cvs) analyzed using the same set of molecular markers. This study also provides basic information for the development of core collections to maximise the representativeness of olive genetic diversity. Our results represent an essential step towards optimised conservation of olive genetic resources and subsequently for genetic association studies to detect quantitative trait loci (QTL) of adaptive and agronomic interest .http://dx.doi.org/10.1155/2014/296590).Young leaves were harvested from 489 olive trees growing in the germplasm collection of CRA-OLI, located along the Ionian coasts near Mirto-Crosia . The analyzed olive plants are all autochthonous and representative of the seventeen regions of Italy: Abruzzo (23 varieties), Basilicata (29), Calabria (36), Campania (43), Emilia-Romagna (12), Friuli-Venezia-Giulia (3), Lazio (25), Liguria (16), Lombardy (2), Marche (19), Molise (24), Apulia (41), Sardinia (20), Sicily (70), Tuscan (101), Umbria (22), and Veneto (3) . DNA quality was checked on 0.9% agarose gel and the DNA concentration was estimated using NanoDrop ND2000 spectrophotometer .The olive trees were genotyped at 11 nuclear SSRs, selected among those available in literature, and proven to be suitable for the characterization and identification of olive varieties in previous papers , 14, 18:μL containing 25 ng of DNA, 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM forward and reverse primers, and 0.05 units of Taq DNA polymerase  as reported in Muzzalupo et al. [The PCR was conducted in a final volume of 25 o et al. . SSR ampo et al. . The ampo et al. .Na), effective number of alleles (Ne), observed (Ho) and expected (He) heterozygosity, and fixation index (F) were computed with GenALEx version 6.5 software [Number of alleles  and absence (0) of bands . Genetical distance based on the Nei coefficient and genetic similarity based on the Simple Matching (SM) coefficient among 489 olive varieties were estimated using the NTSYSpc program version 2.02 . FinallyThe frequency of null alleles was estimated per locus and per region, using the software FreeNA .Principal Coordinate Analysis (PCoA), also available in version 6.5 of the GenALEx program, was conducted using Nei's unbiased genetic distance pairwise population matrix to determine whether observed patterns in molecular data support the partitioning of the olive tree samples into specific groupings.To study the genetic structure of the Italian olive germplasm, a model-based analysis was performed using BAPS 5.3 . This prPT, FST, and RST estimators among BAPS groups identified in this study. Statistical significance of all the ΦPT, FST, and RST estimators were tested using 10,000 permutations.In addition, an Analysis of Molecular Variance (AMOVA) , 29 was P values of the admixture estimates to 0.05.Finally, we used the GraphViz 2.28 package installed in BAPS 5.3, to estimate and draw the gene flows among the clusters identified. In the graph, gene flows were shown by weighted arrows, so that the weights relating to the amounts of ancestry in the source cluster were assigned to the target cluster. This step was performed using the result file from the dataset admixture analysis and by setting the threshold for the significance of The Maximisation strategy –32 impleEleven published primer pairs flanking nuclear microsatellites were employed to investigate the level of genetic variation among the 489 Italian olive varieties analyzed in this study and present in the olive germplasm collection of the CRA-OLI. A total of 84 alleles over 11 loci were detected, ranging from 3 at UDO01 locus to 12 alleles at both UDO39 and DCA9 loci. The shortest allele among the 11 polymorphic loci was allele 108 base pairs (bp) at UDO39, whereas the longest was 259 bp at GAPU71A (Table S2). The most common size variant, namely, the allele 214 bp at locus GAPU71A, was found with a frequency of 0.485 and showed the highest value in the varieties within Molise region (0.813). Three out of 84 alleles were considered private alleles, since they were present only in “Cellina di Nardò” , in “Arancino,” “Ciliegino,” “Emilia,” “Grappolo,” “Gremignolo,” “Gremignolo di Bolgheri,” “Lastrino,” “Lazzera reale,” “Salicino,” “Santa Caterina,” “Borgiona,” and “Corniolo” , and in “Filare”  with a frequency <1%.Ne) ranged from 2.3 to 6.7 with a mean of 4.3  was lower than expected (mean He = 0.664). The difference determines a significant positive value for the mean fixation index (F = 0.114) that could be attributed to the presence of null alleles , UDO03 (0.295), and UDO39 (0.214) loci, where null allele frequencies higher than 0.20 were found  obtained utilizing the UPGMA method that elaborates a matrix of similarity obtained using NTSYSpc program version 2.02  was testParent-offspring relations were found for “Giarraffa” and “Pizzo di Corvo,” “Nera di Oliena” and “Paschixedda,” “Cazzarella” and “Sperone di Gallo,” “Grossa di Venafro” and “Paesana Nera,” “Paesana Bianca” and “Rosciola di Rotello,” “Racemo 1” and “Coratina,” “Rossina” and “Selvatico,” and “Toccolana” and “Olivetta nera,” and these eight cultivar pairs differ by only one allele . Parent-Furthermore, 22 cases of homonymy were identified. These 22 cases of homonymy can be divided into two groups according to the number of different alleles. The first group is represented by plants that have a number of different alleles less than ten. The list of homologies is shown in Finally, SSR analysis allowed the classification of the CRA-OLI olive germplasm into 439 unique molecular profiles corresponding to well-defined genotypes, whereas, for the remaining molecular profiles, they reveal the presence of accessions considered as clones or possible synonyms of the same genotype.Genetic structure was tested using two different approaches. First, the PCoA, performed on Nei's unbiased genetic distance matrix and, based on 62 different size variants, showed that the 439 olive varieties were separated into five main groups .Group I contains olive varieties of Molise (23), Tuscany (79), Abruzzo (23), Basilicata (28), Apulia (35), and Sicily (64). Group II comprises Calabria (34) and Veneto (3) varieties. Group III include the olive varieties present in the regions of Lombardy (1), Lazio (24), Liguria (12), Marche (19), Umbria (21), and Emilia-Romagna (12), while Group IV contains varieties of Campania (42) and Sardinia (16). Group V only contains varieties from Friuli-Venezia-Giulia (3) region and is slightly more distant from genetic groups previously described. In the PCoA analysis, the first two principal axes explain a total of 68.9% of unbiased genetic distance, with 54.4% and 14.5% for coordinates 1 and 2, respectively .Bayesian clustering algorithms, such as those implemented in BAPS 5.3 program, were used for inferring olive Italian germplasm structure . Seven gOur analysis showed that all the olive varieties present in Northern and Central Italy were grouped in a single genetic cluster, with the exception of the varieties present in the regions of Tuscany, Abruzzo, and Molise that were grouped into two separate clusters . The BAPHo in each genetic cluster identified and ranged from 0.680 to 0.852 with a mean of 0.755, than He levels (mean 0.724) producing a negative value of F (mean −0.045)  .FST and RST estimators among BAPS groups (FST = 5.7 and RST = 5.2), with a higher ΦPT estimator value (ΦPT = 10.9) than those reported in literature for Olea [In addition, the AMOVA analysis revealed comparable values of for Olea . All of The analysis of the CRA-OLI Italian olive germplasm collection performed with a Bayesian clustering software, demonstrated a good network of gene flows between the clusters identified in this study. This analysis also revealed how only the clusters that grouped the olive varieties present in Sicily (64), Tuscany (79), Sardinia (16), Apulia (35), Basilicata (28), and Calabria (34) and those included in Northern and Central Italy were more susceptible to the identified gene flows, with a consequent transfer of genetic material . The anaA core collection was herein assembled for Italian olive germplasm, aiming to represent the entire genetic diversity identified in this study. The COREFINDER analysis based on M-strategy showed that, for Italian olive germplasm, 100% of the SSR alleles found in this study could be represented by a core collection of 23 accessions  further provided support for the existence of genetic structure in CRA-OLI germplasm collection and separated the Italian olive varieties into seven main clusters. The Bayesian model-based analysis highlights the real structure and distribution of Italian olive germplasm gene pools, separating the major genetic cluster 6 that grouped olive varieties in Northern and Central Italy, from the other six gene pools found. In addition, BAPS analysis results show the genetic relationship, represented by gene flows, among the seven clusters identified, confirming that the current gene pools and distribution of Italian olive germplasm are due to geographic and cultural aspects mainly involving human activity in the past.PT estimator value (ΦPT = 10.9) than those reported in literature for Olea europaea [Moreover, the AMOVA results surprisingly revealed a higher Φeuropaea , showingHo and He detected in BAPS clusters, with a mean negative value of F that clearly highlights the good levels of genetic diversity, maintained costant in each BAPS group identified in this study.This result was confirmed by higher levels of Olea [The final aim of the study was to construct a valid core collection for cultivated olives in the CRA-OLI germplasm collection, sampling the minimum number of entries that maximize the representativeness of allelic diversity. The core collection that we proposed in this study consists of 23 varieties that capture 100% of the base collection. It was found that only a small number of olive varieties, compared with other values reported in literature for Olea . Van HinOlea  suggesteex situ collection could be considered representative of Italian olive germplasm because each genetic group defined in BAPS reflects geographic distribution and confirms that the Italian olive germplasm is a peculiar gene pool present in the Mediterranean basin.Finally, the result of our BAPS analysis supports both NTSYS and PCoA results, demonstrating how all olive accessions analyzed in this study and maintained in CRA-OLI ex situ germplasm collection.Concluding, the use of molecular markers, like microsatellite, is imperative to build a database for cultivar analysis, for the traceability of processed food, and for the appropriate management of olive germplasm collections. Moreover, the results presented here regarding clustering and core collection are extremely useful for the selections of parents to be used for breeding programs and thus ensuring an optimal management of the CRA-OLI Italian olive germplasm collection. This work is the first study with such a large number of Italian olive varieties analyzed using the same set of molecular markers which allowed characterisation of the genetic structure and identification of a core collection in the largest Italian Olea europaea) germplasm analyzed by SSR markers” by Muzzalupo et al. is composed of three tables.The electronic supplementary material of the research article “Genetic biodiversity of Italian olives  germplasm collection and analyzed by eleven microsatellite markers. The Italian olive varieties included in the core collection are indicated in bold.Table S1: List of the Table S2: Alleles detected at the eleven SSR loci in the 489 olive trees analyzed. For each allele, the size and the frequency are reported.Table S3: Genotype profiles obtained from the combination of the eleven microsatellite markers on 489 olive varieties. The Italian olive varieties included in the core collection are indicated in bold.Click here for additional data file.


Chlamydia trachomatis infection. Suriname, a former Dutch colony, also has a high prevalence of C. trachomatis. Surinamese migrants travel extensively between the Netherlands and Suriname. Our objective was to assess whether the Surinamese migrants in the Netherlands form a bridge population facilitating transmission of C. trachomatis between Suriname and the Netherlands. If so, joint prevention campaigns involving both countries might be required.The large Surinamese migrant population in the Netherlands is a major risk group for urogenital C. trachomatis positive samples were typed through multilocus sequence typing (MLST). A minimum spanning tree of samples from 426 participants showed four MLST clusters. The MLST strain distribution of Surinamese migrants differed significantly from both the native Surinamese and Dutch populations, but was not an intermediate state between these two populations. Sexual mixing between the Surinamese migrants and the Dutch and Surinamese natives occurred frequently. Yet, the MLST cluster distribution did not differ significantly between participants who mixed and those who did not. Between March 2008 and July 2010, participants were recruited at clinics in Paramaribo, Suriname and in Amsterdam, the Netherlands. Participants were grouped as native Surinamese, native Dutch, Surinamese migrant, Dutch migrant, or Other, based on country of residence and country of birth of the participant and of their parents. Risk behavior, such as sexual mixing between ethnic groups, was recorded and C. trachomatis transmission between the native populations. Although our data do not seem to justify the need for joint campaigns to reduce the transmission of C. trachomatis strains between both countries, intensified preventive campaigns to decrease the C. trachomatis burden are required, both in Suriname and in the Netherlands.Sexual mixing occurred between Surinamese migrants in Amsterdam and the native populations of Suriname and the Netherlands. These migrants, however, did not seem to form an effective bridge population for  Chlamydia trachomatis infections occur endemically among the general population of the Netherlands, but most infections are found in defined risk groups, such as adolescents and men who have sex with men (MSM) C. trachomatis infections C. trachomatis has been reported in this population. In 2011, the C. trachomatis prevalence among clients of the Dutch sexually transmitted infection (STI) clinics was 18% for heterosexual Surinamese migrants compared with 11% for native Dutch heterosexuals C. trachomatis infections and other STIs are still lacking for the majority of its inhabitants C. trachomatis infections is high; one study demonstrated around 10% prevalence in a low-risk population (clients of a birth control clinic) and 21% in a high-risk population (clients of an STI clinic) Suriname is a former colony of the Netherlands in the Caribbean region. It is a multi-ethnic society consisting of Creoles, Maroons, Hindustani, Javanese, Chinese, Caucasians, and indigenous Amerindians, as well as a mixed race population. Although considered an upper middle-income country by World Bank standards, Suriname is an emerging economy in which reliable diagnostics to detect Since the independence of Suriname in 1975, a large proportion of the Surinamese population migrated to the Netherlands and today almost as many people of Surinamese origin live in the Netherlands as in Suriname itself. Amsterdam has the largest number of Surinamese inhabitants outside of Suriname. As a result, traveling between the two countries is common. A study in 2008 found that more than half of the population of Surinamese descent living in the Netherlands had visited friends and relatives in Suriname during the preceding five years Discordant sexual mixing is defined as sex between partners from two different groups (e.g. with different age or ethnicity) C. trachomatis between Suriname and the Netherlands. If this were the case, this would be reflected by:We hypothesized that Surinamese migrants constitute a bridge population for the transmission of A high degree of sexual mixing with native Surinamese and native Dutch partners, andC. trachomatis genotypes that is more similar to the native Surinamese or native Dutch population among the Surinamese migrants who report sexual mixing with the native Surinamese or native Dutch population respectively, compared with those that do not report sexual mixing.A distribution of C. trachomatis positive urogenital samples in the two capital cities: Paramaribo, Suriname; and Amsterdam, the Netherlands, and genotyped the C. trachomatis strains with a high-resolution multilocus sequence typing (MLST) method C. trachomatis strains on a population level in a previous study C. trachomatis strain clusters. In a previous study, this approach successfully demonstrated the distinct transmission of C. trachomatis strains in MSM and heterosexuals We collected Participants were recruited at two sites in Paramaribo, Suriname and at one site in Amsterdam, the Netherlands:The Dermatological Service in Paramaribo, an integrated outpatient clinic that offers free-of-charge examination and treatment of STIs and infectious skin diseases such as leprosy and leishmaniasis. Recruitment took place between March 2008 and July 2010.The Lobi Foundation, a center for birth control and sexual health in Paramaribo. Recruitment took place between July 2009 and April 2010.The STI Outpatient Clinic of the Public Health Service of Amsterdam, which is a low threshold clinic serving over 30,000 clients annually. Individuals were prioritized based on a short questionnaire to estimate the risk of having an STI Bom et al. (2013) Van der Helm et al. (2012) Exclusion criteria were: age younger than 18 years, antibiotic use in the previous 7 days, men having sex with men in the past 6 months, and previous participation in this study. After written informed consent, participants were given a unique code to participate anonymously. Participants were interviewed about demographic characteristics, including place of birth, place of birth of both parents, ethnicity of sexual partners, number of sexual partners, and place of residence of their sexual partners. The ethics committees of the Ministry of Health of the Republic of Suriname (VG010-2007) and the Academic Medical Center, University of Amsterdam, the Netherlands (MEC07/127) approved the study. Part of the data collected in the Netherlands has been described previously by Chlamydia assay for the detection of C. trachomatis rRNA  at the Public Health Laboratory in Amsterdam. The urine samples were tested within 40 days, and the vaginal swabs within 50 days after collection. A more detailed description has been reported in a previous study C. trachomatis positive sample was selected. For female participants, vaginal samples were preferred; if vaginal samples were not available, cervical samples were selected. A more detailed description has been reported in a previous study In Paramaribo, urine samples from men and nurse-collected vaginal swabs were obtained, shipped and tested with the Aptima C. trachomatis-positive clinical samples were extracted and tested for the presence of chlamydial DNA ompA, CT046 (hctB), CT058, CT144, CT172, and CT682 (pbpB) Chlamydia trachomatis MLST database (mlstdb.bmc.uu.se). Only samples of which all 6 loci were successfully amplified, sequenced, and identified, and therefore had obtained a full sequence type (ST) or MLST profile, were included in the analyses. As ompA is part of the MLST scheme, genovars could be assigned for all included samples. A minimum spanning tree was generated using MLST profiles. As the number of STs was too large to be used in statistical analyses, cluster analysis was performed, allowing single locus variance using BioNumerics 7.0 . A cluster was defined as a group of STs differing by not more than 1 locus from another ST within that group. Only clusters of at least 25 samples were included in the cluster analysis as these clusters were large enough for statistical analyses. The remaining samples were compiled in a residual group, which was used in the analyses as an additional cluster.Nucleic acids from Participants were classified into 5 groups based on country of residence of the participant, and country of birth of the participant, and of his or her parents Figure 1Native Surinamese group: Participants residing in Suriname; neither the participant, nor the parents were born in the Netherlands, and at least one was born in Suriname;Native Dutch group: Participants residing in the Netherlands; neither the participant, nor the parents were born in Suriname, and at least one was born in the Netherlands;Surinamese Migrant group: Participants residing in the Netherlands; the participant and/or his or her parents (one or both) were born in Suriname;Dutch Migrant group: Participants residing in Suriname; the participant and/or his or her parents (one or both) were born in the Netherlands;Other groups: A participant neither residing in Suriname nor in the Netherlands; or neither the participant nor his or her parents were born in Suriname or the Netherlands.Sexual partners in the past 12 months were classified into three groups based on country of residence of the sexual partner, and the ethnicity of the sexual partner, as perceived by the participant:Native Surinamese partner: sexual partner residing in Suriname and of perceived Surinamese ethnicity;Native Dutch partner: sexual partner residing in the Netherlands and of perceived Dutch ethnicity;Surinamese Migrant partner: sexual partner residing in the Netherlands and of perceived Surinamese ethnicity.χ2 tests for categorical data. Fisher's exact tests were used when the expected cell count was less than one. For continuous data, Mann-Whitney U tests and Kruskal-Wallis tests were used. A p-value of <0.05 was considered statistically significant. Analyses were performed with SPSS package version 19.0 . To test whether the distribution of C. trachomatis clusters found within the Surinamese migrant population was an intermediate state between the distributions of the native Surinamese and native Dutch populations, a range of hypothetical intermediate cluster distributions was calculated. The proportion of every cluster within a hypothetical intermediate state (hyp_ip) was calculated by:SUR_ip is the proportion of cluster i in the native Surinamese population, NL_ip is the proportion of cluster i in the native Dutch population and x ranges from 0% to 100% with a step size of 1%. With x = 0%, the hypothetical distribution would be identical to the distribution found in the native Surinamese population, whereas x = 100% would result in a distribution identical to the one found among the native Dutch. To test whether these hypothetical intermediate distributions differed significantly from the distribution found among the Surinamese migrants, the proportions of each cluster were multiplied by the total number of Surinamese migrants and compared to the actual distribution of clusters found among the Surinamese migrants using Pearson's 2χ tests.A partner of perceived Surinamese ethnicity was defined as a partner with a Creole, Hindustani, Javanese, Chinese, Maroon, Amerindian, or mixed race ethnicity. A partner of perceived Dutch ethnicity was defined as a partner with a Caucasian ethnicity. Differences between clusters were tested using Pearson's C. trachomatis. A total of 508 (96%) C. trachomatis positive samples were available for further testing, of which 219 originated from Paramaribo and 289 from Amsterdam. In 450 samples (89%), the presence of genomic DNA could be demonstrated by qPCR. Of these 450 samples, 426 (95%) could be completely typed by MLST, of which 170 originated from Paramaribo and 256 originated from Amsterdam.A total of 415 men and 1093 women participated in Paramaribo and 449 men and 1051 women participated in Amsterdam. Of those, 95 (23%) men and 129 (12%) women in Paramaribo and 100 (22%) men and 204 (19%) women in Amsterdam tested positive for C. trachomatis positive participants, with a complete MLST profile by city. The median age of the participants in Paramaribo was higher : 22–30 years) compared with those from Amsterdam . In addition, participants from Paramaribo usually had received less education (p<0.001) and reported fewer sexual partners (p = 0.011).ompA is part of the MLST scheme, genovars could be assigned for all typed samples. In both cities we found 9 different genovars, being B, and D through K. The genovar distribution found in Paramaribo and Amsterdam was not significantly different (p = 0.59) with genovars D, E, F and I being most common (As t common Table 1.Table S1). Of these STs, 29 had multiple representatives (n = 2–28) and 36 were found in only a single isolate (singleton). Among the 256 Amsterdam samples, 124 different STs were found (Table S1). Multiple representatives were seen for 34 STs (n = 2–28) and 90 singletons were found. In the total population of the 2 cities, we identified 166 STs (n = 1–46) of which 106 were singletons.Among the 170 MLST profiles from Paramaribo there were 65 different STs . Cluster 1 included 105 individuals and consisted mainly of genovar F samples (77%), but also of genovar D (19%) and J (4%). Cluster 2 (n = 76) and cluster 3 (n = 62) included solely genovar E samples. Cluster 4 (n = 53) consisted predominantly of genovar I (89%), but also of genovar J (11%). Among the residual samples (n = 130), all genovars were present, but genovars D (31%), G (19%), E (14%), K (12%) and J (10%) were most common.From the MLST profiles of these 426 samples, a minimum spanning tree was generated Figure 3p<0.001, In contrast to the genovar distribution, the distribution of samples in the MLST clusters was strongly associated with the city of sampling p<0.001, Figure 2p = 0.003) and ethnic groups (p<0.001) were observed between the clusters (Table S2). The participants in cluster 2 had received more education, whereas participants in clusters 3 and 4 had received less education; these differences are largely due to the city of origin of the participants. Clusters 3 and 4 had more native Surinamese participants than the other clusters and cluster 2 had more native Dutch participants. Cluster 4 had more Surinamese migrant participants compared with the other clusters.Demographic characteristics were compared for the four large clusters and the residual group. Gender, age, and number of sexual partners in the past 12 months did not differ significantly between the clusters. However, significant differences in education . In the analysis for the samples from Paramaribo, no significant differences were found between the clusters and any of the variables tested . For the Amsterdam samples, no significant differences were found between the clusters and gender, age, education, or number of sexual partners, but a very significant difference was found for ethnic group (p<0.001): 73% of samples in cluster 4 were from migrant Surinamese participants, while this group represented only 24% of the participants from Amsterdam (Table S3).Because the distribution of Table S4).When we analyzed the data stratified for three main ethnic groups, no significant differences were found between the clusters and any of the variables tested for the native Surinamese, the native Dutch, or the Surinamese migrants , Surinamese migrant participants (n = 64), and native Dutch participants  reported having had sex with a native Surinamese partner in the past 12 months, and 13% (n = 20/153) of the native Surinamese participants reported having had sex with a Surinamese migrant partner Figure 4p = 0.002). Compared with the native Dutch participants, samples from the Surinamese migrant participants more often belonged to cluster 4 and less often to cluster 2 and the residual group (p<0.001). In addition, the distribution of the clusters found among the Surinamese migrants differed significantly from any intermediate state between the distribution found among the native Surinamese and native Dutch (figure S1).Clear differences were found in the distribution of the clusters, including the residual group, between the three main ethnic groups: native Surinamese, Surinamese migrants and native Dutch Figure 5C. trachomatis strains found among the Surinamese migrant participants did not differ significantly between those who reported sex with a native Surinamese partner and those who did not . The same was observed for the Surinamese migrant participants: no significant differences were found in C. trachomatis strain distribution between participants who reported and those who did not report sex with native Dutch partners (p = 0.66). Also among native Dutch participants no significant differences were found in C. trachomatis strain distribution between those who reported sex with Surinamese migrant partners and those who did not (p = 0.34).The distribution of C. trachomatis strains between infected persons in Paramaribo and Amsterdam, and between Surinamese migrants and native Dutch participants within Amsterdam using the high-resolution MLST genotyping technique. These differences would have been largely obscured if conventional ompA typing had been used. For example, cluster 2 infections were more prevalent in Amsterdam and cluster 3 infections were more prevalent in Paramaribo, but both clusters were composed of MLST sequence types with an identical ompA type, genovar E, which is the most prevalent genovar type worldwide In this study, we showed differences in the distribution of C. trachomatis strains between inhabitants in Suriname and the Netherlands. We demonstrated that sexual mixing among Surinamese migrants occurred, both with native Surinamese and with native Dutch partners (13% and 30%). Sexual mixing between the native Dutch and native Surinamese groups occurred infrequently (1–3%). This indicates, based on reported sexual mixing, that Surinamese migrants potentially constituted a bridge population for STI between the Dutch and Surinamese populations. However, the distribution of C. trachomatis strains found in the Surinamese migrant population differed from both the native Surinamese and Dutch distributions, but was not an intermediate state between the native groups. This is most strikingly illustrated by the overrepresentation of Surinamese migrants in cluster 4, whereas cluster 4 strains are less prevalent among the native Surinamese population and rare in the native Dutch population. Therefore we conclude that limited transmission of C. trachomatis strains occurred between the native Surinamese and Surinamese migrant populations, and between the native Dutch and Surinamese migrant populations. In addition, when we examined the distribution of C. trachomatis strains among the participants that reported sexual mixing and those who did not, no significant differences could be found between the two in the distribution of C. trachomatis clusters. Although the power to show differences between these groups was limited, transmission patterns that could be expected were not observed. For example, among native Dutch participants who mix with Surinamese migrants more cluster 4 strains were expected compared to those who do not mix, as these strains were highly prevalent among the Surinamese migrant participants. However, just 5 Dutch participants were diagnosed with a cluster 4 strain and only 1 of them reported sex with Surinamese migrant partner past 12 months. Consequently, the high prevalence among the Surinamese migrants compared with the native Dutch cannot be explained by sexual mixing with native Surinamese.We hypothesized that the Surinamese migrant population residing in the Netherlands, who travel frequently between the two countries, acted as a bridge population for the transmission of C. trachomatis strains among the Surinamese migrants might be explained by the absence of effective sexual mixing with the native Dutch and Surinamese populations. When subpopulations do not mix effectively, infections cannot spread to other risk populations, and differences in prevalence are sustained C. trachomatis infections is associated with socio-economic status and partnership structures C. trachomatis strains can be explained assuming that the chlamydial populations in communities that do not mix are more susceptible to stochastic effects.In fact, the opposite may be true; both the high prevalence and the divergent distribution of C. trachomatis strains circulating in both countries. Participants completed a questionnaire that was designed to study sexual networks, so detailed epidemiological data were available and these could be linked to high-resolution typing results of the pathogen. Together, these factors enabled us to study the determinants of the specific groups for the transmission of C. trachomatis. Although the data of the participants' ethnic origin was precise (based on their own assessment), the data of their sexual partners was limited to ‘perceived ethnicity’. Therefore the amount of sexual mixing between the groups may be overreported, especially for native Dutch and Surinamese migrant partners. The native Dutch partners were defined as a partner of a perceived Caucasian ethnicity living in the Netherlands. Likely, this group encompasses more than only the native Dutch, as Caucasian minorities also reside in Amsterdam. More biased are the Surinamese migrant partners, as they were defined as a partner of a perceived Creole, Hindustani, Javanese, Chinese, Maroon, Amerindian, or ‘mixed race’ ethnicity living in the Netherlands. Whereas these ethnic groups are specific for native and migrant Suriname individuals, some of these ethnicities are not distinguishable for native Dutch participants, especially the ‘mixed race’ ethnicity. In addition, native Dutch individuals might be unaware of Surinamese ethnic groups and therefore underreport them.We were able to include a large number of participants from Suriname and the Netherlands, within a timespan of just over 2 years. Consequently we obtained an accurate representation of C. trachomatis strains circulating in Suriname and the Netherlands, and the limited transmission of these strains between the two countries. Although our data do not seem to justify the need for joint campaigns to reduce the transmission of C. trachomatis strains between both countries, intensified preventive campaigns to decrease the C. trachomatis burden are required, both in Suriname and in the Netherlands. Moreover, informing travelers about the risks of unprotected sex abroad is still relevant. In addition, the high prevalence of C. trachomatis within the Surinamese migrant population justifies further studies into risk factors and transmission networks of C. trachomatis within this group. This will enable more effective tailored prevention programs to reduce the C. trachomatis burden.Despite the limitations, these data provide a good view of the Figure S1Test for intermediacy. Depicted are the p-values of the C. trachomatis strain distribution found among the Surinamese migrants compared with the distributions of the hypothetical intermediate states, using Pearson's χ2 tests. These intermediate states ranged from 100% identical to the native Surinamese distribution to 100% identical to the native Dutch distribution. None of these intermediate states had a p-value of ≥0.05, therefore the distribution of C. trachomatis strains found among the Surinamese migrants was not an intermediate state between the distributions found among the native Surinamese and native Dutch populations.(TIF)Click here for additional data file.Table S1MLST-data of the 426 samples collected in Paramaribo, Suriname and Amsterdam, the Netherlands, 2008–10. Coding is according to the Chlamydia trachomatis MLST database (mlstdb.bmc.uu.se). Samples S1001 to S1170 were collected in Paramaribo, and samples 3003 to 3329 were collected in Amsterdam.(DOC)Click here for additional data file.Table S2Characteristics of Chlamydia trachomatis-positive participants, by C. trachomatis cluster, from Paramaribo, Suriname and Amsterdam, the Netherlands, 2008–10.(DOC)Click here for additional data file.Table S3Characteristics of Chlamydia trachomatis-positive participants, by C. trachomatis cluster. From Paramaribo, Suriname, 2008–10 (A), and from Amsterdam, the Netherlands, 2009–10 (B).(DOCX)Click here for additional data file.Table S4Chlamydia trachomatis-positive native Surinamese participants, by C. trachomatis cluster, 2008–10 (A). Characteristics of Chlamydia trachomatis-positive native Dutch participants, by C. trachomatis cluster, 2009–10 (B). Characteristics of Chlamydia trachomatis-positive Surinamese migrant participants, by C. trachomatis cluster, 2009–10 (C).Characteristics of (DOCX)Click here for additional data file.


For human immunodeficiency virus, however, a live attenuated strain could present substantial safety concerns. We have used the live attenuated rubella vaccine strain RA27/3 as a vector to express SIV and HIV vaccine antigens because its safety and immunogenicity have been demonstrated in millions of children. One dose protects for life against rubella infection. In previous studies, rubella vectors replicated to high titers in cell culture while stably expressing SIV and HIV antigens. Their viability in vivo, and the protein inserts were highly immunogenic. Antibody titers elicited by the SIV Gag vector were greater than or equal to those elicited by natural SIV infection. The antibodies were long lasting, and they were boosted by a second dose of replication-competent rubella vectors given six months later, indicating the induction of memory B cells.This paper reports the first successful trial of rubella vectors in rhesus macaques, in combination with DNA vaccines in a prime and boost strategy. The vectors grew robustly Rubella vectors can serve as a vaccine platform for safe delivery and expression of SIV and HIV antigens. By presenting these antigens in the context of an acute infection, at a high level and for a prolonged duration, these vectors can stimulate a strong and persistent immune response, including maturation of memory B cells. Rhesus macaques will provide an ideal animal model for demonstrating immunogenicity of novel vectors and protection against SIV or SHIV challenge. Despite the urgent need for a safe and potent vaccine against HIV, efforts to produce a vaccine have been thwarted by antigenic variation, weak immunogenicity of critical epitopes, and short duration of the immune response to HIV vaccine antigens -3. For oWe have recently identified two insertion sites in the rubella genome, where a foreign gene could be inserted without compromising rubella replication in cell culture -19. The in vivo was compromised by the Not I deletion. New generation vectors, with structural site inserts and the Not I deletion restored, grew robustly in vivo, while expressing SIV and HIV vaccine antigens at high levels.The second insertion site was located in the structural region, between envelope proteins E2 and E1 . This siThe rubella vaccine strain RA27/3 readily infects rhesus macaques . The preThe rubella genome is a 9.7 kb positive sense single-stranded RNA Figure A. Genes Three types of vectors were produced, with MPER or Gag sequences inserted into the RA27/3 rubella vaccine background. Type 1 vectors had an insert at the structural site and a compensatory deletion at the Not I site, as described previously . Type 2 Vector replication in Vero cells was monitored by Western blot with antibodies to the rubella structural proteins C and E1  with a panel of five macaques infected with SIVmac251 Figure C. In eacThe kinetics of the response to SIV Gag are shown for individual macaques Figure  to illusIn contrast, macaques CL6V Figure C and CL6The antibody response to the HIV MPER insert was measured by ELISA on plates coated with gp140 SOSIP trimers Figure . High-tiWe measured the persistence of anti-Gag antibodies in group 3 macaques for nine months after immunization. We compared this to the persistence of anti-rubella antibodies in the same animals Figure . Anti-ruWe measured anti-Gag antibodies in the same macaques at the same time points  or 6 months (group 1), we boosted the macaques with rubella vectors expressing MPER and SIV Gag antigens (week 57 of the study). All five animals showed a prompt rise in antibodies to rubella (data not shown).We measured the anti-SIV Gag antibody response four weeks after the boost ppp(5’)G cap analog . To generate rubella virus, the RNA was transfected into Vero cells, using DMRIEC reagent . After 6–9 days of infection at 37°C, culture supernatant containing virus (passage P0) was collected. Infected cells were transferred onto fresh Vero cells to start a new passage. After several passages, passaging was done with cell-free culture supernatant. To produce virus stock, virus production was scaled up in T75 flasks after 5–6 passages. The supernatant was collected, titered by quantitative RT-PCR and the vector insert was sequenced. Purification of viral RNA from viral supernatants and reverse transcription were performed using QIAamp UltraSens Virus kit (QIAGEN) and High Capacity RNA-to-cDNA Kit (Applied Biosystems), respectively. The cDNA was PCR amplified with illustra PuReTaq Ready-To-Go PCR beads  and primers specific for rubella sequences flanking an insertion site, then purified in agarose gel and sequenced using the same primers.The protocols for generating infectious rubella RNA from plasmid DNA, transfecting cells, passaging virus in Vero cells, viral stock expansion, sequencing the inserts, determining viral RNA content of rubella vector stocks and their titers were the same as described previously ,18. BrieRubella structural proteins were detected by Western blot with goat anti-rubella antibodies specific for capsid protein C and envelope protein E1, as described previously ,18. ExprGuide for the Care and Use of Laboratory Animals. To be selected for the study, all animals passed a screening complete physical exam and a veterinary evaluation of their medical history including serology. Animals were negative for antibodies to rubella, herpes B, STLV-1, SIV, SRV  1, 2 and 3. During the study period, the animals were negative for Shigella, Salmonella, Yersinia, Campylobacter and tuberculosis (TB) and free of intestinal pathogenic parasites.Rhesus macaques, between 3 and 16 years of age were obtained from the CBER NHP colony on the NIH campus. All but two were of Indian origin; the two of unknown origin were V200 and V584. The CBER animal research program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All experimental procedures were approved by the CBER Institutional Animal Care and Use Committee and were done in compliance with the current edition of the Depending on their size, the macaques were housed in 6–12 sq ft cages and were habituated for more than 3 months. Before entry into the study, all macaques were weighed and tissue typed. They were confirmed seronegative for SIV Gag and rubella antibodies prior to receiving the vectors. All animals received ketamine anesthesia (10 mg/kg/im) for restraint during immunization, bleeding and taking oral swabs.The 12 macaques were divided into three vaccine groups of three to four each and a control group of two animals Figure . Group 3in vivo electroporation using the Elgen 1000 device .For group 1 macaques, the first two doses of DNA vaccine consisted of four plasmid DNAs: 1 mg DNA coding for SIV gag, 1 mg env DNA (gp140 and gp160), and 200 μg coding for monkey IL-12 . The firEach rubella dose consisted of a pair of rubella vectors of the same type, expressing HIV MPER and SIV Gag determinants. For group 3, the first rubella dose consisted of type 1 vectors, given IM at a dose of 10,000 PFU, followed by a second dose of 30,000 PFU. The next dose was a type 2 vector at 50,000 PFU. Finally, the type 3 vectors were given IM at a dose of 50,000 PFU. Group 1 animals received three doses of DNA vaccine, followed by a single dose of type 3 vectors at 50,000 PFU. This corresponds to about ten times the typical human dose of rubella vaccine.Blood samples were taken before each vaccine dose and one, two and six weeks after the dose. Mouth swabs were also taken before each dose of rubella vector and one and two weeks later. The blood samples were analyzed by ELISA for antibodies to rubella, SIV Gag and HIV MPER. They were also analyzed by tetramer staining for T cells specific for the CM9 epitope. Mouth swabs were analyzed by RT-PCR using primers specific for the gag and MPER inserts.To determine whether rubella virus inoculated in the quadriceps muscle could propagate throughout the body, we used a reverse transcription semi-nested PCR method to detect rubella sequences in oral fluid specimens from immunized rhesus macaques. The oral fluid specimens were obtained as gingival mouth swabs, using cotton swabs prior to each dose of vectors, and again one and two weeks later, using the procedure recommended by CDC ,47. AfteMonkey antibodies to rubella were detected by ELISA in duplicate wells, as measured on plates coated with rubella antigens  at the recommended serum dilution of 1:40. Macaque antibodies to SIV Gag were detected on plates coated with recombinant p55 Gag protein. For this assay, soft plastic plates were coated with 1 μg/ml recombinant p55 SIV Gag overnight at 4°. They were blocked for 5 minutes with BSA (10 mg/ml). The antibodies were then titered on the plates in PBS with 0.1 mg/ml BSA and 0.05% Triton X100. Macaque antibodies to HIV MPER were detected on plates coated overnight at 4° with recombinant gp140 SOSIP trimers at 1 ug/ml.450 was determined in an ELISA plate reader (Thermo Scientific).Macaque antibodies to MPER peptides were titered on plates coated overnight with two peptides of 14 to 16 amino acids containing the 2F5 and 4E10 epitopes. For each antigen, the plates were incubated with antibodies for 1 hour at 37°, washed twice with the above buffer, and developed with goat anti-monkey IgG conjugated to horseradish peroxidase at a 1:5000 dilution . After 30 minutes at 37°, the plates were washed four times and developed with TMB substrate . After ten minutes at room temperature, the reaction was stopped with 1M HCl, and ODThe authors declare that they have no competing interests.KV designed the vectors and experiments, performed experiments, helped coordinating the study wrote the manuscript. MH planned and performed experiments and helped with revision of the manuscript. YN performed experiments and helped with revision of the manuscript. JB helped design the study, performed animal procedures and helped with revision of the manuscript. GNP and BKF helped designing and coordinating the study, provided DNA vaccine, helped with writing of the manuscript. IB designed the study and experiments, performed experiments, wrote the manuscript, coordinated and supervised the study. All authors read and approved the final manuscript.


There are limited number of studies investigating extrapulmonary manifestations of bronchiectasis. The purpose of this study was to compare peripheral muscle function, exercise capacity, fatigue, and health status between patients with bronchiectasis and healthy subjects in order to provide documented differences in these characteristics for individuals with and without bronchiectasis.Twenty patients with bronchiectasis (43.5 ± 14.1 years) and 20 healthy subjects (43.0 ± 10.9 years) participated in the study. Pulmonary function, respiratory muscle strength , and dyspnea perception using the Modified Medical Research Council Dyspnea Scale (MMRC) were determined. A six-minute walk test (6MWT) was performed. Quadriceps muscle, shoulder abductor, and hand grip strength  using a hand held dynamometer and peripheral muscle endurance by a squat test were measured. Fatigue perception and health status were determined using the Fatigue Severity Scale (FSS) and the Leicester Cough Questionnaire (LCQ), respectively.p < 0.05). In bronchiectasis patients, QMS was significantly associated with HGS, MIP and MEP (p < 0.05). The 6MWT distance was significantly correlated to LCQ psychological score (p < 0.05). The FSS score was significantly associated with LCQ physical and total and MMRC scores (p < 0.05). The LCQ psychological score was significantly associated with MEP and 6MWT distance (p < 0.05).Number of squats, 6MWT distance, and LCQ scores as well as lung function testing values and respiratory muscle strength were significantly lower and MMRC and FSS scores were significantly higher in patients with bronchiectasis than those of healthy subjects (Peripheral muscle endurance, exercise capacity, fatigue and health status were adversely affected by the presence of bronchiectasis. Fatigue was associated with dyspnea and health status. Respiratory muscle strength was related to peripheral muscle strength and health status, but not to fatigue, peripheral muscle endurance or exercise capacity. These findings may provide insight for outcome measures for pulmonary rehabilitation programs for patients with bronchiectasis. Bronchiectasis is a chronic pulmonary disease which is caused by the destruction of elastic tissue and smooth muscles on bronchial walls due to repeated severe infection or inflammation and characterized by abnormal permanent dilatation . PreviouBronchiectasis is a common health problem for adult populations in developing countries ; howeverThe study included 20 adult patients with bronchiectasis diagnosed in the Department of Chest Medicine by high resolution computed tomography  and 20 healthy subjects . Consecutive patients who were referred to the Cardiopulmonary Rehabilitation Unit participated in the study. The mean (± SD) number of lobes showing changes of bronchiectasis was 2.9 ± 1.0 . The mean (± SD) time from the diagnosis was 12.8 ± 11.8 years . Individuals who were using antibiotics, had neurologic or orthopedic disease, advanced heart failure, or an acute exacerbation within the last three weeks were not included in the study. The control group was composed of individuals who had no known systemic, orthopedic or neurologic disease, aged between 18–64 years, could walk and cooperate, and were voluntary subjects among the relatives of researchers and academic personnel. Participants were informed about the aim and scope of the study, and written consent was obtained from each participant. This prospective study was approved by Ethical Committee of Hacettepe University.2 (kg/m2). Fat free mass (FFM) was determined using the skinfold method  from biceps, triceps, subscapular, and suprailiac regions. Measurements were repeated three times and the mean of three measurements was used [Physical, physiological and socio-demographic characteristics of the subjects were recorded. Body Mass Index (BMI) was calculated by the formula of weight/heightwas used .1), FEV1/FVC, peak expiratory flow (PEF) and forced expiratory flow 25–75% (FEF25–75%) values were recorded. Parameters of the respiratory function test were expressed as percentages of the expected values in accordance with the subject’s age, height, body weight and sex [The pulmonary function test was performed in sitting position using a spirometer . Forced vital capacity (FVC), forced expiratory volume in one second . Maximal inspiratory and expiratory mouth pressures (MIP and MEP) were recorded. MIP was measured at residual volume, while MEP was determined at total lung capacity. Measurements were repeated three times to avoid any difference larger than 10% or 10 cm H and sex .Perception of dyspnea was evaluated by the Modified Medical Research Council (MMRC) dyspnea scale. The MMRC is a 0–4 point category scale which selects the best expression to define the dyspnea levels among five expressions related to dyspnea .Peripheral muscle strength of the participants  was evaluated by digital dynamometer . Measurements were repeated three times for the dominant side . The higFor the six-minute walk test (6MWT), subjects were asked to walk along a flat corridor at their walking speed as quickly as possible for six minutes . In pre-The Fatigue Severity Scale (FSS) was used to estimate the fatigue level of the subjects . The FSSHealth status was assessed using the Leicester Cough Questionnaire (LCQ). The LCQ is composed of 19 items and includes physical (8 items), psychological (7 items) and social (4 items) sub-dimensions. Each item is scored between 1  and 7 (never). The score of each sub-dimension ranges between 1 and 7. Total score ranges between 3 and 21. Low scores on the LCQ indicate a higher effect of coughing on the subject [t-test was used for the comparison of variables suitable for normal distribution. Mann–Whitney u-test was used to compare nonparametric variables not suitable for normal distribution. Comparison of the variables determined by counting was performed by the Chi-Square test. Corrected correlations  were used to investigate association among the variables. The level of significance was set to p < 0.05.SPSS 15.0 packet software was used for the statistical analysis . Variablp > 0.05, Table 1, FEV1/FVC, PEF, FEF25-75%, MIP, and MEP were significantly lower in patients with bronchiectasis compared to healthy subjects , MIP  and MEP  in patients with bronchiectasis. The SAS was significantly correlated with MEP , and HGS was significantly associated with MIP .Although no significant difference was found between subjects with bronchiectasis and healthy subjects regarding peripheral muscle strength measures .The number of squats was significantly lower in subjects with bronchiectasis compared to healthy subjects . The 6MWT distance was significantly related to LCQ psychological score  in patients with bronchiectasis.The 6MWT distance and 6MWT% distance were significantly lower in subjects with bronchiectasis when compared to the healthy group, while exercise dyspnea perception was significantly higher in subjects with bronchiectasis , LCQ total score  and MMRC score .Thirteen patients (65%) with bronchiectasis reported having severe fatigue. The FSS value was significantly higher in the bronchiectasis compared to the healthy group , and physical score was significantly and inversely related to FSS . The LCQ psychological score was significantly associated with MEP  and 6MWT distance .The LCQ total score and the scores of physical, psychological and social sub-dimensions were significantly lower in the bronchiectasis group than in healthy subjects , Newall et al. , and Murray et al.  [In the present study, MIP and MEP were decreased in bronchiectasis. The MIP was lower than 80% of the expected value in four subjects, and MEP was lower in 15 subjects. This finding is considered significant as it demonstrates the presence of respiratory muscle weakness which is especially evident for expiratory muscles in subjects with bronchiectasis. Respiratory muscle strength has been measured for adult patients with bronchiectasis in a limited number of studies in literature ,25,26. I5 cmH2O) ,25,26 prDyspnea is seen in 60% of patients with bronchiectasis ,24. We uIn a comprehensive evaluation of skeletal muscle function, peripheral muscle strength and peripheral muscle endurance must be considered together . In thisIn previous studies investigating exercise capacity of adult patients with bronchiectasis, exercise performance was reported to be reduced -9. In thIn recent years, fatigue has been reported to be commonly present in chronic pulmonary disease. At clinical investigation, it was determined to be present in as many as 74% of patients ,31. In a1 and daily amount of produced mucus are the factors independently affecting health status in patients with bronchiectasis [In the limited number of studies examining health status of patients with bronchiectasis, impaired health status was found using a disease-specific standard questionnaire ,25,32-36iectasis . In the iectasis . Accordivs. without bronchiectasis. In our study, healthy subjects provided data concerning deviation from the normal values. A search of similar articles in the literature revealed that they did not include any controls [In this study, we used healthy controls for the comparison of extrapulmonary features of muscle function, exercise capacity, fatigue, and health status in patients with bronchiectasis. Although skeletal muscles involvement is frequent in COPD patients, we did not use a control group consisting of COPD patients with the same obstruction but preferred to use healthy subjects, since investigations that lack a healthy control group are not able to provide documented differences in the extrapulmonary characteristics between individuals with controls -8,31-35.In conclusion, we found that perceptions of dyspnea and fatigue, peripheral muscle endurance, exercise capacity and health status are affected by the disease in subjects with bronchiectasis in addition to pulmonary function, inspiratory and expiratory respiration muscle strength. These extrapulmonary features of bronchiectasis may provide insight for outcome measures for pulmonary rehabilitation programs in patients with bronchiectasis.The authors declare that they have no competing interests.


Lactogenesis includes two stages. Stage I begins a few weeks before parturition. Stage II is initiated around the time of parturition and extends for several days afterwards.To better understand the molecular events underlying these changes, genome-wide gene expression profiling was conducted using digital gene expression (DGE) on bovine mammary tissue at three time points (on approximately day 35 before parturition (−35 d), day 7 before parturition (−7 d) and day 3 after parturition (+3 d)). Approximately 6.2 million (M), 5.8 million (M) and 6.1 million (M) 21-nt cDNA tags were sequenced in the three cDNA libraries , respectively. After aligning to the reference sequences, the three cDNA libraries included 8,662, 8,363 and 8,359 genes, respectively. With a fold change cutoff criteria of ≥2 or ≤−2 and a false discovery rate (FDR) of ≤0.001, a total of 812 genes were significantly differentially expressed at −7 d compared with −35 d (stage I). Gene ontology analysis showed that those significantly differentially expressed genes were mainly associated with cell cycle, lipid metabolism, immune response and biological adhesion. A total of 1,189 genes were significantly differentially expressed at +3 d compared with −7 d (stage II), and these genes were mainly associated with the immune response and cell cycle. Moreover, there were 1,672 genes significantly differentially expressed at +3 d compared with −35 d. Gene ontology analysis showed that the main differentially expressed genes were those associated with metabolic processes.The results suggest that the mammary gland begins to lactate not only by a gain of function but also by a broad suppression of function to effectively push most of the cell's resources towards lactation. Lactating cows are generally dried off simply by stopping the milking process approximately two months before the next parturition. The mammary gland then undergoes an involution process, which is marked by the cessation of secretory activity and the reabsorption of milk residue, followed by a relatively static period. The dry period has been proven important for dairy cows. Omitting or shortening the dry period imposes negative effects on mammary health and milk yield in the next lactation Currently, little is known concerning the molecular events underlying lactogenesis. Comparisons of the transcriptomes of pre- and post-parturition mammary glands of dairy cows by Kiera et al Our study had been approved by the Institutional Animal Care and Use Committee of Shandong Agricultural University and performed in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology .Mammary gland samples were collected from 18 Holstein cows reared in dairy farm No. 1 of the Jiabao dairy company, Jinan, China. They were all in the second parity with the same age. There were 6 cows in each group. Mammary gland tissue samples were collected from cows at three different time-points wherein distinct differences exist in the internal status of the gland: one, 35±2 days before parturition (−35 d) which is a totally dry period, two, 7±2 days before parturition (−7 d) characterized by accelerated cell proliferation and pre-colostrum secretion (stage I of lactogenesis) and three, 3 days after parturition (+3 d) characterized by colostrum production (stage II of lactogenesis). Mammary biopsy was performed using Bard Magnum biopsy system . The collected samples were immediately frozen and stored in liquid nitrogen until further analysis.To reduce the number of mammary samples needed for DGE analysis, samples from cows in the same group were pooled together for RNA isolation. Total RNA was extracted with Trizol reagent  in accordance with the manufacturer's instructions. The quantity and quality of the extracted RNA was determined using a spectrophotometer , with RNA quality being evaluated by the absorbance ratio at 260 nm/280 nm.High-throughput sequencing of mRNA was performed on an Illumina HiSeq 2000 platform. All sample preparation and sequencing procedures were executed according to the manufacturer's instructions . Briefly, mRNA from 6 µg of total RNA extracted from samples at each time point was purified by adsorbing to Oligo (dT) magnetic beads, and then use Oligo (dT) as primer to synthesize the first and second-strand cDNA. The bead-bound cDNA was digested with restriction enzyme NlaIII, which recognized and cut off the CATG sites. The fragments apart from the 3′ cDNA fragments connected to Oligo (dT) beads were washed away and the Illumina adaptor 1 was ligated to the sticky 5′ end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site was the recognition site of Mmel, which was a type of Endonuclease with separated recognition sites and digestion sites. It cut at 17 bp downstream of the CATG site, producing tags with adaptor 1. After removing 3′ fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3′ ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After 15 cycles of linear PCR amplification, 105 bp fragments are purified by 6% TBE PAGE Gel electrophoresis. After denaturation, the single-stranded cDNA was anchored on Illumina flowcells of the Illumina HiSeq 2000 system and sequenced. Raw data (tag sequences) were deposited in NCBI's Gene Expression Omnibus (GEO) database under submission number GSE44796.For the raw data, we filtered adaptor sequences, low quality tags (tags with unknown nucleotides N), empty reads and tags that were too short or too long, and tags with only one copy to get clean tags.A virtual library containing all the possible CATG+17 bases length sequences of the bovine gene sequences were constructed by BGI-shenzhen 1, the total number of clean tags in sample 2 is N2, and there are x tags in sample 1 and y tags in sample 2 for gene A. False Discovery Rate (FDR) analysis was applied to determine the threshold P value in multiple tests and analyses A rigorous algorithm has been developed to identify genes expressed differentially between samples. Because every gene's expression occupies only a small part of the library, p(x) was within the Poisson distribution. The probability of a given gene being expressed equally between two samples can be calculated with the following equation The selected genes with significant modification in their expression were subjected to further analysis, which were GO enrichment analysis, pathway enrichment analysis and functional annotation clustering.GO enrichment analysis of functional significance applies hypergeometric test to map all differentially expressed genes (DEGs) to terms in Gene Ontology (GO) database (Version 1.1.1631) Pathway enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Version 58) can identify significantly enriched biological pathways in DEGs compared with the whole genome background The differentially expressed genes detected in stage I and stage II of lactogenesis were further analyzed using DAVID functional annotation clustering tool QRT-PCR analysis was used to verify the DGE results. The RNA samples used for the qRT-PCR assay were both the same as for the DGE experiments and independent RNA extractions from biological replicates. 13 candidate genes were selected and detected using qRT-PCR, including caseinβ, CIDEA, GLYCAM, BGN, ELL3, PAH, GP2, LOC525947, SLC14A1, SST, PNMT, caseinα-S2 and integrinβ. qRT-PCR was performed according to the TaKaRa manufacturer specifications . SYBR Green PCR cycling was denatured using a program of 95°C for 10 s, and 40 cycles of 95°C for 5 s and 60°C for 40 s, and performed on an ABI 7500 instrument . Data were reported as values normalized to the housekeeping gene β-actin, and they were subjected to one-way ANOVA analysis using Statistical Analysis Systems statistical software package . Means were considered significantly different at p<0.05.Characterization of the three cDNA libraries  was listed in A reference database of the bovine genome includes 35,945 transcripts At the cutoff criteria of FDR≤0.001 and |log2 Ratio|≥1, many genes were differentially expressed; these results were shown in The differentially expressed genes were analyzed in the context of gene ontology (GO) biological processes. This analysis revealed that the upregulated genes in DEGs were mainly associated with the cell cycle and lipid metabolism, and the downregulated genes were mainly associated with the immune response and biological adhesion at −7 d compared with −35 d. And the upregulated genes in DEGs at +3 d compared with −7 d were mainly associated with the immune response, and the downregulated genes were mainly associated with the cell cycle phase. The most significantly enriched biological processes in DEGs were metabolic processes at +3 d compared with −35 d.Moreover, during the onset of lactation, some genes were completely turned on or off. There were 305 genes turned on at −7 d compared with −35 d. Gene ontology analysis showed that these genes were associated with signal transduction , transport , the cell cycle , and other processes. In addition, 12 genes encoding hypothetical proteins  were also identified in stage I of lactogenesis. 604 genes were turned off at −7 d compared with −35 d. Gene ontology analysis showed that these genes were associated with ion transport  and protein and lipid metabolism . There were 421 genes turned on at +3 d compared with −7 d, including genes involved in transport , the immune response , and signal transduction . 425 genes were turned off at +3 d compared with −7 d, including cell cycle-related genes  and signal transduction genes .After pathway enrichment analysis, the top 10 significantly enriched pathways were listed in The expression level of many milk protein genes increased significantly during stage I of lactogenesis and kept increasing during stage II. These genes include casein kappa (CSN3), lactalbumin alpha (LALBA), casein beta (CSN2), casein alpha s1 (CSN1S1), and casein alpha-S2 (CSN1S2). The fold changes of these genes were listed in There were 231 genes differentially expressed in the two stages of lactogenesis. Among these genes, 92 genes were downregulated in stage I and upregulated in stage II, 111 genes were upregulated in stage I and downregulated in stage II, and only 11 genes continued to increase in both stages. After analysis of functional annotation clustering, 92 genes fell into 24 clusters. Each term in Cluster 4 was associated with the immune response, and each in cluster two was associated with the extracellular matrix. The top 5 enriched clusters were listed in The results of qRT-PCR were shown in The major goal of this study was to explore genome-wide gene expression profiles during the onset of lactation and to provide information for understanding the underlying molecular mechanisms. Three libraries were constructed from mammary tissues at three different stages . Saturation analysis of the sequences indicated that the three libraries had reached saturation and were therefore complete assessments of all transcripts present in the libraries. The three libraries contained 192,928, 170,427 and 168,233 distinct clean tags, respectively. However, a reference database includes 35,945 transcripts. Theoretically, a tag should be generated by NlaIII digestion from the 3′-most end of a transcript. However, many transcripts contain more than one CATG site, so tags from other NlaIII sites were also generated in our libraries. Because only one tag can be generated per transcript from a given NlaIII site in a cDNA, these extra NlaIII tags represent genes redundantly present in the expression profile. Thus, the number of unique tags generated was greater than that of the annotated bovine genome.To validate the DGE method, the levels of 13 genes were analyzed using qRT-PCR. Although the differences in the expression of some genes did not match the magnitude of those detected using the DGE method, the trends of upregulation and downregulation were similar. Compared with microarray and qRT-PCR methods, the sequencing method has been documented to be more sensitive for the estimation of gene expression, especially for low-abundance transcripts During the transition from pregnancy to lactation in the dairy cow, the mammary gland undergoes dramatic functional and metabolic changes, including the morphogenesis of mammary ducts during early pregnancy and differentiation of the mammary alveolus during late pregnancy Candida albicans, Gram-negative bacteria, and Gram-positive bacteria At −7 d compared with −35 d, genes associated with the immune response accounted for 14.5% of differentially expressed genes, and genes associated with the defense response accounted for 8.20% of differentially expressed genes. Most of these genes were downregulated; for example, Chemokine (C-C motif) ligand 28 (CCL28) was completed turned off at −7 d. CCL28 is selectively expressed in certain mucosal tissues such as the exocrine glands, trachea, and colon and has a potent antimicrobial activity against In this study, many milk protein genes were upregulated in stage I of lactogenesis. It has been shown previously in mouse The main regulator of milk protein expression in non-ruminant mammary glands appears to be the Jak-Stat5 signaling pathway It has been speculated that there are two modes of regulation to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in SCAP deletion mice fed a high-fat diet, leading to alterations to enzyme protein It is well known that the onset of lactation includes two different stages. And in stage I of lactogenesis, the mammary epithelial cells proliferate and differentiate, the extracellular matrix diminishes, and the communication between the two becomes weak. As milk protein genes and lipogenesis genes are upregulated, genes associated with the immune response are downregulated. In stage II of lactogenesis, the mammary gland restores the communication between mammary epithelial cells and the extracellular matrix. The main result of this process is the synthesis of milk using nutrients from the blood.File S1The details of differentially expressed genes at −7 d compared with −35 d.(XLS)Click here for additional data file.File S2The details of differentially expressed genes at +3 d compared with −7 d.(XLS)Click here for additional data file.File S3The details of differentially expressed genes at +3 d compared with −35 d.(XLS)Click here for additional data file.


Neural oscillations are ubiquitous measurements of cognitive processes anddynamic routing and gating of information. The fundamental and so far unresolvedproblem for neuroscience remains to understand how oscillatory activity in thebrain codes information for human cognition. In a biologically relevantcognitive task, we instructed six human observers to categorize facialexpressions of emotion while we measured the observers' EEG. We combinedstate-of-the-art stimulus control with statistical information theory analysisto quantify how the three parameters of oscillations  code the visual information relevant for behavior in a cognitivetask. We make three points: First, we demonstrate that phase codes considerablymore information (2.4 times) relating to the cognitive task than power. Second,we show that the conjunction of power and phase coding reflects detailed visualfeatures relevant for behavioral response—that is, features of facialexpressions predicted by behavior. Third, we demonstrate, in analogy tocommunication technology, that oscillatory frequencies in the brain multiplexthe coding of visual features, increasing coding capacity. Together, ourfindings about the fundamental coding properties of neural oscillations willredirect the research agenda in neuroscience by establishing the differentialrole of frequency, phase, and amplitude in coding behaviorally relevantinformation in the brain. To recognize visual information rapidly, the brain must continuously codecomplex, high-dimensional information impinging on the retina, not all of whichis relevant, because a low-dimensional code can be sufficient for bothrecognition and behavior (e.g. a fearful expression can be correctly recognizedonly from the wide-opened eyes). The oscillatory networks of the braindynamically reduce the high-dimensional information into a low dimensional code,but it remains unclear which aspects of these oscillations produce the lowdimensional code. Here, we measured the EEG of human observers while wepresented them with samples of visual information from expressive faces . Using statistical information theory, we extracted thelow-dimensional code that is most informative for correct recognition of eachexpression . Next, we measured how the three parameters of brainoscillations  code for low-dimensional features.Surprisingly, we find that phase codes 2.4 times more task information thanpower. We also show that the conjunction of power and phase sufficiently codesthe low-dimensional facial features across brain oscillations. These findingsoffer a new way of thinking about the differential role of frequency, phase andamplitude in coding behaviorally relevant information in the brain. Invasive and noninvasive studies in humans under physiological and pathologicalconditions converged on the suggestion that the amplitude and phase of neuraloscillations implement cognitive processes such as sensory representations,attentional selection, and dynamical routing/gating of information We instructed six observers to categorize faces according to six basic expressions ofemotion . We controlled visual information, by presenting on eachtrial a random sample of face information—smoothly sampled from the imageusing Gaussian apertures at different spatial frequency bands. The Gaussianapertures randomly sampled face parts simultaneously across the two dimensions ofthe image and the third dimension of spatial frequency bands . We recoMI, which measures the mutual dependence between two variables;MI measurements: between sampled pixelinformation and behavioral responses to each emotion category (correct versusincorrect), between EEG responses  and behavior, and finally between sampled pixel information and EEGresponse , the MI between the distribution of grey-level values ofeach image pixel  and equal numbers of correct versus incorrect categorizationresponses. MI illustrates MI on a scalefrom 0 to 0.05 bits. High values indicate the face pixels  representing the visual information that the brain mustprocess to correctly categorize the stimuli  The conjunction of phase and power (phase&power) codes moreinformation about complex categorization tasks than phase and power on theirown. In MI measures the reduction ofuncertainty of the brain response, when the behavioral variable correct versusincorrect categorization is known. We provide the measure for each electrode of thestandard 10–20 position system over the Time × Frequency space. Pz, Oz,P8, and P7 had highest MI values of all electrodes, irrespective ofwhether the brain response considered was power (blue box), phase (green box), orthe phase&power (red box). The adjacent MI scales reveal thatphase&power was 1.25 times more informative of behavior than phase, itself 2.4times more informative than power. Phase&power was 3 times more informative thanpower alone. Henceforth, the analyses focus on these four electrodes and onphase&power, the most informative brain measurement for the cognitive task.(b) Phase&power codes detailed categorization-relevant features ofsensory stimuli. MI revealed that the two eyes and themouth are prominent features of expression discrimination , focusing on Pz, Oz, P8, and P7. Thered box represents, at 4 Hz and 156 ms, following stimulus onset . The scale is the adjacent rainbow colors rangingfrom 0 to 0.03 bits. Electrodes P7  and P8  reveal the highest MI to thecontra-lateral eye . At the same time on Pzand Oz, the highest MI is to both eyes and to the mouth.tion see . As explMI values within each mask, independently for each Time× Frequency cell. We then color-coded MI for each feature inRGB color space—red for “right eye,” green for“mouth,” and blue for “left eye”; see schematic coloredfaces adjacent to the Time × Frequency plot for complete color coding. Thebroad red (versus blue) cloud on electrode P7 (versus P8) denotes highestMI to the right (versus left) eye in this Time ×Frequency region, whereas Pz and Oz demonstrate sensitivity to the two eyes (inpurple) and to the mouth (in green). To conclude, phase&power codes detailedcategorization-relevant features of the sensory input.To generalize across Time × Frequency, for ease of presentation, we computedthree masks extracting pixel locations from the left eye, right eye, and mouth. Weaveraged (c) Phase&power coding is multiplexed across oscillatoryfrequencies. Theta (4 Hz) and low beta (12 Hz) on both Oz and Pzdemonstrate the remarkable multiplexing property of phase&power coding: the ideathat the brain codes different information in different oscillatory bands. In MIvalues critically depend on the joint distribution of variables contribute to the coding of information in the biologically relevant cognitive taskof categorizing facial expressions of emotion. We demonstrated that phase codes 2.4times more information about the task than power. The conjunction of power and phase(itself 3 times more informative than power) codes specific expressive featuresacross different oscillatory bands, a multiplexing that increases coding capacity inthe brain.In general, the relationship between our results on the frequency, power, and phasecoding of neural oscillations cannot straightforwardly be related to the codingproperties of more standard measures of the EEG such as event related potentials(ERP). However, an identical experimental protocol was run on the N170face-sensitive potential In these endeavors, the phase and frequency multiplexing coding properties of neuraloscillations cannot be ignored.Six observers from Glasgow University, UK, were paid to take part in theexperiment. All had normal vision and gave informed consent prior toinvolvement. Glasgow University Faculty of Information and Mathematical SciencesEthics Committee provided ethical approval.Original face stimuli were gray-scale images of five females and five males takenunder standardized illumination, each displaying seven facial expressions. All70 stimuli  complied with theFacial Action Coding System FACS,  and formPrior to testing, observers learned to categorize the 70 original images into theseven expression categories. Upon achieving a 95% correct classificationcriterion of the original images, observers performed a total of 15 sessions of1,400 trials  of the facial expressionscategorization task . Short breaks were permitted every 100 trials of the experiment.In each trial a 500 ms fixation cross  wasimmediately followed by the sampled face information, as described before see. StimuliWe recorded scalp electrical activity of the observers while they performed thetask. We used sintered Ag/AgCl electrodes mounted in a 62-electrode cap(Easy-Cap) at scalp positions including the standard 10–20 systempositions along with intermediate positions and an additional row of lowoccipital electrodes. Linked mastoids served as initial common reference andelectrode AFz as the ground. Vertical electro-oculogram (vEOG) was bipolarlyregistered above and below the dominant eye and the horizontal electro-oculogram(hEOG) at the outer canthi of both eyes. Electrode impedance was maintainedbelow 10 kΩ throughout recording. Electrical activity was continuouslysampled at 1,024 Hz. Analysis epochs were generated off-line, beginning 500 msprior to stimulus onset and lasting for 1,500 ms in total. We rejected EEG andEOG artefacts using a deviation threshold over 200 ms intervals on all electrodes. The EOG rejectionprocedure rejected rotations of the eyeball from 0.9 deg inward to 1.5 degdownward of visual angle—the stimulus spanned 5.36°×3.7° ofvisual angle on the screen. Artifact-free trials were sorted using EEProbe (ANT)software, narrow-band notch filtered at 49–51 Hz, and re-referenced toaverage reference.MI between randomvariables X and Y measures their mutualdependence. When logarithms to the base 2 are used in Equation 1, the unit ofmutual information is expressed in bits.In Information Theory p,the joint probabilities between X and Y. When the variables areindependent, the logarithm term in Equation 1 becomes 0 andMI = 0. In contrast, when X andY are dependentMI returns a valuein bits that quantifies the mutual dependence between X andY. Derived from the measure of uncertainty of a randomvariable X expressed in Equation 2 and the conditionaluncertainty of two random variables X and Y(Equation 3),The critical term is X andY share. It quantifies the reduction of uncertainty aboutone variable that our knowledge of the other variable induces (Equation4),Mutual Information measures how much bits of information Here, we use Mutual Information to measure the mutual dependence between thesampling of input visual information from faces and the oscillatory brainresponses to these samples and between the same input information and behavior. For each expression,we then averaged mutual information values across all six observers,independently for each pixel. To establish statistical thresholds, we repeatedthe computations 500 times for each pixel, after randomly shuffling the order ofresponse—to disrupt the association between pixel values andcategorization responses. For each of the 500 computations, we selected themaximum mutual information value across all pixels. We then chose as statisticalthreshold the 99th percentile of the distribution of maxima. This maximumstatistic implements a correction for multiple comparisons because thepermutation provides the null distribution of the maximum statistical valueacross all considered dimensions MI and MI. MI computed, for each electrode, subject, and expression, themutual information between correct and incorrect trials and the power, phase,and phase&power of the Time × Frequency EEG signal. For thiscomputation, we used the same number of trials as for Behavior MI . As with behavior, foreach electrode and type of EEG measurement, we averaged the mutual informationvalues across subjects and expression. To establish statistical thresholds, werepeated the computations 500 times, permuting the trial order of the EEG Time× Frequency values and identified the 500 maxima each time across theentire Time × Frequency space. We identified the statistical threshold asthe 99th percentile of the distribution of maxima  computed, for each subject,expression, and face pixel (down-sampled to 38×24 pixel maps), the mutualinformation between the distribution of each face pixel grey-level value and themost informative of the brain responses, phase&power Time × Frequencyresponses, for correct trials only. That is, an average of 15,750 trials persubject. To establish statistical thresholds, given the magnitude of thecomputation, we computed z scores using the pre-stimuluspresentation baseline (from −500 to 0 ms) to estimate mean and standarddeviation. In z score of 55.97, soall mutual information values this number of bits (see the level marked with anasterisk in z score of 5).MI in all three measures  at a latency of 140–250 ms in two frequency bands (4 Hzand 12–14 Hz). We averaged the MI measures, for eachcluster, electrode, and subject, and subjected these MIaverages to a two-way ANOVA with factors electrode  andmeasure . Both clusters revealed asignificant main effect of electrode  = 8.38, p<0.001 for 4 Hz andF = 79.34,p<0.001 for 12–14 Hz) and measure = 44.24,p<0.001 for 4 Hz and F = 104.77, p<0.001 for 12–14Hz). Post hoc t test confirmed thatMI(Phase&Power) is significantly higher thanMI(Phase)(p = 0.013), which itself is significantlyhigher than MI(Power)(p = 0.003).Figure S1Illustration of the bubbles sampling procedure. The original stimulus isdecomposed into five non-overlapping bands of Spatial Frequencies (SF) ofone octave each . We sampled information from each SF bandusing a mask punctured with Gaussian apertures. These were randomlypositioned trial by trial to approximate a uniform sampling distribution ofall face regions across trials. We adjusted the size of the apertures foreach SF band so as to maintain constant the total area of the face revealedacross trials . We calibrated thesampling density  on a trial-per-trial basis tomaintain a 75% correct categorization performance independently foreach expression. The stimulus presented on each trial comprised informationfrom each SF band summed together.(TIF)Click here for additional data file.Figure S2MI) Framework. Pixel. Reduced 38 ×24 pixels space used for analysis . Behavior. Oneach of the 3000 trials per expression (illustrated for“happy”), we recorded the observer's correct versusincorrect responses to the sampled information. Computation ofMI. Across the 3,000 trials per expression, for each pixel wesummed the Gaussian apertures across spatial frequency bands and collectedthe distributions of resulting grey-level values associated with correct andincorrect responses. We then computed MI between the pixelvalues reflecting the Gaussian apertures and correct versus incorrectresponses, MI. We also computedMI between behavior and the EEG response,MI, independently for power,phase, and the conjunction of phase&power. Finally, we computedMI between the pixels values and the EEG response,MI.Mutual Information ((TIF)Click here for additional data file.Figure S3MI. For one observer, expression “happy,” we provide thefull computation of mutual information using two face pixels (P1 and P2) andan equal number of correct (c) and incorrect (i) categorization responses.Note that if the computation had been between face pixels and EEGparameters, we would have had four rows  in the matrix of joint probabilities, not two (for correct andincorrect).Detailed Illustration of the Computation of (TIF)Click here for additional data file.