Biological soil crusts are common components of desert ecosystem; they cover ground surface and interact with topsoil that contribute to desertification control and degraded land restoration in arid and semiarid regions.P<0.05); and soil organic carbon and nutrients showed an upward trend across the successional stages of biological soil crusts. Fractal dimensions ranged from 2.1477 to 2.3032, and significantly linear correlated with selected soil properties .To distinguish the changes in topsoil affected by biological soil crusts, we compared topsoil properties across three types of successional biological soil crusts , as well as the referenced sandland in the Mu Us Desert, Northern China. Relationships between fractal dimensions of soil particle size distribution and selected soil properties were discussed as well. The results indicated that biological soil crusts had significant positive effects on soil physical structure (Biological soil crusts cause an important increase in soil fertility, and are beneficial to sand fixation, although the process is rather slow. Fractal dimension proves to be a sensitive and useful index for quantifying changes in soil properties that additionally implies desertification. This study will be essential to provide a firm basis for future policy-making on optimal solutions regarding desertification control and assessment, as well as degraded ecosystem restoration in arid and semiarid regions. Biological soil crusts (BSCs) refer to the cohesive, thin, horizontal layer of soil surface created by soil crust organisms composed of algae, lichens, and mosses, which are widespread throughout the world Unique among all ecosystems, BSCs in deserts not only engage in multitudinous ecological processes, but also serve as the impetus of the habitation of plants, animals, and fragile physical environments Structures and functions of BSCs in deserts have been more thoroughly investigated recently. The first efforts mostly focused on assortment, morphological characteristics and distribution of BSCs under different environmental conditions. Phylogenetic and morphological diversity and microstructure were commonly distinguished D) of soil PSD can be used as a practical index for quantifying variations in soil properties and the implications of desertification.Studies regarding BSCs are numerous and have led to a much greater understanding of their place in ecosystems; however, BSCs have not been explored in full as of yet. More information is needed regarding details of the mutual relationships of BSCs and soil properties. Especially, little is known about an effective index to quantify the effects on soil properties affected by BSCs in the desertification processes. In recent decades, the possibility of characterizing soil particle size distribution (PSD) using fractal theory has been explored The study area is located in Yanchi County  in the central part of southern rim area of the Mu Us Desert in the eastern part of Ningxia Hui Autonomous Region, P. R. China . Yanchi Salix psammophila, Caragana korshinskii, Hedysarum scoparium, Artemisia ordosica, Glycyrrhiza uralensis, Sophora alopecuroides, and Cynanchum komanovii. Additionally, the dominant plantation species include Salix matsudana, and Populus alba.The study area has a continent-typical warm monsoon climate in a temperate zone. The average annual temperature is 7.8°C. The winter-summer and diurnal-nocturnal differences in air temperature are significantly high, with an annual range in temperature of greater than 28 and 20°C, respectively. The average annual precipitation is 292 mm with 62% occurring between July and September. The average annual evaporation is 2024 mm, which is dramatically higher than annual precipitation. The study area is perennially windy with average annual wind velocity of 2.6 m/s and a prevailing northwest wind direction, which results in frequent and strong wind erosion The field sampling sites are located in an enclosed area within Zhouzhuangzi Village, Yanchi County, which belongs to the Yanchi Research Station of Beijing Forestry University of the Chinese Terrestrial Ecosystem Research Network (CTERN).Artemisia ordosica communities in the 1 km range  was measured using the micro-Kjeldahl’s method; (3) soil total phosphorus (PT) was determined by the Mo-Sb colorimetry method; (4) soil total potassium (KT) was measured using the hydrofluoric & perchloric acid (HF-HCLO acid)-flame photometer method; (5) available nitrogen (NAvi) was determined by the alkali diffusion method; (6) soil available phosphorus (PAvi) was measured using the sodium bicarbonate (NaHCO3) digestion-Mo-Sb colorimetry method; (7) soil rapid available potassium (KAvi) was determined by the ammonium acetate digestion-flame photometer method All the soil profile samples were naturally dried in a shaded area in the laboratory; plant stems, small stones, and large animals such as worms were carefully removed. Then, parts of the air-dried soil samples were hand-sieved through 2 and 0.25 mm screens prior to laboratory analysis. To meet the experimental needs, parts of soil samples were ground. The topsoil physicochemical properties were analyzed in the following methods (1) soil organic carbon (SOC) was determined by the potassium dichromate wet combustion method; (2) soil total nitrogen , same below; 1g is the simple weight (g); 2g is simple weight when completely wrapped by wax (g); 3g is the original reading of electronic balance (g); 4g is reading of electronic balance with the sample (g); −3; −3; W is water content of sample.Where −3), −3.Where 2O2 solution  to destroy any organic matter. And then, the soil aggregates were dispersed by adding sodium hexametaphosphate (NaHMP) and sonicating the samples for 30 seconds To measure the topsoil particles and fractal characteristics, the unscreened air-dried soil samples were pretreated in an HFractal dimension of soil PSD was calculated based on the volume distribution of soil particle size r is the soil particle size, iR is the soil particle size of grade i, maxR is the maximum value of soil particle size, i)V procedures. The LSD test (at p<0.05) was used to compare means of soil variables; the results of ANOVA were significant at the level of p<0.05. Simple linear regression and correlation analysis were performed to identify the relationships between D and the selected soil properties. Pearson correlation coefficient and 2-tailed test were used to distinguish the correlation and significant differences.All statistical analyses were conducted using SPSS software (Version 17.0). The differences in soil properties and P<0.05). Uncovered sandland soil had the greatest bulk density (1.71 and 1.69 g·cm−3) compared to the BSCs sites, due to severe wind erosion. With the development of BSCs, soil bulk density decreased from algae (1.64 and 1.68 g·cm−3) to mosses crust (1.25 and 1.48 g·cm−3). Furthermore, soil bulk density within the same type of BSCs increased by 2.4%∼21.2% with increasing soil depth in all sampling sites.Variations in soil bulk density were remarkable , and sigP<0.05). However, as shown in Total porosity showed a clear and significant difference in the top layer among different BSCs types; in the underlying layer, significant difference only occurred between mosses crust and other sites (−1) which differed significantly with the sandland (0.94 and 0.69 g·kg−1) (P<0.05), but not significantly different from lichens (4.54 and 3.11 g·kg−1) and algae (3.15 and 2.45 g·kg−1) crusts (P>0.05). Further, SOC within the same type of BSCs deceased from the top to underlying layer.Accompanying the positive changes in soil physical structure, SOC and soil nutrients increased alike . CompariT and NAvi. However, there was a clear increasing tendency across the three types of successional BSCs, and soil nutrients under BSCs sites were greater than the uncovered sandland. For the content of KT, NAvi and PAvi, no significant differences were found among the different BSCs types (P>0.05). However, there were significant differences in the content of NT, PT, and KAvi at different sites, especially between the uncovered sandland and mosses crust (P<0.05). The spatial variations in soil nutrients in the soil profile deceased from the top to underlying layer as well.Changes in soil nutrients varied significantly, especially the levels of KP<0.05). Clay (0–2 µm) and silt (2–50 µm) contents were much lower than sand particles, especially the clay contents (0.4∼0.7%). However, with the development of BSCs, clay and silt contents increased sharply. Compared with the uncovered sandland, from algae to mosses crust, the clay and silt contents increased by as much as 90.9∼112.1%, 163.9∼241.2% and 27.2∼71.9%, 65.4∼167.2% for the top and underlying layers, respectively. As a result, significant differences occurred between mosses crust and other sites for clay and silt contents (P<0.05). Furthermore, sand contents within the same type of BSCs increased from the top to underlying layer, in addition to the decrease in clay and silt contents. In contrast, clay and silt contents of the uncovered sandland were cumulative with the increase in soil depth.D) were subsequently calculated with Eq. (3) based on the soil PSD data, and the variations in D values for the different BSCs are shown in D values increased progressively from algae to mosses. Meanwhile, the D values of algae, lichens and mosses crust were higher than uncovered sandland in both layers. However, significant differences among the different BSCs types only occurred in the top layer (P<0.05). In the underlying layer only mosses significant differed with uncovered sandland (P<0.05). Additionally, D values observed in the underlying layer were lower than the top layer because the wind-induced fine particles remove surface soil.Volume-based soil fractal dimensions (D) and selected soil properties including soil bulk density, total porosity, SOC, soil nutrients as well as soil PSD . This reverse correlation and the different variations in bulk density and total porosity mutually verified.Linear regression and correlation analysis were applied to identify the relationships between fractal dimensions . However, no significant correlation occurred between D values and KT . Therefore, soils with more fertilizer have higher fractal dimension values; values of D do not appear to be related to KT.D); soils with a greater amount of sand particles had lower D values. D values had significant positive linear correlations with clay and silt contents , and a negative linear correlation with sand particles contents . Therefore, wind-induced removal of fine particles resulted in decreased D values, and D values appeared to be closely related to clay particles rather than silt and sand contents.In In the Mu Us Desert, soil properties can be improved through the addition of plants including the lower plants that introduce BSCs. This information is greatly important for the countries in arid and semiarid regions with a high susceptibility to wind erosion and desertification. BSCs protect sand surface from wind erosion. Erosive force and carriage ability are consumed by BSCs through increasing aerodynamic roughness length and threshold wind velocity 2 exchange is observed between BSCs and atmospheric content. CO2 is sequestrated and accumulated through a combination of BSCs photosynthesis and respiration. Free chlorophyll in algae and chloroplast in mosses capture CO2 through photosynthesis and store carbon in the form of carbohydrates in their tissues. The photosynthetic rate of BSCs has been commonly reported under natural conditions or in the laboratory. However, the values are of great uncertainty, ranging from 0.1∼11.5 μ mol CO2 m−2·s−1 due to the difference in various physical environments and the ability to carbon capture of different crusts species −2·a−1 for the net uptake of carbon by BSCs, accordingly, the total carbon sequestration by BCSs in arid and semiarid regions is approximately 1.0 Pg·a−1. Although, it is much lower in comparison with other processes of the global carbon cycle, millions tons of carbon was sequestrated by BSCs and stored in soil ecosystem, which helps soils prosper. It is of significant importance for soil environmental restoration, and can’t be ignored of understated.With the presence of BSCs, a significant amount of CO−2·a−1; the total nitrogen fixation by BCSs in arid and semiarid region is approximately 30 Tg·a−1Nitrogen turnover is as complex as the carbon cycle; it combines nitrogen mineralization, ammonia volatilization, nitrification and denitrification. The presence of dinitrogenase in alga, and bacterium of BSCs play a significant role in this process; these biochemical processes that lead to nutrients fixation Finally, carbohydrates and organic nitrogen introduced by BSCs are input into soils in various ways. The most important way is through the decomposition of BSCs. At the end of BCS lifecycle, remains decompose through enzymatic activities by the combined action of soil bacterium and enzymes. Furthermore, some algal crusts can secrete polysaccharide into soil to increase carbon pool; similarly, nitrogen micromolecules are released into soil by BSCs as well. Meanwhile, SOC and soil nitrogen accumulation is beneficial to the growth of tracheophyte; successions of vascular plants help soil properties prosper. Additionally, some beetles used BSCs as habitat; these BSCs-feeding beetles are also considered as an important nutrients resource in arid and semiarid regions due to the decomposition of their bodies.T is an expectation. In this study, a poor relationship between D and KT was observed which corresponded with a lack of differences in KT among the different types of BSCs and differing degrees of desertified soils. In fact, although potassium is one of the most essential macronutrients for plant growth and development, scholars pay little attention on the conversion due to its substantial deposit in soil system of northern China. In nature, potassium of different forms can affect and transform to each other; however the rate of this conversion is rather slow. Among all the forms, potassium mainly exists in a number of inorganic substances, especially the inorganic mineral T is related to soil mineralogy, and unlikely to be affected by BSCs. Besides, we suspect that KT may lose due to the decrease of pH  induced by combination efforts of vegetation recovery and wind erosion.With carbon sequestration and transport at its core, BSCs increase nutrient contents and make soil more fertile. However, unique among other soil nutrients, soil KVariations in soil properties differ between successional stages of BSCs. Complex and evolutional BSCs achieve greater activity and ability as results of diverse community composition in numbers, species, and functions Characterizing soil organization and functions with a single parameter is a question of great interest when monitoring soil degradation and desertification. In former studies, individual fractions  textual analysis and soil organic matter were the commonly used methods to characterize soil quality. However, soil is an irregular and complex system in which many biological and physical components interact across all space and time scales p<0.01). Estimation of soil fractal dimension under different BSCs is helpful in determining changes in soil properties and vulnerability of desertification. More specifically, lower values indicate fine particles removal in soil coarsening process due to wind erosion in desertification-vulnerable regions. With the loss of fine particles, SOC and soil nutrients decreased concomitantly. Accordingly, higher D values suggest sand particles disintegration, fine particles accumulation and soil properties recovery as affected by BSCs. Furthermore, D values indicate the potential of desertification as well. The higher D values implies the greater possibility of wind erosion due to the bigger proportion of fine particles. Once the sandland loses the protection of BSCs or the actual wind velocity exceeds the threshold, accumulative fine particles will be eroded by destructive wind in a short term. Therefore, fractal dimension is sensitive and useful index for quantifying soil properties variations in the evolution of a desert, and significantly implies for soil degradation and desertification induced by wind erosion.Soil PSD is closely related with soil functions and they are interdependent. It is generally believed that the rich fine fractions (clay and silt) mean fertile, hydrophilic, and biodiversity-rich soil system. Therefore, when wind-induced erosion occurs and soils are lost, we lose far more than just the fractions that they contain, but immeasurable ecological services. Wind erosion not only causes the transport of soil particles, but also nutrient and function losses, and further resulting in the decrease of water-holding capacity, depletion of soil structure and biological properties D values had significant linear relationships with soil bulk density, total porosity, SOC, soil nutrients (except KT) as well as soil PSD with R2 values ranging from 0.494 to 0.955 (p<0.01). Fractal dimension is sensitive to soil coarsening and nutrients losses process in the evolution of desertification, and it is now proven to be a sensitive and practical index for quantifying changes in soil properties and additionally implies desertification and vulnerability to desertification.In the Mu Us desert, the establishment and succession of BSCs caused positive changes in topsoil properties. There was a clear tendency to increase from sandland to BSCs sites in soil total porosity, SOC, soil nutrients, clay and silt particle contents, accompanying decrease in soil bulk density, and sand particle contents. With the succession of BSCs, significant difference occurred between mosses crust and uncovered sandland. Linear regression and correlation analysis showed that


Sand flies include a group of insects that are of medical importance and that vary in geographic distribution, ecology, and pathogen transmission. Approximately 163 species of sand flies have been reported in Colombia. Surveillance of the presence of sand fly species and the actualization of species distribution are important for predicting risks for and monitoring the expansion of diseases which sand flies can transmit. Currently, the identification of phlebotomine sand flies is based on morphological characters. However, morphological identification requires considerable skills and taxonomic expertise. In addition, significant morphological similarity between some species, especially among females, may cause difficulties during the identification process. DNA-based approaches have become increasingly useful and promising tools for estimating sand fly diversity and for ensuring the rapid and accurate identification of species. A partial sequence of the mitochondrial cytochrome oxidase gene subunit I (COI) is currently being used to differentiate species in different animal taxa, including insects, and it is referred as a barcoding sequence. The present study explored the utility of the DNA barcode approach for the identification of phlebotomine sand flies in Colombia. We sequenced 700 bp of the COI gene from 36 species collected from different geographic localities. The COI barcode sequence divergence within a single species was <2% in most cases, whereas this divergence ranged from 9% to 26.6% among different species. These results indicated that the barcoding gene correctly discriminated among the previously morphologically identified species with an efficacy of nearly 100%. Analyses of the generated sequences indicated that the observed species groupings were consistent with the morphological identifications. In conclusion, the barcoding gene was useful for species discrimination in sand flies from Colombia. Lutzomyia França, 1924, Brumptomyia França and Parrot, 1921, and Warileya Hertig, 1948 Lutzomyia (L.) species are known for their role as vectors of medically important pathogens, such as the kinetoplastida protozoan of the genus Leishmania Ross, 1903 Bartonella bacilliformis , and viruses of the families Bunyaviridae, Reoviridae, and RhabdoviridaeThe subfamily Phlebotominae Rondani, 1840, is represented in America by more than 500 species distributed among the genera http://wrbu.si.edu/) are essential tools for studying the systematics, taxonomy, and geographic distribution of this group of medically important insects in the Americas.Although phlebotomine sand flies have been studied more than most other groups of insects because of their role as vectors of pathogens, the taxonomic knowledge of these insects is far from complete. Three major reviews Lutzomyia, 8 Brumptomyia, and 2 Warileya species) have been recorded. This list was obtained from taxonomic studies conducted in Colombia in different natural and endemic environments, with a primary focus on areas of leishmaniasis transmission To date, in Colombia, 163 species of sand flies  and endosymbionts. Also, standardization of the methodology is necessary for each group studied. Furthermore, the use of a single marker for the taxonomy of a wide range of taxa is controversial Despite many demonstrations of the effectiveness of DNA barcoding in other taxa, few studies on phlebotomine sand flies in Colombia have attempted to use this methodology as a valid molecular tool for identifying species of phlebotomine sand flies In the present study, haplotypes of the barcode sequence were assigned to 148 individuals belonging to 36 phlebotomine sand fly species collected in Colombia, some of which are involved in the transmission of leishmaniasis. Specimens were obtained from sites where outbreaks of the disease have occurred. The present study evaluated the usefulness of the barcode sequence in species differentiation compared with previous morphology-based identifications.http://190.147.213.68:8080/HOMEPAGE/ALEGIS_INTER/LEYES_Y_DECRETOS/2013/DECRETO_1376_de_2013.pdf which regulates the permit for collection of specimens of wild species of biological diversity for non-commercial research. No specific permits were required for this study. The sand flies were collected on private property, and permission was received from land owners prior to sampling.Collection of sand flies was done according to the parameters of Colombian decree number 1376 Phlebotomine sand flies were collected using CDC light traps and Shannon traps from different locations in Colombia between 2008 and 2012. Areas selected for the collections were based on previous epidemiological studies of leishmaniasis transmissionmade by the Program for the Study and Control of Tropical Diseases (PECET) .The thor4SO4), 5 µL of 2.5 mM MgCl2, 5 µL of 2 mmol/L dNTP, 2 µl of 10 µmol/L of each oligonucleotide (forward and reverse), 4 µL of the DNA template, and ddH2O to bring the volume to 50 µl. The oligonucleotides used to amplify the 700 bp COI fragment were LCO1490 and HCO2198 Total DNA (50 µL) was extracted from each sample using the Qiagen® DNeasy Blood & Tissue kit . To amplify the mitochondrial 5′ COI gene region, PCR was performed using the following thermal profile: an initial denaturation of 5 min (94°C); followed by 35 cycles at 94°C for 1 min (denaturation), 45°C for 1 min , and 72°C for 1 min (extension); and a final extension at 72°C for 10 min. Each PCR cocktail had a final reaction volume of 50 µl and contained the following: 0.3 µL of Taq DNA polymerase (5 U/µL) (Biolase®), 5 µL of 10× PCR buffer (NHHTS Vacuum Manifold (Millipore). Subsequently, the PCR products of the amplified fragments were sent for sequencing in both chain directions to Macrogen Inc. (Korea) or to the CDC .The amplified fragments were run on an agarose gel at 1% in 1× TBE buffer  to verify the integrity of the fragments, after which the PCR products were purified using a MultiScreenhttp://www.mbio.ncsu.edu/bioedit/bioedit.html) http://blast.ncbi.nlm.nih.gov/Blast.cgi) http://www.ub.edu/dnasp) The chromatograms obtained were edited using BioEdit  http://www.ncbi.nlm.nih.gov/genbank/). The collection locations and other information regarding the specimens, such as the GenBank accession numbers, will be available on the BOLD Systems (http://www.boldsystems.org) platform, as the “Phlebotominae Barcoding Initiative of the Americas - PBIN” project.All of the sequences obtained in the present study have been submitted to GenBank , Lutzomyia evansi , Lutzomyia gomezi , Lutzomyia hartmanni , Lutzomyia longiflocosa Osorno, Morales Osorno and Muñoz, 1970, Lutzomyia longipalpis , Lutzomyia nuneztovari (Ortiz 1954), Lutzomyia panamensis , Lutzomyia pia , Lutzomyia shannoni , Lutzomyia trapidoi , Lutzomyia youngi Feliciangeli & Murillo, 1987, and Lutzomyia yuilli yuilli Young & Porter, 1972.A total of 36 phlebotomine species belonging to 3 genera were collected from 11 Colombian departments . Some ofAedes aegypti (NC_010241.1), which was used as the reference genome. This segment also corresponded with that proposed by Hebert et al. The 5′ fragment of the COI mitochondrial gene was amplified and sequenced for each sample (n = 148), and the total size was 700 bp. The identities of the obtained sequences corresponded with positions 16 to 716 of the COI mitochondrial gene on During alignment at the nucleotide level, a total of 417 conserved sites and 283 variable sites were observed. The obtained COI sequences contained a large number of A+T pairs , particularly in the third codon position (87.6%) .Brumptomyia mesai, Brumptomyia beaupertuyi, Lutzomyia bifoliata, Lutzomyia carpenteri, Lutzomyia nuneztovari, Lutzomyia sordellii, Lutzomyia walkeri, and Lutzomyia (Helcocyrtomyia) sp. 1, which exhibited unique haplotypes for different specimens. The largest numbers of haplotypes were observed in Lutzomyia gomezi (9 haplotypes), Lutzomyia scorzai (7), Lutzomyia trapidoi (9), Lutzomyia panamensis (6), Lutzomyia trinidadensis (6), Lutzomyia triramula (6), Lutzomyia columbiana (5), and Lutzomyia carrerai thula (5)  .Regarding the numbers and types of substitutions observed among different haplotypes of the same species, the results showed between 2 to 40 variable positions , and nucThe set of sequences obtained was used to evaluate efficacy of the barcoding method to delineate species. Genetic distances, as determined by the K2P intraspecies values, ranged between 0 and 6% , and maximum intraspecific divergence was observed in 8 of the 36 species . BetweenGiven the presence of the “barcode gap” as a delimiting criterion and an indicator of species differentiation NJ dendrogram , shows tUsing the divergence of K2P distances, NJ dendrogram bootstrapping, and traditional morphological approaches, we found COI sequence always corresponded with species' identifications derived from morphologic characters .DNA barcode is a system designed to provide rapid, accurate, and automatable species-identification based on the standard analysis of short regions of genes to produce species tag The 5′ region of the mitochondrial COI gene is flanked by highly conserved regions, as noted by Hebert & Gregory The nucleotide lengths of amplified sequences of the COI gene (700 bp) in the present study were longer than those reported in previous studies performed in Phlebotominae: 549 bp reported by Azpurua et al. Phlebotomus and Sergentomyia in studies examining COI Within the DNA barcode sequences in phlebotomine sand flies, high A+T contents were observed, which were within average values (68.1 to 70.7%) reported for species of the order Diptera, including suborders Nematocera and Brachycera Lutzomyia, for which different mitochondrial gene fragments have been analyzed, including Cytochrome b (A+T = 73.09%), tRNAserine (A+T = 81.2%), and ND4 (A+T = 72.5%) In terms of codon usage, a preference for A or T termination within the triplets was clearly observed. This finding is supported by the fact that the base composition of mitochondrial DNA is highly correlated with codon usage because genes encoding mitochondrial proteins exhibit a preference for the use of codons rich in A+T. This phenomenon appears to be a characteristic of the Insecta mitochondrial genome Regarding differences in the numbers and types of substitutions among different haplotypes from the same species, the results revealed 2 to 40 variable positions changes were largely synonymous substitutions of the transitional type (purine by purine and pyrimidine by pyrimidine) at third positions, which do not affect the amino acid composition of the protein. In contrast, between species, nucleotide substitution rates gradually increased in relation to genetic distances. Blouin et al. Aliabadian et al. The use of DNA barcoding can delineate the boundaries between species and assign taxonomic status to unknown individuals from known species. The analysis of genetic distances is one of the criteria for acquiring this information L. gomezi (6% maximum intraspecific variability), L. panamensis (4.6%), and L. trinidadensis (3.4%)   could suL. gomezi and this species' extensive geographic distribution  suggests the present morphospecies may be comprised of two or more cryptic species.The low intraspecific variability observed may be the effect of sampling In the present study, we found a nucleotide divergence range between 9% and 26.7% for differentiation of the 36 species of phlebotomine sand flies identified.In general, our results revealed that there was no overlap between the levels of intra- and interspecific divergence, the previously mentioned barcode gap, even though higher values of intraspecific divergence were observed. This separation confirms the validity and partial effectiveness of the DNA barcode sequence as a molecular identifier of species The geographical scale of sampling has a critical impact on the global application of DNA barcoding The NJ method is conceptually related to grouping, but does not involve a phylogenetic tool, the natural history, or the behavior of a molecular clock L. Longiflocosa, L. youngi, and L. spinicrassa.In the present study, all MOTUs, including individuals belonging to the same species, were supported by the high bootstrap value of 100%, thus validating the consistency and usefulness of the COI barcode sequence to assign individuals to groups that corresponded to the species previously defined by specialists based on morphological features. Haplotypes from single species formed barcode groups that were clearly distinguishable from related species. As expected, some species that were represented by single individuals were not grouped with any other species . IncludiThe COI profile obtained for Phlebotominae confirmed the large variation that occurs in the mitochondrial genomes of these insects. Assigning species in this group required a combination of nucleotide divergence values and conventional taxonomic studies.Lutzomyia genus and most of them are present in other South American countries. This is important since the increased availability of sequences for phlebotomine sand fly species will provide a more comprehensive and explicit view regarding the possibility of using the COI sequence as a molecular marker in this region.This study is specific to Colombia and limited to the specimens collected, it allowed for the assignment of haplotypes corresponding to species within the study area. The species included in this study represent 13 of 27 groupings proposed for Young & Duncan Further study on genetic patterns and evolutionary processes will improve the ability to create a barcode-based identification system in phlebotomine sand flies and, consequently, facilitate future studies related to the biology of these insects.Table S1List of sand fly species, collection sites and number of genotypes per species used in the study.(DOCX)Click here for additional data file.Table S2Sequence divergence and nucleotide composition for the sand flies genera. The frequencies of nucleotides in sequence are presented as the total average values for all Condon positions and for each condon position separately with the accuracy to tenths of a percent.(DOCX)Click here for additional data file.


IQ contributed unique variance to mathematics in preschool and in primary school . A significant interaction of group with IQ  showed that IQ contributed unique variance to attention problems as rated by teachers, but that effects were stronger for very preterm than for term infants. Over and above IQ, EF contributed unique variance to mathematics in primary school , to parent rated inattention in preschool and in primary school , and to teacher rated inattention in primary school . In conclusion, impaired EF is, over and above impaired IQ, an important predictor for poor mathematics and attention problems following very preterm birth.Objective of this study was to examine the impact of executive function (EF) on mathematical and attention problems in very preterm  children. Participants were 200 very preterm (mean age 8.2 ± 2.5 years) and 230 term children (mean age 8.3 ± 2.3 years) without severe disabilities, born between 1996 and 2004. EFs assessed included verbal fluency, verbal working memory, visuospatial span, planning, and impulse control. Mathematics was assessed with the Dutch Pupil Monitoring System and parents and teachers rated attention problems using standardized behavior questionnaires. The impact of EF was calculated over and above processing speed indices and IQ. Interactions with group (very preterm versus term birth status) were examined. Analyses were conducted separately for two subsamples: children in preschool and children in primary school. Very preterm children performed poorer on tests for mathematics and had more parent and teacher rated attention problems than term controls (ß Most very preterm  infants survive without major disabilities.A large body of literature on term children has demonstrated that higher-order neurocognitive processes, the so-called executive functions (EF) are crucial in explaining academic difficulties and behavior problems.Research has consistently described that very preterm birth affects EF.Given the ongoing debate on whether IQ and EF are related or distinct concepts,Aim of this study was to capture the unique contribution of EF to mathematical and attention problems in very preterm children over and above that of processing speed indices and IQ. EF’s were selected on which our very preterm sample had deficits [>.3 standardized mean difference [SMD]) compared to term control children.s<.8; all Ps>.6). The term control group (n = 230) was recruited from three regular schools located in the same neighbourhoods as schools attended by the very preterm children, and included children without histories of prematurity , perinatal complications, and neurological disorders. The present study was carried out in the years 2007 and 2008. Parents of all participating children provided informed consent. The medical ethics review board of the Erasmus University Medical Centre Rotterdam approved the study protocol.The sample of 200 very preterm  children was derived from all (n = 1260) very preterm infants admitted between 1996–2004 to the neonatal intensive care unit (NICU) of the Erasmus University Medical Centre, Sophia Children's Hospital Rotterdam, The Netherlands. 252 infants died. Twins (n = 302) were excluded as inclusion of these children would violate the assumption of independence of data. Very preterm children with severe disabilities not being able to perform tests as employed in the present study were also excluded (n = 77). Such severe disabilities were classified according to Wood et al,Minor neurosensory dysfunctions as observed in participating children are presented in www.cito.com.In the Netherlands, preschool starts at the child's fourth birthday and constitutes at least two years. Primary school starts with grade 1 in August for children who turn 6 years of age between October of the previous year and the following September. Mathematics in preschool and primary school were assessed using standardized tests that are part of the Dutch National Pupil Monitoring System.Inattention in preschool children was rated by parents and teachers using the Attention Problems scale of the Child Behavior Checklist-Preschool [CBCL/1-5),r<.52).To enhance reliability for these primary school questionnaires, we averaged scores on the parent DBD and CBCL attention scales. The same was done for the teacher DBD and TRF attention scales. Average scores were calculated for parent and teacher ratings separately, since interrater correlations were moderate (EF’s that were measured included: 1) verbal fluency, measured in a test that required children to name as many examples of two specific categories: “animals” and “things you can eat or drink” within a 40-second time frame.Speed and inconsistency in speed were assessed using the correctly executed go-trials of the Stop Signal test,IQ was measured with the subtests Vocabulary and Block Design of the WISC-III Mathematical skills in preschool and in primary school were individually assessed by trained school staff. For very preterm children, completion of behavior questionnaires and assessment of EF and IQ took place at the Erasmus University Medical Centre Rotterdam Sophia Children's Hospital Rotterdam. Term children were assessed at their schools.s>.07), and gender , except that they were younger .Data on mathematics were available for 75.3% (n = 311) of the participating children. For the remaining children, data on mathematics were not available because they were either in special education (n = 24), or their school used a different pupil monitoring system (n = 30), or they were too young (n = 55) to be assessed with the mathematics test at the time of participation in our study.s>.41), although the latter group was on average 3 months younger . Primary school parent rated attention was available for 80.70% of the children and teacher rated attention was available for 74.10% of the children. Gestational age, birthweight, parental education, and gender, did not differ between children with and without these ratings , except that parents of children with teacher ratings available had a higher level of education, .Preschool parent rated attention was available for all children. Preschool teacher rated attention was available for 70.0% of the children. Gestational age, birthweight, parental education, and gender, did not differ between children with and without these teacher ratings  which resulted from either examiner error or child noncompliance. These missing values were replaced by means of maximum likelihood estimation (Expectation Maximization).2) of each step in the analyses were evaluated. Steps that did not reach the threshold for significance (P<.05) were not incorporated in further analyses. In step 1, the predictor group (very preterm versus term birth status) was entered, adjusted for grade  and standardized regression coefficients (β) with values of.10, .30, and .50, referring to small, medium, and large effects, respectively.Whether poor mathematics and attention problems could be predicted from EF was examined using hierarchal linear regression analyses. These analyses were conducted separately for children in preschool and primary school. Raw scores were used in all analyses and P-values of <.05 (two-tailed) were considered statistically significant. R-square change values . In Step 4, impulse control significantly predicted parent rated attention problems over and above speed and IQ, but EFs did not significantly predict mathematical problems nor teacher rated attention problems .In preschool, very preterm versus term birth status (i.e. group) significantly predicted poor mathematics and parent and teacher rated inattention. Step 2 and 3 yielded significant effects of IQ on mathematics. Processing speed indices were not significantly predictive for mathematical nor for attention problems  indicated that very preterm boys had the highest teacher ratings of inattention. Step 2 and 3 yielded significant effects of low IQ on parent and teacher rated inattention . A significant interaction with group  indicated that the association between IQ and teacher rated inattention was significantly stronger for very preterm than for term children. Processing speed indices did not significantly predict parent nor teacher rated inattention . Of the EFs, entered in Step 4, poor verbal working memory and visuospatial span significantly predicted parent rated attention problems and poor visuospatial span and impulse control significantly predicted teacher rated attention problems. None of the remaining interactions with group were significant.In primary school, group significantly predicted poor mathematics. Step 2 and 3 yielded significant effects of processing speed and IQ on mathematics, although effects of processing speed disappeared , with EFs entered into the analyses in Step 4. Of the EFs entered in Step 4, poor visuospatial span significantly predicted poor mathematics. Group also significantly predicted parent rated and teacher rated inattention, and a significant interaction between group and gender In our study, effects of EF were calculated while adjusting for IQ, whereas in earlier studies, e.g.Very preterm birth is associated with deficits in mathematics and symptoms of inattention. This study showed that impaired EF was, over and above impaired IQ, an important predictor for these adverse outcomes. Given the increasing body of researchTable S1Correlation Coefficients for Associations Between IQ, Processing Speed Indices, EF, Mathematics, and Attention Ratings in Preschool and Primary School.(DOC)Click here for additional data file.


In women with ischemia and no obstructive coronary artery disease, the Women's Ischemic Syndrome Evaluation (WISE) observed that microvascular coronary dysfunction (MCD) is the best independent predictor of adverse cardiovascular events. Since coronary microvascular tone is regulated in part by endothelium, we hypothesized that circulating endothelial cells (CEC), which reflect endothelial injury, and the number and function of bone-marrow derived angiogenic cells (BMDAC), which could help repair damaged endothelium, may serve as biomarkers for decreased coronary flow reserve (CFR) and MCD.We studied 32 women from the WISE cohort. CFR measurements in response to intracoronary adenosine were taken as an index of MCD. We enumerated BMDAC colonies and CEC in peripheral blood samples. BMDAC function was assessed by assay of migration of CD34+ cells toward SDF-1 and measurement of bioavailable nitric oxide (NO). These findings were compared with a healthy reference group and also entered into a multivariable model with CFR as the dependent variable.2 = 0.45; P<0.05).Compared with a healthy reference group, women with MCD had lower numbers of BMDAC colonies  and NO . Multivariable regression analysis showed strong correlation of CFR to the combination of BMDAC colony count and CD34+ cell function (migration and NO) (RThe BMDAC function and numbers of BMDAC colonies are decreased in symptomatic women with MCD and are independently associated with CFR. These circulating cells may provide mechanistic insights into MCD in women with ischemia. Ischemic heart disease (IHD) remains the leading cause of death and a major cause of disability among women. However, the majority of women with symptoms and other findings suggesting ischemia referred to coronary angiography have no severe coronary obstruction Recently, circulating endothelial cells (CEC) We hypothesized that the number of CEC, which reflect endothelial injury, and the number and function of circulating BMDAC, which potentially participate in repair of injured endothelium, might serve as noninvasive biomarkers for MCD.All study participants gave written informed consent before undergoing evaluation. These studies conform to the ethical principles outlined in the 1975 Declaration of Helsinki and were approved by the University of Florida Institutional Review Board.http://www.clinicaltrials.gov/ct2/show/NCT00000554) is an NHLBI-sponsored study aimed at improving diagnostic evaluation and understanding pathological mechanisms of IHD in women. The design was a prospective cohort, and protocol details, including selection criteria, have been previously published in detail The Women's Ischemia Syndrome Evaluation (WISE)  masked to patient data; detailed methodology has been published Coronary reactivity testing was performed in a stenosis free area of the left anterior descending coronary artery when possible, with the left circumflex artery as a secondary choice. A Doppler-tipped guidewire  was advanced through the diagnostic catheter, and when a stable velocity signal was obtained, baseline recordings were made. Intracoronary bolus injections of 18 mcg of adenosine , a predominantly non–endothelium-dependent microvascular dilator, were administered into the left main coronary artery. At least 3 injections were done to assure a stable average peak blood flow velocity was obtained, with return to baseline velocity documented before each bolus. Pulsed-wave Doppler flow spectra were used to calculate time-averaged peak velocity (APV). Recordings were analyzed at the WISE CFR Core Lab (University of Florida) masked to all other data, and CFR was defined as the ratio of APV after adenosine to average baseline velocity just before adenosine. In prior WISE studies, this measure correlated closely  with volumetric flow A minimum of 8 ml of peripheral blood collected in CPT tubes with heparin  was centrifuged. The mononuclear cells were pelleted, washed twice and then resuspended in 100 µl PBS supplemented with 2 mmol/L EDTA, to which 33 µl of FcR-blocking reagent and 33 µl of magnetic microbeads conjugated with an anti-CD34 antibody  were added. After incubation for 30 min at 4°C, the cells were diluted in 10× volume of PBS supplemented with 2 mmol/L EDTA supplemented with 0.1% BSA. The CD34+ cells were positively selected by automated magnetic selection . Selected cells were confirmed to be CD34+ cells staining with phycoerythrin-conjugated anti-CD34 (Miltenyi Biotec) and flow cytometry.The BMDAC were incubated with 5 µM 4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) diacetate for 30 min at 37°C in the dark. Excess extracellular probe was removed by washing in Hanks balanced salt solution followed by incubation for 10 min at room temperature to allow for probe de-esterification. Green fluorescence was measured using an inverted microscope Axiovert 200 (Carl Zeiss) equipped with CCD camera and image acquisition/analysis software AxioVision (Version 4.5). Fluorescence intensity was measured in 20 to 30 cells per field in at 3–4 fields per experiment.Chemotaxis was assessed by staining the CD34+ cells with CellVue Burgundy  before loading onto a Boyden Chamber. SDF-1 was loaded in the bottom chamber onto which a polycarbonate membrane  coated with 10% bovine collagen was overlaid and the cells were loaded in the top chamber. After 4 h at 5% CO2 at 37°C, the percentage of cells that migrated was determined by collecting media in the lower chamber and determining relative fluorescence using Odyssey® Infrared Imaging System  at excitation of 683 nm and an emission of 707 nm. Percentage increase in migration was calculated as maximum increase in migration in response to SDF as compared to baseline (no SDF).Peripheral blood mononuclear cells were isolated from 8 ml of peripheral blood collected in CPT tubes . Cells were enumerated, resuspended in Endocult medium , and cultured on 6-well plates precoated with human fibronectin (Becton Dickinson). After 48 hours, new medium was added, and nonadherent cells were transferred to a new fibronectin-coated plate to remove monocytes and mature endothelial cells. After 3 days of culture, BMDAC colony–forming units (CFU) were enumerated by light microscopy.The mononuclear cell fraction from the CPT tubes was incubated with immunomagnetic beads conjugated with P1H12, a murine, monoclonal antibody specific for human endothelial cells. The rosetted cells were cytospun onto poly-L-lysine coated slides. After drying overnight, slides were fixed with 1% paraformaldehyde and stained with propidium iodine in phosphate-buffered saline (PBS), prior to mounting the slides in Vectashield with DAPI . Quantitation of CEC was performed by identification of the cells using a Zeiss Axiophot microscope  and expressed as number per ml of blood.2 to satisfy the assumptions of homogeneity, linearity, and Gaussian distribution of errors. The data were transformed as follows: log2CEC = log2(CEC/10); log2Migration = log2(% increase in migration/10+0.01); log2NO = log2(NO/100); log2 BMDAC  = log2(BMDAC/10+0.01). We compared the log transformed BMDAC number and function between the WISE cohort and healthy reference group using the Satterthwaite two-sided t-test. Multivariable linear regression analysis was conducted with CFR as the dependent variable. Pearson's correlation between variables and condition index of each variable were checked using a linear regression model to detect collinearity. Cell covariates include BMDAC colony number, maximum % increase in migration of BMDAC to SDF, NO in BMDAC, CEC, with clinical covariates including diabetes, hypertension, hyperlipidemia, age and double product of HR×SBP. A backward model selection was employed, and CEC, diabetes, hypertension and hyperlipidemia were dropped sequentially from the model because of their insignificant effects. Age, maximum % increase in migration of BMDAC to SDF, and NO in BMDAC were retained in the model to improve overall model fit. A P-value <0.05 was considered statistically significant.The statistical analyses were conducted in SAS 9.3 . Clinical characteristics were presented as either mean ± SD for continuous variables or % for categorical variables. Number of BMDAC colonies, NO, and migration were expressed as median (range), and transformed to log base Pertinent baseline characteristics of the WISE women studied are summarized in Compared with the age, BMI, and sex-matched healthy reference group, the WISE cohort had fewer BMDAC colonies . The intracellular NO in BMDAC was lower in the WISE cohort than in the healthy reference group . The BMDAC function, as measured by migration toward SDF, was not significantly different between the WISE cohort and the healthy reference group . The box plots comparing the cell variables and the results of the t-test comparing the WISE cohort to the healthy reference group are shown in 2CFR and Log2BMDAC (r = 0.50 p = 0.02). Traditional risk factors for atherosclerosis such as diabetes, hypertension, and hyperlipidemia did not show a significant correlation to CFR. Although BMDAC function by itself was not associated with CFR, in multivariable regression the combination of BMDAC colonies and function significantly correlated with CFR , when controlled for cardiac work (HR×SBP) and age. The model is summarized in A total of 23 patients with complete data were included in the regression analysis; patients with incomplete data were excluded. Univariate analysis showed a significant correlation between LogThere was no difference in the number of CEC between the WISE group and healthy reference group . We did not observe a significant contribution of CEC to CFR.Recent studies indicate that the number and functionality of BMDAC may predict clinical outcomes in patients with CAD. Vasa et al reported that number and migratory capacity of BMDAC (as CD34+KDR+ cells) are impaired in patients with CAD compared with age-matched healthy controls We are aware of only two published reports among patients with suspected MCD dealing with BMDAC number and function: both are relatively small in sample size with few women and coronary microvascular function was not directly measured. Shmilovich et al Both studies relied on angina, evidence of ischemia on clinical non-invasive testing, and absence of obstructive CAD on a clinical coronary angiogram for a diagnosis of suspected MCD, and they also included very few women . Our study included 32 women, a prospectively defined protocol which required that angiograms and CFR recordings be read by core labs masked to other patient data, and determination of MCD by direct intracoronary blood flow measurements in response to adenosine to define CFR. We found that the ability of BMDAC to form colonies was not only markedly reduced in the WISE cohort, but the number and function of these cells were significantly associated with measured CFR.Although traditional risk factors such as diabetes, hypertension, and hyperlipidemia are associated with decreased BMDAC number and function, consistent with our previously published work in a larger cohort of patients We did not find any difference in migration between WISE and healthy women. The production of NO in CD34 cells was reduced in the WISE cohort . NO is thought to be a key mediator of BMDAC dysfunction in diabetes Surprisingly, the numbers of circulating CEC, a marker of endothelial injury, were not significantly different between the WISE cohort and healthy reference group. However the role of endothelial injury in MCD is not entirely clear. Bellamy and colleagues, as well as others, have shown that patients with Syndrome X have reduced flow-mediated vasodilatation, a surrogate marker for endothelial dysfunction The main limitation of our study was the small number of subjects, which limited subgroup analysis and the ability to assess more complex interactions between CFR and the determinant variables. Nevertheless, this is the largest number of women with directly measured MCD and cell number and function analyses yet to be reported. Since it would have been unethical to subject our reference group to coronary angiography, we chose a population that was free from known CAD and risk factors for atherosclerosis. This reference group, though closely matched to study subjects, was slightly younger  and had lower triglyceride levels. Moreover, our study only included women, most of whom were postmenopausal. However, our results are consistent with the current understanding of BMDAC function. The model appears sensitive to changes in cardiac work load approximated by the double product (HR X SBP). The ability of cardiac work to predict CFR has been substantiated in previous studies In conclusion, the decreased number and function of BMDAC in the WISE cohort, the correlation of BMDAC number and function with CFR compared with the healthy reference group, and the independent association of number of BMDAC colonies with CFR suggest that these cells might have a role in the pathogenesis of MCD. These circulating cells provide mechanistic insights into the pathophysiology of MCD, and interventions designed to correct functional abnormalities of BMDAC might have therapeutic potential. Further studies are needed to confirm these findings in a larger cohort and to identify if these findings are applicable in other conditions with a reduced CFR.


The compliance with influenza vaccination is poor among health care workers (HCWs) due to misconceptions about safety and effectiveness of influenza vaccine. We proposed an educational prospective study to demonstrate to HCWs that influenza vaccine is safe and that other respiratory viruses (RV) are the cause of respiratory symptoms in the months following influenza vaccination. 398 HCWs were surveyed for adverse events (AE) occurring within 48 h of vaccination. AE were reported by 30% of the HCWs. No severe AE was observed. A subset of 337 HCWs was followed up during four months, twice a week, for the detection of respiratory symptoms. RV was diagnosed by direct immunofluorescent assay (DFA) and real time PCR in symptomatic HCWs. Influenza A was detected in five episodes of respiratory symptoms (5.3%) and other RV in 26 (27.9%) episodes. The incidence density of influenza and other RV was 4.3 and 10.8 episodes per 100 HCW-month, respectively. The educational nature of the present study may persuade HCWs to develop a more positive attitude to influenza vaccination. The compliance with influenza vaccination has been historically poor among health care workers (HCWs) varying from 2 to 36% around the world At Hospital das Clinicas, University of São Paulo School of Medical Sciences, a previous study showed a 34% compliance with influenza vaccination among HCWs. In the mentioned study, the main reasons for non-compliance were the perception of vaccine inefficacy and the fear of adverse reactions Respiratory symptoms occurring after vaccination are frequently misinterpreted as vaccine failure which reinforces the HCW's skepticism on vaccine efficacy. To overcome these false beliefs, we proposed a prospective study in a cohort of HCWs to demonstrate that influenza vaccine is safe and other respiratory viruses (but not influenza) are generally the cause of respiratory symptoms in the months following influenza vaccination.This study was conducted at Hospital das Clinicas, University of São Paulo School of Medical Sciences (HC-FMUSP) from May to October 2006. The Hospital das Clinicas is a 2,000-bed tertiary teaching hospital consisting of 5 buildings attached to the University of São Paulo. The main building has approximately 900 beds and contains most of the surgical and clinical wards and 12 intensive care units. Hospital das Clinicas has an estimated 15,000 HCWs, including permanent and casual staff, employees, students, and volunteers.Since 1999, annual influenza vaccination has been offered free of charge to all HCWs. Vaccination usually takes place at the hospital's Immunization Center during working hours, from Monday to Friday. In 2006, as a strategy to increase compliance with influenza vaccination, vaccine was offered at places of easy access during expanded hours, as suggested by 61% of the interviewed in previous survey During vaccination campaign, HCWs were invited to participate in the present study which had two steps: 1) evaluation of vaccine safety and, 2) cohort study to evaluate which respiratory viruses were more frequently detected in HCWs presenting respiratory symptoms in the four-month period following vaccination. Sample size was estimated taking into account an expected frequency of 10% of adverse events in adult population. Considering an acceptable frequency rate of up to 13%, we estimated to enroll at least 377 HCWs (EpiInfo version 6).Three hundred and ninety eight vaccinated HCWs were surveyed for adverse events occurring within the first 48 h after influenza vaccination. A subset of 337 HCWs participated in the follow-up phase of the study. To assure that all hospital sectors were represented, the cohort was defined during the assessment of adverse events which was performed by a hospital epidemiologist nurse, through visits in all hospital floors and sectors. Afterward, these HCWs were actively surveyed twice a week, at work place, during four months, to check for the occurrence of respiratory symptoms and nasal wash sampling which was done in 93 of the participants. The following adverse events were actively surveyed: fever, headache, malaise, myalgia, local pain, local edema and allergic reaction. Other adverse events spontaneously reported were also registered. During follow-up visits, participants were asked about the presence of the following symptoms: fever, coryza, blocked nose, sneeze, cough, watery eyes, headache, myalgia, sore throat, hoarseness, sibilance, and dyspnea. Allergy was ruled out in those with sneezing as the only symptom. Influenza like illness (ILI) was defined by the presence of fever and cough and/or sore throat according to the CDC definition Respiratory syncytial virus (RSV), influenza (INF) A and B, adenovirus (ADV) and parainfluenza virus (PIV) were diagnosed by direct immunofluorescent assay (DFA) according to the manufacturer's instructions . Aliquots of NW samples were stored at −80°C for later PCR and real time PCR processing. PCR was used to detect coronavirus and picornavirus. RT-PCR products for picornavirus were subsequently sequenced to differentiate rhinovirus from enteroviruses. Real time PCR (Taqman assay) was used to diagnose human metapneumovirus (hMPV). To increase the sensitivity of influenza diagnosis, a real time PCR (Taqman assay), was added to the diagnostic tools. Similarly, a nested adenovirus PCR was used along with DFA due to the low sensitivity of the latter in diagnosing ADV. The PCR protocols used in the present study have been published elsewhere A Comissão de Ética para análise de projetos de Pesquisa - CAPPEsq da Diretoria Clínica do Hospital das Clínicas e da Faculdade de Medicina da Universidade de São Paulo, em sessão de 12/04/2006, aprovou o Protocolo de Pesquisa no. 241/06, intitulado Viroses respiratórias após vacinação contra influenza em profissionais de saúde, apresentado pelo Departamento de Moléstias Infecciosas e Parasitárias, inclusive o Termo de Consentimento Livre e Esclarecido. CAPPesq, 12/04/2006”. Written informed consent was obtained from all participants.The study was approved on 04/12/2006 by local Ethics Committee with the following statement: “χ2 test or Fisher's exact test for discrete variables, and Student's t test or the Mann-Whitney U test for continuous variables. Tests of significance were two sided, and p<0.05 was considered to be statistically significant. Incidence density (ID) of respiratory symptoms, influenza virus infections and other respiratory virus infections were calculated by the formula described below. ID results were expressed per 100 HCW-month SPSS 15.0  was used with the p = 0.017). The sectors with highest frequency of adverse events were the Virology Laboratory (87.5% of the subjects), Burn unit (54.5%), Nephrology (52.4%) and Pneumology (45.5%). In the remaining sectors, AE were reported by less than 40% of the subjects. Those surveyed in the first five days after vaccination were more likely to report such events . Local AE were reported by 18.3% of the participants, systemic AE by 71.6%, and 10% of the participants reported both local and systemic AEs. Headache, myalgia and malaise were more frequently reported . Local pain and local edema was reported by 17.5% and 5% of the HCWs. No severe adverse event was observed.The interviews for adverse events occurring within 48 h of vaccination were made from one to 10 days after vaccination. The majority of the participants (81.4%) were surveyed within the first week of vaccination. All hospital sectors were represented . One hunper HCW, ranging from one to 25 visits).During the 4-month period following influenza vaccination, respiratory symptoms were evaluated in 337 HCWs. A total of 4,182 follow-up visits were performed (median 12 per 100 HCW-month.One hundred and twenty-one HCWs (36%) developed 192 episodes of respiratory symptoms. Coryza, cough, sore throat and myalgia were reported by 36.3%, 25%, 17.4% and 13.7% of the participants, respectively. Seventy-one of them (58.7%) presented more than one episode suggestive of upper respiratory infection (URI). ILI was observed in 17 of the 192 episodes (8.8%). Mean time to the occurrence of respiratory symptoms was 2.9 (0.7 to 5.2) months. The incidence density of respiratory symptoms was 12.4 episodes versus 41.2%, p = 0.37)  no respiratory virus was found, even though 82% had coryza, 53% had cough and 49% had sore throat. The frequency of ILI was similar among HCWs who agreed with sampling and those who did not agree (58.8%  = 0.37) .per 100 HCW-month.Influenza A virus was detected in 5 of 93 episodes (5.4%), considering both techniques. DFA diagnosed two cases and real time PCR detected three additional ones. Respiratory symptoms in HCWs with influenza are described on per 100 HCW-month. Other RV infections occurred significantly later than influenza cases during follow-up (p = 0.04).Other respiratory viruses were diagnosed in 26 of the 93 episodes (28.3%) of URI. Rhinovirus and coronavirus were the RV more frequently detected . Mean tiMisconceptions about influenza vaccine, be it about its safety or its effectiveness, have been identified in all studies included in a recent review of attitudes and predictors of influenza vaccination among HCWs, highlighting the importance of education efforts Initial symptoms of RV infections are often unspecific such as fever, malaise, or myalgia. As influenza vaccine is offered when other RV are circulating , vaccinated HCWs developing symptoms within 48 h of vaccination misinterpret those signs as vaccine adverse events. In addition, the occurrence of respiratory symptoms in the months following vaccination is mistaken as vaccine failure. Other respiratory infections as the cause of such symptoms are hardly ever considered.To diminish the arguments of fear of adverse events or perception of vaccine inefficacy, this prospective study was conducted to demonstrate to a subset of HCWs from our hospital, that severe adverse events following influenza vaccination are rare and the episodes of respiratory symptoms occurring in the first four months after vaccination are generally caused by other respiratory viruses and not by influenza virus.As expected, no severe adverse event was observed in the present study, and the events more frequently reported, such as headache, myalgia and malaise could be related to influenza vaccine itself as well as to other causes, given their unspecificity.In adults, the adverse event more frequently reported after intramuscular administration of inactivate vaccines is local pain, affecting 10% to 64% of the vaccinated Systemic reactions like fever, malaise and myalgia can also occur after inactivate vaccines. In the present study, the frequency of systemic AEs (over 70%) was higher than reported in previous studies. Recent publications have shown rates of systemic adverse events ranging from 30% to 59% in HWCs Anaphylaxis and neurological reactions are rare As HCWs are aware of vaccine adverse events and fear its consequences, it is comprehensible that these events will be more frequently reported by them than by general population. Another study conducted in the same hospital demonstrated that HCWs reported significantly more adverse events (52.9%) than the elderly (25.3%) Interestingly, we observed that some sectors showed significantly higher rates of AE than others, supporting the subjectivity of the information. Also, this data may suggest a mouth to mouth effect among sector coworkers influencing the self-report of AE. Among HCWs, the belief that coworkers take influenza vaccine influences the vaccine uptake. Thus, it is possible that the same occurs concerning to adverse events.Continued education of health professionals is essential to highlight not only the epidemiological importance of the vaccine, but also its safety and the low risk of severe adverse events.Our study also demonstrated that the respiratory symptoms occurring in the months following influenza vaccination were more frequently caused by other respiratory viruses and generally do not mean vaccine failures.p = 0.37).One limitation of our study is that in only 93 of the 192 episodes of respiratory symptoms (48.4%) the subjects agreed with NW sampling. NW sampling is a simple but uncomfortable procedure and this fact may explain why some HCWs preferred not to get tested during working hours. One could argue that influenza cases could be missed among those not tested. However, we believe that this loss has not affected our results as the frequency of ILI was similar between those who agreed with sampling and those who did not , which has been used worldwide for influenza surveillance purposes. However, the sensitivity and positive predictive value of such definition can vary greatly depending on the co-circulation of other respiratory viruses in the community Influenza infection is characterized by the abrupt occurrence of fever, headache, myalgia, and dry cough. During influenza season, the presence of these symptoms is highly predictive of influenza infection and summarizes the case definition of In our series, influenza cases in vaccinated HCWs were mild and occurred significantly earlier following vaccination in comparison to other respiratory viruses. This finding may be explained by the marked seasonality of influenza in São Paulo city as reported previously The effectiveness of influenza vaccines is related predominantly to the age and immune competence of the vaccinee and the degree of similarity between the viruses in the vaccine and those in circulation. Vaccine effectiveness in preventing laboratory-confirmed influenza illness when the vaccine strains are well matched to circulating strains is 70–90% in randomized, placebo-controlled trials conducted among children and young healthy adults, but is lower among elderly or immunocompromised persons Trials that measure laboratory-confirmed influenza virus infections as the outcome are the most persuasive evidence of vaccine efficacy In accordance with the educational nature of our study, we considered all cases of influenza as vaccine failures, since vaccinated health personnel look forward to be protected against influenza. Molecular characterization of influenza cases was not performed to check for possible mismatches between circulating viruses and vaccine strains, which could possibly justify those failures.Our study demonstrated that the fear of severe adverse events seems unjustified as well as the perception of vaccine inefficacy. URI following influenza vaccination were generally caused by other respiratory viruses and not by influenza.In times of pandemic influenza A H1N1 and widespread vaccination, healthcare and emergency medical services personnel are among the priority groups recommended to receive the H1N1 influenza vaccine. It is time to overcome definitively the misconceptions about the vaccine as well as the fear of adverse events. So far, the vast majority (93%) of adverse events reported to VAERS after receiving the trivalent 2010–2011 influenza vaccine, were classified as “non serious”, e.g., soreness at the vaccine injection site We believe that the educational nature of the present study may persuade HCWs to develop a more positive attitude to influenza vaccination.


As a major alternative therapy in Traditional Chinese Medicine, it has been demonstrated that moxibustion could generate a series of molecular events in blood, spleen, and brain, and so forth. However, what would happen at the moxibustioned site remained unclear. To answer this question, we performed a microarray analysis with skin tissue taken from the moxibustioned site also Zusanli acupoint (ST36) where 15-minute moxibustion stimulation was administrated. The results exhibited 145 upregulated and 72 downregulated genes which responded immediately under physiological conditions, and 255 upregulated and 243 downregulated genes under pathological conditions. Interestingly, most of the pathways and biological processes of the differentially expressed genes (DEGs) under pathological conditions get involved in immunity, while those under physiological conditions are involved in metabolism. In acupuncture research, microarray analysis has been widely employed to uncover gene expression profiles at different tissues or organs –19. Basead libitum and randomly assigned to the various experimental groups . The experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals, and all experimental protocols were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine.Adult male Sprague-Dawley rats weighing 200–220 g obtained from Chengdu University of Traditional Chinese Medicine, Experimental Animal Centre, were utilized in this study. Maintained in animal room of automatically controlled day cycles (12 : 12 = light : dark cycle) at 24 ± 2°C, all rats were allowed to freely take food and water In this study, we aimed to explore what would take place at the moxibustioned site in the view of potential molecular target under physiological and pathological conditions. Thus, we designed two different parts of microarray experiment. One is designed for uncovering the gene expression profile at physiological condition in which it consisted of 2 group: healthy control group (C) and healthy control with moxibustion stimulation group (CM). The other one is designed for revealing the gene expression profile at pathological condition in which it composed of model control group (M) and model control with moxibustion stimulation group (MM).Under physiological condition, the rats in CM group received moxibustion at the left acupoint Zusanli (ST36), at the depression below the knee from the anterior crest of the tibia  for 15 mFirstly, the pathological condition was established by injecting subcutaneously with 0.1 mL Freund's Complete Adjuvant  into the plantar surface of the left hind paw of the rat . The CFA2 inhalation. The cutaneous tissue (0.5 cm × 0.5 cm × 0.2 cm) located at moxibustioned site were immediately removed and preserved in RNAlater  to prevent RNA degradation. Total RNA was extracted using TRIzol Reagent  and purified with RNA clean-up Kit  following the instructions of manufacturers, respectively. Total RNA was quantitated by spectrophotometry, and the integrity was assessed by formaldehyde denatured agarose gel electrophoresis. Two hours after one time of moxibustion stimulation was completed, rats was euthanized by COThe microarray analysis service provided by CapitalBio Corporation  was performed as described , 31. BriThe detected signal intensities of all probes on the chip ≥400 were included for comparison analysis. We applied two-class unpaired algorithm in the Significant Analysis of Microarray software  to identify significantly differentially expressed genes between CM and C groups, and MM and M groups. DEGs were determined with the threshold of false discovery rate, FDR ≤5% and fold change ≥2.0 or ≤0.5.http://bioinfo.capitalbio.com/mas3/) which integrated with KEGG and Gene Ontology (GO) database to perform pathway and GO Biological Process term enrichment analysis and calculate the statistical significance as described [P  value <0.001 was considered statistically significant.We employed the online Molecule Annotation System (MAS) established by CapitalBio Corporation  to detect the expression of four DEGs, Hspa1a, Mcpt8, Slpi, and Clqa, which were randomly selected from the 27K Rat Genome Array. http://dx.doi.org/10.1155/2013/890579). While under pathological condition, the results displayed 255 upregulated and 243 downregulated DEGs (Supplementary Table 2).Different numbers of DEGs at moxibustioned site were obtained from different condition. Under physiological condition, we obtained 145 upregulated and 72 downregulated DEGs  at moxibustioned site. Under physiological condition  at moxibustioned site. Under physiological condition  of the involved pathways at moxibustioned site were related to metabolism under physiological condition . HoweverAccording to the biological processes analysis, we also could find out the difference from different state. Without FCA as the pathological stimulation, the biological progresses following moxibustion administration at moxibustioned site were most composed of oxidation reduction, potassium ion transport, and so forth. However, a variety of biological processes related to immunity, such as immune response, antigen processing and presentation, and antigen processing and presentation of peptide antigen via MHC class I, and were exhibited in this study. Taken together, it seemed to be concluded that a series of molecular events would happen at moxibustioned site. Moreover, different pathways and biological progresses at moxibustioned site would be involved in different condition. However, this conclusion should be seriously taken for granted given the following limitations. Firstly, only one stimulation time point was used in this study. How about the time course or different time points, such as 5, 10, 15, 20, and 30 minutes, which were frequently practiced in moxibustion clinic, which needs to be answered in future study? Secondly, the pathological condition was induced by FCA injection in this study. To our knowledge, FCA injection will generate adjuvant arthritis through a series of immune actions , 34. MorAdditionally, in this study the tissue used for RNA extraction and microarray detection was taken from the skin of the moxibustioned site. So far, the skin has been also regarded as an important immune organ , 38 as wThe results suggested that a set of molecular events would have happened at moxibustioned site. Among those molecular events, different genes and different pathways and biological progresses at moxibustioned site would have got involved under different conditions.Supplementary Table 1: The DEGs list under physiological condition (CM versus C)Supplementary Table 2: The DEGs list under pathological condition (MM versus M)Supplementary Table 3: Co-expressed genes list between physiological and pathological condition.Click here for additional data file.


Glycated hemoglobin (HbA1c) is associated with an increased risk of cardiovascular disease. The aim of this study was to examine the relationship between HbA1c levels and the complexity of coronary artery lesions among the older patients with diabetes mellitus (DM).This retrospective study enrolled a total of 3805 consecutive type 2 DM patients aged 60 years and older who underwent their first elective coronary angiography and had their HbA1c levels measured at the Chinese PLA General Hospital between December 2005 and December 2012.The complexity of the coronary artery lesions was evaluated using the Syntax score, and the subjects were divided into three groups according to their HbA1c levels. Logistic regression and Pearson correlation were used to analyze the association between the measured HbA1c levels and Syntax score.p<0.001). The unadjusted correlation coefficient of HbA1c levels and the Syntax score was 0. 371 (p<0.001). In addition, the higher HbA1c categories were able to independently predict patients with intermediate or high Syntax score (Syntax score ≥23) after adjustment for age, sex, hypertension, smoking, dyslipidemia and creatinine levels in the logistic regression analysis.The mean age was 72.3±10.6 years. The higher HbA1c levels were significantly associated with higher Syntax score (HbA1c is significantly associated with the complexity of coronary lesions among older patients with DM. A higher HbA1c value is an independent predictor of the prevalence of complex coronary lesions. Further prospective multi-centre studies are needed to confirm this finding. Diabetes mellitus (DM) is an important risk factor for coronary heart disease The Syntax score is an angiographic grading system based on the severity and complexity of the characteristics of coronary artery lesions The benefits of intensive therapy in an effort to lower HbA1c level should always be weighed against the greater risk of disabling and unpredictable hypoglycemia among older patients with DM, especially in elderly patients. No clinical trial data that exclusively represents the value of strict glycemic control in the geriatric population are available. Therefore, the aim of the present study was to evaluate the relationship between HbA1c levels and complexity of coronary artery lesions among older patients with DM.We retrospectively reviewed the data of consecutive type 2 DM patients aged 60 years and older who underwent their first elective coronary angiography, to evaluate suspected CAD, at the Chinese PLA General Hospital between December 2005 and December 2012. Their medical history, lipid levels, creatinie levels, smoking status, demographic and angiographic data prior to undergoing coronary angiography were obtained from the hospital database. The indications for angiography in individuals in clinically stable condition were chest pain and/or noninvasive test results consistent with myocardial ischemia. We excluded patients whose HbA1c levels were not available during the hospitalization. The HbA1c of reference range for healthy nondiabetic individuals ranges from 4.1% to 6.5%. HbA1c was measured with an immunoassay  during this time. Subjects with age <60 years, known type 1 DM, any previous history of coronary revascularization, or incomplete laboratory measurements were also excluded, resulting in a cohort of 3805 subjects for the present analysis. Patients were divided into three groups according to their HbA1c levels: 1535 patients with HbA1c levels of <6.4% (group 1), 1195 patients with HbA1c levels of 6.5% to 8.5% (group 2) and 1075 patients with HbA1c levels higher than 8.5% (group 3).The written informed consents were obtained from all subjects or their designated relatives. The Institutional Review Board of the PLA General Hospital approved this retrospective study.The severity of coronary artery lesions was quantified with the Syntax score. From the baseline diagnostic angiogram, each coronary lesion producing ≥50% diameter stenosis in vessels ≥1.5 mm was scored separately and added together to provide the overall Syntax score, which was calculated using the Syntax score algorithm2 tests. Linear regression analysis with Pearson's coefficient was used to assess the strength of association between HbA1c and Syntax score. Logistic regression analysis was used to predict the prevalence of an intermediate or high Syntax score, with adjustment in the age, sex, hypertension, smoking, dyslipidemia, and creatinine levels. All data were processed using the PASW . A p-value <0.05 was considered to be significant.The continuous variables are presented as the means ± SDs. The categorical variables are presented as proportions (percentages). Intergroup differences of continuous variables were analyzed by one-way ANOVA. Categorical variables were compared with χp<0.001; group II (6.5%≤HbA1c≤8.5%); and group III (HbA1c>8.5%). Baseline characteristics and laboratory data of the patients are summarized in p<0.001. In addip<0.001). Moreover, the higher HbA1c categories were able to independently predict patients with intermediate or high Syntax score (Syntax score≥23) after the age, sex, hypertension, smoking, dyslipidemia, and creatinine levels were adjusted in the logistic regression analysis  in older diabetic patients. We established that the HbA1c level is significantly associated with the complexity of coronary lesions, independent of age, sex, and other cardiovascular risk factors such as hypercholesterolemia, hypertension, and smoking. Our findings indicate that the severity of coronary artery lesions is also notably influenced by the HbA1c, irrespective of other cardiovascular risk factors, age, and gender. Even after adjustment for these factors, HbA1c was still significantly associated with Syntax score. Additionally, the multivariate regression analysis showed that HbA1c is independently associated with Syntax score. Our study therefore demonstrated that, among older DM patients, HbA1c values can be a predictor of the prevalence of complex coronary artery lesions. The patients with the lower HbA1c levels have a distinctly lower risk of complex coronary artery lesions. Therefore, diabetic patients with cardiovascular risk factors should pay more attention to their blood glucose levels and potential cardiovascular complications, even among older patients with DM. Furthermore, the relationship between the Syntax score and HbA1c demonstrated in this study supports using HbA1c as a simplified indicator of prognosis. This study suggest that an improvement in glycemic control results in more favorable intermediate or long-term clinical outcomes in diabetic patients aged 60 years and older. However, the relationship between glycemic control and coronary atherosclerosis is not a simple one. There are many unmeasured potentially important covariates that may play a role. Maybe, for example, the duration of the diabetic process leads to both worse coronary atherosclerosis and higher HbA1c. As reported in a study, they found that interwoven actions of poor glycemic control, low grade inflammation and low HDL-cholesterol on atherosclerotic processes in type 2 diabetes There were several limitations in this study which should be considered. This study was a retrospective and non-randomized study. First, our patients are only those who underwent first elective coronary angiography. These patients comprise some patients with CAD and may not represent all patients with CAD. Second, several confounding factors may not have been properly accounted for in the analysis. Consequently, numerous patients were excluded, and selection bias may have affected the results. Third, the present study excluded patients with a history of PCI or CABG, therefore our findings may not apply to these excluded subjects who represent more severe cases. We also did not collect information on severe obesity, peripheral vascular disease, cerebrovascular disease, estimated glomerular filtration rate, or duration of diabetes, which were also associated with the burden of vascular atherosclerosis. Thus, we should interpret the results very carefully. Finally, our study is a single-center observational experience. Most of the patients came from Beijing and its neighboring areas, so their glucose metabolic state may not completely be in coincidence with population of other places. Further prospectively multiple centre studies are required to better quantify this finding. Although caution is necessary for the interpretation of our data, we consider it improbable that these limitations have influenced our main findings.In conclusion, the HbA1c is significantly associated with the complexity of coronary lesions amongst type 2 DM patients aged 60 years and older. The present study reveals that higher HbA1c value is an independent predictor of the prevalence of complex coronary lesions, even after adjusting the age and other coronary atherosclerotic risk factors. HbA1c may be a useful indicator of type 2 DM in patients aged 60 years and older with greatest absolute risk of cardiovascular disease. HbA1c could be used as a simple prognostic value after primary PCI in coronary care unit, and it also helps for the strict control of complications and preventive strategies. Control of glycemic metabolism may play an important role in reducing the HbA1c levels. For patients without hypoglycemia, further study is necessary to evaluate the benefit of intensive treatment of glycometabolic disorder to reduce the HbA1c level.