Nosema ceranae. Newly emerged adult workers were collected and divided into four groups: Group I—no treatment; Group II—inoculated with N. ceranae, Group III—antibiotic treatment, and Group IV—antibiotic treatment after inoculation with N. ceranae. Our study showed that Nosema infection did not cause obvious disruption of the gut bacterial community as there was no significant difference in the density and composition of gut bacteria between Group I and Group II. However, the elimination of gut bacteria by antibiotic (Groups III and IV) negatively impacted the functioning of the honey bees’ immune system as evidenced by the expression of genes encoding antimicrobial peptides abaecin, defensin1, and hymenoptaecin that showed the following ranking: Group I > Group II > Group III > Group IV. In addition, significantly higher Nosema levels were observed in Group IV than in Group II, suggesting that eliminating gut bacteria weakened immune function and made honey bees more susceptible to Nosema infection. Based on Group IV having displayed the highest mortality rate among the four experimental groups indicates that antibiotic treatment in combination with stress, associated with Nosema infection, significantly and negatively impacts honey bee survival. The present study adds new evidence that antibiotic treatment not only leads to the complex problem of antibiotic resistance but can impact honey bee disease resistance. Further studies aimed at specific components of the gut bacterial community will provide new insights into the roles of specific bacteria and possibly new approaches to improving bee health.It has become increasingly clear that gut bacteria play vital roles in the development, nutrition, immunity, and overall fitness of their eukaryotic hosts. We conducted the present study to investigate the effects of gut microbiota disruption on the honey bee’s immune responses to infection by the microsporidian parasite  Gut commensal bacteria, also known as microbiota, are microorganisms that colonize the digestive tracts of different animal species including insects. Some of the microbiota are opportunist species whose quantity and composition vary with food sources, season and other environmental factors –4. OtherApis mellifera [Firmicutes, Proteobacteria, and Actinomycetes) found in the gut of adult worker bees, three Gram-negative species, Gilliamella apicola, Frischella perrara, and Snodgrassella alvi (within the phylum Proteobacteria) dominate in the hindgut ileum, and three Gram-positive species, Bifidobacterium asteroides (within the phylum Actinomycetes), Firm 4 and Firm5 (within the phylum Firmicutes) dominate in the hindgut rectum [Proteobacteria , and the phylum Firmicutes (Lactobacillus kunkeei) dominate in the crop of the foregut are also present in the hive and colony food including nectar, honey and beebread [A. cerana and some bumble bee species[Advances in high-throughput sequencing technology have allowed a more comprehensive investigation of the community and function of gut bacteria in European honey bees ellifera –14. In hellifera , 13, 15.ellifera , 13, 15.beebread , 13. Pree species, reflecte species.Drosophila melanogaster reported that gut bacteria could regulate host innate immunity, and affect susceptibility to pathogens or parasites [apidaecin and hymenoptaecin in gut tissue of honey bees was upregulated when the microbiota was present and higher apidaecin concentrations were found in bees harboring the normal gut microbiota than in bees lacking gut microbiota. Raymann et al. (2017) reported that gut bacteria perturbation by antibiotics could increase the susceptibility to opportunistic bacterial pathogens and decreased the survivorship of honey bees [Insects including honey bees lack acquired immune response but have evolved effective immune responses to combat infection by pathogens –19. A prarasites . A similarasites –23. Recearasites , 25. A sarasites  reportedney bees , providiney bees , 25, 27,Nosema. ceranae is an obligate intracellular fungal pathogen of the honey bee and causing a serious disease known as nosemosis that is characterized by digestive tract dysfunction and consequent metabolic disorders and sometimes immune suppression [A. mellifera, N. ceranae has often been invoked in honeybee colony losses worldwide [Nosema infection by disturbing the microbiome with antibiotic, and then infecting the bees with N. ceranae. We found that the disruption of gut bacteria by antibiotic could cause the inhibition of honey bee immunity and make honey bees more susceptible to Nosema infection, thereby shortening their lifespan.pression , 29. Sinorldwide –33. In tApis mellifera), which is neither an endangered nor protected species.No specific permits were required for the described studies. Observations were made in the USDA-ARS Bee Research Laboratory apiaries, Beltsville, Maryland, USA; therefore, no specific permissions were required for these locations. The apiaries are the property of the USDA-ARS and are not privately-owned or protected. Studies involved the European honey bee  to remove larger fragments, the homogenate was centrifuged for 5 minutes at 3,000 g and the supernatant was discarded. The spore-containing pellet was completely resuspended in sterile water and centrifuged for 10 min at 5,000 g. This process was repeated twice. The spore-containing pellet was suspended in distilled water and stored at room temperature to be used within one week. The concentration of the spore suspension was determined using a hemocytometer under light microscopy as previously described [7 using a 50% sugar solution (W /V) for inoculation.eviously , 35. Infescribed . The spoNosema infection by monthly surveys. Briefly, 30 abdomens of bees collected at the entrance of the colony were dissected and ground up thoroughly in 30 ml of deionized H2O. 10 μl of the homogenate was loaded onto a hemocytometer and the presence of spores was determined under light microscopy following a previously described method [N. ceranae infection were individually placed in a mesh-walled cage and incubated in an insect growth chamber at 34 oC and 55% relative humidity. Newly emerged bees roamed the frame for 48 hours so they could be naturally inoculated by residual gut symbionts on the frame surface. The individual bees were collected and transferred to laboratory bee rearing cages (40 bees per cage) and kept in an insect growth chamber as described [Bee colonies used in the experiment were screened monthly for pathogens and parasites by molecular methods and light microscopy. Frames with emerging brood and food stores were removed from colonies identified as free of d method . Brood fescribed .N. ceranae spores by individually feeding 2 μl spore suspensions in 50% sugar solution using a pipette with a 10 μl filtered pipette tip (1×105 spores/per bee); Group III, bees were fed with 50× dilution of penicillin-streptomycin  in 50% sugar solution (W /V) for the experimental period; and Group IV, bees were inoculated with N. ceranae spores following the same methods described in Group II and then treated with antibiotic following the same methods described for Group III. Each rearing cage was equipped with one feeder which was filled with 50% sugar solution and supplied with patties of bee-collected pollen to provision the bees ad libitum.Rearing cages were divided into four experimental groups (six cages/group) and subjected to the following treatments: Group I, bees were fed with 50% sugar solution (W/V) (negative control); Group II, bees were inoculated with oC freezer for subsequent DNA and RNA extraction.For each treatment group, three cages were used and the number of dead bees were counted daily for determining the life span of the different treatment groups. Observations lasted until all bees were dead. Three cages were used for sampling honey bees at Day 3, Day 7 and Day 11 post treatment. Eight live bees were collected at each time point and immediately placed into a -80 Four bees collected at each time point from each cage were used for individual DNA isolation, (N = 4 bees x 3 cages). DNAzol  was used for DNA isolation from the collected honey bees according to the manufacturer's instructions. Four bees collected at each time point from each cage were used for individual RNA isolation (N = 4 bees X 3 cages). TRIzol  was used for RNA extraction following the manufacturer's instructions. The resultant RNA pellets were resuspended in UltraPure DNase/RNase-Free water with the addition of Ribonuclease Inhibitor . The quantity and purity of both DNA and RNA were measured using a NanoDrop 8000 Spectrophotometer . DNA samples were stored at -20°C and RNA samples were stored at -80°C until used.Nosema in Groups II and IV were measured by qPCR method using N. ceranae rDNA primers to determine the effects of antibiotic treatment on honeybees’ susceptibility to Nosema infection [To determine the diversity and composition of gut bacteria in honey bees not exposed to antibiotics, the relative abundance of gut bacterial taxa in Groups I and II were analyzed by qPCR using bacteria 16s rDNA universal primers and phylotype-specific 16s rDNA primers , 39 Tab. Levels nfection .qPCR was run on a CFX384 Touch Real-Time PCR System . The Brilliant III Ultra-Fast SYBR Green QPCR Mix  was used for setting up the qPCR reactions consisting of 5 μl 2×qPCR mix, 0.25 μl of each forward and reverse primers (20 uM), 0.5 μl DNA, and 4 μl nuclease-free water. PCR amplification consisted of the following steps: 95°C for 3 min, followed by 40 cycles of 95°C for 20 s, 55°C for 25 s, and 72°C for 30 s and incubation at 72°C for 10 min. After amplification, a dissociation curve was constructed using 81 complete cycles of incubation where the temperature was increased by 0.5°C/cycle, beginning at 55°C and ending at 95°C to verify specificity of the primers.abaecin, defensin1, and hymenoptaecin in bees from all experimental groups . The expression of a housekeeping gene, β-actin, in each sample was also measured for normalization of the qRT-PCR results. The primers used for measuring expression of the bacteria 16S rDNA, immune genes, and housekeeping gene were previously reported [To determine the effect of antibiotic treatment on gut bacteria activity, expression levels of bacteria 16S rDNA in bees from Group I and Group III were measured by qRT-PCR with bacteria 16S rDNA universal primers. The qRT-PCR method was also used to measure the expression of genes encoding antimicrobial peptides reported , 38, 40 Nosema and expression of genes encoding antimicrobial peptides at each time point post treatment. For each target gene at each time point post treatment, the quantity or level was quantified based on the value of the cycle threshold (Ct) which represents the number of cycles needed to generate a fluorescent signal above a predefined threshold and was expressed as means + SE. The mean value and standard deviations of the target gene were normalized using the Ct value corresponding to the measurements of endogenous control, β-actin to yield ΔCt. At each time point post treatment, the group that had the minimal expression level of the target gene was chosen as a calibrator. The ΔCt value of each group was subtracted by ΔCt value of the calibrator to yield ΔΔCt. The concentration of the target among different experimental groups relative to the calibrator at each time point was calculated using the formula 2-ΔΔCt and expressed as the fold-difference.The comparative Ct method (ΔΔ Ct Method)  was usedSnodgrassella alvi, Gilliamella apicola, а-Proteobacteria, г-Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria) in bees from Group I and Group II (no antibiotic treatment). Bees from Groups III and IV (with antibiotic treatment) had Ct values from PCR amplification using 16S rDNA universal primers and phylotype-specific 16s rDNA primers that were above 30 and considered as undetectable Ct values. Therefore, the groups of bees treated with antibiotic were given a value of 1 (one) and used as a calibrator for each primer pair. The relative abundance of total bacteria and specific bacterial phylotypes at each time point post treatment was expressed as an n-fold difference relative to the calibrator.The comparative Ct method also was used to interpret the relative abundance of bacteria taxa . One–Way ANOVA was used to test for differences in the expression level of antimicrobial peptides abaecin, defensin1, and hymenoptaecin among the four experimental groups.The statistical significance of relative quantification of each target gene at each time point post treatment among the different groups was analyzed by using SPSS . The independent t-test was used to determine whether the mean difference in concentration of bacterial 16S rRNA or P ≤ 0.05 was considered to be significant.Survivorship among the different treatments was analyzed using the Kaplan-Meier method. Pairwise comparisons were carried out using Log-rank test. In all cases, t = -11.946, df = 11.432, P < 0.001; Day-7, t = -26.854, df = 22, P < 0.001; Day 11, t = -17.654, df = 11.526, P < 0.001)  . Also, t< 0.001)  (S1 Tabl< 0.001) .t-test, t = 0.948, df = 2, P = 0.443). Moreover, there was no statistical difference in the quantity of the seven phylotypes between Group I and Group II at day 3, day 7 and day 11 after eclosion , suggesting that Nosema infection did not significantly impact the abundance and type of bacteria in honey bees   reatment  (One–WayP<0.001) . For eacN. ceranae in infected bees in Group IV was signficantly higher than that in Group II at each time point post treatment   . The Nos ceranae .P <0.001; Group II vs Group IV, Log-rank test: χ2 = 59.528, P <0.001), suggesting that the antibiotic treatment alone could significantly reduce the survivorship of honey bees. Nosema infection influenced the survivorship of the honey bees, as seen by the statistically significant differences between Group I and Group II as well as between Group III and Group IV .Of the four groups, the bees in Group I had the highest survival rate and longest survival time while bees in Group IV had the lowest . Bees inNosema infection is not known in detail. As a result, we investigated the effects of gut bacteria disruption on the honey bees’ susceptibility to Nosema infection. Our results demonstrated that antibiotic treatment in combination with stress associated with Nosema infection could significantly and negatively impact the survivorship of honey bees. We hope the insight gained from this study will contribute to our understanding of the functional capacity of the gut microbiome in honey bees and improve the health of honey bees and other pollinator insects [Honey bees are the most important insect pollinator for agricultural crops worldwide. However, honey bees suffer from a broad range of pathogens and parasites and the population of honey bees has declined considerably in recent years –45. Whil insects .N. cerana infection was demonstrated by the significantly lower Nosema loads at all three time points post treatment in honey bees with undisrupted gut bacteria compared with those treated with antibiotic. The gut microbiota-mediated host immune regulation especially by initially colonized gut bacteria was reported previously in honey bee, mice and humans [Nosema infection. This result is consistent with a previous report and suggests that gut bacteria not only protect hosts from disease infection, but also can impact host life expectancy [The specialized gut microbial community of honey bees has been explored and comprehensively reviewed by Kwong and Moran in 2016 . Our stud humans , 52–56, pectancy .N. ceranae is a unicellular fungal parasite that infects and accumulates in mid-gut epithelial cells of honey bees. The gut bacterial community in the hindgut of honey bee adult workers accounts for ~95% of all gut bacteria, while the crop (honey stomach or foregut) is colonized by a small population of bacteria [Nosema and gut bacteria, which might be the reason why Nosema infection had no obvious effects on gut bacteria quantity and community in our study. Therefore, the increased susceptibility of honey bees to N. ceranae infection after disruption of gut bacteria was likely a result of altered honey bee immunity. While it is unclear if antibiotics exert direct effects on honey bee immune system, it is well documented that antibiotics cause rapid and profound alterations of gut microbiota, which in turn leads impaired immunity and produces long-lasting deleterious effects for the hosts.In general, there are two mechanisms that are utilized by gut bacteria to reduce host susceptibility to microbial infections: direct antagonistic interactions and indirect host immune regulation , 58. N. bacteria , 16. NicAbaecin, defensin1 and hymenoptaecin are families of AMPs that are inducible and controlled by Toll and Imd /JNK signaling pathways and have been described in honey bees [abaecin, defensin1 and hymenoptaecin after validating the successful depletion of gut bacteria by antibiotic treatment, we found gene expression levels of the three AMPs all significantly down-regulated in bees treated with antibiotic, compared to the negative control. This result is consistent with previous studies [AMPs are small molecular weight proteins with broad spectrum antimicrobial activity, thereby are a crucial part of the innate immune response found among all classes of life. Insects are one of the major sources of AMPs which are abundant in hemolymph and serve as a potent innate immune defense against diverse microbial pathogens , 59, 60.ney bees . By dete studies , 28, 29 Nosema is a virulent honey bee pathogen that has been suggested as one of the key contributors to increased honey bee colony losses worldwide [Nosema. The only registered treatment for Nosema disease in North America is fumagillin, whose use is forbidden in Europe because it has no established Maximun Residue Level (MRL). With prolonged use of fumagillin, the issue of Nosema resistance to treatment will certainly arise [Nosema infection on the honey bee immune function and lifespan were exacerbated by antibiotic treatment because of the effects on the gut microbiota.orldwide –33, 61. ly arise –69. Our N. ceranae infection, the risk of the disruption of gut bacteria by antibiotics in altering the proper function of host immune system, and the urgency of developing new disease therapeutic strategies that can help ameliorate the destruction of beneficial bacteria by antibiotics thus promoting honeybee health. A better understanding of how specific commensal microbes interact with the host immune system in the future may lead to potential therapeutic avenues for improving bee health.Collectively, the results of this study emphasize the importance of commensal gut bacteria in regulating honey bees’ susceptibility to S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


LPL gene  with development of acute ischemic stroke in Colombian population. To analyze if there is an association between the presence of polymorphisms in the  In a case control design, 133 acute ischemic stroke patients  and 269 subjects without stroke as controls were studied. PCR -RFLP technique was used to detect rs320, rs285 and rs328 polymorphisms in the LPL gene.In the present research was not found any association between any of the LPL gene polymorphism and acute ischemic stroke in the population studied; the allele and genotypic frequencies of the studied polymorphisms were similar in cases and controls and followed the Hardy-Weinberg equilibrium. The study was approved by the IRB and each subject signed the informed consent. LPL gene polymorphisms are not genetic markers for the development of stroke in the Colombian sample used.  Cerebrovascular diseases are characterized by a neurologic deficit, due to a focal vascular lesion in the central nervous system, it includes cerebral infarction, intracranial hemorrhage and subarachnoid hemorrhage LPL gene located in region 8p22, spanning about 30 Kilobases (kb) and containing 10 exons, 9 of them codify for a protein of 475 amino acids. Genetic studies have revealed almost 100 mutations and single nucleotide polymorphisms in lipoprotein lipase genelipoprotein lipase gene may play a role as risk or protection factors for stroke onset. For the first case, rs320 (8:19961566) result of thymine (T) to guanine (G) transition in the intron 8, and rs285 (8:19957678) result of cytosine (C) to thymine (T) transition in the intron 6, are associated with elevated TG and HDL ,Lipoprotein lipase (LPL) catalyzes triglycerides (TG) hydrolysis from circulating chylomicrons and very low-density lipoproteins (VLDL), producing cholesterol rich lipoprotein remnants and components of high-density lipoprotein (HDL). The LPL polymorphisms rs320, rs285 and rs328 and the study of their distribution among Colombian population becomes the first study in Colombia to evaluate their association with stroke development. We hypothesize that polymorphisms  located in the lipoprotein lipase gene acts as exposure markers for development of stroke in Colombian population.These polymorphisms have been studied in several populations, and it show discrepancies regarding their influence on stroke onset, it depends on the ethnic group and patient origin. The analysis of Case control study. One hundred and thirty three patients, both genders, older than 18 years old, with ischemic stroke with diagnosis supported by computer tomography were recruited from medical centers in Cali , Bogota , Bucaramanga (Fundación Cardiovascular de Colombia) and Medellin , between 2003 and 2007. As a control group, 269 healthy individuals, both genders, older than 18 years old who visited as visitors during the same period, the medical centers were recruited. They did not have any history or family history of stroke. The population was recruited in the frame of a previous study that analyzed risk factors for ischemic stroke in Colombian population The study was approved by ethical committee of the institutions involve in the study, patients signed an inform consent to participate in the study. We excluded patients who recent underwent under a surgery (previous 30 days), with cancer, hematological, hepatic or renal (creatinine higher than 1.5 mg) diseases, patients taking anti-inflammatory drugs (except ASA in doses as antiplatelet 75 to 300 mg/dL), pregnant woman and patient with cardio embolic diseases. Sociodemographic information such as age, gender and exposure factor as lipid profile , lipid-lowering drugs and genotyping were analyzed.® software was used.Sample size calculation was estimated taking into account the frequency of the most frequent allele with lowest prevalence for the polymorphisms rs320, rs285 y rs328 in cases, previously reported in studies. EPIDAT 4et al. Nicklas LPL were determinate using polymerase chain reaction-restriction fragments length polymorphisms (PCR-RFLP)DNA was extracted from the buffy coat from each patient that was stored at -70° C, using phenol-chloroform technique. The three polymorphisms of 2 y 1 U de Taq polymerase (Promega®). The final volume of the PCR mix was 25 µL. Each polymorphism had its own amplification protocol using different annealing temperatures. General amplification protocol was initial denaturation 94° C for 5 minutes, followed by 30 cycles; at 94° C for 5 minutes, annealing  for 1 minute and elongation 77° C for 10 minutes, and a final elongation at 77° C for 10 minutes. Oligonucleotides primers for PCR amplification of the three-studied polymorphisms, enzymes for digestion, electrophoresis gel and resulting fragments are described in Genomic DNA (13uL) was mixed with 200 µmol from each primer, 50 µmol/L from each of the four nucleotides, 2.5 mM de MgCl®), PvuII (New England biolabs®), MnlI (New England biolabs®) for the mutations rs320, rs285 y rs328, respectively. The resulting fragments were resolved by electrophoresis  on 2% agarose gel, stained with ethidium bromide and visualized on UV light.Digestion of PCR product was performed by addition of 5 µL respective restriction enzyme and aqueous bathed at 37° C overnight. Restriction enzymes used were HindIII , if the subpopulation had a different risk of having stroke, cases and controls would not be comparable. Linkage disequilibrium is the tendency of the alleles of two separate but linked locus to occur more frequently that might be expected by chance. We evaluated it by estimating the observed frequencies of the haplotypes, i.e., all possible combinations of alleles at the loci rs320, rs285 y rs328. Population genetic analysis was performed with SNPStats y Arlequin 3.5.1.2We included 402 individuals, 133 cases y 269 controls. Mean age was 69 years in the cases group and 64 years in the controls, distributed equally between males and females. The clinical and biochemical characteristics of the study population are given in A significant difference between both groups was found for age, total cholesterol and HDL levels. Therefore, we performed a logistic regression analysis, it revealed an OR= 1.04 (CI 95%: 1.03-1.05) for age, total cholesterol OR= 1.00 (CI 95%: 0.99-1.00) and HDL cholesterol OR= 0.94 (CI 95%: 0.93-0.95). Polymorphisms allowed making the allele assignation in each sample for each polymorphism .Genotype and allele frequencies for rs320, rs285 y rs328, did not differ significantly between controls and cases. For rs320 polymorphism, the most frequent genotype was TT in both study population 51, and 52% respectively. The most common genotype between cases and controls for rs285 was the CT with a frequency of 45% and 48% respectively. For rs328 polymorphism, G allele had a low frequency in both groups, 9% and 7% respectively, and GG genotype had the lowest frequency in both populations, with a value of 1% .p= 0.05), for rs285 and rs328 polymorphism in cases and controls. In contrast, rs320 polymorphism was in Hardy-Weinberg equilibrium in the controls, but no in the cases. However, we applied the Bonferroni correction, we adjusted the significance value to p= 0.02, this new value allowed that the population reaches Hardy-Weinberg equilibrium.The population was in Hardy-Weinberg equilibrium ; because it has been demonstrated, that certain intronic region contains host elements that regulate gene transcription and translation. Moreover, they act as markers for functional mutations and alter amino acids sequence rs320, rs285 y rs328 polymorphisms in Two limitations could be pointed out in relation to our study: the first was the relatively small sample size. It is possible that the association between the markers and stroke it is low and the sample size is not big enough to detect this association or there are other exposure markers for stroke development. And the second was the statistical power for the results of rs320 and rs285 polymorphisms have a statistical value higher than 86%. In contrast, the statistical power for rs328 was lower than 86%, therefore the association analysis for this polymorphism lacks of enough power. However, we provide prevalence information of this polymorphism in our population.lipoprotein lipase gene, in patients with stroke and healthy population, do not act as statistically significant risk markers for stroke development in the our population. Genetic markers analysis for stroke is a simple and accurate tool to predict disease risk, which provides information that can be used to improve the distribution of health budget, identify people with high risk and recruit them through the health system. It allows the identification of associated risk factor, monitor trends trough the time. Providing a base to design and implement interventions, monitor and evaluate the efficacy of these interventions, creation of research networks, determinate country priorities to prevent and treat stroke in the national plans context to prevent and control chronic diseases and compare data between and within populations. However, the current study shows that the difference in the distribution of rs320, rs285 y rs328 polymorphisms in Future studies should evaluate other SNPs that may influence other risk factors for stroke, such as obesity, diabetes, high blood pressure, taking into account that some environmental factors may affect disease development. With the support of molecular, biological and genetic techniques allows elucidate etiophatogenic mechanisms for stroke.


This transfer of newly synthesised organic compounds was dependent on the distance of heterotrophic Bacteroidetes from filamentous Oscillatoria. We conclude that the spatial organisation within these biofilms promotes efficient transfer and cycling of nutrients. Further, these results support the hypothesis that biofilm formation leads to the accumulation of organic matter on cryoconite minerals, which could influence the surface albedo of glaciers.Glaciers are important constituents in the Earth’s hydrological and carbon cycles, with predicted warming leading to increases in glacial melt and the transport of nutrients to adjacent and downstream aquatic ecosystems. Microbial activity on glacial surfaces has been linked to the biological darkening of cryoconite particles, affecting albedo and increased melt. This phenomenon, however, has only been demonstrated for alpine glaciers and the Greenland Ice Sheet, excluding Antarctica. In this study, we show via confocal laser scanning microscopy that microbial communities on glacial surfaces in Antarctica persist in biofilms. Overall, ~35% of the cryoconite sediment surfaces were covered by biofilm. Nanoscale scale secondary ion mass spectrometry measured significant enrichment of  As the Earth warms, losses in glacial mass will lead to an export of freshwater to marine ecosystems and ultimately a rise in sea level. Not only is the sheer volume of freshwater of concern, but also the release of geochemical constituents in glacial ice. It is well-known that life in icy systems is predominantly microbial, but little is known about the direct role of microorganisms in biogeochemical cycling from these environments. Of particular interest for carbon cycling are glaciological features known as cryoconite holes, which contain both mineralogical and biological materials. Cryoconite holes form as aeolian debris melts into ice surfaces, and may cover a significant proportion of the ablation zone of glaciers (3.2–16%) in the Southern and Northern Hemispheres.5 Biologically driven aggregation of particles is influenced by the quantity, quality and decomposition of organic matter (OM), and strong correlations between granule size and OM content have been reported.6 Binding together these aggregates are living matrices, called biofilms, which enhance cell–cell and cell–particle interactions, leading to a positive-feedback increasing biomass and potentially reducing albedo on glacial surfaces. While granules, dominated by cyanobacteria and extracellular polymeric substances (EPS), are evident in Arctic and higher latitude cryoconite ecosystems,6 granules in Antarctic cryoconite have not been observed7 and information on cell–particle interactions is non-existent. In a recent review, Cook et al.6 emphasise the need for understanding the fundamental mechanisms that control cryoconite holes from different geographical locations, in order to develop general models that are applicable to diverse supraglacial environments. We propose that the biofilm matrix is integral to biological activity in cryoconite holes through enhanced nutrient storage, cell adhesion and the promotion of efficient nutrient transfer between community members. As such, development of the biofilm structure supports entrapment of microbes and OM, stimulating cell adhesion to cryoconite particles. This accumulation of microbes and OM will enlarge these aggregates, potentially contributing to surface darkening and a reduction of glacial albedo.6Although research on the role of biological processes on glacier surfaces is still in its infancy, recent reports have linked biological growth to a reduction in glacial surface albedo through increased surface darkening and enhanced melting.Cyanobacteria (27%), Actinobacteria (24%), Proteobacteria (22%) and Bacteroidetes  within Antarctic cryoconite particles.9 Phylum-specific fluorescent in situ hybridisation probes targeting Bacteroidetes enabled the visualisation of cells within the Oscillatoria phycosphere biofilm (n=10 images) of the cryoconite sediment surface area, while autofluorescent, cellular biomass covered 1.70±0.861% (n=10 images). Calcofluor White, a stain that binds to cellulose and polysaccharides10 was used to identify the presence of EPS in the biofilm. EPS-like material covered 19.2±1.02% (n=11 images) of the total surface area and was imaged as diffuse non-microbial staining, unassociated with cellular-like structures, which is another clear indication of the presence of biofilm. Combined, ~35% of the cryoconite sediment surface was covered by biofilm.A suite of analytical methods was applied to investigate cryoconite particles from a single, ice-lidded cryoconite hole on the Canada Glacier in the McMurdo Dry Valleys, Antarctica, and elucidate the role of biofilms on glacial surfaces. A diverse microbial community associated with these particles was determined from 454 pyrosequencing to be dominated by tes 19%; . The pho biofilm . Particl biofilm . Unlike  biofilm , confirm11  were significantly enriched in 13C and 15N above background compared to the natural isotope abundance of cryoconite (13C atom %=1.074 and 15N atom %=0.366). Incorporation of 13C-labelled bicarbonate and 15N ammonium ranged between 1.2–3.0 fmol C per cell per h and 0.10–0.35 fmol N per cell per h, respectively. Incorporation of newly released cyanobacterial 13C-exudates and 15N by heterotrophic Bacteroidetes cells (n=137) decreased with increasing distance to Oscillatoria biofilms, with cells close to cyanobacteria being significantly more enriched in 13C , F=45.2, P<0.0001) and 15N ,15 the amount of cryoconite sediment on the surface of the Canada Glacier,9 and the experimentally determined cell-specific rate of carbon fixation, we estimated that Oscillatoria cells may fix 1.60 kg C within cryoconite across the Canada Glacier per season. A recent study,16 which was synchronised with the sample collection in this study, showed that cryoconite hole communities exhibited net autotrophy with an estimated total carbon fixation potential of 9.07 kg C per season across the surface of the Canada Glacier. As such, Oscillatoria cells may contribute ~20% of the total seasonal C fixation. Bacterial productivity in glacial environments is strongly influenced by the quality and quantity of fixed OC;13 thus, it is important to consider both bulk and species-specific primary production. However, we acknowledge that these extrapolations are greatly oversimplified with biological processes inferred from selected community members (i.e. Bacteroidetes and Oscillatoria) hosted within a single cryoconite hole.Biofilms have been found in diverse environments and proven to be ecologically advantageous for survival.9 Further, as the transport and deposition of black carbon is less in Antarctica than in the Arctic,17 our data indicate that microbial processes may have a substantial effect on the accumulation of OM on cryoconite particles; pertinent findings considering that OM covering Antarctic cryoconite particles has been shown to reduce the reflectance of visible light.7 Thus, we hypothesise that future increases in temperature and longer melt seasons will stimulate biofilm communities and the accumulation of organic matter on cryoconite particles. The importance of biofilm formation in cryoconite holes merits further research to determine their development, the formation of larger aggregates composed of organic and inorganic material, and thus, their potential for reducing surface albedo of glaciers.Antarctic cryoconite have been shown to be ‘hotspots’ of biological activity; herein we show that cryoconite biofilm may have a significant role in promoting this activity.


Exercise interventions benefit cancer patients. However, only low numbers of patients adhere to these interventions. This review aimed to identify predictors of exercise intervention adherence in patients with cancer, during and after multimodality cancer treatment.A literature search was performed using electronic databases  to identify relevant papers published before February 1, 2017. Papers reporting randomized controlled trials, conducted in adult cancer patients who participated in an exercise intervention during and/or after multimodality cancer treatment, and providing outcome of factors predicting exercise adherence were included. Papers were assessed for methodological quality by using the Physiotherapy Evidence Database scale.The search identified 720 potentially relevant papers, of which 15 fulfilled the eligibility criteria. In these 15 studies, 2279 patients were included and 1383 of these patients were randomized to an exercise intervention. During cancer treatment, the factors predicting exercise adherence were as follows: location of the rehabilitation center, extensive exercise history, high motivation for exercise, and fewer exercise limitations. After cancer treatment, factors that predicted adherence were as follows: less extensive surgery, low alcohol consumption, high previous exercise adherence, family support, feedback by trainers, and knowledge and skills of exercise. Methodological quality of the included papers was rated “high”.The most prominent predictors of adherence to exercise interventions were location of the rehabilitation center, extensive exercise history, high motivation for exercise, and fewer exercise limitations. To increase the number of cancer patients who will benefit, these results should be considered into the development and implementation of future exercise interventions. A cancer diagnosis and subsequent treatment may potentially motivate patients to change their lifestyle .Accumulating data on the negative effects of being overweight on the development of cancer and cancer survival fuel the sense of urgency for successful interventions to enhance a healthy lifestyle.The aim of this review is to identify predictors of adherence to exercise interventions in patients with cancer, during and after multimodality cancer treatment. This knowledge will help optimize implementation strategies and eventually help in improving cancer treatment outcome.22.1A systematic review was performed to identify predictors of adherence to exercise interventions from randomized controlled trials (RCTs) and to discuss the methodological quality and results of included papers. This systematic review was conducted in accordance with Preferred Reporting Items of Systematic Reviews and Meta‐Analyses (PRISMA) guidelines.2.2A literature search was performed using electronic databases  to identify relevant paper published before February 1, 2017. The complete search including Medical Subject Headings (MeSH) terms and keywords is described in Tables 2.3Selected papers were screened based on title and abstract. In cases when titles and abstracts implied that a paper was potentially eligible for inclusion, a full paper copy of the report was obtained and evaluated for inclusion.2.42 or area under the curve [AUC]). Two investigators conducted the search and data extraction in collaboration (G.S. and H.O.). The two investigators scored the methodological quality of included papers independently (G.S. and H.O.) using the Physiotherapy Evidence Database (PEDro) scale.Data were extracted using a predetermined extraction form and in accordance with PRISMA guidelines.33.1The primary search strategy identified 720 potentially relevant papers, of which 502 remained after discarding duplicates Figure . After s3.2In total, 2279 cancer patients were included in the 15 studies analyzed.Applied timing of exercise interventions varied. Five studies applied their exercise intervention after systemic (neo‐) adjuvant treatment,In 5 studies, the exercise intervention was performed at a rehabilitation center (center based)2 peak percentage]). All studies conducted the exercise interventions with progressive intensity, and in nearly all studies, physiotherapists or exercise physiologists tailored the exercise interventions to the patient's health by modifying exercise prescriptions. Additionally, patients' adherence to exercise was facilitated in all studies.Intensity of the exercise interventions differed from low to high  agreed on 147 of the maximal 160 points (91.9%). Kappa statistics calculated for agreement of the methodological quality assessment between the 2 investigators was 0.82, corresponding with an excellent agreement. Methodological quality ranged from 4 to 8 as rated on the PEDro scale with a median score of 7 of 10, confirming “high” methodological quality. All studies were rated as high qualitative studies3.4In 7 studies, adherence to exercise intervention was measured using an exercise log.Outcome of adherence to exercise intervention was defined by 7 studies as percentage of scheduled minutes of weekly moderate to vigorous physical exercise.3.5A wide range of predictive factors were investigated, which were classified as socio‐demographic , medical , physical and physiological  and behavioral factors . Study results are depicted in Tables P ≤ .01) and significant (P < .05) or borderline significant (P < .10) associations between exercise intervention adherence and various factors were identified in univariable2, defined as the percentage of variance explained by the model, was reported in 9 studiesP values of univariable and multivariable analyses are summarized in Table Highly significant . This facilitates the comparison of studies investigating predictors of exercise intervention adherence during and after multimodality cancer treatment.The authors have no funding or conflicts of interest to disclose.As a systematic review, no ethical approval was sought for this article.Table S1. Search stringClick here for additional data file.Table S2. Keywords and phrasesClick here for additional data file.Table S3. Main results of the 15 studies included in the systematic reviewClick here for additional data file.


RUNX2, and in the present study we investigated the association between craniofacial length and width and RUNX2 across New World bats (Phyllostomidae) and primates (Catarrhini and Platyrrhini). Our results showed contrasting patterns of association between the glutamate/alanine ratios (Q/A ratio) and palate shape in these highly diverse groups. In phyllostomid bats, we found an association between shorter/broader faces and increase of the Q/A ratio. In New World monkeys (NWM) there was a positive correlation of increasing Q/A ratios to more elongated faces. Our findings reinforced the role of the Q/A ratio as a flexible genetic mechanism that would rapidly change the time of skull ossification throughout development. However, we propose a scenario in which the influence of this genetic adjustment system is indirect. The Q/A ratio would not lead to a specific phenotype, but throughout the history of a lineage, would act along with evolutionary constraints, as well as other genes, as a facilitator for adaptive morphological changes.Establishing the genetic basis that underlies craniofacial variability in natural populations is one of the main topics of evolutionary and developmental studies. One of the genes associated with mammal craniofacial variability is  Until recently, broad phenotypic differences were thought to be the result of several gene mutations, each one with relatively small effects6. This proposal was based on extrapolations from studies with housekeeping genes in model species, as well as on the high capacity of population genetics to predict the results of allelic frequency changes in natural populations7. However, through the use of large-scale DNA sequencing, current studies of genes that either regulate the development process or code for receptors and signalling complex metabolic networks indicate that some mutations can have a large effect on morphology9.An understanding of the genetic basis associated with craniofacial variability in animal natural populations is a major focus of modern evolutionary and developmental studiesRUNX2 (Runt-related transcription factor 2), was suggested to have a large effect on mammals’ morphological variation, specifically on the craniofacial variability12. RUNX2 is one of the most important transcription factors associated with bone tissue formation, and is responsible for promoting cell transitions among cell lineages14 being essential for osteogenesis16. The RUNX family is composed by three genes that share the same DNA binding motif (Runt Domain): RUNX1, RUNX2, and RUNX3. However, RUNX2 is the only one that contains a tandem amino acid repeat region that precedes this domain17. The indicated repeat motif presents one segment rich in glutamate residues (polyQ), followed by a segment rich in alanine (polyA)10. This region appears to play an important role in the repression and activation of essential proteins for craniofacial development18. Clinical genetic studies have demonstrated that haplo-insufficiency of the RUNX2 gene is associated with cleidocranial dysplasia (CCD) in humans, an autosomal dominant disease characterized by systemic skeletal anomalies, causing shortening of the face, abnormalities in the permanent dentition, as well as stature effects in affected individuals24.One of these genes, 25 evaluated the correlation between different amino acid repeat motifs present in developmental genes and their effects across dog breeds with diverse skeletal and craniofacial phenotypes. They found a positive correlation between the ratio of polyglutamines (Q) to polyalanines (A) in the Q/A repeat domain of the RUNX2 gene and craniofacial length. Similar patterns of significant positive correlation between the RUNX2 repeat and facial length was also observed in other mammals belonging to the order Carnivora10. In the same study, based on functional tests, Sears et al.10 found significant correlations between the ratios of polyglutamines to polyalanines in the RUNX2 tandem repeat region and RUNX2 transcriptional activity. Other studies also provided evidence for the polyQ and polyA regions as being a mechanism of fine tuning in the activation and repression of the RUNX2 protein16. Conversely, Pointer et al.11 found no correlation between RUNX2 sequence and face length across a variety of non-carnivoran placental mammals. Also, recently, Newton et al.12 demonstrated that RUNX2 does not drive craniofacial diversity in marsupials. Taken together, these previous studies suggest that Q/A repeat domain of RUNX2 might play a role in craniofacial evolution in some mammalian lineages but not others. Therefore, the role of the Q/A repeat domain of RUNX2 in craniofacial evolution lineage must be investigated independently for each lineage.Fondon and GardnerRUNX2.In the present study we combine phylogenetic comparative methods and morphological integration analysis to investigate the association between craniofacial length and width with RUNX2 Q/A ratios across two mammalian lineages displaying large variation in skeletal terms, the New World leaf-nosed bats (Family Phyllostomidae) and Primates (Catarrhini and Platyrhini). The investigation of these taxa is of interest to understand the developmental mechanism involved in the expression of 26 and represents one of the most ecologically diverse families of mammals27. They present a marked diversity in feeding specialisations, including species that consume insects, blood, small vertebrates, fruits, nectar and pollen. Feeding specialisation is accompanied by morphological adaptations, such as conspicuous craniofacial variability  and apes, including humans. Catarrhines exhibit a diverse range of life histories, habitat use, and dietary strategies, and their geographic distribution includes tropical ecosystems in Africa and Asia44. The evolutionary diversification of the Platyrrhini (NWM) occurred over a period of more than 30 million years, driven by diet and size-related selection46. Species from both Catarrhini and Platyrrhini display diverse cranial morphologies and remarkable variation in body size. Among the studies with hominids, Green et al.47 hypothesized that an evolutionary change in RUNX2 affected the upper body and cranial morphology of modern humans. Additionally, as demonstrated by Lindskog et al.48, humans and macaques showed differences in protein and RNA expression levels in the RUNX2 functional network genes . Other genomic studies showed a correlation between RUNX2 and nose bridge breadth in current human populations49. However, in several other primate lineages, such as the NWM, little is known about the genetic basis that controls the fine-tuning of craniofacial phenotypic variation.From a comparative perspective, primates show remarkable morphological variation Fig. . The CatAnoura caudifer and A. geoffroyi; Glossophaga commissarisi and G. soricina; Lonchophylla thomasi) presented the longest (0.83–0.90 length/GM) and narrower face (0.51–0.54 cm width/GM), what is expected as a morphological adaptation to a diet based on nectar. Alternatively, the hematophagous (Desmodus rotundus and Diphylla ecaudata) showed the shortest (0.44–0.51 length/GM), but broader (0.60–0.62 width/GM) face, revealing a more robust cranium. Glyphonycteris sylvestris, an insectivorous species, also presented a long face . Interestingly, frugivorous species presented the greatest variation of palate shape . The strictly frugivorous Pygoderma bilabiatum presented the shortest (0.54 length/GM) and a moderately broad (0.69 width/GM) palate among the frugivorous lineage. We observed all major forms of palate shape ; in phyllostomids. Primates also presented variation in craniofacial morphology .Craniofacial variation is a conspicuous trait of phyllostomid bats. We observed marked differences in palate shape across species representing all feeding strategies  and significant positive correlation between palate width and Q/A ratio, r = 0.55 (p < 0.01) Fig. . Spearma = 0.94), and a negative correlation between Q/A ratio and palate length  for phyllostomid bats . For primates  the PGLS results showed a phylogenetic signal associated with palate length and width. In that case, we found no significant association between phenotypic traits and Q/A ratios  associated with palate length and width, and with zygomatic width Table . The regos Table . For NMWos Table . Finallyos Table .Table 3RThe morphological integration index (ICV), obtained for phyllostomid bats, exhibited strong negative correlation with Q/A ratios  evolved in nested clades among ancestral animalivorous species, and previous studies have proposed that the insectivorous ancestor of all phyllostomids also fed on plants52. In the present study, the largest variation in the Q/A ratios was found in frugivorous phyllostomids  to obligate frugivory . The origin of the Stenodermatinae clade is associated with a significant increase in the species diversification rate and a high bite performance skull53. The anatomical form of the palate in frugivorous phyllostomids (broad structure50) likely favoured a mechanical advantage for this group. When frugivorous vertebrates emerged ca. 25–38 Myr ago, among all other lineages of bats, only phyllostomids fed on fruits31. Thus, they explored a new adaptive zone that likely required remarkable cranial and dental adaptations. Sorensen et al.50 suggested that the diverse palate shapes of phyllostomids are the result of subtle evolutionary changes in later developmental events.The Phyllostomidae family includes subfamilies that are recognised mainly by their feeding habits and associated morphological adaptations57 the magnitudes of inter-trait association, estimated by the average squared phenotypic correlation between traits (r2), are highly variable56. For the same cranial measurements, primates  and Chiroptera  have r2 values under 0.10, whereas Carnivora  display values on the order of 0.12–0.20, and marsupials have r2 values of 0.30–0.5058.In contrast to the remarkable stability observed in the patterns of covariation in the mammalian skull59. Higher magnitudes of skull integration are associated with constrained evolutionary responses to selection, which would reduce adaptive flexibility to evolve in directions other than those driven by the tight inter-trait associations, at least on short-term60. Alternatively, lower magnitudes indicate that most traits are weakly connected and that there is more potential to evolve across several axes of variation. Species with lower magnitudes of trait association could evolve in more directions, therefore presenting less potential evolutionary constraint and greater evolutionary flexibility59. Thus, the different integration magnitudes in phyllostomid bats in relation to the RUNX2 gene  lists several known skeleton phenotypes compiled so far and associated with RUNX2 mutants. In this context, we could assume that delayed or increased bone ossification  might explain the opposed association of RUNX2 variation and/or integration and facial forms we observe in different groups of mammals. By affecting bone ossification the locus potentially can impact allometric size variation within population, which in turn would usually impact mostly facial traits due to its late development . Consequently it would lead to an increase or decrease in correlation among facial traits and thus to a larger or smaller morphological integration. Thus, the strong positive correlation found between evolutionary flexibility index and the RUNX2 gene  a non-association of these traits12. Sears et al.10 suggested a pattern of increase in face length of carnivores due to a higher Q/A ratio. In contrast, the results obtained in our study point to a reduction in length and a widening of the maxilla, which would lead to shorter and broader faces in phyllostomid bats. However, in NWM, the result is consistent with that described for the order Carnivora. Overall, our results support the main idea proposed by Sears et al.10, reinforcing the role of the Q/A ratio as a flexible genetic mechanism that would rapidly change the time of ossification of the skull throughout development.Our results clearly showed contrasting patterns of association between tandem repeats of RUNX2 gene is indirect, and does not lead to a specific phenotype. However, throughout the history of a lineage (at the species or family level), RUNX2 variability acts with external factors and evolutionary constraints, together with other genes, as a facilitator for the adaptive morphological changes currently found. In the case of phyllostomids, these changes seem to have been selected by dietary constraints, corroborating the phenotype–environment correlation63. Overall, it should be noted that many genetic, epigenetic, and environmental attributes modulates the expression of genetic variance and interact with developmental networks to produce the available phenotypic variation. Therefore, the results of the present study seem to agree with Alberch’s64 view on the conceptualisation of genotype-phenotype association, i.e. genes do not directly specify the development or the shape of an organism; they are only one of the several causal factors that jointly determine the phenotype. Thus, developmental events are able to ambiguously affecting, or be affected, by gene expression.In this context, the findings of the present study propose a complex scenario, where the permissiveness of this genetic adjustment system created by the variability in the Q/A ratios of the Leontopithecus rosalia, Spajus robustus, Brachytheles arachnoides, Callithrix hummelifera, Callithrix pygmaea, Callithrix geoffroyi), from the Laboratório de Evolução Molecular of the Departamento de Genética, Universidade Federal do Rio Grande do Sul, were used. Five species of Catarrhini  were considered using the ENSEMBL  and NCBI (http://www.ncbi.nlm.nih.gov/genbank/[Benson]) genomic databases.Samples from 31 phyllostomid bat species from a wide range of feeding habits and craniofacial morphologies were surveyed in this study  and Neandertal Ensembl  genomes were accessed to obtain modern human and Neandertal data. The DNeasy Blood & Tissue Kit  was used to extract and isolate genomic DNA from muscle samples for the 37 species. The polymerase chain reaction (PCR) was carried out to amplify the target RUNX2 exon, using primers and conditions described by Pointer et al.11, standardising the PCR conditions for the different species surveyed. Shrimp alkaline phosphatase and exonuclease I enzymes  were used to purify the PCR products. Amplicons were sequenced in an ABI3730xl  sequencer, using the same primers employed in the PCR for both DNA strands. Sequences were analysed using the ClustalW algorithm, implemented in the Codon Code Aligner software 65. A prior screening test was performed, which considered only the sequences presenting conserved regions flanking the Q/A motif. These regions were then used as reference markers for the Q/A motif. The Q/A ratio for each specimen was calculated by dividing the number of repeated glutamine residues by the number of repeated alanine residues in tandem11  using a digital calliper, and obtained the primate data set employing a combination of craniometric data recorded by Marroig and Cheverud50. We calculated the geometric mean for all measurements and standardised the palate length and width for each specimen by dividing each measurement by the geometric mean (GM)  Table . Only skRUNX2 tandem repeat ratios and palate length and width. Additionally to this traditional statistical method, a Bayesian phylogenetic comparative method (PCM) approach was implemented in CoEvol 1.4 software68 to investigate the correlation between RUNX2 Q/A ratio and palate length and width, taking into account phylogenetic relationships, evolutionary rates, and divergence time under a Brownian motion model of evolution. In the CoEvol results, a posterior probability (pp) close to 1 represents a strong statistical support for a positive correlation, and a pp close to 0 indicates a supported negative correlation. Two data sub-sets for each taxonomic group (bats and primates) were analysed: I, morphometric measures for each species, corrected by geometric mean of all measures and II, phylogenetic relationships estimated from neutral markers . We ran CoEvol for 30,000 MCMC  and considered the analysis finished when all effective sample size values reached at least 30068.In an initial set of analysis, we performed Spearman’s correlation test to investigate the association between 69 to investigate the magnitude of phylogenetic signal associated with phenotypic  and genotypic (Q/A region - independent variable) changes. This function fits a linear model controlling for phylogenetic non-independence between data. We also used this parameter for comparative analyses with the results of previous studies on carnivores10 and other placental mammals11. We assume that all the three analytical methods employed in our study are integrative and represents different levels of complexity, ranging from a more general exploratory analysis (Spearman’s correlation) to a more complex phylogenetic comparative methods (PGLS and Bayesian).Finally, we performed a phylogenetic generalised linear model (PGLS) using the R function (pgls) of the “caper” R package70;). This index was calculated for primates59 and phyllostomids  as the standard deviation of the eigenvalues (σ(λ)) divided by the average of the eigenvalues (λ). The flexibility and constraints index were also provided from the author’s cited above. The evolutionary flexibility index captures the ability of a species to respond in direction of selection, while the evolutionary constraints index captures the relative influence of the line of least resistance  on the direction of the evolutionary responses59. These measurements are presented in the electronic supplementary material, Table 70 for a detailed description of these index estimates). We performed Spearman’s correlation test in order to investigate the association between RUNX2 tandem repeat ratios and ICV, flexibility and constraints index.The magnitudes of integration among traits were obtained by using the coefficient of variation of the eigenvalues (ICVSupplementary Material


Purpose. This study aimed to examine whether plasma levels of cathepsin S or its inhibitor, cystatin C, may serve as biomarkers for COPD. Patients and Methods. We measured anthropometrics and performed pulmonary function tests and chest CT scans on 94 patients with COPD and 31 subjects with productive cough but no airflow obstruction . In these subjects and in 52 healthy nonsmokers (NS) and 66 healthy smokers (HS) we measured plasma concentrations of cathepsin S and cystatin C using an ELISA. Data were analyzed using simple and logistic regression and receiver operating characteristic analyses. Results. Cathepsin S and cystatin C plasma levels were significantly higher in the COPD and AR groups than in the NS and HS groups (p < 0.01). Among the COPD patients and AR subjects, plasma cathepsin S levels and cathepsin S/cystatin C ratios, but not cystatin C levels, were negatively related to severe airflow limitation  and severe emphysema as assessed by low attenuation area (LAA) score on chest CT scans . Conclusion. Plasma cathepsin S and cathepsin S/cystatin C ratios may serve as potential biomarkers for COPD. Consequently, there is an urgent need to identify and validate sensitive and reliable biomarkers for COPD in compartments that are easy to sample, such as blood, to more quickly assess the efficacy of novel therapies for COPD in clinical trials. In particular, it will be crucially important to identify reliable biomarkers for early stage COPD, the stage at which novel therapies might have the most impact on successfully modifying the course of the disease and preserving lung function.COPD is characterized by narrowing of small airways and lung parenchymal destruction, partly caused by chronic lung inflammation including increased numbers of neutrophils, macrophages, and CD8-positive T lymphocytes, oxidative stress, and alveolar septal cell apoptosis –3. ChangRecent research has identified a number of potential molecules and pathways that could serve as potential biomarkers and be targeted to develop novel therapies . One sucCO) measurements.We hypothesized that plasma concentrations of cathepsin S and cystatin C would differ between COPD patients, subjects with cough and sputum, and healthy smokers and nonsmokers. To test this hypothesis, we measured the plasma concentrations of cathepsin S and cystatin C and explored the association between levels of these biomarkers with degree of airflow obstruction and emphysema severity as assessed by low attenuation areas on chest CT scans and diffusing capacity of the lung for carbon monoxide (DLn = 94) were diagnosed based on airflow obstruction as defined by a ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) lower than 0.7 and an % FEV1 predicted value lower than the 95% confidence interval for this population by PFT. One group of 31 subjects  was defined as having chronic respiratory symptoms  but no airflow limitation as assessed by PFT. Enrolled healthy volunteers were classified into two groups by smoking history: 52 healthy nonsmokers (NS) and 66 healthy smokers (HS). This study was approved by the Ethics Committee of Keio University Hospital and informed consent was obtained from each subject.This study subjects included 125 patients suggestive of COPD consecutively visiting the outpatient clinic of Keio University Hospital from 2000 through 2003 and also included 118 healthy volunteers (age ≥ 50 years). All patients underwent pulmonary function tests (PFT) and chest CT. The patients suspected of having clinically stable COPD fulfilled the following criteria: (1) smoking history of ≥ 10 pack-years or more; (2) age ≥ 40 years old; and (3) having chronic respiratory symptoms including cough, sputum, and dyspnea on exertion. The patients were excluded if they were (1) having any pulmonary disease other than COPD; (2) having serum creatinine level > 1.5 mg/dL with renal dysfunction; (3) having serious comorbidities including unstable cardiovascular or cerebral diseases and treated malignant tumors; (4) having self-reported asthma; and (5) having an exacerbation for at least one month prior to recruitment or being treated with oral corticosteroids. The COPD patients  kit  after diluting the samples 1 : 100 using the diluent supplied by the kit manufacturer. The lower detectable limit of cathepsin S using these kits was 15.6 pg/mL. Plasma concentrations of cystatin C were determined in duplicate using a sandwich ELISA as previously described . GT 13 wCO was estimated by 10 s breath holding in most patients of the COPD (n = 83) and AR (n = 20) groups (n = 103) . Chest CT was also performed in all patients of the COPD and AR groups .Spirometry was performed in all patients of the COPD and AR groups using an electronic spirometer . Regular treatment was not changed prior to spirometric testing. DLTo calculate emphysema severity, the entire lung was divided into six zones . Low attenuation areas (LAA) were visually scored in each zone on a scale from 0 to 4: 0, no LAA; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%. The total (0–24) was assessed by three pulmonologists in a blinded manner and the mean score was defined as the LAA score, a quantitative indicator of emphysematous change , 15. Dia1 predicted < 50%) or emphysematous change (LAA ≥ 8.0) were determined using logistic regression analysis. Logistic regression analysis models were prepared to avoid the influence of potential confounders, including age, sex, body mass index (BMI), smoking status, serum creatinine levels, the presence of hypertension, and treatment with inhaled corticosteroid (ICS). Inclusion of variables was based on existing knowledge of risk factors for COPD and on the factors related to plasma cystatin C levels according to previous reports . Also, the cathepsin S/cystatin C ratio  and age  correlated to severe airway obstruction (p = 0.001. OR = 0.28) and the cathepsin S/cystatin C ratio  were the strongest factors associated inversely with the presence of severe emphysema  when the threshold was set at ≥ 12.1 ng/mL. The specificity improved from 62% to 80% if the threshold was set at ≥ 16.2 ng/mL but the sensitivity decreased to 74%. These additional analyses suggest that plasma cathepsin S levels may serve as a useful biomarker for COPD. Rokadia et al. also reported that active smokers with COPD had significantly higher plasma levels of cystatin C than healthy controls in accordance with our results . These o1/FVC and % FEV1 predicted is positively associated with BMI and malnutrition in a large number of COPD patients [An unexpected result from our study was that, among the COPD patients and AR subjects, plasma cathepsin S and cathepsin S/cystatin C ratios were inversely correlated with the extent of airflow obstruction and emphysema severity, as assessed by LAA on CT scans, even after normalizing for various confounding factors including age, gender, BMI, renal function, smoking history, the presence of hypertension, and treatment with ICS. Thus, plasma cathepsin S levels, but not cystatin C levels, were significantly increased in patients with AR and with mild COPD when compared with patients with severe airflow limitation and emphysema. One possible explanation for the inverse correlation between plasma cathepsin S and severity of disease among the COPD patients is that cathepsin S is produced in lung tissue including activated macrophages , and as patients , and plapatients . Also, ppatients . Additiopatients . A priorpatients .Taken together, we speculate that the inverse correlation between plasma cathepsin S and cathepsin S/cystatin C ratios and severity of airflow limitation and emphysema in patients suggestive of COPD that we observed in this study might reflect these complex processes in the pathophysiology of this disease. However, additional cohorts of COPD and asthma will be needed to confirm these findings. Symptomatic smokers without pulmonary function decline are classified as Stage 0 disease and reported to have an increased susceptibility to COPD in GOLD 2001. AR, that is Stage 0 disease, develops COPD in 50% . In addiThere are several limitations in our study. First, it was impossible to obtain pulmonary function data from subjects in the NS and HS groups. The healthy volunteers (NS and HS) were differentiated from the patients (COPD and AR) since they had no chronic respiratory symptoms leading to visiting hospitals. Second, the mean age of the COPD group was significantly higher than AR and the healthy subjects. However, our results indicate that cathepsin S levels are not affected by age . Third, We have demonstrated significantly elevated plasma levels of cathepsin S and cystatin C, and plasma cathepsin S/cystatin C ratios, in COPD patients versus healthy subjects. Our results suggest that plasma cathepsin S concentrations and cathepsin S/cystatin C ratios may serve as potential biomarkers for COPD.


Mercury contamination in food can pose serious health risks to consumers and coal-fired power plants have been identified as the major source of mercury emissions. To assess the current state of mercury pollution in food crops grown near coal-fired power plants, we measured the total mercury concentration in vegetables and grain crops collected from farms located near two coal-fired power plants. We found that 79% of vegetable samples and 67% of grain samples exceeded the PTWI’s food safety standards. The mercury concentrations of soil samples were negatively correlated with distances from the studied coal-fired power plants, and the mercury contents in lettuce, amaranth, water spinach, cowpea and rice samples were correlated with the mercury contents in soil samples, respectively. Also, the mercury concentrations in vegetable leaves were much higher than those in roots and the mercury content of vegetable leaves decreased significantly after water rinses. Our calculation suggests that probable weekly intake of mercury for local residents, assuming all of their vegetables and grains are from their own farmland, may exceed the toxicologically tolerable values allowed, and therefore long-term consumptions of these contaminated vegetables and grains may pose serious health risks. Rapid industrial development in China is highly dependent on coal energy. Coal comprises ~70% of the energy supply in China, which is much higher than the global average of 28%Mercury is a particularly important heavy metal to consider when examining the environmental consequences of coal burning. Although mercury is released into the environment from natural and anthropogenic sources, coal-fired power plants have been identified as the largest source of mercury emissionsMercury emitted from coal-fired boilers in power plants has increased mercury pollution in neighboring areas. It has been reported that in the atmosphere, mercury is in gaseous and particulate forms457878111315Mercury can be harmful at very low concentrations because of its high toxicity and ability to bioaccumulate171815Other researchers have investigated mercury concentrations in vegetables and grains cultivated near various sources of mercury pollutions, including mercury mines22In this study, we investigated the mercury contamination in grains and popular vegetables cultivated near two coal-fired power plants, estimated the mercury intake of local residents, and discussed potential health risks associated with mercury consumption. This is the first study to investigate mercury concentrations in vegetables and grains cultivated near coal-fired power plants.et al.3 who examined spatial distribution of soil Hg as a function of distance from a coal-fired power plant in the US.We measured mercury concentrations in soil samples collected from six field locations  and the background soil mercury concentrations in China is about 37 μg/kgSoil mercury contents may be reduced naturally via vaporization and run-off. However, rates of these processes are not well studied. There are many factors3637We investigated the mercury content of the edible parts of ten types of vegetable and grain crops from the selected locations in The mercury contents in 79% of vegetable samples and 67% of grain samples exceeded the maximum allowed mercury levels defined by the Food Safety Standards in China Comparing the mercury concentrations in different types of vegetables and grains, we found that the mercury contents in the edible parts of lettuce, amaranth, water spinach, tomato, eggplant, pepper, cucumber, and cowpea were 2.1, 2.9, 5.4, 7.6, 4.3, 6.2, 1.8, and 5.7 fold greater than the maximum allowed mercury levels, respectivelyTo investigate mercury accumulation in different plant organs, we measured the mercury concentrations in leaves, fruit, stems, and roots of tomatoes grown in Location B3. The mercury concentrations were 116.17 ± 14.69 μg/kg in leaves, 29.07 ± 1.45 μg/kg in fruits, 18.35 ± 0.83 μg/kg in stems, and 13.64 ± 1.37 μg/kg in roots. Mercury concentrations were much higher in leaves than in fruits, and the mercury concentrations in the aboveground organs were higher than those in roots .Previous studies have demonstrated that plants can absorb mercury from both air and soil. When plants absorb mercury mainly from the soil, the mercury content should be higher in roots, while the mercury contents should be higher in shoots and leaves tissues if air mercury is the main source of mercury in plants40Results published previously by others show that mercury concentrations in vegetables and grains from coal-fired power plant regions are higher than those of samples from zinc plant, oil well, and fluorescent lamp factory regions . In Chin2It has been reported that the mercury in fly ash is predominantly in the form of particulate mercury that can be deposited on plant leaf surfaces due to wet and dry deposition plants4The majority of China’s coal-fired power plants uses limestone-gypsum wet flue gas desulfurization systems to remove sulfur from the flue gasBecause elemental mercury can easily evaporate at elevated temperatures, cooking may therefore reduce mercury in food. However, studies have shown that cooking reduced mercury contents in mushroom by 10% and had no effect on the mercury contents in fish454617th percentile of mercury concentrations found in vegetable and grain samples were used in the calculation222425th percentile of Hg concentration, that were 71 ng/g FW for vegetables and 62 ng/g FW for rice samples. Because rice is the main food grain in China, while maize is mainly used as livestock feed2450http://faostat.fao.org) show that the total vegetable consumption in China was 332.20 kg/person. In addition, the average bodyweight of Chinese adults is 55.9 kg51We estimated probable weekly intake (PWI) of mercury by residents who rely on locally produced produces using Numerous studies have shown that vegetables and grains cultivated near various mercury sources can be contaminated. Coal-fired power plants are the largest sources of mercury released to the environment in China, and we reported that soil, vegetable, and grain samples collected from field locations within 10 km distance from Power Plant A and B had significantly higher mercury concentrations than the samples purchased from a grocery store away from any power plant, with 79% of vegetable samples and 67% of grain samples exceeded the upper limit of allowable mercury level The average Hg concentration in coal used in power plants from this area is 120 μg/kgPower Plant B began operation in 2013. From the data from the company’s website, the main generating units are two 660 MW coal-fired generating units that use 290.78 g coal per kWh electricity, and in 2013, Power Plant B produced 7.7 billion kWh of electricity. Approximately 268.7 kg of mercury was emitted from the coal burned (~2.239 Mt) in the year 2013.Vegetables, grains, and soil (0–15 cm deep) samples were collected in six open field locations  close to Power Plants A and B  in 2015.The fresh vegetable and grain samples were stored in plastic bags for transport. Samples were treated in the laboratory as described previously3 was added to each sample. Dried, pulverized soil samples were placed into Teflon vessels, and 9 mL of HCl and 3 mL of HNO3 were added to each sample. The sample digestions were based on EPA Method 7473Dried, pulverized vegetable or grain samples were placed into Teflon vessels, and 10 mL of HNOAfter digestion, the vessels were opened carefully. The final solutions were cooled and transferred into 50-mL calibrated flasks, and their volumes were completed with deionized water. All sample processing was performed in a laminar flow fume cupboard to avoid external contamination. The mercury concentrations of all soil and vegetable samples were analyzed using atomic fluorescence spectrophotometry  after pre-concentration and dilution56To estimate local residents’ potential exposure to mercury via vegetable and grain consumption, th percentile of mercury concentrations in a statistical analysis of all vegetable and grain sample data, respectively245051Hg concentration of vegetable and grain samples used in the two equations were the 95How to cite this article: Li, R. et al. Mercury pollutions in vegetables, grains and soils from areas surrounding coal-fired power plants. Sci. Rep.7, 46545; doi: 10.1038/srep46545 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Nilaparvata lugens and Nephotettix spp. However, our knowledge of their diversity is still very limited, due to their small size and the lack of available morphological information. Classifying these parasitoids is the first step to properly understanding their role in the rice agroecosystem. We used traditional morphological identification, as well as DNA sequencing of the 28S rRNA and the COI genes, to investigate the diversity of four important hopper egg parasitoid genera in the Philippines. Parasitoids of the genera Anagrus, Oligosita, Gonatocerus, and Paracentrobia were collected in eight study landscapes located in Luzon. Our findings illustrate that characterization of species diversity using morphological and molecular analyses were concordant only for the genus Paracentrobia. The genera Anagrus and Gonatocerus exhibited more genetic diversity than estimated with the morphological analysis, while the opposite was observed for Oligosita. This is the first study investigating the molecular diversity of rice hopper parasitoids in the Philippines. More research combining morphological, behavioral, and molecular methods, as well as the establishment of a comprehensive DNA database, are urgently needed to assess the performance and suitability of these organisms as biocontrol agents.Ongoing intensification of rice production systems in Southeast Asia is causing devastating yield losses each year due to rice hoppers. Their continuing development of immunity to resistant rice varieties and pesticide applications further complicates this problem. Hence, there is a high demand for biological control agents of rice hoppers. Egg parasitoid wasps are among the most important natural enemies of rice hoppers, such as  Nilaparvata lugens, Stål 1854) and the green leafhopper  are among the economically most important rice pests. These insects cause immense damage in the Asian rice paddies through xylem sap feeding, resulting in wilting, and subsequently, the death of the rice crops [Rice is the main food resource for more than half of the world’s population ,2. This ce crops . So far,ce crops ,8. The mce crops ,9,10.As an alternative, integrated pest management (IPM) strategies can be used to maximize the pest’s mortality by enhancing the role of their biological control agents. BPH and GLH are typically attacked by a wide range of natural enemies, such as spiders, predatory bugs, dragonflies, and egg parasitoid wasps from the families Mymaridae and Trichogrammatidae . Among tDespite their importance, knowledge about species composition and diversity of parasitoid wasps in the tropics is still limited  and Nephotettix spp. (GLH) in eight rice production landscapes located in Luzon, Philippines [Anagrus and Oligosita, while GLH was parasitized by the chalcidoid genera Gonatocerus and Paracentrobia. In the present study, we analyzed the diversity of these genera with traditional morphological and molecular techniques. The present study provides the first molecular identification of parasitoid wasps in rice paddies in the Philippines, combined with a morphological identification based on dichotomous keys.In a previous study, we investigated parasitoid wasps of lippines . We founNilaparvata lugens, while the GLH culture consisted of Nephotettix virescens (Distant) and Nephotettix nigropictus (Ståhl). Both hopper populations originated from wild individuals caught in the rice fields of the Laguna Province. Four gravid females of either BPH or GLH were released into each cage for 48 h to lay eggs. Subsequently, the plants were transferred to three different plots per study site to cover the naturally occurring landscape diversity in the Laguna province. Three plants infested with BPH eggs and three plants infested with GLH eggs were distributed randomly within each plot. After 72 h, all plants were returned to the greenhouse and placed back inside separate insect cages. The parasitoids emerged 13–17 days after the field exposure of the eggs. Due to positive phototaxis behavior, parasitoids aggregated in small transparent glass vials, which were placed upside down on top of the sleeved cages. Adult parasitoids were collected daily within this period and immediately stored at −80 °C. The dead parasitoids were identified to genus under a stereo microscope using morphological keys [Paracentrobia, Gonatocerus, Oligosita, and Anagrus [This study was embedded in the project LEGATO, which focused on sustainable rice production . Parasitcal keys ,30,31. I Anagrus .Paracentrobia, Gonatocerus, Oligosita, and Anagrus) were randomly selected. The 200 parasitoids were permanently slide-mounted using Faure’s medium, and identified to species level using a microscope. A specialist at the International Rice Research Institute (IRRI) helped with the identification using the key published in Heong and Hardy [Fifty parasitoids from each of the four identified genera (nd Hardy .−1) was added. Parasitoid samples were carefully homogenized with sterile micro pestles, and incubated at room temperature overnight.Total genomic DNA was extracted from 267 newly selected whole single female parasitoid individuals  employin2, 2.5 mM of each of the four dNTPs (deoxynucleotide triphosphates), 10 µM of each primer, 1 U TaKaRa Ex Taq polymerase (Takara Biotechnology), and approximately 25 ng of parasitoid DNA. The following thermal cycling scheme was used: initial denaturation at 94 °C for 3 min, 5 cycles of denaturation at 94 °C for 45 s, annealing at 45 °C for 45 s, followed by elongation at 72 °C for 1 min, and 25 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s, followed by another elongation at 72 °C for 1 min. The final extension was carried out at 72 °C for 5 min.Two independent gene fragments were amplified from the extracted DNA: one located on the COI subunit I  and the other one on the expansion regions D2–D3 of the 28S ribosomal subunit . The COI region was amplified using the primer pair HCO2198/LCO1490 . The PCRThe D2–D3 region of the 28S rRNA gene was amplified as described for the PCR above, using the primers D2-3549  and D2-4Sequencing was performed at the Göttingen Genomics Laboratory using an ABI 3730xl system and BigDye terminator chemistry version 3.1 (Thermo Fisher Scientific).http://www.boldsystems.org)  . The besThe genetic diversity between and within species was estimated using the reference sequences from the superfamily Chalcidoidea . The appvegan [drc [To estimate, whether the genetic diversity of the parasitoids was covered by the sample size used in the analyses, we performed a rarefaction analysis  in R vervegan  and drc gan [drc .All sequence data was deposited in the NCBI GenBank under accession number MG785407–MG785510, MG904875–MG904939, MG911990–MG912010.Gonatocerus and Paracentrobia, while three species were found for the genera Anagrus and Oligosita: Anagrus flaveolus (11 specimens), Anagrus optabilis (6 specimens), Anagrus frequens (33 specimens), Oligosita aesopi (41 specimens), Oligosita naias (7 specimens), Oligosita shibuyae (2 specimens), Gonatocerus orientalis (50 specimens), and Paracentrobia andoi (50 specimens)  .Of the 267 specimens used for the molecular analysis, we successfully sequenced a total of 105 parasitoid samples . We failRarefaction analysis for the local hopper egg parasitoids genetic diversity revealed that the majority of the 28S rRNA gene haplotypes was recovered by the surveying effort . By contOligosita exhibited the lowest genetic diversity, with three highly similar sequences in the COI gene analysis and only one haplotype in the 28S rRNA gene analysis. In contrast to that, three Oligosita species were identified using the morphological approach.The molecular analyses based on COI and 28S rRNA genes revealed that the sequences clearly segregated according to the morphologically pre-assigned genera . OligosiParacentrobia sequences varied only slightly, with two highly similar haplotypes in the 28S rRNA gene analysis and three highly similar haplotypes in the COI gene analysis  compared to the other two clusters  and 0.050 ± 0.009 SE (COI gene) for Anagrus, and 0.069 ± 0.012 SE (28S rRNA gene) and 0.100 ± 0.016 SE (COI gene) for Gonatocerus. By contrast, within group pairwise distances were 0.000 ± 0.000 (28S rRNA gene) and 0.005 ± 0.003 SE (COI gene) for Oligosita, and 0.001 ± 0.001 SE (28S rRNA gene) and 0.009 ± 0.004 SE (COI gene) for Paracentrobia . The 28S rRNA gene is valuable to distinguish among closely related species of Chalcidoidea [The molecular analyses of the diversity within the four Chalcidoidea genera studied showed discrepancies with the morphological analysis. This finding is in line with previous studies on hymenopteran parasitoids ,43,44. Fly exist ,47, the ly exist ,49, and cidoidea ,51,52. Icidoidea ,53. ThisOnce a reliable and comprehensive DNA database is established, the identification of hopper parasitoids using DNA sequencing will be a vital step towards assessing the performance and suitability of these organisms as biocontrol agents ,55. Our Anagrus species are described as generalist parasitoids that can switch between alternative hopper species in rice agroecosystems [Anagrus genus is a complex of cryptic species that may be host specialists. This distinction is of high importance to control the hoppers effectively. In addition, the discovery of unknown cryptic species could expand the list of potential biological control agents [For instance, to date, osystems ,58. Yet,l agents .Anagrus and Gonatocerus by mating tests and/or further morphometric analyses, our data strongly indicates that the two genera include unknown species. The level of within-genus genetic difference, for both 28S rRNA and COI gene sequences among samples from the Anagrus and Gonatocerus genera, exceeded the threshold of 0.17–2% within-sequence variation, which is generally accepted to delineate individuals of the same species [Anagrus and Gonatocerus suggests that these two genera were composed of individuals from different species. When comparing the genetic analyses to the morphological results, we found strong evidence that there was at least one species of Anagrus and Gonatocerus that was not detected by the morphological analysis. Interestingly, we observed the opposite for Oligosita samples. In the latter genus, it is possible that one or more species were not detected due to the low success rate of DNA amplification. It is also possible that we observed a discrepancy between morphological and molecular analyses, because they were performed on a different set of specimens. As one Oligosita species was much more prevalent than the other two in the morphological dataset, we cannot exclude that we analyzed the most common species only, in the molecular analysis.Although we were not able to validate the existence of new species of  species ,59. Acco species ,59, geneIn conclusion, our results clearly demonstrate that molecular identification should be used in combination with morphological methods and mating tests for assessing the diversity of rice hopper parasitoids. However, further studies using an integrative approach are needed to cover the whole diversity of parasitoids, as well as to find sustainable solutions to problems caused by BPH and GLH. To validate the potential application of parasitoid wasps as biocontrol agents, it is of crucial importance to have a comprehensive knowledge on their ecology and diversity. We hope that this study will encourage further research by providing the first barcodes for egg parasitoid species from rice paddies in Southeast Asia.