Metastatic adenocarcinoma to the breast from an extramammary site is extremely rare. In the literature, the most current estimate is that extramammary metastases account for only 0.43% of all breast malignancies and that, of these extramammary sites, colon cancer metastases form a very small subset. Most commonly seen metastasis in breast is from a contralateral breast carcinoma, followed by metastasis from hematopoietic neoplasms, malignant melanoma, sarcoma, lung, prostate, and ovary and gastric neoplasms. Here we present two rare cases, in which colonic adenocarcinomas were found to metastasize to the breast. In both cases, core biopsies were obtained from the suspicious areas identified on mammogram. Histopathology revealed neoplastic proliferation of atypical glandular components within benign breast parenchyma which were morphologically consistent with metastatic adenocarcinoma. By immunohistochemical staining, it was confirmed that the neoplastic components were immunoreactive to colonic markers and nonreactive to breast markers, thus further supporting the morphologic findings. It is extremely important to make this distinction between primary breast cancer and a metastatic process, in order to provide the most effective and appropriate treatment for the patient and to avoid any harmful or unnecessary surgical procedures. Colorectal cancer is the third most common type of cancer globally, and most frequently it spreads to the regional lymph nodes, liver, lungs, and bone, in descending order . It is vObtaining a detailed past medical history, appropriate radiologic work-up, followed by a tissue biopsy with histologic and immunohistochemical work-up, has proven to be extremely helpful in determining the source of the malignancy. Anti-cytokeratin 7 (CK 7) and anti-cytokeratin 20 (CK 20) antibodies have been used to differentiate between cancers of a primary or metastatic source , 6. HereA 45-year-old woman with history of stage 4 colon cancer, status after right hemicolectomy in 2012, presented to the outpatient clinic for a follow-up visit and biannual work-up. The patient had received Oxaliplatin with 5-fluorouracil and folinic acid (FOLFOX) and Avastin (bevacizumab) chemotherapy following surgery. Although the CT scans of her lungs and liver did not show any evidence of metastatic disease, the mammogram showed an abnormal density associated with two clusters of microcalcifications in the upper medial aspect of the right breast. The anterior cluster measured approximately 6.8 mm × 5.3 mm in size and was located 3.64 cm from the nipple . The posThe patient was subsequently sent for a stereotactic guided core biopsy of two areas of these microcalcifications. The procedure was carried out using a petite biopsy needle and a Suros EVIVA vacuum to obtain core samples. The core biopsy samples were radiographed with iCAD 7.2, which confirmed the presence of microcalcifications in the samples. Surgical microclips under stereotactic guidance were placed at the biopsy sites.The morphology of all four core biopsy samples revealed benign appearing mammary tissue, interspersed by proliferation of atypical glandular elements, morphologically consistent with adenocarcinoma. A few of the glands showed intraductal microcalcifications, compatible with prior mammogram. In some glands, microcalcifications were accompanied by necrotic cellular debris. A few of the normal appearing breast ducts also showed intraductal microcalcifications. Nuclei of these malignant appearing cells showed hyperchromasia, nuclear crowding with pseudostratification. Considering the prior history of colonic adenocarcinoma, additional immunohistochemical markers were tested, besides the usual hormone receptor studies. The neoplastic glands that are associated with microcalcifications and necrotic cellular debris were positive for CDX2 and CK 20 and negative for CD7, ER, PR, and Her2-Neu. In routine histology, these atypical glands were not surrounded by a convincing myoepithelial layer, as shown by the negative markers for calponin, CD10, and p63. Thus, the diagnosis of metastatic colonic adenocarcinoma was confirmed. The patient was discharged home with outpatient appointments with medical oncologist for further management.A 56-year-old female with history of stage 4 colonic adenocarcinoma with metastasis to the liver, status after resection in 2009, presented to the emergency department with complaints of nausea, vomiting, and abdominal pain. The patient had received radiotherapy and completed six cycles of chemotherapy consisting of Oxaliplatin, 5-fluorouracil and folinic acid (FOLFOX), and Avastin (bevacizumab). The last cycle of chemotherapy was seven weeks prior to presentation.In the emergency room, during the physical examination, the patient stated that she noticed a lump in her right breast. On palpation, a small mass was felt deep in the inferior medial aspect of the right breast at 4 o'clock and 9.7 cm from the nipple. No discharge was noted from the nipple. A bilateral digital diagnostic mammogram study was obtained with views in the craniocaudal and mediolateral oblique positions and with additional spot compression views due to the deep nature of the mass. It was found to be highly vascular, speculated, and hypoechoic mass measuring 1.35 cm × 1.46 cm × 1.22 cm . Some beA right breast ultrasound was performed at the same time and showed the palpable mass to be an irregularly shaped, spiculated, hypoechoic lesion with central necrotic changes and high vascularity . No lympColorectal carcinoma is the third most common cancer among men and women with an estimated number of 71,830 reported new cases in men and 65,000 new cases in women . Ou. Ou11]. Further confirmation of the above morphologic findings by immunohistochemical (IHC) staining is always recommended to establish an accurate final diagnosis. Cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) are the most widely used initial IHC panel , 6, 12. The management of patients with metastatic breast cancer is extremely complex and depends upon multiple factors such as age, functional status, comorbidities, distant metastasis, and lymph node status . Since fUnfortunately, the presence of metastatic tumor in the breast carries a dismal prognosis, since it is usually indicative of disseminated disease , 4, 6. TMetastatic colonic adenocarcinoma to the breast is an extremely rare clinical entity , 6. Ever


N-methylglycine) is an exogenous amino acid that acts as a glycine transporter inhibitor. It modulates glutamatergic transmission by increasing glycine concentration around NMDA (N-methyl-d-aspartate) receptors. In patients with schizophrenia, the function of the glutamatergic system in the prefrontal cortex is impaired, which may promote negative and cognitive symptoms. Proton nuclear magnetic resonance (1H-NMR) spectroscopy is a non-invasive imaging method enabling the evaluation of brain metabolite concentration, which can be applied to assess pharmacologically induced changes. The aim of the study was to evaluate the influence of a six-month course of sarcosine therapy on the concentration of metabolites ; mI, myo-inositol; Cr, creatine; Cho, choline) in the left dorso-lateral prefrontal cortex (DLPFC) in patients with stable schizophrenia. Fifty patients with schizophrenia, treated with constant antipsychotics doses, in stable clinical condition were randomly assigned to administration of sarcosine (25 patients) or placebo (25 patients) for six months. Metabolite concentrations in DLPFC were assessed with 1.5 Tesla 1H-NMR spectroscopy. Clinical symptoms were evaluated with the Positive and Negative Syndrome Scale (PANSS). The first spectroscopy revealed no differences in metabolite concentrations between groups. After six months, NAA/Cho, mI/Cr and mI/Cho ratios in the left DLPFC were significantly higher in the sarcosine than the placebo group. In the sarcosine group, NAA/Cr, NAA/Cho, mI/Cr, mI/Cho ratios also significantly increased compared to baseline values. In the placebo group, only the NAA/Cr ratio increased. The addition of sarcosine to antipsychotic therapy for six months increased markers of neurons viability (NAA) and neurogilal activity (mI) with simultaneous improvement of clinical symptoms. Sarcosine, two grams administered daily, seems to be an effective adjuvant in the pharmacotherapy of schizophrenia.The glutamatergic system is a key point in pathogenesis of schizophrenia. Sarcosine ( Schizophrenia is one of the most devastating mental diseases, with lifetime prevalence from 0.30% to 0.66% and incidence between 10.2 and 22.0 per 100,000 person-years ,5. DLPFCN-methylglycine) is an exogenous amino acid that acts as an inhibitor of glycine transporter type 1 (GlyT-1) [From the neurochemical perspective, negative and cognitive symptoms are associated with impairment of the glutamatergic system, especially in the PFC, where ionotropic NMDA receptors are abundant ,11,12. G(GlyT-1) . Thus, s(GlyT-1) ,16,17,18(GlyT-1)  or treat(GlyT-1) .N-acetylaspartate (NAA), a marker of neuron viability and integrity, are commonly observed [In schizophrenia, there is no consensus on the association between changes in CNS metabolites and exacerbation of symptoms, phase of the disease, treatment strategy or analyzed brain region ,22,23,24observed , and refobserved ,27. Howeobserved ,22 foundobserved  and had observed . Concentobserved ,34,35,36observed ,38,39,40Although the influence of medications was also evaluated spectroscopically, the findings are ambiguous. In some studies, antipsychotics increased levels of NAA after treatment ,41, whilThe aim of the study is to evaluate the influence of sarcosine therapy on the concentrations of NAA, Glx , mI (myo-inositol), Cho (choline-containing compounds) and Cr (creatine plus phosphocreatine) in the DLPFC of the left frontal lobe in patients with schizophrenia. Our experiment can support new data on the pharmacokinetics, pharmacodynamics and psychopharmacological value of sarcosine, as well as glutamatergic agents in general. It can also reveal new aspects of the role played by the glutamatergic system in the pathogenesis of schizophrenia.At baseline, spectroscopy revealed no significant differences in metabolite concentrations between the groups .In a second spectroscopy NAA/Cho, mI/Cr and mI/Cho ratios were significantly higher in patients receiving sarcosine. Moreover in experimental group after the therapy NAA/Cr, NAA/Cho, mI/Cr, mI/Cho ratios increased significantly, comparing to baseline values.vs. 18%).Only NAA/Cr ratio increased after therapy in the placebo group, although to a lesser extent than in the sarcosine group . However, at the end of the experiment, patients treated with sarcosine had significantly lower results . Changes in the negative PANSS subscale followed the trends of the total PANSS subscale. At the beginning of the study there was no significant difference between groups . While the negative PANSS score decreased significantly in both groups , this decrease was greater in the sarcosine group . The difference in metabolite ratios and negative PANSS subscale scores were calculated between the start-point and end-point of the experiment. Correlations between these differences are presented in At the beginning of the study, no significant difference was noted between groups with regard to PANSS score  suggest that these ratios might quantitatively correspond with clinical outcomes of therapeutic intervention.N-acetylaspartate is one of the most common amino acid in the human brain. It is synthesized in neuronal mitochondria and its production closely correlates with glucose metabolism. Due to the fact that it is not present in glial cells, it reflects neuronal activity well [ity well .In our study, both NAA ratios (NAA/Cr and NAA/Cho) in the sarcosine group were significantly higher after six months, indicating an increase of neuronal viability in the DLPFC. In the placebo group, the NAA/Cr ratio was also significantly raised, however, the change was less distinct. Our findings indicate that sarcosine (and probably other GlyT1 inhibitors) might normalize disturbances in brain metabolism and reverse the tendency for NAA levels to decline in schizophrenia. Increased NAA concentrations were also described as an effect of the antipsychotic drugs , which mAn evaluation of Glx level was performed instead of separate glutamine, glutamate and GABA evaluations, as their peaks closely overlap in 1.5 Tesla spectroscopy. Glx is a surrogate of glutamatergic transmission in grey matter as the concentration of glutamate is five times higher than that of glutamine, and 10 times higher than GABA .In schizophrenia, hypofunction of the NMDA receptor may involve GABAergic interneurons, which would result in disturbed glutamatergic transmission . Moreoveet al. showed no significant differences in Glx concentrations between healthy volunteers and groups of medicated and unmedicated patients with schizophrenia [et al. report a decrease in glutamine levels after 14 weeks of antipsychotic therapy [et al. note decreased Glx levels in patients after six months of treatment with second-generation antipsychotics [There is no consensus on the glutamate concentration in the DLPFC of patients with schizophrenia . Kegelesophrenia . Only th therapy , and Gotychotics . Researcychotics ,63.On the other hand, the administration of ketamine, an NMDA receptor antagonist whose effect is opposite to sarcosine, resulted in increased glutamatergic transmission in ACC ,65.et al. report that PANSS positive symptoms subscale scores significantly correlated with levels of GABA and Glx only in MPFC but not in DLPFC [Most studies have failed to find any significant correlation between glutamatergic parameters and PANSS score ,66,67,68in DLPFC .In the present study, a trend was observed towards a decrease of Glx/Cr ratio in both groups. Although it was more expressed in the sarcosine group, the differences were not significant. Further studies using discreet analysis with a stronger magnetic field are required to support more reliable conclusions.Myoinositol is a precursor in the transmission of phosphatidylinositol, which is a widely accepted glial marker . In neurSignificant increases of mI/Cr and mI/Cho ratios in the sarcosine group between two spectroscopies, and in comparison with the placebo group, might indicate unfavourable changes. However, some researchers report greater mI concentrations to be associated with treatment ,70. ThusDue to the limited number of patients and application of 1.5 Tesla magnetic resonance, conclusions should be formulated moderately, as precise separation of glutamate, glutamine and GABA spectra requires a 3 Tesla magnetic field, or higher. Analysis of GABA concentration could be of special interest, because sarcosine indirectly acts on the NMDA receptors located also on GABAergic interneurons. A few studies have found that GABA concentrations varied depending on the analyzed region, including different parts of the frontal cortex ,72. On tAnother important limitation of this work is its application of ratios of metabolites concentrations instead of exact concentrations. Despite changes of Cr and Cho concentrations, depending on duration of schizophrenia, it has previously be demonstrated that treatment with either atypical or typical medication does not alter Cr or Cho levels . Thus, rFinally, it should be noted that applied statistical methods did not protect against Type I errors associated with multiple testing. However, although significant differences in particular metabolites could be obtained by chance, the clinical improvement seems to confirm their relevance.Subjects with schizophrenia, aged 18–60 years who were physically, neurologically and endocrinologically healthy and had normal laboratory values  and electrocardiogram were eligible to enter the study. Patients in acute psychosis, on clozapine treatment or declaring suicidal tendencies were excluded from the study. This study is a part of the Polish Sarcosine Study in Schizophrenia (PULSAR); for further details, please see acknowledgments.1H-NMR spectroscopy was performed according to the protocol described below at the beginning of the study and six months later. Sarcosine or placebo were added to the ongoing antipsychotic treatment in a double-blind manner. Patients in the study group were given plastic capsules containing 2 grams of the amino acid, while subjects in the placebo group . The severity of schizophrenia symptoms was assessed with the Positive and Negative Syndrome Scale (PANSS) [Fifty right-handed patients diagnosed with schizophrenia  criteria) with dominant negative symptoms, and who were in a stable clinical condition, were randomly assigned to a sarcosine or placebo group.  (PANSS) .Subjects were recruited from the outpatient clinic. All patients included in the study have been informed about the aims and methods of the study, and had expressed their written informed consent for participation in this study. The study protocol was approved by the Bioethics Committee of the Medical University of Łódź . There was no financial involvement from industry.Imaging was performed using 1.5 Tesla MR scanner  equipped with a standard head coil.NMR acquisition:(1) FLAIR sequences in axial plane with following parameters: Repetition Time (TR), 9000 ms; Echo Time (TE), 105 ms; inversion time (TI), 2500 ms; flip angle, 150°; voxel size 1.4 mm × 1.3 mm × 3 mm.(2) T2-weighted sequences were obtained in coronal plane with following parameters: TR = 5000 ms; TE = 100 ms; flip angle, 50°; voxel size 0.6 mm × 0.6 mm × 5.0 mm.(3) T1-weighted sequences in transverse plane with following parameters: TR = 400 ms; TE = 7.8 ms; flip angle, 90°; voxel size 0.9 mm × 0.9 mm × 0.5 mm.1H-MRS data was acquired by single voxel spectroscopy (SVS) using a point resolved spin echo (PRESS) sequence 128 averages; TR, 3000 ms; TE, 30 ms; voxel size was 15 mm × 15 mm × 15 mm. The region of interest was placed in the left DLPFC by the neuroradiologist . The Shapiro-Wilk test was used to determine the normality of the data. As the distribution was skewed in one or both compared groups in all cases, the Mann-Whitney test was employed to compare the ratios of substance concentrations between groups, and the Wilcoxon sign-rank test was used for comparisons within the same group. To evaluate the association between changes in concentrations ratios and differences in PANSS score, the Spearman’s rank correlation test was applied. Statistical analysis was performed using Statistica for Windows . A Our findings demonstrate that addition of sarcosine to antipsychotic treatment can cause increases of NAA and mI in DLPFC. These changes were associated with clinical improvement. It indicates that sarcosine improves neuron viability and integrity, and may activate neuroglial cells in brain regions essential for the pathogenesis of schizophrenia. It highlights the role of glutamatergic transmission in the pathogenesis of schizophrenia and confirms that two grams of sarcosine administered daily may become an effective adjuvant in the management of schizophrenia.


Zea mays L.) is the staple diet of the native peasants in the Quechua region of the Peruvian Andes who continue growing it in small plots called chacras following ancestral traditions. The abundance and structure of bacterial communities associated with the roots of amilaceous maize has not been studied in Andean chacras. Accordingly, the main objective of this study was to describe the rhizospheric bacterial diversity of amilaceous maize grown either in the presence or the absence of bur clover cultivated in soils from the Quechua maize belt. Three 16S rRNA gene libraries, one corresponding to sequences of bacteria from bulk soil of a chacra maintained under fallow conditions, the second from the rhizosphere of maize-cultivated soils, and the third prepared from rhizospheric soil of maize cultivated in intercropping with bur clover were examined using pyrosequencing tags spanning the V4 and V5 hypervariable regions of the gene. A total of 26031 sequences were found that grouped into 5955 distinct operational taxonomic units which distributed in 309 genera. The numbers of OTUs in the libraries from the maize-cultivated soils were significantly higher than those found in the libraries from bulk soil. One hundred ninety seven genera were found in the bulk soil library and 234 and 203 were in those from the maize and maize/bur clover-cultivated soils. Sixteen out of the 309 genera had a relative abundance higher than 0.5% and the were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis. Genera Gp4 and Gp6 of the Acidobacteria, Gemmatimonas and Rhodoferax were the most abundant in bulk soil, whereas Flavobacterium, Dechloromonas and Ohtaekwangia were the main genera in the rhizosphere of maize intercropped with bur clover, and Gp4, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Gp6 and Rhodoferax were the main genera in the rhizosphere of maize plants. Taken together, our results suggest that bur clover produces specific changes in rhizospheric bacterial diversity of amilaceous maize plants.Maize ( Zea mays L.) reached the southern Andean highlands after its domestication in Mexico some 8700 years before the present , an annual shrub of the Fabaceae family within the tribe Trifolieae which, though native to the Mediterranean basin, is distributed worldwide. In the Peruvian Andes bur clover grows up to almost 4000 m above sea level where it is also widely used as a pasture and as a cover crop, greatly contributing to the fodder units required for feeding of the national cattle. The microsymbiont from nodules of bur clover has been isolated and identified as Ensifer medicae, and the rhizobial-M. hispida symbiotic association has been suggested to be the main N source for growth of maize plants in the Andean chacras  used by Quechua peasants to grow maize, quinoa, wheat, potatoes, and others cereals and vegetables. Soils were taken from three chacras within the same soil plot located near Allpas , a village in the province of Acobamba . At sampling time, one chacra had been cultivated with amilaceous maize (M soil) for 3 years, other with maize and bur clover (MT soil) for 5 years, and the third was bulk soil from a chacra under fallow conditions (B soil) for at least 3 years. Seeds of Z. mays L. morphotype Qarway were planted by mid October 2012 and soil samples collected 120 days later, when the plants were at the grain filling stage. At the second hilling stage, 75–90 days after planting, bur clover looms up without previous sowing and has to be removed by hand to maintain the chacras free of the legume. Lateral roots  were taken from maize plants grown at four different sites within each chacra and four replicates were sampled for each site. After cleaning of the soil attached to the roots, the remaining adhering rhizospheric soil was carefully removed and pooled together to obtain 2 g. Bulk soil samples  were taken from the chacra under fallow conditions. Soil samples were kept at -20°C until further processing. The three chacras belong to the same soil plot, which has a sandy-loam texture ; the average pH was 6.3 and that of the soil organic matter was 28.7 g/soil kg.Chacras are small parcels of soil  and 10 ng of the DNA template. The PCR program consisted of an initial denaturation step at 94°C for 4 min, 25 cycles of denaturation at 94°C for 15 s, primer annealing at 55°C for 45 s and extension at 72°C for 1 min, followed by a final step of heating at 72°C for 10 min. Amplicons of the same treatment were pooled to reduce per-PCR variability and purified using the ultracentrifugal filters Ultracel-100 K membranes (Amicon) according to the manufacturer’s instructions. After quantification by Nanodrop ND1000 and visualization of the DNA by agarose electrophoresis, the samples were combined in equimolar amounts and pyrosequenced in a Roche Genome Sequencer FLX system using 454 Titanium chemistry at LifeSequencing S.L. .Polymerase chain reaction (PCR) amplification of the hypervariable V4–V5 regions of the 16S rRNA gene was performed over each individual DNA extraction from soils using universal primers U519F and U926R  joined t1 release 11 (Jclass and Jabund) were used to analyze the alpha- and beta-diversity, respectively  pyrosequencing pipelinelease 11 . Sequenclease 11  and remolease 11 . Alignedlease 11 . The RDPlease 11 , was useectively .G-test (w/Yates’) + Fischer’s test in the STAMP software , which also contained 575 (9.15%), 1793 (15.75%), and 1460 (17.47%) unclassified sequences, respectively . The remaining valid sequences distributed into 21, 23, and 21 phyla, respectively (Table 2), of which phyla Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, Gemmatimonadetes, Planctomycetes, Chloroflexi, Armatimonadetes, Parcubacteria, Nitrospirae, Candidatus Saccharibacteria, candidate division WPS-2, candidate division WPS-1, Latescibacteria, BRC1 and Microgenomates (in decreasing abundance) were found in all the three libraries . Phyla Spirochaetes and Chlamydiae were found in MTG and MG and so were Ignavibacteriae and Cyanobacteria in libraries MG and BG, respectively . Hydrogenedentes was detected only in MTG, Deinococcus–Thermus in MG and Elusimicrobia in BG . Numbers of classes, orders, families and genera are also shown in Table 2.A total of 38786 sequences were obtained from the three 16S rDNA samples sent to pyrosequencing, of which 26031 were retained after filtering and removing chimeras. The mean number of total retained sequences per library was 8677, ranging from 6286 to 11387. Average length of retained sequences was 374 ± 5 base pair (mean ± SD). All the sequences aligned correctly in the expected position of the 16S rDNA sequence of Table 1), the Shannon index of 5.13 for OTUs in the MTG clone library was lower than those of 7.06 and 7.21 for the FG and MG libraries, respectively. The Simpson index, however, showed clear differences among the OTUs in each library, with values of 0.0016, 0.0031 and 0.0056 for the MTG, MG and BG clone libraries, respectively (Table 1).With values of the Good’s coverage index higher than 80% at 90% confidence interval (Table 2). Of the total number of genera, the BG library shared 132 and 155 with the MTG and MG libraries, respectively, and libraries MTG and MG had 159 genera in common (Table 3). Values of the classJ index were similar between the BG and MG libraries (0.56) and the MTR and MG libraries (0.57), values that were higher than that (0.49) between the MTG and BG libraries (Table 3). The abundJ indexes for the multiple comparisons among genomic libraries were similar (Table 3).Sequences in library BG distributed in 197 genera, a value lower than those of 203 and 234 found in libraries MTG and MG, respectively (Supplementary Table S1). Sixteen (16) out of the 309 genera had a relative abundance higher than 0.5%, and represented the 64.23% of the total number of sequences classified at the genus level. Altogether, these genera were (in decreasing order of abundance) Gp4, Gp6, Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Gemmatimonas, Dechloromonas, Ohtaekwangia, Rhodoferax, Gaiella, Opitutus, Gp7, Spartobacteria genera incertae sedis, Terrimonas, Gp5, Steroidobacter and Parcubacteria genera incertae sedis (Supplementary Table S1) .From the total 26031 sequences, only 12784 could be assigned to 309 different genera . On the other hand, the relative abundance of genera Gp4, Gp6, Subdivision3 genera incertae sedis of phylum Verrucomicrobia, Rhodoferax, Gaiella and Spartobacteria in MTG were significantly lower than those in the MG library . Differences in the relative abundance of sequences corresponding to genera Gemmatimonas, Ohtaekwangia, Opitutus, Gp7, Terrimonas, Gp5, Steroidobacter and Parcubacteria were not found between the MTG and MG libraries .Numbers of sequences of genera Using 454 next generation sequencing we assessed the composition and abundance of bacterial rhizospheric communities of amilaceous maize plants grown in Andean chacras following Quechua traditional practices. The total number of genera found in our genomic libraries were higher than those obtained in previous studies based on culture-dependent and culture-independent methods , and simAltogether, a 12.71% of the total sequences found inside roots corresponded to unclassified bacteria, which indicates the presence of hitherto uncultured bacterial groups. A high percentage of unclassified sequences have also been reported in the rhizosphere of maize plants when pyrosequencing was used to study bacterial biodiversity .Figure 1). Although the amount of bacterial phyla found in the three genomic libraries was similar (Table 2), the number of OTUs and values of the Shannon and Simpson indexes for the MTG library were lower than those for the BG library (Table 1), which suggests that intercropping reduced richness of the community. These results agree with those previously published that show that microbial diversity in maize rhizospheric soils was lower than in bulk soils . It is possible that the absence of bur clover promts the plants to increase bacterial diversity around the roots to improve their nutrition. Alternatively, it is also possible that more time is required for the maize crop to become well established in soils, which, in turn, could alter the soil microbiota. On the other hand, clear dominance of specific rhizospheric bacteria was not found, and multiple comparisons analyses of the values of the β-diversity indexes indicated the three genomic libraries have a similar bacterial community core. The absence of specific phylogenetic groups between bulk and rhizospheric soil from an oak forest after pyrosequencing of the 16S rRNA gene has been reported (A variety of bacteria have been reported to be rhizospheric, among them mostly Proteobacteria, Firmicutes, Actinobacteria, Acidobacteria and Bacteroidetes . In our lk soils . This re medicae  free-livreported .Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Dechloromonas, Rhodoferax, Spartobacteria and Parcubacteria incertae sedis, the maize-bur clover consortium increased relative abundance of Flavobacterium, Subdivision3 genera incertae sedis of the Verrucomicrobia phylum, Dechloromonas and Parcubacteria incertae sedis.Plants influence the abundance and composition of the bacterial rhizospheric community by secreting a variety of compounds through their roots into the surrounding soil to feed and manipulate the microbes that live there . If one It is well established that plant genotype plays a significant role in shaping plant-associated microbial communities and in determining the biological outcome of such associations . In thisConceived and designed the experiments: DC-G, EJB, GJA. Performed the experiments: DC-G, GJA. Analyzed the data: DC-G, EJB. Contributed reagents/materials/analysis tools: AJF-G, MF-L. Improve the paper: AJF-G, MF-L, GJA. Wrote the paper: DC-G, EJB.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacteroides genus was lower in men than in women  when BMI was > 33. In fact, the abundance of this genus decreased in men with an increase in BMI . However, in women, it remained unchanged within the different ranges of BMI. We observed a higher presence of Veillonella  and Methanobrevibacter genera  in fecal samples in men compared to women. We also observed that the abundance of Bilophila was lower in men compared to women regardless of BMI . Additionally, after correcting for age and sex, 66 bacterial taxa at the genus level were found to be associated with BMI and plasma lipids. Microbiota explained at P = 0.001, 31.17% variation in BMI, 29.04% in triglycerides, 33.70% in high-density lipoproteins, 46.86% in low-density lipoproteins, and 28.55% in total cholesterol. Our results suggest that gut microbiota may differ between men and women, and that these differences may be influenced by the grade of obesity. The divergence in gut microbiota observed between men and women might have a dominant role in the definition of gender differences in the prevalence of metabolic and intestinal inflammatory diseases.Intestinal microbiota changes are associated with the development of obesity. However, studies in humans have generated conflicting results due to high inter-individual heterogeneity in terms of diet, age, and hormonal factors, and the largely unexplored influence of gender. In this work, we aimed to identify differential gut microbiota signatures associated with obesity, as a function of gender and changes in body mass index (BMI). Differences in the bacterial community structure were analyzed by 16S sequencing in 39 men and 36 post-menopausal women, who had similar dietary background, matched by age and stratified according to the BMI. We observed that the abundance of the  Firmicutes/Bacteroidetes ratio [Gut microbiota acts collectively as an organ fully integrated in host’s metabolism, and is involved in energy extraction from nutrients, regulating innate and adaptative immunity, and participating in the control of the energy balance . Howeveres ratio . Studieses ratio . In contes ratio . In factes ratio , 10.In line with this, studies in humans have shown that gut microbiota seems to have coevolved with dietary habit , 12. RecIn addition, alteration of the intestinal microbiota has been demonstrated to be a key player in the protracted course of inflammation in inflammatory bowel diseases (IBD) . This diThe influence of gut microbiota in the incidence of metabolic and intestinal diseases is rather complex due to the inter-individual heterogeneity. Furthermore, the proportion of Firmicutes/Bacteroidetes in lean and obese humans has yielded contradictory results, as well as studies describing gender-related differences in the gut microbiome , 26. In This current study was conducted in a subgroup of 75 patients (39 men and 36 women) within the CORDIOPREV study , an ongoing prospective, randomized, opened, and controlled trial in patients with coronary heart disease (CHD), who had their last coronary event over six months before enrolling in two different dietary models (Mediterranean and low-fat) over a period of five years, in addition to conventional treatment for CHD .We analyzed the baseline fecal samples of 75 patients (39 men and 36 women), who were also divided into three groups, according to their BMI: 13 men and 13 women with BMI < 30; 13 men and 10 women with 30 ≤ BMI ≤ 33; and 13 men and 13 women with BMI > 33. The metabolic characteristics of the subjects in the study are shown in The trial protocol and all amendments were approved by the Reina University Hospital Ethics Committee, following the Helsinki declaration and good clinical practice. However, all patients gave written informed consent to participate in the study.We performed a validated 14-item questionnaire to assess adherence to the Mediterranean Diet  and a siBlood was collected in tubes containing EDTA to give a final concentration of 0.1% EDTA. The plasma was separated from the red cells by centrifugation at 1500 X g for 15 min at 4°C. Analytes determined in frozen samples were analyzed centrally by the laboratory investigators of the Lipid and Atherosclerosis Unit at the Reina Sofia University Hospital, who were unaware of the interventions. Lipid variables were assessed with a DDPPII Hitachi modular analyzer (Roche) using specific reagents (Boehringer-Mannheim). Plasma triglycerides (TG) and cholesterol concentrations were assayed by enzymatic procedures , 31. Hig®, Cambridge, UK) and the samples were stored at -20°C.To collect the fecal samples, we gave the patients a box with carbonic snow and a sterile plastic bottle with a screw cap to keep the frozen sample. Once it was delivered to the laboratory staff, the sample was stored at -80°C until microbial DNA was extracted. This was performed using the QIAamp DNA kit Stool Mini Kit Handbook  following the manufacturer’s instructions. This protocol is optimized for a 180–220 mg sample. DNA was quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer  were used for statistical analyses of data. The normal distribution of variables was assessed using the Kolmogorov-Smirnov test. The statistical differences in the main metabolic variables between groups were evaluated using One-way ANOVA. In order to assess whether specific differences occurred in some bacterial taxa between genders and bacterial species, we compared the abundance of taxa present at least in the 75% of the human fecal DNA samples from men and at least in 75% of the total human fecal DNA samples from women. The specific differences in bacterial taxa between genders and bacterial species were evaluated by the Mann-Whitney U test. The specific differences in bacterial taxa by BMI were evaluated by the Kruskal-Wallis test. EPIDAT program v4.1 was used to evaluate power and sample size calculations ; Universidad CES, Colombia.). Furthermore, we also analyzed the frequency of occurrence of taxa identified at least in 25% of the total human fecal DNA samples from men and at least in 25% of the total human fecal DNA samples from women. The X2 test was applied to establish differences in bacterial prevalence between the studied groups. Results were adjusted by False Discovery Rate (FDR) using Benjamini and Hochberg method. FDR adjusted p-value  of 0.05 was considered statistically significant.All the data presented in this study are expressed as mean±SEM. PASW statistical software, version 20.0  and R software, version 3.0.2  units adjusting for age and gender was performed as described by Fu et al. . This apanalysis .P value (ranging from 0.001 to 0.1). Also, the risk (rm) of the gut microbiome on BMI or lipids for each individual using an additive model was estimated according to Fu et al. [rm, after correcting for age and gender.Finally, the proportion of variation in BMI and lipids could be explained by the gut microbiome, based on significantly associated OTUs identified in the two-part model at a certain u et al. . The varP = 0.022) and men higher diastolic blood pressure (P = 0.035)  . In addi= 0.035)  and in m= 0.035)  between For the bacterial community analyses of the 75 samples, after screening our data for poor quality sequences, we recovered 1,296,641 high-quality 16S rRNA gene sequences with 706,186 and 590,455 sequences for men and women, respectively, with an average of 17,469 sequences per sample . There were no significant differences in bacterial diversity between males and females with any of the alpha diversity estimators used and at a rarefaction level of 2,000 sequences per sample. With this depth we reached satisfactory coverage of the diversity for all samples since all Good’s coverage values ranged between 98.43 and 99.77 (data not shown). Similarly, Principal Coordinate Analysis (PCoA) or UPGMA clustering based on unweighted and weighted UniFrac distances did not show significant differences in microbiota composition between men and women  Figs.Bilophila genus was higher in women than in men . Higher presence of Veillonella  and Methanobrevibacter genera  was observed in fecal samples from men compared to women.Although diversity and overall community composition were not significantly different between males and females, we investigated whether the relative abundance of specific taxa might differ among groups. Regardless of BMI, we did not find any differences at phylum level between men and women. However, at the genera level, we observed that the abundance of the Bacteroides caccae was higher in women than in men . On the other hand, the abundance of Bacteroides plebeius was higher in men than in women . Moreover, we observed a higher presence of Coprococcus catus  in fecal samples from men compared to women.In addition, at the bacterial species level, we observed that the abundance of Firmicutes/Bacteriodetes ratio between men and women when considered independently of the BMI. However, when we stratified men and women according to their BMI, we observed that men had higher Firmicutes/Bacteriodetes ratio under a BMI of 33. By contrast, men had a significantly lower Firmicutes/Bacteriodetes ratio than women in the BMI > 33 group (P = 0.018)  .Bacteroides genus in women than in men  with a BMI > 33, whereas we did not find any difference between genders in the abundance of this genus when the BMI was < 33 , whereas in women, it remained unchanged in the different ranges of BMI  .In addition, we observed that the abundance of = 0.041) . Thus, tC. catus , in fecal samples from men compared to women when the BMI was < 30. In fact, the prevalence of C. catus tended to increase with the BMI in women , whereas it remained unchanged in men at the different BMI ranges.We also observed a higher presence of Bifidobacterium adolescentis , Eubacterium biforme , and Oxalobacter formigenes  in fecal samples from men compared to women when the BMI was < 30. However, the prevalence of these bacterial species was not statistically different between men and women when the BMI was > 30.Moreover, we observed a higher presence of In addition, we study the relationship between gut microbiota, BMI, and plasma lipid levels. After adjusting for age and sex, a total of 1428 associated OTUs were detected at FDR = 0.05. Out of them, 299 OTUs were associated with BMI, 312 with TG, 335 with HDL, 276 with LDL-c, and 223 with total cholesterol ; S3A FigAt the genus taxonomy level, we identified 90 significant associations for 66 unique taxonomies at FDR = 0.05: 23 were associated with BMI, 18 with triglycerides, 17 with HDL, 14 with LDL, and 18 with cholesterol ; S3B FigP = 0.001 level explained 31.17% variation in BMI, 29.04% in TG, 33.70% in HDL, 46.86% in LDL-c, and 28.55% in total cholesterol. As the significance level increases, the risk model included a higher number of OTUs increasing the proportion of the explained variance. Thus, the OTUs identified at P = 0.05 explained 64.06% variation in TG, 55.97% in HDL, 68.98% in LDL-c, and 58.97% in total cholesterol. However, this is with the exception of BMI for which the proportion of the explained variation decreased with the increase in the significance level, being estimated in 19.40% at P = 0.05 , at the genus level , and at the species level . In fact, our results suggest that in this cohort of age- and diet-matched obese subjects, microbiota composition can be affected by gender in a BMI-specific manner.Our study shows that the gut microbiota differs in men and women at the bacterial phyla level  was higher in men [Bacteroidetes [2 (mean±SD) as the mean of BMIand therefore including include lean and mostly overweight people, but not obese people. This was such that a presumably too short range of BMI was analyzed. Our approach by NGS Illumina platform also test for the global pattern of the bacterial community which confirmed the lower abundance of Bacteroidetes in women as compared with men when BMI is around 25 Kg/m2 as was previously shown [Our study reveals that the  obesity , changed obesity , 39. How obesity , or evensubjects . Furthersubjects , 43, andsubjects . Moreover in men . Howeverroidetes . The latly shown . Consequly shown . Additioly shown , dietaryly shown , 15.Firmicutes was found in women regardless of the BMI. Interestingly, a higher proportion of Firmicutes was found in men under a BMI of 33, whereas a lower proportion was detected when BMI was > 33. Thus, this reflects a potential sexual dimorphism in gut microbiota composition that is variably influenced by BMI. In addition, we observed that the abundance of the Bacteroides genus was lower in men than in women when BMI was > 33. This is presumably a consequence of the decrease in the abundance of this genus in men with the increase of the BMI. Nevertheless, in women, it remained unchanged in the different ranges of BMI. Gender differences in fat distribution have been previously reported, and these are related to the differences in sex hormone levels [In this study, we comparatively analyzed the microbiota of lean and obese men and women under a similar nutritional background, matched by age (with a mean age of 60 years) and stratified according to BMI. A higher proportion of e levels . Yet, liThe composition of the gut microbiota may determine how excess energy is stored in the body, and this effect might be sex dependent. Animal experiments have provided solid proof for this phenomenon, as the sexual dimorphism in total body fat content seen in rodents  has shown a fade away in germ free animals, thus suggesting a role for the gut microbiota . Thus, iBilophila and Blautia, two IBD-related genera, were more abundant in women than in men. Bilophila wadsworthia, a sulphite-reducing bacteria, has been associated with an increased incidence of colitis [Blautia, recently reorganized to refer to several misclassified species belonging to the Clostridium cluster XIVa, which according to our study is more abundant in women than in men under obesity condition, display a high incidence in patients with IBD [Bilophila and Blautia between men and women was influenced by the BMI. Thus, while the differences in Bilophila were more evident below 33 of BMI, we observed a significant trend of the differences in Blautia only above 33 of BMI. These findings support the possibility that obesity, by influencing microbiota composition, might influence the development of IBD. Although admittedly, the pathogenic role of overweight in IBD remain poorly understood.In addition, increasing evidence suggests that the intestinal microbiota may play a role in the development of IBD , 49. Mor colitis , and eme colitis . Moreovewith IBD , 54. Of In a recent study by Fu et al. , it has Our study shows gender-related differences analyzing the full microbiome by using a NGS method. Our experimental design included a broad BMI range, including lean, overweight, obese, and morbidly obese people which allowed us to find out gut microbiota signatures associated with obesity as a function of changes in gender and the BMI. Consequently, this could open a new hypothesis to be tested in bigger populations as one limitation of this study is the reduced sample size, although large enough to detect relative gender-related changes in gut microbiota. However, small differences in taxa with high inter-individual variability may not have been detected, or we may not have had a sufficient sample size to detect small differences in taxa between groups. Further investigations in larger populations are needed to confirm these results and extend the knowledge about the gender differences in the gut microbiota.In conclusion, our results suggest that gut microbiota may differ between men and women, and that these differences may be influenced by the grade of obesity. Thus, these results might be relevant for the proper understanding of the basis of gender differences in the prevalence of metabolic and intestinal inflammatory diseases. Further studies will however be needed to unveil the specific mechanisms, such as sex steroid milieu, gonadal status, or genetic factors, underlying this phenomenon, and to what extent this may play a role in the sexual dimorphism in cardiovascular disease.S1 FigRarefaction curves were generated using phylogenetic metrics  and non-phylogenetic metrics . Horizontal and vertical axes represent rarefaction depth and alpha diversity values, respectively. Error bars correspond to standard deviation for alpha diversity values at each rarefaction depth. Rarefaction curves for gut microbiome richness estimated by gender in all subject , in subject with a BMI lower than 30 , in subject with BMI equal or greater than 30 and equal or less than 33 , and in subject with a BMI greater than 33 .(PPTX)Click here for additional data file.S2 Fig3D PCoA Plots were generated using quantitative measures (unweighted unifrac) and qualitative measures (weighted unifrac). Proportion of variance explained by each principal coordinate axis is denoted in the corresponding axis label. Beta diversity was estimated by gender in all subject , in subject with a BMI lower than 30 , in subject with BMI equal or greater than 30 and equal or less than 33 , and in subject with a BMI greater than 33 .(PPTX)Click here for additional data file.S3 FigBMI indicates body mass index; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein; and TC, total cholesterol.(PPTX)Click here for additional data file.S4 FigVariation explained by gut microbes at different levels of significance. BMI indicates body mass index; TG, triglycerides; HDL, high-density lipoprotein; LDL, low-density lipoprotein; and TC, total cholesterol.(PPTX)Click here for additional data file.S1 TableValues correspond to the mean±SEM of the main metabolic variables. The statistical differences between groups were evaluated by One-way ANOVA. N, 39 men and 36 women. BMI < 30 group, 13 men and 13 women; 30 ≤ BMI ≤ 33 group, 13 men and 10 women; and BMI > 33 group, 13 men and 13 women.(DOCX)Click here for additional data file.S2 TableValues correspond to the mean±SEM of a 14-item questionnaire to assess adherence to the Mediterranean Diet and a 9-point score to assess adherence to low-fat diet. Fiber intake was calculated using the Spanish food composition tables. The statistical differences between groups were evaluated by One-way ANOVA. N, 39 men and 36 women. BMI < 30 group, 13 men and 13 women; 30 ≤ BMI ≤ 33 group, 13 men and 10 women; and BMI > 33 group, 13 men and 13 women.(DOCX)Click here for additional data file.S3 TableValues correspond to the mean±SEM. Macronutrient percentage from total energy (E) intake was calculated using the Spanish food composition tables and food frequency questionnaires. The statistical differences between groups were evaluated by One-way ANOVA. N, 39 men and 36 women. BMI < 30 group, 13 men and 13 women; 30 < BMI < 33 group, 13 men and 10 women; and BMI > 33 group, 13 men and 13 women.(DOCX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.


Imprinting effects can lead to parent-of-origin patterns in many complex human diseases. For hypertension, previous studies revealed the possible involvement of imprinted genes. Genetic Analysis Workshop 18 real data, with hypertensive phenotype and genotype of more than 1000 individuals from 20 pedigrees, provided us an opportunity to further substantiate such findings. To test for imprinting effects, we developed a pedigree-parental-asymmetry test taking both affected and unaffected offspring into consideration (PPATu). We carried out a simulation study based on the Genetic Analysis Workshop 18 pedigrees to show that PPATu has well-controlled type I error and is indeed more powerful than the pedigree-parental-asymmetry test (PPAT), an existing method that does not utilize information from unaffected offspring. We then applied PPATu to Genetic Analysis Workshop 18 genome-wide association study data from 20 pedigrees. We identified a number of single-nucleotide polymorphisms showing significant imprinting effects that are within genomic regions that have been previously implicated to be associated with hypertension. Among these imprinted genes, Beckwith-Wiedemann syndrome, Silver-Russell syndrome, Angelman syndrome, and Prader-Willi syndrome are the best known.Genomic imprinting refers to the phenomenon of unequal expression of a heterozygous genotype depending on which parent (father or mother) the imprinted variant is inherited from. It is estimated that approximately 1% of all mammalian genes are imprinted  that considers imbalance of parental origins of the variant allele is simple and powerful. A series of generalizations of PAT, such as the pedigree-parental-asymmetry test (PPAT) for general pedigree data, widen its practical range . However1 and M2, and the disease allele is more likely to be associated with marker allele M1. Let 0, 1, and 2 represent the marker genotypes M2M2, M1M2, and M1M1, respectively. For a child-parents trio, let F, M, and C denote the marker genotypes of the father, mother, and child, respectively. Throughout this article, mating symmetry is assumed; that is, P=P for all f, m = 0,1,2. We also assume that there is no maternal effect; that is, the maternal genotype does not confer additional risk on the child's phenotype.Suppose that the marker of interest has 2 alleles, MN independent pedigrees, and for the ith pedigree, we have i nuunaffected and i naaffected offspring. Define Suppose we have I is the usual indicator function. We can prove that under the null hypothesis of no imprinting effect, E(S) = 0. The unbiased estimator of the variance of S iswhere S statistic; that is, without utilizing information on trios with unaffected offspring. More specifically, The standardized test statistic To evaluate the power of the proposed statistic and to compare with PPAT, we carried out a simulation study under 9 different settings, combinations of 3 sets of haplotype frequencies H1, H2, H3) and 3 imprinting models   genotypes on odd chromosomes and phenotype data, including systolic and diastolic blood pressure (SBP and DBP). In our study, we use a hypertensive binary phenotype; an individual is classified as affected if SBP > 140 mm Hg, or DBP > 90 mm Hg, or on antihypertensive medication at the first examination. There are 20 pedigrees; the sizes range from 27 to 107 individuals. In total, there are only 157 affected offspring, while there are 709 unaffected ones. Hence, based on the experience gained in our simulation, we expect a substantial gain in information for PPATu that makes use of information from both affected and unaffected individuals when compared to PPAT. To reduce the effect of multiple testing, we first used pedigree disequilibrium test (PDT [p values than PPAT. For SNPs detected by both methods, the results for PPATu are more significant. Table Table p values from PDT. The analysis has resulted in larger sets with p values smaller than 0.05, which we call "significant"for easy reference. A search in the NCBI database found 49 genes implicated to be associated with hypertension in previous studies. Figure PLEKHA7 that has been implicated to be associated with hypertension in previous studies [To gain a more global view of the extent of the role of imprinting in hypertension, we also carried out genome-wide testing for imprinting effects without restricting to SNPs with small  studies ,7.In this article, we propose an imprinting test that utilizes both affected and unaffected individuals from general pedigrees, the type of data provided by GAW18. We expect PPATu to be more powerful than the existing test PPAT [The authors declare that they have no competing interests.LS designed the overall study; FZ conducted statistical analyses and drafted the manuscript. Both authors wrote, read, and approved the final manuscript.


Although anxiety and depression have been classified as distinct traits of affective disorders, previous studies have reported their co-occurrence in subjects with migraine. However, few reports are available on the clinical implications of this comorbidity. This study is to assess the comorbidity of anxiety and depression in subjects with migraine and its clinical implications in a population-based sample from Korea.We selected Korean subjects aged 19–69 years by the stratified random sampling method, and evaluated them using a semi-structured interview, designed to identify headache type, anxiety, and depression. We used Goldberg Anxiety Scale questions and Patient Health Questionnnaire-9 for the diagnosis of anxiety and depression, respectively.p = 0.003), migraine with depression alone , and migraine without anxiety or depression . The migraine with anxiety alone  group and migraine with anxiety and depression  group showed higher Visual Analogue Scale scores for pain intensity compare to migraine without anxiety or depression (6.0 [5.0-7.0]) group.Of the 2,762 participants who completed the interview, 147 subjects (5.4%) were classified as having a migraine during the previous year. Among these 147 subjects, 17 (11.6%) had anxiety and depression, 28 (19.0%) had anxiety alone, 9 (6.1%) had depression alone, and 93 (63.3%) had neither anxiety nor depression. Headache frequency per month was remarkably higher in subjects having migraine with anxiety and depression  than in those having migraine with anxiety alone  contains supplementary material, which is available to authorized users. The symsability .Epidemiological and clinical studies have shown that migraine is comorbid with a number of psychiatric disorders such as anxiety and depression -9. HeadaAnxiety is the most common psychiatric comorbidity among patients with migraine, showing a prevalence of 25.5–57.6% in population-based studies ,12,13. TDepression is another Axis-1 disorder in the DSM-IV TR . A closeAlthough anxiety and depression have been classified as distinct traits of affective disorders, concurrence of anxiety and depression has been observed in clinical and epidemiological studies ,23,24. MHowever, the comorbidity of anxiety and depression in migraineurs and its clinical significance has rarely been reported. In the present study, we will: 1) describe the prevalence of anxiety, depression, and migraine in a general Korean population; 2) assess the comorbidity of anxiety and depression among subjects with migraine; 3) examine the clinical characteristics of subjects having migraine according to the diagnosis of anxiety and depression.This study provides a nation-wide, cross-sectional survey of headache and anxiety in the Korean population. Trained interviewers conducted structured interviews, using a questionnaire to diagnose headache disorders and anxiety in adults aged 19–69 years. The interview included questions on the symptoms and impact of headache and anxiety. Further, the socioeconomic, demographic, and geographic characteristics of participants were evaluated. This study was undertaken from November 2011 to January 2012, and was approved by the institutional review board/ethics committee of Hallym University Sacred Heart Hospital , except Jeju-do, and each administrative division is further divided into “si,” “gun,” or “gu” as the basic administrative units. In total, there are 77 si, 88 gun, and 69 gu. The estimated population of Korea in 2010 was 48,580,293, of which approximately 32,356,747 people were aged 19–69 years, as per data from the 2010 population and housing census by the National Statistical Office . This stTo determine the prevalence rates of and analyze the demographic factors affecting common primary headache disorders, we planned to sample 2,750 individuals based on the population structure. We adopted a 2-stage systematic random sampling method. The 15 administrative divisions were designated as the primary sampling unit. We assigned appropriate sample numbers for each primary sampling unit according to the population distribution. In the second stage, we further selected representative basic administrative units  for each primary sampling unit. Overall, 60 representative basic administrative units were selected for this study. For each representative basic administrative unit, we assigned a target sample number regarding age, gender, and occupation. Estimated sampling error of our study is ±1.8% with a 95% confidence interval  the aims of the present study, 2) the meaning of each question, 3) instructions for interpreting the subjects’ responses, and 4) other details that were relevant to conducting a proper interview. All interviewers were employed by Gallup Korea and had previous socials survey interviewing experience. The interviewers were not medical personnel. The survey was conducted by door-to-door visits and face-to-face interviews.We diagnosed migraine, anxiety, and depression using a questionnaire composed of three parts  for diagnosing anxiety. GAS is composed of four screening questions and five supplementary questions ,30. If aThe Patient Health Questionnaire-9 (PHQ-9) was used for diagnosing depression . If a paBased on the definitions of migraine and anxiety, the 1-year prevalence was presented as the number of cases per 100 persons. Age- and gender-specific prevalence was also calculated. The Kolmogorov–Smirnov test was used to test for normality of the distribution.We calculated the odds ratios  for occurrence anxiety or depression with migraine compared to that without migraine using univariate and multivariate logistic regression analyses. In univariable analyses, we modeled the ORs for migraine versus non-migraine without adjusting for covariates. In multivariable-adjusted analyses for anxiety, sociodemographic variables  and depression were used as covariates. In multivariate analyses for depression, sociodemographic variables and anxiety were used as covariates.We classified subjects with migraine into four groups according to their diagnoses for anxiety and depression: the migraine without anxiety or depression, migraine with anxiety alone, migraine with depression alone, and migraine with anxiety and depression. We compared headache days per month, visual analogue scale (VAS) score of headache intensity, and HIT-6 score, among these four groups using the Kruskal–Wallis one-way analysis of variance test. If the median values were significantly different, post-hoc tests were carried out using Bonferroni’s method. In all statistical analyses, the significance level was 0.05, unless otherwise specified. The results were analyzed using the Statistical Package for the Social Sciences 21.0 .As with most survey sampling designs, missing data resulting from non-response occurred in several variables. The data reported are based on the available data. Sample sizes of some variables diverge from the sample size of n = 2,762 because of non-responses on that particular variable. Imputation techniques were not employed to minimize non-response effects .Our interviewers approached 7,430 individuals and 3,114 of them accepted the survey (rejection rate of 58.1%). After 352 individuals suspended the interview, 2,762 subjects completed the survey  subjects had had at least one attack of headache, and 147 subjects (5.4%) were classified as having migraine during the previous year, 274 (10.0%) were classified as having anxiety, and 124 (4.5%) were classified as having depression  had anxiety alone, 9 (6.1%) had depression alone, and 17 (11.6%) had anxiety and depression. The remaining 93 (63.3%) had neither anxiety nor depression , migraineurs with anxiety alone (p = 0.001), and migraineurs with anxiety and depression (p = 0.012).We investigated demographics, headache characteristics, associated symptoms, headache frequency per month, VAS score for pain intensity, and HIT-6 score of subjects with migraine grouped according to presence of anxiety and depression. Demographic distributions, headache characteristics and associated symptoms of migraineurs were not significantly different according to anxiety or depression except photophobia Table . Photophp = 0.003), migraine with depression alone , and migraine without anxiety or depression  groups (Table p = 0.011). The VAS score of the migraine with anxiety and depression group was not significantly different from that of the migraine with anxiety alone group . The HIT-6 score was higher for the migraine with depression alone group than for the migraine without anxiety or depression group . This score did not significantly differ between the migraine with anxiety and depression and migraine with depression alone groups .Headache frequency was remarkably higher in the migraine with anxiety and depression group  compared to that in the migraine with anxiety alone  The prevalence of anxiety, depression, and migraine in the Korean population were 10.0%, 4.5% and 5.4%, respectively; 2) Among subjects with migraine, 19.0% had anxiety alone, 6.1% had depression alone, and 11.6% had both anxiety and depression; 3) Headache frequency was markedly increased in subjects having migraine with anxiety and depression.The 1-year migraine prevalence rate (5.4%) in the present study was somewhat lower than those previously done in European 10-25%) and North American (9-16%) countries 5% and No,36. The The prevalence of anxiety and depression in this study were similar to those previously reported, ranging from 5.6% to 19.3% for anxiety -39 and 3Previous studies have reported an association between migraine and psychiatric comorbidities such as anxiety and depression -7. The cNumerous studies have shown a close association between depression and migraine. As per population-based studies, subjects with migraine are 2.2–3.5 times more likely to develop depressive disorders than those without migraine ,21,44. APrevious epidemiological and clinical studies have reported that anxiety and depression commonly co-occur in individuals with migraine -7. BreslThe negative effect of anxiety and depression on headache-related disability and the impact of headache on quality of life have been documented ,9,15,18.In the current study, we investigated the relation of headache frequency, headache severity, and impact of headache with the presence of anxiety and depression Table . HeadachAlthough the response rate is not high, we used 2-stage clustered random sampling pool of Gallup Korea, which showed a low sampling error . The socIn this study, we did not attempt to examine the presence of aura because the diagnosis of aura is difficult to verify using questionnaire . AccordiWe did not try to diagnose CM in the present study because the diagnostic criteria for CM were currently under amendment and investigating CM in epidemiological study was very difficult -52. The p < 0.001); 3) We observed a quantitative relationship between migraine clinical characteristics and anxiety, and a higher VAS score in migraine with anxiety. Secondly, although this is a population-based study with a low sampling error, its statistical power for examining subgroups was limited. Thus, some results might have not reached statistical significance merely because of the limited sample numbers.This study has some limitations. Firstly, we used GAS to diagnose anxiety. GAS is composed of nine questions on symptoms of anxiety, and one question about a participant’s headaches or neck aches. Based on these questions, subjects with migraine may have obtained higher GAS scores, and hence, be more likely to be diagnosed as having anxiety. However, we think that our results for comorbidity of anxiety and migraine are reliable because of the following reasons: 1) The usefulness of the Korean version GAS for anxiety diagnosis has been validated, with the Korean GAS showing high sensitivity and specificity;  2) SubjeOur study has several strengths. Firstly, we used clustered random sampling proportional to the Korean population and the estimated sampling error was low. Secondly, we investigated both anxiety and depression, which are common comorbid conditions among both the general population and individuals with migraine, and assessed clinical characteristics of migraine according to the status of anxiety and depression. Balancing limitations and strengths, we think that this study successfully assessed the association of anxiety and depression in migraine with their clinical implications.Approximately 1/3 and 1/6 of migraineurs had anxiety and depression in a Korean population-based sample, respectively. One-third of migraineurs with anxiety had depression and 2/3 of migraineurs with depression had anxiety. The concurrence of anxiety and depression was associated with higher headache frequency in migraineurs. Occurrence of anxiety was associated with exacerbation of headache intensity. Impact of headache increased with the accompaniment of anxiety and/or depression. The presence of anxiety and depression should be carefully evaluated in patients with migraine, in order to reduce the impact of headache as well as provide better treatment for these patients.


Cauda equina secondary to metastatic follicular thyroid cancer of the lumbosacral area is a rare entity. We report an unusual case of a 52-year-old male who presented with backache, lower limb weakness, and perianal numbness. A CT-scan of the lumbosacral area showed an enhancing mass at the L4, L5 and S1 vertebrae. Histopathology after excision revealed a metastatic thyroid cancer. Hence, a CT scan of the neck and chest was performed which showed a nodule in the left lobe of the thyroid and a mass in the left chest wall. A total thyroidectomy and excision of the chest wall lesion was undergone, which was diagnosed as a follicular carcinoma of the thyroid. Metastatic workup of spinal metastasis should include evaluation of the thyroid gland. Thyroid cancer is an entity that accounts for approximately 1% of all newly occurring malignant cancers. It accounts for 0.5% of cancers in men and 1.5% in women . ThyroidThis is further categorized into papillary thyroid cancer (PTC) (70-75%) and follicular thyroid cancer (FTC) (15-20%). Undifferentiated carcinomas, which are anaplastic cancers, account for <5% of thyroid cancers. Medullary carcinoma of the thyroid accounts for 5-10% of thyroid cancers ,4.FTC is a slowly growing tumor, which is common in older age groups, with a peak incidence in the fifth decade. Distant metastasis has been reported to occur more commonly in the bones, brain, and lungs . The incWe present a case of metastatic FTC, whose initial presentation was cauda equina secondary to compression by a metastatic mass in the lumbar region.A 52-year-old male was presented to the neurosurgery clinic of our hospital with complaints of backache for 3 months, numbness of the legs for 2 months, and weakness of the lower limbs for the last 2 days. Neurological examination revealed a power of 4/5 in both limbs with diminished reflexes, decreased anal tone, and diminished perianal sensation.All laboratory workups, including prostate specific antigen, were within normal limits. Due to the neurological status, an urgent CT-scan of the lumbosacral spine with contrast was carried out, which showed a soft tissue density enhancing expansile lytic lesion (55x44 mm) involving the spinous process of the L5 vertebra that was causing almost complete erosion of the spinous process and part of the lamina of the L5 vertebra .Further workup was carried out, which included an ultrasound of the abdomen that was reported as normal and a chest x-ray that showed a well defined soft tissue density in the pleura of the left upper lung zone with rib erosion. The patient was sent to the operating room on same day as admission and underwent an L4-L5-S1 Laminectomy, excision of the tumor, and pedicle screw fixation. Intraoperatively, a mass was observed that was soft to firm in consistency, vascular, and was eroding the spinous process of L4 and L5. Final histopathology revealed a metastatic carcinoma most likely of thyroidal origin as the specimen showed thyroid follicles and was positive for the immunohistochemical marker, Thyroid transcription factor 1 (TTF1) ,3. Therefore, serum thyroglobulin marker and thyroid profile were carried out. Only thyroglobulin levels were raised i.e 103 . A CT-scan of the neck and chest was also carried out, which showed a heterogeneously enhancing nodule in the left lobe of the thyroid. It measured 4.3 X 3 cm and another expansile lytic lesion involving the left third rib posterolaterally was also seen measuring 5 X 4 cm .rd rib mass  followed by cervical lymphadeno- pathy (5%) and the rest in the lung, bone, liver, etc…,8. The i0% followFTC is known for its hematogenous spread secondary to vascular invasion. The most frequent site that it can metastasize to is the lung followed by bone and lymph nodes ,12. BoneIn literature, there are few reports that have described distant metastasis as the initial presentation of thyroid cancer. Shaha et al , from SlComplete surgical removal of the tumor has been reported to offer prolonged survival . Demura Diagnosis of metastatic tumors of the vertebrae need a thorough work up that should include assessment of the thyroid gland entailing a detailed clinical history and a physical examination. In conclusion, for these kinds of rare cases, early presentation, proper examination, early diagnosis, prompt initiation of treatment and follow-up can possibly prolong the patient’s life and improve quality of life.


Methanol poisoning is on the rise and has been associated with high morbidity and mortality; it has resulted in growing research in the field of toxicology. The aim of this study was to reveal underlying patterns in scientific outputs related to methanol poisoning at the global level by evaluating different bibliometric indices.We searched for publications that contained specific words regarding methanol poisoning in Scopus database.Clinical Toxicology, whereas 18 (2.0%) were published in The Lancet.A total of 912 articles, with 8,317 citations and with an average of 9.1 citations per document, were retrieved on methanol poisoning, and the bulk of the articles were published from the USA (20.9%), followed by Spain (4.4%), Canada (4.3%), India (3.1%), and France (3.0%). The articles were published belonging to 57 countries. No data related to methanol poisoning were published from 155 (73.1%) out of 212 countries. Twenty-one documents (2.3%) were published in Scientific production related to methanol poisoning is increasing. articles have been published in a wide range of journals with a variety of subject areas, most notably clinical toxicology; and the country with the greatest production was the USA. Methanol, also known as methyl alcohol or carbinol, is a highly flammable colourless liquid that may be found in antifreeze solutions, model engine fuel, and solvents including methylated spirits. Methanol may also be produced as a secondary contaminant during the manufacture of ethanol-containing beverages, and high concentrations may be found in alcoholic drinks are by illicit distillation processes that are subject to poor quality control. Accidental methanol exposure may occur in large outbreaks as a result of contaminated alcoholic drinks, and several major outbreaks in developing countries have been associated with high morbidity and mortality ,2.Methanol is metabolised slowly by the liver. Toxicity is attributable to its metabolites and, therefore, may be delayed for up to several hours after ingestion. Early clinical features within the first few hours of ingestion include ataxia, dysarthria, nystagmus and reduced conscious level, and may resemble ethanol intoxiciation. Other features include headache, delirium, and vertigo . As meta A number of key strategies exist for managing acute methanol poisoning. Firstly, ethanol competitively inhibits the metabolism of methanol by alcohol dehydrogenase, thereby limiting the formation of toxic metabolites. Alternatively, fomepizole is capable of inhibiting alcohol dehydrogenase and may allow more effective prevention of metabolite formation than ethanol administration. In addition, haemodialysis is an important strategy for removal of methanol and its toxic metabolites, whilst also allowing correction of metabolic acidosis .Methanol poisoning is on the rise -10. TodaScopus online database, a database is owned by Elsevier, was conducted. The Scopus database is an online scientific indexing service containing abstracts and citations for academic journal articles [For this analysis, a search of the SciVerse articles .Scopus database focuses on journal activity rather than investigating books and book chapter (s) about methanol poisoning through Scopus might encompass some false negative results. All analysis of citations was completed on 9th November, 2014. The resulting search was as follows: ((TITLE (methanol) OR TITLE  OR TITLE  OR TITLE (“wood spirits”)) AND (TITLE (poison) OR TITLE (poisoning) OR TITLE (intoxication) OR TITLE (toxicity) OR TITLE (toxic) OR TITLE (toxicology) OR TITLE (toxicities)) AND PUBYEAR < 2013) AND  OR EXCLUDE  OR EXCLUDE ). The keywords used to accomplish the purposes of our study were elected from previous review studies related to methanol poisoning ,3,36,37.h-index [SCImago Journal Rank (SJR) Rank, and the impact factor (IF). The SJR or IF of a publication is not the ideal quality indicator, neither are the citations [The extracted data were used to generate the following information: year of publication, institutions, collaboration patterns, subject categories, names of publishing journals, and citations pattern. Bibliometric indicators were presented as rank order using the standard competition ranking (SCR). We consider only the ten top-ranked. The visibility and/or scientific impact of research output was assessed using h-index . Furtheritations . Both faitations . Data weitations ,24,44,23All statistical analyses were run with Microsoft Excel and version 15 of Statistical Package for Social Sciences (SPSS) program for analysis. Data presented as median, with interquartile range (IQR) in parentheses, or numbers, with percentages in parentheses. In order to assess the growth pattern of research output we performed a linear and an exponential regression for the trend in publication. Reliability of our method was assessed on a pilot sample (n = 100 documents) by two different researchers to check documents type and compare it with the primary sources . The information regarding the 100 selected documents were independently assessed by SZ and SA. The Cohen’s kappa value between the two researchers was 0.942. It is suggested that more than 90% reliability should be reached -47. This2 = 0.77) relative to linear (R2 = 0.66) models. A total of 2,088,219 documents were retrieved by Scopus, which represents the total number of documents published globally in leading pharmacology, toxicology and pharmaceutical journals during the study period (1902–2012) according to the methodology stated. This means that the contribution of methanol poisoning to global pharmacology, toxicology and pharmaceutical research output is 0.04%. There was a statistically significant and strong positive correlation between the absolute numbers of published articles in methanol poisoning and numbers of published articles in pharmacology, toxicology and pharmaceutical science during the years of the study period (1902 – 2012); . The first article related to methanol poisoning in Scopus was published in The Lancet in 1902 [From 1902 to 2012, 912 methanol poisoning-related articles were published and indexed in the Scopus database. After screening, nineteen documents were found not related to methanol poisoning, one document was a book and two documents were errata and were excluded. The included documents were comprised of 680 (74.6%) original articles, 69 (7.6%) letters to the editor, 41 (4.5%) review articles, and 122 (13.4%) documents that were categorised as other types of publications such as conference paper, editorial, note, review and short survey. The percentage share of global methanol poisoning research output showed that research output was 11.1% in 1902 to 1962, 37.6% in 1963 to 1992, 21.3% in 1993–2002, and 30% in 2003–2012 , Canada (4.3%), India (3.1%), and France (3.0%); (Table h-index of the retrieved articles was 42 (i.e. 42 articles had been cited at least 42 times). The highest h-index was 34 for the USA, followed by 14 for Canada. In addition, the highest number of collaborations for each country was achieved by the USA, with 6 countries, followed by 5 countries for Norway  of 2 (0.0–10.0) and a mean of 9.1 citations. The highest median number (IQR) of citations was 13 (5–30) for Belgium, followed by 12 (2–27) for the USA and 12 (1–41.3) for Norway. The Clinical Toxicology, whereas 18 (2.0%) were published in The Lancet. Five journals from the top 10 ranking journals had SJR < 1. Only one journal in the top 10 ranking journals was not listed in the JCR 2013.Table Medical Toxicology and Adverse Drug Experience in 1986, has received 227 citations, and the second article, was published in Journal of Toxicology-Clinical Toxicology in 2002, has got 222 citations. The number of articles without citations was 336, which corresponds to 36.8% of the total. The most cited documents in methanol poisoning were review articles. Table Uniwersytet Medyczny w Bialymstoku, Poland , and University of Iowa, USA (0.8%), followed by Cliniques Universitaires Saint-Luc, Brussels, Belgium (1.6%), and Ulleval University Hospital, Norway (1.6%).Table Scopus database, which is commonly used in bibliometric researches investigating scientific activity, was preferred and used in our study. The Scopus has numerous advantages over others, as it has a relatively large database that indexes a larger number of journals than PubMed and Web of Science [The present study has demonstrated a number of characteristics of methanol poisoning research: the progressive growth of publications worldwide; research productivity of the most prolific institutions; the publication of articles in a wide variety of journals in various subject areas; scientific research productivity and collaboration patterns by country; and the citations received by the publications during the period 1902–2012. To analyse the research on methanol poisoning, the  Science ,22,43,58Scopus database between 1902 and 2012 showed that research productivity was low in the first decades but demonstrated an apparent increase in the late 1980s and early 1990s, with peak publications in 2012. Our study reveals evidence that research productivity related to methanol poisoning have followed the general development in scientific research output related to toxicology field [The total publications linked to methanol poisoning were available in gy field -19,21-24gy field -62,10,8.gy field .Some of the results are similar to those found in previous bibliometric studies in other fields ,44,24, mThe most interesting finding was that international collaboration networks in our study are somewhat lesser than that found in previous bibliometric studies with different field ,44. PrevThe ten most prolific countries that were published in methanol poisoning includes new nations different from the familiar other scientific productivity ranking . ParticuClinical Toxicology, has published the greatest number of publications related methanol poisoning, which is logical because it is specific for the poisoning. In addition, it is notable that most of articles have been published in high-impact journals, such as Lancet. As shown, the papers were published both in toxicological and non-toxicological subject areas, such as emergency medicine; ophthalmology; medicine, general and internal; and pharmacology and pharmacy journals, which reveals the contribution and collaboration of many researchers from different subject areas. This type of collaboration is essential in a poisoning treatment such as methanol poisoning, which needs integrated, multidisciplinary research areas among various biomedical scientists. Regarding the visibility and/or scientific impact of publications, in current study the number of documents without citations represents 36.8% of the total. The percentage of documents without citations found in other bibliometric studies varies widely. A previous study documented that in cardiovascular research, 34.3% of documents remain without citations [The clinical toxicology Journal, formerly itations . Moreoveitations . In our itations ,81.Scopus were not included. However, the advantages of Scopus database (i.e. most reliable service for publications and citations) should not be forgotten. Another limitation of this study is that we included only terms as keywords in the title, thus the results being incomprehensible. An original article might have been presented as conference abstracts and it was not possible to exclude these duplicates. In addition, our study does not focus particularly on research articles only, which some would argue to have been favorable, because we suppose our search approach gives a better indication of overall interest in the field of methanol poisoning.The present study is not without limitations, most of which were acknowledged by the authors in previous similar studies ,23,22,43The most imperative conclusions in this study are: 1) there has been an obvious increase in the total number of papers published in the field of methanol poisoning, verifying the importance of worldwide research on this topic; 2) articles have been published in a wide range of journals with a variety of subject areas, mostly in clinical toxicology; and finally 3) internationally collaborated articles were more prevalent with the USA. An increase in international collaboration would lead to increase quality of publications due to the sharing of ideas and workloads. Gaps from most countries have been identified in the literature that has implications for future research. The potential for more collaboration is argued to gain a better understanding of the status of methanol poisoning in most worldwide countries from the viewpoint of epidemiological data, and treatment practices.


Restoration is important for biodiversity conservation worldwide, but surprisingly little is known about its efficiency in a long-term perspective. In this study, we re-examined Swedish semi-natural grasslands 12–20 years after the restoration, by comparing field inventories of vascular plant species diversity made in 2001 with follow-up inventories in 2012. We also analysed restoration effect in relation to six environmental factors and used continuously managed semi-natural grasslands as references of desired state after restoration. We found that total species richness increased over time but not to reference levels, while there were no significant changes in species density or number of grassland specialists. However, the overall species composition in the restored sites, as well as grassland specialist composition, now largely resembled reference conditions. Fertilisation and time between abandonment and restoration were the only environmental variables that affected total species composition change, while site area affected change in grassland specialist composition. Our results show that restoration of semi-natural grasslands can contribute to conservation of semi-natural habitats and their associated biodiversity. Yet, due to the vague restoration goals for these sites, it is difficult to evaluate the restoration success, which emphasise the general need for clear and measurable goals. Humans have altered practically every natural ecosystem on the Earth ,2, some The societal benefits of ecological restoration in farmlands have mostly been discussed in terms of enhancement of biodiversity and ecosystem services ,14,15. S2-scale [Plant species richness in semi-natural grasslands has been shown to increase with elapsed time since restoration e.g. ,30,31). ,31. 30,32-scale ,37. Alth2-scale , it is o2-scale ,30,39,402-scale ,34, but 2-scale ).Several factors are important for the successful outcome of semi-natural grassland restoration ,41,42. SHigh Nature Value farming, where conservation of biodiversity is maintained by low intensity farming, with semi-natural grasslands as a key feature [2) and composition for both all plants and grassland specialist species changed since restoration, 2) the similarities between restored and reference sites, and 3) how the environmental factors; restored site area, time between abandonment of grazing and restoration, time since restoration, abundance of trees and shrubs at the restored site, vegetation height and degree of fertilisation affected potential change in diversity and species composition.One of the priorities in the EU Rural Development Programme is the so-called  feature ,55. Rest feature ,23, espe feature . In thisWe compared data from a plant survey in 2001 of 16 abandoned and restored semi-natural grasslands  with a rIn addition to the 16 restored grasslands, we also surveyed five continuously-managed semi-natural grasslands, which were used as references of the desired state after restoration , with respect to plant richness and composition. They were of similar size  and located in the vicinity to the restored sites. All sites were inventoried with permission from the land owner.2 plots equally distributed in two 40m transects per site both in 2001  and 2012 . The inventories took place during the summer months July and August both years. It is known that year-to-year weather fluctuations could largely affect the vegetation dynamics  and (2) total number of semi-natural grassland specialist species recorded in ten 1m2 plots  were analysed. On plot scale, (3) average number of species/m2 , (4) average number of grassland specialists/m2 , (5) frequency of species/m2, and (6) frequency of grassland specialists/m2 were analysed. Grassland specialist species were defined as having their optimum occurrence in traditionally managed meadows and pastures and decrease in frequency in early to intermediate successional phase [Data collection of plant species richness and abundance followed the same protocol in the restored sites and the reference sites, using ten 1mcs e.g. ,58), how, how2 plal phase ,60.Six explanatory factors were included in the analysis; (1) restored site area, (2) the time elapsed since restoration started, (3) time between abandonment of grazing and restoration, (4) abundance of trees and shrubs at the restored site, (5) vegetation height and (6) degree of fertilisation. The site areas varied between 3 and 25 ha and restoration started between 12 and 20 years ago (average = 14.8 years). The abandonment time was divided into three groups; (1) sites that had low intensity grazing, insufficient to fully prevent succession, during the last 50 years, (2) sites abandoned (i.e. not grazed) for 10–15 years, and (3) sites abandoned for >15 years. Information about site area, time since restoration and abandonment time were acquired from the farmers and county boards. The number of trees and shrubs were counted within a circle with a radius of 20 m both in 2001 & 2012, but since it did not significantly differ between years , an average (ranging from 1 to 60 trees) for each site was used in the analyses. As a measure of grazing intensity , vegetatThe statistical analyses were performed using R 2.15.0 , CANOCO The composition of all species and grassland specialists were analysed in CANOCO 5 and PAST, using detrended correspondence analysis (DCA) and analysis of similarity  to examine if the species composition had changed in the restored sites between T1 and T2, as well as to compare with species composition in the reference sites. Further, constrained partial canonical correspondence analysis (CCA) with ‘Time’ set as a co-variable was used to analyse which environmental factors contributed to the species composition (down-weighted rare species) and grassland specialist composition. One site was not included in these analyses due to missing environmental data. SIMPER-analyses with Bray-Curtis distance measure  were use2) did not differ significantly from T1 to T2, neither did the grassland specialist density , as well as a higher number of grassland specialists per m2  in the reference sites than the restored sites . There were also significantly more grassland specialists , a higher number of species per med sites . Out of ed sites .Composition of both the entire community and grasslands specialists only, changed significantly  in the restored sites between 2001 and 2012. Out of the 246 species recorded in the restored sites, 146  increased in frequency, while 91  decreased. The species’ contributions to the dissimilarity in species composition were evenly distributed  and the continuously managed reference sites  .2 in the reference sites, compared to the restored grasslands. This is consistent with the other studies that have compared restored sites with reference sites in a shorter time perspective . A similar trend was found regarding the number of grassland specialists. Since we focused on restoration effects both on plant species diversity and composition, the restoration progress in our study sites could partly be described as successful. However, the increased species richness was more related to an increase of generalist species, than of grassland specialists. Furthermore, even though species richness has increased over time, this did not result in an increased species density. Moreover, there were still significant differences between the restored sites and reference sites regarding all the tested species richness and density measures. For instance, there were on average 37% more grassland specialists and more than twice as many specialists per mrs, e.g. ,30,39).Species richness in restored semi-natural grasslands has been found to recover more quickly than species density , perhapsAlthough species richness is the most commonly used measure to assess restoration success, it will neither reveal if the species are representative of the native community , nor disA species composition similar to species-rich permanent semi-natural grasslands has only been reported by Schrautzer et al. , but sevOverall, the local factors had low, if any, effect on changes in species richness and composition. This could be due to the lack of precise data for some of our tested variables, but it could also be explained by the relatively small environmental differences between the sites. Nevertheless, the total species composition change was affected by abandonment time and fertilisation, while grassland area affected the specialist species composition, indicating that the local conditions could have an effect on some vegetation aspects following restoration.Earlier studies show that a long time between abandonment and restoration, as well as high level of nutrients, have a negative effect on restoration potential in grasslands ,79, resuMany management programmes today include some kind of ecological restoration, yet the definition of restoration success may not always be clear. Similarly, the reasons for restoration in our selected sites were in most cases not described. The goals were vague, relating mostly to the conservation and increase of biodiversity and cultural values, corresponding to the overarching goals for High Nature Value farmlands in EU. Having clear restoration goals is fundamental for the evaluation of restoration projects, irrespective of habitat type. The goals can be dynamic and have several possible end points , but theWhile broad overarching goals are important for habitat restoration in general there is still a need for measurable goals that can be evaluated through follow-up studies. These goals may specifically target different aspects of biodiversity conservation or be of interest for recreational or historical reasons. If the former is the main goal, we suggest that data of species frequency should be collected to follow the development of species composition over time, since the actual diversity could be missed when using only species richness or red-listed species ,88. AlthEven if the restoration of semi-natural grasslands is seen as successful, it still depends on continued management to maintain high biodiversity ,90. SincWe have shown that the biodiversity measurements used to evaluate grassland restoration can have important implications for whether restoration might be considered a success. Although the over-all species richness increased, it was still higher in reference grasslands, even 12–20 years after restoration. Further, no significant changes were detected in number of grassland specialists or species density. The overall species composition and grassland specialist composition, on the other hand, had changed and now resembled reference grassland sites, indicating the importance of long-term follow-up studies. We suggest that resemblance to reference grasslands regarding species composition should be clearly included in the restoration aims for semi-natural grasslands, since it is a better indicator of restoration success. In that case these restorations could be considered successful, even though species richness did not resemble reference conditions. Having well defined restoration goals related to clear biological measures is hence the key to reach and evaluate restoration success.S1 AppendixClassification of species into ‘Semi-natural grassland specialist species’ (S) or ‘Non-specialist’ (N). Mean abundance in reference sites at T2  and restored sites at T1  and T2. Differences in species abundance in the restored sites between T1 and T2 indicated as increasing (+) or decreasing (-) abundance. Species contribution to the dissimilarity in over-all species composition (i.e. species that are primarily responsible for the observed differences) in the restored sites between T1 and T2 (in %). Nomenclature according to Krok and Almquist.(DOCX)Click here for additional data file.