P > 0.05); however, sectors 2, 3, 4, 8, 9, and 11 of the papilla showed thinning, while in sectors 1, 5, 6, 7, and 10 there was thickening. Total macular volume and RNFL thickness of the fovea in all four inner quadrants and in the outer temporal quadrants proved to be significantly decreased (P < 0.01). Despite the fact that peripapillary RNFL thickness did not statistically differ in comparison to control eyes, the increase in peripapillary thickness in our mild-AD patients could correspond to an early neurodegeneration stage and may entail the existence of an inflammatory process that could lead to progressive peripapillary fiber damage.Decreased thickness of the retinal nerve fiber layer (RNFL) may reflect retinal neuronal-ganglion cell death. A decrease in the RNFL has been demonstrated in Alzheimer's disease (AD) in addition to aging by optical coherence tomography (OCT). Twenty-three mild-AD patients and 28 age-matched control subjects with mean Mini-Mental State Examination 23.3 and 28.2, respectively, with no ocular disease or systemic disorders affecting vision, were considered for study. OCT peripapillary and macular segmentation thickness were examined in the right eye of each patient. Compared to controls, eyes of patients with mild-AD patients showed no statistical difference in peripapillary RNFL thickness ( Alzheimer's disease (AD), the most common cause of dementia, afflicts 67 of every 1000 persons over age 65. Its prevalence and incidence increase exponentially with age . In 2006AD is characterized by a decline in cognitive function, loss of learning and memory, and the formation of neuritic plaques and neurofibrillary tangles, primarily in the cerebral cortex , 4.The retina is a projection of the brain, and a number of similarities between AD pathology and several distinct retinal degenerations have been described , 6. The The RNFL reportedly thins with aging , 10. Som in vivo [Currently it is thought that retinal ganglion cell (RGC) loss in AD might result from amyloid deposits in the eye and/or retina. Amyloid-beta plaques as well as oligomers have been reported in postmortem retinal tissue from patients with AD and in a mouse model of AD, as well as in human retinal tissue in vivo . Therefo in vivo . in vivo.Diagnosis and progression of AD, especially early cases, are complicated because of imprecise neuropsychological testing, sophisticated but expensive neuroimaging techniques, and invasive sampling of cerebrospinal fluid , 31. ImpOCT is a reliable noninvasive technique, routinely used in ophthalmology to visualize and quantify the layers of the retina. OCT enables quantitative cross-sectional imaging of the RNFL and macular volume. A recent study published by our group  has showThe goal of the present study was to examine in detail peripapillary and macular segmentation in order to determine which is the earliest thinned area in patients with mild AD which may be used, in the future, as a predictive tool.To select patients, we reviewed the Database of the Memory Unit of the Hospital Clinico San Carlos in Madrid (Spain), consisting of a total of 2635 patients. First, we excluded the patients with a Global Deterioration Scale (GDS) over 4 and then those with a mood or psychiatric disorder. Next, we took into account 87 patients with mild AD. These patients, according to the National Institute of Neurological and Communicative Disorders and Stroke-AD and Related Disorders Association and the Diagnostic and Statistical Manual of Mental Disorders IV, had mild cognitive impairment according to the Clinical Dementia Rating scale. Then ophthalmic medical records of these patients were reviewed, excluding patients who were previously diagnosed with an ophthalmological pathology . After this analysis, 29 patients with AD satisfied all the requirements to participate in the study . Of the 29 mild-AD patients and 37 age-matched control subjects selected , 6 mild-AD patients and 9 age-matched control subjects were subsequently excluded due to posterior pole pathology including macular degeneration, drusen, suspicion of glaucoma, glaucoma, epiretinal membrane, or cataract that prevented ocular examination. Because of this selection, 23 patients with mild AD and 28 age-matched control subjects were considered for the study. Informed consent was obtained from both groups. The research followed the tenets of the Declaration of Helsinki, and the protocol was approved by the local ethics committee.For the ophthalmological part of the study, the right eye of each patient was analyzed. All participants met the following inclusion criteria: being free of ocular disease, AREDS Clinical Lens Standards <2, retinal drusen, and systemic disorders affecting vision; having a best corrected VA of 20/40; having a ±5 spherocylindrical refractive error; and having intraocular pressure of less than 20 mmHg. For screening, all AD patients and control subjects underwent a complete ophthalmologic examination, including assessment of VA, refraction, anterior segment biomicroscopy, applanation tonometry , dilated fundus examination, and OCT. The RNFL thickness and macular thickness were measured by OCT Model 3D OCT-1000  after pupil dilatation. The RNFL thickness was scanned 3 consecutive times per patient in each area studied. The mean values were considered for statistical analysis. All tests were performed by the same optometrist (ESG) under the same conditions. These tests were selected considering that in this developmental stage of the disease the results were not influenced by the patient's cognitive impairment.3.The peripapillary RNFL thickness parameters evaluated in this study were average thickness (360° measurement), thickness for each 12-o'clock hour position with the 3-o'clock position as nasal, 6-o'clock position as inferior, 9-o'clock position as temporal, and 12-o'clock position as superior. Macular RNFL thickness data were displayed in three concentric rings centered in the foveola that were distributed as follows: a central macular ring, 1 mm away from the fovea; an inner macular ring, 3 mm away from the fovea; and an outer macular ring, 6 mm away from the fovea. As a result, the total area studied made up a 6 mm macular map. In addition, the inner and outer rings were each divided into four quadrants  . The totP value of <0.05 was considered statistically significant.The data are reported as mean values ± SD. The differences between mild AD and control eyes were analyzed using the Mann-Whitney test. Data for the statistical analysis were introduced and processed in a SPSS 19.0 . A Demographic and clinical data for the mild-AD patients and control group are shown in Peripapillary RNFL Segmentation Thickness. Peripapillary RNFL thickness values    . The RNF U test)  in patie U test) .P < 0.05; Mann-Whitney U test)  .Alzheimer's dementia syndromes, like all neurodegenerative diseases, lack objective disease- and stage-specific biomarkers . As a paThinning of the RNFL has been found in several neurological diseases, such as Parkinson's disease , 37–39, In the present work, we compare the peripapillary RNFL segmentation thickness, macular thickness, and the total macular volume in mild-AD patients and age-matched control subjects. One of the relevant issues of the study was that the sample analyzed here was homogeneous in that (i) all patients had recently been diagnosed as having mild AD  with me In vivo studies using different methodologies have confirmed optic-nerve-fiber damage in AD when compared with controls. Optic disc pallor, pathologic disc cupping, and thinning of the neuroretinal rim and the RNFL have been reported in studies based on the subjective evaluation of fundus photographs [Widespread axonal degeneration in the optic nerve was found in a postmortem study of patients with AD . Morphomtographs , 42 and There is controversy on the reduction of the peripapillary RNFL thickness measured by OCT in AD. A reduction in the thickness of all peripapillary RNFL quadrants as measured by OCT has been reported , 43, andMost authors, although working in more advanced stages of disease (MMSE > 23.7), agree that the peripapillary RNFL thinning is significant in the superior and inferior sectors , 37, 44.Reactive astrogliosis in the brain is a well-known feature of AD, but its role in AD is not well understood. Reactive astrogliosis tends to be focal in AD. Reactive astrocytes are intimately associated with amyloid plaques or diffuse amyloid deposits. Astrocytes surround them with dense layers of processes as if forming miniature scars around them, perhaps to wall them off and act as neuroprotective barriers . It is pIn the same way, retinal neurodegenerative diseases are also associated with chronic microglial activation and neuroinflammation. In the degenerating retina, endogenous signals activate microglial cells, leading to their local proliferation, migration, enhanced phagocytosis, and secretion of cytokines, chemokines, and neurotoxins. These immunological responses and the loss of limiting control mechanisms may contribute significantly to retinal tissue damage and proapoptotic events in retinal neurodegeneration –59. A liIn the present study, the analysis of the OCT values of both peripapillary and macular RNFL thickness in patients with mild AD (MMSE = 23.7) showed that only in the macula was there a significant thickness reduction compared to aged-matched controls. Our data, taken together with those reported in the literature, move us to propose the hypothesis that the first affected area of the retina in mild AD is the macular area, where, due to the arrangement of the multilayer bodies of the ganglion cells, the decrease is easier to detect.Subsequently, as the neurodegeneration progresses, a significant decline in peripapillary RNFL thickness will become apparent. The study of the peripapillary segmentation reveals, in a more accurate way, the changes that occur in RNFL thickness in relation to the macular-thickness changes. In this sense, our patients with mild AD differed with respect to controls, although without reaching statistical significance; perhaps due to the early stage of the disease. In addition, the increase in peripapillary thickness in our mild-AD patients may indicate the existence of an inflammatory process that would lead to neurodegeneration of the peripapillary fibers. More extensive studies should be conducted to test these findings.


According to the dual-process theory, coping flexibility is defined as the ability to produce and implement a new coping strategy in place of an ineffective coping strategy. Specifically, coping flexibility includes two processes: evaluation coping and adaptive coping. Evaluation coping refers to sensitivity to feedback about the efficacy of a coping strategy, and adaptive coping involves the willingness to implement alternative coping strategies. The coping flexibility hypothesis (CFH) postulates that more flexible coping will be associated with more adaptive outcomes; importantly, there are numerous theories and studies that support the CFH. The main purpose of this study was to test the CFH based on dual-process theory.A total of 1,770 Japanese college students participated and, completed a set of questionnaires that measured coping flexibility  and depressive symptoms. Depressive symptoms were measured via the Center for Epidemiologic Studies Depression Scale.The proportions of women and men who reported depressive symptoms were 58.69%  and 54.17% , respectively when a cut-off score of 16 on the CES-D was used. A multivariable logistic regression analysis revealed that evaluation coping  and adaptive coping  were significantly associated with lower levels of depressive symptoms.The results of the present study indicated that the CFH based on dual-process theory was supported in a Japanese sample. Depression is a leading cause of disability worldwide. In interviews conducted by the World Health Organization (WHO)  with a cThe WHO  states tCoping flexibility refers to one’s ability to effectively modify one’s coping strategies according to the demands of different stressful situations . For exaevaluation and adaptation processes. Finally, in the feedback state, the efficacy of a chosen strategy is monitored. In Cheng et al.’s model of coping flexibility, three meta-coping skills—evaluation, adaptation, and monitoring—play a major role in the execution and feedback stages. Cheng’s model represents an integration of multiple concepts and theories of coping flexibility. Specifically, Kato , k = 108, N = 28,145). For example, evaluation coping and adaptive coping were more strongly associated with lower depressive symptoms longitudinally ). An analysis using a score of 27 as the cut-off point revealed that women  reported a significantly higher risk of depressive symptoms than men  did, B = 0.28, SE = 0.11, Wald = 6.64, OR = 1.33, p = 0.010, 95% CIs .Means, standard deviations, and Cronbach’s alphas for all variables are presented in p< .001) and adaptive coping  were significantly associated with a low risk of depressive symptoms, after controlling for gender (p< .001) and adaptive coping  were significantly associated with a low risk of depressive symptoms, after adjusting for gender  associated with depressive symptoms, and the prevalence of depressive symptoms was computed with 95% CIs. The multivariable logistic regression analysis with a CES-D score of 16 used as the cut-off point revealed that evaluation coping  out of 100 in a list of the most-cited papers of all time by Nature [There are known racial/ethnicity-specific response patterns on the CES-D . Specifiy Nature  in 2014.The findings from the current study that support the CFH could contribute to the development of stress management strategies that aid in attenuating depressive symptoms. This seems possible given that global prevention-oriented stress management programs are designed to aid in the acquisition of a repertoire of coping strategies, teach when and where strategies will be effective, and facilitate the selection of an appropriate strategy for a particular situation . This tyCoping flexibility has received considerable attention in recent decades ,9, and rDespite the strengths of the current study, several limitations should be described, and some caution should be used in the interpretation of the findings. First, the findings cannot be generalized to other populations beyond Japanese college students. Indeed, Cheng et al.’s  meta-anaAlthough there were several limitations of the present study, the data from Japanese college students provide evidence that evaluation coping and adaptive coping were associated with a low risk of depressive symptoms. That is, the CFH based on dual-process theory was supported.S1 Dataset(XLSX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Dittrichia viscosa is a Mediterranean ruderal species that over the last decades has expanded into new habitats, including coastal salt marshes, ecosystems that are per se fragile and threatened by human activities. To assess the potential risk that this native-invasive species represents for the genuine salt marsh vegetation, we compared its distribution with that of Inula crithmoides, a taxonomically related halophyte, in three salt marshes located in “La Albufera” Natural Park, near the city of Valencia (East Spain). The presence of D. viscosa was restricted to areas of low and moderate salinity, while I. crithmoides was also present in the most saline zones of the salt marshes. Analyses of the responses of the two species to salt and water stress treatments in controlled experiments revealed that both activate the same physiological stress tolerance mechanisms, based essentially on the transport of toxic ions to the leaves—where they are presumably compartmentalized in vacuoles—and the accumulation of specific osmolytes for osmotic adjustment. The two species differ in the efficiency of those mechanisms: salt-induced increases in Na+ and Cl− contents were higher in I. crithmoides than in D. viscosa, and the osmolytes  accumulated at higher levels in the former species. This explains the (slightly) higher stress tolerance of I. crithmoides, as compared to D. viscosa, established from growth inhibition measurements and their distribution in nature. The possible activation of K+ transport to the leaves under high salinity conditions may also contribute to salt tolerance in I. crithmoides. Oxidative stress level—estimated from malondialdehyde accumulation—was higher in the less tolerant D. viscosa, which consequently activated antioxidant responses as a defense mechanism against stress; these responses were weaker or absent in the more tolerant I. crithmoides. Based on these results, we concluded that although D. viscosa cannot directly compete with true halophytes in highly saline environments, it is nevertheless quite stress tolerant and therefore represents a threat for the vegetation located on the salt marshes borders, where several endemic and threatened species are found in the area of study. Limonietalia), intrinsically related to the salt marshes, are considered priority habitats by the European legislation  species which have the ability to spread aggressively outside their natural range and are potentially dangerous for the environment, economy, or human health  Greuter, formerly ascribed to the genus Inula as I. viscosa (L.) Aiton, is a perennial, 40-130 cm high plant, very common in the western Mediterranean but with penetration also in its eastern part. Its primary habitats are gravel riverbeds, mountain screes, and sandy and rocky coasts, but it is frequent mostly in secondary habitats as roadsides and abandoned fields, and sometimes in croplands as a weed , where it invades especially sensitive environments of high ecologic value  Dumort]. This latter species is a small shrub 30-100 cm high, frequent on cliffs and salt marshes, with a Mediterranean-Atlantic distribution, reaching its northern limit in Scotland. The field work was complemented studying the responses of the two species to salt and water stress under controlled experimental conditions in a greenhouse, to establish the relative resistance to stress of the two species and the underlying tolerance mechanisms. For this, some specific biochemical stress markers associated with basic, conserved response pathways were determined in young plants of D. viscosa and I. crithmoides subjected to salt and water stress treatments. We quantified, specifically: (1) growth parameters, that would clearly indicate their relative tolerance to stress, (2) photosynthetic pigments , (3) the major putative osmolytes , (4) monovalent ions , (5) levels of malondialdehyde (MDA), as an indicator of oxidative stress, (6) total antioxidant activity, by the DPPH radical scavenging assay, and total phenolics and flavonoids as examples of non-enzymatic antioxidants, and (7) antioxidant enzyme activities .The main aim of this work was to evaluate the potential risk that D. viscosa and I. crithmoides plants in relation to soil electric conductivity (hereafter EC) and soil moisture was registered in three salt marshes located in “La Albufera” Natural Park  near Valencia, in eastern Spain. La Albufera is the biggest lake in the Iberian Peninsula, formed by the gradual closure of an ancient marine gulf. The salt marshes appear in small inter-dune depressions located in the narrow land strip between the sea and the lake  were analyzed with a WET-2 Sensor  which allows direct in situ non-destructive measurements in the root zones of the plants. Coordinates of these points were established with a GPS (Garmin GPSMAP 76CSx). All field data were taken in spring 2015.The presence of the two species was evaluated along a linear transect in the first salt marsh, since it is much extended and there is a high density of D. viscosa and I. crithmoides were sampled in one of the salt marshes, in October 2014, and well-shaped achenes were directly sown on a mixture of commercial peat and vermiculite (3:1). After 20 days, seedlings homogeneous in size were selected and placed in individual pots. Three weeks later, when young plants were robust enough, treatments were initiated. For the salt treatments, plants were watered twice a week with Hoagland nutritive solution  of the leaves of each sample was expressed as percentage of the FW of the corresponding non-treated control; the absolute FW values of the controls are indicated in the legend to Figure 3. Part of the leaf material was dried at 65°C until constant weight, to calculate the water content of the plants (WC), in percentage, as WC (%) = [(FW – DW)/ FW] × 100.Mature floral heads of 1:5) was measured with a Crison Conductivity-meter 522 and expressed in dS m−1.Electrical conductivity of the substrate was measured after 3 and 6 week-treatments. Soil samples from five pots per treatment were air-dried and then passed through a 2-mm sieve. For each sample, a soil:water (1:5) suspension was prepared in deionized water and mixed for 1 h at 600 rpm, and 21°C. Electric conductivity  and chlorophyll (Chl b) were measured following Lichtenthaler and Wellburn : 100 mg −1 DW.Glycine betaine (GB) was determined in dried leaf tissue according to Grieve and Grattan . The sam−1 DW.Proline (Pro) content was quantified using fresh leaf material, according to the ninhydrin-acetic acid method of Bates et al. . Pro was2. Analysis was carried out injecting 20 μL aliquots with a Waters 717 auto-sampler into a Prontosil 120-3-amino column  maintained at room temperature. An isocratic flux (1 mL/min) of 85% acetronitrile (J.T. Baker) during 25 min was applied in each run. Standards of glucose, fructose, and arabinose served to identify peaks by co-injection. Sugars were quantified by peak integration using the Waters Empower software and comparison with glucose, fructose, and arabinose standard calibration curves.The water-soluble sugar fraction (mono and oligosaccharides) was analyzed using a Waters 1525 High Performance Liquid Chromatography system, coupled to a 2424 evaporative light scattering detector (ELSD). The source parameters of ELSD were the following: gain 75, data rate 1 point per second, nebulizer heating 60%, drift tube 50°C, and gas pressure 2.8 Kg/cmExtractions were performed according to Weimberg , by incuDried leaf material was extracted in 80% methanol, in a rocker shaker, for 24–48 h. Malondialdehyde (MDA) content in the extracts was determined according to the method described by Hodges et al. . The samx/A0) × 100], where Ax is the absorbance of the DPPH solution in presence of the plant extract and A0 the absorbance of the control DPPH solution without the plant sample.Total antioxidant activity in the extracts, measured by their ability to quench the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), was determined spectrophotometrically according to Falchi et al. . The met−1 DW).Total phenolic compounds (TPC) were quantified according to Blainski et al. , by reac2, followed by AlCl3 and NaOH, and the absorbance of the sample was measured at 510 nm. This protocol is often described to detect “total flavonoids” in the sample, although this is not strictly true. The method is based on the nitration of aromatic rings bearing a catechol group and only detects those phenolic compounds containing this chemical structure, which include several subclasses of flavonoids—such as flavonols or flavanols—but also other non-flavonoid phenolics, such as caffeic acid and derivatives. Nevertheless, the method was chosen since the metabolites determined by the reaction with AlCl3 are all antioxidants and there is a good correlation between their levels and the total antioxidant activity of the samples , and express their contents as equivalents of catechin (mg eq. C g−1 DW).Total “antioxidant flavonoids” (TF) were measured following the method described by Zhishen et al. . The metCrude protein extracts were prepared from plant material stored frozen at −80°C, following the procedure described in Gil et al. . Protein2O2 (Δε = 39.4 mM−1 cm−1) upon the addition of the plant extracts  activity was quantified according to Connell and Mullet  was determined following the decrease in absorbance at 240 nm which accompanied the consumption of Hts Aebi, . One CATd Mullet , followid Mullet  by monitd Mullet .©, Instituto Geográfico Nacional de España - Institut Cartogràfic Valencià,” with the programme ArcGIS 2011.The location of the two species in the three salt marshes was established on the orthophotography Terrasit—72258-ecw provided by the “PNOApost-hoc comparisons were made using the Tukey HSD test. Differences between the two species in each treatment were assessed by a t-test for the aforementioned confidence level.Statistical analyses were performed using the program Statgraphics Centurion XVI. Before the analysis of variance, the Shapiro-Wilk test was used to check for validity of normality assumption and Levene test for the homogeneity of variance. If ANOVA requirements were accomplished, the significance of the differences among treatments was tested by one-way ANOVA at a 95% confidence level and I. crithmoides and D. viscosa. All means throughout the text include the SD.All measured parameters in plants submitted to salt stress were correlated using principal component analysis (PCA), for 3 and 6 weeks in both Inula crithmoides and the native-invasive Dittrichia viscosa was registered in three salt marshes in “La Albufera” Natural Park  than in I. crithmoides (twofold), and the same qualitative pattern was observed for chlorophyll b and total carotenoid contents , and total carotenoids by 54% , GB levels increased about twofold in D. viscosa, but nearly eight-fold in I. crithmoides  in their leaves, as a response to the treatment with increasing NaCl concentrations, but reaching much higher levels in  Figures . GB conts Figure . The higD. viscosa plants after 3 weeks of water stress treatment; in I. crithmoides, on the contrary, water stress did induce the accumulation of this compound, albeit at much lower levels than those measured in salt-stressed plants, only a twofold increase over the control, approximately  and I. crithmoides (4-fold increase) plants  was also measured in s Figure . Pro levs Figure ; when ths Figure . Water ss Figure . Yet, itD. viscosa plants grown in the presence of salt for 6 weeks, relative increases of ca. 1.5-, 2.8-, and 5-fold in the levels of Ara, Fru, and Glu, respectively, were observed when comparing the highest NaCl concentration tested (600 mM) and the non-stressed controls. Sugar contents in I. crithmoides control plants were in most cases lower than in D. viscosa, even not detectable in the case of Fru (after the 3-week growth period) and Glu (in samples collected at 3 and 6 weeks); however, the salt-induced increase in the levels of the three sugars was relatively larger in the former species, so that in the presence of 600 mM NaCl the contents of Ara, Fru, and Glu were significantly higher in I. crithmoides than in D. viscosa , could contribute significantly to osmotic adjustment, especially in D. viscosa, which accumulates much lower levels of GB.Spectrophotometric determination of total soluble sugars, after reaction with phenol and sulfuric acid , 5.4-fold (Fru), or 8-fold (Glu) higher than in the control plants. However, these sugars were not detected in the HPLC chromatograms of the leaf samples of I. crithmoides water-stressed plants , chloride (Cl−), and potassium (K+) contents were measured in leaves of D. viscosa and I. crithmoides plants, following the treatments with NaCl for 3 and 6 weeks .I. crithmoides, whereas significant differences with the control were already detected at 300 mM external NaCl concentration in D. viscosa plants , toxic compounds that oxidize amino acid residues in proteins, unsaturated fatty acids in cell membranes, and DNA molecules, thus causing cellular damage , although the differences between the two species were not extremely large  contents increased in salt-stressed plants of  Figures . In I. c Figures . Water ss Figure .D. viscosa than in I. crithmoides under all conditions tested. No significant changes were observed, in general, after 3 weeks of salt treatments , in response to increasing salt concentration, were similar to those of TF, although their absolute levels were clearly higher in s Figure , while i Figures . Regardis Figure .I. crithmoides, catalase (CAT) activity did not vary in the presence of salt, under all conditions tested. The specific activities of glutathione reductase (GR) fluctuated in response to increasing salt concentrations, but the differences between the control plants and those watered with 600 mM NaCl were again non-significant. Only superoxide dismutase (SOD) activity increased significantly, albeit slightly (up to 1.5-fold), in the 6-week treatment with high NaCl concentrations. Regarding the I. crithmoides water-stressed plants, no significant changes were detected in any of the antioxidant activities tested  induced by increasing salt concentrations. Contrary to I. crithmoides, water stress led to significant  variations of antioxidant enzyme activities in D. viscosa, reducing CAT and GR, and increasing SOD, as compared to the non-stressed controls  levels, osmolyte  contents, MDA, total antioxidant activity (DPPH), non-enzymatic antioxidants (TPC and TF), or SOD activity. Although similar correlation patterns were observed for each species, regardless of the duration of the treatment, the variation explained by the first component increased from about 67% at 3 weeks to 79% at 6 weeks, in both D. viscosa and I. crithmoides. This is in agreement with the fact that, in general, the loading vectors of the analyzed variables presented smaller angles with the X-axis—that is, higher correlation with salinity—in the PCAs corresponding the 6 weeks of treatment, due to the prolonged salt stress effects. The joint analysis of all variables indicated that the responses of the two species to salt stress are the same or very similar, qualitatively, and that quantitative differences are most clearly manifested after a longer treatment.Principal component analyses (PCAs) were performed independently for s Figure . In the s Figure , and wasin situ measurements with a portable sensor.Salt marsh ecosystems are characterized by quick changes in their environmental conditions Chapman, . In MediDittrichia viscosa is a species adapted to a wide range of environmental stresses , and carotenoid contents have been reported to correlate closely with the degree of salinity or drought affecting plants of several species  in their leaves, in response to increasing NaCl concentrations in the pots, although significantly higher levels were measured in I. crithmoides; this species is succulent, which facilitates the accumulation of ions in the vacuoles. These results point to a higher efficiency in the mechanism of transport of toxic ions from the roots to the leaves, which would be extremely meaningful in conditions of high salinity, conferring I. crithmoides a clear advantage over D. viscosa in colonizing saline environments.A general response to salt stress in plants, which may contribute to tolerance, is based on the control of ion transport. Accumulation of inorganic ions in the aerial part of the plants is an advantageous mechanism to increase osmotic pressure, more economical in terms of energy consumption than only the synthesis of organic solutes for osmotic adjustment Raven, . Toxic N+ interferes with K+ transport by using its physiological transport systems  NaCl concentrations—under lower salinity conditions, a decrease in K+ was also detected in this species, as expected—suggesting the activation of potassium transport to the leaves. This could partly compensate the accumulation of sodium, thus contributing to salt tolerance in I. crithmoides by avoiding a drastic reduction of K+/Na+ ratios.The accumulation of sodium is generally associated with a decrease of potassium levels in plants, mostly due to the competition for the same binding sites. NaI. crithmoides, as compared to D. viscosa, appear to be partly dependent on the accumulation of higher levels of specific osmolytes, providing better osmotic adjustment under stress. Glycine betaine has been reported as the major osmolyte in I. crithmoides, accumulating at very high concentrations in response to controlled salt treatments in the laboratory  , all of which showed significantly higher values in D. viscosa than in I. crithmoides under all tested conditions. These results partly confirm previous (field) data from our laboratory, indicating that in their natural habitat highly salt-tolerant species, including I. crithmoides, use very efficient mechanisms of response to salinity—based on the control of ion transport and the accumulation of specific osmolytes—thus avoiding the generation of oxidative stress and the need to activate antioxidant systems  for osmotic adjustment. Differences between the two species are quantitative: ion transport and compartmentalization in vacuoles is more efficient I. crithmoides than in D. viscosa, and the osmolytes  accumulate at higher levels in the former species, thus explaining the (slightly) higher stress tolerance of I. crithmoides. The possible activation of K+ transport to the leaves under strong salinity conditions may also contribute to the higher salt tolerance in this species. The results obtained in controlled experiments in the greenhouse corresponded to the relative distribution of the two species in nature, as only I. crithmoides was found in areas with the highest salinity. Still, D. viscosa proved to be quite resistant to salt stress, tolerating low and moderate salinities in the field.Summarizing, determination of growth parameters and several biochemical stress markers in D. viscosa may be a threat for Mediterranean salt marsh species, the answer is negative, if we refer only to true halophytes growing in areas with high soil salinity—such as I. crithmoides—because its mechanisms of adaptation to salinity are less efficient. Yet D. viscosa can endanger less competitive taxa, since it is quite resistant to lower salinities. From an ecological point of view, the lower salinity makes D. viscosa more competitive, as it characterizes an advanced stage of anthropically influenced or degraded communities (Inulo viscosae-Oryzopsietum miliaceae). I. crithmoides is a characteristic species from salt marsh communities (Inulo crithmoidis-Tamaricetum boveanae) with possible sub-nitrophillous behaviour, so it coexists in the salt marshes with D. viscosa, but cannot leave the salt marsh. The consequence is that, although D. viscosa could not directly cause the disappearance of I. crithmoides, it is a powerful chamaephyte from a serial scrub that will be stronger in this habitat when suffering land degradation. However, salt marshes in the area of study shelter only a few rare halophyte species, including several endemics of the genus Limonium. Much more diverse flora, with a higher rate of endemicity, is located on the borders of the salt marshes, where D. viscosa is extremely competitive. Therefore, management programs of protected areas in the region should include not only the control of alien invasive species, but also the native-invasive D. viscosa. Moreover, the invasive character of D. viscosa in disturbed environments should be taken into account when using this species to restore degraded and polluted areas, or as an intermediate species after invasions by exotic taxa. In relation to habitat preservation, measures should not go only in the direction of removing certain species, but in the sense of preserving soil quality and establishing a buffer zone around the selected area. In the light of our results, the use of D. viscosa should be limited, as it could be stabilized as invasive and cause loss of biodiversity and degradation of plant communities. All the aforementioned problems should be considered, especially in areas included within the priority habitat of Coastal lagoons and Mediterranean salt steppes , under the European legislation, where this study has been carried out.Coming back to the main issue of this paper, whether As mentioned above, native-invasive species pose a new challenge for land management in the context of global conservation, so that more detailed studies should be undertaken on this and other native species that may be compromising the conservation of biodiversity.MA, JC performed the biochemical assays and the analysis of the data, contributing also to manuscript preparation. OB carried out ion measurements and collaborated in the elaboration of the maps. ED was in charge of the greenhouse work. ML was responsible for sugar quantification by HPLC and, together with MA, performed the DPPH-quenching activity measurements. MD was responsible for the field work and contributed to the elaboration of the maps and figures. OM contributed to the field work and manuscript preparation. OV participated in the general organization and supervision of the work and the interpretation of the results, and was responsible for the final version of the manuscript. MB conceived and designed the study, and participated in writing the manuscript. All authors have read and approved the final version of the manuscript.Work in the UPV laboratories was partly funded by a grant to OV from the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution from the European Regional Development Fund.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.


Whilst many studies show sex differences in cerebral asymmetry, their mechanisms are still unknown. This report describes the potential impact of sex hormones and sex chromosomes by comparing MR data from 39 male and 47 female controls and 33 men with an extra X-chromosome .Methods: Regional asymmetry in gray and white matter volumes (GMV and WMV) was calculated using voxel based moprhometry (SPM5), by contrasting the unflipped and flipped individual GMV and WMV images. In addition, structural volumes were calculated for the thalamus, caudate, putamen, amygdala, and hippocampus, using the FreeSurfer software. Effects of plasma testosterone and estrogen on the GMV and WMV, as well on the right/left ratios of the subcortical volumes were tested by multi-regression analysis.Results: All three groups showed a leftward asymmetry in the motor cortex and the planum temporale, and a rightward asymmetry of the middle occipital cortex. Both asymmetries were more pronounced in 46,XY males than 46,XX females and 47,XXY males, and were positively correlated with testosterone levels. There was also a rightward asymmetry of the vermis and leftward GMV asymmetry in the cerebellar hemispheres in all groups. Notably, cerebellar asymmetries were larger in 46,XX females and 47,XXY males, but were not related to sex hormone levels. No asymmetry differences between 46,XX females and 47,XXY males, and no overall effects of brain size were detected.Conclusion: The asymmetry in the planum temporale area and the occipital cortex seem related to processes associated with testosterone, whereas the observed cerebellar asymmetries suggest a link with X-chromosome escapee genes. Sex differences in cerebral asymmetry are moderated by sex hormones and X-chromosome genes, in a regionally differentiated manner. Laterality of the human cerebral hemispheres has profound implications for higher cognitive functions and behavior making both functional and structural asymmetries of the brain utterly intriguing  and the planum temporale (Wernicke's area)  escape this process and will be expressed from both X-chromosomes. Because only a few of the escapee genes have homologs on the Y-chromosome  and the z-transformed estrogen and testosterone levels, as described previously , or represent downstream effects of this type of sex-chromosome aneuploidy.The population consisted of 33 47,XXY males , 39 46,XY males , and 47 46,XX females , and has also partly been described in our previous studies , and having a heredity for, history of, or current psychosis. Furthermore, we excluded subjects with a neurological disease, ADHD, personality disorder, major or bipolar depression, alcohol or substance abuse problems. Mild dyslexia was present in about 60% of the 47, XXY men, but no controls.Two of the 47,XXY males were diagnosed as adults during the course of infertility investigation, the others were diagnosed in early adolescence. All but two (who were deemed to not need testosterone) had been receiving testosterone supplementation subsequent to the diagnosis and were in treatment at the time of the study. The starting age of testosterone treatment ranged from 15 to 40 years. The Ethics Committee of the Karolinska Institutet Stockholm approved the study, and prior to study all participants signed informed consent forms.p < 0.05).The 2D:4D ratios of both hands were measured using a steel vernier caliper. Measurements were carried out directly on the fingers, on the ventral side of the hand between the basal crease and the fingertip , 17β-estradiol (pmol/L) , and the sex hormone binding globulin (SHBG) were analyzed in the Chemical Diagnostics Laboratory at the Karolinska University Hospital. The levels of bioavailable testosterone (nmol/L) were calculated using an equation developed by Sodergard et al. . Before TE = 56 ms, TR = 2500 ms, FOV = 24 cm, 23 slices of 3 mm thickness). The latter images were not used in the present report.All the subjects were investigated in a whole-body 1.5-Tesla MRI medical scanner  equipped with 8-channel phased array receiving. The MRI protocol included the following scans: (1) 3D-weighted T1 SPGR images with 1 mm isotropic voxel size according to a previously described protocol  with SPM 5  and Matlab 7.3 , as described in several related studies, and using The Diffeomorphic Anatomical Registration Through Lie Algebra toolbox . All the segmented were modulated (multiplied) by the Jacobian determinants, which allowed direct comparisons of regional differences in the volume of each tissue type.Voxel-based morphometry . Next, both the original and the flipped images were imported to DARTEL to create templates (GM and WM). The GM and WM templates of the entire study group were then normalized to MNI space using the 12-parameter transformation and then flipped vertically in the midsagittal plane (x = 0), and a mean image of the original and of the flipped GM templates was created . The individual images were finally normalized to the symmetrical template and finally smoothed with a 8 mm FWHM kernel.All the T1-weighted MR images were segmented into gray matter (GM) and white matter (WM), and CSF. The segmented images were then flipped vertically in the midsagittal plane , comparing the individual, MNI normalized, and segmented GM and WM unflipped images with the corresponding flipped images. Significant clusters were calculated for each subject group with peak threshold at p = 0.001, FDR corrected at p < 0.01 (the more restrictive significance level was used to avoid large confluent clusters).Within-group asymmetries in regional GMV and WMV were tested through paired p = 0.001, FDR corrected at p < 0.05). TV was calculated as the sum of individual WMV and GMV. The following contrasts were investigated:Between-group asymmetries were tested with a full-factorial design using each group's  unflipped and flipped normalized images as the factor of variance, and using age and total tissue volume (TV) as the covariates of no interest  and ;  and ;  and . Based on previous observations from a similar population, it was assumed that the pattern and degree of asymmetry would be similar among 46,XX females and 47,XXY males and different from that 46,XY males. Therefore, the threshold level of p < 0.05 uncorrected was used when testing conjunction  and , and p < 0.05 corrected was used for the remaining two conjunctional tests.To investigate possible commonalities between two groups vs. the third, conjunctional analyses was used. The following conjunctions were investigated: , according to standard procedure, Fischl, t-tests . Each subcortical volume was divided with the individual intracranial volume, retrieved from the FreeSurfer software. Possible differences in the VOI/total brain volume as well as in the asymmetry index (right/left) of each measured volume between the three groups were tested with One Way ANOVA, after ensuring that the data were normally distributed. Group was used as the between factor (p < 0.05). Tukey's statistical test was employed for post hoc analyses because the sample sizes were unequal.Possible right/left asymmetries were tested for each separate structure with paired Delineation of the right and left cerebral hemispheres. The volumes of interest (VOIs) representing the entire right and left cerebral hemispheres were delineated on every second coronal slice of the individual MR images as described previously , and in cases of significant group difference, the separate groups were further compared with each other using the Mann–Whitney test . Age was employed as the nuisance variable. Based on previous reports about the effects of sex hormones  with age as nuisance variable .F = 14.1: p < 0.001). According to post-hoc analysis (Gabriel's procedure), this difference was constituted by the lower education among in the 47,XXY group compared to both male (p < 0.001) and female controls (p < 0.001), whereas no difference was detected between male and female controls (p = 0.91). There were no group differences in regard to handedness or sexual orientation. No gross anatomical abnormalities were found according to an experienced neuroradiologist.Between-subject One-Way ANOVA showed that there was a significant main effect of group on the number of years of education . The results presented here were based on measurements from rater one, as rater two performed ratings for only a portion of the study group. A significant group difference was found for the 2D:4D ratio of the right hand  but not the left hand . 47,XXY males and 46,XX females had higher ratios than 46,XY males, while the ratios between 46,XX females and 46,XY males did not differ  was shared between 46,XX females and 47,XXY males. The rightward symmetry in the GMV in the superior temporal gyrus and the GMV of the cerebellar vermis was significantly more pronounced in 46,XY males when compared with both 46,XX females and 47,XXY males. No common clusters were detected with respect to asymmetry of male and female controls in relation to 47,XXY males, nor with respect to both male groups in relation to female controls. Thus, the asymmetry pattern in 47,XXY males was more similar to that of 46,XX females.z = 3.6 cluster sizes 2.0 cm3, co-ordinate −42 −8 12 and −40 −42 4 for the GMV, −37 −48 34 for the WMV). Also the rightward asymmetry of the GMV and WMV in the middle occipital lobe was correlated with z-testosterone .The explorative multifactorial regression analysis revealed a significant positive correlation between z-transformed levels of bioactive testosterone and the leftward GMV and WMV asymmetry in the planum temporale/motor cortex . The putamen volumes, on the other hand, showed a significant leftward asymmetry in all three groups . Left caudate volume was larger than the right in 46,XY males (p = 0.016), whereas no asymmetry was detected in the two other groups. The right hippocampus was larger than the left in 47,XXY males (p = 0.005) and 46,XY males (p = 0.013), and no hippocampal asymmetry was found in 46,XX females (p = 0.26); finally, no asymmetries could be detected in the amygdala volumes in any of the groups. Subcortical values are presented in Table The volumes, as well as their right/left asymmetry, were normally distributed (Shapiro–Wilk test). Paired p = 0.001, F = 6.9) and the hippocampus . The rightward thalamus asymmetry was more pronounced in 47,XXY males than in both 46,XY males (p = 0.013) and 46,XX females (p = 0.002), without any difference among the two control groups. The right > left asymmetry of the hippocampus was significantly greater in 47,XXY males (p = 0.013) as well as in 46,XY males (p = 0.041) compared to 46,XX females. No asymmetry differences were detected in other structural volumes.A significant group difference was detected with regard to the asymmetries of the thalamus . Group difference was detected for the left thalamus , as a significantly smaller VOI/TBV ratio was found among 47,XXY males compared to both 46,XY and 46,XX groups (p < 0.001 for both). Thus, the greater right/left asymmetry in 47,XXY males was probably due to their reduced left thalamus volume. Other group differences were bilateral. They were constituted by lower relative volumes in 46,XXY men in relation to both control groups for the putamen, , hippocampus  and the amygdala , P was < 0.001, for all the post hoc comparisons.The ratio of each side's structural VOI to total brain volume (TBV) was calculated t-test showed a significant difference (asymmetry) between the right and left hemispheric volumes in the male controls  nor in the 46,XXY patients , ,  and 46,XY males (p = 0.025) than in 46,XX females, whereas no difference was detected between 47,XXY males and 46,XY males (p = 0.87).The paired-sample p = 0.08, all three groups included), indicating that higher exposure to fetal testosterone was associated with more overall rightward hemispheric asymmetry.Because the distribution of the hemispheric data could not be assumed to be normal, Kendall's tau test, a non-parametric correlation analysis, was used. There was a trend, albeit without a statistical significance, toward an inverse correlation between the digit ratio and the right/left hemispheric ratio (None of the calculated subcortical asymmetries were found to be correlated with z-testosterone, z-estrogen, or right hand digit ratio.right superior temporal lobe, the cuneus, the anterior cingulate, and cerebellar vermis as well as by a more prominent leftward asymmetry in parts of the precentral gyrus and the planum temporale, and in the left occipital lobe and the cerebellar semilunar lobes (Table The present study was designed to explore possible sex differences in regard to the asymmetry of subcortical structural volumes, and regional gray and white matter volumes. A further purpose was to investigate if and how such asymmetries might be related to sex hormone levels and sex chromosome gene dosage. As in the related studies on the underpinnings of cerebral sex differences, specific comparisons were carried out between 46,XX females, 46 XY males, and 47,XXY males, and included evaluations of possible correlations with sex hormone levels and digit ratios. Asymmetries were detected in all three groups of subjects. They were characterized by greater GMV and WMV in the es Table . Similares Table , albeit index of early testosterone exposure rather than prenatal exposure. The failure to find a correlation with digit ratio in the present study should therefore not be taken as an argument against the influence of fetal testosterone on regional cerebral asymmetry.The detected leftward asymmetry of the motor cortex and planum temporale is congruent with the well-known lateralization of language functions, and with the previous reports about sex differences in this asymmetry (Geschwind and Levitsky, anterior rightward asymmetry was found in males and a more prominent lateral leftward asymmetry in females, deserve a comment. These were not simply based on lateralized group differences; rather, they seem to reflect asymmetry as a distinct factor. Several previous region-of-interest-based volumetric studies have shown right-greater-than-left anterior volume asymmetry and left-greater-than-right posterior asymmetry in a normal cerebellum (e.g., Lawson et al., The detected cerebellar asymmetries, where a more pronounced Unlike the cerebellar and cortical asymmetries, those in subcortical volumes showed no correlation with hormone levels or digit ratios, and no pattern of group differences was found to indicate X-chromosome dosage effects. Differences in relation to 47,XXY males detected in the thalamus could be attributed to the left thalamus atrophy in this group that was detected in the present study and the related previous studies Savic, . The facThe volume of gray matter is mainly affected by the number and size of neurons and glia cells. Asymmetric areas have fewer interhemispheric connections than symmetric areas, perhaps as a result of increased axonal pruning (Galaburda et al., Among the more specific issues pertaining to the methods used is that the same structure might have a slightly different spatial location in the two hemispheres.The effect of between hemisphere differences in spatial homology was reduced by the use of a symmetrical template, linear transformations to this template, and by smoothing of the data, as described previously (Good et al., Another important methodological issue pertains to the interpretation of findings from 47,XXY males. Although androgen levels become notably reduced for these individuals during puberty, when the majority of the subjects started testosterone supplementation, it cannot be excluded that the similarities to 46,XX controls could, to a certain extent, be attributed to early androgen deficits.The 47,XXY population had significantly lower education, which, theoretically, could have affected the results. However, adding education as nuisance covariate did not change the results.The fact that hormone levels were, as in several previous studies (Neufang et al., White matter asymmetries largely followed those of the gray matter, which seems biologically plausible. This observation may, however, also reflect an edge effect—if the interface of one tissue compartment were to be displaced, the difference could be appreciated in both compartments. Such effects were not detected by visual inspection of significance maps.The present study expands the previous neuroimaging literature on cerebral asymmetries by proposing that processes linked to X-chromosome gene dosage affect the pattern of cerebellar asymmetries, whereas processes primarily linked to testosterone levels seem to influence the asymmetry in the planum temporale and part of the motorcortex as well as in the occipital cortex. By identifying brain areas that seem to exhibit the effects of X-chromosome genes, the present results add to the animal data concerning the genes located on X- and Y-chromosomes that could contribute to sex differences in regional asymmetry. Brain asymmetry is usually regarded to be a developmentally adaptive and aiming at improving the computational efficiency by promoting intrahemispheric processes in larger brains (Hutchinson et al., The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With an expected sensitivity (Se) of 96% and specificity (Sp) of 98%, the immunofluorescence antibody test (IFAT) is frequently used as a reference test to validate new diagnostic methods and estimate the canine leihmaniasis (CanL) true prevalence in the Mediterranean basin. To review the diagnostic accuracy of IFAT to diagnose CanL in this area with reference to its Se and Sp and elucidate the potential causes of their variations, a systematic review was conducted (31 studies for the 26-year period). Three IFAT validation methods stood out: the classical contingency table method, methods based on statistical models and those based on experimental studies. A variation in the IFAT Se and Sp values and cut-off values was observed. For the classical validation method based on a meta-analysis, the Se of IFAT was estimated in this area as 89.86% and 31.25% in symptomatic and asymptomatic dogs, respectively. The Sp of IFAT was estimated in non-endemic and endemic areas as 98.12% and 96.57%, respectively. IFAT can be considered as a good standard test in non-endemic areas for CanL, but its accuracy declines in endemic areas due to the complexity of the disease. Indeed, the accuracy of IFAT is due to the negative results obtained in non-infected dogs from non-endemic areas and to the positive results obtained in sera of symptomatic dogs living in endemic areas. But IFAT results are not unequivocal when it comes to determining CanL infection on asymptomatic dogs living in endemic areas. Statistical methods might be a solution to overcome the lack of gold standard, to better categorize groups of animals investigated, to assess optimal cut-off values and to allow a better estimate of the true prevalence aiming information on preventive/control measures for CanL. Leishmania infantum and is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anemia. If left untreated, the fatality rate within two years can be as high as 100% in developing countries [Leishmaniases are parasitic diseases ranked second in mortality and fourth in morbidity among tropical diseases, with about 2 million disability-adjusted life years . It was ountries –5.Phlebotomus [In the Middle East, Mediterranean countries, Iran, Pakistan, Afghanistan, Brazil, and China, visceral leishmaniasis (VL) is a zoonotic disease transmitted by female sand flies belonging to the genus ebotomus . In the ebotomus –9. Despiebotomus ; probablebotomus .Dogs are considered the major host for this parasite and the main reservoir for human infection . The cliSince both symptomatic and asymptomatic dogs are infectious to sand fly vectors, this allows the transmission of the parasite to other dogs and humans –19. TherEpidemiological studies on CanL are regularly conducted with the aim to estimate the true prevalence of the disease. It is in this regard important to note that the characteristics of the diagnostic technique(s) used may have a considerable influence on the estimate obtained for the true prevalence –23. SensAccording to the World Organisation for Animal Health , serologi.e. it diagnoses all of the specific diseased individuals that exist and misdiagnoses none [It is important here to be precise about the so-called gold standard (perfect test): it is a test or procedure that is absolutely accurate, ses none . Howeverses none . Sensitises none –42.The aim of the present study was to conduct a systematic literature review on the accuracy of IFAT for the estimation of CanL prevalence in the countries of the Mediterranean basin. In addition, we sought potential causes of variation in the Se and the Sp of IFAT among different epidemiological surveys with the aim to better inform preventive and control measures for CanL. Finally, to estimate the Se and Sp of IFAT in different contexts, a meta-analysis was conducted based on selected available classical studies.Fig 1). Studies were identified using a combination of keywords. The terms searched for were (insensitive): (canine leishmaniasis) AND (immunofluorescence antibody test OR ifat OR ifa OR ifi) AND .This systematic review was conducted in the PubMed database on all papers published before July 31, 2014 . With the aim to ensure rigorous and transparent reporting, the Preferred Reporting of Systematic Reviews and Meta-Analysis (PRISMA) guidelines –44 were First, we checked the lists of titles and abstracts identified by this search to determine the relevance of the articles. Literature review studies were left out. We also excluded studies conducted in countries other than the Mediterranean basin and studies using IFAT as a gold standard to assess other diagnostic tests.The selected articles were read in full to confirm eligibility. Thereafter, the following data were extracted and gathered into a structured table: reference, objectives of the study, type of study, study period, area of origin of the dogs under study, sample size, inclusion criteria for dogs, type of IFAT used , test threshold, type of validation and, lastly, the main findings related to IFAT.http://www.r-project.org/). A random effects meta-analysis model was used in order to better account for heterogeneities (within and between study variability) among the different studies considered [Meta-analysis used to determine diagnostic test accuracy is a statistical technique, which combines findings from independent studies . A meta-nsidered .Of the 89 articles flagged by the search related to the validation of IFAT, 21 did not meet the aim of this review, 30 were carried out in Brazil, 6 were in Portuguese and one literature review study was in German. Thus, a total of 31 articles met the inclusion criteria  for canine leishmaniasis in the Mediterranean basin) for the 26-year period available.S1 Table). Dogs sampled in the studies were from both endemic and non-endemic regions and sample sizes ranged from 22 to 1,035 dogs. Furthermore, the IFAT accuracy was explored in three groups: non-infected dogs, subclinically infected dogs and diseased dogs. The IFAT threshold varied from 1/20 to 1/200 with a mode of 1/80. In one study, the IFAT cut-off was not mentioned [Validation of IFAT in the 31 selected articles was conducted both north and south of the Mediterranean basin  (26 studies), statistical validation (2 studies) and experimental validation (3 studies).Three methods of validation were used: classical contingency table analysis  including 22 studies that used parasitological examinations and/or culture as reference test and 4 other studies that used as reference test respectively an immunoblotting and PCR , a westeAmong these 22 studies, four studies established IFAT as 100% sensitive and 100% specific irrespective of the dog’s clinical status –56.Although Otranto et al. , MancianIn addition, IFAT Se decreased down to 29.4% in asymptomatic dogs from 90% in symptomatic diseased dogs . MoreoveSome studies did not estimate Se and/or Sp but showed a discrepancy between the results of IFAT (positive or negative) and the reference test used , 61–63.Leishmania and other canine pathogens, showed that out of 57 dog samples tested, 11 tested falsely positive for IFAT [Furthermore, a study carried out to evaluate the serological cross-reactivity between for IFAT . Howeverfor IFAT .In the statistical validation, two types of techniques were used: the latent class analysis (LCA) and the Bayesian approach , 66.Validity analysis for three CanL serological tests including IFAT with respect to parasitology and disease, were compared with latent class analysis . This suA Bayesian approach was used to evaluate three serological tests including IFAT for CanL in three groups of dogs according to their functional type  in Algeria . The anaLeishmania [Experimental validation of IFAT was assessed using naturally infected dogs, i.e. with positive culture of lymph nodes or bone marrow aspiration or chancre biopsies  or usingishmania –71. In tishmania . For theishmania –71, respishmania , 71, wheishmania  had a biFig 2). The Se of IFAT was estimated to be 89.86 (95% CI: 83.63–93.89) and 31.25% (95% CI: 18.09–48.35) for symptomatic and asymptomatic dogs respectively. In addition, the Sp of IFAT was estimated as 98.12 (95% CI: 93.69–99.46) and 96.57 (89.06–98.98) in non-endemic and endemic areas respectively.For classical validation method only and based on the selected available studies in the Mediterranean basin after systematic review, the sensitivity of IFAT was estimated using a random effects meta-analysis in symptomatic and asymptomatic dogs , as well as chronic pyoderma, chronic hepatitis and renal disorders were considered and no cross-reactions were observed. The absence of cross-reactions between L. infantum and Ehrlichia canis was later also found by Liéra et al. [eactions –42. For a et al. .Trypanosoma cruzi [Ehrlichia canis [Demodex canis [Toxoplasma gondii [T. cruzi is the only parasite that is not an issue in the Mediterranean basin, being exotic to this area.In contrast, Otranto et al.  reportedma cruzi , Ehrlichia canis , Demodexex canis  and Toxoa gondii  in BraziReference populations. With respect to reference populations, IFAT was shown to be 100% sensitive and 100% specific when used on clinically suspected expatriate dogs [ate dogs . The chaate dogs .Also, in a longitudinal survey carried out in Southern France, an endemic area for CanL, IFAT Se and Sp were estimated at 84.6% and 76.5% respectively .Clinical status. CanL has a disease spectrum in which clinical disease represents one pole of the infection and asymptomatic subclinical infection the other [he other .Leishmania DNA [Thus, IFAT is a good test when used in a group of symptomatic dogs , but its efficiency is markedly lower when the dogs are asymptomatic, having therea Se of 29.4% . Also, wania DNA . Iniestaania DNA  also repania DNA , suffereIn addition, the detection of the T-cell mediated immunity against leishmaniasis in asymptomatic dogs showed an increase of the prevalence of infection, compared with those obtained by IFAT. Hence, when combining IFAT with a leishmanin skin test (LST) in parallel, 27 dogs were considered positive out of 58 asymptomatic dogs . AccordWhen compared to positive results obtained by PCR in blood, nested-PCR (nPCR) in bone marrow or conjunctival swab (CS), the number of positive dogs detected by IFAT ranged from none to 50% , 37, 63.Life cycle stage. Variation in Se and Sp was shown to be dependent on the life-cycle stage (amastigotes versus promastigotes) [tigotes) . Indeed,Cut-off value. Test-results are dependent on the diagnostic cut-off value [ff value . The cutff value –88. Furtff value .L. infantum in a group of dogs sampled at random (DSR) and in a group of dogs with symptomatology compatible with CanL (DSCCanL), both the DSR and the DSCCanL groups produced high percentages of animals with doubtful antibody titres  [By comparing antibody titres against Therefore, the dependence of the diagnostic Se and Sp on the selected cut-off value must be considered for a full test evaluation and for test comparisons; these problems might be addressed by the receiver-operating characteristic (ROC) analysis . The thrA full explanation of the statistical methods in use is found in , 91. TheAdel et al.  found IFThe “gold standard” status of IFAT was challenged by experimental studies –70. TheiLeishmania infection in animal models will depend not only on host immunity but also on a combination of factors, such as inoculated species, virulence of the strain, nature of the inoculum, number of parasites and route of inoculation [Experimental conditions are of course not comparable to natural field infections since the outcome of culation .Another finding related to another carefully observed cohort of naturally infected dogs is a seasonal variation in IFAT Se and Sp. For example, monthly changes in Se and Sp were shown by Dye et al. . This stFor classical validation method only and even though the difference estimated Sp in non-endemic and endemic areas was not statistically significant, this study revealed that the estimation of Se in endemic Mediterranean basin was quite different in function of the symptomatic status of the dogs. Only 2 studies estimated IFAT Se in non-endemic areas and found it 100% , 55.A discrepancy in estimating sensitivity and specificity of IFAT was also demonstrated in another meta-analysis study carried out in Brazil . In factTable 1). IFAT can be considered as a good standard test in non-endemic areas for CanL, but its accuracy declines in endemic areas due to the complexity of the disease. In other words, the accuracy of IFAT is due to the negative results obtained in non-infected dogs from non-endemic areas and to the positive results obtained in sera of symptomatic dogs living in endemic areas. But IFAT results are not unequivocal when it comes to determining CanL infection on asymptomatic dogs living in endemic areas. Indeed, both symptomatic and asymptomatic dogs represent a risk of infection to other dogs and humans and IFAT shows considerable difficulties detecting asymptomatic dogs . Additionally, these studies do not always describe the target population and do not follow proper sampling methods sometimes employing very small sample sizes.We have outlined several issues involved in IFAT validation. In the classical validation, we observed that IFAT was validated against parasitological exams as reference tests, known not to be very sensitive, hence hampering the validity of the analysis. Throughout the selected studies a great variation in the IFAT sensitivity and specificity values was observed in function of many parameters Click here for additional data file.


Depression is a common and mostly undertreated problem in patients with chronic diseases. However, population-based studies on the association between chronic obstructive pulmonary disease (COPD) and subsequent depression are limited in Asian populations. This study evaluated the incidence and risk factors of depression for patients with COPD in Taiwan.Using the claims data from the National Health Insurance of Taiwan, we identified 38,010 COPD patients newly diagnosed in 2000–2004 and 38,010 subjects without COPD frequency, matched by sex, age and index date. The incidence rate and hazard ratio for depression were estimated by the end of 2008.The incidence rate of depression was 1.88 folds higher in the COPD cohort than in the non-COPD cohort . The depression risk was the greatest within the first year following COPD diagnosis and tended to decline with follow-up time. Among COPD patients, multivariate analysis showed that younger women and low-income patients were at higher risk of depression. Hospitalization and comorbidities such as hypertension, arthritis, cancer, and heart disease were also significant predictors for depression risk.This population-based cohort study demonstrated a strong relationship between COPD and subsequent depression. These findings could assist healthcare providers to pinpoint individuals with a higher predisposition to having depression, which could then facilitate the provision of culturally appropriate rehabilitation within the first year after the diagnosis of COPD. Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction, which leads to the slow, progressive symptoms of persistent cough, exertional dyspnea, wheezing, and eventually functional impairment . World HCOPD does not only cause enormous economic burden, it also triggers subsequent illnesses. The risk of lung cancer for COPD patients is more than two folds higher than for those without the disease . CurkendStudies on COPD-related depression have been conducted mainly on Western populations but rarely for people of Asian descent . ChineseIn order to remove financial barriers to medical care for all residents, Taiwan Department of Health launched a single-payer NHI Program in 1995. At the end of 2008, over 99% of Taiwan’s population was enrolled in this program [Diagnoses in the insurance claims data were coded with International Classification of Disease, 9th Revision, Clinical Modification (ICD-9-CM). From records in LHID, we identified patients with COPD among adults newly diagnosed during 2000–2004 with the ICD codes of 491, 492, and 496 as the COPD cohort. To reduce the misclassification, we selected patients with at least two diagnosis of COPD in outpatient visits within 12 months or patients being admitted to hospital with a primary diagnosis of COPD during the 5-year period ,21.A comparison cohort was randomly selected from the remaining insured population without COPD. For each COPD patient, one person free of COPD was selected frequency matched with sex, age, and the index date of COPD. After excluding subjects with a history of depression at the baseline, we identified 38,010 patients with COPD and 38,010 subjects in non-COPD cohort for data analysis. All subjects were followed up to the end of 2008 to measure the incidence of depression. We identified subjects as having depression if they had at least two treatment claims for depression in outpatient visits or hospitalization with depression for ICD-9-CM codes of 296.2, 296.3, 300.4, or 311 during the follow-up period. Follow-up person-years (PYs) were determined by calculating the time interval from the entry date to the earliest of one of the following: a diagnosis of depression, the date of withdrawal from the insurance, or the date of December 31, 2008.The demographic variables used in this study included age, gender, income for estimating insurance payment, and urbanization of the subject’s residential area. The monthly incomes were divided into three levels: ≤NT$17,880, NT$17,881–$43,900, and ≥NT$43,901. An income of NT$17,880 was the government-stipulated minimum for full-time employees in Taiwan. Urbanization levels were stratified into three strata, namely urban (levels 1–2), suburban (levels 3–4) and rural (levels 5–7) areas, based on population density. Level 1 refers to the “most urbanized” and level 7 refers to the “least urbanized” communities . The bas2 test to examine the differences in demographic characteristics and comorbidities between the COPD and comparison cohorts. The incidence rate of depression for the two cohorts was presented with the number of cases per 1,000 PYs. COPD patients were further divided into two groups based on initial way of medical care seeking, and examined the incidence rate of depression among them. Cox proportional hazards regression analysis was applied to compute the hazard ratio (HR) and 95% confidence intervals (CI) of depression for COPD compared with the comparison cohort. We also used multivariate Cox proportional hazards model to determine the risk factors that might predict depression and their adjusted hazard ratio (aHR) within the COPD cohort. We further evaluated whether the relation between COPD and depression risk differed over time by sex. All analyses were conducted using SAS version 9.3 , and p < 0.05 was considered significant.We performed the χTable Of the total sample of 76,020 patients, 4,109 had incident depression during the follow-up period. The incidence of depression in the COPD cohort was nearly two folds higher than that in the comparisons  with an aHR of 1.88  in year one. The aHR decreased with follow-up time to 1.40 (95% CI: 1.22–1.60) after year 5 (Table Previous studies have shown that emotional impairments, depression in particular, are common among COPD patients, but mainly in Western populations. To the best of our knowledge, the present study is the first population-based cohort study addressing the association between COPD and the risk of subsequent depression in an Asian population. The results are consistent with findings in previous studies on Western populations ,23,24. TAdditionally, the same debilitating and continuous physical symptoms may also trigger cognitions of “learned helplessness”, such that one’s inability to successfully cope with physical deterioration and life threatening situations result in increased levels of uncertainty, unpredictability and ultimately emotional distress and depression . On the The depression incidence among COPD patients in this study is somewhat lower than that in a previous report . The difOur study indicated that women are at a 1.19-fold greater risk of depression than men in the COPD cohort, which supports the findings of previous studies ,24. Two We found a lower risk of depression in patients with higher monthly incomes than in those with lower monthly incomes, which is in line with previous studies ,32. PatiConsistent with previous studies ,18,31, wCOPD patients with other comorbidities, such as hypertension, arthritis, heart disease, or cancer had a significantly higher risk of depression, whereas those with diabetes mellitus and chronic kidney disease were at borderline significance. Our findings are partially paralleled with those of a previous study , indicatSeveral limitations in this study should be mentioned. First, information on tobacco use, social network relationships, religious belief, and education level was unavailable from the claims data, which may be related to depression. Further study linking administrative data and the above mentioned information is warranted to examine the impact of these potential confounding variables on depression. Second, inaccurate diagnoses may occur. Therefore, we selected only those subjects with COPD or depression with at least two consistent outpatient visits or one inpatient admission to minimize this error. Furthermore, NHI has randomly sampled claims from hospitals and randomly interviewed patients, as well as reviewed medical charts to verify the accuracy of medical records. Finally, data regarding the severity of COPD, such as lung function data, were not available in the database. We were unable to estimate the dose–response relations between COPD severity and depression. Despite these methodological concerns, this population-based cohort study has been successful in examining the relationship between COPD and the subsequent risk of depression among Chinese patients. The retrospective cohort study design per se provided stronger evidence on the relationship between COPD and depression risk compared with previous studies.Findings from this study reveal that the depression risk is nearly two folds higher for COPD patients than for general population. The risk of depression is related to being female and young, as well as with lower monthly income and comorbidity, such as hypertension, arthritis, cancer, and heart disease. Healthcare providers could be able to better understand the demographic and diseases characteristics that may contribute to the depression risk from this population-based study. The need to routinely screen COPD patients for depression and institute culturally appropriate interventions should be emphasized, especially within the first year since COPD diagnosis. Healthcare providers must be cognizant of the existence of depression among COPD patients and inform them by standard care process to avoid potential embarrassment when discussing psychiatric-related issues; this will help to achieve better therapeutic outcomes for them.The authors declare that they have no competing interests.Conceived and designed the experiments: TYT HL MCL FCS PCC. Performed the experiments: TYT FCS PCC. Analyzed the data: TYT PYT. Wrote the paper: TYT FCS PCC. All authors approved the final version of the manuscript. PCC is the co-corresponding author.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/976/prepub


Objective. This clinical trial was designed to investigate whether goshajinkigan reduces the onset of diabetic complications or not. Materials and Methods. A total of 332 type 2 diabetic mellitus patients were registered from 9 clinical centers from March 2000 to August 2007. Patients were randomly assigned to take goshajinkigan extract powder, 2.5 grams for 3 times a day or no kampo therapy, additionally to the regular treatment. The primary endpoints were the onset of macrovascular diseases or progression of nephropathy or retinopathy. Statistical analysis was performed by the intention-to-treat method. Results. After 5 years of observation, 116 patients were submitted to analysis. Among them, no macrovascular events were observed in both groups. Although 43 participants had upstaging of retinopathy or nephropathy in total, there was no significant difference between goshajinkigan group and control group. Deterioration of ankle reflex was suppressed in goshajinkigan group. Also glycated hemoglobin, and fasting plasma glucose were decreased in the goshajinkigan group. Conclusion. Although the power of analysis was too low to demonstrate any effects of goshajinkigan on the progression of macrovascular diseases, retinopathy or nephropathy, goshajinkigan may be beneficial for diabetic neuropathy and glycemic control. Diabetes mellitus has become a big problem all over the world. Also in Japan, the number of patients with diabetes mellitus has steadily increased on an annual basis. In 1998, the Ministry of Health and Welfare estimated that there were 6.9 million diabetics in Japan. Afterwards, this estimate was increased to 7.4 million and 8.9 million in 2002 and 2007, respectively [Several studies have shown that the progression of diabetic microvascular complications, such as retinopathy, nephropathy, and neuropathy, can be inhibited by a good glycemic control –5. HowevClinical conditions similar to diabetes were treated with the administration of hachimijiogan in the Jingui Yaolue, a Chinese classic written approximately 1,800 years ago. Goshajinkigan, a kampo product, contains 10 herbal ingredients adding two herbs, that is, Achyranthis radix and Plantaginis semen, to hachimijiogan and is applied to patients with lumbago, edema of the lower legs, dysuria, and so forth. In an animal model of diabetes, goshajinkigan showed inhibition of weight gain, inhibitory effects on blood glucose, suppression of microalbuminuria , preventThis trial was specifically designed to determine whether a kampo formulation of goshajinkigan would reduce the rate of cardiovascular events, microvascular complications, and laboratory abnormalities, as compared with a control group without this kampo formulation in middle-aged and older people with type 2 diabetes mellitus and additional cardiovascular risk factors. Here, we report the effects of the kampo formulation on the primary composite outcome of major cardiovascular events and microvascular complications in middle-aged and older people with type 2 diabetes mellitus.2, 2 or more digestive system symptoms , and 3 or more symptoms or activities indicative of sensitivity to heat, including a preference for dressing lightly, sweating upwards from the neck, a tendency to drink cold water, a flushed face, congestion of the eyeballs, and a high body temperature (not less than 36.7°C).This multicenter clinical study was conducted in 9 clinical centers in Japan. We recruited outpatients of type 2 diabetes mellitus of age between 40 and 75 years from March 2000 to August 2007. In order to investigate the progression of diabetic complications, glycated hemoglobin level of ≥6.5% was selected in the inclusion criteria. Exclusion criteria were a past macrovascular events, like cereberal stroke, myocardial infarction, angina pectoris, foot gangrene, or atherosclerotic obstruction. Other exclusion criteria were nephropathy with macroalbuminuria or with a serum creatinine levels of 1 mg/dL or more and proliferative and preproliferative retinopathy. Additional exclusion criteria which were related to goshajinkigan pattern were a body mass index of ≥30 kg/mAll 116 patients were randomly assigned to the goshajinkigan group or control group. Randomization was accomplished by a study controller, and allocations were provided in sealed envelopes. Because of the long-term clinical study, we anticipated that the dropout rate is high in goshajinkigan group and allocation to goshajinkigan group was planned to be double of control group. The trial was open once patients were randomized. Only the readers of the fundus photographs were blinded. No placebo treatments were given.2; targeted total cholesterol was lower than 220 mg/dL; targeted high-density lipoprotein was higher than 40 mg/dL, according to the Japanese Diabetes Society Guide of 1999 for the treatment of diabetes, the Japanese Society of Hypertension Guidelines of 2000 for the management of hypertension, and the Japan Atherosclerosis Society Guidelines of 1997 for the diagnosis and treatment of hyperlipidemia.As a background treatment, all participants received nutrition and physical activity counseling every year upon entering the study. Nutrition counseling included a recommendation that the caloric intake should not exceed the number of calories derived from multiplying the estimated body weight (kg) by a factor of 25. Current smokers and drinkers received smoking and drinking cessation counseling. All participants received glucose-lowering therapy, the same as the one before entering the study, as well as lipid-lowering therapy and/or blood pressure-lowering therapy. No restrictions were made regarding the choice of medications, with the exception that no kampo formula other than goshajinkigan could be used. The targeted glycated hemoglobin was lower than 6.9% ; targeted blood pressure was lower than 130/85; targeted body mass index was lower than 22.0 kg/mPatients received instructional materials and behavioral counseling regarding diabetes care and were provided with goshajinkigan. Therapeutic regimens were individualized at the discretion of the investigators and patients on the basis of study-group assignments and response to therapy. The adverse effects of therapy were carefully audited both locally and centrally to ensure the safety of the patients. Rehmanniae radix (5g), Achyranthis radix (3g), Corni fructus (3g), Dioscoreae rhizoma (3g), Hoelen (3g), Plantaginis semen (3g), Alismatis rhizoma (3g), Moutan cortex (3g), Cinnamomi cortex (1g) and heat-processed Aconiti radix (1g). The extract product Goshajinkigan  is a standardized spray-dried water extract, which includes magnesium stearate, lactose, and fructose fatty acid esters as diluents. The manufacturing process meets all requirements of the Japanese and international GMP guidelines.Patients in the goshajinkigan treatment group received 2.5 g of goshajinkigan extract  preprandially which included 4.5 g of the compound extracts of 10 herbal medicines:The prespecified primary endpoints were the first occurrence of nonfatal myocardial infarction or nonfatal stroke or worsening of diabetic nephropathy (DN) or retinopathy (DR). The progression of DN was evaluated by the new onset of renal failure by dialysis or an increase in the amount of urinary protein. The urinary protein was calculated by dividing the morning urinary albumin by the urinary creatinine not related to any acute intercurrent illness. The amount of urinary protein was divided into 3 stages: <30 mg/gCr, 30–300 mg/gCr, and >300 mg/gCr.The progression of DR was evaluated by standardized eye examinations conducted by ophthalmologists or optometrists, along with fundus photography of 4 standard stereoscopic fields at baseline and every year. The photographs were gathered at the Fundus Photograph Reading Center, located at the University of Kyorin , and were graded by trained personnel masked to the treatment assignments of the participants. The presence of capillary aneurysms, retinal bleeding, hard exudate, ring-like hard exudate, soft exudate, intraretinal microvascular abnormalities, venous abnormalities, and new blood vessels was evaluated. The DR stage was divided into 8 steps from normal to proliferative (3 classifications each pertained to the simple and preproliferative steps).The study investigators also measured the effect of the intervention on body weight, blood pressure, fasting blood glucose, glycated hemoglobin, serum insulin (c-peptide reactivity in the insulin user), total cholesterol, triglyceride, high-density lipoprotein, serum creatinine, urea nitrogen, and DN as secondary outcomes. DN was evaluated by using two-way analysis of variance. The grade of the ankle reflex was classified into 3 steps: normal, decreased, and absent. Moreover, the results of the questionnaire on 10 subjective symptoms were evaluated by 4 steps, and 4 of these 10 symptoms were also evaluated with a visual analogue scale (graded from 0 to 100). The questionnaire included lightheadedness, abnormal sweating, occurrence of constipation and diarrhea, impotence, abnormal feeling like pater stuck to the soles, uncomfortable heat sensation of hands and feet, pain of hands and feet, abnormal sensation of hands and feet, cold sensation of hands and feet, and muscle cramp.For monitoring side effects, aspartate aminotransferase, alanine aminotransferase, serum creatinine, urea nitrogen, uric acid, urinary blood, glucose, and ketone were investigated every 6 months. When critical side effects were suspected because of goshajinkigan, the treatment was stopped immediately, and suitable treatment was performed. Concomitantly, a report was promptly made to the research center.We aimed to recruit 1000 cases for the goshajinkigan group and 500 for the control group. The sample size was calculated for an event rate of 6% per year, an effect of 20%, an alpha error of 5%, and a power level of 80%.t-tests. At each assessment visit, glycated hemoglobin levels and fasting plasma glucose levels were summarized with the use of medians and interquartile ranges. Exposure to glucose-lowering drugs was summarized according to the study group as the number of patients who received a prescription for a medication. Analysis of the primary outcome was performed with the use of the log-rank method according to the intention-to-treat principle, and the occurrences of this outcome in the 2 study groups were compared with the use of hazard ratios and 95% confidence intervals. Kaplan-Meier estimates were used to obtain the proportion of patients who had an event during follow-up. Analyses of the secondary outcomes were performed with the use of log-rank methods, two-sample Wilcoxon's test, and Fisher's exact test. We report here all nominal P values, unadjusted for the multiplicity associated with the various tests performed for this study or the monitoring of the primary and mortality endpoints by the data and safety monitoring committee.All statistical analyses were conducted at the coordinating center with the use of SAS software, version 9.1 (SAS Institute). Baseline characteristics were compared in the 2 study groups with the use of chi-square tests and two-sample n = 100) to control group (n = 49).A total of 332 patients were registered for this clinical trial , among wA total of 116 patients were submitted for analysis of this study. P = 0.244) (P = 0.816)  . In term= 0.816) .P value was 0.040 as determined by the log-rank test , and the ank test .Stable median levels of glycated hemoglobin of 7.8%  and 7.9%  were maintained in the goshajinkigan and control groups, respectively, throughout the follow-up period. According to an exploratory examination, the glycated hemoglobin of the goshajinkigan group decreased significantly in the 60th month as compared to the control group . FastingMost patients had no subjective symptoms through the trial period. An analysis of only the patients who had a change in subjective symptoms was performed, in addition to an analysis of the entire set of patients. For the patients who experienced a change in symptoms, the occurrence of constipation and diarrhea was significantly improved in the goshajinkigan group. For the entire set of patients, no significant changes occurred for any of the issues addressed in the questionnaire. There was no dropout because of the side effects of goshajinkigan.Other outcomes, including body mass index and laboratory test results, showed no significant differences.Goshajinkigan extract (TJ-107) is a pharmaceutical drug covered by national health insurance program. The indications include pain in low extremities, back pain, numbness, blurred vision, dysuria, pollakiuria, and edema. It is mainly used for the senile problems. Goshajinkigan is a medicine for kidney function deficiency in terms of traditional medicine. Kidney function deficiency means a loss in congenital energy. With aging, this function is deteriorated. In this case, kidney function is not the organ kidney function. Kidney keeps one's congenital energy and, with aging, this function is deteriorated. Goshajinkigan is the derivative of hachimijiogan and two additional herbs, Achyranthis Radix and Plantaginis Semen, are added.There are studies to show either hachimijiogan or goshajinkigan is effective for diabetic complications. Yokozawa et al. investigated that hachimijiogan had a protective effect against the diabetic nephropathy in the animal models –19. TheyFor retinopathy, Cameron-Schaefer et al. showed the protective effect of goshajinkigan against the diabetic retinopathy . They shAlthough it has not been shown that goshajinkigan prevented diabetic macroangiopathy, Suzuki et al. evaluated the effects of goshajinkigan on platelet aggregation in streptozotocin-induced diabetic rats. They concluded that goshajinkigan could improve platelet aggregation in diabetes through increased production of nitric oxide via bradykinin B2-receptors and muscarinic acetylcholine receptors .This clinical trial was designed based on these publications. However, we found a difference between animal models and complex clinical trials. The chosen endpoint, macroangiopathy, might have been inappropriate for clinical setting in such a short period. Whilst changes of microangiopathy might not be visible anatomically on fundus photography, goshajinkigan might influence the metabolism in the retina and thus have effects on a functional level—an effect which cannot easily be studied in humans in vivo. There had been no cases of macrovascular events. And there were no differences in the progression of retinopathy and nephropathy. Because the progression of retinopathy and nephropathy takes time, we enrolled the elder type 2 diabetes patients with moderate hyperglycemia. However, 5 years follow-up time is long for patients and the final number for the analysis was only 116 cases. The power was very low compared to the original calculation. So we could not conclude any definite effects of goshajinkigan on preventing macrovascular events such as myocardial infarction or stroke, nor could we state effects on diabetic nephropathy. Recently, some surrogate markers of macroangiopathy were proposed, like flow mediated dilation, ankle brachial pressure index, or pulse wave velocity. These are covered by the Japanese national health insurance and could be in consideration as surrogate markers.On the other hand, our results support a beneficial effect of goshajinkigan on diabetic neuropathy, which we chose as secondary endpoint. There have been a lot of manuscripts showing that goshajinkigan had a beneficial effect on diabetic neuropathy in the animal model  or in thThe mechanism of action of goshajinkigan on diabetic neuropathy was speculated partially by the inhibition of the aldose reductase , 26.Moreover, recently, goshajinkigan has been reported to relieve the peripheral neuropathy due to oxaliplatin in patients with advanced or recurrent colorectal cancer that were receiving FOLFOX therapy . The autOur data has also shown that goshajinkigan has some beneficial effect on serum glucose and glycated hemoglobin. When we have compared the medications in two groups, the patients in the control group had more progressed medications in 5 years (nonmedication to medication or medication to insulin). Body mass index was similar at the beginning of this study, but in 5 years, GJG group kept BMI and the control group lost weight. These two facts support that GJG reduces the serum glucose compared to the control group. Because the difference of the blood serum glucose or glycated hemoglobin was observed only in the late years, we assumed that GJG itself does not affect the insulin secretion itself. An improvement of insulin resistance may be involved to decrease the blood glucose level. This speculation was supported by the previous studies , 29.In large RCTs such as the Action to Control Cardiovascular Risk in Diabetes trial and the Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation trial , 31, hyp