ACE2) gene and the pathogenesis of pre-eclampsia (PE) in Guangxi, China.The aim of this study was to investigate the relationship between potential functional single-nucleotide polymorphisms (SNPs) of the angiotensin-converting enzyme 2  were selected and genotyped using kompetitive allele-specific PCR. The strength of the associations between the studied genetic variants and the risk of PE were evaluated using odds ratios (ORs) and corresponding 95% confidence intervals (CIs).A case–control study was conducted involving 327 PE cases and 591 age-matched, normal, singleton pregnant women. Potential functional vs. AA, OR = 1.43, 95% CI = 1.03–1.99, p = 0.034; AG/GG vs. AA, OR = 1.45, 95% CI = 1.06–1.99, p = 0.019), especially with severe PE . Further stratified analysis showed that rs2106809 was even more pronounced in subjects in the pre-pregnancy BMI (pre-BMI) >23 kg/m2  and triglyceride (TG) >2.84 mmol/L subgroups  under the dominant genetic model. We also found that rs2106809 interacted with pre-BMI (pinteraction = 0.040), thereby affecting an individual’s genetic susceptibility to PE. Multiple dimension reduction analysis demonstrated that rs2106809 made the best one-locus model, and the three-locus model was the best interaction model for predicting PE risk. Functional analysis suggested that rs2106809 A>G causes a change in the reliability of classifications of two putative splice sites in the ACE2 gene, potentially regulating the expression of functional genes  in multiple tissues and cell lines (p< 0.05).After adjusting for age and body mass index (BMI), unconditional logistic regression analysis showed that rs2106809 A>G was significantly associated with PE risk (AG ACE2 gene rs2106809 A>G variant is significantly associated with the risk of PE via individual locus effects and/or complex gene–gene and gene–environment interactions. Regulating the expression of functional genes such as PIR, ACE2, and CLTRN may be the molecular mechanism by which rs2106809 increases an individual’s susceptibility to PE.The  Pre-eclampsia (PE), a common disease that occurs during pregnancy, has a global incidence of 3%–5% and approximately 2.9% in China . PE is cPE may be affected by conditions such as advanced pregnancy, chronic hypertension, inflammatory immune overactivation, vascular endothelial cell damage, nutritional deficiency or obesity, and insulin resistance . In addiACE2 gene plays a key role in the RAS system. ACE2 can induce angiotensin II (Ang II) to produce angiotensin 1–7 (Ang1–7), which dilate blood vessels and thereby lower body blood pressure  is involved in vascular remodeling and blood pressure regulation during pregnancy –17. The pressure . Tamannaications . SeveralACE2 gene variants and susceptibility to PE. We therefore conducted a case–control study involving 327 PE patients and 591 healthy pregnant women to explore the associations between three candidate variants in the ACE2 gene  and the pathogenesis of PE.To date, no studies have been published detailing the relationship between A total of 918 subjects with 327 cases with PE (118 women with mild PE and 61 women with severe PE) and 591 controls were recruited from the First Affiliated Hospital of Guilin University of Medical Sciences between September 2014 and April 2016. Participants who met the diagnostic criteria for PE (first presentation after 20 weeks of gestation with systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg and urine protein ≥0.3 g/24 h) were included as PE cases. Age-matched (± 3 years) healthy pregnant women were recruited during the same period. All subjects were singleton pregnancies and unrelated. Pregnant women with diabetes, hypertension, congenital genetic diseases, cardiovascular diseases, or other pregnancy-related diseases were excluded. Study participants signed an informed consent form before the study, and the Ethics Committee Review Board of Guilin Medical University and the Affiliated Hospital of Guilin Medical University approved the study protocol.Clinical and biological characteristics were obtained from unified questionnaires and patient medical records, including weight, height, pre-pregnancy BMI (pre-BMI), diastolic blood pressure (DBP), systolic blood pressure (SBP), fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), and high-density lipoprotein cholesterol (HDL-c).The genomic DNA was extracted from EDTA-treated whole blood using a DNA extraction kit  and stored in a refrigerator at −20°C prior to PCR.ACE2 gene were screened using the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and SNPinfo Web Server (http://snpinfo.niehs.nih.gov/). The minor allele frequency (MAF) of candidate SNPs reported in HapMap-based Han Chinese in Beijing (HCB) was >0.05, and the linkage disequilibrium (LD) coefficient between SNPs was<0.8 (r2< 0.8). Finally, three genetic variants  located in the ACE2 gene were selected for further study.Potential functional SNPs of the ACE2 gene variants were genotyped using a kompetitive allele-specific PCR (KASP) method, and the corresponding specific PCR primers were designed and synthesized by Sangon Biotech Co., Ltd.  , 61–55°C for 60 s , and 30 cycles of 95°C for 15 s and 55°C for 60 s, with a final incubation at 30°C for 30 s. Candidate , China) Table 1.χ2) test was used to assess the differences of genotypes between cases and controls. A χ2 goodness-of-fit test was performed to determine whether the distribution about genotypes of SNPs in controls conformed to Hardy–Weinberg equilibrium (HWE). Logistic regression model was used to evaluate the associations between genotypes of each variant with PE by computing the odds ratios (ORs) and their 95% confidence intervals (CIs). A p-value< 0.05 was considered as statistically significant and all statistical tests were two-tailed.Statistical analysis was performed with IBM SPSS Statistics 28 for Windows . Clinical and biochemical features were compared by calculating the mean ± standard deviation  was estimated using the method described by Wacholder et al.  to assesThe multiple dimension reduction (MDR) software (version 3.0.2) was used to investigate the joint effect of SNPs. A 100-fold cross-validation and 1,000-fold permutation testing were adopted under the null hypothesis of no association. The cross-validation consistency (CVC) and the testing balanced accuracy helped to identify the best model among all possibilities considered .http://www.mulinlab.org/gwas4d/)  tool . In the gwas4d/)  based onp< 0.001), as shown in A total of 918 participants were selected, including 327 women diagnosed with PE and 591 healthy controls. Analysis of the general population data revealed that the indexes of pre-BMI, SBP, DBP, FPG, TG, and HDL-C were higher among PE cases than controls . The genotype frequencies of rs2106809, rs6632677, and rs2074192 among controls obeyed HWE . After adjustment for age and pre-BMI, logistic regression analysis showed that the associations remained significant in the heterozygote comparison  and in the dominant model . However, compared with AA genotype carriers, the associations in GG genotype carriers exhibited marginal statistical significance . We failed to find significant associations between ACE2 rs6632677 C>G and rs2074192 C>T and susceptibility to PE in the present study (p > 0.05), as shown in The candidate SNPs were genotyped using the KASP method. The D and 2 subgroup  and the TG >2.84 mmol/L subgroup . However, using the same method, a stratified analysis shows that compared with the AA genotype, the AG/GG genotype exhibited a marginal statistical association with PE among subjects ≤29 years of age  and those with TC >5.71 mmol/L . In the dominant genetic model, we also found that rs2106809 interacted with pre-BMI (pinteraction = 0.040) to affect susceptibility to PE, as shown in We also estimated the effect of rs2106809 A>G on PE risk using a stratified analysis under the dominant genetic model. After adjusting for age and pre-BMI, this association remained in the pre-BMI >23 kg/mACE2 rs2106809 and severe PE under the heterozygote comparison  and the dominant model . Furthermore, the rs2106809 GG genotype had no association with severe PE compared with the AA genotype but with marginal p-value (p = 0.052). However, no significant association was found between this variant and the onset of mild PE, p > 0.05. We also did not detect a significant association between variants of ACE2 rs6632677 and rs2074192 with PE risk of different severity , with alt. isoform/cryptic activations of 0.811 and 0.818 and constitutive values of 0.183 and 0.174 for the rs2106809 A allele and alt. isoform/cryptic activations of 0.806 and 0.813 and constitutive values of 0.188 and 0.180 for the rs2106809 G allele, respectively. Thus, the regulation of putative splice sites may be changed, as shown in PIR , ACE2 (44 studies involved), and CLTRN (collectrin) (46 studies involved), which were identified as eQTL, as shown in Furthermore, using the GTEx database, which incorporates 127 tissue/cell type–specific epigenomes datasets, the GWAS4D online tool was used to analyze eQTL between rs2106809 and the regulation of gene transcription. This analysis indicated that rs2106809 could potentially regulate the expression of functional genes associated with PE risk in a variety of specific tissues and cells, including ACE2 rs879922 C>T variant and late-onset PE susceptibility in a Chinese population. These findings suggest that ACE2 plays an important regulatory role in the occurrence and development of PE.PE is a pregnancy-related syndrome linked to the death of more than 70,000 women and 500,000 fetuses worldwide each year , 27. SevACE2 functional genetic variants  and PE risk in a southern Chinese population. Demographic data showed that BMI, SBP, DBP, FPG, TG, TC, and HDL-C were much higher in PE cases than healthy controls, indicating that obesity, hypertension, hyperglycemia, and hyperlipidemia are involved in the pathogenesis of PE as risk factors. Analyses of associations between candidate SNPs and PE risk showed that the ACE2 rs2106809 variant was significantly associated with the risk of PE, and this association was even more pronounced in subjects with higher pre-BMI (>23 kg/m2), TG (>2.84 mmol/L), and TC (>2.71 mmol/L) levels. Moreover, an interaction between rs2106809 and pre-BMI was also detected. This result was consistent with the results of comparisons of general data. It is likely that overweight or obesity before pregnancy or excessive weight gain during pregnancy can lead to abnormal metabolism, accompanied by fat accumulation in the body, thereby aggravating inflammation in placental blood vessels and trophoblasts resulting from the effects of genetic variants in initiating PE  in certain tissues and cell lines. Studies of collectrin levels and PE onset showed that serum collectrin levels play a significant role in controlling blood pressure in pregnant women, with a significant inverse correlation between serum collectrin concentrations and blood pressure , which is a tightly regulated molecule also related to inflammation and the control of innate and adaptive immune responses during onset of labor. Early activation of NF-κB may have an adverse effect by inducing premature termination of pregnancy, with bad outcomes for the mother and the fetus, including pregnancy loss  for the publication of any potentially identifiable images or data included in this article.XY: protocol/project development and manuscript editing. GH and YW: data collection and analysis, and manuscript writing. GH and LQ: data analysis and manuscript writing. BH: data collection and management. All authors contributed to the article and approved the submitted version.This study was supported by the Guangxi Young and middle-aged teachers’ basic ability improvement project (2020KY12028), Guangxi Natural Science Foundation of China (2020GXNSFAA238025), National Natural Science Foundation of China (81760614), the Key Research and Development Program (2018AB62004), and College Students’ Innovation Project (201810601031) of Guangxi, China.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hemileia vastatrix, over 1500 strains were isolated, either as endophytes from healthy tissues of Coffea species or as mycoparasites growing on rust pustules. Based on morphological data, eight isolates—three isolated from wild or semiwild coffee and five from Hemileia species on coffee, all from Africa—were provisionally assigned to the genus Clonostachys. A polyphasic study of their morphological, cultural and molecular characteristics—including the Tef1 , RPB1 (largest subunit of RNA polymerase II), TUB (β-tubulin) and ACL1 (ATP citrate lyase) regions—confirmed these isolates as belonging to three species of the genus Clonostachys: namely C. byssicola, C. rhizophaga and C. rosea f. rosea. Preliminary assays were also conducted to test the potential of the Clonostachys isolates to reduce CLR severity on coffee under greenhouse conditions. Foliar and soil applications indicated that seven of the isolates had a significant effect (p < 0.05) in reducing CLR severity. In parallel, in vitro tests that involved conidia suspensions of each of the isolates together with urediniospores of H. vastatrix resulted in high levels of inhibition of urediniospore germination. All eight isolates showed their ability to establish as endophytes in C. arabica during this study, and some proved to be mycoparasites of H. vastatrix. In addition to reporting the first records of Clonostachys associated with healthy coffee tissues and with Hemileia rusts of coffee, this work provides the first evidence that Clonostachys isolates have potential as biological control agents against CLR.During surveys conducted in South America and Africa to identify natural fungal enemies of coffee leaf rust (CLR),  C. arabica and C. canephora (Rubiaceae). Although Africa—the center of origin of the genus Coffea—plays a relatively small role in world coffee production, this crop is of great social relevance in that continent, as well as worldwide, for the income and employment it generates. It is produced mostly by smallholder communities [Coffee is ranked as the most economically valuable agricultural commodity . Only twmunities . Howevermunities ,4.Hemileia vastatrix , a biotrophic fungus [H. vastatrix via planting coffee in highland situations. Biological control of H. vastatrix has, thus far, received relatively little attention.Amongst these limitations of coffee production is its most devastating disease: coffee leaf rust (CLR) caused by c fungus . The recc fungus . The seac fungus ,8. Outbrc fungus , have caH. vastatrix [Coffea in Africa as a source of antagonists of CLR, as well as of other pathogens that attack the crop, have been poorly studied. A notable exception is that of Mulaw [C. arabica in Ethiopia and focused on their antagonism to Fusarium xylarioides, the causal agent of tracheomycosis.Although there are several studies on potential antagonists of CLR, such as endophytic fungi that grow inside healthy coffee tissues ,11,12,13astatrix ,16,17,18of Mulaw : a studyH. vastatrix, and an unexpectedly high diversity of fungi was revealed [Clonostachys . Here, we report on taxonomic studies to elucidate their identity, as well as on their potential as antagonists of H. vastatrix.In 2015, surveys were initiated in Africa for fungal antagonists of revealed ,25,26,27H. vastatrix were concentrated in Africa, focusing on Cameroon and Ethiopia—representing regions within the centers of origin of C. canephora and C. arabica, respectively—and Coffea species growing in wild or semiwild situations were targeted. Other Hemileia species, in addition to H. vastatrix, were also found on Coffea in these ecosystems. Details of the strategy and the isolation methodology employed to isolate endophytic fungi and mycoparasites are given in detail in previous publications [Surveys for fungal antagonists of ications ,22. PureIsolates were mounted in 60% lactic acid and observed under a light microscope  fitted with a differential interference contrast light and a digital image capture system .Additionally, a slide-culture method  was usedClonostachys and related taxa—shapes of conidiophores, sizes of stipe and penicillus and shapes and sizes of phialides and conidia, among others—were recorded for at least thirty representative structures. Photo images were prepared in Inkscape 1.2.2 (https://inkscape.org/pt/ (accessed on 1 August 2019)).Morphological data of relevance for species delimitation in Colony characters were recorded after 10 days of growth on potato dextrose agar (PDA), OA and 3% malt extract agar (MEA) , at 25 ±Eight out of over 1500 isolates obtained during the surveys were assigned as Clonostachys-like. These isolates were either stored on potato carrot agar (PCA) slants at 4 °C for short-term use or, for long-term storage, kept at −80 °C in cryotubes with 10% glycerol, as described in the literature . SelecteClonostachys spp., pieces of samples of coffee leaves obtained at the end of the in planta antagonism study (described below) and bearing CLR pustules seemingly colonized by Clonostachys were selected and dried in a plant press. Selected pieces of leaves containing mycoparsitized uredinia were further dried via mounting on stubs with double-sided adhesive tape and leaving overnight in a desiccator. These specimens were gold-coated using a Balzer’s FDU 010 sputter coater. A Carl-Zeiss Model LEO VP 1430 scanning electron microscope (SEM) was used, operating at 10 Kv and with a working distance ranging from 10 to 30 mm, to analyze the specimens and generate representative electromicrographs of the colonization events.In order to document the colonization of uredinia by Clonostachys were further analyzed molecularly. Four gene regions were sequenced for those isolates, following the methodology described above: namely, TEF1-α  [Isolates recognized morphologically as belonging to 1 alpha) ,34, ACL1−1) of BSA, 0.5 µL (0.01 µg/µL) of DMSO, 0.5 µL of each primer, 2.5 µL of Water MilliQ and 6 µL of Dream Taq. The cycling conditions used during PCR for β-tubulin were set according to the published description [http://www.macrogen.com, (accessed on 1 August 2019)).The isolates were grown on PDA and then seeded in small plates containing 5 mL of potato dextrose (PD) broth each and incubated for 5 days at 25 °C under a 12/12 h daily light/dark regime. After that period, the mycelial material was removed from the plates and air-dried on sterile filter paper at room temperature for 24 h. Dried mycelial material from each isolate was then transferred to sterile tubes for DNA extraction. Extraction was performed with the Wizard Genomic DNA Purification Kit . Manufacturer’s guidelines were strictly followed. The primer pair of EF1-728F/EF2 was used for amplification and sequencing of the TEF1-α region, acl1-230up/acl1-1220low was used for ACL1 , Fa/R8 wcription . Howeverhttps://www.sequentix.de/ (accessed on 19 March 2020)) was utilized [https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 19 March 2020)). Sequences of reference for species with high identity percentages were selected for alignment according to the same gene region used to amplify the gDNA of the respective isolate. The Muscle algorithm, from the software Aliview, version 1.26 [In order to assemble and edit the nucleotide sequences, DNA Dragon 1.7.3-DNA Sequence Contig Assembler Software developed by SequentiX-Digital DNA Processing ) and Inkscape.Bayesian inference (BI) analyses were launched after definition of the best nucleotide substitution model for each gene. JModeltest 2.1.10 software  was applhttp://www.ncbi.nlm.nih.gov/genbank (accessed on 19 March 2020)), and those from different genes  retrieved from GenBank, are listed in Sequences of this study, partially deposited in the NCBI database (GenBank) , with 4 to 5 pairs of fully differentiated leaves each were utilized. The substrate used for cultivation of test plants was a mixture of heat-disinfected soil and rice colonized by the antagonist (and prepared as described below).Healthy four-month-old shade-house-grown coffee plants, Clonostachys isolates against H. vastatrix was tested between Dec 2019 and May 2020 (assay I) and between Jun and Nov 2020 (assay II). The assays were each composed of nine treatments, including the control (plants untreated with any antagonist). Each treatment had five repetitions, and each repetition was represented by one young coffee plant grown in a black polyethylene bag containing 50 g of rice, colonized by an isolate of Clonostachys spp., mixed with heat-disinfected soil. Both assays I and II included a total of 45 coffee plants each.In planta antagonism of Hemileia vastatrix—Fresh, viable urediniospores were obtained as described by Salcedo-Sarmiento [H. vastatrix (1 × 105 spores/mL), suspended in 0.05% Tween 20, until runoff and placed in a dew chamber . After armiento  or used armiento .Clonostachys spp.—All isolates were grown on OA plates at 25 °C under a 12 h light regime until the plates were fully colonized. Ten milliliters of sterile distilled water (SDW) were then added to each of the plates, and the surface of the colonies was scraped with a rubber spatula in order to produce a concentrated suspension of mycelium and conidia. The contents of the plates were then aseptically transferred to 125 mL flasks, each containing 20 mL of 2% malt extract broth (MEB). Each flask was seeded with a single Clonostachys isolate. The flasks were left on a lab bench at room temperature for three days and manually agitated daily. After this period, 3 mL of the colonized liquid medium was aseptically transferred to a polypropylene bag (12 × 25 cm) containing 50 g of parboiled rice, 0.22 g of CaCO3 and 40 mL of distilled water, which had previously been autoclaved at 121 °C for 20 min. For each treatment (an isolate), five polypropylene plastic bags were prepared. The bags were then placed in a growth room at ca. 22 °C under a 12 h light regime  and incubated for 10 days. Every two days, the bags were rolled, and their contents were hand-squeezed to avoid formation of large rice/mycelial aggregates and to allow for good aeration in order to stimulate sporulation. Bags containing only autoclave-sterilized, noninoculated parboiled rice were used to treat control plants.Clonostachys inoculum, each isolate was grown on OA plates in an incubator adjusted to 25 °C and with a 12 h light/12 h daily regime for five days. The bags were then aseptically seeded in a laminar flow cabinet with 5 mm-diameter plugs (six per bag) taken from the margins of the actively growing colonies.In the second round of production of Clonostachys isolate, six to ten grams of colonized rice were suspended in 0.05% Tween 20 and placed in 125 mL flasks on a shaker at 25 °C at 130 rpm for 20 min in order to release the conidia from the substrate. The contents of the flasks were then filtered through cheesecloth, and the concentrations of conidial suspension were then adjusted with a haemocytometer to 106–108 conidia/mL before use.Depending on the intensity of the sporulation observed for each Clonostachys (prepared as previously described), with an estimated 107–109 conidia/g of rice, were thoroughly mixed separately with heat-disinfected soil. This inoculum–substrate mixture was then transferred to five 24 × 17 × 10 cm plastic bags, each containing one healthy young coffee plant. Controls consisted of plants treated as previously described but receiving only a mixture of uncolonized rice.Inoculation procedures—In total, 250 g of parboiled rice colonized by each isolate of Spraying of the antagonists on the aerial parts of the coffee plants was conducted one month after soil inoculation with colonized rice (as described above). The conidial suspensions of each isolate were applied separately, until runoff, using a handheld sprayer connected to an air compressor. The foliar sprayings were repeated three times at monthly intervals.5 spores/mL until runoff. Inoculation with H. vastatrix occurred 72 h after the last spray-inoculation with the isolates of Clonostachys (3 months after transplantation) and was conducted in the late evening in order to favor spore germination and host infection.Inoculation of the rust fungus was through spray-inoculation of urediniospores with 2 × 10Clonostachys, originally obtained as endophytes (three) and mycoparasites (five), were used. In order to further confirm their ability to grow as endophytes, coffee plants in the in planta antagonism experiment were also used to evaluate the endophytic ability of all isolates. Two similar (pseudorepeat) assays were then conducted to verify (a) the establishment of the isolates as endophytes in Arabica coffee after inoculation and (b) if an anti-CLR antagonistic “bodyguard-effect” resulted from each isolate application.Isolates of C. arabica cv. Catuaí-vermelho with each Clonostachys isolate involved a combination of soil application and a conidial suspension spray of the aerial parts (as described previously). Thirty days after transplantation of the plants, the aerial part of each treated group was separately spray-inoculated with a suspension of each Clonostachys isolate (106–108 conidia/mL).Inoculation of young Assay I: endophytic colonization. Thirty days after transplantation of the coffee plants, one plant was arbitrarily selected from each isolate-treated group and one from the untreated control group for the endophyte-isolation protocol. This was repeated at 30-day intervals, prior to the round of spraying of antagonists on the aerial part of each plant group. The final round of endophyte isolation was conducted after the final assessment of CLR severity . The plants that showed the lowest degrees of CLR severity in each treatment were selected.The endophyte-isolation protocol was as follows: each individual plant was thoroughly washed under a tap to remove all debris and treated according to a modified version of the protocol of Rodríguez . Each coClonostachys colonies. Whenever colonies similar to Clonostachys appeared, the plates were further checked under a dissecting microscope (Olympus SZX7). If needed, the plates were opened in a laminar flow and samples of a colony were mounted on microscope slides in a drop of lactofuchsin and examined under a light microscope (Olympus BX-51). The final results were ranked either as positive (+) for recovery of Clonostachys growing endophytically in tissues or negative (−) for not a single sample of that tissue producing a Clonostachys colony.The plates were observed every two days in order to monitor the presence or absence of typical Assay II—endophytic colonization. This assay was repeated with all eight isolates of Clonostachys. Procedures were mostly as described for assay I. The main difference between assays I and II was the limitation of attempts to confirm the endophytic colonization of Clonostachys isolates in a single round in assay II. This was performed at the end of the assessment of CLR severity, five months after the first inoculation of the antagonists and at the end of the greenhouse experiment.Hemileia vastatrix (as reflected by the effect on CLR severity)Assay I—in planta antagonism to . Antagonism of different isolates to H. vastatrix was tested on coffee plants, in parallel with the test described above. These assays followed the steps described above, but the procedures for assays I and II differed. In order to assess the effects of different isolates, all plants (including the controls) were inoculated with H. vastatrix and grown as described above. CLR inoculation was conducted only once, at the third month after the transplantation and 72 h after the third foliar application of the conidial suspensions of Clonostachys isolates. This assay involved all 16 remaining plants, representing the eight Clonostachys treatments, and two plants for the control treatment (two plants for each treatment).H. vastatrix, eight leaves per plant, which showed typical pale yellow spots  and were identified with plastic tags, were arbitrarily selected. A total of sixteen leaves from each Clonostachys-isolate-treated plant and sixteen control leaves not treated with Clonostachys isolates were labeled. Each individual plant represented an experimental unit. The median of the CLR severities evaluated from the eight leaves per plant was recorded as the CLR severity of the plant. One month after urediniospore application, the first CLR severity assessment of individual leaves was performed. CLR severity on individual leaves was assessed three times at two-week intervals. Each leaf was given a note following the standard protocol described by Belan [Three weeks after inoculation with by Belan , which cHemileia vastatrix (as reflected by effect on CLR severity)Assay II: in planta antagonism to . As previously mentioned, no attempt at recovering Clonostachys from roots, stems or leaves was attempted in assay II until the end. Therefore, all five of each Clonostachys-isolate-treated coffee plant and five control plants were included in the CLR severity evaluation. Evaluation of disease severity followed the same procedure as described above. A total of 40 leaves, in the control and each Clonostachys-treated plant were marked and evaluated. Evaluation of disease severity for individually marked leaves was conducted three times, as described for assay I. Nevertheless, only the data collected from the third disease severity assessment were statistically analyzed in this study due to the single cycle of the disease under our experimental conditions.Clonostachys spp. that showed the highest levels of reduction in CLR severity in both experiments, as compared to both the control and other Clonostachys isolates (COAD 2982 and COAD 2981 (C. rhizophaga) and COAD 2984 (C. rosea)), were included in one additional test. This test consisted of calculating the in vitro inhibition of germination of urediniospores. Suspensions of spores and filtrates of each isolate were tested.Isolates of Clonostachys isolates were also grown separately in 125 mL flasks containing 75 mL of oatmeal broth and seeded with five plugs of the corresponding isolate taken from five-day-old colonies on OA plates. The flasks were placed in a shaker at 29 ± 1 °C and 196 rpm for one week. Subsequently, the supernatants were filtered through sterile filter paper in a Büchner funnel. Each filtrate was used in the test, as described below. A suspension of H. vastatrix urediniospores was prepared from a stock, collected and stored as previously described. Urediniospores were suspended in a 0.05% Tween 20 solution, and the concentration of urediniospores was calibrated to 1 × 105 spores/mL using a haemocytometer. A suspension of each of three Clonostachys isolates was prepared from colonized rice suspended in a 0.05% Tween 20 solution, and the conidial concentration was calibrated to 106 conidia/mL with a haemocytometer.Clonostachys isolate. All 24 boxes were kept in the dark for six hours at 22 °C, after which urediniospore germination was interrupted via adding a 15 μL drop of lactofuschin to each drop and then observed under a light microscope (Olympus BX-51). This protocol was used to test conidial suspensions of each of the selected Clonostachys isolates. The number of germinated vs. nongerminated urediniospores was estimated via observing the first 100 urediniospores on each slide. Urediniospores were considered to have germinated when the germ tubes had a length equal to or longer than the spore diameter. Germination inhibition (% GI) was calculated via following this equation:Two microscope slides were cleaned with 70% ethanol and placed inside polypropylene boxes (11 × 11 × 3.5 cm) that had also been fumigated with 70% ethanol. The boxes were lined with a layer of sterilized paper towel saturated with sterile distilled water (SDW). One 15 μL drop of the urediniospore suspension was transferred, with a micropipette, to the center of each slide; a second 15 μL drop of filtrate of a specific isolate was placed over one of the drops of urediniospore suspension on one of the slides inside the box but not on the other; and the two drops were gently mixed with the tip of the micropipette and the box was covered. Any slide with only one drop of urediniospore suspension served as a control. This study involved four replicates for each tagonist .p = 0.05), using JASP software, version 0.16.0 .The mean values of the percent of severity of CLR were calculated. Data were submitted to assumptions of normality and homogeneity of variance using the Shapiro–Wilk and Levene tests before the proceeding of the independent one-way analysis of variance (ANOVA). The distribution of the estimated CLR severities was represented in the form of boxplots. The mean values of the treatments were compared to the mean value of the control with Dunnett’s post hoc test (Clonostachys (with a threshold of identity ≥ 96%). Amongst these eight isolates, three were isolated as endophytes from stems of C. arabica, four were isolated from colonies growing on rust uredinia on C. canephora and one was from a colony growing on uredinia on C. arabica  of species of the genus Clonostachys had equivalent topologies. The BI tree is shown in Clonostachys isolates from coffee and Hemileia uredinia belonged to Clonostachys byssicola, C. rhizophaga and C. rosea. These are well-known taxa that have already been described and discussed in detail in the literature [BLASTn searches applied to the sequences from the different amplified regions revealed that all isolates belonged to the genus  arabica . The BI terature ,45,46.Hemileia uredinia formed a clade with C. rhizophaga. Two endophytic isolates grouped within the C. rosea clade. The other two isolates—one an endophyte and the other a mycoparasite—formed a clade with C. byssicola. The topologies of the single-gene trees (trees not shown) were not all equivalent to that of the concatenated tree. For ACL1 and RPB1, all species strains were monophyletically grouped in both ML and BI analyses. The four isolates in the monophyletic group of C. rhizophaga were always divided into two groups; however, in the concatenated tree, they were well-grouped and formed a sister group with the strains already identified as C. rhizophaga. In the TEF1 tree, the strains already identified as C. rhizophaga appeared in two separate clades. The same was seen in the TUB tree for the C. byssicola strains, which also appeared in two separate clades in the ML and BI analyses. However, all isolates of the three species formed monophyletic clades in the concatenated tree, with support values ranging from 70 to 100% ML bootstrap support and BI posterior probabilities of ≥ 0.98  [Bionectria byssicola (Berk. and Broome), Schroers and Samuels, Z. Mykol. 63(2): 152 (1997).0 (2001) . Sexual Hemileia cf. coffeicola (COAD 2983) and as an endophyte in stems of wild Coffea arabica (COAD 2986), 25 November 2015, Evans H.C. and Bekele K.B.Material examined: Ethiopia, Southern Nations, Nationalities and Peoples Region, Kaffa Zone, Bonga District, Komba Wild Forest Reserve, cloud forest, 2000 m; isolated as a mycoparasite on uredinia of For a detailed description and illustrations, see Schroers .Dimorphic conidiophores: verticillate primary conidiophores; divergent phialides (2–4 whorls), 19–54 × 2–3 µm; stipes, 10–63 µm long; penicillus, 19–55.5 µm long; adpressed secondary conidiophores, bi- to triverticillate; adpressed phialides (4–7 whorls), 10–32.5 × 1–2.5 µm. Aseptate conidia: cylindrical and/or slightly curved, 2.5–10 (–14) × 1.5–4 µm .In culture : low, coNotes—When compared with COAD 2983, some morphological details given in [C. byssicola were different. Primary conidiophore phialides were shorter (12.4–48 × 1.4–2.8 µm), stipes and the penicillus were longer ; secondary conidiophores had more verticils (2–5) and phialides were in smaller whorls (3–5) and were shorter (7.6–27.8 µm). Such morphological discrepancies between COAD 2983 and the data given in [given in  for C. bgiven in  were notClonostachys rhizophaga: Schroers, Stud. Mycol. 46: 85 (2001) [5 (2001) .Hemileia vastatrix/coffeicola on leaves of Coffea canephora, 22 November 2015, Evans H.C. .Material examined: Cameroon, Eastern Province, Somalomo, 700 m; isolated as mycoparasites of uredinia of For a complete description and illustrations, see Tehon and Jacobs .Dimorphic conidiophores: verticillate primary conidiophores; divergent phialides (2–5 whorls), (9.5–) 11–35 × 1–3 µm; stipes, 20–92 µm long; penicillus, 19–87 µm long; penicillate secondary conidiophores, mono- to quaterverticillate; adpressed phialides (3–6 whorls), (5−) 14.5–17(−28) × 1 − 2.5(−7) µm. Conidia: aseptate, ellipsoidal/cylindrical, minutely curved to curved, with laterally displaced hilum, 3.5–9 (–11) × 2–5.5 (−7.5) µm .In culture : flat orNotes—When compared with COAD 2979, some morphological details given in [C. rhizophaga differed: primary conidiophores’ penicilli were longer (30–100 µm), and phialides in the secondary conidiophores were narrower (2.2–3.2 µm) in Schroers’ description [given in  for C. rcription . Here, wClonostachys rosea: (Link) Schroers, Samuels, Seifert and W. Gams, Mycologia 91(2): 369 (1999) [f. rosea.9 (1999)  f. roseaCoffea arabica, 25 November 2015, Evans H.C. (Cultures COAD 2984 and 2985).Material examined: Ethiopia, Southern Nations, Nationalities and Peoples Region, Kaffa Zone, Bonga District, Komba Wild Forest Reserve, cloud forest, 2000 m; isolated as endophyte from stems of For a complete description and illustrations, see Schroers .Dimorphic conidiophores: verticillate primary conidiophores; divergent phialides (2–5 whorls), 20.5–40.5 × 1.5–3 µm; stipes, 20–122 µm long; penicillus, 25.5–73.5 µm long; adpressed secondary conidiophores, bi- to quaterverticillate; adpressed phialides (5–7 whorls), 10–19 × 1–3 µm. Conidia: aseptate; cylindrical; less curved; distally broadly rounded, with one slightly flattened side; inconspicuous hilum; conidia size, 4–10 × 1.5–3.5 µm .In culture : radiallNotes—A comparison of the morphology of C. rosea f. rosea, as described by Schroers [Schroers , with thSchroers , the priClonostachys isolates was confirmed, albeit with inconsistent results  may be behind such inconsistencies. Consistency in recovery was only observed with plants treated with COAD 2979 in each attempt in assay I and with all treatments, except COAD 2981, two months after the assessment of CLR severity. Conversely, four of the eight treatments  yielded a consistent recovery of tagonist . No ClonClonostachys species, i.e., four belonging to C. rhizophaga, two to C. rosea and two to C. byssicola, were screened in planta  and C. rosea (COAD 2984), at p < 0.01, and C. byssicola (COAD 2986), C. rhizophaga  and C. rosea (COAD 2985), at p < 0.05, had significantly reduced CLR severity when compared to the control (C. byssicola) and the control.In assay I, an independent one-way ANOVA showed a significant effect of treatments on reduction in CLR severity after two months of inoculation of plants with the pathogen;  control . There wp < 0.001. Post hoc testing using Dunnett’s correction revealed that C. rhizophaga , C. byssicola (COAD 2986) and C. rosea (COAD 2984) were significantly different from the control, at p ˂ 0.001, whilst COAD 2985 (C. rosea) was significantly different, at p ˂ 0.05, from the control in reduction in CLR severity. No significant difference was found between COAD 2983 (C. byssicola) and the control  did not reduce the CLR severity in both experiments , COAD 2984 (C. rosea) and COAD 2981 (C. rhizophaga) inhibited urediniospore germination by 94, 99 and 100%, respectively. Exposure to filtrates of the same isolates resulted in urediniospore germination dropping to 21–40%, depending on the Clonostachys isolate involved. COAD 2984 (C. rosea), COAD 2982 (C. rhizophaga) and COAD 2981 (C. rhizophaga) inhibited urediniospore germination to 60, 67 and 79%, respectively. Similarly, to what was shown by Salcedo-Sarmiento [H. vastatrix urediniospores were exposed to both conidial suspensions and filtrates of Clonostachys  that had been isolated from uredinia of Hemileia rusts. The results from the SEM study also confirmed the mycoparasitic status of Clonostachys, showed its ability to colonize pustules of CLR and urediniospores  [C. rosea is marketed for control of various soil and seed-borne pathogens [Research involving cinerea)  in Braziathogens , and thiathogens .Clonostachys as mycoparasites of Hemileia rusts of coffee: namely, C. rhizophaga from parasitized uredinia of H. vastatrix and H. coffeicola on leaves of C. canephora in Cameroon and C. byssicola from uredinia of Hemileia spp. on C. arabica from Ethiopia. In addition, the only previous report of a member of Clonostachys occurring as an endophyte in coffee tissues was of Clonostachys cf. rosea isolated from a coffee leaf in Colombia [C. byssicola and C. rosea f. rosea from the inner healthy tissues of coffee stems of C. arabica in Ethiopia  and C. rosea (COAD 2984 and 2985) proved to be monophyletic, as evidenced by the four genes studied. Nevertheless, some inconsistencies were found for the strains, based on sequences already deposited in GenBank, in the phylogenetic trees of regions such as TUB for C. byssicola and TEF1 for C. rhizophaga. Trees for these genes (not shown) generated polyphyletic topologies. Similar results  have already been reported in TUB for C. byssicola [TEF1 for C. rhizophaga [C. byssicola and C. rhizophaga, monophyly was evident in the single-gene trees for ACL1 and RPB1. More importantly, a monophyletic topology was found in the concatenate/multilocus study  and sporulated poorly when cultivated on autoclaved rice. On OA and PDA, all four isolates released a yellow pigment, which was intense and darker, into the media for COAD 2981 and COAD 2980  as representing a feature of a “wild form” of C. rhizophaga. In that study, CBS 100004 appeared in the same clade as C. rhizophaga CBS 361.77T, and the latter is included in our study. The production of abundant secondary conidiophores as seen in our isolates, as well as their phylogenetic affinity with CBS 361.77T, also suggests a “wild state” for our isolates and may reflect their recent isolation from natural forest situations. One observation that could be of relevance for a better understanding of C. rhizophaga is the finding of spores that were morphologically similar to the ascospores of the sexual stage of Clonostachys in an OA culture of COAD 2980  covered in this study since the plants were first exposed to Clonostachys. Evans [Trichoderma isolates.There is published evidence that some mycoparasites can establish long-lasting endophytic associations with plants. For example, Nemec  providednd Mejía  reporteds. Evans  also repClonostachys tested here were capable of colonizing coffee as endophytes without inducing disease symptoms, indicating that these isolates are nonpathogenic endophytes that may interact with C. arabica in a symbiotic association. This is of particular relevance, since there are records of Clonostachys species, including isolates belonging to C. rhizophaga and C. rosea, being etiological agents of crop diseases: for example, C. rhizophaga causing chickpea wilt in Syria [C. rosea causing root rot of soybeans in the USA [In summary, all isolates of in Syria  and infein Syria  and C. r the USA  and root the USA .Clonostachys species have already been reported as potential biocontrol agents against numerous plant pathogens. Some examples are C. rosea and C. byssicola strains showing evidence of control in vitro of Moniliophthora roreri, the causal agent of frosty pod rot of cacao [C. rosea reducing sporulation of M. roreri on cacao pods [Clonostachys spp. reducing the severity of potato early blight caused by Alternaria grandis [of cacao ; C. rosecao pods ; and Clo grandis .C. arabica produces changes in the expressions of some genes involved in its defense pathways and the release of potential antimicrobials, such as hydrogen peroxide, peroxidases, ascorbate peroxidase, jasmonic-acid components, pathogenesis-related protein, chitinases, beta glucanases and endochitinases [Clonostachys species are known to produce metabolites and enzymes with a broad spectrum of biological activities, such as antimicrobial/mycoparasitic roles and resistance inducers [Clonostachys rosea has also been reported to produce cyclopeptides, piperazines, pyranones, sorbicillinoids and clonostach acids [C. rhizophaga) and with COAD 2984 (C. rosea).It is likely that benign penetration and establishment of endophytes inside itinases ,74,75,76inducers ,78. For ch acids . FurtherClonostachys isolates when they were applied to coffee plants before H. vastatrix suggests that various mechanisms may be involved: competition for space or nutrients, induced resistance or antibiosis. Guzzo [Bacillus thuringiensis (Thuricide HP). Therefore, further studies would be useful to allow a better understanding of the results reported herein.The antagonistic potential shown in the endophytic s. Guzzo  demonstrC. rhizophaga); COAD 2984 and COAD 2985 (C. rosea); and COAD 2986 (C. byssicola) form a promising assemblage for further investigation as potential biocontrol agents to be deployed against CLR. However, the combination of the results of the two assays indicates that the list could be further reduced to “potential elites”, prioritizing C. rhizophaga COAD 2981 and COAD 2982 and C. rosea COAD 2984.Nemec  has mentClonostachys isolates obtained from coffee are capable of growing as endophytes in C. arabica and also that some isolates deserve the attention of biocontrol workers who deal with management of CLR. Strictly organized and analyzed experiments, to be performed both under controlled conditions and in the field, are needed and justifiable now. It is hoped that in future studies, some of selected isolates will prove COAD 2981, COAD 2982 and COAD 2984 to be effective as coevolved coffee “bodyguards” with high endophyte competence—as suggested by Muvea [Although the present work is to be regarded as mostly preliminary and exploratory, it has yielded convincing evidence that by Muvea —or as hi


Alzheimer’s disease (AD) is a neurodegenerative disease associated with memory impairment and other central nervous system (CNS) symptoms. Two myrtenal–adamantane conjugates (MACs) showed excellent CNS potential against Alzheimer’s models. Adamantane is a common pharmacophore for drug design, and myrtenal (M) demonstrated neuroprotective effects in our previous studies. The aim of this study is to evaluate the MACs’ neuroprotective properties in dementia. Methods: Scopolamine (Scop) was applied intraperitoneally in Wistar rats for 11 days, simultaneously with MACs or M as a referent, respectively. Brain acetylcholine esterase (AChE) activity, noradrenaline and serotonin levels, and oxidative brain status determination followed behavioral tests on memory abilities. Molecular descriptors and docking analyses for AChE activity center affinity were performed. Results: M derivatives have favorable physicochemical parameters to enter the CNS. Both MACs restored memory damaged by Scop, showing significant AChE-inhibitory activity in the cortex, in contrast to M, supported by the modeling analysis. Moderate antioxidant properties were manifested by glutathione elevation and catalase activity modulation. MACs also altered noradrenaline and serotonin content in the hippocampus. Conclusion: For the first time, neuroprotective properties of two MACs in a rat dementia model were observed. They were stronger than the natural M effects, which makes the substances promising candidates for AD treatment. Neurodegenerative diseases are a large group of pathological conditions—hereditary or sporadic—with a similar etiology. They are characterized by progressive loss of neuronal structures and functions, as well as neuronal death. The most common neurodegenerative condition is Alzheimer’s disease (AD), which is associated with changes in memory, thinking, orientation, and behavior . The infMyrtenal (M)  is a bicM is a volatile, lipophilic compound, and therefore it is difficult to formulate emulsions containing it. Furthermore, monoterpenes and monoterpenoids often lack selectivity and suffer from low metabolic stability. Thus, chemical modification of a monoterpene can modulate its biological effects, including selectivity, by changing its physicochemical characteristics such as lipophilicity and volatility.Adamantane is a well-established pharmacophore based on known pharmacological agents . It is uBecause M exerts neuroprotective properties and the adamantine structure is established for drug development, myrtenal–adamantane conjugates (MACs) have been synthesized as reported by Teplov et al. , followeThe aim of this study is to examine comparatively the neuroprotective potential of these two MACs in scopolamine-induced Alzheimer’s-type dementia in laboratory rats.p (water-octanol partition coefficient), HD (hydrogen bond donors), HA (hydrogen bond acceptors), PSA , and RB (rotatable bonds) of the ligands are given in p; MAC-197 and MAC-198 have relatively high log p values in the upper end of drug-like chemical space due to their adamantine moiety  pyridiunum-1-yl}methoxy) methyl]pydidinium (HI6)  [The docking center for the AChE crystal structure  was defined as the position of the nitrogen in the pyridine ring substituted with the oxime of the co-crystallized ligand 4-(aminocarbonyl)-1-[({2-[(−24.154) . Fifty d−24.154) , ChemSco−24.154) ,74, Chem−24.154) , and ASP−24.154)  scoring 2A, this equation was used: =(KDI MW + KDI LogP + KDI HD + KDI HA + KDI RB + KDI PSA). For KDI2B, this equation was used: =(KDI MW × KDI LogP × KDI HD × KDI HA × KDI RB × KDI PSA).The QikProp 6.2 (2009) software package was used to calculate the molecular descriptors. The reliability of QikProp was established for the calculated descriptors . The KnoThe experiments were carried out on male adult Wistar rats (180 ÷ 220 g). The animals were kept under standard laboratory conditions in plastic cells, with a 12-h light/dark cycle, drinking water and food for rodents ad libitum, and optimal temperature, humidity, and indoor ventilation. The experimental protocols were carried out following the rules of the Committee on Ethics of the Bulgarian Food Safety Agency and in compliance with national laws and rules , based on the European Directive and in accordance with the rules of the Ethics Committee of the Institute of Neurobiology at the Bulgarian Academy of Sciences.n = 10): (1) Controls , (2) Scopolamine (Scop), (3) Scopolamine and Myrtenal (Scop + M), (4) Scopolamine and MAC-197 (Scop + MAC-197), and 5) Scopolamine and MAC-198 (Scop + MAC-198).Male adult Wistar rats were separated into five groups  for 11 consecutive days as a solution of scopolamine hydrobromide dissolved in distilled water. The other substances were applied simultaneously with scopolamine in different intraperitoneal inoculations. The two myrtenal–adamantane conjugates (MACs) with code names MAC-197 and MAC-198 were injected in an effective dose of 1 mg/kg b.wt. M was used as a referent in an effective dose of 40 mg/kg b.wt., as established in our previous work . M, as w2 inhalation. Their brains were quickly removed, and two main brain structures related to memory—the cortex and hippocampus—were separated for biochemical analysis. Brain acetylcholinesterase (AChE) activity, noradrenaline and serotonin levels, and oxidative status parameters were evaluated.All animal groups were submitted to a behavioral test for memory ability status (passive avoidance test). The animals were then euthanized via mild COThe step-through/passive avoidance test  was usedInitial latency (IL) was conducted before the treatment with the tested substances. Each animal was placed in the light half of the staging, leaving the door between the light and the dark part open. When a rat entered the dark (with four paws), the door closed, and a weak electrical shock was released through the floor (0.7 mA for 3 s).The experimental phase included first testing 1 h after the first application and final testing at 24 h after the last one (on day 12); no electrical current was applied again. The latency time (up to 180 s), i.e., the time after which the animal moved into the dark room, was measured. As a memory status indicator, the difference in latency time vs. IL was recorded.AChE activity in the cortex and hippocampus was determined according to the Ellman protocol . Brain s3COOH, whereas for 5-HT, o-phthaldehyde must be added. Monoamines were determined at λ = 385/485 nm for NA and λ = 360/470 nm for serotonin, calculated based upon standard solution fluorescence, and expressed as µg/g of fresh tissue.The concentration of the monoamines noradrenalin (NA) and serotonin (5-HT) in the brain cortex and hippocampus were measured via fluorescence reaction by the methods of Jacobowitz and Richardson . Noradre−1cm−1 [v/v) for color developing. The absorbance was read at 532 nm against an appropriate blank.The following biochemical parameters were measured: total glutathione (GSH), measured according to Tietze (expressed as ng/mg protein) and with oxidized glutathione (GSSG) as a reference standard . Lipid p−1cm−1 . The pos−1cm−1.Catalase (CAT) activity was determined according to Aebi . The enzProtein content was measured by the method of Lowry et al. .p < 0.05.Results are expressed as means ± the standard error of the mean (SEM). Data statistical analyses were performed by one-way analysis of variance (ANOVA) using the GraphPad Prism 7.0 software . Differences were considered significant at For the first time, the neuroprotective properties of two originally synthesized myrtenal–adamantane conjugates were revealed in the rat dementia model. The new MAC- conjugates restored the impaired memory of demented rats via complex mechanisms. In both brain structures related to memory—the cortex and hippocampus—they decreased AChE activity, modulated monoamine (NA and 5-HT) levels, and exhibited antioxidant potential. The neuroprotective effects of MACs were stronger than those of natural myrtenal, which makes them potential therapeutic candidates for Alzheimer’s-type dementia.


Traditional performance-based measurements of mobility fail to recognize the interaction between the individual and their environment. Life-space (LS) forms a central element in the broader context of mobility and has received growing attention in gerontology. Still, knowledge on LS in the nursing home (NH) remains sparse. The aim of this study was to identify LS trajectories in people with dementia from time of NH admission, and explore characteristics associated with LS over time.In total, 583 people with dementia were included at NH admission and assessed biannually for 3 years. LS was assessed using the Nursing Home Life-Space Diameter. Association with individual  and environmental  characterises was assessed. We used a growth mixture model to identify LS trajectories and linear mixed model was used to explore characteristics associated with LS over time.n = 19, 3.5%), Group 2 , Group 3 , Group 4 . Being younger, having good compared to poor general medical health, less severe dementia, more agitation, less apathy, better physical performance and living in a smaller unit were associated with a wider LS throughout the study period.We identified four groups of residents with distinct LS trajectories, labelled Group 1 (From NH admission most NH residents’ LS trajectory remained stable (Group 2), and their daily lives unfolded within their unit. Better physical performance and less apathy emerged as potentially modifiable characteristics associated with wider LS over time. Future studies are encouraged to determine whether LS trajectories in NH residents are modifiable, and we suggest that future research further explore the impact of environmental characteristics.The online version contains supplementary material available at 10.1186/s12877-021-02345-0. Limitations in mobility is associated with an abundance of adverse health outcomes and identifying and preventing mobility impairments is a global public-health priority . TraditiSeveral studies have examined LS and its associated characteristics in community-dwelling older adults. These indicate that restricted LS is associated with older age, more severe depression, apathy, comorbidity, cognitive impairment, impairments in physical performance, and environmental barriers , 11–15. Influenced by the increased proportion of older adults in society and growing numbers of dependent older adults, the demand for NH care will remain high . NH resiThe evidence of LS in NH residents is sparse, but cross-sectional studies report high levels of LS restrictions , 29 and This is a longitudinal multicentre study based on data from the Resource Use and Disease Course in dementia-Nursing Home cohort (REDIC-NH). The study procedure, methods, attrition, and eligible-but-not-included analyses have been reported in detail previously . BrieflyAt admission, based on all data collected, two physicians (SB & GS) independently diagnosed dementia according to the International Classification of Diseases, version 10, research criteria (ICD-10) . If consdiameter 1 × frequency 1) + 2(diameter 2 × frequency 2) + 3(diameter 3 × frequency 3) + 4(diameter 4 × frequency 4), and the score ranges from 0 to 50, 0 signifying being bedbound and 50 signifying leaving the facility daily [LS was assessed using the Nursing Home Life-Space Diameter (NHLSD) . The NHLty daily  and was Individual characteristics including age, sex, and education were assessed at admission, and general medical health, number of medications, physical performance, pain, dementia severity, and NPS were assessed at admission and follow-ups.General medical health was assessed with the General Medical Health Rating (GMHR) , dichotoEnvironmental characteristics including staff-to-resident daytime ratio, unit size (unit defined as a group of residents living in a common area and having their own care staff during the day), and quality of the physical environment were assessed at one time point between October 2013 and December 2014. The collection of environmental characteristics in REDIC-NH has been described in detail previously . Staff-tIndividual and environmental characteristics were described as means and standard deviations (SD) or as frequencies and percentages. If one of the three items on the SPPB was missing, the total score was calculated as the sum of the two non-missing scores plus their average . MissingA growth mixture model (GMM) was estimated to identify potential groups of residents following distinct LS trajectories. Cases with missing NHLSD information at baseline were excluded from analyses. The number of groups was determined with Akaike’s Information Criterion. In addition, we aimed for reasonable group sizes, non-overlapping confidence intervals of trajectories, and average within-group probabilities of at least 0.80. Next, bivariate and multiple nominal regression analysis for hierarchical data was performed to assess whether individual and environmental characteristics measured at baseline were associated with group-belonging.To identify characteristics associated with overall LS trend, a linear mixed model (LMM) was estimated. The model with fixed effects for non-linear time was estimated first. Next, characteristics were included one at a time, as additional fixed effects, together with the interaction between time and characteristic. Lastly, a multiple model including all characteristics and corresponding interactions was estimated and reduced for excessive interactions using Akaike’s Information Criterion. Significant interactions are illustrated in Supplementary Figure Sp-values below 0.05 were considered statistically significant. The statistical analyses were performed using IBM SPSS V25, SAS V9.4, and STATA V14.All models contained random effects for residents nested within NH units. Only cases with no missing values on covariates were included in the regression analyses. In contrast to complete case models with respect to outcome variable, both GMM and LMM include data from all participants as well as from dropouts. Results with At admission, the mean age of residents with dementia was 84.1 years (SD 7.5), 64.5% were female, and daytime staff-to-resident ratio was 0.32 (SD 0.10) ; Group 2 ; Group 3 ; Group 4   or Group 4 (p = 0.005) compared to Group 2. Additionally, better physical performance (SPPB) at admission was associated with higher odds of being in Group 3 (p = 0.001) or Group 4 (p = 0.001) compared to Group 2. Fewer medications (p = 0.04), less severe dementia (p = 0.002), and more agitation (p = 0.002) at baseline were associated with higher odds of being in Group 4 compared to Group 2 (Table In the multiple model older age at admission was associated with lower odds of being in Group 3 (p < 0.001), having good compared to poor general medical health (p = 0.008), better physical performance , more agitation (NPI agitation) (p = 0.005), less apathy (NPI apathy) (p < 0.001), and living in a smaller unit (p = 0.003) were associated with on average wider LS throughout the study period (Table p = 0.004), i.e., the association became stronger from admission onward but levelled out toward the end of the follow-up period , corresponding to moving outside the unit less than weekly at admission to never after 3 years. Thus, most NH residents’ daily lives unfolded within their unit. This finding aligns with the average LS of NH residents reported in cross-sectional studies , 28, 29.Residents in Group 1 had the most restricted LS at admission, which continued to decline. After 3 years, this group’s LS corresponded with that of residents never moving outside their room. No characteristics measured at admission were significantly associated with belonging to Group 1 compared to belonging to Group 2. Not leaving the room could be associated with characteristics not measured in this study, such as specific diseases or conditions characterized by severe physical impairments and being bedridden. Being confined, this group stands out as especially vulnerable for inactivity. Lack of physical activity is associated with several risk factors for multiple negative health outcomes and reduced quality of life , underpiOf the four groups, Group 3 residents started with the widest LS but progressed to a substantial decline over time. Compared to Group 2, residents in Group 3 were younger and had better physical performance at baseline, characteristics positively associated with LS in NH residents , 28, 29 Residents in Group 4 were the only ones who demonstrated increased LS following admission and maintained the widest LS of the four groups. Group 4 residents were younger, had better physical performance and less severe dementia at admission compared to Group 2 residents. These three characteristics are associated with wider LS levels across populations , 28, 29.Younger age, having good compared to poor general medical health, less severe dementia, better physical performance, less apathy, more agitation, and living in a smaller unit were associated with on average wider LS throughout the study period. Associations with age, comorbidity, and dementia severity are supported by previous studies’ findings of community-dwelling older adults , 12, 14.Lack of initiative and motivation is an expression of apathy; thus, these symptoms are likely to negatively influence LS. Non-pharmacological interventions are recommended for treating NPS , and phyEnvironmental characteristics are accessible and potentially modifiable features that influence mobility within NHs, and higher-quality physical environments have been associated with improved activities of daily life and reduction in NPS , 27. HowFirstly, mortality in this population is high and the major reason for loss to follow-up Fig. . The staFrom NH admission most NH residents’ LS trajectory remained stable, and their daily lives unfolded within their unit. Better physical performance and less apathy emerged as potentially modifiable characteristics associated with wider LS over time. Future studies are encouraged to determine whether LS trajectories in NH residents are modifiable, and we suggest that future research further explore the impact of environmental characteristics beyond the physical environment.Additional file 1: Figure S1. Unadjusted overall trend in life-space (A) and change in association in time between life-space and CDR-SOB (B). NHLSD: Nursing Home Life-Space Diameter (0–50); CDR-SOB: Clinical Dementia Rating Scale Sum of Boxes. Table S1. Estimated life-space trajectories (N = 541). Table S2. Nursing Home Life-Space Diameter (NHLSD) score ranges.


To analyze the possible causes, treatment and outcomes of postoperative pneumothorax in patients with Gross type C esophageal atresia/tracheoesophageal fistula (EA/TEF).Medical records of patients with Gross type C EA/TEF who were diagnosed and treated in Beijing Children’s Hospital from January 2007 to January 2020 were retrospectively collected. They were divided into 2 groups according to whether postoperative pneumothorax occurred. Univariate and multivariate logistic regression analysis were performed to identify risk factors for pneumothorax.P < 0.001, OR 3.516 ] and mechanical ventilation  were independent risk factors for pneumothorax after EA/TEF repair. Further analysis of main parameters of mechanical ventilation after surgery showed that none of them were clearly related to the occurrence of pneumothorax. Among the 85 patients with pneumothorax, 33 gave up after surgery and 52 received further treatment . All of the 52 patients were cured of pneumothorax at discharge.A total of 188 patients were included, including 85 (45 %) in the pneumothorax group and 103 (55 %) in the non-pneumothorax group. Multivariate logistic regression analysis showed that postoperative anastomotic leakage [Anastomotic leakage and postoperative mechanical ventilation were risk factors for pneumothorax after repair of Gross type C EA/TEF, but the main parameters of mechanical ventilation had no clear correlation with pneumothorax. After symptomatic treatment, the prognosis of pneumothorax was good.The online version contains supplementary material available at 10.1186/s12887-021-02948-x. Esophageal atresia/tracheoesophageal fistula (EA/TEF) is a relatively common neonatal gastrointestinal tract malformation with an incidence of 1/4000-1/2500 , recurreThe medical records of patients with Gross type C EA/TEF diagnosed and treated in Beijing Children’s Hospital from January 2007 to January 2020 were retrospectively collected, and all of them underwent one-stage surgical repair (the patients with long-gap type C EA/TEF were excluded). Relevant information, including age, sex, birth weight, combined deformities, perioperative conditions, mechanical ventilation parameters, postoperative complications were extracted from electronic medical records and follow-up was performed. In this study, pneumothorax was defined as clinically significant pneumothorax within 2 weeks after the surgery, which was diagnosed by clinicians in combination with the patients’ symptoms , signs  and chest radiographs. Patients were divided into 2 groups according to whether pneumothorax occurred, and the differences in clinical characteristics and perioperative conditions between the 2 groups were compared. This study was approved by the Medical Ethics Committee of Beijing Children’s Hospital (2019-K-333) and waived patients from the requirement of informed consent.P < 0.05 indicated a statistically significant difference.SPSS 23.0 statistical software was used for analysis. Continuous variables were presented as the mean with standard deviation or median and interquartile range if the normality hypothesis test rejected the null hypothesis of normal distribution. Categorical variables were reported as counts and percentages. Pearson’s χ2 test, Fisher’s exact test, two independent samples t-tests and the non-parametric Mann-Whitney U test were used to compare characteristics between the groups. A total of 188 patients with Gross type C EA/TEF were included in this study, including 130 males (69 %) and 58 females (31 %). The median age at operation was 4  days, and the mean birth weight was (2.92 ± 0.50) kg. One hundred and fifty-four patients had other malformations . Fifty-five patients (29 %) and 127 patients (68 %) underwent thoracoscopic and open surgery, with 98 patients (52 %) and 90 patients (48 %) underwent extrapleural and intrapleural approach respectively (as shown in Supplementary Table P < 0.001) and mechanical ventilation (P = 0.025) were statistically significant, while there were no significant differences in other clinical features between the 2 groups (P > 0.05). Multivariate logistic regression analysis showed that postoperative anastomotic leakage  and mechanical ventilation  were independent risk factors for pneumothorax after EA/TEF repair  and non-pneumothorax group 103, 55 %), and the clinical characteristics and perioperative conditions of the 2 groups were compared. As shown in Table 03, 55 %,P = 0.005) and peak inspiratory pressure  were higher in the non-pneumothorax group. However there were no significant differences in positive end-expiratory pressure  and fraction of inspiration O2  between the 2 groups. In patients with anastomotic leakage, there were no statistically significant differences in ventilator parameters between the 2 groups . Therefore, these results did not support the correlation between mechanical ventilation parameters and occurrence of pneumothorax after EA/TEF repair.In order to explore the relationship between mechanical ventilation and pneumothorax, 78 patients who entered intensive care unit (ICU) after surgery and received mechanical ventilation who did not give up treatment were enrolled into the further analysis of ventilator parameters when pneumothorax appeared. These patients were divided into 2 groups as combined with anastomotic leakage (n = 18) and without anastomotic leakage (n = 60). As shown in Table Among the 85 patients with pneumothorax in this study, 33 gave up treatment after surgery , 52 patients received further treatment . All of the 52 patients were cured of pneumothorax at discharge, 45 patients survived during follow-up, 1 patient died out of hospital due to respiratory failure, and 6 patients were lost to follow-up.Pneumothorax is a common complication after EA/TEF repair, the incidence of pneumothorax in this cohort is 45 %. Severe pneumothorax can affect the respiratory and circulatory system, even endanger life. The treatment strategies of pneumothorax include conservative observation, thorax puncture, and thoracic drainage, and most patients can often be cured. However, the pathogenesis of pneumothorax after EA/TEF repair has rarely been discussed in previous reports. In this study, we reviewed the clinical data of 188 patients with Gross type C EA/TEF and found that anastomotic leakage and mechanical ventilation were independent risk factors for pneumothorax. However, mechanical ventilator parameters could not be considered as being related to pneumothorax.Anastomotic leakage, one of the complications with a high incidence after EA/TEF repair , is closPrevious studies have shown that there is a certain relationship between mechanical ventilation and pneumothorax , 18, but2 were not provided. Diez [2O respectively. The authors believed that some children needed CPAP for respiratory support after extubation, but the PEEP value should not be too high [2O, respectively, which were lower than the former report [Some scholars  believe ed. Diez  reportedtoo high . High PEr report . In addir report  believedThere are some limitations in this study. Surgical details, and perioperative management  changed over time, and access to surgical details and postoperative complications information was limited. Furthermore, the sample size in a single center and the high proportion of patients who gave up and who were lost to follow-up also limited the conclusions in this study. Thus, the relationship between mechanical ventilation and pneumothorax still needs to be further explored in future multi-center studies with a large sample size.The incidence of pneumothorax after EA/TEF repair is about 45 %, and postoperative anastomotic leakage and mechanical ventilation are independent risk factors for the occurrence of pneumothorax. However, the main parameters of mechanical ventilation have no clear correlation with the occurrence of pneumothorax. After timely symptomatic treatment, patients with pneumothorax can have a good prognosis.Additional file 1.Additional file 2.


LVV was diagnosed in five patients, two with aortitis and three with temporal arteritis, corresponding to a total prevalence of 5.0% [95% CI 1.6–11.2%]. This value was significantly higher than the estimated prevalence of LVV in the normal Swiss population . All five patients had GPA, whereas no cases with EGPA or MPA were identified. Anti-PR3 antibodies were detected in four out of five patients, anti-MPO in one patient. Since LVV can occur in a significant proportion of patients with GPA, evaluation for LVV may be considered systematically in the diagnostic workup of AAV.ANCA-associated vasculitis (AAV) in general involves small blood vessels and includes granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA), and microscopic polyangiitis (MPA). Although reported in a few studies, the prevalence of large vessel vasculitis (LVV) in patients with AAV remains to be further explored. The goal of the present study was to assess the prevalence of LVV in a cohort of patients with AAV and to characterize this population. We conducted a ten-year retrospective study of a single-center cohort of AAV, including 101 patients with GPA ( According to the 2012 revised international Chapel Hill consensus conference nomenclature of vasculitides, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) are small vessels vasculitis, encompassing granulomatosis with polyangiitis , microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis , and single-organ AAV   of our overall cohort, or in 5/58 (9%) patients with GPA. These values are clearly higher compared to the prevalence of GCA in a general population close to Switzerland (Freiburg im Breisgau in Germany with 222 cases per million inhabitants ), resultCase 1: A 66-year-old woman presented with chronic bloody nasal discharge, sero-mucous otitis and acute arthritis of the right elbow. With the onset of episcleritis and the finding of cANCA-PR3, a diagnosis of GPA was made. The patient was treated with prednisone and cyclophosphamide, subsequently replaced by mycophenolate mofetil because of hemorrhagic cystitis. The evolution was favorable. Due to weight loss of 10 kg a thoraco-abdominal CT was performed 10 years later revealing hyperdense spontaneous thickening with enhancement around the ascending and horizontal aorta. In addition pulmonary opacity of the right lower lobe was detected and a diagnosis of mononeuropathy with falling foot was made. A PET-CT showed hypermetabolism (SUV max 8.1) at the aortic arch and the ascending aorta. cANCA-PR3 were present at all times. In this context, immunosuppression by corticosteroids and mycophenolate mofetil was intensified. PET-CT scanning 18 months later showed a clear decrease in hypercaptation.Case 2: A 52-year-old woman known for intracranial hypertension for 7 years, who was hospitalized for the investigation of headaches with tonic epileptic seizures. A brain MRI showed diffuse pachymeningitis. Due to unusual headaches located in the temporal area associated with hyperesthesia of the scalp and inflammatory syndrome, a temporal artery biopsy was performed and showed inflammatory lymphocytic infiltrate with intimal fibrosis, disappearance of the elastic lamina, and vasa vasorum granulomas. Nasal biopsy revealed mixed inflammation with rough granuloma. With MRI pansinusitis and positive cANCA-PR3, the diagnosis of GPA was made, and the patient was treated with corticosteroids and rituximab with an improvement in inflammatory syndrome and symptoms.Case 3: A 76-year-old woman presented with progressive bilateral headaches and jaw claudication. The laboratory results revealed inflammation with a CRP of 120 mg/l and a sedimentation rate of 67 mm/h. A temporal biopsy showed a granulomatous and giant cell vasculitis  with normalization of erythrocyte sedimentation rate and methotrexate for a total duration of 4 years. Following the appearance of an orbital pseudotumor with diplopia, treatment with rituximab was able to induce and maintain remission.Case 5: A 46-year-old woman, known for GPA with nasal polyps, lower limb neuropathy, and optic neuritis with granulomatous ocular infiltration, presented with aortic dissection of type A Fig. A. She beThe clinical characteristics of the five AAV patients with LVV are summarized in Table p = 0.65) . Among the five AAV patients with LVV, all having GPA, ENT manifestations seemed to be more predominant (100%), with less renal, pulmonary, neurological, and joint involvement. In the AAV group without LVV, lung manifestations (73%) were more frequent than of ENT manifestations (60%), and renal (50%) impairment (Table p = 0.39), dyslipidemia (OR 1.70 [0.29–8.67] p = 0.62), and smoking (OR 6.39 [0.97–79.13] p = 0.16). Finally, there was more cardiac disease of any cause  (OR 6.37 [0.97–79.13] p = 0.15), and more cancer (OR 3.09 [0.55–18.20] p = 0.21) in the group with large vessel involvement .Table 65) Fig. A, and thtations 7% were moent Fig. B. There PET-CT was performed in 12 of the 101 patients and positive for LVV in one case (patient 1). However, most patients (74/101) had an injected thoracic CT-scan which makes it possible to detect vasculitis of the large vessels by wall thickening and uptake of contrast medium of the arterial wall. Of the remaining patients, 12 of the 27 patients had a cardiac ultrasound. Three temporal artery biopsies were performed when typical clinical symptoms of temporal arteritis were present (2/3 positive). In one case, histological examination was performed following aortic replacement (patient 5).The overlap between LVV and AAV is rare. Nevertheless, we found a prevalence of 5.0% [95% CI 1.6–11.2%] in our cohort of 101 patients. Beside several case reports, there is little systematic data available. Nishino et al. reported five cases of temporal arteritis in a cohort of 345 patients with GPA, corresponding to a prevalence of only 1.4% . FurtherSkeik N et al. report ten cases of aortitis related to AAV, in addition to their case report, and suggested using rituximab for treatment . By studThe prevalence of LVV in our study was significantly higher than that of the southern German population. However, it is important to emphasize that this hospital-based prevalence is not directly comparable to a population-based prevalence.The pathophysiologic mechanisms of large vessels involvement in AAV are largely unknown. While some authors speculate on invasive inflammatory cells and granulomas causing aortitis, others believe that inflammation results from necrotizing vasculitis involving the vasa vasorum of the aortic wall (like in Takayasu disease) . But thep = 0.15). This could be explained either by a link between GCA and cardiovascular risk factors, there was no age difference. The trend for a higher slightly higher prevalence of cancers (3.09 [0.55–18.20] p = 0.21) can also not be explained by age. Finally, we note that LVV patients have slightly more relapse. Unfortunately, our study did not have the statistical power to detect statistically significant differences between the groups with and without involvement of the large vessels. Thus, it is difficult to draw firm conclusions and studies with more cases are needed to corroborate our results.Analysis of the demographic data of AAV patients with large-vessel involvement does not show any clear differences. ENT manifestations seemed to be more common, not surprisingly since all cases with LVV were identified among patients with GPA. Investigation of cardiovascular risk factors revealed a trend towards a higher prevalence of hypertension, dyslipidemia, and tobacco in the LVV group. Moreover, we found more cardiac disease of any type  in the LVV group  or to request additional investigations in the search for LVV. However, this question needs to be addressed in a large multi-center study.Large-vessels involvement was found in five AAV patients  in our cohort of 101 patients with GPA, EGPA, MPA, resulting in a hospital-based prevalence of 5.0%. According to the clinical importance of this co-existing diseases, evaluation for LVV may be considered systematically in the future diagnostic workup of AAV.


Multiple regression analyses showed a significant negative association between the control network CV and the severity of irritability. Mediation analysis showed that IQ partially mediated the association between the control network CV and the severity of irritability. Follow-up analysis on subcortical volume (SCV) showed that adolescents with high irritability had reduced bilateral SCV within the amygdala relative to adolescents with low irritability. Reduced CV within bilateral control and default networks and reduced SCV within bilateral amygdala may represent core features of the pathophysiology of irritability. The current data also indicate the potential importance of a patient’s IQ in determining how pathophysiology related to the control network is expressed.Previous studies examining structural brain correlates of irritability have taken a region-specific approach and have been relatively inconsistent. In a sample of adolescents with and without clinically impairing irritability, the current study examines: (i) cortical volume (CV) in canonical functional networks; (ii) the association between the CV of functional networks and severity of irritability; and (iii) the extent to which IQ mediates the association between structural abnormalities and severity of irritability. Structural MRI and IQ data were collected from 130 adolescents with high irritability  and 119 adolescents with low irritability . Subject-specific network-wise CV was estimated after parcellating the whole brain into 17 previously reported functional networks. Our Multivariate Analysis of Covariance (MANCOVA) revealed that adolescents with high irritability had significantly reduced CV of the bilateral control and default-mode networks ( Irritability is the propensity to initiate an angry response to frustration, threat, and social provocation and can be defined as “mood (trait) and behavioral (reactive state) dysregulation” that results in a propensity to react and respond angrily when an individual’s ability to attain a goal is blocked –3. It caFunctional neuroimaging work has relatively consistently related irritability to dysfunctions in both response control –9 and emcortical networks, therefore, in our main analysis, we used CV as our primary parameter to quantify brain morphometry of each of the 17 cortical networks, whereas in follow-up analysis, we repeated our analysis and explored network-wise thickness and surface area, as well as subcortical volume (SCV) of the six main subcortical structures .The first two goals of the current study were to determine network-wise cortical volume (CV) differences between adolescents with high irritability and adolescents with low irritability, and to further investigate associations between CV of identified networks (and their component regions) and irritability symptom severity. We measured CV from 17 cortical networks using one of the most advanced analytical methods, which does not over- or under-represent tissue according to the cortical convolutions . SpecifiThe third goal of this study was to determine the extent to which IQ mediates the association between the network-wise CV and irritability symptom severity. Previous work has shown strong associations between the cortical structure and IQ –23. SpecThe goals of the current study were to (i) identify brain networks that show differences in CV between adolescents with high irritability and adolescents with low irritability; (ii) investigate the association between CV of identified brain networks (and their component regions) and irritability; and (iii) determine the extent to which IQ mediates the association between the altered network-wise cortical structure and greater levels of irritability. Given the prior neuroimaging work on irritability and dysfunctions in response control and emotional responding –11 and mMage = 15.33±1.89 years, 97 females). Participants were recruited from a residential care facility at Boys Town National Research Hospital (BTNRH) and from the surrounding community. Participants recruited from the residential facility had been referred for behavioral and emotional problems. Participants were divided into high and low irritability groups based on their irritability scores and were matched across sex and age. Specifically, following guideline from previous work  of the Wechsler Abbreviated Scale of Intelligence II (WASI-II)  was usedThe ARI is a seven-item self-report questionnaire  that assesses the youth’s irritability during the preceding 6 months . ParticiConners ADHD scale  and hemispheric mean CV of identified networks significantly predicted irritability scores. To deal with multicollinearity, the regression analyses were performed for each measure of psychopathology separately.Sex differences in irritability scores were determined used two-sample All the main analyses  performed for CV were repeated for cortical thickness and cortical surface area. For cortical thickness analysis, the potential covariates were sex, age, and IQ, whereas for cortical surface area analysis, the potential covariates were sex, age, IQ, and ICV.All the main analyses  performed for CV were repeated for subcortical volume for six subcortical areas i.e., thalamus, caudate, putamen, pallidum, hippocampus, and amygdala. For subcortical volume analysis, the potential covariates were sex, age, IQ, and ICV.Based on age and ARI scores, none of the participants were identified as outliers. However, two participants (one from each irritability group) had IQ more than 1.5 interquartile range above the upper quartile and were, therefore, excluded from further analysis.χ2 = 3.67, p = 0.06) and age (t (245) = −1.85, p = 0.065; Mhigh irritability group = 15.56, SD = 1.83; Mlow irritability group =15.12, SD = 1.93). However, there were significant group differences in IQ (t(245) = 5.49, p < 0.001; Mhigh irritability group = 99.14, SD = 12.13; Mlow irritability group =107.64, SD = 12.21) and ICV (t(245) = 2.45, p = 0.01; Mhigh irritability group = 1.47 × 106 mm3, SD = 0.15 × 106; Mlow irritability group =1.52 × 106 mm3, SD = 0.15 × 106); see Table There were no group differences in sex  = 1.55, p = 0.03; pη2 = 0.20; Wilk’s lambda = 0.80]. There were significant differences bilaterally in CV for the control B network  = 6.79 & 8.59, p = 0.01 & 0.004, pη2 = 0.03 & 0.03, respectively for left and right hemisphere]; Fig. F  = 9.83 & 7.22, p = 0.002 & 0.01, pη2 = 0.04 & 0.03, respectively for left and right hemisphere]; Fig. p < 0.05  = 11.91; p = 0.001]. Adjusted R2 was 0.15. Significant predictors for irritability scores were CBN CV  and IQ . DBN CV, sex, age, and ICV were nonsignificant predictors for irritability scores .Our stepwise multiple regression analysis revealed a significant regression equation for irritability scores  and cortical surface area .Our MANCOVA showed nonsignificant group differences in cortical thickness . No bilateral significant differences in SCV  were seen in any of the subcortical structures at p < 0.05  = 3.76, p = 0.05 and right amygdala: F  = 4.12, p = 0.04). The amygdala also showed a significant negative partial association between hemispheric mean SCV and irritability symptom severity .Our MANCOVA showed significant group differences in SCV [specifically the caudal middle frontal cortices, inferior temporal cortex, and inferior parietal cortex) and amygdala SCV with irritability symptom severity. Moreover, IQ partially mediated the association between the CBN CV and irritability symptom severity.The goals of the current study were to identify the following: (i) networks showing CV differences in adolescents with high irritability relative to adolescents with low irritability; (ii) the extent to which network-wise CV and their component regions predicted irritability symptom severity; and (iii) the extent to which IQ mediated the association between CV of identified networks/regions and irritability symptom severity. Our data indicated that, relative to participants with low irritability, adolescents with high irritability had reduced CV within the control B and the default B networks (CBN and DBN) and reduced bilateral SCV within the amygdala. There was a negative association of CBN CV . While there were group differences in network volumes and IQ, IQ could not account for these differences—given the results of our stepwise multiple regression with IQ as a covariate. To our knowledge, these results are the first to show network-wise structural alterations (at a whole-brain level) associated with elevated irritability. Prior structural MRI studies have mostly reported region-specific structural alterations associated with irritability –14, 17. In contrast to our hypotheses, there were no significant group differences within the limbic network. Given strong empirical claims that irritability is associated with atypical emotional responding , 55, thiCV within the CBN, but not the DBN, was negatively associated with irritability symptom severity. With respect to specific anatomical regions within the CBN, there were significant negative associations between CV of the caudal middle frontal cortex, inferior temporal cortex, and the inferior parietal cortex and irritability  and irritability. Given the cross-sectional nature of the current study, the mediation findings are purely based on correlation analyses and should not be interpreted or viewed as “causal” in nature. These results are consistent with our predictions generated from previous work indicating that IQ  is inversely associated with the risk for the expression of a variety of psychiatric disorders, including ADHD , depressThe current findings must be viewed considering the following three caveats. First, there were group differences in IQ and ICV. However, mitigating this concern, the effect of IQ and ICV was controlled for within our group analyses. Second, networks of interest were constrained only if there were bilateral significant differences in morphometric measures. Therefore, the hemispheric laterality was not analyzed in the current study. Third, the participants with high irritability showed significant psychiatric comorbidities and some were receiving psychiatric medications. However, the follow-up analyses that we conducted with psychiatric comorbidities and psychiatric medications as additional predictors in our multiple regression analyses mirrored our results. This suggested that psychiatric comorbidities and psychiatric medications did not confound the current results and that CBN CV was still one of the significant predictors of irritability.In summary, we found significantly lower CV within the control and default-mode networks in adolescents with high irritability as compared to adolescents with low irritability. Moreover, control network CV showed a significant negative association with irritability. The association between control network CV and irritability was partially mediated by IQ. The current findings enhance our fundamental understanding of cortical structure and irritability at the level of large-scale brain networks that have been specified by considerable prior functional and structural MRI work –74, and Supplementary InformationSupplementary MaterialSupplementary TableSupplementary Figure