BRCA2) is a tumor suppressor gene. The protein encoded by this gene plays an important role in homologous recombination (HR)-mediated DNA repair. Deleterious mutations in BRCA2 and downregulation of its expression have been associated with tumorigenesis in dogs and humans. Thus, regulation of BRCA2 expression level is important for maintaining homeostasis in homologous recombination.Breast cancer 2, early onset (BRCA2 were proposed. Novel splicing variants were identified in the 5′ untranslated region (UTR) of canine and human BRCA2 in canine testis, canine ovary, and canine and human cultured cell lines. In cultured cells, the ratio of BRCA2 splicing variants at the 5′ UTR was altered by serum starvation. These novel splicing variants, excluding one of the canine splicing variants, were found to reduce the translational efficiency. Additionally, the DNA sequence in human BRCA2 intron 1 harbored novel cis-regulatory elements. Three silencer and two enhancer cis-regulatory elements were identified in human BRCA2 intron 1.In this study, the mechanisms that regulate the expression of BRCA2 intron 1 region harbors cis-regulatory elements.This study demonstrates that BRCA2 expression level is regulated via 5′ UTR splicing variants and that the  BRCA2) is a tumor suppressor gene encoding a protein that contributes to homologous recombination (HR)-mediated DNA repair [BRCA2 were both associated with canine mammary tumors [BRCA2 mutation [BRCA2 is one of the causes of tumorigenesis. In cells, reduced or excess BRCA2 expression creates an imbalance between BRCA2 and RAD51. RAD51 is a DNA recombinase that interacts with BRCA2 and is recruited to DNA damage sites to carry out strand invasion, which is a vital step of homologous recombination. The imbalance between these gene products affects HR activity [Breast cancer 2 early onset  should also be associated with silencer elements. To avoid translational effects, the human BRCA2 intron 1 region without promoter sequence (− 187 − + 310 bp) was inserted upstream of human BRCA2 promoter . Next, we attempted to identify where the silencer element was located within the 500 bp sequence. Sequential deletion mutants in 5′ and 3′ ends were compared with the promoter activity . Unexpectedly, the region around + 643 − + 742 bp harbored an enhancer element, and the + 543 − + 642 bp region contained a silencer element . We also speculated that these splicing variants contributed to the protein expression after X-ray irradiation, as greater amounts of transcripts of splicing variants were induced in response to DNA damage [The expression ratios of these splicing variants were altered by the condition of the cells. In CNM-p and CHM-p canine cultured cells, the ratio of transcripts without the intron 1 region compared to total BRCA2 was decreased after serum starvation, which induced G1 phase arrest. Especially in CNH-p and CHM-p cells, the splicing variant pattern was also shifted to favor the long form after serum starvation. Splicing variants containing the intron 1 region, excluding canine splicing variant I, showed decreased translational activity. Thus, in these cells, BRCA2 protein levels were decreased via suppression of translation. MDCK and CIP-p canine cells demonstrated an increased ratio of splicing variants without the intron 1 region after serum starvation. In addition to this, the decreased level of A damage , 24. HowBRCA2 possesses 5–8 start codons which potentially translate short peptides or truncated proteins. As the reference sequence does not contain a start codon in the 5′ UTR, these upstream start codons in the 5′ UTR potentially prevent translation efficiency of the primary open reading frame. The second possibility involves the RNA secondary structure. Both novel splicing variants showed complex and bulky structures according to a secondary RNA structure prediction program. This type of structure obstructs ribosome sliding and scanning. Some genes are regulated by the RNA secondary structure of splicing variants at the 5′ UTR [BRCA2 still possessed similar translational activity compared to the registered sequence, canine and human BRCA2 are potentially regulated by these mechanisms.The mechanisms underlying the suppression of expression by these novel splicing variants are unknown, but there are two possibilities based on the reports on the regulation of other genes . First, e 5′ UTR . AlthougBRCA2 promoter silencer elements, but this is the first study to show that this region, which is a part of the BRCA2 intron 1, functions as cis-regulatory elements [BRCA2 intron 1, accumulation of histone H3K27Ac, which indicates the presence of active promoter and enhancer regions, were reported by the UCSC genome browser (https://genome.ucsc.edu). We did not investigate which trans-regulatory elements interact with this novel cis-regulatory element. This point will be the next important research issue to understand how BRCA2 expression is regulated.The sequence of the splicing variants affected transcription efficiency as well as translation. Thus, canine and human intron 1 regions in the genomic DNA act as cis-regulatory elements. A few studies have described elements –17, 26. BRCA2 was identified more than a twenty-five year ago and many studies of this gene have been reported in humans, canine and mice, the regulatory system identified in this study has not been previously reported. Both canine and human BRCA2 had similar splicing variants, thus it seemed that mammalian BRCA2 employs splicing variants at the 5′ UTR as an expression regulation system. Parts of the intron 1 region were conserved between human and canine, thus transcriptional regulation by the novel cis-regulatory element also seemed to be conserved in mammals. These points are interesting when considering the evolution of BRCA2.Although BRCA2 in canine ovary and testis and canine and human cell lines. These splicing variants reduced the translation efficiency of the gene. The expression ratio of splicing variants was altered depending on the conditions in which the cells were maintained. Unexpectedly, the intron 1 sequence in BRCA2 also suppressed transcription efficiency. This led to the identification of silencer and enhancer cis-regulatory elements in BRCA2 intron 1 region. Although further studies are needed to determine how the splicing variants regulate translation and how novel cis-regulatory elements affect transcription, our data suggests that the BRCA2 expression level was regulated by the novel splicing variants at the 5′ UTR and cis-regulatory elements in intron 1 in canine and human.In this study, we identified novel splicing variants at the 5′ UTR of canine and human 2–95% air.All cultured cells were grown in 100 mm plastic dishes containing 10 mL of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under humidified 5% COTwo ovary and one testis samples from three 1-year-old mixed breed dogs were obtained from the Honda Animal Hospital. This sampling was done as part of contraception or castration under general anesthesia by veterinarians. These tissue samples in RNAlater solution  and all cultured cell pellets were stored at − 80 °C until they were processed for RNA extraction using TRIzol RNA Isolation Reagents  as previously described . All expBRCA2, each reaction mixture contained 100 ng of genomic DNA as a template, and 300 nM forward and reverse primers  according to the manufacturer’s instructions. To amplify canine and human 5 HeLa cells were transiently co-transfected with 20 ng canine or human BRCA2 5′ UTR and firefly luciferase cloned pcDNA3 (Invitrogen) or pGL4 (Promega) vectors and 10 ng pRL-TK (Promega) using FuGENE HD Transfection Reagent . Forty-eight hours after transfection, the cells were lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s instructions. Transfection efficiency was normalized by measuring Renilla luciferase activity.Approximately 2 × 10RPS18 was used as an internal reference for normalization. The human BRCA2 exon 27 region and firefly luciferase from genomic DNA samples were used to normalize the transfection efficiency.RNA was extracted from the transfected cells using the CellAmp™ Direct RNA Prep Kit for RT-PCR  , and cDNA synthesis was performed as described above. Genomic DNA was also prepared using a Gentra Puregene Cell Kit . Quantitative real-time polymerase chain reaction (qPCR) was carried out in a StepOnePlus Real-Time PCR System  using a KAPA SYBR FAST qPCR Kit  and 200 nM of each primer Table . The PCR5 HeLa cells were seeded into 4-well plates. After 24 h, irradiation of HeLa cells in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum was carried out in a MX-80Labo  for a total dose of 10 Gy  at room temperature. Irradiated cells were incubated at 37 °C under humidified 5% CO2–95% air and after the indicated time, the cells were harvested and the RNA was extracted.Approximately 2 × 10t-test. To compare more than three groups, one-way analysis of variance with Dunnett’s test or F test followed by Student’s t-test correcting with Holm’s method was performed.To compare two groups, statistical analyses were performed using an F test followed by Student’s Additional file 1: Fig. S1. Messenger RNA base sequences encoded by exon 1-intron 1-exon 2 region of canine and human BRCA2. Bold characters indicate possible start codons and bold AUG with box indicates actual start sites in exon 2. Underlined characters indicate the GU-AG sites for splicing.


SHOX haploinsufficiency causes 70–90% of Léri-Weill dyschondrosteosis (LWD) and 2–10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5′UTR variants , were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5′UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5′UTR. SHOX), is located within the pseudoautosomal region 1 (PAR1) on Xp22.33/Yp11.32 and encodes a transcription factor that regulates chondrocyte proliferation and differentiation in the growth plate [SHOX is controlled by seven cis-acting conserved noncoding elements (CNEs), residing both upstream and downstream of the gene that exhibits enhancer function in chicken limb buds and neural tubes [The short-stature homeobox gene , results in a wide variety of short stature phenotypes. Molecular defects in SHOX were frequently reported to be responsible for short stature in patients with Leri-Weill syndrome , characterized by skeletal dysplasia with disproportionate short stature, mesomelia  and characteristic Madelung wrist deformity [SHOX mutations were also identified as the cause of growth retardation in 2–10% of individuals with idiopathic short stature  [SHOX expression results in an extreme phenotype of osteodysplasia called Langer mesomelic dysplasia (MIM #249700), which is characterized by extreme short stature, severe shortening or aplasia of the ulna and fibula, and both thickening and curvature of the radius and tibia [eformity . SHOX mu#300582) –11. On tSHOX alterations exhibit considerable phenotypic heterogeneity, and in several cases the proband’s relatives carrying the causative variants are apparently asymptomatic with normal height.Individuals carrying SHOX defects are represented by deletions of different sizes encompassing the whole gene and/or the CNEs, whereas intragenic microdeletions and point mutations occur in the rest of the cases [SHOX coding region [SHOX and/or its enhancers have been reported in patients [Approximately 75% of the he cases . The enhg region  and idenpatients –18 althoThe wide spectrum of phenotypes is likely the result of different degrees of SHOX deficiency attributable to variations that differentially alter the expression levels in combination with different genetic backgrounds.The correct SHOX function is strictly dose-dependent, as demonstrated by the observation that ~25–50% of the causative deletions encompass only the cis-acting enhancer, leaving the coding region intact , 19, 20.SHOX is directed by two alternative promoters, P1 (upstream promoter) and P2 (intragenic promoter), generating transcripts with a 5′UTR of different lengths encoding the same protein. The molecular mechanism underlying the preferential usage of one promoter over the other remains unknown. There is evidence that the transcripts generated from P1 exhibit significant translation inhibitory effects due to seven AUG codons upstream of the main open reading frame [2 are translated with higher efficiency, probably in situations of immediate need of high SHOX amounts. Using several reporter constructs, Blaschke et al. [2 to a region of 300 bp upstream of the AUG start codon. This region contains a canonical TATA box (c.-137) and a CAAT box (c.-257), indicating the core promoter characteristics of P2.The short-range transcription of ng frame  while the et al.  narrowedSHOX performed in the last 8 years on ISS/LWD patients, we identified several individuals carrying different noncoding variants in exon 2 within the 5′UTR of both the transcripts that were classified as VUS (Variants of Uncertain Significance).During the diagnostic screening of SHOX haploinsufficiency either through a reduced gene expression or by affecting the correct RNA splicing.This work aimed to investigate the pathogenic role of these variants by testing their ability to interfere with the correct gene expression and consequently to alter the dosage of the SHOX protein. Our results showed that these noncoding variants might be responsible for SHOX diagnostic testing included 1036 unrelated individuals recruited from multiple Italian centers. Informed written consent was obtained from all the patients or their parents. The patients presented with different phenotypes ranging from ISS to extreme disproportionate short stature to the most severe form of LWD.The For every patient, height, weight, and BMI were stratified according to the Italian growth charts . MeasureSHOX haploinsufficiency such as: cubitus valgus, short forearm, bowing of the forearm, muscular hypertrophy, dislocation of ulna and BMI percentile. A combination of these signs together with sitting height/height ratio and arm span/height ratio was used retrospectively to calculate the Rappold Score [Particular attention was given to clinical sign characteristic of ld Score  (RS) thaA panel of 759 normal statured (−0.5 ≥ SDS ≤ 2.2) healthy individuals matched for sex and geographical origin was also screened for the exon 2 variations.SHOX coding region (exon 2–exon 6a/6b), 5′UTR (exon 1–exon 2) and intron–exon boundaries were amplified by PCR . The entire UTR exon –exon 2 aSearch for deletions/duplications of the single exons, of the entire gene, and of the upstream and downstream enhancers was performed by an MLPA assay using an MLPA Commercial Kit  following the manufacturer’s instructions.SHOX was NG_009385.2, GRCh37/hg19 assembly. Transcript references NM_000451.3 and NM_006883.2 were used for the transcript variants SHOXa and SHOXb, respectively. The two transcripts, identical at the 5′ end (exons 1–5), differ in the final exon (6a vs 6b) at the 3′ end .The variants were submitted to Leiden Open Variation DatabaseSHOX were predicted using the computational tool Human Splicing Finder version 3.1 [http://www.umd.be/HSF/). The potential miRNA target sites were searched in the SHOX 5′UTR using miRWalk 2.0 [http://mirwalk.umm.uni-heidelberg.de/). VarSome [www.varsome.com) was used to predict the pathogenicity of the identified variants according to the ACMG guidelines [Splicing regulatory sequences in sion 3.1   was cloned immediately upstream of the Firefly cDNA. The pGL3-basic construct bearing wild-type 5′-UTR fragment was used as the template into which the variants c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A and c.-9del were introduced by Q5®site-directed mutagenesis kit according to the procedure recommended by the supplier . All the variations in the plasmids were confirmed by bidirectional sequence analysis.2OS cells were maintained in DMEM High Glucose (Gibco-Life Technologies) supplemented with 10% fetal calf serum and 1% Penicillin/Streptomycin in 5% CO2 at 37 °C. A day before transfection 1 × 105 cells were seeded into each well of a 24-well tissue culture plate in 500 μl CGM (Complete Growth Medium). The wells were previously treated with 1:10 dilution Poly-L-lysine solution (Sigma Aldrich) to allow the cells to completely adhere to the plate surface. At 70–90% confluency, cells were transfected with wild-type or mutagenized reporter plasmid constructs and internal control pRL-TK constructs (expressing renilla luciferase gene) with Lipofectamine 2000 transfection reagent (Life Technologies).Ut test was used to determine significant differences of each mutated construct compared with the wild-type construct.Two days after transfection, growth media were removed, and cells were washed gently with phosphate buffered saline. Passive lysis buffer  100 µl/well was added and with gentle rocking for 15 min at room temperature cell lysates were harvested for DLR assay. The activities of firefly and Renilla luciferase were measured using the dual-luciferase reporter assay system  according to the manual of the manufacturer. For each luminescence reading, after injector dispensing assay reagents into each well, there would be a 2-s pre-measurement delay, followed by a 10-s measurement period. In total, 10 µl of cell lysate were transferred in white opaque 96-well plate. The luminescence obtained for the mutated and wild-type constructs were normalized with the internal control Renilla luciferase signal and the activity of the mutated constructs was reported as percentage with respect to the wild-type. Each experiment was performed in triplicate, and three independent experiments were performed. Quantitative data of the reporter gene assay are calculated as mean ± SEM. Student’s 2OS cells were seeded into six-well plates and transfected at 70–80% confluence with 2 µg of the pGL3-based luciferase vectors containing the wild-type or the mutagenized SHOX 5′-UTR. Twenty-four hours after transfection, cells were harvested and washed once with phosphate buffer saline. Total RNA was extracted using the QIAGEN RNA Mini Kit . Purity and concentration of RNA and genomic DNA were evaluated using a NanoDrop ND1000 spectrophotometer. cDNA was generated from 1 µg of RNA by using the GoScript™ Reverse Transcriptase . Real-time PCR was performed using a cDNA aliquot equivalent to 50 ng of converted RNA using a CFX-96 Real-Time System thermal cycler  and the GoTaq® qPCR Master Mix . Relative mRNA quantification was obtained using the ΔCt method, considering the efficiency of cDNA synthesis by the quantification of the Beta2Microglobulin (β2M) housekeeping gene from the same cDNA.USHOX exon 2 flanked by 123 bp of the 3′region intron 1 and 168 bp of the 5′region of intron 2 of SHOX were amplified  were seeded in a six-well culture plate and incubated at 37 °C in a 5% CO2 atmosphere in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum . On the following day, 2 μg of wild-type or mutant vectors were transfected using Lipofectamin 2000 transfection reagent (Life Technologies). The culture medium was changed after 4 h. After 48 h, the cells were harvested and total RNA was isolated using the QIAGEN RNA mini kit (QIAGEN). cDNA was synthesized from 1 µg of RNA by the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to the manufacturer’s instructions. Using vector exon-specific primers, cDNAs produced from the mini-gene constructs were specifically PCR amplified and Sanger sequenced.The U2O-S cells , placed upstream of the firefly luciferase reporter gene (pGL3-SHOX), was created. The transcription efficiency of the plasmid carrying the five variants, including c.-55C > T as a potential negative control, was measured and compared with the wild-type by transfecting a U2OS cell line. Plasmids carrying c.-55C > T and c.-58G > T did not show any significant reduction in the level of luciferase activity compared with the wild-type , 35% (p = 0.02147), and 40% (p = 0.0262), respectively.To evaluate whether the P gene pGL-SHOX, wa gene pGL-SHOX, waSHOX expression, mainly at the post-transcriptional level  predicted that the variant c.-19G > A alters the correct splicing between exons 1 and 2 in mRNA1 by creating a consensus sequence for a novel putative branch point within exon 2. The consequence might be the usage of an alternative cryptic acceptor splice site located downstream of the canonical AUG . Conversely, the plasmid carrying c.-19A, in addition to the wild-type 971 bp fragment, produced a smaller product of 509 bp. The additional 509 bp band is originated by the creation of the strong branch site at c.-19 that promotes the usage of the cryptic acceptor splice site located 27 bp downstream of the canonical AUG start codon  of the probands carrying SHOX alterations. Based on the current knowledge, it was not possible to attribute a pathogenic role to these variations that were thus classified as VUS.During the SHOX-related disorders unless carriers either clearly manifest the clinical signs or the variant arose de novo.Analysis of the family members of the patients Fig.  revealedSHOX 5′UTR might influence gene expression and correct splicing and might thus lead to insufficiency of the SHOX protein and thus to related disorders.Thus, the establishment of functional studies is of crucial importance to gain insight into the significance of these noncoding variants. The 5′UTRs are the sites that serve ribosomes for scanning the mRNA for a suitable translational start codon at which the translation initiation complex can be assembled. This scanning can be influenced by several cis elements, including mRNA secondary structures, the presence of upstream initiation codons and in frame stop codons that act as cis regulatory elements , 32. PreSHOX pathogenic alterations affecting the CNEs regulatory regions are present at the heterozygous state showing an extreme variable expressivity, that might be the consequence of different level of expression in different individuals of the mutated allele that not is completely silenced.The luciferase activity displayed by c.-51G > A, c.-19G > A, and c.-9del was reduced by 60, 35, and 40% under the in vitro experimental condition. In vivo these variations were carried at the heterozygous state and the overall impact on SHOX expression might be different with respect to what observed in vitro. However several previous studies demonstrated that 5′UTR variants that did not completely abolish the luciferase activity were causative of disorders –40. On tTo test whether the reduced luciferase activity was the consequence of a lower transcription efficiency or a decreased translation, the luciferase mRNA levels were quantified by real-time PCR. The plasmid carrying c.-51G > A and c.-9del generated mRNA levels similar to the wild-type, thus suggesting that the reduced luciferase activity Fig.  could beThe variant c.-58G > T did not show any functional effect, it was inherited from a normal height father and although absent in the gnomAD and in the 1518 alleles from our cohort of normal stature individuals Table , it mighBased on the frequency reported in gnomAD Table  and the Although the pedigree is too small to draw solid conclusion, the patients carrying c.-51G > A showed severe short stature (-2.9 SDS) and inherited the variant from a short stature father (-2.3 SDS), which is suggestive of cosegregation of the variant and the phenotype. By combining this information with the experimental results obtained in vitro, we can now refer c.-51G > A as a variant of mild effect which might contribute to affect SHOX expression.Although the variant c.-19G > A showed a mild influence on the transcriptional activity (65% compared with the wild-type), we clearly demonstrated that the main impact of this variant is on mRNA splicing through the creation of a novel branch site that leads to an aberrant splicing between exon 1 and 2, removing part of the 5′UTR of mRNA1 and the AUG start codon Fig. . The usaBased on the functional results, population frequency and screening of a normal stature control population, it is now possible to attribute a likely pathogenic significance to at least the c.-51 G > A and c.-19G > A variations detected in 4 pedigrees (5 patients) out of 1036 (0.0038) short stature unrelated individuals as they exhibited a functional significance in vitro, were absent in our control population and showed a frequency <0.001% in the public database. The presence of these two variations in gnomAD can be explained by the prevalence of short stature individuals (-2 SDS) that represent 3% of the population. Considering that c.-19G > A and c.-51 G > A represent 0.38% of the variants identified in our cohort of short stature individuals it is not unexpected that these variations show a frequency in the gnomAD database of 0.000008 and 0.000004, respectively.In conclusion, this study allowed us to reclassify some of the new variants of uncertain significance identified during diagnostic screening as likely pathogenic. In particular, variants in the 5′UTR might affect the splicing in a subtle way and might represent an important source of SHOX haploinsufficiency.Supplementary Information


Plasmodium falciparum lactate dehydrogenase (PfLDH). The proposed biosensor can specifically detect PfLDH in spiked whole blood down to 10 pM (0.3 ng/mL) without any sample pretreatment. The combination of simple and scalable fabrication, potentially high-throughput analysis, and excellent sensing performance provides a new approach to biosensing with significant advantages compared to conventional fluorescence immunoassays.A plasmon-enhanced fluorescence-based antibody-aptamer biosensor — consisting of gold nanoparticles randomly immobilized onto a glass substrate via electrostatic self-assembly — is described for specific detection of proteins in whole blood. Analyte recognition is realized through a sandwich scheme with a capture bioreceptor layer of antibodies — covalently immobilized onto the gold nanoparticle surface in upright orientation and close-packed configuration by photochemical immobilization technique (PIT) — and a top bioreceptor layer of fluorescently labelled aptamers. Such a sandwich configuration warrants not only extremely high specificity, but also an ideal fluorophore-nanostructure distance (approximately 10–15 nm) for achieving strong fluorescence amplification. For a specific application, we tested the biosensor performance in a case study for the detection of malaria-related marker The online version contains supplementary material available at 10.1007/s00604-021-04746-9. Fluorescence-based techniques are widely employed and rapidly emerging as a leading methodology in biotechnology, biomedicine, and life sciences , 2. High5-fold) [Plasmodium falciparum lactate dehydrogenase (PfLDH) in spiked human blood without any pretreatment.Plenty of plasmonic substrates consisting of two-dimensional (2D) arrays of metal nanostructures have been developed as PEF-based biosensing platforms , 7–9. In5-fold)  and limi5-fold) . 2D arra5-fold) . In a pr5-fold) . Althoug5-fold) . DespitePfLDH concentrations at the nanomolar level in red blood cells with picomolar levels in serum [PfLDH detection in pretreated blood. In these tests, red blood cells are lysed both to increase the PfLDH concentration and to facilitate blood flow along the dipstick. However, such malaria RDTs are not appropriate for early diagnosis and mass screening because of their poor LOD and difficulties of pre-functionalized device transportation and storage in a tropical environment [Malaria-infected individuals exhibit in serum ; very loin serum . Gold stin serum  or polymin serum . Both meironment , 18.Other colorimetric approaches have been explored, but the impossibility to carry out the measurement directly in blood or whole serum strongly limits the practical application of these biosensors –21. In tPfLDH on the top thereby warranting extremely high specificity.The PEF-based assay described here combines the plasmonic features of AuNPs with a unique photochemical functionalization technique giving rise to a strong fluorescence amplification and high sensitivity. The photochemical immobilization technique (PIT) offers a fast and simple strategy (only a UV lamp is required) to covalently tether Abs onto gold surfaces in such a way that one fragment antigen-binding (Fab) is exposed to the surrounding environment , 31. TheFull details are displayed in Section Citrate-stabilized AuNPs were synthesized by chemical reduction of gold(III) chloride trihydrate with sodium citrate  more de in ESM.2 glass coverslips—allowing the random immobilization of citrate-stabilized AuNPs by simple adsorption via electrostatic interactions [Silanization process by (3-aminopropyl)triethoxysilane (APTES) was employed to chemically modify the substrate surface—consisting of 10 × 8 mmractions . The proThe substrate was optically and morphologically characterized to retrieve information concerning the plasmon resonance wavelength and the shape and size of nanoparticles as well as the interparticle distance, respectively  method was adopted to simulate the optical response of the fabricated 2D AuNP array  was achieved by PIT. A volume of 1 mL aqueous solution of anti-PLDH (50 μg/mL) was irradiated by Trylight UV lamp for 30 s and flowed onto the substrate  to prevent coagulation. Moreover, we had to dilute the blood to reduce the solution turbidity. A dilution of 1:100 in 1 mL of 25 mM Tris buffer was used throughout the experiment as good tradeoff for retrieving high signal as well as treatable solution.The desired amount of The functionalized substrate was incubated with 1 mL of contaminated blood and the system was gently shaken for 2 h by a tilting laboratory shaker to improve the analyte diffusion and hence the probability to be captured by immobilized Abs (the incubation time could be reduced to 50 min in view of the results obtained in the binding kinetic study). Then, the sample was abundantly rinsed by Tris buffer (25 mM) and ultrapure water to remove blood residues and unbound proteins.PfLDH-Apt* sandwich was realized (the incubation time could be reduced to 30 min in view of the results obtained in the binding kinetic study). Afterwards, the sample was copiously rinsed by PBS (10 mM) and ultrapure water to remove unbound Apts*.Thus, the substrate was transferred into 1 mL of PBS solution (10 mM) containing 0.1 μM of malaria Apts*. The system was gently shaken for 2 h in dark condition so that the Ab-PfLDH concentration—rather than investigating additive concentrations on the same substrate—to inherently test the reproducibility of the detection procedure.In order to carry out a robust analysis, the fluorescence intensity corresponding to a single measurement was estimated by averaging the signals delivered by ten images randomly recorded on the substrate  [PfLDH with discrimination from Plasmodium vivax LDH (PvLDH) [The Ab-analyte-Apt* sandwich used in this work consisted of anti-e (PLDH) , whereas (PvLDH) .The colloidal solution of AuNPs was characterized by UV-Vis spectroscopy and scanning transmission electron microscopy (STEM) . SEM micrographs show spherical nanoparticles of approximately 30-nm diameter randomly distributed onto the substrate as both single nanoparticles and clusters Fig. . The pred > 3/2D) [The substrate was also optically characterized through UV-Vis spectroscopy by measuring its extinction spectrum. Figure  > 3/2D)  contribu > 3/2D) , whereas > 3/2D) . The covPfLDH-Apt* sandwich warrants a mean fluorophore-nanoparticle distance of approximately 10 nm. The consistent agreement between the experimental extinction spectrum (red continuous line) and that worked out by numerical simulation (gold continuous line) provides assurance about the holding at the macroscopic level of the observed micrometric morphology.The superposition of the plasmonic resonance to the emission peak of 5-carboxyfluorescein (5-FAM) (green dashed line)—the fluorophore used in this experiment—provides an ideal condition for achieving a strong PEF amplification since the Ab-PfLDH concentration (1 μM) as a function of incubation time for both binding processes: (1) Ab-analyte and (2) Apt*-analyte. As it concerns the process (1), PfLDH-spiked blood was preliminarily mixed with a solution containing malaria Apts* so that the resulting PfLDH-Apt* complexes were fluorescently visible while the small size of Apts* did not significantly affect the diffusion and binding kinetics. Thus, functionalized substrates were incubated with PfLDH-Apt* solution and fluorescence images were recorded at different incubation times —and then with the solution containing the malaria Apts*  and (2) exhibit similar kinetics with time constants of 50 ± 10 min and 30 ± 5 min, respectively. Thus, if a commercial fluorescence reader is used to record the signal, the whole analysis could be carried out within few hours, very attractive feature in terms of no time-consuming assay. The slight discrepancy in the asymptotic values Feq(1) < Feq(2) is ascribable to the less effective binding among the PfLDH-Apt* complexes and the immobilized Abs since the Apts* might have targeted all analyte binding sites during their pre-incubation [Firstly, the assay was kinetically characterized by measuring the fluorescence signal at a high es Fig. Sa. The prs* Fig. Sb. Figurecubation .PfLDH concentrations—from 1 fM to 1 μM referring to undiluted whole blood—that is of interest from a medical point of view [PfLDH concentrations, in which the number of green spots is strikingly higher as compared to the control down to 1 pM (33 pg/mL)  and kins an instrumental constant depending on the acquisition parameters. On the other hand, in the presence of the plasmonic substrate, the fluorescence arose from separated emitters; hence, the whole intensity is given by FPEF = kinsNPEFIPEF, where NPEF is the number of fluorescence spots while IPEF is their mean intensity. Since the FE factor is defined as G = IPEF/I0, our plasmonic substrate yielded a fluorescence amplification of 340.Aiming at measuring the FE factor delivered by such a plasmonic substrate, 100 μL of a solution containing 250 fmol of Apts* was drop-casted onto a bare microscope slide in order to estimate the fluorescence on Fig. S. Inner rF as a function of PfLDH concentration in whole blood. The data are well fitted by the four-parameter Hill equation [F1 = 6 ± 2 arb. units, F2 = 88 ± 6 arb. units, K = (1.7 ± 0.9) × 103 pM, the Hill coefficient n = 0.32 ± 0.06, and χ2 = 1.4. The detection range extends over 5 decades with a LOD of 10 pM (0.3 ng/mL)—estimated as three standard deviations above the control value  to obtain a concentration of 10 nM (1 μM referring to undiluted whole blood). In Fig. PfLDH, PvLDH, and control are compared revealing no significant cross-reaction detected with the main PfLDH competitor. In fact, malaria Apts* used as the top bioreceptor layer warranted extremely high specificity only against PfLDH protein [PLDH malaria biomarkers.To determine the specificity of the th PfLDH . The PvL protein , despiteCompared with existing malaria diagnostic test, our present apta-immunosensor is among the most sensitive optical-based sensors reported so far in combination with a broad dynamic range (DR) and extremely high specificity inherently ensured by the double biorecognition Table . MoreoveThe 2D array of AuNPs herein described was implemented as a fluorescence enhancer in an apta-immunosensor for detecting malaria marker in human blood. Extremely high specificity, competitive LOD—better than that achieved by ELISA—and scalable fabrication are the main strengths of the proposed device. In addition, the gold surface biofunctionalization carried out by PIT contributes to increase the detection efficiency as well as the long-term stability. Although there are still some areas that need further optimization , such a plasmonic substrate may constitute the bottom of a multiwell plates paving the way to high-throughput analysis, a feature that makes the device promising in several biological applications since the extension to other analytes can be immediately achieved by properly adapting the sandwich scheme. Moreover, it is expected that the reported LOD lying in the picomolar range may be pushed down to femtomolar level if a transparent matrix  rather than whole blood is analysed.ESM 1(PDF 2460 kb)


Redox control is lost when the antioxidant defense system cannot remove abnormally high concentrations of signaling molecules, such as reactive oxygen species (ROS). Chronically elevated levels of ROS cause oxidative stress that may eventually lead to cancer and cardiovascular and neurodegenerative diseases. In this review, we focus on redox effects in the vascular system. We pay close attention to the subcompartments of the vascular system  and give an overview of how redox changes influence those different compartments. We also review the core aspects of redox biology, cardiovascular physiology, and pathophysiology. Moreover, the topic‐specific knowledgebase DES‐RedoxVasc was used to develop two case studies, one focused on endothelial cells and the other on the vascular smooth muscle cells, as a starting point to possibly extend our knowledge of redox control in vascular biology. Ang IIangiotensin IIAP‐1activator proteinCVDcardiovascular diseaseECendothelial cellsEGFRepidermal growth factor receptorET‐1endothelin‐12O2Hhydrogen peroxideMMPmatrix metalloproteinaseNF‐κBnuclear factor‐kappaBNOnitric oxide−ONOOperoxynitriteOxSoxidative stressPDGFplatelet‐derived growth factorPDGFR‐βplatelet‐derived growth factor receptor betaPTKprotein tyrosine kinasesPTPprotein tyrosine phosphataseROSreactive oxygen speciesSHRspontaneously hypertensive ratsSM‐MHCsmooth muscle‐myosin heavy chainTFtissue factorTF‐FVIIatissue factor‐FVIIaTFPItissue factor pathway inhibitort‐PAtissue‐type plasminogen activatoru‐PAurokinase‐type plasminogen activatorVSMCvascular smooth muscle cellsXOxanthine oxidase1Reactive oxygen species (ROS) and reactive nitrogen species (RNS) stimulate redox homeostasis mechanisms, and thus, they are considered critical signaling molecules for maintaining cellular homeostasis. However, these signaling molecules can be harmful, as well. That is, when ROS/RNS formation outpaces the ROS/RNS removal processes, oxidative stress (OxS) occurs. Chronic OxS leads to several pathologies.1.12•−), singlet oxygen (1O2), hydroxyl radical (OH•─), and perhydroxyl radical (HO2•), which are yielded by the reduction of the oxygen molecule (O2) with two uncoupled electrons.•), peroxyl radical (LOO⁃), lipid hydroperoxide (LOOH), peroxynitrite anion (ONOO─), hypochlorous acid (HOCl), and ozone (O3),2O2), ONOO─, HOCl, and O3), and as such, they are not free radicals.Reactive species are molecules interacting with various biomolecules, including nucleic acids, lipids, and proteins, and generating electron‐deficient species, leading to cell and tissue damages. Different endogenous metabolic and biochemical reactions and exogenous sources, such as ionizing radiation, generate these reactive species. The term “reactive oxygen species” refers to oxygen metabolites, such as superoxide anion radical  that supply oxygen and nutrients to every tissue to maintain cellular homeostasis. Endothelium lines the interior surface of blood vessels—a single layer of homogenous EC layer being in connection with blood and lymph from the circulation and represents a surface (blood/endothelium interface) with roughly 300–1,000 mEC have a vital function in tissue homeostasis by the regulation of solute and macromolecule transport through the vessels. A glycocalyx layer on the EC surface provides a locally charged barrier to cell and protein movement from the blood through endothelium under physiological conditions.Furthermore, the endothelium is responsible for the reaction to pathophysiological conditions such as infection or trauma of the neighboring tissues, vessel remodeling, and growth,The fibrinolytic system is a system that restores a blood vessel when the blood clot is not needed, and it includes an inactive proenzyme, the plasminogen, and an active form, plasmin that is responsible for fibrin degradation. The activity of plasminogen activators (PA) such as tissue‐type PA (t‐PA) and urokinase‐type PA (u‐PA) regulates the fibrinolytic potential of the vasculature.Furthermore, the inhibition of the tissue factor‐FVIIa (TF‐FVIIa) complex occurs by its interaction with a specific polypeptide, the tissue factor pathway inhibitor (TFPI), and formation of a stable quaternary complex TFPI‐TF‐FVIIa‐FXa.VSMC represent one of the most frequent types of cells in arteries. VSMC are also essential for the homeostasis of the vasculature, as well as for the vasculature contractions and relaxations, which are responsible for the blood vessel luminal diameter alterations to maintain blood pressure within the normal range. VSMC contractility is controlled regularly by exchanging two different phenotypes.22H) or sulfonic (─SO3H) acid.2+ ([Ca2+]i) by the activation of ion channels, and ROS upregulates the expression of proinflammatory and proto‐oncogene genes  and NAD(P)H oxidase. NAD(P)H oxidase regulates the conversion of xanthine dehydrogenase (XDH) to XO. The modulation of XDH to XO reduces oxygen to OThe increase in XO activity has been associated with endothelial dysfunction in rats overexpressing renin and angiotensinogen from implanted human genes.2 and consequent formation of superoxide. The membrane‐bound p22phox subunit is crucial for the NAD(P)H oxidase complex activation and is accompanied by an elevated production of O2− in humans with coronary artery disease.NAD(P)H oxidase is an enzyme complex that uses NADH/NADPH as the donor of an electron in the reduction of O2− production after treatment with angiotensin II (Ang II) or ET‐1 when compared with controls.− synthesis and hypertension in mice, supports this finding.2− and the blunted effect of Ang II on the blood pressure were both found in Nox1‐deficient mice.The mononuclear cells isolated from the blood of patients with essential hypertension exhibited increased O2− formation, VSMC hypertrophy, and hypertension in transgenic mice.phox subunit‐deficient mice, the endothelial dysfunction, O2− production, and Ang II‐induced hypertensive response were blunted.2− production in the arterial tissue of SHR.On the other hand, the overexpression of Nox1 enhances Ang II‐induced O2− generated from NAD(P)H oxidase reacts with NO, forming peroxynitrite (ONOO−), which consequently induces changes in the NOS enzyme subunits resulting in further O2− formation and the declination of NO bioavailability, which impairs endothelium‐dependent relaxation in a model of salt‐induced hypertensive rats.The produced ROS from NAD(P)H can react with products of nitric oxide synthase (NOS), which may further amplify vascular function. For example, O91phox‐containing NAD(P)H oxidase is identified as a source of excessive O2− linked with elevated activity of the renin–angiotensin system as shown in renovascular91phox NAD(P)H oxidase inhibitor restrained the elevated O2− aortic generation and the expression of the proinflammatory molecules in a model of hypertensive DSS rats. Importantly, ROS‐induced injury was shown to lead to further ROS formation while proinflammatory cytokines activated the system of NAD(P)H oxidase.phox inhibits Ang II‐induced activation of JAK/STAT signaling and interleukin‐6, which suggests that the NAD(P)H oxidase activity generates the ROS involved in this pathway.The membrane‐bound gp2−. The extracellular O2− moves through chloride channel 3 into cells, where it triggers Ca2+ mobilization and mitochondrial O2− production. Thus, ROS generated from endothelial NAD(P)H oxidase mediates intracellular signaling.Furthermore, ROS produced at one subcellular locus can initiate ROS synthesis in another locus by signal transmission. For example, mitochondria‐derived ROS production activates ROS formation by NAD(P)H oxidase.2− production in the DOCA‐salt hypertensive rat model, while the ETA receptor inhibition decreases the production of vascular superoxide.The ET‐1 also induces an increase in ROS generation by the activation of NAD(P)H oxidase pathway. ET‐1 via ETA receptor activation increases O2− in the endothelium and within the aortic wall. ROS synthesized by the NAD(P)H oxidase activity facilitate the inflammatory process in the aortic endothelium and stimulate the extracellular matrix impairment through the MMP‐2 activation and apoptosis of VSMC. A murine aneurysm model was used to show that inhibition of ROS mitigates the formation of the aneurysm.phox subunit of NAD(P)H oxidase, thus implying the function of ROS generated by the NAD(P)H oxidase in MMP‐2 signaling in VSMC.phox subunit.Activation of NAD(P)H oxidase is also involved in the pathogenesis of abdominal aortic aneurysm. That is, samples obtained from subjects suffering from aneurysms of the abdominal aorta exhibit overexpressed NADPH oxidase and raised level of OVarious growth factors (such as PDGF and transforming growth factor β) and hormones are involved in the modulation of NAD(P)H oxidase activity/expression, while ROS regulates the activity of many signaling enzymes  in the vasculature.3.22O2 generation is mandatory for signal transmission through PDGFR‐β.2O2‐dependent Ang II‐stimulated EGFR transactivation.ROS regulates the tyrosine kinases (receptor and nonreceptor forms) in the vasculature. Receptor tyrosine kinases, PDGF receptor beta (PDGFR‐β), and the epidermal growth factor receptor (EGFR) require ligand‐stimulated signal transduction. It appears that H2O2 and O2− activate MAP kinases in VSMC.2O2 stimulates p38 MAP kinase, required for redox‐sensitive signal transduction initiated by Ang II in VSMC.PDGF and Ang II activate MAP kinases in the vascular tissue.2− and H2O2. Exposure of cells to ROS inactivates PTP through oxidation of Cys residue in its structure and increases protein phosphorylation.2− generation.Protein tyrosine phosphorylation is also affected by ROS. Protein tyrosine kinases (PTK) and PTPs regulate tyrosine phosphorylation. The complex of PTP enzymes dephosphorylates PTK substrate proteins and counteracts PTK activity. Both receptor and nonreceptor PTPs are susceptible to O3.32− production by NAD(P)H oxidase.2− and H2O2) generated through NF‐κB signaling is NAD(P)H oxidase. The NF‐κB cis‐acting elements regulate the promoter of p22phoxNAD(P)H oxidase gene in human aortic smooth muscle cells.2− suggest the nontranscriptional role of NF‐κB in ROS production.The activation of ROS by NF‐κB occurs through the activation of inhibitory κB kinases. The NF‐κB signaling modulates the expression of genes involved in inflammation and stimulates the ROS generation. In diabetic mice, the activation of NF‐κB increased the generation of the proinflammatory cytokine, the tumor necrosis factor‐alpha, which in turn elevated O42− and H2O2 in intact blood vessels. In VSMCs, shear stress was demonstrated to induce ROS generation.2− production in the vasculature affected by oscillatory shear was found in cells lacking NAD(P)H oxidase activity, or by application of oxypurinol, a specific blocker of XO.2− and H2O2, can diminish the efficacy of the antioxidant system, leading to the promotion of OxS. These signaling molecules play a significant role in the pathophysiology of the vasculature.Redox signaling likely also influences the mechanical forces acting on the vascular wall. That is, elevated intraluminal flow stimulates the generation of O5Data‐mining and text‐mining techniques have been used to explore the information contained in published biomedical literature. Advancements in these techniques have led to the development of several topic‐specific knowledgebases (KB),Case Study 1: Here, we characterize the biological functions of VSMC relative to chemical substances in the literature in connection with both VSMC and gene ontology biological functions that were associated with VSMC. As such, Figure https://www.cbrc.kaust.edu.sa/des-rv/) in this context. For instance, it would be interesting to look at the relationship between “H2O2” and “Ang II,” as Ang II and other chemotactic factors are dependent on H2O2 for their release.2O2 and Ang II. In the DES‐RedoxVasc KB, they are linked to vasodilation, vasoconstriction, macrophage activation, as well as VSMC that were the starting point of this exploration.The following redox‐related molecules discussed in this manuscript were retrieved: “oxygen radical,” “superoxide,” and “hydrogen peroxide.” However, DES‐RedoxVasc points out additional molecules that should be explored  retrieved by DES‐RedoxVasc are modulated by PUFAs. That is, three different PUFA‐treated glioblastoma cell lines consistently exhibited an increase in the levels of miR‐20b, and decreased levels of microRNA (miR‐16 and miR‐17) that induces apoptosis‐specific expression changes.This report by Figure 6Endothelial ROS production has a prominent role in regulating the vascular redox homeostatic mechanisms in the vascular system. By understanding the ROS/RNS removal processes, we may influence the overall perturbing homeostatic functioning of the vascular endothelial system and the underlining consequence of a disbalance portraying as OxS in which it elevates inflammation and vascular remolding, activating the events for CVD.The most prevailing players in vascular ROS pathology are XO and NAD(P)H oxidase, with links to various models of heart failure, ischemia injury, and DNA damage in patients with arterial hypertension, and so on. Interestingly, NAD(P)H was found to be in an ROS MMP‐2 signaling pathway in VSMC, suggesting some interwoven pathways of redox signaling, PTK and phosphatase activation, growth factors, vasoactive hormones, and transcription factors. To broaden our understanding of the redox control of the vascular endothelial system, we used text mining and data mining techniques to explore new intricacies of information embedded in the biomedical literature that we may not be aware of at first glance. By retrieving redox relating molecules from the DES‐RedoxVasc, we established new links and relationships between different components, such as with hydrogen peroxide and with Ang II. The results also suggest an overlapping interplay of processes that connect mitochondrial biogenesis and antioxidant systems with miR‐3178 and miR‐937. This shows that a cell expresses a quality control mechanism in the Redox homeostatic mechanisms of the vascular endothelial system. By using data mining in a review process, we have also deepened our understanding to our view the intricacies of how ROS is utilized in the vascular endothelial system redox system and also easier to see new pathways for improved treatment and more adequate prevention of cardiovascular‐related diseases.The authors declare no potential conflict of interest.


To investigate the association between pineal gland volume and symptoms of rapid eye movement (REM) sleep behavior disorder (RBD) in Alzheimer’s disease (AD) patients without any feature of dementia with Lewy bodies.18F-florbetaben amyloid brain positron emission tomography. We measured RBD symptoms using the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) and defined probable RBD (pRBD) as the RBDSQ of 5 or higher. We manually segmented pineal gland on 3T structural T1-weighted brain magnetic resonance imaging.We enrolled 296 community-dwelling probable AD patients who did not meet the diagnostic criteria for possible or probable dementia with Lewy bodies. Among them, 93 were amyloid beta (Aβ) positive on p <  0.001). The smaller the VPP, the more severe the RBD symptoms (p <  0.001). VPP was inversely associated with risk of prevalent pRBD . Area under the receiver operator characteristic curve for pRBD of VPP was 0.80 . These results were not changed when we analyzed the 93 participants with Aβ-positive AD separately.The participants with pRBD had smaller pineal parenchyma volume (VPP) than those without pRBD (In AD patients, reduced pineal gland volume may be associated with RBD. Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by loss of normal skeletal muscle atonia accompanied by dream-enacting behaviors . A largeIn cognitively normal older adults, the smaller pineal gland was associated with more RBD symptoms and higher risk of incident RBD symptoms, suggesting that reduction of melatonin secretion associated with the reduction of pineal gland volume may be a potential cause of RBD . AD patiIn this study, we investigated the association between pineal gland volume and RBD symptoms in probable AD patients who did not meet the diagnostic criteria of possible and probable dementia with Lewy bodies (DLB) .18F-florbetaben amyloid brain positron emission tomography (PET) scan, and 93 were found to be amyloid beta (Aβ)-positive.We enrolled 296 community-dwelling probable AD from the visitors to the Dementia Clinic of the Seoul National University Bundang Hospital (SNUBH) from 2011 to 2020. Among them, 104 underwent a We excluded the participants with following conditions: possible or probable DLB or Parkinson’s disease dementia (PDD); any major psychiatric and/or neurological disorders that could affect cognitive function other than AD; any history of brain tumors, substance abuse or dependence, and use of medications such as clonazepam or exogenous melatonin that may influence RBD symptom; any serious medical conditions that could affect the structure and/or function of the pineal gland or abnormalities in pineal gland morphology such as neoplastic lesions or extremely large cystic gland (diameter greater than 15.0 mm) ; and thoAll participants were fully informed with the protocol of this study, and provided written informed consents signed by themselves or their legal guardians. This study was approved by the Institutional Review Board of the SNUBH.Geriatric psychiatrists with expertise in dementia research conducted in person standardized diagnostic interviews, detailed medical histories, and physical/neurological examinations using the Korean version of the Consortium to Establish a Registry for Alzheimer’s Disease Assessment Packet Clinical Assessment Battery (CERAD-K)  and the Trained research nurses collected data on age, sex, years of education, duration of AD (months), intracranial volume (ICV), history of head injury, amount of smoking (packs/day) and alcohol drinking (standard units/week) over the past 12-month period, and use of drugs influencing sleep or motor activity, including cholinesterase inhibitors , antidepressants , carbamazepine, triazolam, zopiclone, quetiapine, clozapine, and sodium oxybate to each participant. We diagnosed dementia according to the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders Text Revision criteria . Global 18F-florbetaben amyloid brain PET imaging using a Discovery VCT scanner  in three-dimensional (3D) acquisition mode. The participants were injected with 8.1 mCi (300 MBq) of 18F-florbetaben (Neuraceq) as a slow single intravenous bolus (6 s/mL) in a total volume of up to 10 mL. After a 90-min uptake period, we obtained 20-min PET images comprising four 5-min dynamic frames. The determination was based on the visual interpretation of tracer uptake in the gray matter of the following four brain regions: the temporal lobes, frontal lobes, posterior cingulate cortex/precuneus, and parietal lobes. Participants were considered Aβ positive if smaller areas of tracer uptake were equal to or higher than those present in the white matter extending beyond the white matter rim to the outer cortical margin involving the majority of the slices within at least one of the four brain regions (“moderate” Aβ deposition) or a large confluent area of tracer uptake  was equal to or higher than that present in the white matter extending beyond the white matter rim to the outer cortical margin and involving the entire region including the majority of slices within at least one of the four brain regions (“pronounced” Aβ deposition). Participants were considered Aβ negative if tracer uptake in the gray matter is lower than that in the white matter in all four brain regions (no β-amyloid deposition).We performed We evaluated behavioral features of RBD using the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) . The RBDx-, y-, and z-dimensions. We implemented bias field correction to remove the signal intensity inhomogeneity artifacts of MR images using Statistical Parametric Mapping software . We resliced the MR images into an isotropic voxel size of 1.0 × 1.0 × 1.0 mm3. We measured ICV using FreeSurfer software  to adjust for interindividual variabilities in brain volume. We assessed pineal gland volume as described in our previous work [3 using the ITK-SNAP software . We measured pineal gland volume and pineal cysts volume and estimated the volume of pineal parenchyma (VPP) by subtracting the pineal cysts volume from the pineal gland volume  images using a Philips 3.0 Tesla Achieva scanner  within 3 months of clinical assessments with the following parameters: acquisition voxel size = 1.0 × 0.5 × 0.5 mm; sagittal slice thickness = 1.0 mm; repetition time = 4.61 ms; echo time = 8.15 ms; number of excitations = 1; flip angle = 8°; field of view = 240 × 240 mm; and acquisition matrix size = 175 × 256 × 256 mm in the ous work . In briep <  0.001) and 0.934 , respectively.The intra-rater and inter-rater intraclass correlation coefficient were 0.983  analysis. We calculated the optimal cutoff value and area under the curve (AUC) using Youden index maximum (sensitivity + specificity − 1). We examined the association of VPP with RBDSQ total score (RBDSQ-T) and the item-6 score of the RBDSQ (RBDSQ-6) using multiple linear regression model adjusted for the covariates stated above. The RBDSQ item 6 comprises four subitems on the core symptoms of RBD: nocturnal vocalization (6.1), sudden limb movements (6.2), complex movements (6.3), or bedside items that fall down (6.4). We conducted the same analyses only on the Aβ-positive AD patients.We compared the continuous variables using the independent samples p value less than 0.05 as statistically significant and employed Bonferroni corrections to reduce type I error when multiple comparisons were conducted. We performed ROC analyses using MedCalc for Windows version 18.11.3  and all the other statistical analyses using the Statistical Package for the Social Sciences for Windows version 20.0 .For all analyses, we considered a two-tailed p < 0.001). VPP was inversely associated with the risk of pRBD , indicating that the AD patients with smaller VPP may be more likely to have pRBD than those with smaller VPP. The AUC of VPP for pRBD was 0.80 . VPP was also inversely associated with the RBDSQ-T  and the RBDSQ-6 . VPP was inversely associated with the risk of pRBD , and AUC of VPP for pRBD was 0.81 . VPP also showed significant inverse association with the RBDSQ-T  and the RBDSQ-6  had pRBD. The Aβ-positive AD patients with pRBD showed smaller VPP than those without pRBD p = 0.00. VPP wasIn this cross-sectional study, we found that smaller pineal parenchyma volume was associated with more RBD symptoms in AD patients, which is in line with our previous observation that smaller pineal parenchyma volume was associated with the more RBD symptoms and the higher risk of future pRBD in cognitively normal older adults .It is now well established that RBD is a strong predictor of neurodegeneration, in particular α-synucleinopathies . AccordiRBD was common in clinically diagnosed AD , 6 and 3In our previous and the current works, we demonstrated the association of smaller pineal gland with the risk of pRBD in both cognitively normal older adults without any symptom or sign of neurodegenerative disorders including synucleinopathies  and in AOur study has several methodological limitations. First, we used a questionnaire to determine if a participant was at a high risk of RBD, whereas video PSG is required to establish the definitive diagnosis of RBD . This coIn conclusion, the current study suggests that smaller pineal gland may be associated with the risk and/or severity of RBD in AD patients.


Primary immunodeficiences (PIDs) are a group of chronic, serious disorders in which the immune response is insufficient. In consequence, it leads to an increased susceptibility to infections. Up to date, there are about 350 different disorders classified in that group. There are also patients suffering from recurrent respiratory tract infections (RRTI), however that group doesn’t present any abnormalities in terms of conducted immunological tests. Many factors, including medical, can have an impact on physical development of a child. Data such as birth weight and length, also weight, height, BMI during admission to the hospital were collected from 195 patients’ medical histories from their hospitalization at Clinical Immunology and Paediatrics Ward of J. Gromkowski Hospital in Wrocław. Investigated groups included patients with PIDs, RRTI and a control group of healthy children. Our purpose was to evaluate the physical growth of children with PID and children with RRTI by assessment of their height and weight. All of parameters were evaluated using centile charts, suitable best for the Polish population.The lowest mean birth weight and height was found among the PIDs patients group. Children with PIDs during hospitalization had statistically relevant lower mean weight than the control group and almost 18% of them had their height situated below 3rd percentile. The statistically relevant differences have been found between them and RRTI group in terms of weight, height and nutritional status. The statistically significant difference was detected between the nutritional status of PID and control group.There is a higher percentage of PID patients with physical growth abnormalities in comparison to healthy children. Our findings indicate a need for further investigation of immune system irregularities and their influence on physical growth of children. Primary immunodeficiencies (PIDs) are uncommon, chronic and severe disorders of the immune system in which patients cannot mount a sufficiently protective immune response, leading to an increased susceptibility to infections, as well as autoimmune or neoplastic diseases . About 3More than six serious diseases a year are defined as recurrent respiratory tract infections (RRI) . During Although care standards of children with PIDs were elaborated, still physical abnormalities in their physical development, such as depressed weight or height gain, can be observed, which according to The Jeffrey Modell Foundation, are also among the warning signs of PIDs , 7. We dWhile many studies have investigated higher frequency of malnutrition and anthropometric values abnormalities in some of diseases from the PID spectrum, we have not come across a paper that deals with assessment of nutritional status of patients with PID beside patients with recurrent respiratory tract infections and healthy children.p = 0,75). 26.92% (n = 14) of children with PID were born with weight under 2500 g, while in the RRTI group only 5.08%  and in the control group 2.86%  of children had the same low weight score. There were no statistically significant differences between males and females in the PID group . There was a significant statistical difference between PID group and RRTI group , as well as between PID and control group . Such difference was not observed between RRTI group and control group . 10 of 14 children, who where born with weight under 2500 g, were diagnosed with predominantly antibody deficiencies.Birth weight values of 185 patients were available for analysis. The highest standard deviation (SD = 738) and the lowest mean birth weight (mean 2860.1 g) referred to patients with PID  and the highest standard deviation among all groups (SD = 6.69); when dividing into groups with or without substitution of immunoglobulin, the highest SD (7.31) and the lowest mean birth length (h = 50.4 cm) related to PID Ig + therapy . They were assigned to certain centiles, based on the Polish growth standards  had weight under 3rd percentile, 1,5% (n = 1) of the RRTI group. There was a statistically significant difference in weight centiles between these 2 groups . Relevantly more often weight under 3rd percentile affected children with PID Ig + therapy (n = 10) than those without substitution , although there were no statistically significant differences between those groups . A significant statistical difference was also found between weight centiles of children with PID and control group . Such difference was not found between RRTI and control group . There were no statistically significant differences between males and females, both in the PID group  and the RRTI group . In case of PID group, weight under 3rd percentile most often affected patients between 6 and 12 years old.Weight during hospitalization data  were available for all the patients . They were classified to certain centiles according to both Polish  and 9% RRTI patients (n = 6) were classified in the < 3 centile compartment. In the 3–10 percentile interval there were 6 patients in the PID group (11%) and 3 in the RRTI group (4%). There was a statistical difference between height assigned to certain centiles of PID and RRTI group  with the Polish standards. According to WHO’s, 10 patients  from the PID group were situated below 3rd percentile of height, same with the z-score < −2SD  than to patients without substitution (n = 3), although it wasn’t statistically relevant . There was a statistically relevant difference detected between female and male height Z-score from the PID group , but no in the RRTI group . In the PID group, there were 5 girls with height between -2SD and -3SD z-score and 0 girls with height under -3SD z-score, while 3 boys with PID had height between -2SD and -3SD z-score and 2 had height under -3SD z-score. 6 patients (8.7%) from the RRTI group had their height below Z-score < −2SD. There were no statistically significant differences between PID and RRTI group in terms of height Z-score  or between PID and the control group both in the height centiles of Polish standards  and Z-score .Height during hospitalization data were available as well for all the patients . It was within normal for most of the patients . 9 patients over 3 years old from the PID group were underweight . In the RRTI group 3 patients over 3 years old were underweight . 4 PID patients and 10 RRTI were diagnosed with overweight. Obesity was observed in one person in the RRTI group, whereas in the PID group no sign of obesity was found. The difference between these 2 groups was statistically significant . The statistically significant difference was also observed between the nutritional status of PID patients and the control group , but was not detected between RRTI and control group   than boys (n = 1).Based on data collected from all of the patients the BMIs were calculated and classified to certain nutritional status of Polish centile charts standards Fig. . Nutritin = 2 z-score < − 3; n = 1–3 < z-score < − 2) with no other already diagnosed conditions. Z-score between − 1 and -2SD included 10 patients with PID (17.86%). 14 patients (25%) had a z-score between + 1 and + 2SD and only one patient (1.64%) had BMI >2SD.BMI was also assessed using the z-score based on WHO standards for all participants Fig. . Z-scorep = 0,307), as well as between PID and RRTI .In RRTI group 2 patients (2.9%) had Z-score < −2SD, 7 patients (10.14%) had Z-score between -2SD and -1SD, 13 people (18.84%) 1SD < Z-score < 2SD and 11 patients (15.94%) > 2SD. There was no statistically relevant difference in z-score BMI between PID and control group , than to Ig-therapy  , although it’s worth noting that underweight more frequently referred to Ig + therapy patients , 4 people had been diagnosed with ataxia-telangiectasia, 2 with Nijmegen breakage syndrome, 1 with Edwards syndrome, 1 with main classes immunoglobulin deficiency and 1 with C-1 esterase inhibitor and partial C4 component deficiency. Among patients under 3 years old, three with main classes immunoglobulin deficiency had BMI Z-score below -2SD.Nutritional status is a valuable factor, which has an impact on immunological response . The relImproper nutritional status has been seen in the patients with common variable immunodeficiency (CVID) , 13. Addn = 1) didn’t allow for any clear conclusions about nutritional status of them.1 patient with chronic granulomatous disease took part in our study. During hospitalization his weight, height and BMI remained normal. (− 2 < Z-score < + 2). Cole et al. study compared patients with the disease after hematopoietic stem cell transplantation and without this treatment . Among tn = 2) BMI < -1 was noticed, at 28.6% (n = 2) BMI < -2SD. A small number of patients affected with disease, as well as high frequency of nutritional status irregularities within the group implies the necessity to conduct further in-depth researches on anthropometric parameters of patients being under constant care of each health centre. Stewart E et al., reported 101 patients specifically on their progressive growth failure and even recommended early proactive consideration of percutaneous endoscopic gastrostomy (PEG) from age 8 years onwards to prevent it [A special group of patients with PID are the ones of chromosomal instability group (ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS). Apart from genetically lowered capability to DNA reparation, these two syndromes are also connected by higher tendency to immune system disorders. Because of the rarity of ataxia-telangiectasia, there are few available sources evaluating physical growth and nutritional status of this disease. In Ehlayel et al. study on 13 patients with AT, 38% were too short, based on the height standard deviation score (HtSDS) < − 2SD. Patients’ BMI was low in 31% of them (BMI SD < − 2) . In our event it . In our n = 2), boys were within norm. Both females were also born with birth weight under 2500 g, while boys > 2500 g. In case of that disease, growth retardation, as well as microcephaly are in its symptoms spectrum. Differences between sexes and small numbers of current publications about growth and physical development of children with Nijmegen breakage syndrome suggest the need for further studies of this topic.Nijmegen breakage syndrome seems to occur worldwide, but with a distinctly higher prevalence among Central European and Eastern European populations , 19. TheEvaluation of physical growth presented in our study on heterogenous group of diseases, which are primary immunodeficiency disorders, shows higher percentage of children with PID improperly developed, compared to the control group and RRTI group. Almost every 5th patient with PID had weight and height < 3 centile, moreover 23% over the age of 3 were underweight. It is worth to mention that improper nutritional status involves not only specific syndromes like Nijmegen breakage syndrome or ataxia-telangiectasia but also predominantly antibody deficiencies. We wanted to pay attention to higher risk of malnutrition in all PID patients, not only in particular conditions, where patients are known to have growth retardation primary or secondary to their disease.In Adirmaz et al. study 11.2% of children with PID were retarded in their height and physical development and in the study on Egyptian children interrupted physical development and growth were detected among 28% of patients diagnosed with PID , 22. In The study was performed as Students Research Circle’s activity. Main limitations of the study were relatively short time-span and lack of data about gestational age of patients.Knowledge about primary immunodeficiency disorders have expanded widely during the last few years because of assigning over 200 genes to a certain PID types . In manyIn our study statistically significant differences were detected between RRTI and PID group in the field of birth weight, as well as weight, height and nutritional status during hospitalization. No statistically significant differences were found between RRTI and control group. Heterogenous etiology of RRTI makes implementation of universal procedures for these patients difficult. Each clinical case should be investigated individually, and if possible, the cause of disease should be removed. Presence of physical growth abnormalities in both groups indicates the need to increase awareness  of factors which may influence the parameters mentioned above , as well as immune system irregularities among children.The study was conducted utilizing retrospective analysis. Primary research material were medical histories of patients hospitalized in Clinical Immunology and Pediatrics Ward, J. Gromkowski Provincial Clinical Hospital in Wrocław from October 2014 up to March 2015.n = 96 female, n = 99 male), whose age ranged from 4 weeks old up to 18 years old. The group was divided into 3, which is shown at Table S1 [supplementary file n = 56); largest group were patients with predominantly antibody deficiencies (n = 41). Most of the patients were diagnosed with IgG deficiency  and IgG subclass deficiency . There was one patient with X-linked agammaglobulinemia (XLA). No patients with combined immunodeficiency were hospitalized in our clinic during data collection. Furthermore, PID was divided in terms of applied treatment - with immunoglobulin replacement therapy  and without  (Table Sn = 5).□ PID , no abnormalities in terms of immunological tests were found, but had a history of mild recurrent upper respiratory tract infections at least 8 times in 12 months and required immunological diagnostics. These patients were admitted to our clinic at the same time as PID group to conduct immunological tests. Patients with chronic conditions , which may have an impact on recurrent respiratory tract infections, were excluded from the study.□ Among recurrent respiratory tract infections group ; children with occasional infections, their cause of hospitalization were: random poisoning (31%), acute pharyngitis (19%), acute gastroenteritis (10%) and other causes (40%). No history of recurrent infections (at least 8 times in 12 months) was detected.□ Control group (The study group numbered 195 people (Patients with PID treated with immunoglobulin (PID Ig + therapy) were admitted to the clinic every month or every 3 months. For adequate groups comparison, data were collected at the same time as PID patients were hospitalized.□ Birth length and weight□ Height and weight during admission to the hospital□ Body mass index (BMI)Assessed parameters2. The BMI was defined as: underweight, normal weight, overweight and obese, based on the latest development standards of BMI published in Poland for age 3–18 years old [Due to insufficient amount of patients’ data about gestational age at birth, we have not assessed birth length and weight with use of centiles. We have acknowledged a cut-off point at 2500 g. We decided to exclude patients with Nijmegen breakage syndrome from birth length and weight analysis. Analyzing height and weight during the admission, each parameter was assigned to corresponding centile intervals: < 3, 3–10, 10–25, 25–50, 50–75, 75–90, 90–97, > 97 centile. These values were assessed using development standards analyzed and published for the Polish children population . Polish ears old , whereasears old , 30. Furears old  for all Statistical analysis of data was conducted using the spreadsheet of the Microsoft Office Excel 2010  and Statistica 12 – for categorical data – Pearson’s chi-squared; for continuous data – Student’s t-test and Mann-Whitney U test.The study had a consent of a bioethical commission.Additional file 1: Supplementary file 1. Tables and figures.Additional file 2: Supplementary file 2. Data generated and analysed during the study.


Concomitantly, the power spectrum changed in frontal, bilateral central, and bilateral parietal clusters. RmANOVAs revealed significant main effects of task difficulty for theta band power in the frontal  and both central clusters  as well as for alpha-2 band power in both parietal clusters  and in the central right cluster . Increases in theta band power  and decreases in alpha-2 power  with increasing balance task difficulty may reflect increased attentional processes and/or error monitoring as well as increased sensory information processing due to increasing postural demands. In general, our findings are mostly in agreement with studies conducted in adults. Similar to adult studies, our data with adolescents indicated the involvement of frontoparietal brain areas in the regulation of postural control. In addition, we detected that activity of selected brain areas  changed with increasing postural demands.Electroencephalographic (EEG) research indicates changes in adults’ low frequency bands of frontoparietal brain areas executing different balance tasks with increasing postural demands. However, this issue is unsolved for adolescents when performing the same balance task with increasing difficulty. Therefore, we examined the effects of a progressively increasing balance task difficulty on balance performance and brain activity in adolescents. Thirteen healthy adolescents aged 16–17 year performed tests in bipedal upright stance on a balance board with six progressively increasing levels of task difficulty. Postural sway and cortical activity were recorded simultaneously using a pressure sensitive measuring system and EEG. The power spectrum was analyzed for theta (4–7 Hz) and alpha-2 (10–12 Hz) frequency bands in pre-defined frontal, central, and parietal clusters of electrocortical sources. Repeated measures analysis of variance (rmANOVA) showed a significant main effect of task difficulty for postural sway ( Postural control requires the complex interaction of different structures within the somatosensory system to maintain and recover balance during the performance of sport and everyday activities , by reducing the base-of-support , by changing the characteristics of the surface , or a combination of these modalities. Considering the variety of modifying factors, it is difficult to establish how these multimodal factors contribute to task difficulty. Previous research  on postural sway and frequency band power by means of ICA-based source space analyses in healthy adolescents. Based on the relevant literature, we expected that increasing postural demands result in increased postural sway  of base of support on theta frequency band power reported by Hülsdünker et al.  using a single group repeated measures analysis of variance (rmANOVA) design with 7 levels (baseline and 6 levels of task difficulty) was calculated. The analysis revealed that a total sample size of N = 8 would be sufficient to find significant and large-sized main effects of difficulty level , with an actual power of 0.85 . A physical education class including 13  healthy high-school students aged 16–17 years volunteered to participate in this study. Anthropometrics as well as the results on postural sway and electromyographic activity of the leg muscles have been reported previously  and its six difficulty levels. Thereafter, participants performed three sets of six balance tasks. Each set consisted of a randomized order of the six levels of balance task difficulty. Overall, testing of one participant comprised 18 trials (3 × level 1–6) with each trial lasting 30 s per level. Continuous EEG activity was recorded during every trial, while participants performed respective balance tasks on the balance board. Furthermore, we recorded a separate 3 min EEG baseline condition during quiet bipedal stance prior to the first set and another separate baseline measure after the third set. Anthropometric data  were assessed using a stadiometer  and a bioimpedance analysis system , respectively.©, Artzt GmbH, Dornburg, Germany) which allows to tilt in every direction. The pivot attached to the board platform has an integrated mechanism to increase task difficulty. By gradual clockwise rotation, the pivot can be adjusted at six different positions (level 1–6). The change in position elevates the platform progressively from 6.5 cm (level 1) to 8 cm (level 6) and simultaneously reduces the pivots base-of-support from approximately 14–4 cm . For this purpose, two pressure-sensitive sensor mats  were fixed on the balance board to record CoP trajectories at the maximum sampling rate of 40 Hz using the Posturo 32 Expert software . EEG and CoP data were synchronized at the start of the CoP recordings by sending a continuous 5 V signal from the Pedar© system  to the EEG system  with 64 Ag/AgCl passive electrodes implemented in an elastic sensor cap . Electrode positions were set according to the extended 10–20 system of electrode placement. Channels were referenced to the CPz electrode and electrode impedance was kept below 5 kΩ to provide a high signal-to-noise ratio. The analog EEG signals were amplified and then digitized using a 24-bit analog-to-digital converter . Digitized EEG signals were recorded with a sampling frequency of 1,024 Hz using the eego™ software .k means algorithm was applied to cluster the remaining ICs across all participants. ICs were assigned to a cluster if they were located within two standard deviations of the respective cluster. Clusters that contained components from less than 11 participants (< 80% of the sample) were excluded Overall, 170 ICs were used for cluster analysis with an average of 13 ICs (± 4) per participant. For frequency specific analyses, EEG data were merged for all three trials within a level of task difficulty. This was done for each participant separately. After artefact rejection and IC identification, the average length of the merged trials was 86.2 s (± 6.2 s) per level. Absolute spectral power was calculated for two predefined frequency bands  and for each IC using a fast Fourier transformation with a spectral resolution of 1 Hz and a 10% Hanning window. For analyses, absolute spectral power for each frequency band was averaged across individual ICs within the respective clusters.The acquired EEG data were processed offline using MATLAB  and the EEGLAB 13.5.4b toolbox  were converted into Cohen’s d and interpreted according to Cohen . Model residuals of CoP and EEG data were tested using the Shapiro–Wilk test to verify normality assumption for repeated measures analyses of variance (rmANOVA). To control if balance performance was affected by task difficulty, a single rmANOVA was computed for postural sway (absolute CoP displacements) with the six levels of task difficulty as repeating factors. Furthermore, seven separate rmANOVAs were computed for the absolute spectral power of predefined frequency bands  within the respective clusters. The factor task difficulty comprised six increasing difficulty levels together with baseline measures. If significant main effects of task difficulty were registered for balance performance or cortical activity, post-hoc tests were applied using Bonferroni-corrected paired to Cohen  with ≥ 0F = 121.6, p < 0.001; d = 6.36). Post-hoc tests showed a significant increase in CoP displacements with increasing task difficulty. A more detailed report on the post-hoc results including figures can be found in a previous study , bilateral central  and bilateral parietal  areas  = 16.137, p < 0.001; d = 1.80). Post hoc tests identified a significant increase in absolute theta power , between level 1 and levels 4–6 , between level 2 and levels 5 and 6  and between level 3 and level 5 (p = 0.029). Effect sizes of the applied post-hoc tests ranged between d = 0.07–0.17.The rmANOVA revealed a significant large-sized main effect of balance task difficulty for absolute frontal theta frequency band power  = 11.594, p < 0.001; d = 1.49) and in the central right  = 15.637, p < 0.001; d = 1.42) cluster as well. Post-hoc tests revealed significant increases in absolute theta power for the central left cluster , except level 4 which differed significantly from level 1 . Significant increments in power were found for the central right cluster , between level 1 and levels 4–6 , as well as between levels 2 to 4 and level 6 .A significant main effect of task difficulty was found for absolute theta frequency band power in the central left  = 6.632, p = 0.005; d = 0.92). All applied post-hoc tests did not reach the level of significance  = 2.755, p = 0.087; d = 0.72)  = 13.614, p < 0.001; d = 1.39; parietalR F = 6.885, p < 0.001; d = 1.05). For the parietal cluster . For the parietalR cluster .The statistical analyses for central brain areas showed a significant large-sized main effect of balance task difficulty for absolute alpha-2 frequency band power in the centralR cluster  increased and cortical activity changed with increasing balance task difficulty. In terms of cortical activity, theta frequency band power in frontal and bilateral central (left and right) areas increased with increasing balance task difficulty. Furthermore, we found significant decreases in alpha-2 frequency band power with increasing instability in bilateral parietal (left and right) areas. Alpha-2 power in bilateral central areas decreased as well but we observed no significant differences between levels of task difficulty.In accordance with our hypothesis, we observed increased postural sway when task difficulty increased. Our findings in adolescents are consistent with previous studies  as well as frequency characteristics of cortical activity on basis of ICA-based source space analyses evoked by a continuous increase in balance task difficulty in healthy adolescents. Consistent with previous adult studies, we found increased theta frequency band power in frontal and central clusters reflecting attentional and error-based processes as well as decreased alpha-2 frequency band power, mainly in parietal areas, reflecting sensory information processing as a function of increasing postural demands and task difficulty. These findings support the notion that frontal, bilateral central as well as parietal areas are involved in postural control processes with increasing postural demands which may reflect the activity of cortical balance network. Furthermore, we demonstrated that EEG source localization can be applied during a continuous balance task with increasing level of difficulty. Moreover, we were able to show that postural control strategies involve the activity of frontal, bilateral central as well as parietal brain areas and that activity of these areas change with increasing postural demands. Therefore, future studies may use high-density EEG systems to specify these functional areas and their time–frequency characteristics during increasing instability as well as cortico-muscular coherence analysis to link cortical to muscle activation patterns during increasing postural demands.