Scatophagus argus was employed in the present study. Renal expression profiles of S. argus at different salinity levels were characterized using RNA-sequencing, and an integrated approach of combining molecular tools with physiological and biochemical techniques was utilized to reveal renal osmoregulatory mechanisms in vivo and in vitro.To control the osmotic pressure in the body, physiological adjustments to salinity fluctuations require the fish to regulate body fluid homeostasis in relation to environmental change via osmoregulation. Previous studies related to osmoregulation were focused primarily on the gill; however, little is known about another organ involved in osmoregulation, the kidney. The salinity adaptation of marine fish involves complex physiological traits, metabolic pathways and molecular and gene networks in osmoregulatory organs. To further explore of the salinity adaptation of marine fish with regard to the role of the kidney, the euryhaline fish S. argus renal transcriptomes from the hyposaline stress , hypersaline stress  and control groups (25‰) were compared to elucidate potential osmoregulatory mechanisms. In total, 19,012 and 36,253 differentially expressed genes (DEGs) were obtained from the FW and HW groups, respectively. Based on the functional classification of DEGs, the renal dopamine system-induced Na+ transport was demonstrated to play a fundamental role in osmoregulation. In addition, for the first time in fish, many candidate genes associated with the dopamine system were identified. Furthermore, changes in environmental salinity affected renal dopamine release/reuptake by regulating the expression of genes related to dopamine reuptake (dat and nkaα1), vesicular traffic-mediated dopamine release , DAT phosphorylation (CaMKIIα and pkcβ) and internalization (akt1). The associated transcriptional regulation ensured appropriate extracellular dopamine abundance in the S. argus kidney, and fluctuations in extracellular dopamine produced a direct influence on Na+/K+-ATPase (NKA) expression and activity, which is associated with Na+ homeostasis.S. argus. Significantly, the results of this study revealed the mechanism of renal dopamine system-induced Na+ transport is essential in fish osmoregulation.These transcriptomic data provided insight into the molecular basis of renal osmoregulation in The online version of this article (10.1186/s12864-019-5795-x) contains supplementary material, which is available to authorized users. Scatophagus argus is a euryhaline fish distributed widely throughout coastal and estuarine habitats in the Indian-Pacific Ocean ) group , hypersaline water acclimation (50‰ SW) group  and control group . Fish from the FW and HW groups were transferred from the 25‰ SW tank to FW and 50‰ SW tanks, respectively, where they remained for another 4 weeks. Simultaneously, fish from the control group were transferred from their original 25‰ SW tank to a new SW tank with the same salinity. After 4 weeks, fish were anaesthetized with a bath of 50 mg/L Tricaine methane sulfonate  and then euthanized by a sharp blow to the head. Fifteen fish in each group were divided into three sets. Each set (five fish) represented one biological replicate. Plasma samples were harvested and kept at 4 °C overnight. Serum was separated from blood cells by centrifugation  and assayed to determine the dopamine concentration. Fresh kidney tissues from each set were sampled, frozen in liquid nitrogen immediately and then stored at − 80 °C until needed. Animal welfare and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals  and were approved by the Animal Ethics Committee of Shenzhen University (Reference No. 2014–162).Total RNA was extracted using TRIzol Reagent  following the manufacturer’s instructions. RNA was treated with DNase I  and quantified using a Qubit RNA Assay Kit and Qubit™ 4 Fluorometer . RNA samples (4 μg) were employed to construct an RNA-Seq library using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina . The indexed libraries were pooled and sequenced on HiSeq™ 2500 sequencing platform  to generate 150-bp paired-end reads.http://bowtie-bio.sourceforge.net/index.shtml) [https://github.com/trinityrnaseq/trinityrnaseq/wiki). To investigate the accuracy of the assembled transcriptomes, raw reads were mapped to the assembled transcriptomes using Bwa-0.7.9a with the BWA-MEM algorithm, and mapping statistics were calculated using SAMtools 0.1.19 (http://samtools.sourceforge.net/samtools.shtml) with the SAMtools flagstat command [http://www.ncbi.nlm.nih.gov) and the manually annotated and reviewed protein sequence database  using a cutoff E-value threshold (< 1e-6). The BLAST results were extended to the Functional classifications of Gene Ontology , Clusters of Orthologous Groups of proteins  and Pathway Annotation of the Kyoto Encyclopedia of Genes and Genomes . GO annotation was performed using Blast2GO [Raw reads from all three groups were combined and quality-filtered using the Trimmomatic read trimming tool . Reads cx.shtml) . Transcrx.shtml) , fragmen command , 34. To  command  against Blast2GO . The staBlast2GO .p < 0.001. The FPKM values of the DEGs were normalized by log2 transformation and median centered. Hierarchical cluster analysis was performed based on Euclidean distance using a Trinity Perl script [p-value < 0.001 and log2|fold-change| ≥ 1 were considered significant. GO and KEGG enrichment analysis were performed to identify the biological functions of DEGs. The transcriptomic analysis workflow used in the present study is shown in Fig. The filtered transcriptome generated via de novo assembly was used as a reference transcriptome for RNA-Seq expression analysis. Raw reads generated from each sample were mapped to the reference transcriptome, and the relative expression level of each unigene was determined using FPKM values calculated using RSEM software . The resl script . Genes w260/A280 ratio in the range of 1.8 to 2.0 were employed. Complimentary DNA (cDNA) was synthesized via the reverse transcription (RT) of 1 μg of total RNA with oligo (dT) primers using a PrimeScript RT Reagent Kit with gDNA Eraser  and then stored at − 20 °C.The total RNA of each sample was extracted using TRIzol Reagent . RNA integrity was determined using agarose gel electrophoresis and quantified using a Nanodrop-2000 spectrophotometer . Only RNA samples with an Ahttp://bioinfo.ut.ee/primer3-0.4.0/primer3/input.htm) was used to design gene-specific primers according to the assembled transcriptome sequences. The β-actin gene was used as an internal control to normalize the expression value of the target transcript based on the variation in cDNA abundance, as reported previously [2O, 0.2 μL of forward and reverse primers (10 μM each) and 1 μL of cDNA. The reaction was performed under the following conditions: 34 s at 95 °C; 40 cycles of 5 s at 95 °C and 30 s at 60 °C; 15 s at 95 °C; 1 min at 60 °C; and 15 s at 95 °C . The data were collected at 60 °C. The RT-qPCR primer sequences used in this study are listed in Table Subsequently, RNA samples were re-analyzed using quantitative PCR (qPCR) to validate the RNA-Seq results. The analysis focused on genes related to dopamine transport. Primer 3  - Ct . All data were analyzed by the 2-ΔΔCt method as described in Mu et al. [Each reaction had a single peak in the melt curve that corresponded to a single product. The amplification efficiencies of both the target and reference genes ranged from 99.6 to 100.3%. The relative transcript levels of different genes were determined by subtracting the cycle threshold (Ct) of S. argus fish as described in Lakra et al. [2 cell culture flasks containing 8 mL of growth medium with 20% fetal bovine serum (FBS). The primary cells were incubated at 28 °C, and one-half of the medium was changed every 3 days for 2 weeks. When the migrating cells formed a complete monolayer, the cells were trypsinized with a 0.25%-trypsin solution and transferred into a fresh T25 flask with fresh medium containing only 10% FBS. The cell cultures were then maintained at 28 °C.Kidney tissue was obtained for primary cell culture from a healthy 25‰ SW-acclimated a et al. . In brieFor osmotic stress experiments, primary cells were subjected to hypoosmotic (100 mOsmol/L) and hyperosmotic (600 mOsmol/L) media, whereas control cultures were exposed to fresh isosmotic medium (300 mOsmol/L). The culture media and cells were collected 24 h after salinity challenge.https://www.cusabio.com/) according to the manufacturer’s instructions. The 96-well microtiter plate provided in this kit was pre-coated with anti-dopamine monoclonal antibody, and no signification cross-reactivity or interference between dopamine and its analogs was observed. The precision of the assay was determined by the repeated measurement of control or experiment samples. Both positive and negative controls (phosphate buffer solution (PBS)-only and blank, respectively) were analyzed in this assay. After all reactions, the optical density of each well was measured at 450 nm within 10 min of reaction termination using a Synergy H4 Hybrid Multi-Mode Microplate Reader . The dopamine concentration of each sample was determined according to a standard curve.The dopamine concentrations in serum and culture media were measured using the Dopamine ELISA Kit specific for fish  in PBS containing 0.5% Triton X-100 (PBST) overnight at 4 °C, incubated with fluorescein isothiocyanate- (FITC-) conjugated secondary antibody  (1:1000 dilution) in PBST for 3–4 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole  in PBS for 5 min. Slices were visualized with a LSM510 confocal laser scanning microscope . Negative controls were incubated with normal rabbit serum.The kidney tissue from a healthy 25‰ SW-acclimated +/K+ ATPase alpha 1 antibody  (1:5000 dilution) in PBS overnight at 4 °C. Membranes were washed and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody  (1:5000 dilution) for 2 h at room temperature. Chemiluminescence was used to quantify secondary antibody conjugation using a Universal Hood II gel documentation system .The total protein of each kidney sample was extracted using a Protein Extraction Kit , and the protein concentration was then determined using a BCA Protein Assay Kit . Total protein (75 μg) was separated on 12% sodium dodecyl sulfate- (SDS-) polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Following blocking in a 5%-skim milk/PBS solution , membranes were incubated with anti- NaTotal protein from primary renal cells and kidney tissues was isolated and quantified using a BCA Protein Assay Kit . Subsequently, NKA activity was measured using a Sodium-Potassium-ATPase Kit  according to the manufacturer’s instructions. Activity was expressed in μmolPi/mgprot/h.Culture medium samples were diluted with deionized water  and filtered with Nalgene syringe filters . Analyses were conducted using a Dionex™ ICS-1500 ion chromatography system  with an AS-DV automated sampler.t-test with SigmaStat software . p < 0.05 was considered statistically significant .All data were analyzed for normality (Kolmogorov-Smirnov’s test) and homoscedasticity of variance (Levene’s test). Statistically significant differences between the treatment and control groups were determined using one-way analysis of variance (ANOVA), followed by Student’s S. argus kidney subjected to different salinity acclimation conditions to produce a large-scale S. argus transcriptome. A total of 1,023,047,770 paired-end reads (150 bp each) were obtained. After removing low-quality reads, 575,312,926 clean reads remained and were used for de novo transcriptome assembly, resulting in 186,397 unigenes (> 250 bp). By selecting transcripts with FPKM ≥ 1, 43,933 unigenes were determined as viable for downstream analysis. The N50 value of these unigenes was 3118 bp, with an average length of 2022 bp , Stegastes partitus (16.45%), Notothenia coriiceps (7.51%), Oreochromis niloticus (5.74%) and Oncorhynchus mykiss (5.43%)  and 8360 unigenes were functionally annotated by NCBI-BLAST searches against protein databases (NR and SWISS-PROT), respectively (Table mykiss 5.% (Additip < 0.001 and log2|fold-change| ≥ 1) . The results showed that the number of DEGs in the HW group was greater than that in the FW group. This finding suggests hypersaline stress causes greater changes in renal gene expression than FW stress, indicating elevated environmental salinity might have a significant impact on renal function in S. argus.In comparison with the control group, 19,012 DEGs (9956 up-regulated and 9056 down-regulated) were identified in the FW group. Of those DEGs, 1439 DEGs were significantly expressed in the kidney (p < 0.001) in the DEGs included the following: sodium/chloride symporter activity (GO:0015378), ion channel activity (GO:0005216), neurotransmitter secretion (GO:0007269), synaptic transmission involved in micturition (GO:0060084), regulation of ion transmembrane transport (GO:0034765) and synaptic vesicle (GO:0008021). GO enrichment analysis indicated hormone secretion and ion transport in the S. argus kidney were significantly influenced by exposure to fluctuating salinity levels. In various organisms, hormones are used to communicate between organs and tissues for physiological regulation, including regulation of ion balance. Dopamine can inhibit renal Na+ transporters, channels, and pumps, resulting in a decrease in tubular sodium reabsorption and eventually an increase in sodium excretion. The results of this study identified the GO terms related to the dopamine system  (p < 0.001). Similar to the GO annotation results, ‘endocrine system’ is also the primary classification in the KEGG analysis, which contained ‘renin-angiotensin system’. For ‘nervous system’ and ‘excretory system’, the ‘synaptic vesicle cycle’ and ‘aldosterone-regulated sodium reabsorption’ pathways were identified. Interestingly, the KEGG pathway enrichment analysis showed the ‘Parkinson’s disease’ pathway related to dopamine dysfunction was significantly enriched for DEGs , an inhibitor of dopamine transporter (DAT) internalization, was significantly up-regulated in the kidney after FW acclimation. In addition, a significant increase in angiotensin-converting enzyme (ace), which promotes dopamine release, was observed. These results suggest long-term hypoosmotic stress inhibits DAT internalization, which can directly promote dopamine reuptake, although ACE-induced dopamine release may be enhanced.The RT-qPCR results showed RAC-alpha serine/threonine-protein kinase (dat), DAT phosphorylation , DAT internalization (akt1) and vesicular traffic-mediated dopamine release  was all significantly reduced. These results indicate phosphorylated DAT- and vesicular traffic-mediated dopamine release was weakened in the kidney of S. argus during chronic hypersaline stress, and the decreased expression of DAT fundamentally reduced the influx of extracellular dopamine.In the HW fish kidney, expression of the genes involved in dopamine reuptake  in the dopamine concentration were observed in the serum (from 0.21 ± 0.04 ng/ml to 0.39 ± 0.05 ng/ml) and kidney tissues (from 2.30 ± 0.13 ng/g to 4.39 ± 0.21 ng/g) of HW fish  of primary renal cells exposed to hypoosmotic shock (100 mOsmol/L) decreased significantly (from 68.37 ± 6.87 pg/106 cells to 46.90 ± 3.09 pg/106 cells) at 24 h post-treatment (*p < 0.05) but increased in the hypertonic medium (600 mOsmol/L) (from 68.37 ± 6.87 pg/106 cells to 97.01 ± 2.84 pg/106 cells) (**p < 0.01)  when compared with cells pre-treatment.The in vitro dopamine concentration in the culture medium . In the HW group kidneys, both NKAα1 expression and NKA activity (6.53 ± 0.37 μmolPi/mgprot/h) were significantly inhibited (**p < 0.01)  slightly altered the mRNA and protein expression of NKAα1 and significantly increased NKA activity (from 8.61 ± 0.20 μmolPi/mgprot/h to 10.84 ± 0.79 μmolPi/mgprot/h)  that outflowed under isosmotic conditions (86.22 ± 4.23 μg/106 cells). In addition, K+ was pumped out of cells (14.46 ± 1.94 μg/106 cells) during chronic hypotonic stress, although an influx of K+ was observed under isosmotic conditions (16.17 ± 2.91 μg/106 cells). In contrast, exposure of renal primary cells to hypertonic medium promoted Na+ (35.50 ± 1.04 μg/106 cells) and K+ (46.59 ± 5.24 μg/106 cells) influx to maintain the homeostasis of kidney cells.The concentrations of NaS. argus harvested from the HW and FW groups.Salinity adaptation in euryhaline teleosts is a complex process involving physiological responses in several osmoregulatory organs. In fish, the kidney is an important osmoregulatory organ involved in maintaining osmotic homeostasis , 45. Fun+ transporters via its specific receptors [2|fold-change| < 1). Thus, the dopamine availability at dopamine receptors and the timing and magnitude of dopamine signaling were dependent on the regulation of extracellular dopamine levels. This finding was consistent with the assumption that chronic salinity tolerance has a considerable influence on dopamine signaling by interfering with its transport, which directly affects the dopamine-mediated regulation of the Na+ balance.Similar to other vertebrates, the regulation of salinity adaptation in teleosts is dependent on the endocrine system, which mediates many physiological processes to maintain a salt and water balance , 47. Dopeceptors . In thisS. argus increased significantly during hypersaline tolerance but remained relatively stable under hyposaline conditions  of extracellular dopamine was observed after the exposure to hyperosmotic medium, but its concentration decreased significantly (p < 0.05) in hypoosmotic medium. These results indicated changes of osmotic pressure in the culture medium greatly influenced extracellular dopamine levels. To avoid cytotoxicity, intracellular dopamine levels remained relatively stable regardless of whether cells were exposed to hypoosmotic or hyperosmotic stress. Apart from regulating the expression and activity of Na+ channels via dopamine receptors, dopamine can also promote natriuresis via increasing renal blood flow and the glomerular filtration rate in vivo [ELISA assay results showed the dopamine levels in serum and the kidney tissues of ons Fig. . In addi in vivo . This shTwo major regulators of extracellular dopamine are vesicular traffic-mediated release and DAT-mediated reuptake . The pripink1, lrrk2, ace and apn) were significantly reduced . LRRK2 is a member of the leucine-rich repeat kinase family. Notably, mutations in lrrk2 are the most common genetic cause of Parkinson’s disease, a disease in which imbalanced synaptic transmission has been implicated as a causal factor [lrrk2 increases striatal dopamine release [lrrk2 was decreased by hypersaline stress. In addition, pink1, which is intimately involved with mitochondrial quality control by identifying damaged mitochondria and targeting specific mitochondria for degradation, was inhibited under the hypersaline condition. Mitochondria play a crucial role in synaptic function, consistent with the high energy demand of synaptic terminals, by restoring ionic gradients following synaptic transmission and reloading synaptic vesicles with neurotransmitters [+/water homeostasis [pink1, lrrk2, ace and apn) associated with vesicular traffic-mediated dopamine release was inhibited.In the present study, the expression levels of vesicular traffic-mediated dopamine release-related genes  and calcium/calmodulin-dependent protein kinase , 70. Preakt1 gene, a member of the AKT subfamily of genes, was significantly up-regulated. This finding suggested hypo-salinity acclimation may promote extracellular dopamine clearance by increasing DAT activity and inhibiting DAT internalization by AKT1. In addition, an increase in the mRNA expression of ace-induced dopamine release was observed in FW fish. This phenomenon may be a compensatory mechanism for the increase in extracellular dopamine clearance by DATs.Cell-surface DAT levels are sensitive to changes in the cell membrane potential, which can rapidly drive DAT internalization, which can alter the capacity of DATs to increase extracellular dopamine levels. Known also as protein kinase B, AKT participates in multiple cellular processes and plays an essential role in the homeostatic regulation of DAT activity in mammals . The inh+ ions and 1 Cl− ion [+ ions are major determinants of renal dopamine transport [+ gradient, which is generated by NKA. NKA consists of α-catalytic and β-glycoprotein subunits and plays a crucial role in maintaining osmotic homeostasis [+ concentration dramatically impairs or completely suppresses DAT-mediated extracellular dopamine clearance [nkaα1 was identified from the renal transcript library of S. argus. The α-subunit of NKA is a transmembrane protein that cleaves high-energy phosphate bonds and exchanges intracellular Na+ for extracellular K+ [+ influx enhanced the osmotic pressure of renal cells, which is required for adaptation to hypertonic environmental conditions. The Na+ influx resulted in an observed negative effect on DAT-mediated extracellular dopamine reuptake. In contrast, the increase in NKA activity in the FW group was accompanied by an increase in Na+ efflux, which induced extracellular dopamine clearance. These results indicated osmosis can be regulated by the renal dopamine system by altering dopamine release/reuptake and maintaining Na+ homeostasis during chronic salinity stress.The physiological functions of DAT in dopamine removal are coupled with the translocation of 2 Na Cl− ion . Thereforansport . DATs paeostasis , 74. Blolearance . Herein,lular K+ . InhibitS. argus exposed to different environmental salinities was conducted. Differences in transcriptomic levels under hyposaline and hypersaline conditions were revealed. The transcriptomic analyses indicated the renal dopamine system is essential in osmoregulation. The differential expression levels of dopamine release/reuptake-related genes, which ensured appropriate extracellular dopamine abundance, were reflected by changes in kidney function, including Na+ transport. The results of this study indicated the efficiency of extracellular dopamine clearance mediated by DAT was higher than that of dopamine release mediated by vesicular transport and phosphorylated DATs. Therefore, the concentration of extracellular dopamine decreased under hyposaline stress, which reduced the inhibition of NKA on the membrane, resulting in Na+ efflux. In contrast, increased dopamine levels were accompanied by the inhibition of NKA activity and expression, preventing the loss of Na+ , whereas unigenes with an average length of 1590 bp were the least abundant (2735). Sequences longer than 2000 bp were grouped together. The number of sequences decreased as length increased. (TIF 1027 kb)Additional file 2:Figure S2. Characteristics of the homology search for Illumina sequences against the NCBI-NR database. Species distribution is shown as a percentage of the total homologous sequences with an E-value ≤1e-6. All proteins in the NCBI-NR database were used in the homology search. The best sequence hits were selected for analysis. (TIF 87320 kb)Additional file 3:Figure S3. GO classification of unigenes. The results for ‘biological process (BP)’, ‘cellular component (CC)’ and ‘molecular function (MF)’ terms were summarized. (TIF 1925 kb)Additional file 4:Figure S4. Eukaryotic orthologous group (KOG) classification of the assembled unigenes in S. argus. (TIF 22535 kb)


Our observations are consistent with simplified molecular dynamics calculations that revealed that the molecules are aligned with the laser polarization axis by their cooperative motion after photoisomerization. This insight advances the fundamental chemistry of photoresponsive molecules in soft matter as well as their ultrafast photomechanical applications.The photoisomerization of molecules is widely used to control the structure of soft matter in both natural and synthetic systems. However, the structural dynamics of the molecules during isomerization and their subsequent response are difficult to elucidate due to their complex and ultrafast nature. Herein, we describe the ultrafast formation of higher-orientation of liquid-crystalline (LC) azobenzene molecules via linearly polarized ultraviolet light (UV) using ultrafast time-resolved electron diffraction. The ultrafast orientation is caused by the  Photoisomerization of molecules is used to control the structure of soft matter but the response of molecules during isomerization is difficult to elucidate. Here the authors describe ultrafast formation of higher-orientation of liquid-crystalline azobenzene molecules via linearly polarized UV light using time-resolved electron diffraction. In synthetic systems, the drastic structural changes induced by isomerization are used to control the structures of soft materials, such as liquid crystals2, micelles4, vesicles6, thin films8 and surfactant systems9, via order-to-disorder transitions. The photoisomerization of a single molecule occurs on the timescale of hundreds of femtoseconds15, and the subsequent response of soft matter may initiate in a few picoseconds. However, to the best of our knowledge, neither the ultrafast structural dynamics of the photoisomerization of molecules in the liquid-crystalline (LC) phase nor the subsequent responses have been directly observed. Here, we report the ultrafast orientation in a single direction of LC azobenzene molecules in a smectic B phase using picosecond time-resolved electron diffraction and model calculations based on molecular dynamics.The isomerization of photochromic molecules can induce dynamic structural changes in soft matter. The isomerization of retinal in the photoreceptor protein rhodopsin, which enables vision, is an example of this type of system in nature16. The ordered states of liquid crystals can be controlled by photostimuli if photochromic molecules are incorporated into the material25. Azobenzene molecules are representative photochromic molecules that change from straight trans-forms to bent cis-forms via photoisomerization28. LC molecular assemblies containing azobenzene molecules have various applications, e.g., photomechanical materials18, photoswitching devices21, storage devices22 and molecular photoalignment processes25.Liquid crystals combine the fluidity of liquids and the order of solid crystals30. Great advances in ultrashort-pulsed and ultrabright electron sources have been made recently, and time-resolved electron diffraction can now be used to elucidate the structural dynamics of organic molecular crystals31. Very recently, this technique has been applied to more complex soft materials32. The difference between the diffraction patterns before and after photoexcitation reveals the molecular-level structural change of interest in soft matter.Ultrafast time-resolved diffraction provides structural information about the atomic rearrangements during the phase transitions of materials or chemical reactions between molecules, making it an ideal methodology for investigating photoinduced collective molecular motionstrans-to-cis isomerization induced in the azobenzene-molecule-based LC assembly via linearly polarized ultraviolet (UV) light induces ultrafast higher orientation.In the present study, we examine the molecular motion of photoresponsive LC azobenzene molecules by picosecond time-resolved electron diffraction measurements and molecular dynamics calculations. Our findings show that the 1 are irradiated with a pulse of linearly polarized UV light. A fraction of the homeotropically aligned LC azobenzene molecules in the smectic B phase undergo trans-to-cis photoisomerization. In the LC phase, the transition dipole moments of azobenzene discs  and cis-azobenzene molecules (n–π* transition), the wavelengths of the probe light were set to 266 and 400 nm. As shown in Fig. trans- and cis-azobenzene molecules decrease and increase, respectively, under UV photoexcitation. The fitting curves in Fig. t, t0, A0, y0 and τ are the time delay, deviation at time zero, amplitude of ΔT/T, offset and time constant, respectively, used as fitting parameters. Figure τ = 180–190 ps). The processes of photoisomerization can typically be separated into three parts, i.e., the electronic π–π* transition, the electronic transition to the conical intersection and the structural change from the trans-form to the cis-form. The electronic transitions occur quite rapidly (~1 ps) and the time constant of the structural change strongly depends on the surrounding conditions. The structural changes of azobenzene molecules in n-hexane and ethylene glycol occur in 0.5–1 ps and >10 ps, respectively34. The slow photoresponse observed in azobenzene molecules in the LC phase . As shown in Fig. Q-value of the diffraction spots is 1.4 (2π × 0.22) Å−1, which means that the plane distance is 4.5 Å. The plane distance and molecular distance have relation of 37 . The diffraction pattern from the azobenzene molecules in the crystalline phase did not change upon UV photoexcitation  of 0.50 mJ cm−2  occurs in a few milliseconds, since we observed the time-resolved changes at a laser repetition rate of 200 Hz (5 ms) and could not observe the changes at a repetition rate of 1 kHz (1 ms) due to signal accumulation  of the excitation laser, the single photon energy  and the absorptivity (A = 23%) of the sample, the number of absorbed photons per unit area (Nex) was calculated to be 1.5 × 1014 photons cm−2 using the following equation:Based on the incident fluence (Nmol) was determined by the sample thickness (d = ~60 nm), volume density (ρ), molecular weight (m = 518.86 g mol−1) and Avogadro constant (NA) according to the following equation:The number of molecules per unit area  is calculated to be ~1% by the following equation:ηi is the photoisomerization quantum efficiency (50%)40. The ability of that such a small fraction of cis-azobenzene molecules within the predominantly trans-azobenzene molecules to induce a higher orientation was surprising. The trans-to-cis isomerization was expected to disorder the system; however, the opposite was observed, i.e., a small fraction of cis-azobenzene molecules oriented in the same direction rotate the molecular system and induce a higher orientation through the very nature of the molecular interactions in the LC phase. The change in the intensities of the positive spots observed in Fig. The typical volume density value of LC materials is ~1 g cm42. Figure trans-to-cis photoisomerization via UV photoexcitation; for example, the molecules shown in an ellipse in Fig. cis-form with a bent structure. The bent molecules push against the adjacent molecules due to steric forces, which decreases the free space in the plane in the direction of the bend (laser polarization axis). This motion rotates and aligns the primary and/or secondary neighbouring molecules into the sixfold symmetric pattern. Because of the lower free space in the plane, the molecular system with bent molecules  was measured by a commercial spectrometer (JASCO V-670) at room temperature. The same sample was subsequently heated and melted on a hotplate at 80 °C while being continuously irradiated with UV light (~20 W m−2) for 3 min. The sample was quenched to room temperature while still being irradiated with irradiated UV light, and the static optical spectrum of the sample (cis-azobenzene molecules) was measured by the spectrometer at room temperature. This procedure was performed because the sample does not undergo trans–cis or cis–trans isomerization in the crystalline phase, but only in the liquid or LC phase. The optical spectra of trans- and cis-azobenzene molecules are shown in Fig. Azobenzene molecules in toluene solution (2 w/w%) were spin-coated on bulk sapphire substrates at 2000 rpm. The resulting thin-film sample was heated and melted on a hotplate at a temperature of 80 °C in 3 min; the temperature of the surface of the hotplate was measured with a K-type thermocouple. The sample was quenched to room temperature, and the static optical spectrum (transmission and reflection) of the sample  was obtained by a lock-in amplifier. A solution of the sample in toluene (2 w/w%) was spin-coated onto a bulk sapphire substrate.Transient transmission spectroscopy was performed on the azobenzene molecules in the LC (55 °C) phases in air. The experimental setup is shown in the supplementary information  membranes.The experimental setup of the compact DC-accelerated electron diffraction system is provided elsewhere47. As indicated in ref. 16, the correlation among the interlayer molecules is much weaker than that among the intralayer molecules in the smectic B (Hex) phase; therefore, two-dimensional MD simulation should be appropriate. Azobenzene molecules were simplified to spheroids in the model, and the ratios of the three axes of the simplified trans- and cis-azobenzene molecules were 1:1:10 and 1:3:8, respectively , and the density of the close-packed condition was set to 1.00. One hundred azobenzene molecules were contained in a unit system, and photoexcitation could change an azobenzene molecule from the straight form to the bent form (1%). The calculated diffraction pattern was generated using CrystalMaker® and SingleCrystal® software48 using molecular coordinates obtained from the molecular dynamics calculations.Two-dimensional molecular dynamics calculations were performed based on the LAMMPS Molecular Dynamics SimulatorSupplementary InformationPeer Review FileSource Data


Due to the structural similarity between phosphinothricin and some bisphosphonates (BPs), this study focuses on investigating the possibility of repurposing phosphinothricin as a bisphosphonate analogue, particularly in two medicine-related activities: image probing and as an anti-cancer drug. As BP is a competitive inhibitor of human farnesyl pyrophosphate synthase (HFPPS), in silico molecular docking and dynamic simulations studies were established to evaluate the binding and stability of phosphinothricin with HFPPS, while the results showed good binding and stability in the active site of the enzyme in relation to alendronate. For the purpose of inspecting bone-tissue accumulation of phosphinothricin, a technetium (99mTc)–phosphinothricin complex was developed and its stability and tissue distribution were scrutinized. The radioactive complex showed rapid, high and sustained uptake into bone tissues. Finally, the cytotoxic activity of phosphinothricin was tested against breast and lung cancer cells, with the results indicating cytotoxic activity in relation to alendronate. All the above results provide support for the use of phosphinothricin as a potential anti-cancer drug and of its technetium complex as an imaging probe. Bone-seekers are a group of complexes of radioisotopes that tend to accumulate in bones when they are introduced into the body. Bone-seekers have been used for more than 30 years in nuclear medicine to provide convenient and effective means for monitoring disease progression, improving the quality of life for patients with diseases, such as cancer, or for radio-imaging purposes . The mec99mTc. The examples of 99mTc-BP complexes are 99mTc-methylene diphosphonate (99mTc-MDP), 99mTc-3,3-diphosphono-1,2-propane dicarboxylic acid (99mTc -DPD), 99mTc-3,3-1,2-ethanediylbis[nitrilobis-(methylene)]tetrakis-phosphonic acid (99mTc-EDTMP) and 99mTc-TEDP -phosphinic acid or glufosinate) is the active ingredient of the non-selective herbicide BASTA. Phosphinothricin, which inhibits glutamine synthetase  × 100% where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the survival curve of each tumor cell line after treatment. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose response curve for each conc. using Graphpad Prism software . The biological evaluation was done at the Regional Center for Mycology & Biotechnology, Al-Azhar University.For cytotoxicity assays against breast cancer MCF-cell line and lung cancer A-549 cell line the cells were obtained from the VACSERA culture unit . The cells were seeded in 96-well plates at a cell concentration of 1 × 1050 of 32.6 μg/mL (180.1 nM) and IC50 of 45.8 μg/mL (253 nM) against breast cancer MCF-7 and lung cancer cell line A-549, respectively. The study confirmed the repurposing of phosphinothricin as a novel technetium-99m imaging probe with potential anti-cancer activity.Phosphinothricin was repurposed as an anti-cancer drug because of its structural similarity to alendronate. The docking results revealed that phosphinothricin possessed a ΔG of −25.65 kcal/mol with a pki of 37.54, while its Tc-complex showed a ΔG of −10.12 kcal/mol with a pki of 21.74, which confirmed its high affinity when compared to alendronate. The radiolabeling of phosphinothricin showed that bones displayed high and sustained uptake of the radioactivity starting 10–90 min after administration , which is a another confirmation of its affinity to bone cells. The in vitro anti-cancer screening of phosphinothricin had an IC


This study was conducted in order to investigate the role of gray-scale ultrasound (US) and optoacoustic imaging combined with gray-scale ultrasound (OA/US) to better differentiate between breast cancer molecular subtypes.All 67 malignant masses included in the Maestro trial were retrospectively reviewed to compare US and OA/US feature scores and histopathological findings. Kruskal–Wallis tests were used to analyze the relationship between US and OA/US features and molecular subtypes of breast cancer. If a significant relationship was found, additional Wilcoxon–Mann–Whitney tests were used to identify the differences between molecular subtype groups.p values < 0.05). Regarding OA/US features, the sum of internal features helped to differentiate between TNBC and HER2-enriched subtypes (p = 0.049). Internal vessels (p = 0.025), sum of all internal features (p = 0.019), and sum of internal and external features (p = 0.028) helped to differentiate between LUMA and LUMB. All internal features, the sum of all internal features, the sum of all internal and external features, and the ratio of internal and external features helped to differentiate between LUMA and TNBC. The same features also helped to differentiate between LUMA and TNBC from other molecular subtypes .US sound transmission helped to differentiate between LUMA and LUMB, LUMB and TNBC, and LUMB and all other molecular subtypes combined  combined with gray-scale US helped to differentiate between breast cancer molecular subtypes.The online version of this article (10.1007/s00330-019-06262-0) contains supplementary material, which is available to authorized users. Breast cancer is the most frequently occurring malignancy and most common cause of cancer-related death in women worldwide . HoweverImaging plays a crucial role in detection, diagnosis, guiding biopsies and interventions, monitoring response to therapy, and surveillance of breast cancer . MammogrAngiogenesis has been recognized as one of the hallmarks of breast cancer. The production of new blood vessels is essential to support the development of malignant lesions once they become larger than 2 mm. Judah Folkman described the essential role of angiogenesis to provide nutrients and oxygen for tumor growth. He characterized malignant tumors as being basically “hot and bloody”, illustrating the typical flush perfusion and hyperemia found in these lesions –10. BaseThe goal of our study was to retrospectively determine the relationship between prospectively defined US and OA/US characteristics and histopathological prognostic indicators of breast masses, including the following: histologic grade, each of the three individual components of histologic grading , and with secondary prognostic biomarkers such as continuous number of mitoses, HER2 receptor status, hormone receptor status, and Ki-67 proliferative index. We also assessed the relationships between US and OA/US feature scoring and molecular subtypes.In this study, all malignant masses included in the Maestro  trial weWe used a handheld US device that could perform both conventional gray-scale US alone and OA/US (fusion of US and laser light). The laser light is transmitted into the breast from the handheld duplex probe (OA and US) at two different wavelengths, which are used to image primarily oxygenated and deoxygenated hemoglobin  were obtained from surgical specimens to further facilitate the comparison between histologic characteristics and OA/US internal and external features in the external boundary and peripheral zones. The central pathology histopathologic diagnosis was the reference standard for OA/US comparison.p values reported in this study can be considered as descriptive statistics in this context.Given the non-normality of the distributions and small sample size, we chose nonparametric tests to analyze the data. Jonckheere–Terpstra tests were performed to evaluate if OA/US features helped differentiate between histologic grades of invasive carcinomas  and between each of the three components of histologic grading: tubule formation, nuclear pleomorphism, and number of mitoses scores. Spearman correlation was used to analyze the relationship between OA/US features and continuous number of mitoses index, percentages of estrogen receptor (ER) and progesterone receptor (PR) status, and continuous Ki-67 proliferative index. Kruskal–Wallis tests were used to analyze the relationship between OA/US feature scores and tumor margins  and the relationship between OA/US feature scores and HER2 status . The same statistical method was used to analyze the relationship between US and OA/US feature scores and molecular subtypes of breast cancer. If a significant relationship was found, additional Wilcoxon–Mann–Whitney tests were used to identify the differences between molecular subtype groups. Significance testing was performed for these supportive analyses without adjustment for multiple testing. Therefore, the Based on the St. Gallen International Expert Consensus of 2013 , breast All statistical analysis was performed using SPSS, version 24.0 (IBM Corp).Of the 215 biopsied masses enrolled in our study, histopathology was benign in 146, high risk in 2, and malignant in 67 . The ages of patients with malignant lesions ranged from 30 to 84 (mean 57) years and ages of those with benign lesions ranged from 20 to 82 (mean 46) years. The mean maximum diameters were 1.43 for benign masses and 1.36 cm for malignant masses.p values, medians, the 25th and 75th percentiles, and the interquartile ranges (IQRs) for the comparisons between OA/US feature scores and histopathological results of invasive breast carcinomas, including tubule formation, nuclear pleomorphism, number of mitoses scores, tumor margins, HER2 receptor status, ER and PR status, and continuous Ki-67 index. Table p values obtained by the Kruskal–Wallis tests when comparing the performance of US vs OA/US in accessing breast cancer molecular subtypes. Table p values, medians, the 25th and 75th percentiles, and the IQRs for the pairwise comparisons between US and OA/US feature scores and breast cancer molecular subtypes. The pairwise comparisons were only obtained for features that were found to be significant by the Kruskal–Wallis tests , ER status (p = 0.033), and Ki-67 (p = 0.009) percentages (Table p = 0.028), as well as LUMB and TNBC (p = 0.006) and LUMB and all other molecular subtypes combined (p = 0.0069). Black and white asterisks in Figs. p = 0.025), sum of all three internal feature scores (p = 0.019), and the sum of total internal and external feature scores (p = 0.028) helped to differentiate between LUMA and LUMB molecular subtypes. Figures p = 0.003), internal blush scores (p = 0.004), total internal hemoglobin scores (p = 0.005), sum of three internal feature scores (p = 0.001), and the ratio between the sum of the three internal and the sum of the two external feature scores (p = 0.006), as well as the sum of all five internal and external feature scores (p = 0.010) helped to differentiate between LUMA and TNBC subtypes. Figures 1, 2, 3, and 4 show examples of typical OA/US and histopathological differences between LUMAs and TNBCs. TNBCs show rich internal findings at OA/US and relative lack of external peripheral zone findings  did not help to distinguish between tubule formation, nuclear pleomorphism, number of mitoses scores, or histologic grades of invasive carcinomas Table . HER2 regs Figs. , while Lp = 0.049). Figure p = 0.003), deoxygenated blush scores (p = 0.010), internal hemoglobin score (p = 0.005), sum of three internal feature scores (p = 0.001), the ratio of the sum of three internal and the sum of the two external feature scores (p = 0.013), and the sum of all five internal and external feature scores (p = 0.008) helped to differentiate between LUMAs and other molecular subtypes. The very same features also helped to differentiate between TNBCs and other molecular subtypes , higher blood microvessel density, and higher numbers of mitoses per 10 high power fields –50. ThisOur results showed that OA/US feature scores assigned to LUMB subtypes were not significantly different than those assigned to TNBCs. Although LUMB tumors present lower cellularity and lower grade and less extensive necrosis than TNBCs, these differences are not as pronounced when comparing LUMAs vs TNBC –25.Another interesting finding was the significant difference between TNBC and HER2-enriched regarding total internal features. These two types of tumors are known to have many overlapping characteristics: both of them are usually high grade, have low cell cohesion, and present with more extensive necrosis , 31, 51.Our findings shed new light on the use of OA/US technology to help clinicians to better differentiate between breast cancer molecular subtypes. Molecular analysis requires specialized equipment and technical expertise, consequently increasing healthcare costs. Recent studies with small number of patients using MRI to better differentiate between breast cancer molecular subtypes presented reasonable results –55. Howep values reported in this study.The generalizability of these results is subject to certain limitations. First, the scope of this study was limited in terms of the number of patients, and the number of malignancies for each molecular subtype group was relatively low. Second, our statistical analysis was performed without adjustment for multiple testing, and future studies are needed to confirm the Notwithstanding these limitations, the study suggests that both the functional and morphologic information provided by OA/US might help radiologists to better differentiate between breast cancer molecular subtypes. Nevertheless, this emerging technique is in its infancy and more studies with larger sample sizes are needed to confirm these preliminary results.ESM 1(DOCX 906 kb)


The growing threat posed by antimicrobial resistance (AMR) continues to attract scientific and policy attention ,2. A numRochford and others recently called for an international inter-agency effort to strengthen global governance of AMR . They suFollowing previous observations in Ethiopia of a possible reduction in childhood mortality resulting from antibiotic interventions tackling trachoma, the New England Journal of Medicine published details of a multi-country cluster-randomised trial in Sub-Saharan Africa, providing twice-yearly mass administration of azithromycin vs placebo to children aged 1 to 59 months . A totalLancet Infectious Diseases highlighted that the mortality reduction was highest in Niger at 18.1%  while there were non-significant reductions of 5.7%  in Malawi and 3.4%  in Tanzania [An editorial in the Tanzania , the nutat risk of wasting (<-1 WHZ) this further increased to 35% [Epidemiological data has shown that malnutrition increases the risk of infectious morbidity and mortality in childhood. Our examd to 35% . Our finWe acknowledge that the relationship between infection and malnutrition is complex  offer nutritional education, to tackle low awareness or cultural barriers ; 2) provMobile phone-based  tools have potential to support the delivery of such holistic interventions as well as to collect data on nutritional and infection indicators to guide the targeting of interventions and enable smarter AMR management . While hWe strongly recommend that nutrition be considered as an essential element of strategies to provide antibiotic stewardship and security. Active and preventative, patient-centred nutritional interventions within primary health care and communities have a key role to play in addressing the global challenge of AMR, supported by further research to unpick the complex relationship between nutrition, infection and AMR.


Double aneuploidies - especially in combination with structural aberrations - are extremely rare among liveborns. The most frequent association is that of Down (DS) and Klinefelter syndromes (KS). We present the case of a male newborn with a unique 47,XY,+ 21[80%]/48,XY,+i(X)(q10),+ 21[20%] karyotype, hypothesize about his future phenotype, discuss the aspects of management and review the literature.The additional association of isochromosome Xq (i(X)(q10)) could be the result of a threefold non-disjunction event. 47,XY,+i(X)(q10) KS is not common and its symptoms differ from the classical KS phenotype. In combined DS and i(X)(q10) KS, the anticipatory phenotype is not simply the sum of the individual syndromic characteristics. This genotype is associated with higher risk for several diseases and certain conditions with more pronounced appearance: emotional and behavioral disorders; poorer mental and physical quality of life; lower muscle mass/tone/strength; connective tissue weakness; muscle hypotonia and feeding difficulties; osteopenia/−porosis with earlier beginning and faster progression; different types of congenital heart diseases; more common occurrence of hypertension; increased susceptibility to infections and female predominant autoimmune diseases; higher risk for hematological malignancies and testicular tumors.In multiple aneuploidies, the alterations have the potential to weaken or enhance each other, or they may not have modifying effects at all. Prenatal ultrasound signs are not obligatory symptoms of numerous chromosomal anomalies , therefore combined prenatal screening has pertinence in uncomplicated pregnancies as well. Double aneuploidies - especially in combination with structural aberrations - are extremely rare among liveborns. The most frequent co-occurrence is of Down (DS) and Klinefelter syndromes (KS) (coincidence 0.098%) , becauseThe 2nd child of a healthy, non-consanguineous Caucasian couple  was first referred to genetic counseling as a male newborn exclusively with signs and symptoms of DS: flat occiput, small and low-set ears, epicanthal folds, upslanting palpebral fissures, rectus diasthasis, umbilical hernia, cutis marmorata, sandal gaps on feet, moderate appetite and feeding difficulties because of generalized muscle hypotonia and a relatively large tongue. During the event-free pregnancy none of the prenatal ultrasound examinations showed any abnormalities. The child was born naturally at 39 gestational weeks with 3170 g, 54 cm, 9/10 Apgar scores and his postnatal adaptation was uneventful. Family history was negative for genetic diseases and developmental disorders and no teratogenic damage was reported. Postnatal analysis of his 15 Giemsa-stained metaphases from standard 72-h peripheral blood lymphocyte cultures led to the 47,XY,+ 21[80%]/48,XY,+i(X)(q10),+ 21[20%] genotype. The proportion of mosaicism was confirmed to be 47,XY,+ 21[80%]/48,XY,+i(X)(q10),+ 21[20%] by fluorescent in situ hybridization (FISH) of 200 interphase cells with 21q22 region , Xp and Xq  specific probes  arise from chromosome abnormalities. In the case of stillbirths this rate is 6–13% , where t− 2) than expected (1.298 × 10− 6) [The incidences of various chromosome aneuploidies differ between livebirths and spontaneous abortions or stillbirths. While autosomal trisomies in combination with X monosomy are more common in the latter group, the association of DS and KS are more dominant in the former  . Co-occu× 10− 6) . This hy× 10− 6) . The simCumulative cell division defects may be explainable by the presence of different epigenetic and other risk factors. The risk is higher in instances of increased maternal (≥35 years) or paternal (≥45 years) age , cousin 88% of DS cases arise from the 2nd maternal and the 1st grandmaternal meiotic cell division, 8% have paternal origin and 4% occur after fertilization , 10. MeiIn the presented newborn the excess of chromosome 21, as it is non-mosaic, could have originated from either a paternal or maternal germ cell, or from an early postzygotic cell division defect. In contrast, the i(X)(q10) is not present in all the lymphocytes of the patient. Some authors suggest that 47,XY,+i(X)(q10) could be maternally inherited , but on The immune system’s dysregulation and its consequences, as well as the higher risk for infections and some types of leukemias are characteristic for DS , 14. A fChildren with DS have a 20-fold greater risk for acute lymphoid leukemia (ALL) and a 500-fold higher risk for myeloid leukemia (ML) compared to children without DS. Overall survival (OS) in DS-ALL (70%) is below the group of patients without DS (OS: 89%). The increased rate of treatment-related toxicity and infectious deaths in DS-ALL can be explained by the patients’ altered metabolic profile and immunodeficient state. The long-distance outcomes are further worsened by the high risk for relapse even after hematopoietic stem cell transplantation. Minimal residual disease response can differentiate between low and high relapse risk DS-ALL patients, and indicate the necessary and safe intensity of treatment .GATA Binding Protein 1 (GATA1) gene mutations in 30% of DS newborns. 25% of TMD in DS (DS-TMD) undergo spontaneous regression (in the first 3 months of life), while the remainder will go on to develop myeloid leukemia of DS (ML-DS). Most cases of DS-TMD are diagnosed 3–7 days after birth, but almost all of them present signs of TMD by age 2 months. 20–30% of TMD  will go on to develop ML-DS  or, less commonly, ALL. ML-DS involves two entities: myelodysplastic syndrome (MDS) and acute megakaryoblastic leukemia (AMKL). MDS often precedes AMKL [5–10% of DS newborns have manifest, and 10–31% have silent transient myeloproliferative disorder (TMD). TMD is a preleukemia characterised by des AMKL .GATA1 mutations in patients with DS, but international guidelines recommend at least one full blood count (FBC) for newborns. Patients who have documented DS-TMD require regular monitoring every 3 months until 4 years of age with FBC and physical examination, due to their higher risk for developing ML-DS. ML-DS patients have significantly better disease-free survival (DFS: 88–89%) compared to children without DS (DFS: 42%), because ML-DS shows better response to less intense therapy. The better chemosensitivity of AMKL could be explained by alteration of cytarabine metabolism. Patients with DS – who develop AMKL – have better 5-years event-free survival (5yEFS: 81%) under the age of 4 years than older children (5yEFS: 33%). Children over 4 years of age with DS and AMKL are more likely to have cytogenetic aberrations similar to sporadic AML, therefore older children with AMKL need more intense therapy [There is no standard for screening  therapy .short stature homeobox (SHOX) gene on Xp [47,XY,+i(X)(q10) is very rare among patients with KS. Including our patient, 23 cases have been reported so far. Although the KS encompasses a wide spectrum of features, the classical phenotype includes consequences of hypergonadotropic hypogonadism. Height can exceed the average, presumably because of delayed epiphyseal plate closure and the extra copy of ne on Xp –23.X inactive specific transcript (XIST) gene on the long arm of the active X. This mechanism regulates X-chromosomal gene dosages in relation to the number of autosomes, therefore in cases of tri-, tetra- or higher polyploidy, there are more than one active X chromosomes [Several studies have dealt with the distinct phenotypic effect of the supernumerary X chromosome in KS. It has emerged that the X inactivation pattern of KS patients is similar to that of healthy women, which extends not only to the extra X chromosome(s), but also to the autosomes. Protection against inactivation is ensured by the expression of omosomes . Therefoomosomes , 23. In omosomes .Skakkebæk et al. analysed the genome-wide transcriptome and methylome of peripheral blood leukocytes in KS patients, male and female controls and detected a unique, KS-specific genetic and epigenetic landscape. They identified candidate genes (coding and non-coding) and regions responsible for the diverse KS features and comorbidities .In 47,XY,+i(X)(q10) some traits, but not all, could easily be originated from the altered dosages of the X chromosomal arms. The excess of Xq is responsible for most of the typical KS symptoms, and the relative lack of Xp for normal height and intellect .Forkhead Box P3 (FOXP3) gene on Xp11.23, which encodes a transcription factor responsible for regulatory T-cell differentiation, leads to insufficient inhibition of autoreactive T-cells. Deletion of FOXP3 causes the IPEX syndrome, which is characterized by immune-dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance. Further anti- (Interleukin 10 (IL10), Transforming Growth Factor Beta 2 (TGFβ2)) and pro-inflammatory (Interleukin 6 (IL6), Transforming Growth Factor Beta 1 (TGFβ1)) cytokine genes are located on the X chromosome, altered dosages of which influence the immune system’s regulation in the opposite way. The decreased dosages of anti-inflammatory cytokine genes on Xp and the increased copies of pro-inflammatory cytokine genes on Xq lead to higher risk for autoimmune diseases. This data is supported by the fact that some disorders (e.g. Hashimoto’s disease) occur more commonly among patients with 45,X,i(X)(q10) Turner syndrome [The frequency of various autoimmune diseases distributes differently between the sexes and different types of chromosome abnormalities, which illustrates the dosage-dependent effect of X-chromosomal genes. Approximately 15% of them remain active (30% of the Xp and < 3% of the Xq genes) . More prsyndrome . A simileukaryotic translation initiation factor 2 subunit gamma (EIF2S3), which is associated with diabetes, cognitive dysfunction and altered brain volumes, hypogonadism and azoospermia in typical KS [EIF2S3 is not altered in i(X)(q10) KS, the aforementioned symptoms should be not present. This hypothesis is supported by the fact that as of yet all reported i(X)(q10) KS patients have normal intellect. On the other hand, these patients are affected by hypogonadism and azoospermia, which further complicates the proper understanding of genotype-phenotype correlations.It is not yet known how inactivation is affected in structural X abnormalities, notably in the 47,XY,+i(X)(q10) genotype. Considering the results of Skakkebæk et al., the expression of candidate Xp genes presumably involved in the KS phenotype may not be altered in the case of i(X)(q10) KS, therefore those features should be lacking. One of those genes is pical KS . If the Since the first description of double 48,XXY,+ 21 trisomy , 82 postIn cases of combined DS and KS the incidence of congenital heart disease could be higher compared to the isolated forms of these aneuploidies. In 12 previously described 48,XXY,+ 21 cases atrial and atrioventricular septal defects were the most frequent abnormalities .In addition, the spectrum of different mental problems could be wider, and their risk could be higher in this group of patients.The phenotype of 48,XXY,+ 21 double aneuploidy is characterized by classical DS features in children, who then develop additional KS symptoms from the age of puberty. If we experience the following symptoms, we should suspect the presence of KS beside DS: male infant with developmental abnormalities of the genitalia , delay or absence of puberty, signs of hypergonadotropic hypogonadism , oligo−/azoospermia, tall stature (>97th percentile).The combined DS and i(X)(q10) KS is associated with higher risk for several diseases and certain conditions with more pronounced appearance: emotional and behavioral disorders; poorer mental and physical quality of life; lower muscle mass/tone/strength; connective tissue weakness; muscle hypotonia and feeding difficulties; osteopenia/−porosis with earlier beginning and faster progression; different types of congenital heart diseases; more common occurrence of hypertension; increased susceptibility to infections and female predominant autoimmune diseases; higher risk for hematological malignancies and testicular tumors.In multiple aneuploidies and compound chromosomal abnormalities the anticipatory phenotype is not simply the sum of the individual syndromic characteristics. The alterations have the potential to weaken or enhance each other, or they may not have modifying effects at all. Prenatal ultrasound signs are not obligatory symptoms of numerous chromosomal anomalies , therefore combined prenatal screening has pertinence in uncomplicated pregnancies as well. This might also suggest that only chromosomal anomalies that usually lead to pronounced anatomical malformations (e.g. 21/13/18 trisomies) may be detected during the 18- to 22-week fetal anatomical ultrasound. In cases of i(X)(q10) KS long-term follow-up, transcriptome and methylome analyses are needed to clarify the role of the X-chromosomal regions in KS traits, the regulation of the immune system and autoimmune diseases.


Scientific Reports 10.1038/s41598-018-33294-1, published online 17 October 2018Correction to: The original version of this Article contained errors in the Abstract.muta, yet growth models supported rapid growth; Indo-Pacific sponges were over twice as old as published estimates of comparably sized X. muta , although extrapolation errors are likely to increase with sponge size.”“Interestingly, SGRs were slightly slower than that of X. now reads:muta, yet growth models supported rapid growth; published estimates of comparably sized X. muta were over twice as old as Indo-Pacific sponges , although extrapolation errors are likely to increase with sponge size.”“Interestingly, SGRs were slightly slower than that of X. These errors have now been corrected in the PDF and HTML versions of the Article.


Piezoelectric poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) nanofibers fabricated by electrospinning have drawn increasing levels of attention in the fields of flexible sensors and nanogenerators. However, the directional dependence of piezoelectricity of electrospun nanofibers remains elusive. In this study, the piezoelectric performances of individual nanofibers are characterized by piezoresponse force microscopy (PFM), while the effects of annealing on β-phase crystallinities are investigated by X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The experimental results reveal that the as-spun P(VDF-TrFE) nanofibers form higher content of β-phase compared with spin-coated films, and the content of β-phase increases by annealing. The annealed P(VDF-TrFE) nanofiber exhibits distinct vertical polarization switching characteristics. The high piezoelectric output in the thickness direction and low piezoelectric output in the longitudinal direction of the nanofiber mats further confirm that the preferential dipole orientation of electrospun P(VDF-TrFE) nanofibers is normal to the surface of the substrate. Highly aligned P(VDF-TrFE) nanofibers show directional strain sensing ability due to the piezoelectric and mechanical anisotropy. Initially, PVDF and P(VDF-TrFE) devices that were processed into films as the sensing materials in micro-electro-mechanical systems (MEMS) [Piezoelectric polymers and associated nanostructured materials are widely used in mechanical sensing and energy harvesting for their abilities to accumulate electrical charges in response to applied mechanical stresses ,2,3,4. Ps (MEMS) ,7,8,9,10s (MEMS) ,13,14,15s (MEMS) ,20,21,22s (MEMS) . A wave-s (MEMS) . Though 2 dipoles in the P(VDF-TrFE) nanofibers web prefer to be oriented perpendicular to the nanoweb surface; this preference has been verified by polarized Fourier transform infrared spectroscopy (FTIR) as well as piezoelectric signal detection in a nano-pressure sensor [The piezoelectric performance of a P(VDF-TrFE) nanofiber is determined by its fabrication process and morphology. Compared with randomly oriented nanofibers, well aligned arrangements of such nanofibers can improve piezoelectric performance. Lin et al. proposed a near-field electrospinning (NFES) method for the fabrication of aligned PVDF and P(VDF-TrFE) nanofibers ,24. The e sensor . HoweverP(VDF-TrFE)-copolymer powder with a 75/25 mol % VDF/TrFE ratio, was purchased from Kureha Chemical Industries . Dimethylformamide (DMF) which was used as solvent for the P(VDF-TrFE), was obtained from Tianjin Guangfu Science and Technology Co., Ltd . Acetone was supplied by Beijing Chemical Works  and was used to dilute the mixture of P(VDF-TrFE) and DMF.w/w; DMF/acetone) by mechanical stirring for 12 hours at room temperature. The weight fraction of P(VDF-TrFE) in the solvent was 21 wt %. A typical far-field electrospinning setup is shown in The P(VDF-TrFE) powder was dissolved in dimethylformamide/acetone (3:2 The morphologies of the P(VDF-TrFE) nanofibers were examined by scanning electron microscopy  at an accelerator voltage of 15 kV. Fast Fourier transforms (FFT) was performed on SEM images using the ImageJ software  to determine the degrees of the nanofibers alignment. In order to analyze the compositions and crystallinities of the P(VDF-TrFE) nanofibers, Fourier transform infrared spectroscopy  in absorbance mode, and X-ray diffraction  over the 15–25° 2θ range were employed. A P(VDF-TrFE) spin-coated film was fabricated to study its diversity compared with the electrospun nanofibers. In addition, the P(VDF-TrFE) nanofibers were annealed at 130 and 140 °C for 2 h to investigate the variations in β-phase crystallinity.The local piezoelectric response of a single P(VDF-TrFE) nanofiber was evaluated using piezoresponse force microscope (PFM) . A conductive Ti/Ir coated Si cantilever  with a force constant of 2 N/m and a free air resonance frequency of 70 kHz was used during the measurements. P(VDF-TrFE) nanofibers were sparsely collected on an ITO-coated glass substrate using the electrospinning method. The vertical piezoresponse hysteresis loops were measured by dual ac resonance tracking PFM (DART-PFM) mode. The contact frequency varied from 275 to 285 kHz due to slightly changes on sample surface from point to point. An alternate voltage of 2 V was applied between the PFM cantilever tip and the bottom electrode. The nanodomain switching characteristics of individual nanofibers with nanometric resolution were studied by switching spectroscopy PFM (SS-PFM) measurement. For SS-PFM, a sequence of pulses DC bias voltage (Vdc) was utilized to offer the nanofiber with essential electric field. The amplitude of the DC pulses varied stepwise from −50 to 50 V. The PFM amplitude and phase measurements were carried out in the “off state” of the pulses, so that the electrostatic effect was eliminated from the hysteresis. In the process, at least three different locations along the individual fiber were examined.The far-field electrospinning method using a rotating-drum collector can produce well-aligned P(VDF-TrFE) nanofibers. The SEM images displayed in −1) are highlighted in the FTIR spectra in The piezoelectricity of P(VDF-TrFE) depends on the concentration of the piezoactive β-phase, which exhibits the best piezoelectric properties because of its large spontaneous polarization. Ferroelectric-polymer polymorphs are generally identified and quantified by XRD and FTIR. X-ray diffraction reveals the changes that occur in the crystalline structure of the P(VDF-TrFE) as a result of annealing, while FTIR is an important method for the evaluation of the polar β-phase content, α to β transitions, as well as the dipolar orientation in the material. XRD and FTIR spectra confirmed the formation of the β-phase crystalline structure in the P(VDF-TrFE) nanofibers as shown in The piezoelectricity of the P(VDF-TrFE) nanofiber was characterized by the PFM method. As shown in The piezoelectric output of the P(VDF-TrFE) nanofibers in the longitudinal direction was determined by applying tensional stress in the same direction. As shown in 2 and 65%, respectively. As a result, the piezoelectric constant in the nanofiber’s longitudinal direction is calculated to be 0.04 pC/N. The weak piezoelectric effect in the nanofiber’s longitudinal direction further confirms that the direction of polarization of the P(VDF-TrFE) nanofibers fabricated by the far-field electrospinning process is normal to the surface of the substrate.The piezoelectric output was measured for nanofibers with different heat treatments and stretching conditions. As shown in A strain sensing device composed of P(VDF-TrFE) nanofibers sandwiched by electrodes was fabricated. The upper and lower electrodes are both 7 mm square. The thickness of the P(VDF-TrFE) nanofibers mat is approximately 50 µm. The sensor device was positioned with two clamps that were driven by a piezoelectric vibrator as shown in As shown in In order to further demonstrate the directional sensing performance of the nanofibers, two P(VDF-TrFE) nanofiber mats with identical dimensions were embedded into a polydimethylsiloxane (PDMS) cantilever with their fiber alignment directions parallel and perpendicular to the cantilever length direction, respectively a. While The PFM results presented here indicates that the electrospinning process for P(VDF-TrFE) nanofibers yields high population of aligned electrical dipoles in the direction normal to the substrate surface, thereby demonstrating excellent piezoelectric response without further electrical poling process. Compared with spin-coated P(VDF-TrFE) films, which require additional electrical poling to achieve high piezoelectricity , the P nanofibers. XRD and FTIR spectra confirmed that the as-spun P(VDF-TrFE) nanofibers  were composed of high proportion of piezoactive β-phase and could be further enhanced by annealing. Specifically, the PFM experiments and the piezoelectric output in the nanofiber’s longitudinal direction demonstrated that the electrical dipoles preferred to orient themselves in the direction normal to the nanofiber mat surface. We developed well-aligned P(VDF-TrFE) nanofibers mats that exhibited enhanced directional strain sensing properties. These findings provide a new strategy for optimizing the design and fabrication of biomimetic sensors using P(VDF-TrFE) nanofibers.


A real time control facility consisting of a batch reactor, data acquisition software “LabVIEW” and a PC for monitoring and control is used to implement these operating conditions. The resulting product is analyzed by gel permeation chromatography (GPC). The data generated can be used by researchers, academicians and industry for generating control strategies, automation and scale up of polymerization reactors.This work describes an approach towards experimental implementation of real time control studies conducted on a batch polymerization reactor. The information is related to the controlling of molecular weight for styrene acrylonitrile copolymer polymerization system in a batch reactor generated under a varied range of temperatures, reactant concentrations and retention times. The operating conditions of 6 hrs and temperature of 343 K for yielding a molecular weight in the range of 39,900–40,000 gmol Real time temperature monitoring using DAQ with desired setpoint is shown in 2M1, M2), solvent and initiator (I). The mathematical model of the process is given as follows:In this work, styrene acrylonitrile copolymer is chosen where it is also modeled prior to experimentation ,3. The fThe moment equations for dead polymers are given by three moments:The rate constants, reported .3Experiments were conducted for styrene acrylonitrile polymerization process in a batch reactor with the conditions mentioned in 4The data was monitored using a data aquisition software “LabVIEW” which is a commercial software package installed in the personnel computer to acquire, monitor, handle, analyze and log the data. All the control logic and the program for data logging is written using functions in the block diagram. All the controls and indicators are displayed in the front panel where we can also have online monitoring of the process in the form of charts at runtime. In the block diagram the process parameters are read using the input DAQ Assistant function. These values are logged into text files using the write to measurement file function. The process variable is given to the PI control function and the obtained controller output is fed back to the O/P modules using the output DAQ Assistant function. The required process values are given to a chart to have an online trend while running the process which can be seen on the front panel.5Free radical polymerization experiments were carried out in 250 ml glass reactor to establish and optimize the process conditions. The experiment is conducted with varying temperatures  is listed in Real time temperature monitoring using DAQ with desired set point is shown in 6separates sample components from one another with rapid analyses. Detector used is refractive index to monitor molecular weight distribution which is directly proportional to concentration. In this procedure the molecular weight of the sample is determined based on the standards obtained by plotting the retention time against the log of molecular weight. The operating conditions for these methods are established as flow rate of 0.05 ml/min, detectors were refractive index, the temperature is 24 °C and the column porosities are10000 Å. The molecular weight of SAN copolymer obtained through this method is 30,000.The polymer synthesized at the end of the reaction time is subjected to GPC analysis for determination of molecular weight. The sample is first dissolved in solvent (THF-tetra hydro furan) and is injected into a continually flowing stream of tetrahydrofuran (THF) without disturbing the continuous mobile phase flow at a flow rate of 0.05 ml/min. The mobile phase flows through millions of highly porous, rigid particles tightly packed together in a column. The column 7The operating conditions of reaction time for 6 hrs and temperature of 343 K yield a molecular weight of 39,900 g mol-1 which is nearer to the desired molecular weight of 40,000 g mol-1 is selected as the best operating conditions. The experiment with optimal operating conditions is repeated in a 2 L batch reactor having a PC for online monitoring with data acquisition software. The experimental setup consists of a stainless-steel reactor (Parr) of 2 L capacity with an external jacket with an electric coil for heating, and also with an internal cooling coil. A total volume of 1200 ml of mixture (70% of styrene and 30% of acrylonitrile) was used to carry out the experiments. The experiments were carried out by monitoring and controlling the reactor temperature using the manipulated heater with the aid PID controller algorithms accordingly. The optimum operating conditions that are used in pilot scale experiments were temperature: 343K, styrene: 160ml, acrylonitrile:160ml,xylene:800ml and azobisisobutyronitrile: 1.6 g.


IGF2 and CDKN1C and their associated pathways, and thereby, cause growth disturbances as key features of both diseases. The increase of knowledge has become possible with the development and implementation of new and comprehensive assays. Whereas, in the beginning molecular testing was restricted to single chromosomal loci, many tests now address numerous loci in the same run, and the diagnostic implementation of (epi)genome wide assays is only a question of time. These high-throughput approaches will be complemented by the analysis of other omic datasets , and it can be expected that the integration of these data will massively improve the understanding of the pathobiology of imprinting disorders and their diagnostics. Especially long-read sequencing methods, e.g., nanopore sequencing, allowing direct detection of native DNA modification, will strongly contribute to a better understanding of genomic imprinting in the near future. Thereby, new genomic loci and types of pathogenic variants will be identified, resulting in more precise discrimination into different molecular subgroups. These subgroups serve as the basis for (epi)genotype–phenotype correlations, allowing a more directed prognosis, counseling, and therapy. By deciphering the pathophysiological consequences of SRS and BWS and their molecular disturbances, future therapies will be available targeting the basic cause of the disease and respective pathomechanisms and will complement conventional therapeutic strategies.Silver–Russell and Beckwith–Wiedemann syndromes  are rare congenital human disorders characterized by opposite growth disturbances. With the increasing knowledge on the molecular basis of SRS and BWS, it has become obvious that the disorders mirror opposite alterations at the same genomic loci in 11p15.5. In fact, these changes directly or indirectly affect the expression of  Silver–Russell and Beckwith–Wiedemann syndromes  are rare congenital human disorders characterized by opposite growth disturbances and molecular alterations , Table 1SRS is associated with pre- and postnatal growth retardation, relative macrocephaly, prominent forehead, asymmetry, feeding difficulties, and further rather heterogeneous features . The cliBWS is also characterized by a variable phenotype that may include macroglossia, anterior abdominal wall defects , pre- and/or postnatal overgrowth, neonatal hypoglycemia, hemihypertrophy, and predisposition to embryonal tumors (particularly Wilms tumor) . Its incBoth syndromes are associated with alterations affecting the chromosomal region 11p15.5. This region harbors two clusters of imprinted genes, i.e., genes which are monoallelically expressed either from the maternal or paternal allele  4]. The. The4]. Like the majority of the other known imprinting disorders, SRS and BWS are associated with similar types of molecular alterations, comprising both genomic variants affecting the coding DNA sequence and structure of imprinted genes and the regulation regions, as well as altered methylation of the respective ICs . WhereasThe most common underlying molecular change in SRS in loss of methylation of the imprinting center 1 in chromosome 11p15 (IC1 LOM) in 40% to 50% of cases . In 10% In more than 70% of BWS patients, molecular alterations of the imprinting centers 1 and 2 in 11p15 can be observed, with IC2 LOM and paternal UPD 11p15 (upd(11p15)pat) as the major findings  [IGF2 and CDKN1C has been suggested to be associated with growth disturbances  and new personalized approaches.In this review, we aim to review the current knowledge on pathobiology and pathophysiology of SRS and BWS as models for opposite congenital phenotypes associated with contrary molecular disturbances. By understanding the functional (epi)genotype–phenotype links and the affected physiological pathways, therapeutic strategies can be improved and developed.Up to now, molecular testing in SRS and BWS has been restricted to evidence-based molecular alterations, comprising disturbances of the imprinted regions in 11p15.5 and upd(7)mat in case of SRS. With the commercial availability of methylation-specific (MS) multiplex ligation-dependent probe amplification (MLPA) based tests targeting several imprinted loci on different chromosomes, the number of tested patients with the suspicious diagnosis of SRS or BWS has increased. However, there is a growing demand for molecular testing to confirm or exclude the clinical diagnosis. In fact, it should be emphasized that, as in other genetic disorders, exclusion of an imprinting disorder is hardly possible. This is due to the widespread mosaic distribution of epimutations in an individual, the targeted analysis of specific genomic regions leaving out others , the unspecificity of symptoms and the genetic heterogeneity of SRS, BWS, and the other imprinting disorders.In case of the 11p15.5 associated disorders, the application of MS MLPA allows the identification of three different molecular alterations , affecting the IC1 and IC2, in the same run . MeanwhiAs mentioned before, MS MLPA is suitable to identify the majority of characteristic molecular alterations in 11p15.5, but it also detects unexpected and even molecular opposite (epi)genetic variants. Hence, several molecular alterations which do not fit with clinical features of SRS or BWS but are consistent with the other 11p15.5 associated imprinting disorder, have been reported, e.g., patients referred as SRS with IC2 LOM or those diagnosed as BWS with IC1 LOM [As recent data show, methylation testing in BWS and SRS should not be restricted to the 11p15.5 loci (and those on chromosome 7 in case of SRS), because a broad spectrum of alterations at imprinted loci others than in 11p15.5 can be detected in such patients. Two causes are responsible for this observation: (a) Overlap between clinical phenotypes of BWS, SRS, and other imprinting disorders, and (b) a significant number of patients with LOM of either IC1 or IC2 carry so-called multilocus imprinting disturbances (MLID), harboring varying genome-wide methylation alterations of imprinted loci.(a) The overlapping phenotypes of imprinting disorders make a clear clinical classification sometimes difficult. Thus, it is not surprising that molecular diagnostics sometimes detect genomic alterations known for another imprinting disorder than the one which has been initially suspected . In the (b) In the majority of imprinting disorder patients, only the disease-specific loci are affected, but an increasing number of individuals has been identified to carry MLID . It is cIn addition to the occurrence of disturbed imprinting patterns, there is a growing number of patients with molecular and clinical features of SRS or BWS exhibiting genomic alterations, i.e., insertions, deletions, duplications. Currently, three different groups of genomic variants can be discriminated:IGF2 and CDKN1C, their central role in the pathophysiology of the 11p15.5 associated imprinting disorders has become obvious (KCNQ1 in IC2) do not only affect the function of the encoded protein itself but also the methylation of the respective DMR [The development of high throughput and high-resolution DNA analyzing techniques ) has made the detailed characterization of the structure and sequence of the two ICs in 11p15 (and other regions) and the genes under their control possible ,35,36. F obvious . Finallytive DMR ,34.NLRP2, NLRP5, NLRP7; [The second group of variants in families with imprinting disorders is associated with an uncommon mode of inheritance, the so-called maternal effect variants. This mode of inheritance describes the observation that the mothers in these families are carriers of pathogenic variants without clinical features but which cause aberrant imprinting in the offspring, i.e., MLID and LOM of the IC2 in 11p15.5 . These v, NLRP7; ). Matern, NLRP7; , but it , NLRP7; ,40.HMGA2 and PLAG1, new genes causing SRS have been found [With the application of whole exome sequencing (WES), whole genome sequencing (WGS), and DNA microarrays in genetic diagnostics of SRS and BWS, not only alterations within imprinted regions are detected, but also an increasing number of pathogenic genomic changes in other chromosomal regions could be identified. In fact, with en found , therebyomic approaches  which will allow the parallel determination of the functional relevance of (epi)genetic alterations on the expression of some imprinted genes [In future, these (epi)genome tests should be complemented by further ed genes . Therebyed genes ), this nHowever, the majority of genomic variants are detected in genes associated with differential diagnosis of SRS and BWS ,2,10,22.One major reason to apply broad tests is the chance to identify the molecular causes in case of clinically and genetically heterogeneous disorders, as well as their cost efficiency and velocity in comparison to stepwise approaches. As a result, assays covering multiple genetic loci or even whole exomes (and genomes) are increasingly applied in routine diagnostics.The identification of pathogenic variants in imprinted chromosomal regions or genes causing aberrant imprinting as well as in genes associated with overlapping clinical pictures does not only increase the diagnostic yield but it also contributes to the understanding of the pathobiology of imprinting disorders and their functional consequences.In the last years, it has become obvious that many imprinted genes interact in a so-called imprinted genes network (IGN) . EvidencH19, IGF2, and CDKN1C as members of the imprinting clusters in 11p15.5 [As outlined earlier, there is considerable clinical and molecular overlap between different imprinting disorders, which can be explained by the IGN regulating zinc-finger TF PLAGL1. PLAGL1 is co-expressed with hundreds of genes, many of which are imprinted  among th 11p15.5 ,44. ChanThis complex network of interactions between multiple genes and pathways is also highlighted by the role of noncoding RNAs encoded by the 11p15.5 region and other imprinted regions  which act as microRNAs. In fact, the molecular function of these ncRNAs is often unclear, but a role for the attenuation of imprinted genes expression in specific cells or tissues  or in fuThe coordinated control of imprinting at different loci is corroborated by the identification of multilocus imprinting disturbances (MLID) in a significant number of patients with specific imprinting disorders. This occurrence of aberrant methylation at different chromosomal loci indicates that common genomic alterations localized in trans are involved in the etiology of MLID. As preliminary data show, these alterations affect the maintenance of imprinting marks in the developing embryo, and the factors mediating this process are either expressed from the embryonal genome or are already expressed in the oocyte .ZFP57 mutations [An example of a factor expressed from the embryonic genome is ZFP57, loss of function mutations occurs in TNDM cases with MLID . In the utations ,50,51.As aforementioned, mutations in factors expressed in the oocyte also contribute to aberrant imprints and comprise maternal-effect variants. Several members of the subcortical maternal complex in the oocyte have been identified to carry maternal effect variants , with NLAltogether, these observations contribute to the understanding of the mechanisms of imprinting regulation and its disturbances but also help to understand phenotypic outcomes, comorbidities, and clinical overlaps. Many imprinted genes are functionally related and part of common physiological pathways. These pathways and the IGN comprise both imprinted as well as non-imprinted networks, showing the complexity of the regulation of genomic imprinting.IGF2, CDKN1C, HMGA2, and PLAG1, but also genes mutated in patients with differential diagnoses of SRS or BWS . In addition to the upstream factors, variants in genes downstream of IGF2 in the pathways are also associated with disturbed growth and features reminiscent of SRS and BWS [PIK3R1, which cause autosomal-dominant SHORT syndrome [PIK3R1, there are also patients with activating mutations in the catalytic subunit PIK3CA of PI3K. These mutations lead to an overgrowth phenotype in contrast to patients with PIK3R1 mutations resulting in growth retardation and reduced PI3K/AKT-signaling [The major growth-related pathways affected in patients with 11p15.5 associated imprinting disorders include IGF2 . In fact and BWS . An examsyndrome . PIK3R1 syndrome . These psyndrome . The actsyndrome . In addiignaling .KCNQ1 variants predisposing to LongQT disorder and IC2 LOM [In parallel to the identification of the heterogeneous molecular alterations associated with SRS and BWS features, numerous studies aimed to determine the correlation between these changes and clinical features as the basis for a more directed therapy . Though  IC2 LOM . Future In SRS, clinical differences are less obvious between the major molecular subgroups, IC1 LOM in 11p15, upd(7)mat and changes affecting the 14q32 DMRs ,64, but Particular care should be given to patients with 14q32 alterations, as  showed aThe cognitive development of patients with the clinical diagnosis of SRS and BWS is reported to be in the normal range in the majority of patients. However, specific molecular subtypes predispose developmental delay in some individuals. In SRS, neurocognitive problems are more frequent in patients with upd(7)mat  than in Though SRS and BWS are, meanwhile, well-established entities and huge clinical datasets are available in the neonate and childhood period, systematic follow-up studies to estimate the impact on life-long health and quality of life are outstanding. In fact, the first studies on metabolism and psychosocial situation in adults with SRS have been published ,68, but In addition to the need to precisely determine the molecular subtypes of chromosomes 7 and 11 disturbances, the identification of the molecular cause in patients with SRS or BWS features but without the characteristic (epi)mutation might also have a massive impact on the clinical management. Several differential diagnoses of SRS and BWS are associated with specific features requiring special attention and treatment . ExampleIn the future, understanding of the pathobiology of imprinting disorders will broaden the current conventional approaches by precise therapeutic strategies directly targeting the molecular cause of the disease. The first potential therapeutic interventions have been suggested for Angelman syndrome by reducing the UBE3A antisense transcript with antisense oligonucleotides (ASO) . ExperieFinally, the identification of the molecular cause in patients with SRS or BWS is required to estimate the recurrence risk in their families. Whereas, the majority of epimutations in both disorders occurs sporadically and is, therefore, not associated with a recurrence probability, in particular, CNVs can be associated with a significantly increased recurrence risk of up to 50%. In the case of maternal-effect mutations, this risk can even be higher.Furthermore, in context with the use of WES or WGS tests, it has to be considered that incidental findings might be identified. These variants are not functionally related to the disorder itself but might have a predictive value for other disorders. In fact, this aspect has to be discussed with the patients and their families before genetic testing. If possible, a molecular diagnostic workup should, therefore, rather start with targeted assays addressing disease-specific genetic loci to circumvent the detection of incidental findings and/or variants of unknown significance, which might cause uncertainty and anxiety in the patients and their families. Therefore, the use of diagnostic tools covering whole exomes or genomes should only be used after careful weighing of the pros and cons.With the increasing knowledge on the molecular basis of SRS and BWS, it has become obvious that the disorders mirror opposite alterations at the same genomic loci by contradictory phenotypic outcomes. In fact, these changes directly or indirectly affect the expression of IGF2 and its associated pathways and thereby, cause growth disturbances as key features of both diseases.omic datasets , and it can be expected that the integration of these data will massively improve the understanding of the pathobiology of imprinting disorders and their diagnostics. Especially long-read sequencing methods, e.g., nanopore sequencing, allowing direct detection of native DNA modification, will strongly contribute to a better understanding of genomic imprinting in the near future. Thereby, new genomic loci and types of pathogenic variants could be identified, resulting in more precise discrimination into different molecular subgroups. These subgroups serve as the basis for (epi)genotype–phenotype correlations, allowing a more directed prognosis, counseling, and therapy. By deciphering the pathophysiological consequences of SRS and BWS and their molecular disturbances, future therapies will be available targeting the basic cause of the disease and respective pathomechanisms, and will thereby, complement conventional therapeutic strategies.The increase of knowledge has become possible with the development and implementation of new and comprehensive assays. Whereas, in the beginning, molecular testing was restricted to single chromosomal loci, many tests now address numerous loci in the same run, and the diagnostic implementation of (epi)genome-wide assays is only a question of time. These high-throughput approaches will be complemented by the analysis of other In addition to the benefit to the patients, these improvements will also support their families in decision making, by providing them with more information on genetic risks and therapeutic options.