Here, we tested if the hot vapor equilibration method originally developed for cellulose is applicable for NSC, verified by comparison with the traditional nitration method. We set up a detailed analytical protocol and applied the method to plant extracts of leaves from species with different photosynthetic pathways . δ2Hne of commercial sugars and starch from different classes and sources, ranging from −157.8 to +6.4‰, were reproducibly analysed with precision between 0.2‰ and 7.7‰. Mean δ2Hne values of sugar are lowest in C3 (−92.0‰), intermediate in C4 (−32.5‰) and highest in CAM plants (6.0‰), with NSC being 2H‐depleted compared to cellulose and sugar being generally more 2H‐enriched than starch. Our results suggest that our method can be used in future studies to disentangle 2H‐fractionation processes, for improving mechanistic δ2Hne models for leaf and tree‐ring cellulose and for further development of δ2Hne in plant carbohydrates as a potential proxy for climate, hydrology, plant metabolism and physiology.The analysis of the non‐exchangeable hydrogen isotope ratio (δ 2Hne analysis of sugar and starch. Additionally, we show first insights into differences of δ2Hne values within and between C3, C4 and CAM plant species growing under the same constant climatic conditions.Here, we present a hot water‐vapor equilibration method that is suitable for high‐throughput δ The interference of Hex greatly affects the analysis of δ2Hne, which retains useful information on climate, hydrology, metabolism and physiology. The oldest method of measuring δ2Hne is to derivatize hydroxyl groups with nitrate esters, using a mixture of either H2SO4 or H3PO4 with HNO3   grown under the same controlled climatic conditions.The difficulty of establishing reliable methods for δ22.1n = 4; spruce cellulose, Fluka, Honeywell International Inc., Morristown, New Jersey, U.S.A., Prod. No. 22181; IAEA‐CH‐3, International Atomic Energy Agency (IAEA), Vienna, Austria; Merck cellulose , Wei Ming , and in‐house produced cellulose standards , commercially available starch standards , commercially available standards for sugars of different classes ‐glucose ≥99.5%, SIGMA Life Science, St. Louis, Missouri, U.S.A., Prod. No. 49139; d‐(−)‐fructose ≥99%, Fluka, Prod. No. 47739; d‐(+)‐raffinose pentahydrate ≥99%, Fluka, Prod. No. 83400; d‐(+)‐trehalose dihydrate ≥99%, SIGMA Life Science, Prod. No. T9449 and myo‐Inositol ≥99.5%, Sigma Life Science, Prod. No. 57569) and two household sugars . All reference materials were oven‐dried at 60°C for 48 hr and stored in an exicator at low relative humidity (2–5%) until further use.As reference materials, we used both commercially available  Moench, Cannabis sativa L., Hordeum vulgare L., Salvia hispanica L. and Solanum cheesmaniae (L. Riley) Fosberg), C4 grasses (Sorghum bicolor (L.) Moench, Zea mays L.) and CAM plants . Seeds or plantlets were sown or planted in 3 L pots containing potting soil . The orchid Phalaenopsis was bought in a local supermarket and grown in a special substrate based on bark mulch. The climate chamber conditions were set to 16 daytime hours (30°C and 40% relative humidity), 8 nighttime hours (15°C and 60% relative humidity) and photosynthetically active radiation of 110 μmol m−2 s−1 at plant height with uniform fluorescent tubes . All plants were regularly watered to field capacity with tap water  to avoid any water limitation, except for Phalaenopsis that was watered with 50 ml twice a week to keep the substrate moist but prevent root rot due to excess water. The plants were grown for three months to ensure ample leaf material was grown for harvest.Ten plant species with different photosynthetic pathways grown under controlled conditions in walk‐in climate chambers  were used to apply the new method, and compare δH. vulgare, were sampled after 7 hr of light to allow the plants to synthesize enough sugars and starch on the day of harvest and to guarantee steady‐state leaf water enrichment conditions  for a first offset correction and the USGS62, USGS63 and USGS64 caffeine standards   are calculated as given in Equation  Coplen,:(1)δ=R2H/1H of the sample (RSample) and of Vienna Standard Mean Ocean Water  as the standard defining the international isotope scale. To express the resulting δ values in permil (‰), results have been multiplied by 1,000.R = 2.42Hne values  was extracted once per sample. Cellulose (hemicellulose) was extracted from 100 mg of the fragmented leaf material in F57 fibre filter bags . In brief, the samples were washed twice in a 5% sodium hydroxide solution at 60°C, rinsed with deionized water, washed 3 times for 10 hr in a 7% sodium chlorite solution, which was adjusted with 96% acetic acid to a pH between 4 and 5, and subsequently rinsed with boiling hot deionized water, and dried overnight in a drying oven at 60°C. The neutral sugar fraction , which led to a total peak area between 20 and 30‐V seconds (Vs) of each IRMS analysis. For sugar standards, one mg was transferred first into a 5.5 × 9 mm silver foil capsule (IVA), and additionally packed in a second capsule of the same size and folded again. The reason for the double packing was the observation that sugar samples became liquefied and rinsed out of single‐packed capsules during the hot water vapor equilibration, which led to a loss of sample and to negative impacts on the analysis of δe sugars . The dours Table . All pac−1) into the equilibration chamber. The temperature setpoint of the preheated oven was set to a constant 130°C, ensuring immediate evaporation of water after entering the equilibration chamber. The end of the outlet metal tube was inserted into a glass vessel and checked for vapor flow and condensation of the blown‐out vapor. After 2 hr of equilibration, the feeding capillary was switched to a capillary delivering dry nitrogen gas  with a pressure of one bar for 2 hr to ensure complete removal of gaseous water in the chamber, which was still kept at 130°C. The duration of equilibration and drying, as well as the equilibration temperature, were step‐wise tested for cellulose to ensure maximum equilibration and no residual vapor. However, the high equilibration temperature of 130°C might be important to break down the crystalline structure of sugars and gelatinize starch to enable the access of water vapor  and three times with Water 2 (δ2H = −428‰). For starch and digested starch, triplicates were equilibrated only once with Water 1 and once with Water 2.All samples were equilibrated in a home‐built offline equilibration system , consistPlus XP IRMS . It is crucial to transfer the samples as fast as possible and still hot from the equilibration chamber to the autosampler to avoid any isotopic re‐equilibration of the sample with air moisture and water absorption. The autosampler carousel was evacuated to 0.01 mbar and afterwards filled with dry helium gas to 1.5 bar to avoid any contact with ambient water (vapor). The samples were pyrolysed in a reactor according to Gehre, Geilmann, Richter, Werner, and Brand (−1) to the IRMS. Raw δ2H values of standard material  were immediately transferred into a Zero Blank Autosampler (N.C. Technologies S.r.l.), which was installed on a sample port of a high‐temperature elemental analyser system. The latter was coupled via a ConFlo III interface to a Deltand Brand , and car2H values of plant‐derived compounds were offset corrected using PEF. The calculated δ2Hne of plant extracted sugar and sugar derived from starch :δEquation  using onStatistical analyses (one‐way ANOVA and Tukey posthoc test) were performed using R version 3.6.3  ranging between 0.9‰ and 4.1‰ for both equilibration waters. In addition, high accuracy was found, as indicated by a deviation of −1.0 to +5.7‰ between the δ2Hne value of the hot water vapor equilibration and the δ2H value of the corresponding cellulose nitrate (δ2Hne‐ δ2Hnitro), except for two of the commercial cellulose samples from Fluka and Wei Ming, with a deviation of −18.8 and +7.7‰, respectively. For the samples with high accuracy, the calculated xe ranged between 19.3 and 22.1% compared to a theoretical xe.pot of 30%. These xe values are comparable to those 20.5 ± 0.1% observed in the original implementation of the hot water vapor equilibration for cellulose  and xe.pot of pure glucose (41.7%). This lower xe of starch‐derived sugar compared to glucose could be explained by incomplete digestion of the starch to glucose monomers, leading to a mixture of mono‐ and oligosaccharides.The δ2Hne of NSC in plants or in other environmental or biological samples.Overall, our results show that sugars of different classes, as well as sugar derived from digested starch, can be measured with high precision, accuracy and reproducibility. On a daily routine, we were able to measure up to 66 NSC samples and 32 standards. This proves that a method is now a reliable tool that enables high‐throughput analysis of δ3.22H‐depleted compound, followed by sugar, while cellulose was the most 2H‐enriched compound. In C3 plants, all compounds were significantly different from each other and showed the strongest 2H‐depletion of all photosynthetic types, with a mean δ2Hne of −121.7‰ for starch, −92.0‰ for sugar and −61.4‰ for cellulose. In C4 plants, mean δ2Hne values of −60.9‰ for starch were significantly lower compared to those of −32.5‰ and −16.5‰ for sugar and cellulose and thus reflect intermediate δ2Hne values compared to C3 and CAM plants. In CAM plants, only δ2Hne values of starch and cellulose differed significantly and showed the strongest 2H‐enrichment of all photosynthetic types, with a mean δ2Hne of −10.2‰ for starch, 6.0‰ for sugar and 10.9‰ for cellulose. The comparison of the δ2Hne of the same compound between the photosynthetic types resulted in significant differences between C3 and C4 and between C3 and CAM plants. The difference in sugar and cellulose between C4 and CAM plants was only slightly significant and not significant for starch. Our results go along with studies on δ2Hne values of organic matter and cellulose, showing also a 2H‐enrichment in C4 and CAM plants compared to C3 plants  such as sugar and starch. By applying the method for compounds from different plant species, we demonstrated that this analytical method can now be used to estimate 2H‐fractionation among structural and NSC and to distinguish plant material from plants with different photosynthetic pathways. It should be noted that the method presented herein enables analysis of δ2Hne of bulk sugar and sugar derived from digested starch and is therefore not compound‐specific nor position‐specific. Yet, our δ2Hne method allows us to measure NSC samples in high‐throughput and we thus expect that it will help to identify important 2H‐fractionation processes. These findings could then eventually be studied in more detail with compound‐specific methods (GC‐IRMS [Abrahim et al., 2H distributions in organic material (Roden, Lin, & Ehleringer, In conclusion, we show that a hot water vapor equilibration method originally developed for cellulose can be adapted for accurate, precise and reproducible analyses of δThe authors declare no conflicts of interest.Appendix S1. Supporting Information.Click here for additional data file.


Human Parainfluenza Virus (HPIV) Type-1, which is an anti-sense ribonucleic acid (RNA) virus belonging to the paramyxoviridae family, induces upper and lower respiratory tract infections. The infections caused by the HPIV Type-1 virus are usually confined to northwestern regions of America. HPIV-1 causes infections through the virulence of the hemagglutinin-neuraminidase (HN) protein, which plays a key role in the attachment of the viral particle with the host’s receptor cells. To the best of our knowledge, there is no effective antiviral drugs or vaccines being developed to combat the infection caused by HPIV-1. In the current study, a multiple epitope-based vaccine was designed against HPIV-1 by taking the viral HN protein as a probable vaccine candidate. The multiple epitopes were selected in accordance with their allergenicity, antigenicity and toxicity scoring. The determined epitopes of the HN protein were connected simultaneously using specific conjugates along with an adjuvant to construct the subunit vaccine, with an antigenicity score of 0.6406. The constructed vaccine model was docked with various Toll-like Receptors (TLRs) and was computationally cloned in a pET28a (+) vector to analyze the expression of vaccine sequence in the biological system. Immune stimulations carried out by the C-ImmSim Server showed an excellent result of the body’s defense system against the constructed vaccine model. The AllerTop tool predicted that the construct was non-allergen with and without the adjuvant sequence, and the VaxiJen 2.0 with 0.4 threshold predicted that the construct was antigenic, while the Toxinpred predicted that the construct was non-toxic. Protparam results showed that the selected protein was stable with 36.48 instability index (II) scores. The Grand average of Hydropathicity or GRAVY score indicated that the constructed protein was hydrophilic in nature. Aliphatic index values (93.53) confirmed that the construct was thermostable. This integrated computational approach shows that the constructed vaccine model has a potential to combat laryngotracheobronchitis infections caused by HPIV-I. HPIVs are non-segmented and enclosed viruses, having anti-sense RNA that infect the respiratory tract, especially its ciliated epithelial cells , of any In the United States of America (USA), HPIV-1 is proved to be the most common reason for laryngotracheobronchitis or simply known as croup, which is the reason for the hospitalization of more than 30,000 victims every year in the USA ,10,11. ITo overcome or more specifically reduce any kind of allergic reactions, researchers are adapting to peptide-based vaccine designed by the use of recent advancements in computational biology ,14. VaccThe establishment of epitope-based vaccines involves the attachment of predicted epitopes and adjuvant through linkers. In epitope-based vaccines, the antigenic effect is limited as compared to live attenuated vaccines. In case of a purified epitope vaccine, the adjuvant is an essential component that can enhance the magnitude of antigenicity of antigens (epitopes) in adaptive immune responses. This magnification of antigens by an adjuvant can enhance antibody production and affects the longevity of the vaccine through the co-administration of epitopes. A limited number of reported adjuvants are available that are incorporated into human vaccines . Here, aTo the best of our knowledge, there is no vaccine of HPIV-1 using HN protein that has been reported yet. Epitope-based vaccines hold promise over other types because of their ability to induce a substantial immune response in a lower concentration. The current study was designed to identify the highly antigenic peptides having a tendency to induce the immune cells  and interferons. The construct was screened for antigenicity, allergenicity, and physicochemical properties along with protein–protein docking with TLR receptors. Immune simulations were performed to predict vaccine efficiency.https://www.uniprot.org/ (accessed on 25 July 2021)) having UniProt IDs P12605, Q01427, P16071, J7FXM0, and P26590, respectively. VaxiJen score of five selected proteins along with their UniProt IDs has been mentioned in the The FASTA format sequences of fusion glycoprotein (F), membrane (M) protein, hemagglutinin-neuraminidase (HN) protein, nucleocapsid (N) protein, and nucleoprotein of several HPIV-1 strains were fetched from the UniPort database (http://www.ddg-pharmfac.net/vaxijen/ (accessed on 25 July 2021).The retrieved proteins were screened for vaccine candidacy on the VaxiJen v2.0 server to evaluate their antigenicity values ; the serhttps://web.expasy.org/protparam/ (accessed on 25 July 2021) include but are not limited to atomic composition, amino acid composition, instability and aliphatic index, extinction coefficient, Grand average of Hydropathicity (GRAVY), and theoretical PI.The physicochemical characteristics of the selected protein were computed via ProtParam, which is available at the ExPasy server . The parSecondary structure analyses of the protein were rendered by PSIPRED , while Ihttp://tools.iedb.org/bcell/ (accessed on 26 July 2021) calculated the linear epitopes confined to B cells. The Bepipred Linear Epitope Prediction 2.0 method of prediction was selected with a threshold of 0.500 and 4 center positions; this method predicts B-cell epitopes from a protein sequence, using a Random Forest algorithm trained on epitopes and non-epitope amino acids. It determines the results from crystal structures, which follows by the sequential prediction later.The B-cell Antigen Sequence Properties tool of IEDB  (http://http://tools.iedb.org/mhci/ (accessed on 26 July 2021) and II (http://tools.iedb.org/mhcii/ (accessed on 26 July 2021) of the major histocompatibility complex [Tools of the IEDB T-cell database were utilized to analyze the epitopes restricted by both types, type I ( complex .http://tools.iedb.org/population/ (accessed on 27 July 2021). In this tool, interaction of the selective alleles of the major histocompatibility complex with the selected epitopes was examined, screening the ones that cover the maximum population .To check which epitopes cover the maximum world population, we used the population coverage analysis integrated in the IEDB server , which ihttps://webs.iiitd.edu.in/raghava/toxinpred/algo.php (accessed on 27 July 2021) [https://www.ddg-pharmfac.net/AllerTOP/method.html (accessed on 27 July 2021), were employed for toxicity, antigenicity, and allergenicity profiling sequentially.The finalization of the suitable epitopes was completed by further filtration of predicted epitopes. To this end, Toxinpred analysis (ly 2021) ), VaxiJely 2021) , availabA vaccine construct consists of four main components namely, an adjuvant, linkers, B and T cell epitopes, and 6X histidine molecules. All epitopes with the target were linked using specific linkers along with adjuvant Human Beta Defensin 3 with UniProt ID: Q5U7J2 . AntigenThe Disulfide tool on Design 2.0 (DbD2) was employed to perform the Disulfide concoction of the constructed vaccine ; this tohttp://raptorx.uchicago.edu/ (accessed on 28 July 2021). ProSA-web, introduced by [https://prosa.services.came.sbg.ac.at/prosa.php (accessed on 28 July 2021), and Ramachandran plot assessment was executed via PROCHECK v6.0 [https://saves.mbi.ucla.edu/ (accessed on 28 July 2021), for the validation and stability confirmation of the constructed vaccine model. GalaxyRefine [http://galaxy.seoklab.org/cgi-bin/submit.cgi?type=REFINE (accessed on 28 July 2021) refined the 3D vaccine model.RaptorX was employed to investigate the tertiary structure of the fabricated vaccine   [https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php (accessed on 29 July 2021). The refined vaccine model and different Toll-like receptors  were docked using both online servers. TLRs are the main human body receptors recognizing and binding to various epitopes to generate an immune response against them. The vaccine was docked against these receptors to evaluate whether it fits in the binding pocket and interacts enough to elicit a response. Molecular dynamic simulations of the docked complexes were performed using the iMODS server [Molecular docking was performed by online docking servers, i.e., ClusPro (ly 2021)  and Patcly 2021)  (https:/S server .https://www.jcat.de/ (accessed on 31 July 2021), and in silico cloning was performed after optimization through the SnapGene software introduced by Insightful Science (snapgene.com (accessed on 31 July 2021)).The Java Codon Adaptation Tool was applied to perfect the codons of refined vaccine construct for enhanced expression  (https:/https://kraken.iac.rm.cnr.it/C-IMMSIM/ (accessed on 31 July 2021) [The C-IMMSIM server produced immune simulations (ly 2021) , as it eThe antigenicity scores-based analysis of all the candidate proteins showed that the HN protein was a viable candidate for vaccine construction, and the physicochemical parameters of selected protein are given in Residues of the HN protein having scores above the threshold of 0.500 of the tools (IEDB’s Bepipred Linear Epitope Prediction 2.0 method) were predicted to be a member of an epitope. The tool predicted a total number of 19 B-cell epitopes, but for further analysis, only the epitopes with the length of 9–50 amino acids were picked. B-cell-restricted epitope predictions of HN protein objectively allowed the finding of potential antigens that would interact efficiently with B-lymphocytes. B-cell-restricted epitopes of HN protein will generate rich immune responses against viral infection upon recognition by the surface receptors of B-lymphocytes. The stimulation by these epitopes will trigger the differentiation of B-cells to plasma cells, generating the antibodies able to recognize the epitopes of the viral HN protein. The More than 30,000 possible MHC-I binding epitopes were predicted; out of these, the top 50 epitopes, based on the IC50 value, were filtered and analyzed for the vaccine construct. Similarly, epitopes from the viral core peptides binding to MHC-II molecules were selected and analyzed. The prediction of epitopes restricted to MHC-I and MHC-II using the viral HN protein is crucial, as the MHCs  are the coordinators between innate and adaptive immune defense, which help in response transition. Both classes, MHC-I and MHC-II, are restricted to CD8+ cytotoxic T-lymphocytes (CTLs) and CD4+ helper T-lymphocytes, respectively, and both play a vital role in viral infections. The antigen-presenting cells (APCs) from the innate immune response deliver the viral epitopes to MHC molecules, which activate the adaptive immune response, i.e., activation of B-cells and production of antibodies. T-cell-restricted epitopes of HN protein will just stimulate the MHC molecules available at T-cells and create immunity against HPIV-1 viral infection. Reference set alleles for MHC-1 and MHC-II molecules has been shown in Out of 19, only three epitopes of the B-cell were picked to become part of the final vaccine construct; similarly, 16 epitopes restricted by MHC-I and 15 epitopes for MHC-II passed the filtration tests and were selected for the construct. All epitopes that passed the filters are given in A cumulative coverage of the selected epitopes illustrated 98.55% and 72.18% against MHC-I and II molecules sequentially, advocating the efficiency of the construct. The individual epitope coverage for each MHC class is depicted in The assembly started by joining all the B-cell epitopes to the adjuvant sequence using EAAAK linkers, which were then connected to the MHC-I epitopes, using the GPGPG linkers. Furthermore, MHC-II was linked to the chain via AYY linkers. The 6xHis tag was utilized at the carbon and the amino-terminus of the sequence for protein identification and purification during the cloning process. The construct is provided in AllerTop predicted the construct is non-allergen with and without the adjuvant sequence, and the VaxiJen 2.0 predicted that the construct is antigenic with 0.6406 and 0.6510 scores, respectively, with and without the adjuvant. According to Toxinpred predictions, the construct was evaluated to be non-toxic in nature.The construct composed of 641 amino acids having 69661.70 Da of molecular weight, and the molecular formula exhibited the exact number of atoms present in the vaccine peptide, while the other physicochemical properties are shown in Raptor X demonstrated that the protein has 34% helices, 4% beta sheets, and 61% coils. More than half (59%) of the protein residues were categorized as exposed (E), 19% were found to be medium exposed (M), and 20% of the residues were considered buried (B). There was a total of 50 residues, constituting 7% of total protein content, that were found to be disordered. The three-dimensional vaccine construct with the Highest CNS-solve program score was further processed for refinement and validation analysis.The GalaxyRefine server considered different parameters in the refinement process. It was noted from the Ramachandran plot that 90.5% residue of the refined protein was in the highly acceptable region, 8.3% residues were found in the moderately allowed region, 0.8% were present in the scarcely allowed sector of the plot, and 0.4% residues were in the outlier or unfavorable region. The predicted z-score was −5.06, validating the refined 3D structure. Scores of different parameters used by GalaxyRefine for the refinement of 3-D model has been shown in The disulfide engineering was completed using a DbD2 server between the 12 selected residue pairs, selected on the basis of energy level between them. The mutated structure with possible predicted disulfide bonds in yellow color is given in MD simulations results were almost immediately available for the provided complex . The models revealed little contortion at each residue’s capacity level. The NMA-calculated B-factor scores were the same as the RMS values. An elasticated network model, shown in The refined codon series was 1923 nucleotides in length, with a CAI of 0.97 and a GC content of 53.25% . The impH, TR, TC) population within 50 days and an increase in the NK cell population  and CD8+ (helper T-cells), respectively . MeanwhiThe selected epitopes in this study were combined together via linkers  as discussed in a previous study by Naveed, Tehreem, Arshad, Bukhari, Shabbir, Essa, Ali, Zaib, Khan, Al-Harrasi and Khan ; the conThe vaccine 3D structure predicted, refined and validated so we can perform docking with TLRs, because TLRs play an important role in the initiation of innate immune response . TLRs arStudy limitations: The current study was performed in silico, and no wet lab experiments were carried out to empower the reported outcomes. Although excellent results were observed in the B-cell and T-cell epitope analysis, problems such as the non-availability of most HLAs in the IEDB, the 9–14 AA-specific immune response that may lead to variable results when tested in vivo and in vitro due to length restriction, the poorly defined motifs in the epitopes, and the TLR–vaccine complex could not be addressed in the study. It is therefore recommended for future researchers to find alternative for these limitations, especially providing wet-lab evidence wherever possible.HPIV-1 is not catered by any of the existing preventive measures, leading to limits regarding the consistent increase in its incidences. The current study construct provides a clinical breakthrough in response against HPIV infections with hope to limit its spread and pathogenesis by designing an epitope-based vaccine using the HN protein as an immunogenic target by an immunoinformatic approach. The protein–protein interaction showed that the construct has the potential to be recognized by the immune cell receptors. The cloning of a construct in the PET28a (+) plasmid using snapgene showed that the expression level of the vaccine peptide was excellent. Immune simulations illustrated that this construct can generate a good immune response by the significant production of T-cells, B-cells, and other immune molecules. This integrated computational approach saves time, is cost effective, and guides experiments with less chance of errors and with fewer trials. However, the efficacy of any drug or vaccine can be ensured via experimental validations (both in vitro and in vivo). At clinical trials levels, the other peptide vaccines had shown good results with much better immune response, so the current vaccine construct could be considered a good candidate against Human Parainfluenza Virus Type-1.


The prepared glass-zirconia was analyzed with scanning electron microscopes (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction spectroscopy (XRD). The wettability, roughness and 3D morphology of zirconia were altered, and shear bonding strength (SBS) test demonstrated almost double increase in SBS values of the nanostructured glass-zirconia structure. The failure modes and microstructure characteristics also verified the improved interfacial stability. This investigation provides a promising method for enhancing the structural stability of bilayer zirconia restorations.Bilayer zirconia restoration is one of the most commonly used restorations in dental practice, but the high frequency of the cohesive/adhesive fracture of veneered porcelain is still a problem. This paper focuses on the development of nanostructured glass-zirconia to improve the interface stability of dental zirconia substrate and veneered porcelain. A novel SiO With the increasing demand for aesthetics, ceramic restorations have become a preferable option in clinical practice . In partHowever, the clinical research results show that the bilayer zirconia restoration has a high probability of cohesive/adhesive fracture of veneered porcelain, which has become an important reason affecting the clinical longevity of restorations ,6. Many Among these treatments, sandblasting is a traditional method to roughen the surface of zirconia , which hAnother commonly used method is adding a liner layer between zirconia and porcelain to improve the bond strength. Nevertheless, there are different interpretations—some studies reported that the liner enhanced the bond strength, while others reported that it weakened the bond strength after long-term thermal cycling ,18,19. OThe chemically inertness of zirconia was considered to attribute to the weak bonding strength between dental zirconia and veneering porcelain . AccordiRecent literatures have reported some novel methods of surface modification of zirconia, such as fusion sputtering and glass-ceramic spray deposition, which can be easily implemented in the dental laboratory ,23,24. TThe major structural difference between zirconia polycrystalline ceramics and glass ceramics is the absence of the glass phase, which makes zirconia chemically inert. A series of studies on graded zirconia by Zhang et al. ,27,28,29In this study, a novel nanoscale Si-Li-Al glass was developed to infiltrate on a commercial dental zirconia to construct nanostructured glass-zirconia structure. The roughness, wettability, microstructure, and phase composition of the nanostructured glass-zirconia were analyzed. Shear bonding strength and the interface characteristics of porcelain and nanostructured glass-zirconia were evaluated.2-Li2O-Al2O3 (SLA) glass was prepared following a melt-diffusion strategy. The starting materials and constituent ratios are shown in −1. After holding for 2 h, the molten mixture was quenched in deionized water and then ball-milled with alcohol for 24 h to obtain the SLA glass powder with nanoscale particles. The size distribution was analyzed by the Nano Measurer 1.2 Software  from the scanning electron microscopy (SEM) images.The SiO−1 and held for 1.5 h. Then, the nanostructured glass-zirconia (ngZir) was obtained. Some sintered zirconia specimens without any further treatments were also made as non-treat control (Zir) for comparison.The zirconia specimens were prepared from commercial dental zirconia blocks  using CAD/CAM technique and fully sintered following the manufacturer’s recommended procedure . The sinThe morphology and microstructure of glass powder, non-treat zirconia (Zir) and nanostructured glass-zirconia (ngZir) were observed with SEM . The distribution of glass in ngZir specimens was detected. All specimens were ultrasonically cleaned and sputter-coated with platinum .−1. The percentage of monoclinic phase of ngZir was determined with grazing incidence X-ray diffraction (GIXRD). The angle of incidence was set at 1° to measure the glass-zirconia layer. The percentage of monoclinic phase (mV) was determined as follows [mI and tI are the peak intensities of monoclinic and tetragonal zirconia that could be read directly. Since the ngZir contains only tetragonal and monoclinic zirconia, the percentage of tetragonal phase (tV) can be calculated by tV = l − mV.Zir and ngZir specimens were analyzed with XRD  to examine the phase composition. The powder diffraction patterns from the International Center for Diffraction Data (ICDD) were used to compare and analyze the XRD peaks of the specimens. The scans were carried out with Cu Kα radiation from 10° to 80° with a scan rate of 10°·min follows ,31: (1)V−1 and frequency of 100 Hz). The average surface roughness (Ra) and arithmetical mean height of the scale limited surface (Sa) were analyzed and calculated with professional 3D software .Specimens of both groups were ultrasonically cleaned with ethanol for 10 min and dried at 80 °C for 2 h before the roughness assessment. A non-contact optical profilometer  was used to obtain the surface morphology . Distilled water droplets (~10 μL) were dropped onto the surface of each specimen. The image of water droplet shape was captured using an image acquisition device when the water droplet stabilizes. The image in JPEG format was analyzed with ImageJ for the determination of the contact angle. Ten specimens of each group were evaluated to obtain the average value of contact angles.Forty sintered zirconia specimens were prepared and divided into two parts. Twenty zirconia specimens were glass infiltrated with SLA glass powder to construct nanostructured glass-zirconia, and the other twenty zirconia specimens were sandblasted with 27 μm aluminum oxide particles in a fine sand-blaster . Two veneering techniques were used including the hand-layering method and the heat-pressing method. For the hand-layering method, a fluorapatite porcelain  was applied on the surface of specimens via the silicone mold with the attempt to keep identical contact surface and dimension of the specimens (a cube with sides of length about 4 mm). For the heat pressing method, polymethyl methacrylate (PMMA) blocks (a cube with sides of length about 4 mm) were fixed on top of the zirconia and invested using rapid investment materials . Consecutively, the invested molds were subjected to the burn out processes and the space created by the burn-out of the PMMA pattern was then filled by the veneering porcelain  in the specific furnace  following the manufacture’s guidelines. The grouping situation is shown in −1. All specimens were tested and the fracture loads were recorded. The bond strength was calculated as follows:2).The specimens were tested using a universal testing machine  with a vertical compressive load at the junction of zirconia and porcelain. The crosshead speed was set at 0.5 mm·minThe failure mode between zirconia and the porcelain was inspected with stereomicroscope  and classified as adhesive failure, cohesive failure, and mixed failure, according to the location of failure.The bilayer zirconia-porcelain specimens were cut with a diamond saw to expose the cross-sectional surface of bonding interface between zirconia and porcelain. The cut marks were removed using sandpapers of 400, 800, 1200, 2000 grit and diamond slurry of 2.5, 1, and 0.25 μm successively. The SEM and backscattered electron (BSE) were used to observe the microscopic morphology of the interface, and energy dispersive spectroscopy (EDS) was used to perform semi-quantitative analysis of elemental distribution at the interface.p < 0.05. One-way analysis of variance (ANOVA) and post hoc Tukey tests was used to investigate the statistical difference.In this study, the data of different groups were analyzed with SPSS 27.0 , and the significance level was set at The morphology and particle size distribution of SLA glass powders are shown in −3 and an open porosity of approximately 0. According to the literature ,33, the The dense structure and stable chemical properties of zirconia make it difficult to form an effective mechanical embedding effect or chemical bond with veneered porcelain. The formation of pores makes it possible to form mechanical interlocking between porcelain and zirconia, and the introduction of nano-glass improves the chemical activity of zirconia. Therefore, the formation of nanostructured glass-zirconia is beneficial to enhancing the bonding strength between zirconia and veneered porcelain ,34,35.t-phase) (Zir in t-phase (ICDD 70-4427) and monoclinic (m) phase (ICDD 81-1314) zirconia. The surface layer of the ngZir specimen is composed of 90.8 vol% m-ZrO2 and 9.2 vol% t-ZrO2.The XRD patterns of SLA glass powder, zirconia and nanostructured glass-zirconia are shown in ) Zir in b. After p < 0.05). The results indicated that the glass infiltration in ngZir group altered the morphology and improved the roughness. It is reported that the improvement of roughness could enhance the SBS values between zirconia and porcelain [orcelain ,14,15. Tp < 0.05). Based on Young and Neumann’s equation, the contact angle of the material is inversely proportional to the surface energy [During the fabrication of bilayer zirconia restorations in clinical practice, dental porcelain is veneered to zirconia at a temperature above its melting point, and the melted porcelain contacts zirconia surface in the liquid form. Therefore, the wettability of the zirconia surface is crucial to the interfacial stability of porcelain and zirconia ,21. Figue energy . As a sm(a)Adhesive failure: the failure occurred at interface of zirconia and porcelain.(b)Cohesive failure: the failure occurred inside the porcelain.(c)Mixed failure: both a and b failure modes exist simultaneously.After the SBS test, the fracture patterns were recorded and classified according to the following criteria ,16,37:(aAll three types of fracture patterns were observed in all groups. The proportion of adhesive failure was lower in the ngZirL and ngZirP groups, which also indicates better interface stability.For ngZirL and sZirL groups, the same veneering methods were adopted, therefore the higher SBS values and lower occurrence of adhesive failure in ngZirL group were due to the development of nanostructured glass-zirconia structure. The same results can also be found in the ngZirP and sZirP groups, indicating that the nanostructured glass-zirconia surface significantly improved the bond strength between zirconia and porcelain. This conclusion is consistent with the results in previous literatures ,39.The bonding interface between porcelain and zirconia was analyzed with SEM and EDS. In the sZirL group, the interface tended to be more of a straight line a,b, wherIn this study, nanostructured glass-zirconia was constructed on fully sintered commercial dental zirconia by infiltrating a novel SLA glass. The nanostructured glass-zirconia has higher chemical activity due to the introduction of glass components. After modification, ngZir has higher roughness and better wettability. Due to the extensive mechanical interlocking between ngZir and veneer porcelain, a wider range of elemental transition area is formed at the interface. The above factors lead to the significant increase in the SBS value. Analysis of the fracture patterns also showed that the proportion of adhesive failures was significantly reduced. Therefore, it can be concluded from the above results that the construction of nanostructured glass-zirconia significantly improves the structural durability of zirconia bilayer restorations. In addition, the zirconia modification method used in this study can be operated simply and conveniently, and can be easily implemented in the dental laboratory.In conclusion, the construction of nanostructured glass-zirconia on dental zirconia ceramics significantly improves the chemical activity, roughness and wettability of zirconia. Compared with sandblasting, nanostructured glass-zirconia broadens the elemental transition region and increases the bond strength between zirconia and porcelain by approximately double. This study provides a practical, effective and promising method for improving the interface stability of bilayer zirconia restorations.


This research aimed to explore feasibility and the time required when erbium-doped yttrium aluminum garnet (Er:YAG) laser as a non-invasive treatment modality to retrieve different thicknesses of zirconia material bonded by two dental cements from titanium implant abutments.Prepared 80 titanium blocks  and square zirconia sheets (length: 10 mm) with different thicknesses  were 20 pieces each. Resin modified glass ionomer cement  and resin cement  were used to bond zirconia sheet and titanium block. Specimens were kept in 100% humidity for 48 h. Er:YAG laser was used to retrieve the zirconia sheet and recorded the time. Universal testing machine was used to measure the residual adhesion of the samples that did not retrieve after 5 min of laser irradiation. Shear bond strength (MPa) and the time data (s) were analyzed using Kruskal–Wallis Test. The bonding surface and the irradiation surface of the zirconia sheet was examined with the scanning electron microscopy (SEM).Within 5 min of laser irradiation, RXL group: 1 mm group all fell off, 2 mm group had 3 specimens did not fall off, there was no statistical difference in the average time between the two groups; CSL group: half of the 1 mm group fell off. Shear bond strength test results: there was no statistical difference between 1 and 2 mm in RXL group and 1 mm in CSL group, there was no statistical difference between 3 mm in RXL group and 2 mm in CSL group, and there were significant differences statistically in comparison between any two groups in the rest. SEM inspection showed that the bonding surface and the irradiation surface of the zirconia sheet had changes.In this vitro study, the following could be concluded: it is faster to remove zirconia crowns with thickness less than 2 mm from titanium abutment when luted with RelyX Luting 2 compared to Clearfil SA luting. Increasing patient’s demands and expectations in esthetics have driven the modern dental practice into all-ceramic restorations, zirconia restorative materials due to its excellent mechanical property and biocompatibility have become one of the most used materials in restorative dentistry , 2. ZircErbium-doped yttrium aluminum garnet (Er:YAG) laser with a wavelength of 2940 nm, theoretically, the wavelengths of these lasers operate with the mid-infrared spectrum, which coincides with the range for water absorption spectrum, the transferred energy can efficiently act on the molecules of water or related groups and is rarely absorbed by other molecules, so that it can precisely act on the water-containing substances to avoid damage to other substances –9. It maThe aims of this study were to examine the feasibility of a non-invasive retrieval of zirconia crowns from titanium implant abutments by Er:YAG laser in vitro experiment, comparison of the removal effect of two cements and different thicknesses of zirconia materials. The hypothesis was that it was no difference of removal time between composite resin adhesive and resin modified glass ionomer cement. It was also hypothesized that there was no difference between required time from 1 to 4 mm of zirconia material, to provide reference for restoration removal from implant abutment clinically.The specimen preparation for the experiment was adapted from methodology by Barbara et al. . In thisThe specimens were irradiated by laser Er:YAG  using the R14 handpiece at the following parameters: 300 mJ, 15 Hz, 4.5 W, operation mode: MSP1, spot 0.9 mm, air/water spray at 4/4 with noncontact mode, 1 mm from the surface of the zirconia specimen. Irradiation with the Er:YAG was directed perpendicular to the surface, irradiate on the surface according to the "Z" shape, continuous motion of the laser handpiece on the surface was done to ensure even distribution of laser beam without stagnation, the air/water spray was used throughout the irradiation process. The irrigating solution was distilled water, 20 ml/min. During the irradiation process, if the zirconia specimen was debonded, the total irradiation time was recorded. If the zirconia did not come off after 5 min of irradiation, the specimen was subjected to a shear bond strength test. All experiments were done by the same operator.2) to provide results in units of stress (MPa), record the maximum load for debonding. Statistical analysis was performed using the Kruskal–Wallis Test for dismantling time and shear bond strength.The test specimen was placed on the universal testing machine, and the loading head is evenly contacted with the upper edge of the zirconia sheet through the micro-adjustment of the fixture. Shear bond strength was using the Universal Testing Machine  at a crosshead speed of 0.5 mm/min Fig. . The forZirconia sheet specimen after laser irradiation were selected to examine the bonding surface and the irradiation surface with a SEM . Representative morphology of the bonding surface of the zirconia sheet were examined in SEM with an acceleration voltage of 5 kV after sputtering using a gold–palladium alloy conductive layer. The flow chart of the experimental process is shown in Fig. P < 0.05). Within 5 min of laser irradiation, RXL group: 1 mm group all fell off, 2 mm group had 3 specimens did not fall off, the average time for the two groups were 3.3 min and 3.5 min respectively, not statistically different, 3 mm and 4 mm groups did not fall off; the CSL group, only half of the 1 mm specimens fell off, and the average removal time was 4.9 min. For shear bond strength test results, there was no statistical difference between the 1 mm and 2 mm of the RXL group and the 1 mm of the CSL group, and there was no statistical difference between the 3 mm of the RXL group and the 2 mm of the CSL group, there are significant differences statistically in comparison between any two groups.The Kruskal–Wallis test showed that the comparison results of the removal time and shear bond strength values of the two adhesives and four thicknesses of zirconia sheets are summarized in Table Visual inspection showed that the zirconia sheets and titanium blocks did not have obvious fractures, and only black burning spots were visible on the surface of some zirconia specimens. SEM observation was performed to test the bonding surface of zirconia sheets. Visual inspection showed that the zirconia sheets and titanium blocks did not have obvious fractures, and black burning spots were visible on the surface of some zirconia specimens. Scanning electron microscope (SEM) observation was performed to test the surface damage of zirconia sheets, compare to the zirconia sheet without any treatment, the inspection showed that the surface of 1 mm or 2 mm with shorter laser irradiation time had minor changes, but the surface of 3 mm or 4 mm with longer laser irradiation time 5 min) had burning pits appeared  or about 4–5 min (composite resin cement) , 12, 16.This in vitro study demonstrated the feasibility of Er:YAG laser to retrieve zirconia crowns from the titanium abutments, however, it still has some limitations. First of all, the zirconia sheets and titanium blocks which were used in this experiment are different from the crowns and abutments used in actual clinical work, and more time may be required to retrieve a crown in the clinical application; secondly, in this vitro study, the laser can better contact the surface of the restoration, avoiding the obstruction of soft and hard tissues (such as adjacent teeth and gingiva), so that the laser can better act on the surface and edge of the restoration; third, in this study, only resin-reinforced glass ionomer adhesives (RXL) and composite resin-based adhesives (CSL) were used, and the proportions of water and monomer molecules in different adhesives are different, the resulting demolition results will also be different; fourth, with the rapid development of zirconia materials, many zirconia materials have appeared clinically, different zirconia materials may have different reactions to laser retrieval due to their different physical properties (such as light transmittance); fifth, the skill of the operator affects the success rate and time required for irradiation. Finally, it is important to note that future clinical in vivo studies with varieties of abutment/crown materials, cements and prosthetic designs, and clinician skills will be needed to further optimize and understand the clinical applications of Er:YAG laser.Within the limitations of this in vitro experimental study, it can be concluded that:Atraumatic decementation of a cement-retained implant prosthesis using Er:YAG laser is a viable method for debonding zirconia crowns from titanium abutments. Er:YAG laser removal is recommended for zirconia crown with thickness less than 2 mm, and resin-reinforced glass ionomer cement is easier to remove than resin cement.


Urethral reconstruction in complex hypospadias poses a significant challenge. We report our 10-year experience with buccal mucosa graft (BMG) in the two-stage repair of complex hypospadias and compare its results to the skin graft.We retrieved the data of 15 patients with complex hypospadias who underwent two-stage repair using the BMG at our institution. The data were compared to 13 patients who underwent skin graft during the same period.p > 0.05). On the other hand, the incidence of meatal stenosis and urethral stricture was significantly lower in the BMG group (0% each) compared to the skin graft group . At the same time, there were no reported cases of graft contracture. The frequency of donor site morbidity was significantly higher in the skin graft group compared to the BMG group (p = 0.003). The BMG led to a lower incidence of postoperative straining than the skin graft . Only one patient needed revision surgery after skin graft, compared to no case in the BMG (p = 0.27).The median follow-up duration was 14 (12–17) months in the BMG group and 16 (13.5–22.5) months in the skin graft group. Patients in the BMG had a numerically lower incidence of the diverticulum, wound dehiscence, fistula, and infection than the skin graft group, however, without statistically significant difference (The present study demonstrates the feasibility and durable outcomes of the BMG in the setting of two-stage repair of complex hypospadias. Hypospadias affects up to 0.4% of the global male population, which is expected to rise in the upcoming years. Previous epidemiological figures highlighted a notable geographic variation in the incidence of hypospadias, being highest in North America and lowest in Asia , 2. The Two-stage hypospadias repair is commonly indicated for patients needing revision surgery due to one-stage repair’s shortcomings in terms of scarred/replaced urethral plate and slower healing rate . The twoThe protocol of the current trial was approved by the local ethics committee of Quality Education Assurance Unit, Al-Azhar Faculty of Medicine, Nasr city, Cairo, Egypt (Registration Number: ped_1. 16Med._00016). All procedures run in compliance with the standards of the Declaration of Helsinki . Due to In this retrospective chart review, the total number of hypospadias repairs in the study period were 1652 patients. BMG grafts in total were 82. We retrieved the data of all 15 patients with complex hypospadias who underwent two-stage repair using the BMG at Al-Azhar University Hospitals through the period from January 2010 to December 2020. In addition, we retrieved the data of all 13 patients who underwent skin graft during the same period. Patients with incomplete records of postoperative data or follow-up duration of less than 6 months were excluded. Patients from both groups were chosen consecutively from all available data records.The following data were retrieved from the patients’ records: age, previous surgeries, presence of preoperative penile torsion, type of hypospadias at previous surgery, previous graft, graft characteristics , site of the meatus, type of the second layer, pre and postoperative dripping and straining, pre and postoperative penile curvature or chordae, pre and postoperative meatal stenosis, postoperative spraying of micturition, diverticulum, postoperative wound dehiscence, fistula infection, urethral stricture, graft contraction, need for revision surgery, type of the second layer.All patients underwent general anesthesia and procedures were performed in supine position. The choice of the surgical technique was per the surgeon’s preference. A total of 15 patients underwent a two-stage repair with BMG graft in the first stage followed by second stage urethroplasty later with local tissue flap  as a second layer coverage of the new urethra. Tunica vaginalis flap was used in five cases, and subcutaneous dartos flap was used in the remaining ten cases. The BMG was harvested in all cases from the inner check by the technique described by Eppley et al. . BrieflyThe primary outcome of the present study was to compare the rate of late and postoperative complications between BMG and skin graft. The secondary outcome was to compare the two grafts in terms of success and donor site complications.p-value < 0.05 was regarded as statistically significant.Retrieved data were summarized and processed with IBM SPSS statistical software (version 25). Frequencies and summary statistics were used to describe continuous and categorical data, respectively. The hypothesize of significant difference between the two types of graft in terms of postoperative complications was tested using the Chi-square test, with Fisher exact whenever needed. The association between the type of graft and continuous data was tested using the Mann–Whitney test. p = 0.52). The majority of the patients in both groups had two previous surgeries . The hypospadias was mostly mid-shaft or penoscrotal in both groups (p = 0.81). Patients in the skin graft group had numerically higher rates of penile torsion (30.8%) and meatal stenosis (30.8%); however, without significant difference (p > 0.05). On the other hand, 60% of the BMG group had penile curvature or chordae, compared to 30.8% in the skin graft group (p = 0.12). The frequency of preoperative urination difficulties was comparable between both groups. Likewise, the graft length and width were similar between the BMG and skin graft groups (Table The mean age of the included patients was comparable between BMG and skin graft groups (s p = 0.8. Patientp = 0.34). Concerning early complications, patients in the BMG had a numerically lower incidence of the diverticulum (0% vs. 7.7%), wound dehiscence (0% vs. 15.4%), fistula (13.3% vs. 23.1%), and infection (13.3% vs. 30.8%) than the skin graft group, however, without statistically significant difference (p > 0.05). On the other hand, the incidence of meatal stenosis and urethral stricture was significantly lower in the BMG group (0% each) compared to the skin graft group . At the same time, there were no reported cases of graft contracture. The frequency of donor site morbidity was significantly higher in the skin graft group compared to the BMG group (P = 0.003). The BMG led to a lower incidence of postoperative straining than the skin graft . Only one patient needed revision surgery after skin graft, compared to no case in the BMG (p = 0.27), Table The median follow-up duration was 14 12–17) months in the BMG group and 16 (13.5–22.5) months in the skin graft group . To our knowledge, this is the first study that compared the incidence of acute postoperative outcomes between BMG and skin graft two-staged hypospadias repair. However, our findings concerning the rate of acute postoperative outcomes following BMG run in line with previous reports demonstrating low incidence of infection, wound dehiscence, fistula, and bleeding among patients with repeated surgery [Patients undergoing hypospadias repair are prone to wide range of early and postoperative complications, whose risks are significantly increased in patients undergoing multiple surgeries. Previous reports demonstrated that patients with hypospadias repair can develop acute postoperative complications in the form of infection, wound dehiscence, fistula, penile malformation, hemorrhage, and flap necrosis . Such co surgery –19.p = 0.003).According to Bracka, the use of extra-genital skin graft in the setting of two-stage hypospadias repair should be discouraged due to the possibility of hair growth and high incidence of donor site morbidity, including keloid scars . On the The present study poses certain limitations. The study was retrospective in nature, which increases the risk of ascertainment and selection bias. Besides, the study was limited to a single center only with a small sample size, which may affect the generalizability of our findings. The follow-up duration was also limited to a maximum of 2 years, and there were no available data to assess the long-term outcomes of the BMG and skin graft beyond the 2 years of follow-up. Lastly, the BMG was performed by more than one surgeon; it was previously reported that the outcome of the BMG depends on the surgeon’s skills and years of experience with the technique . The stuIn conclusion, the present study demonstrates the feasibility and durable outcomes of the BMG in the setting of two-stage repair of complex hypospadias. Our results showed the onlay BMG urethroplasty was associated with lower incidence of postoperative complications and donor site morbidity than the skin graft urethroplasty. Besides, the onlay BMG urethroplasty significantly improved the symptoms of urination difficulty. Thus, onlay BMG urethroplasty should be preferred over the skin graft in centers with experience in onlay BMG urethroplasty. Further studies are warranted to confirm our findings and identify the subgroup of patients who are suitable candidate for onlay BMG urethroplasty.


Interestingly, ferromagnetism is stronger at a hole doped peak and at an upper Hubbard band of O with spin-polarization degree as high as 40%, revealing strong ferromagnetism of Mottness. For in-ab-plane spin-polarizations, the spin of doped holes in O2p–Cu3d–O2p is a triplet state yielding strong ferromagnetism. For out-of-ab-plane spin-polarization, while the spins of doped holes in both O2p–O2p and Cu3d–Cu3d are triplet states, the spin of doped holes in Cu3d–O2p is a singlet state yielding ferrimagnetism. A ferromagnetic-(002) Bragg-peak of the doped holes is observed and enhanced as a function of d revealing strong ferromagnetism coupling between Cu-O layers along the c-axis.We report strong ferromagnetism of quasiparticle doped holes both within the  Long-range magnetic order of quasiparticle doped holes is important for understanding the physics of cuprate superconductors, albeit difficult to probe in experiments. Ong et al. observe ferromagnetism of quasiparticle doped holes in a cuprate heterostructure and discuss implications for cuprates in the ground state. The cuprates have shown a rich exotic phase diagram in which, as a function of temperature and hole-doping, various exotic phases occur such as antiferromagnetic insulator, pseudogap, spin and charge density waves, spin-glass, strange metal, and superconductivity5. It is widely regarded that such a rich and diverse phase diagram is induced by quasiparticle holes doped into the antiferromagnetic insulator. Note that the quasiparticle doped hole is attributed to the extra charge provided by Ba-substitution and is thus not the same as the intrinsic holes from Cu2+ provided by the parent compound La2CuO4. Soon after the discovery of the unconventional superconductivity, ferromagnetism has been theoretically predicted to occur in the hole-doped cuprates2. In recent progress, theoretical calculations on one hand suggest that ferromagnetism competes with unconventional superconductivity; while on another hand, other theoretical calculations suggest that ferromagnetism is a precursor or intertwined with the unconventional superconductivity8. Nevertheless, these theoretical studies have all agreed that ferromagnetism plays an important role in the cuprates and is, therefore, the key to understanding the nature of normal states of the quasiparticle doped holes and their pairing mechanisms responsible for the rich phase diagram in the cuprates. It has been very challenging, however, to directly probe the long-range order of spin of the quasiparticle doped holes, given the overwhelming contribution from the antiferromagnetic ordering of localized Cu spins in the Cu-O plane8. Hence, it is a general consensus that this fundamental problem should be experimentally addressed before one can move forward in establishing a reliable model to describe the magnetic and electronic structures of doped holes in Cu-O plane of the cuprates7. Therefore, an experimental method capable of probing simultaneously long-range magnetic and electronic correlations of the quasiparticle doped holes is critical in addressing this fundamental problem.One of the unresolved problems in condensed matter physics is elucidating the interplay between long- and short-range interactions of spin of quasiparticle charge carriers, i.e. hole or electron doping, that determines magnetic and electronic structures in strongly correlated electron systems. Of particular interest is copper-oxides (cuprates) of La2-xBaxCuO4, which serves as a model system consisting of a few holes doped into a two-dimensional-like Cu-O plane separated by two La-O layers , soft X-ray absorption spectroscopy (XAS) measurements have shown that the lowest unoccupied state mostly consists of Cu3d hybridized with O2p, known as the upper Hubbard band (UHB), while the first excitation state (or the highest occupied state near the Fermi level) is mostly O2p in character, placing the cuprates as a charge transfer system in a Zaanen-Sawatzky-Allen picture , the Cu-O layer is doped with holes and the electronic structure changes dramatically due to many-body interactions. Measurements with XAS have shown that the doped hole goes into O2p creating an unoccupied state17, namely a hole-doped peak (HDP), just above the Fermi level with a few eV below the UHB. As a function of hole doping, the HDP increases and is accompanied by spectral weight transfer between the UHB and the HDP, thus revealing the importance of many-body electronic correlations in the cuprates  heterostructures  using newly developed spin- and site-selective resonant soft X-ray and magnetic scattering (RSXMS). The d is varied from 4, 8 to 12 unit-cells (uc) to show the roles played by Cu-O layers. Interestingly, ferromagnetism of the doped holes is stronger at O and increases for thicker d. The low-dimensional Au, which consists of highly oriented single-crystalline gold quantum dots (HOSG-QDs), is a Mott-like insulator with an optical band gap of ~1.5–2 eV, and is different from bulk, thin-film gold, or gold nanoparticles,23. The observation of ferromagnetism is further supported with zero-field XMCD at Cu L3,2 and O K edge.Here, we experimentally observe strong ferromagnetism of the quasiparticle doped holes in the new low-dimensional-Au/d-LBCO/LAO heterostructure with d = 4uc as a representative. Evidence from in-situ RHEED specular spot intensity oscillations  and (002) substrate peaks are at 23.44° and 47.92° , (006), and (008) film peaks are at 26.59°, 40.80° and 55.16°, respectively. Results of the XRD and RHEED analyses reveal LBCO belonging to the I4/mmm phase27  substrate has been realized with the following relationship given by {LBCO(001)//LAO(001) and LBCO < 001 > //LAO < 001 > }. Kiessig fringes are also observed in the XRD θ−2θ scans, and from the analysis of LBCO(006) associated fringes, the epitaxial LBCO film thickness is extracted to be ~5.5 ± 0.5 nm, which is equivalent to 4uc of LBCO. At this thickness, the surface has a root mean square (rms) roughness of ~0.53 nm, which is comparable to the initial rms surface roughness of LAO  = Re χ(ω) + Im χ(ω) of HOSG-QDs/4uc LBCO/LAO and 4uc LBCO/LAO is obtained at the O K edge , n(ω) and K(ω) are the complex refraction index, refractive index, and extinction coefficient respectively, as a function of photon energy ω. From μ(ω), we calculate K(ω) and perform a Kramers-Kronig Transformation (KKT) to get n(ω), then compute χ(ω). The XAS are measured in total electron yield mode and a broad energy range from 3.7 eV to 1500 eV covering O K, Ba M4,5, La M4,5, and Cu L3,2 edges to achieve stabilized KKT. The K(ω) shows that the chemical composition and stoichiometry for all cuprate heterostructures are all the same with x ~0.20 and no impurity is detected to the limit of the XAS  shows that HOSG-QDs/d-LBCO/LAO  and 4uc LBCO/LAO have similar electronic structures at Cu L3,2 edges but some differences at the O K edge. The XAS at the O K edge exhibits two pre-peaks: (i) the HDP at ~528.9 eV, which corresponds to a transition from O1s to the hole-doped peak in O2p, and (ii) the UHB at ~530.1 eV, which corresponds to a transition from O1s to Cu3d hybridized with O2p , the XAS at O K edge shows only the UHB peak17. Upon hole doping, the hole-doped peak grows accompanied by spectral weight transfer between UHB and HDP21. Therefore, both pre-peaks contain strong doped hole (charge carrier) and magnetic characters. As a function of d, the overall HDP and UHB increases. By fitting the HDP and UHB using a Gaussian profile , which is a transition from Cu2p to Cu3dL, where L is a ligand hole35.The complex dielectric susceptibility, dge Fig.  and Cu Lges Fig.  as a repO2p Fig. . Both prely Fig. . The holL3’) of the Cu-O-planes, we construct a spin- and site-selective RSXMS, which is a combination of magnetic scattering and valence spectroscopy . The RSXMS is measured over an almost full range of θ from ~10° to ~88° corresponding to Q intervals  →  at Cu L3’ and  →  at HDP and UHB of O K edge and enabling the in-plane (at lower incident angles) and out-of-plane (at higher incident angles) magnetism to be probed. Ferromagnetism occurs when magnetic dichroism, which is defined as the difference between LCP and RCP, exists. To complement RSXMS, X-ray magnetic dichroism (XMCD) measurements at O K edge and Cu L3,2 edge are also performed. To further demonstrate the resonance effects, we also perform RSXMS at ~927.9 eV, which is an off (magnetic) resonance. It is worth highlighting that the scattering matrix element of RSXMS, In order to reveal long-range order of magnetic and electronic correlations of the doped holes in O (~528.9 eV of HDP) and in Cu  are long-range order ferromagnetic  is the magnetic easy-axis. For the in-plane spin-polarization, the strong magnetic dichroism at ~528.9 eV (~530.1 eV) shows that the spin of the doped hole (UHB) is coupled ferromagnetically between O2p–O2p (Cu3d–Cu3d) as seen in Fig. p‒O2p, Cu3d–O2p, and Cu3d–Cu3d. Such a strong ferromagnetism highlights the importance of the spin-triplet state of the doped holes between Cu3d‒O2p  in both Cu3d‒Cu3d and O2p–O2p, the spin of doped holes and UHB is coupled antiferromagnetically (spin singlet) in O2p‒Cu3d. This suggests that both spin triplet and singlet states are present for out-of-plane spin-polarization yielding ferrimagnetism. Such an interplay of spin-triplet and singlet states results in anisotropy between in- and out-of-plane ferromagnetism in the lightly hole-doped cuprate.A key observation is the presence of strong magnetic dichroism occurring at these resonance photon energies and even at a high temperature of 300 K for all cuprate heterostructures , at ~931.9 eV  as illustrated in Fig. d, the intensity of ferromagnetic-(002) Bragg peak of the quasiparticle doped holes enhances, and the full width at half maximum of the hole-doped ferromagnetic-(002) Bragg peak decreases following the film thickness, i.e., when the thickness of the film increases, the Bragg peak becomes sharper  Bragg peak arising from the entire film, i.e. a bulk property. Thus, ferromagnetism is a distinct and unique property in the phase diagram of cuprates and occurs as a broader phenomenology of the Cu-O plane.Another significant result is that we observe a new ferromagnetic-(002) Bragg peak of the doped holes at higher  eV Fig. . Interesper Fig. . Upon he layers ~uc, at ~9d‒O2p‒Cu3d is dominating, yielding stronger ferromagnetism. Upon cooling to a lower temperature (36 K), while the spin-triplet state remains, the spin-singlet state of the doped holes in Cu3d‒O2p is enhanced yielding ferrimagnetism. As a function of d and temperature, the in-Cu-O-plane ferromagnetism of the doped holes decreases but remains significantly large  and it occurs remarkably even at 350 K  is proposed3. On the other hand, a model of three-spin polaron is proposed where the doped hole in oxygen can promote local ferromagnetic fluctuations of Cu2+ spins surrounding the doped hole in an otherwise antiferromagnetic background.2 Our new experimental data reveal that the ground state of the cuprates is a strong mixture of spin triplet-and-singlet wavefunction resulting in a remarkably strong degree of spin-polarization and ferromagnetic correlations between Cu-spins near the doped holes . This shows that the ground state of the cuprates is a strong combination of spin triplet-and-singlet wavefunction yielding ferromagnetism. HOSG-QDs further enhance the spin-triplet state, and consequently increase the ferromagnetic long-range order strength of the quasiparticle doped holes in the Cu-O plane. Note that since HOSG-QDs are ferromagnetic Mott-like insulators with a spin-polarized Mott-like state around 1.5–2 eV32 which is located within the charge transfer gap in the cuprates36, spin and electronic correlations may, in turn, be enhanced between HOSG-QDs and LBCO. We further perform zero-field XMCD and find that the XMCD signals are indeed observed at both Cu L3,2 edges and at O K edge, particularly at HDP and UHB, therefore fully supporting the findings of ferromagnetism of the doped holes in the Cu-O planes  heterostructures using a unique RSXMS technique combined with XMCD. Ferromagnetism of the doped holes persistent in 4uc La1.8Ba0.2CuO4/LaAlO3(001) reveals that the ground state of Cu-O planes consists of a strong mixture of spin triplet- and singlet-states. The increase in ferromagnetism in HOSG-QDs/4uc La1.8Ba0.2CuO4/LaAlO3(001) suggests that the low-dimensional HOSG-QDs enhance the spin-triplet state through the spin and electronic correlation effects, and in turn strengthens the long-range order of spin of the doped holes. Furthermore, it is also further enhanced in thicker LBCO films for HOSG-QDs/d-La1.8Ba0.2CuO4/LaAlO3(001) heterostructures (with d up to 12uc) suggesting a strong ferromagnetic coupling between Cu-O layers along the c-axis. Our result also opens new possibilities to study the relationship between ferromagnetism and unconventional superconductivity and demonstrates a unique strategy in utilizing RSXMS to reveal spin, charge, and orbital degrees of freedom in strongly correlated electron systems.In conclusion, we experimentally observe ferromagnetism of the quasiparticle doped holes with a high degree of spin-polarization in the Cu-O planes of new low-dimensional Au/−9 Torr. The substrate is first outgassed in a vacuum at 310 °C for 45 mins before being annealed at 750 °C for 60 mins under 50 mTorr O2 to obtain a clean LAO(001) surface as verified using in-situ RHEED  sample is first reduced to the growth temperature of 450 °C. Au is then pulse-laser deposited at the growth temperature under 10 mTorr O2. The laser energy is fixed at about 3.25 J cm−2 for all depositions. The sample is then cooled down to room temperature under 10 mTorr O2 at 25 °C min−1, while all other temperature ramps are fixed at 50 °C min−1. Prior to each deposition stage, the LBCO and Au targets are pre-ablated at 10 Hz with 2000 pulses. Transport measurement suggests that LBCO/LAO films with thicknesses of up to 12uc are in the regime of insulator-metal transition as a function of temperature and thickness31.Highly oriented single-crystal gold quantum dots (HOSG-QDs) and LBCO films are prepared on 1 cm × 1 cm LAO(001) substrates by a unique ultrahigh vacuum molecular beam epitaxy pulsed laser deposition (UHV-MBE PLD system) equipped with a solid-state ablation Nd:YAG laser (laser output wavelength 266 nm) and growth monitoring using in-situ reflection high-energy electron diffraction (in-situ RHEED). Each LAO(001) substrate is rinsed in isopropanol and blown-dried in the air before being loaded into the UHV MBE PLD system with a base pressure of 5 × 10−9 mbar at the soft X-ray-ultraviolet (SUV) beamline of the Singapore Synchrotron Light Source33. For RSXMS, the scattered photon is collected using a photodiode to get the total fluorescence yield (TFY) from the sample. X-ray photons with selectively left- and right-circular polarizations (LCP and RCP) are used with a degree of circular polarization of ~80% and the measurements are done at reflectivity mode in which the angle of incidence of the sample (the angle of the detector) is scanned in almost a full range from 10° to 88° (20° to 176°) as shown schematically in Fig. Io). We find that at the range of θ ~50°−60°, which are quite far from the ferromagnetic-(002) Bragg peak at the high θ and magnetic dichroism at low θ, the RSXMS of LCP and RCP at on- and off-resonance are naturally lined up and thus we set this range of θ as the reference for the RSXMS. This background is then confirmed with XMCD, where there is no dichroism beyond resonance edges. We find that this approach of normalization is qualitatively solid for this case.Resonant soft X-ray and magnetic scattering (RSXMS), soft XAS, and X-ray magnetic circular dichroism (XMCD) measurements are carried out in an ultrahigh vacuum (UHV) five-circle geometry of a magnetic scattering chamber with based pressure of 10Supplementary Information