A new strategy to generate nanobodies for the coumaphos based on the synthesized nanobody libraries.

To break through the bottleneck in preparation of nanobody (Nb) for chemical contaminants induced by the difficulties in the synthesis of immunogen, complexity and unexpectable efficiency of immunization, a novel strategy to generate Nbs based on the designed synthetic Nb libraries with final size up to 109 cfu/mL was adopted and succeeded in selection of anti-coumaphos Nb A4. Furthermore, an affinity-matured mutant Nb 3G was obtained from the secondary library. Finally, an ic-ELISA was established with the limit of detection for coumaphos low to 1.90 ng/mL, 6.4-fold improved than the parent Nb A4, and the detection range from 3.06 to 15.77 ng/mL. Meanwhile, the recovery rate of vegetable samples was from 89.9% to 98.5%. Finally, the accuracy was testified by the standard UPLC-MS/MS method with R2 up to 0.99. Overall, fully synthetic Nb libraries constructed in this work provided an alternative possibility to generate the specific Nbs for chemical contaminants.

Enhancing stock volatility prediction with the AO-GARCH-MIDAS model.

Research has substantiated that the presence of outliers in data usually introduces additional errors and biases, which typically leads to a degradation in the precision of volatility forecasts. However, correcting outliers can mitigate these adverse effects. This study corrects the additive outliers through a weighting method and let these corrected values to replace the original outliers. Then, the model parameters are re-estimated based on this new return series. This approach reduces the extent to which outliers distort volatility estimates, allowing the model to better adapt to market conditions and improving the accuracy of volatility forecasts. This study introduces this approach for the first time to generalized autoregressive conditional heteroskedasticity mixed data sampling (GARCH-MIDAS) models, so as to establish an additional outliers corrected GARCH-MIDAS model (AO-GARCH-MIDAS). This pioneering approach marks a unique innovation. The research employs a diverse array of evaluation methods to validate the model's robustness and consistently demonstrates its dependable performance. Findings unequivocally reveal the substantial influence of outliers on the model's predictive capacity, with the AO-GARCH-MIDAS model exhibiting consistent superiority across all evaluation criteria. Additionally, while the GARCH model showcases stronger estimation capabilities compared to the GARCH-MIDAS model, the latter demonstrates heightened predictive prowess. Notably, regarding variable selection, the results underscore the greater predictive informational value inherent in realized volatility over other low-frequency factors.

Effects of Separation and Purification Methods on Antioxidation, Hypoglycemic and DNA Protection Activity of Fenugreek Polysaccharide.

Six low molecular weight fenugreek polysaccharides (FP) were isolated and purified by ethanol stepwise precipitation (EFP-20, EFP-40, and EFP-60) and DEAE-52 cellulose column method (DFP-0, DFP-0.15, and DFP-0.3), respectively. The effects of different separation and purification techniques on the preliminary properties and biological activities of fenugreek polysaccharides were compared. The results showed that the DEAE-52 cellulose-eluted fractions had a higher total sugar content and displayed a looser structure. The molecular weights of all six fractions were in the range of 4-19 kDa, with significant changes in the ratio of galactose to mannose. All six fractions contained α-D-galactopyranose and ß-D-mannopyranose structures. Activity tests showed that all six fractions possessed antioxidant, hypoglycemic and DNA-protective activities. Among them, the DFP-0 fraction showed the highest activity. Overall, different isolation and purification methods lead to changes in the properties and bioactivities of FP, which provides a theoretical basis for the development and application of FP in functional foods and drugs.

Subcellular localization of viral proteins after porcine epidemic diarrhea virus infection and their roles in the viral life cycle.

Porcine epidemic diarrhea virus (PEDV) is one of the most devastating diseases affecting the pig industry globally. Due to the emergence of novel strains, no effective vaccines are available for prevention and control. Investigating the pathogenic mechanisms of PEDV may provide insights for creating clinical interventions. This study constructed and expressed eukaryotic expression vectors containing PEDV proteins (except NSP11) with a 3' HA tag in Vero cells. The subcellular localization of PEDV proteins was examined using endogenous protein antibodies to investigate their involvement in the viral life cycle, including endocytosis, intracellular trafficking, genome replication, energy metabolism, budding, and release. We systematically analyzed the potential roles of all PEDV viral proteins in the virus life cycle. We found that the endosome sorting complex required for transport (ESCRT) machinery may be involved in the replication and budding processes of PEDV. Our study provides insight into the molecular mechanisms underlying PEDV infection. IMPORTANCE: The global swine industry has suffered immense losses due to the spread of PEDV. Currently, there are no effective vaccines available for clinical protection. Exploring the pathogenic mechanisms of PEDV may provide valuable insights for clinical interventions. This study investigated the involvement of viral proteins in various stages of the PEDV lifecycle in the state of viral infection and identified several previously unreported interactions between viral and host proteins. These findings contribute to a better understanding of the pathogenic mechanisms underlying PEDV infection and may serve as a basis for further research and development of therapeutic strategies.

Monoclonal antibody development for early detection of ASFV I73R protein: Identification of a linear antigenic epitope.

African swine fever virus (ASFV), which was first identified in northern China in 2018, causes high mortality in pigs. Since the I73R protein in ASFV is abundantly expressed during the early phase of virus replication, it can be used as a target protein for early diagnosis. In this study, the I73R protein of ASFV was expressed, and we successfully prepared a novel monoclonal antibody (mAb), 8G11D7, that recognizes this protein. Through both indirect immunofluorescence and Western blotting assays, we demonstrated that 8G11D7 can detect ASFV strains. By evaluating the binding of the antibody to a series of I73R-truncated peptides, the definitive epitope recognized by the monoclonal antibody 8G11D7 was determined to be 58 DKTNTIYPP 66. Bioinformatic analysis revealed that the antigenic epitope had a high antigenic index and conservatism. This study contributes to a deeper understanding of ASFV protein structure and function, helping establish ASFV-specific detection method.

The Potential Mechanisms of Arrhythmia in Coronavirus disease-2019.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) leads to coronavirus disease-2019 (COVID-19) which can cause severe cardiovascular complications including myocardial injury, arrhythmias, acute coronary syndrome and others. Among these complications, arrhythmias are considered serious and life-threatening. Although arrhythmias have been associated with factors such as direct virus invasion leading to myocardial injury, myocarditis, immune response disorder, cytokine storms, myocardial ischemia/hypoxia, electrolyte abnormalities, intravascular volume imbalances, drug interactions, side effects of COVID-19 vaccines and autonomic nervous system dysfunction, the exact mechanisms of arrhythmic complications in patients with COVID-19 are complex and not well understood. In the present review, the literature was extensively searched to investigate the potential mechanisms of arrhythmias in patients with COVID-19. The aim of the current review is to provide clinicians with a comprehensive foundation for the prevention and treatment of arrhythmias associated with long COVID-19.

Artificial kagome lattices of Shockley surface states patterned by halogen hydrogen-bonded organic frameworks.

Artificial electronic kagome lattices may emerge from electronic potential landscapes using customized structures with exotic supersymmetries, benefiting from the confinement of Shockley surface-state electrons on coinage metals, which offers a flexible approach to realizing intriguing quantum phases of matter that are highly desired but scarce in available kagome materials. Here, we devise a general strategy to construct varieties of electronic kagome lattices by utilizing the on-surface synthesis of halogen hydrogen-bonded organic frameworks (XHOFs). As a proof of concept, we demonstrate three XHOFs on Ag(111) and Au(111) surfaces, which correspondingly deliver regular, breathing, and chiral breathing diatomic-kagome lattices with patterned potential landscapes, showing evident topological edge states at the interfaces. The combination of scanning tunnelling microscopy and noncontact atomic force microscopy, complemented by density functional theory and tight-binding calculations, directly substantiates our method as a reliable and effective way to achieve electronic kagome lattices for engineering quantum states.

ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses.

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection.

Porcine alveolar macrophages host proteins interacting with African swine fever virus p72.

Introduction: African swine fever virus (ASFV) is a highly contagious virus that spreads rapidly and has a mortality rate of up to 100% in domestic pigs, leading to significant economic losses in the pig industry. The major capsid protein p72 of ASFV plays a critical role in viral invasion and immune evasion. Methods: In this study, we used yeast two-hybrid screening to identify host proteins interacting with p72 in porcine alveolar macrophages (PAMs) and verified these proteins using confocal microscopy and immunoprecipitation techniques. Results and Discussion: We validated 13 proteins that interact with p72, including CD63, B2M, YTHDF2, FTH1, SHFL, CDK5RAP3, VIM, PELO, TIMP2, PHYH, C1QC, CMAS, and ERCC1. Enrichment analysis and protein-protein interaction network analysis of these interacting proteins revealed their involvement in virus attachment, invasion, replication, assembly, and immune regulation. These findings provide new insights into the function of p72 and valuable information for future research on the interaction between ASFV and host proteins.

Scleral remodeling during myopia development in mice eyes: a potential role of thrombospondin-1.

BACKGROUND: Scleral extracellular matrix (ECM) remodeling plays a crucial role in the development of myopia, particularly in ocular axial elongation. Thrombospondin-1 (THBS1), also known as TSP-1, is a significant cellular protein involved in matrix remodeling in various tissues. However, the specific role of THBS1 in myopia development remains unclear. METHOD: We employed the HumanNet database to predict genes related to myopic sclera remodeling, followed by screening and visualization of the predicted genes using bioinformatics tools. To investigate the potential target gene Thbs1, we utilized lens-induced myopia models in male C57BL/6J mice and performed Western blot analysis to detect the expression level of scleral THBS1 during myopia development. Additionally, we evaluated the effects of scleral THBS1 knockdown on myopia development through AAV sub-Tenon's injection. The refractive status and axial length were measured using a refractometer and SD-OCT system. RESULTS: During lens-induced myopia, THBS1 protein expression in the sclera was downregulated, particularly in the early stages of myopia induction. Moreover, the mice in the THBS1 knockdown group exhibited alterations in myopia development in both refraction and axial length changed compared to the control group. Western blotting analysis confirmed the effectiveness of AAV-mediated knockdown, demonstrating a decrease in COLA1 expression and an increase in MMP9 levels in the sclera. CONCLUSION: Our findings indicate that sclera THBS1 levels decreased during myopia development and subsequent THBS1 knockdown showed a decrease in scleral COLA1 expression. Taken together, these results suggest that THBS1 plays a role in maintaining the homeostasis of scleral extracellular matrix, and the reduction of THBS1 may promote the remodeling process and then affect ocular axial elongation during myopia progression.

A breakthrough of immunoassay format for hapten: recent insights into noncompetitive immunoassays to detect small molecules.

Immunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection.

Two-stage sequencing batch reactors with added iron shavings for nutrient removal and aerobic sludge granulation treating real wastewater with low carbon to nitrogen ratios.

In response to the challenges of limited nutrient removal and the difficulty in forming aerobic granular sludge (AGS) with low carbon to nitrogen (C/N) ratios, a novel two-stage sequencing batch reactors (SBRs) (R1 and R2) system with added iron shavings was proposed and established. The results showed that AGS was developed and nitrogen (82.8 %) and phosphorus (94.7 %) were effectively removed under a C/N ratio at 1.7 ± 0.5. The average size of R1 and R2 increased from 45.3 µm to 138.7 µm and 132.8 µm. Under high biological selective pressure, phosphorus accumulating organisms like Comamonadaceae (14.8 %) and Chitinophagales (5.7 %) experienced enrichment in R1. Furthermore, R2 exhibited an increased abundance of nitrifying bacteria (2.3 %) and a higher proportion of nitrogen removal through autotrophic denitrification (＞17.5 %). Overall, this study introduces an innovative two-stage SBRs with added iron shavings, offering a novel approach for the treatment of low C/N ratios wastewater.

Enhanced sandwich immunoassay based on bivalent nanobody as an efficient immobilization approach for foodborne pathogens detection.

BACKGROUND: Nanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation. RESULTS: Here, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM KD, as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 103 CFU mL-1 for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %. SIGNIFICANCE AND NOVELTY: In this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens.

Case Report: Osteosclerotic metaphyseal dysplasia with optic nerve involvement and progressive osteonecrosis of the jaw due to a novel LRRK1 mutation.

Background: Osteosclerotic metaphyseal dysplasia (OSMD, OMIM 615198) is an extremely rare autosomal recessive osteopetrosis disorder resulting in a distinctive pattern of osteosclerosis of the metaphyseal margins of long tubular bones. To date, only thirteen cases have been reported (eight molecularly confirmed). Five homozygous sequence variants in the leucine-rich repeat kinase 1 (LRRK1) gene have been identified to cause OSMD. We present two male siblings with OSMD with a novel LRRK1 variant. Cases: The index case, now aged 6 years, was referred aged 9 months when diffuse sclerosis of the ribs and vertebral bodies, suggestive of osteopetrosis, was incidentally identified on a chest radiograph for suspected lower respiratory tract infection. Parents were consanguineous and of Pakistani origin. Further evaluation revealed developmental delay, nystagmus with bilateral optic nerve hypoplasia and severe visual impairment. Skeletal survey confirmed typical changes of OSMD, with widespread diffuse sclerosis and Erlenmeyer flask deformity of long bones. His older sibling, now aged 12 years, was 7 years at the time of referral and had similar clinical course and skeletal findings. Additionally, he had a chronic progressive osteonecrosis of the left mandible that required debridement, debulking and long-term antibiotics. Skeletal survey revealed findings similar to his sibling. Neither sibling had significant skeletal fractures or seizures. Unlike most previous reports suggesting sparing of the skull and lack of visual impairment, our patients had evidence of osteosclerosis of the cranium. Genetic screening for the common autosomal recessive and dominant pathogenic variants of osteopetrosis was negative. Whole Exome Sequencing (WES) followed by Sanger sequencing, identified a novel homozygous LRRK1 c.2506C>T p. (Gln836Ter) nonsense variant predicted to result in premature truncation of LRRK1 transcript. Conclusion: Our cases confirm the autosomal recessive inheritance and expand the spectrum of genotype and phenotype of OSMD reported in the literature. Increasing reports of LRRK1 variants in this phenotype raise the question of whether LRRK1 should be included in targeted osteopetrosis panels. Bone histology in previous cases has shown this to be an osteoclast rich form of osteopetrosis raising the possibility that haematopoietic stem cell transplantation may be an appropriate treatment modality.

Regional impact of aging population on carbon dioxide emissions in China: Evidence from panel threshold regression (PTR).

Carbon dioxide emission is a high-profile issue that can affect both the human economy and human existence, but few scholars have studied the relationship between these two. Therefore, this study constructs the panel threshold regression (PTR) based on the National Bureau of Statistics of China's panel data from 2002 to 2019 in 19 regions. One of the advantages of PTR is to leverage segmented functions, allowing for a more detailed analysis of the data. Besides, by introducing the idea of a threshold, PTR can effectively avoid structural changes in the data. The different between this study and other research is that this study divides 19 regions into two parts: Eastern China and Central China. Based on that, this study researches and compares the different influences of the aging population on carbon emissions in these two regions. The results show that although the Environment Kuznets Curve has been confirmed in both Eastern China and Central China, with the development of the economy, the carbon emissions will increase in Eastern China and decrease in Central China, respectively. In addition, the key factors affecting carbon emissions in Eastern China and Central China are trade dependence and urbanization rate separately. Hence, this study suggests that the regional governments in Eastern China may guide and encourage more international trading companies to move to Central China, and at the same time, the regional governments in Central China should issue more policies to attract these companies, such as: reducing land lease fees and building better transportation infrastructure. Apart from that, the governments in Central China should vigorously increase the rate of urbanization to reduce energy consumption and improve energy efficiency.

Molecular Evolution of Antiparathion Nanobody with Enhanced Sensitivity and Specificity Based on Structural Analysis.

Nanobody (Nb) has gained significant attention in immunoassays owing to its numerous advantages, particularly its ease of molecular evolution. However, the limited understanding of how high sensitivity and specificity attained for antihapten Nbs hamper the development of high-performance Nbs. Herein, the antiparathion Nb (Nb9) we prepared previously was chosen as the model, and an approach based on X-ray crystallography, molecular docking, and rational site-directed saturation mutation for constructing a rapid and effective platform for nanobody evolution was described. Based on the structural analysis, two mutants, namely Nb-D5 (IC50 = 2.4 ± 0.2 ng/mL) and Nb-D12 (IC50 = 2.7 ± 0.1 ng/mL), were selected out from a six-sites directed saturation mutation library, 3.5-fold and 3.1-fold sensitivity enhancement over Nb9 to parathion, respectively. Besides, Nb-D12 exhibited improved sensitivity for quinalphos, triazophos, and coumaphos (5.4-35.4 ng/mL), indicating its broader detection potential. Overall, our study advances an effective strategy for the future rational evolution of Nbs with desirable performance.

Design of an Antigen-Triggered Nanobody-Based Fluorescence Probe for PET Immunoassay to Detect Quinalphos in Food Samples.

Photoinduced electron-transfer (PET) immunoassay based on a fluorescence site-specifically labeled nanobody, also called mini Quenchbody (Q-body), exhibits extraordinary sensitivity and saves much time in the homogeneous noncompetitive mode and is therefore regarded as a valuable method. However, limited by the efficiency of both quenching and dequenching of the fluorescence signal before and after antigen binding associated with the PET principle, not all original nanobodies can be used as candidates for mini Q-bodies. Herein, with the anti-quinalphos nanobody 11A (Nb-11A) as the model, we, for the first time, adopt a strategy by combining X-ray structural analysis with site-directed mutagenesis to design and produce a mutant Nb-R29W, and then successfully generate a mini Q-body by labeling with ATTO520 fluorescein. Based on this, a novel PET immunoassay is established, which exhibits a limit of detection of 0.007 µg/mL with a detection time of only 15 min, 25-fold improved sensitivity, and faster by 5-fold compared to the competitive immunoassay. Meanwhile, the recovery test of vegetable samples and validation by the standard ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) both demonstrated that the established PET immunoassay is a novel, sensitive, and accurate detection method for quinalphos. Ultimately, the findings of this work will provide valuable insights into the development of triggered PET fluorescence probes by using existing antibody resources.

Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods.

Nanobodies (Nbs) have great potential in immunoassays due to their exceptional physicochemical properties. With the immortal nature of Nbs and the ability to manipulate their structures using protein engineering, it will become increasingly valuable to understand what structural features of Nbs drive high stability, affinity, and selectivity. Here, we employed an anti-quinalphos Nb as a model to illustrate the structural basis of Nbs' distinctive physicochemical properties and the recognition mechanism. The results indicated that the Nb-11A-ligand complexes exhibit a "tunnel" binding mode formed by CDR1, CDR2, and FR3. The orientation and hydrophobicity of small ligands are the primary determinants of their diverse affinities to Nb-11A. In addition, the primary factors contributing to Nb-11A's limited stability at high temperatures and in organic solvents are the rearrangement of the hydrogen bonding network and the enlargement of the binding cavity. Importantly, Ala 97 and Ala 34 at the active cavity's bottom and Arg 29 and Leu 73 at its entrance play vital roles in hapten recognition, which were further confirmed by mutant Nb-F3. Thus, our findings contribute to a deeper understanding of the recognition and stability mechanisms of anti-hapten Nbs and shed new light on the rational design of novel haptens and directed evolution to produce high-performance antibodies.

A municipal wastewater treatment plant "drinking beer" for reduction of cost and carbon emission.

In wastewater treatment plants (WWTPs), external carbon sources are often required due to low C/N influent. However, the use of external carbon sources can increase treatment costs and cause large carbon emissions. Beer wastewater, which contains a substantial amount of carbon, is often treated separately in China, consuming significant energy and cost. However, most studies using beer wastewater as an external carbon source are still on a laboratory scale. To address this issue, this study proposes using beer wastewater as an external carbon source in an actual WWTP to reduce operating costs and carbon emissions while achieving a win-win situation. The denitrification rate of beer wastewater was found to be higher than that of sodium acetate , resulting in improved treatment efficiency of the WWTP. Specifically, COD, BOD5, TN, NH4+-N and TP increased by 3.4%, 1.6%, 10.8%, 1.1%, and 1.7%, respectively. Additionally, the treatment cost and carbon emission per 10 000 tons of wastewater treated were reduced by 537.31 yuan and 2.27 t CO2, respectively. These results indicate that beer wastewater has significant utilization potential and provide a reference for using different types of production wastewater in WWTPs. This study's findings demonstrate the feasibility of implementing this approach in an actual WWTP setting.

Self-Limited Embedding Alternating 585-Ringed Divacancies and Metal Atoms into Graphene Nanoribbons.

Because of their theoretically predicted intriguing properties, it is interesting to embed periodic 585-ringed divacancies into graphene nanoribbons (GNRs), but it remains a great challenge. Here, we develop an on-surface cascade reaction from periodic hydrogenated divacancies to alternating 585-ringed divacancies and Ag atoms via intramolecular cyclodehydrogenation in a seven-carbon-wide armchair GNR on the Ag(111) surface. Combining scanning tunneling microscopy/spectroscopy and noncontact atomic force microscopy combined with first-principles calculations, we in-situ-monitor the evolution of the distinct structural and electronic properties in the reaction intermediates. The observation of embedded Ag atoms and further nudged elastic band calculations provide unambiguous evidence for Ag adatom-mediated C-H activation in the intramolecular cyclodehydrogenation pathway, where the strain-induced self-limiting effect contributes to the formation of the GNR superlattice with alternating 585-ringed divacancies and Ag atoms, which shows a band gap of about 1.4 eV. Our findings open an avenue to introducing periodic impurities of single metal atoms and nonhexagonal rings in on-surface synthesis, which may provide a novel route for multifunctional graphene nanostructures.