Autophagy, a highly conserved cellular self‐degradation pathway, has emerged with novel roles in the realms of immunity and inflammation. Genome‐wide association studies have unveiled a correlation between genetic variations in autophagy‐related genes and heightened susceptibility to autoimmune and inflammatory diseases. Subsequently, substantial progress has been made in unraveling the intricate involvement of autophagy in immunity and inflammation through functional studies. The autophagy pathway plays a crucial role in both innate and adaptive immunity, encompassing various key functions such as pathogen clearance, antigen processing and presentation, cytokine production, and lymphocyte differentiation and survival. Recent research has identified novel approaches in which the autophagy pathway and its associated proteins modulate the immune response, including noncanonical autophagy. This review provides an overview of the latest advancements in understanding the regulation of immunity and inflammation through autophagy. It summarizes the genetic associations between variants in autophagy‐related genes and a range of autoimmune and inflammatory diseases, while also examining studies utilizing transgenic animal models to uncover the in vivo functions of autophagy. Furthermore, the review delves into the mechanisms by which autophagy dysregulation contributes to the development of three common autoimmune and inflammatory diseases and highlights the potential for autophagy‐targeted therapies. This review presents an overview of the autophagy machinery and summarizes the roles of autophagy and autophagy proteins in regulating immunity and inflammation. The authors discuss how dysregulation of autophagy contributes to the pathogenesis of three common autoimmune and inflammatory diseases and emphasize autophagy as a promising therapeutic target. It presents the genetic links between variants in autophagy‐related genes (ATGs) and a range of autoimmune and inflammatory diseases. Additionally, it explores the in vivo function of autophagy through the utilization of transgenic animal models. Furthermore, this review delves into the underlying mechanisms by which autophagy dysregulation contributes to the pathogenesis of three prevalent autoimmune and inflammatory disorders: inflammatory bowel diseases (IBD), systemic lupus erythematosus (SLE), and multiple sclerosis (MS). Finally, it summarizes the findings of clinical trials using autophagy‐targeted drugs and biological agents and explores the prospects of therapies aimed at modulating autophagy.2Autophagy is a conservative mechanism of self‐degradation, which transports cellular materials to lysosomes for decomposition. The process of autophagy encompasses three distinct mechanisms: macroautophagy, microautophagy, and chaperone‐mediated autophagy, each employing unique strategies for transporting cargo to the lysosome.Although macro‐, micro‐, and chaperone‐mediated autophagy are all considered to be genuine forms of autophagy, as they transport cytoplasmic materials to lysosomes,3Although initially discovered for its role in degrading unwanted or damaged cytoplasmic material to maintain cellular homeostasis, recent studies also suggest that autophagy has broader functions in immunity and inflammation.Atg5, renders mice more susceptible to infections and disrupts normal inflammatory responses.Autophagy contributes to innate immunity by assisting in pathogen clearance and inflammation regulation. Two autophagy‐associated pathways, xenophagy and LAP, are utilized by innate immune phagocytes to promote degradation of microbes within lysosomes.Atg5.Atg5 in distinct subsets of immune cells in murine models has been shown to result in notable impairments in various aspects of immune function, such as compromised B lymphocyte maturation, impaired differentiation of plasma cells, diminished T cell survival, and reduced T cell proliferation.Autophagy plays a pivotal role in bridging innate and adaptive immunity by facilitating antigen presentation. Recent studies have provided emerging evidence that autophagy plays a facilitating role in the presentation of antigens on major histocompatibility complex (MHC) class II molecules.In addition to its aforementioned immunomodulatory functions, autophagy's role in onco‐immunology is also increasingly recognized. The involvement of autophagy in tumor development is influenced by multiple variables, such as the specific tumor subtype, stage of progression, and the characteristics of the tumor microenvironment. Autophagy plays a critical role in maintaining homeostasis, activating, differentiating, and promoting the survival of immune cells, which in turn can have both promotive and inhibitory effects on tumor development. Thus, autophagy functions as a double‐edged sword in the context of tumorigenesis. Since the topic of autophagy and tumor immunity has been covered comprehensively in recent reviews,4The association between mutations in ATGs and heightened susceptibility to autoimmune and inflammatory diseases has piqued interest in the involvement of autophagy in disease mechanisms. Nevertheless, as indicated in Table 4.1ATG16L1. This discovery has generated significant interest in unraveling the role of autophagy in the context of IBD. Moreover, GWAS have revealed additional single‐nucleotide polymorphisms (SNPs) in ATG genes that exhibit a strong association with IBD, as shown in Table IBD, encompassing CD and ulcerative colitis, manifests as a chronic gastrointestinal disorder marked by severe inflammation and mucosal damage.ATG16L1 T300A variant, our discussion will focus primarily on ATG16L1, supplemented by other ATGs. The T300A variant (Thr300Ala) causes the substitution of the evolutionarily conserved polar threonine residue at position 300 of the WD repeat domain in ATG16L1 with a nonpolar alanine. During the process of autophagosome formation, the ATG16L1 protein engages in interactions with the ATG12–ATG5 conjugate, leading to the formation of a large molecular complex. This complex facilitates the lipidation of LC3/ATG8, thereby contributing to autophagosome development.Atg16l1 display dysregulated autophagy and intestinal homeostasis, and the relevant mechanisms are summarized in Figure Considering that most of the evidence linking autophagy‐related genetic variants to IBD is derived from functional studies utilizing the 4.1.1Atg16l1 in epithelial cells and found that autophagy‐deficient mice had systemic translocation of bacteria and an exaggerated inflammatory response compared with their wild‐type counterparts. Under normal conditions, autophagosomes can engulf invaded S Typhimurium in IECs. However, mice with Atg16l1 deletion in IECs showed cell‐specific disruptions of autophagy and failed to control the spread of intestinal pathogens. In addition, these mice displayed abnormal morphology of Paneth cells, specialized epithelial cells that play a crucial role in the secretion of antimicrobial peptides.Irgm1Atg4b,Atg16l1T300A/T300A mice caused a defect in mucin secretion in their goblet cells.In a healthy state, the intestinal epithelial cells (IECs) establish a strong mucosal barrier capable of withstanding intestinal pathogens. Additionally, specialized epithelial cells, including Paneth and goblet cells, secrete defensins and mucus to limit bacterial adhesion and infiltration.4.1.2Atg16l1‐deficient macrophages exhibited heightened production of the proinflammatory cytokines IL‐1β and IL‐18 upon stimulation with lipopolysaccharide, owing to the activation of caspase‐1.Atg16l1 T300A variant, introduced via knock‐in, exhibited defective antibacterial autophagy and an increased cytokine response.IBD are chronic gastrointestinal inflammatory conditions, and autophagy has been underscored in recent research as a vital mechanism for regulating inflammatory responses.4.1.3Atg5 knockout display an altered gut microbiota composition and structure, characterized by an upsurge of proinflammatory bacteria and a decrease in alpha diversity.Atg7 in colonic epithelial cells also exhibit comparable outcomes.Atg16l1T300A/T300A mutation exhibit an altered gut microbiota composition, characterized by a notable increase in the prevalence of bacteria linked to IBD, such as Ruminococcaceae.The gut microbiota comprises trillions of bacteria inhabiting the mammalian intestinal lumen, and their interaction with the host is essential for maintaining physiological metabolism and intestinal immune equilibrium. The importance of autophagy in regulating the gut microbiota has been underscored by recent investigations. By eliminating intracellular pathogens, autophagy restricts the replication and dissemination of pathogenic microorganisms. In addition, autophagy helps regulate the gut microbiota composition by maintaining mucosal barrier stability. It is involved in secreting antimicrobial peptides and mucus into the intestinal lumen, which safeguards against pathogen invasion and infection. Dysfunctional autophagy has been associated with dysbiosis of the gut microbiota, which heightens the susceptibility to developing IBD.4.2SLE is distinguished by an excessive immune response and the breakdown of immune tolerance toward self‐antigens.4.2.1Cybb and Ncf1, displayed deficient uptake and degradation of necrotic material, increased production of inflammatory mediators, or exacerbated lupus.One of the key factors in the development of SLE is the imbalance between the accumulation of material from dead cells and the disposal of apoptotic debris, which contributes to increased autoantigen exposure. Under normal circumstances, professional phagocytes like macrophages clear apoptotic debris, which remains sequestered from the immune system. However, impaired clearance of dead cell debris can activate nucleic acid recognition receptors, triggering the production of proinflammatory cytokines. Recent research has emphasized the crucial function of LAP, a distinct form of noncanonical autophagy, in the efficient removal of dead cells.4.2.2Rubcn has been found to provide survival advantages for SLE‐prone mice, including B6.Sle1.Yaa and MRL.Faslpr mice, by reducing autoantibody production and protecting them from renal disease.Rubcn promotes autoimmunity in murine lupus by activating autoreactive B cells, promoting autoreactive germinal center reactions and plasmablast response. Autophagy also plays a critical role in the differentiation of plasma cells and the survival of memory B cells.Atg5 or Atg7 impairs the terminal differentiation of B cells into plasma cells.Atg5 are more vulnerable to cell death because they lack the protective effects provided by autophagy.One prominent characteristic of SLE is the generation of autoantibodies by self‐reactive B cells.4.2.3Tlr7.1 transgenic mice with and without B cell‐specific Atg5 knockout, researchers observed that Tlr7.1 transgenic Atg5 knockout mice did not exhibit an IFN‐α signature in their serum.Atg5 knockout mice also exhibited reduced IFNα and increased lifespan,Exposure of the body to autoantigens containing nucleic acid can trigger an immune response, leading to the production of autoantibodies. The subsequent interaction between these autoantibodies and autoantigens can result in the formation of immune complexes, which possess the ability to initiate inflammation and cause tissue damage. Plasmacytoid dendritic cells (pDCs) take up immune complexes by binding to Fcγ receptors (FcγRs) and transport them to endosomal compartments where TLR7 and TLR9 are located.4.2.4lpr/lpr mice suffering from nephritis and in renal biopsies from LN patients, signifying the activation and participation of autophagy in the development of LN.Lupus nephritis (LN) is a serious complication and a primary contributor to mortality among SLE patients.4.3CALCOCO2/NDP52, which seems to be linked to autophagy and MS.ATG5 in both the blood and brain tissue of MS patients.MS is a disorder that impacts the central nervous system (CNS) and is distinguished by inflammation and demyelination.4.3.1Atg7, which is necessary for both canonical autophagy and LAP, is specifically deleted in the microglia of mice. This deletion leads to the development of a progressive MS‐like disease characterized by impaired autophagy‐associated phagocytosis.Atg5 in microglia have demonstrated that the absence of Atg5 in these cells results in increased intracellular accumulation of myelin and inflammatory debris. This accumulation subsequently contributes to persistent neuroinflammation and neurotoxicity.Ulk1 specifically deleted, a gene required only for canonical autophagy, did not exhibit a similar disease phenotype, suggesting that the disease phenotype was mainly dependent on noncanonical autophagy rather than canonical autophagy.In microglia, the primary CNS‐resident macrophages, the machinery of autophagy performs an essential function in regulating phagocytosis, the process of engulfing and digesting unwanted substances, including cellular debris and protein aggregates, to promote tissue repair and homeostasis.4.3.2Atg7 or Pik3c3 specifically in myeloid cells of mice leads to a decrease in EAE severity,Pik3c3, Rb1cc1/Fip200, Atg5, or Atg7 in dendritic cells leads to a reduced occurrence and gravity of EAE,Rubcn, a gene essential for LAP but not canonical autophagy, in dendritic cells, displayed wild‐type levels of EAE susceptibility, which differs significantly from DC‐specific Pik3c3‐deficient or Rb1cc1‐deficient mice.Experimental autoimmune encephalomyelitis (EAE) is an extensively used animal model to investigate MS, and many investigations have shown that modulation of autophagy leads to a significant improvement in the severity of EAE. Autophagy participates in the regulation of antigen loading onto MHC molecules and the subsequent antigen presentation by APCs, including dendritic cells and macrophages. Selective deletion of 4.3.3Pik3c3 or Becn1 displayed resistance to EAE induction and exhibited impaired ability to induce autoreactive T cell responses.Pik3c3 exhibited suppressed oxidative respiration and diminished glycolysis,Becn1‐deficient T cells experience a significant increase in cell death upon stimulation.The hallmark of MS lies in the activation and proliferation of autoreactive CD4 T cells.5Autophagy‐targeted drugs and biological agents have shown great potential as effective therapeutic options in autoimmune and inflammatory diseases. Several drugs have been used in clinical treatment, including rapamycin and hydroxychloroquine (HCQ). Clinical trials investigating various autophagy modulators are summarized in Table Moreover, recent investigations have focused on the modulation of autophagy to enhance immune responses and facilitate antitumor effects. Clinical trials targeting autophagy, either alone or in combination, in cancer treatment have mostly relied on HCQ, and the outcomes of these trials have been summarized in some recent reviews.While certain drugs mentioned above, including rapamycin and HCQ, have been approved for human use, their clinical application was not specifically targeted toward autophagy regulation.6This review emphasizes the essential role of autophagy in both innate and adaptive immune responses, encompassing crucial functions such as clearance of pathogens, processing and presentation of antigens, production of cytokines, and the survival and differentiation of lymphocytes. Malfunctioning of autophagy in humans can lead to various immune disorders, such as inflammatory, autoimmune or general immunity disorders. Inhibiting autophagy can alleviate diseases like SLE and MS by interfering with antigen presentation and lymphocyte survival, but it may also exacerbate diseases such as IBD. Dysfunctional autophagy can disrupt intestinal epithelial homeostasis, change gut microbiota composition, and amplify intestinal inflammation in IBD. These findings demonstrate that autophagy regulation varies across different diseases, tissues, and cells. Therefore, Therefore, future investigations should focus on elucidating the distinct roles of autophagy in different tissues and cell types, aiming to gain a comprehensive understanding of disease‐specific mechanisms.While many autophagy modulators are currently undergoing clinical trials, there are still several unresolved clinical questions. First, current pharmacological agents like rapamycin and HCQ indirectly modulate autophagy, and there is a lack of approved pharmaceuticals that specifically address autophagy as a therapeutic target. Second, the development of targeted therapies necessitates not only a drug with exceptional specificity but also a comprehensive understanding of the optimal timing and administration protocols. Methods for accurately quantifying the activity of the autophagy pathway in vivo are currently inadequate. Consequently, assessing the extent of autophagy modulation in patients undergoing therapy with autophagy modulators presents a significant challenge. Last, despite the promising therapeutic opportunities that autophagy has presented for effective cancer treatment, significant challenges and obstacles exist. Autophagy in tumor development exhibits a complex and multifaceted nature, encompassing both advantageous and detrimental contributions. Although the suppression of autophagy in certain scenarios can bolster the immune response and bolster the antitumor effects of immunotherapy, it can also contribute to immune evasion by tumor cells and foster resistance to treatment. Therefore, identifying the dominant role of autophagy (antitumorigenic or protumorigenic) in particular cancers and deciding when to target autophagy for treatment during tumor development are crucial questions.ATG16L1T300A/T300A genotype. In addition to conventional treatment options, gene therapy emerges as a potential avenue for tackling IBD. Excitingly, several miRNAs have been discovered to effectively suppress autophagy.In conclusion, autophagy is integral to the maintenance of immune homeostasis and serves as a protective mechanism against inflammatory and autoimmune diseases. The suppression or enhancement of autophagy has been shown to have significant effects in various cell culture and animal models, impacting self‐immune responses, inflammatory conditions, and a variety of tumors. These discoveries have provided invaluable knowledge regarding the underlying mechanisms of these diseases and have paved the way for the emergence of groundbreaking therapeutic approaches. For instance, research has shown that effectively inhibiting autophagy effectively reduces the onset and severity of actively induced EAE, providing a compelling intervention strategy for autoimmune diseases informed by an animal model.Ting Gan conceptualized this review and drafted the manuscript. Shu Qu drew the figures and edited the language. Xu‐jie Zhou and Hong Zhang provided helpful suggestions on the structure and content of this review. All authors revised the manuscript. All authors have read and approved the article.All authors have approved of the manuscript and declare no potential conflict of interest.Not applicable.


Herein, we report the improvement of the easy-to-make magnetic silver nanoparticle-based sensor system for cyanide determination in an extended calibration range with higher selectivity and precision. As far as our knowledge is concerned, the detectable linear range from 1.0 nM to 160 μM (0.026 ng mL−1 to 4.16 μg mL−1) of the improved simple highly precise technique represents the widest assay that has been reported so far. The method is based on strong enhancement of scattered light of the plasmonic nanoparticles and simultaneously cyanide fluorescence quenching. Although the fluorescence of cyanide is highly selective and precise, its intensity is poor. On the other hand, the strongly enhanced Rayleigh signal has a low repeatability. We proposed a method to remove the interference and obtained an effective factor that is directly proportional to cyanide concentration utilizing both above signals simultaneously. In this work, Ag/Fe3O4 NPs have been synthesized easily using a green preparation method and the NPs were consequently characterized using powder XRD, UV-Vis absorption spectroscopy, transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). A combination of absorption, Rayleigh and fluorescence characteristics were used for detection of cyanide in real samples and an overview of recently reported sensors for cyanide was also provided.In the line of our previous studies, we have reported a developed sensitive and selective probe for cyanide detection based on Ag/Fe 3O4 nanoparticles (NPs) with an extremely low limit of detection at the level of ng per milliliter.In the line of our previous studies, we have reported a developed sensitive and selective probe for cyanide detection based on Ag/Fe Lethal doses (LD50) of cyanide ion (CN−) are about 0.5–3.5 mg kg−1 of body weight.3 Numerous approaches for cyanide sensing have been studied for more than 10 years.1,2,4–12 Different methods have been widely used to analyze cyanide such as titrimetric, voltammetric, potentiometric, ion chromatography and universal spectrophotometric techniques.10,13–15 A comparison of the performances of the earlier reported works for CN− ion sensing using different detection systems has been reported recently.16 Here, Table 1S17 despite cyanide's widespread presence in many industrial and municipal wastewaters. In relation to the increased interest in the subject of cyanide poison analysis, researches aim to offer analytical techniques that improves the sensitivity and selectivity with rapidly and simply implementation. Previously, we succeeded to introduce a direct probe for simple detection of trace cyanide concentration in solutions.15 The method was based on the large affinity of cyanide in complex formation by Ag NPs and decrement of enhanced Rayleigh signal due to the Ag etching. The use of the strong Ag–CN interaction is an attractive idea for a label free cyanide detection.15,18–20 To improve the selectivity and precision of the sensor in an essential unique wide concentration range of cyanide, we, in this report, proposed a new precise dual Rayleigh–fluorescence assay based on a simplest and most basic titrimetric method.Considerable research into the development of methods for cyanide detection is of prime importance due to the lethal toxicity of cyanide in physiological systems, as well as environmental concerns.21–23 H. Ju group have been reviewed optical sensing of cyanide anions by noble metal nanomaterials.24 Metal NPs, especially silver and gold NPs, with the distinct optical and electronic properties have received significant attention for the fabrication of plasmonic sensors.25–27 The collective excitations of electrons in the conduction band in resonance with an electromagnetic field of incoming light  and consequently an intense excitation signal can enhance weak multi-photon processes leading to surface enhanced spectroscopy that improve the sensitivity dramatically. Surface enhanced spectroscopy, refers to the enhancement of any well-known spectroscopic signals from the molecules at the surface, such as strong plasmon absorption, surface enhanced luminescence, surface enhanced Raman scattering, and surface enhanced Rayleigh scattering.15 NP made of noble metals specially in hybrid forms with other nanostructures has been used based on surface enhanced spectroscopies for a variety of applications.15 As examples Au/Fe3O4 dumbbell-shaped heterostructures were used as dual optical/MRI agents during in vitro studies of epithelial cells by Xu et al.29 The Wei group30 reported Au/Fe3O4 usage to make plasmon-resonant nanoparticles and nanostars for magnetomotive imaging. High performance photocatalytic hydrogen production and degradation of levofloxacin by wide spectrum responsive Ag/Fe3O4 bridged SrTiO3/g-C3N4 plasmonic nano-junctions and Joint effect of Ag and Fe3O4 were proposed by Kumar in 2018.31 Amplification effect of plasmon NPs was suggested widely for detection as sensors and biosensors.32–35Over the last decade, several types of sensors have been developed on the basis of the plasmonic optical properties of NPs made of noble metals for a wide range of applications in catalysis, optics, measurement of chemical and biological quantities and medical therapeutics.27,36–38 The most SERS active metal is silver, while SERS enhancement factors as high as 1014–1015 have been described for silver NPs,39,40 resulted in detection limits as low as a single molecule.39,41 Also, the immense sensitivity achieved in SERS permits the ultrasensitive and selective detection of biomolecules38,42 as well as concomitants.37,43Surface enhanced Raman scattering (SERS) is probably one of the most powerful related techniques currently available for chemical and biological sensing analysis, biomedical applications, food and environmental safety control and also for molecular structural detection of specific molecules.15 Strong plasmonic enhancement of light scattering by gold or silver nanoparticles, made nanotechnology capable to detect and sense molecular information even in single living cells44,45 that improve medical diagnosis. Beside the plenty useful usage of Rayleigh scattering, there have been studies on particle size measurement as well.45,46 Rayleigh and Raman  scattering are closely similar processes in which light is scattered by atoms or molecules. Mentionable that Rayleigh scattering is stronger and more sensitive than Raman vibrations which are usually weak. The efficiency of Rayleigh scattering for silver NPs is about 106 times higher than the fluorescence efficiency of a fluorescent dye molecule with the distinct advantage that silver NPs neither blink nor bleach against fluorescent probes.47In recent years Rayleigh scattering as a highly sensitive, simple and quick analytical technique has also shown its broad applications.48–50 MNPs compared with noble metal and semiconductor NPs, have neither no fluorescence nor surface plasmon absorption.51 But, due to their high refractive indices and molecular weights despite their small sizes, make them a powerful enhancer for plasmonic responses.52 Therefore, the use of MNPs as SPR amplification factor was the subject of many researches.52,53 Also, it is particularly beneficial to design hybrid NPs that combine the magnetic properties together with plasmonic activity54 which allow simultaneous monitoring of analytes and magnetic control of the NPs (to avoid from their probably further pollution).19,37,55,56 The research of metal nano-based fluorescent sensors is a particularly active research field due to their intrinsic advantages of the fluorescent methods.19,30,57 According to Zhai et al.,19 bifunctional Au–Fe3O4 NPs for sensitive and selective turn-on fluorescent detection of cyanide based on the inner filter effect field provided a sensitive strategy. Song, et al. in 2020,30 gathered magneto-optical properties of Ag modified Fe3O4 NPs with all-in-one sensing platforms.At the same time, the integration of magnetic nanoparticles (MNPs) with analytical methods has opened a new path for sensing, purification, and quantitative analysis.15 we have proposed an improved hybrid Rayleigh–fluorescence method for cyanide monitoring using dual-functional magnetic-optical nanosensor. The prepared silver magnetite-based nanocomposite (Ag/Fe3O4) showed a remarkable optical property, enabling a significant improvement in the sensitivity due to the surface plasmon enhancing of both Ag and magnetic NPs. The sensor also has the unique property of strong rapid interaction of nanocomposite with cyanide ions, providing resulting to fabricate a low cost label-free sensor for fast, selective and highly precise detection of ultra-trace amounts of cyanide. The proposed method showed the widest linear concentration range for cyanide detection compared to those reported previously . Transmission electron microscopy was performed on a JEOL, JEM-2100 electron microscope. The chemical compositions were examined using energy-dispersive spectroscopy (EDS). X-ray diffraction (XRD) studies were carried out on a Philips X'Pert-MPD X-ray diffractometer at the ambient temperature. A Metrohm 713 pH-meter and a 40 kHz RoHS universal ultrasonic cleaner bath 35 W were used for pH adjustments and cleaning, respectively. Fourier transform infrared (FTIR) spectra of the samples were obtained using a Nicolet, IR 100 FT-IR spectrometer in the KBr matrix.3O4 NPs were synthesized through alkaline precipitation of ferric and ferrous iron salts from their acidic aqueous solutions as before.58,59 The Ag/Fe3O4 nanoparticles were prepared, using glucose as a mild green reducing agent and AgNO3 in water, similar to our previously work.15Magnetic Fe3O4 nanoparticles (0 to 0.5 mg L−1) were added to cyanide solution and spectroscopically analyzed using the main bands at 313, 400 and 625 nm (excitation wavelength of 300 nm) which can be assigned to strong resonance Rayleigh scattering, cyanide fluorescence, and second-order Rayleigh scattering (2Rayleigh scattering) bands, respectively. The enhancement in Rayleigh scattering signal (ΔIR) is proportional to the nanoparticles and cyanide concentration in a wide range. For greater impact of present assay, the applicability of the new rapid, selective and highly precise method in real samples has been investigated. CAUTION: care should be taken during contact with any solutions containing cyanide.20 Prior studies demonstrated that Ag NPs and iron oxide both in the hybrid nanostructure, amplify the Rayleigh signals and enhance the sensitivity of the cyanide probe whereas separately usage, which emphasize the role of iron oxides besides Ag NPs enhancement effect in addition to the reusability capability of the nanocomposite , (200), (220) and (311) planes of the face-centered cubic lattice of silver.61 The diffraction peaks at 30°, 35.5°, 43.1°, 53.4°, 57° and 62.8° should be related to Fe3O4, corresponds to the cubic inverse spinel structure (ICDD No. 88-0315). The optical properties of repeatedly prepared magnetic nanoparticles were examined and the results almost were the same. Therefore, based on our observations, the reproducibility for the preparation of the silver magnetite nanoparticles was satisfactory here and in our previous studies.15Transmission electron microscopy (TEM) shows an average particle size of below 20 nm for the Fearticles . Using tspectrum  a crystaO4 peaks  are ascrications  and EDX ications  of the rications  shows thications  and also.60,61 T−1 can be belong to the C–O bond stretching mode. Also for Ag NPs, the FTIR indicated the presence of peak nearby 1700, which may correspond to stretching vibrations of glucose, as reductant. Comparing the FTIR spectra of Fe3O4 and Ag NPs with the Ag/Fe3O4, the presence of Fe3O4 is clearly indicated by presence of peaks in the 400–700 cm−1 region. The peaks corresponding to O–H and C–O stretching vibration are also present in the nanocomposite indicating the presence Ag NPs and its strong interaction with Fe3O4 as seen in 60Besides, the peaks around 1050 cm3O4 NPs with different amounts (ranging from 0 to 0.5 mg L−1) were mixed gradually with exact concentrations of cyanide solutions . Resonance Rayleigh scattering, cyanide fluorescence as well as second-order Rayleigh scattering (2Rayleigh scattering) bands in the visible range of the solutions were then analyzed immediately for cyanide sensing (λ = 87 nm for Rayleigh and Δλ = 225 nm for 2Rayleigh), which contributes to the accurate measurement of the intensities of the two peaks.Ag/Fe sensing . The fluBased on our observations these signals were proportional to the cyanide concentrations; therefore, we concluded that, in order to improve the sensing assay, both fluorescence and Rayleigh scattering signals for cyanide determination should be used. The cyanide fluorescence signals were not appearing to be so strong, and in spite of high selectivity, its sensitivity  was limited.3O4 NPs to cyanide solution, the fluorescence emission of cyanide decreased and at the same time the Rayleigh scattering was proportionally increased.Also in the presence of interferences or matrix effects, despite the fluorescence of special concentration of cyanide remains constant, the Rayleigh scattering was slightly increased and its reproducibility and precision was relatively poor. Considering these two factors is a key for effective cyanide sensing. That means by addition of Ag/Feλem = 400 nm, while the ΔF remains constant with tolerance t ≤ ±5%. Under such condition, the enhancement in Rayleigh scattering signal was linearly proportional to the cyanide concentration. In overall, besides highly enhancing in Rayleigh scattering signal because of the presence of small amount of Ag and iron oxides NPs in the probe, selective fluorescence quenching could serve as the basis for direct, sensitive and highly selective cyanide determination in a wide concentration range. In particular, iron oxide parts could be recovered due to the reproducible properties under an external magnetic force which in turn helps to remove their probably pollution effects. Also, the iron oxide parts could be simply re-used in the preparation of nanostructures with Ag. We should note that the iron oxides were highly magnetic, even after they formed a nanostructure with silver.52 As the reversibility parameters could have established to term the composite as a good probe, the recyclable magnetic nanoparticles particles were employed again by extraction with a magnet and washing, to create silver iron oxide nanostructures in order to use in a separate experiment for cyanide detection. The results showed approximately the same efficiencies  (note: the magnetic particles loss is little due to their high strong magnetic property and collected simply for future use). Also the re-used NPs were gathered for another cycle repeatability after the first sensing experiment, by separation and washing the composite without any silver modification. The results showed that the efficiency decreases dramatically.Ag could be etched by cyanide selectively, liberated from the NPs; thus, rapid complexation and decreasing in fluorescence is occurred. Fluorescence signal quenching brings a good selectivity and reproducibility for cyanide detection. Meanwhile, increasing the enhanced Rayleigh intensity related to the cyanide fluorescence decreasing, resulted in high sensitivity. By addition of the NPs to some extent to cyanide solution titrimetrically, the fluorescence intensity of cyanide disappears. The first zero point was achieved by monitoring the instrumental fluorescence signal at This can be attributed as a result from etching of Ag by cyanide ion that liberate the silver NPs from the nanostructure to form a stable complex by cyanide and therefore decreasing the Rayleigh scattering.IF) was determined using the data obtained from the spectra  and the NPs concentration. IR was increased in some orders of magnitude relative to the decrease of fluorescence signal, resulted in improving sensitivity of the method. Considering the formula relation IR = KI0cM,67 the scattering intensity (IR) will be proportional to the concentration of the solution (c), when the incident light intensity (I0), the molecular weight of the particle (M) and K are constant.In addition, results have also shown a relationship between the enhanced Rayleigh intensity  − IR0(313 nm)} and the cyanide concentration was obtained in the range of 1.0 nM to 160 μM . The reproducibility of the present method was evaluated. The relative standard deviation (RSD) for six replicate measurements of a typically solution containing 80 μM CN− was 1.2%, illustrated that the response toward cyanide was highly precise. In order to appraising the potential application of our proposed method, quantitative determination of cyanide in tap and waste water real samples was investigated. Credible recoveries  with slope about −0.8).However, after 15 min, Rayleigh scattering is decreased gently. To identify the results, the Rayleigh scattering and the fluorescence spectra were repeated at least 3 times for more than one batch of nanoparticles and the relative standard deviation of the changes was below ±1%. Also the result revealed that the fast interaction between the composite and cyanide ion can be completed immediately after the addition of NPs (one of the method advantages is a quick response time that mean decreasing in fluorescence and increasing in Rayleigh scattering were occurred simultaneously in seconds ). Fig. 815Therefore, spectral data could be recorded immediately upon the addition of NPs, indicated that the method proved a rapid capability for cyanide determination. Furthermore, since the fluorescence quenching was time independent , this st−, NO3−, SO32−, H2PO4−, CrO4−, OH−, Cl−, Br−, S2−, S2O32−, Zn2+, Na+ and K+ to the mixtures of 3 mg L−1 of cyanide solution and 0.08 mg L−1 of Ag/Fe3O4 solution at room temperature were studied  and the reason may be due to the lower solubility product constant, Ksp for silver halide preparation . That means that using higher halides concentration, Ag+–halide formation could compete with Ag+–CN− interaction. Therefore, it proved that the method of cyanide sensing that we employed, provided an excellent selectivity over the environmentally common interface ions because of the highly selective Ag+–CN− complexation formation and so on , for [Ag(CN)2]− at 25 °C is about 1.0 × 1021). As scrutiny relative to our previous work, higher selectivity over competitive interferences and much better precision due to the reproducible selective fluorescent signal was achieved.The results confirmed that the most species that we investigated did not interfere even when present 1000-fold excess than CN3O4 NPs as an approved probe for analysis and recognition of different levels of cyanide contamination in a wide toxic concentration range of 1.0 nM to 160 μM. The new probe displays remarkable dual changes in fluorescence and highly analytical signal of surface-enhanced Rayleigh scattering by nanoparticles addition. ΔIR increment proportional to the fluorescence decreasing was used, as a beneficial improvement in the precision, accuracy and selectivity of cyanide sensing.In this research work, we have reported an easy “green” preparation of plasmonic–magnetic of Ag/FeWe also reported an improvement of the easy-to-make magnetic silver nanoparticle-based sensor system for cyanide determination in an extended calibration range with higher selectivity and precision. We proposed a method to remove the interference and obtained an effective factor that is directly proportional to cyanide concentration utilizing both above signals simultaneously.3O4 NPs have been synthesized easily by green preparation method and the NPs were consequently characterized using powder XRD, UV-Vis absorption spectroscopy, transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDX). Combinations of absorption, Rayleigh and fluorescence characteristics were used for the detection of cyanide in real samples.We also demonstrated that the sensor exhibited more effective applicability in real sample that can be used for detecting lethal cyanide in practical water systems. The new strategy led to other advantages of minimal pH dependence, fast response time, low detection limit (as low as 1 nM)/high sensitivity, high precision and selectivity in presence of other competitive environmental ions and also low cost preparation. Therefore, improved performance  is achieved in this improved study. In summary, in this work, Ag/FeRazieh Moosavi: conceptualization; methodology; formal analysis; investigation; writing – review & editing and Ramin Zibaseresht: project administration; resources; supervision; writing – review & editing.There are no conflicts to declare.RA-013-D3RA06654A-s001


Scandium (Sc) is a high value Critical Material thatis most commonlyused in advanced alloys. Due to current and potential supply limitations,there has been an international effort to find new and improved waysto extract Sc from existing and novel resources. Solid-phase extraction(SPE) is one promising approach for Sc recovery, particularly foruse with low-grade feedstocks. Here, unfunctionalized, powdered hierarchicallyporous silica monoliths from DPS Inc. (DPS) are used for Sc extractionin batch and semicontinuous flow systems at model conditions. Thesorbent exhibits excellent mass transfer properties, much like thewhole monoliths, which should permit Sc to be rapidly recovered fromlarge volumes of feedstock. The Sc adsorption capacity of the materialis ∼142.7 mg/g at pH 6, dropping to ∼12.0 mg/g at pH3, and adsorption is furthermore highly selective for Sc comparedwith the other rare earth elements (REEs). Under semicontinuous flowconditions, recovery efficiency is limited by a kinetic process. Theprimary mechanism responsible for the system’s slow approachto equilibrium is the Sc adsorption reaction kinetics rather thaninter- or intraparticle diffusion. Overall, this unmodified hierarchicallyporous silica powder from DPS shows great promise for the selectiveextraction of Sc from various feedstocks. Powder derived from hierarchically porous silica monolithswas used to selectively extract scandium from aqueous feedstocks atmodel conditions. Scandium is used primarilyfor lightweight alloys, for example in the aerospace industry, andfor solid oxide fuel cells, as well as more minor applications inceramics and electronics.3 These uses have drivenits price up, and Sc is presently the most valuable of the REEs.3 Indeed, its value is high enough that the economicpotential of some possible future REE resources may be largely determinedby the presence of Sc alone.4 However,despite the value and practical utility of Sc, meaningful productionof this metal is limited to only a few major sources around the world,with 66% coming from China in 2020.1 Sincethese sources have been and continue to be vulnerable to various geopoliticalcircumstances, there has been a significant push in many regions toidentify new potential Sc feedstocks and to develop new and improvedtechniques for Sc extraction and purification. Among these emergingnovel recovery systems, solid-phase extraction (SPE) is one broadclass of metal recovery techniques which employs a solid materialto selectively recover a specific component of an aqueous feedstock, generally through adsorption. The purifiedaqueous metal is then typically precipitated and roasted to producea salable solid product, such as a metal oxide. SPE can offer advantages,particularly in the area of environmental impact, as these systemstypically require limited or no hazardous organic solvents for synthesisor use, unlike traditional liquid–liquid extraction approaches.They are also highly reusable and can function well with low-gradefeedstocks like recycled materials or mining wastes,5 which represent valuable potential new resources for thismetal.6 Numerous studies have examineda wide range of materials for the solid-phase extraction of Sc andother REEs from many different feedstocks.Scandium (Sc) is commonly classified asone of the 17 rare earthelements (REEs), and is one of the metals considered to be a CriticalMaterial (CM) by governments around the world, particularly in NorthAmerica and Europe.7 Silica is particularlypromising for Sc extraction, as silica surfaces are inherently ableto selectively adsorb Sc without prior modification.9 The mechanism of this selective Sc adsorption is not yet fully understood,but it is attributed to the silanol functional groups present on thesurface of amorphous silica, and a specific structure or conformationsof silanol groups may be required for adsorption to occur.8 Optimal Sc extraction occurs at a specific pHrange (∼3–5) because, at lower pH, the silica surfacebecomes positively charged, repulsing the Sc3+ ion, whileat higher pH, Sc precipitation becomes an issue.8 Unmodified silica can adsorb certain other metals, suchas thorium,9 so this effect is not entirelyexclusive to Sc, which must be taken into account for some futurefeedstocks. The monolithic silica structures, in particular, generallyexhibit a high surface area for adsorption, and the silica monolithsspecifically display exceptional adaptability, due to the range ofoptions for functionalization and excellent tunability in terms ofcharacteristics like pore size, pore volume, and framework structure.10 Perhaps most importantly, however, the hierarchically porous monolithshave superior mass transport properties compared to traditional particulateadsorbents.11 This attribute would, in principle,permit large volumes of a given feedstock to be rapidly processedthrough the sorbent, greatly elevating the potential scalability ofthe system and circumventing common issues, such as pressure buildupand column clogging. That significant advantage, combined with theother promising characteristics of hierarchically porous silica monoliths,has generated widespread interest in these sorbents for solid-phaseextraction applications.Monolithic structureswith both macropore and mesopore regimes,known as hierarchically porous monoliths, have long been an especiallytempting target for use as solid-phase extractants for specific metals,such as Sc. The monoliths have so far been comprised of various differentmaterials, including carbon and titania, although silica monolithsare perhaps the most common, as they offer several key advantagesfor SPE systems.12 Significant progress has been made since this originalstudy; however, even 30 years later and despite their advantages,whole silica monoliths have, to our knowledge, not yet been appliedfor selective metal extraction on anything approaching the industrialscale. The primary obstacle to their use appears to be scalability.To establish a point of reference, if one were to assume a relativelylow desired single-batch system output of 100 g of Sc and a relativelyhigh sorbent recovery capacity of 100 mg of Sc/g of monolith, theprocess would require 1 kg of sorbent, which would be approximatelyequivalent to one 5 L cylindrical monolith. This size is far largerthan the largest silica monoliths that have been reported thus far(∼1.1 L),13 and there are key obstaclespreventing scale-up to that level. First, larger monoliths are highlyprone to cracking during drying due to the surface tension exertedby water evaporating in the mesopores.14 This issue can be mitigated by enlarging the mesoporesize, for example, through the inclusion of a hydrothermal posttreatmentstep in the synthesis procedure;14 however, it remains to be seen whether this solution would be sufficientto prevent breakage of even larger monoliths. There are other, moreniche, methods to address the cracking problem, such as drying themonoliths through a coating of paraffin oil,15 but none present a clear solution for very large monoliths withoutsignificant drawbacks. The second issue is that the monolith’sphysical characteristics are quite sensitive to temperature duringgelation and aging.14 For monoliths with a large diameter, it is difficult to maintaina uniform temperature across the width of the mold during synthesis,which could result in material heterogeneity within the monolith,which is not ideal for highly controlled metal extraction operations.13 A theoretical workaround would be to producemany smaller monoliths to use in parallel for Sc extraction. However,this solution has its own drawbacks, namely monolith containment,which is itself a nontrivial obstacle to monolith scale-up. Each individualmonolith, regardless of size, must be strictly contained within sometype of external sleeve 17 to retain the liquid feedstockwithin the system without leaking. If the containment system is notprecisely fitted to the monolith, the recovery process will sufferfrom wall effects and/or channeling and exhibit reduced extractionefficiency. The costs and risks associated with monolith containmentare expected to increase with the use of many small monoliths comparedwith fewer, larger monoliths. Despite decades of research, these scalabilityissues continue to prevent the use of hierarchically porous silicamonoliths for selective metal recovery on the industrial scale.Silica monoliths with the macropore–mesoporestructure werefirst reported in 1991 by Nakanishi and colleagues.Powdered silica monoliths may offer an alternative monolith-basedsorbent, largely retaining the advantages of single-piece monolithswhile avoiding many of their disadvantages. Essentially, a whole monolithis ground and passed through a series of sieves to achieve a powderwith a homogeneous particle size. These powders have the same surfacechemistry and macropore–mesopore framework  as the whole monoliths, so they are readily functionalized,and their structure can still be easily tuned during the normal monolithsynthesis procedure prior to powdering. Furthermore, since the particlesstill retain the hierarchically porous framework of the whole monoliths,they are also expected to exhibit improved mass transport propertiescompared to more traditional particulate silica sorbents. The useof powdered monoliths makes the possibility of monolith cracking anonissue, given that the monoliths will be broken up into small particlesanyway. It also minimizes the impact of any monolith heterogeneitygiven that the fine powder will be mixed and homogenized during grinding.Adsorbent containment is also much more straightforward since thepowder can just be packed into a column of any size and shape likethe traditional SPE sorbents that have been used for decades. Withall of these expected advantages, powdered silica monoliths presenta promising material for the recovery of Sc and other Critical Materials.In this study, the characteristics and selective Sc extractionperformance of powdered silica monoliths were assessed. First, thephysical attributes of the material are reported and its mass transportbehavior is analyzed. Then, the Sc recovery capabilities of the powderare discussed in the context of a series of batch and semicontinuousflow column extraction tests at various environmental conditions.Throughout, these findings are evaluated in the context of the potentialof this SPE sorbent for future large-scale metal recovery operations.N-morpholino)ethanesulfonicacid) buffer and HomoPIPES ) buffer were acquired from Sigma-Aldrich, and ammonium oxalateand reagent-grade nitric acid were acquired from Alfa Aesar. For columnstudies, the powder was packed in empty flash columns from SantaiScience. The dimensions of the metal extraction columns were 2.2 cm× 12.6 cm, and the dimensions of the mass transport test columnswere 4.6 mm × 100 mm.Hierarchically porous silicamonolith powders were obtained fromDPS Inc. in Kyoto, Japan. For the experiment testing column pressureas a function of flow rate, 106–212 μm diameter particleswere used, and for all other experiments, 63–105 μm diameterparticles were used. There are many options for tuning the physicalparameters of these powders, and the characteristics of the selectedpowder are listed in To determine the mass transport characteristics ofthe monolithpowders compared to traditional spherical sorbents, isopropanol waspassed through the fixed-bed columns at 0–6 mm/s. The pumpused was a Shimazu LC-20AD. The pressure was measured at the inletand outlet of the column, and the difference between them was reportedas a pressure drop. The reported values are the median of 120 measurementsmade with the pressure gauge (SMC PSE560-01). Throughout these tests,the temperature was kept at 25 °C and the humidity was kept at50%. Scanning electron microscopy (SEM) images of the DPS and YMCsorbents were taken on a JEOL Ltd. JSM-6510 instrument.Batchexperiments for this study included an adsorption capacitytest at various feedstock concentrations, a kinetics test, and a REEselectivity test, as well as tests at different pH and temperatureconditions. To construct an adsorption isotherm, 10 mg aliquots ofthe DPS powder were exposed to 14.8 mL of a Sc solution from 0 to100 ppm. The feedstock solutions were all buffered to pH 4 using a10 mM HomoPIPES buffer and kept at room temperature. For the pH test,10 mg aliquots of the DPS powder were exposed to 14.8 mL of Sc solution(100 ppm) at room temperature and the pH was varied from 3 to 6. AtpH 3, the feedstock was composed of 1 mM nitric acid and was not buffered.At pH 4 and 5, the feedstock was buffered with 10 mM HomoPIPES buffer,and at pH 6, the feedstock was buffered with 10 mM MES buffer. Forthe temperature test, 10 mg aliquots of the DPS powder were exposedto 14.8 mL of Sc solution (100 ppm) at pH 4 (10 mM HomoPIPES buffer)and placed in an oven or freezer at 5, 25, 40, 50, or 60 °C.Each of these assays was left for 1 week before being sampled foranalysis. For the kinetics assay, 10 mg aliquots of the DPS powderwere exposed to 14.8 mL of a Sc solution (20 ppm) at pH 4 (10 mM HomoPIPESbuffer). The samples were shaken at room temperature for a set periodof time from 10 to 10080 min (7 days) before the experiment was concluded.For the REE selectivity test, 10 mg aliquots of the DPS powder wereexposed for 1 week to 14.8 mL of a mixed REE solution containing 5ppm of each REE (except radioactive Pm) at pH 4 and room temperature.In every case, samples of the liquid phase were diluted in 3% nitricacid, and their Sc or REE contents were analyzed on an Agilent 7800ICP-MS. All experiments were conducted in triplicate.3+ solution  or ∼0.6 L of bromide (Br–) tracer solution were passed through the column andcollected in 7 mL increments. In both experiments, the fluid flowrate through the column was 0.5 mL/min for every step. Prior to analysis,every sixth sample (for the Sc column) or every second sample (forthe Br column) was diluted in 3% nitric acid. The Sc and Br contentsof the samples were analyzed on an Agilent 7800 ICP-MS.For theSc and Br column tests, 8.0 g of monolith powder (dry weight)was suspended in isopropanol (Sigma-Aldrich) and carefully packedinto an empty flash column. Following packing, the sorbent beds werefirst conditioned with ∼2 L of 0.1 mM nitric acid (pH 4). Then,∼4.5 L of Sc18 The Sc and Br breakthrough behaviors were fittedusing a standard and/or fractal-like Bohart–Adams model.19 These models are discussed in more detail in Theresults for the adsorption isotherm, the adsorption kinetics,and the breakthrough columns were fitted with various models usingnonlinear least-squares curve fitting in Matlab. The Sc adsorptionisotherm data were fitted using the standard Langmuir and Freundlichisotherm models. The Sc adsorption kinetics data were fitted withpseudo-first-order, pseudo-second-order, and Weber and Morris models.13 andthis behavior is expected to be retained in the monolith powders.To test the mass transport characteristics of the powders, the effectof increasing flow rate on internal pressure was observed for twocolumns, one packed with powdered monolith material (DPS), and theother packed with representative mesoporous silica spheres (YMC).The particles in both columns had a similar size (DPS = 106–212μm and YMC = 150 μm) and mesopore diameter (DPS = 8.19nm and YMC = 12 nm). The DPS powder exhibited a typical macroporousstructure (1 μm diameter macropores), while the YMC spheresdid not. At flow rates between 0 and 6 mm/s, the column pressure washigher for the silica spheres than for the monolith powders, and thepressure difference between the two columns became more exaggeratedas the flow rate increased could be modeled using the typical Langmuir and Freundlich isotherms,with the Langmuir model providing a better fit  of each REE were exposedto the DPS powder at pH 4, only Sc was adsorbed , so temperature will typically not be a factorthat presents an issue. The performance of the DPS powder in morerealistic, semicontinuous flow systems will be assessed in the followingsections; however, the fundamental sorption characteristics are promisingfor practical Sc extraction.In the context of actual industrial-scale Sc extraction,the adsorptionbehavior of DPS powder presents certain advantages. That strong selectivityfor Sc, which is characteristic of unmodified silica, is the firstclear benefit. The DPS powder did not adsorb any of the other REEs,and based on the adsorption behavior of other unmodified silica materials,it is not expected to adsorb significant amounts of most abundantnon-REE metals.Larger-scale SPE operations frequently involve continuous or semicontinuousflow of the feedstock through a fixed bed column in order to maximizethe volume of feedstock exposed to a given mass of adsorbent and therate of that exposure, so the DPS powder was tested in a fixed-bedcolumn setup. Eight grams of powder was packed into a 2.2 cm ×12.6 cm column, and ∼4.5 L of 50 ppm of Sc solution (pH 4)was passed through that column at a flow rate of 0.5 mL/min. Scandiumbreakthrough was observed after ∼17 bed volumes ∼680mL; 4. Thein18 External diffusion limits are unlikely to bethe main cause of the observed behavior, given that the majority ofthe particle surface area and therefore functional groups for adsorptionare expected to be contained within the mesopore structure. Internaldiffusion limitations, however, are known to produce similarly asymmetricalbreakthrough patterns in other systems.25 To investigate this possibility, the breakthrough data was modeledusing a fractal-like Bohart–Adams model .Even the fractal-like model that is well-suited to account for intraparticlediffusion effects could not provide a precise fit to this highly asymmetricalbreakthrough pattern (–), a tracerion that is not adsorbed by the silica, exhibited close to ideal breakthroughbehavior , suggesting that diffusion of the Br− ions throughthe available pore volumes did not strongly limit the transport ofBr through the system. Taken together, these modeling results appearto show that internal diffusion may not be the primary limiting factorin this system.Given the extent to which the breakthrough behaviorexhibited bythis system departs from the expected, theoretical behavior, it isclear that Sc recovery was limited by one or more kinetic process.The rate of Sc adsorption onto the silica surfaces could be limitedby (1) external diffusion, which in this system would include Sc iondiffusion in the spaces between particles and diffusion within theparticle macropores (1 μm in diameter), (2) internal diffusionoccurring within the particle mesopores (<10 nm in diameter), and(3) Sc adsorption reaction kinetics on the silanol functional groups.ms model 1, which  pattern 4. Intere18 The pseudo-second-order model provided a betterfit (R2 of 0.9653 compared to 0.8195),which suggests that the adsorption reaction kinetics are likely thelimiting factor rather than intraparticle diffusion of Sc adsorption onto the silica surfaces are complex, potentiallyinvolving van der Waals forces, electrostatic interactions betweenSc3+ cations and the negatively charged silica, and theformation of covalent Si–O–Sc bonds.27 Whilethe former two processes occur rapidly, the Si–O–Sccovalent bonds form more slowly,27 which could explainthe observed behavior of the DPS powder in column and in batch. Overall,Sc extraction from the fixed-bed column is limited to an extent byone or more rate-limiting processes within the system, and the slowSc adsorption reaction kinetics appears to be primarily responsiblefor this limitation.To further explore the breakthrough behaviorexhibited by the DPSsorbent, the Sc adsorption kinetics of the material were analyzedin batch. Aliquots of the DPS powder (10 mg) were gently shaken in∼15 mL of 20 ppm of Sc solution (pH 4) for a set period oftime from 10 min to 7 days 5a,b. Theiffusion 5a.18,26 the data 5b, and tThe DPS powder did function as a fixed-bed sorbentfor Sc recoveryunder semicontinuous flow, though the breakthrough behavior may presentcertain difficulties for real-world SPE operations. The equilibriumadsorption capacity at idealized conditions is quite high (∼142.7mg/g at pH 6), and the selectivity against other REEs is promising.However, if during extraction, the operation must be halted at thebreakthrough point, then a large fraction (>68%) of the total adsorptioncapacity is not used, reducing the practical capacity of the column.If the operation is halted at a later point to take advantage of thisadditional capacity, significant Sc will have escaped through thecolumn as waste, reducing the extraction efficiency of the system.That being said, the adsorption capacity up to breakthrough (∼3.7mg/g at pH 4) may still be adequate for extraction operations in somescenarios, especially given the other advantages of this simple system.Typically, highly engineered sorbents such as the DPS powder are functionalizedwith ligands designed to target specific metals. Eliminating thismodification step decreases costs, minimizes the use of potentiallyhazardous solvents during production, and likely increases materialreusability since there is no ligand that can be leached out of thesorbent over time. These promising characteristics may in some casesoutweigh the reduced practical adsorption capacity due to the slowreaction kinetics.Looking into the future, the basic physicochemicalproperties ofthe DPS powder provide a solid foundation to build upon for a highlyadaptable CM sorbent. In the case of Sc, the DPS powder is expectedto function well with feedstocks such as red mud, bauxite residuefrom aluminum mining that is enriched in Sc, and manufacturing wastefrom the production of Sc-bearing alloys and fuel cells, just to nametwo examples. Additional forthcoming testing will assess the extractionperformance at more realistic conditions that more accurately reflectthe conditions found in leachates from these real-world feedstocks.Further testing to elucidate the nature of the interactions betweensilanol groups and Sc would also be useful in optimizing the Sc recoveryprocess. To target other desirable metals beyond Sc, the DPS powdercan, of course, be functionalized with a wide range of targeted ligands,which could both increase adsorption capacity for a given metal andcompletely circumvent the kinetics issue by changing the species relevantfor the adsorption reactions, though at an increased cost. Other REEs,noble metals, and various other Critical Materials could potentiallybe extracted and purified with this approach. With or without functionalization,the DPS powder does show great promise for future CM extraction applications.1.Mass transfer of fluid feedstocks througha fixed-bed column could occur rapidly without producing significantpressure increases, unlike many powdered SPE sorbents, due to thehierarchical pore structure of the DPS powder.2.The material, even without surfacemodification, achieved a high Sc adsorption capacity, ∼24.9and ∼142.7 mg/g at pH 4 and 6, respectively. Temperature increasesabove 25 °C had minimal effect on Sc adsorption.3.The unmodified silica surfaces hada high selectivity for Sc adsorption compared to the other REEs.4.The DPS powder did functionas an extractantfor Sc in a semicontinuous flow column system, though optimal performancewas reduced by the slow Sc adsorption reaction kinetics, with a potentialminor contribution from intraparticle diffusion within the mesopores.5.The high Sc adsorptioncapacity andpromising selectivity, especially given the lack of surface modification,combined with the excellent mass transfer properties demonstrate thatthe DPS powder has great potential for the SPE of Critical Materials.The performance of DPS powder as an extractantfor batch and semicontinuousflow recovery of scandium was analyzed at conditions relevant forfuture real-world metal recovery operations. The sorbent exhibitedthe following characteristics:


Nanopore sequencing is becoming increasingly commonplace in clinical settings, particularly for diagnostic assessments and outbreak investigations, due to its portability, low cost, and ability to operate in near real-time. Although high sequencing error rates initially hampered the wider implementation of this technology, improvements have been made continually with each iteration of the sequencing hardware and base-calling software. Here, we assess the feasibility of using nanopore sequencing to determine the complete genomes of human cytomegalovirus (HCMV) in high-viral-load clinical samples without viral DNA enrichment, PCR amplification, or prior knowledge of the sequences. We utilised a hybrid bioinformatic approach that involved assembling the reads de novo, improving the consensus sequence by aligning reads to the best-matching genome from a collated set of published sequences, and polishing the improved consensus sequence. The final genomes from a urine sample and a lung sample, the former with an HCMV to human DNA load approximately 50 times greater than the latter, achieved 99.97 and 99.93% identity, respectively, to the benchmark genomes obtained independently by Illumina sequencing. Thus, we demonstrated that nanopore sequencing is capable of determining HCMV genomes directly from high-viral-load clinical samples with a high accuracy. Cytomegalovirus humanbeta5) [Seropositivity to human cytomegalovirus   ranges fanbeta5) ,3. Infecanbeta5) , but in anbeta5) . Antiviranbeta5) . The ideab–UL–b′a′c′–US–ca, in which unique regions UL (193 kbp) and US (35 kbp) are flanked by inverted repeats ab/b′a′ (1 kbp) and a′c′/ca (3 kbp). Early work involving restriction fragment length polymorphism analysis and Sanger sequencing revealed extensive genome variation among HCMV strains, including the existence of multiple discrete genotypes of a subset of hypervariable genes [HCMV has a linear, double-stranded DNA genome of approximately 236 kbp ,9. The ole genes ,11,12, tle genes , and thele genes ,15,16. Tle genes , thus engions UL 3 kbp andle genes ,21,22 anle genes ). Howevehttps://nanoporetech.com/accuracy, accessed on 30 March 2023).In recent years, there has been an acceleration in the development of high-throughput, long-read sequencing. The Oxford Nanopore Technologies (ONT) platform monitors disruptions in ionic current as DNA molecules pass individually through proteinaceous membrane pores in a highly parallel fashion. Read lengths on this platform can exceed 2 Mb ,26, thusNanopore sequencing has been applied to HCMV in several studies. In one study, nanopore data were generated from a laboratory strain (TB40/E) grown in cell culture and assembled de novo. The analysis was complicated by the existence of many variants in the viral stock used, but the accuracy of the consensus sequence (which was not published) in comparison with previously published sequences for this strain was reportedly high . In a seHere, we assessed the utility of direct nanopore sequencing  for determining complete HCMV genomes from high-viral-load clinical samples. Our finding that the nanopore sequences were highly accurate, although inferior in quality to the sequences determined on the Illumina platform, encourages a future investigation into the application of nanopore sequencing to research HCMV, including that in the clinical setting.VIC-CACCCTGCTTTCCGAC-MGB ) and the human FOXP2 gene . The amplification conditions were as follows: an initial step of 50 °C for 2 min, denaturation at 95 °C for 10 min, 40 cycles of 95 °C for 30 s, and annealing and elongation at 60 °C for 1 min. One sample had been extracted from urine and had viral and human DNA loads of 258,199 IU/µL and 323 genome copies/µL, respectively; 1 IU is approximately equivalent to 1 genome copy. The other sample had been extracted from lung tissue and had viral and human DNA loads of 172,259 IU/µL and 9636 genome copies/µL, respectively. The urine sample was therefore approximately 50 times more enriched in viral DNA than the lung sample.Two fully anonymised surplus diagnostic extracts from samples that had been prepared on the NucliSENS easyMAG  or Abbott m2000  platforms were provided by the West of Scotland Specialist Virology Centre. The samples had high ratios of HCMV to human DNA, as determined using qPCR assays for the HCMV UL97 gene  was sheared to an average size of 500 bp using an LE220 sonicator . HCMV DNA enrichment using Agilent SureSelectXT  custom biotinylated RNA baits and a KAPA Biosystems sequencing library preparation kit were carried out as described previously ,21,22 , and FAST5-formatted files were base-called using Guppy v4.0  in high accuracy mode with a minimum quality score of 7. The reads were assessed using MinIONQC v1.3.5 [https://github.com/rrwick/Porechop, accessed on 16 November 2021) (B3). The 10,616,519 and 8,731,192 reads obtained from the urine and lung samples, respectively, were then mapped to a collated set of 265 HCMV genomes representing all HCMV strains that had been sequenced at the time of analysis  was processed into a sequencing library using an SQK-LSK109 ligation sequencing kit . This involved treatment with an NEBNext FFPE DNA repair mix and an NEBNext Ultra II end repair/dA-tailing module , purification using Agencourt AMPure XP beads , ligation of adapters using an NEBNext Quick ligation module (New England Biolabs), enrichment of large (>3 kb) fragments using long fragment buffer, and final purification in elution buffer. The libraries were loaded onto primed R9.4.1 flow cells (ONT) at concentrations of 17.7 and 37.4 ng/µL for the urine and lung samples, respectively, and sequencing was carried out for 72 h on a GridION instrument . The reads were acquired using MinKNOW v4.3.11  (B5). Both programs enumerate reads containing conserved genotype-specific kmers for each hypervariable gene. These kmers were initially defined at one per genotype for 12 genes [The genotypes of 13 hypervariable HCMV genes were determined from the input Illumina data using the genotyping module in the HCMV genome assembly program GRACy v0.4.4  (A4) and12 genes  and were12 genes .https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/, accessed on 10 November 2020) under the parameters --illumina --paired, aligning the filtered reads to the sequences using Bowtie 2 v2.3.1 [The Illumina input data were assembled using GRACy (A5). The draft genomes were assessed by trimming and quality-filtering the reads using Trim Galore v0.4.0  and inverted repeats (ab/b’a’ and a’c’/ac) appropriately . This approach generated an incomplete consensus genome.In the first approach (B6–B8), the nanopore input data for each sample were assembled into contigs using Canu v.1.9  under thhttps://github.com/rjorton/VSensus, accessed on 10 November 2020) with the minimum coverage and minimum base quality parameters set at 20 and 0, respectively (B10). Coverage statistics were generated using weeSAM v1.5 .In the second approach (B9–B10), the best-matching genome (from B7) was used as the reference for read mapping using Minimap2 (B9). BAM and mpileup files were generated from the alignment using Samtools v1.9 , and thehttps://github.com/nanoporetech/medaka, accessed on 10 November 2020), which has been trained to correct for common errors in ONT data (B12). These steps resulted in properly configured HCMV genomes that did not depend at any stage on the corresponding Illumina data (B13).The genomes derived by the two approaches were aligned using MAFFT v7.310, and any gaps in the former were filled with data from the latter (B11). Finally, this draft genome was processed using Medaka v1.0.3  [The coding sequences of genes UL97 and UL54 were analysed using a web-based mutation resistance analyser  .Initial screening of the Illumina data to determine the number of HCMV strains present in the original samples was carried out using GRACy, and that of the nanopore data involved using a program devised for our study, Minion_Genotyper. Neither tool requires a genome but rather analyses the reads using a kmer-based approach, and each identified the same single genotype for each of the 13 hypervariable genes analysed . This anDetermination of the final Illumina genomes was accomplished using GRACy followed by manual amendments and ended with 86% of reads aligning to the urine genome  at an average coverage depth of 2554 reads/nt and 66% of reads aligning to the lung genome  at an average coverage depth of 2368 reads/nt. The final nanopore genomes were determined by a more complex series of steps. De novo read assembly resulted in a single contig  corresponding to the entire HCMV genome and a partial repeat for the urine sample but in 20 contigs for the lung sample, with the four largest originating from the HCMV genome. The three largest of these contigs  represented the whole genome but for one gap of 1849 nt. Subsequent steps led to an intact nanopore genome for both samples, each with complete read coverage and determined independently of the corresponding Illumina genome. The analysis ended with 37,241 reads aligning to the urine genome at an average coverage depth of 203 reads/nt, and with 11,764 reads aligning to the lung genome at an average coverage depth of 26 reads/nt.Comparisons between the Illumina and nanopore genomes revealed differences at 62 nt (0.03%) and 164 nt (0.07%) for the urine and lung samples, respectively . The greIn clinical practice, the sequences of genes UL54 and UL97 are typically examined for resistance mutations when patients fail to respond to antiviral therapy. As a test of the utility of nanopore genomes for such investigations, the coding sequences of these genes were submitted to MRA, which hosts a curated database of published resistance mutations from a range of viruses including HCMV . The UL9To our knowledge, our study is the first to use nanopore technology to determine complete HCMV genomes from clinical samples without involving cell culture, bait-based target enrichment, PCR amplification, incorporation of data for the same genome acquired on other platforms, or amendments based on prior knowledge of the target sequence. We sequenced two high-viral-load samples from urine and lung on the Illumina platform with bait-based target enrichment and independently on the ONT platform without enrichment. We used separate genotyping of the Illumina and nanopore data to demonstrate the presence of a single HCMV strain in each sample. The nanopore genomes were determined using a combination of two approaches, one involving de novo read assembly and the other involving read alignment to the best-matching HCMV genome, with the outputs being incorporated and polished to produce the final sequences. As anticipated from the approximately 50-fold greater ratio of viral to nonviral DNA in the urine sample, the urine genome was assembled more readily and at a greater read depth than the lung genome.https://nanoporetech.com/accuracy, accessed on 30 March 2023), in our study they did not attain the level of accuracy achieved using the Illumina platform. For a completely accurate characterisation of HCMV strains on the ONT platform, supplementary data from another platform would be required. A further implication is that it would be inadvisable to rely on nanopore sequencing alone to determine the sequence of a novel large DNA virus for which no prior sequence information is available.Sequence comparisons suggested that the differences between the Illumina and nanopore genomes were largely or entirely due to errors in the latter, as would be anticipated from the characteristics of the platforms used. Nonetheless, the nanopore genomes were highly accurate , with the errors distributed thinly  and located mostly in polynucleotide tracts in noncoding regions. Thus, although improvements in the ONT hardware and the software for analysing the data have steadily boosted the base accuracy to >99% (The main limitation of our study was that the two clinical samples were chosen for sequencing because they contained a high number of HCMV genomes and a relatively high ratio of HCMV to human DNA, thus meeting the requirements of nanopore sequencing. In a clinical setting, the mainstay sample type for post-transplant monitoring of HCMV reactivation or re-infection is blood, but HCMV genomes in blood are highly fragmented, thus obviating one of the chief advantages of nanopore sequencing; blood also contains high levels of host DNA, which can lead to low efficiency enrichment for HCMV genomes ,48. In cDespite this limitation, nanopore technology potentially offers much to HCMV testing in clinical practice in the context of transplantation, particularly as the focus is usually on one or two genes, at which scale nanopore sequencing is almost as accurate as Illumina sequencing. In the UK, HCMV antiviral resistance testing is referred to specialist reference laboratories in which analysis is based on Sanger sequencing of the PCR-amplified UL97 and UL54 genes applied on a timescale that is not conducive to prompt clinical action. A nanopore protocol similar to our approach could provide a means of resistance testing on a shorter timescale. Recently, an Illumina sequencing pipeline was established that enables whole genome HCMV analysis for resistance mutations, which could facilitate resistance testing against novel drug targets . NanoporIn conclusion, we were able to determine two HCMV genomes by direct nanopore sequencing from high-viral-load clinical samples at an accuracy that was close enough to that of Illumina sequencing to encourage further investigation. We await with interest additional improvements in the hardware and software used in this technology, as well as appropriate target enrichment techniques.


The gut microbiota has been suggested to be involved in the pathogenesis of canine atopic dermatitis (cAD). However, the gut microbiota has not been well characterized in dogs with atopic dermatitis (AD). In addition, the efficacy of fecal microbiota transplantation (FMT) in dogs with AD remains unclear. This research, therefore, aimed to characterize the gut microbiota of dogs with AD and conduct pilot evaluation of the efficacy of a single oral FMT on clinical signs and the gut microbiota of dogs with AD. For these purposes, we used 12 dogs with AD and 20 healthy dogs. The 16S rRNA analysis of the fecal microbiota revealed significant differences between 12 dogs with AD and 20 healthy dogs. Next, a single oral FMT was performed in 12 dogs with AD as a single-arm, open-label clinical trial for 56 days. A single oral FMT significantly decreased Canine Atopic Dermatitis Extent and Severity Index (CADESI)-04 scores from day 0  to day 56 (8) and Pruritus Visual Analog Scale (PVAS) scores from days 0  to day 56 (1). Furthermore, a single oral FMT changed the composition of the fecal microbiota of dogs with AD at the phylum and genus levels. The number of common amplicon sequence variants in the fecal microbiota between donor dogs and dogs with AD was positively correlated with CADESI-04 and PVAS reduction ratios 56 days after FMT. Our findings suggest that the gut microbiota plays a pivotal role in the pathogenesis of cAD, and that oral FMT could be a new therapeutic approach targeting the gut microbiota in cAD. The pathogenesis of cAD involves multiple factors, including genetic predisposition, aberrant immune responses to environmental allergens, and skin barrier dysfunction3. cAD is considered a natural homolog of human atopic dermatitis (AD) because of the similarities in clinical features and the pathogenesis4. A variety of treatment options, such as glucocorticoids, cyclosporin, Janus kinase inhibitor, anti-dog interleukin-31 antibody, and allergen-specific immunotherapy, are available for dogs with AD5. However, it is often difficult to control clinical signs of cAD. To accomplish effective and long-term management of cAD, alternative therapeutic targets based on the pathogenesis need to be explored.Canine atopic dermatitis (cAD) is an allergic inflammatory and pruritic skin disease8. Intestinal dysbiosis is a clear shift of the gut microbiota from the normal outside of a healthy population, and has been reported in various gastrointestinal (GI) and extra-GI diseases, including AD, in humans8. Next-generation sequencing (NGS) analysis revealed intestinal dysbiosis in dogs with several GI and extra-GI diseases, such as acute diarrhea9, inflammatory bowel disease (IBD)10, heart diseases12, and obesity13. A recent study reported that treatment with a nutraceutical improved the index of intestinal dysbiosis, as assessed by seven bacterial taxa in dogs with AD14. However, the gut microbiota has not been characterized in detail by NGS in dogs with AD and has not been compared with healthy dogs. Thus, it remains unclear whether the microbial shift exists in the GI tract of dogs with AD. In addition, the role of the gut microbiota in the development of cAD and as a therapeutic target remains poorly understood.The gut microbiota plays an essential role in maintaining homeostasis in humans and animals, including immunomodulation, physical barrier against intestinal pathogens, provision of nutrients, and regulation of host metabolism16. Although the detailed mechanisms of FMT have not been fully elucidated, it has been shown that it corrects intestinal dysbiosis by increasing the number and variety of beneficial bacteria and restoring the diversity and function of the gut microbiota17. In human medicine, FMT is widely used as an effective treatment for recurrent or refractory Clostridium difficile infection (CDI)18. Meta-analyses have revealed the clinical efficacy of FMT for human GI and extra-GI diseases other than CDI, including IBD, hepatic disorders, metabolic syndrome, and antibiotic-resistant organisms19. Recently, the efficacy and safety of FMT have also been reported in a small population of human AD21. In dogs, FMT was shown to be effective for several GI diseases, including parvovirus infection22, acute diarrhea23, C. difficile-associated diarrhea24, IBD28, and chronic enteropathy29. However, the efficacy of FMT for extra-GI diseases, including cAD, has not been evaluated in dogs. Considering the potential effect of FMT in human AD, FMT could be a promising therapy for cAD.Fecal microbiota transplantation (FMT) is a treatment approach in which feces from a healthy individual is transplanted into the GI tract of a diseased personTherefore, the purposes of this study were to characterize the gut microbiota of 12 dogs with AD using NGS and to perform pilot evaluation of the efficacy of a single oral FMT on clinical signs and the gut microbiota of 12 dogs with AD.Fusobacterium, Megamonas, Prevotella, Roseburia, Sutterella, and Phascolarctobacterium and higher numbers of Escherichia/Shigella, Ruminococcus gnavus group, and Klebsiella -04 scores from day 0  to day 28  and day 56   excreted mild soft feces for 1–3 days after oral FMT.Escherichia/Shigella, Clostridioides, and Lachnoclostridium compared with that after oral FMT   (Table ρ <  − 0.4)  Fig. –S4. At tThe present study demonstrated the significant differences in the fecal microbiota between dogs with AD and healthy dogs. Furthermore, a single oral FMT significantly decreased skin lesions and pruritus scores, and changed the fecal microbiota in dogs with AD. These findings suggest that the gut microbiota plays a pivotal role in the pathogenesis of cAD, and that oral FMT could be a new therapeutic approach targeting the gut microbiota in cAD.30. In contrast, Firmicutes, Bacteroidota, and Proteobacteria occupied the fecal microbiota of dogs with AD. We also showed that Fusobacteriota and Actinobacteriota increased in the fecal microbiota of dogs with AD after oral FMT. Therefore, the lower abundance of Fusobacteriota and Actinobacteriota may be characteristic of the fecal microbiota of cAD at the phylum level. It was also seen that as compared to HC dogs, the fecal microbiota of dogs with AD had lower occupancy of Fusobacterium, Megamonas, Prevotella, Roseburia, Sutterella, and Phascolarctobacterium, and higher numbers of Escherichia/Shigella, Ruminococcus gnavus group, and Klebsiella. These may be a feature of the fecal microbiota of cAD at the genus level. However, similar microbial shifts were reported in dogs with GI diseases or after antibiotic use31. Thus, to characterize the cAD-specific gut microbiota, further analysis would be required using a large population of healthy dogs, AD dogs, dogs with GI diseases, and dogs on broad-spectrum antibiotics.In this study, we found Firmicutes, Fusobacteriota, Bacteroidota, Proteobacteria, and Actinobacteriota to be the major components of the fecal microbiota of healthy dogs, similar to a previous study17. In this study, a single oral FMT significantly decreased CADESI-04 and PVAS scores without changing the medication scores in dogs with AD. It also significantly increased bacterial diversity, as assessed by the number of ASVs (richness). In particular, the number of the common ASVs between donor dogs and dogs with AD was noted to have significantly increased after a single oral FMT. Furthermore, positive correlations between the number of the common ASVs and CADESI-04 and PVAS reduction ratios were detected in dogs with AD. These results suggest that oral FMT may have introduced a large number of donor-derived bacteria into the GI tract of dogs with AD, thereby decreasing clinical scores associated with cAD. Alternatively, oral FMT may have stimulated the gut microbiota of dogs with AD to increase the microbial diversity. We also demonstrated that among the common ASVs increased after oral FMT, 19 bacterial genera were significantly correlated with reduction in clinical scores in dogs with AD. In particular, Fusobacterium and Sutterella were positively correlated with CADESI-04 and PVAS reduction ratios and were negatively correlated with CADESI-04 and PVAS scores. Thus, among the 19 bacterial genera, Fusobacterium and Sutterella might be more crucial toward the reduction in clinical scores associated with cAD. To further determine the importance of these bacterial genera as contributors to the efficacy of oral FMT in cAD, detailed investigations are required.The mechanisms underlying FMT are not fully understood. However, it may be associated with increased microbial diversity, restoration of normal microbiota, and enhanced numbers of beneficial bacteria33. However, there is no established method or route of fecal administration in dogs. Previous studies conducted FMT in dogs via rectal enema27, endoscopic introduction into the small or large intestine28, oral administration of fecal solution24 or frozen feces25, and oral capsules of concentrated lyophilized feces29. Based on our previous report24, in this study, we selected oral administration of fecal solution for dogs with AD. As a result, a single oral FMT significantly decreased clinical scores and changed the gut microbiota in dogs with AD. Our findings thus suggest that even a single oral FMT may be an effective method for treating cAD for at least 56 days. To determine the long-term effects of a single oral FMT on clinical sings and the gut microbiota of dogs with AD, a longer follow-up period for more than 56 days would be needed.Guidelines for FMT have been proposed in dogsEscherichia coli occurred in two human patients after FMT, and one died due to this severe infection34. Thus, safety is an important issue in FMT. In this study, although four of the 12 dogs with AD briefly excreted mild soft feces after oral FMT, no severe adverse events were observed. In addition, we previously reported that oral FMT did not induce any adverse events in a dog with C. difficile-associated diarrhea24. These results suggest that oral FMT might be a safe and well-tolerated treatment for dogs. However, this study was designed as an uncontrolled study. Therefore, to evaluate the safety of oral FMT for cAD, the frequency and severity of adverse events associated with oral FMT should be compared between the study and control groups in future studies.Although FMT has been considered a safe treatment, a recent study reported bacteremia due to drug-resistant 35 and had already received other therapies, excluding antibiotics, against cAD. Therefore, we could not assess the clinical efficacy of a single oral FMT for severe cAD or untreated dogs with AD. Oral administration of fecal solution is a simple procedure that does not require anesthesia, sedation, endoscopic equipment, or specialized capsule, and can be repeated easily. Thus, repeated oral FMT may induce favorable outcomes in dogs with severe AD or untreated dogs with AD.The dogs with AD in this study were categorized as cAD in remission, mild, or moderate cAD according to the CADESI-04In this study, antibiotics had not been administered to the dogs with AD for 2 months prior to the study enrollment and during the study period. However, the histories of antibiotic use in the dogs with AD before more than 2 months prior to the study enrollment could not be traced. Thus, we can’t rule out the possibility that previous antibiotic use might have affected the gut microbiota in the dogs with AD.In conclusion, the present study revealed that a single oral FMT significantly decreased skin lesions and pruritus scores and changed the gut microbiota in dogs with AD. Since this study was designed as a pilot trial with a short observation period (56 days), further studies are needed to clarify the long-term effect of a single or repeated oral FMT on cAD using a large population of dogs with mild to severe AD and appropriate controls. Nevertheless, this study provides evidence for a crucial role of the gut microbiota in the pathogenesis and a therapeutic target of cAD.36 were enrolled in this study . The average total fecal solution administered was 26 mL/dog (range 15–50 mL/dog). Skin lesions, pruritus, medication scores, and fecal microbiota were evaluated in 12 dogs with AD at study inclusion and after oral FMT.A single oral FMT was designed as a single-arm, open-label clinical trial. The study period lasted 56 days. Oral FMT was performed according to a procedure reported previously35 by the same veterinarian at study inclusion (day 0) and 28 and 56 days after oral FMT. The CADESI-04 assessed three lesions  with four severity scores  at 20 body sites, giving a maximum score of 20 × 3 × 3 = 180. According to the CADESI-4, disease severity of cAD is categorized as follows: < 10, normal dogs or cAD in remission; 10–34, mild cAD; 35–59, moderate cAD; ≥ 60, severe cAD35.Skin lesions of dogs with AD were scored using the CADESI-0437 by the owners of dogs with AD at study inclusion (day 0) and 14, 28, and 56 days after oral FMT.The severity of pruritus was scored from 0 (no pruritus) to 10 (extremely severe pruritus) using the PVAS38, with slight adaptation for this study by inclusion of an additional medication [a recombinant Dermatophagoides farinae 2 immunotherapy shot ] (Table The medications administered to dogs with AD were scored as reported previously)] Table . Medicatg at 4 °C for 10 min. The pellets were washed with 35 mL of PBS and resuspended in PBS at a final concentration of 0.5 g/mL of the initial fecal weight. A 400 μL volume of suspension was subjected to genomic DNA extraction using a Chemagic DNA Stool 200 Kit . The V3–V4 regions of the 16S rRNA gene were amplified by PCR and subjected to paired-end sequencing using Illumina MiSeq 40. The sequence data were processed using QIIME 2 version 2020.641. The DADA2 software package version 2020.2.0 incorporated into QIIME 2 was used to correct the amplicon sequence errors and yield high quality reads42. To rarefy the data, we used 5000 high quality reads from each sample. Unique ASVs of the 16S rRNA gene and its abundances were summarized in an ASV abundance table, and the alpha and beta diversities were calculated using QIIME2. Microbial taxonomy was assigned using a Naïve Bayes classifier trained on the SILVA 138 database44.Fecal samples (0.1–0.5 g) were collected from 20 HC dogs and 12 dogs with AD before (day 0) and 28 and 56 days after oral FMT. Fecal samples were suspended in 50 mL of phosphate buffered saline (PBS), filtered using a pluriStrainer 100 μm , and centrifuged at 9000×t test. Other categorical variables were compared using Fisher’s exact test. Clinical scores such as CADESI-04, PVAS, and medication scores were compared using the Friedman test, followed by the Scheffe’s multiple comparison test. The alpha diversity of the fecal microbiota was analyzed using the Mann–Whitney U test or the unpaired t test, depending the normality of the data; the beta diversity was compared using the Permutational Multivariate Analysis of Variance (PERMANOVA). Other fecal microbiota data were compared using the one-way analysis of variance (ANOVA), followed by the Scheffe’s multiple comparison test, or the Kruskal–Wallis test, followed by the Steel–Dwass test. The correlations between two variables were evaluated using the Spearman’s rank correlation coefficient or the Pearson's correlation coefficient, depending on the normality of the variables. Statistical analyses were performed using BellCurve for Excel  software and QIIME 2 version 2020.6. P < 0.05 was considered statistically significant. To determine different taxa between two groups, the linear discriminate analysis (LDA) effect size (LEfSe) on the Galaxy browser was used45. In LEfSe, cut-off value was set at |LDA score|> 3 and P < 0.05.The normality of the data was analyzed using the Shapiro–Wilk test. The age difference between dogs with AD and HC dogs was compared using the unpaired The collection and use of feces from healthy donor dogs were approved by the Institutional Animal Care and Use Committee of Tokyo University of Agriculture and Technology (No. 30-131). Oral FMT was approved by the Research Ethics Committee of Tokyo University of Agriculture and Technology (No. 0016013). Written informed consent was obtained from the owners of the dogs with AD and HC dogs. All experiments were conducted in compliance with the ARRIVE guidelines and in accordance with the relevant guidelines and regulations of Tokyo University of Agriculture and Technology.Supplementary Information.