E. coli, S. enterica, and B. cinerea) microorganisms was evaluated during 10 days of storage at 6 ± 1 °C. Respiration rate and mass loss were also determined. Ozone was applied prior to storage at 0.4, 0.5, 0.6, and 0.7 ppm, for 3 min. The best results were observed with the higher ozone dose, with initial maximum reductions of ~0.5, 1.09, and 0.46 log units for E. coli, S. enterica, and B. cinerea, respectively. For the native microflora, maximum reductions of 1.85, 1.89, and 2.24 log units were achieved on day 1 for the mesophiles, psychrotrophs, and yeasts and molds, respectively, and this effect was maintained throughout storage. In addition, the lower respiration rate and mass loss of the blackberries ozonated at 0.7 ppm indicate that this treatment did not induce physiological damage to the fruit. Gaseous O3 could be effective in maintaining the postharvest quality of blackberries throughout refrigerated storage but higher doses could be advisable to enhance its antimicrobial activity.Andean blackberries are highly perishable due to their susceptibility to water loss, softening, mechanical injuries, and postharvest diseases. In this study, the antimicrobial efficacy of gaseous ozone against spoilage  and pathogenic ( Rubus glaucus Benth) are mostly cultivated in temperate and cold climates in South America and are usually consumed processed as pulp, jams, juices, and desserts [Andean blackberries , chemical , and biological treatments have been studied for decay control in fresh fruit and vegetables. 3, which has a high oxidant capacity and can be used for the inactivation of a wide range of microorganisms and for the degradation of chemical contaminants and off-odors in storage rooms [3 as a sanitizer: it is produced on site and does not require storage, and its precursors are abundant and economically advantageous. In addition, it decomposes almost immediately to oxygen, presenting no safety concerns regarding the consumption of chemical residues [One of these alternatives could be the use of gaseous Oge rooms . There aresidues . In 2001residues .E. coli and Salmonella spp., has been studied in different berries including, among others, strawberries, table grapes, blueberries, blackberries, and raspberries.Due to the lack of a protective skin and berries’ surface roughness and sensitivity, gaseous treatments are preferred for these fruit . MoreoveBotrytis cinerea conidia, and the incidence and severity of gray mold during cold storage of table grapes [Gaseous ozone contributed to reduce sour rot , the gere grapes . Similare grapes ,20, raspe grapes ,22, mulbe grapes , bluebere grapes , and blae grapes . On the e grapes , mulberre grapes , and blue grapes ,29.Even though the highest ozone doses often resulted in the highest antimicrobial activity, low concentrations of ozone are preferred in order to minimize exposure of workers to potentially hazardous concentrations of the gas and to reduce the risk of physiological damage to the treated produce . In effeE. coli, S. enterica, and B. cinerea) on Andean blackberries during refrigerated storage. The respiration rate and mass loss of the fruit were also determined as indicators of possible physiological damage.Therefore, the objective of this study was to investigate the effect of low gaseous ozone doses (0.4–0.7 ppm for 3 min) on the growth of native microflora  and inoculated pathogens (Rubus glaucus Benth), hand-harvested in Tungurahua Province, Ecuador at maturity stage 4 (dark red), according to external color of the fruit and following the color chart of the Ecuadorian Quality Standard for fresh blackberries [The plant material used for this study was Andean blackberries (kberries . ImmediaEscherichia coli (ATCC 25922) and Salmonella enterica (ATCC 9842) were obtained from the American Type Culture Collection  and Botrytis cinerea was kindly provided by BioSeb Organics Ltd. .The strains of 9 CFU mL−1 was reached. Microbial concentration was determined according to the McFarland scale at 600 nm (OD600) [B. cinerea was cultured in a Petri dish on potato dextrose agar . Streptomycin (1.0 mg mL−1) was added to the media to inhibit bacterial growth and the plate was incubated at 25 ± 1 °C for 7 days. The fungi spores were scraped with a sterile loop and diluted with sterile distilled water. Conidia were counted using a hemocytometer and the suspension was adjusted to 107 CFU mL−1. A loopful of each stock bacterial culture was individually transferred into 30 mL of brain heart infusion (BHI) broth  and incubated at 37 ± 2 °C for 24 h prior to experimentation. Cells were used when a concentration of 10 (OD600) . An isol4 conidia g−1 of Botrytis cinerea and 104 CFU g−1 of E. coli and S. enterica by placing the inoculum on the surface of the fruit with a calibrated micropipette. Three containers/pathogen were prepared for each treatment and evaluation date.The blackberries were distributed in transparent polyethylene terephthalate (PET) plastic containers with perforated lids. The fruit was placed in a single layer, with each box containing 100 ± 5 g of fruit. The blackberries of each box were inoculated to reach 10B. cinerea, the fruit were disinfected with an ethanol solution  for 10 s, washed with sterile distilled water, and dried at room temperature. Inoculated blackberries were air dried for 1 h at room temperature (20 ± 2 °C) in a biosafety cabinet to allow the attachment of the microorganisms to the fruit surfaces. Thereafter, the fruit was stored under refrigeration (6 ± 1 °C) for 24 h until ozone treatment.In order to avoid the growth of native fungi and guarantee the growth only of the inoculated microorganism, before the inoculation with −1 and ozone concentration was continuously monitored and controlled by circulating air from the chamber through an ultraviolet absorption ozone analyzer , calibrated in the range of 0–2000 ppm connected to a computer. The software integrated the concentration and time data and when the appropriate dose (concentration × time) was reached, the ozone generator was stopped.Ozone was generated in situ utilizing a surface discharge ozone generator  and synthetic air as the feeding gas . A fan iTwo independent ozonation processes were performed, one for the inoculated fruit and the second for the non-inoculated blackberries. In both cases, the fruit was divided in five groups: untreated berries represented the control sample, whereas the remaining four groups were subjected to gaseous ozone at 0.4, 0.5, 0.6, and 0.7 ppm, for 3 min.The inoculated fruit was ozonized one day after the inoculation. For this purpose, the containers with the fruit were placed in the treatment chamber, with previous retirement of the lids. For each pathogen, three samples/evaluation date were treated with each of the ozone doses studied.3 dose.The effect of ozonation on the native microflora of the blackberries was evaluated on non-inoculated samples. Around 2 kg of blackberries were placed on stainless steel mesh trays and taken inside the chamber for the treatment with each O3 treatments, the fruit for the native microflora  studies were packaged in the same transparent polyethylene terephthalate (PET) plastic containers (100 ± 5 g) as those used for the inoculated fruit. All the samples were stored at 6 ± 1 °C.After the O2 and CO2 were determined after 8 h with the use of an O2/CO2 gas analyzer . The results were expressed as mg of CO2 produced per kilogram per hour (mg kg−1 h−1).The respiration rate of the blackberries was measured using the closed system method . SamplesFor mass loss, three trays were randomly selected and individually weighed at the beginning of the experiment, and every two days during the storage period. Results were expressed as percentage of mass loss relative to the initial mass.Microbiological analyses were performed on days 1, 4, 7, and 10 of storage, considering day 1 as the day of ozonation. In addition, the containers were visually controlled daily in order to detect symptoms of microbial development. At each evaluation date, 3 samples/treatment were analyzed.B. cinerea, E. coli, and S. enterica, respectively. All the culture media were from Acumedia . Culture conditions were as follows: 37 ± 2 °C for 48 h for E. coli and S. enterica and 25 ± 1 °C for 5 days in the case of B. cinerea.For the analyses, 5 g of fruit inoculated with each of the pathogens under study was aseptically transferred to an individual filter stomacher bag and homogenized in 45 mL sterile buffered peptone water  for 120 s at 200 rpm, using a Stomacher 400 circulator . Serial decimal dilutions of each homogenized sample were made in peptone water. From each dilution, 0.1 mL aliquots were aseptically surface-plated on the following media: Sabouraud dextrose agar plus chloramphenicol, Eosin Methylene Blue (EMB) Agar Levine, and Salmonella Shigella (SS) Agar, for 10 (cfu g−1) of fruit.Serial dilutions for these microbial groups were prepared as described above. From each dilution, 1 mL aliquots were aseptically pour-plated for mesophiles and psychrothrophs and 0.1 mL was surface-plated for molds and yeasts analyses. The following media and culture conditions were used: (1) plate count agar  incubated at 35 ± 2 °C for 48 h and at 7 °C for 7 days, for total mesophilic and psychrotrophic microorganisms, respectively, and (2) Sabouraud dextrose agar plus chloramphenicol media  incubated at 25 °C ± 2 for 5 days for yeasts and molds. All the samples were analyzed in duplicate, and microbial counts were expressed as logThe analyses were conducted in triplicate, considering each container as the experimental unit. Data were subjected to a one-way analysis of variance (α = 0.05) using the IBM SPSS Statistics Version 27 software . When significant differences were observed, mean treatments were compared using Tukey’s test.3-treated blackberries is shown in Respiration rate (RR) is an indicator of the metabolic activity of fruit and vegetables and thus, an indicator of postharvest shelf-life. The RR of the control and the O3-treated blackberries) and 11.98 ± 0.51 (control) mg CO2 kg−1 h−1, with no significant differences among treatments (p > 0.05). Similar results were observed in cantaloupes [2 production of broccoli treated with high doses of this gas (7 ppm), which was attributed to physiological damage caused to the florets. During the storage period, the RR of all the fruit increased continuously. However, the highest CO2 production occurred in the control and those blackberries treated with 0.4 ppm O3. In effect, after 10 days, the RR of these fruit was significantly higher than in the blackberries ozonized with the highest doses. Chen et al. [2 production observed in the present study, which represented an increase of around 55% and 75%, in the control and the 0.4 ppm O3-treated fruit, respectively, could be related to the highest microbial growth observed in these treatments by the end of the storage period.Initially, the respiration rate of the blackberries ranged between 11.30 ± 0.26  to 3.49 ± 0.06 log units in the control samples, whilst Salmonella was not detected on day 10 in the blackberries exposed to 0.6 and 0.7 ppm O3. Daş et al. [3-treated cherry tomatoes inoculated with Salmonella after 6 days of cold storage. Both blackberries and tomatoes are acidic fruit and Salmonella is very susceptible to acid environments. On the contrary, some strains of E. coli remained viable even at pH 2.5 [During the storage period, the counts for both pathogens progressively decreased in all the treatments. The final counts of ş et al.  reportedt pH 2.5 .Botrytis on non-ozonated blackberry samples or O3-treated fruit  are shown in Botrytis counts for the control reached 3.84 ± 0.05 log units and the maximum reduction (0.46 log units) was observed in the blackberries treated with the highest O3 dose. In effect, only this treatment significantly reduced Botrytis counts initially and following the storage period  when compared with the control. Furthermore, the extent of the reductions increased with increasing O3 dose, ranging from 0.27 to 1.85, 0.49 to 1.89, and 0.37 to 2.24 log units for the mesophiles, psychrotrophs, and molds and yeasts, respectively. During the cold storage period, the microbial populations gradually increased regardless of the treatment. Yet, in all the ozone-treated blackberries, the reductions achieved were maintained throughout the storage period. Among the O3 treatments, the best results were observed when the blackberries were exposed to 0.7 ppm O3 with counts on day 10 for total aerobic mesophiles and psychrotrophs even lower than the initial counts of the control samples.In all the O−3/30 min and 0.5 to 2 mg L−1/60 min reduced the total mesophile counts in juniper berries [−1 gaseous ozone applied to strawberries for 30 min were effective in controlling aerobic mesophiles and molds and yeasts, respectively, during 4 days of storage. In studies involving the application of ozone during storage, the exposure of sweet cherries to 2 ppm gaseous ozone for 30 min every 6 days delayed decay development and lowered decay incidence on the fruit for up to 18 days [3 in cycles of 30 min once every 12 h reduced the aerobic mesophilic bacteria and fungi counts on this fruit during 3 days of storage at room temperature [3 applications were only partially effective in preventing fungal growth on table grapes and strawberries, respectively [Gaseous ozone can be applied either as a pre-storage treatment or continuously or intermittently during the storage period. Similar to our results, the application of gaseous ozone at doses of 130 g m berries  and gree berries , with th berries  found th 18 days . As wellperature . On the ectively ,57.These variable and somehow contradictory results could be explained by differences in the type of product, differences in the ozone application methods and in the doses used (time and concentration), the microbial type and microbial load, as well as variations in environmental conditions , all of which can affect the efficacy of ozone as a sanitizer .3 for 3 min. This treatment slowed down the respiration and mass loss rates, indicating that no physiological damage occurred in the treated fruit. Moreover, it was proven effective in reducing both the native microflora and the inoculated pathogens on the fruit throughout the storage period. Therefore, gaseous ozone could be considered a promising processing technology for prolonging the postharvest life of fresh Andean blackberries during refrigerated storage. However, as the log reduction in microbial populations observed in this study may not be enough to ensure the safety of the product, further studies would be necessary to determine the optimal treatment conditions for this fruit. As well, the effects of the treatment on the bioactive compounds and the physicochemical and sensory quality of the blackberries would be assessed.The application of low doses of gaseous ozone prior to storage was studied as an environmental-friendly alternative to guarantee Andean blackberries’ quality and safety. The best results were obtained after the exposure of the fruit to 0.7 ppm gaseous O


The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed across cancer genomes, associating with single-stranded (ss) DNA formed during DNA replication and hairpin-forming sequences. The biochemical and cellular factors that influence these specificities are unclear. We measured A3A’s cytidine deaminase activity in vitro on substrates that model potential sources of ssDNA in the cell and found that A3A is more active on hairpins containing 4 nt ssDNA loops compared to hairpins with larger loops, bubble structures, replication fork mimics, ssDNA gaps, or linear DNA. Despite pre-bent ssDNAs being expected to fit better in the A3A active site, we determined A3A favors a 4 nt hairpin substrate only 2- to fivefold over linear ssDNA substrates. Addition of whole cell lysates or purified RPA to cytidine deaminase assays more severely reduced A3A activity on linear ssDNA (45 nt) compared to hairpin substrates. These results indicate that the large enrichment of A3A-driven mutations in hairpin-forming sequences in tumor genomes is likely driven in part by other proteins that preferentially bind longer ssDNA regions, which limit A3A’s access. Furthermore, A3A activity is reduced at ssDNA associated with a stalled T7 RNA polymerase, suggesting that potential protein occlusion by RNA polymerase also limits A3A activity. These results help explain the small transcriptional strand bias for APOBEC mutation signatures in cancer genomes and the general targeting of hairpin-forming sequences in the lagging strand template during DNA replication. APOBECs convert cytidine (C) in single-stranded DNA (ssDNA) to deoxyuridine (dU), which either templates for adenosine causing a C to thymidine (T) mutation or is subsequently converted to an abasic site that leads to an C to T or C to guanosine (G) mutation depending on the polymerase used for bypass6. These enzymes become dysregulated during tumorigenesis, especially in cervical, bladder, breast, lung, and head and neck cancers, and aberrantly mutate the human genome11. Consequently, APOBEC3 enzymes are the second-most prominent cause of mutation in sequenced tumors12.APOBEC3  is observed in many primary human tumors16. The number of APOBEC-signature mutations significantly correlates with A3A expression in both primary tumors and breast cancer cell lines17. In addition, A3A often provides the majority of cytidine deaminase activity and APOBEC-signature mutations in breast cancer cell lines (even in the presence of A3B and A3H-I) in part due to its stronger cytidine deaminase activity and unique resistance to RNA inhibition22.Of the seven APOBEC3 proteins, A3A, APOBEC3B (A3B), and APOBEC3H haplotype I (A3H-I) have been implicated in contributing to cancer mutations27. These distributions of A3A-induced mutations indicate that substrate characteristics beyond the already defined sequence preferences32 modulate A3A activity. Biochemical studies of substrate preferences have been instrumental in understanding the targeting of mutations caused by the related APOBEC, AID, to transcription intermediates formed in immunoglobulin loci. These efforts initially highlighted the structural loops required for AID WRC sequence specificity33, determined that AID (and by extension most other APOBECs) is ssDNA specific36, and identified roles for transcription37, R-loops38, G-quadruplexes39, RPA40, and RNA exosome complex activity in allowing AID to deaminate both strands of a transcription unit41. Biochemical activities of A3A have been similarly characterized in vitro. These activities include TC sequence specificity31 even with damage directly upstream of the deaminated C42, ssDNA specificity43, activity at 5-methyl C44, and ability to deaminate transcribed DNA45, DNA synthesis intermediates45, as well as RNA substrates48. Moreover, crystal structure analysis of A3A has revealed how the enzyme’s preferred sequence is mediated by specific amino acids which also contribute to the bending of ssDNA into the A3A active site50. As a consequence, A3A prefers hairpin substrates in vitro51, presumably due to greater binding affinity to the already bent ssDNA30. This known specificity likely underlies recurrent mutations at hairpin-forming sequences in cancer genomes that have been predicted to be deaminated by APOBECs 38- to 265-fold over non-hairpin-forming sequences25. However, A3A has been shown to deaminate ssDNA within replication intermediates in cells53 and related APOBECs can deaminate transcription intermediates58, raising the question as to why replicative asymmetries dominate transcriptional asymmetries among APOBEC-signature mutations in cancer genomes.Within tumor genomes, APOBEC-signature mutations are non-uniformly distributed, showing a strong DNA strand bias associated with replication direction and enrichment in hairpin-forming sequences and tRNA genes30. However, highly purified A3A only favored hairpin-forming sequences 2- to fivefold (depending on A3A concentration) over linear ssDNA. A3A activity on linear ssDNA was more severely reduced than on hairpin substrates when incubated in the presence of whole-cell lysate or RPA, indicating cellular ssDNA binding proteins likely out-compete A3A for longer ssDNA substrates. This result highlights that substrate specificities for other ssDNA binding proteins likely enhance the preference of A3A-induced mutations for hairpin-forming sequences in tumors. Additionally, we determined that A3A deamination activity is low on ssDNA bubble substrates, like those found during transcription, using an in vitro transcription assay. These results indicate that DNA structure and competition with other ssDNA binding proteins are important factors in predicting at-risk locations in the human genome for A3A-induced mutagenesis.To determine how DNA substrate characteristics influence the distribution of A3A-induced mutations observed in vivo, we measured activity of A3A on several ssDNA substrate types. We recapitulated the previously reported preference for hairpins over linear ssDNA substrates25 likely due to A3A’s affinity for U-shaped ssDNA present in hairpin structures50. We first incubated A3A at a range of concentrations with a constant concentration of substrate to see how deaminase activity would titrate on these substrates  and a substrate with the 3′ arm of the stem loop replaced with poly-A to prevent hairpin formation (NUP93-noHP)13 13 Fig. . A3A dis)13 Fig. . This in30, upstream of the target site towards the 5’ end of the loop. When incubated with 20 nM A3A, hairpin substrate deamination was decreased fourfold, sixfold, and fivefold on the 8 nt loop, 11 nt loop, and 14 nt loop, respectively  is a likely candidate, as it is known to inhibit A3A activity on linear ssDNA substratesRPA Fig. B, a heteRPA Fig. C, indicaRPA Fig. D, indica58. However, little evidence for transcriptional asymmetry of APOBEC-induced mutations in cancer genomes exists26, indicating that ssDNA within transcription bubbles may be protected from APOBEC cytidine deaminase activity. Our previous data indicates that the small ssDNA bubbles themselves reduce A3A activity. Moreover, transcription bubbles are frequently 14–22 nt in length63, indicating that most ssDNA present will be near the synthesizing RNA polymerase, which may further reduce A3A activity. To better understand whether RNA polymerase itself blocks A3A activity, we generated an in vitro transcription system similar to previously described systems utilizing T7 RNA polymerase45, a 90 bp dsDNA substrate, and our purified A3A. To enhance the stability of transcription-associated ssDNA, we designed the substrate to only contain T bases in the transcribed DNA strand downstream of the TTCA deamination target on the non-transcribed strand and excluded rATP from the transcription reaction. The lack of rATP stalls T7 RNA polymerase across from the TTCA deamination target sequence, causing it to remain single stranded as long as T7 RNA polymerase is bound. 0, 20, 50, or 100 nM of purified A3A were incubated with 20 nM dsDNA substrate and 50 units of T7 RNA polymerase at 37 °C for 24 h. A3A was incapable of deaminating the dsDNA alone , we believe this impact would be small. Supporting this assertion, un-tagged A3A and the c-terminal strep-tagged A3A display similar levels of cytidine deaminase activity on a hairpin substrate with a 4 bp loop  would allow binding of ssDNA-binding proteins whose footprint would be unable to bind to the shorter 4 nt ssDNA contained within the hairpin substrate. However, A3A activity towards a 4 nt ssDNA gap substrate was also reduced by SKBR3 whole cell extracts, indicating that the bent conformation of the 4 nt loop of the hairpin may be the primary barrier to the binding of putative competitor proteins.Surprisingly, A3A only preferred hairpin ssDNA 2- to fivefold over fully linear ssDNA, a less drastic fold difference than has been previously reported67, and binds to a relatively large stretch of ssDNA (~ 30 nt)62, suggesting that it may be unable to inhibit A3A activity on smaller, structured ssDNA regions. Supporting this, RPA efficiently inhibits A3A activity at near equimolar ratios of RPA to a 40 nt linear ssDNA substrate, while impacting A3A activity on a hairpin substrate with a 4 nt loop to a lesser extent. Thus, the binding of longer ssDNAs, formed during replication or in extended DNA R-loops, may provide protection from mutation, enhancing the preferential mutagenesis on hairpin-forming sequences. RPA typically binds and prevents the formation of DNA secondary structures, protecting the ssDNA from DNA damaging agents like A3A. In cancers, replication stress-induced ssDNA formation can exhaust RPA levels68, leaving sections of ssDNA in replication forks unprotected. These long regions of ssDNA can then form hairpins that would otherwise be repressed by RPA binding and further make themselves targetable by A3A. As a result, A3A-induced mutations are enriched at hairpin-forming regions of cancer genomes and show strand bias for replication intermediates.The identity of specific protein factors that influence the distribution of A3A-induced mutation are unknown. RPA is one likely candidate as it inhibits the activity of multiple APOBECs, including A3A, on linear ssDNA69. The large size of this complex likely occludes A3A and prevents A3A from deaminating and eventually mutating transcription intermediates. Hypothetically, the non-transcribed strand would be single-stranded for a period during transcription and could be deaminated by APOBEC3s. Indeed, there is experimental evidence for A3A deaminating transcription intermediates in vitro45. However, only a small enrichment has been found for APOBEC-signature mutations on the non-transcribed strand in human cancers26. Consistent with this result, we saw nearly undetectable A3A activity on the non-transcribed strand during stalled T7 RNA polymerase transcription. Additionally, A3A activity was also reduced towards a ssDNA bubble substrate that mimicked the static structure of a transcription intermediate. These data suggest that A3A is unlikely to be active at transcription bubbles, especially those directly blocked by the presence of the elongating RNA polymerase. Instead, transcription would likely require the creation of an elongated ssDNA bubble by the formation of an R-loop, allowing A3A sufficient access to the ssDNA for deamination. Consequently, transcriptional strand asymmetry of A3A-induced mutations in human cancers may only be present in DNA regions prone to R-loop formation.It is also possible that larger ssDNA binding proteins, like RNA Polymerase occlude A3A binding. In our in vitro transcription assay, a 99 kDa T7 RNA Polymerase is stalled across from the TTCA target site and its proximity likely inhibits the smaller 23 kDa A3A by blocking access to the target site. Transcription of protein coding genes in human cells is carried out by the RNA Polymerase II complex, which is comprised of 10 subunits that together constitute a mass of ~ 500 kDa17. Briefly, HEK293T cells were transduced with lentiviral vectors encoding the APOBEC3A gene under control of the Tet repressor. Cell populations were expanded to approximately sixty 10 cm dishes, after which A3A protein expression was induced with doxycycline for approximately 84 h. A3A-expressing cells were then harvested via trypsinization and centrifugation. Lysates were prepared in buffer containing protease inhibitors and DTT, sonicated, and treated with Benzonase. Insoluble materials were removed via centrifugation and filtering. The Strep-tagged A3A was batch bound to Strep-tactin resin and put over a glass econo-column. The column was washed to remove any non-specific binding proteins and then the Strep-tagged A3A was eluted using buffer containing 20 nM d-desthiobiotin. Eluted A3A was further purified through an 1 mL Enrich-Q (BioRad) column. A3A-containing fractions were pooled, concentrated, dialyzed, frozen in aliquots in liquid nitrogen and stored at − 80 °C. Aliquots were not used past 5 freeze/thaw cycles.A3A was purified as inE. coli . Human RPA was purified following protocols previously described in70, using chromatography on subsequent 5 mL HiTrap Blue HP (Cytiva lifesciences), 2 mL hydroxyapatite CHT-type1 (BioRad), and 1 mL Enrich-Q (BioRad) columns.Three subunit human RPA was expressed from plasmid p11d-tRPA(123), , in T7 express 17. The coding sequence of A3A with its natural stop codon (no tag) and A3A intron 3 was cloned into the HincII and EcoRV sites of pENTR1A no ccDB . An LR-Clonase reaction between the resulting plasmid and pTM-63717 was used to create pTM-925, a lentiviral vector for inducible expression of untagged A3A. HEK293T-TETR cells were transfected with plasmids psPAX2 , pMD2.G , and either pTM-664 or pTM-925 to produce lentiviral particles, which were subsequently used to transduce HEK-293T-TETR cells using LentiBlast (OZ Biosciences). Following selection of stable cell populations with 1 μg/ml puromycin, expression of A3A was induced with doxycycline at 50 μg/ml for 72 h prior to harvesting for whole cell lysates. Whole cell lysates were produced by resuspending cell pellets in 500 µL of M-PER lysis buffer and 2 × protease inhibitors then incubating at 4 °C for 30 min with agitation. Lysates were sonicated four times for 15 s. Debris was removed from lysates by centrifugation at 18,000 rcf for 20 min at 4 °C then glycerol was added to 10% (v/v) supernatant. 20 µg whole cell protein lysate was electrophoresed on a Bio-Rad Mini-Protean TGX SDS-PAGE Precast gel before transferring onto a 0.2 µm PVDF membrane via semi-dry transfer on a Trans-Blot Turbo transfer system. Membranes were blocked with 2% ECL-blocking agent (GE Biosciences) in TBST at room temperature for 1 h before probing with either mouse anti-A3A21 (gift from Dr. John Maciejowski) or rabbit anti-alpha tubulin (Ab4074) diluted in 1% ECL-blocking agent in TBST overnight at 4 °C. Membranes were probed with a secondary goat anti-mouse IgG HRP (Ab205719) or goat anti-rabbit IgG HRP (Ab97051) antibody. Blots were developed with ECL prime and imaged on a ChemiDoc MP.Creation of HEK293T-TETR cells and pTM-664, a lentiviral vector for inducible expression of Strep-tagged A3A, were described previouslyThe oligonucleotides used were obtained from IDT technologies and sequences are listed in Table Substrates with complementary oligonucleotides were annealed by mixing the corresponding oligos in buffer containing 50 mM Tris, pH 7.5 and 100 mM NaCl before heating at 95 °C for 5 min and then slowly cooling (1 °C per minute) in a BioRad Thermocycler. Annealing reactions were then concentrated by EtOH precipitation and run out on a large native 5% polyacrylamide gel (1 × TBE), bands imaged on the Biorad ChemiDoc for Cy5, and excised. DNA was isolated from excised bands using the freeze/squeeze method. Briefly, bands were minced and resuspended in 200 µL of 10 mM Tris, 0.1 mM EDTA buffer, and put through 3 cycles of vortexing (20 s), flash-froze in liquid nitrogen, thawed (65 °C) before incubating at 37 °C, and rotated overnight. Gel debris was separated from DNA-containing supernatant using a 1 mL filter column and spinning at 13 k rpm for 10 min at 4 °C, then DNA was EtOH precipitated from the supernatant. The band with the correct annealing product was determined by restriction digest, as double-stranded sections would have a combination of PacI-HF, KpnI or DraI sites, and would only cut if annealed properly Fig. .All deaminase activity reactions in this manuscript were conducted with a single preparation of purified A3A. Deaminase activity assays with A3A (with concentrations as indicated), 5 units of Uracil DNA glycosylase (NEB), 20 nM oligonucleotide substrate, 20 mM Tris HCl pH 7.5, 1 mM DTT, and 1 mM EDTA, in a 20 μL volume were incubated for 30 min (unless otherwise indicated) at 37 °C and terminated by the addition of 7 µL stop buffer . Proteinase K  was then added and incubated at 37 °C for another 30 min to limit protein binding to substrates during electrophoresis. Alternatively, cytidine deaminase reactions to test the impact of the strep-tag on A3A activity contained 20 µg cell extract from un-transduced HEK293T cells or HEK293T cells expressing un-tagged A3A or C-terminally Strep-tagged A3A and 1 µM oTM-814 and were incubated for 5 min at 37 °C prior to termination as conducted for assays described above with purified A3A. We then added 23.2 µL formamide buffer , and 4.8 µL of 1 M NaOH  and incubated at 95 °C for 10 min to break abasic sites present in the substrates. Reactions used to evaluate the impact of RPA on A3A activity also contained 10 nM of unlabeled ssDNA that lacks a cytidine. Deaminase activity products were separated from undigested substrate on pre-warmed 15% polyacrylamide gels with 7.9 M urea (7 cm) in 1xTBE buffer at 15 W for 15 min. Gels were imaged on a BioRad Chemidoc using the Cy5 setting. Percent substrate cleaved was determined by quantification of the intensity of the substrate band and cleavage product bands using the Volume tool on BioRad’s Image Lab software version 5.2, and by using the following equation = (Product intensity)/(substrate + product intensity) × 100.17) or buffer were pre-incubated at 37 °C for 30 min with the 20 nM substrate in 20 mM Tris HCl pH 7.5, 1 mM DTT, and 1 mM EDTA with 5 units of UDG, in a 19 µL volume prior to the addition of A3A. After pre-incubation, 1 µL of A3A was added to 20 nM final concentration followed by incubation for an additional 30 min at 37 °C and was processed like the other deaminase assays. Similarly, RPA-containing deaminase assays, involved an initial 30 min pre-incubation of the deamination substrate with a twofold excess of RPA relative to substrate at 37 °C, prior to the addition of A3A.For deaminase assays with the addition of whole cell lysate, 40 µg of SKBR3 whole cell lysate , 20 mM Tris–HCl pH 7.5, 1 mM DTT, 1 mM EDTA, with 5 units of UDG. Binding reactions were incubated at room temperature for 15 min, before adding glycerol to 30%, and electrophoresing through a 6% native polyacrylamide gel on ice at 80 V for 1 h. Gels were imaged on a BioRad Chemidoc using the Cy5 setting. Percent substrate bound was determined by quantification of the intensity of size-shifted (bound) band relative to unbound substrate, using the "Volume" tool on BioRad’s Image Lab software version 6.0.1.The dsDNA substrate was prepared and purified as above using the oligos ALH287 and ALH288 , 50 units of T7 RNA Polymerase (NEB), 5 units of Uracil DNA glycosylase (NEB), 20 nM oligonucleotide substrate, 1 mM each of GTP, CTP, and UTP, 40 mM Tris HCl pH 8, 6 mM MgClon Figs. –S8.Supplementary Information 1.Supplementary Information 2.


The effective conduction of action potential in the peripheral nervous system depends on the structural and functional integrity of the node of Ranvier and paranode. Neurofascin (NF) plays an important role in the conduction of action potential in a saltatory manner. Two subtypes of NF, NF186, and NF155, are involved in the structure of the node of Ranvier. In patients with chronic inflammatory demyelinating polyneuropathy (CIDP), anti-NF antibodies are produced when immunomodulatory dysfunction occurs, which interferes with the conduction of action potential and is considered the main pathogenic factor of CIDP. In this study, we describe the assembling mechanism and anatomical structure of the node of Ranvier and the necessary cell adhesion molecules for its physiological function. The main points of this study are that we summarized the recent studies on the role of anti-NF antibodies in the changes in the node of Ranvier function and its impact on clinical manifestations and analyzed the possible mechanisms underlying the pathogenesis of CIDP. The typical clinical manifestations of CIDP include a progressive relapsing-remitting pattern in the extremities, symmetric paresthesias and weakness, areflexia, cranial nerve involvement, autonomic symptoms, and less commonly neuropathic pain. Chemical analysis of the cerebrospinal fluid of patients reveals the cell-protein separation phenomenon and electromyography indicates demyelinating or axon damage. Atypical manifestations of CIDP include simple motor dysfunction, simple sensory dysfunction, and Lewis–Sumner syndrome , for which conventional immune therapy is not effective , 2019. TThe structure of the nervous system is similar to that of a cable transmission system. With respect to the myelinated axons, the nodes of Ranvier act as repeaters to regenerate the action potential, as they propagate in a saltatory manner along the axon to the terminal nerve and significantly increase the velocity of action potential conduction . NF playNeurofascin comprises two subtypes such as NF186 and NF155. Due to the diverse functions and structures of each subtype of immunoglobulin (Ig) and the different anatomical features of the paranode and node, the manifestation and therapy of anti-NF155 antibody-positive CIDP are different from those of anti-NF186 antibody-positive CIDP . In thisIn humans, myelin is applied to most nerve fibers in the PNS by Schwann cells. To some extent, the involved nerves in CIDP are influenced by the anatomical differences in the peripheral nerves. A study of 9 patients with anti-NF155 antibody-positive CIDP showed that the median and ulnar nerves are more vulnerable than the sural sensory nerves, which are consistent with their different structures. Moreover, conduction studies on the median and ulnar nerves show that NF autoantibodies affect the properties of the nerve terminals, while those on the sural nerves show that NF autoantibodies affect the intermediate nerve segment . These aAll the types of glial cells found in the PNS originate from neural crest cells, with gliogenesis starting at embryonic day 11 in the mouse . Two typThe paranode begins to assemble after the node cluster NaV and then the potassium ion channel concentrates on the juxtaparanode . In the Neurofascin, a neural-cell surface protein, is part of the L1 group of the immunoglobulin superfamily, including L1, close homolog of L1 (CHL1), and NrCAM . DiffereThe PAT domain is thought to confer an extended and more flexible structure of NF186, which can enhance the interactions of NF186 with gliomedin and NrCAM and enable NF186 to target at the node and guide NaV and AnkG located at proper locations. When NF186 is absent, the nodal gap shortens progressively and AnkG and Nav disappear from the axon, resulting in the deduction of conduction velocity. Moreover, NF186 coordinates nodal organization and the enrichment of both the neuron-specific proteins and glial-specific proteins to nodes in PNS myelinated axons and acts as a barrier to restrict the invasion of flanking paranodal domains in myelinated axons .Paranode stability requires interactions between glial NF155 and the CNTN1-CASPR1 complex, which is expressed on the axonal membrane . There aIn summary, NF is indispensable for the structural integrity of the node of Ranvier and paranode, which is the basis for saltatory conduction of action potentials. Both the NF186 and NF155 play separate roles in the myelin sheath: NF155 is a component of the paranode that stabilizes ion channels located in two flanks of the paranode, whereas NF186 acts as a barrier to restrict the invasion of flanking paranodal domains in myelinated axons.Some patients with CIDP have infections after the onset of neurological symptoms, but so far, no causative pathogens have been found to be related to the occurrence of the disease. CIDP is considered an autoimmune disease and may be treated and improved by effective targeting of the autoimmune response and the therapeutic methods include intravenous immunoglobulin (IVIg), plasma exchange, and corticosteroids, which have been proven to inhibit the inflammatory response in the blood circulatory system and peripheral nerves . There aThe isoform of NF155 is more vulnerable to attack in immune modulatory dysfunction than other molecules in CIDP . The IgGFN3 is considered an antigenic determinant of NF155 in CIDP and other inflammatory demyelinating polyneuropathies . HoweverThe BNB directly communicates with the circulating blood and nerve lumen and consists of simple endoneurial microvascular endothelium cells, which are connected by intercellular tight junction. It is a selectively permeable barrier that increases transendothelial electrical resistance, regulates the diffusion of molecules and nutrients, and restricts hematogenous cells from invading nerves . The endin situ/systematic inflammation), molecules involved in Schwann cell-axon interaction, such as NF, CNTN1, and CASPR1, are accessible to the immune system and act as putative antigens, which are presented by antigen-presenting cells through the expression of the costimulatory molecules CD80 and CD86 to T cells; in effect, T cells activate and release cytokines, including interleukin-4 (IL-4) and IL-6  than in the control Spanish populations (HLA-DRB1*15, -DRB1*15:01, -DQB1*06:01/06:02, -DQB1*06:02, and -DRB1*15:01-DQB1*06:02 (Immunoglobulin G4 exists in a monovalent bispecific form through a process termed Fab-arm exchange , which rulations . HoweverB1*06:02 .in vivo (Patients with immunoglobulin G4-predominant anti-NF155 antibody-positive CIDP are often refractory to treatment with IVIg, but they partially respond to rituximab and corticosteroid treatment because IgG4 does not fix complements or bind to Ig receptors in a monovalent bispecific form in vivo . Most anin vivo . Anotherin vivo . IgG4 anin vivo . The inhin vivo . This diThe clinical features of anti-NF155 antibody-positive CIDP include younger age at onset, predominant distal limb weakness, high-amplitude and low-frequency tremors, ataxia with cerebellar features, and a higher prevalence of poor response to IVIG when compared with seronegative patients . AlthougRecently, an increasing number of studies have revealed that the assembly and maintenance of the node of Ranvier depends on the normal functions of various molecules on the node, paranode, and juxtaparanode including NrCAM, gliomedin, CNTN1/2, CASPR1/2, MAG, and NF. These molecules keep the ion channels in proper positions to ensure that the action potential is conducted in a saltatory manner. The BNB dysfunction is considered the initiation of pathology of CIDP and passive transfer of anti-NF antibodies to EAN could aggravate and delay ongoing neuritis. Antigenic targets should be accessible to antibodies, which are realized by cytokines and immune cell infiltration, resulting in opening the BNB and providing access to autoantibodies. The immune response is activated by the exposure of autoantigens or foreign antigens, resulting in the production of cytokines and antibodies to resist the invasion of the “foreigner.” These immune mediators could disrupt the physiological effects of the node of Ranvier and lead to the occurrence of diseases such as CIDP, AIDP, and combined central and peripheral demyelination. Antibodies to both the NF155 and NF186 are involved in the pathogenesis of CIDP, but Ig subclasses and clinical manifestations are significantly different. The positive rate of the anti-NF155 antibody was higher than that of NF186 in CIDP probably because NF155 can partly compensate for the function of NF186. The mechanism by which autoantibodies belonging to the same IgG4 subclass can cause IgG4 antibody-specific disease features and different responses to conventional immunotherapy requires further study and it could guide the development of more efficient treatments and avoid unnecessary therapy. The sequential order of Ig indicates that the appearance of IgG4 results from long “foreign” stimulation and the production of IgG3 and IgM is an acute immune response. Unique diagnosis and treatment strategies are required for IgG4-related neuropathy. Previous studies indicate that IgG4-related CIDP is poorly responsive to IVIg, but no multicenter studies have focused on the effect of immunosuppressors in patients with CIDP. Further multicenter studies are necessary to clarify the clinical characteristics of the autoantibody subtypes, which assist diagnosis and the choice of therapeutic strategies. Further, studies are needed to reveal the mechanism of the different responses of anti-NF186/-NF155 IgG4-positive CIDP to IVIg. Although we have discussed in depth the mechanism underlying the pathogenesis of NF in CIDP, many problems remain unsolved in this field. Further study will be essential for understanding the specific function of each domain in the different isoforms of NF, how the NFs interact with their corresponding receptors/ligands, the factors that trigger dysfunction of the BNB, the role of the pericytes in pathology of CIDP, and the different modifications of the BNB that occur in the various subtypes of CIDP.YG conceived and prepared the manuscript. LK, SL, and KL prepared the manuscript and the figures. JZ helped to conceive and reviewed the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Herein we report a simple four-step process to recycle the Nd2Fe14B sludge containing ~ 10% of contaminants. Sludge was leached in H2SO4 and selectively co-precipitated in two steps. In the first co-precipitation, Al3+ and Cu2+ were removed at pH 6. Thereafter, in the second co-precipitation Fe2+ and RE3+ sulfates were converted to the Fe and RE hydroxides. By annealing at 800 °C RE and Fe hydroxides precipitates were converted to the oxides and residual carbon was oxidized to CO2. After the addition of boric acid, Fe and RE oxides were reduced and diffused to the (Nd-RE)2Fe14B by calciothermic reduction diffusion. Removal of CaO by washing with D.I. water in glove box reduced the oxygen content (~ 0.7%), improved crystallinity and enhanced the magnetic properties significantly. Coercivity increased more than three times (from 242.71 to 800.55 kA/m) and Mr value was also enhanced up to more than 20% (from 0.481 to 0.605 T). In this green process Na2SO4 and Ca(OH)2 were produced as by-product those are non-hazardous and were removed conveniently.Complete recycling of Nd They have drawn attention due to their applications in modern appliances which lead to large market demand and a rapid increase in their production11. A huge amount (21%) of rare earth elements (RE) are being consumed for the synthesis of permanent magnets. RE resources are depleting and the cost of RE extraction from the ores is continuously soaring, hence recycling of the Nd2Fe14B sludge is becoming an important area of modern research.Among permanent magnets, Nd2Fe14B sludge is produced in the cutting and grinding process and more than 95 wt% of it, is recyclable12. However, because of costly physical recycling processes and high level of contamination, usually, recycling magnet sludge is not economically feasible in most part of the world. Nd2Fe14B sludge mainly consists of oxidized particles of Nd2Fe14B with different RE, C, Al, and d-block transition metals . Al, Zn, Mn, Cr, and Ni come from the protective coatings, those are applied to avoid the corrosion of bulk magnet surface. Cu is added to the sintered Nd2Fe14B magnets to decouple the magnetic grains to stop the fast flip-over of the magnetic domains and enhance the coercivity11. Co addition to enhances the Mr value and curie temperature. Sludge can have a high quantity of carbon because of the mixing of machine oil and lubricant during the cutting process. All these contaminants reduce the value of sludge.A large quantity (~ 30%) of the Nd2Fe14B sludge is a multi-step process which requires a huge amount of chemicals, energy, and produces hazardous wastes as byproduct13. These wastes include oxides , dangerous metals (e.g. As), organic solvents, RE vapors and electrolytes. Several chemical methods have been introduced for the recovery of RE from Nd2Fe14B magnets scrap/sludge26 but complete recycling of Nd2Fe14B, via chemical route is relatively new field. Haider et al.12 and Yin et al.13 have recently introduced chemical methods. These methods are very innovative and useful, but they deal with sludge with a low level of contamination. It is very difficult to control the contamination in the sludge, hence it was required to introduce a new method to recycle this kind of sludge with good magnetic properties.Commonly used physical method for recycling of Nd2Fe14B sludge which is equally useful for sludge with high contamination and variable composition. Na2SO4 and Ca(OH)2 are produced as byproducts, those are non-hazardous and easy to remove. Magnetic properties are further enhanced by the removal of contamination and reduction of oxygen content. A comparison of between our method and the common physico-chemical12 method used for the recycling of Nd2Fe14B sludge is given in the Fig. Herein we propose a time and energy efficient-recycling method for recycling of Nd2(SO4)3·6H2O), sodium hydroxide (NaOH), boric acid (H3BO3), calcium hydride (CaH2), sulfuric acid (H2SO4), ethyl alcohol (C2H5OH) and acetone (CH3COCH3) were analytical grade and obtained from Sigma-Aldrich Co. Magnet sludge was obtained during the cutting process of Nd2Fe14B.All chemicals used in this work, including neodymium (III) chloride hexahydrate  of the leachate showed that it consisted of Nd, Pr, Tb, Fe, Cu, Al, and C 3·6H2O was added to the leachate solution.Magnet sludge obtained during the cutting of Nd2SO4 and NaOH and then annealed at 800 °C to convert all Fe and RE hydroxides to oxides. The annealing was done for 30 min while the air was flowing in the furnace. Produced oxide particles were mixed with boric acid and CaH2 in glove box and the mixture was pressed to the pellet. Pellet increases the physical contact between the constituents e.g. calcium hydride, boric acid, oxides of RE and Fe, which is useful for the efficient R-D (reduction-diffusion). Boric acid was added in such a way that the molar ratio of RE:Fe:B was kept as 15:77:8. Oxides (+ boric acid):CaH2 weight ratio was fixed as 1:1.Co-precipitation of leachate was performed with a 3.5M NaOH solution which was added drop-by-drop, to raise the pH of the leachate solution up to 6. At pH 6, Al, Zn, and Cu chlorides were converted to their respective hydroxide precipitates. These precipitates were removed from the reaction mixture by centrifugation. The left-over reaction mixture was co-precipitated by further addition of NaOH solution. By maintaining the pH at 13, the solution was stirred for 30 min, which lead to the formation of RE and Fe hydroxides precipitates. The precipitates were washed thrice with de-ionized water to remove the Na2) were reduced and diffused by annealing at 1000 °C for 3 h. Products obtained after R-D were washed with water in the glove box repeatedly to remove CaO and rinsed with acetone twice. Finally, vacuum drying was done and recycled (Nd-RE)2Fe14B powder was stored in inert conditions. Flow diagram of experimental process and summary of chemical reactions during the process are provided as Fig. RE and Fe oxides . Crystal structure and phases were determined by X-ray diffraction (XRD) patterns using a Rigaku Diffractometer . The morphology, size, and elemental distribution were observed with field emission scanning electron microscope , conventional transmission electron microscopy , and aberration-corrected TEM (ARM-200F) with energy- dispersive X-ray spectroscopy (EDS). TEM was operated at the accelerating voltage of 200 kV. Magnetic properties (M-H curves) of final product were measured by Physical Property Measurement System  in the vibrating sample magnetometer mode. LECO ON-736 analyzer was used to determine the oxygen content in (Nd-RE)27 and orientation of the sample along the required zone axis was confirmed by using electron backscatter diffraction (EBSD) by TEAM™ Pegasus, Ametek Co. Ltd. USA.Specimens for TEM were prepared by focused ion beam  using the lift-out technique. For TEM measurement, the sample was treated as the same process reported by Kim et al.2Fe14B was dissolved in the H2SO4. Overall composition of the sludge before and after the precipitation is provided in Fig. 2Fe14B produced from them. After leaching of sludge in the H2SO4, next step was the selective precipitation to remove Al3+ and Cu2+. Precipitation of multivalent (e.g. Al3+ and Cu2+) ions from the solution depends on many factors e.g. oxidation state, Ksp value, the concentration of other ions, precipitating agent, and temperature. Precipitation of Fe3+, Al3+, and Cu2+ from their chloride solution occurred at pH values of 3.5, 5.0, and 6.028. However, different results were observed, when the solution of acid mine drainage containing Fe3+, Al3+, and Cu2+ was co-precipitated28. In the acid mine drainage experiment, Fe3+, Al3+, and Cu2+ chlorides were precipitated at pH 3.5, 4.5, and 5.5, respectively28. Precipitation of Al3+ and Cu2+ at pH value of 5.5 was also observed29. Different pH of the precipitation for Al3+ and Cu2+ in the previous studies indicated is a complicated process. It is commonly observed that Fe3+ precipitates completely at pH value ~ 3, however, Fe2+ precipitates at pH value of ~ 730. In our study, when pH approached 4, Al (Ksp constant = 1.9 × 10−33) started to precipitate.Magnet sludge produced during the cutting of Nd2 is 1.6 × 10−19 and it was next to be precipitated hence precipitated out between the pH values of 5–6. Almost ~ 90% hydroxides of Al and Cu were separated in the form of precipitates at pH 6, they were removed from the leachate solution by centrifugation. Separated Al and Cu hydroxides were analyzed, and analysis details are provided in the supporting information on Figs. , S4.2+ which does not precipitate below pH 6 but slightly (~ 2%) precipitates at pH 6  started to precipitate as Fe(OH)2. Soe et al.31 reported that Nd3+, Pr3+, Dy3+, and Tb3+ also start to precipitate at pH value of ~ 7 and similar was observed in our study.Fe in the leachate exists as FeH 6 Fig. . HoweverCo-precipitation was stopped at pH values of 10, 11, 12 and 13 in four different experiments. The maximum percentage yield was obtained at pH value of 13 Fig. . At pH 1Hydroxides obtained by co-precipitation were aqua complexes of RE and other metals. Being amorphous, these hydroxides could not be detected by XRD Fig. e analysi2. Meanwhile, annealing also converted hydroxide precipitates to the oxides. SEM analysis revealed that the average particle size of the oxide particles was ~ 150 nm 2Fe14(AlCu)B and (Nd-RE)2Fe14B. Both of these products contained CaO byproduct, and were washed with water to remove it. (Nd-RE)2Fe14(AlCu)B was washed in the air while (Nd-RE)2Fe14B was divided into two parts. One part was washed in the air and other part was washed in the glove box to minimize the exposure to oxygen. In this way three different products (Nd-RE)2Fe14(AlCu)B, (Nd-RE)2Fe14B and (Nd-RE)2Fe14B (low oxygen), were obtained. To determine the detailed structure of the products XRD, SEM, SEM–EDS, TEM, TEM-EDS, and HRTEM analysis were performed. Nd2Fe14B, Dy2Fe14B, Tb2Fe14B, and Pr2Fe14B have similar crystal structures, hence their XRD patterns are also very similar. It was very hard to distinguish between all four RE2Fe14B phases with the help of XRD. Hence all these phases are marked as Nd2Fe14B (JCPSD #36-1296) in the XRD patterns shown in Fig. 2Fe14(AlCu)B. To evaluate the crystallinity of the of (Nd-RE)2Fe14(AlCu)B, SAED patterns 2Fe14B was detected. Any peak of Al or Al alloy was not detected in the XRD, however, in TEM-EDS image, Al was detectable 2Fe14(AlCu)B confirmed the homogeneous mixing of all RE, indicate the substitution of Dy, Pr, and Tb in the Nd2Fe14B crystal lattice 2Fe14B (low oxygen) are provided in supporting information as Figs. SEM images in Fig. 2Fe14B powders is ~ 0.4%. This one of the reasons that commercial (Nd-RE)2Fe14B powders exhibit excellent magnetic properties. In the rare earth based magnetic particles produced by the R-D, washing with the water is employed to remove the CaO, byproduct of the R-D process44. On average, (Nd-RE)2Fe14B magnetic particles produced by the reduction diffusion process contains ~ 2% of oxygen. It was concluded that oxygen content reduces the crystallinity Nd2Fe14B because no oxide is detected in the XRD patterns. To solve the oxidation problem, (Nd-RE)2Fe14B was washed in the glove box. Before washing the (Nd-RE)2Fe14B with the water in the glove box, nitrogen gas was blown in the water, which further removed the dissolved oxygen in the water. N2 was simply purged into the water at the rate of 25 ml/s for 40 min. This condition was taken from the work by Butler et al.45 as they have reported that more than 60% of the oxygen can be removed at this optimum condition. The glove box was filled with the Ar, with the oxygen level reduced to the ~ 50 ppm. By taking these preventive measures, the oxygen content of (Nd-RE)2Fe14B was reduced to ~ 0.7%. Figure 2Fe14B was washed in air. This further confirms that washing with the water reduces the crystallinity of the Nd too. It is well-known fact that the addition of the Nd phase (up to a certain limit) enhances the magnetic properties especially, coercivity12.Oxygen content in the commercial (Nd-RE)2Fe14B, hence their EDS mapping was not studied to avoid the complexity of data. It was found that oxygen content suddenly increased near the grain boundary because these parts of the grain are directly exposed to the water during the washing process. Pb and Sn detected in TEM-EDS come from the solder.LAADF-STEM images Fig. b–d were 2Fe14B crystal is yet unknown but most probably Cu and Al interfere during the diffusion of RE, Fe, and B. Poor crystallization after reduction-diffusion is the major factor that reduced the magnetic properties of (Nd-RE)2Fe14(AlCu)B. The second reason for the poor magnetic properties of (Nd-RE)2Fe14(AlCu)B is the non-magnetic behavior of Cu. Cu has an electronic configuration of [Ar] 3d10 4s1 which indicates the absence of unpaired electrons. Hence Cu and its alloys tend to be non-magnetic. In the bulk sintered (Nd-RE)2Fe14B magnets, Cu is used to decouple the large magnetic grains. Nd-Cu reduces the effect of the surge of domain wall removal in bulk magnets and enhances the coercivity. But in the case of magnetic particles, the grain decoupling phenomenon does not work efficiently. Hence non-magnetic Nd-Cu phase reduces the overall magnetic properties of the (Nd-RE)2Fe14(Cu-Al)B. Al is detectable in the final product but it is in the amorphous form. Effect of amorphous Al or its possible amorphous alloy on the magnetic properties is not clear.It is evident from the XRD patterns Fig. e that Cu2Fe14(AlCu)B > (Nd-RE)2Fe14B-(low oxygen) > (Nd-RE)2Fe14B. (Nd-RE)2Fe14(AlCu)B has highest Ms value, which indicates the presence of amorphous Fe or low anisotropy field. (Nd-RE)2Fe14B-(low oxygen) has slightly higher Ms value as compared to the (Nd-RE)2Fe14B that refers to the enhanced Mr value of (Nd-RE)2Fe14B-(low oxygen), which slightly affected the Ms. However, this enhancement in the Ms value is much higher as compared to the Ms value, which is also evident in the squareness ratio graph .Decreasing order of the Ms value in all the products is given as (Nd-RE)aph Fig. a showing2Fe14(AlCu)B exhibit the highest magnetic moment (21.6 μB) and Ms (1.057 T) value among all three products. Higher magnetic moment and low Mr value of (Nd-RE)2Fe14(AlCu)B refer to the low anisotropic field or presence of amorphous soft magnetic phase (e.g. Fe). Higher Mr value of (Nd-RE)2Fe14B-(low oxygen) contributes to its highest squareness ratio among all three products B, (Nd-RE)2Fe14B, and (Nd-RE)2Fe14B-(low oxygen) were determined as 21.06, 20.5, and 18.4 μB, respectively. These values of magnetic moments were determined by the Ms values from hysteresis loops nd Ms 1.07 T valuend Ms 1.07 T value2Fe14(AlCu)B, (Nd-RE)2Fe14B, and (Nd-RE)2Fe14B-(low oxygen) were determined as 242.71, 568.13 and 800.55 kA/m, respectively. (Nd-RE)2Fe14B-(low oxygen) showed the highest coercivity due to low oxygen content, better crystallinity, and least magnetic moment. Oxygen content values of (Nd-RE)2Fe14(AlCu)B, (Nd-RE)2Fe14B and (Nd-RE)2Fe14B-(low oxygen) are recorded as 2.2, 2.1 and 0.7%. The increasing order of coercivity is (Nd-RE)2Fe14(AlCu)B < (Nd-RE)2Fe14B < (Nd-RE)2Fe14B-(low oxygen) and the decreasing order of magnetic moment is (Nd-RE)2Fe14(AlCu)B > (Nd-RE)2Fe14B-(low oxygen) > (Nd-RE)2Fe14B. Individual Mr values of (Nd-RE)2Fe14(AlCu)B, (Nd-RE)2Fe14B, and (Nd-RE)2Fe14B-(low oxygen) were determined as 0.481, 0.489, 0.605 T, respectively. Mr (emu/g), Ms (emu/g), squareness ratio (Sq), magnetic moment (μB), and coercivity (Hc), for the all the products are shown in Fig. Reduction in magnetic moment enhanced the coercivity. From the hysteresis loops, coercivity values of (Nd-RE)2Fe14B sludge was recycled by four-step chemical process. The process consisted of leaching of sludge in H2SO4, removal of impurities by selective co-precipitation, annealing, and calciothermic reduction diffusion. Al3+ and Cu3+ were removed by co-precipitation at pH 6 and residual carbon was removed by annealing at 800 °C. CaO byproduct was separated by washing in the glove box, in presence of very low level of oxygen that reduced the oxidation of (Nd-RE)2Fe14B produced. Removal of impurities and low oxygen content (~ 50 ppm) tripled the coercivity (over 800 kA/m) and enhanced the Mr value up to 0.605 T. The Method reported in this study is simple, eco-friendly, and energy efficient.Contaminated NdSupplementary Information.


The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV.In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV.The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 μl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 μl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively.The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV. HoweverThe present study aimed to establish AC-ELISA and ICT strip methods using two MAbs to detect H9N2 AIV.All the viruses used in this study are described in Table Madin-Darby canine kidney (MDCK) cells and SP2/0 mouse myeloma cells were maintained in our laboratory. Purified HA protein from the H9N2 (A/chicken/Zhejiang/329/2011) subtype AIV was purchased from Sino Biological  .BALB/c mice (9 weeks old) were immunized with the purified H9N2 subtype AIV HA protein mixed Freund’s adjuvant  intramuscularly, twice at 3 weeks apart. After 6 weeks, the mice were immunized once more with HA protein by tail vein injection. After 3 days, the spleen lymphocytes of the selected mice were fused with SP2/0 cells , 44. TheIsotyping of the MAbs was performed using a Monoclonal Antibody Isotyping Kit . The affinities of each MAb were measured using ELISA, as described previously . In brieAn immunofluorescence assay (IFA) was used to visualize the binding of the MAbs to the virus-infected MDCK cells , 47. AftThe procedure for AC-ELISA Fig.  was descThe procedure for using the ICT strip is shown in Fig. The ICT strip contained an absorbent pad, nitrocellulose (NC) membranes, a MAb-gold conjugated, pad and a sample pad. The NC membranes were coated with MAb 2G7 as the test line and with goat anti-mouse IgG  as the control line.The specificity of the assays was tested using different subtypes of AIV (19 H9N2 AIVs and 25 other AIV subtypes), and other viruses . To determine the sensitivity, twofold serial dilutions of H9N2 AIV allantoic fluid and purified H9N2 proteins were used. A twofold serial dilution of H10N7 AIV allantoic fluid was used as the negative control. To evaluate repeatability, all samples were tested in triplicate, and all assays were repeated three times.To assess the clinical application of the two methods, 200 cloacal swabs (100 from chickens and 100 from ducks) collected from 10 poultry farms in Zhejiang Province were detected using multiplex qRT-PCR, AC-ELISA, and ICT strip methods. There were 20 samples for every farm. An Influenza A virus universal PCR kit  was used to perform the qRT-PCR assay .Five murine MAbs  were screened using ELISA. MAbs 3G4, 2G7, 1B12, and 1C12 belonged to the IgG1 subclass, and Mab 2F1 belonged to the IgG2a subclass. Among these MAbs, MAbs 3G4 and 2G7 were chosen to develop the detection method because of their high affinity . In the intra- and inter-batch repeatability tests, the coefficient of variation (CV%) was < 5% Table , which sThe specificity of the ICT strip was tested using H9N2 viruses and other non-H9N2 viruses, as described above. Only the H9N2 subtype AIV samples showed positive results Fig. a. The re3.15 -infected chicken eggs was used to test the sensitivity of the strip. The detection limit of the ICT strip was 100 μl of allantoic fluid containing a 4 HA titer of the virus or a median tissue culture infectious dose (TCID50) of 10To verify their stability, the ICT strips were tested after being assembled for 30 and 60 days, and the results revealed comparable specificity and sensitivity to newly assembled ICT strips.To assess the actual clinical application of the methods, cloacal swabs were collected from poultry in farms and subjected to analysis by qRT-PCR, AC-ELISA, and ICT strips Table . We usedEpidemic of H9N2 subtype AIVs have caused direct financial losses to the poultry industry and threatened human health . Since tThe AC-ELISA and ICT strip methods were developed to rapidly detect the H9N2 AIVs based on suitable MAbs. MAbs 3G4 and 2G7 were selected because of their high affinity. Although MAbs 3G4 and 2G7 did not show HI and VN activities, they reacted specifically with H9 HA antigens. This indicated that the MAbs might bind to a linear epitope on the HA antigen, which meets the diagnostic requirements . TherefoAn ICT strip to detect one strain of H9N2 virus was developed previously and showed good sensitivity and specificity . HoweverIn the detection of actual poultry samples, the AC-ELISA method achieved slightly higher specificity and sensitivity than the ICT strips . This miIn conclusion, the AC-ELISA and ICT strip methods were established using two MAbs to detect H9N2 subtype AIVs rapidly. The two methods have good specificity and sensitivity, and possessing important application value for the rapid detection of viral diseases, and could be further applied in clinical practice.


Neurodegenerative–neuroinflammatory disorders of the retina seriously hamper human vision. In searching for key factors that contribute to the development of these pathologies, we considered potential interactions among purinergic neuromodulation, glycinergic neurotransmission, and microglia activity in the retina. Energy deprivation at cellular levels is mainly due to impaired blood circulation leading to increased release of ATP and adenosine as well as glutamate and glycine. Interactions between these modulators and neurotransmitters are manifold. First, P2Y purinoceptor agonists facilitate reuptake of glycine by glycine transporter 1, while its inhibitors reduce reverse-mode operation; these events may lower extracellular glycine levels. The consequential changes in extracellular glycine concentration can lead to parallel changes in the activity of NR1/NR2B type NMDA receptors of which glycine is a mandatory agonist, and thereby may reduce neurodegenerative events in the retina. Second, P2Y purinoceptor agonists and glycine transporter 1 inhibitors may indirectly inhibit microglia activity by decreasing neuronal or glial glycine release in energy-compromised retina. These inhibitions may have a role in microglia activation, which is present during development and progression of neurodegenerative disorders such as glaucomatous and diabetic retinopathies and age-related macular degeneration or loss of retinal neurons caused by thromboembolic events. We have hypothesized that glycine transporter 1 inhibitors and P2Y purinoceptor agonists may have therapeutic importance in neurodegenerative–neuroinflammatory disorders of the retina by decreasing NR1/NR2B NMDA receptor activity and production and release of a series of proinflammatory cytokines from microglial cells. Neurodegenerative disorders occur with a high incidence in the elderly. These pathologies affect the motor system in Parkinson’s or Huntington’s diseases, memory in Alzheimer’s disease, or other additional critically important neural functions after impairments in cerebral blood circulation. The retina, which is commonly considered as an outside part of the central nervous system, is also affected by neurodegenerative disorders. Most of them may lead to loss of neurons in the retinal circuitry and consecutive impaired vision or blindness. Among the neurodegenerative diseases, we mention retinal hypoxia/ischemia, glaucomatous and diabetic neuropathies, and age-related macular degeneration and those that appear less frequently, such as human recessive retinitis pigmentosa or inherited photoreceptor degeneration. These disorders may occur as a sudden pathological event or show a progressively declining clinical course, but vision may be seriously hampered or lost in all disorders. Despite different clinical symptoms, there are a number of common factors in their pathogenesis, like neuroinflammation associated with neurodegeneration. Retinal hypoxia, as a persisting insult, may induce enhanced purinergic modulation and glutamatergic–glycinergic neurotransmission. Activation of the two systems may evoke sustained release of inflammatory mediators from activated microglia and the resultant chronic proinflammatory environment may induce development or exacerbation of retinal neurodegenerative disorders. There are several excellent review articles published on different aspects of the neurodegenerative pathologies of the retina ,2,3.In spite of the extended research effort, the currently used therapeutics only slow down the progression of retinal neurodegenerative disorders, whereas normalization of impaired visual functions can be rarely achieved. It is, therefore, mandatory to develop novel therapeutic interventions. This goal, however, cannot be reached without unfolding the pathophysiology of retinal neurodegenerative diseases. The aim of this review is to highlight a possible interaction between purinergic and glycinergic signaling systems in neuroinflammatory–neurodegenerative disorders of the retina. This interaction is, however, further complicated by the findings that microglia, the resident immune cells of the central nervous system and retina, are involved not only in neuronal cell destructions but also in compensatory neural repair following insulting influences ,5.Besides the neural circuits, the retina also contains numerous glia cells termed macroglia (Müller cells and astrocytes) and microglial cells. The principal macroglia Müller cells span all retinal layers, whereas astrocytes are mostly located close to the nerve fiber layer and the ganglion cell layer ,7,12. MüThe other major type of retinal glia cell is microglia. They originate from macrophage or monocyte precursors and are considered as the major immunocompetent cell type of the retina . In healMicroglia are the resident immune cells of the central nervous system as well as the retina. Microglia exist in different forms. Resting microglia are derived from macrophage-like cells and they can be characterized by small cell bodies and multiple branched processes arising from the cell bodies with down-regulated macrophage-like properties . MicroglThe shape of microglial cells turns to be amoeboid during microglial activation in retinopathological events and cellular hypertrophy and process retraction can be also observed . The amo+ channel expression: Resting microglial cells do not express voltage-activated K+ channels, their expression together with P2Y purinoceptors can be observed following microglia activation glutamate and [3H]glycine release in the retina ensuring excess availability of the agonist and co-agonist at NR1/NR2B-type NMDA receptors glycine release in isolated rat retina and this effect was due to an inhibition of the reverse-mode operation of GlyT-1. Besides inhibition of the reverse mode operation of GlyT-1, stimulation of normal mode operation also leads to a decrease of extracellular glycine concentrations and consequently to inhibition of overactivated NMDA receptors.Besides these self-strengthening mechanisms, attenuative regulations can also be observed in the proposed tripartite interaction. Accordingly, activated microglia are altered in order to express purinoceptors of the P2Y type and their activation decreases the production and release of proinflammatory cytokines and progress of neuroinflammation may slow down . Moreove1,12,13 purinoceptor agonist 2-MeS-ADP stimulated the uptake  mode operation of GlyT-1 in rat brainstem primary neuronal cultures. This effect was partially reversed by the P2Y1 antagonist MRS2179, the P2Y13 antagonist MRS2211, and the non-selective P2 purinoceptor antagonist suramin (1 and Gi protein-coupled P2Y13 purinoceptors in the regulation of GlyT-1 activity with an apparent P2Y1 purinoceptor dominance. Thus, besides GlyT-1 inhibition, P2 purinoceptor stimulation may also decrease extracellular glycine concentrations by stimulation of the normal-mode operation of GlyT-1. As shown in 3H]glycine release from rat retina. While we consider our finding preliminary and in need of further confirmation using more selective receptor ligands, we speculated that ATP stimulating P2Y purinoceptors forces GlyT-1 operation into the uptake mode. The resulting decrease in extracellular glycine concentrations may then prevent overactivation of NMDA receptors and thereby attenuate the exocytotic cascade. The biological substrate for the cross-talk between purinoceptors and GlyT-1 might be the astrocytes, which express P2 type purinoceptors as well as GlyT-1 immunoreactivity in the retina [Jiménez  and cowo suramin . This fie retina ,79.The third participant in this tripartite interaction is microglia. Concerning retinal pathologies, in which glutamatergic–glycinergic neurotransmission and ATP/adenosine-mediated signaling are functional, the presence of activated microglia has also been demonstrated. Induction of proinflammatory cytokine production may be deleterious to injured cells after ischemic insults as was shown in retinal vascular occlusion and reperfusion . MicroglThe tripartite interaction between purinergic modulation, glutamatergic–glycinergic neurotransmission, and microglia activation provides three therapeutic target pathways in neuroinflammatory–neurodegenerative disorders of the retina. First, GlyT-1 inhibitors ,113 bloc1 adenosine receptor agonists and A2 adenosine receptor antagonists [Second, P2Y purinoceptor agonists and P2X purinoceptor antagonists inhibit micoglial activity and decrease the production and release of TNFα and other proinflammatory biological substances. Thus, modulation of microglial reactivity by P2Y agonists and P2X antagonists has emerged as a possible therapeutic intervention to maintain neurodegenerative courses at a low level. A similar effect might be expected from Aagonists .Third, microglia activation inhibited by minocycline may suppress the damage of retinal ganglion cells in ischemia and glaucoma . Moreove


The odds ratio of hearing loss was significantly higher in subjects with four components of metabolic syndrome: waist circumference, blood pressure, and triglyceride and fasting blood sugar concentrations (p < 0.0001). Conclusions: The number of components of the metabolic syndrome is positively correlated with the rate of sensorineural hearing loss.The prevalence of sensorineural hearing loss has increased along with increases in life expectancy and exposure to noisy environments. Metabolic syndrome (MetS) is a cluster of co-occurring conditions that increase the risk of heart disease, stroke and type 2 diabetes, along with other conditions that affect the blood vessels. Components of MetS include insulin resistance, body weight, lipid concentration, blood pressure, and blood glucose concentration, as well as other features of insulin resistance such as microalbuminuria. MetS has become a major public health problem affecting 20–30% of the global population. This study utilized health examination to investigate whether metabolic syndrome was related to hearing loss. Methods: A total of 94,223 people who underwent health check-ups, including hearing tests, from January 2010 to December 2020 were evaluated. Subjects were divided into two groups, with and without metabolic syndrome. In addition, Scopus, Embase, PubMed, and Cochrane libraries were systematically searched, using keywords such as “hearing loss” and “metabolic syndrome”, for studies that evaluated the relationship between the two. Results: Of the 94,223 subjects, 11,414 (12.1%) had metabolic syndrome and 82,809 did not. The mean ages of subjects in the two groups were 46.1 and 43.9 years, respectively. A comparison of hearing thresholds by age in subjects with and without metabolic syndrome showed that the average pure tone hearing thresholds were significantly higher in subjects with metabolic syndrome than in subjects without it in all age groups. ( Sensorineural hearing loss is an important public health problem whose prevalence has increased as life expectancy has become longer . For exaMetabolic syndrome is a disease that includes hypertension, central obesity, hyperlipidemia, and diabetes . MetabolThis study included a total of 94,223 people, ranging in age from their 20s to their 70s, who underwent health checkups, including hearing tests, at a tertiary university hospital from January 2010 to December 2020. Height, weight, body mass index, waist circumference and blood pressure (both systolic and diastolic) were measured in all subjects, and all subjects underwent hearing and blood tests and pulmonary function tests (PFT). Subjects without hearing test results and those with a ≥ 20 dB difference between the two hearing thresholds, a history of surgery for otitis media, or suspected central disease were excluded. The study protocol was approved by the Institutional Review Board of Kyung Hee University Medical Center (KMC 2019-07-065).The criteria for metabolic syndrome were defined according to the revised National Cholesterol Education Program Adult Treatment Panel III:  (1) waisHearing threshold tests were performed at frequencies of 500, 1000, 2000, 3000, 4000, and 6000 Hz, measured in that order, followed by a retest at 1000 Hz, with air conduction measured at a frequency of 500 Hz. Pure tone audiometry was performed using the standard hexadecimal method, by measuring the air conduction values of each ear at four frequencies  and calculating their sum using the equation: (500 Hz + 2 × 1000 Hz + 2 × 2000 Hz + 4000 Hz)/6 ,15,16,17To systematically review the results of studies on the correlation between hearing loss and metabolic syndrome, the Scopus, Embase, PubMed, and Cochrane library databases were searched using the keywords “hearing loss” and “metabolic syndrome”. A total of 1373 papers were identified initially, including 390, 612, 364, and seven, respectively, in these databases. Of these, 1300 papers were excluded due to lack of relevance in the title, and an additional 44 papers were excluded due to duplication of papers. Of the 29 papers reviewed in full, 17 were included in the systematic analysis .p values < 0.05 defined as statistically significant.Subjects were divided into two groups, those with and without metabolic syndrome, for analysis of clinical results, and into six groups by age, 20s, 30s, 40s, 50s, 60s, and 70s and over, for analysis of average hearing threshold and degree of hearing loss. To evaluate risk factors related to hearing loss, subgroups were classified according to the number of factors corresponding to the diagnostic criteria for metabolic syndrome. Continuous variables were compared by t-tests and categorical variables by chi-square tests and Fisher’s exact tests. Normal and adjusted odds ratios (ORs) of the relationships of hearing loss with age and number of factors of metabolic syndrome were analyzed by univariate and multivariate logistic regression analyses. All statistical analyses were performed using IBM SPSS version 22 , with p < 0.001). Waist circumference, BP, and triglyceride and fasting blood glucose concentrations were all significantly higher in subjects with metabolic syndrome than in subjects without it. (p < 0.001)  had metabolic syndrome and 82,809 (87.9%) did not. The mean ages of these two subgroups were 46.1 and 43.9 years, respectively. Of the 11,414 subjects with metabolic syndrome, 8255 (72.3%) were men, whereas, of the 82,809 subjects without metabolic syndrome, 45,200 (54.5%) were men, making the percentage of men significantly higher in subjects with metabolic syndrome (< 0.001) .p < 0.001) (p < 0.05)  . In addi < 0.05) .p < 0.0001)  .p < 0.0001). Relative to subjects with 0 components of metabolic syndrome, the odds ratios of hearing loss in subjects with 3, 4, and 5 components of metabolic syndrome were 1.06 (p = 0.1119), 1.29. (p < 0.0001), and 1.21 (p = 0.1410), respectively (To correct for the effects of age and gender on the relationship between BMI (Body Mass Indx) and hearing loss, multivariate analysis was performed to determine the normal and corrected odds ratios of hearing loss. The odds ratio of hearing loss was 1.89-fold higher in women than in men, and it was 1.13-fold higher as age increased by 1 year (ectively .p < 0.0001)  .This cross-sectional study involving a large population assessed the relationship between metabolic syndrome and hearing loss. This study is based on health examination data for relatively healthy patients. In this study, 12.1% or 11,413 subjects met the diagnostic criteria for metabolic disease. First, due to the nature of this study, we cannot rule out the possibility that the study population was biased toward people interested in health. In addition, it is possible that there were fewer subjects who met the criteria for metabolic syndrome because at the time of assessment subjects may have been taking medications to control blood pressure, blood sugar, and/or other blood levels. However, the results of this study are meaningful because sufficient number of subjects, 94,223 was included.To compare the incidence of hearing loss in adults with and without metabolic syndrome after adjustment for age, the rates of hearing loss were analyzed in subjects with 3, 4, and 5 of the components of metabolic syndrome, and the factors most closely related to hearing loss were identified.The result of this study demonstrated an association between hearing loss and metabolic syndrome. The threshold of PTA was significantly higher in the group with metabolic syndrome in Although the mechanism(s) underlying the correlation between metabolic syndrome and hearing impairment remain undetermined, peripheral vascular disorders may be associated with both of these conditions.Analysis of data from the US National Nutrition Survey showed a link between hearing loss and high blood pressure . HyperteDiabetes is associated with microvascular and neuropathy complications affecting the retina, kidneys, peripheral arteries, and peripheral nerves ,32. PathRecent studies have shown that increases in triglyceride concentrations are closely associated with reduced hearing function . VacuolaObesity has been associated with hearing loss in both humans and animals. Central obesity, increased waist circumference, and increased hearing threshold after BMI correction of the content of visceral adipose tissue were found to be related . ObesitySeventeen previous studies have examined the relationship between metabolic diseases and hearing loss. Nine of these studies analyzed the correlation between the overall components of metabolic syndrome and hearing loss; four analyzed the correlations between indices such as BMI, WHR, and factors relevant to abdominal fats (FRA) and hearing loss; three analyzed the correlations of diabetes DM and HbA1c concentration with hearing loss, and one analyzed the correlation of TG/HDL ratio with hearing loss. Of these 17 studies, 12 were cross-sectional in design, two were prospective studies, two were retrospective studies, and one as a case-control study. Most of the study subjects were middle-aged.One study reported that metabolic syndrome was related to high-frequency hearing loss in a noise-exposed population . Two stuMany studies have found a correlation between HDL and hearing loss. For example, one study in US adults found that low HDL correlated with hearing loss, with low HDL being most responsible for the correlation between hearing loss and metabolic syndrome.  Two studSeveral studies also observed relationships between obesity-related anthropometric indices and hearing loss. For example, one study found that WHR may be a surrogate marker for predicting the risk of hearing loss,  whereas This study had several limitations. First, because it was a cross-sectional study, the causative relationships between hearing loss and components of metabolic syndrome could not be determined. Second, because these data were from health examinations, the correlation between the prevalence of metabolic syndrome and hearing loss could not be accurately determined. Third, this study did not evaluate subjects who received medical treatment for hearing loss, nor did it evaluate factors contributing to hearing loss, such as noise, ototoxic drugs, otitis media, and family history of hearing loss.This cross-sectional study involving a large population analyzed the association between metabolic syndrome and hearing loss. Hearing loss showed a positive correlation with the number of diagnostic factors for metabolic syndrome, especially in subjects with four specific factors: high waist circumference, blood pressure, triglyceride concentration, and fasting blood glucose concentration.This study highlights the importance of control of metabolic syndrome in management of hearing loss. Subjects with metabolic diseases should therefore undergo regular hearing tests and, if necessary, hearing rehabilitation along with the management and treatment of their metabolic diseases. In addition, patients and health professionals may not be aware of this information regarding association between metabolic syndrome and hearing loss; hence, this can be included in part of health education.