Apis mellifera) viruses at the colony level, the dynamics of virus infection within colonies remains poorly explored. In this study, the two main variants of the ubiquitous honey bee virus DWV as well as three major honey bee viruses  were analyzed from Varroa-destructor-parasitized pupae. More precisely, RT-qPCR was used to quantify and compare virus genome copies across honey bee pupae at the individual and subfamily levels . Additionally, virus genome copies were compared in cells parasitized by reproducing and non-reproducing mite foundresses to assess the role of this vector. Only DWV was detected in the samples, and the two variants of this virus significantly differed when comparing the sampling period, colonies and patrilines. Moreover, DWV-A and DWV-B exhibited different infection patterns, reflecting contrasting dynamics. Altogether, these results provide new insight into honey bee diseases and stress the need for more studies about the mechanisms of intra-colonial disease variation in social insects.RNA viruses play a significant role in the current high losses of pollinators. Although many studies have focused on the epidemiology of western honey bee ( RNA viruses stand amongst the most diverse and widespread disease-causing agents ,2. In thApis mellifera) [Varroa destructor. In fact, while feeding on honey bees, Varroa can act as a vector for highly virulent viruses [V. destructor also influences the immune status of its host, triggering uncontrolled replication through immunosuppression [V. destructor, including the ubiquitous Deformed wing virus (DWV), Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute bee paralysis virus (IAPV)  and Sacbrood virus (SBV) are also often detected in A. mellifera colonies [RNA viruses play a major role in the pollinator losses currently observed across the globe ,12. Manyllifera) ,14. In t viruses ,16. V. dpression ,18. As apression ,20,21,22e et al. ). Due toe et al. , Black qcolonies ,26.A. mellifera colonies display genetic heterogeneity across individuals emerging from the same queen, which may be of high relevance when studying pathogens [In honey bee epidemiology, the colony is often considered the basic unit, suggesting that viral loads are uniform among individuals from the same hive. However, due to their polyandrous mating system, athogens . Thus, wathogens , queens athogens ,30. Thisathogens . HoweverA. mellifera pupae of known patrilines across distinct periods and under different V. destructor infestation statuses. The results documented here reveal new insights into the ecology of honey bee viruses and stress the need to further consider intra-colonial heterogeneity in disease infection for social insect research.The aim of this study was to analyze the patterns of infection of some of the major honey bee RNA viruses at the intra-colonial level. This was achieved by comparing virus infection levels between individual A. mellifera were sampled in the INRAE institute’s apiary  from the end of August to the end of October 2018 to study the relationships between mite infestation and worker subfamilies .Colonies of ies (see ). In briThe phenotyped individuals were dissected directly upon sampling. DNA from 486 collected pupae (purple eye stage) was isolated using a hind leg per individual according to the Chelex extraction protocol described by Walsh et al. . The patV. destructor, originated from three colonies with a total of 36 samples per colony. Accordingly, the 36 selected pupae per colony belonged to 3–4 distinct subfamilies  was used according to the manufacturer’s protocol.Each pupa was crushed in 800 µL of QIAzol  containing a 9 mm silver bead using a TissueLyser (Qiagen) at 30 Hz for 30 s with 30 s intervals and repeated four times, according to the protocol described in Dalmon et al. . The tub® spectrophotometer  and normalized at 500 ng/µL. Reverse transcription was performed with the high-capacity RNA to cDNA kit  using 1 µg RNA in 20 µL total reaction volume containing random primers according to the manufacturer’s protocol.The RNA was quantified using a Nanodrop®, Thermo Scientific, Illkirch, France). Three microliters of ten-fold diluted cDNA were added to 5 µL of SYBR Green master mix (Applied Biosystems). Five reactions were conducted independently, each containing 10 µM of forward and reverse virus and variant-specific primers [The number of virus genome copies of each virus was determined by quantitative PCR using a StepOne-Plus Real-Time PCR System  were collected during the sampling, virus genome copies were compared between the different age categories using a linear regression to test whether virus infection slopes differed significantly from zero. These tests were non-significant, thereby enabling the grouping of samples of different ages for subsequent analyses . As the Differences of virus genome copies among the three colonies and among the patrilines within each colony were tested using non-parametric tests: Wilcoxon–Mann–Whitney tests were used when comparing two groups, Kruskal–Wallis tests were used to compare >2 groups together and Dunn tests were used in the case of significant differences for subsequent pairwise comparisons. Notably, only groups with at least six samples were considered for these statistical analyses.Finally, the number of virus genome copies of the colonies was compared to the mite infestation level in the brood in the same matching period. Moreover, as all pupae chosen for the experiment were infested with a single foundress mite, successfully reproducing or otherwise, additional Wilcoxon–Mann–Whitney tests were applied to assess the effect of mite reproduction on the virus genome copies.10 genome copies/pupa) or strongly (>1012 genome copies/pupa) infected , only one (DWV) was found to be over the detection thresholds . All saminfected . Interesinfected .The comparison of the DWV variants genome copies across the two collection periods revealed contrasting temporal patterns in the two colonies tested. Significant increases in DWV-A were detected in both colonies, while the levels of DWV-B did not change significantly over time . Given tWhen analyzing the DWV-A and DWV-B infection levels across the colonies, significant differences were found during the first sampling period between colony B and the other two colonies .Comparing the levels of DWV variants between the different subfamilies within colonies and sampling periods revealed some significant differences across the groups tested. More precisely, significant differences were found when comparing two and three patrilines for their infection levels with DWV-A and DWV-B, respectively . Interesp > 0.05, When comparing the level of mite infestations of the colonies with the number of virus genome copies of DWV-A and DWV-B, similar patterns were found across the colonies and time periods. More precisely, virus genome copies of both variants appeared to be positively correlated to levels of mite infestations in the brood for two colonies (B and E), but not for the third colony (D) . FinallyA. mellifera colonies. Although four common viruses were screened for, only one (DWV) was detected. Investigating two variants of this virus (DWV-A and DWV-B) revealed novel insights into the biology of this major bee pathogen by unravelling new features of its epidemiology across time, colonies, subfamilies and individuals.In this study, patterns of infections by honey bee viruses were investigated within A. mellifera pupae sampled in fall in France was also documented in another study [To date, the natural infection of honey bee viruses has been principally studied in adults, and very rarely in the brood . In the er study . Given ter study ,39, reco10 genome copies/pupae) or strongly (>1012 genome copies/pupae) infected. Furthermore, investigations of the relationships between the two variants of DWV at the individual level revealed four distinct groups of pupae: high infections with both variants, moderate infections with both variants and both cases of high infection with one strain and moderate infection with the other. Such puzzling patterns have been described in previous studies, with DWV-A and DWV-B showing a bimodal distribution between symptomatic and asymptomatic adult honey bees [6–8 DWV-A copies/bee and 109–10 DWV-B copies/bee [V. destructor foundress and from the absence of treatments against this DWV-vector for more than one year in the colonies prior to sampling.First, the analysis of DWV variant infection levels overall, as well as between the analyzed colonies, revealed that the pupae were either moderately  display lower virus infection levels in the brood [A. mellifera population displays tolerance or resistance to virus infections.Two of the colonies analyzed here (colonies D and E) originated from a Varroa-surviving population , while the brood . HoweverV. destructor, the vector was probably not responsible for the differences observed. This suggests that other factors governing the dynamics of the two DWV variants might explain the temporal differences observed when comparing DWV-A and DWV-B over the sampling periods. For instance, differential mortality associated with the two variants [As DWV variant levels differed across colonies, the temporal dynamics of virus genome copies were analyzed at the colony level. This analysis revealed a variation of temporal infection between the two DWV variants, with significant differences in virus genome copies detected between the two sampling periods when comparing the levels of DWV-A, though not of DWV-B. As similar patterns were detected when comparing the genome copies of the two variants to the colony-level brood infestation with variants ,47 mightvariants .V. destructor foundress and were collected on the same frame). Moreover, when individuals were grouped by subfamilies, significant differences in DWV-A and/or DWV-B genome copies were found. Notably, the significant variation of virus genome copies across subfamilies is in line with previous work showing differential infection in honey bee subfamilies with two other pathogens: Paenibacillus larvae [Ascosphaera apis [in ovo virus infection’ showed that virus resistance has a strong genetic component in A. mellifera [A. mellifera pupae and stresses the need for more studies to identify the source of this variation.The results of this study also showed that patterns of DWV-A and DWV-B infections can differ significantly across honey bee pupae, even when these hosts belong to the same colony and share the same environment . Colonies are often considered an epidemiological unit, but this might fail to account for the variability described here, leading to erroneous conclusions. In this study, potential co-evolutionary interactions between hosts and virus genotypes may have caused the differential interactions between the two variants of DWV and the individual and subfamily levels of infections. If parental genetic components are indeed responsible for variations in virus infection levels, new avenues for the breeding and selection of more disease-resilient honey bee colonies can be explored. This could contribute to ensuring more sustainable beekeeping globally, as well as providing new knowledge on host–pathogen interactions and the importance of genetic diversity in disease epidemiology.


Results: We found that MLIV patients had prominently increased cytokine levels compared to familial controls and identified profiles of cytokines correlated with motor dysfunction, including IFN-γ, IFN-α2, and IP-10. We found that IP-10 was a key differentiating factor separating MLIV cases from controls based on data from human plasma, mouse plasma, and mouse brain. Conclusions: Our data indicate that MLIV is characterized by increased blood cytokines, which are strongly related to underlying neurological and functional deficits in MLIV patients. Moreover, our data identify the interferon pro-inflammatory axis in both human and mouse signatures, suggesting that interferon signaling is an important aspect of MLIV pathology.Background: Mucolipidosis IV (MLIV) is an autosomal recessive pediatric disease that leads to motor and cognitive deficits and loss of vision. It is caused by a loss of function of the lysosomal channel transient receptor potential mucolipin-1 and is associated with an early pro-inflammatory brain phenotype, including increased cytokine expression. The goal of the current study was to determine whether blood cytokines are linked to motor dysfunction in patients with MLIV and reflect brain inflammatory changes observed in an MLIV mouse model. Methods: To determine the relationship between blood cytokines and motor function, we collected plasma from MLIV patients and parental controls concomitantly with assessment of motor function using the Brief Assessment of Motor Function and Modified Ashworth scales. We then compared these profiles with cytokine profiles in brain and plasma samples collected from the  MCOLN1 gene. Patients typically present with delayed developmental milestones in the first year of life and reach a plateau in psychomotor function equivalent to the 18-to-20-month range [Mcoln1 knockout mice  [Mucolipidosis type IV (MLIV) is a neurodevelopmental and neurodegenerative disorder caused by the loss of function of mucolipin 1 (TRPML1), a lysosomal channel encoded by the −/−, KO) ,4,5. AtyMcoln1−/− mice demonstrate a paucity of subcortical white matter, hypoplasia/dysgenesis of the corpus callosum, and variable white matter lesions [Mcoln1−/− mice primarily manifest in the form of reduced myelination, astrocytosis, microgliosis, and partial loss of cerebellar Purkinje cells, while cortical neuron populations are largely unaffected [MLIV results in a hypomyelinating leukodystrophy with iron accumulation in the basal ganglia and progressive cerebellar atrophy. Brain imaging in MLIV patients and histopathological evaluation of  lesions . In pati lesions . In paraaffected ,8,9.Mcoln1−/− mice early in the course of disease, before the functional symptoms first emerge [Mcoln1−/− mice, suggesting that the interferon pathway may be strongly linked to disease progression in MLIV.We previously found that signs of neuroinflammation, including astrocytosis, microgliosis, and increased expression of numerous pro-inflammatory cytokines/chemokines, are present in the brains of t emerge . Becauset emerge ,12 and dt emerge , we reasMcoln1−/− mice. We hypothesized that interferon levels would be strongly linked to motor dysfunction in human MLIV patients. To test this, we collected blood plasma from MLIV patients and familial controls while simultaneously measuring gross and fine motor scores in MLIV patients. By quantifying 41 cytokines/chemokines in the plasma, we found that cytokines were broadly upregulated in MLIV patients compared to familial heterozygous controls. Moreover, among MLIV patients, those with reduced fine or gross motor function had robustly increased blood cytokines, including TNF-α, IFN-γ, and IFN-α2. To test whether these findings would translate to mice, we also analyzed 32 cytokines in blood plasma from Mcoln1−/− mice at one, two, and six months of age, corresponding to the presymptomatic, early-symptomatic, and late stages of the disease in mice. Our analysis revealed a robust proinflammatory cytokine profile that changed with age, including trending or significantly increased IP-10 at all time points. Finally, we found that several blood cytokines from human or mouse plasma overlapped with cytokines expressed in the MLIV mouse brain, including IP-10, suggesting that these cytokines may be candidate biomarkers of brain pathology in MLIV. In total, our findings reveal that MLIV blood cytokine signatures are strongly associated with the severity of motor dysfunction in patients and suggest that peripheral cytokine signature may reflect the brain’s neuroinflammatory milieu.Because interferons are known to pass through the blood–brain barrier, we set out to identify cytokine/chemokine signatures in plasma from MLIV patients and N = 18, F = 9 and M = 9) and familial controls  were recruited through the Mucolipidosis Type IV Foundation. Written informed consent was obtained from legal guardians for participation in our approved natural history and biomarkers studies according to protocols approved by the Massachusetts General Hospital Institutional Review Board. Written informed consent or assent were obtained from patients if their neurological capacity allowed. Inclusion criteria for patients included a documented diagnosis of MLIV by (1) clinical or research-based sequencing of MCOLN1 and identification of two pathological MCOLN1 alleles or (2) presence of the expected constellation of clinical symptoms associated with MLIV and documentation of at least one of the following: one pathological MCOLN1 allele, elevated gastrin levels, or a tissue biopsy with evidence of lysosomal inclusions consistent with MLIV. One familial control was excluded from cytokine analysis when it was later revealed that this subject had undergone a surgical procedure in the week prior to sample collection, which could affect cytokine levels. MLIV cases were assigned to either typical (N = 15) or mild (N = 3) presentation. Based on our clinical experience, we classified patients as mild if they had attained independent ambulation at some point in life. All patients underwent a full examination by a single board-certified pediatric neurologist, and their function was scored with the Brief Assessment of Motor Function (BAMF) scales  and Modified Ashworth scale. Due to limitations associated with patient access and cost, repeat scoring by independent evaluators was not possible.Patients with MLIV (2EDTA-coated Purple/Lavender top vacutainers (BD 368047). Plasma was isolated within 4 h from collected blood samples by centrifugation at 1000 g for 10 min at 4 °C. Isolated plasma was stored at −80 °C.Blood was collected in KMcoln1−/− knockout (KO) mice (on a C57Bl/6J background) were maintained and genotyped as previously described [−/−Mcoln1 (KO) and littermate +/+Mcoln1 (WT) controls at 1 , 2 , and 6  months of age, and plasma was isolated within 4 h from collected blood samples by centrifugation at 1000g for 10 min at 4 °C. Isolated plasma was stored at −80 °C. All experiments were performed according to the U.S. National Institutes of Health guidelines and approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.escribed ,8,9. Theescribed , and no Plasma was stored at −80 °C. For human cytokine analysis, samples were diluted to 8% in Milliplex assay buffer and analyzed using the Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel—Premixed 41 Plex kit . Mouse plasma was diluted to 70% in assay buffer and analyzed using the Milliplex MAP Mouse Cytokine/Chemokine Multiplex assay . Assays were read out with a MAGPIX Luminex instrument .Dunn’s test was performed in R  and adjusted using a Bonferroni correction for multiple comparisons. Wilcoxon rank sum test was performed in R. Partial least squares regressions (PLSRs) and discriminant PLSRs (D-PLSRs) were performed in R using the ropls package v1.4.2. The data were z-scored before input into the function. Cytokine measurements were used as the independent variables, and the discrete regression variable in all D-PLSR analyses was genotype/phenotype. Orthogonal rotations were applied to the sample scores and analyte weightings to obtain consistent separation of each group along the LV1 and LV2 axes. Error bars for LV loadings were calculated by iteratively excluding K samples without replacement 100 times , and regenerating the D-PLSR model each time. Error bars in the LV1 plots report the mean and SD computed across the models generated to provide an indication of the variability within each cytokine among the models generated. Sample outliers were checked for and removed in the mouse cytokine data by performing a principal component analysis on the data and iteratively removing data points that fell outside of a 99.5% confidence ellipse .Mcoln1−/− mice [Having previously identified robust neuroinflammatory changes in the CNS of −/− mice , here, w−/− mice . The D-PGiven the group-wise differences between typical and mild MLIV patients, we next asked if plasma cytokine levels were related to clinical disease severity. To test this, we performed an analysis of plasma cytokine levels and motor function scores for each patient. We used the Brief Assessment of Motor Function (BAMF) ,15,16,17To identify the cytokines that were most strongly associated with the BAMF and Modified Ashworth scores, we next used partial least squares regression (PLSR) analysis to separate patients with better motor scores to the right and with reduced motor scores to the left C,D. InteMcoln−/− mice exhibit a similar relationship between plasma cytokines and progression of disease. Identification of a relationship between cytokine levels and disease state in mice will enable the use of cytokines as biomarkers for pre-clinical testing. To test this relationship, we collected plasma from mice at 1, 2, and 6 months of age in male  from wild-type mice based on the LV1 profiles, including the 26 cytokines that overlapped between the human and mouse panels. The coincident cytokines, IP-10, IL-9, Eotaxin, IL-17, IL-13, VEGF, and IL-1α, were selected based on having mean values on LV1 (≥0.2) and coefficients of variation (<1)  B. This rion (<1) D. Additiion (<1) C to analthe left E. We conthe left . This anMcoln1−/− mouse brain and demonstrated profoundly altered peripheral cytokine levels in the plasma of Mcoln1−/− mice and MLIV patients. Based on previous evidence of neuroinflammation in the brains of early symptomatic Mcoln1−/− mice and the ability of cytokines to pass through the blood–brain barrier, we aimed here to establish peripheral cytokine profiles in MLIV patients and Mcoln1−/− mice and to test whether peripheral cytokines correlate with disease progression. Indeed, our data showed that a profile of cytokines in blood plasma correlated with a loss of gross and fine motor function as well as increased muscle tone. Further, we found an overlap between cytokine signatures in plasma collected from MLIV patients and Mcoln1−/− mice and between plasma signatures and the Mcoln1−/− mouse brain. Taken together, our data suggest that plasma cytokine profiles reflect neuroinflammation in the CNS of MLIV patients. Further understanding of the roles of these cytokine alterations in disease pathogenesis may provide an accessible means of monitoring disease progression or suggest new targets for therapeutic intervention.In this study, we extended upon our previous finding of disrupted cytokine homeostasis in the Mcoln1−/− mice, including presymptomatic (1 month of age), early-symptomatic (2 months) and late (6 months) time points, and identified the top dysregulated cytokines for each of them  and Modified Ashworth scales. These signatures included elevations of IL-2, TNF-α, MIP-1β, IFN-γ, and IFN-α2 C, which  of them . We did with age . Startinrod test  and worsrod test . Motor drod test ,26. Thernd IP-10 C) have bTRPML1 has a well-established role in regulating a broad range of lysosomal functions ,30, and Though several studies demonstrate the role of TRPML1 in regulating immune cell functions in in vitro systems ,32,33,35Overexpression of cytokines, including interferons, in the brain during neurodegenerative process has led to studies determining whether interferons and other cytokines can pass through the blood–brain barrier, as well as whether cytokines may serve as a blood biomarker of neuroinflammatory dysfunction or neurodegeneration, e.g., in Alzheimer’s disease .Collectively, our mouse and human data suggest an intricate relationship among neuroinflammation, peripheral cytokines, and loss of motor function in MLIV. Although our data do not reveal whether the commonalities between the brain and periphery are due to cytokines crossing the blood–brain barrier or due to a common mechanism between the brain and peripheral compartments, they do reveal that the peripheral cytokine signature is strongly associated with motor dysfunction in MLIV. Given this robust relationship and the likelihood that new therapies will simultaneously target the brain and peripheral compartments, cytokines hold strong potential to serve as an early indication of patient response to forthcoming therapeutic strategies for this devastating disease.


Our study underlines the need for active monitoring and surveillance of PEDV in herds and their environments, along with the coordinated means, to eliminate the virus and maintain a negative herd. The tools described in this study will serve as a framework for regional and national PED eradication programs.Porcine epidemic diarrhea virus (PEDV) has negatively affected the welfare of animals and their productivity in South Korea for three decades. A shortage of effective control measures has led to the virus becoming endemic in domestic pig populations. This study aimed to describe how our intervention measures were implemented for PEDV elimination in an enzootically infected farm. We operated a risk assessment model of PEDV recurrence to obtain information about the virus itself, herd immunity, virus circulation, and biosecurity at the farm. Next, we conducted a four-pillar-based two-track strategy to heighten sow immunity and eradicate the virus, with longitudinal monitoring of immunity and virus circulation, involving strict biosecurity, prime-boost pre-farrow L/K/K immunization, all-in-all-out and disinfection practices in farrowing houses, and disinfection and gilt management in wean-to-finish barns. In particular, we observed a high prevalence and long-term survival of PEDV in slurries, posing a critical challenge to PED eradication and highlighting the necessity for consecutive testing of barn slurry samples and for the management of infected manure to control PEDV. Genetic analysis of PEDVs in this farm indicated that genetic drift continued in the spike gene, with a substitution rate of 1.683 × 10 Alphacoronavirus within the family Coronaviridae of the order Nidovirales. The virus possesses a single-stranded, positive-sense RNA genome of ~28 kb that encodes 16 nonstructural proteins (nsp1–16); 4 canonical coronaviral structural proteins, including the spike (S) glycoprotein; and a single accessory gene, ORF3 [Porcine epidemic diarrhea virus (PEDV), a highly infectious swine coronavirus, is the causative agent of porcine epidemic diarrhea (PED), which has a deadly effect on neonatal pigs in the first week of life, causing watery diarrhea, vomiting, fatal dehydration, and high mortality ,2. PEDV PEDV was first described in South Korea three decades ago and it has persisted as an endemic pathogen, shifting from seasonal (winter) PED to a year-round disease and causing small- to large-scale outbreaks throughout the nation ,2,5. TheVaccination or intentional virus-exposure (feedback) practices have been implemented nationwide to manage PED by producing herd immunity, but PEDV has continued to impair swine health, leading to substantial financial losses within the domestic pork business. A large number of PED-affected swine farms have experienced recurrent outbreaks within one or two years, a situation which indicates that endemic or chronic infection of resident PEDV has become established on the farms, increasing the economic damage done to South Korea. When a disease becomes endemic, it is important to implement coordinated control measures (four pillars) to interrupt the chain of disease transmission at the farm level. These measures involve: 1) tightening biosecurity protocols and disinfection practices; 2) testing populations for serological and virus monitoring to check herd immunity levels in sows and to identify growing or finishing pigs with exposure to circulating PEDV; (3) vaccinating gilts and sows if necessary; and (4) improving herd management [ tighteni testing We first carried out nucleic acid and antibody-based monitoring and surveillance (MoS) of PEDV in a commercial farm affected by recurrent PED. All stool samples collected were positive for PEDV detected by rRT-PCR, and no other viral pathogens that cause diarrhea were detected. Next, the full S gene and the complete genome of the representative isolate with the lowest Ct value 15.6) were independently sequenced. The sequencing analysis revealed that the recurrent strain, designated as KOR/KNU-1907/2019 (referred to as KNU-1907), carried a 4161-bp segment of the S gene, predicted to encode a 1386-amino acid protein. It contained the genetic signature of the G2 field epidemic strains, including S insertions-deletions (S INDELs), compared with the prototype G1a CV777 strain [5.6 were Based on genetic information about the S genes of the Korean PEDV strains collected in our laboratory, the barcode pattern of KNU-1907 is clearly distinguishable from that of the 2018–19 representative strains, KNU-1836, KNU-1904, and KNU-1911, which were identified in December 2018–March 2019 and were temporally and spatially similar to KNU-1907. However, KNU-1907 was analogous to the 2017 isolates, KNU-1704 and KNU-1708, collected in December 2017 that came from the same region where KNU-1907 was isolated . These dWe also conducted serological examinations using VNT and ELISA to evaluate the immunity of sows against PEDV and to screen pigs with past exposure to the PEDV endemically circulating in the farm. All sows, including primiparous sows, developed PEDV-specific NAbs with titers ranging from 32 to 256 and anti-PEDV IgA antibodies, probably arising from natural or intentional infection or both  and spread  of pathogens such as PEDV. The farm used in this research received the best score, 5, following on-site evaluation of the external and internal biosecurity practices at the yard, staff, and barn levels. However, despite full biosecurity management, the overall risk assessment indicated that this farm is considered to be at a medium risk of recurrence of PED due to relatively low sow immunity (score 3) and continual virus circulation (score 5) in the herd  NAb and IgA antibody kinetics in sows compared with those in the previous month  . These rPen-side slurry samples collected weekly or monthly from 20 farrowing rooms, 24 nursery rooms, and 8 finisher barns were subjected to rRT-PCR. The MoS conducted on 26 June found that seven farrowing rooms (35%) were severely contaminated with PEDV, the S gene sequence of which was 100% identical to that of the virus strain collected on 18 June . HoweverWhile reinforcing the internal biosecurity and disinfection practices to prevent the spread of the virus within the nursery barns as well as to the farrowing houses and to clean the virus from wean-to-finish barns, MoS involving molecular and serological screening was continued. The slurry-based rRT-PCR revealed that until the middle of September, 30–50% of the nursery rooms were positive for PEDV, although the number repeatedly decreased and increased . The serDespite retaining PEDV-free status in the farrowing houses, the number of PEDV-positive nursery rooms (13 of 24) alarmingly increased again on 11 November , and fouOn 3 December, a suspected infection of PED appeared simultaneously in several replacement gilts that were purchased from external conventional pig breeding herds and had been acclimated in finisher barns. The affected gilts started showing mild diarrhea and tested positive for PEDV. Genetic analysis revealed that the virus (KNU-1907-8) that infected the gilts was 100% equal to that collected in the nursery barns on 25 November (KNU-1907-7) , suggestThe challenge of eliminating the virus continued through the disinfecting and remodeling of the nursery barns, followed by the implementation of active MoS. As a result, the number of PEDV-contaminated rooms was significantly reduced by 10 February 2020, even though four rooms remained positive for PEDV. This reduction was transient, and all nursery rooms, except for two, were recontaminated with PEDV on 29 April, again posing a potential risk of PED recurrence on this farm . Howevern = 20) and finishing (n = 20) pigs in December 2020 through February 2021 and subjected to VNT. No animals seroconverted against PEDV, suggesting the absence of virus circulation  were determined in this farm during this study . We were−4 substitutions/site/year and 2.239 × 10−3 substitutions/site/year at nucleotide and amino acid levels, respectively  biosecurity, (2) diagnostics and MoS, (3) vaccination, and (4) herd management . In the We first conducted PEDV diagnostic testing using RT-PCR and nucleotide sequencing to obtain timely genetic information about the virus, which is of primary importance in recurrent outbreaks. The genetic details of PEDV collected at the time of recurrence on the farm indicated that the virus might have persisted since the first occurrence. Next, we executed an assessment model system that evaluates the status of biosecurity, herd immunity, and virus circulation through on-site data collection and laboratory examination to estimate the risk of PED recurrence in the farm. Although this farm followed all external and internal biosecurity procedures strictly, it was at risk of a recurrence of PEDV. This susceptibility resulted from low herd immunity and ongoing virus circulation on the farm, as shown by the serological evidence in this study. The first evidence was obtained using VNT and IgA ELISA to measure the immunity of sows, which pass on lactogenic maternal antibodies to suckling piglets for protection against PEDV. The second line of investigation used only VNT to detect PEDV-seropositive pigs that had been exposed to the virus circulating in wean-to-finish barns. Therefore, the serology must be performed bidirectionally in sows and their offspring from farrowing houses as well as in grow/finish pigs from the nursery and finisher barns to obtain valuable information from PED-affected farms. Our data also suggested that adopting a feedback practice in this farm at the emergence of the disease might succeed in minimizing the time period of neonatal mortality but appeared to fail at assuring immunity and the cessation of shedding of PEDV within the population, thereby leading to recurrence within a year. Given the drawbacks of a long-term view, feedback that may help mitigate PED and induce immunity through controlled oral exposure should be contemplated only in urgent explosive epidemic cases with very high mortality and morbidity.Based on our initial MoS information, a two-track control strategy was introduced in this farm to elevate the immunity of sows and to break the chain of virus circulation. This strategy was evaluated using longitudinal laboratory tests to monitor its effects. Colostral antibodies received from immune dams enable their offspring to combat PED. Sow immunity is therefore a vital parameter required for the prevention and control of PED . In SoutThe second strategy focused on virus screening in feces from individual pigs and pen slurries from the environment. rRT-PCR diagnostics can detect PEDV in rectal swabs of infected pigs for up to two months after infection ,19. PEDVPEDV recurred in June and genetic data collected at this time demonstrated that PEDV infection spread in the farrowing houses. However, unlike the first recurrence, PEDV reinfection had little effect on newborn piglets but caused preweaning scours in piglets aged ≥ 18 days, with no mortality in the affected pigs, although the severity and mortality of PEDV is age dependent and occurs almost exclusively in neonatal piglets less than 1 week old , the virAt five months postrecurrence on this farm, PEDV was eliminated from the farrowing houses in August 2019 as a result of coordinated active monitoring and management efforts. However, viral and serological screening indicated that there was survival and spread of PEDV in wean-to-finish barns. In particular, early rRT-PCR-based MoS on 26 June detected virus circulation throughout nearly all nursery barns. As environmental monitoring of PEDV in wean-to-finish barns first began at this time, we cannot exclude the possibility that there had been environmental contamination by PEDV since the recurrence. However, we suspected another factor, such as the management of infected piglets. In the PEDV reinfection on 18 June, early weaning was performed to remove piglets that became infected. It was anticipated that the movement of infected piglets to weaning pens would be the main source of magnifying the extent of virus contamination in nursery barns. Thus, it is recommended for pig farmers to employ depopulation by euthanizing affected piglets to avoid disease transmission to other barns within the farm instead of early weaning in enzootically infected farms.Cooperative efforts to decontaminate virus circulation resulted in a decrease in PEDV from the nursey barns until the end of September 2019, when only two barns remained contaminated. Virus circulation was reproduced in November, caused by unknown factors, which suddenly increased the number of PEDV-contaminated barns from November to December 2019, potentially threatening the on-farm health status. During this period, active MoS was strengthened to monitor all herds closely and to quickly cope with the situation when pigs that showed clinical signs of PEDV were observed. At the end of November, we found purchased replacement gilts, which became infected with PEDV during acclimation in finisher barns. However, the incidence of the virus was limited to the gilts, and further disease transmission did not occur. Therefore, this risk category was named the first potential recurrence but did not lead to an actual outbreak of PED. In this instance, the virus might be hidden in the environment of the nursery barns that were PEDV-positive according to the last monitoring and may play a role in a “Phantom Menace (PM)” that can stealthily infect pigs, amplify, contaminate, and spread to other pigs. As the subclinical spread of PEDV occurs in grow/finish pigs, with the animals developing a humoral immune response against the virus, it is necessary to monitor swine populations in affected farms using complementary virus and serological screening to identify the PM virus itself or the pigs with exposure to the virus in their environment and to further intervene in the chain of virus circulation.PEDV infection of gilts indicates that virus contamination in wean-to-finish barns must be removed, or the farm faces a risk of recurrence. It is generally acknowledged that PEDV infection is self-limiting or asymptomatic in weaner to finisher pigs; however, the virus is shed in their feces, contaminates their barns, and replicates in a subclinical manner in these populations . Thus, PAlthough a second fade-out of PEDV from the contaminated nursery barns was observed in February 2020, virus contamination was transmitted throughout most nursery rooms from April to May 2020. However, no typical PEDV infection was reported on this farm during this period. The monitoring data collected in December 2019 indicated that the nursery barns were seriously contaminated, whereas the finisher barns tested as virus free. The data obtained in February 2020 show that the nursery barns were largely decontaminated, whereas the finisher barns were significantly recontaminated . These r−4 substitutions/site/year and 14.80 × 10−4 substitutions/site/year in the S gene, respectively, indicating that genetic evolution proceeds at a faster rate on Jeju Island than on the South Korean mainland [−4 substitutions/site/year in the S gene and 4.921 × 10−7 substitutions/site/year in the whole genome, which is lower than the results reported by previous studies [The full S sequences of PEDVs were temporally collected and further analyzed to determine the evolution rate of KNU-1907. Previous studies estimated that the substitution rates for the PEDV G2b mainland and Jeju Island strains are 7.18 × 10mainland . However studies . While p studies . The dev studies . The preA 1500-sow commercial farrow-to-finish farm was selected for this study. The farm is located in the Gyeongbuk Province in southeastern South Korea and has no neighboring swine farms within 5 km. The farm contains 17 main buildings, including one breeding barn; two gestation barns; three farrowing houses with separate rooms 1–8, 9–16, and 17–20; three nursery barns with separate rooms 1–11, 12–19, and 20–24; and eight finisher barns .In March 2018, PEDV first appeared in this farm, causing acute PED infection with lethal watery diarrhea and >90% mortality in newborn piglets. To control PED, sows were intentionally exposed to PEDV via oral exposure in the form of homogenized intestines from infected pigs in the early phase of the outbreak. This practice was used to initiate and maintain herd immunity and it could gradually mitigate the significant losses on this farm. Since then, no further PED outbreaks occurred on the farm until early 2019. At the end of February 2019, PED recurred on this farm, primarily affecting first-parity sows and their offspring and producing 50–60% mortality. Fecal samples collected from diarrheic pigs were submitted to our laboratory for diagnosis and further management.g . The clarified supernatants were tested by PEDV-specific real-time RT-PCR and were further subjected to nucleotide sequence analysis, if necessary. Blood was periodically taken from first-parity (primiparous) and second-parity or greater (multiparous) sows and pigs of different ages, as described in the legends to Collection of individual pig samples  and pen-side samples (slurry) was started on 11 March 2019, two weeks after the PED recurrence in the farm, and was continued until February 2021. Stool samples from diarrheic pigs were collected using 16-inch cotton-tipped swabs. Pig slurry specimens were obtained over time using syringes from slurry pits in 20 farrowing rooms, 24 nursery rooms, and 8 finisher barns. Fecal and slurry samples were diluted with phosphate-buffered saline (PBS) to produce 10% (wt/vol) suspensions. The suspensions were vortexed and centrifuged for 10 min at 4500× Viral RNA was extracted from fecal samples prepared as described earlier, using i-TGE/PED Detection Kits  according to the manufacturer’s protocol. PEDV S gene-based quantitative rRT-PCR was performed using One-Step SYBR PrimeScript RT-PCR Kits  with the forward primer 5′-ACGTCCCTTTACTTTCAATTCACA-3′, reverse primer 5′-TATACTTGGTACACACATCCAGAGTCA-3′, and a probe 5′-FAM-TGAGTTGATTACTGGCACGCCTAAACCAC-BHQ1-3′, as previously described ,24,25,26The S glycoprotein gene sequences of PEDV-positive samples were determined using traditional Sanger methods. Two overlapping cDNA fragments spanning the entire S gene of each isolate were amplified by RT-PCR, as previously described . The indThe sequences of fully sequenced S genes of global PEDV isolates were aligned using the ClustalX 2.0 program , and theBayesian analyses were performed with BEAST v2.6.3 to estimate the rate of evolution of the full S and whole-genome sequences . BEAST p4 cells/well were grown in 96-well tissue culture plates for 24 h. KNU-141112-P5 virus stock was diluted in serum-free α-MEM to achieve 200 TCID50 in a 50 μL volume. Next, the diluted virus was mixed with 50 μL of 2-fold serially diluted (1:2 to 1:512) inactivated serum or colostrum samples and incubated at 37 °C for 1 h. The mixture was added to Vero cells and incubated at 37 °C for 1 h. After removing the mixture, the cells were rinsed with PBS five times and maintained in a virus growth medium [2 incubator for 2 days. The neutralizing endpoint titers were calculated as the reciprocal of the highest serum dilution that inhibited the virus-specific cytopathic effects by ≥80% relative to the controls in duplicate wells. The serum samples with neutralizing endpoint titers of ≥1:4 were considered to be positive for the PEDV-neutralizing antibody.The presence of PEDV-specific neutralizing antibodies (NAbs) in serum and colostrum samples collected from the pigs was determined using a conventional virus neutralization test (VNT) in 96-well microtiter plates with PEDV isolate KNU-141112 as previously described ,33, withh medium ,25 at 37in-house PEDV G2b S1-based indirect ELISA as described previously [Recombinant PEDV S1 protein was purified from PK-rS1-Ig cells as described previously . Anti-PEeviously ,34,35, whttps://www.manitobapork.com/images/producers/pdfs/biosecurity/PEDv-Biosecurity-Quick-Facts.pdf, accessed on 16 January 2021), with some modifications to adjust to the circumstances of domestic pig farming, as described previously [Subjective monitoring encompassed the classic approach based on the data collected from farms and surveys and verification of the checklists of established tasks. External and internal biosecurity protocols were reviewed by a herd veterinarian using on-site inspection and in-person interviews and were evaluated at the yard, staff, and barn levels using purpose-designed checklists with a 1–5 scoring scale, wherein 1 = worst and 5 = best, which is based on the PEDV Biosecurity Quick Fact Sheet , the measurement level of sow immunity , and the endemic status , as described previously . The sub®, CAVAC, Daejeon, Korea) once, using drenching in accordance with the manufacturer’s instructions, and were then intramuscularly (IM) boosted twice with a commercial inactivated G2b PEDV vaccine  at 6, 4, and 2 weeks prepartum or prebreeding, respectively.Live prime-killed/killed boost (L/K/K) multiple-dose PED vaccination at two-week intervals starting prior to farrowing has been implemented using commercial second-generation vaccines since March 2019. All sows, including gilts, were orally administered a G2b live oral vaccine  clet-test. p values of less than 0.05 were considered to be statistically significant.All values are expressed as mean ± standard deviation of mean (SDM). Statistical analyses were conducted using Student’s The present study is the first to report a case of PEDV elimination in an endemically infected farrow-to-finish pig farm. To accomplish the final goal of the eradication of PEDV, conducting a risk assessment of PED recurrence is critical for the timely gathering of information about pathogen exposure and immune responses in PED-affected farms. This assessment involves: (1) molecular and genetic diagnostics (virus screening) of PEDV to obtain information on whether the virus has remained inside the farm after the outbreak or was newly introduced from outside the farm; (2) serological screening in farrowing herds to obtain information about sow protective immunity against PEDV; (3) viral and serological screening in growing and finishing herds to obtain information on virus circulation within the farm; and (4) subjective biosecurity monitoring to obtain information on how well the farmer follows biosecurity protocols. According to the results of the risk assessment, the four-pillar-based coordinated intervention measures are actively carried out to enhance the immunity of the hosts and diminish the infectivity of PEDV in the farrow-to-finish farm suffering from endemic virus. These measures include: (1) strict internal and external biosecurity; (2) prime-boost prefarrow L/K/K vaccination and longitudinal monitoring of protective immunity in sows and their offspring; (3) secure cleaning/disinfection practices, combined with the AIAO management, if needed, in farrow houses and longitudinal MoS of PEDV in the herd and the environment (slurry); and (4) disinfection and gilt management in wean-to-finish barns, in parallel with longitudinal MoS of PEDV in the herd and the environment (slurry).


The correct surname for the tenth author, Cláudio Tadeu Daniel-Ribeiro, is Daniel-Ribeiro (not Tadeu Daniel-Ribeiro). The name "Tadeu" is part of the compounding first name (Cláudio Tadeu) and not of the family name.The author’s name should also be abbreviated as follows: Daniel-Ribeiro, CT.


To perform a post hoc analysis of patient-incurred costs in a randomized controlled clinical trial comparing prostatic artery embolization (PAE) and transurethral resection of the prostate (TURP).Patients older than 60 years with indication of TURP were randomized to PAE or TURP procedure. After intervention and hospital discharge, patients were follow-up during 12 months The associated patient costs were categorized according to the study period: pre-intervention, intervention, hospitalization, and follow-up. Several items for both groups were analyzed within each study period.(p = 0.026). For most evaluated items, the mean costs were significantly higher for TURP. No significant differences were observed in the mean costs of PAE  and TURP  procedures (p = 0.061). However, the histopathology analysis, recovery room stay, and intraoperative laboratory analysis increased the interventional costs for TURP  compared with PAE  (p < 0.001). No cost differences were observed between PAE (€ 725.26) and TURP (€ 556.22) during the 12 months of follow-up (p = 0.605). None of patients required a repeat intervention during the study period.The mean total costs per patient were lower for PAE  than for TURP , with this difference of € 781.70 being significant Considering the short-term follow-up, PAE was associated with significantly lower costs compared with TURP. Future investigations in the context of routine clinical practice should be aimed at comparing the long-term effectiveness of both procedures and determining their cost-effectiveness.Level of evidence: Level 1 (a-c) Benign prostatic hyperplasia (BPH) is characterized by the nonmalignant overgrowth of prostatic tissue surrounding the urethra, and is a common cause of lower urinary tract symptoms (LUTS) in men. This condition is present in approximately 8% of men aged between 31 and 40 years. This prevalence increases significantly with age, to approximately 90% by the eighth decade of life .Prostatic artery embolization (PAE) has emerged as a minimally invasive therapy for symptomatic patients due to BPH and as an alternative to TURP, with similar clinical outcomes, fewer complications, and shorter recovery time –9. The NConsidering the substantial differences between the technical aspects of PAE and TURP, there may also be differences in their costs, which is why we believe that a cost analysis comparing each procedure seems to be of particular interest. To date, two cost analysis are available: Bagla et al.  and MüllThe data were obtained from an unblinded, single-center, non-inferiority, randomized, controlled trial performed in the Urology and Vascular and Interventional Radiology departments of the study site . The triThe costs of each participant were categorized according to study the period: pre-intervention (from the informed consent to the operation), intervention (PAE or TURP), inpatient stay  and follow-up . All costs were covered by Spanish National Health System and analyzed from a health care provider’s perspective.During the pre-intervention and follow-up periods, patients received urology and interventional clinic , primary care clinic, as well as emergency care, when required. A computed tomography (CT) angiography was performed on all patients during the pre-intervention period as part of the trial’s selection criteria. The intervention costs were those associated with the procedure, histopathology and the stay in the recovery room. The costs of the PAE procedure item included: the professional fees of the interventional radiologist, the costs of the operation facilities , and the medical supplies required for PAE . The costs of the TURP procedure item included: the professional fees of the urological surgeons, the costs of the operation facilities , the medical supplies required for TURP, and the costs of the anesthesia . The costs of the inpatient stay included the physician’s professional fees, the nursing service fees, the medical supplies, the hospital pharmacy costs, the laboratory costs. Following hospital discharge, the patients from both groups were followed through scheduled visits at 1, 3, 6, and 12 months. Complications were graded using the Clavien classification system  and theiStaffing services costs; imaging studies, hospital room stays, the medical supplies, the hospital pharmacy costs, the laboratory costs and clinic visits were provided by the hospital’s Accounting Department. Administrative and accommodation  expenses were also included. The calculations were performed based on mean personal costs per hour, charges for room costs per hour, and the proportionate depreciation of equipment.T test and with the mid-p exact test in the case of complications. All comparisons were two-sided and a significance level of 0.05 was considered. Analyses were performed using software SPSS® Statistics version 22.0.The resulting costs were summarized using means and standard deviations for continuous variables and frequencies and percentages for categorical variables. Differences between PAE and TURP in terms of their baseline characteristics, preoperative data, postoperative data, and costs were compared using Student’s (p = 0.026). Detailed itemized costs arising from PAE and TURP are shown in Table A total of 61 patients were randomized in the trial: 31 to PAE group and 30 to TURP group. After patient exclusion due to not receiving the allocated intervention or discontinuing it, a final total of 54 patients completed the trial protocol and were included in the cost analysis: 27 of PAE group and 27 of TURP group (bipolar resection). The baseline characteristics of the study patients, as well as the preoperative and postoperative data, are described in Table p = 0.015). The differences were observed for the pre-anesthesia items required prior to TURP procedure. No significant differences were observed in the mean costs of PAE  and TURP  procedures (p = 0.061). However, the recovery room stay and the histopathology substantially increased the total interventional itemized costs for TURP  compared with PAE  (p < 0.001). Significant differences in costs were also observed during the post-intervention hospital stay: € 591.79 for PAE and € 849.43 for TURP (p = 0.004). The mean total in-hospital costs, that included intervention and inpatient stay, was found to be significantly greater for TURP population compared with PAE population .Significant cost differences between PAE and TURP were observed during the pre-intervention period  and no significant differences were observed in the mean costs. During this period, complications appeared in both groups (17 for PAE and 47 for TURP) and accounted for 49.64% of the total follow-up cost in PAE population and for 34.98% in TURP population. The differences between both groups in this respect were not significant. The outpatient prescriptions for the urological process consisted of α1-blockers, 5α-reductase inhibitors, urinary antispasmodics, analgesics, antibiotics, low-molecular-weight heparin (LMWH), and incontinence pads or urine collection bags. The costs of the outpatient prescriptions were significantly higher in TURP group .The mean follow-up was 376.33 days for PAE and 360.00 days for TURP  , 4, 17. To date, two comparative in-hospital cost analyses between PAE and TURP have been published: Bagla et al.  carried p = 0.003). Nevertheless, the mean length of hospitalization for TURP observed in our study is shorter than that published in the literature [Inpatient stay costs were also significantly higher for TURP. The excess costs could be related to longer hospital stay compared with PAE . Another limitation of the study is that thromboprophylaxis with LMWH is site routine practice but is not endorsed by the European Association of Urology (EAU) guidelines, so may differ from that applied in other healthcare frameworks. Although the costs related to the depreciation of equipment, electricity, security, medical records, and housekeeping were included, those of lost productivity, unpaid work (domestic), or lost leisure time were not. Some studies suggest that re-intervention rates in PAE are clearly higher than in TURP due to persistent LUTS. According to the data of the UK Registry of Prostate Embolization (UK-ROPE) study [It is essential to bear in mind that this study has several limitations, including the fact that it was performed as a post hoc analysis, which has inherent limitations. In addition, the data were obtained from a randomized clinical trial carried out with patients who were protocol-linked and operated on in a single site. To accurately reflect routine clinical practice, costs of CT angiography of TURP group and PAE inpatient room should not be considered. PAE is currently performed as outpatient settings, which results in considerably lower costs by removing the need for hospitalization. Considering these conditions, significant differences in mean total costs per patient were still significantly lower for PAE (€ 2,904.68 vs € 3,846.72; For most items, the analyzed costs related to TURP were significantly higher than those of PAE in the context of this clinical trial with a short-term follow-up. The most relevant costs for both procedures were those arising during the interventional period, with significant differences being found between them. However, no significant differences were observed during the 12-month follow-up, including in terms of the secondary complications. Future investigations in the context of routine clinical practice should be aimed at comparing the long-term effectiveness of both procedures and determining their cost-effectiveness.


Hericium erinaceus is the softest without obvious granular texture, following Lentinus edodes, while Ganoderma lucidum and Grifola frondosa have a relatively hard texture. The yield of four kinds of IDF from high to low came from Ganoderma lucidum, Hericium erinaceus, Lentinus edodes, and Grifola frondosa. Fourier transform IR (FTIR) and X-ray diffraction (XRD) spectra showed that the four IDFs had similar functional groups and all of them contained a large amount of cellulose. Physical and chemical analysis showed that all the four IDFs had certain water holding capacity, water binding capacity, and oil holding capacity. In-vitro digestion experiments showed that the four IDFs could inhibit the digestion of starch and fat to a certain extent. By-products of edible fungi are an ideal material for the recovery of IDFs, which have the potential to be processed into functional food materials due to their physicochemical properties and physiological functions.With the rapid development of the edible fungi industry in the world, especially in China, the resource utilization of edible fungi by-products has become an urgent problem for the industry's sustainable development. The waste residue of edible fungi after polysaccharide extraction by water accounts for a large proportion, which contains a large amount of water-insoluble dietary fiber (IDF). At present, the extracted residue is generally treated as fertilizer or solid waste, which not only pollutes the environment, but wastes resources too. In order to develop these by-products, expand their potential utilization in the food industry, the structure characterization, physicochemical properties, and the influence of IDF on dietary energy intake were studied. The IDF from the residues of polysaccharides extracted from four edible fungi was extracted using the Association of Official Analytical Chemists (AOAC) method. The results showed that IDF in the four kinds of edible fungi residues was similar in composition but different in texture. Cellulose and hemicellulose are the main IDF extracted from four kinds of edible fungi. Among them,  DF can be shown . In genebe shown . DF is abe shown . Based oModern dietary habits have resulted in a serious lack of nutrients obtained from food, especially the “seventh nutrient”—DF. Although DF does not provide energy, it can effectively improve the nutritional metabolism of the human body. DFs play a significant role in improving human health and are known as potential formulations in human health due to their beneficial effects in the control of life-threatening chronic diseases, e.g., diabetes mellitus, cardiovascular disease, obesity, and cancer . DF is bThe different physiological functions of DF are closely related to their structures and components. DF with strong water holding capacity, oil holding capacity, cholesterol adsorption capacity, and antioxidant properties can modify the texture of food, stabilize high-fat food and emulsions, and improve the shelf-life of foods . IDF plaAstraeus odoratus (77.1%), Schizophyllum commune (68.2%), and Lentinus polychrous (60.6%). The SDF and IDF content in all the mushrooms ranged from 24.8 to 72.4% DW and 1.63 to 13.9% DW, respectively (Lentinus edodes (IDF-Len), Hericium erinaceus (IDF-Her), Grifola frondosa (IDF-Gri), and Ganoderma lucidum (IDF-Gans) by combining enzyme extraction method and chemical extraction method. The structure, hydration properties, and adsorption capacity of IDFs from these four sources were studied and the effects of starch and fat digestion models in vitro were studied. The indexes of IDF measured in this experiment provide a theoretical basis for future research on the properties of IDF and its influence on human energy intake, which is conducive to the development and utilization of IDF in edible fungus waste residue, and achieve the purpose of turning waste into treasure.In order to achieve the best activity of DF, it is necessary to consume 50–75% IDF in a daily diet, so the role of IDF cannot be ignored in inhuman health . At presectively . The extLentinus edodes, Grifola frondosa, and Hericium erinaceus were purchased from the Gouzhuang Farmers' Market in Hangzhou and pulverized into powder. Ganoderma lucidum extract residue was provided by Zhejiang WisePlus Health Technology Corporation Ltd. NaOH, HCl, Na2HPO4·12H2O, NaHPO4·H2O, sodium taurocholate, sodium carboxymethyl cellulose, DNS, and soluble starch were purchased from Sinopharm Chemical Reagent Corporation Ltd. . Neutral protease, alpha amylase, and lipase were purchased from Beijing Gao Ruisen Technology Corporation Ltd. Corn oil was purchased from Shanghai Maclean Reagent Corporation Ltd. All the other chemicals used in this study were of analytical grade.The fresh Extraction of the IDF was carried out following the Association of Official Analytical Chemists (AOAC) 985.29 method. Fresh raw materials were washed and fully dried in a constant temperature air blowing drying oven. The dried raw materials were crushed through an 80-mesh sieve and then stored in sealed bags. Then, 1 g of crushed edible fungus powder was added to the beaker, and then 10 ml 0.25 mol/l NaOH solution was added for hydrolysis under alkaline conditions at 55°C for 2 h in a constant temperature culture oscillator, centrifuged in a centrifugal tube, removed the upper solution, and retained the lower solid for subsequent processing. The first step was to adjust the pH to between 6.3 and 6.5 with phosphoric acid buffer solution and 0.35 mol/l HCl. Then, 0.1 ml 0.4% α-amylase hydrolyzed solution was added to remove impurities, e.g., starch. The required conditions were vibration extraction at 120 rpm at 65°C for 40 min. After the supernate was removed by centrifugation, the lower residue was put into a beaker with 0.1 ml of 0.4% neutral protease solution and shaken at 120 rpm at 60°C for 90 min to remove the protein. The supernatant was removed by centrifugation again at 4,000 rpm for 15 min. Rinsed with distilled water for three times and centrifuged at 4,000 rpm to remove the supernatant from the upper layer and obtain the residue from the lower layer. In total, 280 ml of 95% ethanol was added to the prepared residue and precipitated at room temperature for 60 min to remove other impurities. The supernatant was removed by centrifugation at 4,000 rpm for 15 min, followed by decolorization with 78% ethanol for three times, and eluting with 95% ethanol until the eluent became colorless. Next, eluted IDF were pre-cooled in a ultra-low temperature refrigerator for 24 h, dried for 48 h in a vacuum freeze-dryer, crushed, weighed, calculated the yield, and stored in a refrigerator at 4°C .The yield was calculated after preparation, and the formula was as follows:Lentinus edodes, Hericium erinaceus, Grifola frondosa, and Ganoderma lucidum were laid on the sample table for adhesion and gold plating and then the gold-plated samples were put into the SEM for scanning. The microscopic morphology of IDF was observed under an accelerated voltage of 5 kV and representative photos were taken to obtain SEM images of 500 and 1,000X.The microstructure and morphology of four IDFs were observed by a SEM . A small amount of dried and constant weight IDF samples of Lentinus edodes, Hericium erinaceus, Grifola frondosa, and Ganoderma lucidum were measured by a Nicolet iS5 Fourier transform infrared spectrometer . First, 100 mg KBr was mixed with four different 5 mg IDF samples, and the mixture was ground and pressed. After the compression, the sample was analyzed with a resolution of 4 cm−1. The scanning time of the sample was 32 s and the scanning range was 400–4,000 cm−1. Meanwhile, the scan results of four IDFs were compared with the blank KBr background.The FTIR spectroscopy spectra of IDF samples of The crystal structures of four IDF powders were determined by an X-ray diffractometer . The DF samples from different edible mushrooms were dried to constant weights and evaluated according to the previous method . X-ray dS) was mixed with 25 ml distilled water in a centrifuge tube at room temperature for 2 h. After centrifugation at 3,000 rpm for 10 min, the excess was absorbed with filter paper and weighed . The wet sample was then dried in an oven at 110°C to constant weight and weighed .According to the previous method , the holThe calculation formula of holding power is as follows:The calculation formula of binding power is as follows:S as the sample weight) was accurately measured into the centrifugal tube, and 5 g of corn oil was added into it, shaken at 37°C for 1 h, and centrifuged at 8,000 rpm for 30 min. Next, the supernatant was removed and the residue was collected for weighing (W0).The oil holding capacity of four IDFs was measured by the method of Japan Gan with appropriate modifications . First oThe calculation formula of oil holding capacity is calculated according to the formula below:In-vitro fat digestion experiment was carried out according to previous methods  released:  methods , 22 within vitro was conducted according to the methods , and the yield of IDF-Gri was the lowest and significantly lower than that of IDF-Gan and IDF-Her (p < 0.05), which may be related to the high content of IDF in Ganoderma lucidum and low content of IDF in Grifola frondosa.The yield of the four IDFs is shown in Agrocybe cylindracea was compact or porous structures and contained porous surface, and filiform structures were mostly presented on its surface , i.e., their water holding capacity was similar. In the process of significant difference analysis of combined hydraulic power, the significant difference was only found between IDF-Gri and the other three IDFs (p < 0.05), i.e., the water holding capacity of IDF-Gri was the strongest. There were no significant differences among IDF-Len, IDF-Her, and IDF-Gan (p > 0.05), i.e., there was no difference in water binding power between them. The comparable water binding capacities among four SDFs might be attributed to the nature of the water-binding sites, structure, and chemical composition, which is worth further study.It can be concluded from p > 0.05) between IDF-Gan and IDF-Gri, and between IDF-Len and IDF-Her, while the oil holding capacity of IDF-Gan and IDF-Gri was significantly higher than that of IDF-Len and IDF-Her.The chemical structure of DF contains a large number of hydrophilic groups, but fewer lipophilic groups, which is closely associated with the high water holding capacity, high oil holding capacity, and high swelling capacity of DF . DF with3 or =CH2), ethers (C–O), etc. A large number of hydrophobic groups were exposed during the IDF preparation via the enzymatic method . Except for blank, IDF-Gan digestible emulsion was the fastest, followed by IDF-Gri and IDF-Len, and IDF-Her. The inhibitory effect of IDF-Len and IDF-Her on lipase was significantly higher than that of IDF-Gan and IDF-Gri (p < 0.05). In addition, the inhibitory effect of IDF-Gri was significantly higher than that of IDF-Gan (p < 0.05). Different DFs have different degrees of inhibition on fat digestion. It may be that the addition of IDFs blocks the effect of lipase on triacylglycerol and inhibits the effect of the enzyme, leading to the reduction of the separation of FFAs. Among them, IDF-Len and IDF-Her have the most obvious inhibitory effect, which may be because their materials are relatively soft, loose, and porous, which can combine to affect more oil and inhibit the decomposition of fat. The degree of lipid digestion was calculated by the accumulation of FFA and then titrated with NaOH, while the added IDF would limit the contact between lipase and triacylglycerol .in vitro shown in p < 0.05) compared with the control group. The inhibition rate of IDF-Gan on starch was significantly higher than that of the other three IDFs (p < 0.05), and the starch inhibition rate of IDF-Gri was significantly higher than IDF-Her (p < 0.05). The mechanism of DF inhibiting α-amylase activity has been reported. According to previous reports, α-amylase non-specifically binds on the cellulose surface, and cellulose can inhibit α-amylase activity against starch (Actinidia deliciosa) inhibited starch hydrolysis in a concentration-dependent manner, and IDF shows higher inhibition of α-amylase than SDF at the same concentration. Moreover, there are also studies that contradict our findings that three IDF samples  had no effect on the digestibility of starch and would not delay the digestion of starch . The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.BY was employed by Zhejiang WisePlus Health Technology Co., Ltd. The remaining 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.


The adsorption mechanism involves replacement of CaII by PbII ions coordinated to the carboxylate groups of the alginate backbone. After treatment with a Na2EDTA solution, the beads can be reused, without significant loss of activity for at least two times. X-Ca-alginate aerogels can also uptake organic solvents and oil from seawater; the volume of the adsorbate can be as high as the total pore volume of the aerogel (6.0 mL/g−1), and the absorption is complete within seconds. X-Ca alginate aerogels are suitable for the decontamination of aquatic environments from a broader range of inorganic and organic pollutants.Polyurea-crosslinked Ca-alginate  aerogel beads (diameter: 3.3 mm) were evaluated as adsorbents of metal ions, organic solvents, and oils. They were prepared via reaction of an aromatic triisocyanate (Desmodur RE) with pre-formed Ca-alginate wet gels and consisted of 54% polyurea and 2% calcium. X-Ca-alginate aerogels are hydrophobic nanoporous materials (90% v/v porosity), with a high BET surface area (459 m Water pollution is one of the most serious problems facing humanity, becoming even more severe over the last century because of industrialization. Pollutants can be roughly categorized as inorganic  and organic , all posing a serious threat to human health. For example, lead causes encephalopathy, neuropathy, reduced intellectual capacity in children, and kidney damage ; cadmiumSeveral technologies have been developed for the capture, removal, and sometimes recovery of pollutants, including chemical/electrochemical and physical methods, such as chemical/electrochemical precipitation, membrane separation/electrolysis, liquid extraction, adsorption, coagulation, flotation, crystallization, and others ,5,6,7. AAmong the technologies developed for water decontamination, adsorption is one of the most preferable methods because it is easy to apply and cost-efficient, and because there is usually a wide range of materials that can be used as adsorbents for a specific pollutant or class of pollutants . Such ma2) with a suitable monomer . The reaction leads to formation and accumulation of a nano-thin conformal polymer coating over the entire inorganic or biopolymer skeletal framework. The distinguishing feature of X-aerogels is their greatly enhanced mechanical strength compared to that of native aerogels, while other properties related to the nanoporous structure, e.g., high surface area, low thermal conductivity, low dielectric constant, and high acoustic attenuation, are affected only to a small extent. In addition, X-alginate aerogels are hydrophobic, while native alginate aerogels are extremely hydrophilic [Expanding the polymer-crosslinked (X-aerogel) technology that was initially developed with silica ,31,32,33rophilic . Use of rophilic ,41. OverIn line with the above, in this paper, we report an initial evaluation of X-Ca-alginate aerogels prepared from Ca-alginate and an aromatic triisocyanate ; 2 groups by water remaining adsorbed onto the surface of the alginate network. The –NH2 groups react further with still free triisocyanate in the pores, forming urea groups and eventually polyurea. X-Ca-alginate wet-gel beads were solvent-exchanged with acetonitrile and were dried into aerogels with liquid CO2 taken out at the end as a supercritical fluid. An optical photograph, size distribution, and mean diameter of X-Μ-alginate aerogel beads are shown in X-Ca-alginate aerogel beads were prepared as outlined in 13C NMR  crosslinking polymer and agreed with those reported previously [w/w (0.047 mol per 100 g of aerogel). The PUA content, calculated from the skeletal density of X-Ca-alginate aerogel beads (−3) [w/w.The chemical identity of the X-Ca-alginate aerogel beads was confirmed with attenuated total reflection Fourier transform IR (ATR-FTIR) , solid-s13C NMR , and atoeviously . The calel beads  and the ads (−3) , was fou−3), high BET (Brunauer–Emmett–Teller) surface area (459 m2/g−1), and high porosity (90% v/v). The N2-sorption isotherm  is larger than V1.7–300nm (from N2-sorption). Some microporosity , as indicated by the early rise of the isotherm at low partial pressures, is attributed to the rigid aromatic core of TIPM [Selected material properties of X-Ca-alginate aerogel beads are summarized in isotherm  showed aace area 9 m2/g−1, of TIPM ,47,48,49−1 of each of the following metal ions: AlIII, AsIII, VIII, CrIII, MnII, FeIII, CoII, NiII, CuII, ZnII, CdII, RuII, SrII, CsI, BaII, and PbII. Determination of the equilibrium concentration of each metal ion showed that PbII was selectively adsorbed from the above solution, in agreement with the expected relative affinity of alginates to metal ions, as has been reported in the literature [II was studied more thoroughly, using standard solutions of various concentrations in the range of 0.01 to 60 mg/L−1 in ultrapure water (pH = 5.63) and in seawater (pH = 8.46). II uptake from aqueous solutions of various concentrations after 24 h. Such a long incubation time was chosen in order to ensure that equilibrium was established, as determined by the plateau in the uptake-versus-time curve. For the PbII uptake from ultrapure water solutions  corresponds to the replacement of only one-third of the calcium initially contained in the X-Ca-alginate aerogels .X-Ca-alginate aerogel beads were tested for the adsorption of heavy metal ions from aqueous solutions. First, the beads were kept in a solution containing 100 mg/Lterature . Therefoolutions A, the add in 6 h . The maxII from seawater collected from an unpolluted marine area was also tested . The maximum adsorption capacity (13 mg PbII or 0.06 m/mol PbII per g of aerogel) was about half of that from ultrapure water solutions. This finding is not surprising, because the higher ionic strength of seawater is expected to affect the adsorption capacity [+, K+, Mg2+, Ca2+) competes with PbII toward complexation to the alginate network. In addition, native alginate aerogels cannot be used in seawater; they shrink within minutes  at equilibrium, Ceq is the PbII concentration in the solution at equilibrium (in mg/L−1), Qmax is the adsorption capacity (in mg/g−1), KL is the Langmuir equilibrium constant (in L/mg−1), KF is the Freundlich equilibrium constant, and n is the empirical adsorption intensity of the Freundlich model that represents a measure of nonlinearity. The corresponding plots and calculated parameters are shown in II uptake vs. the initial PbII concentration . That maximum adsorption capacity could not be achieved experimentally, because of the low value of the calculated equilibrium constant (KL = 0.13 L/mg−1). The Freundlich model was also fitted  was found to be equal to 1.9, indicating that the adsorption was favored (n > 1). The Freundlich model fitted the experimental data )  and the ntration A,C, the o fitted , and theII adsorption capacity after 13 h of agitation at 40 mg/L−1 of the initial PbII concentration. Interestingly, X-Ca-alginate aerogels could be recycled twice without significant loss of their adsorption capacity  were prepared and the corresponding amount of X-alginate aerogel beads was added .v/v porosity), with a high BET surface area (459 m2/g−1), consisting of 54% w/w polyurea and 2% w/w calcium, and they have been tested for potential application in environmental remediation as adsorbents of metal ions and organic solvents. X-Ca-alginate aerogels selectively adsorb PbII from ultrapure water . Adsorption of PbII from seawater  can also be achieved, although the adsorption capacity is lower compared to solutions of ultrapure water, presumably because of the higher ionic strength of seawater. PbII replaces CaII in the alginate network and coordinates to the carboxylate groups. PbII can be removed by placing the beads in a solution of Na2EDTA; afterward, the beads can be used again for PbII adsorption from new contaminated water samples, without significant loss of activity for at least two cycles. X-Ca-alginate aerogels can also adsorb organic solvents and oil from seawater; the volume of the adsorbent can be as high as the total pore volume of the aerogel, and the absorption process can be complete in less than 1 min.Polyurea-crosslinked Ca-alginate  aerogel beads (diameter: 3.3 mm) were prepared via reaction of an aromatic triisocyanate (Desmodur RE) with pre-formed Ca-alginate wet gels. They are hydrophobic nanoporous materials  from a broader range of inorganic and organic pollutants.2, CoCl2·6H2O, toluene, mesitylene, and bromoethane were purchased from Sigma . Desmodur RE  solution in ethyl acetate (EA)) was generously provided by Covestro AG . MeCN (HPLC grade), chloroform, hexane, cyclohexane, and tetrahydrofuran were purchased from Fisher Scientific . Acetone and Na2EDTA were purchased from Lach-Ner . Chlorobenzene was purchased from Chem-Lab Analytical . All solvents were used as received.Sodium alginate PROTANAL LF 240 D (G/M = 0.43–0.54) was used as starting material. CaCl2 was allowed in the autoclave; acetone was drained out as it was being displaced by liquid CO2 . Subsequently, the autoclave temperature was raised to 45 °C, and it was maintained for 1 h. Finally, the pressure was gradually released, allowing SCF CO2 to escape as a gas, leaving aerogels.Supercritical fluid (SCF) drying was carried out in an autoclave . Wet gels were placed in the autoclave at 12 °C and were covered with acetone. Liquid CO13C NMR spectra were obtained with a 600 MHz Varian spectrometer  operating at 150.80 MHz for 13C. Attenuated total reflection Fourier transform IR (ATR-FTIR) spectra were obtained with a Perkin Elmer Spectrum 100 Spectrometer.Solid-state cross-polarization magic angle spinning (CPMAS) 2-sorption measurements were performed on a Micromeritics Tristar II 3020 surface area and porosity analyzer . Skeletal densities (sρ) were determined by He pycnometry using a Micromeritics AccuPyc II 1340 pycnometer . Bulk densities (bρ) of the samples were calculated from their weight and natural dimensions.N3 65% .The calcium content of X-Ca-alginate aerogel beads was determined with atomic emission spectrometry (AES) employing a Varian SpectrAA 200 instrument , following by wet digestion of the beads with supra-pure HNOw/w) was added dropwise, using a 25 mL burette, to a 0.2 M solution of a metal salt (CaCl2) under mild magnetic stirring. Spherical hydrogel alginate beads were formed instantly, and they were left to age for 24 h; after that time, they were stepwise solvent-exchanged with MeCN/H2O mixtures  and finally with MeCN (4×). Subsequently, the wet-gel beads were kept in a solution of a triisocyanate in EA/MeCN (0.75 M) for 24 h at room temperature and for 72 h at 70 °C in order to complete the crosslinking reaction. Afterward, crosslinked Ca-alginate  wet-gel beads were solvent-exchanged with acetone (3×) and were dried with SCF CO2 to the corresponding aerogels.X-Ca-alginate aerogel beads were prepared according to a literature procedure . In brieII solutions, prepared from a stock standard PbII solution (Merck) of either ultrapure water (Millipore) or seawater, at given concentrations ranging from 0.01 to 60 mg/L−1. The vials had been previously cleaned with supra-pure HNO3 10% (Merck) and rinsed with ultrapure water of 18.2 MΩ/cm . For the preparation of all required solutions, class A volumetric glassware was used. The beads remained in each solution under continuous stirring for a given time. Subsequently, concentrations of remaining PbII in ultrapure water solutions were measured with graphite furnace atomic absorption spectrometry (GFAAS), employing a Varian SpectrAA 640Z with Zeeman background correction (Varian) (limit of detection (LOD) equal to 0.87 μg/L−1), for solutions with the given concentrations of 0.01 and 0.1 mg/L−1 and with flame atomic absorption spectrometry (FAAS), employing a Varian SpectrAA 200 instrument (Varian) , following appropriate dilution of the samples for the rest of PbII solutions. In the case of seawater solutions, corresponding direct measurements were performed electrochemically using a μAutolab type III  instrument connected to a three-electrode cell  with a static mercury drop electrode (SMDE) as the working electrode. The reference electrode was a Ag/AgCl (3 M KCl) electrode, while a carbon-rod electrode served as the auxiliary electrode. Throughout the experiment, the temperature was maintained equal to 20 °C. The uptake of PbII  was calculated as the ratio of the mass of PbII ions adsorbed to the mass of X-Ca-alginate aerogel beads used, according to Equation (3).Accurately weighed quantities of X-Ca-alginate aerogel beads were added to plastic vials containing Pbq; mL/g−1) was calculated as the ratio of the volume of the solvent adsorbed to the mass of the X-Ca-alginate aerogel beads used, according to Equation (4).Accurately weighed quantities of X-Ca-alginate aerogel beads were kept in a tube containing an organic solvent, an oil, or water under mild stirring. At selected time intervals, the beads were taken out of the tube and weighed. Afterward, the beads were re-immersed in the respective solvent, and the above steps were repeated for 1 h. The temperature was 20 °C. The solvent uptake (v/v). Sudan blue was used as a dye to visualize the adsorption of the organic solvent from the beads. The wet beads were removed after they had adsorbed the whole quantity of the solvent, and the time required for that was measured. The temperature was 20 °C.X-Ca-alginate aerogel beads were weighed and then added to a tube containing a mixture of seawater and an organic solvent or diesel oil (3%