Silver nanoparticles (AgNPs) have been studied worldwide for their potential biomedical applications. Specifically, they are proposed as a novel alternative for cancer treatment. However, the determination of their cytotoxic and genotoxic effects continues to limit their application. The commercially available silver nanoparticle Argovit™ has shown antineoplastic, antiviral, antibacterial, and tissue regenerative properties, activities triggered by its capacity to promote the overproduction of reactive oxygen species (ROS). Therefore, in this work, we evaluated the genotoxic and cytotoxic potential of the Argovit™ formulation (average size: 35 nm) on BALB/c mice using the micronucleus in a peripheral blood erythrocytes model. Besides, we evaluated the capability of AgNPs to modulate the genotoxic effect induced by cyclophosphamide (CP) after the administration of the oncologic agent. To achieve this, 5–6-week-old male mice with a mean weight of 20.11 ± 2.38 g were treated with water as negative control (Group 1), an single intraperitoneal dose of CP , a daily oral dose of AgNPs  for three consecutive days, or a combination of these treatment schemes: one day of CP doses (50 mg/kg of body weight) followed by three doses of AgNPs  and three alternate doses of CP and AgNPs . Blood samples were taken just before the first administration (0 h) and every 24 h for seven days. Our results show that Argovit™ AgNPs induced no significant cytotoxic or acute genotoxic damage. The observed cumulative genotoxic damage in this model could be caused by the accumulation of AgNPs due to administered consecutive doses. Furthermore, the administration of AgNPs after 24 h of CP seems to have a protective effect on bone marrow and reduces by up to 50% the acute genotoxic damage induced by CP. However, this protection is not enough to counteract several doses of CP. To our knowledge, this is the first time that the exceptional chemoprotective capacity produced by a non-cytotoxic silver nanoparticle formulation against CP genotoxic damage has been reported. These findings raise the possibility of using AgNPs as an adjuvant agent with current treatments, reducing adverse effects. The increasing use of silver nanoparticles (AgNPs) in many fields and especially in biomedical applications requires the expansion of studies on their biocompatibility and biosecurity, especially concerning cytotoxicity and genotoxicity . ResearcAllium test [Allium cepa model, a concentration range of 5–100 μg/mL did not induce cytotoxic or genotoxic damage. It was suggested that the lack of damage was due to the polyvinylpyrrolidone (PVP)/silver ratio in the formulation. The promising results obtained with Argovit™ demonstrate the need for the completion of its toxicity studies.Since 2004, Argovit™, a commercial formulation of silver nanoparticles, has been studied in vitro and in vivo for its multiple biomedical applications  ,12,13,14ium test ,18. In tIn this context, an in vivo micronucleus test is commonly used to assess and identify genetic material damage produced by several materials and substances that do not necessarily kill the cells . MicronuThe antineoplastic compound cyclophosphamide is widely used as a positive control of myelosuppression (cytotoxicity) and irreversible DNA damage (genotoxicity) in the micronucleus assay in mouse peripheral blood erythrocytes ,22. Its Therefore, the present work explores the cytotoxicity and genotoxicity of Argovit™ AgNPs based on the micronucleus technique in peripheral blood erythrocytes of BALB/c mice. The Argovit™ AgNP formulation was administered under different treatment schemes that include combination with the antineoplastic agent cyclophosphamide to identify its toxicity and protective potency.w/w) of PVP, the concentration of metallic silver is 1.2% (w/w), and the rest of the formulation is distilled water. The surface plasmon resonance absorbance was found at 420 nm, with a zeta potential of −15 mV, and a hydrodynamic diameter of 70 nm [Argovit™ AgNPs were donated by the Scientific and Production Center Vector Vita , and their physicochemical characteristics have been previously reported by our group . The metof 70 nm . The plaCyclophosphamide (CP) was applied intraperitoneally (i.p.) as a positive control. The i.p. dose of 50 mg/kg guarantees the induction of MN ,27.Distilled water .AgNPs (6 mg/kg) were orally administered considering the results observed in other antiviral and antitumor in vivo models ,14,28.The application of positive and negative controls through different administration routes is accepted for this type of study . All dosThe study was carried out on male BALB/c mice of 5–6 weeks of age and a mean weight of 20.11 ± 2.38 g obtained from the Centro de Investigación Biomédica de Occidente (CIBO) of Instituto Mexicano del Seguro Social (IMSS), Guadalajara, Jalisco, Mexico. The animals were placed inside polycarbonate cages bedded with sawdust powder, were acclimated to the laboratory conditions for two weeks before the treatment application, and were fed ad libitum with standard rodent pellets and water. The mice management was carried out as established in the Technical Specifications for Production, Care, and Use of Laboratory Animals . For all groups, clinical signs, including skin and fur appearance, eyes, membranes, conduct, respiratory pattern, the presence of nausea, vomiting, diarrhea, tremors, convulsions, and lethargy, were carefully registered throughout the experiment. Animals were weighed at the beginning and the end of the experiment. Obtained values were compared with averaged weight values reported by the provider according to the age of the animal.Five random groups of six male mice were formed, as suggested by the OECD Guidelines for the Testing of Chemicals . Before Just before the first administration and during the treatment, a drop of peripheral blood from each mice tail were obtained each 24 h until 168 h, smeared on a sample holder, and air-dried for 10 min. After that, the sample was fixated with ethanol  for 10 min and then stained with acridine orange . All slides were air-dried and stored in the dark . The prep < 0.0001) using Statistica 8.0  software. The graphics were generated with GraphPad Prism 9.0.0 .Statistical analysis was performed through two-way ANOVA and the post-hoc Tukey test  and immature (MNPCE) erythrocytes are used as genotoxicity biomarkers . MNE frep < 0.0001, letter E) than in organisms treated only with CP. However, the cytoprotective effect is not maintained when applying CP for more days, since the acute genotoxic damage is evident, as observed in Group 5, even with the myelosuppression observed in In the case of AgNPs administered in three consecutive doses (Group 3), the frequency of MNPCE was similar over time, suggesting that the AgNPs are not genotoxic. Even when administered after the application of CP (Group 4), the nanoparticles show a cytoprotective effect since they reduce the acute genotoxic effect of CP, which is corroborated with the frequency of MNPCE being approximately 50% lower . The acute genotoxic damage in Group 5 is remarkable considering the myelosuppression observed since the 72 h point  in both.The increase in MNE frequency observed at 24 and 48 h, despite noteworthy, was not taken into account on the genotoxic damage analysis following the recommendation of OECD guideline 474 . On the This work investigated the genotoxic potential of the commercially available Argovit™ AgNP formulation on BALB/c mice. This formulation has shown remarkable results as an antiviral ,14,34,35This study found no evidence of myelosuppression Group 3,  or acute2 induce DNA breaks and hence, the formation of MN [As observed in on of MN . On the on of MN ,53, whicThe great stability of the Argovit™ formulation and its low cytotoxicity could be the reason why myelosuppression or acute genotoxic damage is not observed. Some 20 nm AgNPs formulations have been reported to compromise viability (>10 µg/mL) and enzymatic and non-enzymatic antioxidant systems (>0.4 µg/mL) of erythrocytes in a concentration-dependent manner, with the released silver ions being responsible for those effects ,55. SilvOn the other hand, the administration of a single dose of CP produces remarkable myelosuppression Group 2,  and acutS. officinalis extract through feeding for 7 days [S. officinalis.A new question arises: do AgNPs serve as adjuvants for CP? The antiproliferative activity of Argovit™ AgNPs against several human tumor cell lines has been previously reported ,61. Therr 7 days  results Several natural products have shown an ability to decrease MN frequency provoked by CP, although practically all compounds must be administered before CP to show their chemoprotective effect ,66,67,68The buffering capacity of Argovit™ against acute and accumulative genotoxic damage at earlier stages seems not sufficient to counteract consecutive doses of CP. However, it is important to remember that the chosen CP dose of 50 mg/kg used in this work guarantees an MNPCE increase. We found that a single dose of 6 mg/kg of the AgNP formulation can reduce by up to 50% the MNPCE frequency elicited by a single dose of CP, delaying MNPCE production after multiple dose administrations of CP of a genotoxic concentration. Although the administration of AgNPs cannot reverse the myelosuppression caused by CP, it helps to reduce nuclear damage in PCEs. More studies must be performed to identify the events that lead to accumulative genotoxic damage without evidence of myelosuppression and acute genotoxic damage. Additionally, further experiments are needed to recognize the administration scheme that could be effective as adjuvant on antitumor treatments.Despite the accumulative genotoxic damage observed after three consecutive doses of AgNPs, the above results prove that bone marrow incurs no damage after the oral administration of an effective dose of AgNPs Argovit™, as no changes were presented on immature erythrocyte population compared with the negative control. The Argovit™ silver nanoparticle formulation presents neither cytotoxic nor acute genotoxic damage in peripheral blood erythrocytes of BALB/c mice treated with a 6 mg/kg scheme every 24 h for three days. Although cumulative genotoxic damage was observed, the effect disappeared over time without decreasing the PCE frequency. These results indicate the significant biocompatibility of these AgNPs on the murine model BALB/c, compared with CP that induces myelosuppression. To our knowledge, this is the first time that the modulation of genotoxic damage produced by cyclophosphamide has been reported using a non-cytotoxic silver nanoparticle formulation. Argovit™ can reduce by up to 50% the micronucleated polychromatic erythrocyte frequency (acute genotoxicity) when administered 24 h after a genotoxic dose of CP (50 mg/kg) and delay the appearance of micronuclei until 24 h after multiple cyclophosphamide administrations. These results suggest a silver nanoparticle-modulating effect on the damage that cyclophosphamide can generate in healthy cells. In addition to this genotoxic modulating effect, it seems that AgNPs reduce the cytotoxic damage caused by the previously applied antineoplastic agents.These results will undoubtedly contribute to the design of new chemotherapy treatments that will diminish the adverse effects caused by the current treatments taking advantage of the exceptional antigenotoxic capacity of the Argovit™ silver nanoparticle formulation.


Nonrandom multiple respiratory virus (RV) detection provides evidence for viral interference among respiratory viruses. However, little is known as to whether it occurs randomly.The prevalence of dual RV detection (DRVD) in patients with acute respiratory illnesses (ARIs) at 4 academic medical centers was investigated; data about the prevalence of 8 RVs were collected from the Korean national RV surveillance dataset. Linear regression analysis was performed to assess the correlation between observed and estimated prevalence of each type of DRVD.In total, 108 patients with ARIs showing DRVD were included in this study between 2011 and 2017. In several types of regression analysis, a strong correlation was observed between the observed and estimated prevalence of each type of DRVD. Excluding three DRVD types , the slope of the regression line was higher than that of the line of random occurrence (1.231 > 1.000) and the 95% confidence interval of the regression line was located above the line of random occurrence.Contrary to the results of previous epidemiologic studies, most types of DRVD occur more frequently than expected from the prevalence rates of individual RV, except for three underrepresented pairs above. After performing multiplex polymerase chain reaction (PCR) test for the detection of common respiratory viruses (RVs), simultaneous detection of two different RVs—dual RV detection (DRVD)—is frequently observed in patients with acute respiratory illness (ARI). Clinicians are interested in the two aspects of DRVD. First, patients with ARI showing DRVD may present more severe clinical manifestations or poorer clinical outcomes than those showing single RV detection. Many clinical studies have been conducted for this subject , 2. SecoTherefore, we estimated the frequency of DRVD on a weekly basis from the national RV surveillance dataset that included the weekly prevalence of each RV in the community over a much longer study period of about 6 years (the surveillance dataset) and summed the frequencies according to each type of DRVD. Moreover, we collected the clinical data of adult hospitalized patients with ARI showing every type of DRVD from multiple academic medical centers . Subsequently, we compared the observed frequency of each type of DRVD from the clinical dataset to the summed weekly estimated frequency of each corresponding DRVD type from the surveillance dataset to identify if DRVD occurs randomly or not.This study was performed at 4 academic medical centers in South Korea: 2 in Seoul  and 2 in the Province of Gyeonggi . We identified adult patients (aged ≥ 16 years) with ARI who underwent multiplex PCR test for RVs and showed DRVD during the following periods at each study center: Chung-Ang University Hospital, first week of 2012–14th week of 2016; Kyung Hee University Medical Center, first week of 2011–13th week of 2017; Inje University Ilsan Paik Hospital, first week of 2012–14th week of 2017; and Soonchunhyang University Bucheon Hospital, first week of 2012–43rd week of 2015. Infectious disease physicians at each center retrospectively reviewed the patients’ medical records and chest radiographs. Demographic characteristics, date of multiplex PCR test, illness-related symptoms, and chest radiography findings were investigated.RVs were classified into the following 8 categories: adenovirus (ADV), human bocavirus (hBoV), human coronavirus , influenza virus , human metapneumovirus (hMPV), parainfluenza virus , picornavirus , and respiratory syncytial virus . ARI was defined as the development of at least one of the following respiratory symptoms: cough, sputum production, rhinorrhea, sore throat, and dyspnea.The national RV surveillance dataset of South Korea, that is, the data from the Korea Influenza and Respiratory Surveillance System established by the Korean Center for Disease Control and Prevention (KCDC), was used to determine the viral activity of each of the 8 RVs in the community. The dataset included weekly reports of positivity rates for each of the 8 RVs: ADV, hBoV, hCoV, IFV, hMPV, PIV, RHV, and RSV. Detailed information about the dataset can be found elsewhere . Data pek and P[B]k) was obtained from the surveillance dataset, number of multiplex PCR tests performed for RVs during k week (Nk) was available from the study centers, and conversion constants for each RV (C[A] and C[B]) were obtained from a previous study conducted by our group, which showed the relationship between the surveillance dataset and the data of adult hospitalized patients with ARI during 2 years [Estimation of the number of patients who showed DRVD of RVs A and B during the 327 weeks was performed as described in Fig.  2 years . The conY , and an independent variable, X (estimated frequency according to the surveillance dataset). P value < 0.05 was considered statistically significant. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software .Linear regression analysis was performed to assess the correlation between the observed and estimated prevalence rates of each type of DRVD. We used the standard model of linear regression, assuming the presence of a dependent variable, During the study period, 5,476 RV-related multiplex PCR tests were performed at the 4 study centers, and the median number of tests performed each week was 14.0 . A total of 134 adult hospitalized patients showed multiple RV detection. Of these patients, 10 had no symptoms and signs of ARI. Of the remaining 124 patients, 10 showed DRVD involving the same RV groups , and 6 showed detection of 3 RVs . After excluding these patients, 108 adult hospitalized patients with ARIs showing DRVD were finally included in our analyses. The median age of the study patients was 68.5 years (ranged from 16 to 94 years) and more than a half of them were male . The most common underlying disease was diabetes mellitus , followed by chronic lung disease , chronic renal disease , use of immunosuppressants , cerebrovascular disease , heart failure , solid tumor , and so on. The majority of them had community-acquired ARI . Pneumonia, severe pneumonia (pneumonia requiring the use of ventilator therapy or vasopressor), co-infection with non-RV pathogens, and in-hospital death were observed in 87 (80.6%), 35 (32.4%), 48 (44.4%), and 18 (17.5% of 103), respectively. Table Figure R2 = 0.532, P < 0.0001).Figure R2 = 0.662, P < 0.0001). The line showing direct proportional relationship  is within the 95% confidence intervals of the regression line (Y = 0.814X + 0.862). Figure Figure R2 = 0.862, P < 0.0001). After the removal of the above 3 DRVD types from Fig. Y = 1.173X + 0.205) was higher than that (Y = 0.814X + 0.862) in Fig. Y = X) lies below and outside the 95% confidence intervals of the regression line.Figure The observed frequencies of the 28 types of DRVD from several hospitals and the estimated frequencies of each corresponding DRVD type from the national weekly RV surveillance dataset were strongly positively correlated. Most of the DRVD types occurred more frequently than expected from the prevalence rates of individual RVs except for the 3 DRVD types , which occurred far less frequently than expected.Viral interaction between RVs has been suggested largely through four types of studies . First, Most DRVD types were found more frequently than expected from the prevalence rates of individual RVs. These findings are contradictory to the findings of the previous epidemiological studies, which reported that many types of DRVD were underrepresented. Brunstein et al. reported that IFV-B/RSV-A, hMPV/RHV, RSV-A/hMPV, and RSV-B/hMPV were the underrepresented pairs . Greer eThree DRVD types  were removed from Fig. This study has important limitations. Surveillance dataset and clinical dataset were collected with different purposes, objects, and methods. Because of the differences, care should be taken when interpreting the results. However, methodological limitations of the previous studies may not be easily overcome without such vast resources of surveillance dataset. Additionally, we used the data of the previous study of our group to address this problem—the conversion constants were calculated from the data of the study . This stWe found that, in the era of multiplex PCR test for RVs, most DRVD types significantly occurred more frequently than expected from the prevalence rates of individual RVs, except for a number of underrepresented pairs such as IFV/Picor, IFV/hMPV, and ADV/RSV.


Composting is a promising source of mesophilic and thermophilic microorganisms directly involved in the decay of organic matter. However, there is a paucity of information related to bacterial and fungal diversity in compost and their enzymatic activities during the composting process. In this work, bacterial and fungal diversity during the mesophilic and thermophilic phases of textile waste composting was investigated as a way to explain the physical–chemical results obtained during the composting process. This was accomplished using a next-generation sequencing approach that targets either the 16S rRNA or ITS genomic regions of bacteria and fungi, respectively. It was observed that Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant bacterial phyla present at the mesophilic phase but not at the thermophilic one. Composting textile waste exhibits a sustained thermophilic profile (above 55 °C) that usually precludes fungal activity. Nonetheless, the presence of fungi at the thermophilic phase was observed. Rozellomycota, Basidiomycota, and Ascomycota were the most dominant phyla during both composting phases. Such thermophilic fungi with great ability to decay organic matter could be isolated as pure cultures and used for the bioaugmentation of textile waste composting to achieve an advanced maturity level of textile waste compost. It is a highly dynamic process that consists of three phases: mesophilic, thermophilic, and maturation2. Alternating mesophilic and thermophilic microbial consortia perform the bio-decomposition of organic matter according to distinct requirements and tolerances that are compatible with the continuously changing environment3. Microbial metabolic processes play a significant role in decaying recalcitrant carbon- and nitrogen-containing molecules such as cellulose, lignocellulose, and proteins present in the feedstock, resulting in a product that can be used as a fertilizer for plants or as a soil conditioner4.Composting is a self-heating treatment through which the microbial metabolism raises the temperature above 50 °C, followed by a gradual cooling towards the end of process5. This waste is a fountain of organic matter, which could be recovered biologically through composting into bio-fertilizer, thus providing the nutrients necessary for plant growth.In Morocco, the textile industries generate around 4 tons of solid waste annually. This waste is disposed of into the environment without any form of treatment, and thus represents a significant threat to the functioning of many ecosystems1. In line with their outstanding role in composting, microorganisms have aroused the interest of researchers6. Despite the number of studies concerning the identification of mesophilic and thermophilic microorganisms during composting, a deeper understanding of specific taxonomic and functional groups is still required3. Several groups of microorganisms are related to the composting process, with bacteria and fungi being the most prominent6. Bacteria are considered the most dominant species during the transformation processes due to their higher thermal tolerance7. However, fungi play a significant role in composting by releasing the enzymes  that destroy recalcitrant molecules, which are not easily decomposed by other microorganisms6. In addition, they can tolerate change even under the harshest environmental conditions. Indeed, during composting, fungal diversity is influenced by changes in physical–chemical parameters such as temperature, moisture, and nutritional properties according to different stages8.Composting is considered a promising source of new mesophilic and thermophilic bacteria and fungi, especially those involved in the degradation of biomass6. Next-generation sequencing is considered a practical approach to expanding the repertoire of known biodegrading microorganisms and elucidating their metabolic potential during the different phases of composting.However, two different methodological perspectives could be applied to the study of the microbiota associated with composting: culture-dependent and culture-independent methods. Owing to their potential to provide complete information on the microbiome during composting, thus avoiding the problems associated with cultivation, molecular techniques have recently become increasingly popularSeveral studies have focused on the maturity of compost, but only a handful have been devoted to the alternation between mesophilic and thermophilic microbial communities. As outlined above, the main objective of this study was to establish the relationships between specific microorganisms  and composting phases using next-generation sequencing. Additionally, this study aimed to identify new mesophilic and thermophilic microbial genera throughout the composting of textile waste, which could be beneficial for performing processes that are more efficient and improving final products.4+/NO3− ratios depended on temperature  and NH4+/NO3− ratios (p = 0.0006). According to several authors, a C/N ratio between 15 and 20 and an NH4+/NO3− ratio below 1 reflects a good level of organic matter degradation, thus proving a very advanced degree of maturation of textile waste compost10. The temperature increased significantly, (p = 0.02) from 30 °C on week 9 to 53 °C on week 28, and decreased to 39 °C by the end of composting. Together with the temperature increase, the moisture decreased significantly, (p = 0.0004) from 71.2 to 10.8%, and the pH (p = 0.0001) decreased from 7.6 to 6.4 by the end of composting  sequencing of mesophilic and thermophilic compost samples generated by NGS. Table Analysis of Fig. Cellulomonas (Actinobacteria), Steroidobacter (γ-proteobacteria), Mycobacterium (Actinobacteria), Streptomyces (Actinobacteria), and Paenibacillus (Firmicutes) were the most abundant throughout the mesophilic phase . Other genera were also identified during this investigation as organic matter degraders, although in minor abundance—namely Chitinophaga (Bacteroidetes), Nitrosomonas (Proteobacteria), and Nitrobacter (Proteobacteria).In total, 21 genera were detected in the mesophilic and thermophilic phases of textile waste composting. They are presented in Table Cellulomonas in the thermophilic phase, which was entirely absent (Table Devosia (α-proteobacteria), Flavobacterium (Bacteroidetes), Pseudoxanthomonas (Proteobacteria), Pseudomonas (γ-proteobacteria), and Achromobacter (β-proteobacteria) were the most abundant genera during the thermophilic phase. The abundance of these genera was substantially lower in the mesophilic phase. Additionally, using the UniProt database, the most common enzymatic activities in the mesophilic and thermophilic phases were predicted, thus forecasting the presence of cellulase, hemicellulase, xylanase, pectin depolymerase, and phosphatases  in both phases  sequencing of mesophilic and thermophilic compost samples generated by NGS. Table The relative abundances of fungi phyla in compost samples were examined at different phases of composting. Figure At the class level, Sordariomycetes, Tremellomycetes, Agaricomycetes, Eurotiomycetes, Saccharomycetes, Orbiliomycetes, Pezizomycetes, and Dothideomyctes were present in both phases. Indeed, Sordariomycetes, Tremellomycetes, Saccharomycetes, Orbiliomycetes, and Dothideomyctes  were the most abundant fungal classes in the mesophilic phase. In contrast, Agaricomycetes, Eurotiomycetes, and Pezizomycetes  prevailed in the thermophilic phase.Fusarium, Acremonium, Penicillium, and Rhizopus were detected as the most frequently identified genera during composting, but with a generally higher abundance in the thermophilic phase compared to the mesophilic. In the mesophilic phase of composting, Aspergillus and Cyphellophora were present, while for the thermophilic phase, Fusarium, Acremonium, Penicillium, Rhizopus, and Phialophora were the most abundant genera. Still, some exceptions were noted with some genera from the list of abundant fungi present in the mesophilic phase that were not present in the thermophilic phase, and vice-versa  through their enzymatic system: Devosia, Flavobacterium, Pseudoxanthomonas, Pseudomonas, and Achromobacter20. Aside from this, the Devosia genus was described as a degrader of organic sulfur, phosphorus, and aromatic compounds according to Chandni et al.21. Several authors have demonstrated that Paenibacillus (belonging to the Firmicutes) could utilize different enzymatic activities to destroy the lignocellulosic compounds such as cellulose, xylose, and hemicelluloses22. This is highlighted using the UniProt database  during the composting of textile waste. They also reflect the level of diversity of the microbial community in the feedstock and the high degree of functional redundancy known to exist in these microbial communities. In this regard, different metabolic pathways lead to significant changes in the physical–chemical parameters of the substrate, which in turn lead to community changes and influence microbial abundance and the succession of microorganisms. Several phases occur during the composting of textile waste. At the beginning of composting, temperatures below 45 °C characterize the mesophilic phase Fig. . During Chitinophaga, Nitrosomonas, and Nitrobacter. Chitinophaga (belonging to Bacteroidetes) was recently identified as cellulose-degrading species during the mesophilic phase, and was found to be able to grow on solubilized forms of cellulose from green-waste compost2. Interestingly, three genera associated with the nitrification phenomenon—namely Steroidobacter, Nitrosomonas, and Nitrobacter—were identified. The numbers of nitrate- (Nitrobacter) and ammonium-oxidizing bacteria (Nitrosomonas) rose markedly through the composting processes. Steroidobacter is capable of denitrification using only a narrow range of organic substrates and nitrate as the electron acceptor23. All of these genera are nitrogen transformers, and can degrade organic matter into products capable of supporting plant growth by providing the necessary nitrogen. Our findings could be used to explain the changes in the NH4+/NO3− ratio observed during this investigation. Ultimately, using the rarefaction curves  are abundant in the thermophilic stages of composting, and have demonstrated their ability to metabolize complex organic compounds—except in the case of Cellulomonas, which is not conspicuous in the thermophilic phase. This could be assigned to the fact that bacteria of this genus cannot survive at high temperatures. The enzymatic activities  of Cellulomonas were more remarkable in the mesophilic phase (37 °C).Additionally, actinomycetes are multicellular filamentous bacteria acting at the later stages of composting. An increase in the concentration of actinomycetes during the thermophilic phase has previously been reported18. The high concentration of cellulolytic microorganisms during the thermophilic phase could be explained by high temperatures that favor the degradation of cellulose26, thus proving the major role played by thermophilic microorganisms in decaying cellulose during the composting of textile waste. Notably, sequences associated with Escherichia, Salmonella, or Listeria were not detected, which demonstrates that composting is an effective process for the destruction of pathogens and pathogen indicators owing to multiple heating cycles during composting18.All of the genera detected during both phases possess several enzymes Table  involved27. Variations in fungal communities corresponding to different phases (mesophilic and thermophilic) could be explained by changes in the physical–chemical characteristics of the compost highlighted during this investigation, such as pH, temperature, and moisture content. Temperature is the most critical factor affecting fungal growth. Most fungi are mesophilic, and thrive between 5 and 37 °C, with an optimum temperature of 25–30 °C29, but some also grow at higher temperatures. For example, Penicillium and Fusarium, which have been the subject of numerous studies, have demonstrated the ability to produce enzymes active in cellulose and lignin degradation even at high temperatures of 46–49 °C8. The presence of Penicillium, Aspergillus, Acremonium, and Fusarium in compost samples was expected. Penicillium is ubiquitous in the environment, and plays a vital role in decaying organic matter 31. Aspergillus, Acremonium, and Fusarium are associated with the degradation of organic matter . These findings are supported by the prediction obtained from the UniProt database , and corroborates the results of the current study regarding the C/N and NH4+/NO3− ratios.Interestingly, fungi can decay the organic matter not transformed by bacteria. Several authors found that Sordariomycetes and Eurotiomycetes are the most abundant eukaryotes in both phases of the composting process29. Remarkably, the added value of this study lies in the presence of a high abundance of thermophilic fungi in this phase . A mixture was established for composting shredded waste, labelled ‘Mix A’ (with a 40%/30%/30% ratio of textile/green/paper and cardboard waste). After the appropriate components were mixed, the silo was turned at least three times per week for 44 weeks. Samples were collected according to the four cardinal positions  from three depths  in triplicate each week , and were then placed into polythene bags and stored at 4 °C until further analysis was conducted.The preparation of the mixture was performed according to a previously established procedure32. The C/N ratio for the initial mix and the end products was calculated by analyzing the total C and N percentages using a TOC analyzer (Shimadzu-V CSN)33. The total ammonium and nitrate ion (NH4+/NO3−) ratios for the pre-composted mix and the final products were calculated from the percentage of NH4+ and NO3−. This was performed using the method based on alkaline distillation for ammonium ions (NH4+), and using reduction by Dewarda alloying for nitrate ions (NO3−)9.A calibrated pH meter was used to measure pH (Hanna Hl-10530 pH meter). Moisture was analyzed according to the protocol described by the French Association for Standardization—AFNORTemperature was measured in triplicate each week. The silo was divided into three depths. After turning, measurements were taken from each of these three areas to allow for the equilibration of silo temperature after the incorporation of cooler air by inversion. The temperature was monitored using an all-sun ETP109B Digital Thermometer. To measure the inner temperature, the probe was pushed to the maximum depth of the silo, while the outer temperature was measured close to the surface. Both readings were then averaged.DNA was extracted from compost samples which were taken from the silo during both the mesophilic (week 9) and thermophilic phases (week 28) of the process. This was done using the PureLink Microbiome DNA Purification Kit following the manufacturers’ instructions, and was stored at − 20 °C until use. The quantity and quality of the extracted DNA were initially checked using agarose gel electrophoresis, and the samples later passed quality control at Macrogen before amplification and sequencing.The sequencing and amplification of 16S rRNA or Internal Transcribed Spacer (ITS) was performed at the Genomic Analysis Platform Macrogen (Republic of Korea), using the Illumina sequencing platform.The following oligonucleotide sequences were used for the amplification of the 16S rRNA gene, targeting the V3–V4 region from 16S rRNA primers specific to bacteria:Forward:5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG.Reverse:5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC.For the fungi, the internal transcribed ITS2 spacer region was amplified using forward primer ITS3 (5′-GCATCGATGAAGAACGCAGC-3′) and reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).34, the files generated from the paired-end sequencing were filtered and aligned with the SILVA v132 database. Fungal sequences were then trimmed using CUTADAPT to remove primer sequences and, finally, sorted according to barcode. The selected region of fungal origin was then filtered using DADA2, an open-source pipeline that uses a different algorithm for sequence clustering, which was then aligned with the UNITE 8.0 database35. Rarefaction curves were created to see the bacterial and fungal diversity of the samples; these curves were added in the supplementary materials. Enzymatic activities were predicted using the UniProt  database, a comprehensive resource for protein sequence and annotation data that allows different protein sequences to be searched for, coding for a wide range of enzymatic proteins. In this way, bacterial and fungal genera found in samples were searched for in the UniProt database. The proteins encoding genes were also searched for in sequenced species from the genera found. All of these results were analyzed using ANOVA , carried out using the GraphPad Prism.Bioinformatics analysis and data annotation were performed by using Mothur to analyze 16S rRNA gene fragments for bacteria and DADA2 to analyze ITS regions for fungi. After demultiplexing the raw FASTQ files using Mothur v 1.44.3Supplementary Figures.


Our results showed that artificial shade decreased community biomass accumulation whereas fertilization increased it. In contrast, both shade and fertilization reduced species diversity. Extinction of non-Gramineae species  was the main reason for species diversity decline. Species loss can be explained by the limitation of light availability and predicted by species traits associated with light acquisition capability under fertilization and low light tolerance under artificial shade. Specifically, fertilization eliminated species with lower stature and artificial shade exterminated species with the higher light compensation point (LCP). The findings suggest that light availability is consistently important for plant growth and that low competitiveness for light under fertilization and intolerance of low light conditions under artificial shade trigger species loss process in the alpine meadow. Our experiment helps clarify the mechanisms of how artificial shade and fertilization decreased species diversity and highlight that LCP, which tends to be neglected by most of the studies, is one of the vital drivers in determining species coexistence.Fertilization is an effective management strategy to promote community biomass but can simultaneously reduce species diversity in many grassland systems. Shifts in competition for resources have been proposed to explain the decline in plant species diversity due to fertilization, yet the underlying mechanism driving species loss remains controversial. This uncertainty may be driven by variation in aboveground and belowground resource availability. However, experiments simultaneously manipulating both light availability and soil nutrients are rare. Using a 6-year field experiment to manipulate light availability ( Human activities, such as industrial agriculture, are altering terrestrial nutrient availability worldwide by widespread fertilizer application . FertiliThe decline of plant species diversity at high soil fertility or high productivity is thought to be a result of increased competition for light . The ligWhile previous studies have observed species decline due to a reduction in light availability after fertilizer application , there iWhile the majority of experimental approaches used to investigate the relationship between light competition and diversity loss are derived from community-level studies , fewer svia increasing soil resource availability and directly via shade cloth in an alpine meadow of the Tibetan Plateau. We compared the effects of nutrient addition and artificial shade on species diversity and community composition and then, explored the relationships between species relative abundance (SRA) and functional traits to address the following three questions. (1) How does plant species diversity respond to changes in light and nutrient availability? (2) Do changes in community composition within the artificial shade and fertilization treatment convergent or divergent along a temporal scale? and (3) Are the functional traits associated with light acquisition capability and the minimum light level required for photosynthesis good indicators to predict species loss in the alpine meadow of the Tibetan Plateau?Alpine meadows within the Tibetan Plateau are a climate-sensitive ecosystem and currently face the selection pressures of increasing nutrient loading ,b. UnderKobresia graminifolia), grasses , species of Compositae , and other broad-leaved species . The average aboveground biomass within the experimental site (450 m × 220 m) ranges from 280 to 400 g m–2 dry weight, which corresponds to the median value of global grassland productivity; and the species richness ranges from 20 to 35 per 0.25 m2, at the upper limit of species diversity among the global grassland ecosystems , eastern Tibetan Plateau, China. The annual average temperature during the sampling period (2008–2012) ranged from −8.2°C in winter to 11.7°C in summer, with the annual average precipitation of 706 mm, mainly distributed during the short, cool summer . The vegosystems .–2 year–1 (no fertilizer addition) and 45 g m–2 year–1 of a slow-release pelletized fertilizer [(NH4)2HPO4] manufactured by Tianjin International Trading Company, Tianjin, China, corresponding to 9.5 g N m–2 and 10.6 g P m–2. The nutrient treatment was nested within the light availability treatment block, resulting in a total of four treatments: fertilizer addition and ambient light (F); no fertilizer addition and shade (S); synchronous fertilizer addition and shade (F+S); and no fertilizer addition and ambient light (CK). Thirty-two permanent 2 × 2 m plots were established with 16 plots under the shade shed and the other 16 plots outside the shed, arranged in a regular 4 × 4 matrix with a 2-m buffer zone between plots. Half of the plots both inside and outside of the shade shed were randomly selected to add fertilizer, which means that each treatment had eight replications (refer to n = 12) to evaluate the entire experimental community characteristics of the location site on August 25, 2007. Shade and fertilization treatments were applied annually at the beginning of each growing season (end of May) from 2008 to 2012. Artificial shade shed was dismissed annually during the nongrowing season  to avoid destruction by local strong wind and heavy snow.To assess the impact of light and soil nutrient availability on species diversity, two blocks (25 m × 30 m) were established within the fenced exclosure (450 m × 220 m) to exclude herbivores. Light availability was manipulated by covering one block with a black polypropylene mesh shade cloth, which was permeable to air and water, to reduce 70 ± 2% (mean ± SD) of PAR. The shade cloth was suspended 1.3 m above the ground surface by wooden stakes attached to the corners of one block and fastened to the ground on all sides. The other block remained uncovered, corresponding to ambient light availability. Nutrient availability was manipulated by two fertilizer addition levels: 0 g m–2 g). We separated all species into two functional groups: Gramineae and non-Gramineae groups, which generally correspond with their appearance in the canopy and understory within the quadrats, respectively. Within the non-Gramineae group, species belonging to Cyperaceae and Fabaceae families were analyzed as separate subgroups since these species are known to be sensitive to light resource availability  was randomly selected in each plot at the end of each growing season (September), approximately 0.5 m away from the edge to avoid the edge effect. Within each quadrat, species were identified, and the number of all individuals was recorded. For clonal species, we regarded a ramet as an individual . To quanlability .–2 g). Soil characteristics  were analyzed using the protocol described by To assess belowground biomass and soil nutrients, we employed the root in-growth method (3.5 cm diameter × 20 cm deep) to estimate the belowground biomass from each quadrat post aboveground harvesting in 2012. Soil samples were brought to the laboratory in air-tight plastic bags, and then, roots were washed from the soil cores with a 2-mm mesh sieve after air-drying. Root samples were dried at 60°C for 72 h and weighed  in August 2012. Light readings were taken on a cloudless day between 11:00 and 13:00 h. The ceptometer was placed north-south across each plot, and PAR was recorded at 0, 10, and 40 cm above the soil surface (below vegetation canopy). Additionally, PAR above the vegetation canopy was also recorded in each plot as a measure of full sunlight. The soil temperature was not monitored during the experimental period for the reason that the shade shed was permeable to air and water and suspended 1.3 m above the ground, resulting in no significant difference with fertilization plots in soil moisture and understory PAR .–2 s–1 were provided by red light-emitting diodes. Ambient air in the leaf chamber was maintained at 20°C, and the CO2 concentration of the incoming air was controlled at 400 μmol L–1. Parameters of photosynthesis-light response curve were simulated by the nonrectangular hyperbola equation using SPSS software (version 22.0). The model is given as follows:Prior to the community biomass collection in 2012, we selected 14 common species in fertilization plots, 17 species in shade plots, 7 species in synchronous shade and fertilization plots, and 21 species in control plots to measure functional traits . These sA is the net leaf photosynthesis rate (μmol m–2 s–1), Amax is the light-saturated net leaf photosynthesis rate (μmol m–2 s–1), a is the light-limited quantum efficiency (μmol CO2 μmol–1 photons), k is the curvature parameter, and Rday is the dark respiration rate. When A is zero, the PAR is the LCP.where H), which is calculated as:Two plant diversity indexes are calculated to describe the species diversity within each treatment plot. One is species richness, which is defined as the number of species present in a quadrat and the other one is the Shannon–Weiner index  for each treatment plot.where t-test (2-tailed) to test for differences of plant functional traits of Gramineae and non-Gramineae groups among the treatment plots at the community level. All the above statistical analyses were performed using SPSS version 22.0 .Prior to analysis, all response variables were tested to meet the assumptions of normality and heterogeneity of variance. Any variable not meeting these assumptions was log-transformed. To account for differences in light resource availability at different levels of the canopy, the scores of the first principle component (PC1) of principal component analysis (PCA) were viewed as a proxy of light availability . The PC1To assess the differences in community composition between the treatment plots, we computed the dissimilarities of plant species composition among different treatment plots along temporal scale by nonmetric multidimensional scaling (NMDS) with Bray-Curtis distance using the “metaMDS” function of the R package “Vegan” . The staGramineae and non-Gramineae groups, respectively. The pathways through which fertilization and shade influence SRA were also assessed  was employed to examine the relationships of fertilization, shade, and species functional traits and SRA among assessed . Two catassessed . All SEMF = 581.3, P < 0.001) and were significantly affected by fertilization, shade, and their interactions . On average, light intensities (0–40 cm heights above the ground) were 2,529 ± 74 μmol m–2 s–1 in the control plots. Fertilization, shade, and F+S treatments significantly reduced light intensity by 51.4, 76.3, and 76.9%, respectively , respectively. In addition, shade treatment had no significant effects on either SAN or SAP (both P > 0.05). There was a significant interactive effect of shade and fertilization on SAN (P = 0.001), yet the interaction effect was not detected on SAP (P > 0.05). Neither fertilization nor shade treatment significantly affected soil moisture, soil pH, and soil organic carbon , shade (49.2%), and F+S treatment (142.8%), and the SRA of non-Gramineae inevitably decreased in response to fertilization (27.3%), shade (14.6%), and F+S treatment (42.2%)  . The tree group, .Trigonella ruthenica (Fabaceae), Kobresia capillifolia (Cyperaceae), and E. nutans (Gramineae) in control plots to Gramineae species with high height (Poa poophagorum and Koeleria cristata) and non-Gramineae shade-tolerant species (Anemone obtusiloba) in fertilization plots and shade plots , but the relationship was not detected in the control, fertilization, or F+S plots , whereas no such relationship was observed in the control, shade, or F+S treatment plots  on the variation of SRA and found that the optimal SEM model explained 59% of the variations in SRA of ramineae . For Grasing LCP . For nonsing LCP .via simultaneously manipulating the light availability and soil nutrient conditions revealed that artificial shade could reduce species diversity as same as fertilization in the alpine meadow. Reduction in light availability triggered species loss process both in the artificial shade and fertilization plots but through different mechanisms. Specifically, fertilization facilitated the survival of species with tall stature, whereas artificial shade promoted the performance of species with low LCP. Our results confirmed that species height and LCP were effective indicators of species coexistence patterns in the light limitation habitat of the Tibetan Plateau.Overall, our 6-year field experiment via increasing aboveground biomass 2HPO4], a slow-release pelletized fertilizer, to manipulate the soil nutrient input. The fertilizer is widely used in the local agricultural cultivation and showed weak alkalinity, while other studies involving fertilization usually employed ammonium nitrate (NH4NO3), strong acid and weak base salt, as fertilizer and showed that fertilization induced soil acidification and reduced species diversity, which differed with our experimental design. Overall, to the best of our knowledge, the result that artificial shade performed the same negative effect on species diversity as fertilization had not been reported in previous studies. Our results provide novel evidence that artificial shade had the same negative effect on species diversity as increased nutrient availability and that species loss can be simply triggered by light limitation, independent of nutrient addition within alpine meadow systems.Our results also mirrored the commonly observed negative relationship between plant diversity and nutrient availability , 2015. IGramineae species, such as P. poophagorum and K. cristata, rapidly increased and dominated in the fertilization plots and shade plots instead of the former dominant species (T. ruthenica and K. capillifolia) of the initial communities  . The reamunities . Fabaceant plots .T. ruthenica (8.22%) dominated the control community but absent from fertilization plots and only covered 0.53% in the shade plots . In contrast, artificial shade decreased aboveground and belowground biomass synchronously, resulting in a neutral —change in the root:shoot ratio  remarkably increased in shade plots .The original contributions presented in the study are included in the article/JL and ZY contributed to the conception and design of the study. JL organized the database and wrote the first draft of the manuscript. JL, ZY, and LC performed the statistical analysis and wrote the draft of the manuscript. GD and SF provided the funding and revised the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


To improve the bone regeneration ability of pure polymer, varieties of bioactive components were incorporated to a biomolecular scaffold with different structures. In this study, polysilsesquioxane (POSS), pearl powder and dexamethasone loaded porous carbon nanofibers (DEX@PCNFs) were incorporated into polylactic (PLA) nanofibrous scaffold via electrospinning for the application of bone tissue regeneration. The morphology observation showed that the nanofibers were well formed through electrospinning process. The mineralization test of incubation in simulated body fluid (SBF) revealed that POSS incorporated scaffold obtained faster hydroxyapatite depositing ability than pristine PLA nanofibers. Importantly, benefitting from the bioactive components of pearl powder like bone morphogenetic protein (BMP), bone mesenchymal stem cells (BMSCs) cultured on the composite scaffold presented higher proliferation rate. In addition, by further incorporating with DEX@PCNFs, the alkaline phosphatase (ALP) level and calcium deposition were a little higher based on pearl powder. Consequently, the novel POSS, pearl powder and DEX@PCNFs multi-incorporated PLA nanofibrous scaffold can provide better ability to enhance the biocompatibility and accelerate osteogenic differentiation of BMSCs, which has potential applications in bone tissue regeneration. Bone tissue engineering has developed to be an essential method for therapy of bone defects caused by mechanical injury, osteoporosis and other diseases ,2,3. As 3 and organic matters including BMP-2, which makes it show better bone regeneration activity than hydroxyapatite (HA), the main inorganic component of natural bone [Some growth factors will attend the regulation of bone formation process, such as bone morphogenetic protein (BMP), transforming growth factor β (TGF-β) and insulin like growth factor (IGF) ,17,18. Pral bone ,25,26,27To further enhance the capacity of bone regeneration, some drugs have been introduced with growth factors to perform a combined therapy, for instance, dexamethasone (DEX) is proven to be an osteogenic inducer to bone marrow stromal cells (BMSCs) ,29,30,31Moreover, except for the stimulation of cell activity, calcium deposition is another crucial property to new bone formation. Generally, pure polymer cannot quickly deposit calcium unless incorporated with some additives, which can play a role as nucleus or obtain special groups to catch calcium. Silica based bioceramics have been proven to chemically combine with bone tissue and promote new bone healing and growth ,36,37. PIn this study, a multi incorporated composite PLA nanofibrous scaffold was prepared with POSS, DEX and pearl powder by electrospinning, and the controlled release of DEX was realized by dexamethasone loaded porous carbon nanofibers (DEX@PCNFs). The effects of these components on mineralization, cytotoxicity and osteogenic differentiation of BMSCs were investigated to evaluate the potential application in bone tissue engineering.6, Guanghua Weiye Co., Ltd., Shenzhen, China) nanofibers were fabricated by electrospinning as reported in our previous work [The polysilsesquioxane  incorporated polylactic  prepared as the reference  for a ce2SO4 and HNO3 (volume ratio is 3:1) was used to treat the surface of PCNFs under 70 °C to make it hydrophilic and able to disperse in a water environment. For drug loading, 1 g dexamethasone (DEX) and 1 g PCNFs were added in 10 mL deionized water; the solution was ultrasonicated and kept stirring for 24 h under dark condition. Afterwards, the solution was centrifuged, and the solid was washed with deionized water to obtain DEX@PCNFs. The supernatant and washing solution were collected to determine the DEX loading amount by using UV–VIS spectra at wavelength of 242 nm. Before calculating the loading efficacy of DEX on PCNFs, a DEX standard curve of adsorption and concentration was studied. Subsequently, DEX solutions before and after adsorption were scanned by UV–VIS to obtain adsorption value, and corresponding concentrations were calculated according to the standard curve. Finally, the mass change, loading amount was further calculated.Firstly, porous carbon nanofibers (PCNFs) were prepared from polyacrylonitrile (PAN) and polymethylmethacrylate (PMMA) via electrospinning with the same procedures and factors as Part 2.1, followed by pre-oxidation 280 °C for 2 h and carbonization under 800 °C for 2 h of PAN/PMMA blend nanofibers. Then, a mixed acid solution of HBased on the fabrication of PS-NF in Part 2.1, pearl powder  and DEX@PCNFs were further added in the electrospinning to prepare multi-incorporated nanofibrous scaffold. To guarantee the success of electrospinning process, the adding amount of DEX@PCNFs was 1 wt% to PLA, and pearl powder was 5–15 wt%. Due to the amount of POSS and DEX@PCNFs was determined, the as-prepared multi-incorporated scaffold was marked as PSCP-NF according to the variation of pearl powder amount, for instance, PSCP5-NF mean that pearl powder is 5 wt% to PLA. The morphology and distribution of pearl powder and DEX@PCNFs were observed by SEM and transmission electron microscope .2 for normal proliferation cytotoxicity assay. When it came to the osteoconducting test, the normal medium was supplemented with 10 mM β-glycerol phosphate and 50 μg/mL of L-ascorbic acid. During all the culture period, the medium would be replaced every 3 days.Bone mesenchymal stem cells  of fourth to sixth generation were selected to complete all the in vitro experiments in this work. The cells were cultured in normal complete medium of low glucose DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO4 cells per well. After incubating for pre-determined time point, the samples were washed with PBS and continued to be incubated with 360 μL fresh low glucose DMEM and 40 μL MTT solution (5 mg/mL) per well for another 4 h. Then, the solution was removed, and 400 μL dimethylsulfoxide (DMSO) per well was added for further 0.5 h incubation in a 37 °C shaker for 10 min under dark condition. A volume of 100 μL of the supernatant per well was transferred to 96-well plate for the absorbance measurement at wavelength of 570 nm with a microplate reader . Every scaffold would set at least 3 parallel samples, and the average and standard deviation (SD) of the absorbance data were analyzed.The cytotoxicity of different samples was measured by MTT assay. Briefly, BMSCs were seeded on the surface of sanitized samples in 24-well plate with 400 μL normal complete medium at a density of 2 × 10For cell adhesion evaluation via confocal laser scanning microscopy (CLSM), BMSCs were seeded on the scaffolds and cultured for 3 days, then washed with PBS and fixed with 4% glutaraldehyde for 30 min at 4 °C. Afterwards, the cells were washed with PBS and soaked in 0.1% Triton X-100 solution for 5 min. After washing with PBS again, the cells were stained in turn with Alexa Flour@ 488 phalloidin solution (165 nM) and DAPI solution (100 nM) to label cytoplasm and nucleus, respectively. Finally, the samples were washed with PBS for CLSM observing.The alkaline phosphate (ALP) activity of BMSCs was tested by corresponding assay kit . Briefly, after incubating with different samples in conducting medium at pre-determined time points, BMSCs were lysed by cell lysis buffer, and 50 μL cell lysate was transferred into 96-well plate with 50 μL substrate solution for 30 min incubation at 37 °C, followed by adding 100 μL stop solution to terminate the reaction for evaluating the absorbance at wavelength of 405 nm via plate reader. In addition, another 10 μL cell lysate was used to measure the total protein concentration by protein assay kit .w/v) alizarin red S (ARS) solution with adjusted pH value of 4.1–4.3 for 10 min at room temperature. Then, ARS solution was removed, the sample was washed with deionized water until there was no residual ARS to obtain the stained sample, which was photographed with a camera.After incubating in conducting medium for pre-determined time point, BMSCs were fixed with 4% glutaraldehyde for 30 min and washed with PBS. Deionized water was further used to wash the samples to remove residual salt for the purpose of staining BMSCs with 2%  and Scheffe’s post hoc test. The statistical significance for all tests were considered at * p < 0.01 ,44.To improve the mineralization ability of PLA nanofibrous scaffold, POSS was added with different amount. The surface of as-prepared nanofibers was observed with SEM and showed in 2+ from SBF accelerate HA deposition [Mineralization property is an important aspect of the scaffold for the application of bone regeneration field. In this study, HA depositing capacity of the scaffold was investigated to reveal the effect of POSS incorporation. After incubating with SBF for 3 days, the morphology change of PLA-NF and PS2-NF was observed with SEM to find deposited HA particles. As shown in position .2O and CO2 [PCNFs were applied as drug carriers in this study, which were prepared through electrospinning of PAN and PMMA, followed by pre-oxidation and carbonization. As reported in our previous work, due to the different physical and chemical behavior of these two polymers during the preparing process, PAN would transform into carbon framework, while PMMA would be decomposed into H and CO2 . As showBesides the applied POSS and DEX@PCNFs, pearl powder was also incorporated in the nanofibrous scaffold. As shown in The investigation of the cytotoxicity of the as-prepared scaffold should be carried out before the cell differentiation test. BMSCs were applied to this in vitro experiment with MTT assay. The O.D. value of various scaffolds was presented in In this study, POSS was applied to accelerate HA deposition, which is the main inorganic component of natural bone tissue and could raise osteoconduction ability to BMSCs. DEX, a bioactive agent to improve the osteogenic differentiation activity of BMSCs, was loaded on PCNFs to construct drug-controlled release system. Pearl powder in nanosize was also used in bone tissue regeneration due to the bioactive component including BMP-2, which is a peptide benefiting to enhance BMSCs differentiation. Based on the above advantages of these materials, the multi-incorporated scaffold was expected to obtain much better osteoblastic differentiation activity comparing with pristine PLA. As a typical mark of differentiation, ALP activity of BMSCs incubated with different scaffold was measured, the upregulation of which is a key signal at the early period of bone regeneration. The results were showed in BMSCs would synthesize calcium deposition as mineralized product during the osteoconducting incubation period, which could be stained to red with ARS solution. After 2 weeks of differentiation, the ARS-stained samples were observed under optical microscopy and presented in In summary, a multi-incorporated PLA nanofibrous scaffold with different functional components was prepared through electrospinning for the application of bone tissue engineering. The mineralization result showed the POSS in the scaffold could accelerate HA deposition, which is beneficial to reconstruct bone tissue structure. Moreover, PCNFs were prepared and used to load DEX to obtained drug delivery system of DEX@PCNFs, which was co-incorporated with pearl powder to the scaffold. The in vitro experiment demonstrated that the DEX and bioactive component from pearl powder would contribute to the improving of biocompatibility, upregulating ALP activity and increasing calcium deposition of BMSCs based on pure PLA. Therefore, the multi-incorporated PLA nanofibrous scaffold obtained better osteogenic differentiation ability and showed bright potential for bone tissue regeneration.


Our recent study showed instability of orally administered egg yolk IgY in chicken GI tract, as demonstrated by both in vivo and ex vivo evidence. To better understand the magnitude and dynamics of instability of egg yolk IgY in vivo, in this study, we conducted comprehensive ex vivo analyses by spiking hyperimmune egg yolk IgY in fresh GI contents collected from five broilers at each sampling age . The pH in gizzard slightly increased with age from 2.4 to 3.0, while the pH in the small intestine was around 5.8. ELISA analysis indicated that a short time of treatment (30 or 60 min) of IgY with the gizzard contents from the chickens at 2, 4, and 6 weeks of age greatly reduced specific IgY titer by over 8, 6, and 5 log2 units, respectively, when compared with saline control. However, small intestine content only had a mild effect on egg yolk IgY, leading to 1 log2 unit of reduction in IgY titer upon 30 min of treatment. Consistent with these findings, SDS-PAGE and immunoblotting analyses provided direct evidence demonstrating that egg yolk IgY could be drastically degraded to undetectable level in gizzard content upon as short as 5 min of treatment; however, the IgY was only slightly degraded in small intestine content. Immunoblotting also showed that treatment of IgY with HCl (pH 3.0) for 60 min did not affect its integrity at all, further supporting the enzymatic degradation of IgY in gizzard. Collectively, egg yolk IgY could be substantially degraded in chicken gizzard, highly warranting the development of effective approaches, such as encapsulation, for the controlled release and protection of orally administered egg yolk IgY in livestock.Egg yolk antibody , due to its unique features , is emerging as a promising passive immune agent and alternative to antibiotics to combat infectious diseases, particularly in livestock. Oral administration of egg yolk IgY is the most common and convenient route that has been extensively investigated for controlling enteric pathogens. However, the  Chicken immunoglobulin Y (IgY) is the functional equivalent of IgG in mammals and can be transferred from serum to yolk during egg formation. Egg yolk contains a large quantity of IgY that can confer passive immunity for chicks against pathogens, either at embryonic or post-hatching stage . Immunizvia different routes, depending on the infection sites of targeted pathogens. Oral administration through feed or drinking water is the most common and convenient approach, especially against enteric pathogens  tract have not been examined in most previous studies and are still largely unknown to date. Addressing this issue is critical for appropriately assessing the efficacy of orally administrated egg yolk IgY and for developing effective egg yolk IgY-based passive immune intervention strategies. Limited in vitro data are available concerning the stability of IgY in response to pepsin (a gastric protease), trypsin , and low pH alone , slightly processed with saline, and subsequently subjected to comprehensive ex vivo analyses using multiple approaches .In a recent chicken trial , we haveThe chicken trial performed in this study was approved by the Institutional Animal Care and Use Committee at The University of Tennessee, Knoxville, with protocol number 1387-0219. Briefly, 15 newly hatched broiler chicks (Hubbard Efficiency Plus) were fed with regular feed for 6 weeks. At 2, 4, or 6 weeks of age, five chickens were randomly selected and euthanized. The pH in gizzard and small intestine (the junction site between the duodenum and jejunum) was measured on site using a pH spear tester . Subsequently, the fresh GI contents (5 ml per sample) were collected using clean stainless steel laboratory scoops, transferred into 15 ml sterile plastic tubes, stored on ice, and immediately subjected to downstream work as detailed below.ex vivo IgY stability assays were performed as described previously , followed by centrifugation at 13,000g for 2 min. The supernatant containing egg yolk IgY was used as stock to spike various GI samples. Each of the fresh gizzard and small intestine content samples was gently mixed with same volume of sterile saline and centrifuged at 5,000g for 5 min. The supernatant was kept and used as corresponding GI content matrix system for ex vivo IgY stability assay. The supernatant was measured for pH and spiked with 5% (v/v) of the egg yolk IgY solution, while saline or HCl  served as a control. The mixtures were incubated at 42°C (chicken body temperature) for different lengths of time. Subsequently, the mixtures were subjected to ELISA or SDS-PAGE analysis. If needed, some mixtures containing gizzard content or HCl were neutralized to pH 7.0 using less than 2% volume of NaOH upon completion of treatment.The eviously , 7 with eviously , the lyo® One-component Peroxidase Substrate (SeraCare), and the reaction was stopped after 30 min by adding 100 μl of stopping solution (1% SDS). The absorbance was measured at optical density of 405 nm (OD405) using a microplate reader  and data were collected using Gen5 software (BioTek Instruments). The wells without the addition of primary antibody served as a blank control. Endpoint titer was defined as the last dilution at which OD405 of sample wells exceeded the cutoff value (0.1). Duplicate measurements were performed for each sample.Indirect ELISA was conducted as described previously  to deterg for 20 min. Subsequently, the supernatant was added with 12% (w/v) of PEG 6000 for IgY precipitation and the mixture was incubated at room temperature for 10 min. Following centrifugation at 5,000g for 20 min, the precipitate was dissolved in PBS and added with 12% (w/v) of PEG 6000 again for another round of IgY precipitation. After centrifugation, the precipitate was dissolved in a small volume of PBS, added with equal volume of precooled (−20°C) ethanol, and centrifuged at 5,000g and 4°C for 20 min to remove PEG 6000. Lastly, the precipitate containing a high proportion of IgY was dissolved in a small volume of PBS and the solution was dialyzed against PBS. Initial egg yolk, the supernatant and precipitate fractions obtained from the centrifugation of 12% PEG 6000 mixture, and the final dialyzed IgY extract were subjected to SDS-PAGE analysis.The hyperimmune egg yolk generated in our recent study  was usedex vivo assay samples were subjected to SDS-PAGE analysis  and stained with Coomassie blue R-250 dye . For immunoblotting, the proteins on polyacrylamide gel were electrotransferred to a nitrocellulose membrane, which was subsequently blocked in blocking buffer (PBS + 0.05% Tween 20 + 5% skim milk) at 4°C overnight. Then the membrane was incubated with goat anti-chicken IgY-horseradish peroxidase  at room temperature for 1 h. After the washing procedure (PBS + 0.05% Tween 20), the membrane was developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate .All P <0.05 was considered as a statistically significant difference.Statistical analyses were conducted using Mann–Whitney test in GraphPad Prism 8. Data were presented as mean ± standard deviation (SD). A probability level of in vivo pH at these three time points. After dilution of the gizzard content using saline, the supernatant obtained for ex vivo assays had slightly elevated pH, which was approximately 0.57 higher than the corresponding original gizzard content  between ex vivo sample pH and the reduction of specific IgY titer in response to 30 min of treatment in gizzard content  of reduction when compared with saline control.As shown in in vivo and ex vivo findings  or HCl solution (pH 3.0). The egg yolk treated in the matrixes of control saline, HCl for 60 min, and the gizzard content for 0 min consistently displayed multiple protein bands with similar pattern  and provex vivo sample had much higher pH (4.62) than other samples   for roteases . Pepsin,roteases . Other droteases . Second,, cecum) . Therefo, cecum) , 16. Dur, cecum) . Consequ, cecum) . It is w, cecum) . Therefoex vivo assay to better mimic in vivo conditions, with emphasis on reaction time and pH. Under physiological condition, the retention of feed in chicken gizzard ranges from 30 to 80 min, while the retention time in the upper small intestine is approximately 30 min , which may be the major factor leading to a slight reduction (two-fold) in IgY titer  to the antimicrobial effect resulting from orally administered egg yolk IgY. Thus, the findings from previous studies need to be revisited and confirmed in conjunction with examination of the in vivo stability and level of the specific egg yolk IgY administered via the oral route.Egg yolk IgY is increasingly recognized as a promising passive immune agent for the control of various microbial infections . To conts agents , 2. NotaWe expect that the findings from this study can also be extrapolated to other animals and even humans. For monogastric organisms , pepsins are produced in their stomachs as the case in chicken gizzard. The low pH in these stomachs  can fulfill the requirement of pepsins to exert degradation on various peptides and proteins –21. Rumiin vitro system, i.e., the buffer supplemented with digestive enzymes, was used to evaluate the efficacy of encapsulation in IgY protection and release in these studies  Award No. 2018-67015-28295.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.


Fomitopsis pinicola (Sw.) P. Karst.  are connected with the character of the forests and/or the degree of decay (DD) of the fruiting bodies. The research was conducted at Białowieża National Park (BNP), in forests close to natural ones and in Karkonosze National Park (KNP) which was affected by a large-scale forest dieback in the 1980s. Eighty fruiting bodies (40 at each study site) of F. pinicola belonging to four DD categories were collected. In total, 4,345 individuals of 120 mite species were recorded at BNP, and 13,912 individuals of 96 species were recorded at KNP. Analyses revealed that the sample dispersion at each study site was comparable, nevertheless the samples from each study site were clearly grouped into slightly overlapping sets which allow observation of the differences between them. In the less decayed fungi (DD 1 and 2) there were fewer mite species and individual mites than in the more decayed samples (DD 3 and 4). There were also significant differences between the fauna of the fungi in each particular DD: the fauna of DD 1 differed from all others, whereas the fauna of heavily decayed fungi (DD 3 and 4) was more comparable.The fruiting bodies of bracket fungi are a specific microhabitat colonized by various invertebrates of which mites (Acari) are rarely studied, and if they are, the study is usually faunistic. The aim of the research was to determine whether the diversification of mite assemblages  inhabiting the fruiting bodies of The online version contains supplementary material available at 10.1007/s10493-021-00635-1. Bracket fungi play a crucial role in the forest environment, they decompose wood and contribute to the effective processing of energy and matter flow in ecosystems Niemelä . GeneralHoploseius  P. Karst. , is a widespread and a very common species of fungus which occurs worldwide in temperate and boreal forests in the Northern Hemisphere , pine (Pinus spp.) or fir (Abies spp.), and less frequently deciduous trees, e.g., birch (Betula spp.) and beech (Fagus spp.). It is known to cause a strong and fast-spreading brown rot which generates significant economic losses , chosen for the study, is the largest area of natural forest in the North European Plain , constitutes an example of an area that has experienced a strong anthropogenic impact, particularly due to forest management, settlements, herding, and the exploitation of minerals  the structure of mite assemblages colonizing fruiting bodies of F. pinicola varied depending on their degree of decay (DD).In this research focused on biodiversity in the two types of ecosystems, we tested the following hypotheses. (1) There are differences in density and structure of mite communities inhabiting the fruiting bodies of Tilio-Carpinetum . In the park there are also marsh communities: valleys in a primeval forest, which are periodically flooded, covered with an ash-alder riparian forest Circaeo-Alnetum (8.4%), whereas peatland valleys and marshy interior basins are covered with alder swamp forests Ribeso nigri-Alnetum or Sphagno squarrosi-Alnetum (5.9%). Moreover, the boreal spruce forest on peatland Sphagno girgensohnii-Piceetum (1.6%) and the sub-boreal swampy birch forest Thelypteridi-Betuletum pubescentis (5.3%) are other communities that can be found there. The poorest habitats growing on sands are covered with pine forests: the subcontinental fresh coniferous forest Vaccinio vitis-idaeae-Pinetum, midland dry pine forests Cladonio-Pinetum (3.8%), swampy coniferous forest Vaccinio uliginosi-Pinetum and raised-bog community Ledo-Sphagnetum magellanici (1.7%)  was established in 1947 which makes it the oldest Polish national park. BNP is located in the northeast of Poland , near the village of Białowieża Fig. . The larGalio sylvatici-Carpinetum betuli and riverine alder forest Alnetum incane in foothill zone, acidophilic montane beech forest Luzulo luzuloidis-fagetum as well as lower mountain fir-spruce forest Abieti-Piceetum (montanum) in the lower mountain zone, and the upper mountain spruce forest  in the upper mountain zone. In the subalpine zone there are Sudety dwarf-pine thickets Pinetum mugo sudeticum and downy willow shrub Salicetum lapponum  is a park in the Karkonosze Mountains in the Sudetes located in south-western Poland , along the border with the Czech Republic in the highest part of the Sudetes , the samples collected in each of the analyzed national parks may be considered as pseudoreplicates of one site.F. pinicola were collected on 18 and 19 July 2014 at the KNP and between 4 and 9 August 2014 at the BNP. In total, 80 fruiting bodies at different degrees of decay were collected on both locations (40 in BNP and 40 in KNP). An axe was used to remove them from tree trunks. The fruiting bodies were collected from the trees where F. pinicola occurred most often in the studied location, i.e., Norway spruce, Picea abies, which is a common woody species in both parks. In order to reflect the specificity of the studied locations, the samples were taken randomly according to the diameter at breast height and the age of the trees. The fruiting bodies were collected from tree trunks at the height between 0 cm (ground level) to 210 cm above ground level, taking into consideration their degree of decay (DD). A single fruiting body equals one sample.The fruiting bodies of The classification of the DDs of the fruiting bodies used in this study is borrowed from Gdula et al.  and, likIn order to extract mesofauna from the bracket fungi they were placed for 72 h in Tullgren funnels. The collected mites were preserved in 70% ethanol, and divided into two taxonomic groups: mesostigmatid and oribatid mites. In order to identify the Mesostigmata, semi-permanent (using lactic acid) and permanent (using Hoyers medium) microslides were prepared. All adult and immature mesostigmatid mite individuals were examined using light microscope (Zeiss Axioskope 2) and identified using taxonomical literature  were identified with a light microscope (Nikon Eclipse E600). Prior to the examination, the internal tissue had been removed using concentrated lactic acid, 60% lactic acid or lactophenol as a clearing agent, but with weakly sclerotized forms diluted lactic acid was also appropriate (Norton 1990). Oribatid mites were determined to the species level by following keys and original species descriptions  and Institute of Biology, Biotechnology and Environmental Protection, University of Silesia, Poland (Oribatida).The assessment of statistical significance of the examined factors was based on: raw data collected in the box-plot graphs, the level of significance  obtained using the Mann–Whitney test (when comparing two species) and Kruskal–Wallis test (to compare DDs). A horseshoe effect has not been observed in the analysed experiment; hence, the samples collected at BNP and KNP together with the samples belonging to each particular DD from each study site were compared using principal coordinate analysis (PCoA). Bray–Curtis dissimilarity matrix was adopted as the distance matrix for PCoA. The weights that were used were a sum of observations for each particular site and divided by the sum of all observations . To present the deployment of the DDs and parks on the graph, the sum of observations for each particular DD and park was used, and community data were analysed by two-way permutational ANOVA. Indicator species analysis was used to provide specific species for each park and DD. Cluster analysis following the Ward method for Bray–Curtis dissimilarity matrix was used for the parks and DDs. Next, the PCoA analysis was performed separately for Mesostigmata and Oribatida mites, which was followed by the permutation test of species selection and is most strongly correlated with PCoA1 and PCoA2 (multiple regression). This test pointed at the specific species for each given park. All testing was conducted at a significance level of 0.05. All analyses were conducted in the R environment using the procedures of the vegan package , Dendrolaelaps pini (268) and Carabodes subarcticus (261). Among the 10 most numerous mite species in BNP, eight  belonged to the Mesostigmata and two  belonged to Oribatida.The total number of collected mite individuals in BNP was 4,345 , and the total number of species was 120 (37 mesostigmatid species and 83 oribatid species). The number of mite individuals per sample was between 1 and 622 (mean ± SE = 108.63 ± 22.63) – the number of Mesostigmata was between 0 and 101 (20.60 ± 3.61), whereas the number of Oribatida was between 0 and 580 (88.03 ± 21.06). The number of mite species per sample was between 1 and 38 (9.63 ± 1.26); the number of Mesostigmata species was between 0 and 10 (3.18 ± 0.38), and Oribatida species was between 0 and 28 (6.45 ± 1.00). The most numerous mite species in BNP were: Carabodes femoralis , Carabodes areolatus (1030) and Zerconopsis remiger (256). The total share of Mesostigmata among the most numerous species in KNP was lower than in BNP: only four out of the 10 most numerous species  belonged to the Mesostigmata, and six  belonged to the Oribatida , and the total number of mite species was lower: 96 (34 mesostigmatid and 62 oribatid species). The number of mite individuals per sample was between 1 and 2056 (mean ± SE = 347.80 ± 62.94), the number of Mesostigmata was between 0 and 126 (23.90 ± 4.96), whereas the number of Oribatida was between 0 and 2000 (323.90 ± 60.63). The number of mite species per sample was between 1 and 33 (11.98 ± 0.89); the number of Mesostigmata species was between 0 and 10 (3.05 ± 0.35), and the number of Oribatida was between 0 and 25 (8.93 ± 0.68). The most numerous mite species in KNP were: Carabodes femoralis); nevertheless, there were considerably fewer individuals (2766) in the samples from BNP than from KNP . Moreover, the composition of dominant species in each of the analysed mite group  in both national parks differed from one another. For instance, some of the most numerous Mesostigmata in BNP (like Dendrolaelaps pini) were significantly less numerous in KNP and outnumbered other species from their group , whereas oribatid species such as the third most numerous species in BNP, Carabodes subarcticus, did not occur in KNP at all; Carabodes areolatus, the second most numerous species in KNP, occurred only rarely in the samples from BNP (Online Appendix).The fungi collected at BNP contained considerably fewer mite individuals than at KNP. However, there was not only a greater number of species in the BNP sample, but also a different species composition. At both studied locations, the same species occurred in highest numbers , there were clearly fewer mite individuals, the numbers were slightly higher for the DD 2 samples, whereas the highest numbers of individuals were in the DD 3 and 4 samples. The lowest species richness in KNP was also noted in the least decayed bracket fungi (DD 1); however, the highest number of species occurred in DD 3 .Differences were also evident in species composition of the mite assemblages at each of the study sites depending on the DD. The most numerous species of mesostigmatid mites in the bracket fungi of DD 1 (BNP) was Carabodes femoralis, but in the case of other species there were differences between the particular DDs. In the bracket fungi from DD 2, 3, and 4, the second most numerous species was Carabodes areolatus, which was considerably less frequent in the least decayed fungi. The second most numerous species in DD 1 was Hoploseius oblongus, which rarely occurred in the other DDs. In DD 1, nine out of 10 of the most numerous mite species was an Oribatida; among the 10 most numerous species in DD 2 bracket fungi, there were five species each of Mesostigmata and Oribatida. In DD 3 and 4, seven out of 10 most numerous mite species were Oribatida (Online Appendix).The most numerous mite species in the samples of all DDs from KNP was Carabodes areolatus, Zerconopsis remiger, Chamobates borealis, Cepheus dentatus, Oribatella calcarata, Scheloribates pallidulus, Phthiracarus longulus, Dendrolaelaps cornutus, Carabodes femoralis, Zercon storkani, Trachytes aegrota, Steganacarus (Atropacarus) striculus, Lasioseius zerconoides, Parasitidae, Oribatella quadricornuta, Carabodes reticulatus, Liebstadia pannonica, Carabodes tenuis, Pergamasus sp.; for BNP they were: Carabodes subarcticus, Zercon curiosus, Trichouropoda ovalis, Chamobates cuspidatus, Damaeus sp., Dinychus perforatus, Sejus togatus, Microgynium rectangulatum, Dendrolaelaps pini, Dinychus arcuatus. Six species were selected in KNP. The DDs differed regarding five species: in DD 1, there were two indicator species: Hoploseius oblongus, and Scheloribates pallidulus; DD 2 also had two indicator species Lasioseius ometes and Thenargamasus sp.; in DD 4 one species was selected, namely Parasitidae. Cepheus cepheiformis was related to two groups: DD 3 and 4. The analysis conducted in BPN selected four indicator species, with Parachipteria punctata and Lagenobates lagenulus for DD 1, Dinychus perforatus for DD 4 and Hoploseius oblongus for DD 1 and 2.The differences between the samples belonging to each particular DD group in the two parks were indicated by PCoA, whose total Inertia was 0.29. The analysis revealed that samples from particular study sites constituted concentrations which were dispersed to different degrees. In the case of bracket fungi from KNP, the set was less dispersed and matches the lower values on the PCoA1 axis, whereas the BNP set was more dispersed and shifted in the direction of higher values on the PCoA1 axis. The analysis also showed that the bracket fungi which were most similar were those from DD 3 and DD 4. Samples from DD 1 were further on the axis than others which signifies that they differ from the bracket fungi belonging to the higher DDs. PCoA also revealed some similarities in the manner of sample groupings in particular DDs in both parks; there was a trend in both KNP and BNP displaying that the least decayed bracket fungi, which differed from the samples of higher DDs, were located in the range of higher values on the PCoA2 axis. The bracket fungi from DD 3 and 4 in both study sites were similar to one another and group closely to the higher values on the axis Fig. . The twoCluster analysis (Ward method) from Bray–Curtis dissimilarity matrix led to similar grouping of the bracket fungi as that based on PCoA. The most similar bracket fungi from BNP belonging to DD 3 and 4 constituted a cluster, to which the bracket fungi from DD 2 and 1 could be added provided they came from the same park. A separate cluster was created out of bracket fungi from KNP, among which the DD 3 and 4 bracket fungi were characterised by the greatest similarity, to which bracket fungi from DD 2 and fungi from DD 1 could be added Fig. .Fig. 8ClA separate analysis of the Mesostigmata and the Oribatida revealed similar trends, and confirmed the differences between the fruiting bodies belonging to various DDs in both locations. In the case of the mean number of individuals and the mean number of species for a single bracket fungus in BNP, in each DD oribatid mites outnumbered mesostigmatid mites. Only the samples for DD 1 were an exception as there were more Mesostigmata than Oribatida (mean ± SE = 21.25 ± 5.33 vs. 18.25 ± 6.3 per sample). However, there were more individuals and species of Oribatida in the samples from KNP in all DDs  in both study sites.Our hypothesis that mite groupings would differ among fruiting bodies in various DDs was supported – the least decayed samples were characterised by lower species richness and mite individuals than the more decayed samples. The increase in the number of species and individuals with the degree of decay of fruiting bodies was non-linear. Particular mite species are characterized by different microhabitat preferences, the scale of tolerance and their requirements  between the substrates. For instance, fresh fruiting bodies (DD 1) can create favourable conditions for presumably fungivorous Our results can be compared to the results obtained in a similar microhabitat such as decaying wood. Similar to our results, Braccia and Batzer , SkubałaCarabodes spp. This genus is a dominant element of the fauna in other habitats as well, such as soil, bark of trees, and wood  of species inhabited this microhabitat exclusively , tree-hollows , or soil (D. trapezoides) , magpie (Pica pica) and rook (Corvus frugilegus)  magnus, or Damaeus (Paradamaeus) clavipes, can be considered as fungivorous Below is the link to the electronic supplementary material.