Due its ability to provide a global snapshot of kidney physiology, urine has emerged as a highly promising, non-invasive source in the search for new molecular indicators of disease diagnosis, prognosis, and surveillance. In particular, proteomics represents an ideal strategy for the identification of urinary protein markers; thus, a urinomic approach could also represent a powerful tool in the investigation of the most common kidney cancer, which is clear cell Renal Cell Carcinoma (ccRCC). Currently, these tumors are classified after surgical removal using the TNM and nuclear grading systems and prognosis is usually predicted based upon staging. However, the aggressiveness and clinical outcomes of ccRCC remain heterogeneous within each stratified group, highlighting the need for novel molecular indicators that can predict the progression of these tumors. In our study, we explored the association between the urinary proteome and the ccRCC staging and grading classification. The urine proteome of 44 ccRCC patients with lesions of varying severity was analyzed via label-free proteomics. MS data revealed several proteins with altered abundance according to clinicopathological stratification. Specifically, we determined a panel of dysregulated proteins strictly related to stage and grade, suggesting the potential utility of MS-based urinomics as a complementary tool in the staging process of ccRCC. Renal Cell Carcinoma (RCC) comprises a heterogeneous group of tumors of which the most frequent (70–80%) and aggressive morphotype is clear cell RCC (ccRCC) ,2. In coccRCC is a malignancy of particular interest for clinical investigations because of its associated inter-tumor and intra-tumor heterogeneity , high reRoutinely, ccRCC lesions are classified by considering two principal clinical features according to the recommendation of the World Health Organisation (WHO) classification . One is Although this system is well established and represents the key tool in the armory of pathologists, the need for new molecular markers that are able to assist in ccRCC diagnosis and prognosis remains essential. In particular, this is due to both grading and staging stratifications employing descriptive parameters that rely on the subjective evaluation of the physician . ConsequAdditionally, regarding the molecular mechanisms underpinning the progression of ccRCC, they are still unclear and studies that can provide a more in-depth understanding of the biological process involved in this advancement will be beneficial for its management.In this context, proteomics strategies have been widely used and biofluids have represented the sample type of choice due to them being easily and noninvasively obtainable . The newHowever, excluding some exceptions ,22, mostForty-four patients from San Gerardo hospital  were recruited between 2011 and 2016, and all subjects had signed informed consent. The local ethical committee  approved all protocols and procedures. Analysis was performed in agreement with the Declaration of Helsinki. The diagnosis was performed after careful histopathological evaluation, including tumor size and position (pT), grading (ISUP classification system), sarcomatoid features, vascular invasion, tumor necrosis, presence of distant metastasis, and involvement of lymph nodes (stage).g for 10 min at 4 °C and stored at −80 °C until the day of the analysis. An amount of 6 mL of the stored supernatant was concentrated by centrifugation on a 30 kDa cut-off filter  for 10 min at 4 °C.The second urine sample of the morning, before total or partial nephrectomy, was collected into sterile urine tubes. The samples were then centrifuged at 1000× g (10 min) and acidified. The resulting peptide solution was desalted and concentrated using μ-C18 ZiptipTM pipette tips  following the standard protocol provided by Millipore. Purified samples were resuspended in 98% H2O: 2% acetonatrile: 0.1% trifluoroacetic acid and analyzed by nUHPLC-MS/MS.Protein concentration was determined with the BCA method  and 200 μg of protein from each of the urine samples was used for tryptic digestion ,25. Briem/z 300–2000 range) with a charge state between +2 and +5 and with at least 1575 counts (fixed cycle time of 5 s) for fragmentation, obtained by collision-induced dissociation. MS scans as well and MS/MS data were recorded in centroid. Raw MS and MS/MS data were corrected using both an internal calibration (lock mass m/z 1221.9906) and a calibration segment, based on a 10 mM sodium formiate cluster solution (15 min before each run), prior to being converted and deconvoluted to XML file format using DataAnalysis software .LC-ESI-MSMS analysis was performed using a Dionex UltiMate3000 rapid separation (RS) LC nano system  coupled with an UHR-nESI-qTOF . Approximately 1.4 μg of protein was loaded onto μ-precolumn  and then separated by an analytical C18 column . A multistep gradient with phase A (0.1% formic acid) and phase B (0.1% formic acid/80% acetonitrile) from 4% to 66% was run for 240 min. Eluted peptides were analyzed in Data-Dependent Acquisition mode, and MS/MS data were acquired by targeting precursors . Trypsip-value yielded by the Mann–Whitney test was less than 0.05. Due to the high degree of biological variability, no further statistical correction was performed. Functional analysis was carried out for the proteins of interest: KEGG pathway term clustering using ClueGO v2.5, Clupedia v1.5, and Cytoscape network analysis framework was performed [Protein abundances were calculated using the three most abundant unique peptides, normalized against the total ion current and only those proteins detected with at least two unique peptides and in at least 50% of the samples were used for protein quantification. Files containing information regarding protein abundance in the form of statistical matrices (samples as rows and proteins as columns) were exported from PEAKS Studio 8.5 and imported into an in-house developed software. For each protein, statistical evaluation was performed as follows in order to retrieve a list of differentially expressed proteins. First, patients were grouped according to the levels of a chosen outcome attribute , then proteins were considered to have a different abundance (ratio > 1.5 and <1.5) when the erformed .Conventional histopathological evaluation of tumors, such as stage and grade of the lesions, has prognostic significance. However, the molecular mechanisms involved in tumor development and progression are not well understood. Therefore, the possibility to obtain information regarding molecular changes associated with tumor lesions has been investigated using easily accessible urine samples.Urine samples were collected from 44 patients  with a proven diagnosis of ccRCC . All diaFollowing the FASP protocol, samples were analyzed by nUHPLC-MS/MS and a total of 21,640 peptides sequences, corresponding to 1609 proteins, were identified with an FDR on PSM of 1% and at least one unique peptide . The proPrimarily, GO term enrichment analysis highlighted glycolysis/gluconeogenesis, the pentose phosphate pathway, and pyruvate metabolism as the chiefly represented processes with a total of 52 identified proteins found to be associated with them. A number of urinary proteins coded by hub genes involved in the cellular reactions of carbohydrates , lipids , and amino acids  were also detected. Additionally, functional annotation analysis indicated the presence of proteins involved in immune response and coagulation cascade in urine.We further investigated the correlation between urinary protein levels and tumor features (stage and grade), focusing on urinary proteome alterations that possibly reflected morphological changes of the lesions.Initially, the influence of the grade on the urinary proteome was evaluated. Patients with different tumor grades were divided into two groups, HG (G3-G4) and LG (G1-G2) tumors, and, between them, 39 proteins were identified as differentially expressed. Among them, 10 were down and 29 upregulated in HG compared to the LG group .Moreover, in order to highlight alterations of the urinary proteome related to different stages, we also evaluated the differences between stage 1 and stage > 1. As result, 79 proteins with a statistically significant variation in their abundances were detected: 16 proteins were downregulated while 63 were upregulated in stage > 1 versus stage 1 patients .Urinary proteins altered according to the stage (stage 1 versus stage > 1), and grade (LG versus HG) were also compared A and theIn order to better understand which of the urinary protein alterations are specifically correlated with these clinical hallmarks of disease (grade and stage), we proceeded to sub-classify our cohort of patients by considering each feature individually.Initially, we primarily focused on the influence of the stage stratification on the urinary proteome. We compared patients with LG and stage 1 lesions with those with LG and stage > 1 lesions (groups A and B); then, compared patients with stage 1 and stage > 1 lesions at HG (groups C and D). We determined six proteins that were upregulated in advanced stage with respect to stage 1 when grade classification was excluded as a confounding factor A,B.Subsequently, we investigated how the expression of urinary proteins reflected tumor grade stratification. Firstly, we compared patients with lesions at different grades: LG (group A) versus HG (group C) at stage 1 and LG (group B) versus HG (group D) at stage > 1 A,B. ThisProteomics studies of ccRCC using different MS technologies have been applied to a variety sample types, including tumor cell lines and different types of human samples, such as serum, tissue, and urine ,30,31,32In light of these previous findings, we hypothesized that the urinary proteome may reflect both the morphological features (stage and grade) used by the pathologist to define the progression of the tumor. For this purpose, we analyzed the urine of ccRCC patients, stratified according to grade (LG and HG) and stage (stage 1 and stage > 1). Firstly, we identified more than 1600 urinary proteins, of which 500 were previously detected in ccRCC tissues, supporting the hypothesis that urine also has the potential to provide key information regarding the functionality and pathological state of the kidney.GO network analysis of these common proteins highlighted the involvement of various pathways, including glucose oxidation and the pentose phosphate pathway A,B. It iMoreover, information provided by urine and tissue had a considerable overlap: proteins detected in both ccRCC urine and tissue samples are also involved in the activation of immune response in line with the data present in the literature. For instance, Tian et al. identified components of complement activation pathway, such as C1QA, C1QB, and C1QC, as key enriched genes . The samIn order to enlighten the possible correlation between urine proteome and histopathological tumor classification, we performed a quantitative label-free analysis. Firstly, the urine samples were grouped according to grade stratification. Thirty-nine proteins with a statistically significant differential abundance between the HG and LG classes were observed . SecondlHowever, evaluating both the stage and grade simultaneously could act as a confounding factor. In order to examine this aspect in greater detail, the entire cohort of patients was reclassified, considering a single morphological feature at a time. Therefore, four additional comparisons were performed: patients with the same grade were compared to each other by varying only the stage feature, and patients with the same stage were compared to each other considering as variable only the grade feature .It was noteworthy that nine of the fifteen proteins, initially found to be commonly altered between stage and grade, reappeared in these further comparisons. As such, this panel of proteins could represent a good indicator of ccRCC progression. For instance, GDF15 and IGLV1-40 were upregulated when stage features (early and advanced) were compared, even when adjusted for different grades. Accordingly, we can speculate that their expression primarily reflected the stage, but the influence of the grade still remains relevant. In fact, these proteins are statistically significant even in the general comparison HG vs. LG . The samTo the best of our knowledge, there are no studies investigating protein changes in urine associated with ccRCC lesions at different stages and grades simultaneously. However, some of the proteins that we found to be quantitatively varied in the urine of ccRCC affected patients have been reported as altered in diverse cancers types .p-value 0.003) and HG groups . Moreover, when patients were reclassified, fixing one histopathological feature at a time, LAMC1 was once again upregulated in stage > 1 and in LG  , a core structural protein present in the basement membrane of several organs, including the kidney. Immunohistochemical analysis of kidney specimens from patients with Chronic Kidney Disease (CKD) indicated an increased presence of LAMC1 in the glomerular basement membrane that could reflect an acceleration of its remodelling. A fragment of LAMC1 has also been detected in the in serum and urine of CKD patients with a high risk of progression . Furtherue 0.03) .p-value 0.005) and HG . The same pattern of overexpression was confirmed for the stage > 1 when we considered only the LG sub-cohort  (Another important player in cancer cell proliferation has been demonstrated to be the glutamylcyclotransferase enzyme (GGCT). Involved in glutathione metabolism, it was found to be overexpressed in a variety of cancers, including uterine cervix, lung, and colon . Our datue 0.04) .p-value 0.0002) and an HG . In addition, the protein was once again upregulated in stage > 1 patients, even when the cohort was divided into HG  and LG groups  , a stress-inducible cytokine, also has important implications in the context of ccRCC. It plays key roles in prenatal development, inflammation, regulation of cellular responses to stress signals, and tissue repair ,42. GDF-ue 0.03) .Once a panel of urinary proteins for which its variation could be linked to the ccRCC stage was determined, each clinical parameter was separately considered. In performing this, many of the dysregulated proteins were present only in initial comparisons .However, the additional statistical analysis , stage >Notwithstanding, a subset of proteins, such as ADGRF5, PATJ, PRG2, and SFN, also showed a complementary expression trend and suggested a more complex molecular scenario. They were upregulated in LG of the stage > 1 subpopulation and in the stage > 1 of the LG subpopulation . We specOur results are in accordance with those already reported in the literature. For instance, Pals1-Associated Tight Junction protein (PATJ) is a highly expressed protein in kidney epithelia and is involved in the maintenance of cell polarity . By usinThe hypothesis that the urine proteome reflects all the theoretical combinations of stage and grade could also explain the characteristic quantitative pattern of GOLM1 and LAIR1. Both were upregulated in stage > 1 when considering only the HG sub-cohort and up-regulated in HG, when considering only the stage > 1 sub-cohort . Golgi MMoreover, a further two proteins with possible implication are THBS4 and XPNPEP2. They were both determined to be downregulated in HG in the general comparison of HG vs. LG but upregulated in the HG group when the stage 1 feature was fixed . In thisp-value 0.02) but upregulated in the HG lesion group  when the sub-cohort of stage 1 patients was evaluated. We, therefore, suggest that THBS4 plays a role in advanced ccRCC grade only when HG and early stage lesions are co-present . It is an adhesive glycoprotein, part of the extracellular matrix, and it is involved in proliferation, attachment, adhesion, and cell migration as well as spreading in various types of malignancies . For ins-present .p-value 0.016) and in stage > 1 when the LG lesion is fixed   . In accoIn this study, we highlight that the urinary proteome could represent a precious source of information regarding the molecular understanding of ccRCC progression. Collectively, we revealed a set of proteins with altered urinary expression according to tumor metastasis. Several of them were already known to be involved in carcinogenesis, tumor progression, and aggressiveness in RCC and in other cancer types. These findings suggest that such proteins profiles could be helpful in characterizing this malignancy in a non-invasive manner.Indeed, the urinary proteome of ccRCC patients primarily mirrors stage classification and partially provides information regarding the grade of the tumor, operating as a liquid biopsy of kidney cancer progression. As such, it was possible to generate a panel of proteins for which its abundance was specifically linked to each of these tumor features. Taken together, these findings represent a valuable starting point when seeking to develop a complementary molecular tool to support TNM and nuclear grading systems, improving individual prognostic evaluations of ccRCC patients.In the future, we plan to enlarge the patient cohort in order to assess the possible association with comorbidities and to collect urine from post-surgery subjects in order to enforce the robustness of our results.


Mycobacterium vanbaalenii PRY-1 and Mycobactetrium sp. KMS and the anaerobic one, by the facultative bacteria anaerobe Pseudomonas sp. JP1 and Klebsiella sp. LZ6 are reviewed and presented, to describe the complete and integrated degradation mechanism pathway of pyrene. The different microbial strains with the ability to degrade pyrene are listed, and the degradation of pyrene by consortium is also discussed. The future studies on the anaerobic degradation of pyrene would be a great initiative to understand and address the degradation mechanism pathway, since, although some strains are identified to degrade pyrene in reduced or total absence of oxygen, the degradation pathway of more than 90% remains unclear and incomplete. Additionally, the present review recommends the use of the combination of various strains of anaerobic fungi and a fungi consortium and anaerobic bacteria to achieve maximum efficiency of the pyrene biodegradation mechanism.Microbial biodegradation is one of the acceptable technologies to remediate and control the pollution by polycyclic aromatic hydrocarbon (PAH). Several bacteria, fungi, and cyanobacteria strains have been isolated and used for bioremediation purpose. This review paper is intended to provide key information on the various steps and actors involved in the bacterial and fungal aerobic and anaerobic degradation of pyrene, a high molecular weight PAH, including catabolic genes and enzymes, in order to expand our understanding on pyrene degradation. The aerobic degradation pathway by  Among the most prevalent and persistent contaminants, polycyclic aromatic hydrocarbons (PAHs) have attracted increasing attention. They are a ubiquitous group of organic pollutants, consisting of two or more single or fused aromatics rings . They arVarious physical and biochemical, environmentally sustainable, and acceptable methods have been developed together for this purpose. The decontamination techniques involve a substantial reduction in the quantity (volume and mass) of pollutants, by using appropriate methods such as bioremediation. PAHs are biologically eliminated or degraded under controlled conditions to a harmless state or to levels below the concentration limits established by regulatory authorities ,11. The Mycobacterium vanbaalenii PYR-1 is reported as the first isolated bacteria capable of totally mineralizing pyrene by mono- and dioxygenase reactions  Rieske center and mononuclear iron binding domain, permitting the transfer of electron to dioxygen, which constitutes the final acceptor [vanbaalenii PYR-1 culture, pyrene oxide was identified as an intermediate product [trans 4,5-dihydroxy-4,5-dihydropyrene by epoxide hydrolase. The enzyme dihydrodiol dehydrogenase transforms trans 4,5-dihydroxy-4,5-dihydropyrene to 4,5-dihydroxypyrene, which is metabolized by following the same route as in PYR-1, as shown in Figure 1As in many other strains, in the acceptor ,53. Diox product ,55. This product  demonstr2) is reduced or even almost absent. Under such anoxic conditions, the metabolism of PAHs in general and pyrene in particular is questionable. However, it has been found that thermodynamically, anoxic mineralization of pyrene in various reducing environments is achievable, despite its lower energy efficiency than aerobic degradation [Clostridium sp. ER9, a strict rod-shaped Gram-positive bacterium, anaerobically metabolized pyrene up to 93% within 49 days of incubation [Paracoccus denitrificans M-1, a facultative anaerobic bacterium, showed a capacity to degrade pyrene under anoxic conditions with different nitrate/nitrite (NO3−/NO2−) ratios within 5–6 days of incubation [Hydrogenophaga sp. PYR1 could anaerobically remove pyrene under iron reduction conditions with an efficiency of 22%.In many natural habitats, such as groundwater, topsoil, marine sludge, and sediments, dioxygen  and carbon-hydrogen (C-H) bonds in the aromatic rings. This is explain why the activation of the compounds in the anoxic medium can occur, by carboxylation, methylation, or the addition of fumarate. During the pyrene mineralization process, with a succession of decarboxylation and hydroxylation, phenanthrene is formed. The mechanism of degradation of phenanthrene obtained from pyrene is anaerobic, consisting of hydroxylation and methylation reactions in the first ring and thus continuing in the other aromatic rings until all the constituent rings of the compound are fully cleaved. Metabolites observed during the degradation of pyrene include 1,2,3,4-tetrahydro-4-methyl-4-phenanthrol, phenol, 2-methyl-5-hydroxy-benzaldehyde, p-cresol, 1-propenyl-benzene, anthraquinone, and 1-anthraquinone-carboxylix acid ,62,63,64Pseudomonas sp. JP1 and Klebsiella sp. LZ6, are used as models to describe the anaerobic degradation mechanism pathway of pyrene.Diverse paths are proposed by various studies using strict and optional anaerobic microorganisms metabolizing low and high PAHs. In the following, two facultative different bacterial strains, Klebsiella sp. LZ6 strain was reported to degrade not more than 33% of 50 mg/L of pyrene, different metabolites were observed and have permitted to propose a degradation pathway for this strain , lignin peroxidase (EC1.11.1.14), and manganese peroxidase (EC1.11.1.13), leading to the complete degradation of PAHs into CO2 . Basical enzymes . Indeed, enzymes  reported 18 days . The effa et al. . The optTrichoderma harzianum, Penicillum simplicissiimum, P. janthinellum, P. funiculosum, and P. terrestrial are able to use pyrene as the sole carbon and energy source. It was reported that, when the concentration of pyrene in the test medium was 50 mg/L, 65% of pyrene was degraded by T. harzianum, while, at 100 mg/L, 33% was metabolized. Among the five species, P. terrestrial showed the best results in pyrene degradation, by metabolizing 75% at 50 mg/L and 67% at 100 mg/L in 28 days. However, P. janthinellum exhibited the lowest performance with only 57% of degradation rate at 50 mg/L and 31.5% at 100 mg/L [Pseudotrametes gibbasa was reported to degrade 28.33% of pyrene (10 mg/L) in 18 days [Armillaria sp. FO22 degraded 63% of pyrene (5 mg/L) [Rhizoctonia zeae SOL3. Overall, 64% of pyrene at 100 mg/L was degraded by Scopulariopsis brevicaulis in 30 days [Pleurotus pulmonarius FO43 degraded 99% at 10 mg/L of pyrene in 30 days of incubation [Marasmiellus sp. CBMAI 1062 has been reported to degrade almost 100% at 0.08 mg/mL of pyrene in just 48 h under saline condition [Peniophora incarnate strain KUC8836 after two weeks of incubation recorded a higher degradation rate for phenanthrene (95.3%), fluoranthene (95.0%), and pyrene (97.9%). This high performance was explained by the capacity of this strain to produce laccase, LiP, and MnP in higher quantities [Phlebia brevispora KUC9045 was used to investigate the degradation of phenanthrene, anthracene, fluoranthene, and pyrene and the production of extracellular laccase and MnP during degradation process. This strain was effective in metabolizing the four PAHs with a degradation rate of 66.3%, 67.4%, 61.6%, and 63.3%, respectively, for phenanthrene, anthracene, fluoranthene, and pyrene [Peniophora incarnata KUC8836 and Mycoaciella bispora KUC8201 were reported to have significant rates of pyrene removal (82.6%) after 2 weeks of incubation [ 18 days . In 30 d(5 mg/L) . Khudhai(5 mg/L)  stated t 30 days . The strcubation . The Basondition . The Pend pyrene . SimilarAgrocybe dura FBCC478, Agrocybe praecox FBCC587, Gymnopilus luteofolius FBCC466, Irpex lacteus FBCC1012, Mycena galericulata FBCC598, Phanerochaete velutina FBCC941, Stropharia aeruginosa FBCC521, Stropharia rugosoannulata FBCC475, and Trametes ochracea FBCC1011 showed significant growth on plates with a combination of 16 PAHs [Phanerochaete velutina showed a degradation rate of 96% and 39%, respectively, for PAHs with 4, 5, and 6 rings [Rhizochaete sp. KUC8364 and Dentipellis dissita. KUC8613 developed perfectly well in the media rich in fluoranthene and pyrene, with a growth rate ranging 90–100%, while that of the strains Phanerochate calotricha KUC8040 and KUC8003 in the same media ranged 70–90% [A species tolerance to stress in general, and pollutants in particular, is reflected in its ability to survive and grow under changing conditions. Microorganisms get their nutrients from the environment where they live. Although they are supposed to be toxic, organic pollutants can serve as a source of carbon and energy for organisms which present the required genetic characteristics and adaptation capacity. The survival of organisms among so many others is sufficient proof that they present criteria to wonder if they are capable of using these pollutants as the only source of survival and opens the subject to the optimum conditions for the metabolism of PAHs. The strains  16 PAHs . Similar 6 rings . On the d 70–90% ,82,83.2) in order to create arene oxide and water is the initial transformation of PAHs in general and pyrene in particular. It is crucial here to notify that both lignolytic and non-lignolytic fungi such as Aspergillus, Penicilium, Phanaerochate, Pleurotus, Tramates, and Cunninghamella are reported to have the ability to produce cytochrome P450 monooxygenase during the degradation of PAHs having 2-5 aromatic rings [P. ostrestus can metabolize several PAHs including pyrene. The extracellular enzymes laccase, LiP, and MnP oxidize organic compounds and create PAH transient diphenols, which are automatically oxidized to quinone thereafter [2 [The ability of aerobic fungi to break down PAHs relies on oxygenase enzymes which reduce elemental oxygen in order to activate the fungi, which then led them to enter the substrates. The aerobic oxidation of pyrene by fungi starts with the configuration of dihydrodiol by an enzymatic cascade of dioxygenases, which is later used either by the meta or ortho pathway, resulting in the formation of intermediates such as catechol and protocatechuate . Aerobicic rings  The lignereafter ,96. The ereafter ,98. The after [2   with a strain of fungi (Fusarium sp.). These microorganisms are reported each having the ability to use pyrene as a carbon source and energy. However, in their consortium, the authors showed that the initial concentration of pyrene (100 mg/kg) was degraded by more than 96% in the liquid medium and 87.2% in the contaminated soil. The synergy between different strains in the metabolism of PAHs is explained in the sense that each of these different strains breaks down PAHs. For example, in a bacteria–fungi consortium, it is obvious to imagine that the fungi initiate the first stage of oxidations and then the bacteria perform other oxidations, as shown in [Rhodococcus wratislaviensi strain 9 and Chlorella sp. strain MM3), the bacteria strain is used as a heterotrophic partner and that of the microalgae as a phototrophic partner. The bacterial strain Mycobacterium sp. B2 individually was effective in the degradation of pyrene up to 82.2% and 83.2% for free and immobilized cells after 30 days of incubation. Thus, although the Mucor sp. F2 strain was not as efficient as the B2 strain, their consortium was much more efficient. The degradation rate in the consortium was increased by 51.6% compared to the cumulative rate of pyrene degraded by each individually in an alkaline medium [2 released by bacteria during respiration can be used by microalgae, without of course omitting the important role that the surface of microalgae can play as a stable habitat for bacteria. Consequently, in a perspective that each strain can individually degrade PAHs, their mixture will have more interesting results compared to their individual effect. Luo et al. [Selenastrum capicornutum) and a bacterium (Mycobacterium sp. strain A1-PYR) to evaluate the degradation of pyrene. The result reveal that the joint and mutualistic effect of the consortium was significant, not only in the degradation of pyrene but also in the growth of bacteria [Bioremediation involving bacterial strains, fungi, or microalgae capable of degrading organic compounds is a globally accepted reality. Several strains of microorganisms have been shown to be effective in the bioremediation of organic pollutants and many have been extensively studied to establish the different mechanism pathways for certain compounds such as pyrene. Strains have individually shown impressive efficacy; however, it is obvious and more interesting that, when two or more microbial populations  are in consortium, the result would be more significant compared to when they are involved individually. Liu et al.  studied shown in . In a coe medium . In a coo et al.  realizedbacteria .A better understanding of bioremediation requires a great mastery of interactions and important ecological relationships between different microorganisms. As astonishing as it may seem, it is not easy to study dynamic microbial interactions in real time because of the numerous uncultivable microorganisms and limited molecular tools. However, as pointed out by Professor Gu, in a microbial culture, a target pollutant under investigation is not the only source of carbon and energy for the development of microorganisms. Such results would be the work of a biochemical process called co-metabolism. In the present case, co-metabolism is an efficient and common route for the natural biodegradation of pyrene, whether in a monoculture or a microbial consortium. Technology and science have increasingly developed powerful tools such as multi-omics techniques including metagenomics, transcriptomics, and proteomics for the reconstruction of interactive networks of microorganisms in purpose of PAHs biodegradation. However, for a true construction of a mechanism and degradation pathway, the culture medium must be controlled so that the constituent compounds do not interfere with the degradation process. For this, a defined mineral medium with a chemical composition known to achieve the objective of degradation by microorganisms capable of using the test chemical as the sole source of carbon and energy is the key motor. Therefore, using the appropriate technological means such as RT-qPCR, microarray, PCR-based fingerprints, and biosensors, the different actors of degradation including catabolic genes, enzymes, and intermediates can be determined and identified. Besides, to understand the medium for a real reconstruction of pathway degradation, future studies on the anaerobic degradation of pyrene with strains already known to have degradation capacity would be a unique initiative to propose a complete and integrated pathway on the mechanism of degradation of PAHs in general and pyrene in particular. It is evident that anaerobic fungi have shown some potential for the metabolism of organic pollutants including PAHs. It is therefore recommended to use the combination of various strains of anaerobic fungi and anaerobic fungi–bacteria consortium to achieve maximum efficiency of the biodegradation mechanism of pyrene.


This retrospective observational study aimed to examine the correlation and correspondence between skeletal maturation indicators (SMI), cervical vertebral maturation indicators (CVMI), and radius-ulna-short bones (RUS) skeletal maturity scores in Korean adolescents, and to determine whether easily obtainable SMI or CVMI can replace the RUS skeletal maturity score. A total of 1017 participants were included with both hand-wrist radiograph and lateral cephalogram acquired concurrently. From the lateral cephalogram, CVMI was determined; through the hand-wrist radiograph, SMI was categorized, and the RUS skeletal maturity score was evaluated as well. Associations were examined using the Mann–Whitney U test, Spearman’s rank-order correlation analysis, and multiple correspondence analysis. There was no statistically significant difference in chronological age between males and females; however, the SMI, CVMI, and RUS skeletal maturity scores were significantly higher in females. The SMI, CVMI, and RUS skeletal maturity scores showed a statistically significant strong degree of both positive correlation and correspondence. However, a precisely corresponding RUS skeletal maturity score was difficult to obtain for a specific CVMI and SMI stage, implying the absence of a quantitative correlation. In conclusion, detailed evaluation should be conducted using the RUS skeletal maturity score, preferably in cases that require bone age determination or residual growth estimation. Orthodontics requires accurate predictions of the growth of adolescent patients to determine the timing of treatment. In this context, to account for individual diversity, the developmental age is used rather than chronological age as the indicator of interest . SkeletaIn other contexts, such as in pediatrics or growth clinics, methodologies other than SMI are mainly used to evaluate skeletal maturity from hand-wrist radiographs ,5, incluThe RUS skeletal maturity score allows the calculation of bone age using a conversion table of the TW3 method and also the prediction of height at growth completion . ConsequIn practice, many patients and guardians who visit the clinic for orthodontic treatment often inquire after the expected height at the time of growth completion or the residual amount of statural growth. Provided that inferring the RUS skeletal maturity score from the SMI and CVMI is possible based on a correspondence between these factors, orthodontists may be able to report more instructive statural growth-related information to patients and guardians. However, studies into the correlation and correspondence between the SMI, CVMI, and RUS skeletal maturity scores in Korean adolescents are limited.Thus, this retrospective observational study aimed to examine the correlation and correspondence between the SMI, CVMI, and RUS skeletal maturity scores in Korean adolescents, and to determine whether easily obtainable SMI or CVMI can replace the RUS skeletal maturity score with high confidence. The null hypothesis was that RUS skeletal maturity score can be logically deduced from the SMI or CVMI.This study included 1017  participants with a mean age of 11.9 ± 2.5  years who visited private clinics and for whom both hand-wrist radiograph and lateral cephalogram examinations were concurrently performed between August 2019 and February 2021 .Exclusion criteria were as follows: (1) age of >19 years, (2) severe dentofacial anomalies such as a cleft lip or palate, (3) previous history of growth hormone therapy, (4) presence of chronic disease or history of medication, and (5) radiographs with quality precluding skeletal maturity evaluation.The study protocol adhered to the tenets of the Declaration of Helsinki (2013) and was approved by the Institutional Review Board of Yonsei Dental Hospital (IRB No. 2-2021-0071). The ethical approval and informed consent were waived in view of the retrospective nature of the study; all performed procedures were part of routine care. p-values of <0.05, a power of 80%, and an effect size of 0.1; the minimum sample size was 612 to enable the assessment.The effect size for the analyses was determined via G*Power ver. 3.1.9.2  with Both lateral cephalograms and hand-wrist radiographs were obtained with a multipurpose imaging system . Hand-wrist radiographs were obtained following the manufacturer’s instructions, placing a patient’s left hand on the carpus plate without displaying the nasal positioner. The radius and ulna of the wrist were then exposed, and exposure factors  were defined.CVMI was determined using the Hassel’s classification method by evaluating the contour of the lower border of the second, third, and fourth cervical vertebral bodies and the height of the cervical vertebrae in the lateral cephalogram and classifying them from stage 1 to stage 6 [SMI was categorized from stage 1 to stage 11 according to Fishman’s SMI method by evaluating the thumb, third finger, fifth finger, and radius through a hand-wrist radiograph . To deteAll assessments of skeletal maturity were performed by one orthodontist with 23 years of clinical experience. Intra-observer reproducibility was investigated by comparing the values of the original with those of re-evaluated variables 2 weeks after the first measurement in a randomly chosen set of 50 subjects. The intraclass correlation coefficient was of >0.97 for all evaluation values.p-values of <0.05. The Shapiro–Wilk test was used to verify the normality of data distribution in the RUS skeletal maturity score; the data were non-normally distributed and thus a nonparametric test was used.All statistical analyses were performed in IBM SPSS Statistics for Windows , and the results were considered statistically significant at r of <0.4, weak correlation; 0.4 < r < 0.6, moderate correlation; r of >0.6, strong correlation) [The Mann–Whitney U test was used to compare chronological age, the SMI, CVMI, and RUS skeletal maturity scores between males and females. Spearman’s rank-order correlation analysis was used to evaluate correlations between the SMI, CVMI, and RUS skeletal maturity score (elation) . In addip = 0.053); however, the SMI, CVMI, and RUS skeletal maturity scores were significantly higher in females than in males (p < 0.001). The descriptive statistics of participants’ SMI, CVMI, and RUS skeletal maturity scores are presented in Spearman’s rank-order correlation analysis showed a statistically significant strong degree of positive correlation between the SMI, CVMI, and RUS skeletal maturity scores in the overall sample and in both male and female subgroups .The two-dimension multiple correspondence analysis in all subjects revealed that the first and second dimensions had eigenvalues of 2.956 and 2.793, inertia of 0.985 and 0.931, and Cronbach’s alpha of 0.993 and 0.963, respectively. Regarding sex, the corresponding values presented in males were 2.977 and 2.831, 0.992 and 0.944, 0.996 and 0.970, respectively. The corresponding values presented in females were 2.956 and 2.844, 0.985 and 0.948, 0.993 and 0.973, respectively. Since all measurements conducted in this study were related to the skeletal maturity index, Cronbach’s alpha was very high [The observed correspondence was CVMI 1-SMI 1, CVMI 4-SMI 7, CVMI 5-SMI 9, and CVMI 6-SMI 11 in males, and CVMI 1–SMI 1, CVMI 2–SMI 4, CVMI 3–SMI 6, CVMI 4–SMI 7, CVMI 5–SMI 9, and CVMI 6–SMI 11 in females . The corn = 1017) aimed to determine the correlation and correspondence between the SMI, CVMI, and RUS skeletal maturity scores obtained from hand-wrist radiographs and lateral cephalograms.This study (Patient’s skeletal maturity may determine the treatment strategy in orthodontics, which depends on the growth status and residual growth of the maxillofacial skeleton. Although information on chronological age and secondary sexual characteristics can be easily obtained using questionnaires, these methods have a limited capacity to accurately evaluate bone maturity. Therefore, previous studies aimed to evaluate skeletal maturity using radiographs such as SMI, CVMI, and dental age, all of which are associated with maxillofacial growth ,11,12,13Fishman’s SMI, which evaluates 6 parts with 2–3 stages in the hand-wrist radiograph, is a much more simplified method compared to the TW3 method, which weights and evaluates 13 parts, respectively. Therefore, this method is commonly used at the chairside to determine the growth status of growing patients presenting at orthodontic clinics. This method is particularly useful for evaluating the pubertal growth spurt or growth termination with the emergence of the sesamoid bone or the fusion of the ulnar bone, which relatively limits disagreement between observers. However, an accurate assessment of bone maturity remains a challenge due to the likelihood of interobserver disagreement in evaluating the width and the capping of the phalanx at each finger. In addition, considering the fact that the development of the hand and wrist bones follows the Scammon’s general body growth curve , SMI hasCVMI, which can be obtained by analyzing a lateral cephalogram, determines skeletal maturity as stage 1–6 by evaluating three cervical vertebrae in 2–3 stages, and may be used as an alternative to Fishman’s SMI, as the two measures have a high correlation ,16. The In the present study, a statistically significant strong degree of positive correlations were observed between the SMI, CVMI, and RUS skeletal maturity scores; these findings are consistent with those of previous studies ,23. HoweIn addition, to the best of our knowledge, this study is first to show a sex-based difference in the relationship between SMI and CVMI. In the observed correspondence between CVMI and SMI, CVMI 1–SMI 1, CVMI 4–SMI 7, CVMI 5–SMI 9, and CVMI 6–SMI 11 showed strong associations in males b, whereaThis study had some limitations. First, this was a retrospective clinical study. Second, despite its large sample size, the distribution of patients was somewhat unequal, particularly in terms of males. To overcome these limitations and to confirm the correlation between various skeletal maturity indices and other clinical variables, further case-controlled studies are required. The null hypothesis of this study is rejected. The SMI and RUS skeletal maturity scores obtained from hand-wrist radiographs and the CVMI obtained from lateral cephalograms had a statistically significant strong degree of positive correlations in both sexes, and all three indicators can be used effectively used in orthodontic clinics. However, because the simplified methodology of evaluating skeletal maturity in 6 or 11 stages has a limited accuracy, the precision of evaluation should be improved using the RUS skeletal maturity score, preferably in specific cases that require the bone age determination or residual growth estimation. Therefore, it is recommended that clinicians should acquire a hand-wrist radiograph and perform a detailed evaluation as circumstances demand.


Objectives: The aim of this study was to characterize multiple sclerosis (MS) patients exposed to dimethyl fumarate (DMF) and to evaluate the predictors of therapeutic response. In addition, the study offers a picture of how DMF use has changed over the past few years in naive or switcher patients.Methods: In this observational monocentric study, we examined the prescription flow of DMF in MS patients categorized as naive or switchers  from 2015 to 2019. Clinical and magnetic resonance imaging data of DMF-treated patients were analyzed, and NEDA-3 status at 24 months was evaluated by the three assessment components . Determinants of therapeutic response were also evaluated using regression analysis.Results: The sample included 595 MS patients exposed to DMF categorized as naive  and switchers for reasons of safety/tolerability , inefficacy , and de-escalation strategy . A 15% increase in DMF use in naive and horizontal shift groups was observed in the last 3 years of observation, whereas there was a drop, with prescription passed from ~20% to <5%, as an exit strategy from second-line therapies. NEDA-3 status was calculated for 340 patients after 24 months of DMF treatment and achieved in 188 (55.3%) of these. Analyzing the predictors of DMF response, we observed that lower annualized relapse rate (ARR) in 2 years pretreatment  and being naive patients  were associated with achievement of NEDA-3. Analogously, ARR in 2 years pretreatment affected the NEDA-3 achievement at 24 months in patients of the de-escalation group , also indicating an effect related to the DMF initiation within 3 months .Conclusion: Our findings confirm DMF as a handy drug with broad clinical utility, with greater benefits for naive patients and horizontal switchers. Additionally, an increase in the flow of DMF prescriptions in these two groups of patients was also observed in our cohort. In the last decade, many changes have marked the therapeutic scenario of multiple sclerosis (MS), with the introduction of new disease-modifying therapies with different mechanisms of action, efficacy, and safety profile, resulting in improved choice and steps toward a personalized therapy . DimethyBased on these considerations, the present study aimed to (i) define demographic and clinical features of MS patients undergoing DMF therapy categorized as naive or switchers , also describing how DMF prescription flow has changed in these four patient categories over the past 5 years, and (ii) evaluate the efficacy data in the different DMF patient groups, also evaluating the predictors of therapeutic response.This is an observational monocentric study that included MS patients diagnosed with the revised McDonald criteria , who stat-tests for quantitative variables and χ2-tests for qualitative variables. Mann–Whitney U tests were used to compare the ARR calculated 2 years before DMF therapy and at 24 months following DMF therapy for the four groups of patients, naive and switchers, also categorized in relation to the last disease-modifying therapy. Therefore, the achievement of NEDA-3 status at 24 months was calculated as a percentage of patients with no clinical relapses, EDSS progression, and radiological activity, and the predictors of NEDA-3 status were investigated using binary regression analysis. For all assays, statistical significance was set at p < 0.05.SPSS for Mac version 20.0  was used to perform the statistical analysis. Descriptive statistics are presented using mean, SDs, and frequencies (absolute and relative). First, the percentage of naive patients and switchers initiated to DMF therapy was assessed for the years 2015–2019. Thereafter, demographic  and clinical differences  among patients exposed to DMF categorized as naive or switchers  were evaluated using independent-samples The sample included 595 MS patients exposed to DMF categorized as naive  and switchers for reasons of safety/tolerability , inefficacy , and de-escalation strategy . Of the patient group, the mean DMF exposure was 28.7 (SD = ±18) months, while the median was 27 months (±32 months of IQR).2 and independent-samples t-tests, showing lower age, MS duration, and EDSS score (p < 0.005) for naive patients. The percentage of naive MS patients who initiated DMF use is detailed in p = 0.014), switchers for inefficacy , and switchers for safety/tolerability . No difference in ARR before and after 24 months of DMF therapy was found in the de-escalation group, which continued DMF treatment. However, nine de-escalating patients  discontinued DMF within the first year, whereas 10 patients  discontinued DMF between the first and second year, mainly due to ineffectiveness (72.2%).p = 0.001)] and being naive patients  were associated with achievement of NEDA-3 (p = 0.041), also indicating an effect related to the DMF initiation within 3 months  of these. In detail, relapse-free status was observed in 229 patients (67.5%), no disability progression in 299 (87.9%), and MRI NEDA-3 status in 283 patients (83.2%) . Analyzif NEDA-3 . AnalogoThe overall discontinuation rate was of 17.9% (107/595 patients); of these, 60 of 595 patients (10%) discontinued DMF due to ineffectiveness, 34 (5.7%) of whom within the first year of treatment [nine de-escalating from second-line Disease Modifying Treatments (DMTs)]. Analogously, of 21 patients (3.5%) who discontinued DMF between the first and second year, 10 were after de-escalation strategy.Finally, 47 of 595 patients (7.9%) in our cohort discontinued DMF for safety/tolerability reasons, mainly during the first year of treatment (72% of cases). Of these, gastrointestinal (GI) symptoms were reported as the primary side effect, accounting for 4.6% of drug suspension, followed by flushing (3.1%) and laboratory testing abnormalities (hypertransaminasemia for 0.1% and prolonged lymphopenia for 0.1%). A shift to oral teriflunomide was reported in 18 (38.3%) of these patients, to glatiramer acetate (Copaxone) in 15 (31.9%), and to interferon in 11 (23.4%), whereas 3 patients (6.4%) did not undertake other immunotherapies.Previous studies have evaluated the effectiveness of DMF with analysis of the real-world data. Our data can be differentiated from previous studies in several respects, including data source, cohort , method of analysis , and the evaluation of the results with the examination of predictors of MS outcomes.In line with other studies that have shown a good efficacy profile of DMF both in naive and horizontal switchers , 5, 7, wThe reduction of DMF use as an exit strategy observed in our cohort may be attributable to a better selection of patients to be initiated on natalizumab therapy based on JC virus serostatus , as wellAnalyzing the predictors of NEDA-3 status, clinical activity in the 2 years preceding DMF and being naive patients emerged as significant determinants confirming, as previously demonstrated by Lanzillo et al., the utility of this oral agent from the earliest stages of the disease . AnalogoOverall, safety data confirmed a favorable profile for DMF, with 7.9% patients dropped out due to safety or tolerability issues, the most frequent being GI tolerability 4.6%). Grade III lymphopenia, which other studies reported as an infrequent event ranging between 3 and 15% of patients .6%. Grad, 25. TheAnother important safety issue linked to the increasingly widespread use of DMF in young women is related to pregnancy, on which preliminary data would have shown safe outcomes . FurtherThe present study has several limitations mainly due to its retrospective nature. However, compared to other studies on this topic, the monocentric nature of our study allowed limiting the variability in radiological and clinical data collection. Furthermore, the study focused exclusively on evaluating DMF efficacy outcomes, considering the composite evaluation of clinical relapses, EDSS progression, and neuroradiological activity (NEDA-3) as disease outcome. Safety aspects of DMF and the possible predictors of safety outcomes have not been deliberately explored.Our findings confirm DMF as a handy drug with broad clinical utility. DMF use has progressively increased in clinical practice, showing greater benefits for naive patients and horizontal switchers. Further studies are needed to better investigate the predictors of efficacy, as well as the predictive biomarkers, for the best identification of patients to be initiated on DMF treatment, in the modern perspective of an effective, early, and personalized therapy .The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Cagliari. The patients/participants provided their written informed consent to participate in this study.LL and ECo conceptualized the study and wrote, reviewed, and edited the manuscript. ECa, MF, JF, GF, GC, SP, and MB were responsible for resources and data curation. All authors contributed to the article and approved the submitted version.LL, JF, GF, GC, and ECo received honoraria for consultancy or speaking from Biogen, Novartis, Sanofi, Genzyme, Serono and Teva, and Almirall. 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.


Apis cerana cerana, we selected an OBP gene, AcerOBP6, which is highly expressed in the antennae of worker bees, acquired a purified protein via a prokaryotic expression system, and analyzed its function using bioinformatics, molecular biology, and electrophysiology. According to the result, AcerOBP6 was a protein with extensive binding affinity, and we speculated that its function was chiefly related to foraging. Overall, this research not only explains the essential role of OBPs in ligand binding, but also provides valuable resources to help researchers further understand the nature and mechanism of the olfactory system.The olfactory sensillum, which is located in the antenna of insects, is the basic unit of the olfactory organ. Olfactory-related genes are expressed in the sensillum. It is believed that the process of olfaction recognition is mainly mediated by two gene families, odorant binding proteins (OBPs) and olfactory receptors (ORs). The honeybee possesses a large numbers of ORs, but few OBPs. Up to now, the function of OBPs in the honeybee has not yet been fully elucidated. In order to reveal the specific role of OBPs from Apis cerana cerana. Beyond that, the mRNA expression profiles and the binding affinity of AcerOBP6 were researched. According to qRT-PCR analysis, AcerOBP6 transcripts were mainly expressed in the antennae of forager bees. In addition, we found that the expression level of AcerOBP6 was higher than that of AmelOBP6. The fluorescence competitive binding assay indicated that the AcerOBP6 protein had binding affinity with most of the tested odors, including queen pheromone, worker pheromone, and floral volatiles, among which the strongest one was linolenic acid . However, AcerOBP6 was not sensitive to the brood pheromones. A further study based on EAG assay revealed that the antennae had the strongest response to 2-heptanone. The EAG recording values of the selected ligands were all reduced after AcerOBP6 was silenced, with 8 of 14 declining significantly (p < 0.01) given that these odors could specifically bind to AcerOBP6. As revealed in our current study, AcerOBP6 might be a crucial protein involved in olfactory recognition for foraging. Overall, the research provides a foundation for exploring the olfactory mechanism of A. cerana cerana.Olfaction is essential in some behaviors of honeybee, such as nursing, foraging, attracting a mate, social communication, and kin recognition. OBPs (odorant binding proteins) play a key role in the first step of olfactory perception. Here, we focused on a classic OBP with a PBP-GOBP domain from the Asian honeybee,  Olfaction plays a significant role in the survival and reproduction of insects by modulating several of their biological behaviors. Additionally, the antennae are the main olfactory organs, with a large number of olfactory sensilla located on them ,2,3. OBPApis mellifera and Apis cerana, are the major honeybees raised in China. Compared with A. mellifera, A. cerana possesses a more sensitive olfactory system, meaning that it has a good foraging ability for the use of sporadic nectar and pollen sources  represents the concentration of the competitor reduced to half-maximal fluorescence intensity, [1–NPN] represents the free concentration of 1–NPN, and K1–NPN represents the dissociation constant of the AcerOBP6/1–NPN complex.Taking 1–NPN as the fluorescent reporter, we measured the binding affinity of each odorant by competitive binding assays using. All the 33 ligands . The odorants were dissolved in liquid paraffin to final concentrations of 300 μg/μL, and liquid paraffin was adopted as a blank control. A filter paper strip (3 cm × 1cm) filled with 10 μL tested solution was prepared and put into the tube of Pasteur Pipette, which was connected with the gas feeder equipment. The stimulation time of each odor was 0.5 s and the interval was 30 s. Three technical repetitions were carried out, and ten antennae of female adults were tested for each compound. Finally, the EAGPro software (Syntech) was used to analyze the EAG response recordings.t-test. A two-sided p-value of 0.05 was considered to be statistically significant.All statistical tests were conduct using SPSS version 26.0. The normality and homoscedasticity of data were verified using Shapiro-Wilk and Homogeneity of variance test. Data from multiple groups were compared using One-Way ANOVA, followed by Bonferroni multiple comparison test for paired comparison. Data from two groups were compared using Student’s The open reading frame (ORF) of AcerOBP6 was 441 bp, encoding 146 amino acid residues of 16.95 kD. A hydrophobic signal peptide with 24 amino acids was identified and the deduced amino acid sequence had six conserved cysteines, which was a typical motif of classical OBPs .The results of homology analysis for homologous proteins showed that AcerOBP6 had a high identity with its sibling species , such asF = 15.54, p < 0.01), which had the lowest level in the abdomen , and the homologous gene was expressed more highly in A. c. cerana than in A. mellifera ligustica   c.The recombinant protein of AcerOBP6 was constructed and purified. On this basis, we found that the band size in the SDS-PAGE electrophoresis gel was consistent with the expected molecular weight , and the50 and the dissociation constants (Ki) were listed in First of all, the binding affinity of 1-NPN to AcerOBP6 was tested a. BeyondHowever, there were 11 compounds that could not bind or had a weak binding affinity with AcerOBP6, including seven brood pheromones , four plant volatiles , and one alarm pheromone (isoamyl acetate).p < 0.05). Furthermore, the silencing effect was better at 48 h after feeding  than at 0, 24, or 96 h. In addition, the expression levels of the dsGFP groups did not change significantly compared to the sugar water group (p > 0.05)  , indicatp < 0.05). Notably, there was no significant difference in response between the negative control and dsGFP groups. From the results, we also found that the EAG value would not necessarily change greatly after RNAi, although the odors had a strong binding affinity in the fluorescence competitive binding assay.According to the results, we selected the dsOBP6-2 dsRNA to feed to the workers. Then, after 48 h the bees were used in the following EAG assay. A total of 14 ligands with relatively low Ki values were used in the EAG assay. As shown in A. cerana cerana. According to the sequence analysis, we observed that AcerOBP6 had a small molecular weight, six highly conserved cysteines, and contained a signal peptide, indicating that AcerOBP6 belongs to a typical ‘classic’ OBP family [Insect OBPs are thought to be the pioneer proteins that are involved in the process of odor discrimination, binding, and delivery of chemical stimuli to ORs. In this research, we determined the sequence and expression features and the binding affinity of an OBP gene, AcerOBP6, from the antennae of P family .The potential biological significance of genes could be derived from their transcript expression profiles . It is wA. cerana cerana including AcerOBP1, AcerOBP10, AcerOBP11, and AcerOBP15, in which AcerOBP1 and AcerOBP15 were the proteins that were mainly involved in pheromone binding [trans-cinnamate is isolated from lodgepole pine, Pinus contorta, which has been bioassayed as an antifeedant for pine weevils [A. cerana cerana. Meanwhile, AcerOBP6 had a very weak binding affinity with brood pheromones. This might be because these ester pheromones are the non-volatile components, while insect OBPs mainly bind with volatile molecules [Competitive fluorescence binding assays have been widely used in the binding affinity studies of insect OBPs . In all  binding ,21,33,34 binding . Notably binding ,37. It w binding ,39. Ethy weevils  but it i weevils . Therefoolecules .Bemisia tabaci also reported the similar viewpoint [In order to further confirm whether AcerOBP6 participates in binding of those volatiles screened in the competitive fluorescence binding assay, RNAi combined with EAG methods were performed. Similar to other previous research, we used the feeding method to accomplish RNAi ,43,44. Aiewpoint .A. cerana cerana was sensitive to 2-heptanone and the chemical compound was likely the specific binding ligand of AcerOBP6. 2-Heptanone is an alarm pheromone that delivers dangerous signals in the colony to trigger the defensive response of bees [In addition, 2-heptanone elicited strong EAG responses to workers’ antennae and displayed a significant change after the bees dealt with dsRNA. In other words,  of bees . What’s  of bees ,48.E. coli expression system was developed to obtain an odorant binding protein of A. cerana cerana. The amino acid sequence of this protein was highly homologous with AmelOBP6. AcerOBP6 was mainly expressed in the antennae of forager bees. This protein showed a good affinity with most floral volatiles and some bee pheromones, except for the larval pheromones. Linolenic acid was the strongest binding odorants among the selected compounds, which is an odorant compound in some floral pollen. Using the combined methods of EAG and RNAi assays, we further investigated the special ligands of AcerOBP6 including 2-heptanone, hexyl acetate, eugenol, β-ocimene, 1-nonanol, myrcene, (+) -cinene, and linalool. The findings of this study suggested that AcerOBP6 plays an important role in the odorant binding process, especially in modulating pollen foraging behavior of workers.To conclude, an


Objective: This study aimed to demonstrate the safety and the efficacy of the self-gripping mesh (ProgripTM) for inguinal hernia repair in morbid patients of the higher American Society of Anesthesiologists (ASA) classification (ASA III and IV). The incidence of chronic pain, postoperative complications, and hernia recurrence was evaluated.Methods: Data were collected retrospectively from the files of the patient and were analyzed for 198 hernias in 147 patients. All the patients included in this study had undergone inguinal hernia repair by Lichtenstein approach with the self-gripping mesh (ProgripTM) in the same clinical center. Preoperative, perioperative, and postoperative data were collected and a long-term follow-up of 31.8 ± 19.5 m (5–60 m) was performed. Complications, pain scored on a 0–10 numeric rating scale (NRS), and hernia recurrence were assessed.Results: During the past 5 years, 198 hernias in 147 patients were repaired with the Lichtenstein procedure with the self-gripping mesh (ProgripTM). The majority of the patients were high level of the ASA classification (ASA III and IV) (95.9%), with ASA III (10.2%) and IV (85.7%). The mean operation time was 71.2 ± 23.8 min. The mean length of postoperative stay was 2.5 ± 2.1 days. There were no intraoperative complications. About 14 cases (7.1%) suffered from postoperative surgical wound complications, which were limited to the skin and subcutaneous tissue and were cured with the conservative methods successfully; there was no mesh infection, the acute postoperative pain was low or mild [visual analog scale (VAS) score ≤ 4] and the chronic postoperative pain was reported in three patients (1.5%) and tolerable, hernia recurrence  occurred in one patient half a year after during the follow-up period.Conclusion: This study demonstrated the advantages of the self-gripping mesh in hernia repair of the high-risk patients with inguinal hernia (ASA III and IV) by Lichtenstein procedure under local anesthesia. Inguinal hernia repair is the most common procedure in general surgery, with more than 20 million patients being repaired globally each year , 2. The TM) has been developed to avoid the use of additional fixation  grips indicated for inguinal hernia repair. Recent studies have demonstrated a reduction in operating time and costs of a hernia repair for the Lichtenstein technique with Parietex ProGripTM self-gripping mesh compared to a classically fixed mesh for relatively inexperienced surgeons  classification (ASA III and IV) has not yet been studied. In addition, this study aimed to evaluate the long-term results of the use of ProGripTM mesh in this group of patients (ASA III and IV).In this study, we routinely used the Lichtenstein technique for inguinal hernia repair with the self-gripping mesh, if the patients are not suitable for laparoscopic repairs as judged by surgeons. Although the short-term results of a large comparative randomized study have demonstrated the safety and the efficacy of the use of ProGripapproach , the lonTM) were used in all the procedures. Approval of the patient was obtained from each patient before operation, and ethical approval was not required for this retrospective type of study. Preoperative, perioperative, and postoperative data were collected. Perioperative data collected included patient demography , type and location of the hernia, and medical risk factors . Postoperative data collected at discharge were assessment of postoperative medical and surgical complications, acute and chronic pain, and hernia recurrence. The pain was assessed by using a visual analog scale (VAS) score (0–10), score 0 is no pain and score 10 is worse pain. If the patient reported hernia recurrence symptoms, the patient was recalled for a visit, and a physical examination was performed to confirm recurrence. Hernia recurrence was defined as a clinical manifest bulge or protrusion exacerbated by a Valsalva maneuver in the operated groin region, a CT scan or sonography was ordered, if necessary.A total of 198 inguinal hernias were operated on with the Lichtenstein procedure between January 2015 and June 2020 in 147 patients . Each prTM Self-Gripping Polyester Mesh, Covidien, Trevoux, France) was used in each patient, the ProgripTM mesh is a lightweight, self-gripping mesh composed of monofilament polyester and PLA grips indicated for inguinal hernia repair. A meta-analysis has demonstrated the reduction in operating time of hernia repair for the Lichtenstein technique with Parietex ProGripTM self-gripping mesh compared to a classically fixed mesh . In all the patients, a skin incision 4–6 cm parallel to the inguinal ligament was performed. The three inguinal nerves  were intraoperatively identified and preserved, the iliohypogastric nerve may be resected if it preventing mesh placement. The fibers of the cremaster muscle were divided, but not resected. The small direct hernia sac was not ligated, but returned to its normal course. The large indirect hernia sac was transected in middle, and the distal sac was left  Chastan , the pos Chastan , with sp Chastan , make su Chastan , the wou2 at the surgery. Among the 147 patients, 92.5% of them were male (n = 136), 7.5% were female (n = 11), and 37 patients with only left-side hernias (25.2%), 59 cases with only right-side hernias (40.2%), and 51 cases were bilateral hernias (34.7), and with 45 hernias were direct-type hernias, 120 hernias were indirect type, and 32 hernias were combined hernias. Therefore, 198 hernias were repaired with the Lichtenstein procedure.Patient demography and perioperative data are presented in n = 126) and 10.2% of them were the ASA classification of III patients (n = 15), the total number of ASA III and IV were 141 (95.9%).Of note, this group of patients were all morbidity patients, 85.7% of them were the ASA classification of IV patients (The most frequent risk factors were hypertension (53.7%) followed by cardiac disease (30.6%), smoking (23.1%), obesity (18.4%), pulmonary disease (9.5%), and cirrhosis (4.8%), 29.3% of the patients were taking antiplatelet at the time of surgery, and 4.1% were taking an anticoagulant, also 2.7% were taking heparin. Emergent operations due to incarcerated hernias were operated in six patients.There were 88 hernias located on the left side and 110 hernias located on the right side. Of the 198 hernias, 45 of them were medial (direct) hernias, 120 were indirect hernias, and 32 were combined hernias. The mean operation time was 71.2 ± 23.8 min. The mean length of postoperative stay was 2.5 ± 2.1 days .There were no intraoperative complications. About 14 cases (7.1%) suffered from postoperative surgical wound complications; as illustrated in All patients reported no or low to mild acute pain (VAS score ≤ 4), there was no patient experienced severe acute pain during the early period of postoperative time. Chronic postoperative pain over 3 months after surgery was reported in three patients (1.5%), but the pain level was mild and tolerable and the pain level decreased over time. At the mean follow-up of 31.8 ± 19.5 m (5–60 m), one patient had a hernia recurrence, and this recurrence occurred as a femoral hernia recurrence half a year after the initial operation, a redo operation was successfully performed by open preperitoneal repair. The follow-up rate was 91.2% (134/147). Eight patients died during the follow-up period; however, the death was not related to hernia repair.Inguinal hernia repair is one of the most commonly performed operations worldwide and the repair methods can be performed under a variety of approaches, namely, open, laparoscopic, or robotic . In 2018Laparoendoscopic inguinal hernia repair is superior to open in terms of lower postoperative pain incidence , 9; howeInguinal hernia is feasible to be repaired under local, regional, or general anesthesia ; usuallyTM) was developed, and this mesh was associated with shorter operative time compared with the conventional suture mesh and provided comparable perioperative and long-term outcomes  or Lichtenstein in selected cases and we agree with others that nonviable intestine cannot be regarded as an absolute contraindication for prosthetic repair , 35. TheThis study has several limitations; first, this is a retrospective study, second, there was no comparative group of the conventional suture mesh; however, this study aimed to evaluate the efficacy of self-gripping mesh and the safety of this repair in morbidity patients, rather than to compare two different techniques.TM in exclusive the patients with high comorbidities  under local anesthesia (92.5%), there was no perioperative death, and with a low incidence of postoperative complication rate and low recurrence rate  (0.5%). This study demonstrated the several advantages of the self-gripping mesh Lichtenstein procedure in high-risk inguinal hernia patients (ASA III and IV), namely, short operative time, safe and simple manipulation, less overall and surgical postoperative complications, and low recurrence rate.To the best of our knowledge, this report is the first study evaluating the use of ProgripThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements.WZ, YZ, ZJ, and JL: conception and design of the study. WZ, YZ, XS, and JL: acquisition of data. WZ, YZ, TC, ZJ, and JL: data analysis. WZ, YZ, XS, TC, ZJ, and JL: drafting of the manuscript and/or critical revision and approval of a final version of the manuscript. All the named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article.This work was supported by the Medical Science and Technology Development Foundation, Nanjing Municipality Health Bureau (ZKX18052).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.


However, submicrometer particles (SMPs) with spherical shape can be experimentally fabricated, even for hard carbides, via instantaneous pulsed laser heating of raw particles dispersed in a liquid (pulsed laser melting in liquid). The spherical shape of the particles is important for mechanical applications as it can directly transfer the mechanical force without any loss from one side to the other. To evaluate the potential of such particles for mechanical applications, SMPs were compressed on various substrates using a diamond tip in a scanning electron microscope. The mechanical behaviors of SMPs were then examined from the obtained load–displacement curves. Particles were fractured on hard substrates, such as SiC, and fracture strength was estimated to be in the GPa range, which is larger than their corresponding bulk bending strength and is 10–40% of their ideal strength, as calculated using the density-functional theory. Contrarily, particles can be embedded into soft substrates, such as Si and Al, and the local hardness of the substrate can be estimated from the load–displacement curves as a nanoscale Brinell hardness measurement.Generally, hard ceramic carbide particles, such as B Spherical submicrometer particles (SMPs) are larger than well-studied nanoparticles (NPs) but have been an interesting research topic owing to their mechanical applications. The spherical shape of the particles is important, as it can directly transfer the mechanical force without any loss from one side to the other. Submicrometer size is another important factor because suitable fabrication techniques via a top-down approach, such as milling, or a bottom-up approach, such as the nucleation process, are currently unavailable. However, the use of spacers, milling agents, lubricant fillers, etc., has been proposed and is in demand ,2.2 [Ductile SMPs made of polymers or glasses are relatively easy to fabricate and are commercially available. Spherical SMPs composed of NP aggregates have also been reported for TiO2 . However2 .Our group reported a new fabrication technique for various spherical SMPs, which is called pulsed laser melting in liquid (PLML) ,6,7,8,9.2, and ZnO, can be fabricated via a simple transient melting process, and B4C SPMs can be reactively fabricated via laser melting of raw B particles in ethanol (as a carbon source) [PLML appears similar to the well-studied pulsed laser ablation in liquid (PLAL) for NP fabrication ,13,14, a source) ,6. Thus,4C and TiO2 were fabricated via PLML, and their mechanical properties were measured using a nanoindenter equipped in a SEM [4C and TiO2. Thus, SMPs obtained via PLML were harder compared with those obtained using other techniques, such as chemical methods, and are promising for various mechanical applications.However, only a few studies on mechanical properties of hard, brittle, crystalline SMPs have been reported so far ,17. In o4C, B and newly fabricated TiC were used as hard and brittle SMPs. Not only a hard substrate, such as SiC, but also softer substrates, such as Si and Al, were used to compare the mechanical behaviors using an embedment process at submicrometer scale. In particular, the possibilities of nano-Brinell hardness measurement and local surface enforcement were explored by analyzing the particle embedment process.Here, by extending our previous studies on the mechanical property measurements of SMPs fabricated via PLML , indenta4C, B, and TiC were fabricated via PLML and used for mechanical tests. The fabrication procedures of B4C and B have been previously reported [4C SMPs and then irradiated using a Nd:YAG laser  with a fluence of 200 mJ pulse−1 cm−2 for 10 min at room temperature. During laser irradiation, a magnetic stirrer was used to stir the liquid for agitation to disperse the raw particles. For B4C SMP fabrication, unreacted B was dissolved in nitric acid and removed. Common byproducts of boric acid for both processes were dissolved in water and removed. For TiC SMPs, TiC NPs  were used as raw particles. A 532 nm Nd:YAG laser was irradiated at 150 mJ pulse−1 cm−2 in ethanol for 10 min and toluene for 120 min. The particles in toluene required a longer ultrasonication time for pre-dispersion (60 min) compared with those in ethanol.Brittle SMPs of Breported ,6. BriefThe morphology of the obtained spherical SMPs following laser irradiation was observed using a field emission scanning electron microscope . The crystallinity of the obtained particles was analyzed from X-ray diffraction patterns . The composition profile within individual particles was analyzed via energy-dispersive X-ray analysis (EDX) equipped with an aberration-corrected scanning transmission electron microscope . The crystallinities of individual particles were measured via the electron diffraction technique using a high-resolution TEM .−1. Further detailed experimental conditions and the typical arrangement of the particles, substrate, and indenter tip were the same as those described in a previous report [2, and SrTiO3, and polycrystals of Al (A5052), which have to be sufficiently flat and electroconductive for SEM observation during indentation. The particles must be sparsely distributed on the substrate to avoid compression of multiple particles with the indenter tip during measurements.An indentation device (Hysitron PI SEM PicoIndenter) installed in a SEM JEOL JIB-4600 F) was utilized for compressive fracture and embedment tests of the SMPs. The indenter speed for all compression tests was fixed at 12.5 nm s600 F was2 rutile phase, which was probably induced by reaction with oxygen in ethanol. 4C SMPs on SiC substrates using diamond tip indentation [4C SMPs was calculated to be 6–12 GPa, assuming tensile fracture at the center of a particle. This value was rather large compared with the typical bending strength by tensile fracture of a B4C sintered body (0.3–0.9 GPa) and was 18–38% of the ideal strength calculated by first-principles DFT. This high strength is due to the less defective nature of SMPs obtained via the PLML process.We previously reported fracture tests of BSt (N m−2) denotes the fracture strength; F (N), the failure load of a spherical particle; and D (m), the particle diameter. The values indicate that the particles obtained in toluene  have higher fracture strength than those obtained in ethanol , although the average size obtained in toluene (396 nm) is larger than that in ethanol (301 nm), which is due to the difference in the permittivity of liquid, which controls the aggregation behavior of raw particles [4C and TiO2 SMPs obtained via PLML ranged from 18% to 38% and 10% to 40% of their respective ideal tensile strengths, which is similar to the TiC SMPs obtained in toluene. In relation to bulk fracture strength, TiC SPMs obtained in toluene were greater by one order of magnitude, as in the case of B4C [The above data for the SMP fracture strength measurements were obtained using hard SiC substrates, and the particles were interlaid between the substrate and a diamond tip to be fractured. Si substrates can also be used for the indenter test of TiC, as shown in When soft Al substrates were used, the particles did not fracture but were gradually embedded, as shown in 4C particles into Si single-crystalline substrates with SEM images before and after the load application. Nearly the entire particle was embedded after the load application, and only the top part of the particle can be observed after embedment. Plateau ranges in the load–displacement curves began when the displacement exceeded half of the particle size, suggesting the embedding process described in HB, was calculated from the applied load, F; particle size, D; and measured indentation diameter, d, of the specimen in overhead view after unloading:With regard to the tip shape to push the substrate, the experimental arrangement in this study is similar to common Brinell hardness measurements using a macroscopic spherical tip, mostly made of a diamond of millimeter order in size and, recently, with micrometer range ,32,33,34h, can be adopted as the indentation depth, and the Brinell hardness can be calculated as follows:In our experimental setup, a particle was continuously pushed during particle embedment. Thus, the displacement of the tip from the surface, d/D range: 0.66–0.87), the calculated Brinell hardness values were close, irrespective of the particle size used to push the Si substrate.Fp, in h/D, is 0.5 and, therefore, Equation (3) can be simplified as follows:The load value at the plateau range, 4C SMPs on a Si substrate. The solid black line is a curve fitted with a parabolic curve based on Equation (4) and is well fitted to the experimental data, indicated by red circles, without pop-in. The fit is also good for the data with pop-in behavior (blue triangles)—for particles larger than 450 nm. The Brinell hardness estimated from the fitting parameter in Nanoindentation techniques for hardness estimation have been developed to measure the mechanical properties of nano-objects. However, most nanoindentation devices use Vickers tips based on a pyramidal shape. Nanosized, sphere-shaped tips have seldom been utilized, probably owing to the unavailability of hard and small spherical particles. Spherical SMPs are a possible candidate for nano-Brinell tips to evaluate the hardness of nanosized objects.hc; the particle size, D; embedded volume, Vh; and particle volume, VD,  into Si substrates, irrespective of the particle size. The average volume fractions, indicated by lines with corresponding colors, were nearly the same.2, Si, SrTiO3, and Al substrates. The embedded volume fractions do not have a clear size dependence in the case of Si substrates, though the embedded volume fraction increased with the decrease in Vickers hardness of the substrates, as presented in 4C, B, or TiC particles must be embedded, substrates with a Vickers hardness smaller than 12 GPa have to be used. Thus, site-selective surface enforcement is considered to be possible simply by spreading mechanically hard SPMs on a soft material surface and applying local force.4C, TiC, and B, were experimentally fabricated via the PLML process, which employs instantaneous pulsed laser heating of raw particles dispersed in a liquid. To explore and evaluate their potential for mechanical applications, these hard SMPs were compressed using a diamond tip on various substrates. The hard particles were fractured on hard substrates, such as SiC, and embedded into soft substrates, such as Si or Al. From the fracture process, particle strength can be estimated to be 5–12 GPa, which is larger by one order of magnitude than the bulk bending strength and 10–40% of the ideal strength of 24.4 GPa from the DFT calculations. From the embedment process, the submicrometer-scale Brinell hardness of 7.01 GPa of the Si substrate can be estimated by analyzing the load–displacement curves. The relationship of particle embedding depth and the hardness of substrates was elucidated from various combinations of SMPs and substrates.Spherical non-porous SMPs of hard and brittle ceramics, such as B


Coleophora therinella Tengström, 1848 is the first report for the family Coleophoridae in Lepidoptera. The 15,539-bp long complete genome has an arrangement identical to that observed in most lepidopteran genomes. COI had the atypical CGA codon that is frequently found in the start region of the lepidopteran COI, and COII had the GTG codon found previously in Drosophila yakuba ND5 and Rattus norvegicus ND1. The 457-bp long A + T-rich region was the second largest, next to Blastobasis lacticolella, which belongs to Blastobasidae in the superfamily Gelechioidea. The A/T content of the whole mitogenome was 80.7%; however, it varied among the regions/genes as follows: A + T-rich region, 94.8%; srRNA, 85.0%; lrRNA, 84.3%; tRNAs, 81.5%; and PCGs, 78.9%. Phylogenetic analyses with concatenated sequences of the 13 PCGs and two RNA genes using the maximum likelihood method, placed Coleophoridae, represented only by C. therinella, as the most basal lineage of the Gelechioidea families consisted of Stathmopodidae, Scythrididae, Blastobasidae, Autostichidae, and Oecophoridae, but nodal support for this grouping was very low (27%). Currently, several families of Gelechioidea are represented by a single species. Thus, extended sampling is required for further reasonable inference for the relationships of these families.The mitochondrial genome (mitogenome) of  Coleophora therinella Tengström, 1848 belongs to Coleophoridae in Lepidoptera. The genus Coleophora, which is composed of 400–500 species is commonly known as ‘casebearers’ because the larvae of most species construct distinctive portable protective silken cases , South Korea in 2012. This voucher specimen and DNA were deposited at the Chonnam National University, Gwangju, Korea, under the accession no. CNU6202 . Using DNA extracted from the hind legs, three long overlapping fragments  were amplified and used as templates for the amplification of 26 short overlapping fragments using the primers reported in Kim et al.  method that is implemented in CIPRES Portal v. 3.1  and two rRNA genes of 20 mitogenome sequences available from Gelechioidea in Lepidoptera, including that of C. therinella was composed of typical sets of genes  and a major non-coding 457 bp A + T-rich region (GenBank acc. no. MH473596), with the gene arrangement being identical to that observed in most lepidopteran genomes . Eleven PCGs had the typical ATN start codon, whereas COI had an atypical CGA codon that is frequently found in the start region of the lepidopteran COI, and COII had the GTG codon found previously in Drosophila yakuba ND5 and Rattus norvegicus ND1 . Strong support was obtained only for the monophylies of Stathmopodidae (100%) and Gelechiidae (91%), respectively. Currently, only 20 species belonging to nine families are available for their mitogenome sequences in Gelechioidea, including that of C. therinella. Thus, most families are represented by one-three mitogenome sequences, excluding the family Gelechiidae. Therefore, an inference of phylogenetic relationships among the families of Gelechioidea is unavoidably limiting. Thus, more mitogenome sequences from a diverse taxonomic group are required for further reasonable inference for the relationships of the families in Gelechioidea.Phylogenetic analysis placed Coleophoridae, represented by the current


Ninety-seven suspected cases of PRV infection were collected from sick pigs vaccinated with Bartha-K61 to isolate PRV. The results showed that the average positive rate of the PRV gE antibody decreased from 38.20% in 2018 to 18.12% in 2020, but there was a high positive rate in sows. The isolation rate of PRV was 13.40% (13/97), and four strains were purified through plaque assay . The homology and genetic evolution of four PRV strains based on gE, gC, gI, and TK genes were analyzed and showed that these four strains shared more than 99.0% nucleotide homology with the variant PRV XJ5 strain, and they clustered in the same sub-branch with the domestic variant PRV strains, including JS-2012 and XJ5. Furthermore, the pathogenicity of the isolated variant strain was assessed by intranasal infection of 16-week-old pigs with 1 mL PRV-SD1 strain. The results of the animal experiment demonstrated that the PRV-SD1–infected pigs exhibited obvious clinical symptoms as early as 2 days post inoculation (dpi), and all infected pigs died within 1 week. The severe hyperemia of meninges and swelling of lungs and tonsils were observed. Histopathology analysis showed the obvious lymphocytes necrosis of tonsils, interstitial pneumonia, and viral encephalitis. Many positive staining cells were observed in tonsils and brains through immunohistochemistry staining assay. Viral shedding in oropharyngeal and rectal swabs were detected at 2 dpi, reached a peak at 3 dpi, and then gradually decreased. The detection of viral loads in the tissues showed that tonsils had the highest virus titer, further proving it may be the target organ of variant PRV infection. In conclusion, variant PRV strains were still highly prevalent in Shandong Province, and they had a strong pathogenicity in pigs.Pseudorabies (PR) is a disease that is seriously endangering the pig industry in China. To understand the current prevalence of pseudorabies virus (PRV) in Shandong Province, China, 19,292 serum samples were collected from 16 locations in Shandong from 2018 to 2020. The  Pseudorabies, also known as Aujeszky's disease, is caused by the pseudorabies virus (PRV) and is characterized by anorexia, respiratory distress, and neurological disorders in pigs. This disease can be transmitted by saliva, nasal discharge, and airborne particles. It has been a major viral disease in pigs, resulting in great economic losses to the pig industry worldwide . AlthougAlphaherpesvirinae subfamily within the family Herpesvirida and possesses a double-stranded DNA genome approximately 145 kb in length, containing at least 72 genes and coding more than 100 proteins (gB), gC, and gD have been studied in depth and play key roles in virus entry, virulence, and immunity induction. In particular, gE is a major virulence factor of PRV and determines tropism for the central nervous system , according to the manufacturer's instructions. The results were determined by the formula S/N = OD560 test serum/OD650 negative control serum. S/N <0.6 was considered to be positive, 0.6 < S/N <0.7 was suspected, and S/N > 0.7 was negative.All serum samples were detected for gE gene primer (The mixed samples were homogenized in 1 ml of DMEM containing penicillin 100 U/mL) and streptomycin (100 μg/mL) and centrifuged at 3,000 rpm for 10 min. The supernatant was collected and used for DNA extraction according to the manufacturer's instructions for the TIANamp Genomic DNA Kit . Then, the extracted DNA was used for PRV detection through polymerase chain reaction (PCR) using the 0 U/mL an−1 to 10−6) of PRV. An uninfected control was included in the experiment. The plates were incubated at 37°C for 1 h, after which the supernatants were removed, and DMEM containing 1% low-melting agarose was added to each well, and the plates were placed in an incubator to allow plaque formation. Plaques of a suitable size were selected and inoculated into 1 mL DMEM, frozen, and thawed three times. The virus stock was inoculated into Vero cells for propagation, and titers were calculated as median tissue culture infective dose (TCID50) by the Reed–Muench method . In group II, all pigs were injected with equal sterile PBS in the same manner, as a negative control group.Ten healthy growing pigs (Duroc × Landrace × Yorkshire hybrid) were bought from a pig farm without PRV vaccination and outbreaks of field strains in Liaocheng city, Shandong Province. PRV, porcine reproduction and respiratory syndrome virus, classical swine fever virus, and porcine circovirus two were tested by PCR methods as described , and all50 method as described above. At 14 dpi, all pigs were euthanized with an overdose of pentobarbital by intracardial injection, and the gross lesions were observed. Meanwhile, the lymph glands, lungs, tonsils and brains were collected, and part of the tissues were fixed with 4% paraformaldehyde solution for histopathological examination. Other tissues were stored at −80°C for viral titers analyses using TCID50. During the experiment period, all pigs were housed in the isolators and had ad libitum access to feed and water.After the challenge, the pigs were observed continuously for 14 days and clinical symptoms, such as depression, anorexia, dyspnea, and ataxia were recorded. The rectal temperatures were measured. Oropharyngeal and rectal swabs of each pig were collected from 2 to 7 days post-inoculation (dpi) and were tested for viral shedding by the TCIDThe tissue samples  were fixed with 4% paraformaldehyde and embedded in paraffin. The sections of the fixed tissues were cut to 3 μm. Some sections were stained with hematoxylin and eosin (H&E) and others were used for immunohistochemistry staining. H&E staining sections were observed by light microscopy for histopathological analysis.The immunohistochemistry staining was performed as previous described , but thet-test was used to analyze the statistical difference by SPSS 19.0 . P < 0.05 was considered statistically significant.All data were expressed as means ± standard deviations (SD) and processed by GraphPad Prism 5.0 . Student's gE antibody had decreased from 38.20% in 2018 to 18.12% in 2020, displaying a declining trend. However, the positive rates varied greatly in the different pig herds, with the positive rate of gE antibody in sows being about 46.0–61.8% in the past 3 years, which was significantly higher than those from other herds .To analyze the pathogenicity of the variant PRV strain in pigs, animal experiments were carried out. The results showed that all the pigs in group I died within 1 week after the challenge, while the control pigs in group II survived and remained healthy throughout the experiment. Specifically, at 2 dpi, the PRV-infected pigs showed symptoms of shaking head and sneezing. At 3–4 dpi, the symptoms of depression, loss of appetite, frequent sneezing, and shaking were observed in all five infected pigs. The sick pigs showed dyspnea, one pig developed ataxia , and then died at 5 dpi. Two pigs each died at 6 dpi and 7 dpi, with symptoms similar to the dead pig at 5 dpi. The PRV-infected pigs developed fever at 2 dpi and lasted for consecutive 5 days, and their body temperature was significantly higher than that of the control group  were resistant to the infection of variant PRV , the Novel Agricultural Research Program of Jiangsu Province (SXGC[2017] 231), the Key R&D Program of Jiangsu Province (BE2020320), the funding from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Science and Technology Support Program of Jiangsu Province (BE2014355) as well as the earmarked fund for Jiangsu Agricultural Industry Technology System (JATS[2018]221).HR was employed by the company DSM Vitamin Co., Ltd. (Shandong). 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.