Virus-like particles (VLPs) are nanostructures assemble from viral proteins. Besides widely used for vaccine development, VLPs have also been explored as nanocarriers for cargo delivery as they combine the key advantages of viral and non-viral vectors. While it protects cargo molecules from degradation, the VLP has good cell penetrating property to mediate cargo passing the cell membrane and released into cells, making the VLP an ideal tool for intracellular delivery of biomolecules and drugs. Great progresses have been achieved and multiple challenges are still on the way for broad applications of VLP as delivery vectors. Here we summarize current advances and applications in VLP as a delivery vector. Progresses on delivery of different types of biomolecules as well as drugs by VLPs are introduced, and the strategies for cargo packaging are highlighted which is one of the key steps for VLP mediated intracellular delivery. Production and applications of VLPs are also briefly reviewed, with a discussion on future challenges in this rapidly developing field. The delivery of genes and their products (nucleic acids and proteins) into cells or tissues covers a broad spectrum of applications with respect to vaccines against viruses or cancers, gene therapy and diagnostic imaging ,2,3,4. DVLPs are natural or artificial nanostructures mimicking viruses but without enough viral genetic materials to support replication. VLPs can be naturally produced as defective virus and are observed in normal virus infection . The facThe three-dimensional structure and cell invasion property of VLPs also makes them an excellent candidate for intracellular delivery of biomolecules. Proteins, nucleic acids or chemical compounds may be carried by the VLP and delivered into different types of cells. The cargo molecules are protected by the nanocarrier from degradation or undesired binding in vivo, and genetic engineering of the VLPs can guide the carrier to specific cells or organs, achieving targeted delivery of cargo. Moreover, diverse cargo loading methods have been developed, some of which are able to release the cargo in a controlled way, enabling precise delivery of biomolecules. With the increasing advances in gene therapy, genome editing and precise drug administration, development and application of VLPs for intracellular delivery of diverse biomolecules and drugs have grown extensively . This reIn this review, we first describe the general progress on delivery of biomolecules and drugs by VLPs, then highlight the cargo loading strategies since loading is one of the key steps for molecule delivery. Next, frequently used VLPs derived from multiple virus species will be discussed more specifically on their properties, production and applications, and finally we provide a short future perspective for VLPs as delivery tools.At present, common methods for biomolecule or compound delivery include physical methods, chemical methods and virus-based delivery systems. Physical methods deliver biomolecules into cells either by physical force  ,10,11,12Viruses are natural vectors that deliver the viral protein and genome into permissive cells; therefore, viral vectors were initially developed for delivery of foreign genes. Lentivirus, adenovirus and adeno-associated virus vectors are most frequently used for gene therapy , but ineProteins participate in almost all biological activities and are begin explored as important therapeutics in recent years. It is difficult for proteins to enter the target cells directly because large molecular proteins are challenging to cross the cell membrane barrier and might elicit immune responses in vivo. Several delivery methods, such as cell-penetrating peptides (CPPs) , extraceVLPs developed from both enveloped viruses such as murine leukemia virus (MLV), avian sarcoma leukosis virus (ASLVs), paramyxovirus  and human immunodeficiency virus-1 (HIV-1) and non-enveloped viruses like bluetongue virus (BTV) have been used for the targeted delivery of proteins ,35,36,37There are, however, still several bottlenecks for VLPs to deliver proteins, such as the limitation of the amount of loading cargo, correct release of proteins inside the target cells, precise localization of cargo at the cytoplasm and cell nucleus, as well as engineered targeting specificity in vivo. Further improvements would greatly help the applicability of VLP for protein delivery.Nucleic acids are desired cargo for therapeutic applications. However, direct delivery of nucleic acids is limited by the physical and chemical features that they are negatively charged, typically with large linear structures, and poor stability for RNA molecules. Virions are naturally nanocarriers for viral genomic DNA or RNA, therefore, VLPs could be engineered conveniently for intracellular delivery of nucleic acids. Delivery of DNA molecules goes back two decades ago when non-replicative adenovirus, which indeed is a VLP, was tested together with replicative adenovirus vectors to deliver DNA encoding human p53 protein into tumor cells for therapeutic purposes . Since tAlthough therapeutic DNA delivery was developed first, VLPs are favored for RNA delivery in recent years as RNA in general has better biosafety than DNA, and different types of RNAs have been delivered with VLPs. mRNAs can be delivered into the cytoplasm directly and translated into functional proteins without the risk of genome integration. In addition, technologies for in vitro transcription (IVT) or chemical synthesis of mRNA are mature and cost-effective and suitable for large-scale mRNA production. However, the mRNA is easily degraded by RNases due to its intrinsic biochemical nature, thus efficient delivery of mRNA is rather challenging and improved delivery methods are required for broad applications of mRNA therapeutics. The VLP can protect mRNA from the environment and stabilize mRNA for better delivery efficiency, and VLPs derived from bacteriophage Qβ were shown to encapsulate mRNA fusing to the Qβ stem-loop RNA structures . In addiIn addition to the regular VLP derived from active virus, the Liu lab developed a new RNA delivery strategy which uses mouse endogenous retroviral gene products to assemble virus-like particles. The system can transfer mRNA of interest such as Cre mRNA and Cas9 mRNA into targeted cells . The resArtificially synthesized small interfering RNAs (siRNAs) or microRNA (miRNA) can destabilize targeting mRNA and exert gene silencing effects , thus arIn some cases, both mRNAs and noncoding RNAs need to be co-delivered via VLPs. To achieve Cas9-sgRNA mediated genome editing, Yadav et al. adopted the strategy of VLPs co-packaging Cas9 mRNA and sgRNA . Cas9 mRRibonucleoproteins (RNPs) are complexes formed by protein and RNA, which exist widely in cells and play many different roles. The nuclease active Cas9 in CRISPR-Cas systems is a RNP composed of the Cas9 protein and sgRNA, and how to effectively deliver the RNP for genome editing or targeting is one key issue for gene therapy of genetic diseases. Currently, retroviral vectors and bacteriophage-like particles have been modified for delivering Cas RNPs. The Cas RNPs could be packaged with direct fusion method and physical interaction method . More spWhile most chemical drugs, like small compounds, are permeable to cells and thus can diffuse into cells randomly, it is desired to concentrate the drug in its target cells more specifically so the drug efficacy can be enhanced and potential side effects can be reduced. For some drugs, such as cell-killing drugs for tumor therapy, precise administration of the drug is required to kill the tumor cells specifically. VLPs are one of the ideal choices for drug delivery and has many advantages. First, VLPs could be engineered for targeted intracellular delivery of the drug, accumulating active drugs within the desired tissues, thereby promoting treatment effects . Second,With these advantages, drugs for tumor therapy and antimicrobial infection have been delivered into cells with VLPs via different targeting strategies. In spite of the natural tropism of VLP, the function of cell targeting is typically achieved either by chemical modification on its surface to display ligand or antibody, or by genetic modification to insert peptides into the coat proteins . VLP froCargo loading is one of the most important steps for VLP-based delivery systems, and the approaches for loading cargo into VLPs include chemical, biological and physical strategies, which are summarized in The feature that many scaffold proteins for VLPs could maintain the capability of self-assembly when fused to foreign proteins or peptides enables direct fusion of protein cargo to the scaffold for cargo loading. Scaffold proteins of many viruses, such as MLV, HIV-1, and paramyxovirus have shown tolerance to cargo fusions. Protein cargo, depending on the conformation and structure of the scaffold-cargo fusion, could be caged inside of the particle or presented on the exterior surface of the particle, suitable for applications ranging from intracellular delivery of functional proteins or presentation of antigen proteins to immune cells. As each of the VLPs is assembled from multiple units of the scaffold protein, multiple number of cargo proteins could also be assembled into the same particle with the fusion strategy. Therefore, several types of proteins could be fused and delivered simultaneously, allowing for combinatorial delivery of several proteins and offering a powerful approach for combination and multivalent vaccine development. Chimeric VLPs which are generated by insertion of antigen peptides from other viruses or subtypes into VLPs have been widely used for new vaccine development ,84,85. INucleic acids are natural cargos of VLPs as infectious virions are formed by packaging of viral genomic DNA or RNA into the viral particles. Therefore, nucleic acids with certain sequences or structure properties could be recognized by the capsomere subunits and assembled into the VLP de novo. Mixing the subunit protein of some VLPs with certain nucleic acid molecules under certain conditions, the nucleic acid molecules could recruit capsomere proteins and facilitate VLP formation with encapsulated nucleic acids, shielding the desired nuclei acid molecules from the environment and allowing for intracellular delivery of the molecules .Proteins, nucleic acids or drugs could also be loaded into the pre-formed VLPs, and one of the common ways is loading by osmotic shock. Pre-assembled VLPs are placed in buffers with low ionic strength, and the space distance between the surface subunits of VLPs would increase to accommodate the entry of cargo molecules such as nucleic acid molecules . PhysicaThrough a direct electrostatic interaction with the positively charged coatings or complexes such as PEI and poly L-lysine, the negatively charged nucleic acids can be loaded with VLPs . Some vi2 putatively enhances electrostatic repulsions between the protein monomers and facilitates reassembly of VLP containing nucleic acids [Disassembly and re-assembly of the VLP in order to encapsulation different kinds of cargo can be achieved by chemical treatment with urea/NaCl or DTT/CaCl2 reagents . The priic acids ,98. Chemical linking is a classical approach for biomolecule coupling. On the one hand, cargo could be loaded by formation of covalent bonds between the VLP and cargo via chemical reactions. Multiple amino acid residues, such as lysine (amino group), cysteine (sulfhydryl group), aspartate and glutamate residues (carboxyl group) could be functionalized and form covalent bonds between the VLP and cargo via chemical reactions ,99. On tSeveral studies have found that the physical interaction force between capsids, protein and the others (CP−CP interaction) could drive the assembly of virions, and the packaging process of genome could also promote their assembly ,107,108.Due to the diversity of the VLPs, multiple platforms based on bacterial-, yeast-, insect-, plant- and mammalian-cell expression systems have been developed for production of VLPs. Some VLPs with simple structures could assemble spontaneously when expressed in the prokaryotic or eukaryotic systems, or even formed in a cell-free system. Therefore, purification of these VLPs could be achieved by simple ultracentrifugation. But for more complicated VLPs, such as many enveloped VLPs, mammalian cell or baculovirus-insect expression systems are required to offer the lipid membrane for VLP formation. For some VLPs, such as VLPs obtained by non-secreted systems like bacteria, yeast and plant cells need to go through processes like cell lysis and purification, then assemble under suitable conditions to allow for formation of VLP structures. More specific details on production of VLPs have been summarized by recent review papers ,111. AccThe stability of VLP is an important issue as the special nanostructure is the basis for successful loading and delivery of biomolecules. The stability of VLPs could be affected by temperature, pH, or ion concentration, for example , and it Notably, unlike the VLP for vaccine, which generally requires stable structure and good immunogenicity, ideal VLPs for other types of cargo delivery should have variable stability and low immunogenicity. On the one hand, the VLP should be stable enough to protect the cargo in vitro or in the body. On the other hand, once engulfed into the target cells, stability of the VLP could be a hurdle as the cargo needs to be released either naturally (such as endosome pH responsive) or in an artificially controlled way. Therefore, thermo-induced or near-infrared light responsive VLPs are designed, which could facilitate VLP disassembly and cargo release upon induction ,121. ForAs mentioned above, the VLP has a wide range of applications such as vaccination, diagnostic imaging, and delivery of biomolecules and compounds. Plenty of VLP-based vaccines are in preclinical or clinical phases at present, which was summarized by a recent review from Mohsen and Bachmann , but VLPThe first vaccine based on VLP was approved in 1986, and since then investigations of VLPs relative to vaccines have sprung up. With the advances of biological technologies in cutting-edge fields, VLPs have gradually become one of the most promising delivery vectors in biology and biomedicine areas, and multiple VLPs and various cargo loading methods have been developed for intracellular delivery of biomolecules and compounds.Although with fascinating progress, many concerns and limitations need to be stressed. Importantly, further investigations on the tropism, target-cell specific VLPs and controlled release of the cargo are required for precise delivery of molecules into proper tissues and cells, which could significantly enhance the applicability of the method and still is one of the major weak points. Generalized cargo loading strategies and VLP platforms which could carry different types of cargo without rebuilding the system should be established. These efforts could lower the requirement for thoroughly optimizing cargo/VLP production conditions, resulting in a more practicable technology. For example, a generalized mRNA delivery platform for antigen presenting would greatly help to develop new RNA vaccines in case of a new virus emerging. For RNA delivery and RNA vaccine development, VLPs stabilizing the cargo RNAs would be of special interest as the current lipid-based RNA vaccine requires strict conditions during transportation and distribution, which increases the cost and makes it impracticable for remote areas. As virus generated delivery tools, the immunogenicity of VLPs could not be neglected, undesired immune response or preexisting neutralizing antibodies could lead to failure of molecular delivery or gene therapy. Therefore, how to escape from the surveillance of the immune system and reduce immunogenicity to avoid adverse cellular responses would be one vital topic for future research. Endogenous virus-derived VLPs  or artif


Nowadays, with numerous sensors placed everywhere around us, we can obtain signals collected from a variety of environment-based sensors, including the ones placed on the ground, cased in the air or water, etc. These sensors can produce various signals, such as audio, image, video, infrared data, and so on. The obtained signals are usually processed using dedicated software, utilizing the output of these sensors. The construction of optimized sensors, the development of the theory that can he applied for signal representation and analysis, as well as the design of software for the processing of the obtained data represent broadly explored research topics. Therefore, recent advances in these areas are of interest for many researchers, and for the users of sensing techniques.The Special Issue entitled “Analytics and Applications of Audio and Image Sensing Techniques” is focused on original research involving the use of various audio and image sensing devices, both simultaneously and separately. It collected a diverse set of papers that span a wide range of analyses and possible applications.Digital signal processing;Audio signal analysis;Image analysis;Pattern recognition.Topics for the Special Issue included the following:Altogether, 17 papers have been included in this Special Issue.The papers presented in this Special Issue cover a broad range of topics, from the theory of signal analysis, to practical applications of audio and image sensing techniques. Audio signals discussed in these papers cover both speech and music, including measurements of airflow in organ pipes. Image and video data analyzed in the presented papers include microscope images and infrared images, as well as face images. All papers are briefly presented below.The paper  by A. StSix contributions in this Special Issue are dedicated to audio signal, including both music and speech.The paper  by M. BlP. Wrzeciono in his paper  presentsThe sound of organ pipes is also addressed in the paper  by D. WęP. Odya, J. Kotus, A. Kurowski, and B. Kostek in  analyzedIn the next paper , A. KuroK. Szklanny and J. Lachowicz in  describeFive papers in this Special Issue deal with image processing, including face images, microscopic images, and infrared images.A. E. Mahdaoui, A. Ouahabi, and M. S. Moulay in  propose M. Geremek and K. Szklanny in  investigIn , E. KubeThe next paper , writtenL. C. M. Dafico, E. Barreira, R. M. S. F. Almeida, and H. Carasek in  use infrFive papers in this Special Issue present techniques related to the processing and usage of video signal and audiovisual data.G. Canet Tarrés and M. Pardàs in  propose In paper , F. VossK. Hirayama, S. Chen, S. Saiki, and M. Nakamura in  describeThe study presented in  by J. QuK. Szklanny, M. Wichrowski, and A. Wieczorkowska in  present Sensors will find many of them interesting and inspiring.The papers collected in this Special Issue present a wide range of topics in audio, image, and video domains. I do hope the readership of Sensors, for their hard work in successful preparation of this Special Issue.I would like to thank all the authors who submitted papers to this Special Issue, the reviewers for their efforts in improving the papers, and the editors of


Microsoft Office Excel (2019) was used for descriptive statistical analysis. VOSviewer (1.6.18) was used to perform and visualize co-authorship analysis and co-occurrence analysis.On the 1The Lancet published the largest number of papers (n=12), followed by Gynecologic Oncology (n=11). Most of the papers were from the United States (n=58), followed by Italy (n=8) and Netherlands (n=7). Duke University, Johns Hopkins University, University of California San Francisco and University of Southern California  contributed the most papers . Nicoletta Colombo contributed the most papers (n=3) as the corresponding author. The co-occurrence keywords were classified into three clusters: cluster 1 (epidemiology study), cluster 2 (molecular biology study) and cluster 3 . Early research that was published prior to 2005 in this field was mainly focused on epidemiology and molecular biology; the mean publication year for keywords in cluster 3 was later than other clusters. The keywords “external-beam radiotherapy,” “uterine serous carcinoma,” and “intermediate-risk” showed relatively later mean publication year and lower mean frequency of occurrence.These 100 papers were cited a total of 45, 685 times, and the mean number of citations was 456.85 . Most papers were published between 1996 and 2000, and between 2006 and 2010. This study provides medical researchers with bibliometric information relating to endometrial carcinoma. Our results show that the United States is a clear leader in this field. The clinical treatment of endometrial carcinoma has received increasing levels of attention over recent years and is likely to remain a major area of research in the future. Meanwhile, it is recommended to pay attention to potential research hotspots, such as external-beam radiotherapy, uterine serous carcinoma and intermediate-risk. Endometrial carcinoma, an epithelial tumor of the endometrium, is one of the three main malignant tumors of the female reproductive tract. The global incidence of endometrial carcinoma is increasing annual and the age of onset is becoming younger . AccordiBibliometric analysis is defined as a statistical evaluation of published scientific papers, books, or book chapters –6. The ast of March 2022, papers regarding endometrial carcinoma published from 1971 to 2021 were retrieved through search of the Web of Science Core Collection database. (https://www.webofscience.com/wos/woscc/basic-search). The literature retrieval strategy was as follows: . The time frame for publications was set from 1971 to 2021 as papers published prior to 1971 may have been outdated. The search was limited to articles or reviews and excluded other types of papers such as editorials, meeting abstracts, letters and news reports. We also excluded papers in which endometrial carcinoma was only listed in a group of diseases and papers that focused on other topics. We only included papers that had been written in English. Two investigators (PX and CY) independently screened the title, abstract and whole text of papers to identify the top 100 most cited papers relating directly to endometrial carcinoma. Next, the title, corresponding author, journal, citation count, publication year, impact factor (IF), country, institution and research type of the top 100 most cited papers were extracted. The country and institution affiliated with each of the included articles were recorded based on the corresponding author. Microsoft Office Excel (2019) was used for descriptive statistical analysis and IFs were defined according to the Journal Citation Report (2020).On the 1VOSviewer (1.6.18) was used to perform and visualize co-authorship analysis and co-occurrence analysis of keywords . The visUsing our specific search terms, literature screening identified 14, 227 papers. The top 100 most cited papers are listed in The year of publication for the top 100 most cited papers were divided into 10 five-year intervals Figure 1With regards to research type, the top 100 most cited papers were divided into five groups: (1) observational studies (OS) including epidemiology, case-control and cohort studies; (2) basic science studies (BS) including pathology, experimental and animal studies; (3) randomized controlled trials (RCTs); (4) clinical guidelines, and (5) review articles including meta-analysis and systematic reviews. The respective number of papers in each of these five groups were as follows: OS (n=34), BS (n=31), RCTs (n=14), review articles (n=18) and clinical guidelines (n=3).The Lancet published the largest number of papers (n=12), followed by Gynecologic Oncology (n=11). Of the journals with more than one paper, The Lancet had the largest mean number of citations per paper (653.67 citations), followed by Obstetrics and Gynecology (577.00 citations) and The Journal of the National Cancer Institute (567.80 citations).The top 100 most cited papers were published in 33 different journals. We ranked the journals with more than one paper and their impact factors (2020/last five years) in descending order by the number of papers published Table 1.The corresponding authors of the top 100 most cited papers were from 13 different countries or regions Figure 3A total of 92 authors had been listed as the corresponding author. The corresponding authors with more than one paper are listed in VOSviewer identified a total of 487 keywords for the top 100 most cited papers. When limiting the minimum number of occurrences of the keywords to 3, sixty-six items were obtained. As shown in Finally, we constructed an overlay visualization map for the 66 co-occurrence keywords according to the mean publication year .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.


Drosophila neurons extends female lifespan but does not improve negative geotaxis senescence and has a detrimental effect on exploratory walking senescence in both sexes. We hypothesize that individual neuronal subtypes respond differently to IIS changes, thus the behavioral outcomes of pan-neuronal IIS reduction are the balance of positive, negative and neutral functional effects. In order to further understand how reduced IIS in neurons independently modulates lifespan and locomotor behavioral senescence we expressed a dominant negative Insulin receptor transgene selectively in individual neuronal subtypes and measured the effects on lifespan and two measures of locomotor senescence, negative geotaxis and exploratory walking. IIS reduction in cholinergic, GABAergic, dopaminergic, glutamatergic, and octopaminergic neurons was found to have either no affect or a detrimental effect on lifespan and locomotor senescence. However, reduction of IIS selectively in serotonergic neurons resulted in extension of lifespan in females with no effect on locomotor senescence. These data indicate that individual neuronal subtypes respond differently to IIS changes in the modulation of lifespan and locomotor senescence, and identify a specific role for the insulin receptor in serotonergic neurons in the modulation of lifespan.Reduced Insulin/IGF-like signaling (IIS) plays an evolutionarily conserved role in improving longevity and some measures of health-span in model organisms. Recent studies, however, have found a disconnection between lifespan extension and behavioral health-span. We have previously shown that reduction of IIS in  Alteration of IIS can have pleiotropic effects on growth, development, metabolic homoeostasis, reproduction and aging . There iDrosophila we have previously shown that the insulin receptor in neurons independently modulates lifespan and different types of locomotor senescence  outbred background, as previously described (DN is described in DN) was obtained from the Bloomington Drosophila Stock Centre . The UAS-InRDN transgene causes an amino acid substitution in the kinase domain (R1409A) of the Drosophila insulin receptor (dInR), resulting in its dominant negative activity. Targeting of the UAS-InRDN transgene was achieved using the following neuronal subtype-specific GAL4 lines: Th-GAL4 (dopaminergic neurons), Vglut-GAL4 (glutamatergic neurons), Chat-GAL4 (cholinergic neurons), Gad1-GAL4 (GABAergic neurons) trhGAL4 (serotonergic), and TdcGAL4 (octopaminergic). The following GAL4 lines were obtained from the Bloomington Drosophila Stock Centre: elavGAL4C155 (#458), TrhGAL4 (#38389), TdcGAL4 (#9313), ThGAL4 (#8848), VglutGAL4 (#26160), ChATGAL4 (#6798), and Gad1GAL4 (#51630). Stocks were maintained and experiments conducted at 25°C on a 12 h:12 h light:dark cycle at constant humidity using standard sugar/yeast medium   was carried out at 25°C in parallel with survival analysis of separate cohorts of flies generated and maintained under the same conditions.ntop), the mean number of flies at the bottom (nbottom) and the total number of flies assessed (ntot) were recorded. Performance index was calculated as 1/2(ntot + ntop −nbottom)/ntot, as described in Negative geotaxis of males and females was measured as described in Exploratory walking behavior of individual male or female flies was measured in 4 cm diameter/1 cm height circular Perspex arenas as described in p < 0.05. Data are presented as means of raw data ± SEM and * denotes significant difference from controls.Statistical analyses were performed using JMP (version 8) software (SAS Institute). Lifespan data were subjected to survival analysis (Log Rank tests) and presented as survival curves. Other data (QPCR and locomotor behavior) were tested for normality using the Shapiro-Wilk W test on studentized residuals  and apprDN transgene restricted to specific neuronal subtypes resulted in varying effects on lifespan  significantly reduced the lifespan of both male and female flies (p < 0.001) (DN (VglutGAL4/UAS-InRDN) resulted in a significant reduction in lifespan of approximately 10% in females (p = 0.0008 to VglutGAL4/ + and p < 0.0001 to InRDN/ +) and 25% in males (p < 0.0001) (DN/ + control group. Reduced IIS in cholinergic neurons (ChATGAL4/UAS-InRDN) had no significant effect on female lifespan (P < 0.0001)   by appro 0.0001) . Howeverlifespan  and resu 0.0001) . HoweverDrosophila resulting from pan-neural IIS reduction is due to the sum of varying effects of reduced IIS in different neuronal subtypes. Furthermore, the data show that reduced IIS specifically in serotonergic neurons is sufficient to extend female lifespan, identifying a specific role for these neurons in the extension of lifespan in response to reduced IIS.Together, these data suggest that the lifespan extension of female In contrast to its beneficial effect on female lifespan, reduced IIS in serotonergic neurons had no effect on the normal age-related declines (senescence) of negative geotaxis locomotor behavior  and all DN) was normal at 10 days old but declined sooner than controls, showing a significantly lower PI than controls at 20 days old  were unaffected, but ChAT-GAL4/UAS-InRDN males performed fewer changes in direction (rotations) at 10 days old compared to controls, and their rotation frequency then largely recovered and was not significantly different to controls at older ages  modulate walking speed under various conditions  or a detrimental effect on a specific function that limits lifespan. In addition, each of the lifespan and behavioral experiments in the different neuronal subtypes in this study were performed at different times because the size of the experiments precluded running all in parallel. Thus, there is some variability in median lifespan of the UAS-InRDN/ + control genotype between experiments. Such lifespan variation is common in Drosophila and the median lifespan of the wDah genetic background has been shown to vary from 32 to 75 days between experiments under defined control conditions  or cholinergic neuron-specific (ChATGAL4/UAS-InRDN) reduction in IIS. For instance, negative geotaxis senescence in flies with a pan-neural IIS reduction was normal  resulting in a stronger or weaker reduction in IIS and/or compensatory effects in elavGAL4/UAS-InRDN flies of reduced IIS in other neuronal subtypes . These findings further highlight the complexity of IIS in the nervous system in aging. They also highlight that using lifespan as an indicator of the effects of a treatment on organismal aging, although useful, does not provide information about the animal’s health with age. Moreover, under controlled laboratory conditions animals can have normal or extended lifespans despite compromised health or function. In this study, we used two locomotor assays as measures of locomotor health and brain function with age in addition to measuring effects on aging using lifespan. Interestingly, for all neuronal subtypes other than cholinergic there was little correlation between lifespan and locomotor behavior effects of reduced neuronal IIS. Locomotor behaviors were largely unaffected by reduced IIS in dopaminergic, glutamatergic, GABAergic and serotonergic neurons despite the variable lifespan effects of reduced IIS in these neuronal subtypes. For instance, long-lived trhGAL4/UAS-InRDN females (serotonergic) and short-lived ThGAL4/UAS-InRDN (dopaminergic) and VglutGAL4/UAS-InRDN (glutamatergic) flies, all showed a normal senescence of negative geotaxis and exploratory walking. IIS reduction in cholinergic neurons resulted in detrimental effects on locomotor behaviors and possibly shortened lifespan, the only instance of a similar effect on both lifespan and locomotor behavior. However, as mentioned above, although the short-lived ChATGAL4/UAS-InRDN (cholinergic) experimental flies showed an exacerbated senescence of locomotor behavior compared to both controls (DN/ + control flies. Taken together, the data show that the disconnection between IIS modulated lifespan and behavioral health-span suggested in The phenotypes of flies with reduced IIS in cholinergic neurons seen here raises the possibility that the detrimental behavioral effects of pan-neural IIS reduction  were dues normal  but redulifespan  whereas controls , the ChADrosophila. In particular, we have identified a specific role for IIS in serotonergic neurons in extending lifespan, and IIS in cholinergic neurons in having negative effects on locomotor behavior. It is still unclear as to whether or not neuronal aging is altered in response to changes in IIS in each neuronal subtype. The data in other neuronal subtypes show neutral or detrimental effects on locomotor behavior in long or normally lived flies, which suggests that the aging of these neurons is not slowed or delayed by reduced IIS. However, there are detrimental behavioral effects of reduced IIS in these neuronal subtypes. Further studies are needed to elucidate the role of IIS in neuronal aging, and to identify the physiological, molecular, cellular, and possibly developmental, mechanisms mediating the detrimental effect of reduced IIS on behavioral function and the beneficial effect of reduced IIS in serotonergic neurons on longevity.This study has shown that individual neuronal subtypes play specific and independent roles in the modulation of lifespan and locomotor behavioral senescence in response to altered IIS in The original contributions presented in this study are included in the article/SB conceived and designed the experiments. SB, ND, TS, ID, CB, LO, GV, TW, and TT performed the experiments. SB and ND analyzed the data. SB, ND, and AS wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Since 17th January 2022, the co-author GV has been employed by Frontiers Media SA. GV declared his/her affiliation with Frontiers, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The study is aimed at analyzing the predictive value of serum Ig A, Ig G, and TNF-α level by enzyme-linked immunosorbent assay. The data obtained in this study were analyzed by univariate analysis to choose the factors with difference in statistical significance to draw the ROC curve, and the areas under the curve (AUC) were recorded to analyze the potential mechanism of Ig A, Ig G, and TNF-α in predicting the recurrence of MM.  136 patients with MM treated in our hospital from January 2010 to January 2017 were followed up for 5 years. Finally, 100 patients who met the inclusion and exclusion criteria and had the complete follow-up visit were selected as the study subjects, with the recurrence of MM as endpoint event, and the observation was taken until the occurrence of endpoint event in patients or the termination of this study. They were divided into the recurrence group (RG) and the nonrecurrence group (NRG) according to whether the endpoint event occurred. The venous blood of patients was collected at the first diagnosis and subsequent visit (at the time of recurrence or termination of the study) to measure the Ig A and Ig G using a full automatic special protein analyzer and the TNF-P > 0.05). Compared with the NRG, the levels of soluble interleukin-2 receptor (sIL-2R) and β2-microglobulin (β2-MG) in the RG at the first diagnosis were distinctly higher (P < 0.001); the levels of Ig A, Ig G, and TNF-α in the RG at the first diagnosis were visibly higher (P < 0.05); and the levels of Ig A, Ig G, and TNF-α in the RG at the subsequent visit were clearly higher (P < 0.05). There was a correlation between Ig G, Ig A, and TNF-α and β2-MG at the first diagnosis and the subsequent visit (P < 0.05); there was a correlation between Ig G and TNF-α, and sIL-2R at the first diagnosis and the subsequent visit (P < 0.05); and there was a correlation between Ig A and sIL-2R at the subsequent visit (P < 0.05). The AUC of Ig G, Ig A, and TNF-α in predicting the MM at the first diagnosis were 0.772, 0.776, and 0.778, respectively.  After follow-up visit, there were 62 patients with recurrence (62.0%) and 38 patients without recurrence (38.0%), with no obvious difference in gender, age, body weight, and immune classification between the two groups (α had a predictive value in the recurrence of MM, and TNF-α was correlated with sIL-2R and β2-MG, with the highest AUC and the best predictive value. The serum Ig A, Ig G, and TNF- Ig A and Ig G are synthesized by plasma cells differentiated from B cells, and TNF-α can promote B cell differentiation, which plays an important role in osteolytic destruction in patients with MM. Based on this, 100 patients with MM in complete remission after treatment were followed up in this study to analyze the relationship between the levels of Ig A, Ig G, and TNF-α and the recurrence of MM to establish a good predictive mechanism in clinic.Multiple myeloma (MM), as a malignant proliferative disease of bone marrow plasma cells, belongs to the category of B cell lymphoma, characterized by abnormal proliferation of plasma cells with the overproduction of monoclonal immunoglobulin or light chain (M protein) , 2, withain type , 6. Therce of MM . At presce of MM , 9, so tα in the recurrence of MM  , and patβ2-MG, Ig A, Ig G, and TNF-α. The fasting venous blood of patients (3 ml) was taken in the morning at the first diagnosis to obtain the serum after centrifugation to determine the levels of serum sIL-2R and TNF-α by enzyme-linked immunosorbent assay , the β2-MG level by radioimmunoassay method , and the levels of Ig A and Ig G by a full automatic special protein analyzer  20182220158).100 patients with MM in complete remission after treatment who were treated in our hospital from January 2010 to January 2017 were selected to collect the data of social demography and clinical manifestation at the first diagnosis. The data of social demography included gender, age, and body weight of patients, and the data of clinical manifestation included immune classification, clinical stage, and the levels of serum sIL-2R, α, and then, the univariate analysis was performed on the levels of Ig A, Ig G, and TNF-α at the first diagnosis and subsequent visit. For the factors with difference in statistical significance, the occurrence of endpoint event was assigned to 1, and no occurrence of endpoint event was assigned to 0. The ROC curve was drawn by SPSS20.0 to record the areas under the curve (AUC) to analyze the value of Ig A, Ig G, and TNF-α in predicting the recurrence of MM.All patients were followed up for 5 years, mainly outpatient follow-up, supplemented by telephone follow-up, with the recurrence of MM as the endpoint event (recurrence referred to the recurrence after complete remission of primary treatment), and the observation was taken until the occurrence of endpoint event in patients or the termination of study. They were divided into the recurrence group (RG) and the non-recurrence group (NRG) according to whether the endpoint event occurred. The fasting venous blood of patients was taken again at the subsequent visit (at the time of recurrence or termination of the study) in the morning to determine the levels of Ig A, Ig G, and TNF-X2 test and t test. P < 0.05 indicated that the difference was statistically significant.In this study, the data processing software was SPSS20.0, and the GraphPad Prism 7  was used to draw the pictures. The items included in the study were enumeration data and measurement data tested by β2-MG at the first diagnosis, there was no significant difference in other baseline data between the two groups (P > 0.05), as shown in Except for the level values of sIL-2R and α in the RG at the first diagnosis were visibly higher , and the levels of Ig G, Ig A, and TNF-α at the subsequent visit in the RG were clearly higher . See the levels of Ig A, Ig G, and TNF-α in patients with different immune classifications at the first diagnosis and subsequent visit in Compared with the NRG, the levels of Ig G, Ig A, and TNF-α, and β2-MG at the first diagnosis and the subsequent visit (P < 0.05); there was a correlation between Ig G and TNF-α, and sIL-2R at the first diagnosis and the subsequent visit (P < 0.05); and there was a correlation between Ig A and sIL-2R at the subsequent visit (P < 0.05), as shown in There was a correlation between Ig G, Ig A, and TNF-α in predicting the MM at the first diagnosis were 0.772, 0.776, and 0.778, respectively. See variable assignment in The AUC of Ig G, Ig A, and TNF-β2-MG, Ig A, Ig G, and TNF-α in the RG at the first diagnosis were signally higher than those in the NRG (P < 0.001). β2-MG, as a classical marker for evaluating the clinical stage of patients with MM in the ISS, exists on the serous membrane of all karyocytes, and it involves in the surface identification of lymphocytes and the killing of cell receptors [β2-MG can reflect the proliferation rate of myeloma cells, some studies use it as a state variable to test the value of related indicators in evaluating prognosis, and indicators that are significantly related to β2-MG can be used to test the severity of patients with MM [α, and β2-MG at the first diagnosis (P < 0.05); that is, high levels of Ig G, Ig A, and TNF-α were independently correlated with high β2-MG level. The higher the β2-MG level at the first diagnosis, the higher the levels of Ig G, Ig A, and TNF-α, and the higher the possibility of recurrence after complete remission, with a consistency of the overall trend and the results of related factors analysis. Ig G, as a classical indicator to reflect the B cell status, is commonly used in the studies related to immune [α and sIL-2R at the first diagnosis and the subsequent visit (P < 0.05), with no correlation between Ig A and sIL-2R at the first diagnosis. sIL-2R, as an immunosuppressive factor that reflects the tumor burden, can compete with the membrane to bind interleukin-2 (IL-2), hindering the important biological response regulated by IL-2, making the cellular immune dysfunction of body, and eventually leading to the cells with malignant clone escaping from immune surveillance to have an excessive proliferation. In this study, sIL-2R is also a state variable, which has been confirmed to be associated with the recurrence of MM. Therefore, the determination of Ig G, Ig A, and TNF-α at a specific time can be used to infer the level changes of β2-MG and sIL-2R, thus evaluating the prognosis of patients.B cells are derived from pluripotent stem cells of bone marrow, which can be differentiated into plasma cells under antigen stimulation, and plasma cells can synthesize and secrete the antibody (immunoglobulin), affecting the immunoglobulin levels in patients , so thateceptors , 18. Bec with MM , 20. R ao immune , 22. Liuo immune . Ig A acα in predicting the MM at the first diagnosis were 0.772, 0.776, and 0.778, respectively, suggesting that the predictive value of TNF-α is higher than that of Ig G and Ig A, and the relationship between TNF-α and B cells has been confirmed in the literature. This marker can promote the proliferation of B cells and lymphocytes and accelerate the rate of B cells differentiation into plasma cells [α level in patients with MM is signally higher than that in normal control group, and with the increase of clinical stages, the TNF-α level shows a progressive tendency in ladder type, suggesting that TNF-α is related to tumor load in patients with MM, and this marker reflects the biological parameters of tumor load [α, and MM can be treated in clinic by inhibiting TNF-α to act on related signaling pathways. The above results showed that TNF-α was an important serum marker in the diagnosis, treatment, and prognosis of MM and had a high clinical reference value.ROC analysis further showed that the AUC of Ig G, Ig A, and TNF-ma cells . Moreovemor load . Yuhua hIt is worth noting that this study only included patients with MM who had complete remission after initial treatment. Affected by practical factors, only 100 patients received the complete follow-up visit, with small study samples. Subsequent studies need to further increase the sample size to support the above conclusions, and enrich the system of immune factors and inflammatory factors related to B cells to predict the MM.α between recurrent and non-recurrent patients, and the three indicators were correlated with the classical index (β2-MG) of MM, with the AUC ≥ 0.7. Therefore, the serum Ig A, Ig G, and TNF-α have a predictive value in the recurrence of MM, and TNF-α has the highest AUC and the best predictive value. In practice, the three indicators could be combined to evaluate the prognosis of patients comprehensively.In recent years, the application of new targeted drugs like proteasome inhibitor and immunomodulator has prolonged the complete remission rate of patients with MM, but patients are still unable to cure, and most patients still relapse after systematic treatment, even ushering in a death ending. Since the recurrence mechanism of MM is not clear, it is important to establish an effective prediction mechanism of recurrence. In this study, 100 patients with MM were followed up, founding that there was an obvious difference in the levels of serum Ig A, Ig G, and TNF-


Hansenula polymorpha expression system developed by our laboratory, two kinds of recombinant HPV B19 VLPs were expressed, i.e., the VLP co-assembled by VP1 and VP2 (VP1/VP2 VLP) and the VLP whose VP1 content was improved (VP1h/VP2 VLP). The expression, purity, and morphology of these two VLPs were characterized, and then their immunogenic properties were investigated and compared with those of the VLP containing VP2 alone (VP2 VLP) previously developed by our group. Furthermore, the location of the VP1 unique region in the VLPs was determined by using the immunogold electron microscopy (IGEM). Our experimental results show that the VP1h/VP2 VLP elicits a stronger neutralization against the HPV B19 than VP2 and VP1/VP2 VLPs, which implies that the increase of VP1 content significantly improves the level of neutralizing antibodies. In addition, the IGEM observations suggest that the unique region of VP1 may be located inside the recombinant VLP. The VLPs recombinantly expressed by our Hansenula polymorpha system may serve as a promising candidate immunogen for HPV B19 vaccine development.Human parvovirus B19 (HPV B19) is pathogenic to human, which can cause fifth disease, transient aplastic crisis, arthritis, myocarditis, autoimmune disorders, hydrops fetalis, and so on. Currently, no approved vaccines or antiviral drugs are available against HPV B19, and thus the development of effective vaccines is needed. The capsid of HPV B19 is composed of two types of proteins, i.e., the major capsid protein VP2 and the minor protein VP1. Previous experimental studies have shown that the dominant immune responses against HPV B19 are elicited by VP1, especially the unique region on the N-terminus of VP1. It has been found that VP2 alone or VP2 and VP1 together can assemble into virus-like particle (VLP). The VLP structure formed by VP2 has been resolved, however, the location of VP1 in the capsid, especially the location of VP1 unique region with strong immunogenicity, is still not clear. In the present work, using the  Human parvovirus B19 (HPV B19) was first discovered in the sera of blood-donors and patients by Cossart and his colleagues in 1975 . The subErythrovirus genus of Parvoviridae family [HPV B19 is a nonenveloped, single-stranded linear DNA virus, as a member of e family . HPV B19e family . Saccharomyces cerevisiae, and a new promoter was constructed to regulate the expression ratio of VP1 to VP2 [HPV B19 cannot yet be cultured in vitro, and the production of recombinant virus-like particles (VLPs) is a promising strategy for the development of effective vaccines against the virus. Several experimental studies have shown that either VP2 alone or VP2 and VP1 together can form VLPs, but VP1 alone cannot self-assemble into VLPs. Although VP2 is necessary for the formation of VLPs, VP1 plays critical roles in eliciting the neutralizing immune responses against HPV B19 and many neutralizing antigenic epitopes have been found to be located on VP1u ,15,16. K1 to VP2 . Suzuki 1 to VP2 . The three-dimensional structure of HPV B19 VLP formed by VP2 alone has been determined by X-ray crystallography with the resolution of 3.5 Å . HoweverHansenula polymorpha expression system developed by our laboratory, VP1 and VP2 were co-expressed to produce the two-component HPV B19 VLP. Furthermore, a stronger expression promoter was adopted to improve the relative proportion of VP1 in the VLP. In our study, two types of VLPs were produced, i.e., the VLP co-assembled by VP1 and VP2 (VP1/VP2 VLP) and the VLP with an improved VP1 content (VP1h/VP2 VLP). The expression, purity, and morphology of these two VLPs were characterized, and then their immunogenicity was evaluated and compared with that of the VLP formed by VP2 alone (VP2 VLP). In addition, the location of the immunodominant VP1u in the recombinant VLPs was determined by using the immunogold electron microscopy (IGEM).In the present work, using the Hansenula polymorpha  developed by our laboratory in which the key gene elements of the synthesis of uracil and leucine were deleted by UV irradiation and genetic engineering methods [In the present study, two recombinant HPV B19 VLPs were produced, i.e., VP1/VP2 and VP1h/VP2 VLPs. For the expression of these two-component VLPs, the plasmids for VP2 and VP1 were constructed, respectively. In the VP2 plasmid construction, the gene codons of HPV B19 VP2, which was truncated from the VP1 gene (Genbank accession No. AY386330.1), were codon-optimized and then cloned into the “HP-MU” shuttle plasmid constructed by our laboratory. This plasmid contains a URA3 selectable marker, a MOX promoter and terminator, as well as a 18S rDNA homologous recombination arm responsible for the integration of the target gene into the yeast genome , as show methods  to co-exHansenula polymorpha to produce VP1h/VP2 VLP.Furthermore, in order to improve the expression level of VP1 to generate the VP1h/VP2 VLP, the DAS promoter and terminator in the VP1 plasmid were replaced by a stronger MOX promoter and terminator to construct the VP1h plasmid called “HP-ML” in which the VP1 gene was inserted between the MOX promoter and terminator C. Then, Hansenula polymorph yeast strains with high-level expressions were selected to produce the recombinant VLPs by high-density fermentation. In our study, to select the Hansenula polymorph strains with high-level protein expressions, the plasmid vector encoding VP2 was firstly linearized and transformed by electroporation into the Hansenula polymorpha, and the strains with high expression of VP2 were screened. Then, the plasmid encoding VP1 or VP1h was also transformed into the cells to realize the co-expression of VP1 and VP2, and the highly expressing strain was finally selected to produce the two-component VLPs. During the strain screening process, the VP2 protein expression level was evaluated by enzyme linked immunosorbent assay (ELISA) using the VP2-binding antibody MAB8293 (purchased from Millipore Co.) as the detection antibody, and the VP1 expression level was determined using the VP1u-specific polyclonal antibodies (PcAbs) obtained by using VP1u to immunize rabbits. The strain with the highest optical density (OD) value at 450/630 nm in the ELISA assay was selected as the high-expression strain.The Hansenula polymorpha, the highly expressing strain was inoculated in 20 mL MD medium . After incubation at 37 °C and 230 rpm for 24 h, it was transferred to 100 mL MD medium and incubated at 37 °C and 230 rpm for an additional 24 h. After amplification, 100 mL of the Hansenula polymorpha solution was added into the bioreactor (Bioengineering AG KLF2000-3) containing yeast culture medium. The temperature was set to 37 °C, and the stirring speed gradually increased to 800 rpm with 100% aeration. After incubation for approximately 24 h and the rise of the dissolved oxygen to more than 100%, the feed pump was opened for glucose de-repression so that the dissolved oxygen in the reactor was maintained at approximately 60%. After glucose de-repression for 24 h, methanol was added to trigger the expression of the target protein, and the yeast solution was collected after continuous induction culture for 72 h.For the high-density fermentation of the Hansenula polymorpha cells was crushed by high pressure cracker in the buffer solution , and the supernatant was separated by centrifugation. Then, the supernatant sample was roughly purified by two-aqueous phase extraction, followed by the purification using the gel column chromatography in the buffer solution . The collected protein sample was further purified using the anionic-exchange chromatography in the buffer solution . Subsequently, the purified VLPs were obtained by elution with the buffer solution .The recombinant VLPs were purified using the gel column chromatography  followed by the anionic-exchange chromatography . Specifically, during the purification of the recombinant VLPs, the The purified recombinant HPV B19 VLPs were analyzed by SDS-PAGE and Western blot to verify the expression of VP1 and VP2 proteins. In Western blot experiments, a monoclonal antibody (mAb) , which can bind to both VP1 and VP2, was used to detect the presence of VP1 and VP2, and the polyclonal antibodies (PcAbs) specific to VP1u (produced by our laboratory) were also applied to detect the presence of VP1. The purity of the VLPs was measured by using the size exclusion chromatography combined with the high-performance liquid chromatography (SEC-HPLC) method. The relative contents of VP1 and VP2 in the recombinant VLPs were determined by SDS-PAGE combined with the gray scanning software ImageQuant TL. The integrity, homogeneity and morphology of the recombinant VLPs were examined by transmission electron microscopy (TEM). In TEM, the VLP samples were dropped onto the carbon-coated copper grids. After the absorption for 5 min, the samples were negatively stained with 2% phosphotungstic acid solution and subsequently observed by TEM (FEI Tecnai 12).The immunogold electron microscopy (IGEM) was utilized to determine whether the VP1u is located inside or outside of the capsid in the VP1/VP2 and VP1h/VP2 VLPs. The VP1u-specific PcAbs were used to detect the exposed VP1u in the VLP samples. If VP1u is located in the interior of the capsid, the PcAbs cannot bind to the VLP, whereas if VP1u is exposed on the surface of the capsid, the PcAbs can attach to the VLP and can then be detected by IGEM. In the experiment, the VLPs were firstly absorbed on the sample carrier of the TEM and blocked with 1% bovine serum albumin (BSA) for 10 min, which then floated on the droplets of VP1u-specific PcAbs. After incubation at room temperature for 2 h, the carrier was washed three times with 1% BSA/Phosphate Buffered Saline (PBS) for 10 min. Subsequently, the carrier floated on the droplets of the secondary antibody labeled by colloidal gold and incubated at room temperature for 1 h. After washing three times with 1% BSA/PBS for 10 min, the carrier was placed on the droplets of 0.5% glutaraldehyde for 10 min. Then, the carrier was washed three times by ultrapure water and negatively stained with phosphotungstic acid. After dyeing, the carrier was dried and observed by TEM (FEI Tecnai 12). The expressed HPV B19 VP1/VP2 and VP1h/VP2 VLPs, along with the VP2 VLP produced previously by our group , were re2PO4, Na2HPO4·12H2O, NaCl, KCl, Tween-20) buffer. The serum samples from the immunized mice were firstly diluted to 1:200, which is followed by 5-fold serial dilutions. The diluted sera were added into the well with 100 μL per well and incubated at 37 °C for 1 h. After washing the plates three times with PBST, the secondary antibody, i.e., HRP-labeled goat anti-mouse IgG (purchased from ZSGB-Bio), at the dilution of 1:16,000 was added into the well with 100 μL per well, which continued to be incubated at 37 °C for 1 h. Subsequently, the plate was washed three times with PBST, and 50 µL tetramethylbenzidine and 50 µL hydrogen peroxide solutions were added to the well. After color development for 5 min, the reaction stop solution (0.2 M sulfuric acid) was added into the well with 50 μL per well, and the value of optical density at 450/630 nm (OD450/630nm) was obtained from the plate reader. The OD450/630nm value measured for the well without the addition of serum was taken as the blank control, and the cutoff value of OD450/630nm was set to be 2.5 times of the blank value. The titer of IgG antibodies was determined as the reciprocal of the maximum dilution of the serum whose OD450/630nm is equal to or greater than the cutoff value.The titer of IgG antibodies in the sera of the immunized mice were evaluated by enzyme linked immunosorbent assay (ELISA) using the VP2 VLP to coat the plate. The purified VP2 VLP produced in our previous study  were dilDue to the fact that HPV B19 cannot be cultured in vitro, the serum neutralizing antibodies cannot be directly evaluated by microneutralization assay. The P-antigen (globoside) on the membrane of human red blood cells is the receptor for HPV B19 virus, and HPV B19 VLPs can specifically bind to the receptor, which causes the agglutination of the red blood cells (RBCs) ,28,29,30g under 2–8 °C for 10 min. The supernatant was abandoned and the RBCs were collected. The RBCs were then washed three times with PBS-BSA buffer , 0.1% BSA(w/v)). The washed RBCs were diluted by PBS-BSA buffer to produce the RBCs suspension with the concentration of 50%, which was then stored at 2–8 °C for the following experiments. The VP1/VP2 VLP sample with a starting concentration of 100 μg/mL was diluted by 2-fold serial dilutions and added into the well of the U-type 96-well plate. Another well was added by PBS-BSA buffer as a control. Subsequently, 1% RBCs suspension produced above was added into the wells to observe the hemagglutination of the RBCs. Base on this RBCs hemagglutination experiment, the optimal VLP concentration causing the hemagglutination of the RBCs was determined to be 12.5 μg/mL, which was then used in the following HI assays.Before the HI assay, we first detected the hemagglutination of RBCs induced by the HPV B19 VLPs in which the recombinant VP1/VP2 VLP was used. In this experiment, 4 mL fresh human blood was mixed with 4 mL Alsever’s solution, which was subsequently centrifuged at 1000In the HI assay, the serum samples from the immunized mice were diluted by 2-fold serial dilutions starting with 1:10 and the diluted sera were added into the wells of the plate. Then, 25 μL VP1/VP2 VLPs with the concentration of 12.5 μg/mL were added into the wells and incubated with the sera at room temperature for 1 h. Subsequently, 1% RBC solution was added into the wells and incubated at 4 °C for 2 h. Finally, the inhibition of the hemagglutination of the RBCs was observed, and the titer of HI antibodies was evaluated as the reciprocal of the maximum dilution of the sera that completely inhibits RBCs hemagglutination. Hansenula polymorpha  developed by our laboratory. The expressed VLP was purified by the gel column chromatography and the anionic-exchange chromatography. Then, the SDS-PAGE and Western blot experiments were carried out to detect the expression of the component proteins of the recombinant VP1/VP2 VLP. The experiment results are shown in In this study, the recombinant B19 VLP co-assembled by VP1 and VP2 (VP1/VP2 VLP) was expressed by the URA3/LEU2 auxotrophic It has been revealed that VP1, especially VP1u, plays important roles in mediating the attachment of HPV B19 to the host cells, and many immunodominant neutralizing epitopes have been identified to be located at VP1u ,15,16. SThe amount of VP1 in the two recombinant VLPs, i.e., VP1/VP2 and VP1h/VP2 VLPs, was determined, respectively, by SDS-PAGE combined with the gray scanning software ImageQuant TL. In the VP1/VP2 VLP, the proportion of VP1 was measured to be approximately 5.11%, where the value is consistent with that of the natural HPV B19 virus . WhereasThe purity of these two recombinant VLPs, i.e., VP1/VP2 and VP1h/VP2 VLPs, was analyzed by the SEC-HPLC method. The experimental results are shown in Hansenula polymorpha expression system in our previous study [The integrity, homogeneity and morphology of the recombinant VLPs were observed by negative staining TEM, which were compared with those of the VP2 VLP produced by using the same us study . The TEMThe immunogenicity of the recombinant VP1/VP2 and VP1h/VP2 VLPs was evaluated and compared with that of the VP2 VLP produced previously by our laboratory using the same expression system. For each recombinant VLP, 6 BALB/c female mice were immunized and the serum was collected. Then, the titer of IgG antibodies in the sera was measured by using ELISA, and furthermore the titer of neutralizing antibodies was evaluated by using HI assay. The ELISA results show that both the VP1/VP2 and VP1h/VP2 VLPs elicit a high level of IgG antibodies, as displayed in Then, the levels of neutralizing antibodies in the sera from the mice immunized with different VLPs were evaluated by using HI assays, and the detection results are displayed in Hansenula polymorpha. The above experimental results demonstrate that VP1u plays an important role in the immune activity of the recombinant HPV B19 VLPs. However, the location of VP1u in the capsid structure is still not clear, and two seemingly conflicting views have been proposed. For the recombinant VLPs expressed by the insect cell, VP1u was detected to be exposed on the surface of the capsid ,22, wherHansenula polymorpha expression platform developed by our laboratory in which two plasmids respectively encoding VP1 and VP2 were constructed to co-express the target proteins. The relative amount of VP1 to VP2 was regulated by changing the protomers, and the recombinant yeast strains were screened to optimize the expression level with a stable VP1:VP2 ratio. Our expression platform has the advantage of low cost, which is practical for large scale manufacturing. The study results show that the VLPs produced by Hansenula polymorpha are efficiently expressed, highly purified, morphologically homogenous, and good immunogenic, which may serve as promising antigen candidates for developing VLP-based vaccines. As appropriate culture systems are still not available for HPV B19 infection, the recombinant VLPs are considered promising candidates for developing vaccines against the virus. Our experimental results and several previous studies have demonstrated that the containing of VP1 component in the VLP and improving its amount to a certain level are critical for eliciting a high neutralizing antibody response ,19,20,21Hansenula polymorpha. Immunogenicity evaluation showed that the containing of ~5% VP1 in the VLP significantly improved the IgG antibody response but did not distinctly enhance the neutralizing antibody level. Then, the relative amount of VP1 in the VLP was improved to ~25% by using a stronger protomer . It was found that the VLP with this higher content of VP1 exhibited significantly improved neutralizing antibody response. These results are consistent with other studies [HPV B19 natural virions are composed of approximately 5% VP1 and 95% VP2. We produced the two-component VLP with the similar composition to natural virions  using  studies ,19. Our Hansenula polymorpha, it is not known whether VP1u could be exposed from the capsid by changing expression, purification or environment conditions, which will be investigated in the follow-up studies.It has been revealed that VP1u plays a crucial role in eliciting immune responses against HPV B19. However, the location of VP1u in the capsid is not clear. Two seemingly conflicting results have been obtained by immunological experiments. In the recombinant VLPs expressed by insect cells, VP1u was found to be exposed on the capsid surface ,22, wherHansenula polymorpha expression system developed by our laboratory, the VP1 and VP2 were co-expressed to produce the two-component HPV B19 VLPs. Furthermore, the promoter for the expression of VP1 was optimized to improve the amount of VP1 in the VLPs. The recombinant VLPs produced by Hansenula polymorpha exhibit high concentrations, morphological homogeneity, and good immunogenic effects. It is also found that the containing of VP1 and increasing its content are important for the recombinant VLPs to elicit high-level neutralizing antibodies against HPV B19. Moreover, IGEM observations indicate that the VP1u with predominant neutralizing activity may be located inside the capsid, similar to that in the capsid of a natural virus. In addition, our expression system has the property of low cost. Our studies provide a potential candidate and an efficient expression system for the development of anti-HPV B19 vaccines.HPV B19 is a highly infectious virus that can result in serious consequences for the fetus and the immunocompromised persons. At present, no approved vaccine or drug is available against the virus and thus the development of effective vaccines is needed. HPV B19 cannot yet be cultured in vitro, and the recombinant VLPs are promising candidates for vaccine design. In the present work, by using the


Metabolic reprogramming is a hallmark of cancer, alteration of nucleotide metabolism of hepatocellular carcinoma (HCC) is not well-understood. MYBL2 regulates cell cycle progression and hepatocarcinogenesis, its role in metabolic regulation remains elusive.Copy number, mRNA and protein level of MYBL2 and IMPDH1 were analyzed in HCC, and correlated with patient survival. Chromatin Immunoprecipitation sequencing (Chip-seq) and Chromatin Immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) were used to explore the relationship between MYBL2 and IMPDH1. Metabolomics were used to analyze how MYBL2 affected purine metabolism. The regulating effect of MYBL2 in HCC was further validated in vivo using xenograft models.The Results showed that copy-number alterations of MYBL2 occur in about 10% of human HCC. Expression of MYBL2, IMPDH1, or combination of both were significantly upregulated and associated with poor prognosis in HCC. Correlation, ChIP-seq and ChIP-qPCR analysis revealed that MYBL2 activates transcription of IMPDH1, while knock-out of MYBL2 retarded IMPDH1 expression and inhibited proliferation of HCC cells. Metabolomic analysis post knocking-out of MYBL2 demonstrated that it was essential in de novo purine synthesis, especially guanine nucleotides. In vivo analysis using xenograft tumors also revealed MYBL2 regulated purine synthesis by regulating IMPDH1, and thus, influencing tumor progression.MYBL2 is a key regulator of purine synthesis and promotes HCC progression by transcriptionally activating IMPDH1, it could be a potential candidate for targeted therapy for HCC.The online version contains supplementary material available at 10.1186/s12885-022-10354-4. Liver cancer is the fifth common malignancy and the second leading cause of mortality worldwide. Hepatocellular carcinoma (HCC) accounts for around 90% of liver cancer, the rest are cholangiocarcinoma (CCA) or mixed forms . HCC hasRecently, metabolic reprogramming, especially nucleotide metabolism, is considered an initiating factor of tumorigenesis and tumor progression , 6. PuriOncogenic transformed cancer cells are characterized by altered metabolism, including heightened nutrients uptake, enhanced glycolysis, glutaminolysis and fatty acid synthesis , 20. TheWe systematically investigated genetic alterations, mRNA and protein expression of MYBL2 in HCC human tumors, and explored possible downstream pathways controlled by MYBL2 using bioinformatic tools. Furthermore, using a combination of metabolomics and genomic analysis, we demonstrate that MYBL2 promotes de novo purine anabolism by up-regulating IMPDH1 at transcriptional level, facilitating tumor growth in vivo. These data provided a potential mechanism of HCC growth and progression from the perspective of nucleotide metabolism.2.HepG2 cell line was purchased from ATCC. Cells cultured in DMEM supplemented with 10% fetal bovine serum , 1% penicillin and streptomycin at 37 °C under 5% COTotal protein from cultured cells was extracted in RIPA buffer, and quantified with BCA assay. Protein samples were separated by 4–20% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies against β-actin , MYBL2 , IMPDH1  and c-Myc , subsequently with corresponding HRP-conjugated secondary antibodies . The immunoreactive bands were visualized by chemiluminescence assay.The lentiviral CRISPR MYC or MYBL2 knockout vectors were constructed by cloning of MYC or MYBL2 guide RNA (gRNA) sequences into the site of BsmBI of lentiCRISPRv2 vector as described . LentiCRhttp://www.metaboanalyst.ca).Cells were incubated with fresh media for 2 h before collection. Cells were washed with ice-cold saline, lysed with 80% methanol in water and quickly scraped into an Eppendorf tube followed by three freeze–thaw cycles in liquid nitrogen at the time of collection. The supernatant was obtained and transferred to a new tube after insoluble material was pelleted in a 4 °C centrifuge, then evaporated to dryness using a SpeedVac concentrator (Thermo Savant). In order to move debris, metabolites were reconstituted in 100 µl of 0.03% formic acid in LC-MS-grade water, vortex-mixed and centrifuged. LC-MS/MS was performed by using an AB QTRAP 5500 liquid chromatography/triple quadrupole mass spectrometer (Applied Biosystems SCIEX) as described previously with an injection volume of 20 µL . MultiQuTotal RNA was extracted by Trizol agent  in accordance with the manufacturer’s instructions. Total RNA samples were reversely transcribed by 5 × All-In-One RT MasterMix (abmGood). GAPDH was used as internal control for quantification of target genes. Real-time PCR was performed with UltraSYBR Mixture  on Bio-Rad IQ5. Primers for IMPDH1  and GAPDH  were used.6 live cells/mouse in a final volume of 100 µl. Subcutaneous tumor size was measured twice a week with calipers until any tumor reached 2 cm in its largest diameter, calculated by 0.5 x short diameter2 x long diameter.The establishment of HCC xenograft model was described previously . All aniParaffin-embedded tissue sections were deparaffinized in xylene and rehydrated in graded alcohol after fixing with formalin. 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity for 10 min. Antigen retrieval was carried out by autoclave sterilization in sodium citrate buffer for 3 min. Slides were incubated with 10% normal goat serum solution for 20 min so as to reduce background non-specific staining. The rabbit polyclonal antibody against IMPDH1, MYBL2 was applied at a concentration of 1:100 and incubated at 4 °C overnight. HRP-conjugated secondary antibody was carried out according to the manufacturer’s instructions. The slides were subsequently incubated with DAB to visualize expression of IMPDH1, MYBL2 and followed by hematoxylin counterstaining. Image capture was performed by using a RGB JVC solid-state camera connected to an Olympus BH2 microscope at 10- and 20-fold objective magnification fitted with a motorized stage.Immunohistochemical analysis was performed in accordance with standard procedures in a blinded fashion by two pathologists independently. The expressions level was accessed semi-quantitatively based on proportion and staining intensity of positive stained cells. Proportion was scored using semiquantitative criterion: 0 (no staining); 1, minimal (< 10%); 2, moderate (10–50%); and 3, diffuse (> 50%) staining cells. Staining intensity was also classified as 0 (negative); +1 (weak); +2 (moderate); and + 3 (strong). Taken two scores, composed of both proportion and staining intensity together, were added to give each case final expression scores as (0), negative; 1+ (1 or 2), weakly positive; 2+ (3 or 4), moderately positive; 3+ (5 or 6), strongly positive.p < 0.05 was considered significant. Analysis of the relationship between MYBL2 mRNA expression and pathological stages in Fig. Data were shown as means ± S.D. Statistical analysis was performed using a software package . Analysis of the clinic pathological features and data were carried out with Person’s Chi-Square and Likelihood Ratio tests. A level of Recent studies reported genetic alterations in MYBL2 gene locus, and contributes to tumorigenesis in colorectal, prostate and breast cancer. To understand the role of MYBL2 in HCC, we looked into the liver cancer cohort of the TCGA database. While genetic mutations are rare in MYBL2 gene locus, we found copy number gain in about 10% of HCC tumors, which led to increased mRNA expression  analysis using tissue microarray. Protein abundance were measured by IHC score ranging 1 + to 3+, as shown by representative images of different staining intensity (Fig. Following investigation of expression profile of MYBL2 in HCC, we subsequently explored functional cues of this potentially important gene. We checked the relationship of MYBL2 expression with clinical and phenotypic parameters, while no correlation with clinical stages and pathological features were seen, MYBL2 aligned well with molecular subtypes of HCC based on unsupervised clustering of transcriptomic data (Fig. MYBL2 is a known transcription factor playing pluripotent roles in cell proliferation, cell cycle regulation and cell differentiation. We were interested in the function of MYBL2 in purine metabolism, which is not reported before. Based on our analysis, the metabolic gene most positively correlated with MYBL2 was IMPDH1, in contrast, the functional redundant isoform IMPDH2 was not that positively correlated (Fig. Upregulation of IMPDH is reported in different tumor types. In liver cancer cohort of TCGA database, overall survival analysis showed high mRNA abundance of IMPHD1 represented worse clinical outcome Fig. A. Taken To understand the specific function of MYBL2 in HCC, we generated functional knockout of MYBL2 by CRISPR/Cas9 system in HepG2 cells, and implanted the MYBL2 knockout and parental cells in the flanks of NSG mice for 3 weeks, tumor growth were retarded after loss of MYBL2 Fig. A, B. NexIMPDH1 is an enzyme that catalyzes the synthesis of XMP from IMP, the rate-limiting step in the de novo synthesis of guanine nucleotides. Detailed relationship between MYBL2 and IMPDH1 is not revealed yet, Chip-seq analysis from published dataset  showed MHCC remains a challenging disease worldwide due to poor prognosis and lack of treatments . OncogenCancer is characterized by reprogramming of cellular metabolism under both direct and indirect regulation of oncogenic mutations . NucleotIMPDH catalyzes the oxidative reaction of IMP to XMP, a rate-limiting step in guanine nucleotide biosynthesis and hence IMPDH is an essential enzyme that maintain the stability of cellular pool of guanine nucleotides. Given this, IMPDH represents an important therapeutic target that has attracted much attention due to its pluripotent functions such as the immune response  or cell Our results were in line with limited evidence that IMPDH1 expression represent worse clinical outcomes, while combining MYBL2 and IMPDH1 together, prognosis gap is larger, indicting they were important in HCC progression, and there is a synergy between the two. Correlation analysis showed MYBL2 was positively associated with IMPDH1. Chip-seq showed MYBL2 is a transcription factor for IMPDH1, so it might regulate IMPDH1 at transcriptional level, which linked MYBL2 to purine anabolism through key enzyme control. RT-PCR validated suppressed MYBL2 impaired IMPDH1 transcriptional level, in line with the prediction.While CRISPR/Cas9 is a new tool for gene editing, its application in metabolomics made metabolic reprogramming alteration clearer, especially critical molecular in regulation of cancer metabolism. In this study, MYBL2 is knocked out by the same method, alteration of metabolic profiles revealed MYBL2 positively correlated with GMP, while in process of GMP generation, and IMPDH1 is essential and a rate-limiting enzyme. This also illustrated the regulation of MYBL2 on IMPDH1 in HCC cells. GMP is the precursor of GDP and GTP, which is an energy mole particularly important for protein synthesis in rapidly dividing cells, such as tumor cells , 48. So,IMPDH1 is an enzyme catalyzes a crucial step in de novo purine synthesis, especially for GMP. Knocking out of MYBL2 in HCC cells by CRISPR/Cas9, which made a dramatic alteration in nucleotides metabolic profiles in HCC, especially in purine anabolism as described in our study. Tumor bearing models by HCC cells also exhibited suppression in purine anabolism with MYBL2 knocking out. Together, these results illustrated guanine synthesis is controlled by MYBL2 in both vivo and vitro, thus influenced proliferation in HCC cells.Here in our study, we first demonstrated IMPDH1 expression is regulated by transcription factor MYBL2, and thus linking oncogenic transformation and purine catabolism together. Of note, this is the first study illustrated MYBL2 regulated purine anabolism and contributed to HCC tumorigenesis. The study also provides evidence that MYBL2 is a promising target for cancer treatments and proposed a possible mechanism of HCC progression.Additional file 1.Additional file 2.Additional file 3.Additional file 4.Additional file 5.


Varicocele is one of the main causes of infertility in men, thus representing an important clinical problem worldwide. Inflammation contributes mainly to its pathogenesis, even if the exact pathophysiological mechanisms that correlate varicocele and infertility are still unknown. In addition, oxidative stress, apoptosis, hypoxia, and scrotal hyperthermia seem to play important roles. So far, the treatment of varicocele and the care of the fertility-associated problems still represent an area of interest for researchers, although many advances have occurred over the past few years. Recent experimental animal studies, as well as the current epidemiological evidence in humans, demonstrated that many functional foods of natural origin and nutraceuticals that are particularly abundant in the Mediterranean diet showed anti-inflammatory effects in varicocele. The aim of the present narrative review is to mainly evaluate recent experimental animal studies regarding the molecular mechanisms of varicocele and the state of the art about possible therapeutic approaches. As the current literature demonstrates convincing associations between diet, food components and fertility, the rational intake of nutraceuticals, which are particularly abundant in foods typical of plant-based eating patterns, may be a reliable therapeutic supportive care against varicocele and, consequently, could be very useful in the cure of fertility-associated problems in patients. Varicocele is a pathological venous dilation of an internal testicular vein and of the pampiniform plexus, which is located in the spermatic cord, inducing blood reflux . It is cMoreover, the left internal spermatic vein is about 8–10 cm longer than the right one. This, in addition to the anatomical arrangement of the blood vessel, causes an increase of the hydrostatic pressure on the left side, and consequently, the dilation of the pampiniform plexus (hydrostatic pressure theory) . Among tEven if varicocele is usually asymptomatic , the patConsequently, the damage of testicular function is linked to physio-pathological events, including testicular hypoxia and hyperthermia, inflammation, oxidative stress, hormonal imbalance and cellular death ,11,12,13A correct diagnosis of varicocele is particularly important to avoid a worsening of the pathology and to be able to combine traditional surgery with treatment with natural substances capable of modulating the pathogenetic mechanisms involved in the disease, with particular regard to the inflammatory cascade .We consulted the following databases: MEDLINE-PubMed and Google Scholar. The keywords “varicocele and pathophysiology”, “varicocele and epidemiology”, “varicocele and animal models”, “varicocele and inflammation”, “varicocele and inflammatory nutraceuticals”, and “varicocele and diet” were selected for data extraction, also including the main references of the chosen articles. In the first search, we found 370 articles, which were selected by reading their titles and abstracts, when present. If the articles were considered relevant for the review, the full text was read. We included reviews and original papers from 1981 to 2022, but only 71 were chosen on the basis of their topic. We excluded some papers dealing with clinical features on varicocele, as our review was based mainly on animal models. Other papers were excluded, as they examined only different features of the pathophysiology of varicocele, such as oxidative stress and apoptosis, or because the considered molecules showed effects different from anti-inflammatory, which was the main topic of our review.Even if varicocele is almost exclusively observed in humans, owing to the upright posture, animal models have been suggested, all based on the incomplete occlusion of the left renal vein after the insert of the internal spermatic vein. In particular, in rats, the first surgical approach was proposed by Saypol et al. . Since tAfter induction of anesthesia, through an abdominal midline incision, the left renal vein, the inferior vena cava, and the left spermatic vein are identified, and a clamp is passed behind the left renal vein just distal to the spermatic vein insertion. A silk ligature is loosely placed around the left renal vein at this site and a rigid probe is placed on the left renal vein; then, the ligature is tied around the vein over the top of the probe. The latter is withdrawn, and the vein is allowed to expand to the limits of the ligature . Therefore, renal and spermatic veins dilate immediately .Besides the classical method of varicocele induction, another microsurgical model was proposed in rats . In addiVaricocele triggers inflammation through infiltration and activation of macrophages and lymphocytes, which leads to higher quantities of inflammatory cytokines . Among tAmong the other cytokines involved in the testicular tissue during varicocele, IL-6 and tumor necrosis factor-α (TNF-α) play an important pro-inflammatory role. As for the IL-6, Leydig and Sertoli cells and testicular macrophages are considered at the site of secretion . Its oveTNF-α is another cytokine implicated at physiological levels in diverse functional activities in testes, such as androgen receptor expression in Sertoli cells and lactate providing to spermatid and spermatozoa through Sertoli cells . HoweverIn addition, the increased production of TNF-α, together with augmented Transforming Growth Factor (TGF)-β, a pleiotropic cytokine with strong regulatory and inflammatory activity, impairs the normal organization of the blood-testis barrier (BTB) . In fact2, thus further amplifying inflammatory reactions. In testis, COX2 is constitutively expressed in germinal and in Sertoli and Leydig cells [Another mediator involved in varicocele-induced inflammation can be considered cyclooxygenases (COX), the enzymes which convert polyunsaturated fatty acids and arachidonic acid to prostaglandins (PGs). In particular, COX2 induces the production of PGEig cells . Furtherig cells .During inflammation, an activation of inflammasomes was also observed. These multiprotein complexes are formed by different members of nucleotide oligomerization domain (NOD)-like receptor family pyrin domain  and absent in melanoma (AIM), but in varicocele pathogenesis, the NLRP3 inflammasome was shown to play an important role. Once activated, it splits procaspase-1 to caspase-1, which converts the pro-interleukin-1β and 18 to their active forms. The consequent increase of these pro-inflammatory cytokines in the testis could cause damages in testicular function, spermatogenesis and androgen production ,28.In fact, varicoceles are also characterized by a decrease of serum testosterone levels, which is related to a down-regulation of the Steroidogenic acute regulatory protein (StAR) protein, whose role in promoting the transport of cholesterol into the mitochondria for testosterone synthesis was demonstrated . The incThe care of fertility-associated problems in varicoceles is strongly concentrated over the last few years, when the role of inflammation was demonstrated, owing to the increased levels of cytokines correlated with lower sperm motility, augmented sperm necrosis, and altered spermatogenesis .Consequently, the dietary pattern and the components of the diet and nutrients have been studied, particularly from a molecular point of view, as possible bases of sperm function and/or fertility . The “WeBy contrast, MD provides a low level of saturated and trans fatty acids with adequate levels of nutrients, such as omega-3 fatty acids, antioxidant molecules, and vitamins. Moreover, higher intakes of fruit, cereals, and vegetables, as well as the consumption of extra virgin olive oil, were positively related to sperm motility and concentration ,47,48. TSurprisingly, the role of a VD in the preservation of sperm quality is controversial. As a matter of fact, it was indicated that this dietary pattern reduced sperm concentration and spermatozoa motility, probably because the occurrence of environmental estrogenic compounds in the VD, which could have a negative effect on sperm parameters .Overall, the differential impacts of WD, MD, and/or VD on male fertility depend on the amount and quality of the nutrients introduced . TherefoThus far, as suggested in the accurate review by Ferramosca and coworkers , diets rstudies proposed to evaluate the possible role of different substances, including natural functional foods and nutraceuticals, on the different mechanisms implicated in the pathogenesis of the varicocele. However, an accurate analysis of the literature showed that only a few papers took into account the role of natural functional foods and nutraceuticals to counteract inflammation in varicocele testes  seeds, was evaluated. Its biological effects are exerted on vascular endothelial cells, improving venous contraction and increasing blood return, and on the protection of collagen fibers of vein walls and prevention of varicosities. Another important role of aescin is to decrease the activation and migration of leucocytes, thereby reducing inflammation. In experimental varicoceles, lower interstitial edema and reduced polymorphonuclear leucocytes density were observed when aescin was administered [Among these studies, the role of aescin, an extract from nistered .Morinda officinalis How plant (MOP), used as a traditional Chinese medicine, were evaluated in experimental varicoceles in adolescent rats [The effects of the polysaccharide of the ent rats . MOP resOther substances whose role in the regulation of structural and functional damages induced by varicoceles were examined are resveratrol and N-Palmitoylethanolamide (PEA). Resveratrol (Res)  is present in some plants, such as mulberries, peanuts, and grapes, that demonstrate a well-known anti-inflammatory activity. In experimental varicoceles, Res significantly down-regulated the gene expression of NLRP3, a component of the inflammasome complex, modulating the inflammatory pattern .A similar mechanism was demonstrated in PEA, an endogenous compound of living organisms synthesized from precursor phospholipid and found in the seeds of some legumes, in some vegetables, and in milk. In murine varicocele, PEA reduces NLRP3 expression; in addition, its anti-inflammatory mechanism in varicoceles can be explained by the concomitant down-regulation of pErk 1/2 and TGF- β3, thus improving the functional and structural features .Silybum marianum; it contains a combination of flavonolignanes, of which silibinin is the principal component. Mazhari et al. [The effects of silymarin (SMN) in a rat model of varicoceles were also considered. SMN is an extract from the ripe seeds of i et al.  showed tCuscuta chinensis Lamark, which is able to improve male reproductive function [Allium cepa L. (Liliaceae), recovered spermatogenesis in experimental varicoceles . A treaCoptis chinensis and Hydrastis canadensis [Another natural substance with an anti-inflammatory role in experimental varicoceles was shown to be berberine (BBR), an isoquinoline alkaloid found in nadensis . Treatmenadensis .Gryllus bimarculatus, has shown pharmacological activity, owing to the presence of an anti-inflammatory glycosaminoglycan [Gui-A-Gra, a commercial powder that has recently been obtained from the edible insect noglycan . In expenoglycan .Furthermore, the effects of an herbal combination in experimental varicoceles following varicocelectomy were examined, and a reduction of IL-6, IL-1β, and TNF-α was demonstrated .Selenium (Se), an essential element, plays several favorable roles in the human body, among which, an anti-inflammatory effect was proposed , as it iAdditionally, many other favorable compounds have been studied in varicoceles. Among them, vitamin C ,63, vitaRecent experimental animal studies and current clinical trials suggest the positive effects of dietary habits on fertility-associated problems in varicoceles. Dietary habits could be related to the presence of functional foods and/or specifical substances with nutraceutical activity that positively impact a series of molecular pathways directly or indirectly linked to exaggerated inflammatory response and deeply demodulated in varicoceles.Probably, in our opinion, a better experimental knowledge of the mechanisms of actions involved could help to better define a healthy diet and/or specific functional foods and/or clinically useful nutraceuticals, in addition to surgery, for the therapeutic management of fertility-associated problems in varicoceles. Therefore, future research appears fascinating but also complex. In our humble opinion, the development of an appropriate meta-analysis could be useful to improve our knowledge about the intriguing crosstalk between fertility and foods in varicocele patients.


The historical scaling down of electronics devices is no longer the main goal of the International Roadmap for Devices and Systems , but theNanotechnology for Electronic Materials and Devices”, is to collect dedicated papers in several nanotechnological fields. The issue consists of eleven selected regular papers focusing on the latest developments in nanomaterials and nanotechnologies for electronic devices and sensors. Thus, topics such as approaches to synthesis, advanced characterization methods and device fabrication techniques have been covered in the present issue.In this context, the aim of this Special Issue, entitled “(i)Nanoscaled materials and their properties: several papers in this issue focus on nanolaminated oxide combinations (such as hafnium oxide/iron oxide  or tungs(ii)Moreover, innovative synthesis approaches have been described in some papers for nanomaterials and thin films, such as aluminium nitrides , silicon(iii)Other paper focus on advanced nanoscale characterization, mainly based on scanning-probe methods (scanning non linear dielectric microscopy  and high(iv)Finally, some papers are dedicated to device performances  and circWe editors are aware that, due to the many topics related to the use of nanotechnology for electronics, the present issue cannot provide a comprehensive presentation of the arguments; however, we are confident that the main general areas have been discussed and can be summarized in the following research topics:Nanomaterials readership will find interesting imputs in the wider scenario of electronical applications of nanotechnology.We editors are grateful to all the authors for submitting their scientific results to the present Special Issue and we hope that