Virus-like particles (VLPs) are virus-derived structures made up of one or more different molecules with the ability to self-assemble, mimicking the form and size of a virus particle but lacking the genetic material so they are not capable of infecting the host cell. Expression and self-assembly of the viral structural proteins can take place in various living or cell-free expression systems after which the viral structures can be assembled and reconstructed. VLPs are gaining in popularity in the field of preventive medicine and to date, a wide range of VLP-based candidate vaccines have been developed for immunization against various infectious agents, the latest of which is the vaccine against SARS-CoV-2, the efficacy of which is being evaluated. VLPs are highly immunogenic and are able to elicit both the antibody- and cell-mediated immune responses by pathways different from those elicited by conventional inactivated viral vaccines. However, there are still many challenges to this surface display system that need to be addressed in the future. VLPs that are classified as subunit vaccines are subdivided into enveloped and non- enveloped subtypes both of which are discussed in this review article. VLPs have also recently received attention for their successful applications in targeted drug delivery and for use in gene therapy. The development of more effective and targeted forms of VLP by modification of the surface of the particles in such a way that they can be introduced into specific cells or tissues or increase their half-life in the host is likely to expand their use in the future. Recent advances in the production and fabrication of VLPs including the exploration of different types of expression systems for their development, as well as their applications as vaccines in the prevention of infectious diseases and cancers resulting from their interaction with, and mechanism of activation of, the humoral and cellular immune systems are discussed in this review.  Viral-like particles (VLPs) are nanoscale structures made up of assembled viral proteins that lack viral genetic material and are therefore non-infectious . VLPs arStructural proteins from viruses, such as human immunodeficiency virus (HIV), adeno-associated virus, Hepatitis B virus (HBV), Hepatitis C virus (HCV) and bacteriophages have been used to produce VLPs –11. ThesA largely exploited application of VLPs is their potential in vaccinology where they can offer several advantages over conventional vaccine approaches –20. BecaVLPs are formed by spontaneous interaction between one or more viral structural capsid proteins to form the final structure. VLPs are structurally and visually similar to live viruses but lack either a complete virus genome or lack the entire virus genome. The variety of structures adopted by different VLPs makes them structurally and functionally attractive. Spontaneous polymerization of different viral capsid proteins can produce VLPs with geometrical symmetry, usually in the form of icosahedral, spherical, or rod-like structures, depending on which virus from which they were derived. VLPs can generally be divided into different groups based on their structural complexity. Capsid proteins can be arranged in one, two or three layers. Other single layer VLPs can contain more than one structural protein. While single-protein VLPs have a relatively simple structure, multi-protein VLPs contain unique structural components such as presence of several distinct capsid layers Fig. a. Other Non-enveloped VLPs are further classified as single or multi-capsid protein VLPs and also as single-layer, double-layer, and triple layer VLPs. The simplest available non-enveloped model of VLP consists of single capsid VLP structure like human papillomavirus (HPV) VLP vaccines. These simple VLPs are composed of a single capsid protein that can be expressed in both eukaryotic and prokaryotic systems. In some cases of the simple VLPs, capsid proteins can also be formed in a cell-free system. In these cases to form soluble homogeneous VLP capsomer the proteins can be expressed first in a cell-based expression system and then assembled in a cell-free environment for proper folding –37. In cEnveloped VLPs are also sub-divided into single-layer, double-layer and multi-layer internal structures that lie below the lipid envelope. Enveloped VLPs obtain their lipid membrane from the cell in which they are expressed during the assembly and budding of VLPs from the cell. One or more glycoprotein anchors can be inserted into the lipid membrane, and these glycoproteins are commonly the main target antigens detected by the immune system for the production of neutralizing antibodies. The precise nature, origin and composition of the envelope are different for various enveloped VLPs and the precise detail of their nature depends on the virus from which the VLP is derived which in turn determines the process of assembling and budding of the VLP from the host cell line that is used for their production.Formation of fully infectious virus particles requires the presence of all of the necessary components including the viral genome, internal structural proteins, and, if present, the glycoproteins, but the minimum requirements for eVLP formation are not well understood . TypicalThe generic manufacturing process for a VLP-based vaccine generally consists of three main sections: (A) upstream processing (production), (B) downstream processing (purification), and (C) formulation. The first step in VLP production is to clone the viral structural genes of interest. Next, viral structural proteins with self-assembling ability are expressed in prokaryotic  or eukaryotic  expression systems. After harvesting and lysing the cells, to ensure removal of contaminating cell debris and aggregates a clarification step is performed , 60. To baculovirus/insect cell (B/IC) system [Various expression platforms including prokaryotic, and eukaryotic systems can be used for producing VLP vaccines , 63. Euk) system , mammali) system  and plan) system . In addi) system . The qua) system . Each exBacteria are one of the most widely used expression systems for the production of recombinant proteins and are also used to produce many VLPs. However, due to various factors such as lack of PTM system, incomplete disulfide bond formation and protein solubility problems, they are not suitable platforms for producing enveloped VLPs . HoweverEscherichia coli is the most common bacterial host cell for VLP production [E. coli expression system has many advantages including low production cost, rapid cell growth, high protein expression level, and simplicity of scaling-up. The E. coli expression system is commonly suggested for producing of small proteins with limited PTM [E. coli expression systems have entered clinical trials for use against infectious and non-infectious diseases. Hecolin, a Hepatitis E vaccine manufactured by Xiamen in the form of a p239 VLP-based vaccine, was the first Hepatitis E virus (HEV) approved vaccine using an E. coli expression system [E. coli expression platform to produce a bivalent vaccine against HPV 16/18 L1 VLPs has also been shown to be safe and immunogenic [Plasmodium falciparum, has been successfully produced in E. coli [E. coli expression system for self-assembly has shown complete protection in mice against influenza [E. coli expression system against various infectious such as West Nile virus (WNV), foot-and-mouth disease (FMS) virus, and HCV have also entered preclinical trials [E. coli the successful formation of VLP has been observed in some other bacterial species. Self-assembly of HPV-16 L1 protein VLPs was successfully carried out in Lactobacillus casei using a lactose-inducible promoter system for L1 protein expression [Pseudomonas fluorescens [oduction . An E. cited PTM . Variousn system . An E. cunogenic , 70. Mal E. coli –73. In anfluenza . Many otl trials . In addipression . The coworescens .E. coli expression platform [Furthermore, several chimeric VLPs vaccines against non-infectious diseases including hypertension, allergies, diabetes, cancer, and Alzheimer's have been sufficiently developed by antigen conjugation with bacteriophage Qβ RNA in platform .Saccharomyces cerevisiae and Pichia pastoris, are most favored due to advantages such as rapid cell growth, high yield of expression proteins, scalability, cost-effective production, and providing a degree of PTM processes [Pichia pastoris has been reported [Yeast cells are frequently used for recombinant proteins expression and has also been used for VLPs production . Yeast erocesses , 77. Tworocesses , 79. Recreported . Despitereported .Spodoptera frugiperda (Sf9/Sf21) and Trichoplusia ni (Tn5) [Baculovirus/insect cell platform has also been used for obtaining prophylactic vaccine candidates against several infectious diseases such as HIV 1, influenza virus A, Chikungunya virus, severe acute respiratory syndrome (SARS), Ebola virus, dengue fever virus, Rift Valley fever virus (RVFV), Norwalk virus and HCV [The B/IC expression system is the most commonly used expression system for production of both enveloped- and non-enveloped-VLPs . Due to ni (Tn5) . Cervari and HCV , 82. The and HCV . However and HCV . Therefo and HCV ..Plants expression systems have several advantages over conventional approaches, including high expression levels of up to 80% total soluble protein, low refining costs, and high-performance expression processing. Using MagnICON and CPMV-HT technology, plant-based vaccine has become a versatile and promising platform bringing the cost of protein production to less than $50 per gram for the production of several proteins and antibodies used in the human and veterinary pharmaceutical industry which –87. The Detection of 34 different plant viruses from the human gastrointestinal tract and monitoring of some plant viruses in human feces showed that plant viruses such as CPMV and pepper mild mottle virus (PMMoV) are structurally very stable in intestinal conditions . AntigenPlant systems have also been used to generate VLPs based on animal and human viruses. Induction of Norwalk virus (NV)-specific intestinal mucosal antibody requires administration of an oral vaccine. Non-enveloped Norwalk virus capsid (NVCP) VLP has been successfully expressed in tomatoes, potatoes, tobacco and lettuce with a structure similar to native NV particles replicated in the human gastrointestinal tract. However, these systems display slow and very low expression and accumulation of NVCP VLP which has been a major hurdle to their use. To overcome these limitations, new developments in an expression platform based on the BeYDV and MagnICON replication systems have led to an increase of more than 80-fold in the accumulation of NVCP VLPs in transgenic tobacco and tomato .Nicotina bentamiana [More recently the use of plant-based systems to express heteromultimeric protein complexes which resemble influenza VLPs has been explored with positive indications that good yields can be achieved from plants in a matter of days.The cowpea mosaic virus-Hyper Translatable (CPMV-HT) platform, which is based on the rapid transient expression of recombinant proteins in plants through Agrobacterium-mediated infiltration, has been used to produce 10 M dose of VLP H1N1 (swine flu) vaccine in just 30 days compared to the 9–12 months required for more common approaches in current use , 86. In ntamiana . These VPlasmodium falciparum parasite conjugated to the AIMV has been generated. This plant-based VLP, Pfs25 VLP-FhCMB, was produced by transient expression in N. bentamiana and its safety and immunogenicity in phase I clinical study was successfully evaluated [There are several licensed Yeast and Chinese hamster ovary (CHO) cells-derived VLP-based vaccine against hepatitis B virus surface antigens (HBsAg) . In addivaluated . Medicagvaluated .Animal cell expression systems remain valuable and attractive platforms which can be used for producing multiple structural proteins of non-enveloped and enveloped VLPs , 77. AniIntracellular assembly of HBsAg VLPs using the CHO cell line is an example of a very successful production system and both dengue virus VLP and Hantavirus VLP have also been generated in CHO cells , 105. ThCell-free protein synthesis (CFPS) systems provide an additional option for the in vitro expression express the recombinant proteins for VLPs production . These sDownstream processing for VLP purification is a crucial step to ensure suitable efficacy and safety for clinical use . After cTo reduce the steps and costs of the purification process, a clarification step is performed to remove whole cell debris and aggregates from primary VLP preparations. Capturing and concentration of VLPs is a critical step that significantly reduces the bulk volume and increases the ratio of VLP concentration to other cellular impurities. A variety of methods is used for clarification, including cell sedimentation, depth filtration and microfiltration tangential flow filtration (TFF). For separation of the VLPs from host cell contamination like cell debris, digested DNA or components of media, ultrafiltration/diafiltration (UF/DF) and TFF with membranes or hollow fibers methods are commonly used. Affinity chromatography, ion-exchange chromatography, and hydrophobic interaction chromatography may also be used for capturing VLPs in bind-and-elute approach , 111. OtDisassembly and reassembly is an optional step which is performed to increase stability, homogeneity and immunogenicity of VLPs product. Titration or cross-flow filtration is a chosen option for this step . In the l-histidine, sodium borate/phosphate and 2-phenoxyethanol are often used in the formulation of VLP-based vaccines as a surfactant stabilizer, buffering agent and preservative, respectively [Vaccine formulation focuses on improving the stability, efficacy, and safety of the vaccine during storage and shipping until the point of administration. For optimizing the efficacy of VLPs adjuvants and authorized excipients are added in most vaccine formulations , 112. InMany VLPs have molecular and structural properties that can spontaneously stimulate the immune system, thus eliminating the need to use adjuvants. Nevertheless, using adjuvants in VLPs vaccine formulations may increase the immunogenicity of the vaccine and stimulate a specific type of immune response . SeveralPRR agonist adjuvants such as AS04, AS01/AS02, Poly I:C/Poly ICLC , CpG oligonucleotide (ODN), flagellin, Toll like receptors 7 (TLR7), and TLR7/8 agonists, which are compounds derived from pathogen-associated molecular patterns, are the amongst the most commonly used adjuvants for VLP formulation , 112. ThChitosan is a mucosal adjuvant that can effectively deliver the vaccine to local phagocyte cells and thus has the ability to stimulate strong induction of both systemic and mucosal immune responses . IntranaE. coli as an adjuvant with a rotavirus VLP vaccine efficiently enhanced specific immune responses [One of the important cytokines involved in immounoregulation which is produced primarily by antigen-presenting cells is IL-12 . In an aesponses , 118.Biochemical, biophysical and biological characterization of purified VLPs is an essential component of producing VLP-based vaccines to be able to determine the functionality, potency and stability of the product , 112. ExBiochemical characterization of VLPs involves a determination of properties including primary amino acid sequence, molecular mass, isoelectric point, and purity. Mass spectrometry (MS) can be used to analyze the molecular mass of the assembled proteins, their protein sequences and their amino acid composition . The molBiophysical parameters of VLPs such as morphology, size and polydispersity contribute to the potency and safety of VLPs vaccines. The morphology of VLPs can be detected by visualization techniques such as transmission electron microscopy (TEM), cryo-electron microscopy (Cryo-EM) and atomic force microscopy (AFM) , 112. TEThe biological characterization of VLPs are often conducted by analyzing the binding of functional epitopes of VLPs to a panel of specific monoclonal antibodies. This binding activity is a preferred indicator of the safety and efficacy of the vaccine in vivo. Various immunoassay methods such as surface plasmon resonance (SRP), and enzyme-linked immunosorbent assay (ELISA), have been used to analyze antibody binding affinity to VLPs , 131.To date 110 viral proteins from 35 viral families have been shown to be capable of assembly into the VLPs . From thHepadnaviridae, is the major causative agent of hepatitis B. HBV infection can lead to acute and chronic hepatitis and significantly increase complications and mortality [HBV, a virus in the family ortality . Epidemiortality . Three gortality . The thiortality , 134.4 adjuvant and the TLR4 MPL agonist which directly stimulates APCs [Persistent HPV infection is the major cause of the cervical cancer and genital warts . There ates APCs , 140. DuRecently, a recombinant HPV type 16/18 vaccine (Solulin) was developed using the Escherichia coli expression system. This system has shown acceptable safety and performance in phase 3 clinical trials . It has E. coli expression system, vaccine production costs is greatly reduced and it can therefore be useful for vaccination in developing countries. In addition to Hecolin, two other HEV vaccines based on amino acids 112–607 and amino acid s439-617, p179 of pORF2 have been entered into clinical trials [Hecolin, the first VLP-based vaccine for HEV launched and produced in China, has shown significant benefits in preventing HEV infection . HEV caul trials .The B/IC expression system has been using to generate influenza VLPs. Sf9 cells were infected using three different baculoviruses each encoding one of the genes of the influenza hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins. The HA and NA glycoproteins are the major antigens of the virus. Simultaneous expression of these three proteins leads to the formation of a VLP, which can be harvested from the culture supernatant. These VLPs produce a broader immune response compared to an inactivated virus or recombinant hemagglutinin protein alone. Transgenic plant technology has also been using to produce influenza VLPs that have shown promising results in the preclinical stages , 146.S. cerevisiae have now reached the clinical trials [HIV VLPs have been generated using various expression platforms. HIV VLPs composed of p17 and p24 structural proteins and produced by l trials , 147. Sel trials .Human parvovirus has two main structural proteins, VP1 and VP2. Human parvovirus B19 (HPVB19) VLPs composed of VP1 and VP2 proteins have reached clinical trials. These have been produced in the B/IC system in which sf9 cells are infected with two baculoviruses, leading to the production and self-assembly of immunogenic VLPs .Norovirus (NV) encodes a large protein that is broken into structural proteins VP1 and VP2 and regulatory NS1/2 to NS7 proteins. The NV VLP form of the NV VLP-based vaccine used in clinical trials is composed of the VP1 capsid protein that is expressed in the B/IC system (Sf-9) has been promisingly evaluated in clinical trials. VP1-based NV VLPs have also been produced using transgenic plants and these have also undergone early clinical trials .Lassa fever virus (LASV) is a rodent-borne arenavirus that causes severe hemorrhagic fever. The LASV genome consists of two fragments of RNA (S and N). The S fragment encodes the virus nucleocapsid protein and the precursor glycoprotein (GPC). The L fragment encodes the viral polymerase (L) and the zinc finger matrix protein (Z). The major immunogenic targets of the virus are the GP1 and GP2 glycoproteins that are produced by post-translational cleavage of GPC. GP1 functions as a receptor binding protein, while GP2 is a transmembrane protein . LASV VLBunyaviridae includes 5 genera including Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus that infect animals and humans and Tospovirus that infect plants. The Bunyaviridae genome consists of three RNA-negative segments including the large segment (L), the middle segment (M), and the small segment (S). Glycoproteins within the lipid envelope of the virion usually consist of two heterodimers, GN and GC that interact to form surface spike structures. A number of Bunyavirus VLP vaccines have been developed. Hantavirus VLPs can be obtained by expressing the virus Gn, Gc and NP genes in CHO cells. Animal experiments have shown that Hantavirus VLPs increase CD8+ T cell activity and induce antibody responses comparable to those seen with inactivated vaccines [The family vaccines . Zhou e vaccines .+ and CD8+ T cells and protected mice from deadly challenges [The filoviruses VP40 matrix protein plays a critical role in the structure and assembly of these highly infectious and dangerous viruses. However, expression of VP40 alone results in poor VLP production . An Ebolallenges .+ T cell response. Neutralizing antibodies have also been observed in pigs vaccinated with NiP VLPs, but in these animals no CD8+ T cell responses were detected [Paramyxovirus VLPs can be assembled following the simultaneous expression of viral matrix and glycoproteins. Nipah virus VLPs can be formed in HEK293T cells expressing the virus attachment glycoprotein (G), fusion (F) glycoprotein and matrix (M) protein . Mice vadetected . VLPs gedetected .Several animal coronaviruses have led to serious diseases in humans. These include SARS-CoV-1), Middle east respiratory syndrome-CoV (MERS-CoV) and most recently SARS-CoV-2 that has led to a global pandemic Coronavirus particles are composed of 4 structural proteins including spike (S) protein, envelope (E) protein, membrane (M) protein, and nucleoprotein (N). Experiments on SARS-CoV-1 VLPs showed that expression of SARS-CoV-1 M and E proteins in a BVES generated a smooth VLP without spikes while the simultaneous expression of M, E and S yields a structure that mimic the native SARS . These r+ T-helper cells and T-helper cells are required for both B and T cell proliferation and differentiation processes [+ T cells by vaccines also reduces the severity of the disease [+ cell epitope, and a CD4+ cell epitope of Toxoplasma gondii stimulated both humoral and cellular immunity with greatly increased levels of IgG and IFN-ɣ, respectively [+ T cell knockout mice animals were vaccinated with simian/human immunodeficiency virus (SHIV) VLPs or a chimeric of influenza HA/SHIV VLPs formulation. The results showed that the chimeric VLP-based system was capable of strongly stimulating humoral immunity via a T-helper cell-independent pathway. A second type of T cells, CD8+ cytotoxic T cells, typically detect intracellular pathogens only through the MHC class I-antigen complex [+ T cell pathway without the involvement of extracellular antigen. In one such study a mannosylated rabbit hemorrhagic disease virus (RHDV) VLP was presented as MHC class I-exogenous antigen and was able to directly activate cytotoxic T cells [− DCs and transferred to a secondary lymphoid organ [− and CD8alpha+ DCs via macropinocytosis and stimulated CD8+ T cells through the endosome-to-cytosol processing pathway [Virus capsids typically contain a repetitive protein structure that can stimulate innate immunity and induce B cells directly to produce neutralizing antibodies –160. DCsrocesses –172. In rocesses , 173–175rocesses . In gene disease –179. Simectively . The desectively . There a complex , 182. Ho T cells . The mecid organ . Antigenid organ  -dependeA strong cytotoxic T lymphocyte (CTL)-mediated immune response is a major factor in eradicating tumor cells and the ability of VLPs to induce these responses by delivering antigen to the cytosol and activating the MHC class I pathway makes them excellent candidates for development of vaccines in the treatment of cancer. Several important elements of the immune system play a role in targeting cancer cells. First, DC cells must receive sufficient signals to mature and stimulate adaptive immunity. This is important to ensure that the immune system does not become tolerant to the antigen by activating regulatory T cells (Tregs) and suppressing the immune system. Second, most cancer antigens are related or identical to self-antigens. Finally, T cells must be able to overcome the immune-suppressive signals generated by tumor cells. VLPs have features that allow them to address these challenges. In this section, we consider the VLP-based vaccines that have been explored as potential anti-tumor treatments .Some viral infections can lead to cancer. For example, HPV is one of these viruses, especially the two types HPV16 and HPV18, which cause 70% of cervical cancer cases . The virhttps://www.who.int/). 20–30% of cases of invasive breast cancer is associated with overexpression of human epidermal growth factor receptor 2 (HER2) that is involved in the proliferation and inhibition of programmed cell death [Acinetobacter phage AP205 coat protein, with surface displayed HER2 protein, was evaluated in FVB mice which had been transplanted with human HER2-positive breast cancer cells. This showed that vaccination was able to inhibit tumor growth in the mice. This approach induced strong humoral immunity and also overcame immune system tolerance [Breast cancer is the most common cancer in women and affects men as well , is overexpressed in many cancers, including pancreatic cancer, and is seen used as a potential anti-cancer drug target. This glycoprotein is involved in cell adhesion and causes cancer cell masses to attach to mesothelial cells. A VLP-based on SHIV VLPs with murine MSLN displayed on the surface of the particles was used to immunize mice harboring pancreatic tumor cells . Followi +, CD8+ , and the natural killer cell population. Moreover, the population of Treg and myeloid‐derived suppressor cells in the tumor microenvironment was decreased. Decreased expression of immunosuppressive cytokines such as IL-10 and TGF-β confirmed that the treatment inhibited tumor-suppressing immune signals induced by the tumor cells [An alternative therapeutic target for pancreatic cancer treatment is the transmembrane glycoprotein Trop2, which is also overexpressed in other cancers . This pror cells .+ T cells specific for the B16F10 murine melanoma significantly increased and tumor progression was inhibited leading to increased survival of the mice. All three types of VLPs were able to provide a degree of protection but the mixture of the two provided significant protection and prevented the progression and invasion of B16f10 cells changed the tumor microenvironment by increasing Ly6G+ granulocytic cells and decreasing Ly6C+ monocytic population [+ T cell mediated immune response is essential for vaccine efficacy in cancer prevention. In a study to analyze the importance of adjuvant size in stimulating T cell immunity and to evaluate the immune response in a mice model of melanoma a VLP-based platform derived from CMV and incorporating tetanus toxoid epitope TT830–843 (CMVTT-VLP) was established. The p33 peptide epitopes as a model antigen, derived from Lymphocytic choriomeningitis virus, was displayed on the particle surface and the CuMVTT-p33 VLP vaccine was formulated with micron-sized microcrystalline tyrosine (MCT) adjuvant. Comparison of the results with commercial adjuvants, Alum and B type CpGs showed that the micron-sized adjuvant stimulated CD8 + T cell immunity as much as CpG but more potent than Alum. The VLP-based vaccine showed notable antitumor effects against the B16F10 murine tumor cells [Melanoma is the cause of 10% of all skin tumors and more than 90% of deaths due to skin cancer . A VLP-bpulation . These ror cells . VLP-basAlthough VLPs are best known for their immunogenic properties, other capabilities of these nanoparticles have recently been considered, including their applications in drug delivery and gene therapy. In addition to carrying peptides/proteins or other active molecules displayed on the surface of the VLPs, they have the ability to entrap proteins, nucleic acids, or other small molecules. Therefore, they can be used as a means of delivering these molecules to specific cells, tissues, or organs .Cells use receptor-mediated endocytosis for uptake of VLPs. During endocytosis, the plasma membrane surrounds the VLPs and buds off inside the cell as a vesicle. Then, the vesicle is separated from the membrane and enters the cytosol. The released vesicles are transported along the cytoskeleton to combine with the primary endosomes. Eventually, the endosomal vesicles separate from the primary endosome, mature as the final endosome and merging with the pre-lysosomal vesicles containing acidic hydrolase to form lysosomes. The foreign materials are broken down inside the lysosomes and made available to the cells. However, lysosomal degradation prevents proper drug delivery, so that about 40% of newly produced drugs are disapproved due to poor bioavailability. The use of drug nanocarriers is a good strategy to overcome this limitation. So far, there are several nanocarriers that have been developed using different methods. Among nanocarriers, VLPs are highly suitable for drug delivery purposes due to their ability to escape endosomes before lysosomal degradation . VLPs haMore specific targeting is usually achieved by displaying receptor-binding domain on the VLP surface. Target domains can be chemically or genetically attached to the surface of VLPs, which allows the VLPs to selectively bind to cancer cells that express a specific receptor and enhance the therapeutic effects of drugs .VLPs can also be used to deliver nucleic acids. For example, one study showed that systematic delivery of miR-146a, a known gene silencer, via bacteriophage MS2 -derived VLPs, is an effective treatment for reducing inflammatory cytokines in mice susceptible to systemic lupus erythematosus . ExampleWith rapid advances in nanotechnology and protein engineering, interesting capabilities have been developed for the development and improvement of vaccine carriers as well as packaging and delivery tools for drugs. In recent years, viruses have been considered as not only to be the cause of disease, but also as functional NPs that are useful for a variety of applications. Self-assembled VLPs are one of these nanostructures with the ability to deliver antigens and drugs to various targets within tissues and organs. Traditional vaccines are often produced by inactivating or attenuating viral strains and genome-free VLPs offer new and safer alternatives. The variety of VLPs makes them structurally attractive and functionally diverse and VLPs can be designed to carry polyvalent antigenic structures that can also deliver antigenic compounds to specific target tissues. As well as being immunogens in their own right. VLPs have also been successfully used as adjuvants to elicit a strong immune response. By choosing appropriate types of VLPs, it is possible to stimulate both the innate and adaptive immune systems and in some cases VLP-based vaccines without any adjuvants have been shown to stimulate humoral and cellular immunity through the MHC class I and II pathway.VLPs can be used not only as preventive vaccines by displaying the foreign antigens on their surface to stimulate the immune system and to prevent infectious diseases, but also as therapeutic vaccines to present patient’s own antigens and to help them fighting against chronic and metabolic diseases or different types of cancers. Several VLP-derived vaccines are commercially licensed or are under evaluation in clinical trials. However, more evidence is needed to fully assess the potential efficacy, side effects, challenges and benefits of VLP-based vaccines in the treatment or prevention of various types of cancers.Technical challenges such as getting molecules to display on the particle surface properly remain but the continuing study of these structures is providing a considerable body of information to successfully address these problems. A potential solution to overcome this problem, which has been developed in our lab, is the insertion of a sortase recognition motif (LPXTG) into different parts of VLP that are likely to be exposed on the particle surface. In this way, it is possible to retain the integrity of the VLP and make it possible to conjugate any protein on the VLP surface –223. Oth


A citation study of a sample of earth science projects in citizen science from the FedCats Catalog was undertaken to assess whether citizen science projects are as productive and as impactful as conventional research that does not employ volunteer participation as a part of their data gathering and analysis protocols. From the 783 peer-reviewed papers produced by 48 projects identified from project bibliographies, 12,380 citations were identified using the Web of Science archive and their citation search engine to the end of 2018. Various conventional productivity and impact measures were applied including the Impact Factor, H and M-indices, and entry into the Top-1000 papers in cited research. The earth science projects tend to under-perform in terms of Impact Factor (IF = 14–20) and the M-index (M<0.5) but perform at the level of a ‘tenured professor’ with <H> = 23. When compared to non-citizen science research in general, there is a ten-fold higher probability of the earth science papers reaching the Top-1000 threshold of most-cited papers in natural science research. Some of the reasons for the lower performance by some indicators may have to do with the down-turn in published papers after 2010 for the majority of the earth science projects, which itself could be related to the fact that 52% of these projects only became operational after 2010 compared to the more successful ‘Top-3’ projects, whose impacts resemble the general population of non-citizen science research. Among the many concerns that scientists raise to the citizen science (hereafter CS) approach for conducting scientific research are whether the results will actually be meaningful to the scientific community at large. This consideration relates to questions about whether novice, non-professional participants are capable of delivering high-quality data to the Principle Investigator or project team ,2. LaborContemporary bibliometric studies use a variety of indices to gauge productivity and impact including counts of publications and citations . This isThis paper is a follow-on to the previous investigation in space science, but this time within the project areas of earth science. The objective as in the space science study is to use traditional citation analysis methodology to investigate the impact of earth science CS (ESCS) projects compared to non-ESCS projects.One of the easiest indices to create is simply the cumulative number of published papers, P, over a particular span of years. This ‘productivity’ index has been used since at least the 1960’s and its historical applications to academic research has been discussed by . HoweverA second measure of productive output is the number of times a paper is cited by others in the community described historically by . A citatAlthough a bibliography of published works can be current up to the present year, citations of these papers may not appear until several years after publication. The consequence of this lag is that the citations to recent publications in a bibliography will be under-counted relative to older papers. Awareness of this effect will be taken into consideration when the various metrics are evaluated and interpreted. In general, the citation trends of projects will be carefully scrutinized for papers published since 2015 where this effect will be the most prominent.A more worrisome issue is that the citation database itself may be incomplete. A study by  found thcaveat emptor (e.g. Let the buyer beware). Although the number of publications can be considered to be complete up to the current year, the number of citations can be seriously incomplete for younger papers compared to papers that have been in circulation for many years. As the citation counts approach the current year, their numbers will invariably start to decline as recent publications have not had enough time to circulate and be cited.In addition to the citation index CI, one straight-forward secondary index is the citation per paper ratio also called the Impact Factor, IF, ,16,17,18An additional higher-order measure of impact is the H-index originally suggested by  and lateth ranked publications has 4 citations. A number of criticisms of the H-index have been offered such as those reviewed by [The H-index is defined for an author as having published ‘H’ papers that have each been cited at least ‘H’ times. It is computed by taking an author’s rank-ordered citation count among the author’s publication list and finding the H-th rank such that the rank matches the number of citations. For example, if a project, institution or author has 5 publications with citations of 34, 12, 8, 4, and 2, the H-index will be 4 because the 4iewed by ,25. For iewed by , and it iewed by . Also, tiewed by ,28. The iewed by ,29.As a means for testing the hypothesis that ESCS projects have similar impact and productivity to non-CS projects, I will use the indices P, CI, CPY, IF and the H and M-indices to compare the ESCS, and non-ESCS research to determine whether CS research is statistically different from conventional research in earth science and in what specific ways. I will also compare ESCS projects with SSCS projects to see if earth science and space science CS projects have statistically similar or different impacts given that they both involve the CS approach in analyzing data.Generally, citation studies identify a group of papers within a uniform subject matter area and then proceed to use various tools such as the WoS to tabulate the citations to these papers. In a previous study of SSCS projects by  this steTo narrow the list to a manageable number, I used the Federal Crowdsourcing and Citizen Science Catalog   and other similar documents and books.The 108 active ESCS projects, representing the efforts of some 4 million participants, reported 5001 publications including scientific research papers, conference abstracts, popular articles in newspapers and magazines, books, and dissertations. Virtually all of the ‘Education’ publications were from 10 of the number of participants. Column 7 is the total number documents found for the project including research publications (column 8), reports (column 9), popular essays (P: column 10), conference abstracts (C: column 11), books (B: column 12), educational reports (E: column 13), and dissertations. Across the 108 active projects, the median age of these active-CS projects (column 5) is 6 years, and the median number of participants is about 1,000. About half of the projects (48) report a combined 783 articles (column 8) in peer-reviewed journals for a median of about two papers published per project among these programs. The total number of participants among the active projects is about 4.3 million. There have also been a total of 110 books and 42 Masters/PhD dissertations generated by this cohort of projects. Note that the large number of documents for the Smithsonian Digital Volunteers project reflects the fact that each document transcribed by volunteers becomes part of the permanent Smithsonian archive and so represents a single ‘published’ document. The table is ordered by decreasing numbers of peer-reviewed publications (col. 8).Christmas Bird Count (306), eBird (196), and The GLOBE Project (61) that collectively account for 72% of the 783 peer-reviewed research papers. The second group, which I will call the ‘Lower-45’, consists of publications contributed by the remaining 45 projects totaling 219 papers. This investigation will treat these two groups separately to avoid biasing the discussion of the more typical project performance data from the more successful projects.Although there were 108 earth science projects in the FedCat, only 48 were found to have peer-reviewed publications. The remaining 60 programs in The Silent-ESCS projects with no identifiable publications are listed in For convenience, The citation frequency for each of the 782 peer-reviewed papers was extracted from the online data base provided by WoS using the standard search tools provided on the website. On the Advanced Search page, I entered the Boolean string for the first-author’s last name and the journal. I then selected the year range spanning the year of the article publication. The search provided a page of ‘hits’ and from these I selected the specific article by the author being studied. A Citation Report was then produced by WoS for that article, which included the total number of citations accumulated from the time of the publication to the end of 2018, which is the end year of the current study.In the majority of the cases, it was only necessary to search under the first-author’s surname and initials for the year of the publication. The aggregated results for the citations of all peer-reviewed papers published for a given project are provided in By this measure, the Top-3 projects shown in For the typical projects of SKYWARN and Did you feel it?, have participation levels comparable to those of the Top-3 projects yet produced significantly less output, so among this second group of projects, the level of public participation is in detail not a good predictor of the project’s productivity.Although the Top-3 projects had a median of 100,000 participants  col. 2, 2 < 0.5, which indicates no correlation and that 99.5% of the variation is caused by random error. The two outlier points at PPY = 2.0 (Landslide Reporter) and 5.3 (Snow Surveyors) are exceptional given the small number of participants.The conclusion from this study is that, as for the SSCS projects studied by , the ESCThe cumulative citations for each of the 782 papers in this study were identified using the WoS online citation catalog. The full ensemble of 48 projects with peer-reviewed papers generated a total of 12,380 citations, with the specific sub-totals by the Top-3 projects shown in eBird project, which continues to generate papers between 2010–2018 that are producing significant numbers of citations. This suggests that there are either fewer papers being published by the Lower-45 cohort after 2010, or those papers that are published are being under-cited. The former possibility can be discounted because in Although the citations generally increase for both groups through ca 2015, there is a progressive fall-off after ca 2015. Since it takes up to five years or longer before a paper begins to be cited, this lag probably explains the 2015–2018 fall off. These distributions are, however, generally what would be expected since, all other factors being equal, older papers published before ca 2010 ought to have accumulated more citations than more recent ones. According to , a studyThe ensemble of 783 peer-reviewed papers included 266 that were not cited by the end of 2018. Given that the publication record ends in 2018, if we apply a grace period of two years before we expect the first citations to appear, we find that papers published in 2017 and 2018 account for 84 of these cases and have not as yet begun to have citations to them. Correcting the percentages for the four project categories, the zero-citation rates are Else = 12%, eBird = 14%, GLOBE = 18% and Christmas = 0%. While these no-citation rates of 12–18% appear high, the study by  suggestsAccording to , “It is Ultimately, the goal of this investigation is to compare ESCS projects against non-CS research approaches to assess whether ESCS projects are as productive based upon standard bibliographic indices as conventional research approaches. This comparison was performed for space science projects by  who founThe Astronomical Journal, the Monthly Notices of the Royal Astronomical Society and The Astrophysical Journal. The previous strategy was to identify all papers published in these three journals for the year 2000, and then follow the collective history of the citations for these papers through 2018. In contrast, the ESCS papers were published in over 352 different journals whose audiences are specialized, and with none accounting for more than about 10% of the peer-reviewed publications in this study. Among the Top-3 programs, which are primarily in the field of ornithology, the most popular journals were Ecology and Biological Conservation. The most popular journals for the Lower-45 papers were Frontiers in Ecology and the Environment, Global Change Biology, and the International Journal of Biometeorology. Similarly to the previous space science study, we selected a reference year, in this case 2003, because this was the first year of publication for the Frontiers in Ecology and the Environment, and it represents the start year of the majority (80%) of the ESCS projects. The counting via WoS was performed over the interval 2003–2018. A total of 980 papers were identified in these five journals and yielded 78,575 citations over the 15-year period.In the previous study, the non-CS, space science papers used as a reference appeared in only three main journals: GLOBE program with 422 citations, [Christmas Bird Count project with 320 citations, [Christmas Bird Count project with 303 citations, [GLOBE project with 277 citations, and finally [The annual citation history of these non-CS, earth science papers has been normalized by dividing each journal’s citations by the total number during the 2003–2018 reference interval. The resulting normalized history was then multiplied by an arbitrary factor of 300 to place the curves on the same scale as the ESCS papers. The resulting hypothetical non-CS citation history curves for each journal are shown in tations,  with 336tations,  from thetations,  also frotations,  from the finally  with 270 finally  with 427 finally  with 268What is seen in The Impact Factor is a ratio of the number of citations over a specific span of time divided by the number of papers published over the same time interval. In the case of CS projects, the appropriate time interval is the difference between the inception year of the project and 2018 when this study was concluded. For example in A study by  has inveAlthough comparing CS with non-CS papers is problematical because of the different methodologies employed to gather and analyze data, we can investigate whether CS projects in two distinct disciplines have similar impacts. A detailed comparison between the IF distributions for the Lower-45 ESCS and Lower-23 SSCS projects can determine whether these projects using similar citizen science approaches also have similar impacts.2 = 144. The T-score is computed from the formula1/2 = 2.52 for 5 degrees of freedom. The corresponding T-value is 2.05 for rejecting the null hypothesis at the 5%-level (i.e. accepting the null hypothesis at the 95%-level). Since the T-value is greater than the calculated 2.52, the SSCS and ESCS IF distributions are, therefore, statistically different. Judging from the histograms in We can check the null hypothesis that there is no significant difference between the ES and SS groups by performing a one-sided, Student’s T-test using the N = 6 categories in Christmas Bird Count (H = 31), eBird (H = 23), and The GLOBE (H = 17). In space science the corresponding projects are Einstein@Home, Planet Hunters and Galaxy Zoo. The large dispersion in H-indices especially for the most recent years since 1990, indicates that there is no significant correlation with project age after 1990. The ESCS and SSCS groups each yield a flat regression (slope ±0.05) with R2 <0.005, which indicates that no correlated behavior exists in this data to a significance exceeding 99%. However as expected, the projects with the largest number of publications, at least within the limitations of small-number statistics, tend to have the highest H-indices.For the ensemble of 48 ESCS projects, the combined, ranked project citations in Tables The age-corrected M-index has been suggested by  as a betAccording to an assessment of the M-index by , M = 1  achieved more than 1000 citations by 2014. For space science papers in particular [Another measure of impact proposed by  is the nrticular  found theBird); 33 in 1000 (GLOBE); and 7 in 1000 (Christmas Bird Count), while the Lower-45 had a rate of 10 in 1000. These rates are substantially higher than that found by [A similar analysis was performed for all 48 ESCS projects, which resulted in five papers having more than 257 citations after five years, which I call the P257-index, for an equivalent of 10 papers per 1000. Taken separately, the Top-3 papers had a rate of 5 in 1000 , eBird (196), and The GLOBE Project (61), which collectively account for 563 papers (72%). The second ‘Lower-45’ group, contributed the remaining 219 papers. Although 80% of the projects have only been in existence since 2000, the Top-3 projects have a nearly ten-fold higher rate of paper production (2.6 papers per year) compared to the Lower-45 projects. Although the difference cannot be attributed to the median number of authors per paper in each group, which is nearly the same (e.g. 3–4) for each group. The Top-3 projects have a median of nearly 100 times the number of participants than the Lower-45 projects, which suggests a higher throughput of data from which papers could be generated. However, within the Lower-45 group, there is no commensurate correlation between annual paper output and participatory scale, although these projects tend to be significantly younger than the Top-3 projects.A total of 783 peer-reviewed citizen science research papers in the area of earth science were identified through the end of 2018. The Top-3 projects were The full ensemble of 48 projects with peer-reviewed papers generated a total of 12,380 citations by the end of 2018, with the Top-3 projects contributing 7,854 citations and the Lower-45 with 4,426 citations. In terms of annual paper citations, for most years between 1975–2010 the papers from the Lower-45 and Top-3 are well-mixed during any given year, which indicates that papers from diverse projects are being cited about equally. After 2010, and especially after 2012 there is a sharp decline for all groups in the citations for the Lower-45 papers but not for the Top-3 projects, which continue to generate papers between 2010–2017 that are producing significant numbers of citations. This suggests that there are either fewer papers being published by the Lower-45 cohort after 2010, or those papers that are published are being under-cited. The former possibility can be discounted because there was no decline in papers-per-year for this group during this time interval. This suggests that more recent papers among these projects are not being cited at the same rate as papers in this cohort before ca 2010. This is supported by the discovery that in terms of the number of papers from each project that are not being cited at all, the Lower-45 rate of 12% is comparable to the median rate of 14% for the Top-3 projects.When the annual citation histories of the earth science projects are compared to non-citizen science projects in earth science, the profiles of the most-cited CS papers do follow the history trend of the average non-ES papers with annual citations that gradually increase between 1–5 years after publication, and then level off with a long sustained annual citation rate lasting in some instances 15 years. The most-successful of these projects enjoy a more prolonged growth to Year-8 before they, too, appear to reach a plateau.n = 25 are common with considerable sub-discipline variation. Both the earth and space science citizen science papers fall below IFn, and only three of the Top-3 projects equal or exceed this level of impact.A comparison of the earth science and space science citizen science projects which use similar data gathering and analysis methodologies suggests that the Top-3 space science projects tend to have higher impacts (IF = 24) than the corresponding earth science projects (IF = 13), but that the general populations of these projects excluding the Top-3 favor the earth science projects (IF = 21) over the space science projects (IF = 10) in impact. Studies by other investigators of natural science research papers between 1990 and 2010 find that typical impact factors near IFThe CS projects in both earth and space science achieve aggregated H-indices that suggest they are as impactful as the publication efforts of equivalent individual scientists applying for a tenure decision. Generally, only the Top-3 citizen science projects in earth and space science have H-indices that place them above the threshold for ‘tenure’. The H-index is known to be flawed in that it is age-dependent favoring scientists and programs that have longer publication histories. The M-index is created by dividing the H-index by the age of the publication record such that M = 1 (e.g. H = 20 after 20 years of research) is the threshold for a ‘successful’ scientist, while M = 2 is the threshold for ‘outstanding scientists’. By this measure, virtually all of the earth science projects fall below the threshold for a ‘successful’ scientist while of the SSCS projects fall above this threshold.A number of variants on the idea of the Top-1000 papers in an entire research domain have been proposed to indicate impact. One method counts the number of papers with more than 257 citations in five years yielding a one-in-1000 rate as a suggested baseline for the most successful papers. A similar analysis on space science citizen science papers and found an equivalent rate of 26 papers per 1000. Taken separately, the Top-3 papers had an average rate of 15 per 1000, while the Lower-45 had a rate of 10 per 1000. These rates are ten-fold higher than found by other investigators for non-CS research impact.Assessing a variety of measures for productivity and impact for the earth science citizen science peer-reviewed research papers, we can tentatively conclude that the majority of the citizen science research tends to fall below the typical impact factors for natural science research, with the majority of the earth science research falling below the level of ‘successful’ research as indicated by the M-index. Nevertheless, when gauged by the presence of this research among the Top-1000 papers being cited, citizen science papers in both earth and space science areas have about 10-times the number of papers making it to this level. Some of the reasons for the lower performance may have to do with the down-turn in published papers after 2010 for the majority of the earth science projects, which itself is related to the fact that 80% of these projects only became operational after 2000 compared to the more successful ‘Top-3’ projects, whose impacts resemble the general population of non-citizen science research.S1 DataThe contents are presented in seven spreadsheets identified as ‘Projects’, ‘Bibliography’, ‘Journals’, ‘Citations’, ‘eBird’, ‘GLOBE’, ‘Christmas’. The Projects tab contains a complete listing of the surveyed projects along with tabulated information about the estimated number of participants and the break down of the kinds of published documents. Bibliography lists the inventoried research papers for each project. Journals is a listing of the peer-reviewed journals in which the research articles appear. Citations is the cumulative number of citations through the end of 2017 for each bibliographic reference. The eBird, GLOBE and Christmas tabs are the separate bibliographies for the eBird, GLOBE and Christmas Bird Count projects. The tabs for Tables (XLSX)Click here for additional data file.


Experimentally infected pigeons that were vaccinated with VLPs also showed no detectable viral titer. The results of the current study demonstrated the potential use of PiCV rCap-VLPs as an effective vaccine candidate against PiCV.Pigeon circovirus (PiCV) is the most recurrent virus diagnosed in pigeons and is among the major causative agents of young pigeon disease syndrome (YPDS). Due to the lack of an established laboratory protocol for PiCV cultivation, development of prophylaxis is hampered. Alternatively, virus-like particles (VLPs), which closely resemble native viruses but lack the viral genetic material, can be generated using a wide range of expression systems and are shown to have strong immunogenicity. Therefore, the use of VLPs provides a promising prospect for vaccine development. In this study, transfected human embryonic kidney (HEK-293) cells, a mammalian expression system, were used to express the PiCV capsid protein (Cap), which is a major component of PiCV and believed to contain antibody epitopes, to obtain self-assembled VLPs. The VLPs were observed to have a spherical morphology with diameters ranging from 12 to 26 nm. Subcutaneous immunization of pigeons with 100 µg PiCV rCap-VLPs supplemented with water-in-oil-in-water (W/O/W) adjuvant induced specific antibodies against PiCV. Observations of the cytokine expression and T-cell proliferation levels in spleen samples showed significantly higher T-cell proliferation and IFN- γ expression in pigeons immunized with VLPs compared to the controls ( The pigeon circovirus (PiCV) is among the major causative agents of a multifactorial disease known as the young pigeon disease syndrome (YPDS) . PiCV geORF C1 encodes a 30 kDa protein that is responsible for the assembly of the viral capsid . On the For more than two decades, one of the common problems in young racing pigeons is YPDS, which is associated with poor racing performance, and high morbidity and mortality rates among pigeons of age 3 to 20 weeks . The strThe capsid protein of circoviruses is believed to display antigenic properties that induce antibodies when the host is infected with the virus. This was verified in the cases of beak and feather disease virus (BFDV), PiCV, and porcine circovirus genotype 2 (PCV2) ,12,13. AVirus-like particles (VLPs) are multiprotein structures that mimic the characteristics of the virus, which are ideal for vaccine development . VLPs laDue to the high prevalence of PiCV and with no available vaccines, severe losses were incurred by the poultry and pigeon racing industry. Because of the persistent threats posed by PiCV, vaccine development would be of great interest. In this study, recombinant capsid protein was generated using a mammalian expression system. Transfected human embryonic kidney (HEK-293) cells were used to produce self-assembled VLPs. Furthermore, challenge tests and qPCR analysis of cytokines were done to determine the immune response post-immunization. The current study is limited to the development of a vaccine against PiCV alone.® CRL-1573™) were cultured and propagated in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and with 50 µg/mL of penicillin-streptomycin  and incubated at 37 °C and 5% CO2.Human embryonic kidney 293 cells (HEK-293)  was codon usage-optimized by Genomics, New Taipei City, Taiwan and synthesized in pGS21-a  with EcoRI and XhoI as restriction sites. The constructed plasmid contains a genomic sequence that encodes PiCV capsid gene with a C-terminal GST tag and an N-terminal His-tag. On the other hand, for the mammalian cell expression system, the same gene sequence (GenBank Accession No. HQ401274.1) was codon usage-optimized  and synthesized in pCDNA3.1  with KpnI and NotI as restriction sites. The constructed plasmid contains a genomic sequence that encodes PiCV capsid gene with His-tag. Verification of the constructed sequence was performed through DNA sequencing. The resulting plasmid was used for transfection and protein expression experiments.For the E. coli (BL21) containing the recombinant pGS21a-Cap plasmid and incubated at 37 °C until O.D. 600 reached 0.6. Afterward, rCap expression was induced by adding 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) to the medium and subsequently incubating for another 6 h at 37 °C. After incubation, the culture was then centrifuged at 10,000 rpm for 10 min. The resulting bacterial pellet was resuspended in 10 mL Tris-buffered saline (TBS), and the mixture was sonicated thrice in 20% pulsing cycles . Cell debris from the sonication was removed by centrifugation at 10,000 rpm for 10 min. The supernatant was subjected to Ni2+ affinity column chromatography to purify the collected recombinant capsid proteins. Purity of the obtained rCap was assessed by SDS-PAGE Analysis and Western blot analysis. The purified rCap was then stored at −80 °C for further use.One liter (1 L) of LB was inoculated with Briefly, 10 μL of protein samples was separated using 12% of SDS-PAGE and was visualized by Coomassie blue dye. For Western blot analysis, separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane. Unwanted sites on the membrane were blocked by incubating in 10 mL of 5% skim milk  with shaking for 1 h at 37 °C. The membrane was washed with PBST (0.1% Tween 20 in 1x PBS) 5 times for 5 min each. After washing, the membrane was incubated with mouse anti-His primary antibody  diluted 3000-fold in 10 mL 5% skim milk with shaking at 4 °C for 12 h to enable the binding of primary antibody with the histidine tag (6x His-tag) of the target protein. After the addition of primary antibody, the membrane was again washed with PBST 5 times for 5 min each. Next, the membrane was incubated with horse radish peroxidase (HRP)-conjugated rabbit anti-mouse IgG  diluted 5000-fold in 10 mL 5% skim milk with shaking for 1 h at 37 °C. Using a chemiluminescent substrate for the detection of HRP , the blot was visualized by X-ray film development.The pCDNA3.1-Cap vector was transfected into HEK-293 cells using lipofectamine  to produce PiCV VLPs. The medium containing the cells was collected after 72 h of incubation at 37 °C, and it was subjected to centrifugation at 1500 rpm for 5 min to separate the cells from the medium. The cells were washed using PBS, and the mixture was centrifuged again for another 5 min at 1500 rpm. Collected cells were mixed with whole cell extract buffer and then placed on ice for about 30 min. Afterward, the mixture was centrifuged at 10,000 rpm for 10 min. The supernatant was precipitated by adding 7% poly-ethylene glycol (PEG) and 2% NaCl solution, and then agitating it at 4 °C for 4 h. After agitation, it was centrifuged with a speed of 9500 rpm at 4 °C for 30 min. The supernatant was disposed, while the pellet containing the PiCV VLPs was resuspended in 4 mL PBS. The resulting VLPs solution was stored in −20 °C for further characterization, and until use for immunization. SDS-PAGE and subsequent Western blotting were performed as described above, but the blot was visualized using a digital imaging system . For size comparison, the protein weight marker captured under visible light was automatically merged with the image of the blot by the imaging program. Electron microscopy was also performed to observe the morphology of the VLPs.The purified PiCV rCap-VLPs were captured on a carbon coated slotted grid, which was then stained with phosphotungstic acid (PTA) for 60 s. After staining, a transmission electron microscope (TEM)  was used to visualize the morphology of the VLPs.Columba livia) obtained from a private hatchery were used in this study. Prior to immunization, the antibody titers of pigeons against PiCV capsid protein were measured by ELISA at 21 and 28 days of age. Pigeons with serum ELISA optical densities (O.D.) higher than that of the 0 day old PiCV-free pigeon serum (negative control) were not selected for use in this study. The pigeons selected for both the control and VLP groups were subcutaneously (S.C.) immunized twice at age 28 and 42 days. The control group was immunized with saline solution supplemented with water-in-oil-in-water (W/O/W) adjuvant , while the VLP group was immunized with 100 μg PiCV rCap-VLPs supplemented with W/O/W adjuvant. On 0, 14, and 21 days post-vaccination (dpv), blood samples were collected from the wing vein of 5 pigeons in each group to measure the antibody titer. Spleen samples were also collected from 5 pigeons from each group at 21 dpv for T-cell proliferation and cytokine assays. Additionally, at 21 dpv, a challenge test was administered to 10 pigeons (5 pigeons per group) by giving the pigeons feed containing 3 g of lymphoid tissues containing 6 × 103.5 copies/g of PiCV. All pigeons were euthanized using CO2 at 28 dpv for histopathological examination and quantification of the viral load.Twenty (20) twenty-one (21) day old pigeons , with IACUC permit number NPUST 107-056.Following the euthanasia of pigeons, tissue samples from the spleen were collected. The extracted tissue samples were fixed in 10% phosphate buffer formalin solution and embedded in paraffin. For histopathological examination, hematoxylin-eosin was used to stain 5 µm thick tissue samples. The samples were examined under a light microscope, and images were obtained using a Nikon DS-L2 camera unit connected to a Nikon Eclipse E-200 microscope. E. coli was used to coat the wells of the ELISA plate. Each of the collected pigeon sera were diluted 120-fold in the assay buffer, and 100 μL of the serum was deposited in each well. Serum from 0 day old PiCV-free pigeons, diluted 512-fold, was used as negative control. For the secondary antibody, horseradish peroxidase (HRP)-labeled goat anti-pigeon IgG  diluted to 6000-fold was used. The assay was carried out with ABTS ) substrate for 15 min. An ELISA plate reader was used to read the optical density (OD) at 405 nm.To determine the PiCV-specific IgG antibodies, the collected sera from the wing vein of the pigeons were each tested by ELISA. Coating buffer  containing 40 μg/mL of purified capsid protein expressed in ® 1077 density gradient medium and centrifuged at 1800 rpm for 20 min at room temperature. Lymphocytes at the interface were collected and washed three times in HBSS. Lymphocytes isolated from the spleen of both groups were seeded into a 96-well plate. Each well was seeded with 1 × 105 cells in 100 μL medium (10% FBS DMEM with penicillin and streptomycin). The cells were then incubated with 10 μg of VLPs for 72 h at 37 °C. Following the incubation, 10 μL of MTT with a concentration of 0.5 mg/mL was added into each well, and the plate was further incubated for another 4 h at room temperature. The medium inside the ELISA plate was collected and centrifuged at 1500 rpm for 15 min. Following centrifugation, the supernatant was discarded, and the cells were lysed in a buffer (0.01 M HCl + 10% SDS). An ELISA reader  was used to measure the extent of color change at 570 nm. T cell proliferation activity was expressed as stimulation index (S.I.).An MTT -2,5-diphenyltetrazolium bromide) assay was used to determine the T cell proliferation activity in the spleen samples. Spleen samples were macerated through a sieve using a syringe plunger to obtain single-cell suspension in Hank’s Balanced Salt Solution (HBSS). The cell suspensions were overlaid onto Histopaque2O) was added to wash out excess salts followed by centrifugation at 8000× g for 2 min. Excess alcohol was drained, and the RNA pellets were air-dried for 30 min and re-suspended in 50 μL of DEPC H2O. RNA concentration was measured using OD at 260 nm, and its quality was evaluated by calculating the OD260/OD280 ratio. Complementary DNA was produced from 100 to 200 ng of RNA per reaction. The RNA was subjected to RT-PCR using a RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s instructions. Furthermore, RT-PCR was carried out using a thermal cycler  with the following conditions: initial denaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and elongation at 72 °C for 30 s. The assay was performed using the StepOne™ Real-Time PCR System . The calibrated cytokine content relative to the control group was evaluated through the comparative Ct method (2−∆∆Ct).Lasergene package  was used to design a RT-PCR primer pair specific for IFN-γ, TGF-β, and IL-8. Pigeon β-actin was used as internal control . Amounts−∆∆Ct).To detect the vial load, qPCR primers used were designed specifically for PiCV and pigeon β-actin  using Lap < 0.05. All data are shown as the mean ± standard deviation ), are used for generating immunogenic VLPs [Virus-like particles (VLPs) are self-assembled macromolecules that mimic the viral protein structure of a target virus but contain no genetic materials of the native viral strain. VLPs exhibit antigenic epitopes that are positioned in a correct conformation and in a highly repetitive manner . Hence, nic VLPs . This renic VLPs . HEK-293nic VLPs . Mammalinic VLPs . Due to nic VLPs ,36.cap (C1) gene is called capsid protein (Cap). This protein is not only responsible for the capsid assembly, but it is also used as antigen for antibody detection during PiCV infections [Mx1 gene and IFN-γ, which proved the antiviral effect of PiIFN-α [The structural protein encoded by PiCV fections . Aside ffections . With nofections . A previfections . Moreovecap C gene is fections . Taking  PiIFN-α .In this study, vaccination with VLPs demonstrated an increase in antibody production of immunized pigeons. Furthermore, the challenge test of experimentally infected pigeons showed no detectable copies of PiCV in spleen samples post-immunization, therefore proving that the VLP treatment can significantly reduce PiCV viral loads in the spleen. Similarly, VLP immunization in mice and guinea pigs was able to significantly induce specific antibody responses to PCV2 Cap . MeanwhiTo further understand the immunogenic response to the PiCV rCap-VLPs treatment in this study, T-cell and cytokines were quantified. It was observed that the immune response caused by the PiCV rCap-VLPs significantly increased the proliferation of various T-cell and cytokines. T-cells are an important part of the immune system for the cell mediated immunity and the activation of immune cells, while cytokines are important as signaling molecules to regulate immunity . SpecifiSince prophylaxis against PiCV is not available, potential approaches were proposed for further development of the vaccine research. To summarize the results of this research, it was demonstrated that PiCV rCap-VLPs was successfully produced using a mammalian expression system. PiCV rCap-VLPs induced antibody titers in immunized pigeons and significantly reduced viral titers in experimentally infected pigeons. The constructed VLPs were proven to be an effective potential candidate vaccine against PiCV. Additional assessments and research on VLPs generated by mammalian expression system are required to further test the effectiveness of this vaccine.The nonavailability of laboratory protocols for culturing pigeon circovirus (PiCV) and prophylaxis against the virus causes severe losses in the pigeon industry. A mammalian expression system was used in this study to obtain PiCV rCap-VLPs. Immunization with PiCV rCap-VLPs was shown to induce antibody response and significantly reduced the viral titer in infected pigeons, which proved its potential as a vaccine candidate. Further assessment of the VLPs generated by mammalian expression system is needed to investigate the efficacy of this potential prophylaxis. Results from this experiment provide important insights into the future development of PiCV vaccine research.


The majority of older patients who develop heart failure (HF), particularly older women, have a preserved left ventricular ejection fraction (HFpEF). Patients with HFpEF have severe symptoms of exercise intolerance, poor quality‐of‐life, frequent hospitalizations, and increased mortality. The prevalence of HFpEF is increasing and its prognosis is worsening. However, despite its importance, our understanding of the pathophysiology of HFpEF is incomplete, and drug development has proved immensely challenging. Currently, there are no universally accepted therapies that alter the clinical course of HFpEF. Originally viewed as a disorder due solely to abnormalities in left ventricular (LV) diastolic function, our understanding has evolved such that HFpEF is now understood as a systemic syndrome, involving multiple organ systems, likely triggered by inflammation and with an important contribution of aging, lifestyle factors, genetic predisposition, and multiple‐comorbidities, features that are typical of a geriatric syndrome. HFpEF is usually progressive due to complex mechanisms of systemic and cardiac adaptation that vary over time, particularly with aging. In this review, we examine evolving data regarding HFpEF that may help explain past challenges and provide future directions to care patients with this highly prevalent, heterogeneous clinical syndrome. She also has peripheral edema; increased jugular venous distention elevated 10 cm above the right atrium. An electrocardiogram did not demonstrate ischemic changes. Her baseline echocardiogram showed mild left ventricular (LV) hypertrophy with an EF of 55% and right ventricular (RV) systolic pressure of 50 mm Hg. During a stress echocardiogram, she exercised for only 3 minutes on a modified Bruce protocol, stopping for extreme shortness of breath. Her resting BP was 160/70 mm Hg and her HR was 76 bpm. At peak exercise, her BP was 196/90 mmHg, with a peak HR of 105 bpm. Echocardiographic images at the end of exercise demonstrated augmentation of contractility of all walls without significant mitral regurgitation. What can be done to improve her symptoms and quality of life?A 79‐year‐old woman with long‐standing hypertension, obesity, and type II diabetes presents with shortness of breath on exertion that began 6 months earlier and has since gradually worsened and interferes with daily activities. She denies exertional chest pain. While she is able to shop in the local supermarket, carrying her packages home has become increasingly difficult. She desires to return to her previously active life. Current medications include amlodipine 10 mg daily, metformin 1000 mg daily, Lisinopril 20 mg daily, atorvastatin 20 mg daily. On exam, her blood pressure (BP) is 160/80 mm Hg, heart rate (HR) is 78/minutes, body mass index (BMI) 36 kg/m2.1Diagnosing HF in older adults poses specific challenges; false‐positive clinical diagnoses are not uncommon.2.22+]i regulation, decreased β‐adrenergic reserve, and physical deconditioning, have been identified as important contributing causes for HFpEF.Cardiac aging is known to affect many, if not all, of the pathophysiological components present in HFpEF. Specific alterations in structural and function in aging, such as ventricular vascular stiffening, vascular dysfunction, impaired [Ca2.3Is HFpEF is a single entity or comprised of several different diseases?Are there inflammatory biomarkers that may help to diagnose HFpEF and better understand its pathophysiology?2.4Tables 2.5Was the lack of definite benefits in pharmacological trials to date caused by a flawed study designs or by ineffective study interventions?Should future HFpEF trials include broader group of subjects or individual subpopulations?2.6Metabolic risk factors: HFpEF patients demonstrate a high prevalence of obesity and diabetes. Increased adiposity promotes inflammation, hypertension, insulin resistance, and dyslipidemia and impairs cardiac, arterial, skeletal muscle, and physical function,Coronary Artery Disease (CAD) Patients with HFpEF and symptoms and signs of ischemia are treated with standard therapy including beta‐blockers and calcium channel blockers.Atrial fibrillation (AF) prevalence has been increasing due to an aging general population and increased longevity. AF in HFpEF associated with impaired LV systolic, diastolic function and functional reserve, larger left atria (LA) with poor LA function, RV dysfunction, more severe neurohumoral activation, and impaired exercise tolerance.Anemia is more prevalent in HFpEF than in HFrEF patients and associated with increased risk of HF hospitalization and overall mortality.Table 2.7Is rate control alone or rhythm control the best strategy for treatment in HFpEF patients?What is the best way to manage comorbidities in HFpEF patients?2.8Recent data support the beneficial impacts of lifestyle modification, including weight reduction, dietary and nutrient consumption, physical activity, and cardiorespiratory fitness on HF risk. In a pooled analysis of 51 000 participants from the Women's Health Initiative, Multiethnic Study of Atherosclerosis, and Cardiovascular Health Study cohorts, the risk for incident HFpEF increased in a dose‐dependent manner as BMI increased and leisure‐time physical activity declined.In 2010, Kitzman et al reported the first randomized controlled trial evaluating ET as a treatment for HFpEF, showing substantial improvement in cardiorespiratory fitness with training  HFrEF.ACC/AHA guidelines recommend moderate, regular physical activity for all HF patients, which seem reasonable. However, in the absence of data regarding effect of ET on clinical events, the Centers for 2.10What is the most effective and safe exercise prescription for older HFpEF patient?2.11In the CHAMPION trial , clinical management guided by physician knowledge of central hemodynamics significantly reduced HF hospitalization Table .27, 68 TGiven that rises in left atrial (LA) pressure and pulmonary venous congestion are shown to herald HF events in patients with HFpEF, creating a controlled left‐to‐right interatrial shunt to allow LA decompression could be a rational non‐pharmacological strategy for alleviating symptoms. The Reduce Elevated LA Pressure in Patients with HF (REDUCE LAP‐HF) study is a multicenter, prospective, non‐randomized, single‐arm phase 1 study designed to assess the safety and performance of the device in patients with HFpEF with NYHA II‐IV despite optimal medical or device therapy and demonstrated reductions in LA pressure during exercise with improvements in functional capacity and health‐related quality of life scores 6 and 12 months after implantation of this device  Diastolic dysfunction by itself is not enough to establish HFpEF. (b) HFpEF is not simply a disease of aging nor does it occur only in females. (c) HFpEF has significant phenotypic and etiologic heterogeneity. (d) Due to its heterogeneity, a “one‐size‐fits‐all” strategy is unlikely to work in HFpEF. (e) HFpEF is associated with multiple comorbidities. (f) HFpEF is a multi‐system disease, with the heart being a major component but with others providing major contributions.2.18Our 79‐year‐old woman should be placed on diuretics at the lowest effective dose for symptomatic relief. She should have a home scale and weigh herself daily. We should provide instruction for steps to take diuretics based on weight changes. Her BP was not well controlled and lisinopril to be adjusted to keep SBP < 130 mm of Hg. She should be advised regarding dietary compliance. She should be encouraged to exercise daily. She should be provided comprehensive HF disease management, including education, diet, exercise therapy, and close follow‐up. Ideally as described earlier, she is recommended to participate in a structured, supervised ET program; however, lack of CMS coverage can be a major barrier to formal cardiac rehab in older HFpEF patients.3HFpEF is the most common form of HF in the community, its prevalence is increasing, and prognosis has not improved or even worsened. It is nearly unique to older adults and is a true geriatric syndrome. Despite a moderate number of clinical trials, therapeutic successes to date have been few, and clinical management is largely empiric. An evolving paradigm suggests that, like other geriatric syndromes, HFpEF is complex and multifactorial, probably systemic, and clinically heterogeneous and has a multifactorial pathophysiology, underlying age‐related changes, frequent multiple chronic comorbidities and multiorgan involvement. Understanding the relationship between HFpEF and aging may help with understanding the biology of HFpEF more generally. In addition, machine learning techniques suggested by Shah et al, when applied to large phenotyped  datasets in combination with clinical outcomes may help to improve our understanding of how biological phenotypes integrate with clinical phenotypes. Efforts are underway to utilize these concepts to identify novel therapeutic targets, improve the design of future clinical trials, and to develop effective clinical management algorithms. Finally, the complexities of these patients demand an approach that is more holistic by addressing not only direct HF‐related conditions but also optimal management of geriatric syndromes, focusing on quality of life.Dr. Kitzman declares the following relationships: Consultant for Abbvie, Bayer, Merck, Medtronic, AstraZeneca, Merck, Corvia Medical, and Actavis, research grant funding from Novartis, Bayer, and AstraZeneca, and stock ownership in Gilead Sciences and Relypsa. Dr. Upadhya has received research funding from Novarits and Corvia.


The purpose of this study is to compare diagnostic performance of Prostate Imaging Reporting and Data System (PI-RADS) version (v) 2.1 and 2.0 for detection of Gleason Score (GS) ≥ 7 prostate cancer on MRI. Three experienced radiologists provided PI-RADS v2.0 scores and at least 12 months later v2.1 scores on lesions in 333 prostate MRI examinations acquired between 2012 and 2015. Diagnostic performance was assessed retrospectively by using MRI/transrectal ultrasound fusion biopsy and 10-core systematic biopsy as the reference. From a total of 359 lesions, GS ≥ 7 tumor was present in 135 lesions (37.60%). Area under the ROC curve (AUC) revealed slightly lower values for peripheral zone (PZ) and transition zone (TZ) scoring in v2.1, but these differences did not reach statistical significance. A significant number of score 2 lesions in the TZ were downgraded to score 1 in v2.1 showing 0% GS ≥ 7 tumor (0/11). The newly introduced diffusion-weighted imaging (DWI) upgrading rule in v2.1 was applied in 6 lesions from a total of 143 TZ lesions (4.2%). In summary, PI-RADS v2.1 showed no statistically significant differences in overall diagnostic performance of TZ and PZ scoring compared to v2.0. Downgraded BPH nodules showed favorable cancer frequencies. The new DWI upgrading rule for TZ lesions was applied in only few cases. The standard is based on the consensus of a group of experts incorporating scientific evidence and their experience in prostate MRI. In 2012 the first version of PI-RADS was published1, and in late 2014 PI-RADS v2.0 was released2. PI-RADS has been rapidly accepted among the radiological and urological community and is being incorporated into clinical care pathways3, as many studies have shown good diagnostic accuracy of the reporting system8.The prostate imaging and reporting data system (PI-RADS) was created to standardize prostate MRI reporting with the goals of improving prostate cancer detection in MRI and achieving a more structured and comparable reporting quality9. PI-RADS v2.1 includes modifications to technical specifications as well as revisions in the scoring of prostate lesions. The scoring of transition zone (TZ) lesions has changed with regards to scores 1–3: typical benign prostatic hyperplasia (BPH) nodules are now scored as T2 weighted (T2W) score 1 as opposed to previously score 2. Atypical nodules, that are mostly encapsulated or homogenous without encapsulation, are now scored as T2W score 2 as opposed to previously score 3. Furthermore, a TZ lesion with a T2W score of 2 can be upgraded to an overall score of 3 when a DWI score of 4 or 5 is assigned. Another important change concerns DWI scores 2 and 3 for all zones: a linear or wedge-shaped ADC-hypointense or DWI-hyperintense lesion should be scored as score 2 now, whereas formerly indistinct ADC-hypointense lesions were scored into this category. The criteria for DWI score of 3 have been slightly modified: in PI-RADS v2.1 focal ADC-hypointense and/or focal DWI hyperintense lesions are scored a 3. Signal intensities can be marked in one of the sequences but should not be marked in both sequences. The definition of “marked” has been clarified as “a more pronounced signal change than any other focus in the same zone”9. With these updates in the scoring system, changes in the diagnostic accuracy of the scoring system are possible.In March 2019, modifications to PI-RADS version 2, termed PI-RADS v2.1, were published with carefully selected improvements and the assertion that further research was required to corroborate the incremental valueThe purpose of this study was to directly compare diagnostic performance of PI-RADS v2.0 and v2.1 for detection of GS ≥ 7 tumor using targeted MRI/transrectal ultrasound fusion biopsy (TB) and systematic 10-core biopsy as a reference standard.16. Patients were assigned randomly to initially four readers, 112 to 114 lesions by each radiologist. One reader was not available for the re-read after 12 months. The remaining 342 patients were re-read, 9 patients were then excluded because of unclear documentation of the targeted lesion’s location in TB (n = 7) or because no lesion was identified by the reader (n = 2), leaving a final study cohort of 333 patients. Figure This retrospective, single-center study was approved by the institutional review board (Ethikkommission der Charité – Universitätsmedizin Berlin) and patient consent was waived by the latter. All methods were carried out in accordance with relevant guidelines and regulations. All patients who received prostate MRI and subsequent MRI/TRUS fusion prostate biopsy in combination with 10-core systematic biopsy at our institution between January 2012 and July 2015 were considered eligible for this study (n = 454). Exclusion criteria were incomplete or non-standard MRI. Subgroups of the same collective have been included in earlier studies with endpoints independent from this study2). Axial T1-weighted imaging  of the whole pelvis and axial dynamic contrast enhanced imaging .MRI was acquired according to relevant ESUR guidelines. All imaging was performed on one of two identical 3T MRI scanners . All patients received at least biparametric MRI including T2W and DW images. In 186 patients (54.4%) DCE was performed additionally. Typical parameters were: Axial and coronal T2W imaging , axial diffusion-weighted imaging . Reviewing was performed in a blinded and randomized setting by using a standardized, in-house built reviewing software. Readers were blinded to histopathological results and all other patient-related data. In the first round, readers were instructed to mark dominant prostate lesions, to assign DWI-, T2W- and DCE-scores according to PI-RADS v2.0 guidelines, and to tag the localization of the lesion according to the segmentation model of PI-RADS v2.0. For MRI datasets without DCE images lesions were scored according to the rules for assessment without adequate DCE as specified in the PI-RADS v2.0 guidelines. At least 12 months later, the previously marked MRI datasets were presented to the same readers. Readers assessed the same patients as in the previous session. They were blinded to their previous assessment and were instructed to assign PI-RADS v2.1 scores to every lesion they had marked before as well as identify the matching v2.1 segments. This led to a 1:1 comparison of PI-RADS v2.0 and v2.1 for the same reader on a per lesion basis. The overall score per lesion was assigned in accordance with the respective algorithm for PI-RADS v2.0 or v2.1, following the dominant sequence and upgrading rules. Furthermore, lesions were attributed to either the peripheral zone (PZ) or the transition zone (TZ). Lesions that extended through PZ as well as TZ and lesions that were located in the anterior stroma (AS) or central zone (CZ) were assigned to either the PZ or the TZ group depending on the most likely zone of origin.All patients had undergone targeted MRI/TRUS fusion biopsy (TB) in combination with a 10-core systematic biopsy in the same session. Histopathological findings of cancerous lesions were classified according to the Gleason grading system. A GS of 3 + 4 or higher on TB or in a matching segment on systematic biopsy was considered positive for clinically significant prostate cancer (csPCa). If no cancerous tissue was found upon TB or systematic biopsy in the segment of the suspicious MR lesion, the respective lesion was considered negative for PCa. Histopathological findings that indicated non-cancerous changes were: no tumor cells, acute prostatitis, chronic prostatitis, granulomatous prostatitis, prostatic intraepithelial neoplasia or benign prostatic hyperplasia, each without the additional mentioning of a neoplastic disease.17 to test differences between the AUC of the two scoring systems and the three readers as well as interactions between readers and scoring systems18. Proportions of cancerous lesions per score were compared using the two-proportions Z-Test. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value were calculated after dichotomizing the PI-RADS assessment categories using a predefined cut-off value: a PI-RADS category of ≥ 3 was defined as positive. Sensitivity and specificity of the two versions were compared using the McNemar’s test. PPV and NPV were compared using the test by Lange and Brunner19.Data was analyzed on a per lesion basis and was modeled in terms of a factorial diagnostic trial involving the factors “scoring system” (PI-RADS v2.0 versus PI-RADS v2.1) and “reader”. The area under the ROC curve (AUC) of each reader and scoring system combination assessed the diagnostic accuracy. Since data are measured on an ordinal scale, we applied the nonparametric ANOVA-type statisticp < 5%. Statistical analysis was performed by F.K. and T.P. using R version 1.1.419 (www.r-project.org) and SAS version 9.4.Results were declared to be significant if The final study cohort consisted of 333 patients with a mean age of 66.8 years and a mean PSA level of 12.8 ng/ml. Patient characteristics are summarized in Table Diagnostic performance of the scoring systems was assessed by ROC curves and AUC. ROC-Curves for the overall score of PZ and TZ lesions are given in Fig. p > 0.05), but the difference did not reach statistical significance. In TZ lesions, the AUC was also slightly higher for PI-RADS v2.0 , again the difference did not reach statistical significance. Furthermore, no significant difference between performances of the three readers and no interactions between readers and scoring systems could be detected using the nonparametric ANOVA-type statistic (p > 0.05)17.For the PZ, ROC analysis revealed a slightly higher AUC for v2.0  while significantly less TZ lesions were assigned an overall score of 2 . In both zones, more lesions were assigned an overall score of 3 in v2.1, but the difference was not statistically significant. The frequency of GS ≥ 7 tumor per score did not differ significantly between the two versions  in v2.1 were marked in 31 of the lesions (9.06%). In all of these lesions neighboring segments were additionally marked.21.This study aimed at comparing the diagnostic performance of the recently released PI-RADS v2.1 to its predecessor version 2 using TB and systematic biopsy results as a reference. Our data demonstrate similar AUC and per-score frequencies of GS ≥ 7 tumor in both versions with no statistically significant differences. The distribution of GS ≥ 7 tumor per score in this cohort are comparable to those reported for PI-RADS v2.0 in other studies22. A reason for this different outcome may be the different study design in which only TZ lesions that were suspicious based on v2.0 were evaluated. With fundamental changes in the definitions of T2W-based scores 1 and 2 for TZ lesions in v2.1, some stirring up in lesion distributions in categories 1 to 3 could be expected. In v2.0 typical BPH-nodules that show a round shape and complete encapsulation were assigned to category 2, while in v2.1 they are scored into category 1. Likewise, in this study a significant number of TZ lesions scored as category 2 in v2.0 were downgraded into category 1 in v2.1, all of which showed no tumor upon biopsy (0/11). This change constitutes an important improvement in the new version; typical BPH nodules are highly unlikely to be GS ≥ 7 cancer and accordingly are now classified as category 1 lesions for which biopsy is typically not advised. Although this change may not yield a change of outcome for the majority of patients, since neither PI-RADS 1 nor 2 lesions are usually biopsied in practice, it improves the conclusiveness of the radiological report and can prevent unnecessary biopsies in patients with high clinical risk factors and without suspicious MR lesions.For the TZ we found very similar AUC for v2.0 and v2.1 with no significant differences. In a recently published study investigating TZ lesions in 58 patients read retrospectively by two radiologists, AUC for v2.1 was slightly higher without statistical significance Another significant change in PI-RADS v2.1 is the newly introduced DWI upgrade of T2W score 2 to an overall score of 3 in TZ lesions. In this study, this new upgrade was applied to only 6 lesions out of a total of 143 TZ lesions (4.2%). The impact of the new upgrading rule could thus be minor. Furthermore, none of the upgraded lesions yielded GS ≥ 7 tumor. These upgraded lesions may have contributed to the fact, that specificity in the TZ was lower for v2.1 in this study, with a higher rate of false positives. These results may, however, not be reliable due to the retrospective nature of the study with the possibility that these lesions might have been missed upon systematic biopsy and the fact that lesions were initially identified using PI-RADS v2.0 as well as the small sample size of 6 lesions.Furthermore, we evaluated a proposed decision rule for TZ scoring: Lesions with a T2W score of 3 and a DWI score of 1 or 2 could be downgraded to an overall score of 2. In this study, 6 lesions were applicable to this decision rule and none of these lesions yielded GS ≥ 6 tumor. Unnecessary biopsies in these patients could have been prevented with this decision rule. With only a small number of cases, this proposed decision rule needs validation in a larger cohort.9. The added specification regarding the shape of these lesions has led to an upgrading of oval or round shaped lesions in the PZ to a score of 3. These specific lesions showed an unfavourably low frequency of GS > 7 tumor (9.1% or 1/11) in this study.In our study, AUC for PZ lesions was slightly lower in v2.1 than in v2.0. To date, there is no literature published concerning diagnostic accuracy of v2.1 in the PZ that our data could be compared to. The modified definition of DWI score 2 might have contributed to the lower AUC of v2.1 found in this study. In v2.0, “indistinct hypointense on ADC” lesions are scored into DWI category 2, whereas in v2.1 lesions of this category should be “linear/wedge shaped hypointense on ADC and/or linear/wedge shaped hyperintense on high b-value DWI”Additionally, we noticed a small inconsistency with the new DWI score 2 definition that is applicable for all zones. The above mentioned, new criteria describe the appearance of prostatitis in the PZ, despite the fact that prostatitis presents differently in the TZ. Additional clarification regarding DWI score 2 in the TZ could thus be provided in the future.Regarding the segment model of the prostate, two new segments have been introduced in PI-RADS v2.1: the left and right posteromedial PZ segment in the base. Nine percent of the investigated lesions in this study extended through this newly defined segment on either side but none of these were limited to this specific segment. While the addition of these segments can further specify the localization of a lesion, we did not identify any lesions in this cohort that would have benefited substantially from a biopsy in this specific segment.23 reports in a large, prospective, multicentre study, that GS ≥ 7 tumor would have been missed in 7.6% (95% CI 4.6–11.6%) of patients, had TB not been done. Furthermore, readers in this study marked lesions on basis of v2.0 so that in the second review session evaluation was limited to lesions marked in the first review. AUC of v2.1 could thus be biased, e.g. the number of BPH nodules with marked restricted diffusion could be underestimated. Moreover, 45.6% of the included patients received only biparametric MRI without DCE, thus limiting reliability of results in the PZ in category 3 and 4. Furthermore, the readers in this study worked at the same institution, and each lesion was assessed once by a single reader. Interobserver agreement was not investigated in this study. It was previously shown to be moderate for PI-RADS v2.024. At the time of publication only two studies have addressed interobserver agreement of v2.1 finding a substantial agreement25. Lastly, although this analysis is based on a large patient cohort, subgroup analysis is underrepresented.There are a number of limitations to this study. The standard of reference was histopathologic results of TB based on the initial read (done prior to the study) and systematic 10-core biopsy. A more reliable standard of reference would be histopathology after surgical prostatectomy, which however would bias the underlying collective towards medium-aggressive cancers. In addition, due to the retrospective design of the study some lesions identified by the readers were not targeted in the biopsies taken ahead of the re-read and could have been missed in the systematic biopsies. Generally, cancer detection rates are higher when TB and systematic biopsy are combined, but the rate of missed GS ≥ 7 tumor in systematic biopsy only is acceptable; Rouvière et al.9. PI-RADS version 2.1 has been mainly created to clarify certain assessment criteria and improve inter-reader agreement while maintaining the framework of version 2. In this study, we find no objections to implementing version 2.1 with regards to its overall diagnostic performance. However, in the TZ, we found significant reduction of category 2 lesions in favor of category 1, corresponding to typical BPH nodules with a favorably low frequency of prostate cancer. Meanwhile, the newly introduced DWI upgrade of T2W score 2 lesions in the TZ was applied only in a few cases, which questions the impact of the new upgrading rule.The adoption of version 2.1 did not yield significant differences in diagnostic performance regarding per-score frequencies of GS ≥ 7 prostate cancer and ROC-AUC. This is in line with the objective of version 2.1 as stated in the original documentSupplementary Information.