Virus-like particles (VLPs) are nanostructures that possess diverse applications in therapeutics, immunization, and diagnostics. With the recent advancements in biomedical engineering technologies, commercially available VLP-based vaccines are being extensively used to combat infectious diseases, whereas many more are in different stages of development in clinical studies. Because of their desired characteristics in terms of efficacy, safety, and diversity, VLP-based approaches might become more recurrent in the years to come. However, some production and fabrication challenges must be addressed before VLP-based approaches can be widely used in therapeutics. This review offers insight into the recent VLP-based vaccines development, with an emphasis on their characteristics, expression systems, and potential applicability as ideal candidates to combat emerging virulent pathogens. Finally, the potential of VLP-based vaccine as viable and efficient immunizing agents to induce immunity against virulent infectious agents, including, SARS-CoV-2 and protein nanoparticle-based vaccines has been elaborated. Thus, VLP vaccines may serve as an effective alternative to conventional vaccine strategies in combating emerging infectious diseases. The majority of currently available vaccines are predominantly based on either inactivated (killed) or live attenuated approaches. Although these traditional vaccines have been used effectively against various infectious diseases, some of these have several limitations, which include their lower potential to induce a stronger immune response and poor efficacy . Recent Virus-like particle (VLP) technology presents an alternative platform for developing effective vaccines to combat infectious diseases of serious concern, and it is moving in parallel with mRNA and viral-vector based vaccines . VLPs arThese bioinspired nanostructures have repetitive and highly dense antigens from different virulent agents that aids in triggering a strong immune response. Moreover, these highly immunogenic molecules have the self-assembling property of viral proteins . They ar® (GlaxoSmithKline) and Recombivax HB® (Merck & Co) against HBV, Gardasil® (Merck & Co) and Cervarix® (GlaxoSmithKline) against HPV, Hecolin® (Xiamen Innovax Biotech Co.) against HEV  and eukaryotic . The functionality of VLPs can be increased through modifying their exterior or interior surface by displaying the heterologous epitopes of interest using different methods like peptide conjugation, genetic fusion, and chemical crosslinking . The VLPBased on their structural complexity VLPs can be classified into two groups: enveloped and non-enveloped VLPs. Both groups display foreign antigens .These VLPs are often made up of single or many self-assembled components of the targeted pathogen or viral protein structures. There is no host cell membrane (lipid envelope) in these newly formed VLPs. The expression of the major viral nucleocapsid proteins is primarily responsible for the formation of these VLPs. Non-enveloped VLPs are still being investigated as preferable candidates for developing subunit vaccines against several pathogenic diseases as they are easier to produce and purify . FurtherIn comparison to non-enveloped VLPs, enveloped VLPs (eVLPs) have a far more complicated composition. These nano-structures consist of a cell membrane acquired from the host cell called an envelope, with viral proteins present on the outer surface . One or in vivo stability is also quite uncertain (Chimeric virus-like particles (cVLPs) are considered an effective tool for developing vaccines that provide broader, more powerful, and comprehensive protection against emerging infectious diseases . The comncertain .+ and CD8+ cells proliferation  signals or homologous interactions, which control the production of cytokines. In order to enhance the efficacy of VLPs, different molecules like Toll-like receptor ligands, biologically active mediators, or other cell receptors can also be attached to VLPs .® (® against human papillomavirus (HPV) vaccine was licensed by the FDA  (HBsAg and HBcAg), and the tobacco mosaic virus (TMV) . The fir® . Later,  the FDA . Followi the FDA . Several the FDA . However the FDA .At Present, VLPs are as effective as conventional vaccines, with the additional benefit of being safer. Nevertheless, several obstacles in the successful development of VLP-based vaccines need to be addressed. The main challenge is to identify issues related to downstream processing in the production of clinically viable VLPs  for theiVLPs are generally considered more stable than subunit vaccines . HoweverSome VLP-based vaccines are much more complex and, as a result, have a higher cost of production. The numerous impurities associated with eVLPs pose a daunting challenge. During downstream processing (DSP) various impurities like host cell debris (HCD), host cell protein (HCP), and host cell DNA (HCD) are co-purified as in the case of enveloped baculovirus particles. These contaminations can cause undesirable side effects in vaccines if not removed properly during DSP . Large sThe genetic fusion of the sequences of epitope into VLPs can sometimes be challenging, as VLPs may lose their self-assembly property or cause particle misfolding . GeneticDifferent expression systems (ESs) were utilized to make VLP vaccines, including plant, mammalian, insect, yeast, and bacteria .Escherichia coli  and demonstrated the similar binding pattern for VLPs assembled in E. coli as for Sf9 cells assembled VLPs. Similarly, in a recent study, the full-length CP of type 2 porcine circovirus and VP2 protein of porcine parvovirus were expressed in E. coli, which were self-assembled into VLPs. The study suggested that the expression of the CP and VP2 in E. coli is possible for the mass development of VLP vaccines , the ORF2 protein region 368–606 aa was purified in vitro from the insoluble E. coli fraction that is assembled into VLPs. This HEV VLP promises 100% effectiveness in clinical trials against symptomatic HEV and is approved as a vaccine for commercial use in China  poor immunogenicity, (b) inability to develop recombinant proteins with mammalian−like post-translational modifications (PTMs), (c) issues of protein solubility, (d) inability to create the correct disulfide bonds, and (e) presence of bacterial endotoxins/or lipopolysaccharides in preparation of recombinant proteins .E. coli strain with a genetically modified LPS which does not elicit an endotoxic reaction in humans, has been developed  and expressed in P. pastoris. The ELISA results for L1-HPV-16 Ab as well as L2-HPV-16 Ab detection indicated a positive reaction with sensitivity comparable to the commercial testing kit. Similarly, in the P. pastoris ES, P. pastoris ES seems to be a superior and safer alternative to the baculovirus (Bv) ES.The FDA has approved some yeast-derived VLP vaccines, including Gardasillciparum . Some stP. pastoris through co-expression of the viral protease 3CD and the structural precursor protein P1.During viral infection, enteroviruses like poliovirus, generate empty capsids that are antigenically indistinguishable from that of mature virions. The recombinant synthesis of such capsids with the help of heterologous systems like yeast has enormous potential as candidates for the VLP vaccine. E. coli. Another disadvantage of the yeast ES is its lack of resemblance to mammalian ESs in protein PTMs, particularly glycosylation  of influenza virus and its utilization in generating an arsenal of influenza-derived VLPs with functional modifications in influenza virus hemagglutinin (HA), which are expected to regulate the VLP-derived immunological response .The insect cell-Bv EVSs have proven to be as effective as conventional egg- and cell-based approaches in influenza virus vaccine production with additional features like high production yields, and short production times. Moreover, rBVs construction has become faster, easier, and more flexible, allowing for the fusion of genes from different types and/or subtypes of influenza viruses inside the same expression vector. The Zaire Ebola virus serotype (ZEBOV) is the most virulent and has the highest mortality rates among other serotypes. ZEBOV-VLPs development have been achieved in insect and mammalian cell lines via co-expression of 3 viral structural proteins, the nucleocapsid protein (NP), the matrix structural protein (VP40), and the glycoprotein (GP). A technique for generating ZEBOV-VLPs in insect cell line was reported by The main limitations of the insect cell ES are (a) protein contamination by enveloped baculoviruses, (b) difficult to scale-up, and (c) simpler N-glycosylation than mammalian cells .For more than two decades, various mammalian cell lines such as murine myeloma , Chinese hamster ovary (CHO), murine C127, baby hamster kidney (BHK21), HT-1080, and HEK293 were established as a possible source of commercialized protein therapeutics for medical purposes, due to their capability to properly fold, assemble, and post-translational modification to proteins . SystemsSeveral studies reported the efficient production of VLPs from mammalian cell cultures. Expression systems based on mammalian cells require large-scale production facilities, like fermentation bioreactors, which are prohibitively expensive to construct. As a result, the high cost of production is a challenging part of the cell-based mammalian ES . The oth“Molecular farming” is a term used to describe the utilization of plants or plant cells to produce recombinant proteins or other biologic drugs for use as cosmeceuticals, biopharmaceuticals, therapeutics, vaccines, and other reagents . Plants Several studies have extensively discussed the concepts for constructing VLPs in various plant ESs, efficient growth of VLPs in plant hosts, and the self-assembly of multiple structural proteins of viruses in plants. Plants for the production of the VLP platform are not entirely acceptable due to comparatively lower production levels of VLP than mammalian ESs and plant-specific N-glycosylation of glycoproteins . NonetheSeveral VLP vaccines have been produced and are being used against different viral and parasitic infections in recent years . The devZika virus (ZIKV) is a small-enveloped mosquito-borne neurotropic positive-strand RNA virus of the family Flaviviridae . ZIKV inZIKV has an RNA genome with only one open reading frame, and a solitary polyprotein is formed. This protein-strand is cut into 7 non-structural proteins  and 3 structural proteins  by cellular and viral proteases. E-protein is engaged in the binding of viral particles as well as its fusion . E-proteMany VLPs for ZIKV have been developed . Garg etIt’s a severe and periodic infectious disease caused by CHIKV (chikungunya-virus) transmitted by a carrier mosquito. Symptoms are high fever, skin rash, etc. No vaccine is available at present, but many VLP-vaccine candidates are under development in different stages . ProtectInfluenza virus (Iv) infections are the leading cause of chronic human respiratory symptoms, leading to severe public health outcomes about endemic and seasonal infections and even result in unpredictable pandemic outbreaks . Iv is aHA, the key antigen involved in infection, attaches to the residues of sialic acid on the cell membrane surface, facilitating the Iv to enter the host cell. NA appears to be less frequent on the viral surface as compared to HA, with a generally observed NA:HA ratio of 1:4. Its enzymatic function is critical in the cleavage of sialic acid, thereby facilitating viral release from the infected host cell surface. NA activation also enables the successful penetration by influenza to mucus through a mechanism involving cleavage of sialic . Kim et Recently, Human papillomaviruses (HPVs) are members of the Papillomaviridae family of tiny, circular, double-stranded DNA viruses that cause cervical cancer, the world’s second most fatal disease in women after breast cancer . Currentin vitro or the basement membrane in vivo and induces a conformational shift  is highly conserved across all HPV serotypes and has long been considered a major potential target antigen for developing an HPV vaccination with broad protection. In contrast to CP (L1), the CP (L2) cannot be used for VLP production and is, therefore, less immunogenic . The reaal shift .Several methods have been applied to improve and enhance the L2 peptides low immunogenicities, such as concatemeric fusion peptides consisting of L2 epitopes of various types of HPV, combined with immune activating TLR agonists or using repeated surface arrays of epitopes of L2 on particulate immunostimulatory platforms, such as VLP of TMV, bacteriophages MS2 or PP7, Adeno-associated virus, Lactobacillus casei, or bacterial thioredoxin .Another promising strategy to improve L2 low immunogenicity is the presentation of L2 epitopes on the surface via VLPs assembled from HPV L1-L2 chimeric proteins. It was achieved through the genetic insertion of highly conserved B cell epitope RG1 of HPV16 L2 into the surface loop (DE) of the protein HPV16 L1 leading to its multivalent (360x) immunogenic display on the surface of VLP, whereas the conformational neutralization antigenic determinants of the HPV16 L1 scaffold remained mostly preserved. Immunizations with this chimeric RG1-VLP caused high type-specific HPV16 titers and extensive cross-neutralization of heterologous mucosal and distantly associated cutaneous HPVs , 2013.® (Gilead Sciences), Paxlovid® (Pfizer), Molnupiravir® (Merck and Co.) have been approved by the FDA and WHO for treatment of patients infected with SARS-CoV-2. Due to the limitations including development, cost, and distribution of targeted drug therapies against the virus, researchers are aiming their focus and efforts on developing vaccines for the long-term fight against COVID-19.With worldwide causalities crossing 2 million people and more than 20 million individuals affected in more than 200 countries this past year, the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has emerged as a nuisance across the globe. The COVID-19 global pandemic resulting from SARS-CoV-2 has impacted billions of individuals and emerged as the first major global catastrophe after the 2009 H1N1-pandemic . CurrentVLPs against SARS-CoV-2 can help combat the widespread pandemic. VLPs can act as therapeutic agents and viable vaccines against this viral disease, with the addition of serving as a feasible diagnostic tool . SARS-CoBefore developing SARS-CoV-2 VLP-based vaccines, previous work on VLPs against the preceding and closely related strains must first be considered. VLPs have previously been demonstrated to be effective in eliciting specific humoral and cellular immune defenses against SARS-CoV in mice by production in rBV ES . Ho et aCurrently, the NVX-CoV2372 is the only commercially available VLPs based vaccine against SARS-CoV-2. This vaccine is manufactured by the display of SARS-CoV-2 S-protein in rBV, which are then used for mass production by infecting moth cell expression system. This vaccine has shown various levels of effectiveness in different trials across separate countries . CurrentDifferent mammalian ESs were utilized for the synthesis of SARS-CoV-2 VLPs. Results indicate that SARS-CoV-2 VLPs produced from Vero E6 cell line are more durable and integrated than those derived from HEK-293T cells . RecentlA capsid VLP centered SARS-CoV-2 vaccine (ABNCoV2) has shown efficient neutralization of SARS-CoV-2 in mice models. The ABNCoV2 vaccine is made by displaying the receptor-binding domain of the SARS-CoV-2 S-protein in insect (Drosophila) cells . ResearcProtein nanoparticles (NPs) can help in the development of vaccines for immuno-evasive pathogens such as HIV, influenza, malaria, and can aid in the fight against emerging virulent strains by either acting alone or acting as carriers for targeted drug delivery . The engin vivo. HA-ferritin NPs showed higher immunogenicity and greater protection against a viral challenge as compared to soluble protein (HA).Among the wide array of platforms available for the display of antigens, ferritin has emerged as a major protein complex that can be combined with NPs for vaccine development . Due to in vivo studies in mice (Recently, a structure-based design of self-assembling protein NP immunogen that produces protective and potent antibody responses against SARS-CoV-2 has demonstrated the development of innate immunity in  in mice . Similar in mice .in vivo applications of NPs are often restricted by several challenges due to their organic nature (in vivo conditions. The surface protein attached to NPs affects their biological behavior and changes their functionality, which occasionally results in gain or loss-of-function (Though protein-NPs can serve as viable candidates for vaccine development, c nature , includic nature . The terfunction . The profunction . Some cafunction .Over the past two decades, resistance in some pathogens against commercially available antimicrobial drugs has increased drastically, mainly due to unregulated over-the-counter sales, misuse and overuse of drugs, and genetic adaptations. The process of drug development is a rigorous task and requires ample time, funding, and repeated trials to commercialize a viable product that could aid in the fight against numerous emerging pathogens. Due to the time-consuming limitation, especially access of patients to appropriate drugs is hindered, whereas the resistance in pathogens is consistently increasing. Therefore, preventive measures are of significant importance to address the emergence of infectious diseases. In the efforts to combat such diseases, VLPs have come to light in the healthcare industry, with multiple applications ranging from vaccine development, drug delivery systems, and molecular diagnostics. VLP based approaches provide an alternative to the available conventional methods for vaccine developments. Currently, some VLP-based vaccines are commercially available against perilous pathogens like ZIKV, HCV, HBV, and HPV. Meanwhile, efforts are underway in the production and designing of efficient VLP vaccines against various emerging virulent pathogens, including SARS-CoV-2. However, there are still some major hurdles that need to be addressed before VLP-based therapeutics can compete with conventional drug therapies in terms of cost and effectiveness. Nonetheless, VLP-based medicinal strategies may be used extensively in the future along with pharmaceutics to aid in the fight against numerous infectious diseases.HT and SB conceptualized and drafted the manuscript. HT, SB, and SA wrote the manuscript. HT was responsible for designing and illustrating the figures in the manuscript. MA reviewed and edited the manuscript. BA supervised and proofread the manuscript. All authors have read and agreed to the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The HFA-PEFF score from the ESC is based on expert consensus and refers to a multi-step evaluation process of patients with dyspnea to diagnose HFpEF. The scoring systems aim to replace current simpler and phenomenological American College of Cardiology/American Heart Association definitions of HFpEF, which relies on signs and symptoms of heart failure, evidence of abnormal diastolic parameters, and preserved ejection fraction. This opinion piece offers concerns over attempts to protocolize a vastly heterogenous group of patients using diagnostic scoring systems.Due to an aging population, heart failure with preserved ejection fraction (HFpEF) is on the rise. Yet this condition remains difficult to characterize and diagnose. There have been two recently proposed risk scores for the evaluation and diagnosis of patients with suspected HFpEF , 2. ThesThe HFA-PEFF algorithm suggests that evaluation of patients with dyspnea begin with ruling out cardiac and non-cardiac comorbid conditions that may mimic heart failure. Specifically, the algorithm targets coronary artery disease, lung disease, and anemia as comorbidities that need to be ruled out, but identifies obesity, diabetes, and atrial fibrillation as common risk factors in patients with HFpEF. However, teasing out the contribution of various comorbidities, including those that either mimic or are consistent with HFpEF, may be difficult in practice and have limited clinical implication .2FPEF score  is identified. Importantly, a thorough evaluation of patient signs/symptoms (such as dyspnea or BNP elevation) and echocardiographic abnormalities  can include specialist referral, ischemic evaluation, strain echocardiography, cardiac MRI, genetic testing, or other indicated testing which can lead to specific management decisions without the need to first establish a general HFpEF diagnosis. The multiple neutral clinical trials in patients with presumed HFpEF further suggest that the approach to diagnosis and management deserves re-evaluation  13–18).–18.13–18Much work is needed to optimize the diagnosis and management of a heterogeneous group of patients presenting for evaluation of dyspnea and volume overload. Future evaluation and management should focus on characterization of patient populations into subgroups based on underlying pathophysiology , 20. UndDA and PP conceived and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Today’s zoos and aquariums claim to be shifting from being entertainment facilities to centers for the conservation of biodiversity. To make this shift, scientific research, including understanding the biology of endangered species and improving the environment of captive animals, is essential. Several studies have examined the trends in the number of research papers published by zoos and aquariums globally and indicated that this number has increased over the past few decades. In this study, we examined the trends in the number of papers published by Japanese zoos and aquariums over the past 62 years to determine whether research activities are also developing in Japan. We found that the number of research papers has significantly increased and that the research fields have diversified since around 1990. However, we also found some problems: about a quarter of the institutions have published no papers, research targets in zoos were biased toward captive mammals and that aquariums conducted little research on animal welfare. Addressing these issues, we would argue, will help Japanese zoos and aquariums to make further progress.With the shift in their social roles, modern zoos and aquariums are required to develop scientific research. Although zoos and aquariums worldwide have reported an increase in the number of papers they publish and the diversification of their fields in recent decades, the specific circumstances in Japan are slightly unclear. We listed peer-reviewed papers authored by Japanese zoos and aquariums using search engines and quantitatively evaluated the changes in the number of papers published over 62 years. Our results showed that papers published in Japan have increased remarkably since the 1990s, and research fields have diversified as in the rest of the world. In particular, joint research with research institutes has seen an upward trend, and the instances of English-language papers have increased. Meanwhile, the content of the research was biased. In zoos, research on animal welfare has been increasing, but the focus was heavily biased toward captive mammals. Aquariums contributed to the understanding of local ecosystems through the fundamental study of wildlife, but there were fewer papers on improving husbandry. Our results indicated that while research by Japanese zoos and aquariums is developing, research on welfare, conservation, and education regarding native endangered species must still be improved. In recent years, zoos have been expected to not only provide entertainment but also to play a social role in biodiversity conservation and environmental education ,2. To acAlthough only a few attempts have been made to evaluate whether zoos and aquariums are contributing to conservation and education through scientific research, several studies have been reported in the past several years that have quantified the growth in the number of peer-reviewed papers ,5,6,7,8.Andrias japonicus), a species endemic to Japan, by Asa Zoological Park. Based on the breeding ecology they discovered in their field research [Journal of Japanese Association of Zoos and Aquariums (JJAZA), an original peer-reviewed journal published by the JAZA, which meant other local governments or conservation organizations in Japan have been able to utilize it, but it has been missed by the international research database. In quantifying the expansion of research activities in zoos and aquariums, it is important to include these articles rendered in the local language.However, it is difficult to determine whether zoos and aquariums are contributing to conservation, as those studies lack some perspectives. First, it is unclear whether the research is led by the zoos. Sometimes zoos become one of the authors simply by providing samples to outside biologists, but if this is the case for most of the published papers from zoos, it cannot be regarded as a substantial contribution by them. This is one way to evaluate whether the first author belongs to zoos, but none of these studies mention it. Second, it is not clearly stated whether the research targets are captive or in the wild. Although important findings can be obtained from research on captive animals, the extent to which research is conducted on wild populations is considered to be an important indicator to evaluate whether the goal of biodiversity conservation is being achieved. Finally, these studies did not count papers in local languages. They used journal databases ,5,6,7,8,research , they prresearch . This zoBased on these points, we clarify the shift in the number of peer-reviewed papers by Japanese zoos and aquariums. Although a declining trend was reported in the number of papers in the JJAZA , the oveThe research contribution of each of the 140 active JAZA member institutions, 90 zoos and 50 aquariums, extant on 1 April 2021, was quantified. We listed peer-reviewed papers in which one or more authors belonged to those JAZA institutions. Specifically, we intended to include original papers, review articles, case reports, short communications, and technical materials published in peer-reviewed scientific journals, including the JJAZA. Non-peer-reviewed papers such as bulletin papers published by universities or museums, dissertations, conference proceedings, Special Issues, book reviews, and book chapters were not counted in the total. Papers published between 1959, when the JJAZA was founded, and 2020, were listed.Previous studies have used databases such as the Thomson Reuters Web of Science or Elsevier Scopus ,5,6,7,8,For all articles, the following elements were recorded: year of publication, institution, first author’s affiliation, language, research target, and research field. The affiliation of the first author was recorded as being a JAZA member or not; this indicates whether the paper is authored mainly by zoo and aquarium staff or co-authored with another institution. The language of the article was identified as Japanese or English. Research targets were classified as captive animals, wild animals, both, or others . “Wild aWe compiled 2789 papers from 103 institutions. The mean per institution was 21.23, and the median was 11. There was a wide variation among institutions, with the top four accounting for 33% of the total, while 37 (27 zoos and 10 aquariums) had no publications at all. The overall number of papers from the JAZA showed an increasing trend . The groThe trends of results differed between 63 zoos and 40 aquariums. On the one hand, in zoos, the number of first-authored papers had decreased compared to the peak year of 1967. However, the number of co-authored papers had increased, and the recent overall trend was upward. On the other hand, in aquariums, the number of papers had increased rapidly from the 1990s, although it had been low until then. Both first- and co-authored papers continued to increase until 2020, despite the decrease in member institutions since 2005.The transition of the number of papers in English and Japanese is illustrated in In the results for zoos only, the ratio of papers written in English began to increase around 1995. For the past 10 years, the number of studies in English and Japanese has been almost the same. Aquariums published a more notable number of papers in English than zoos in recent years, nearly double the number in Japanese.Additionally, the number of first-authored papers in English by JAZA staff is shown in In aquariums, research on captive fish had been the main focus, but the target has diversified since 2000. While studies targeting both captive animals and “others” are increasing, the growth in the number of papers on wild animals was more pronounced, exceeding the total number of studies on captive animals. Initially, most research highlighted issues that had some association with the basic husbandry of zoo and aquarium animals, such as Animal care, Reproduction, and Veterinary science . HoweverA limited amount of the published literature about Animal care and Ecology came from aquariums in the 1900s. However, since 2000, the number of studies in Ecology, Morphology, and Taxonomy has increased greatly compared to other fields. As in zoos, studies in Genetics, Physiology, and Reproduction were also on the increase, but there was almost no research on Animal welfare. Further, similar to zoos, there were few papers on Education, Exhibition, and Nutrition.Our analysis over 60 years showed that JAZA zoos and aquariums have increasingly contributed to scientific research through the publishing of peer-reviewed papers. According to Escribano et al. , over thThis is similar to the global trend in that physiological and genetic methods are becoming more common as biology develops , and theIn contrast, most papers published by JAZA aquariums were descriptive studies of taxonomy, ecology, and morphology , many ofMost papers published until around 1990 were written in Japanese, but the number of English-language papers increased gradually, and in recent years, the number of papers in English has exceeded those in Japanese . To dissMore than half the total papers were in Japanese. Most organizations involved in local ecosystem conservation and the citizens who are our targets of environmental education are native language speakers. Scientific papers written in English may limit the ability to communicate important results to local practitioners and decision-makers, such as environmental managers . Indeed,The social role of zoos and aquariums around the world is changing to that of conservation and environmental education, and the increasing number of peer-reviewed papers may be evidence of their contributions through scientific research. As we have shown, in Japan, JAZA’s scientific research has developed positively. However, this has also clarified that the JAZA is lagging behind the rest of the world in research fields that could contribute to modern goals for the zoos. Most studies by JAZA zoos focused on captive mammals, and it seems that more work needs to be conducted on conservation and improving the welfare of endangered species in other taxonomic groups, though it is also a problem worldwide. Promoting research on animal welfare and behavior should provide a useful scientific background for the formulation of the husbandry guidelines that the JAZA is currently working on. In contrast, the negligible number of welfare papers published is a serious problem for JAZA aquariums. The relative ease of collecting individuals in fishery-rich Japan may have helped promote aquarium research on wildlife while neglecting efforts to care for captive individuals. Discussions on the welfare of domestic aquatic species in the JAZA are still in their infancy; for instance, while a large-scale study is developing worldwide to improve the welfare of cetaceans , breedin


Circulating myeloid precursors are responsible for post-natal osteoclast (OC) differentiation and skeletal health, although the exact human precursors have not been defined. Enhanced osteoclastogenesis contributes to joint destruction in rheumatoid arthritis (RA) and tumour necrosis factor (TNF) is a well-known pro-osteoclastogenic factor. Herein, we investigated the interplay between receptor activator of nuclear factor kappa-Β ligand (RANK-L), indispensable for fusion of myeloid precursors and the normal development of OCs, and TNF in directing the differentiation of diverse pre-OC populations derived from human peripheral blood.Flow cytometric cell sorting and analysis was used to assess the potential of myeloid populations to differentiate into OCs. Transcriptomic, epigenetic analysis, receptor expression and inhibitor experiments were used to unravel RANK-L and TNF signalling hierarchy.+ monocyte (MO) differentiation into OCs by inhibiting osteoclastogenesis to favour macrophage development. In contrast, a distinct previously unidentified CD14−CD16−CD11c+ myeloid pre-OC population was exempt from this negative regulation. In healthy CD14+ MOs, TNF drove epigenetic modification of the RANK promoter via a TNFR1-IKKβ-dependent pathway and halted osteoclastogenesis. In a subset of patients with RA, CD14+ MOs exhibited an altered epigenetic state that resulted in dysregulated TNF-mediated OC homoeostasis.TNF can act as a critical homoeostatic regulator of CD14These findings fundamentally re-define the relationship between RANK-L and TNF. Moreover, they have identified a novel pool of human circulating non-MO OC precursors that unlike MOs are epigenetically preconditioned to ignore TNF-mediated signalling. In RA, this epigenetic preconditioning occurs in the MO compartment providing a pathological consequence of failure of this pathway. OC differentiation and maturation from myeloid precursors relies on receptor activator of nuclear factor kappa-Β (RANK) signalling and murine studies suggest that tumour necrosis factor (TNF) is a potent coactivator.One of the gold standard therapy for rheumatoid arthritis includes blocking TNF although a consistent portion of patients do not respond and display progressive joint destruction.We have discovered a novel non-monocyte (MO) osteoclast (OC) precursor population in the human peripheral blood and demonstrated its epigenetic and transcriptomic imprint towards OC differentiation.+ MOs by inhibiting their differentiation into OCs to favour macrophages and showed how this mechanism is epigenetically perturbated in a portion of patients with rheumatoid arthritis (RA) with active disease.We have demonstrated that TNF can act as a homoeostatic regulator of classical peripheral CD14These data provide an important insight into the cellular and epigenetic heterogeneity in RA and can be used to develop alternative therapeutics for those patients that do not respond to current therapies.Osteoclasts (OCs) are polykarion bone-resorbing cells that can be derived from either yolk sac erythro-myeloid progenitors or bone marrow/circulating monocyte (MO) precursorsMurine studies have shown that in combination with RANK-L, TNF is a direct maturation/activation stimulus of OC precursors.−CD16−CD11c+ myeloid precursor population that is epigenetically predisposed to rapidly differentiate into OCs. Notably, this population is unresponsive to a previously unrecognised homoeostatic TNF-mediated signal that fundamentally governs cell fate decisions in CD14+ MOs that favours Mϕ development. Moreover, we provide evidence that this effect is mediated by TNF via epigenetic regulation of the RANK promoter in circulating CD14+ MOs and that this pathway is perturbed in RA.Herein, we report the existence of a human CD14Detailed experimental procedures and analyses are provided in 10.1136/annrheumdis-2020-219262.supp1Supplementary data+ MOs are regarded as classical OC precursors,−HLA−DR+CD14+CD16−, intermediate Lin−HLA−DR+CD14+CD16+ and non-classical Lin−HLA−DR+CD14dimCD16++) and other myeloid cells (Lin−HLA−DR+CD14−CD16−). Culture of these populations with macrophage-colony stimulating factor (M-CSF) and RANK-L verified that classical MOs differentiate into mature OCs while intermediate and non-classical populations only produced few and variable numbers of small OCs−HLA−DR+CD14−CD16− myeloid cells differentiated into OCs, with numbers comparable to classical CD14+ MOs  and plasmacytoid DCs (CD14−CD16−CD11c−)−CD16−CD11c+ population was able to adhere and differentiate into mature OCs (Circulating CD14D14+ MOs . Lin−HLAture OCs .+ MOs or in vitro differentiated MO-derived pre/immature DCs or Mϕ takes in excess of 7–14 days.+ MOs are preincubated overnight with M-CSF to generate CD14+ pre-OCs. This preincubation leads to upregulation of RANK and CSF1R (M-CSF receptor) transcripts  in RA.+ (or CD1C cells) are present in the inflamed synovium and have the molecular potential to differentiate into OC; although their contribution to RA bone erosion remains elusive.Cell in the myeloid compartment and TNF play key roles in RA pathology. Analysis of circulating CD14 reduced . This is+ MOs controls the inhibitory TNF effect, that RA blood CD14+ MOs may exhibit a distinct epigenetic state. To gain further insight into this dysregulation, genome-wide H3K4me3 histone modification was investigated using ChIPseq. Comparison between patients with biological naïve RA with moderate to severe disease (DAS28=4.44±0.9) and healthy control CD14+ MOs, identified 6764 significantly differential peaks (adjusted p>0.05)  . Pathway p>0.05) .10.1136/annrheumdis-2020-219262.supp2Supplementary data+ MOs at the RANK and TNFR1 loci but not TNFR2 and CSF1R  contribute to the bone-associated OC pool. Unfortunately, these assays are not currently possible. Further studies will need to define a way of identifying Lin−HLA−DR+CD14−CD16−CD11c+—versus MO-derived OCs in situ to better understand this alternative route to mature OCs. Moreover, they should also investigate the global molecular and epigenetic signature of Lin−HLA−DR+CD14−CD16−CD11c+ and CD14+ precursors cells, and their respective mature OCs, to ultimately identify the specific precursor cells and molecular mechanisms involved in inflammatory joint destruction.Based on this work, we suggest that in a normal self-resolving inflammatory event (which can include sterile/acute inflammation), Lin−HLA−DR+CD14−CD16−CD11c+ precursor route to mature OCs represents an unexplored independent mechanism to mature OCs and potentially erosive progression in RA. A detailed understanding of this new pathway could reveal an as yet untargeted pathway in the disease context. In summary, these data support further evaluation of these pathways in RA and other diseases associated with bone pathology to discover their utility as alternative therapeutic strategies to abrogate erosive progression.Anti-TNF therapy is one of the gold standard treatments for multiple inflammatory-mediated diseases including RA. It is paradoxical however, given that TNF inhibitors are known to be antierosive,


AimThe cytokines particularly tumor necrosis factor-alpha (TNF-α) have a substantial role in the pathophysiology of rheumatoid arthritis (RA). The goal of this study was to evaluate the role of serum TNF-α as a competent biomarker of disease activity in RA and to assess the correlation of serum TNF-α with DAS28-ESR (disease activity score-erythrocyte sedimentation rate in 28 joints) and other markers expressed in serum of RA patients.MethodologyThe study was conducted from May 2020 to October 2020 after approval from the Ethical Review Committee of Ziauddin University. This cross-sectional study included 90 diagnosed cases of RA from 30 to 65 years with the complaint of arthralgia. Patients from the rheumatology clinic were enrolled in the study by a non-probability consecutive sampling technique. Informed consent was taken from each patient and they were assessed through a set of questions based upon disability in the performance of daily activities due to RA. Evaluation of serum levels of anti-cyclic citrullinated peptide (ACCP), rheumatoid factor (RF), erythrocyte sedimentation rate, and TNF-α were done by enzyme-linked immunosorbent assay (ELISA). Patients were segregated into groups based upon DAS28-ESR with erythrocyte sedimentation rate as an inflammatory marker. The Kruskal Wallis test was applied for the comparison of different variables in these groups. Spearman correlation was applied for the association between different variables. Multiple variable analysis was performed to assess the predictability of disease activity by serum markers included in the study.ResultsThe results of our study disclosed a significant difference in ACCP, TNF-α, tender joint count of 28 joints (TJ-28), swollen joint count of 28 joints (SJ-28), and health assessment questionnaire-disability index (HAQ-DI) in disease activity groups. A significant correlation of serum TNF-α with DAS28-ESR in RA patients was observed.ConclusionThis study illustrated a significant correlation of serum TNF-α with DAS28-ESR in RA patients. We found that expression of serum TNF-α may intensify the inflammatory activity in early RA, therefore, RA patients must be screened for this cytokine to monitor that disease activity could be useful for patients undergoing anti-TNF therapy. Cytokines belong to a diverse family of proteins associated with the inflammatory activity in autoimmune diseases. They portray a significant role in the preservation of homeostasis. An imbalance in the cytokine network leads to enhanced inflammation. Therefore, these cytokines might be valuable as predictive biomarkers of disease activity ,2. CytokAmong these cytokines tumor necrosis factor-alpha (TNF-α) is a principal cytokine, that is chiefly produced by macrophages . TNF-α iIt has been proposed by many studies that disease activity can be monitored by diagnostic markers for RA, the rheumatoid factor (RF), and anti-cyclic citrullinated peptide (ACPP) ,14. HoweIn RA, the inflammatory activity can be evaluated by a disease activity score with an erythrocyte sedimentation rate (ESR) as the inflammatory marker (DAS28-ESR) in 28 joints. Evaluation of disease activity guides the clinicians and patients toward a standardized treatment approach. The DAS28-ESR is a widely used measure for the clinical assessment of RA patients. It is a score that includes 28 tender and swollen joint counts, along with a patient global assessment, and a physician global assessment. Values are defined by high disease activity (HAD), moderate disease activity (MDA), low disease activity (LDA), or remission. Improvement or worsening of disease is reflected by changes in score values . The goaThis cross-sectional study was conducted from May 2020 to October 2020 after approval from the Ethical Review Committee of Ziauddin University (Refcode: 0920319MIPHY) and included 90 diagnosed cases of RA from 30 to 65 years with the complaint of arthralgia. Patients from the rheumatology clinic were enrolled in this study on the basis of a non-probability consecutive sampling technique. Patients were diagnosed on the basis of 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria for RA . Study pA venous blood sample of 5 ml was taken from each patient by a trained phlebotomist and it was centrifuged at this 3000 rpm to get the serum and stored in multiple aliquots at −80 °C. RF levels were determined using an RF enzyme-linked immunosorbent assay (ELISA) kit . The reference range was from 5.0 to 100 IU/mL, and analytical sensitivity was 1.0 IU/mL. According to the manufacturer’s protocol, the assay sample and buffer were incubated together with RF-horseradish peroxidase (RF-HRP) conjugate in a microtiter plate pre-coated with anti-RF antibody for one hour. The wells were washed and incubated with a substrate for the HRP enzyme. A blue-colored complex was formed and later after the addition of stop solution color changed to yellow. The intensity of color was measured spectrophotometrically at 450 nm in a microplate reader.ACCP levels were determined using an ACCP antibody, ELISA kit . The reference range was from 25 to 500 U/mL and analytical sensitivity of 1.0 U/ml. According to the manufacturer’s protocol, the assay sample and buffer were incubated together with CCP-Ab-HRP conjugate in CCP-Ab pre-coated plate for one hour. The wells were washed and incubated with a substrate for the HRP enzyme. A blue-colored complex of enzyme-substrate reaction was formed. A stop solution was added to terminate the reaction. The intensity of color was measured spectrophotometrically at 450 nm in a microplate reader.Cat.No.E0082Hu; Bioassay Technology Laboratory, Shanghai, China). The reference range was from 3 pg/ml to 900 pg/ml and analytical sensitivity of 1.52 pg/ml. According to the manufacturer’s protocol, TNF-α present in the sample is added and binds to human TNF-α antibodies coated on the wells. Then, streptavidin-HRP is added and binds to the biotinylated TNF-α antibody. After incubation plate wells were washed. Substrate solution was added and color developed in proportion to the amount of human TNF-α. The reaction was terminated by the addition of stop solution and absorbance was measured at 450 nm.Assay of serum TNF-α was done by a sandwich ELISA , version 20 . Median and interquartile ranges were calculated for numeric variables The Kruskal Wallis test was applied for comparison of three groups. Spearman's correlation was applied for finding a correlation among the study variables. Multiple variable analysis was performed to assess the predictability of disease activity by serum markers included in the study.Table Our results regarding correlation of DAS28-ESR and serum TNF-α with biochemical, clinical, and metabolic parameters, showed significant correlation with serum ACCP , ESR , TJ-28 , and serum TNF-α . There was no correlation of BMI , RF , and HAQ-DI  with DAS28-ESR. Significant correlations of serum TNF-α with BMI , and DAS28-ESR  was observed. There was no correlation of TNF-α with RF, ACCP, ESR, TJ28, SJ28, and HAQ-DI  triggers the release of inflammatory cytokine TNFα via the Fcγ R-dependent pathway by macrophages . These cMultivariable analyses revealed that high levels of serum TNF-α shows an increased probability of severity in disease activity in RA patients. However, Dissanayake et al. in their study observed a significant association of DAS28 with the cellular expression of TNF-α .This research highlights not only the importance of TNF-α alpha in the severity of disease in early RA patients but can also contribute to the monitoring of disease activity patients on DMARD treatment. There were certain limitations to our study. It was a single-centered cross-sectional study and patients’ samples were collected only once. They were not followed up after few months of treatment for RA. To validate our results, we propose that, multicenter studies, with a larger sample size and patient progress over a couple of months, are needed in selected RA patient groups.Our study has found a significant correlation of TNF-α with DAS28-ESR in newly diagnosed RA patients. The available therapeutic agents that target this cytokine have excellent clinical effects in RA patients. In future studies on targeting TNF-α in the early stage of RA and its evaluation to monitor the progress of disease could be useful for patients undergoing anti-TNF therapy. If these patients are followed up properly, then serum TNF-α levels together with the assessment of clinical activity of disease by DAS28 could be used as a guide in determining the dose and intervals between dosing of TNF-α inhibitors in order to accomplish the desired therapeutic response. The number of patients in our study was relatively small, however, our findings are highly suggestive and require further large-scale investigations in the future to make use of serum TNF-α levels as a potential biomarker for evaluation of disease activity both before and after the start of anti-TNF therapy.


Limited data are available regarding cardiac expression of molecules involved in heart failure (HF) pathophysiology. The majority of the studies have focused on end‐stage HF with reduced ejection fraction (HFrEF) without comparison with healthy subjects, while no data are available with regard to HF with preserved ejection fraction (HFpEF). HFpEF is a condition whose multiple pathophysiological mechanisms are still not fully defined, with many proposed hypotheses remaining speculative due to limited access to human heart tissue. This study aimed at evaluating cardiac expression levels of key genes of interest in human biopsy samples from patients affected with HFrEF and HFpEF in order to possibly point out distinct phenotypes.n = 6) and HFpEF (n = 7) and healthy subjects (n = 9) undergoing elective cardiac surgery for valvular replacement, mitral valvuloplasty, aortic surgery, or coronary artery bypass. Real‐time PCR was performed to evaluate the mRNA expression levels of genes involved in somatotropic axis regulation , in adrenergic signalling , in myocardial calcium handling (SERCA2), and in TNF‐α. Patients with HFrEF and HFpEF showed reduced serum IGF‐1 circulating levels when compared with controls . At myocardial level, HFrEF showed significant decreased GHR and increased IGF‐1R expressions when compared with HFpEF and controls , while no differences in the local expression of IGF‐1 mRNA were detected among the groups . With regard to calcium handling and adrenergic signalling, HFrEF displayed significant decreased levels of SERCA2  and increased levels of GRK2  and GRK5 , while no significant difference was found in ADRB1  and ADRB2 mRNA expression  when compared with HFpEF and controls. Finally, no changes in the local expression of TNF‐α were detected among groups.Total RNA was extracted from left ventricular cardiac biopsies collected from stable patients with HFrEF (Heart failure with reduced ejection fraction and HFpEF patients with stable clinical condition display a distinct molecular milieu of genes involved in somatotropic axis regulation, calcium handling, and adrenergic derangement at a myocardial level. The unique opportunity to compare these results with a control group, as reference population, may contribute to better understand HF pathophysiology and to identify novel potential therapeutic targets that could be modulated to improve ventricular function in patients with HF. In the last decades, a relevant piece of scientific literature has contributed to increase the knowledge of the molecular mechanisms underlying heart failure (HF) onset and progression, which enabled the identification of novel therapeutic targets. Mounting evidence supports the role of anabolic deficiencies in HF pathophysiology,n = 6) and HFpEF  groups according to 2016 European Society of Cardiology Guidelines,n = 3) and hypertension (n = 6) as major co‐morbidities. For the purpose of the study, patients with ejection fraction between 40% and 50% were intentionally excluded. Nine subjects with normal cardiac structure and function, without symptoms or sign of HF, undergoing thoracic surgery were recruited as healthy controls. At the time of enrolment, all participants underwent a complete clinical examination, including New York Heart Association (NYHA) functional class assessment and transthoracic echocardiography, and peripheral blood samples were collected. Cardiac biopsies were obtained during surgical procedures by highly qualified and expert surgeons following the maximum safety standards. Samples with a minimum size of 200 mm3 were collected from the apical portion of the left ventricle or the junctional portion between the free wall of the left ventricle and the interventricular septum (IVS). Each sample was taken by direct vision, through the aortic valve, and with the scalpel. No full‐thickness samples were taken, and no biopsy needle was used. The techniques and the modalities were exactly the same adopted to remove the muscle spur in the context of obstructive hypertrophic cardiomyopathy. No histopathologic evaluation was performed.Thirteen HF patients undergoing elective cardiac surgery for valvular replacement, mitral valvuloplasty, aortic surgery, or coronary artery bypass, assigned to HFrEF , and GH receptor (GHR)], adrenergic signalling , myocardial calcium handling (SERCA2), and tumour necrosis factor‐alpha (TNF‐α). Each sample was analysed in duplicate. The relative comparative CT method was used to analyse the quantitative PCR data . In the CT method, the amount of target normalized to an endogenous reference and relative to a calibrator sample is given by 2−ΔΔCT. The amount of each transcript was normalized to the amount of the reference gene PPIA (cyclophiline A) expressed in the same sample. The calibrator is the same defined stock sample analysed in triplicates in each of the quantitative PCRs. Thus, the values represent arbitrary but linear relative amounts of each transcript.Afterwards, each sample was transferred in a sterile tube containing RNAlater  and stored at −80°C until assayed. Total RNA was extracted from cardiac biopsies through TRI Reagent kit . Real‐time PCR  was performed using predesigned TaqManP value < 0.05 was considered statistically significant. Statistical analysis was performed using the R statistical programming environment, version 3.5.The distribution of the parameters was tested with the Kolmogorov–Smirnov test. The intergroup differences were tested with the one‐way ANOVA, with Holm correction as appropriate. Correlations between IGF‐1 circulating levels and IGF‐1R mRNA were examined using Spearman rank‐order correlation. A The study was approved by the ethics committee (protocol number 2535) and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. All participants gave written informed consent.Table P < 0.001, respectively; Figure Figure P < 0.05; Figure Figure R = −0.79, P = 0.01; R = −0.92, P > 0.001; and R = −0.85, P = 0.03, respectively).No differences were observed in terms of age, sex, kidney function, and haemoglobin levels among the three study groups. Notably, HFrEF and HFpEF patients displayed comparable stable clinical status, as suggested by the similar NYHA functional class and serum BNP levels, with average values <100 pg/mL Table 1. FurthP < 0.05, Figure P < 0.05, respectively; Figure P < 0.05) and GRK2  were significantly increased in HFrEF when compared with HFpEF and controls, respectively .


Surprisingly, the potency of sennoside B was 5.7-fold higher than that of the synthetic TNF-α inhibitor SPD304. Molecular docking was performed to determine the binding mode of sennoside B to TNF-α. In conclusion, we successfully developed a novel competition binding screening method to discover small molecule TNF-α inhibitors and identified the natural compound sennoside B as having exceptional potency.Natural products (NPs) have played a significant role in drug discovery for diverse diseases, and numerous attempts have been made to discover promising NP inhibitors of tumor necrosis factor α (TNF-α), a major therapeutic target in autoimmune diseases. However, NP inhibitors of TNF-α, which have the potential to be developed as new drugs, have not been reported for over a decade. To facilitate the search for new promising inhibitors of TNF-α, we developed an efficient competitive binding screening assay based on analytical size exclusion chromatography coupled with liquid chromatography-tandem mass spectrometry. Application of this screening method to the NP library led to the discovery of a potent inhibitor of TNF-α, sennoside B, with an IC Tumor necrosis factor-alpha (TNF-α), an important pleiotropic cytokine, is a well-known central biological mediator of critical pro-inflammatory and immunomodulatory effects ,2. Overe50 value of 4.6 µM. X-ray crystallography revealed that SPD304 displaces a subunit of the TNF-α trimer to form a complex with the resultant dimer [Suramin was the first small-molecule to demonstrate inhibitory effect on TNF-α through a direct action on the ligand rather than on its receptors . Later, nt dimer . Severalnt dimer . Ma et ant dimer . Luzi etnt dimer . Cao et nt dimer . Using Tnt dimer . Despitent dimer ,16. TherNatural products (NPs) have played a significant role in the drug discovery and development. According to a recent review paper, among 1394 small-molecule drugs approved over the last four decades, NPs and NP derivatives account for 441 drugs (32%) . The conEncouraged by the fact that natural compounds with high potency and low toxicity that directly target TNF-α have rarely been reported, we developed a competitive binding screening assay coupled with analytical size exclusion chromatography (SEC) and liquid chromatography (LC) tandem mass spectrometry (MS) and applied the screening method to an in-house NP compound library to discover of novel small-molecule TNF-α antagonists. In vitro cell-based assays were performed to validate the inhibitory effect of the NPs and in silico modeling was performed to predict their interactions. Collectively, we observed that sennoside B, a NP small-molecule, effectively inhibited TNF-α and its downstream signaling pathways. Moreover, the binding mode of sennoside B with the TNF-α crystal structure was analyzed using molecular docking, and the key residue interactions between sennoside B and TNF-α were predicted.2 (PGD2) and prostaglandin E2 (PGE2), and internal standards (IS) of d4-prostaglandin D2 (d4-PGD2) and d4-prostaglandin E2 (d4-PGE2) were purchased from Cayman Chemical . Dimethyl sulfoxide (DMSO), actinomycin D (AMD), and SPD304 were purchased from Sigma-Aldrich . L929 and HeLa cells were purchased from ATCC and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS.All HPLC grade solvents were purchased from Fisher Scientific . Centrifugal ultrafiltration filters (Microcon YM10) were obtained from Millipore . Human recombinant TNF-α was obtained from PeproTech . Fetal bovine serum (FBS) was purchased from the American Type Culture Collection . Prostaglandin DTo develop a competitive binding screening assay, the known TNF-α inhibitor SPD304 and the NP compound were incubated with TNF-α in 70 µL buffer (pH 7.5) consisting of 100 mM Tris, 10% glycerol, 50 mM KCl, and 1 mM EDTA at room temperature for 20 min. The final concentration of SPD304 and the NP compound was 3.5 μM each, and of TNF-α was 0.1 μM. After incubation, the mixture solution was loaded into an SP-6 Bio-gel SEC column , followed by spin-down at 1000 g for 4 min at 4 °C. The eluted solution containing the ligand and TNF-α complex was washed three times with 50 mM ammonium acetate (pH 7.5), followed by another centrifugation at 13,000 g for 10 min. Then, the ligands were dissociated using 400 μL of methanol. The ultrafiltrate containing the ligand was dried using a SpeedVac . The sample was reconstituted with 100 µL of 50% methanol, and 2 µL of the sample was injected into the LC–tandem MS system.The samples were analyzed using an Agilent 1290 HPLC system  interfaced with a triple quadrupole mass spectrometer  with multiple reaction monitoring (MRM) scan in electrospray ionization positive mode. The separation was achieved using a Waters ACQUITY BEH  C18 column  fitted to a Waters ACQUITY UHPLC BEH C18 Vanguard pre-column . The mobile phase consisted of mixtures A  and B . The flow rate was set to 0.6 mL/min. The following gradient program was employed: 0–10 min, 5–100% B. Re-equilibration was applied for 3 min between analyses. The oven temperature was set to 40 °C. The mass spectrometer parameters were as follows: voltage, 5.5 kV; probe temperature, 400 °C; gas 1, 35; gas 2, 40; curtain gas, 40; and dwell time, 200 ms. The MRM parameters for the SPD304 were set up as follows: Q1 (548.4), Q3 (274.3), DP (100), EP (10), CE (43), and CXP (15).4 cells/well and cultured overnight. Prepared DMEM containing different concentrations of sennoside B was added to the cells and incubated for 18 h. Subsequently, cells were washed with phosphate-buffered saline three times and cell viability was assessed by adding CCK-8 solution to the treated cells and incubating for 2 h. The optical density in the CCK-8 assay was measured at 450 nm using a microplate reader .A cell viability assay was performed using Cell Counting Kit-8  according to the manufacturer’s instruction. Briefly, mouse L929 and human HeLa cells were seeded in 96-well plates at a density of 2.0 × 104 cells/well and cultured overnight. Prepared DMEM containing different concentrations of sennoside B (6.25–100 µM), 10 ng/mL of TNF-α, and 1 µg/mL of AMD was added to the cells, and incubated for 18 h. The cell viability was assessed by microscopic examination and CCK-8 assay. SPD304 was used as a positive control.Treating mouse L929 cells with TNF-α induces cytotoxicity, and the susceptibility of L929 to TNF-α is greatly increased when cells are exposed to AMD ,19. ThisL929 and HeLa cell lines were used for western blotting analyses. After the treatment, the cells were lysed and the total protein concentration was determined using Bradford reagent . Total protein samples (20 µg) were separated using sodium dodecyl polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride  membranes. The membranes were blocked with 5% BSA in TBST and the protein levels of caspase 3, cleaved caspase 3, inhibitor of kappa B-alpha (IκB-α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using anti-caspase 3 primary antibody , anti-cleaved caspase 3 primary antibody , anti-IκB-α primary antibody , and anti-GAPDH primary antibody , respectively. Densitometric analysis of the bands was performed using the LAS4000 system .5 cells/well and cultured overnight. Prepared DMEM containing different concentrations of sennoside B (25–100 µM), 10 ng/mL TNF-α, and 1 µg/mL AMD was added to the cells and incubated for 18 h. The collected cell medium was centrifuged at 13,000 g at 4 °C to remove dead cells. Then, 2 µL of d4-PGE2 and d4-PGD2  were added to the collected supernatant. The supernatant was cleaned using solid-phase extraction cartridges . The filtrate was dried under nitrogen flow and reconstituted with 100 µL of 50% aqueous methanol, and 2 µL of the reconstituted filtrate was injected directly into the LC-high resolution MS system.L929 cells were seeded in 6-well plates at a density of 5.0 × 102 and PGE2 was performed according to a previous report [m/z 200–500) in negative ion mode at a mass resolving power of 70,000 (FWHM at m/z 200). The electrospray ion source parameters were as follows: spray voltage, 3.5 kV; sheath gas (N2 > 95%), 40; auxiliary gas (N2 > 95%), 10; sweep gas (N2 > 95%), 1; capillary temperature, 320 °C; heater temperature, 300 °C; and S-lens RF level, 50. The full scan mass spectra were acquired using the following parameters: automatic gain control (AGC), 5 × 105 and maximum accumulation time, 100 ms.Analysis of PGDs report  using a The crystal structure TNF-α was retrieved from the Protein Data Bank (ID: 2AZ5). TNF-α and its dimeric form were used as the receptors and sennoside B as the ligand. Molecular docking was performed using the AutoDock 4 . Proteinp < 0.05, after one-way analysis of variance (ANOVA) with Tukey multiple comparison test.All results are presented as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 7.0 software . Statistical significance was considered at The scheme illustrating the overall screening method described in the Materials and Methods section is shown in Using the validated method, a competitive binding screening assay was performed with the natural product compound library listed in 4 at a retention time of 7.2 min. However, when sennoside B was added to the mixture of SPD304 and TNF-α, the signal of SPD304 was significantly attenuated to 0.8 × 104. Additionally, the peak of sennoside B was simultaneously detected at a retention time of 2.8 min. These results showed that sennoside B competes with SPD304 for binding to TNF-α. In the case of acutumidine, the result was not reproducible, which led us to focus on sennoside B for further validation.To further validate whether the two hits, acutumidine and sennoside B, competitively bind to TNF-α with SPD304, a competition binding assay with the two compounds was performed and the final eluate was analyzed using UPLC-Q Exactive hybrid quadrupole-orbitrap MS. As shown in Next, we evaluated the efficacy of sennoside B using a TNF-α dependent L929 cell cytotoxicity assay ,20,26. I2 and PGE2, are involved in diverse cellular responses, including cell proliferation, apoptosis, angiogenesis, and inflammation [2 and PGE2 compared with the control group. This effect was significantly inhibited after treatment with 25, 50, and 100 µM of sennoside B , as observed earlier  [There are several viable strategies to antagonize TNF-α using small molecules. SPD304 directly binds to the inner pocket of the homotrimer of TNF-α and disassembles the homotrimer, resulting in a dimer that has no signaling activity against TNFR . The natHere, we developed a screening assay based on analytical SEC LC–tandem MS to identify small molecule inhibitors of TNF-α, that can compete for the active site with the known inhibitor SPD304. In silico molecular docking-based virtual screening method offers a fast and cost-effective way to screen diverse chemical libraries against the target of interests, although it requires rigorous validation steps. In vitro protein evolution methods, such as phage display, bacterial display, or mRNA display are powerful methods to screen peptides or proteins that have strong affinities against the desired target, but their utilization for small-molecule screening is limited. Other in vitro affinity-based assays, such as ELISA or SPR, can be used for small molecule screening, but require specific antibodies against the target or target immobilization. Our analytical SEC LC–tandem MS screening method utilizes an intact TNF-α homotrimer in solution without any chemical derivatization on the surface, which allows a much more straightforward and high-throughput screening. In our study, application of this screening method to in-house prescreened NP library compounds led to the identification of sennoside B, which showed higher efficacy than the known inhibitor SPD304.Cassia angustifolia, which has been widely used as a natural laxative in traditional medicines [50 value of 0.32 µM in human HeLa cells. To date, the most potent NP TNF-α inhibitor was quinuclidine, as reported by Chan et al. [50 value of 5 µM in TNF-α-induced cell toxicity assays. Although the assayed platform was different, they used SPD304 as a positive control, and upon comparing the biological efficacy of compounds using SPD304, sennoside B showed an IC50 value approximately 5.7 times lower than SPD304, whereas quinuclidine had an IC50 value 1.6 times higher than SPD304. To our knowledge, sennoside B is by far the most potent NP TNF-α inhibitor known to date. The potency of sennoside B is comparable to that of UCB-9060, the most potent synthetic compound TNF-α inhibitor, with an IC50 value of 116 nM in the L929 cell-based assay [Sennoside B, a dianthrone glycoside, is one of the main compounds extracted from edicines . Additioedicines  and inhiedicines . We discn et al. . QuinuclSennoside B is believed to directly bind to the active site of TNF-α, as evidenced by LC-MS detection of both SPD304 and sennoside B. When sennoside B was added to the incubation mixture consisting of SPD304 and TNF-α, and the analytical SEC assay was further processed, the signal of sennoside B appeared, whereas the signal of the positive compound SPD304 was attenuated due to the competitive binding of sennoside B with SPD304 against TNF-α. Molecular docking using the crystal structure (PDB code 2AZ5) of the TNF-α dimer also indicated the possible hydrophobic interactions and hydrogen bonds between sennoside B and the binding site of TNF-α, suggesting that sennoside B effectively binds to and inhibits TNF-α, as specifically described in the results of In this study, we developed and validated a competition binding screening method based on analytical SEC coupled with LC–tandem mass spectrometry for the discovery of small-molecule TNF-α inhibitors. Among the screened natural product library compounds, sennoside B showed an exceptional effect on TNF-α inhibition and is probably the most potent natural product TNF-α inhibitor identified to date. The activity of sennoside B was further validated by a series of in vitro cell assays that showed that sennoside B successfully inhibited TNF-α induced cell death and subsequent downstream signaling pathways in both mouse and human cell lines. Finally, the binding mode of sennoside B with the TNF-α crystal structure was analyzed using AutoDock 4, and the key residue interactions between sennoside B and TNF-α were predicted. Currently, the optimization of sennoside B to enhance its pharmacokinetic and metabolic properties is in progress for drug development.


The purpose of this study was to compare heart rate (HR) and heart rate variability in young endurance athletes during nocturnal sleep and in the morning; and to assess whether changes in these values are associated with changes in submaximal running (SRT) and counter-movement jump (CMJ) performance.During a three-week period of similar training, eleven athletes (16 ± 1 years) determined daily HR and heart rate variability (RMSSD) during sleep utilizing a ballistocardiographic device (Emfit QS), as well as in the morning with a HR monitor (Polar V800). Aerobic fitness and power production were assessed employing SRT and CMJ test.Comparison of the average values for week 1 and week 3 revealed no significant differences with respect to nocturnal RMSSD , morning RMSSD , morning HR , SRT HR , SRT blood lactate , CMJ  or training volume . There was a strong correlation between morning and nocturnal HRs during week 1  and week 3 , as well as between morning and nocturnal RMSSD values .This study concluded that HR and RMSSD obtained during nocturnal sleep and in the morning did not differ significantly. In addition, weekly changes in training and performance were small indicating that fitness was similar throughout the 3-week period of observation. Consequently, daily measurement of HR indices during nocturnal sleep provide a potential tool for long-term monitoring of young endurance athletes. Over the past several decades, the scientific approach to finding a balance between endurance training and recovery has continued to grow , resultiRecovery from endurance training involves a multitude of physiological responses, with the cardiovascular system playing a key role in this context , 7. ThisHRV is regulated by the sympathetic and parasympathetic activity of ANS, and two common methods for measuring HRV are time and frequency domain . RMSSD mTherefore, the purpose of this study was to access the ability of nocturnal HR and RMSSD values determined by ballistocardiography (BCG) to predict the respective validated morning values recorded with a heart rate monitor during orthostatic tests . The aimEleven well-trained young endurance athletes participated in this study. All participants were students at a sports academy high school competing and training for cross-country skiing (10 subjects) and biathlon (1 subject) year-round. Characteristics of the athletes are presented in This study occurred during a 3-week period of normal endurance training in preparation for the upcoming racing season (early November). Preceding participation, practice tests were provided so subjects were familiarized with the CMJ and SRT protocols. Running and jump exercises were already incorporated into the tested individuals’ normal training so a preparatory training period was unnecessary. The SRT and CMJ tests were performed during week 1 and week 3 of the study. During night sleep, ANS state was assessed with nocturnal HRV and HR analysis, collected using a ballistiocardiographic (BCG) sleep-tracking device . In addition, morning values for HRV and HR were also evaluated with orthostatic tests performed using a Polar V800 heart rate monitor  and H10 heart sensor . Morning and nocturnal values for HR and HRV were measured daily throughout the study. At the beginning of the study, body fat percentage and weight were measured using the bioimpedance method . Nocturnal HR and HRV were monitored using a contactless sleep-tracking device. This device employs BCG to numerically and graphically depict repeated movements , such aspm) and morning (RMSSDam) sleep and the whole night average (RMSSDNOC) was considered to be the mean of both values. Although previously the average 3-minute values for the entire night have been used for analysis [Subjects were instructed to place the device under the mattress near the chest area to minimize the distance to the heart and thus, maximize the signal quality. The subjects were unable to detect the device’s presence, but the device sensed when body weight was present and automatically began the recording process. The recording continued at a sampling rate of 100 Hz, stopping when the subject left the bed in the morning. Nocturnal HR and HRV data were collected using continuous 3-minute periods, and disrupted and/or poor signals were excluded from the data. The average HR values for each 3-minute period were calculated and these averages were used to determine whole night daily averages. Nocturnal HRV was interpreted relative to time, by utilizing RMSSD. To determine several different nocturnal RMSSD values during each night, average RMSSD for each 3-minute period was calculated and used to produce a graphical representation of nocturnal RMSSD values. The endpoints of the best linear fit for each night were chosen to represent the average RMSSD values for evening . Test results provide average values for HR and HRV in both the supine and standing position as well as peak HR when standing. Minimal compliance for HRV and HR analysis was set at 3 days each week, therefore, if a subject did not have 3 days in one of the test weeks they were omitted from the study.Morning orthostatic tests were performed using a Polar V800 heart rate monitor and H10 heart sensor. This sensor has previously shown acceptable levels of agreement with a 12-lead electrocardiogram system for recording RMSSD  and the Counter movement jump (CMJ) performance tests  were evaSRT tests were conducted to evaluate aerobic fitness. The SRT performed during this study was designed to elicit heart rates (HR) at approximately 90% of maximal HR so significant changes in HR variation were measurable . The SRTDescriptive statistics were calculated for all variables and all values are reported as means ± SD with the 95% confidence interval (CI). Sample distribution was tested using the Shapiro-Wilk test for nocturnal and morning HR indices  as well as performance test variables. Normal distribution was present for all weekly variables as well as the difference between weeks for nocturnal and morning HR indices. A within-subject approach was applied by utilizing paired sample t-tests to examine the differences between nocturnal versus morning measurements and the differences between the different test weeks. To evaluate the extent of agreement between morning and nocturnal values Pearson’s correlation coefficient (r) was calculated. To assess the difference between measurements made in the morning or during the night, the coefficient of variance and 95% CI for HR (HRCV) and RMSSD were determined each week for all HRV parameters (HRVCV).The extent of correlation among the morning and nocturnal measures for HR and three different nocturnal HRV measures  was tested using Intra-class correlation coefficient (ICC) or reliability coefficient, a measure of the reliability of measurements. ICC estimates and their 95% confidence intervals were calculated by a single-rating, consistency-agreement, 2-way mixed-effects model . Values NOC , morning RMSSD , morning HR , SRT HR , SRT blood lactate , CMJ  or training volume . Individual and group values for HR and RMSSD during morning and sleep are presented in CV and RMSSDCV values are presented in NOC during week 1  and week 3 . The main results from the current study were that HR and RMSSD values obtained with a BCG device under real-life conditions during sleep were in good agreement to morning values derived from orthostatic tests , providiAt the same time, our hypothesis that resting RMSSD and HR would be slightly lower and higher, respectively, than the corresponding nocturnal values turned out to be incorrect, since the former value obtained with orthostatic tests in the morning was higher and HR very similar at both time-points. Previous studies on the HRV of individuals with high levels of stress under real-life conditions revealed a lower variation in the values obtained with orthostatic tests after awakening, but not during nocturnal rest, suggesting that parasympathetic withdrawal occurs upon awakening . In the Previous research has challenged the evaluation of HR and RMSSD to monitor athletes, suggesting that the high day-to-day variation and variability of these values limits their usefulness because small and moderate changes often occur inside differences that are normally expected . HoweverHeart rate variability corresponds to countless stimulus and is influenced by physiological, psychological and environmental factors . As a reAlthough coaches and athletes have followed HR for decades, current research has placed greater emphasis on HRV, considering it a more sensitive tool . ResearcWe hypothesized that changes in HR and RMSSD would be associated to changes in SRT and CMJ tests. However, during the present study, training remained similar and no significant differences were present in SRT or CMJ, suggesting that the above changes are not due to changes in training load or volume . PreviouAdditionally, during normal in-season training, good reliability was shown in both CMJ and SRT with a slight indication that SRT may be a more sensitive monitoring tool to predict performance changes . TherefoOur main concern in connection with our current investigation may be the lack of standardization of the measurement procedure, due to the collection of HR indices in individual home environments. Furthermore, although previous research has shown that resting HRV values do not differ between males and females , 36, theThe present findings indicate that the accuracy and reliability of weekly average values for HR and RMSSD obtained during sleep employing a BCG-based device is acceptable. Thus, day-to-day monitoring of nocturnal HR and RMSSD appears to be a convenient and valid approach for long-term monitoring of young endurance athletes.The application of BCG-based values used in the present study, provides facile monitoring of HR and RMSSD with a fully automatic and contact-free analysis without added time constraints . Based o


Despite significant advances in therapeutic possibilities for the treatment of inflammatory bowel disease (IBD) in recent years, there is still a big room for improvement. In particular, biological treatment can induce not only clinical remission but also mucosal healing of the gastrointestinal tract. Among these therapeutic molecules, anti-tumor necrosis factor-alpha (anti-TNF-α) antibodies were the first to revolutionize treatment algorithms in IBD. However, due to the parenteral route of administration and systemic mode of action, TNF-α blockers are characterised by high rates of immunogenicity-related loss of response and serious adverse events. Moreover, intravenous or subcutaneous therapy is not considered patient-friendly and requires occasional, direct contact with healthcare centres. To overcome these limitations, several attempts have been made to design oral pharmaceutical formulations of these molecules. It is hypothesized that oral anti-TNF-α antibodies therapy can directly provide a targeted and potent anti-inflammatory effect in the inflamed gastrointestinal tissues without significant systemic exposure, improving long-term treatment outcomes and safety. In this review, we discuss the current knowledge and future perspectives regarding different approaches made towards entering a new era of oral anti-TNF-α therapy, namely, the tailoring of biocompatible nanoparticles with anti-TNF-α antibodies for site-specific targeting to IBD. In particular, we discuss the latest concepts applying the achievements of nanotechnology-based drug design in this area. Inflammatory bowel disease (IBD) represents chronic, disabling, and incurable gastrointestinal disorders of unknown origin. There are two main disease entities classified as IBD—Crohn’s disease (CD) and ulcerative colitis (UC). Approximately seven million people suffer from IBD globally . FurtherThe main goal of IBD therapy is to heal the inflamed gastrointestinal tract, achieving steroid-free remission, and protect the patients from irreversible bowel damage and disability . The theDespite the apparent advances in the quality of care in IBD, there is still a broad range of unanswered questions regarding how to improve the therapeutic outcomes further. One of the new strategies undertaken in this area is developing new pharmaceutical formulas of already used and effective drugs to overcome their known limitations, including immunogenicity and adverse events. In this paper, we present the current knowledge and future directions in terms of the oral administration of mAbs, as these biological molecules have been described as the most suitable approach to decorate nanoparticles for site-specific targeting. In particular, we discuss the application of the newest achievements in nanotechnology-based drug design in this area.Infliximab (IFX) and adalimumab (ADM) are the two most widely used TNF-α inhibitors , 6. IFX Both drugs are administered parenterally—IFX intravenously or subcutaneously, ADA—subcutaneously –7. TheseSeveral steps have been undertaken to improve these results. One of the therapeutic strategies adopted depends on a combination of a TNF-α inhibitor together with an immunomodulator (a combo therapy) . MultiplAnother possible limitation of the current forms of anti-TNF-α therapy, associated with the parenteral route of drug administration, is a need for regular and direct contact with healthcare providers, including the need for hospitalizations. This limitation is relevant mainly for patients receiving intravenous IFX injections. While subcutaneous administration of ADM or IFX is relatively easy, and most patients can do it without any assistance, there is still a need to undergo training supervised by a professional healthcare provider and visit the healthcare centre occasionally. This limitation is fundamental in the context of restrictions and recommendations for maintaining social distance related to the coronavirus disease-19 (COVID-19) pandemic , 13, 14.Considering all the limitations of currently available pharmaceutical formulations of anti-TNF-α antibodies discussed above, it is of high importance to search for new solutions to improve therapeutic outcomes and their safety. Developing a unique formula of orally administered TNF-α inhibitors seems to fulfil all criteria for being a significant step forward in the quality of care in IBD. Table Interestingly, there is some evidence on the potential oral application of TNF-α-neutralizing antisense oligonucleotides or small interfering RNAs . HoweverCurrently, mAbs are administered mainly parenterally. Oral delivery of proteins remains a significant challenge. However, due to dynamic advances in drug design and chemistry, the possibilities to develop this specific pharmaceutical formula of different therapeutic molecules have improved significantly. Several approaches have been proposed to increase the stability of orally administered antibodies against the acidic and protease-rich environment of the gastrointestinal tract ; these iOchi et al. were the first to show that oral administration of a mAb—anti-CD3 effectively suppressed experimental autoimmune encephalomyelitis . In 2010As a consequence of these developments, interest in the oral administration of mAbs for targeted intestinal drug delivery in IBD has also increased in recent years , 23. ForThe studies on the efficacy and tolerability of OKT3 in IBD represented a fascinating approach. However, it investigated a novel therapeutic molecule administered in a completely new, experimental way. It seems more feasible and clinically relevant to design a novel orally administered formulation but containing a drug with a known and precisely proven efficacy, like TNF-α inhibitors.Lactococcus lactis secreting monovalent and bivalent TNF-neutralizing nanobodies. The authors demonstrated that oral administration of these bacteria resulted in a local, colonic secretion of anti-TNF molecules, which induced anti-inflammatory effects in a dextran sulfate sodium (DSS) chronic colitis in mice. Bhol et al. [The first attempts to develop an oral form of TNF-α blockers were undertaken by Worledge et al. already in 2000 . The autl et al.  proposedl et al. , 29. Thel et al.  formulatl et al. . Since tAnother concept was proposed by Crowe et al. . In theiNicotiana tabacum (BY2) tobacco plant expressing recombinant TNFR2-Fc fusion protein. In this study, twenty-five UC patients were enrolled in an open-label manner to receive two different doses of OPRX-106 for 8 weeks. At the end of the evaluation, 67% and 28% of patients experienced clinical response and remission, respectively. This was accompanied by the reduction in fecal inflammatory markers and improvement in colonic histological scores. In parallel to clinical assessment, the authors conducted further research on the potential mechanisms of action of the investigated molecule. While they detected no significant absorption of OPRX-106 into the systemic circulation, they were able to show an increase in a CD4+ CD25+ FoxP3 subset of anti-inflammatory, suppressor T lymphocytes. One possible explanation for this phenomenon is the interaction of anti-TNF-α-acting molecule via Fc-receptor with a subgroup of CD14+/HLA-DR+ cells [In 2021 results from a phase 2a clinical trial have been published showing that administration of OPRX-106—a novel oral TNF-α-blocking molecule is effective and safe in patients with mild-to-moderate UC . OPRX-10R+ cells . On the Another important observation was the reduction of IL-6 and interferon-gamma levels after administration of OPRX-106 . IL-6 seThe immunoregulatory properties of OPRX-106 showed in patients with active UC confirmed previous observations made by the authors in animal models of chemically-induced steatohepatitis and colitis , 39. TheMore recently, other attempts have been made to develop oral formulations of anti-TNF-α antibodies by using the discoveries of pharmaceutical nanotechnology. Kim et al.  proposedTable An ideal drug delivery system should combine the ability to overcome anatomical and biological barriers, selectively recognize the target sites through surface ligands, and be stable, biodegradable, and non-toxic . As reflLPNs are obtained from biodegradable lipid materials of physiological nature and of high melting point. The high melting point  ensures that the lipid core of nanoparticles is solid, promoting the sustained release of the loaded drug . The lipThe use of nanoparticles for oral administration is mainly associated with the safety of LNPs and their ability to promote enteral absorption with the increased bioavailability of both hydrophilic and lipophilic drugs. On the other hand, understanding the impact of the size and shape of LNPs on their distribution in the intestine can be used to develop improved drug delivery systems to treat gastrointestinal diseases, such as IBD. The biodegradable lipid matrix of SLN/NLC undergoes enzymatic decomposition into components naturally occurring in the human body . Due to Enteric-coated systems are intended to pursue colon delivery by exploiting differences in the pH of gastrointestinal fluids . LoadingBesides the advantages of LNPs towards drug stability and bioavailability, they must take physicochemical interactions between carriers and loaded proteins (antibodies) into account, to ensure the release of intact and biologically active drugs. Drugs may be located inside the nanoparticles matrix or be adsorbed onto the surface of the nanoparticles. Thus, depending on their location, the antibody-lipid matrix interactions may distinctly affect antibody structure and bioactivity. The intermolecular forces between the protein and the lipid matrix may encompass covalent and electrostatic binding, polarization interaction, dispersion forces, and hydrophobic binding. However, there is a lack of knowledge on how these interactions may affect the 3D structure of proteins and, ultimately, how they influence bioactivity.Till now, several drug-designing protocols have been successfully finalized, and different nanoparticle-based therapeutic formulations containing monoclonal antibodies have been developed. As an example, our group has recently described a new cationic SLN formulation composed of solid lipid (Compritol ATO 888), surfactant (Poloxamer 188), and cetyltrimethylammonium bromide (CTAB) to incorporate perillaldehyde 1,2-epoxide, and surface-tailored with a mAb for site-specific targeting of human epithelial growth receptor 2 (HER2) . PerillaThe concept of loading anti-TNF-α antibodies into novel orally administered formulations can also have some possible limitations—pharmaceutical and clinical. First of all, their administration through the oral route can be compromised by their stability in the gastrointestinal tract. Yadav et al.  have shoRegarding the possible clinical limitations, orally administered TNF-α inhibitors can be insufficient for IBD patients with the most severe forms of the disease. This can be due to a targeted mode of action directly in the gastrointestinal wall with considerably low systemic exposure. This could also be problematic in the case of patients experiencing extraintestinal manifestations. However, individuals with the highest IBD activity usually have to be hospitalized and are preferably treated using intravenous and/or subcutaneous drugs. In this scenario, starting oral anti-inflammatory treatment can be considered after the initial induction of clinical response via the parenteral route. On the other hand, irrespectively of the clinical circumstances, the activity of intestinal inflammation is the driving factor of all symptoms and complications typical for IBD. Thus, a rapid decrease in disease severity in the intestinal tissues induced by orally administered therapeutic molecules can also indirectly result in a systemic response. Therefore, it seems that oral administration of anti-TNF-α antibodies could be considered in the phase of inducing remission in IBD of mild-to-moderate activity and in selected patients with severe disease, as well as in all patients in the maintenance treatment.The main goal of IBD therapy is to treat the inflamed gastrointestinal tract to achieve steroid-free remission and to protect patients from irreversible bowel damage, together with a life-long disability. Among a growing range of therapeutic options, biologic agents, in particular TNF-α inhibitors, have revolutionized treatment strategies in IBD with the highest anti-inflammatory potential. Despite rapid advances in the quality of care in IBD brought by these biologic agents, there are still some significant limitations to consider. Currently, biologic agents are administered parenterally, which results in a systemic mode of action, particularly beneficial for those severely ill. However, this can also lead to immunogenicity and serious adverse events and often has to be performed under professional medical supervision.That is why it is hypothesized that introducing oral anti-TNF-α therapy can revolutionize treatment algorithms and significantly improve clinical outcomes in IBD. Several attempts have been made in this area. The promising results of a growing number of nanotechnology-based scientific protocols focused on developing orally administered formulations of nanoparticles loaded with mAbs are believed to enable entering a new era of orally administered biologic therapy. Great expectations are raised to the newly proposed pharmaceutical formulation of TNF-α inhibitors loaded in LNPs Fig. . The adv