Heart failure with preserved ejection fraction (HFpEF) is a disease for which there is no definite and effective treatment, and the number of patients is more than 50% of heart failure (HF) patients. Gut microbiota (GMB) is a general term for a group of microbiota living in humans’ intestinal tracts, which has been proved to be related to cardiovascular diseases, including HFpEF. In HFpEF patients, the composition of GMB is significantly changed, and there has been a tendency toward dysbacteriosis. Metabolites of GMB, such as trimethylamine N-oxide (TMAO), short-chain fatty acids (SCFAs) and bile acids (BAs) mediate various pathophysiological mechanisms of HFpEF. GMB is a crucial influential factor in inflammation, which is considered to be one of the main causes of HFpEF. The role of GMB in its important comorbidity—metabolic syndrome—also mediates HFpEF. Moreover, HF would aggravate intestinal barrier impairment and microbial translocation, further promoting the disease progression. In view of these mechanisms, drugs targeting GMB may be one of the effective ways to treat HFpEF. This review focuses on the interaction of GMB and HFpEF and analyzes potential therapies. Heart failure (HF) is a group of clinical syndromes caused by pathological changes of cardiac structure and (or) function, often manifested as fatigue, edema , and dyspnea, etc. According to the left ventricular ejection fraction (LVEF), HF is divided into three types: HF with reduced ejection fraction (HFrEF) (LVEF < 40%), HF with mildly reduced ejection fraction (HFmrEF) (40% ≤ LVEF < 50%), and HF with preserved ejection fraction (HFpEF) (LVEF ≥ 50%). HFpEF is highly prevalent, accounting for 50% or more of all HF patients and is associated with significant mortality, mainly chronic morbidity and high frequency of re-hospitalizations ,2,3. HowThere are a large number of microbes living in human intestines, which have a symbiotic relationship with human beings. They can affect human bodies by metabolism, immunity, secretion, or in other ways, while the vivo environment will also affect their growth and function. The interaction between gut microbiota (GMB) and their host has been a research hotspot in recent years. A lot of researchers have demonstrated that the GMB are strongly related to cardiovascular problems, such as atherosclerosis, myocardial infarction, hypertension, arrhythmia, and HF. HF, especially HFrEF, has been extensively studied, but relatively less has been done on HFpEF. This review focused on the possible interaction between GMB and HFpEF and proposed some potential treatments.Although there are not enough direct studies on the GMB composition of HFpEF patients, most of the available results indicated significant differences in the community structure between HFpEF patients and healthy people, with a gut microbial dysbiosis in HFpEF patients. Huang et al.’s research, including 30 HFpEF patients and 30 healthy controls, showed an increasing abundance of flora that could trigger inflammatory response (Lactobacillus and Enterococcus) paralleled by a decrease of anti-inflammatory flora  . Beale eIn addition, an association between the composition of microbiota and some markers of myocardial fibrosis has been reported, such as C-terminal propeptide of procollagen type I (PICP), N-terminal propeptide of pro-collagen type III (PIIINP), and left ventricular extracellular volume (ECV) ,8, whichApparently, existing research on the composition of intestinal flora in HFpEF patients is insufficient. The composition of GMB differs among regions, races and diets. Therefore, larger sample size, multi-region, and multi-center studies on the GMB of HFpEF patients can provide a more reliable theoretical basis for the treatment of microbial adjustment.Unfortunately, HFpEF has great pathophysiological heterogeneity among different patients, so there is no accurate and detailed theory that can explain the pathogenesis of all patients. However, some pathophysiological changes have been shown to be associated with the occurrence and development of HFpEF, including left heart diastolic/systindiolic dysfunction, pulmonary vascular disease, right heart dysfunction, inflammation, cellular and extracellular structural changes, cardiometabolic abnormalities, autonomic dysregulation, cardiac fibrosis, vascular stiffening, myocardial ischemia, endothelial dysfunction, etc. Diastolic dysfunction is a basic component of the pathophysiology ,11. GMB Intestinal barrier dysfunction is always observed in HF patients . The intBacterial translocation is defined as the penetration of a large number of tissues and organs outside the intestine by normal microbiota and their endotoxins, peptidoglycans, or metabolites that initially colonized in the intestine through the intestinal mucosal barrier . It is oA lot of experimental and clinical evidence has demonstrated the critical role of inflammation in the occurrence and development of HEpEF by direct injury or from interacting with other pathophysiological changes. Regardless of the primary etiology, HF is related to both local and systemic inflammatory responses . HFpEF pHigh levels of inflammatory factors could lead to endothelial dysfunction, myocardial fibrosis or other pathological changes that then promote the progress of HFpEF ,27,28,29In addition, recent studies reported that the NLR family pyrin domain containing 3 (NLRP3) inflammasome may be critical for the systemic inflammatory response and cardiac remodeling of HFpEF. Activation of the NLRP3 inflammasome and secondary IL-1β/IL-18 overproduction were found in HFpEF mouse models ,32. NLRPGMB is associated with systemic inflammation levels. There is already evidence of an increase in the abundance of pro-inflammatory bacteria and a decline in anti-inflammatory bacteria in patients with HFpEF . In addiIn conclusion, when intestinal immune homeostasis is disrupted, the inflammatory cascade triggered by GMB, especially its endotoxins, is a complex immune response system composed of intricate cytokine networks and has not been completely studied. If future studies could screen out the immune factors that play the key role in this network and carry out precise immune targeted therapy on them, it will have important therapeutic significance for systemic inflammation and HFpEF. Of course, targeting microbiota and endotoxins in the upstream of the cascade or NLRP3 is a relatively simple approach, so the effect of TLR4 inhibitors or NLRP3 inhibitors on HFpEF is worth studying.TMAO is an amine oxide, which naturally presents in our diets, such as in fish, or that can be generated from its metabolic precursors—food-derived choline, carnitine and other substances. The precursors must first be converted into trimethylamine (TMA) by TMA lyase derived from GMB, then TMA will enter the portal circulation and be oxidized to TMAO by hepatic enzyme flavin-containing monooxygenase 3 in the liver. Diet and endogenous TMAO can be released by the liver and absorbed by extrahepatic tissues or excreted with urine ,45. FirmPatients with HFpEF have more TMAO in the plasma than healthy people . This isTMAO may lead to cardiac diastolic dysfunction, which is the key hemodynamic change of HFpEF, and myocardial fibrosis is a significant reason for diastolic dysfunction. A prospective cohort study of 112 samples presented a positive correlation between TMAO and diastolic dysfunction indices, such as mitral E/septal Ea and LA volume index . Chen etSCFAs are organic fatty acids containing less than six carbon atoms; acetic acid, propionic acid and butyric acid are more common in human bodies. SCFAs are products of the fermentation of various fiber substances in food by the GMB, after which they are taken by clonocytes or enter the portal vein and provide energy to the liver cells; a minor fraction of colon-derived SCFAs enters the systemic circulation and is taken advantage of by other tissues ,59.Most evidence suggests that SCFAs have a protective effect against HF. In the normal adult heart, energy is mainly derived from the oxidation of long-chain fatty acids (LCFAs). In failing hearts, however, LCFAs produce less ATP, while SCFAs normalize contractile function as an effective energy source ,61. AddiSCFAs also have positive effects on intestinal microbial homeostasis and barrier function. Small intestinal bacterial overgrowth (SIBO) is a common manifestation of microbial dysbiosis, which was found in some patients with HFpEF and which increased their risk of cardiovascular death . ButyratBAs can be classified into primary bile acids (PBAs) and secondary bile acids (SBAs), according to their sources. PBAs are synthesized in the liver by cholesterol, then about 5–10% of them reach the colon and undergo biotransformations by certain microbiota and finally into SBAs such as deoxycholic acid, ursodeoxycholic acid (UDCA), and lithocholic acid (LCA) ,71. MoreBAs bind to receptors like farnesoid X-receptor (FXR) and takeda G-protein-coupled receptor 5 (TGR-5), etc., to affect the heart . For exaBAs also modulate the stability of intestinal ecology. In fact, bile salts have a certain degree of antibacterial activity, which provides a selective pressure on the flora, thus regulating the composition of GMB . PBAs prIn conclusion, BAs produce a marked effect, mainly through FXR and TGR-5, to protect cardiomyocytes, improve diastolic function, resist inflammation in HFpEF, and repair intestinal barrier. However, not all BAs have positive effects, such as hydrophobic BAs, so it is necessary to study the impact of different BAs on HFpEF.In addition to TMAOs, SCFAs, and BAs, which have been extensively studied, new potential HFpEF-related metabolites are continually being discovered. For instance, polyphenols are plant-derived antioxidants. Dietary polyphenols are converted into low-molecular-weight, absorbable bioactive metabolites in the small intestine by specific enzymes derived from GMB . In HF mGMB has been shown to be correlated with amino acid (AA) metabolism. Microbes help the host to establish amino acid homeostasis, while AAs regulate the abundance and diversity of AA-fermenting microbiota . The metTimethyl-5-aminovaleric acid (TMAVA) is the metabolite of trimethylysine (TML) through GMB. TML is also the metabolic precursor of TMAO, and TML will be preferentially converted to TMAVA rather than TMAO in mice. Zhao et al. found the relationship between TMAVA and HF in 2022. In a cohort study involving 1647 patients, TMAVA was positively correlated with the risk of HF and the risk of death. In animal experiments, they found that TMAVA could inhibit the synthesis of carnitine, lead to the reduction of fatty acid oxidation, ultimately disrupt the myocardial energy metabolism, and induce the excessive accumulation of myocardial lipids, which is related to the decline of diastolic function .The research on these newly discovered metabolites is still insufficient, and more experimental evidence is needed to verify their association with HFpEF. Of course, searching for new metabolites is an important part of intestinal microbiology and metabolomics. New technologies, like machine learning, can help to screen new metabolic markers.MetS is a series of metabolic disorders including central obesity, insulin resistance, glucose intolerance, hypertension, and dyslipidemia . GMB wasMeanwhile, MetS is a classical hazard factor of cardiovascular diseases, particularly as an important comorbidity of HFpEF. Among various cardiovascular diseases, obesity has the strongest correlation with HF and more significantly raises incidence rate and mortality of HFpEF compared with HFrEF ,101. FroThe interaction between GMB and HFpEF is shown in In the last decade of microbiome research, diet has been identified as a crucial determinant of the structure and function of GMB . The MedAntibiotics, as strong modulators of microbiome, have been tried to regulate gut microbiota and control inflammation in some earlier studies . Some kiProbiotics are live microorganisms that could regulate the composition of GMB. Oral Akkermansia muciniphila supplementation could improve the MetS . Probiotdysmicrobiota [However, the role of probiotics and prebiotics is limited due to the diversity and complexity of GMB, meaning that it can only be used as an adjunctive therapy. In addition, there are researchers pointing out their potential side effects. For instance, strains of probiotics can directly cause bacteremia in ICU patients and inulin induced liver damage in mice with crobiota ,126. HFpFMT, a technique for transplanting functional bacteria from the stool of a healthy person into the intestine of a patient, is mainly used to treat Clostridium difficile infection. It could directly reconstruct healthy GMB and repair the intestinal barrier . There aExercise training is one of the primary and secondary prevention methods for cardiovascular disease. The beneficial effects of exercise on HFpEF are more notable than those on HFrEF . ExercisCompared with the regulation of GBM structure, targeting downstream metabolites or inflammatory cascade may be a more accurate and effective therapeutic idea. TMAO is clearly harmful to HFpEF patients. The non-lethal microbial enzyme inhibitor of choline TMA lyase has been used to attenuate the TMAO production in animal models. For instance, 3,3-dimethyl-1-butanol (DMB) prevented inflammation, cardiac hypertrophy, and fibrosis in HF mice, and mice did not exhibit toxic reactions after the chronic DMB exposure ,138. IodSupplemental SCFAs mainly depend on a higher intake of fiber from diet or prebiotics, or it can also be administered exogenously . Apart fUDCA is a kind of BAs with great medicinal value for cardiovascular system in current research. It improved peripheral blood flow and liver function of patients with CHF in a RCT , and hadAs for the anti-inflammatory treatment, antibiotics are the most commonly used anti-inflammatory drugs, but we do not recommend them. In addition to SCFAs and BAs we have also mentioned, there are other drugs that can reduce systemic inflammatory response. Empagliflozin—an inhibitor of sodium-glucose-cotransporter 2 always used as hypoglycemic medicine—has been approved for the treatment of HFpEF. Kolijn et al. found that an anti-inflammatory effect is also one of the pharmacological effects of empagliflozin. It could inhibit oxidative stress response and reduce myocardial inflammation through preventing the activation of PKGIαoxidation, thus protecting endothelial function and suppressing cardiomyocyte passive stiffness . Anti-cyTCM is a medical system based on clinical experience with a history of thousands of years. TCM herbs can modulate the composition of GMB and GBM also has a conversion effect on TCM components . SeveralIn addition, the heterogeneity of pathophysiological mechanisms in HFpEF is an obstacle in the field of HFpEF therapy. If we could classify patients into mechanistically homogeneous subgroups, the treatment would be more precise and effective . For exaThe association between GMB and various cardiovascular problems is of great concern. However, compared with atherosclerosis, hypertension, HFrEF, etc., the results about HFpEF are insufficient. The incidence of HFpEF is still increasing and the potential of therapeutic pathways targeting GMB cannot be ignored. Therefore, more studies on changes in the GMB of HFpEF are needed and the heterogeneity of GMB in different regions, races, and diets needs attention. Research on the application of FMT in HFpEF is also lacking. Drugs regulating the metabolites and inflammation deserve to be the focus due to the precision and effectiveness of their treatment. TCM has been shown to be beneficial to GMB, and finding ingredients that are effective for HFpEF is also a research direction in the future. Moreover, searching for appropriate criteria to classify HFpEF patients and achieve the goal of personalized treatment may be a practical clinical project.GMB is correlated with HFpEF. The composition of GMB in HFpEF patients significantly changed, which could affect the process of HFpEF via inflammation and metabolites such as TMAO, SCFAs and BAs. Its comorbidity—MetS—also mediates the influence of GMB on HFpEF. Meanwhile, the decrease of cardiac function will undermine the intestinal barrier, result in microbial translocation then disrupt the function of the GMB. Because of these mechanisms, GMB can be used as a potential therapeutic target for HFpEF. Dietary intervention, probiotics, prebiotics, exercise training, drugs targeting metabolites and inflammation as well as TCM have been preliminarily proven to be effective, but more evidence is needed, including animal experiments and clinical studies. But it is important to note that the mechanisms in all patients with HFpEF are still unclear, so these interactions may not be consistent with all patients, which indicates to us that separating HFpEF patients into different phenotypes and looking for different treatments may be helpful.


Zika virus (ZIKV) is a reemerging mosquito-borne flavivirus that causes febrile illness and is also linked to Guillain-Barré syndrome as well as to microcephaly in newborns. Due to the risk of fetuses developing microcephaly, ZIKV is a serious problem for pregnant women. Although different types of vaccine antigens have been investigated, there is still no approved vaccine that prevents ZIKV. The aim of this study was to produce a potential anti-Zika virus vaccine candidate based on virus-like particles (VLPs) in mammalian cells and to analyze the role of dosing regimen and adjuvant type on the immunogenicity of the obtained antigen. Novel recombinant VLPs (F2A) were designed by introducing the optimized signal sequence of prM protein and by adding a self-cleavage peptide 2A between proteins prM and E. These modifications improved the formation of the glycoprotein E dimer. It has been shown that the increasing dosing regimen generates a significantly higher titer of antibodies; however, the adjuvant type does not affect this process. Sera from mice immunized using an increasing dosing schedule also showed higher neutralization activity against both Zika strains . In summary, this is the first report showing the influence of vaccination schedules and adjuvants on the immunogenicity of ZIKV virus-like particles.IMPORTANCE Considering the transmission of ZIKV and the risk of another epidemic as well as the neurological complications that follow ZIKV infection, the virus remains a serious problem for the human population, especially pregnant women. Therefore, there is a great need to develop new effective vaccine candidates. Although different types of vaccine antigens have been used in preclinical studies worldwide, there is still no approved vaccine to prevent ZIKV. VLPs are among the most potent antigens, but to use VLPs, adjuvants must be added to the formulation and appropriate administration must be performed. In this study, we show for the first time the influence of vaccination schedules and adjuvants on the immunogenicity of recombinant ZIKV VLPs. The obtained results can be used in new vaccine designs not only against ZIKV but also against other important viral pathogens. Aedes spp. mosquitoes, although ZIKV can also be transmitted via other routes, i.e., blood transfusion, from mother to child, and via sexual contact. The geographical range of mosquitoes is still expanding due to climate changes, and there are new reports of active transmission of flaviviruses, e.g., West Nile virus, Usutu virus, dengue virus, and Zika virus (–Zika virus (ZIKV) is a mosquito-borne human pathogen that was discovered in 1947 in West Africa , 3. Thenka virus . One of ka virus . Althougka virus –12.Although many efforts have been made to develop an effective vaccine or antiviral therapy, no vaccine has been approved . ConsideVirus-like particles (VLPs) are among the strategies for developing vaccine antigens. Naturally, two envelope glycoproteins of ZIKV, prM and E, are able to assemble into VLPs and are further secreted to the cell culture medium when expressed together in eukaryotic cells . HoweverIn this study, we analyzed the influence of VLPs dosing and immunization regimens on the immunogenicity of ZIKV VLPs. For this purpose, we designed novel recombinant VLPs by modifying the signal sequence of prM and introducing a self-cleavage peptide to increase the secretion of VLPs. VLPs were produced in a mammalian expression system and further purified using a two-step chromatography process. Next, we examined the influence of the dosing regimen—three increasing or decreasing doses of VLPs in combination with a squalene-based oil-in-water nanoemulsion adjuvant—in a mouse model on the antibody response. The selected dosing regimen of VLPs was also tested with another adjuvant, aluminum hydroxide in combination with monophosphoryl lipid A (MPLA). The results show superiority of the increasing dosing regimen in the generation of an antibody response against Zika virus; however, the adjuvant type did not significantly influence the immunogenicity of VLPs. Moreover, despite the numerous studies on the potency of VLPs as vaccine antigens against ZIKV, this is the first report of the influence of vaccination schedules and adjuvants on their immunogenicity.To increase the production of ZIKV VLPs in mammalian cells, novel recombinant VLPs (F2A) were designed by exchanging the native signal sequence of prM protein with an artificial, optimized signal sequence and introducing a self-cleavage peptide 2A from porcine teschovirus-1 between prM and E protein  and B. TMoreover, the immunogenic properties of both types of VLPs were compared in animal studies. For that purpose, VLPs were purified from cell culture medium using polyethylene glycol 6,000 precipitation, followed by size exclusion chromatography. The purified particles were used in combination with AddaVax adjuvant for the immunization of mice . The obtained results demonstrated the superiority of the F2A VLPs in the induction of anti-E antibodies in the sera from immunized animals, as well as the stronger neutralizing activity of immune sera . Therefore, only F2A VLPs were used for further experiments.It has been proven that a 28°C incubation temperature after transfection can also increase ZIKV protein expression and favor the formation of E dimers . However20–In the next step, F2A VLPs were produced using the above protocol and then purified using a simple chromatographic process. The cell culture medium was harvested and clarified via centrifugation and filtration, and then F2A VLPs were subsequently purified using a two-step chromatographic process with anion exchange and multimodal chromatography. The first step was performed using a HiTrap Capto Q column to capture the F2A VLPs, which were then eluted and loaded directly on the second HiScreen Capto Core 700 column. With this column, the F2A VLPs were excluded from the shell of the resin and were recovered in the flow-through fractions while the cell culture proteins remained bound to the resin. The purity of the F2A VLPs and the content of prM/M and E proteins were evaluated in fractions collected during the purification process using Coomassie staining and immunoblotting, respectively  and E. TNext, F2A VLPs were characterized in terms of morphological properties and antigenicity. The presence of F2A VLPs in the final preparation was evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM) . The DLSin vitro studies  and in the DI of E (N154), respectively –25. N-gl– studies . The binn = 6) were immunized via the subcutaneous route (s.c.) three times, 2 weeks apart, with three increasing or decreasing doses of F2A VLPs  formulated with AddaVax adjuvant . This adjuvant is capable of stimulating both cellular and antibody responses (n = 3) received PBS buffer plus adjuvant. Serum samples were collected 2 weeks after each immunization and were used to evaluate the antibody response  and synthetic monophosphoryl lipid A (MPLA) toll-like receptor 4 (TLR4) agonist, contributing to the cellular (Th1- type) response , 31. TheDue to climate change, vector-borne flaviviruses, including Zika virus, are constantly spreading, and new viral strains are emerging. Given the risk of neurological complications in fetuses and newborns after ZIKV infection, the development of a safe and effective vaccine remains a high priority.The main objective of the study was to evaluate the impact of the VLPs dosing regimen and adjuvant type on the antibody response. For this purpose, we designed novel recombinant ZIKV VLPs (F2A VLPs) to facilitate production in eukaryotic cells. We introduced the following changes in the sequence of the prM and E protein cassette: the optimized signal sequence of the prM protein and the inclusion of a self-cleavage peptide (P2A) before a signal sequence of the E protein. Other groups reported the use of various signal sequences, including the JEV prM protein or the IL-2 signal sequence with ZIKV VLPs, to increase VLPs secretion into the culture medium , 32. TheFurthermore, we purified the F2A VLPs using a two-step chromatographic process. The most common methods of flavivirus VLPs purification are polyethylene glycol precipitation, diafiltration, and density gradient ultracentrifugation. However, these methods are laborious and difficult to scale up; thus, other methods need to be explored. Our approach involved a combination of anion-exchange chromatography with multimodal chromatography. In the first step, F2A VLPs were captured from the clarified cell culture medium onto HiTrap Capto Q resin; a high concentration of particles occurred, and most host cell proteins were removed.In the next step, F2A VLPs were further purified with the use of HiScreen CaptoCore 700, in which particles were recovered from the flow-through. A similar strategy was also applied for the purification of YFV wild-type VLPs and other ZIKV VLPs produced in stable HEK293SF-3F6 cell lines; however, the immunogenicity of these particles was not assessed . MoreoveIn the next step, we characterized the antigenic determinants of F2A VLPs. Due to genetic alterations in the sequence of prM and E glycoproteins in the F2A VLPs, the N-glycosylation status was evaluated first. Treatment with PNGase F demonstrated the presence of mature N-glycans on the E protein and the pr fragment. The N-glycosylation of the ZIKV E protein plays a crucial role in the replication cycle, especially in the assembly and secretion of virions, as shown using VLPs as a model . AdditioFurthermore, the presence of certain epitopes on F2A VLPs was determined. The ELISA analysis demonstrated high binding of F2A VLPs to two neutralizing antibodies (ZV48 and ZV67), which recognize epitopes in the DIII of the E protein, and one antibody (ZKA78) which is directed against an epitope in the DI/DII. A weaker interaction was observed with cross-reactive 4G2 antibody, which recognizes the fusion loop epitope in the E protein. The ZV67 antibody should bind to E protein, regardless of the oligomeric status of E protein in contrast, to other antibodies . The epiThe other aspect of this study was to evaluate the impact of adjuvant type on the immune response profile. The following types of adjuvants were compared: MF59-like oil-in water nanoemulsion adjuvant (AddaVax) and aluminum hydroxide/MPLA (Alhydrogel/MPLA). Both adjuvants were used in combination with ZIKV VLPs with different regimens  and were shown to induce a potent antibody response , 36. It To conclude, a novel strategy for the design of ZIKV VLPs and the production protocol in the mammalian expression system was implemented. The recombinant VLPs could be easily purified via chromatographic methods. Antigenic determinants, such as N-glycosylation, and the display of certain epitopes on the surface of particles, were analyzed. The most important observation that follows from our studies is the crucial role of the dosing regimen, but not the adjuvant type, for the immunogenicity of ZIKV VLPs in a mouse model. In our next study we aim to evaluate the protective efficacy of F2A VLPs in the preclinical infection models.AMA12084.1). The wild-type VLPs construct (F) consists of prM and E proteins preceded by the wild-type signal sequence of prM- transmembrane domains of the capsid protein C region 105 to 122 in the amino acid sequence of the Zika virus polyprotein (ss1). The recombinant F2A VLPs construct was designed by introducing the artificial signal sequence (optss) instead of the prM signal sequence and the P2A self-cleavage peptide between prM and E proteins. The signal sequence optss was an optimized artificial 16 amino acid sequence, which was developed based on the ss1 signal sequence. Sequence optimization was carried out by increasing the number of hydrophobic amino acids, such as alanine, valine, glycine and leucine in the ss sequence and shortening the sequence. The amino-acid similarity of both signal sequences was less than 40%. The sequence encoding the P2A peptide was derived from porcine teschovirus-1 . Additionally, the E protein was preceded by a signal sequence derived from the transmembrane domains of the prM protein. The nucleotide sequences of both constructs were chemically synthesized by GeneArt (Thermo Fisher Scientific).The sequences of structural proteins were derived from the ZIKV BeH818995 strain (GenBank: E. coli cells and purified using the Plasmid Midi AX (A&A Biotechnology).The synthesized DNA fragments were subcloned into the plasmid vector pcDNA3.1 (Thermo Fisher Scientific) utilizing the BamHI/EcoRI restriction enzyme sites. Plasmids were amplified in TOP10 competent 2. Both media were supplemented with 2 mM l-glutamine, 8% (vol/vol) heat-inactivated fetal bovine serum (FBS), and 1× Penicillin-Streptomycin  (Gibco).293T cell line (CRL-3216) and Vero E6 cell line  were cultured in Dulbecco’s Modified Eagle’s Medium (d-MEM) . The A549 cell line  was cultured in Minimum Essential Medium Eagle (EMEM) with Earle's Balanced Salt Solution (EBSS) (Lonza). All cells were cultured at 37°C under 5% COThe following reagent was obtained through BEI Resources, NIAID, NIH: Zika Virus, H/PAN/2016/BEI-259634, NR-50210. The following reagent was obtained through BEI Resources, NIAID, NIH, as a part of the WRCEVA program: Zika Virus, MR 766, NR-50065. Both strains of Zika virus were propagated in Vero E6 cells for 3 to 4 days following a low multiplicity of infection (MOI = 0.01).293T cells were transfected with pcDNA3.1 plasmid vectors encoding F or F2A VLPs constructs using Transporter 5 transfection reagent (Polysciences Europe GmbH) according to the manufacturer’s protocol. Transfection was performed at 37°C for 72 h and 96 h posttransfection. Next, samples were collected from the cell culture medium, and VLPs were precipitated using a solution of 10% polyethylene glycol 6000 (PEG6000) supplemented with 300 NaCl overnight at 4°C. Proteins were pelleted and dissolved in 50 mM Tris/100 mM NaCl/50 mM EDTA buffer. The protein solutions were further used to analyze prM/M and E protein expression.g for 10 min at 4°C followed by filtration through a 0.45 μm PVDF filter. The supernatant containing VLPs was stored at 4°C until further analysis or purification.F2A VLPs for further experiments were produced using an optimized protocol. 16 h posttransfection, the cells were supplemented with 2 mM sodium butyrate, 1× MEM Non-Essential Amino Acids Solution (Gibco), and 0.075% of Sodium Bicarbonate 7.5% solution (Gibco) and were further cultured at 28°C or 37°C. Medium from transfected cells was harvested 96 h posttransfection and clarified via centrifugation at 3,500 × The E and prM/M protein contents were analyzed using SDS-PAGE. For all experiments except purification, the same amount of protein in the analyzed samples was used (20 to 40 μg/per well). The total protein content in VLPs samples was quantified using a Quick Start Bradford Protein Assay (Bio-Rad). The samples were incubated at room temperature (RT) in reducing (addition of β-mercaptoethanol) or nonreducing conditions and then loaded on Novex WedgeW 4% to 20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific) in Tris-Glycine-SDS running buffer. After electrophoretic separation, the gels were used for Western blotting or Coomassie staining. For Western blotting, the proteins were transferred from gels onto the ROTI PVDF 0.45 μm membrane (Carl Roth) using wet overnight electrotransfer. The membrane was blocked with 5% nonfat milk in TBS-T (TBS buffer with 0.1% Tween 20 [vol/vol]), and ZIKV proteins were detected with the following specific antibodies: mouse monoclonal anti-Flavivirus group antigen antibody (4G2) (Absolute Antibody)  (used with nonreducing conditions), rabbit monoclonal anti-ZIKV (strain SPH2015) envelope protein (Domain III) antibody   or rabbit polyclonal Zika virus prM protein antibody  , followed by anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) . Next, the blots were developed using Super Signal West Pico Plus Substrate (Thermo Fisher Scientific) and visualized with Chemidoc system Alliance Q9-Series (UVITEC). Coomassie staining of the gel after purification was performed using Imperial Protein Stain (Thermo Fisher Scientific).2SO4, and the absorbance was measured at 450 nm with a microplate reader (TECAN).ELISA plates were coated with clarified cell culture supernatant containing VLPs. After blocking  and washing, rabbit monoclonal anti-ZIKV (strain SPH2015) envelope protein (Domain III) antibody  was added for 2 h at RT. Next, after washing an anti-rabbit HRP-conjugated secondary antibody (Santa Cruz Biotechnology)  was added. TMB Substrate Solution (Thermo Fisher Scientific) was used for reaction visualization. The reaction was stopped with 0.5 M HThe F2A VLPs purification process was based on the procedure designed for the purification of yellow fever virus and VLPs with some modifications , 45. FirThe purification was performed in two chromatographic steps using the anion-exchange chromatography column HiTrap Capto Q, 5 mL (Cytiva) operating in bind-and-elute mode, followed by a multimodal chromatography step using a 4.7 mL HiScreen CaptoCore 700 column (Cytiva) operating in flow-through mode. Both chromatographic steps were performed using a peristaltic pump with a flow rate of 2 mL/min.In the first step, the HiTrap Capto Q column was equilibrated with PBS/50 mM NaCl, and then the diluted retentate with F2A VLPs was loaded to bind the particles to the resin. Next, the column was washed with PBS/50 mM NaCl and PBS/100 mM NaCl, and F2A VLPs were eluted using PBS/350 mM NaCl. Finally, the column was regenerated by washing with PBS/1 M NaCl, followed by washing with 1 M NaOH.The second purification step was performed with a multimodal resin. The HiScreen CaptoCore 700 column was equilibrated with PBS/350 mM NaCl, and the eluate from the first step was loaded. The F2A VLPs were recovered in the flow-through fraction. The column was then regenerated by a wash with 30% isopropanol in 1 M NaOH.The total protein content in the final F2A VLPs preparation was determined using the Bradford method. The purified F2A VLPs were further evaluated by SDS-PAGE, Western blotting, transmission electron microscopy and dynamic light scattering.The F2A VLPs samples were loaded on carbon-coated 200 mesh nickel grids (Electron Microscopy Science). Negative staining was performed with 2% uranyl acetate. Immunogold labeling was performed according to the protocol of Aurion with some modifications. Briefly, the protocol was performed as follows: particle-loaded grids were blocked with Blocking Solution for Goat Gold Conjugates (Aurion). After washing, the grids were incubated with mouse anti-E-protein DIII (LR) (ZV-67) antibody for 1 h at RT. The labeling was performed with goat anti-mouse IgG conjugated with 6 nm gold particles (Aurion) for 1 h at RT, and then the grids were washed again and fixed with 4% paraformaldehyde. Finally, the grids were stained with 2% uranyl acetate. The samples were analyzed using a transmission electron microscope Tecnai G2 Spirit BioTWIN (FEI) .To examine the size and distribution of F2A VLPs, the particles were analyzed by Dynamic Light Scattering using a Malvern Zetasizer Nano Z. F2A VLPs samples were diluted in ultrapure water to a protein concentration of 50 μg/mL and analyzed. The hydrodynamic diameter was measurements were performed in duplicates.The presence of N-glycans on the prM/M and E glycoproteins was analyzed using PNGase F (Thermo Fisher Scientific) and Endo Hf (New England Biolabs) treatment. Two samples of F2A VLPs (2 μg of protein content) were incubated in denaturing buffer, and one sample was treated with PNGase F or Endo Hf, while a second one was an undigested control. The incubation was continued for 16 h at 37°C. After treatment, the samples were analyzed by a mobility shift assay by SDS-PAGE and immunoblotted with anti-domain III or anti-prM antibody as described above.2SO4, and the absorbance was measured at 450 nm with a microplate reader (TECAN).ELISA plates were coated with purified serially diluted F2A VLPs in PBS buffer pH 7.4. After blocking (3% BSA in PBS- 0.05% Tween 20 buffer) and washing, primary antibodies (2 μg/mL) were added for 2 h at RT. The following different primary antibodies were used: mouse monoclonal anti-flavivirus group antigen antibody (4G2) (Absolute Antibody), mouse monoclonal anti-E-protein DIII antibody (C-C’) (ZV48) (Absolute Antibody), mouse monoclonal anti-E-protein DIII antibody (LR) (ZV67) (Absolute Antibody), and mouse monoclonal anti-Envelope protein antibody (ZKA78) (Absolute Antibody). Next, after washing, an anti-mouse HRP-conjugated secondary antibody (Santa Cruz Biotechnology)  was added. TMB Substrate Solution (Thermo Fisher Scientific) was used for reaction visualization. The reaction was stopped with 0.5 M HTwo groups of 6 female BALB/c mice (6 to 8 weeks of age) were immunized subcutaneously with a mixture of F2A VLPs and adjuvant AddaVax (InvivoGen) . Then mice were immunized 3 times in 2-week intervals using the following dosing regimens: decreasing dosing to 15, 10, 5 μg of VLPs or increasing dosing to 5, 10, 15 μg. The total protein content in the VLP antigen for immunization was quantified using a Quick Start Bradford Protein Assay. The mice used as a negative control were immunized with adjuvant and PBS buffer only. Sera were collected on Days 0, 14, 28. On the day 42, the mice were sacrificed, and complete sera were collected for immunological response analysis. For the second study, the following different adjuvants were used: AddaVax and a combination of Alhydrogel 2% (InvivoGen)/Monophosphoryl Lipid A (MPLA) (InvivoGen) using an increasing dosing schedule. The Alhydrogel/MPLA adjuvant was used in the quantity of 200 μg/5 μg. As negative controls, two groups of mice were immunized with PBS and each of the adjuvants. All experiments on animals were conducted by an accredited company  in accordance with the current guidelines for animal experimentation. The protocols were approved by the Local Committee on the Ethics of Animal Experiments of the University of Science and Technology in Bydgoszcz (Permit Number: 35/2016 and 17/2020). All surgeries were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.2SO4, and the absorbance was measured at 450 nm with a microplate reader (TECAN).ELISA plates were coated with recombinant E protein (MyBioSource) or DIII  antigen (2 μg/mL) in PBS buffer pH 7.4. After blocking (3% BSA in PBS- 0.05% Tween 20 buffer) and washing, serially diluted mouse sera were added for 2 h at RT. Next, after washing, anti-mouse IgG, IgG1 and IgG2a HRP-conjugated secondary antibodies  were added (0.5 μg/mL). TMB Substrate Solution (Thermo Fisher Scientific) was used for reaction visualization. The reaction was stopped with 0.5 M HTo measure ZIKV neutralization by the obtained sera, PRNTs were performed. Briefly, 12-well cell culture plates were seeded with A549 cells to near confluence on the day of ZIKV infection. The sera were heat-inactivated at 56°C for 30 min. Sera were diluted 2-fold in fresh medium and mixed with equal volumes of ZIKV strain H/PAN/2016/BEI-259634 or MR766 containing approximately 50 PFU/well. The mixtures were incubated for 1 h at 37°C and used to infect cells. After 1 h, the inoculum was aspirated, and the cells were overlaid with 1% methylcellulose in MEM medium. The plates were incubated for 4 days at 37°C and stained with 0.5% crystal violet solution in 20% ethanol. Plaques were counted, and PRNT90 was calculated as the highest dilution that resulted in at least a 90% reduction in ZIKV plaques.All statistical analyses were performed using GraphPad Prism 5 software. Statistical significance was determined using a two-way ANOVA with Bonferroni posttest.The data generated and/or analyzed during this study are available from the corresponding author upon reasonable request.


Besides reviewing these findings, we discuss the potential problems related to the instability and molecular heterogeneity of NGR-TNF and review the various approaches so far developed to obtain more robust and homogeneous TNF derivatives, as well as the pharmacological properties of other peptide/antibody-TNF fusion products, muteins and nanoparticles that are potentially useful for targeting the blood–brain tumor barrier. Compared to other TNF-related drugs, the administration of extremely low-doses of NGR-TNF or its derivatives appear as promising non-immunogenic approaches to overcome TNF counter-regulatory mechanism and systemic toxicity, thereby enabling safe breaking of the BBTB.The blood–brain tumor barrier represents a major obstacle for anticancer drug delivery to brain tumors. Thus, novel strategies aimed at targeting and breaching this structure are of great experimental and clinical interest. This review is primarily focused on the development and use of a derivative of tumor necrosis factor-α (TNF) that can target and alter the blood–brain-tumor-barrier. This drug, called NGR-TNF, consists of a TNF molecule fused to the Cys-Asn-Gly-Arg-Cys-Gly (CNGRCG) peptide , a ligand of aminopeptidase N (CD13)-positive tumor blood vessels. Results of preclinical studies suggest that this peptide-cytokine fusion product represents a valuable strategy for delivering TNF to tumor vessels in an amount sufficient to break the biological barriers that restrict drug penetration in cancer lesions. Moreover, clinical studies performed in patients with primary central nervous system lymphoma, have shown that an extremely low dose of NGR-TNF (0.8 µg/m The prognosis of patients with tumors of the central nervous system (CNS), such as primary lymphomas, gliomas, or brain metastases from neoplasms that originate in other organs, is still poor. This is mainly due to the presence of the blood–brain barrier (BBB), which represents an important obstacle for anticancer drug delivery to brain tumors. Although tumors in the brain compromise the integrity of the BBB in the affected area, this barrier  is heterogeneously permeable and represents, therefore, a major obstacle for a homogeneous penetration of anticancer drugs in tumor tissues. The description of structure and function of the BBTB is beyond the scope of this paper, and we refer the readers to an excellent review on this subject, recently published . Thus, pE. coli, represents a prototypical example of these class of compounds [TNF is an inflammatory cytokine originally discovered for its capability to cause massive hemorrhagic necrosis of cancer lesions in mice ,5. Unforompounds .+ tumor vasculature, but not other normal tissues that express high levels of CD13 [The CNGRCG peptide was originally identified by panning peptide-phage libraries in tumor-bearing mice . This co of CD13 .The molecular bases of this selective recognition are still unclear. CD13 is a dimeric glycoprotein with an archlike structure and with monomers that can assume closed or open conformations . It is pBecause of their tumor vasculature homing properties, CNGRCG and other NGR-peptides have been exploited for delivering a variety of therapeutic or imaging compounds to neoplastic lesions, including liposomes, chemotherapeutic agents, DNA, viruses, anti-angiogenic compounds, fluorescent molecules, contrast agents and also cytokines . In NGR-Finally, considering the notion that CD13 is expressed in angiogenic vessels, it is important to highlight the fact that besides targeting the angiogenic vasculature of tumors, off-target delivery of NGR-TNF to non-tumoral angiogenic vessels might occur in the case of concomitant diseases with an angiogenic component.Considering that human TNF can efficiently bind murine type-1, but not type-2, TNF receptor , most prStudies on the mechanisms of NGR-TNF anti-tumor activity have shown that targeted delivery of low amounts of this drug to tumor blood vessels  can affect the tumor vasculature and microenvironment, causing the up-regulation of intercellular adhesion molecule (ICAM)-2 and vascular cell adhesion molecule (VCAM)-1 in the endothelial lining of tumor vasculature and the release of various chemokines and cytokines involved in the activation and migration T-cells in the tumor tissue, such as MCP-1/CCL-2, MIP-2, oncostatin-M, MCP-3/CCL-7, and stem cell factor . Low-dosLow-dose NGR-TNF  can increase the response of tumors to chemotherapeutic drugs, as observed with cisplatin, melphalan, doxorubicin, gemcitabine and paclitaxel in various models of transplantable tumors and in an orthotopic model of prostate cancer (TRAMP) ,31,32.Maximal synergism can be obtained with a 2-hour delay between NGR-TNF and chemotherapeutic drug administrations, irrespective of tumor model and drug used. Mechanistic studies performed in mice bearing melanomas or lymphomas have shown that low-dose NGR-TNF can increase the tumor uptake of the anthracycline doxorubicin . This phLow-dose NGR-TNF can promote lymphocyte extravasation in tumors  Figure B. This pHuman NGR-TNF (NGR-hTNF) has been evaluated alone or in combination with chemotherapy in several phase-I and -II studies enrolling patients with various types of solid tumors, and in also in a phase-III study in patients with malignant pleural mesothelioma.2) with a maximum tolerated dose (MTD) of 45 μg/m2 administered in 1 hour. The most frequent observed toxicities were rigors and fever. The terminal half-life of NGR-hTNF was 1 to 2 hours. No objective responses were observed, but 39% of patients had stable disease (SD), with a median duration of response of 12 weeks. Based on the results of another phase-I study, designed to define safety and optimal dose in patients with advanced solid tumors, the dose of 0.8 µg/m2 was chosen for subsequent studies, either as single-agent or in combination with chemotherapeutic drugs, considering the favorable toxicity profile observed with this dose, the disease control obtained (44% of SD for a median time of 5.9 months), the lack of stimulation of TNF-receptors shedding and the significant anti-vascular effects observed [2) and showed that patients did not develop anti-NGR-hTNF antibodies.The first phase I study, conducted on 69 patients with histologic or cytologic confirmation of advanced cancer, showed for the first time in humans that treatment with NGR-hTNF could cause anti-vascular effects in tumors . NGR-hTNobserved . This st2, 1 h infusion, weekly or every 3 weeks) as a single agent were then conducted in pre-treated patients with hepatocellular carcinoma (HCC), malignant pleural mesothelioma (MPM), and colorectal cancer (CRC). The results showed again anti-vascular effects and significant disease control [Thus, phase II clinical studies with low-dose NGR-TNF  [2) [2, combined with a fixed dose of capecitabine-oxaliplatin (XELOX). Both these treatments were safe, but apparently a sign of efficacy was evident only with the lower NGR-hTNF dose [2) seemed to be related to the induction of soluble TNF-receptors shedding in the circulation, i.e., the release of TNF inhibitors. However, the results of a phase I study conducted with doses (60–325 μg/m2) higher than the MTD previously established in 48 patients with refractory solid tumors (45 μg/m2) suggest that these doses are tolerated and can induce anti-tumor effects if a more protracted length of infusion (2 h) and mild premedication with paracetamol are used [Phase I studies on the combination of low-dose NGR-hTNF with doxorubicin (60–75 mg/m2)  or cispl2) [2)  showed t2) [2) . No unex2) [2) . In thesTNF dose . This isare used . This beNGR-hTNF was also combined with a peptide-based vaccination in a pilot phase-I study enrolling patients with metastatic melanoma . Accrual2, weekly) in combination with the best investigator choice have been published in 2018 [n = 200) a significant improvement in progression-free survival (PFS) and OS was observed in patients who progressed more rapidly after first-line treatment , suggesting that there could be a benefit from treating patients with more aggressive MPM with NGR-hTNF.The results of a randomized, double blind, placebo-controlled phase-III clinical trial on 400 previously treated patients with MPM and treated with or without low-dose NGR-hTNF (0.8 μg/m in 2018 . Althoug2 delivered 2 hours before R-CHOP by a 1 h infusion). Per protocol, the first course of R-CHOP was not preceded by NGR-hTNF in the first 10 patients. All subjects who achieved a partial response (PR) or a complete response (CR) at the end of the program, were evaluated for consolidative therapy which, per protocol and accordingly to prior treatments, could be represented by whole-brain radiation therapy (30–36 Gy), carmustine-thiotepa-conditioned autologous stem cell transplantation or oral lenalidomide maintenance. A total of 28 patients were enrolled ; most patients had unfavorable features at trial registration (IELSG risk score intermediate or high in 82%). Tumor response was observed in 21 patients , complete in 11 patients, and the predetermined efficacy threshold (≥12 responses) was achieved, indicating that the combination of NGR-hTNF and RCHOP was efficacious in these patients  of the CNS (PCNSL), a rare extra-nodal non-Hodgkin lymphoma confined to brain, eyes, meninges, and other structures of the CNS, is a paradigmatic example of one of the major challenge in the treatment of CNS tumors: the delivery of drugs across the BBB . PCNSL apatients A. At a mal areas B. NotablStudies on the immunogenicity of the CNGRCG domain of NGR-TNF, carried out in rabbits and mice injected with NGR-TNF or with various compounds consisting of CNGRCG coupled with immunogenic carrier proteins, have shown that the CNGRCG peptide ligand is poorly, or not at all, immunogenic . ExperimEscherichia coli (E. coli) cells, have shown that these products consist of trimers made by subunits with different molecular weights, including subunits with the expected molecular weight  and subunits with −17, +1, +42 and +58 Da than expected [TNF is a homotrimeric protein. Structural studies of human and murine NGR-TNF, produced by genetic engineering technology in expected . The molexpected .While the acetylated-CNGRCG domain can recognize CD13 with a binding strength similar to that of the non-acetylated domain, the −17 Da subunits are likely non-functional, as this modification (which involves the reaction of the cysteine α-amino group with the asparagine side-chain of the CNGRCG domain) destroys the NGR motif . Thus, iThe asparagine deamidation reaction leads to the formation of aspartate and isoaspartate. The consequent NGR-to-isoDGR transition causes receptor switch from CD13 to αvβ3 integrin, isoDGR being an integrin-binding motif . NotablyP. pastoris, instead of E. coli [E. coli of a SCNGRCG-TNF product  more homogeneous and stable than NGR-TNF [Considering the molecular heterogeneity of NGR-TNF subunits and assuming a random association of different subunits in trimer formation, different trimers are expected to be present in NGR-TNF preparations. This may represent a major problem in drug development, particularly in terms of lot-to-lot consistency and quality assurance. Thus, various attempts have been made to generate NGR-TNF derivatives more homogeneous and stable. At this regard, an NGR-TNF product lacking the +58 Da and +42 modifications can be obtained by expressing the cDNA encoding this protein in  E. coli . Further NGR-TNF . Finally NGR-TNF . All the2 in patients) are sufficient to affect the tumor vasculature and to induce anti-tumor effects. This also suggests that the CNGRCG peptide (NGR) and the CD13 receptor represent an efficient ligand–receptor system for delivering TNF to the tumor vasculature. However, besides CNGRCG, other ligands and delivery systems can be exploited for increasing the therapeutic index of TNF [The results of preclinical and clinical studies obtained with NGR-TNF indicate that extremely low doses of this drug  nanoparticles [Other approaches used by different investigators for delivering TNF to tumors are based on the use of nanomaterials capable of exploiting the “passive” targeting mechanism consequent to the “enhanced permeability and retention effects (EPR)” of neoplastic tissues ,73, sucharticles , dendritarticles , lactofearticles , carbon articles , and polarticles . Notablyarticles ,86. Finaarticles ,91,92,93articles .All these strategies are promising, but their clinical utility in safely breaching the BBB in cancer patients is yet to be demonstrated.2) is well-tolerated. The results of these studies have also shown that the use of an extremely low dose of NGR-TNF can overcome, on the one hand, the problem of TNF systemic toxicity, and, on the other hand, systemic counter-regulatory mechanisms that limit the therapeutic efficacy of this cytokine. This is made possible by the excellent accessibility of the CD13 target  and the high avidity of the interactions among NGR-TNF, CD13 and TNF-receptors, co-expressed on the endothelium of the tumor vasculature. The NGR targeting domain, besides delivering TNF effector molecules to CD13-positive tumor vessels, can also induce co-signaling mechanisms that impair the activation of pro-survival pathways by the TNF moiety, a mechanism that may contribute to the biological effects observed with extremely low doses of the fusion protein.The results of studies performed with NGR-TNF in animal models of solid tumors and in PCNSL patients suggest that coupling TNF with the CNGRCG peptide (NGR) is a valuable strategy for delivering an amount of cytokine to tumor blood vasculature sufficient to alter the biological barriers that limit drug penetration in cancer lesions, including the BBTB. Remarkably, the dose of NGR-TNF necessary for BBTB alteration in patients  immunogenicity of the NGR peptide, may represent important advantages compared to other ligands and vehicles so far developed for delivering TNF (protein or gene) to tumors, e.g., those based on antibodies, nanoparticles, viral agents, or genetically engineered cells, particularly in the case of therapies that need repeated treatments. In conclusion, the results achieved so far in animal models and in PCNSL patients suggest that targeted delivery of low amounts of NGR-TNF (or its more homogeneous derivatives) to CD13-positive tumor neovasculature represents a valuable strategy for breaching the BBTB and for enhancing anticancer drug delivery to neoplastic cells in patients with PCNSL. Further studies are warranted to assess the potential use of NGR-TNF (or its more stable and homogeneous derivative S-NGR-TNF), in combination with chemo/immunotherapy, for the treatment of other primary and secondary brain tumors, such as glioblastoma multiforme and brain metastases of breast and lung cancer.


We discuss Heart Rate Variability (HRV) measured during mindful breathing meditation. We provide a pedagogical computation of two commonly used heart rate variability metrics, i.e. the root mean square of successive differences (RMSSD) and the standard deviation of RR intervals (SDRR), in terms of Fourier components. It is shown that the root mean square of successive differences preferentially weights higher frequency Fourier modes, making it unsuitable as a biosignal for mindful breathing meditation which encourages slow breathing. We propose a new metric called the autonomic balance index (ABI) which uses Respiratory Sinus Arrhythmia to quantify the fraction of heart rate variability contributed by the parasympathetic nervous system. We apply this metric to heart rate variability data collected during two different meditation techniques, and show that the autonomic balance index is significantly elevated during mindful breathing, making it a good signal for biofeedback during meditation sessions. Mindfulness has shown promise as a non-pharmaceutical intervention in the management of stress, as well as a variety of other conditions . Meditatd measures of stress . Two specific meditative techniques were investigated by RR interval time series data. Data were collected during meditation and also during the period prior to meditation, which serves as a control. Also included were three additional non-meditating cohorts to serve as an additional control: 3) 14 healthy subjects  following metronomic breathing at 15 min−1, 4) 11 healthy subjects  during sleep, and 5) 9 elite triathlon athletes  during sleep. This is summarized in N5 is the total number of 5-min segments we used in the analysis.The data used for this analysis have been described by Let us now investigate a technique for quantifying the autonomic balance through RSA. There have been multiple methods suggested for quantifying RSA , where RRi = ti−ti−1. The RR time series contains a constant term ⟨RR⟩ and a fluctuating term RR⟩. The differences between successive RR intervals ΔRRi may be computed as:ti = ti−ti−1 = RRi, and we ignored term of quadratic order in HR⟩ is the mean heart rate, and “bpm” stands for beats per minute. The RMSSD is the root mean square of the successive differences ΔRRi, i.e.N/T0 to obtain an N samples . We may expand this in a Fourier series:nF ≤ nmax is the total number of Fourier modes to be included in the approximation, and by Nyquist’s theorem, nmax = N/2. ti are the time intervals at which the RR intervals are calculated. We have assumed bn ≠ 0 and an and bn may be computed as:n = 1, 2, 3, ⋯nF. Let SDRR (nF) and RMSSD (nF) be the values of SDRR and RMSSD estimated using nF ≤ nmax Fourier modes, while SDRR and RMSSD are the exact values. Eq. nF) from Eq. nF) using Eq. Let T0ti+φn,where n= t1, t, t3, ⋯ T0ti+φn,where nRi2⟩1/2.Let us i−1. Plot c) shows the ratios [RMSSD (nF)/RMSSD] and [SDRR (nF)/SDRR], where the values of RMSSD (nF) and SDRR (nF) are calculated from the approximate formula we derived, and include up to nF Fourier modes. RMSSD and SDRR are the exact values. Both RMSSD (nF) and SDRR (nF) approach their true values as nF → nmax, but the RMSSD (nF) computation made some simplifying assumptions, and is hence not as accurate as the SDRR (nF) computation. RMSSD (nF) also converges to the true value with a far larger number of Fourier modes than SDRR (nF) since it preferentially weights high frequency modes. SDRR (nF) requires only 18 Fourier modes (corresponding to a peak frequency of 88/5 = 3.6 min−1) to reach 90% of the true value. This makes intuitive sense since the peak of the PSD was found to be at 3.2 min−1 and there is very little power at higher frequencies. RMSSD (nF) on the other hand, requires 88 Fourier modes (corresponding to a peak frequency of 88/5 = 17.6 min−1) to reach 90% of the true value. Frequencies above 3.6 min−1 contribute ≲ 10% to the SDRR, while contributing ≈35% to the RMSSD. This discussion highlights a major flaw in using the RMSSD to quantify HRV for slow breathing: A small amount of power at high frequencies is preferentially weighted by the RMSSD even though the high frequency Fourier modes in this case are not associated with respiration. The SDRR being an unbiased HRV estimator does not weight Fourier modes differently by frequency. The lack of high frequency content also results in the RMSSD being much lower than the SDRR. For this example, we find RMSSD = 36.8 ms, while the SDRR = 102.6 ms.RR time series may then be simplified as:f is the respiratory rate and A is the amplitude of oscillations. The SDRR is then simply RR as:f is measured in min−1 and ⟨HR⟩ is measured in beats per minute. Taking the root mean square of Eq. −1) and relatively constant, i.e. during sleep. In the example shown in −1, and the mean heart rate is 72 beats per minute. From our approximate analysis  compared to the resting phase (18.5 min−1), 2) Most of the power is contained within the respiratory band during the meditation phase (ABI = .86). In the case of the resting period prior to meditation, there is considerable power at frequencies not associated with respiration {ABI = .57, some of this power is likely due to Mayer Wave Sinus Arrhythmia /metric (rest). Thus, a positive fractional change implies that the metric is higher in the meditation group compared to the rest group. The column for statistical significance tests whether the difference is statistically significant at the p < .05 level for a 2-tailed test (we used the condition |Z| > 1.96). The sample sizes are provided in Table I.d−effect sizes for the respiratory rate are d = −6.1 for Chi, and d = −12.3 for Yoga. The ABI shows a large positive effect size (d = 3.9 for Chi and d = 5.0 for Yoga). Among the other metrics we considered, only the SDRR and LF power are statistically significant for both forms of meditation. For SDRR, we find d = .5 (1.1) for Chi (Yoga), while for LF power, we find d = 1.1 (1.4) for Chi (Yoga). The HR is statistically significant for the Yoga cohort, but not for the Chi cohort. Nevertheless, we do not recommend using HR as a biosignal since it increased during meditation (Yoga cohort) in this particular example. The cohort with the lowest heart rate was “Ironman”, i.e. the elite athletes. We note that the effect size computation for the Yoga (rest) and Yoga (med) cohorts needs to be treated with caution, due to the small sample sizes.The metrics that show the largest difference between rest and meditative states are the respiratory rate and the ABI. Not surprisingly, the respiratory rate decreases during meditation. The Cohen’s We have shown that the ABI is greatly increased during mindful breathing. In this subsection, we discuss how the ABI can potentially be used as a biofeedback tool during meditation, or else, to monitor stress  showed a statistically significant change for both types of meditation.We then applied the ABI to heart rate time series data collected during meditation, and described in LF/HF ≲ 1 indicate parasympathetic dominance. From −1, the HF power should not be interpreted as indicative of PNS activity and the LF/HF ratio should not be considered for situations involving slow breathing. From We do not recommend the use of the LF/HF metric to measure autonomic balance during slow breathing. The LF/HF metric is most useful during sleep when the respiratory rate is typically high enough for vagal activity to manifest in the HF band of the HRV power spectrum. In that case, the LF/HF ratio indeed has useful information, and values of f1, f2) of possible respiratory rates as due to stress, i.e. sympathetic nervous system activity creates low frequency power. However not all power at low frequencies is due to stress, e.g. the Mayer Wave Sinus Arrhythmia (MWSA) at f ≈ 6 per minute is caused by blood pressure oscillations [RR interval data when subjects are moving.There are several limitations to this work. The ABI algorithm interprets power at frequencies outside the chosen range (llations , and a p


Diastereodivergent heterocycle synthesis has been recognized as an important tool for drug discovery in recent years, yet strategies based on nickelacycle formation have not been established. Here, we report a NHC-Ni catalyzed highly 1,3- and 1,4-diastereodivergent heterocycle synthesis from enyne, which is achieved by manipulating the enyne N-substituent . The key to success is the efficient diastereodivergent formation of a nickelacyclopentene, with broad enyne scope at mild conditions, which subsequently provides reductive hydroalkenylation, acylation and silylation products on demand. Diastereoisomers which are sterically hard to distinguish or difficult to access by conventional routes are now accessible easily, including those with very similar 4°, contiguous and skipped stereocenters. Diastereoselective cyclisation of heteroatom-substituted enynes offers a valuable route to bioactive heterocycles. Here, a diastereodivergent nickel-catalysed cyclisation of heteroenynes offers access to diastereomers of 1,3- and 1,4-hydroalkenylation products, controlled by the choice of substituents. Given that different diastereomers may exhibit diverse bioactivities, synthesis of individual diastereomers is of great importance. Multi-substituted hydropyran and piperidine skeletons with exocyclic olefin are key core structures or precursors in many biologically active natural products and drug molecules stereodivergent heterocycle synthesis13. Substituents in 1,2-, 1,3- or 1,4-diastereodivergent relationships could be built by tailor-made ring closure methods efficiently, which are controlled often by selective facial attack either on prochiral sp2 center or on radical acceptors  as well as the high demands of functional groups often limited the scope and the efficiency of the process.Heterocycles bearing multiple stereocenters are common structural motifs in many biologically active moleculesors Fig. 14,15. So24. Highly 1,3-syn-diastereoselective synthesis has been achieved in a few examples according to the steric demands of enyne substituents and metallacycle formation . Moreover, unlike a few examples noted in intramolecular hydroalkenylation of diene  in toluene. This is useful because selective nickelacyclopentene formation is less efficient for those terminal enynes due to highly competitive and undesired [2 + 2 + 2] oligomerization or dimerization. Yet, the most remarkable result is that a highly diastereodivergent piperidine synthesis was achieved by this rarely employed strategy. Also, the role of R1 size in 1,3-syn-:anti-selectivity was found not so crucial (R1 = Ph c.f. Me). A high 1,3-syn-:anti-selectivity (3:3′) was observed by using 1c-d (with propargyl-Z = NH). On the contrary, a high 1,3-anti-selectivity (4′) was noted in 1e-f (with propargyl-Z = NMs). Oxa-enyne 1a-b followed the same reactivity and selectivity pattern as 1c-d did. Moreover, a highly efficient diastereodivergent 1,4-stereotransfer was observed by using enyne 2 with different allyl-Z as substrate (Table 1 = Ph or Me). Heterocycles 5′ and 6 were obtained as major products (Table -:anti-selectivity) was found correlated to the selected choice of Z. Yet, the preferred stereo-configuration directed by allyl-Z was found different from the propargyl-Z cases .Our study commenced with a set of simple 1,7-heteroenynes 1 in both Eqs. (1) and (2) caught our attention. That made us suspected it was not just directed by simple steric interaction, so Z with different steric and electronic property was evaluated next . Similar phenomenon was noted in Table At first, one might consider the steric difference in Z is sufficient to explain the above diastereodivergent synthesis. Yet, very similar efficiency among enynes having different sizes of R 1) and  caught o and  cau (1) and  caught o1a, 1c and 1e as well as 2a, 2c and 2e . Several notable results deserve further comment. First, the diastereodivergent synthesis efficiency is excellent even in cases with substituents as small as Me. It highlighted that the basis of this switching is not primarily on steric repulsion  and (2)). Second, the method can still perform even when interfered by an extra 2-substituent at syn/anti-relative configuration (Set 4). This indicated that such gauche interactions are not competitive enough to the desired preference directed by our strategy ; <5:95 in Eq. (2)). Hence, assortments of densely substituted stereoisomers are now easily accessible by this reductive hydroalkenylation. Third, stereo-defined 4° centers only having marginal steric differences are hard to make selectively . Our approach offers a possible entry to build those very similar products as well as isomers. Overall, it complements to a few shortcomings by the traditional steric approaches, the cyclic templates, the rearrangement of side chains or the stoichiometric organometallic reagents3OH, ArCHO and TESH instead of 1-phenylethanol . It was demonstrated by a reductive hydroalkenylation of enynes using 2° alcohol as reductant. Highly competing side reactions were suppressed and broad scope was achieved simultaneously. Unlike a number of former diastereodivergent approaches that based on facial selection at the latter stage of the catalytic cycle, here the diastereodivergent efficiency is governed by Z directed nickelacyclopentene forming step at the start . Moreover, that can be promoted by choice of NHC ligand (IPr vs SIPr). That feature makes the preferred diastereodivergent outcome highly predicable and tunable, which in turn makes it less dependent on steric interactions among external reaction partners and substituents on the tether. Products packed densely with diastereocenters which is difficult to be made by conventional steric approaches  are now easily accessible expediently, rather than just the sterically favorable ones. Finally, the diastereodivergent results obtained in acylations and silylations imply a great potential of this work in joining many other transformations and cross-coupling reactions that based on nickelacyclopentenes and related species for broader scope. Exploration along this line is now underway.Preparation of hetero-substituted enynes: See Determination of diastereoselectivity: See Acylation procedure: See Sylilation procedure: See Products characterization: See Supplementary Data 2 and IPr (0.05 mmol each) were dissolved in toluene (2 mL) and stirred at 30 °C in glovebox for 1 hr. An enyne and 1-phenylethanol (0.5: 1.5 mmol) toluene solution (1 mL) were added dropwise to the above catalyst in 0.5 h, and was stirred at 30 °C for 3 h. After work up, the yield, structure and selectivity were determined by 1H NMR, NOESY, isolation and GCMS (average of two runs). Mesylation of NH products were carried out for direct comparison of the isomers when necessary ecessary .Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1


Animal models are a useful tool for studying pathogenesis of a wide range of brain disorders, including channelopathies. Complementing multiple well-established rodent models, the zebrafish (Danio rerio) has become a popular translational model organism for neurobiology, psychopharmacology and toxicology research, and for probing mechanisms underlying CNS pathogenesis. Here, we discuss current prospects and challenges of developing genetic, pharmacological and other experimental models of major CNS channelopathies based on zebrafish.Channelopathies are a large group of systemic disorders whose pathogenesis is associated with dysfunctional ion channels. Aberrant transmembrane transport of K Ind. Ind4].  current . The losmigraine . In addimigraine . First, migraine . For insIt is therefore impossible to assume that migraine-like pain models in rodents cause migraines per se, and the same problem applies a priori to any such zebrafish models as well. In addition, when modeling migraine, only that accompanied by the aura lends itself to quantitative and qualitative assessment. In contrast, it seems that migraine without the aura cannot be reliably diagnosed in animal models. Accordingly, it remains unclear whether it is possible to simulate migraine and migraine-like pain in fish, and whether fish are even capable of experiencing headaches similar to those observed clinically. Likewise, we do not know what pharmacological and genetic tools can help model these conditions best in both rodents and fish.In summary, the high global prevalence of diseases directly related to abnormal ion channels necessitates our improved understanding of the pathological mechanisms underlying CNS channelopathies, as well as the development of novel pharmacological agents for their treatment. For this, zebrafish models can be a useful tool for identifying evolutionary-conservative mechanisms underlying CNS channelopathies, and a powerful efficient drug-screening platform, to induce or prevent these conditions in vivo. With multiple conceptual and methodological questions remaining open in the field , this co


Cashew nut kernel oil (CNKO) is an important oil source from tropical crops. The lipid species, composition, and relative content of CNKO were revealed using ultra high performance liquid chromatography time-of-flight tandem mass spectrometry (UPLC-TOF-MS/MS), and the physicochemical properties, functional group structure, and oxidation stability of CNKO at different pressing temperatures were characterized using a near infrared analyzer and other methods. The results showed that CNKO mainly consisted of oleic acid (60.87 ± 0.06%), linoleic acid (17.33 ± 0.28%), stearic acid (10.93 ± 0.31%), and palmitic acid (9.85 ± 0.04%), and a highly unsaturated fatty acid (78.46 ± 0.35%). In addition, 141 lipids, including 102 glycerides and 39 phospholipids, were identified in CNKO. The pressing temperature had a significant effect on the physicochemical properties of cashew kernels, such as acid value, iodine value, and peroxide value, but the change in value was small. The increase in pressing temperature did not lead to changes in the functional group structure of CNKO, but decreased the induction time of CNKO, resulting in a decrease in their oxidative stability. It provided basic data support to guide subsequent cashew kernel processing, quality evaluation, and functional studies. The cashew nut is a genus of plants of the genus Dicotyledonea, order Sapotaceae, family Lacertidae, and genus Cashew . CurrentCashew nut kernels (CNKs) are the kernels of shelled cashew nuts after dehulling, which are mainly composed of 47.0% fat, 21.1% protein, 4.6–11.2% starch, 2.4–8.7% sugar, and other components, as well as a variety of amino acids, vitamins, and trace elements, such as phosphorus, iron, and calcium , and proCNKO is a very important nutritional component of CNKs, accounting for approximately 47% . CNKO coThe aqueous extraction method, squeezing extraction, supercritical carbon dioxide extraction, and solvent extraction method were common extraction methods for oils. Phuong et al.  used enzCNKs are rich in protein and tightly combined with oil, and the aqueous extraction method easily produced the serious emulsification phenomenon, which affects the extraction of oil. The supercritical carbon dioxide extraction method involves a large equipment cost investment, while the pressing method is a simple and low-cost process. However, there are few reports on the lipid composition of CNKO and the effect of pressing temperature on the physicochemical properties, functional group composition, and oxidative stability of CNKO. Therefore, in this study, the lipid species, composition, and relative content were revealed in CNKO using high performance liquid chromatography time-of-flight tandem mass spectrometry, and the physicochemical properties, functional group structure, and oxidation stability of CNKO at different pressing temperatures were characterized using a near infrared analyzer and other methods. This study will provide basic data for the processing and product development of CNKO.Cashew nut kernels (W240) were purchased from Chang-da-Chang Super Shopping Supermarket, Zhanjiang, China. The temperature of the press was adjusted, and then the cashew kernels were placed in the press  for extraction, and the crude oil was collected and centrifuged at 20 °C and 5000 rpm for 10 min, and the upper oil layer was collected and stored at 4 °C for use.The determination of acid values was made with reference to GB5009.229-2016 , and the0. A total of 0.05 mL of ferrous chloride was added to sample solution 1, and the reaction was left for 5 min after vortex shaking to obtain sample solution 2, and the absorbance of sample solution 2 was at 501 nm. The absorbance of sample solution 2 at 501 nm was E2. Without adding oil, the absorbance at 501 nm was E1, and the peroxide value (POV) = (E2 − (E0 + E1))/(55.82 × m).The peroxide value was determined by referring to the method of Akbar et al.  with app232, and the corresponding specific extinction coefficient K232 = A232/ω. The absorbance of the sample solution at 270 nm was A270, and the corresponding specific extinction coefficient K270 = A270/ω.The specific extinction coefficient was determined by referring to the method of HST57-2017 and Yakindra et al.  with appThe fatty acid composition in cashew nut kernel oil was determined by gas chromatography (GC-MS)  coupled with the potassium hydroxide methylation method. The relevant parameters were referred to in Liu et al. .The lipid composition in CNKO oil was performed by a Shimadzu UPLC LC-30A system  equipped with a Phenomenex Kinete C18 column , and the relevant parameters were referred to in Liu et al. .−1 with a resolution of 4 cm−1 and 64 scans, and the infrared spectral data were collected with the OMSNIC software  with an average of 3 parallel acquisitions per sample. The average spectrum was used as the sample spectrum, and the infrared spectral curve was plotted using Origin software .Referring to the method of Yakindra et al.  with somThe oxidative stability of the oil was determined by referring to the method of Xia et al.  with somAll samples were measured 3 times in parallel. LipidView software  was used to undertake qualitative analysis of shotgun-MS data. In the process of data analysis, the relevant parameters of the software were set as follows: the mass tolerance was 0.5, the minimum% intensity was 1, the minimum signal-to-noise ratio was 10, the average flow injection spectrum from the top was 30% TIC, and the total double bond was ≤12. OriginPro , SIMCA , and Photoshop  were used for plotting, data processing, and statistical analysis.The fatty acids of CNKO were palmitic acid (9.85 ± 0.04%), palmitoleic acid (0.26 ± 0.01%), stearic acid (10.93 ± 0.31%), oleic acid (60.87 ± 0.06%), linoleic acid (17.33 ± 0.28%), and arachidonic acid (0.76 ± 0.02%). The content of saturated fatty acids was approximately (21.54 ± 0.37%) and unsaturated fatty acids was approximately (78.46 ± 0.35%), including (61.13 ± 0.07%) of monounsaturated fatty acids.The lipid composition of CNKO was comprehensively profiled using UPLC-TOF-MS/MS, and information on the precise relative molecular masses of lipids, isotopic distribution, and secondary mass spectrometry cleavage fragments were obtained in composite scanning mode. The lipids of CNKO were identified as shown in As shown in As shown in The CNKO obtained at different pressing temperatures was slightly yellow in color, without obvious precipitation, with a slight aroma of cashew nut kernel, and its physicochemical properties are shown in The results of the significance analysis showed that there were different degrees of influence of pressing temperature on acid value, iodine value, peroxide value, refractive index, and specific extinction coefficient. The differences in acid value were not significant at temperatures greater than 100 °C. The differences in peroxide value were not significant at 140 °C and 160 °C, and significant at other temperatures . The differences in iodine value were not significant at 120 and 140 °C, and significant at other temperatures. The differences in the refractive index were not significant at 160°C and 180 °C, and the differences in the refractive index were not significant at 100 °C, 120 °C, 140 °C, and 200 °C, and significant between the two groups. Although the results of the significance analysis showed that the relevant indexes of the oils obtained at different temperatures were affected, the value fluctuated less, indicating that the physicochemical properties of CNKO were relatively stable.The Fourier NIR spectra of CNKO are shown in −1 corresponds to the -CH3 antisymmetric stretching vibration, at 2930.41 cm−1 corresponds to the -CH2 antisymmetric stretching vibration, at 2855.40 cm−1 corresponds to the -CH2 symmetric stretching vibration, and at 1744.57 cm−1 corresponds to the C=O stretching vibration. This could be used to identify ketones, aldehydes, acids, esters, and anhydrides. The possibility that CNKO had an anhydride structure was ruled out because the anhydride would have a double peak due to vibrational coupling. Although 1658.01 cm−1 corresponded to the C=C stretching vibration, 2–4 peaks due to benzene ring skeleton vibration were not found near 1600 cm−1 and 1500 cm−1, so the possibility of the presence of an aromatic ring structure in CNKO was excluded. The peak at 1461.81 cm−1 corresponded to the -CH3 asymmetric deformation vibration and at 1375.25 cm−1 corresponded to the -CH3 symmetric deformation vibration. Although it corresponded to the C-C stretching vibration at 1233.88 cm−1, the position of this absorption band changed with the structure of the compound molecule due to the vibrational coupling effect and weak intensity, so it was not meaningful in the structure identification. The peak at 1164.63 cm−1 corresponded to the C-O stretching vibration in alcohols, which could be used to distinguish between primary, secondary, and tertiary alcohols, and CNKO might be the C-O stretching vibration of tertiary alcohols. The peak at 720.30 cm−1 corresponded to a swinging vibration in the -CH2 plane and had more than four methylene-linked structures. As shown in As shown in Oxidative stability could be a good predictor of the oxidation reaction of oils. The auto-oxidation of oils was divided into the induction phase as well as the oxidation phase, and the length of time required from the induction phase to the oxidation phase could reflect the ability of oils to resist auto-oxidation, which was the oxidation stability of oils ,23. The As shown in Cashew nut kernel oil accounted for approximately 47% of the cashew nut kernel content, which was higher than the oil content of avocado 8–29%) ,25, oliv% ,25, olPhospholipids are the main components of the cell membranes of animal and plant cells and play an important role in maintaining the physiological activity of biological membranes and the normal metabolism of the organism. They have important functions in antioxidation and delaying aging , regulatPhysicochemical properties and oxidative stability were important indicators of the quality of oils ,24. The In addition, Michae et al.  showed tIn this study, 141 lipids, including 102 glycerides and 39 phospholipids, were isolated and identified from cashew nut kernel oil using high performance liquid chromatography time-of-flight tandem mass spectrometry. Cashew nut kernel oil was a high oleic acid oil. The glycerol esters were mainly composed of DG, EtherDG, DGCC, DGGA, TG, EtherTG, and OxTG, and the phospholipids were mainly composed of LPC, LPE, LPI, PC, EtherPC, PE, EtherPE, PG, PI, and EtherPI. The total number of carbon atoms in the side chains of fatty acids in cashew nut kernel oil mass was 16–62, with a double bond number of 0–7. With the increase in pressing temperature, the functional group structure of cashew nut kernel oil was not changed, although the iodine valence, peroxide value, and the specific racemization coefficient increased, decreasing the induction time and reducing the oxidative stability of the cashew nut kernel oil.