Rhiszostoma pulmo, was also successfully applied to other edible jellyfish species  present in the Mediterranean Sea. This study discloses an innovative process for the preparation of jellyfish-based food products for potential future distribution in Europe.Edible jellyfish are a traditional Southeast Asian food, usually prepared as a rehydrated product using a salt and alum mixture, whereas they are uncommon in Western Countries and considered as a novel food in Europe. Here, a recently developed, new approach for jellyfish processing and stabilization with calcium salt brining was upgraded by modifying the pre-treatment step of freshly caught jellyfish and successfully applied to several edible species. Treated jellyfish obtained by the application of the optimized version of this method respected both quality and safety parameters set by EU law, including no pathogenic microorganisms, absence or negligible levels of histamine and of total volatile basic nitrogen, no heavy metals; and the total bacterial, yeast, and mold counts were either negligible or undetectable. Jellyfish treated by the presented method exhibited unique protein content, amino acid and fatty acid profiles, antioxidant activity, and texture. The optimized method, initially set up on  Jellyfish (JF) are mainly available and consumed as food in Asian countries. However, their use in food preparation has recently spread widely worldwide in the form of ready-to-use products, also attributed to the availability on internet market channels ,2. In reJellyfish blooms and the invasive behavior of some species make them a good candidate for potential resources for food and other applications ,6,7.JF for human consumption are generally prepared by separating the umbrella from the oral arms and washing them extensively in order to eliminate mucus, gonads, sand, and superficial microorganisms. Then, these highly perishable JF tissues are treated with mixtures of NaCl and aluminum salts  ,8 in ordThe research on new JF stabilization procedures and treatments for food uses is recently moving to limit the use of alum, due to its toxicity ,12, and i) very high aluminum content of Asian traditionally preserved JF products and (ii) lack of safe stabilization methods for treating and processing JF tissues according to EU safety standards. Consequently, the development of a new, safe, and validated technology for processing JF could encourage regulatory authorities to approve the use and commercialization of edible JF species.At present, JF is a novel food in Europe  and is lIn our previous work, we proposed new parameters for the risk assessment of JF as food in Europe . They weRhizostoma pulmo and successively verified on three other potentially edible JF species . A comparison of the JF treated with the optimized processing method proposed here with JF prepared with the traditional Asian methods was also carried out.In this paper, the above-mentioned method was further optimized by modifying the JF pre-treatment step and including washes with sterile seawater; furthermore, the whole procedure was validated by using several JF species with different characteristics. Two different strategies are described here in order to substitute the use of sterile seawater for JF washing, with this step being quite challenging and not applicable on an industrial scale. Additionally, JF treated with calcium salts were tested for safety and quality aspects—the treated tissues were analyzed for protein, fatty acids, amino acids, element content, and antioxidant activity. The process efficacy was initially tested on the model species Rhizostoma pulmo, Macrì 1778  specimens were hand-collected from a motorboat in the Ionian Sea  using a nylon landing net (3.5 cm mesh size) during samplings in the 2019–2020 summer period and processed by either of the two procedures described below.R. pulmo specimens by cutting the oral arms from the umbrella and removing the gastric content. For Cotylorhiza tuberculata, Phyllorhiza punctata, and Rhopilema nomadica samples, also sampled during the 2019–2020 summer period, whole JF were immediately frozen at −40 °C. At least 5 different specimens from each species were used; for Rhizostoma pulmo, randomized sampling was conducted as in Leone et al. [In the absence of specific slaughter guidelines for cnidarians , the traAll the procedures followed in this study are summarized in the scheme reported in Procedure 1 (JF-B). JF oral arms were separated from the umbrellas, immersed in refrigerated seawater, and transported to the laboratory (max 3–6 h). At the laboratory, jellyfish parts were extensively washed for 3–5 min in drinking water.Procedure 2 (JF-DW). Whole JF were immersed in refrigerated seawater immediately after capture and transported to the laboratory (max 3–6 h). At the laboratory, the jellyfish were immersed in drinking water, the umbrellas were separated from the oral arms, and these parts were extensively washed for 3–5 min.Following both procedures, the umbrellas and oral arms were placed in sterile food-grade plastic bags and stored at −80 °C or immediately treated in the newly formulated brines with a firming agent.Cotylorhiza tuberculata samples harvested from the Gulf of Trieste  and the Ionian Sea , and to Rhopilema nomadica and Phyllorhiza punctata harvested from the Eastern Mediterranean , frozen at −40 °C, and shipped on dry ice to Italy. Frozen material was thawed overnight on ice and then extensively washed with drinking water.Procedure 2 (JF-DW) was also applied to Aliquots of all untreated and treated JF samples (see below) were freeze-dried in order to analyze the elemental, lipid, and amino acid compositions or immediately frozen to evaluate the protein content and antioxidant activity. A commercial product, jellyfish stored in brine, from Japan  was also analyzed and used for comparison.Escherichia coli, Salmonella enteritidis, Coagulase-positive staphylococci, Staphylococcus aureus, Vibrio spp. , Bacillus spp. , Shewanella putrefacens, Aereomonas hydrophila, and Pseudomonas fluorescens was assessed following the procedure described by Bleve et al. [Ten grams from each JF sample were added to 90 mL of buffered peptone water  as a diluent (1:10). For total bacterial counts (TBCs), samples were diluted and plated by the pour plate technique on plate count agar (PCA)  at pH 7.0 and incubated at 30 °C for 72 h; the enumeration of yeast and molds was performed by incubation at 25 °C for 5 days on dichloran Rose–Bengal chloramphenicol agar . The presence of Enterobacteriaceae, e et al. .w/v peptone) and artificial seawater. All samples and their respective serial dilutions were plated in different media dissolved in artificial seawater as described by Bleve et al. [For the determination of halophilic microorganisms, JF samples were homogenized with a sterilized blender, and 25 g of each sample was added to peptone seawater 0.1% , coagulase-positive staphylococci (UNI EN ISO 6888-2:1999), and yeast and molds . For the detection of the pathogenic bacteria Salmonella spp. (UNI EN ISO 6579-1:2017) and Listeria monocytogenes (ISO 11290-1:2017), 25 g of jellyfish samples were suspended in 225 mL of buffered peptone water  and Fraser broth at half concentration , respectively, as diluents.The JF samples were also submitted to an accredited external laboratory for independent analyses . Ten grams of each JF sample were added to 90 mL of buffered peptone water  as a diluent (1:10) and homogenized for 2 min in a Stomacher in accordance with specific standard methods for the total bacterial count (UNI EN ISO 4833-1:2013), coliforms (ISO 4832:2006), β-glucuronidase-positive v/v), all from Sigma-Aldrich . These concentrations were arbitrarily chosen and tested for JF stabilization treatment, as already previously described by Bleve et al. [Solutions of calcium citrate and calcium lactate were prepared using 0.1 M calcium-citrate solution or 0.1 M calcium–lactate hydrate solution and adjusted to pH 5.0 by using the corresponding alpha organic acids, 1 M citric acid or 1 M lactic acid 85%  exerted undesirable effects on JF consistency by damaging (attacking and corroding) the tissue (data not shown).v/v, JF tissue:brine) in food-grade glass or plastic containers. These technological phases were arbitrarily transferred from the conditions generally used for vegetable stabilization treatment and directly tested for the first time for their possible adaptation to JF tissues. The containers were stored at 4 °C for 5 days and then JF tissues  were removed, washed with drinking water to eliminate excess salts, sealed in food-grade plastic bags, and stored at −80 °C for further tests. Aliquots of each sample were also freeze-dried and stored.Pre-treated jellyfish  were immersed in brines at a 1:1 ratio  NaCl and 10% KAl(SO4)2·12H2O  (w/w)  using about 100 g of salt–alum mix per 1 kg of JF biomass and incubated at 4 °C in a food-grade glass container. After 4 days, brines released from the JF tissues were removed and the umbrellas were covered with a salt mixture containing 92.5% (w/w) NaCl and 7.5% alum (w/w). After 4 days, the same procedure was repeated, but the percentage of alum in the salt mix was reduced to 5% (w/w) and finally to 2.5% (w/w). At the end of the process, the salted jellyfish samples were left to dry on a draining rack at room temperature for 4 days, inverting them several times to drain and remove excess water. The entire process took 20 days. Aliquots of each sample were also lyophilized.n method ,22 usingThe histamine concentration in the JF was determined according to the AOAC N° 021402 2014 method . The total volatile base nitrogen (TVBN) was determined by treating each jellyfish sample (100 g) with 0.6 M perchloric acid . After alkalinization, the extract was exposed to steam distillation and an acid receiver absorbed the volatile base components. The TVBN concentration was determined by the titration of the absorbed bases .2 by operating on samples consisting of radial triangular slices of the JF tissues. Firmness values were reported as the means of three different measures, expressed in Newtons (N). All analyses were carried out in triplicate.For salinity and pH determination, 15 g of JF tissue was collected and stored at −80 °C for further analysis. The texture was measured as described in Bleve et al. , using a2O2 and 6 mL of super-pure HNO3 69%, digested at 180 °C for 10 min using a microwave digestion system , Italy), cooled, diluted with super pure water, and filtered through 0.45 μm syringe filters. A spectrometer  was previously calibrated for quantitative analysis with five standard solutions containing known concentrations of the elements . The calibration lines showed correlation coefficients (r) greater than 0.99 for all the measured elements. The analysis results were expressed as the average (+/− standard deviation of three different measurements) element concentrations, expressed in ppm (mg/kg of sample weight). JF supernatants obtained after the centrifugation of the JF samples were also analyzed, corresponding to the lyophilized samples.The elemental composition  of the JF samples was measured using inductively coupled plasma–atomic emission spectroscopy (ICP-AES). Lyophilized JF samples were weighed and mixed with 4 mL of HJellyfish samples were homogenized in a blender and diluted in distilled water until a homogeneous solution was obtained. The total protein content in each sample was evaluated using the Bradford assay , set forIn the diluted JF samples (protein assays), the antioxidant activity (AA) was evaluated using the Trolox Equivalent Antioxidant Capacity (TEAC) method based on the radical cation ABTS•+ and Trolox as a standard. The assay was adapted for an Infinite 200 PRO microplate reader , and both samples and the standard were assayed as described in De Domenico et al. . The resg for 5 min. The lower phase was set aside, and the upper phase was subjected to further extraction with one volume of a solution of chloroform:methanol . After phase separation, the lower phase was isolated and added to the first one, and mixed with one-quarter volume of a solution of methanol:water . The lower phase was dried using nitrogen and analyzed for lipid composition.Total lipids were extracted using a modified Bligh and Dyer method : lyophil3), as follows. The total lipid extract in hexane (200 μL) was saponified at 90 °C for 20 min with 0.5 M KOH in methanol (3 mL) with a known quantity of internal standard (methyl-tricosanoate). Fatty acids were methylated with 2 mL of BF3 in methanol (14%), and the samples were evaporated under a stream of nitrogen and dissolved in 50 μL of hexane, and 1 μL was analyzed by gas chromatography–mass spectrometry (GC-MS). GC–MS analyses were performed using an AGILENT 5977E gas chromatograph  on a VF-WAXms  with the following parameters: the column temperature was maintained at 160 °C for 1 min, programmed at 4 °C/min to 240 °C for 30 min. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The mass spectrometer was operated in the electron impact mode with a scan range of 50–700 m/z. The temperature of the MS source and quadrupole were set at 230 °C and 150 °C, respectively. Analyses were performed in the full-scan mode. Compounds were identified by comparing the retention times of the chromatographic peaks with those of authentic standards  analyzed under the same conditions. The MS fragmentation patterns were compared with those of pure compounds, and a mass spectrum database search was performed using the National Institute of Standards and Technology (NIST) MS 98 spectral database.Fatty acid methyl esters (FAME) were obtained according to Leone et al.  using bo+ ion-exchange column . Eighteen standard amino acids, ammonia, and taurine were quantified from standard curves measured with the amino acid standards. Prior to the analysis, the samples were hydrolyzed in 6 M HCl containing 0.4% mercaptoethanol for 24 h at 110 °C (HCl hydrolysis). Glutamine and asparagine were converted to glutamic and aspartic acid, respectively. Cysteine (Cys) was quantified as cystin (Cys-Cys). The samples were filtered via a micro filter, the pH was adjusted to 2.2, and the samples were further diluted with a citrate buffer (pH 2.2) for HPLC analysis.The amino acid profile in lyophilized samples was analyzed with an HPLC system  coupled with an online post-column derivatization module , using ninhydrin (Trione) as a derivatizing reagent and a Nan = 3). Statistical analysis was based on one-way analysis of variance. Tukey’s post hoc method was applied to establish significant differences among the means . All statistical comparisons were performed using Sigma-Stat, version 3.11 .All data presented are the means of three independent replicates  and data were represented as the mean ± standard deviation (SD). Principal component analysis (PCA) to compare important physical parameters and chemical compounds associated with the samples was carried out using XLSTAT software .Statistical analyses on the protein content and antioxidant activity were performed in Graphpad Prism 6.0 using an analysis of variance (ANOVA) followed by Dunnett’s multiple comparison post hoc test to compare each treatment with the control, and Bonferroni’s multiple comparison post hoc test to compare the samples with each other. Differences were considered statistically significant for Two pre-treatment strategies are described here in order to set up an optimized stabilization method for jellyfish intended for food use. In particular, in the first approach , jellyfish were immediately washed with refrigerated seawater just after being harvested, and their umbrellas were promptly separated from their oral arms, an operation that can be carried out on the freshly caught jellyfish directly on board. This approach can greatly reduce the ashore disposal of possibly large quantities of JF by-products, thus returning unused JF material directly to the sea. In the second approach , whole JF were transported in chilled seawater to the laboratory, where the use of drinking water was tested for washing JF after transport.This study proposes the combination of washing with drinking water, a procedure commonly used in the fishing industry, and subsequent treatment with calcium salts. It was observed that this method helped to stabilize the JF tissues, to improve their texture and nutraceutical traits, and to reduce undesired microorganisms in the processed JF products. Moreover, this approach represents an optimization of the recently proposed method for stabilizing and processing JF as food products for human consumption .The main phases of the two procedures for JF preparation proposed in this study are presented in In both pre-treatments proposed in this article, JF samples were washed with drinking water, although at different times. In the JF-B procedure, this washing step occurred after cleaning, cutting, and storing the animals in seawater, and before placing them in calcium salt solutions in the laboratory; in the JF-DW approach, instead, whole JF were firstly transported to the laboratory in chilled seawater and were then immersed in drinking water for the time necessary to wash and prepare the JF tissues before soaking them in calcium salt solutions.Vibrio spp., Salmonella spp., Listeria monocytogenes, and staphylococci) and spoiling microbial contaminants in both JF-B and JF-DW  , thus inR. pulmo was treated in parallel using mixtures of NaCl and alum for tissue stabilization  as described by Hsieh et al. [Bacillus spp. (4 × 101 CFU/g) and discrete levels of halophilic bacteria (4.4–9.9 × 102 CFU/g) and yeasts (2 × 102–103 CFU/g), although no microbial pathogens were detected  exerted different effects on the texture, pH, and salinity of the obtained samples .The optimized calcium citrate and calcium lactate brine treatments exerted different effects on the chemical–physical features of the JF tissues. The preliminary results reported here showed that salt treatment in the JF-B samples reduced the tissue texture (in terms of penetration force); moreover, salinity values of around 2% were measured and the pH values were very different between the two calcium treatments . In Ca-CLee et al.  already R. pulmo JF-DW, 97.85 ± 0.35 g/100 g FW for Ca-Citrate R. pulmo JF-DW, 97.6 ± 0.2 g/100 g FW for Ca-Lactate R. pulmo JF-DW, 81.94 ± 0.56 g/100 g FW for Salt-Alum R. pulmo JF-DW, and 73 ± 0.61 g/100 g FW for Salt-Alum Jp. These data reveal that there were no statistically significant differences existing between the untreated and Ca-Citrate and Ca-Lactate JF-DW R. pulmo samples, whereas a substantial reduction in moisture was obtained in both alum-treated JF samples, thus revealing a further difference between the final products obtained by the two types of procedures. The different moisture contents were considered during the analyses and comparisons of nutrient compounds.In order to characterize their nutritional values, the JF samples treated by different procedures were analyzed to evaluate their protein content, amino acid composition, antioxidant activity, lipid content, and fatty acids composition. The moisture contents of the different JF samples were: 96.88 ± 1.12 g/100 g FW for the untreated R. pulmo tissues washed with drinking water only (JF-DW) contained 253.2 mg protein per 100 g of fresh weight (FW) (The ght (FW) a. This vght (FW) a, slightR. pulmo samples) were assayed in untreated JF, JF-DW, and in calcium salts-treated JF (Ca-Citrate JF-DW and Ca-Lactate JF-DW). The total content of amino acids increased from the untreated JF (6%) to the JF washed with drinking water (JF-DW) (9.2%), Ca-Citrate JF-DW (15.4 ± 0.7%), and Ca-Lactate JF-DW (15.3 ± 0.8%)  . WashingThe percentages of the taurine, leucine, tyrosine, phenylalanine, and lysine amino acids were higher in the fresh and untreated JF than in the treated JF samples. Increases in the percentages of proline, hydroxyproline, and glycine were also observed in JF-DW and the Ca-treated samples (Ca-Citrate JF-DW and Ca-Lactate JF-DW), with proline and hydroxyproline being abundant in collagen , a proteAntioxidant activity (AA) was also evaluated in the same JF samples and expressed in nanomoles of Trolox equivalent per gram of fresh weight  was much higher than that in the treated mo JF-DW b. This eR. pulmo samples, there was an increase in the SFA percentage, from 50.4% (JF-DW) to 79.3 and 64.4% in Ca-Citrate JF-DW and Ca-Lactate JF-DW, respectively. The SFA content in Salt-Alum JF-DW R. pulmo was 81.3% and that in the commercial ready-to-eat jellyfish was 87%. Moreover, Salt-Alum JF-DW and Salt-Alum Jp samples exhibited a more complex lipid profile, since they contained several SFAs that were absent from JF-DW, such as nonadecanoic acid (C19:0), arachidic acid (C20:0), behenic acid (C22:0), and lignoceric acid (C24:0). These differences should be mainly due to the alum treatment of R. pulmo. The total MUFA content generally increased in all treated JF samples compared with untreated JF-DW, thus indicating that the initial content of oleic acid (C18:1) was preserved, and also that iso-oleic acid (C18:1 trans-10), palmitoleic acid (C16:1), and vaccenic acid (C18:1 cis-11) appeared.In JF-DW, saturated fatty acids (SFAs) accounted for about 50% of total fatty acids (FA), followed by polyunsaturated fatty acids  and a small amount of mono-unsaturated fatty acids  . In R. pThe increase in the saturated fatty acids content of all treated samples could be due to lipid oxidation, which may lead to isomerization events and the production of new SFA and PUFA species when lipid carbon chains break up and unsaturated FAs are converted to SFAs .The total PUFA content decreased after all the salt treatments. In the Ca-Lactate JF-DW and Ca-Citrate JF-DW samples, the PUFA content decreased to 14.9% and 26%, respectively, from the initial value of 45% in untreated JF-DW. Moreover, PUFAs were heavily reduced in both Salt-Alum JF-DW and Salt-Alum Jp to 4.8 and 4.9%, respectively. However, despite the decrease in quantity, the PUFA composition was still preserved in the calcium-salt-treated JF. Linoleic (C18:2), linolenic , and stearidonic (C18:4) acids were detected in the calcium-treated samples, but not in the JF-DW, while the contents of other nutritionally relevant FAs, such as arachidonic (C20:4), eicosapentaenoic , docosapentaenoic , and docosahexaenoic  acids, were maintained or increased. Interestingly, the novel ω3-PUFA stearidonic acid (C18:4) was detected in the Ca-Lactate JF-DW sample. This FA species is the substrate for the conversion of alpha-linolenic acid (ALA) into longer ω3-PUFAs  in humans, and it has attracted great interest in recent years because it is obtained only from plants .Overall, in comparison with the corresponding samples obtained by the same authors following the JF-SW method , JF-DW pIn addition, only the treatments with calcium salts yielded ω6/ω3 ratios less than 1 , which represents a healthy composition, as suggested by the nutritional recommendations. The calcium salt-based treatments increased the total lipids concentration in the samples and improved the ratio of essential fatty acids (EFA) naturally present in the untreated material (ω6/ω3 = 2.9). Conversely, the previous not-optimized method proposed by Bleve et al.  reportedR. pulmo treated with the traditional salt–alum traditional method , a value of Pb corresponding to 9.748 ppm (or mg/Kg) of dry weight was observed. Notably, the Ca-Citrate JF-DW and Ca-Lactate JF-DW showed lower contents of metals compared with the samples produced with the traditional alum-based process . This evidence was important to demonstrate the unique features of the products obtained by the optimized method described here, since the element composition of the food matrix can directly impact human health and is therefore closely related to food safety.The profiles of some elements associated with JF samples treated with calcium citrate and calcium lactate brines, as well as with JF treated with salt–alum, revealed the absence or very low levels of cadmium (Cd), lead (Pb) and mercury (Hg) in JF tissues . In R. pChromium (Cr) is considered an essential element, playing a role in the maintenance of carbohydrates, fats, and protein metabolism. However, the levels of this element in Asian-style produced JF should be supervised, since the European suggested daily intake range for humans is 25–200 mg/day . The samAll the tested JF samples did not contain significant levels of Pb, Cd, and Hg, which are considered critical contaminants in foodstuffs ,37.Notably, very low levels of aluminum were detected in the Ca-Citrate and Ca-Lactate JF-DW samples. In accordance with other studies ,38, the A principal component analysis (PCA) was carried out in order to thoroughly compare the JF products obtained by the different treatment methods. The PCA was applied to many relevant parameters, such as the texture, pH, salinity, protein content, fatty acid content, antioxidant activity, and metal content, that can describe the JF samples: JF-DW, Ca-Lactate JF-DW, Ca-Citrate JF-DW, JF treated by the traditional salt–alum method , and the commercial JF  .A bi-plot is used to show the projection of the variables on the plane defined by the first and second principal components. The total variance of the two main components was 80.3 % . PC1 cluThe application of this unsupervised technique disclosed the considerable difference between the products obtained by the newly proposed method and the traditional methods used to prepare JF for human food uses. The Ca-Lactate-JF-DW and Ca-Citrate JF-DW final products were very close to the fresh untreated JF and were characterized by peculiar food safety and food quality traits.Cotylorhiza tuberculate, Rhopilema nomadica, and Phyllorhiza punctata. These three scyphozoa are consumed by humans as food in different areas of the world [The described JF treatments were also applied to three other putatively edible JF species: he world  and wereFood safety and several product features , together with basic nutritional characteristics  were assessed for these samples to evaluate them as potential sea-derived food.Safety and quality traits. The loads of potential pathogenic species, spoilage microbes, and halophilic microbes were not significant in the untreated samples for any of the analyzed JF  showed protein contents of 176.8 mg/100 g FW and 170.3 mg/100 g FW, respectively,  value  and the PUFA isolinolenic acid  when treated with calcium lactate. P. punctata treated with calcium salts exhibited a decrease in the SFA content and an increase in the PUFA contents, whereas the levels of MUFAs did not vary compared with the untreated sample. Remarkably, the MUFA iso-oleic acid (C18:1) and PUFA eicosadienoic acid (C20:2) appeared in P. punctata Ca-Lactate JF-DW, although they were not initially detectable in the untreated JF sample. However, as shown in P. punctata was less than 1 (ω6/ω3 < 1) and was maintained after the samples were treated with calcium salts . Among all other treatments, C. tuberculata JF-DW treated with calcium citrate was the only species having yielded a favorable ratio of ω6/ω3 < 1 (0.7, R. nomadica and C. tuberculata samples treated with calcium lactate always achieved a ω6/ω3 ratio higher than the recommended score of 1 (The FA profiles, reported as the percentage of total FA, were very different between the three JF species . R. noma< 1 0.7, . On the ore of 1 .R. pulmo JF species, and later successfully applied to other JF species present in the Mediterranean Sea, thus leading to a preliminary validation of the proposed method. The products obtained by the method described here were used for the formulation of new food prototypes. The characterization of the safety, quality, nutritional, and sensory aspects is ongoing. This study can offer a contribution to fill the knowledge gap in the assessment of JF use as human food in Western countries, even though further important evidence needs to be gathered in terms of toxicological and allergen testing. In addition, the technological simplicity of this process will allow its application in poor coastal environments. As a potential future application, a new commercial kit based on the procedure proposed here could be easily developed and retailed by the same fish shops selling freshly harvested edible JF as a helpful tool for consumers interested in preparing homemade and safe JF-based dishes.An optimized method that combines pre-treatment with drinking water followed by a soaking step in calcium salt brine was proposed to stabilize and treat JF for possible food uses in Europe and Western countries. The described procedure for JF-DW pre-treatment improved the fundamental aspects of JF tissue stabilization. The significant reductions in any microbiological growth and undesired enzymatic risks, increased texture values, and desirable antioxidant and fatty acid profiles are some relevant improvements with respect to the very recently proposed JF-SW method . MoreoveThe optimized method for JF treatment and stabilization described in this manuscript is part of the European Patent EP 3763224, deposited on 2020 .


Norovirus is the most important cause of acute gastroenteritis, yet there are still no antivirals, vaccines, or treatments available. Several studies have shown that norovirus-specific monoclonal antibodies, Nanobodies, and natural extracts might function as inhibitors. Therefore, the objective of this study was to determine the antiviral potential of additional natural extracts, honeys, and propolis samples. Norovirus GII.4 and GII.10 virus-like particles (VLPs) were treated with different natural samples and analyzed for their ability to block VLP binding to histo-blood group antigens (HBGAs), which are important norovirus co-factors. Of the 21 natural samples screened, date syrup and one propolis sample showed promising blocking potential. Dynamic light scattering indicated that VLPs treated with the date syrup and propolis caused particle aggregation, which was confirmed using electron microscopy. Several honey samples also showed weaker HBGA blocking potential. Taken together, our results found that natural samples might function as norovirus inhibitors. Norovirus infections can occur in all age groups and results in significant morbidity and mortality, particularly in the very old and very young patients. Preventive measures against norovirus include hand washing, isolation of those infected, meticulous cleaning and disinfection.Human noroviruses typically cause an acute illness of fever, nausea, vomiting, cramping, malaise, and diarrhea that can typically persists for 2 to 5 days. Noroviruses are highly contagious and 10–100 viral particles may be sufficient to infect an individual. The US Centers for Disease Control and Prevention estimates that it is the most common cause of acute gastroenteritis in the United States with 21 million cases each year and an estimated 70,000 hospitalizations and 800 deaths per year. Worldwide, norovirus infections account for ~ 20% of diarrheal illnesses and as many as 200,000 child deaths each year2. Each of these genogroups is further subdivided into numerous genotypes. Human noroviruses often evolve into genetically and antigenically variant strains, and frequent genetic recombination at the RdRp and capsid junction increases the genetic and antigenic diversity.Noroviruses have a single-stranded, positive sense RNA genome of ~ 7.4 to 7.7 kbases. The genome contains three open reading frames (ORFs), where ORF1 encodes the non-structural proteins, which include the protease and RNA dependent RNA polymerase (RdRp), ORF2 encodes the capsid protein (VP1), and ORF3 encodes a minor structural protein. Based on the capsid amino acid sequences, noroviruses can be divided into at least ten genogroups (GI-GX), with GI, GII, and GIV mainly causing infections in human4. Likewise, the GII.10 VLPs exhibited both T = 3 and T = 1 icosahedral symmetry6. The X-ray crystal structure of norovirus VLPs identified two domains, shell (S) and protruding (P) domain. The S domain surrounds the viral RNA, whereas the P domain, which can be further subdivided into P1 and P2 subdomains, and contains determinants for co-factor attachment and antigenicity.Expression of the capsid gene in insect cells leads to the self-assembly of virus-like particles (VLPs) that are antigenically similar to the native virions with a T = 3 icosahedral symmetry. However, recent structural studies have discovered the sizes of VLPs can vary among the genotypes, where GII.4 VLPs were discovered to mainly fold into particles with T = 4 icosahedral symmetry, but also native sized T = 3 and smaller T = 1 particles were observed13. The ABH types are characterized by an α-l-fucose-(1-2)-β-d-galactose connection, whereas the Lewis types contain an α-l-fucose-(1-3)/(1-4)-β-d-N-acetyl-glucosamine. The GII noroviruses primarily interact with the ABH-fucose, Lewis-fucose, or a combination of both. Earlier structural studies showed that noroviruses bind two HBGA molecules per P domain dimer, and the GI and GII noroviruses bind HBGAs in different regions on the capsid. We recently identified two additional fucose-binding pockets on the GII norovirus capsid, which suggested that HBGA binding interactions are likely more complex than previously recognized15. Moreover, both epidemic and rarely detected noroviruses bind multiple HBGA types16.Noroviruses bind histo-blood group antigen (HBGA) co-factors, which are polymorphic carbohydrate structures present as free antigens in saliva and found on the surface of various epithelia. HBGAs are typically grouped into either ABH or Lewis types and at least nine different HBGA co-factors have been recognized to bind to noroviruses20. A number of other studies have also discovered norovirus-specific monoclonal antibodies that can inhibit HBGA binding25. The identification of such compounds blocking HBGA binding are potential treatments for norovirus infections. A number of studies have also examined the HBGA blocking potential of natural extracts, for example Chinese gall, pomegranate juice, and tannic acid26, cranberry juice27, grape seed extract28, persimmon extract and tannins29, and green tea extracts30. Many of these extracts were found to target the HBGA pocket and show great promise as natural therapies.The HBGA- and fucose-binding pockets on the norovirus capsid are one conceivable target for antiviral compounds. Indeed, we found several compounds that bind at the HBGA pocket, i.e., human milk oligosaccharides (HMOs), citrate, and molecules that overlap the HBGA pocket, i.e., norovirus-specific Nanobodies and a norovirus-specific monoclonal antibodyIn this study, we screened seven different natural extracts , ten different honey samples, and four propolis samples to identify inhibitors that block norovirus GII.4 and GII.10 VLPs from binding to HBGAs. Our data showed that several natural samples  inhibited VLPs from binding to HBGAs. Treatment of VLPs with these samples indicated a disruption of VLP integrity and resulted in aggregation of particles. Overall, our results indicated that both epidemic and rarely detected genotypes could be blocked from binding to HBGAs with date syrup and propolis.50 = 0.06% and 0.11%, respectively). Coconut blossom syrup and apple sweetener only weakly inhibited GII.4 VLPs , but not GII.10 VLPs. These three extracts showed a dose-dependent inhibition. Several natural extracts exhibited unusual binding properties with the GII.4 VLPs, i.e., barley malt, royal jelly, and agave nectar showed negative-inhibition effects at low concentrations. At concentrations above 0.78%, barley malt inhibited GII.4 VLPs (IC50 = 0.90%). This unusual GII.4 inhibition event with barley malt was also observed with 2’FL inhibition assays (unpublished data).Seven natural extracts were examined for their ability to block the epidemic GII.4 and the rarely detected GII.10 norovirus VLPs from binding to HBGAs using ELISA , Alpine forest honey (IC50 = 4.78%), Oak tree honey (IC50 = 5.85%), and eucalyptus honey (IC50 = 6.22%). For GII.10 VLPs, a similar finding was observed, i.e., Alpine forest honey (IC50 = 3.38%), raspberry honey (IC50 = 5.60%), eucalyptus honey (IC50 = 5.96%), and Oak tree honey (IC50 = 6.11%). Experiments with only solvent produced results similar to the PBS controls (no treatment), which indicated the solvents did not affect the inhibition. Overall, these findings showed that honey samples from different herbal and regional origins could block norovirus VLPs at varied concentrations and similarly between the two genotypes. We also evaluated the treatment with honey on the stability of VLPs using DLS  and GII.10 (IC50 = 0.57%). A small difference in inhibition levels was observed with DMSO propolis i.e., GII.4 (IC50 = 1.74%) and GII.10 (IC50 = 5.38%). We also evaluated propolis treatment on the stability of GII.4 and GII.10 VLPs using DLS  occurred when treated with the monoclonal antibody and suggested that the VLPs might cross-link which in turn sterically blocked the HBGA binding site. However, the monoclonal antibody did not bind directly to the HBGA pocket and the footprint was located on the side of the P domain. A similar observation was made with several norovirus-specific Nanobodies6. Inhibition studies using natural extracts, such as pomegranate juice, cranberry juice, and green tea extracts have indicated varied levels of HBGA inhibition, although the precise binding site on the capsid remains unknown30. The structural basis other natural samples, i.e., citrate and HMOs showed that these molecules bound precisely at the HBGA pocket on the capsid, which in turn blocked VLP attachment to HBGAs19.A recent study using a norovirus cell culture system, where human enteroids are used to propagate the virus, showed a norovirus-specific monoclonal antibody inhibited norovirus virions in cell culture40.Presently, there is little available for treating a norovirus infection, while vaccines and antivirals are still at the clinical trial phase and development stage, respectively. From a therapeutic point of view, most infected individuals would favor the possibility of reducing the severe symptoms, which can include profuse vomiting, nausea, fever, and diarrhea. In an outbreak setting, norovirus is highly contagious, and virions are stable in the environment for long periods. Disinfection methods include chlorine bleach and hydrogen peroxide. Alcohol based disinfectants were found to have mixed conclusions but are generally regarded as a last line for defense. For practical purposes, chlorine bleach and hydrogen peroxide generally require the closure of the contaminated areas, and this can include hospital wards, schools, cruise ships, and aged-care homes. Another important area of norovirus infections is related to food contamination, which can include oysters, clams, ice, and fruit. Contaminated food is becoming an increasing problem worldwide and decontaminating food is a high priority for the agriculture and seafood sectors. The discovery of an inhibiting, safe, and natural treatment would be of great interest and benefit. So far, decontamination methods have included UV radiation, chemical disinfectants, and high pressure50 = 0.06%) and GII.10 (IC50 = 0.11%) norovirus VLPs using ELISA, which was also confirmed using DLS and EM. Barley malt showed a weaker inhibition potential with GII.4 norovirus VLPs (IC50 = 0.90%), which was confirmed using DLS and EM, whereas for GII.10 norovirus barely malt had little effect at the tested concentrations. Honey samples were less effective at blocking GII.4 and GII.10 VLP attachment to HBGAs compared to date syrup, although treatment caused VLP aggregation. All propolis samples showed inhibition potential for GII.4 and GII.10 VLPs, especially 96% EtOH propolis , which was confirmed using DLS and EM. Our results were comparable to citrate and Nanobody treatments that showed the integrity of norovirus VLPs were compromised and changes in morphology included particle disassembly and aggregation18. Likewise, treatments with other natural extracts observed similar findings. For example, grape seed extract treatment resulted in particle deformation and enlargement28. Several studies using surrogate noroviruses  also found that natural fruits or their components could inhibit or reduce infectivity45.In the current study, we showed date syrup was the strongest inhibitor of GII.4 , ten different honey samples , and two propolis samples (MF and tincture) were purchased from various sources Table . Natural46. Briefly, VLPs were clarified from insect cell medium, pelleted by ultracentrifugation, and then further purified using CsCl gradient ultracentrifugation.Norovirus VP1 of two different GII genotypes, GII.4  and GII.10 , were expressed in insect cells as described previously17. Briefly, 96-well plates were coated with pig gastric mucin type III (PGM), washed three times with PBS containing 0.1% Tween 20 (PBS-T), and subsequently blocked with 5% skimmed milk in PBS. The concentration of untreated GII.4 and GII.10 VLPs were optimized for binding to PGM as previously described47. The GII.4 VLPs (1 μg/ml) or GII.10 VLPs (10 μg/ml) were mixed (1:1) with serially diluted extracts (starting at a concentration from 25%) for 3 h at room temperature. Plates were washed three times with PBS-T, then 100 µl of each VLP/ natural extract mixture was added to triplicate wells for 2 h at room temperature. After washing, 100 µl of GII.4 or GII.10 genotype-specific polyclonal rabbit antibody was added as a primary detection antibody for 1 h at room temperature. Following a wash step, horseradish peroxidase (HRP) conjugated polyclonal anti-rabbit antibody or HRP-conjugated streptavidin was added to the wells and incubated for 1 h at room temperature. All plates were washed and developed with o-phenylenediamine dihydrochloride (OPD) and H2O2 in the dark for 30 min at room temperature. The reaction was stopped with 3 N HCl and absorbance was measured at 490 nm (OD490). Negative controls, i.e., VLPs without inhibitor and no VLPs were used for all plates. The OD490 value of the untreated VLPs was set as the reference value corresponding to 100% binding. The concentrations of the different extracts were defined as 100% pre-dilution. The percentage of inhibition was calculated as [1-(treated VLP mean OD490/mean reference OD490)] × 100. The half-maximal inhibitory concentration (IC50) was determined using Prism software (version 8.0). All experiments were performed with three replicates and the mean and standard deviation calculated.Blocking assays were performed using a method as descried earlier for HMOsThe hydrodynamic diameter of VLPs was analyzed using dynamic light scattering (DLS) with a Zetasizer Nano S . The VLPs (1 mg/ml) and natural extracts  were mixed (1:1) and incubated for 10, 60, and 120 min at room temperature, diluted in 1 ml of distilled water, and then measured. For propolis samples, a 25% working solution was left for 48 h at 4 °C and then briefly centrifuged to remove precipitated beeswax. Intensity measurements were performed at 25 °C in three runs with 15 measurement cycles.6. A 25% stock of natural extracts or honey were mixed (1:1) with 1 mg/ml VLPs and incubated for 1 h at room temperature . Propolis samples were prepared as described for DLS. Samples were diluted 1:40 in distilled water and immediately loaded on carbon coated EM grids. Grids were washed with distilled water, stained with 0.75% uranyl acetate, and then examined on a Zeiss 900 electron microscope. Numerous EM images were analyzed for each sample and one representative was shown.The VLPs (treated and untreated) were analyzed using negative stain electron microscopy (EM) as described


Mycobacterium tuberculosis (Mtb) to resist and tolerate antibiotics complicates the development of improved tuberculosis (TB) chemotherapies. Here we define the Mtb protein CinA as a major determinant of drug tolerance and as a potential target to shorten TB chemotherapy. By reducing the fraction of drug-tolerant persisters, genetic inactivation of cinA accelerated killing of Mtb by four antibiotics in clinical use: isoniazid, ethionamide, delamanid and pretomanid. Mtb ΔcinA was killed rapidly in conditions known to impede the efficacy of isoniazid, such as during nutrient starvation, during persistence in a caseum mimetic, in activated macrophages and during chronic mouse infection. Deletion of CinA also increased in vivo killing of Mtb by BPaL, a combination of pretomanid, bedaquiline and linezolid that is used to treat highly drug-resistant TB. Genetic and drug metabolism studies suggest that CinA mediates drug tolerance via cleavage of NAD-drug adducts.The ability of  et al. show that the protein CinA mediates drug tolerance in Mycobacterium tuberculosis by cleaving NAD-drug adducts, suggesting CinA as a potential target to shorten tuberculosis treatment by potentiating the efficacy of currently used antibiotics.Drug tolerance complicates the treatment of tuberculosis. Here, Kreutzfeldt  Mycobacterium tuberculosis (Mtb) remains the leading cause of death by a single infectious bacterium1. Partly responsible for the lack of success in fighting tuberculosis (TB) is the length and complexity of the required drug regimen, which still consists of at least 6 months of combination therapy with up to four drugs when TB is caused by drug-sensitive Mtb2. The need for such lengthy and complicated regimens is generally believed to be due to Mtb subpopulations that persist in the face of drugs that sterilize actively replicating Mtb in vitro. The mechanisms that enable Mtb to persist are complex. They include poor penetration of drugs into some infected lesions3 and alterations in bacterial physiology leading to drug tolerance, a phenomenon that is distinct from drug resistance. Drug resistance allows bacteria to grow at drug concentrations that kill their susceptible siblings. In contrast, drug tolerance prolongs survival during exposure to a bactericidal drug concentration without changing the minimal concentration of the drug that is required to prevent growth4. In vivo, the most tolerant forms of Mtb are located within the avascularized lung caseum8. Additionally, drug tolerance facilitates the emergence of drug resistance9. Targeting Mtb drug tolerance is thus not only a promising strategy to shorten TB chemotherapy10 but may also slow or prevent the emergence and spread of drug-resistant TB.Killing 1.5 million people last year, Mtb, isoniazid is converted by the catalase-peroxidase KatG into an isoniazid-NAD adduct11 representing its major bioactive species12. Binding of isoniazid-NAD to the NADH-dependent fatty acid synthase II (FAS-II) component enoyl-acyl carrier protein reductase InhA inhibits mycolic acid biosynthesis13. In murine models of Mtb infection, isoniazid shows effective killing in the acute phase of infection, during which bacteria replicate exponentially inside the lungs. This is followed by a protracted chronic phase during which bacterial numbers stabilize as bacterial replication slows due to onset of adaptive immunity, resulting in a marked loss of activity of isoniazid against Mtb16. In addition, treatment of infected humans and guinea pigs with isoniazid results in a biphasic mode of killing that consists in a rapid initial killing of actively replicating Mtb followed by a slower killing of isoniazid-tolerant subpopulations18.Isoniazid is an essential component of the current front-line treatment regimen for drug-sensitive TB which also includes rifampicin, pyrazinamide, and ethambutol. Within competence inducing gene A (cinA), is present in the majority of bacteria and encodes a deamidase that is thought to facilitate the recycling of nicotinamide mononucleotide generated through non-redox utilization of NAD by enzymes such as the DNA ligase LigA19. In Mtb, as is the case for many bacterial species, this deamidase is fused to an N-terminal COG1058 domain that is endowed with pyrophosphatase activity against adenosine nucleotide-containing molecules such as ADP ribose and NADH21.The so-called Mtb CinA protein acts as a modulator of four clinically used TB drugs, namely isoniazid, ethionamide, delamanid, and pretomanid, most likely via a single mechanism, the cleavage of drug-NAD adducts. We report detection of a pretomanid-NAD adduct, which has previously been suggested but not reported to contribute to the mechanism of action of pretomanid22. We show that deletion of CinA results in accelerated killing in vitro via reduction of drug-tolerant Mtb persisting during treatment despite being intrinsically susceptible to these drugs. In vivo, the absence of CinA potentiates not only isoniazid treatment during the chronic phase of mouse infection, when isoniazid kills less effectively, but also enhances killing by the recently developed pretomanid containing regimen BPaL, which showed promising outcome when used to treat patients with multidrug-resistant Mtb in the NIX-TB trial and has been approved by the FDA to treat highly drug-resistant Mtb strains23.Herein, we demonstrate that the pyrophosphatase domain of the Mtb transposon mutants exhibiting increased susceptibility to killing by isoniazid in two conditions in which tolerance to the drug is well documented: adaptation to IFNγ-activated primary mouse bone marrow-derived macrophages (BMDM)7 and starvation in phosphate-buffered saline (PBS)24. In BMDMs, the mutant library was subjected to two cycles of infection. Each time, isoniazid treatment was initiated 24 h post infection at which point the bacteria have been internalized by the macrophages. Comparing the relative abundance of each mutant in the input pool to the mutant pool recovered after the second BMDM infection identified 18 mutants that were significantly underrepresented after intracellular exposure to isoniazid . Similarly, cinA mutants were the only mutants underrepresented with statistical significance after treatment of PBS-starved Mtb with isoniazid  and confirmed loss of CinA by immunoblot  of isoniazid and rifampicin were similar for the ΔMtb Fig. , indicatMtb Fig. .cinA mutant replicated normally during acute infection and survived equally well as wild-type when Mtb entered the chronic phase of infection  while wild-type Mtb decreased less than 10-fold  or the PncC deamidase domain (CinAK323A)  of Mtb Fig. . Two othugs Fig. .Mtb residing in caseum is extremely drug tolerant8. Therefore, we tested the impact of isoniazid and pretomanid on ΔcinA residing in a caseum mimetic30. Following a 4-week incubation in lipid-rich caseum mimetic, non-replicating bacteria were treated with increasing concentrations of isoniazid and pretomanid for 7 days  were 10- and 15-fold lower, respectively, compared to the MBC90 against wild-type Mtb and the complemented strain. Of note, the impact of deletion of cinA on the pretomanid bactericidal activity was concentration dependent. Above 8 μM, the difference in kill kinetics between wild-type and ΔcinA disappeared.ays Fig. . The mincinA also accelerated killing of replicating Mtb by ethionamide, delamanid and, to a lesser extent, pretomanid, but not rifampicin  and 15 × MIC against wild-type (4.5 μg/ml). The difference in pretomanid kill kinetics was concentration dependent similar to what we observed in the caseum mimetic; at 1 μg/ml pretomanid selectively killed ΔcinA but did not affect viability of wild-type Mtb, while increasing the pretomanid concentration to 4.5 μg/ml resulted in a similar killing of wild-type and ΔcinA  was observed indicating increased susceptibility of the mutant which might be a result of CinA contributing to intrinsic resistance to that drug  inducible promoter (cinA-TetON). This resulted in CinA overexpression in both the absence and presence of atc, likely because of leaky transcription by the TetR-controlled promoter . This analysis revealed a molecule that: (i) matched both the predicted mass of the isoniazid-NAD adduct ([M + H] = 771.153) and observed chromatographic retention time and mass of a chemically synthesized isoniazid-NADH standard with a mass error <10 ppm, (ii) was only observed in isoniazid-treated samples; and (iii) exhibited a fragmentation pattern consistent with its predicted chemical structure and observed with a chemical standard synthesized by chemical oxidation of INH with stoichiometric amounts of manganese pyrophosphate and NAD(H)  and adenine ([M + H] = 136.063) moieties of the isoniazid-NAD adduct. The intensity of these peaks increased in a dose-dependent fashion, with the mutant accumulating approximately threefold higher levels of adduct than wild-type  Fig. 31,32. Thype Fig. . As antiype Fig.  and overype Fig. . Mtb expMtb to pretomanid and sought evidence of molecules that met the following criteria: they were specific to pretomanid-treated Mtb, as reported by their absence in both untreated cells and cells treated with INH or ETH; displayed a dose-dependent increase in cells exposed to pretomanid and were structurally consistent with adduction to a NAD molecule. We identified one such ion ([M + H] = 979.1995) that matched these criteria and yielded fragmentation products corresponding to an in silico prediction of a putative pretomanid-NAD adduct33, as a chemical standard could not be synthesized  accumulated pretomanid-NAD similar to ΔcinA, while expression of CinAK323A (with an inactive PncC deamidase domain) resulted in levels similar to those in the complemented strain  had a favorable outcome in 90% of patients with multidrug-resistant TBeks Fig. . This reMtb drug treatment, kills replicating bacilli in vitro and in vivo. However, when Mtb enters a slow- or non-replicating state, for example, due to nutrient starvation, hypoxia, or in response to adaptive immunity, the entire population becomes isoniazid tolerant and persists even in the presence of high isoniazid concentrations34. Little is known about the mechanisms leading to isoniazid tolerance when Mtb ceases to replicate. To identify genetic factors that aid the survival of Mtb in conditions that render the whole population refractory to isoniazid treatment we performed genome-wide screens and identified a single gene, cinA.Isoniazid, a corner stone of standard cinA or inactivation of CinA’s pyrophosphatase domain not only increased killing in conditions where the entire Mtb population exhibits isoniazid tolerance, it also resulted in a substantially smaller persister subpopulation within actively replicating Mtb cultures. During axenic growth isoniazid rapidly kills more than 99% of the replicating Mtb population; however, a small subpopulation of isoniazid persister cells is only killed slowly resulting in a biphasic kill curve35. Stochastic repression of KatG activity, asymmetric cell division, and reversible increases in drug efflux or posttranslational modification can all lead to isoniazid-tolerant persisters40. The fraction of persisters present in replicating Mtb may thus consist of yet smaller, mechanistically distinct subpopulations. These subpopulations refractory to drug treatment are of particular interest as they are thought to contribute to the unusually long TB treatment and targeting these subpopulations has the potential to shorten TB chemotherapy10. The absence of CinA did not prevent the emergence of drug-resistant bacteria, but the drug-resistant population of ΔcinA was smaller than the one that emerged in drug-treated wild-type Mtb. This suggests that CinA inactivation could delay the appearance of drug-resistant Mtb. This work also underscores that drug tolerance does not always rely on slowly or non-growing cells, because the pyrophosphatase activity of CinA impaired drug mediated killing of actively replicating Mtb.Deletion of Mtb and the cinA mutant, indicating that CinA mediates tolerance and not resistance to these drugs. In the case of pretomanid, however, a small MIC shift was observed leaving the possibility that CinA also confers intrinsic resistance to this drug. Moreover, overexpression of CinA increased the MICs of isoniazid and ethionamide but had no impact on the activities of pretomanid or delamanid. All four drugs are pro-drugs whose activation requires a bacterial enzyme. For isoniazid, ethionamide, and delamanid it has been demonstrated that activation leads to NAD-drug adducts; adduct formation of pretomanid has been predicted because of the similarity of this drug to delamanid29. We confirmed this prediction by identifying the pretomanid-NAD adduct in live Mtb. This supports our hypothesis that CinA mediates tolerance through cleavage of formed NAD-drug adducts, which is further substantiated by our finding that in the absence of CinA NAD-drug adducts accumulated inside the bacilli during isoniazid, ethionamide, and pretomanid treatment. The different impact of CinA expression on drug tolerance and resistance suggest that the antibiotics we tested inhibit growth and kill Mtb by more than one mechanism and that the NAD adduct is more important for killing than growth inhibition. Similarly, the concentration-dependent hypersusceptibility of ΔcinA to pretomanid implies that the pretomanid-NAD adduct contributes to its bactericidal activity at low concentrations, while at higher concentrations other mechanisms dominate.CinA mediated tolerance not only to isoniazid but also to ethionamide, delamanid, and pretomanid. The MICs of isoniazid, ethionamide, and delamanid were not different between wild-type 42. Clinical precedents for this strategy exist, for example, in the form of Zavicefta in which the β-lactamase inhibitor avibactam strongly potentiates the activity of the cephalosporin ceftazidime43 to the extent that it is effective against Mtb including highly drug-resistant strains44.In this study we identified a single protein, CinA, as a mediator of tolerance to four TB drugs in clinical use. CinA-mediated tolerance is a direct result of its pyrophosphatase domain facilitating cleavage of NAD-drug adducts. Hence, targeting CinA with inhibitors of its pyrophosphatase domain has the potential to shorten treatment of drug-sensitive TB by potentiating the first-line drug isoniazid or enhancing treatment regimens for multi- and extensively drug-resistant TB by potentiating delamanid and pretomamid. Furthermore, targeting CinA could also complement and extend ongoing efforts to improve the clinical value of ethionamide containing regimensMycobacterium tuberculosis H37Rv was used as the parental strain for all mutants throughout the study. Bacteria were cultured using Middlebrook 7H9 liquid medium supplemented with 0.2% glycerol, 0.05% Tween-80, and ADN  or Middlebrook 7H10 solid agar supplemented with 0.2% glycerol, 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC)  and if necessary supplemented with 0.4% activated charcoal. Antibiotics for selection of genetically modified strains were added at the following concentrations: hygromycin (50 μg/ml) and kanamycin (25 μg/ml).2O.Isoniazid (INH), Rifampicin (RIF), Ethionamide (ETH), and Pretomanid (PMD) were purchased from Sigma; Delamanid (DMD) was procured from MedKoo Biosciences. All antibiotics were dissolved in DMSO with exception of INH, which when used for treatment of mice, was dissolved in H45. Macrophages were seeded at 1 × 107 cells per T75 flask or 1 × 105 per well in 96-well plates in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 10% L929 culture filtrate, and 10 mM HEPES overnight prior to infection. Where indicated macrophages were activated with IFNγ (20 ng/ml) overnight prior to infection and maintained in IFNγ for the remainder of the infection. Bacteria were grown to early log phase, washed in PBS-Tween-80 0.05% prior to being diluted into DMEM, and added to macrophages at a multiplicity of infection (MOI) of 0.1. After 4 h macrophages were washed twice with warm PBS to remove extracellular bacteria. Intracellular bacteria were enumerated by lysing macrophages with 0.01% Triton X-100 and plating serial dilutions of lysates on 7H10 agar supplemented with 0.4% charcoal.Bone marrow cells were harvested from female C57BL/6 mice and differentiated into BMDMs by culturing the cells in the presence of 20% L-cell conditioned mediumMtb H37Rv by himar1 mutagenesis47. Transposon mutant library stocks were cultured in liquid medium for 4 days with agitation (100 rpm) to allow recovery. For PBS starvation experiments cultures were washed twice in PBS containing 0.05% tyloxapol and adjusted to an optical density (OD)580 of 0.2. Libraries were starved for 14 days prior to exposure to 0.5 μg/ml INH or an equal amount of DMSO. After a total of 28 days of starvation libraries were cultured on 7H10 agar supplemented with 0.4% charcoal.Transposon mutant libraries were constructed in wild-type 7 IFNγ-activated BMDMs were infected with transposon mutant libraries at a MOI of 1. At 24 h post infection (p.i.) 0.1 μg/ml INH or an equivalent amount of DMSO was added to infected BMDMs. Macrophages were lysed 120 h p.i. and lysates cultured on 7H10 agar supplemented with charcoal. After 21 days of outgrowth colonies recovered from infected macrophages were collected, transferred into liquid medium and grown for 4 days prior to re-infection of BMDMs as before.For infection of BMDMs 1 × 1048. Sequencing reads were processed and analyzed using TPP and TRANSIT tools from the TRANSIT TnSeq analysis platform, respectively, and transposon–chromosome junctions were mapped to the Mtb H37Rv reference genome (GenBank accession number NC_018143.1)49.Genomic DNA was extracted from the recovered transposon mutant libraries and transposon–chromosome junctions were amplified and sequenced to determine the relative abundance of each transposon mutant in input and INH-treated libraries as described previouslycinA mutant was constructed in Mtb by replacing cinA (rv1901) with a hygromycin cassette through homologous recombination as described elsewhere51.The first 100 bp of the open reading frame of cinA were retained to avoid disruption of the rv1900c promoter. Specifically, nucleotides 101–1193 of cinA were replaced with a hygromycin-resistance cassette. To achieve this, Mtb H37Rv transformed with pNit-RecET-sacB-kanR was grown to mid log phase and expression of the RecET proteins was induced by addition of 10 μM isovaleronitrile. After 8 h of RecET induction the cells were treated with glycine and incubated for 16 h as described51. A DNA fragment containing the hygromycin-resistance cassette flanked by 500 bp of the upstream region including the first 100 bp of cinA and 500 bp of the sequence downstream of cinA was synthesized (GeneScript Biotech) and transformed by electroporation. Knockout candidates were selected on hygromycin-containing agar. Deletion of cinA was confirmed by PCR and western blot analysis. Plasmid pNitET-sacB-kan was cured by counterselection on 10% sucrose containing agar. A complemented strain was obtained by introducing a copy of wild-type cinA into the Mtb genome at the attL5 attachment site under the control of a hsp60 promoter. For this, we constructed pGMCK-Phsp60-cinA-containing a kanamycin resistance gene, the hsp60 promoter upstream of cinA (nucleotides −28 to 1193), the integrase gene, and the phage attP sites for site-specific integration into the mycobacterial chromosome. We used plasmids pDE43-MCK , pEN41A-T02 (ampR multisite Gateway entry plasmid), pEN12A-Phsp60 52, and pEN23A-cinA . Complementation strains expressing cinA harboring point mutations  rendering either the pyrophosphatase or the deamidase domain of the gene inactive were obtained by the same approach27. For overexpression we cloned cinA downstream of the anhydrotetracycline inducible promoter P606 (ref. 53). All DNA constructs were obtained from GenScript Biotech and plasmids were generated using Gateway Cloning Technology (Life Technologies).The ΔcinA mutant and the impact of INH during infection were conducted following guidelines for care and use of laboratory animals provided by the National Institute of Health and with approval from the Institutional Animal Care and Use Committee of Weill Cornell Medicine (IACUC protocol 060-441A). Female 8-week-old C57BL/6 mice (Jackson Laboratory) were infected with ~100–200 CFU/mouse using an Inhalation Exposure System (Glas-Col). The mice were housed in HEPA-filtered cages within the ABSL-3 facility with a 12 h light–dark cycle at 70F ± 2F with 50% humidity (range is 30–70%). At indicated time points bacterial burden in lungs and spleens was determined by culturing serial dilutions of organ homogenates on 7H10 agar. Where indicated, mice were treated with isoniazid delivered through drinking water at the following concentrations: 10, 5, and 2.5 mg/kg/day starting from day 42 p.i. Isoniazid-containing water was replaced every 7 days.The mouse experiments to assess virulence of the ΔcinA H37Rv was conducted following guidelines for care and use of laboratory animals provided by the National Institute of Health and with approval from the Institutional Animal Care and Use Committee of Hackensack Meridian Health (IACUC protocol 265). Female 8-week-old BALB/c mice (Charles River Laboratories) were infected as described above. At 4 weeks p.i., mice received daily oral doses (7 days a week) of BDQ 20  mg/kg, PMD 50 mg/kg, and LZD 100 mg/kg, formulated in 20% HPBCD in 50 mM sodium citrate pH 3, 10% HPBCD/10% lecithin/80% water and 0.5% CMC/0.5% Tween-80, respectively. BDQ and PMD were given in the morning and LZD 4 h later as described in Xu et al.54. After 4 weeks of treatment, bacterial burden in lungs and spleens was determined by culturing serial dilutions of organ homogenates on 7H10 agar. After 1 and 3 weeks of daily drug administration, therapeutic drug monitoring was performed by sampling blood from the tail vein of three to six mice per group, at peak and trough, as indicated. Therapeutic drug monitoring demonstrated consistent drug levels across animals within a group, and across mice infected with wild-type Mtb and ΔcinA excluding pharmacokinetic variability as a potential confounding factor in interpreting the efficacy data –pretomanid (PMD)–linezolid (LZD) in mice infected with wild-type and Δ580 of 0.02 and 50 μl culture was added to 384-well plates containing drugs and incubated for 7–12 days. OD580 values were determined using an M2 SpectraMax Microplate reader and final values were normalized to no-drug controls. Compounds were dispensed using a D300e Digital Dispenser (HP) with all wells normalized to a final DMSO concentrations of 1%. For time-kill assays cell suspensions were adjusted to an OD580 of 0.1 and drugs were added at the following concentrations: INH 0.5 μg/ml, RIF 0.5 μg/ml, ETH 4.5 μg/ml, DMD 50 μg/ml, PMD 1 μg/ml. At indicated time points bacterial survival was determined by culturing serial dilutions on 7H10 agar  or 7H10 agar supplemented with 0.4% charcoal.Bacteria were grown with agitation (100 rpm) to mid log phase and washed once prior to preparation of single-cell suspensions in 7H9 liquid medium supplemented with 0.2% glycerol, 0.05% Tween-80, and ADN . For drug susceptibility profiling cell suspensions were adjusted to an OD580 of 0.05. Cells were starved for 14 or 21 days and then exposed to the following drug concentrations INH 0.5 μg/ml, RIF 0.5 μg/ml, ETH 50 μg/ml, PMD 1 μg/ml, DMD 0.5 μg/ml, and 1 μg/ml or equal volume of DMSO. Colony-forming units were enumerated 7 days later by culturing serial dilutions on 7H10 agar supplemented with 0.4% charcoal.Bacteria were grown with agitation (100 rpm) to mid log phase and washed twice in PBS-Tyloxapol 0.05%. Single-cell suspensions were prepared in PBS-Tyloxapol 0.05% and adjusted to an OD8 CFU/ml Mtb H37Rv , and incubated at 37 °C for 4 weeks in 96-well plates, to allow for physiologic and metabolic adaptation to the lipid-rich environment. The caseum mimetic55 was prepared with the following modifications: to induce lipid droplet formation, PMA-differentiated THP-1 macrophages were infected with irradiated Mtb (BEI Resources) at an approximate MOI of 1:50. The foamy macrophages were washed three times with PBS, followed by three freeze–thaw cycles to lyse the cells and incubation at 75 °C for 20–30 min to denature proteins in the matrix55. The lysate was allowed to rest at 37 °C for 3 days, prior to freezing at −80 °C until inoculation. After 4 weeks of Mtb incubation in the caseum mimetic, drugs were added at concentrations ranging from 0.125 to 512 μM, in fourfold increment, the samples as well as no-drug controls were incubated for 7 days, and serial dilutions were cultured on 7H11 agar medium for 6 weeks prior to CFU enumeration.Bacteria were grown to mid log phase inoculated into caseum mimetic at 10cinA was cloned into pET28b vector and transformed into BL21(DE) E. coli. Bacteria were grown to an OD600 of 0.6 in LB media supplemented with 50 μg/ml kanamycin. Subsequently, cultures were cooled to 16 °C and cinA expression was induced by addition of 0.5 mM IPTG for 16 h. E. coli cells were harvested by centrifugation and lysed by French Press in 20 mM TRIS-HCl pH 7.5, 0.2 M NaCl in the presence of protease inhibitor cocktail (Roche) and DNAase. Lysates containing CinA inclusion bodies were pelleted at 55,000g, washed twice in 20 mM TRIS-HCl pH 7.5, 0.2 M NaCl, 0.1% Tween-80, and dissolved in 20 mM TRIS-HCl pH 7.5 containing 8 M urea. The insoluble fraction of the sample was removed by centrifugation and unfolded protein was purified using a Ni affinity column under denaturing conditions, maintaining 8 M urea throughout all steps. CinA was eluted with 500 mM imidazole. The concentration of the eluted CinA was adjusted to 0.5 mg/ml in 20 mM TRIS-HCl pH 7.5 containing 8 M urea.Full-length 2, 10% glycerol, 4 M urea overnight at 4 °C. The urea concentration was reduced by step dialysis at room temperature, by twofold diluting the dialysis buffer every 2 h in urea-free buffer until a urea concentration of approximately 0.25 M was reached. Subsequently, the eluate was dialyzed against urea-free buffer overnight at 4 °C. Any precipitated protein was pelleted by centrifugation and the soluble fraction was concentrated to 0.5 mg/ml. Rabbit polyclonal antiserum against recombinant CinA was generated by Thermo Fisher Scientific.The eluate was dialyzed against 50 mM Nacitrate-phosphate pH 7.6, 500 mM NaCl, 1 mM MgClTo purify CinA-specific antibody from anti-CinA serum 50 μg of purified CinA was resolved through SDS-page and transferred to nitrocellulose membrane. The membrane was incubated with anti-CinA serum (1:6 dilution) at 4 °C overnight. CinA-specific antibody was eluted with 10 mM glycine pH 2.7 for 2 min and the eluate was immediately neutralized with 300 μl of 1.5 M Tris-HCl pH 8.8. Subsequently, the antibody was dialyzed against PBS at 4 °C.cinA, ΔcinA:: cinA, ΔcinA:: cinAD80A, ΔcinA:: cinAK323A, cinA-TetON strains by mechanical lysis with 0.1 mm zirconia/silica beads in PBS in presence of protease inhibitor cocktail (Roche). Subsequently, lysates were filtered through 0.22 μm Spin-X columns (Corning). Protein concentrations were determined using a DC Protein Assay Kit (Bio-Rad). For immunoblot analysis of CinA, 20 or 40 μg of protein was resolved through SDS-page, transferred to nitrocellulose membrane and probed with anti-CinA serum (1:50 dilution) and anti-PrcB  or anti-PckA . Following incubation with anti-rabbit secondary antibody (LI-COR Biosciences), protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).Protein extracts were prepared from wild-type, ΔMtb were prepared as described56 with some modifications. Briefly, bacteria were grown to an OD580 of 1.0 and 1 ml of culture was inoculated onto 0.22 μm polyvinylidene fluoride membrane filters (Millipore Sigma) using vacuum filtration. Filters were transferred to 7H10 agar and incubated for 5 days to allow for sufficient biomass to accumulate for drug adduct detection. Subsequently, filters were placed on top of inverted 15 ml centrifuge caps containing 7H9 liquid medium for 24 h. After 24 h the medium inside the caps was replaced with 7H9-containing antibiotics at the following concentrations: INH 0.03 μg/ml or 0.3 μg/ml, ETH 3 μg/ml, PMD 3 µg/ml or an equal volume of DMSO. Small molecules were extracted after 24 h of drug exposure by bead-beating in acetonitrile:methanol:water 57. Samples were stored at −80 °C until drug adduct detection by LC-MS/MS.Drug metabolite extracts of drug-treated 32. MnIII pyrophosphate was prepared by stirring an aqueous solution of sodium pyrophosphate decahydrate (200 mM), MnIII acetate dihydrate (50 mM), and pyrophosphoric acid (added to pH 4.5) for 24 h at room temperature59. Next, NAD+  and INH (0.1 ml of a 20 mM stock) were combined in 0.9 ml of phosphate buffer , followed by the addition of MnIII pyrophosphate . Twenty minutes after the last addition, the reaction mixture was clarified by centrifugation and stored at −80 °C.INH-NAD adducts were synthesized by reacting INH and NAD+ in the presence of MnIII pyrophosphate, as described by Nguyen et al.58, using an Agilent 1290 Infinity LC system coupled to a 6545 quadrupole time-of-flight mass spectrometer for high-resolution MS/MS fragmentation analysis and a 6495 triple quadrupole mass spectrometer with iFunnel for quantification (Agilent Technologies). Samples were diluted 10-fold into mobile phase A (see below), injected (10 μl) onto an Accucore C18 , and separated using a 0.2 ml/min gradient of acetonitrile with 0.1% acetic acid (mobile phase B) in water with 0.1% acetic acid and 20 mM ammonium acetate (mobile phase A) as follows: 0–6 min: 3–20% B, 6.01–8 min: 100% B, followed by 4 min re-equilibration at 3% B.Putative NAD adducts of isoniazid and ethionamide were separated based on a previously published chromatography methodAdduct quantification was performed by multiple reaction monitoring in the positive ionization mode  at a collision energy (CE) of 30 and 55 V, respectively, with a cell accelerator voltage of 5, a dwell time of 100 ms and the following source settings: gas temperature: 200 °C, gas flow: 12 l/min, nebulizer: 15 psi, sheath gas temperature: 395 °C, sheath gas flow: 12 l/min, capillary voltage: 3000 V, nozzle voltage: 1500 V.Mtb exposed to a drug at 10× MIC were concentrated twofold (ETH) or 10-fold  by drying under vacuum  without heating (Vacufuge Eppendorf), resuspending in mobile phase A, and centrifuging . Fragmentation spectra were generated using a narrow quad isolation window of 1 amu and subject to CEs of 30 and 55 V. Generated spectra were compared to a synthesized standard (INH) or to predicted fragmentation spectra (ETH and PMD) using an in silico competitive fragmentation modeling algorithm33. All chemicals used for LC-MS/MS experiments were precured from Sigma, unless stated otherwise.For putative identification of the adducts, pooled extracts from P values. Q values were obtained by adjusting for multiple comparisons using the Benjamin–Hochberg correction. Genes with a Q value of <0.05 according to the permutation test were considered to be significant determinants of fitness in the presence of INH. TnSeq fold changes (TnSeq-FC) were computed as log2-transformed ratios of the normalized read counts between INH treated and untreated libraries49. All other statistical analyses were performed using GraphPad Prism 9.3.1 (GraphPad Software). The statistical tests used are indicated within figure legends and the corresponding P values are reported as *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.To determine statistical significance for TnSeq screens a nonparametric permutation test was used to calculate Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The incidence of heart failure with preserved ejection fraction is increasing in patients with obesity, diabetes, hypertension, and in the aging population. However, there is a lack of adequate clinical treatment. Patients with obesity-related heart failure with preserved ejection fraction display unique pathophysiological and phenotypic characteristics, suggesting that obesity could be one of its specific phenotypes. There has been an increasing recognition that overnutrition in obesity causes adipose tissue expansion and local and systemic inflammation, which consequently exacerbates cardiac remodeling and leads to the development of obese heart failure with preserved ejection fraction. Furthermore, overnutrition leads to cellular metabolic reprogramming and activates inflammatory signaling cascades in various cardiac cells, thereby promoting maladaptive cardiac remodeling. Growing evidence indicates that the innate immune response pathway from the NLRP3 inflammasome, to interleukin-1 to interleukin-6, is involved in the generation of obesity-related systemic inflammation and heart failure with preserved ejection fraction. This review established the existence of obese heart failure with preserved ejection fraction based on structural and functional changes, elaborated the inflammation mechanisms of obese heart failure with preserved ejection fraction, proposed that NLRP3 inflammasome activation may play an important role in adiposity-induced inflammation, and summarized the potential therapeutic approaches. The incidence of heart failure (HF) is rapidly increasing. As of 2017, an estimated 64.3 million individuals have been diagnosed with HF worldwide  hypertrophy than the right , 10. AniAlthough relatively rare, right ventricular (RV) thickness and volume can also increase in individuals with obesity. Preliminary studies have suggested that RV hypertrophy and dilatation are secondary to pulmonary hypertension due to obesity-induced LV failure, sleep apnea, and hypoventilation , 17. HowIn addition to macro-anatomical structures, the hearts of patients with obesity often undergo multiple morphological changes at the cellular level. Diffuse cardiomyocyte hypertrophy is the most specific histological feature of obesity-associated cardiac injury. Early clinical research found that compared with patients without obesity but with HF, cardiomyocyte hypertrophy is more evident in patients with obesity and HF . DespiteBoth animal and human studies have demonstrated microvascular rarefaction, interstitial fibrosis, and cellular infiltration in the myocardium of patients with obesity-related HFpEF , 23. MyoStudies have shown that LV diastolic dysfunction, rather than systolic dysfunction, represents the earliest sign of cardiac involvement in patients with obesity , 29. DiaIn recent years, methods to assess systolic function by quantifying myocardial strain have been proposed, including echocardiography to measure circumferential fiber-shortening velocity, tissue Doppler echocardiography, cardiac magnetic resonance tissue labeling, and feature tracking –39. PrecObesity not only leads to LV dysfunction but also RV dysfunction. Studies have found that the RV stroke volume and RV ejection fraction are lower in individuals with overweight and obesity, even if the LV parameters are adjusted, supporting the opinion that RV dysfunction in HFpEF may be parallel to LV dysfunction rather than a consequence of HF deterioration , 43. ThoCompared with patients with HFpEF without obesity, patients with obese HFpEF have significantly different hemodynamics and a unique pattern of myocardial remodeling. At rest, patients with obese HFpEF show higher right atrium (RA) pressure and pulmonary capillary wedge pressure (PCWP). During exercise, obese HFpEF patients had higher left and right heart filling pressures and higher pulmonary artery pressures which induced more severe RV dysfunction. In addition to RV dysfunction, increased left ventricular mass and volume, and the increased ratio of the two indicated greater concentric remodeling in obese HFpEF , 42. ProDespite more severe symptoms and hemodynamic abnormalities, brain natriuretic peptide (BNP) levels, which are a critical diagnostic tool for HF, are lower in patients with obesity than those without obesity. Multiple studies have found an inverse correlation between BMI and BNP levels , 48. TarLow BNP levels in the obese HFpEF phenotype indicate that measurement of natriuretic peptides may be misleading in its diagnosis. Therefore, it is necessary to explore other specific biomarkers of the obese phenotype. Obese HFpEF has been associated with higher levels of inflammatory markers, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and soluble growth stimulation expressed in gene 2 , 54. BasVarious central pathophysiological anomalies have shown indications of being associated with the obese HFpEF phenotype, such as energy substrate metabolism changes, neurohormonal perturbations, and sodium retention . InflammExcessive intake of nutrients by the body increases insulin secretion and induces the transport and storage of adipose and muscle cells in the form of triglycerides and glycogen . AdiposeH2) cells predominate in the adipose tissue and secrete IL-10 to block adipose tissue inflammation. There is a conversion in fat T cells during the onset of obesity, before the influx of macrophages allows CD8+ and TH1 adipose tissue T cells to dominate, and Treg and TH2 cells are depleted. This transformation may initiate the trafficking of macrophages to fat via chemokines such as CCL2, which promotes local and systemic inflammation.It is generally considered that the initial event of the inflammatory process is the imbalance of adipokine secretion from adipocytes, characterized by the reduced secretion of adiponectin and increased secretion of inflammatory mediators, including IL-6, leukotriene B4, and C-C motif chemokine ligand 2 (CCL2), which mediate circulating monocyte trafficking into the adipose tissue , 64, 65.In obesity, the degree of inflammation in the adipose tissue varies in different anatomic regions. The subcutaneous adipose tissue is the largest lipid reservoir that prevents fat accumulation in organs under physiological conditions . Since tPrevious studies have suggested that hemodynamic changes and insulin resistance are leading causes of HFpEF , 57. HowCoronary microcirculation disturbances are caused by the imbalance between endothelial-derived relaxing factors such as nitric oxide (NO) and contractile factors such as endothelin 1 . Pro-infin vitro results, IL-1 receptor type I-deficient mice were protected from aging-exacerbated HFpEF, due to being unresponsive to IL-1  in cardiomyocytes by activating the nuclear receptor peroxisome proliferator-activated receptor α. Once the enhanced FFA uptake exceeds the FAO capacity, excess FFAs are stored as triglycerides, which mediate the ectopic triglyceride accumulation in the heart and lipotoxicity . Increasmyocytes . This lemyocytes , 104.In addition to cardiomyocytes, nutrient overload can directly induce inflammation in other types of non-cardiomyocytes, such as macrophages and endothelial cells. Resident cardiac macrophages play essential roles in maintaining myocardial equilibrium during steady-state and tissue repair in the injured heart. At a steady state, cardiac macrophages resemble alternatively activated anti-inflammatory macrophages and function in homeostasis by helping to remove senescent dead cells and defending against infection without inducing an immune response . These aIn contrast to cardiomyocytes, macrophages are naturally insulin-resistant and take up glucose through insulin-independent GLUT1. Thus, in obesity-related insulin resistance, macrophages can sense glucose availability and dynamically reprogram their core metabolism by shifting toward cytosolic glycolysis and away from mitochondrial respiration, polarizing themselves away from the anti-inflammatory subtype and toward the pro-inflammatory subtype through glycolysis intermediates. For example, lactate has been shown to increase histone lactylation to induce homeostatic gene expression in macrophages . MoreoveThe preference for substrate utilization in endothelial cells, particularly arterial endothelial cells, differs from that of cardiomyocytes and macrophages. Because of its low energy demand, glycolysis is the predominant glucose utilization pathway, whereas FFAs are not an important fuel in coronary endothelial cells in the normal state , 111. ReAltogether, it is clear that the preference of different cells for metabolic substrates leads to different metabolic phenotypes in obese HFpEF. This phenomenon is important and should be considered in therapies targeting myocyte metabolism with unknown effects on other cardiac cells that continue to be prescribed.The innate immune system, which is the first line of defense of the host immune system, detects pathogens or host tissue damages, also known as pathogen-associated molecular patterns or damage-associated molecular patterns (DAMPs), respectively, mediated by pattern recognition receptors (PRRs). Upon recognition of DAMPs, some PRRs, including Nod-like receptors (NLRs) and Toll-like receptors (TLR), can assemble with other proteins to form multi-molecular complexes termed inflammasomes. Currently, the NLRP3 inflammasome is the most well-characterized inflammasome and is composed of the NLRP3, the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), and pro-caspase-1 . The canResting NLRP3 localizes to endoplasmic reticulum (ER) structures. However, a point of confusion is where the NLRP3 assembly occurs. Mitochondrial damage and ROS have been shown to act as upstream mechanisms of NLRP3 activation, implying a role of the mitochondria in NLRP3 inflammasome assembly. An informatics-based structure-function analysis predicted that NLRP3 localizes to mitochondria . SubsequThere has been increasing recognition that NLRP3 inflammasome activation acts as a chief instigator of obesity, contributing to obesity-related systemic inflammation and insulin resistance. A Study by Stienstra et al.  found thIn addition to the production of systemic inflammation originating from inflamed adipose tissue, recent research has shown that activation of the NLRP3 inflammasome is closely related to the severity of the HFpEF phenotype. Mice with HFpEF show NLRP3 inflammasome activation and overproduction of cytokines IL-1β/IL-18 in the heart , 130. CaNLRP3 inflammasome, which is an important mediator in the development of obese HFpEF, is regarded as a promising therapeutic target due to its important role. This section focuses on drugs that directly or indirectly suppress inflammasome assembly and activation.O2) and aerobic exercise capacity (Therapeutic targeting of the NLRP3 inflammasome in patients with HF has commonly used the IL-1 receptor antagonist (IL-1Ra) anakinra, thereby inhibiting signaling of both IL-1α and IL-1β. The D-HART pilot trial in patients with HFpEF showed that short-term IL-1 blockage with anakinra led to a significant reduction in systemic inflammatory mediators and improvement in peak oxygen consumption  trial, 150 mg canakinumab treatment improved peak Vreatment .In addition to IL-1, IL-6 blockade in mice through genetic deletion is associated with decreased cardiac hypertrophy and fibrosis following angiotensin II stimulation . AlthougColchicum autumnale, is a potent drug with anti-inflammatory properties. It acts on inflammation through different mechanisms, with the inhibition of spindle microtubule assembly via copolymer formation being the best characterized  pilot study and LoDoCo2 trial conducted by Nidorf et al. , 146 estcolchicine in acute coronary syndrome . In addiThe effectiveness of low-dose colchicine in atherosclerotic cardiovascular disease, a chronic inflammatory disease, raises the question of whether inhibiting NLRP3 inflammasome-related inflammation with colchicine is effective in the treatment of obese HFpEF. Currently, evidence that colchicine can reduce the risk of HF is limited to a single randomized controlled trial that showed colchicine reduced the circulating inflammatory biomarker levels and had favorable effects on LV remodeling in patients with HFrEF. However, this did not translate into an impact on clinical outcomes, exercise capacity, or improvements in mortality . Consideβ-hydroxybutyrate (BHB) is a ketone metabolite tested by Youm et al.  for NLRPSodium-dependent glucose co-transporter 2 (SGLT-2) inhibitors, which have been found to increase systemic and tissue BHB levels, thereby attenuating NLRP3 inflammasome activation and the development of HFpEF , 154, arAs previously mentioned, although some therapeutic agents that directly or indirectly inhibit the NLRP3-IL-1/IL-6 pathway could turn out to be promising strategies against obese HFpEF, some have intolerable side effects and still need clinical trials. Future studies should focus on the discovery of drugs with specific targets and improved pharmacokinetic properties. Valuable HFpEF animal models that mimic the clinically obese HFpEF phenotype are essential for the successful development of novel drugs. Currently, the lack of suitable preclinical obese HFpEF models that adequately reflect the disease’s clinical complexity has led to the inability to determine the mechanistic models and develop new therapeutic agents.The development of obese HFpEF in humans is the result of multiple comorbidities, including obesity, prediabetes, and pulmonary and systemic arterial hypertension, which are difficult to reproduce in one animal model. Recently, two diagnostic algorithms (H2FPEF and HFA-PEFF scores) have been proposed to standardize the diagnosis of human HFpEF , 159. WiSeveral methods can be used to induce obesity in laboratory animals, including the use of genetically modified mice. Obese mouse models lacking the leptin gene (ob/ob) and leptin receptor (db/db) show signs of HFpEF, including concentric hypertrophy, exercise intolerance, and pulmonary hypertension . ObesityTo avoid potential perturbations based on altered leptin expression and signaling in genetically modified models, numerous dietary treatment regimens have been used to induce obesity in rodents, which has become a standard approach in preclinical studies. HFD usually contains a total fat content of up to 60%, whereas a Western diet typically has a high-fat content and commonly uses sucrose. Cardiac abnormalities in the diet-induced obesity model vary depending on the total fat content and the duration of these dietary treatments, thereby limiting pre-clinical utility . For exaGiven that the development of obese HFpEF in humans is the result of several comorbidities, combinatory models of HFD and other factors have been evaluated. Schiattarella et al.  combinedIn addition to the limitations mentioned above, the hearts of rodent models are not comparable in size, structure, and function to the hearts of humans. Therefore, there is an urgent need for large-animal HFpEF models that can recapitulate the complex features of obese HFpEF in humans. The currently available methods for generating large animal models of HFpEF are summarized in detail elsewhere , 175. A In vitro cardiomyocyte models of humans can overcome the limitations of animal models and species differences. However, in vitro studies of cardiomyocytes have many technical challenges. Current in vitro cardiomyocyte models that can be used to simulate HFpEF include induced pluripotent stem cells (iPSCs), primary cells, and cardiac organoid models  of ventricular myocardium and cultured under mechanical tension and electric field stimulation . This meIn summary, based on the advantages and disadvantages of each model, we advocate that future HFpEF studies that explore molecular mechanisms and screen for potential new drugs should consider the use of multiple HFpEF animal models to narrow the gaps in HFpEF pathophysiology and promote the study of molecular mechanisms and the development of HFpEF therapeutics.in vitro models of human cardiomyocytes could provide a more comprehensive understanding of the complex paradigm of HFpEF and potentially serve as reliable preclinical platforms for testing its therapeutic potential.Obesity is an independent risk factor for myocardial dysfunction and HF. Obese HFpEF has a unique cardiac remodeling pattern and pericardial restraint mechanics. Inflammation is considered the main pathophysiological factor in obese HFpEF. We propose inflammation-related mechanisms of obese HFpEF, as well as potential therapeutic approaches. Currently, treatment options for HFpEF are limited, and the pharmacological inhibition of NLRP3 inflammasome assembly and activation may provide a novel treatment strategy targeting obese HFpEF. However, the discovery of novel therapeutics is hindered by the lack of appropriate HFpEF animal models that recapitulate this complex, comorbidity-laden, heterogeneous disease. Successfully validated large animal models or YB directed the writing and revision of the thesis. CL and DQ wrote the article and generated figures and tables. DH, JH and YY contributed to the mechanisms of the paper. All authors contributed to the article and approved the submitted version.This work was supported in whole or in part by the National Natural Science Foundation of China (No.82170483 to BY and No.82100496 to DH) and the Natural Science Foundation of Hunan Province of China (No.2020JJ4786 to JH).All authors are grateful to Zhenyu Tian for illustrating our 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.