Danio rerio) larva as a vertebrate screening model to study drug disposition. As the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in this study whether 3- to 4-day-old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cutoff of 4.4–7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of organic anion transporter oat (slc22) multidrug resistance-associated protein mrp1 (abcc1), mrp2 (abcc2), mrp4 (abcc4), and zebrafish larva p-glycoprotein analog abcb4 was shown. In addition, nonrenal clearance of fluorescent substrates and plasma protein binding characteristics were assessed in vivo. The results of transporter experiments were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 h postfertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.There is an increasing interest in using zebrafish (NEW & NOTEWORTHY The study of renal function remains a challenge. In vitro cell-based assays are approved to study, e.g., ABC/SLC-mediated drug transport but do not cover other renal functions such as glomerular filtration. Here, in vivo studies combined with in vitro assays are needed, which are time consuming and expensive. In view of these limitations, our proof-of-concept study demonstrates that the zebrafish larva is a translational in vivo test model that allows for mechanistic investigations to study renal function. Here, small molecules such as salts, water, glucose, citrate, and amino acids are exchanged or recovered to sustain homeostasis. Besides this, epithelial cells of the PCT are equipped with energy-dependent transporters, enabling the active secretion of xenobiotics into the tubular lumen. These drug transporters belong to the superfamilies of ATP-binding cassette (ABC) transporters and solute carriers (SLC), which are either apically or basolaterally expressed . Examplerocesses , 4.+-K+-ATPase is highly expressed in proximal tubules of zebrafish (Danio rerio) and other teleosts, e.g., killifish (Fundulus heteroclitus)  , 10.The in vivo study of renal function remains a challenge. Such studies are time consuming and expensive and often rely on in vivo experimentation with higher vertebrates. They are often combined with in vitro or ex vivo transporter assays to provide mechanistic insights at a cellular level. The latter models are reliable and provide a high throughput, but a translation to the in vivo situation remains cumbersome . In viewSince this ex vivo model does not respect all relevant kidney functions and aspects of a living organism, in vivo vertebrate models are desired. ZFL can be used for this purpose. They are transparent, undergo fast embryogenesis in that they already hatch by 3 days postfertilization (dpf), are easy to handle, and are therefore frequently used for toxicological, pharmacological, and developmental studies . Recent Therefore, the aim of this work was to implement ZFL as a translational in vivo vertebrate screening model to study renal function. In contrast to the established ex vivo killifish model mentioned above, the zebrafish model is expected to cover not only proximal tubular secretion but also glomerular filtration and distal tubular reuptake. These processes were studied in this work using fluorescent model compounds including polymers of different molecular weight and substrates of specific transporters in combination with their corresponding inhibitors. Fluorescent test compound distribution within the pronephros and blood system was assessed by confocal microscopy in living ZFL, using recombinant zebrafish lines expressing the fluorescent proteins enhanced green fluorescent protein (eGFP) or mCherry in the endothelium or glomerulus/proximal tubular cells. Finally, tubular secretion of fluorescent substrates was evaluated ex vivo in killifish to compare our results to a well-established tubular secretion model.N-ϵ(4-nitrobenzofurazan-7-yl)-D-Lys8]cyclosporine A (NBD-CsA) was synthesized as previously described . SAMSA fluorescein and NHS-TRITC were purchased from Thermo Fisher Scientific . Agarose, probenecid, erythromycin, verapamil hydrochloride, p-aminohippuric acid, MK-571 sodium salt hydrate, zosuquidar hydrochloride, saquinavir, indinavir sulfate salt hydrate, folic acid, fluorescein sodium salt (Fluo), FITC-dextran , 1-phenyl-2-thiourea (PTU), and ethyl-3-aminobenzoate methanesulfonate  were purchased from Sigma-Aldrich .Sulforhodamine 101 (Sulfo101) was purchased from Chemodex , 8-(2-[fluoresceinyl]aminoethylthio)adenosine-3′,5′-cyclic monophosphate (8-fluo-cAMP) was purchased from Biolog Life Science , a fluorescent cyclosporine A derivative  was determined by fluorescence correlation spectroscopy. An Olympus IX73 inverted microscope  using an immersion Super Apochromat objective  was used to perform the measurements. Emitted photons were filtered with a bandpass filter (512 nm) before detection with a single-photon avalanche diode . The free dye Atto 488 carboxylic acid  was dissolved at a concentration of 10 nM in double distilled water and used to calibrate the confocal volume of the excitation channel at 481 nm. Intensity fluctuations were recorded over 60 s. The experimental autocorrelation curves of the calibration dye Atto 488 and the fluorophore coupled samples were fitted with a one-component triplet state model, as previously described . Chemically reactive compounds  were incubated for 2.5 h in FCS  to neutralize reactive moieties followed by a short centrifugation before injection. For intravenous injections, a micromanipulator , a pneumatic Pico Pump PV830 , and a Leica SAPO microscope  were used. Tail regions were imaged 0.5–9 h postinjection (hpi) using an Olympus FV3000 confocal laser scanning microscope equipped with a ×20 UPIabSApo  objective and a ×30 UPIanSApo  objective. Confocal images were acquired using a sequential line scan, excitation wavelengths of 488 and 561 nm (argon laser), and emission wavelengths of 500–540 and 570–620 nm, respectively. Of note, no signal cross talk was detected between the different channels.The number of larvae in a 25-mL dish did not exceed 100. The formation of pigment cells was suppressed by adding 30 μg/mL PTU to the media. Then, 72 and 96 hpf hatched ZFL were embedded in 0.3% agar containing PTU and tricaine (0.01%). Experiments were carried out at room temperature. Randomly chosen larvae were injected with a calibrated volume of 1–2 nL of 0.1–2 mM stock solutions of test compounds into the cardinal vein (CV) above the heart. Embryos were obtained from the mating of six male and six female adult zebrafish and were randomly assigned to the treatment groups. Water-soluble substances were dissolved in PBS. Lipophilic substances such as NBD-CsA, erythromycin, and probenecid were administered in an up to 15% (vol/vol) DMSO-PBS solution by injection of 2 × 1 nL with a delay of 30 s between injections. FA-PEGn = 5). For the preparation of figures and the corresponding quantitative analysis, data from one representative series of experiments were used.To reduce intraexperimental variability, comparative experiments were performed during the same day by the same operator and using the same stage. Droplet size consistency (sample volume) was permanently checked using a reticle mounted on the eyepiece of a Leica SAPO binocular to ensure that the same volume of compound was injected throughout the course of the experiment. Reproducibility was verified by repeating series of experiments on at least three different days. For each condition of each series of experiments at least five zebrafish larvae were used (https://www.openmicroscopy.org/omero/) as an image processing program. Quantitative signal intensities in distinct organs were evaluated using Fiji software 2.1.0/1.53c (https://imagej.net/software/fiji/) and were done as follows: for drug transport experiments , zebrafish lines were used, which express a fluorescent marker  in vascular endothelia or renal epithelia. This allowed for a localization of fluorescent-labeled compounds within a defined three-dimensional anatomic structure such as the dorsal artery (DA) or kidney tubule. Signals were quantified by measuring mean signal intensities within this selected region of interest. Untreated controls and all corresponding treatment groups were analyzed using the same laser and microscopy settings, which allowed for a direct comparison of treatment groups. Alternatively , regions of interest were selected based on bright-field microscopy images. Analysis of the DA was used to quantify signals of circulating fluorophores within the blood compartment. Quantification of signal intensities was based on maximum intensity projections. Signal intensities are presented as fold changes normalized to the mean of the  control.Obtained confocal microscopy images were analyzed and edited using OMERO software 5.4.10 (F. heteroclitus) proximal kidney tubules were isolated and prepared as previously described  containing 1 μM of the indicated fluorescent substrate in the presence or absence of a 10- to 20-fold excess of a nonfluorescent inhibitor. Incubations were carried out at 8°C. Tubular accumulation of fluorescence signals was monitored using an Olympus FV1000 inverted confocal laser scanning microscope . Signal intensities were quantified using image capture and analysis software .Killifish  using unpaired two-tailed The pronephros of ZFL consists of two nephrons that are combined at the larva’s midline just ventral to the dorsal aorta . For a bDH and purity of these polymers were measured using fluorescence correlation spectroscopy. We obtained a diffusion coefficient of 125 and 48 μm2/s for FITC-PEG5 and FITC-PEG40 samples, respectively. This corresponds to a DH of 3.7 and 9.7 nm , whether a molecular weight cutoff can be established, and how this relates to values obtained from literature for higher vertebrates. The glomerular filtration molecular weight cutoff in the 96-hpf wild-type ZFL was determined using fluorescent dextran and PEG conjugates. FITC-DX was commercially available; larger molecular weight PEG was instead coupled to SAMSA fluorescein in house. The d 9.7 nm A. For FIectively D. Their 2000-FITC . These high concentrations were necessary to allow for detection in the patent luminal space of the distal tubule. To assess FA-mediated tubular reabsorption, a 100-fold molar excess of native FA as a specific inhibitor was preinjected 10 min before administration of 1 nL of 0.1 mM FA-PEG2000-FITC. Intensity within the DA was assessed 1 hpi to evaluate the extent of reabsorption from the tubular lumen back into the bloodstream. A representative confocal image is shown in left, demonstrating higher signal intensities (red > blue) in the DA compared with FA-preinjected ZFL. This is indicative of continuous tubular reuptake of FA-PEG2000-FITC in ZFL. This reuptake is reduced by a factor of 2 in the presence of an excess of native FA, which indicates competitive tubular reabsorption via folr1 (right).Next, distal tubular reuptake of FA by the folr1 homolog transporter was investigated using a fluorescent labeled derivative of FA, i.e., FA-PEG000-FITC A. The puia folr1 D, right.Proximal tubular secretion was investigated based on specific clearance of fluorescent labeled transporter substrates from the bloodstream. The transport substrate/inhibitor pairs selected for this study were selected based on a comprehensive review of the existing literature. p-aminohippurate, did not interfere with transport of the mrp2 substrate Sulfo101  ZFL were preinjected and incubated with a specific inhibitor  followed by the injection of a fluorescently labeled substrate. One hour postinjection, the tail region was imaged and signal intensity within the DA was compared with ZFL that were not preinjected with the corresponding inhibitor. Starting with apically located transporters, the functionality of mrp2 (abcc2) was assessed using Sulfo101 (or Texas red) as a fluorescent substrate and MK-571 as an inhibitor. Inhibitor concentration-dependent inhibition of mrp2 by MK-571 resulted in reduced renal secretion and thus enhanced retention in the bloodstream A. QuantiSulfo101 B. Likewitrations C. Transpippurate D. NBD-Csippurate E.Analysis of the ZFL vasculature after injection of fluorescent labeled substrates of proximal tubule transporters revealed in some instances a punctuated staining pattern at the level of the dorsal CV A and B. To verify the hypothesis that endocytosis of protein-bound fluorescent small molecules gives rise to the observed punctuated staining patterns in ZFL, a chemically reactive fluorescent dye (TRITC-NHS), used as amine-reactive cross-linker for protein labeling, was intravenously injected. Indeed, a punctuated staining pattern within, for example, the PCT was observed A. Qualit7.4 < 0.25) could be visualized based on their appearance within the PCT, their lipophilic counterparts  could not be detected within the PCT but did accumulate within tubular epithelial cells  were used as a renal tubular transport model .To allow for a direct assessment of proximal tubular secretion based on the appearance of fluorescent signals within the tubular lumen, isolated proximal tubules of killifish  and no apparent signs of toxicity.To control chemical exposure within ZFL, all test compounds were injected intravenously into the duct of Cuvier, resulting in a bioavailability of 100%. By this approach, potential interference by intestinal metabolism can be as well excluded. Of note, alternative clearance mechanisms via the liver are not considered to take place since liver associated xenobiotic metabolism is not operational by 5 dpf , 48. InjOf note, blood concentrations of circulating fluorophores were measured within the DA. Signals in the CV are indicative of cellular uptake by nonprofessional phagocytic cells. This process is mediated by endothelial scavenger receptors , as shown in DH threshold for glomerular filtration in the range of 4.4–7.6 nm. This is in line with studies in rats   . FOLR1 ieceptor)  and facieceptor) . Howevereceptor) . In our polymers . The flupolymers . It shoupolymers . In thesTransporter-mediated proximal tubular excretion of fluorescent labeled xenobiotics was studied based on their appearance within the PCT or, alternatively, their disappearance from the central blood compartment. Criteria for the selection of the used fluorescent substrates and nonfluorescent inhibitors as well as information on their selectivity and use by other authors are shown in Although the human homolog p-glycoprotein/MDR1 (ABCB1) gene is absent in ZFL, active secretion of lipophilic, uncharged, or moderately basic substrates in ZFL can be compensated by an abcb4 transporter , 9, 67. We cannot exclude that expression of drug transporters outside of the pronephros may have an impact on the indirect measurements of fluorescent substrates within the blood compartment. This can potentially lead to the accumulation of fluorescent signals in specific organs and tissues such as the brain or developing liver. However, we have not observed such effects e.g., . This caWhen comparing tail images from our transporter experiments, a punctuated staining pattern after injection of NBD-CSA and Sulfo101 was observed within the CV. Experiments with 72-hpf tg(kdrl:mCherry-CAAX) ZFL expressing mCherry in endothelial cells revealed a colocalization of fluorescent signals. This observation is indicative of cellular uptake of fluorescent molecules by scavenger endothelial cells located in the CV. Such endothelial cells with a scavenging function can be found in various organs in teleost fish, sharks, and lampreys . In mammN-hydroxysuccinimide) showed an additional punctuated staining of the PCT. This finding is in line with previous reports of PCT endocytosis of smaller 10-kDa dextran-FITC conjugates  were used as a complementary ex vivo transport model (To confirm the conservation of transport functions in an additional teleost species, freshly isolated and sealed tubuli of killifish (rt model . These trt model , 34. IndThe ZFL is an attractive in vivo vertebrate model that is extensively used, for instance, in toxicological, pharmacological, and nanomedicine research , 58. OurJ.S.B. was supported by the Stiftung zur Förderung des pharmazeutischen Nachwuchses in Basel. J.S.B., G.F., and J.H. were supported by Maine INBRE Grant GM103423, Salisbury Cove Research Foundation, and Ulric Dahlgren Fund.No conflicts of interest, financial or otherwise, are declared by the authors.J.S.B., A.P., C.L.A., and J.H. conceived and designed research; J.S.B., A.P., C.L.A., G.F., and J.H. performed experiments; J.S.B., A.P., C.L.A., and J.H. analyzed data; J.S.B., A.P., C.L.A., G.F., and J.H. interpreted results of experiments; J.S.B., C.L.A., and G.F. prepared figures; J.S.B., C.L.A., and J.H. drafted manuscript; J.S.B., A.P., C.L.A., G.F., and J.H. edited and revised manuscript; J.S.B., A.P., C.L.A., G.F., and J.H. approved final version of manuscript.


There is an increasing number of nutraceutical combinations (NCs) on the market for hypercholesterolemia, although clinical trials to verify their safety and efficacy are scarce. We selected fourteen randomized, placebo-controlled clinical trials (RCTs) on different lipid-lowering NCs in hypercholesterolemic subjects. We described each compound′s mechanism of action and efficacy in the mixtures and summarized the clinical trials settings and NCs safety and efficacy results. Almost all NCs resulted efficient against hypercholesterolemia; only one reported no changes. Interestingly, red yeast rice (RYR) was present in eleven mixtures. It is not clear whether the lipid-lowering efficacy of these combinations derives mainly from the RYR component monacolin K “natural statin” single effect. Up to now, few RCTs have verified the efficacy of every single compound vs. NCs to evaluate possible additive or synergistic effects, probably due to the complexity and the high resources request. In conclusion, to manage the arising nutraceutical tide against hypercholesterolemia, it could be helpful to increase the number and robustness of clinical studies to verify the efficacy and safety of the new NCs. Cardiovascular diseases (CVDs) remain the leading cause of death worldwide . AtherosDespite an incontrovertible efficacy of lipid-lowering therapy in reducing the incidence of cardiovascular events, a significant number of people refuse drug therapy. Nutraceuticals have the potential as an alternative approach and treatment for hypercholesterolemia. Moreover, the European Society of Cardiology and the European Atherosclerosis Society guidelines, as well as the International Lipid Expert Panel, recommend using dietary supplements and a balanced diet to improve lipid profile ,7.Nutraceuticals are bioactive compounds of plant or microbial origin, with possible beneficial effects on human health. Some nutraceuticals are prescribed as lipid-lowering substances. Several meta-analyses highlighted the efficacy of common nutraceuticals with a different mechanism of action  to treat hypercholesterolemia in maintaining the physiological levels of plasma cholesterol ,9. It isWe searched the MEDLINE (PubMed) to identify eligible articles published up to the end of May 2021. The articles investigating the effects of nutraceutical combinations on the hypercholesterolemic populations analyzed by placebo-controlled RCTs were considered. RCTs are the “gold standard” of clinical trials investigating the relationships between an intervention and a health outcome. The following keywords were used: hypercholesterolemia, nutraceutical combinations, dietary supplements, and filtered by a randomized clinical trial. The total articles found manually excluded those that did not correspond to the topic of interest. To cover information of NC effects on the general population, we have excluded: RCTs that observed only particular populations such as subjects with age under 18 or over 75, populations with specific conditions such as hypertension, familial hypercholesterolemia, postmenopausal women, subjects in secondary prevention with history of CVDs. When multiple RCTs for a specific NC were found, we included RCT with more subjects included in the study. The selected nutraceutical combinations are presented in Red yeast rice (Monascus purpureus). The extract of red yeast rice (RYR) containing the active ingredient monacolin K has the most cholesterol-lowering potential on the market. It is reported that in humans, the use of 3 to 10 mg daily of monacolin K reduces LDL cholesterol plasma levels between 15% and 25% after 6 to 8 weeks [ 8 weeks . RYR con 8 weeks . MonacolGerards et al., in a recent meta-analysis, included 20 clinical trials studies to evaluate the efficacy and safety of RYR. The results demonstrated that doses from 1200 mg/day to 4800 mg/day of RYR (4.8 mg to 24 mg of Monacolin K) reduced LDL-cholesterol on average by 1.02 mmol/L  compared with placebo after 2–24 months of its use. These results were similar to the effects of 40 mg of pravastatin, 20 mg of lovastatin, and 10 mg of simvastatin ,27.Vitamins. It is known that elevated plasma total homocysteine (Hcy) has been associated with cardiovascular risk and is considered a risk factor for atherosclerosis. B9 vitamin (folate) leads to coronary plaque regression by lowering Hcy [ring Hcy . A mild ring Hcy . Moreovering Hcy . The mecring Hcy . It is kring Hcy , increasring Hcy . In thisring Hcy . Vitaminring Hcy . It is wring Hcy .p = 0.01) [Satapathy et al., in a recent multi-arm clinical trial, investigated the effects of folic acid and vitamin B12 on glycaemic control, insulin resistance, and serum lipid profile in subjects with type 2 diabetes mellitus. The significant effect of B12 in plasma insulin, insulin resistance, and serum adiponectin have been demonstrated. However, the improvement in the lipid profile was not found . A clini = 0.01) . Ajuluch = 0.01) . However = 0.01) . The sinCoenzyme Q10 (CoQ10). Atherosclerosis is considered a chronic inflammatory disease [ disease . Oxidati disease . Moreove disease . Thus, h disease . The antp = 0.01). However, significant effects of CoQ10 on LDL cholesterol were not found [Jorat et al., in a meta-analysis that included eight clinical trials, researched the effects of coenzyme Q10 supplementation on lipid profiles among patients with coronary artery diseases. The results showed that CoQ10 significantly decreased total-cholesterol (standard mean differences (SMD): −1.07, 95% confidence interval (CI): −1.94, −0.21, ot found .Berberine (Berberis species). Berberine (BBR) is an alkaloid drug extracted from plants that belong to the Berberis species. There are several molecular cholesterol-lowering mechanisms of action of BBR. It has been demonstrated on HepG2 human hepatoma cells that its extracts can inhibit cholesterol synthesis by activating AMP kinase [P kinase . In humaP kinase ,47. ConsP kinase .Dong et al., in a meta-analysis that included eleven clinical trial studies, showed that the administration of berberine produced a significant reduction in total cholesterol , and LDL cholesterol levels  .Policosanols. Policosanols have an inhibitory effect on HMG-CoA reductase and bile acids absorption. Policosanols inhibit cholesterol synthesis in hepatoma cells by activation of AMP-kinase [P-kinase . MoreoveP-kinase .Berthold et al. researched the effects of policosanols on lipid levels. They divided 143 participants into groups treated with 10, 20, 40, and 80 mg of policosanols or placebo. Nobody in the groups showed a >10% LDL cholesterol reduction after 12 weeks . AnotherMinerals. A correlation between abnormality in serum calcium and phosphorous metabolism on lipid profile has been shown, even though the underlying molecular mechanisms are still unknown [ unknown . There iPhytosterols. Phytosterols and their esterified derivates, stanols, are structurally similar to cholesterol. They are poorly absorbed in the gut [ the gut ,56. The  the gut . Talati et al., in a meta-analysis that included 14 clinical studies, highlighted that in doses up to 3 g/day, there were no differences in efficacy on cholesterolemia between stanols and sterols . MoreoveOlive (Olea europaea). The beneficial effects of hydroxytyrosol (HTyr) and tyrosol phenolic antioxidants from Olea Europea have been well described on human health and atherosclerosis [clerosis . LDL choclerosis . HTyr haclerosis .p = 0.002) and LDL cholesterol (−0.19 (±SD 0.56) mmol/L, p = 0.017) [Lockyer et al. demonstrated that the use of phenolic-rich olive leaf extract containing 136 mg of oleuropein and 6 mg of hydroxytyrosol reduces total plasma cholesterol (−0.32 (±SD 0.70) mmol/L, = 0.017) .Artichoke leaf extract (Cynara scolymus). Active ingredients of artichoke that have previously shown a lipid-lowering effect are polyphenols, phytosterols, and fibers [d fibers . The impd fibers ,66. Luted fibers . Moreoved fibers . On the d fibers .p < 0.001), and LDL cholesterol  [A meta-analysis from Sahebkar et al. of data from 9 trials that researched the lipid-lowering effect of artichoke showed a significant decrease in plasma concentrations of total cholesterol  .Garlic (Allium sativum). Biological lipid-lowering mechanisms of action of garlic oil are similar to that of statins. It inhibits cholesterol synthesis by deactivating HMG-CoA reductase through enhanced phosphorylation. Still, it does not change the levels of mRNA of this enzyme in cultured rat hepatocytes [atocytes . Moreoveatocytes .A clinical study on the lipid-lowering effects of time-released garlic powder tablets-Allicor of 600 mg daily, in 42 men with mild hypercholesterolemia, showed a reduction in total cholesterol by 7.6% and LDL cholesterol by 11.8% after 12 weeks of treatment . MoreoveAstaxanthin. The carotenoid astaxanthin has antioxidant activity and anti-inflammatory properties [operties . There ioperties .A meta-analysis of 7 studies that analyzed the lipid profile and glucose changes after supplementation with astaxanthin did not show any significant effect of astaxanthin on plasma concentrations of total cholesterol .Banaba (Lagerstroemia speciose). Banaba has anti-diabetic and anti-obesity properties [operties . The exaoperties . On the operties , contriblagerstroemia speciosa efficacy on lipid profile still needs to be researched by RCT. However, the efficacy of the hypoglycemic effects of banaba is well known. It plays a relevant role in CVD risk prevention [The single effect of evention .Curcumin (Curcuma longa). Curcumin is a polyphenol compound found in Curcuma longa. Preclinical studies have shown that curcumin inhibits cholesterol biosynthesis via transcriptional inhibition of HMG-CoA reductase, independently from acyl-CoA/cholesterol acetyl transferases that regulate the synthesis of cholesteryl esters [l esters . Curcumil esters . Moreovel esters ,84,85.p < 0.05) [Sahebkar et al., in a meta-analysis with five clinical trial studies, investigated the effects of curcumin on blood lipid levels and showed no significant changes . However < 0.05) .Guggul (Commiphora Mukul). Guggul is a resin from Commiphora Mukul tree. It has been demonstrated that E- and Z-guggulsterones were antagonists of the farnesoid X receptor (FXR) [or (FXR) . FXR is or (FXR) ,89.p = 0.047). However, the mean levels of LDL-C did not change significantly.Nohr et al. researched the use of 2160 mg guggul daily or placebo on 43 women and men . After 1Probiotic (Bifidobacterium longum BB536). The intake of probiotics reduces total cholesterol and LDL in mildly hypercholesterolaemic subjects [Bifidobacterium longum BB536 show high biliary salt hydrolase activity [B. longum BB536 contributes to lower circulating total cholesterol and LDL levels by reducing intestinal cholesterol reabsorption [B. longum BB536 can incorporate cholesterol in its cell membranes leading to inhibition of intestinal cholesterol and decreasing serum levels [subjects . Probiotactivity . B. longsorption . There am levels . The incm levels .B. longum BB536 still needs to be investigated by RCT. However, a meta-analysis that researched the effects of other probiotics consumption on lipid profile and CVD risk factors demonstrates that probiotic use is effective in lipid-lowering [The single lipid-lowering effect of lowering .Catechin/theaflavins. Polyphenols in tea include catechins, theaflavins, tannins, and flavonoids [avonoids . The mecavonoids . Catechiavonoids .p = 0.01) and for LDL cholesterol −16.4 ± 1.1% (p = 0.01), respectively, in the tea extract group [p = 0.0004) and LDL cholesterol  [A clinical trial studied 240 subjects on a low-fat diet with mild to moderate hypercholesterolemia the effect of daily use of 375 mg theaflavin-enriched green tea extract or placebo. After 12 weeks, the mean ± SEM changes from baseline in total cholesterol were −11.3 ± 0.9% (ct group . Another 0.0001) .Pine Bark Extract (Pinus Pinaster). Pycnogenol is an extract of Pinus pinaster. The effect of this extract on oxidative stress and lipid profile in humans was studied. Pine bark extract increases plasma antioxidant capacity and HDL while it reduces LDL levels [p = 0.012), and decreased oxidized LDL concentrations by 31.72 U/l (p = 0.015) [L levels . A clini= 0.015) .RCTs are a gold standard for clinical research to verify the efficacy and safety of a novel NC . RCTs stAmong our selected studies, it appeared that twelve of fourteen studies followed the parallel design . From thOne among the selected studies was a crossover type, in which each patient received treatment and placebo but in a different order and timing . The advNutraceuticals have the potential of reducing the incidence of various chronic degenerative diseases since they exert preventive and therapeutic activities ,107. NCsThousands of nutraceuticals are already discovered. An exponentially increasing number of NCs are becoming available on the market against hypercholesterolemia, leading to the research of the effect of their combinations.However, in the literature, there is little evidence of their actual effects on humans. In particular, RCTs on the safety and efficacy of lipid-lowering NCs are scarce. In fact, despite an enormous number of NCs available in the market, we found only fourteen RCTs focused on the use of combined nutraceuticals, in the form of capsules, to lower the total cholesterol in hypercholesterolemic subjects.In our RCTs sample, the study period range was from four ,20 to twRegarding the efficacy of these mixtures, RCTs demonstrated that almost all NCs tested effectively lower lipid levels. In particular, 13 of 14 RCTs verified lipid-lowering effects while only one  reportedInterestingly, RYR has been used in the mixture of eleven among thirteen effective NCs .Monacolin K, the lipid-lowering active compound of the RYR, exists in two forms, lactone and acidic . Its lacUp to now, few studies have verified the efficacy of every single compound in the mixture vs. NCs to evaluate possible additive or synergistic effects. Probably for the time and high resources request depending on the number of nutraceutical candidates and their possible combinations to perform the studies . HoweverScientific opinions of the safety of the monacolin K have been put forward . It is rHowever, it is relevant for the RYR intolerant populations to have the possibility to use NCs without the presence of Monacolin-K. Our sample contained three NCs that did not include RYR ,19,23. TIn conclusion, to manage the arising nutraceutical tide, we strongly suggest increasing the number of clinical studies to evaluate the efficacy and safety of the new NCs. Moreover, in our opinion, robust design such as RCTs should be the preferred choice when planning a clinical trial on NCs in hypercholesterolemia. In addition, to verify possible additive and/or synergistic effects, the new nutraceutical candidates should be individually tested by their use in additional arms of subjects vs. NCs and placebo arms in the RCTs.This review offers a picture of the main scientific RCTs evidence to evaluate NCs as an alternative approach also in subjects that refuse lipid-lowering drug therapy.


Schistosoma mekongi. This disease contributes to human morbidity and mortality in the Mekong region, posing a public health threat to people in the area. Currently, praziquantel (PZQ) is the drug of choice for the treatment of Mekong schistosomiasis. However, the molecular mechanisms of PZQ action remain unclear, and Schistosoma PZQ resistance has been reported occasionally. Through this research, we aimed to use a metabolomic approach to identify the potentially altered metabolic pathways in S. mekongi associated with PZQ treatment.Mekong schistosomiasis is a parasitic disease caused by the blood-dwelling fluke S. mekongi were treated with 0, 20, 40, or 100 μg/mL PZQ in vitro. After an hour of exposure to PZQ, schistosome metabolites were extracted and studied with mass spectrometry. The metabolomic data for the treatment groups were analyzed with the XCMS online platform and compared with data for the no treatment group. After low, medium (IC50), and high doses of PZQ, we found changes in 1,007 metabolites, of which phosphatidylserine and anandamide were the major differential metabolites by multivariate and pairwise analysis. In the pathway analysis, arachidonic acid metabolism was found to be altered following PZQ treatment, indicating that this pathway may be affected by the drug and potentially considered as a novel target for anti-schistosomiasis drug development.Adult stage S. mekongi. Identifying potential targets of the effective drug PZQ provides an interesting viewpoint for the discovery and development of new agents that could enhance the prevention and treatment of schistosomiasis.Our findings suggest that arachidonic acid metabolism is a possible target in the parasiticidal effects of PZQ against  Schistosomiasis is a major neglected tropical disease that is a public health threat in many countries worldwide. Hundreds of millions of people live in endemic areas and are at risk for this disease. PZQ is the drug of choice for many parasitic infections and is the primary drug used to treat schistosomiasis. PZQ resistance is a problem in endemic areas, emphasizing the need for novel drugs in the fight against schistosomiasis. This study aimed to elucidate the molecular mechanisms of how PZQ affects schistosomes and thereby identify novel targets for anthelminthic drug development. Our findings highlighted the anandamide and arachidonic acid metabolism pathways as important targets of the anti-schistosomal effects of PZQ. These pathways might present valid targets for drug development for the treatment of schistosomiasis. Schistosoma. There are six species of Schistosoma fluke that infect humans: S. mansoni, S. japonicum, S. intercalatum, S. guineensis, S. mekongi , and S. haematobium  . P. PS. manignaling , inflammignaling . In mammnic acid ,48. Aracmekongi. . Supplemmekongi. . In vitrtructure . A reductructure . Arachidtructure ,54. ThusSchistosoma muscles, and this paralysis is caused by a rapid Ca2+ influx inside the schistosome. Therefore, schistosome calcium ion channels are currently proposed targets of PZQ [+/Ca2+ exchanger [2+ influx into schistosomes, affecting their muscles. When school children in Ethiopia were treated for S. mansoni, most (80.7%) reported three or more side effects, such as headache, dizziness, nausea, tiredness, weakness, loss of appetite, and vomiting [Schistosoma spp. , anandamide, and arachidonic acid metabolism may be effective approaches to overcome the problem of PZQ-resistance and schistosomiasis in the future.In summary, we treated S1 Fig(A). Control. (B) Low dose PZQ treatment. (C) Medium dose PZQ treatment. (D) High dose PZQ treatment. The worms in treatment groups were bended and coiled comparing to the control group.(TIF)Click here for additional data file.S1 DataThe.wiff files of each sample were generated by LC-MS. (A) Control replication 1. (B) Control replication 2. (C) Control replication 3. (D) Low dose PZQ treatment replication 1. (E) Low dose PZQ treatment replication 2. (F) Low dose PZQ treatment replication 3. (G) Medium dose PZQ treatment replication 1. (H) Medium dose PZQ treatment replication 2. (I) Medium dose PZQ treatment replication 3. (J) High dose PZQ treatment replication 1. (K) High dose PZQ treatment replication 2. (L) High dose PZQ treatment replication 3.(ZIP)Click here for additional data file.S2 DataS. mekongi worms was presented step-by-step.The workflow of metabolite extraction from (DOCX)Click here for additional data file.S3 DataHomo sapiens, Schistosoma mekongi, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma bovis, Fasciola hepatica, Paragonimus westermani, Clonorchis sinensis, and Echinococcus granulosus fatty acid amide hydrolase that were used for alignment were provided.The amino acid sequences of (DOCX)Click here for additional data file.S4 DataThe.clustal_num file result generated from Clustal alignment analysis was provided.(CLUSTAL_NUM)Click here for additional data file.S5 DataThe.txt file result generated from Clustal alignment analysis was provided.(TXT)Click here for additional data file.S1 TableS. mekongi after low-, medium-, and high-dose PZQ treatment.The pairwise comparison was performed on these results. Phosphoserines were highly produced in (DOCX)Click here for additional data file.S2 TableS. mekongi after low-, medium-, and high-dose PZQ treatment.The pairwise comparison was performed on these results. Anandamide was highly produced in (DOCX)Click here for additional data file.S3 TableNumbers in the table represent percentage identity matrices. Fatty acid amide hydrolases among parasites had high percent similarities.(DOCX)Click here for additional data file.


Botrytis cinerea and Trichoderma atroviride are two relevant fungi in agricultural systems. To gain insights into these organisms’ transcriptional gene regulatory networks (GRNs), we generated a manually curated transcription factor (TF) dataset for each of them, followed by a GRN inference utilizing available sequence motifs describing DNA-binding specificity and global gene expression data. As a proof of concept of the usefulness of this resource to pinpoint key transcriptional regulators, we employed publicly available transcriptomics data and a newly generated dual RNA-seq dataset to build context-specific Botrytis and Trichoderma GRNs under two different biological paradigms: exposure to continuous light and Botrytis-Trichoderma confrontation assays. Network analysis of fungal responses to constant light revealed striking differences in the transcriptional landscape of both fungi. On the other hand, we found that the confrontation of both microorganisms elicited a distinct set of differentially expressed genes with changes in T. atroviride exceeding those in B. cinerea. Using our regulatory network data, we were able to determine, in both fungi, central TFs involved in this interaction response, including TFs controlling a large set of extracellular peptidases in the biocontrol agent T. atroviride. In summary, our work provides a comprehensive catalog of transcription factors and regulatory interactions for both organisms. This catalog can now serve as a basis for generating novel hypotheses on transcriptional regulatory circuits in different experimental contexts. Furthermore, external signals must be integrated with internal developmental programs to generate a response. Part of this response is driven by changes at the gene expression level, where transcription factors (TFs) - among other regulatory molecules - play a pivotal role. TFs are proteins characterized by the presence of one or more DNA-binding domains (DBDs) that recognize specific motifs in DNA sequences. Different experimental high-throughput approaches are used to determine the DNA binding preferences of these molecules, such as Protein Binding Microarrays (PBMs), SELEX-Seq, DAP-Seq Genome-scale characterization of regulatory interactions is one of the goals of systems biology. One approach to describe the complex interactions between TFs and their target genes is by building Gene Regulatory Network (GRN) models, which are graphical representations denoting TFs and non-TF genes as nodes connected with edges depicting the regulatory interconnections Saccharomyces cerevisiae, Aspergillus nidulans, and Neurospora crassaUstilago maydis and Penicillium spp. In fungal systems, the reconstruction of GRNs has been limited by the availability of experimentally characterized TF-target interactions and high-throughput gene expression datasets. Thus, most GRNs come from studies in the model species Fusarium graminareumBotrytis cinerea occupies a position of distinction, being ranked as the second most important fungal phytopathogen worldwide Trichoderma has been recently syndicated as the fungal genus with the greatest biocontrol potential Fungal organisms constitute one of the largest groups of plant pathogens, and the emergence of new fungicide-resistant strains is compromising human food security and wildlife biodiversity B. cinerea can infect over 1000 plant species, including numerous crops. It has a predominant necrotrophic lifestyle, co‐opting the host programmed cell death response to achieve infection B. cinerea is troublesome to control in agricultural fields due to its diverse attack modes and a broad range of hosts. Besides, it can survive as mycelia, conidia, or sclerotia under extended unfavorable periods, and the appearance of fungicide‐resistant isolates has been well documented Trichoderma spp., a natural antagonist of B. cinerea and other fungal phytopathogens, could reduce or prevent the use of environmentally unfriendly chemical pesticides, avoiding the upsurge of new fungicide-resistant strains. T. atroviride is a fast-growing ascomycete that can be found in soil as free-living or associated with plants, favoring advantageous outcomes such as plant growth, strengthening abiotic stress tolerance, and enhancing resistance to pathogens T. atroviride obtains nutrients employing CWD enzymes, among diverse strategies that include antimicrobial compounds Biological control agents like B. cinerea and T. atroviride, we constructed reference GRNs employing their latest genome assemblies. We inferred whole-genome TFBS by first compiling a manually curated TF dataset for both fungi. These networks were refined using public high-throughput gene expression data. To assess the relevance and applicability of these GRNs, and considering the major transcriptional impact of light on fungal physiology B. cinerea and T. atroviride grown in constant light and darkness with our reference GRN to build light-dependent GRNs for both organisms. In addition, we carried out fungal confrontation experiments between the biocontroller agent and the phytopathogen to build confrontation-dependent GRNs. These results provide both fungal communities with an unprecedented resource, facilitating a standardized strategy to formulate data-derived hypotheses.To leverage the molecular understanding of the events shaping transcriptional responses in both 22.1B. cinerea and T. atroviride  were retrieved from EnsemblFungi B. cinerea and T. atroviride containing at least one of these IDs were selected for further analysis. In parallel, a custom Hidden Markov Model (HMM) profile for TF DBDs was generated (Supplementary File 2) and used to scan the proteomes using hmmsearch (HMMER V.3.3.1) B. cinerea and T. atroviride, respectively).Protein sequences for 2.2http://cisbp.ccbr.utoronto.ca/TFTools.php) was used to query the full-length protein sequences. The highest scoring motif considered for each TF was selected. Only motifs belonging to fungal TFs were retrieved. Therefore, motifs derived from TFs of non-fungal organisms were discarded. In the case of TFs with more than one DBD, we retrieved PWMs describing both DBD preferences by searching each DBD at the “Protein Scan” web tool (Supplementary File 4).To assign a DNA binding motif to each identified and manually-curated TF, the “Protein Scan” web tool at CisBP , species that harbor the largest dataset of fungal TFs with direct experimental determination of DNA binding preferences [4]. B. cinerea and T. atroviride TFs were classified into different TF orthogroups by first performing an all-against-all BLASTp analysis followed by automatic orthogroup definition carried out by OrthoFinder software (v 2.4.0) For TF sequences with no identified motif in the CisBP “Protein Scan” web tool B. cinerea and T. atroviride is described in Supplementary Files 4 and 5.Finally, PWMs corresponding to each assigned motif were obtained from CisBP. Each TF-PWM pair (when available) for both 2.3B. cinerea and T. atroviride TFs, promoter sequences for both fungi were obtained by extracting a genomic sequence of 1000 bp upstream from the transcription start site (TSS) of each gene. TSS information was obtained from gene annotation files from both fungi, available on EnsemblFungi . PWMs for each TF were then used to scan promoter sequences using the Find Individual Motif Occurrences (FIMO) tool from the MEME Suite (v 4.11.2) -4).To determine putative target genes for the 2.4B. cinerea Pers. Fr. [Botryotinia fuckeliana (de Bary) Whetzel] was originally isolated from Vitis vinifera (Germany) T. atroviride was first isolated from a plum tree in southern Sweden B. cinerea or T. atroviride. To facilitate mycelia harvesting, the culture media was covered with a cellophane overlay. Cultures were incubated for 3 days in Percival incubators  at 20 °C in constant light  until both fungi reached each other . Constant light was used since circadian regulation and light have a significant impact on B. cinerea and T. atroviride physiology and interaction  or the B. cinerea and T. atroviride plates, dried, and snap-frozen in liquid nitrogen. Frozen mycelia were ground to powder, and total RNA was extracted using TRIzol reagent  Approximately 10 mg of tissue was collected from the 2.6https://sourceforge.net/projects/bbmap/) . Filtered reads were mapped to each fungal genome using HiSat2 Low-quality reads and adapter sequences were filtered out from FASTQ files using BBDuk , a widely used expression-based GRN inference algorithm B. cinerea and T. atroviride from data obtained from the NCBI Sequence Read Archive (SRA) (Supplementary File 8). For this purpose, the entire dataset was quality-filtered using BBDuk, as mentioned above. Only samples with at least 1 million reads mapped to each fungal genome were considered for downstream analysis. Gene expression in the datasets was determined by pseudoalignment of the reads to B. cinerea or T. atroviride transcripts using Kallisto (v.0.44.0) To generate a reference GRN for B. cinerea and T. atroviride. In the case of previously published microarray experiments, we retrieved DEGs between each mutant and the wild-type genotype from each publication http://shenlab-sinai.github.io/shenlab-sinai/).To test our reference GRNs, we obtained the list of DEGs for six loss-of-function TF mutants for 2.8B. cinerea and T. atroviride using the BLAST2GO functional annotation pipeline To perform functional term enrichment analyses of the data, we first carried out a whole-genome functional annotation of 2.9T. atroviride, this corresponds to the SRA accession number SRP069026 B. cinerea to accession number SRP235144. The new data reported herein was deposited in SRA with the following accession number: PRJNA756518.Publicly available RNA-Seq data was downloaded from the NCBI SRA database. In the case of the publicly available experiments in constant light for 33.1B. cinerea and T. atroviride, we first established a manually-curated full repertoire of TFs (TFome) for both fungi by de novo annotation of their reported proteins using different protein annotation tools and criteria, as depicted in B. cinerea and T. atroviride, respectively (Supplementary File 3). These numbers significantly differ from previous TF annotations, including the CisBP database  and the work of Shelest, 2017, which reports 411 and 577 TFs, for B. cinerea and T. atroviride, respectively. Thus, a manual inspection of the predicted TF list was undertaken to filter proteins erroneously assigned as TFs, as described in Materials and Methods . Manual inspection of each sequence generated a final curated TF list consisting of 471 and 561 sequences for B. cinerea and T. atroviride, respectively (Supplementary File 5). For B. cinerea, 378 of these TFs were also reported in CisBP, while 93 additional TFs were found by our annotation pipeline   and botrydial (BOT) B. cinerea and 37.5% in T. atroviride), as well as the “Zinc finger C2H2-type” domain (IPR013087), present in all eukaryotes  ; Fig. 2Akaryotes ; Fig. 2AThe remaining identified domains are typically found in fungal genomes As expected, a portion of the TFs displayed two DBDs (approximately 2–3% of the TFs in both fungi) , Fig. 2BN. crassa, A. nidulans, and S. cerevisiae. For B. cinerea, only one TF, Bcin05g07400, has a directly determined binding preference, and for T. atroviride, no direct motifs are available. Nevertheless, a total of 106 and 104 TFs for B. cinerea and T. atroviride, respectively, have an automatically inferred motif derived from homologous TFs, as the DNA binding specificity can be inferred following general rules that depend on each TF family and the degree of similarity among DBDs. Since most TFs from our curated catalog do not possess any automatically inferred DNA binding motif, we assigned a motif for these TFs using the “Protein Scan” web tool from CisBP or by performing an orthogroup classification as detailed in Methods . In the case of dual-specificity TFs, we assigned two different PWMs (one to each DBD) for four out of 16 TFs in B. cinerea and eight out of ten TFs in the biocontroller fungus (Supplementary File 4).With the defined repertoire of TFs for each fungus, we established the DNA binding preference for each of them. The CisBP database  Methods A. We wer3.2B. cinerea or T. atroviride, using FIMO, considering a distance threshold of 1 Kb between the TFBS and the TSS. We found 370B. cinerea TFs and 420 T. atroviride TFs  having at least one binding site in promoters. The network of TF-targets predicted with FIMO comprises 606,188 putative TF-target interactions for B. cinerea and 891,966 potential TF-target interactions in the case of T. atroviride (B. cinerea and 163 samples for T. atroviride (Supplementary File 8). Samples were processed as described in Methods and expression matrices, as well as the list of TFs for both fungi, were given as input for GENIE3. The GENIE3-generated network comprised 5,516,823 gene interactions for B. cinerea and 6,603,528 gene interactions in the case of T. atroviride. To remove unlikely interactions, we constrained the network to the top 20% better scoring GENIE3 TF-target interactions. This represents approximately the top one million interactions, a cutoff that has been previously used for filtering GENIE3-generated GRNs B. cinerea (by 3.8-fold) and Trichoderma (by 4.9-fold) .To identify TFs target genes, we inferred regulatory interactions based on the distance between a given TFBS and the transcription start site (TSS) of each annotated gene of roviride . To imprroviride  and was .9-fold) . The refB. cinerea and 0.083 in T. atroviride). In fact, a relevant proportion of nodes have a clustering coefficient of 0 (see Supplementary Files 9 and 11), indicating neighborhoods of nodes are sparsely connected. Low clustering coefficients have also been reported for other large-scale fungal GRNs To characterize the topology of the reference GRNs, their properties were analyzed. Both reference networks comprised a single connected component, indicating that there is a sequence of nodes and edges (path) connecting each node in the network. However, the average clustering coefficient of the networks is low  has been characterized as a homolog to the Skn7 response regulator of S. cerevisiae, a stress-responsive TF involved in the oxidative stress response, cell cycle, and cell wall biosynthesis and highly conserved among fungi B. cinerea network.For the phytopathogen, the reference network comprises 11,700 nodes and 158,374 edges. The Botrytis network’s top 10 connected nodes (hubs) correspond to TFs from different families , with thTrichoderma, the network was comprised of 11,700 nodes and 181,571 interactions , annotated as a C2H2 domain TF harbors a 90.5% identity with ACEI, a TF encoded by the ace1 gene in Trichoderma reesei[115]. ACEI can bind the promoter of the major cellulase cellobiohydrolase I (cbh1) gene in vitro and in vivo. Consistently, disruption of ace1 in T. reesei alters its growth in cellulose medium In the case of ractions . The mosB. cinerea and T. atroviride, in comparison with their wild-type counterparts To further test the generated reference GRNs, and to determine how these networks allow for the prediction of potential TF-target interactions that can result in changes in gene expression, we gathered global gene expression information from different TF mutants in 3.3N. crassa and A. nidulans, and other relevant models including B. cinerea and T. atroviride, the activation of asexual developmental programs is induced by light B. cinerea, light regulates the production of reproductive structures and virulence T. atroviride. In this biocontroller fungus, the blue light  regulators 1 and 2 (BLR1 and BLR2) N. crassaT. atroviride, light transcriptional signaling has been much less investigated [80]. Moreover, although some TFs involved in light signaling have been reported in both fungi, a more integrated picture of the regulatory interactions shaping fungal responses to light is still lacking. To determine the GRNs underlying light-dependent gene expression in B. cinerea and T. atroviride, we generated light-specific GRNs using available transcriptomics data. For B. cinerea and T. atroviride, we analyzed previously deposited RNA-Seq data from wild-type hyphae exposed to darkness or continuous light .Having a reference GRN allows for constructing context-specific networks by mapping regulatory interactions occurring under particular experimental conditions. In fungi, light is considered a strong cue that impacts several biological processes, such as asexual and sexual developmental programs, secondary metabolism, pathogenicity, and even nutrient acquisition B. cinerea, we determined a total number of 1610 differentially-expressed genes (DEGs) under constant light. From these, 663 genes (42.2%) were induced by the presence of constant light, whereas 947 were down-regulated (58.8%). For T. atroviride, 2286 DEGs were determined. Unlike the phytopathogen, where most genes were down-regulated by constant light, for T. atroviride, 1545 genes (67.6%) were more expressed in the mentioned culture condition (Supplementary File 13). Trichoderma upregulated genes have enriched GO terms associated with five biological processes: metabolic and oxidation–reduction processes, transmembrane transport, and catabolic process related to chitin, a critical structural component of fungal cell walls . We determined six GO biological processes among repressed DEGs, with related functional categories associated with redox activities and transmembrane transport .For B. cinerea DEGs did not reveal any significantly enriched GO term. To determine regulatory interactions driving these changes in gene expression in both fungi, we built GRNs consisting only of DEGs, using the TF-target information from the reference GRNs. Statistics of these context-specific networks are provided in Supplementary Table 3. Remarkably, of the 1610 DEGs in B. cinerea, 57 were TFs, and for 53 of them, it was possible to predict putative target genes, generating a network encompassing 82.5% of DEGs. In the case of T. atroviride, we could infer regulatory interactions for 61 out of 68 TFs among DEGs, with a GRN that contains 75.7% of the total number of DEGs.In contrast, GO analysis of B. cinereaT. atroviride. To delve into this observation, we paid particular attention to the top 10 most connected TFs for both fungi. These can be found in B. cinerea are associated with less expressed (down-regulated) genes in contrast to Trichoderma, in which most transcriptional modules  are light-induced but not repressed B. cinerea is homologous to those in Trichoderma, except for the TEA/ATTS domain TFs Bcin05g04650 and TRIATDRAFT_322845, which show very low sequence identity between them , a key regulator of photomorphogenesis and functional counterpart of BlrA The abovementioned results exemplify the profound transcriptional effect that light has on both organisms, which is beginning to be deciphered in een them . Bcin05gT. atroviride; see Supplementary File 12). The BcSMR1 TF has been characterized in B. cinerea as one of the key regulators of melanin synthesis B. cinerea, melanin biosynthetic genes are encoded in two gene clusters while other genes are in non-cluster genomic locations. Two polyketide synthetases (PKS), bcpks12 and bcpks13, are key enzymes for melanin production for sclerotia and conidia, respectively, and are encoded in two different clusters. bcpks12 is clustered with the bcsmr1 TF, while bcpks13 is clustered with the bcztf1 and bcztf2 TFs and the bcscd1 and bcbrn2 genes bcpks12 is expressed during sclerotia development, which occurs in the dark, and its expression has been reported to be controlled by the bcsmr1 TF bcpks12 and bcsmr1 – members of the first cluster of biosynthetic genes – are repressed by light in our analysis and are nodes in the B. cinerea light-GRN, no regulatory interaction between these genes was found. However, when we analyzed the gene members of the second gene cluster, we observed that bcztf2, bcbrn2, and bcpks13 are more expressed in the presence of constant light, and in this case, the GRN predicts that bcbrn2 is directly regulated by bcztf2. As previously suggested bcpks13, it is expected that some additional yet uncharacterized LTFs may play a role in this process. According to the light-GRN, BcLTF15 may regulate the expression of bcbrn1 as well as the expression of bcpks13 , revealing a new potential link between melanogenesis and light that has not been previously experimentally determined.Other conserved and highly connected TF in both GRNs are BcSMR1 (Bcin02g08760)  and TRIA3.4Trichoderma possesses to counteract different phytopathogens Trichoderma can induce the plant’s activation of complex immune responses when associated with plants. This requires phytohormones and MAP kinases signaling cascades and plant TFs acting as critical regulators of the plant response Trichoderma-fungal interaction scenario is far from being entirely understood. Early efforts employing ESTs sequencing and 454 gene expression analysis contributed evidence of transcriptional changes in Trichoderma induced by B. cinerea cell-wall derivates B. cinereaRhizoctonia solaniIn addition to an array of molecular tools that Trichoderma-Botrytis interaction and to identify candidate key TFs of this process, we first carried out a confrontation assay  and analyzed the changes in the whole transcriptome of both fungi using RNA-Seq. We found that the interaction between B. cinerea and T. atroviride elicited a different response regarding the total number of DEGs in each fungus, being significantly stronger in the biocontroller. We determined 283 upregulated genes and 255 downregulated genes in Trichoderma during the interaction, and only 128 upregulated and five downregulated genes in B. cinerea (Supplementary File 14). During the Trichoderma-Botrytis interaction, enriched GO terms among Trichoderma induced genes include carbohydrate metabolic process, oxidative stress, and proteolysis . Nevertheless, no enriched GO terms were identified among B. cinerea DEGs.To gain insights into the transcriptional response of the T. atroviride, eight modules of DEGs were observed, representing 37.4% of DEGs  B. cinerea mutants deficient in bcbmr1 are more sensitive to iprobenfos (an organic thiophosphate molecule) and polyoxin bccpcA) might control the expression of these genes. In Aspergillus, cpcA encodes a functional orthologue of S. cerevisiae Gcn4p TF, and mutants lacking cpcA are less virulent S. cerevisiae, plays a role in the so-called cross-pathway control in A. nidulans and N. crassabcatrA (Bcin11g04460) bcatrA gene has a low expression during vegetative growth and is induced during the initial stages of B. cinerea infection. When heterologously expressed in S. cerevisiae, bcatrA confers augmented resistance to cycloheximide and catechol. As suggested, in the absence of defined substrate specificity, it could also protect B. cinerea from toxic compounds during saprophytic growth bcatrB that protects B. cinerea against the phytoalexin camalexin during the infection of A. thaliana plants In comparison with ingle TF A. This ningle TF B. In thi4B. cinerea and T. atroviride, including a curated set of TFs for both fungi, based on their latest annotation. We show with two different examples that the reference networks can be integrated with global gene expression data to guide the development of context specific GRNs. The light and interaction networks offer novel hypotheses of transcriptional control of relevant biological processes, including attack and defense strategies in T. atroviride and B. cinerea, respectively, pinpointing key TFs that can be further experimentally validated.In aggregate, our work provides a highly valuable resource of regulatory interactions in This work was supported by ANID – Millennium Science Initiative Program - ICN17_022 to L.F.L., E.A.V., and P.C., ANID-PCI REDI170218 to C.O.Y. and REDES180097 to E.A.V., ANID FONDECYT POSTDOCTORADO N° 3190628 to C.O.Y., ANID FONDECYT POSTDOCTORADO N° 3180328 TO A.S., FONDECYT-Regular 1190611 to P.C., International Research Scholar program of the Howard Hughes Medical Institute to L.F.L. Powered@NLHPC: This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02).Consuelo Olivares-Yañez: Conceptualization, Investigation, Methodology, Data curation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing. Evelyn Sánchez: Investigation, Methodology, Data curation, Formal analysis, Writing – review & editing. Gabriel Pérez-Lara: Investigation, Methodology, Data curation, Formal analysis, Writing – review & editing. Aldo Seguel: Formal analysis, Data curation, Writing – review & editing. Pamela Y. Camejo: Investigation, Methodology, Formal analysis, Writing – review & editing. Luis F. Larrondo: Resources, Funding acquisition, Conceptualization, Writing – review & editing. Elena A. Vidal: Supervision, Resources, Funding acquisition, Project administration, Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing. Paulo Canessa: Supervision, Resources, Funding acquisition, Project administration, Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Fuji is a vital apple cultivar, and has been propagated clonally for nearly a century. The chloroplast genome variation of Fuji apples in China has not been investigated.This study used next-generation high-throughput sequencing and bioinformatics to compare and analyze the chloroplast genome of 24 Red Fuji varieties from nine regions in China.The results showed that the 24 chloroplast genomes were highly conserved in genome size, structure, and organization. The length of the genomes ranged from 160,063 to 160,070 bp, and the GC content was 36.6%. Each of the 24 chloroplast genomes encoded 131 genes, including 84 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The results of repeat sequence detection were consistent; the most common sequence was forward repeats (53.1%), and the least common sequence was complementary repeats (4.1%). The chloroplast genome sequence of Red Fuji was highly conserved. Two indels were detected, but the PI value was 0, and there were no SNP loci. The chloroplast genome variation rate of Red Fuji was low. Malus × domestica) belongs to the genus Malus in the family Rosaceae. It is among the most popular and nutritious fruits  of China Tiangen Biotechnology (Beijing) Co., Ltd. The integrity and purity of the DNA samples were determined by agarose gel electrophoresis. NuoheZhiyuan Technology Co., Ltd.  used the Illumina Hiseq 2000 sequencing platform to perform genome sequencing of quality DNA samples. The read length was 150 bp. A total of 492.617 G of raw data was generated by sequencing, and the filtered clean data was 491.884 G, with an average of 20987.043M per sample .https://github.com/ndierckx/NOVOPlasty), the configuration file config.txt needs to be set before use. Download the published chloroplast genome of Malus yunnanensis (GB: MH394390) from NCBI as the reference file, select its rbc L gene as the seed file, kmer = 39. The preliminary annotation of the assembled plastome was performed using Geseq (https://chlorobox.mpimp-golm.mpg.de/geseq.html). Then, Sequin 16.0 was used to correct the start and stop codons and intron/exon boundaries for the genes with software annotation errors. Finally, the complete cp genome sequence and its annotations were submitted to GenBank. The specific serial numbers are detailed in We chose a reference map for assembly. The obtained clean data were assembled using Novoplasty software , gene composition, base composition and GC (AT) content were calculated using Geneious 11.1.2 software, The characteristics of Red Fuji genomes were analyzed.https://irscope.shinyapps.io/irapp/).Different plant species have different gene sequences in the four junction regions. The change of cp genome length is the main reason for the contraction and expansion of the IR region . The borCodon usage bias (CUB) refers to the phenomenon that some codons are used more than other synonymous codons in the process of gene translation between different species or within the same species . CUB is https://bibiserv.cebitec.uni-bielefeld.de/reputer/) software was used to identify repeats which contain forward, reverse, and complex, repeats as well as palindromes of the cp genome of Red Fuji (https://webblast.ipk-gatersleben.de/misa/index.php), with motif sizes of 1–6 nucleotides (The online REPuters (Red Fuji . The folleotides . All othhttp://genome.lbl.gov/vista/index.shtml) online software. mVISTA is an online tool used for multiple DNA sequence alignments, where sequence similarity can be evaluated by comparing coding regions with non-coding regions, introns, and exons  of Red Fuji was calculated in Dnaspv6 software. SNP and indel detection was carried out using Geneious 11.1.2.The cp genomes of Red Fuji apples from nine regions were compared and visualized by mVISTA . The model is GTRGAMMA+I. Maximum Likelihood (ML) methods were used to construct phylogenetic trees was used to perform the RAxML-HPC BlackBox 8.2.12 in CIPIES (https://www.phylo.org/) (A phylogenetic tree was constructed based on the combined cp genomes. lo.org/)  the ML aThe 24 cp genomes of the Red Fuji apples collected were highly conserved in terms of gene content, gene order, and gene intron number. The genomes ranged in length from 160,063 bp to 160,070 bp, and as expected, the cp genomes contained the LSC and SSC, separated by a pair of IR regions . The lenycf3 and clpP) had two introns. Additionally, 62 protein-coding and 22 tRNA genes were located within the LSC; 12 protein-coding genes, 14 tRNA coding genes, and 8 rRNA coding genes were located within IRs, and 12 protein-coding genes and one tRNA gene were located within SSC. The genes in the LSC region accounted for 64.1% of the cp genome, the two IR regions accounted for 26%, and the genes in the SSC region accounted for 9.9%.Each plastome encoded 131 genes, including 84 protein-coding genes (PCGs), 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. There were 22 introns in the annotated genes. Of the annotated genes, 18 embodied one intron, and two (M. sieversii (MK434920), and one M. sylvestris (MK434924). The results displayed that the boundaries of the four regions were relatively conserved, and the gene types distributed in the boundary region were highly consistent. The genes distributed in the boundary between the LSC and IR region were rps 19 and trn H-GUG. In addition, rp1 2, ycf 1, and ndh F are distributed in the boundary of the SSC/IR region.  were encoded, and the usage frequency of each amino acid ranged from 1.56% to 9.38%. Leucine, serine, and arginine were the most abundant amino acids. Among the encoded amino acids, except methionine and tryptophan, other amino acids were encoded by two to six synonymous codons . For exaA total of 49 repeat sequences were detected in each material, which consisted of 26 forward, 17 palindromic, four reverse, and two complement repeats . Among tM. sieversii (MK434920) as the reference. The nucleotide sequence similarity of the ten cp genomes was extremely high, suggesting that there was no variation in the cp genome of Red Fuji compared with its ancestral species  was 0. Geneious was used to detect nine regions’ single nucleotide polymorphisms (SNPs) of M. sieversii and M. sylvestris, also clustered into a branch, that diverged earlier than Red Fuji, suggesting that they are the ancestors of the cultivated species.Phylogenetic analysis using Maximum likelihood (ML) yielded well-supported tree topologies . All redMalus species regarding genome size, gene content, gene sequence, and GC content , tRNA genes (37), and rRNA genes (8). The GC content was highly conserved (36.6%). However, there was significant diversity in the GC content among the different regions of the cp genome. The GC content in the IR region was significantly higher than that in other regions, which may be due to the distribution of rRNA genes.This study compared the cp genome sequences of 24 Red Fuji apples The genomes were very similar to those of other  content . The lenMoreover, the GC content of the rRNA gene is more abundant. Previous studies have shown that the IR region plays a significant role in preserving essential genes and stabilizing cp DNA structure . In geneAs the linker of nucleic acids and proteins, codons play a significant role in genetic information transmission . Codon uM. sieversii and Red Fuji from the nine different regions showed high sequence identity with species in the Rosaceae. Phylogenetic analysis showed the same results. The coding regions were more conserved than the non-coding regions, and the LSC and SSC regions were more divergent from each other than the IR regions. Mutations in the inverted region, are transformed, reducing this region’s mutation frequency (rps 16 and ndh A genes. The above results further confirm that the variation rate of cp DNA of Red Fuji apples in different regions was very low. A low mutation rate was also found in Previous research has suggested that repetitive sequences significantly influence the sequence differences of the cp genome . We idenrequency . The nucde novo using Illumina high-throughput sequencing data. The genomic structures of the 24 samples were compared and analyzed. On this basis, we concluded that the cp genome structure and gene content of Red Fuji apples in different regions showed little difference. Sequence alignment showed almost no variation in the chloroplast DNA sequence of Red Fuji apples in the different areas. These results are consistent with previous studies reporting slow cp genome evolution. These findings provide a theoretical basis and technical support for the genetic breeding of apples.In the present research, the complete cp genome of Red Fuji was assembled 10.7717/peerj.12927/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.12927/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.12927/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.12927/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.12927/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12927/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12927/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12927/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.12927/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.12927/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.12927/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.12927/supp-12Supplemental Information 12Click here for additional data file.10.7717/peerj.12927/supp-13Supplemental Information 13Click here for additional data file.10.7717/peerj.12927/supp-14Supplemental Information 14Click here for additional data file.


Bifidobacterium bifidum and Bifidobacterium longum possess lacto-N-biosidases with unrelated structural folds to release the disaccharide lacto-N-biose from HMOs, suggesting the convergent evolution of this activity from different ancestral proteins. The crystal structures of enzymes that confer the degradation of glycans from the mucin glycoprotein layer provide a structural basis for the utilization of this sustainable nutrient in the gastrointestinal tract. The utilization of several plant dietary oligosaccharides has been studied in detail, and the prime importance of oligosaccharide-specific ATP-binding cassette (ABC) transporters in glycan utilisations by bifidobacteria has been revealed. The structural elements underpinning the high selectivity and roles of ABC transporter binding proteins in establishing competitive growth on preferred oligosaccharides are discussed. Distinct ABC transporters are conserved across several bifidobacterial species, e.g. those targeting arabinoxylooligosaccharide and α-1,6-galactosides/glucosides. Less prevalent transporters, e.g. targeting β-mannooligosaccharides, may contribute to the metabolic specialisation within Bifidobacterium. Some bifidobacterial species have established symbiotic relationships with humans. Structural studies of carbohydrate-utilizing systems in Bifidobacterium have revealed the interesting history of molecular coevolution with the host, as highlighted by the early selection of bifidobacteria by mucin and breast milk glycans.Bifidobacteria have attracted significant attention because they provide health-promoting effects in the human gut. In this review, we present a current overview of the three-dimensional structures of bifidobacterial proteins involved in carbohydrate uptake, degradation, and metabolism. As predominant early colonizers of the infant's gut, distinct bifidobacterial species are equipped with a panel of transporters and enzymes specific for human milk oligosaccharides (HMOs). Interestingly,  Bifidobacterium genus have attracted significant attention as health-promoting ‘probiotic’ HGM members . Th. ThBifidreported  28]. Th. ThBifidBifidobacterium species, e.g. B. longum subsp. longum (B. longum), B. longum subsp. infantis (B. infantis), B. breve, and Bifidobacterium bifidum are prevalent in infant's guts .,40.N-aceP-GlcNAc  41]. Ga. GaN-aceman host . The bifman host , which rman host . Since tman host .Bacteroides, rely on ABC transporters to import dietary oligosaccharides. The infant gut-associated B. infantis and B. breve use ABC transporters for uptake and subsequent intracellular digestion of HMOs . Tw. TwB. lo (NagBb) , which r (NagBb) . The catBifidobacterium animalis subsp. lactis (B. lactis) and B. longum subsp. longum, are commonly used commercial probiotics. This section focuses on the bifidobacterial proteins that target abundant dietary plant saccharides.Exposure to complex glycans from vegetables and fruits triggers a pronounced expansion of the infant HGM diversity during weaning . The abuBacteroides . Th. ThB. lomulation  91]. Al. AlB. lof HypBA1 .B. longum KACC 91563 for saponin metabolism [B. longum NCC2705 for N-glycan utilization [B. bifidum S17 [Bifidobacterium genus. However, considering their surprisingly broad carbohydrate utilisation capabilities, our structural and biochemical insights into these catabolic pathways remain limited; thus, further investigation of such enzymes and transport proteins is required. Bifidobacteria have been highly successful in establishing symbiotic relationships with different hosts, most notably humans. Therefore, deciphering the structure-based mechanisms of the protein components is instrumental for revealing the interesting molecular coevolution history between bifidobacteria and their hosts.The crystal structures of other bifidobacterial glycosidases were also reported: GH3 β-glucosidase from tabolism , GH5 β-mlization , and GH4lization  and GH43idum S17  for degrImportance to the field: A considerable expansion of pathways targeting host-derived or dietary plant glycans is observed in Bifidobacterium. Although bifidobacteria are considered secondary degraders with few predicted extracellular glycosidases, they are equipped with an arsenal of ABC transporters that are crucial for competitive growth on diverse oligosaccharides.Summary of current thinking: Although some of the transporters of bifidobacteria have been broadly shared within the genus, others have evolved to drive specialization within specific groups. The ability of substrate-binding proteins to capture ligands with high affinity and specificity is likely to be important for efficient cross-feeding.Future directions: Bifidobacteria have been widely used as commercial probiotics, but the molecular mechanisms responsible for their symbiosis with humans and associated health benefits remain underexplored. Insight into the species- and/or strain-specific metabolic signatures will be instrumental for designing personalized mechanism-based dietary interventions to target distinct bifidobacteria and promote human health.


Bacillus spp. isolated from TMK were studied to have anti-cerebrovascular disease activity and probiotic properties. Seventy-three TMK samples from different regions were collected to assess the biogenic amine contents, bacteria composition using next-generation methods, and bacterial functions using Picrust2. Bacillus spp. was isolated from the collected TMK, and their antioxidant, fibrinolytic, and angiotensin I conversion enzyme (ACE) inhibitory activities and probiotic properties were examined. KS TMK had lower sodium contents than the other TMK. There were no significant differences in histamine and tyramine contents among the TMK samples in different provinces. The predominant bacteria in TMK was Bacillus spp., but KS included much less Bacillus spp. and higher Enterococcus and Staphylococcus than the other TMK. Gene expression related to lipopolysaccharide biosynthesis was higher in KS TMK than the other TMK in Picrust2. The predominant Bacillus spp. isolated from TMK was B. subtilis and B. velezensis. B. subtilis SRCM117233, SRCM117245, and SRCM117253 had antioxidant activity, whereas B. subtilis had higher fibrinolytic activity than other Bacillus spp. Only B. velezensis SRCM117254, SRCM117311, SRCM117314, and SRCM117318 had over 10% ACE inhibitory activity. In conclusion, KS had less Bacillus related to lower sodium contents than the other TMK. The specific strains of B. subtilis and B. velezensis had antioxidant, fibrinolytic, and ACE inhibitory activity, and they can be used as a starter culture to produce better quality controlled Kochujang with anti-cerebrovascular disease activities.Traditionally made Kochujang (TMK) is a long-term fermented soybean and rice mixture with red pepper and salts. The ambient bacteria in rice straw and nutrient components of Kochujang influence the bacteria community. We aimed to investigate the bacterial composition and quality of TMK from different provinces of Korea: Chungcheung (CC), Jeolla (JL), Kyungsang (KS), and GeongGee plus Kangwon (GK) provinces, and Jeju island (JJ). Furthermore,  In Korea, primary seasonings or sauces are based on fermented soybeans, called Jang. Jang comprises chungkookjang, doenjang, Kochujang, and kanjang. Chungkookjang is a short-term 2–3 days) fermented soybean without salts, while the other types of Jang are composed mainly of meju, fermenting cooked soybeans with or without grains for more than two months in a dry and cool condition without salts . Kochuja–3 days fBacillus spp. may have different characteristics in different types of Jang because their fermentation condition and ingredient compositions are different .n = 23), Kyungsang , Gyungkang , and Chungcheung provinces , and Jeju island . Seventy-three TMK samples were collected in various areas of Korea in 2019–2020, and the collected TMK samples were stored at 4 °C until further analysis.The scheme of the study design is shown in Sodium contents of TMK samples were measured according to the Korean Food Code method 9.1.2.1.1 and 9.1.2.1.6. As sample preparation for measuring sodium contents, each TMK sample was digested using a microwave digestion system, and the digested TMK sample was diluted into deionized water. Sodium contents were measured using ICP-AES  and ICP-MS  .The DNA extracted from the 0.2 g well-mixed TMK samples using the DNeasy PowerSoil Kit  was amplified using 16S amplicon primers targeting V3 and V4 regions in a polymerase chain reaction (PCR). Libraries of the DNA PCR products were prepared according to the GS FLX plus library prep guide. The DNA was amplified with 16S universal primers in the FastStart High Fidelity PCR System  described elsewhere . BacteriThe 16S amplicon sequences of each TMK were assessed using Mothur v.1.36. Using the Miseq SOP, the sequences were aligned using the Silva reference alignment v.12350, and the taxonomy and bacteria counts in each TMK were determined  × 100, where B is the absorbance with ACE and HHL without the ACE inhibitor component; A is the absorbance with ACE; HHL and C are the absorbance with the HHL without ACE and ACE inhibitor components, respectively.The ACE inhibition activity was measured using the method reported by Ghanbari et al. . The culp values < 0.05 were considered significant.A statistical analysis was conducted using the SPSS version 20.0 . The results were presented as means ± standard deviations or frequency distributions. A one-way analysis of variance (ANOVA) was used to analyze the significant differences in the TMK samples according to the different regions. A Tukey’s test was conducted for multiple comparisons between groups; Bacillus spp. to produce biogenic amines.KS contained sodium contents that were significantly lower than the other TMK. The contents of biogenic amine, including histamine and tyramine, appeared to be different among the Kochujang from five different provinces. Owing to the large variations, they were similar among Kochujang samples . The biop = 0.012; p = 0.016; The α-diversity was determined using the Chao1 and Shannon index representing bacterial richness in TMK. The TMK samples from JL and JJ showed the highest Chao1 index. CC had the lowest index among the groups  than the other TMK in the family level, while TMK in JJ and CC had approximately 90% Bacillaceae, and JL and GK contained approximately 80% A. IntereBacillus B.B. subtilis in all TMK. On the other hand, KS (26%) did not include B. subtilis as much as TMK from other provinces (~40%)  C. Intere confuse C.Acinetobacter baumannii, Staphylococcus aureus, B. cereus, Proteus mirabilis, Pseudomonas aeruginosa, Morganella morganii, and Hafnia alvei. JL contained the highest harmful bacteria among the TMK groups  and fructose and mannose metabolism (p < 0.001) was higher in TMK of the KS area than that of other areas. On the other hand, gene expression related to alanine, aspartate, and glutamate metabolism was higher in TMK of JL and GK than in the other areas. TMK from KS showed lower gene expression involved in cofactor biosynthesis, thiamine metabolism, and biotin metabolism than JL and GK (Gene expression of bacteria in the TMK samples from the different areas showed significant differences in the relative abundance of the Kyoto Encyclopedia of Genes and Genomes Orthology (KO) involved in LPS biosynthesis, alanine, aspartate, glutamate metabolism, cofactor biosynthesis, thiamine metabolism, biotin metabolism, fructose, and mannose metabolism  and B. velezensis (SRCM117254) had a high reactive oxygen species removing capacity (DPPH%) and SOD-like activity  and B. amyloliquefaciens (SRCM117311) had 51.1 and 33.7% adhesion ability to colon cells, respectively  and tyrosine decarboxylase (tdc) gene expression, indicating that they did not produce biogenic amines, histamine, and tyramine  related to B. cereus  contains less salt than doenjang (~12%) . The difBacillus (7.5%), Pantoea (7%), Halomonas (6.4%), Lactobacillus (5.5%), Sphingomonas (5.4%), and Staphylococcus (5%) after a 12-month aging [In Asian countries, there are several types of fermented soybean products, including natto, tempeh, douche, and sufu . They arth aging . It indiBacillus spp., were similar in TMK and doenjang, TMK contained more diverse bacterial communities than doenjang because of the digested rice powder and lower salt contents in TMK than doenjang [Weissella, Tetragenococcus, Oceanobacillus, Debaryomyces, and Lactobacillus [Bacillus and promotes the diversity and abundance of bacteria when its quality is well-controlled and maintained. However, in KS TMK, beneficial bacteria were lower and harmful bacteria were higher than other TMK. JL and CK contained some Lactobacillus. The bacterial community differences in TMK from doenjang might be due to sodium, carbohydrate contents, and environmental temperature in the present study. Therefore, TMK can have high beneficial and diverse bacteria when its production has better control during the fermented and aging periods.Although the dominant bacteria, doenjang . TMK frodoenjang . The lowbacillus . AlthougBacillus spp. (~60%). Moreover, the bacteria distribution in KS TMK was separated from the other TMK except for JJ TMK in β-diversity. Ryu et al. [Bacillus (54.7%), Aerosakkonema (23.8%), and Enterococcus (8.5%), which are abundant bacteria at the genus level after fermentation. Consistent with the Bacillus proportion in the Ryu et al. [Bacillus proportion was much lower in KS TMK than the other TMK; while KS TMK, but not TMK from the other areas, contained approximately 10% Enterococcus and 8% Leuconostoc. TMK from JL and GK contained approximately 8% and 3% Lactobacillus. On the other hand, the bacteria composition at the species level was somewhat different between Ryu et al. and the present studies: B. haynesii, and B. licheniformis are dominant after fermentation, and E. hirae and E. faecium exist in the TMK according to Ryu et al. [B. subtilis was dominant, and B. velezensis and B. coagulans were abundant in TMK.The bacterial communities of TMK in KS were separated significantly from those of TMK in other areas in the β-diversity analysis. KS contained the lowest u et al.  reportedillus 54.%, Aerosaillus 54.%, Aerosatdc and hdc was detected in some TMK. It suggests that the collected TMK did not have a high biogenic amine metabolism. In addition, a large sample size is needed to detect the bacterial distribution because the bacterial composition varies among the TMK samples. In Ryu et al. [The differences in bacterial distribution influenced the biogenic amine contents to influence the TMK quality . Doenjanu et al. , regionaC-hexoside, quercetin-O-rhamnoside, and genistein O-dihexoside), capsaicinoids (capsaicin and dihydrocapsaicin) and capsinoids (dihydrocapsiate), and phospholipids [Bacillus, and Bacillus spp. were isolated from doenjang, chungkookjang, and TMK [Bacillus spp. On the other hand, some Bacillus spp. isolated from doenjang and TMK, have not been used to produce the inoculated doenjang and TMK.TMK has been reported to have anti-obesity, antidiabetic, anti-dyslipidemic activities in cell-based, animal, and human studies ,11,12,32holipids . On the holipids . The dif and TMK . ChungkoB. subtilis strains in TMK had higher antioxidant and high fibrinolytic activity, while some B. velezensis strains had more than 10% ACE inhibitory activity. Previous studies showed that tomato fermented with B. subtilis, sardinelle protein fermented with B. subtilis A26, and B. amyloliquefaciens An6, and Ruditapes philippinarum fermented with B. natto have shown ACE inhibitory and antioxidant activities [Bacillus spp. to have ACE inhibitory activity. The Bacillus spp. to have antioxidant, ACE inhibitory, and fibrinolytic activities did not have biogenic amine and Bacillus cereus-related genes. They can be used to make inoculated Kochujang with anti-cerebrovascular diseases.The bacteria compositions of TMK affect its quality, including taste, odor, and metabolic functions. In the present study, some tivities ,35,36. OB. subtilis, B. amyloliquefaciens, and B. velezensis had anti-cerebrovascular disease activity, including increasing fibrinolytic, antioxidant, and ACE inhibitory activity. The isolated ones can be applied to make functional Kochujang as a starter culture. The inoculated Kochujang can be registered as a functional food having anti-cerebrovascular disease activity in the Korean Food and Drug Administration after its efficacy and mechanism are confirmed in animal and human studies in the near future.Overall, TMK from all areas in Korea exhibited sustainable quality control, but KS TMK must increase beneficial bacteria in the present study. TMK should maintain optimal quality control to increase various beneficial bacteria and to not contain harmful bacteria and biogenic amines. Several Bacillus spp. for producing inoculated Kochujang, B. subtilis, and B. velezensis had probiotic properties, no biogenic amine producing genes, antioxidant, fibrinolytic, and ACE inhibitory activities. These B. subtilis and B. velezensis strains can be used as a starter culture to make inoculated Kochujang protect against cerebrovascular diseases. Further animal and human studies are needed to produce inoculated Kochujang with optimal quality control and investigate its functionality for anti-cerebrovascular diseases.Salt and carbohydrate contents and ambient bacteria from different areas influence the bacterial composition of TMK. TMK must be well-monitored and controlled to have good quality since it is susceptible to growing harmful bacteria due to lower salt and higher carbohydrate contents than doenjang. All TMK contained mostly beneficial bacteria and <0.5% harmful and restricted bacteria, suggesting they were well-managed. However, KS TMK may have a lower quality of bacteria than the other TMK. It is better to produce inoculated Kochujang to include well-controlled bacteria and have specific functionality. Among the isolated