Corynebacterium glutamicum that is used for the industrial production of amino acids including tryptophan lacks tryptophanase. To test if indole is metabolized by C. glutamicum or has a regulatory role, the physiological response to indole by this bacterium was studied. As shown by RNAseq analysis, indole, which inhibited growth at low concentrations, increased expression of genes involved in the metabolism of iron, copper, and aromatic compounds. In part, this may be due to iron reduction as indole was shown to reduce Fe3+ to Fe2+ in the culture medium. Mutants with improved tolerance to indole were selected by adaptive laboratory evolution. Among the mutations identified by genome sequencing, mutations in three transcriptional regulator genes were demonstrated to be causal for increased indole tolerance. These code for the regulator of iron homeostasis DtxR, the regulator of oxidative stress response RosR, and the hitherto uncharacterized Cg3388. Gel mobility shift analysis revealed that Cg3388 binds to the intergenic region between its own gene and the iolT2-rhcM2D2 operon encoding inositol uptake system IolT2, maleylacetate reductase, and catechol 1,2-dioxygenase. Increased RNA levels of rhcM2 in a cg3388 deletion strain indicated that Cg3388 acts as repressor. Indole, hydroquinone, and 1,2,4-trihydroxybenzene may function as inducers of the iolT2-rhcM2D2 operon in vivo as they interfered with DNA binding of Cg3388 at physiological concentrations in vitro. Cg3388 was named IhtR.The aromatic heterocyclic compound indole is widely spread in nature. Due to its floral odor indole finds application in dairy, flavor, and fragrance products. Indole is an inter- and intracellular signaling molecule influencing cell division, sporulation, or virulence in some bacteria that synthesize it from tryptophan by tryptophanase.  Stevia rebaudiana [Indole is a bioactive aromatic compound and used as flavor and fragrance in the cosmetics  and food  industries because of its floral odor which is typical for jasmine . The horbaudiana . Indigo,baudiana .d-glyceraldehyde-3-phosphate [l-tryptophan to indole, pyruvate, and ammonia [In nature, the versatile widely distributed signaling molecule indole has significant roles in bacterial physiology, pathogenesis, animal behavior, and human diseases . In bacthosphate . In bact ammonia .E. coli and Pseudomonas putida, but decreased in Paenibacillus alvei. Similarly, tolerance to antibiotics is decreased by indole in Staphylococcus aureus and Vibrio anguillarum, but increased in Agrobacterium tumefaciens and Vibrio cholera [E. coli, where Mtr [Different physiological responses to extracellular addition or intracellular synthesis of indole have been described for indole-producing as well as for non-indole-producing bacteria affecting growth, biofilm formation, antibiotic resistance, acid and heat resistance, or indole persistence . These e cholera . Thus, where Mtr ,9 and Mthere Mtr  have beehere Mtr .Acinetobacter [Cupriavidus [Aerobic degradation of indole proceeds via the two key intermediates: isatin and anthranilate ,13. Sevetobacter  and Cuprriavidus . A gene riavidus . Indole riavidus ,13.C. glutamicum, which is used for the industrial production of amino acids including the aromatic amino acid l-tryptophan, does not synthesize indole, but is able to utilize a number of other aromatic compounds for growth [C. glutamicum. The degradation of resorcinol and 2,4-dihydroxybenzoate also occurs via this pathway, using 1,2,4-trihydroxybenzene as intermediate [C. glutamicum. In order to test if indole is metabolized by C. glutamicum or exerts a regulatory role as a putative signaling molecule, we determined the physiological and transcriptomic response of C. glutamicum to indole.The knowledge about the genetic mechanism underlying the physiological response in bacteria is limited. Most studies were focused on the visible changes in the physiological response on indole and only a few about the transcriptomic response ,17. C. gr growth ,26,27,28r growth ,29. Thisrmediate ,24,30. Irmediate ,31. Tranrmediate ,33,34,35rmediate ,39,40,41rmediate ,46,47,48E. coli DH5α [C. glutamicum WT and C1* were used for investigation of indole response. Standard molecular genetic techniques were performed as described in [E. coli DH5α [C. glutamicum was performed by electroporation [All bacterial strains used are listed in oli DH5α  was usedribed in . Competeoli DH5α  was perfoli DH5α . Transfoporation .C. glutamicum were performed by two-step homologous recombination [mobsacB [C. glutamicum as described elsewhere [mobsacB by Gibson Assembly resulting in the plasmids listed in mobsacB-ΔdtxR and pK19mobsacB-ΔrosR, the strains ΔdtxR and ΔrosR were used as templates for fragment amplification by PCR. Transfer of the suicide vectors was carried out by transconjugation using E. coli S17 as donor strain [sacB. Suicide vector excision was selected by sucrose resistance. Gene deletions or replacements were verified by PCR and sequencing with respective primers .Chromosomal gene deletions, replacements, and base exchanges in bination  using th[mobsacB . The genlsewhere  using thr strain . For the primers . OverexpE. coli and C. glutamicum were performed in lysogeny broth (LB) at 37 or 30 °C in baffled shake flasks on a rotary shaker (160 or 120 rpm). Cultures were inoculated freshly from LB agar plates. When necessary, kanamycin (25 µg m L−1) or spectinomycin (100 µg m L−1) were added to the medium. For induction of gene expression from vectors pVWEx1 or pEKEx3 1 mM isopropyl-β-d-1-thiogalactopyranoside (IPTG) was added to the medium. For growth or production experiments with C. glutamicum, precultures as described above were harvested , cells were washed with TN-buffer pH 6.3  and inoculated to an optical density at 600 nm (OD600) of 1 in CGXII minimal medium [−1 glucose as sole carbon source if not otherwise noted. C. glutamicum was grown in 500 mL or 100 mL baffled shake flasks or in Micro-Flask microtiter plates  at 30 °C and 120 or 220 rpm, followed by measuring OD600 using V-1200 spectrophotometer  or microbioreactor system Biolector . For growth in the Biolector system, cultures were grown in LB-rich medium overnight and transferred to second preculture of CGXII minimal medium with 40 g L−1 glucose with addition of indole, if required until early exponential phase before inoculating to the main medium of CGXII minimal medium and 40 g L−1 glucose. Growth experiments in the Biolector system were carried out using 48-well flower plates  with a filling volume of 1 mL, at 30 °C, and 1200 rpm shaking frequency. Humidity was kept constant at 85%, and online biomass measurements of scattered light were monitored with backscatter gain of 30.Precultures of l medium  with 40 C. glutamicum WT or C1* were cultivated in triplicates in CGXII minimal medium with 40 g L−1 glucose with addition of either ±2.5 or ±4 mM indole , ±3 mM indole-alanine TFA , ±2.5 mM resorcinol , or ±2.5 mM myo-inositol . Cultivation was performed in 100 mL baffled shake flasks at 220 rpm at 30 °C for all conditions. For the cultivation with ±2.5 mM indole, 500 mL baffled shake flaks were used. For transcription analysis by RNA sequencing, exponentially growing cells  were collected to 50 mL falcons filled with ice and centrifuged at 4 °C for 10 min at 3.220× g. For transcription analysis by qRT-PCR, 2 mL of culture was shortly spin down in precooled tubes at 4 °C at 3.220× g. The resulting cell pellets were frozen in liquid nitrogen and stored at −80 °C until further use. Growth was monitored in independent parallel cultures.For mRNA isolation, C. glutamicum WT was cultivated in triplicates using 100 mL baffled shaking flasks at 120 rpm and 30 °C in CGXII minimal medium with 40 g L−1 glucose with different concentrations of indole (dissolved in ethanol). The culture reaching the highest OD600 after 24 or 72 h was harvested and washed with TN buffer and used to inoculate three new cultures. The indole concentration in the medium was increased when growth OD600 values were observed in the previous culture. In total, 38 transfers were done. Evolved strains were stored as glycerol culture at −80 °C.For adaptive laboratory evolution, C. glutamicum cells, bacterial cell pellets previously harvested and kept at −80 °C were thawed on ice and RNA was extracted individually for each cultivation condition using a NucleoSpin RNA isolation kit . Polymerase chain reactions with Taq polymerase  were performed to detect if contaminating genomic DNA remained in the samples. RNA samples with genomic DNA contamination were treated with RNase-free DNase . Total RNA concentrations were measured using a spectrophotometer . RNA quality was checked by Trinean Xpose  and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer . The extracted RNA samples were either pooled (treatment with indole) or separately treated (treatment with Ind-Ala). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina  was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was free of detectable rRNA. Preparation of cDNA libraries were performed according to the manufacturer’s instructions of the TruSeq stranded mRNA Kit (Illumina). Subsequently, each cDNA library was sequenced on a HiSeq1500 (2 × 70 nt PE rapid v2) Sequencer system (Illumina). The software Bowtie2 [p-value ≤ 0.01) with log2 fold change >1.5 or <−1.5 were used. The transcriptomic data are available via NCBI GEO accession identifiers GSE159887 and GSE159888.In order to isolate total RNA from  Bowtie2 ,62 were  Bowtie2 ,64. Stat® No-ROX One-Step Kit  and the CFX96 cycler system (Bio-Rad). The temperature profile was (1) 45 °C for 10 min (reverse transcription); (2) 95 °C for 2 min; (3) 40 cycles of 95 °C for 5 s, 55 °C for 10 s, and 70 °C for 5 s; (4) melt curve analysis with measures between 65 and 95 °C. The log2 fold change of the negative ΔΔCq (reference Cq–sample Cq) value, using the reference gene parA (cg3427), was used in calculations [All qRT-PCRs were performed according to the manufacturer’s instruction using the SensiFASTTM SYBRulations . For eacC. glutamicum WT and evolved strains were cultivated in triplicates in LB medium 100 mL baffled shake flasks at 120 rpm and 30 °C overnight and the complete culture harvested. Genomic DNA was isolated using the NucleoSpin Microbial DNA kit for DNA, RNA, and protein purification (Macherey-Nagel) according to the manufacturer. Quality of isolated gDNA was analyzed using a spectrophotometer . The complete digestion of the RNA was verified by gelelectrophoresis. Library preparation involved a TruSeq DNA PCR-free high-throughput library prep kit (Illumina) and Illumina genome sequencing was performed with a HiSeq1500 sequencer system 2 × 250 nt PE v2 HT (Illumina). The software snippy (https://github.com/tseemann/snippy) [For isolation of genomic DNA (gDNA), /snippy)  was usedg) for HPLC analysis. Separation was performed with a precolumn (LiChrospher 100 RP18 EC-5µ (40 × 4 mm), CS Chromatographie Service GmbH, Langerwehe, Germany) and a main column (LiChrospher 100 RP18 EC-5µ (125 × 4 mm), CS Chromatographie Service GmbH, Langerwehe, Germany). A mobile phase of buffer A (0.1% trifluoroacetic acid dissolved in water) and buffer B (acetonitrile) was used with a flow rate of 1 mL min−1. The following gradient was applied: 0–1 min 10% B; 1–10 min a linear gradient of B from 10% to 70%; 10–12 min 70% B; 12–14 min a linear gradient of B from 70% to 10%; 14–18 min 10% B [Extracellular amino acids and aromatic compounds were quantified by high-performance liquid chromatography (HPLC) . The culture supernatants collected at different time points were centrifuged , and C1*(pEKEx3-cg2796-cg2797) were analyzed after growth without or in the presence of 2.5 mM indole. Supernatants were collected after 16 h of cultivation and extracted with ethyl acetate. The ethyl acetate extract was washed three times with water to remove traces of CGXII medium components, followed by water removal using a Pasteur pipette plugged with silanized glass wool and Na2SO4. One microliter of each sample was injected into GC–MS. Samples were analyzed using a gas chromatograph  equipped with a 30 m × 0.25 mm × 0.25 µm film thickness column . Helium was used as the carrier gas and the flow rate was adjusted to 1 mL min−1 for GC–MS analysis. The injector was used in splitless mode and inlet temperature was set to 250 °C. The initial oven temperature was 45 °C for 1 min, and increased to 300 °C after 1 min at a rate of 10 °C min−1, which was held for 5 min at 300 °C. The GC was coupled to a Triple-Axis detector .For GC–MS analysis, supernatants of strains C1* (pVWEx1), C1* (pVWEx1-cg3388 in E. coli BL21(DE3) transformants were grown at 37 °C in 500 mL LB medium with 50 µg mL−1 ampicillin to an OD600 of 0.5 before adding IPTG (0.5 mM) for induction of gene expression. After induction, cells were cultivated at 21 °C for an additional 4 h and were harvested by centrifugation at 4 °C. Pellets were stored at −20 °C. Crude extract preparation and protein purification via Ni-NTA chromatography was performed as described elsewhere [After transformation of the pET16b-lsewhere . The purcg3388 and cg3387  in band shift buffer  glycerol, 50 mM KCl, 10 mM MgCl2, 0.5 mM EDTA, pH 7.5) in a total volume of 20 µL. The intergenic region was PCR-amplified and purified with NucleoSpin kit . A 78 bp-fragment of the upstream region of cg2228 was added in every sample as a negative control using oligonucleotides cg2228_EMSA_F and cg2228_EMSA_R. BSA  was used as negative control. After 30 min of incubation at room temperature, gel shift samples were separated on a native 7.5% (w/v) polyacrylamide. Additionally, the binding affinity in the presence of myo-inositol and different aromatic compounds 1,2,4-trihydroxybenzene, hydroquinone, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 2,4-dihydroxybenzoic acid, 34-dihydroxybenzoic acid (protocatechuate), indole, 6-hydroxyindole, 5-hydroxyindole, l-tryptophan, and 5-hydroxy-l-tryptophan as effector was analyzed by incubation of the protein with the effector under buffered conditions for 15 min at room temperature prior to the addition of the intergenic DNA region. Subsequently, the gel shift samples were separated on 7.5% acrylamide retardation gel at 100 V buffered in 44.5 mM Tris, 44.5 mM boric acid and 1 mM EDTA at pH 8.3. Staining of the DNA was achieved with ethidium bromide.To analyze the physical protein–DNA interaction between the Cg3388 protein and their putative native target DNA, band shift assays were performed. The His-tagged Cg3388 protein was mixed in varying molar excess with 45 ng of PCR amplified and purified intergenic fragment between start codon of 2+-specific chelator bathophenanthroline disulfonic acid (BPS) was measured at 534 nm according to Müller et al. 2020 [3+ reduction was determined by an increase of absorbance. The iron reduction assay was performed in 25 mM Tris-HCL buffer (pH 7.4), and 0.42 mM FeCl3  was added from a stock in 10 mM HCl neutralized with 50 mM NaOH immediately before use; 19.5 μM PCA or indole was used. The reaction was started by the addition of 6.5 mM BPS and measured every 30 min. As control, reactions were measured without addition of PCA or indole.The complex formation with the Feal. 2020 . The kin3-solution (50 mL), aqueous HCl , water (100 mL) and saturated NaCl-solution (100 mL). The organic phase was dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was used for the further reaction without purification. Boc-cleavage was performed under acidic conditions and cooling. Thereafter the crude of protected pseudo-Dipeptide was dissolved in DCM (5 mL), cooled to 0 °C with an ice bath, and treated slowly with HCl . After 15 min the ice bath was removed, and the solution was stirred for 2.5 h at ambient temperature. Then the solvent was removed under reduced pressure, the residue was dissolved in water (50 mL) and DCM (50 mL). The aqueous layer was washed with DCM , neutralized with sat. NaHCO3-solution (50 mL) and extracted with DCM . OP2 was dried over MgSO4 and the solvent was removed under reduced pressure. The crude product was purified via reversed phase HPLC (water/ACN/0.1% trifluoracetic acid (TFA)) to receive the desired Indole-Ala-pseudo-Dipeptide  as a colorless solid TFA-salt. The reaction mechanism and the 1H-NMR spectrum of the final product are shown in For the synthesis of Indole-Ala-pseudo-Dipeptide, sodium hydride  was suspended in ice cold N,N-dimethylformamid (DMF) (4 mL) and a solution of Indole  in DMF (8 mL) was added dropwise over 2 min and further stirred for 5 min at 0 °C. Boc-Ala-OH  and 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU)  were dissolved in dimethylformamide (10 mL), followed by the dropwise addition of 4-methylmorpholin (NMM)  and stirred for 40 min at ambient temperature. The Boc-Ala-OH solution was then added to the ice-cold sodium hydride suspension over a period of 2 min. The ice bath was removed after 2.25 h and the reaction was further stirred for 1.3 h under ambient temperature. Afterwards the mixture was diluted with dichlormethane (DCM) (50 mL) and water (100 mL). The aqueous layer was extracted with DCM (3 × 50 mL), the organic layers were combined and washed with water (100 mL), saturated NaHCOC. glutamicum strains WT and C1*. Indole (2 mM) was used to replace either the carbon equivalent of glucose (5.3 mM) or the nitrogen equivalent of the combined nitrogen sources urea and ammonium sulfate . No growth was observed with indole as sole nitrogen or sole carbon source for 24 h, but both strains grew when afterwards either 220 mM glucose or 151 mM (NH4)2SO4 and 83 mM urea were added (data not shown). Thus, indole does neither support growth of C. glutamicum as sole carbon or nitrogen source.First, it was tested whether indole may serve as sole carbon or sole nitrogen source for growth of C. glutamicum strains WT and C1* to indole as additive to glucose minimal medium, different indole concentrations (0 mM to 8 mM) were added to the medium before inoculation and growth in a biolector cultivation was monitored  and reached the same biomass concentration as the control without added indole  in the presence of indole, while no gene showed decreased expression  were performed. The pattern of differential gene expression determined in the RNAseq analysis was confirmed for all nine genes analyzed.To determine the gene expression changes of erformed . RNA waspression . Next, wphe (cg2966), members of the DtxR regulon , copper-related genes , and the operons for p-cresol catabolism and cytochrome bd (cydABCD)  . Since tn source , these ephe and the two DtxR regulated genes cg2796 and cg2797 coding for unknown proteins, it was tested if their deletion or overexpression affected growth of C. glutamicum. However, growth in the presence of indole was comparable to that of the parent strain  make up for PCA as an iron chelator that supported maximal growth rates of 0.40 ± 0.01 h−1 for WT and C1*. Based on the recent finding that PCA reduces Fe3+ to Fe2+ [3+ to Fe2+ in a similar manner as 19.5 µM PCA  C. Precul to Fe2+ , a BPS-d5 µM PCA D, while 5 µM PCA  may be d3+ to Fe2+ by indole  in the presence of indole-alanine, whereas 6 genes showed decreased expression , regulator genes , and dps encoding a starvation-induced DNA protecting protein showed increased RNA levels . A common hallmark in the response to extracellular indole and to an increased intracellular indole concentration due to feeding indole-alanine peptide was increased expression of a subset of six genes: cg0018 (coding for an uncharacterized membrane protein), cg0464 (coding for a putative copper ion transporting P-type ATPase), cg2962 (encoding a putative enzyme synthesizing extracellular polysaccharides), cg3402 (coding for a copper chaperone), phenol oxygenase gene phe, and DNA protection gene dps . The indole concentration added to fresh medium was stepwise increased (4 to 8 mM) in the serial transfers  and one insertion of 7 nucleotides , which was confirmed by PCR (C. glutamicum [pyk [(Asn/Gln) amidotransferase subunit B GatB (D452G) and putative membrane protein Cg3132 (G69D). Amino acid exchange T2I in the regulator of oxidative stress response RosR may not affect the function of the protein. However, the corresponding base change on the DNA level alters one of the two RosR binding sites (binding site rosRb 5′-TAGGTGATATGA(C→T)AACAC-3′) affecting RosR autoregulation [The genome of the ALE strain IVO38 showed an ~82 kbp deletion (~Δd by PCR . Excisioutamicum . Besidesgulation . Two SNPdtxR and cg3388 and their (putative) regulatory targets cg0405 and rhcM2 (cg3386), respectively, differs between C. glutamicum WT and the ALE strains IVO20 and IVO38, a qRT-PCR analysis was performed with RNA isolated from cells growing exponentially in the presence or absence of 4 mM indole  to regulate the divergently transcribed operon iolT2- rhcM2D2.In order to test if expression of M indole . The RNAan in WT C and wasan in WT B,D. Exprmicum WT A,C. ThisG69D) expression of cg3388 was higher than in WT  due to absent or nonfunctional Cg3388. Reverse engineering revealed a major role of Cg3388M1T in indole toleranceThe cg3388 RNA level was comparable in WT and IVO20, in which likely no Cg3388 protein is synthesized due to the SNP mutation of the translational start codon. However, in IVO38  and rosR (0.31 ± 0.00) as compared to the wild type (0.23 ± 0.02), while growth in the absence of indole was comparable , which was comparable to the growth rate observed with C1*cg3388M1T  may result in the complete absence of Cg3388 protein, while the effect of the amino acid change G69D is not known. Therefore, we deliberately chose to delete cg3388 as we consider this the most clear cut genetic modification. Strain ΔiolT2-rhcM2D2. A qRT-PCR analysis of WT and Δcg3388 grown without or with 2.5 mM indole revealed increased RNA levels of rhcM2. This finding is commensurate with a role of Cg3388 as repressor of rhcM2 and, by inference, likely the iolT2-rhcM2D2 operon. UniProtKB analysis predicts an N-terminal helix-turn-helix DNA binding domain (HTH iclR-type) at position 20–81 and a C-terminal effector binding domain (IclR-ED) at position 82–266 for Cg3388 (Q8NL89), a domain structure typical for repressor proteins. As rhcM2 and rhcD2 codes for maleylacetate reductase and catechol 1,2-dioxygenase enzymes involved in resorcinol catabolism [C. glutamicum cell [cg3388 and rhcM2 were comparable to indole addition  was performed C. Purifil-tryptophan, and 5-hydroxy-l-tryptophan (data not shown). By contrast, 1,2,4-trihydroxybenzene, hydroquinone , and indole interfered with binding of Cg3388 to its target DNA at concentrations exceeding 0.05, 0.5, and 4 mM, respectively  and as maleylacetate reductase RolM and catechol 1,2-dioxygenase RolD are homologs of RhcD2 and RhcM2 converting 1,2,4-trihydroxybenzene to β-ketoadipate, 1,2,4-trihydroxybenzene is most likely the main physiological effector of Cg3388/IhtR. However, since hydroquinone and indole affected Cg3388/IhtR in vitro at physiologically relevant concentrations they may also play a role in C. glutamicum in vivo.For identification of effectors of Cg3388, different compounds were added during the incubation of His-tagged Cg3388 protein with target DNA. The binding of Cg3388 to target DNA was hardly affected by the addition of 5 mM of inositol, resorcinol , 1,2-dihydroxybenzene, 2,4-hydroxybencoic acid, PCA , 6-hydroxyindole, 5-hydroxyindole, ectively C. Thus, C. glutamicum to indole, which was shown to reduce Fe3+ to Fe2+ in the culture medium, is governed by increased expression of iron and copper metabolism genes, of phenol 2-monooxygenase gene phe, and the p-cresol catabolic cre operon. Deletion of genes encoding the regulator of oxidative stress response RosR, the iron response regulator DtxR, and Cg3388/IhtR improved indole tolerance.The response of 3+ to Fe2+  in addition to the DtxR regulon . Notably, when the intracellular indole concentration was increased by feeding the dipeptide indole-alanine, only two genes (dps and cg0405) of the DtxR regulon were induced , the cre operon for catabolism of p-cresol, and the transcriptional regulator Cg3388/IhtR. Strain C1* lacks the cre operon and cannot use cresol as sole carbon source . The iolT2-rhcM2D2 operon also encodes catechol 1,2-monooxygenase and maleylacetate reductase enzymes. Paralogs are encoded by the rol operon (cg1112-cg1115), which is negatively regulated by RolR and required for catabolism of resorcinol and 2,4-dihydroxybenzoate [iolT2-rhcM2D2 operon is not required for growth with resorcinol, it is, however, transcribed in the presence of resorcinol [iolT2-rhcM2D2 operon, which is derepressed in the presence of 1,2,4-trihydroxybenzene or aromatic compounds other than the inducers of RolR resorcinol and hydroxyquinol that lead to 1,2,4-trihydroxybenzene. Indole only induced the iolT2-rhcM2D2 operon, but not the rol operon; thus, it is not an inducer of RolR. Induction of the iolT2-rhcM2D2 operon by indole may be a metabolic burden, slowing growth in its presence or indole may be converted to a growth inhibitory compound by catechol 1,2-monooxygenase RhcM2 and/or maleylacetate reductase RhcD2. In the latter case, this compound may be synthesized intracellularly, but we did not obtain evidence for its accumulation in the culture medium. The nature of this putative indole-derived inhibitor remains elusive.Adaptive laboratory evolution to increase indole tolerance led to the discovery of Cg3388/IhtR as indole, hydroquinone, and 1,2,4-trihydroxybenzene responsive transcriptional regulator repressing transcription of the divergently transcribed cum cell ,83, whilcum cell . Transcr product . By contbenzoate ,30. Althsorcinol . Resorci


Lotus seed-derived activated carbon materials display hierarchically porous structures comprised of micropore and mesopore architectures, and exhibited excellent supercapacitance performances. The specific surface areas and pore volumes were found in the ranges 1103.0–1316.7 m2 g−1 and 0.741–0.887 cm3 g−1, respectively. The specific capacitance of the optimum sample was ca. 317.5 F g−1 at 5 mV s−1 and 272.9 F g−1 at 1 A g−1 accompanied by high capacitance retention of 70.49% at a high potential sweep rate of 500 mV s−1. The electrode also showed good rate capability of 52.1% upon increasing current density from 1 to 50 A g−1 with exceptional cyclic stability of 99.2% after 10,000 cycles demonstrating the excellent prospects for agricultural waste stuffs, such as lotus seed, in the production of the high performance porous carbon materials required for supercapacitor applications.Of the available environmentally friendly energy storage devices, supercapacitors are the most promising because of their high energy density, ultra-fast charging-discharging rate, outstanding cycle life, cost-effectiveness, and safety. In this work, nanoporous carbon materials were prepared by applying zinc chloride activation of lotus seed powder from 600 °C to 1000 °C and the electrochemical energy storage (supercapacitance) of the resulting materials in aqueous electrolyte (1M H ActivatWh kg−1) ,13,14. Fatteries 0 Wh kg−1Wh kg−1) ,18,19,20Wh kg−1) . Previouatteries 0 Wh kg−1Wh kg−1) ,22,23,24Wh kg−1) ,26,27,283PO4), basic , or neutral reagents  [2. ZnCl2 is a well-known dehydrating agent and it promotes the breakdown of carbon materials during the carbonization process, and also controls the tar formation [2 is present during carbonization of lignocellulose, it undergoes pyrolytic decomposition and enrichment of porosity due to depolymerization and dehydration of the resulting biochar [In addition to the molecular nanocarbon materials—such as fullerene, graphene, and carbon nanotubes, porous carbon materials derived from biomass—have been comprehensively employed as the electrode materials in supercapacitor applications because of low-cost and extraordinary specific surface area and large porosity ,31,32,33, ZnCl2) ,35. In tormation . Chemicaormation ,41,42,43ormation  with theormation ,45. When biochar .2 activated hierarchically porous carbon materials derived from lotus seed in aqueous 1 M sulfuric acid solution at 25 °C. Biochar was initially prepared by the heat-treatment of lotus seed powder in air at 300 °C, with the product then being chemically activated by its impregnation with ZnCl2 at a weight ratio of 1:1. Subsequent carbonization was carried out at higher temperatures (600–1000 °C). The morphology, structure, and textural properties of the prepared carbon materials were examined by scanning electron microscopy, powder X-ray diffraction, Raman scattering, and analysis of their nitrogen adsorption isotherms.In this contribution, we report the electrochemical energy storage supercapacitance performances of ZnClNelumbo nucifera (Gaertn), a perennial aquatic plant) was collected from a local herbarium market at Kathmandu, Nepal. The Lotus seed was dried at 120 °C in an oven for 10 h and crushed in an electric grinder. The crushed powder was then heat-treated at 300 °C for 3 h in the air, and biochar was obtained. This was followed by mixing with ZnCl2 (1:1 weight ratio), and final carbonizations were carried out at high temperatures (600–1000 °C) under a flow of nitrogen gas (120 cm3 min−1) in a tube furnace  at a heating speed of 5 °C min−1 and a hold time of 3 h. The resulting carbon materials were washed with dilute aqueous hydrochloric acid (0.5 M) to remove unreacted ZnCl2 then also washed with distilled water multiple times until the pH of the washings was neutral. The final products were vacuum dried at 80 °C for 6 h then ground finely to powders. The chemically activated nanoporous carbon samples are denoted as LTSC_Z600, LTSC_Z800, and LTSC_Z1000 based on the carbonization temperature. A control material was prepared by directly carbonizing (without ZnCl2 activation) Lotus seed powder at 800 °C with the product obtained being denoted as LTS_800.Lotus seed , powder X-ray diffraction , Raman scattering , Fourier-transform infrared spectroscopy , and nitrogen adsorption isotherm analysis using an automatic adsorption instrument  at liquid nitrogen temperature (−196.1 °C). Barrett–Joyner–Halenda (BJH) method and density functional theory (DFT) were used to obtain pore size distributions. Note that BJH and DFT theory assumes cylindrical and slit-like pores, respectively.Nanoporous carbon materials obtained from the ZnCl2SO4 aqueous electrolyte solution at 25 °C. Modified glassy carbon electrode  with the material for analysis was used as the working electrode and Ag/AgCl and a platinum wire were used as counter and reference electrodes, respectively. Working electrodes were prepared as follows. First, the GCE was cleaned and mirror polished with a slurry of alumina (Al2O3), cleaned with distilled water, and vacuum dried at 60 °C for 3 h. Separately, the carbon material for analysis was dispersed in a solvent mixture (ethanol-water = 1:4 v/v mixture: concentration of 1 mg mL−1) and sonicated in a bath sonicator  for 1 h. The obtained suspension (3 μL) was dropped onto the GCE surface and dried at 60 °C for 6 h for removal of the solvent. A Nafion solution (5 μL: 5% in ethanol) was added on top of the active materials as a binder and again by dried at 60 °C for 12 h. Electrochemical impedance spectroscopy (EIS) measurements were conducted in 1 M H2SO4 electrolyte at an open-circuit potential. The frequency range was varied from 0.01 Hz to 100 kHz at an amplitude of 5 mV. CV, CD, and EIS measurements were performed on a CHI 660E workstation  at 25 °C.The electrochemical performances  of Lotus seed-derived nanoporous carbon materials was calculated based on cyclic voltammetry (CV) and galvanostatic charge–discharge (CD) measurements. The electrochemical measurements were performed using a three-electrode system in 1 M HCs) was calculated asI, ν, m, and ∆V, respectively denote the current (A), scan rate (V s−1), the mass of active electrode materials (g), and the potential window (V).From the CV curves, specific capacitance , discharge time (s), mass of active electrode materials (g), and the operating potential window , respectively.Similarly, from CD curves, 2: 2 activated carbon materials  of the carbon granules with porous surface structures a,c,e,g.  samples d,f,h, poarticles c,e,g, a −1 and a weak peak at 1631 cm−1 due to aromatic C=C stretching vibrations commonly observed in the activated carbons [−1 indicates C–H stretching indicating the presence of an aromatic ring. The broad vibration at 3446 cm−1 is due to OH (str) suggesting the presence of an alcoholic functional group or from moisture [Oxygenated surface functional groups are generally observed in biomass-derived activated carbons ,53. SurfStructural properties of the Lotus seed-derived nanoporous carbon materials were further characterized by XRD and Raman scattering spectroscopy. D band) and ~1588 cm−1 (G band) can be seen in all the samples, which are typical features of the activated amorphous carbon materials. The D band reveals the disordered structure of carbon induced by impurities present during high-temperature carbonization, whereas the G band reflects an ordered graphitic layer structure. To estimate the graphitization degree, the intensity ratio of G and D bands (IG/ID) was calculated from the obtained raw data. The obtained IG/ID ratio in the range of 1.01 to 1.04 demonstrates amorphous graphitic carbon structure with fewer defects [Raman scattering spectra b further defects .Porosity properties  of the prepared carbon materials were evaluated by nitrogen adsorption measurements. 2-activation pyrolytic decomposition occurs and porosity enhancement is obtained due to depolymerization and dehydration of biochar. The well-developed porosities of the ZnCl2-activated samples can be judged from the mixed Type-I/Type-IV adsorption isotherms. A Type-I adsorption isotherm corresponds to a microporous structure, while a Type-IV isotherm corresponds to a mesoporous form indicating a hierarchically porous structure [0 < 0.03) reveals a microporous structure, which contributes greatly to enhance the specific surface area and charge storage capacity. Clear hysteresis loops caused by capillary condensation at higher relative pressures indicate a mesoporous structure, which promotes electrolyte ion diffusion in supercapacitors electrode materials. The nonporous structure of the LTS_800  results in a Type-III adsorption isotherm. Thermolysis of lotus seed powder at a high temperature (800 °C) without activating agent randomly disrupts the carbon skeleton leading to a macroporous or nonporous structure. In contrast, in case of ZnCltructure . Nitroge2-activated Lotus seed carbons are summarized in 2 g−1) is due to its lack of porosity. In contrast, activated samples show very high surface areas with the optimal sample exhibiting a surface area of 1316.7 m2 g−1 and large pore volume of 0.794 cm3 g−1. The average mesopore diameter of the optimum sample was calculated to be 3.67 nm.The porosity properties of ZnClCs, rate capability, and cycle stability of the electrode materials were also calculated. −1 where the semi-rectangular shape of the CV curves signifies EDLC type behavior of the electrodes [−1, which explains the rapid charge propagation with excellent reversibility at high scan rate. Using Equation (1) Cs were calculated from the CV curves  attained the highest capacity of 317.5 F g−1 at a low scan rate of 5 mV s−1 and retained 70.4% capacitance at a high scan rate of 500 mV s−1. Cs of samples LTSC_Z600 and LTSC_Z1000 were calculated to be 212.0 and 282.4 F g−1, respectively.Inspired by the high specific surface area and well-developed porosity of the lotus seed-derived nanoporous carbon materials, the electrochemical energy storage capacities of the prepared materials were studied by CV and CD measurements. ectrodes . The totectrodes . Simultaectrodes . CV profV curves f. As exp−1. All the samples exhibit semi-triangular shape CD profiles inferring EDLC behavior of the materials [−1) are shown for LTSC_Z600, LTSC_Z800, and LTSC_Z1000 (−1 (LTS_800), 190.6 F g−1 (LTSC_Z600), 272.9 (LTSC_Z800), and 256.4 (LTSC_Z1000) at 1 A g−1. Cs vs. current density curves for these carbon materials. Because of the high surface area (mainly micropore surface area) the LTSC_Z800 showed the highest electrochemical energy storage performance and also a good rate capability of 52.3% at 50 A−1.aterials ,60,61 wiaterials . LTS_800SC_Z1000 b–d. SpecCycle life of supercapacitor devices is a key parameter in real world applications. We have examined the cyclic stability performance of selected electrodes up to 10,000 charging–discharging cycles f. Both eCs of the ZnCl2-activated Lotus seed nanoporous carbon electrodes with similar other biomass-derived nanoporous carbon materials  can be judged from the sharp rise of the curves at the lower frequency region of Nyquist plots. Hierarchically porous structure comprising of both the mesopore and micropore architectures enables fast electrolyte ion transfer within the electrode materials and thus contributes to enhancing the supercapacitance performance. Whereas, poor ion transport in the LTS_800 is caused due to the absence of well-developed porosity. From Nyquist plots the equivalent series resistance (ESR) was ca. 4.16 Ω (LTS_800), 4.17 Ω (LTSC_Z600), 4.37 Ω (LTSC_Z800), and 4.32 Ω (LTSC_Z1000). The ESR values of all the samples are comparable demonstrating that the difference in the specific surface area and pore volume of the materials are mainly responsible for the better supercapacitance performance of the LTSC_Z800.It should be noted that the high surface area, well-developed porosity with large pore volume, narrow and well-defined pore size distributions, and pore network connectivity in porous carbon materials comprising of macro, meso, and micropores architectures are the key parameters in supercapacitors applications of carbon materials . Using c2-activation of lotus seed powder are reported in an aqueous electrolyte in a three-electrode system. ZnCl2 activated lotus seed carbon materials obtained by the high-temperature carbonizations (600–1000 °C) exhibit a hierarchically porous structure comprising a micro-and mesopore architecture. Cyclic voltammetry and chronopotentiometry results indicate the excellent energy storage performances of the materials. The optimal sample exhibited a high specific capacitance of 317.5 F g−1 at a scan rate of 5 mV s−1 and 272.9 F g−1 at a current density of 1 A g−1 accompanied by high capacitance retention of 70.49% at a very high scan rate of 500 mV s−1. The electrode also had excellent rate capability, retaining 52.1% capacity at a high current density of 50 A g−1 accompanied by exceptionally high cyclic stability (99.2%) after 10,000 charging–discharging cycles. The obtained excellent electrochemical performances of the materials can be attributed to the high surface area (1316.7 m2 g−1) with associated large pore volumes (0.887 cm3 g−1). Judging from the surface textural properties (surface areas and porosity) and electrochemical performance, it can be concluded that lotus seed, an agricultural waste product (agro-waste), is a good precursor material for the scalable production of high-performance supercapacitor electrode materials.In summary, the electrochemical energy storage performances of hierarchically porous carbon materials produced by ZnCl


Morus alba) fruits on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were investigated. The ethanol (EtOH) extract of white mulberry fruits and its derived fractions contained adequate total phenolic and flavonoid contents, with good in vitro antioxidant radical scavenging activity. The extract and fractions also markedly inhibited ROS generation and antioxidant activity. After treatment with the EtOH extract and its fractions, LPS stimulation-induced elevated nitric oxide (NO) production was restored, which was primarily mediated by downregulation of inducible NO synthase expression. A total of 20 chemical constituents including flavonoids, steroids, and phenolics were identified in the fractions using ultra-high-performance liquid chromatography (UHPLC)-quadrupole time-of-flight (QTOF) high-resolution mass spectrometry (HRMS). These findings provide experimental evidence of the protective effects of white mulberry fruit extract against oxidative stress and inflammatory responses, suggesting their nutraceutical and pharmaceutical potential as natural antioxidant and anti-inflammatory agents.In this study, the protective effects of white mulberry ( Inflammation, a major mechanism mediating innate and adaptive immunity, is a complex physiological response that protects the organism against foreign harmful stimuli such as pathogens, particles, and viruses. Inflammation is primarily classified as acute and chronic based on the underlying mechanisms and processes. Cellular and molecular processes of chronic inflammation are varied and depend on the organ involved and, thus, are closely associated with the development and deterioration of many chronic diseases including cardiovascular, neurological, pulmonary, metabolic, endocrine, and autoimmune disorders as well as cancer . FollowiMacrophages have been found to play a key role in the host defense system, where they are involved in many immunologic functions including inflammatory modulation and removal of apoptotic cells. Macrophages are activated by exogenous mediators such as lipopolysaccharide (LPS), an endotoxin expressed in the cell walls of gram-negative bacteria. This phenomenon is considered the first step in the inflammatory process, and many studies of protective effects mediated by anti-inflammatory, immune-modulating, and antioxidant activities have been performed using LPS-treated macrophage cells ,6.Morus alba L.), a perennial plant belonging to the Moraceae family, is used in traditional medicine and widely known as an important food source for the silkworm [The white mulberry tree -quadrupole time-of-flight (QTOF)-high-resolution mass spectrometry (HRMS).n-butanol (BuOH) fraction (204.3 ± 4.7 mg GAE/g), the highest flavonoid content was in the ethyl acetate (EA) fraction (74.9 ± 4.7 mg CAE/g), while the hexane (HX) fraction exhibited the lowest values of both contents (The results showed that the ethanol (EtOH) extract of the white mulberry fruits and its derived fractions contained adequate total phenolic /g) and flavonoid /g) contents. Furthermore, the highest total phenolic content was found in the contents .p < 0.001, p < 0.01).50), 133.6 ± 4.7 μg/mL). The EtOH crude extract and HX fraction did not exhibit DPPH radical scavenging activity at the concentration range tested (IC50 > 1000 μg/mL).Comparative results of the in vitro antioxidant assays are presented in 50, 216.6 ± 28.8 and 218.1 ± 22.6 μg/mL, respectively) than the extract and other fractions. The ferric reducing antioxidant power (FRAP) values of the studied samples ranged from 0.505 to 3.727 mmol Fe2+/g. Similar to the results of the DPPH and ABTS assay, those of the FRAP assay showed that the EA fraction (3.727 ± 0.055 mmol Fe2+/g) exhibited the highest value of all tested samples.The extract and all fractions of white mulberry fruits except for the HX fraction, exhibited 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities at the concentration range tested. The EA and MC fractions showed higher activity (ICp < 0.001).Intracellular ROS levels of LPS-stimulated RAW 264.7 macrophages were 5-fold higher than those of the control, whereas co-treatment with white mulberry fruit extract and fractions significantly inhibited LPS-induced ROS generation. Even at the lowest concentration, the EtOH extract and EA and MC fractions inhibited ROS production  more than the positive control  . After tp < 0.01), and MC fraction had the highest GPx activity (2.700 ± 0.044 nmol/min), reaching the significant levels compared to BuOH (p < 0.05) and HX (p <0.01) fraction.The antioxidant activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) were also significantly enhanced by treatment with white mulberry fruits extract and fractions at the lowest studied concentrations, as described in p < 0.05).As shown in p < 0.01 for EA vs. HX, p < 0.001 for others).M. alba fruit extracts were qualitatively analyzed using UHPLC-QTOF-HRMS. In total, 20 chemical constituents were detected in the fractions of the crude extract  generated by reacting the parent compound with a strong oxidizing agent such as potassium persulfate. It can be used over a wide pH range in both aqueous and organic solvent systems [3+-TPTZ) complex is reduced to the ferrous form (Fe2+) at low pH, with an intense blue color [50 values for DPPH and ABTS assays over the upper concentration limit tested in this study (IC50 > 1000 μg/mL)  are simple and most commonly used in the early screening of antioxidant properties of vegetable and fruit extracts and products . DPPH is systems . The FRAue color . In this0 μg/mL) . These fIntracellular levels of free radicals can damage various cell constituents and activate specific signaling pathways, which both affect numerous cellular process linked to aging and the development of related diseases . Overpro2−) by catalyzing their breakdown to oxygen and H2O2 [2O2 undergoes further degradation [Antioxidant enzymes such as SOD, GPx, and CAT stabilize or inactivate the detrimental effects of free radicals on cellular components. They also inhibit the oxidizing chain reaction to minimize free radical-induced cellular and molecular damages. By reducing cellular exposure to free radicals, antioxidant enzymes contribute to decreasing the risk for various associated health problems including the physiological manifestations of aging, cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer . SOD estand H2O2 . This ROradation . We founM. alba fruit.NO is a free radical widely distributed in the body that regulates various biological functions including vasodilation, smooth muscle contraction, neuronal signaling, platelet aggregation inhibition, immunological regulation, and inflammatory responses ,32. LPS-M. alba fruits [a fruits . Odisolaa fruits ,48. It wThe limitation of this study is that the signaling pathway associated with anti-inflammatory effects of white mulberry fruits were not fully identified. Nuclear factor (NF)-κB induces pro-inflammatory cytokines, chemokines, and adhesion molecules that are essential for both innate and adaptive immune responses . NF-κB h(±)-Catechin, copper (II) chloride, 2′,7′-dichlorofluorescin diacetate (DCF-DA), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), Folin-Ciocalteu’s phenol reagent, gallic acid, glutathione (GSH), GSH reductase, hydrogen peroxide, 3--2,5-diphenyltetrazolium bromide (MTT), LPS, nicotinamide adenine dinucleotide phosphate (NADPH), nitroblue tetrazolium (NBT), phosphate-buffered saline (PBS), quercetin, xanthine, and xanthine oxidase were purchased from Sigma-Aldrich .The Griess reagent system kit was purchased from Promega . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Thermo Fisher Scientific . Primary antibodies for iNOS and β-actin were purchased from Cell Signaling Technology . Goat anti-rabbit IgG secondary antibody and other chemical reagents were purchased from Merck Millipore .M. alba) were acquired from the Kyungdong Market (Woori Herb), Seoul, Korea, in January 2014. The material was verified by one of the authors (K.H.K.), and a voucher specimen (MA 1414) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea. The M. alba fruits (0.9 kg) were dried in a hot air oven at 60 °C and the dried materials were extracted with 70% aqueous EtOH and filtered using Whatman filter paper No. 42 three times at room temperature. The filtrate was evaporated in vacuo to obtain the crude EtOH extract (140 g). The extract was dissolved in deionized water and then solvent-partitioned with 800 mL each of HX, MC, EA, and BuOH three times, yielding 2.8 8.5, 3.3, and 13.9 g of the fractions, respectively. Concentrated extracts and fractions were subsequently lyophilized and stored at −20 °C prior to analysis.Mulberry fruits (2CO3), the mixtures were incubated for 60 min at room temperature in the dark. The absorbance was measured at a wavelength of 725 nm using a microplate spectrophotometer . Gallic acid was used as the standard, and the total phenolic content were determined from calibration curves for gallic acid . Results were expressed as milligrams of gallic acid equivalents per gram of sample (mg GAE/g).The total phenolic content was determined using the Folin-Ciocalteu method with some modifications . Each sa3) method with some modifications [2) and allowed to react for 5 min. Then, 300 μL 10% AlCl3 solution and 1 mL 1 M sodium hydroxide were added, and the absorbance was measured at a wavelength of 510 nm using a microplate spectrophotometer at 510 nm. (±)-Catechin was used as the standard, and the flavonoid content were determined from calibration curves for (±)-catechin . Results were expressed as milligrams of catechin equivalents per gram of sample (mg CAE/g).The flavonoid content was determined using the aluminum chloride (AlClications . Each sa2) at 37 °C.Murine RAW 264.7 macrophage cells were cultured in DMEM containing 4 mM L-glutamine, 4.5 g/L glucose, and sodium pyruvate supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere with 5% carbon dioxide (CO4 cell/well) and treated with 0.5% DMSO (control), 10 μM quercetin (positive control), or different concentrations of the crude extract of white mulberry fruit (50–150 μg/mL) and the EA (5–20 μg/mL), BuOH (10–50 μg/mL), HX, (5–20 μg/mL), and MC (5–30 μg/mL) fractions in the absence or presence of LPS (2 μg/mL in PBS) for 24 h. Following treatment, 20 μL MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated at 37 °C in an atmosphere of 5% CO2 for 2 h. Then, the supernatant was removed, and 100 μL DMSO was added to each well to dissolve any formazan crystals that developed. The absorbance of each well was measured at 570 nm using a microplate spectrophotometer.Cell viability was assessed using the MTT assay with some modifications ,57,58,590 − Ac)/A0) × 100 .The DPPH radical scavenging activity was determined using the method of Blois  with som0 − Ac)/A0) × 100 .The ABTS radical scavenging activity was determined using the method of Arts et al.  using AB3∙6H2O) at a 10:1:1 ratio. Then, 175 μL FRAP reagent was added to 25 μL of each test sample at a concentration of 1000 μg/mL and incubated at 37 °C for 4 min. Ferrous sulfate (FeSO4∙7H2O) was used as the standard, and the absorbance was measured at a wavelength of 593 nm using a microplate spectrophotometer. The results are expressed as millimoles (mmol) of FeSO4∙7H2O equivalents per gram of sample (mmol Fe2+/g).The FRAP assay was performed using the method of Benzie and Strain  with som4 cell/well), treated with the positive control or different concentrations of crude extract and fractions of white mulberry fruits for 2 h, and then incubated with LPS for 20 h. Then, the supernatant was discarded, and 20 μM DCF-DA in serum-free DMEM was added, followed by further incubation at 37 °C for 30 min, protected from light. The supernatant was removed and washed with PBS twice, and then 100 μL PBS was added to each well. The fluorescence intensity was detected at excitation and emission wavelengths of 485 and 535 nm, respectively, using a multi-mode microplate reader .Intracellular ROS levels were measured using the DCF-DA assay as described by Sittisart and Chitsomboon . Cell we5 cell/well), treated with the positive control or white mulberry fruits extract and fractions for 2 h, and then they were incubated with LPS for 20 h. The culture medium was removed, and the cells were washed twice and then scraped with 1 mL PBS. Cell suspension were centrifuged at 14,000 rpm at 4 °C for 5 min.The antioxidant enzyme capacity was assayed in accordance with the methods previously described by Lee et al. . BrieflyFor the determination of SOD activity, cell homogenates were prepared by homogenizing cell suspensions with 0.05 M sodium carbonate buffer (pH 10.2). The final reaction mixture consisted of 50 μL cell homogenate and 0.05 M sodium carbonate buffer containing 3 mM xanthine, 0.75 mM NBT, 3 mM EDTA, and 1.5 mg/mL bovine serum bovine albumin (BSA). The reaction was initiated by adding 50 μL xanthine oxidase (0.1 mg/mL) and incubating at room temperature for 30 min, and then it was stopped by adding 6 mM copper (II) chloride and centrifuging at 1500 rpm for 10 min. The absorbance of blue formazan in the supernatant was determined at a wavelength of 560 nm.2O2) was added to each sample at a final concentration of 1 mM, followed by the measurement of activity at a wavelength of 340 nm.The assay mixture for determining GPx activity contained 0.1 M phosphate buffer (pH 7.0), 1 mM EDTA, 1.5 mM NADPH, 1 mM sodium azide, 1 unit of GSH reductase, 10 mM GSH, and 100 μL cell lysates. This mixture was incubated at 37 °C for 10 min, and then hydrogen peroxide (H2O2 and 100 μL cell lysates in 50 mM phosphate buffer (pH 7.0), and samples were incubated at 37 °C for 2 min. The absorbance of the samples was measured for 5 min at a wavelength of 240 nm. The variation in absorbance is proportional to the breakdown of H2O2.The assay mixture for CAT activity contained 12 μL 3% H4 cell/well) and treated with the positive control or different concentrations of white mulberry fruits crude extract and fractions for 2 h, followed by LPS for 24 h. Then, 50 μL samples of the supernatant from the treated culture medium was mixed with 50 µL 1% sulfanilamide in 5% phosphoric acid and incubated at room temperature for 10 min, protected from light. Then, 50 µL 0.1% N-1-napthylethylenediamine dihydrochloride in water was added, followed by incubation at room temperature for 10 min, protected from light. The absorbance was measured at a wavelength of 540 nm using a microplate spectrophotometer. The NO level of each experimental sample was calculated using a NaNO2 (0–100 µM) standard curve.The concentration of nitrite, a stable oxidized product of NO, in the cell culture medium was determined using a Griess reagent system kit (Promega). Cells were seeded in 96-well plates (2 × 105 cells/well) and pre-incubated for 2 h with the positive control and different concentrations of the crude extract and each fraction. After LPS stimulation at 37 °C for 20 h in a humidified atmosphere of 5% CO2, cells were washed with PBS, homogenized with lysis buffer containing a protease inhibitor cocktail, and centrifuged at 14,000 rpm . Each supernatant sample containing an equal total protein amount (20 μg) was loaded for separation using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane.For Western blot analysis, cells were seeded in 12-well plates  at 4 °C overnight, followed by a horseradish peroxidase-conjugated secondary antibody (1:2000 dilution in 5% skim milk) at room temperature for 1 h. The membrane was washed, and immunoreactive bands were detected using the ChemiDoc imaging system with an enhanced chemiluminescence solution kit (Bio-Rad).n-BuOH soluble fractions were dissolved in the respective extraction solvents and filtered through 0.45-μm filters before injection. The identified compounds were separated using an Agilent EclipsePlus C18 column  maintained at 20 °C. The mobile phrase consisted of 0.1% formic acid in water (solvent A) and 100% acetonitrile (solvent B). The gradient elution was performed on the following schedule: 90% A → 100% B (0–10 min), 100% B (11–16 min), and 90% A (16–20 min) for equilibration before the next injection. The samples were monitored at 210 and 254 nm during the chromatographic run. The mass spectral analysis was performed using the MassHunter software , and the mass spectrometer conditions were as follows: ionization mode, electrospray ionization (ESI (+)); MS scan range, m/z 100–1700; nebulizer gas (N2) pressure, 35 psi; dry gas (N2) flow rate, 8 L/min; drying gas temperature, 225 °C; sheath gas temperature, 320 °C; capillary voltage, 3.5 kV; fragmentor voltage, 100 V; collision energy, 3.0 eV [expmass and calcmass are the experimental mass and calculated mass from previously published molecular formulas, respectively [Extracts of white mulberry fruits were chemically profiled using an Agilent 1290 Infinity II HPLC instrument  coupled to a G6545B Q-TOF mass spectrometer (Agilent Technologies). HX, MC, EA, and , 3.0 eV . The exa, 100% B –16 min, , 3.0 eV ,14,15,1650 value of the DPPH and ABTS radical scavenging assays was defined as the concentration of sample scavenging 50% of the free radicals. The significance of the difference in mean values between each mulberry fruit sample and control or LPS treatment sample were analyzed using the Student’s t-test, and the difference in mean values among mulberry fruit fractions at the lowest concentration  were analyzed using the one-way analysis of variance (ANOVA) with post-hoc Tukey multiple comparison test. p-values < 0.05 were considered statistically significant.All experiments were replicated three or five times, and assay results are expressed as the means ± SD. The ICIn conclusion, we demonstrated that white mulberry fruits contain adequate amounts of total phenolics and flavonoids and exhibited beneficial antioxidant and anti-inflammatory properties without cytotoxicity. The present study also reported the chemical constituents of white mulberry fruit extracts, including some associated with observed biological activities of antioxidant and anti-inflammatory agents. Our findings indicate that white mulberry fruits have protective effects against oxidative stress and inflammatory responses, suggesting their nutraceutical and pharmaceutical potential to be developed as natural antioxidant and anti-inflammatory agents.


Twenty males joined either the CrossFit® group  or the resistance training (RT) group  thrice a week, for 8 weeks. Salivary levels of cortisol, interleukin 1-beta (IL-1β), and uric acid were assessed via enzyme-linked immunosorbent assays before (PRE) and 30-min after (POST) SESSION 1 and SESSION 24. Variables’ percentual changes were computed as (POST-PRE)/PRE*100 in each session (Δ%). CrossFit® acutely increased cortisol levels in both sessions, with a significant decrease in Δ%cortisol from SESSION 1 to 24. In the RT group, cortisol values decreased in both sessions, only acutely. A significant decrease in IL-1β levels was registered acutely in both groups, in both sessions, whereas Δ%IL-1β was not different between the two groups. While uric acid levels increased in both groups acutely, a chronic downregulation of Δ%uric acid, from SESSION 1 to 24, was appreciated for the RT group only. Overall, CrossFit® appeared to induce more intense effects than the RT program as to the investigated catabolic responses. Given the wide variety of conditioning program trainings employed, the present study compared the catabolic effects induced by CrossFit Successful sports performance entails an optimal combination of aerobic and anaerobic metabolism, muscular power and strength, speed and agility, according to specific tasks . Amongst® is one of the new modality of HIFT that have emerged in the last few years. A typical CrossFit® training is organized into daily sessions called “workouts of the day” (WOD), including metabolic exercises , gymnastic movements , and weightlifting  and performed at an intensity close to 95% of the maximum heart rate (HRmax) [CrossFit (HRmax) ,5. WODs  (HRmax)  or as qu (HRmax) .® has been demonstrated to improve body composition and physical fitness [® training has been shown to induce an immunosuppressive effect [®) may produce premature fatigue and additional oxidative stress level in athletes [® training a greater acute cortisol response [The undoubted beneficial effects of exercise have been underlined in a number of studies. Nevertheless, exercise is a stress situation that challenges homeostasis , and the fitness ,7, also  fitness , inflamm fitness ,9,10, an fitness . Furthere effect  and acute effect . Particue effect . On the e effect  analyzedathletes . The immathletes . Moreoveathletes . With reathletes . Lastly,athletes . Studiesathletes  or reducathletes . Elevatiathletes , while iresponse  and a loresponse . Assessi2max in non-athlete subjects [Yet, it is well established that training can alter host defense, leading to changes in disease susceptibility and severity . Both aesubjects . In anotsubjects . ® program [Finally, the antioxidant defense is activated by exercise, preferably via low molecular weight non-enzymatic antioxidants, i.e., uric acid . It is k program , induce ® program and of a classical gym RT program in moderately trained subjects. Based on previous studies, the primary hypothesis was that these conditioning training programs would be accompanied by exacerbated stress responses. As a secondary endpoint, magnifying the extent of these different training-induced changes would be likewise of interest.The present study aimed at shedding light on the stress responses elicited by two strength/power programs in order to bring useful indications in the monitoring and scheduling of these two different training regimens, including their respective periodization plans. Therefore, the purpose of this study was to examine the oxidative stress, the hormonal and the immunological acute and chronic responses following 8 weeks of a CrossFit® and Resistance Training, were enrolled for the study. They were divided into two groups, with a convenience sampling: the CrossFit® group (n = 10) and the RT group (n = 10). Before and after the intervention period, participants underwent to the maximum repetition test (1RM test), in order to assess their individual maximum load lifted (see 1RM test paragraph for a description of the tests). The two groups were homogeneous regarding age  = −1.09, p = 0.29), and practice experience (1 year). Twenty male young adults, classified as moderately trained according to Sheppard et al. , with 1 All subjects were fully informed about the study aims and procedures and gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Università degli Studi di Milano . All participants were instructed not to change their diet and physical activity practices throughout the intervention period. Subjects were also recommended not to consume alcohol, caffeine, theine, hot drinks, or smoke within 24 h of the session. None of the participants were using medications or performance-enhancing drugs during the study.Exclusion criteria were muscle or joint injuries, orthopedic problems, severe visual impairment, or any other contraindication within three months before the commencement of the study.® group and 10 in the RT group, to allow for drop-out during the intervention period [Estimation of sample size was performed using the GPower software  applying ANOVA repeated measures (F Test). This calculation generated a desired sample size of at least 18 subjects. However, we recruited 20 participants, 10 in the CrossFitn period . Before and after the intervention period, participants underwent (a) the maximum repetition test (1RM test), in order to assess their individual maximum load lifted, and (b) body composition assessment. Intervention period lasted 8 weeks. Throughout the 8-week training program, participants performed 24 sessions, 3 times/week, and all the training sessions were held at the same time of day (5 pm). Before (PRE) and 30 min after (POST) the end of both the first training session (SESSION 1) and the last training session (SESSION 24), salivary levels of cortisol, IL-1 beta, and uric acid were ascertained . Prior to 1RM tests, subjects performed 5 min of warm-up including low-intensity functional movements for the joints. The maximum repetition test was recorded as the maximum load lifted (1RM) for one repetition. Participants established one-repetition maximums (1RMs) on military press, squat clean, and power clean. Before attempting a 1RM, subjects performed a progressive series of five submaximal sets of 1 to 2 repetitions with moderate to heavy loads (50–90%) of the estimated 1RM. If a weight was properly lifted during a 1RM trial, the subsequent 1RM weight attempt was increased by 2.5 to 7 kg, and the participant attempted another 1RM trial with 3 min of rest between efforts. Each 1RM was determined within 3 to 5 trials ,34. ® session consisted of 20 min of warm-up (running at low intensity and joint mobility exercises), 30 min of WOD (metabolic + gymnastic + weightlifting), and 10 min of cool down. The WOD was characterized by metabolic , gymnastics , and weightlifting  exercises [Each CrossFitxercises . ® participant. Templates were individually prescribed and recorded for each CrossFit® protocol was as follows: The CrossFitWeeks 1–4: (metabolic): 4 min of running, 2 min of rest, 4 min of jumping rope; (gymnastic): 4 min of pull-ups and squats, 2 min of rest, 4 min of pull-ups and squats; (weightlifting): 4 min of front squats and kettlebell swings at 50–60% 1RM, 2 min of rest, 4 min of front squats and kettlebell swings at 50–60% 1RM. Weeks 5–8: (metabolic): 4 min of running, 2 min of rest, 4 min of jumping rope; (gymnastic): 4 min of pull-ups and squats, 2 min of rest, 4 min of pull-ups and squats; (weightlifting): 4 min of front squats and kettlebell swings at 65–75% 1RM, 2 min of rest, 4 min of front squats and kettlebell swings at 65–75% 1RM. ® sessions were held in a CrossFit® training center and supervised by a CrossFit® Level 1 certified trainer. Each training session lasted approximately 60 min, including warm-up and cool-down.The WODs were performed as quickly as possible with no rest period or “as many repetitions as possible” (AMRAP) fashion. All CrossFitResistance training sessions lasted approximately 60 min, with one minute of rest between all sets and exercises. Sessions always included 3 sets of 15 crunches and other exercises varied from Monday  to Wednesday  to Friday . Participants performed three sets of each exercise, separated by 1-min rest, using the following progressive repetition scheme: Weeks 1–2: 15 reps at 50% 1RM; Weeks 3–4: 12 reps at 55% 1RM; Week 5: 10 reps at 60% 1RM; Week 6: 10 reps at 65% 1RM; Week 7: 8 reps at 70% 1RM; Week 8: 8 reps at 75% 1RM .Saliva samples were collected before (PRE) and 30 min after (POST) the end of the first training session (SESSION 1) and the last training session (SESSION 24). Saliva samples were obtained by means of cotton swabs and saliva collecting tubes . The athletes were instructed to place the cotton swab into their mouths, under the tongue, for 2 min. The absence of blood contamination was checked with a salivary blood contamination kit . The saliva collecting tubes were centrifuged at 3000 rev/min for 15 min at 4 °C. Samplings were stored at −80 °C until they were assayed. To exclude inter-assay variance, all samples were thawed once and analyzed in triplicate in the same assay run. As said, subjects refrained from consuming any food, drinking hot fluids, or brushing their teeth for two hours prior to their arrival. Upon their arrival, before starting the training session, participants were asked to remain seated for 15 min before providing their resting sample (PRE), and subsequently, they completed the training session. At the end of the training session, they were asked to remain in a relaxed position  for 30 min until the collection time point (POST). The participants could drink water freely during resting time but were asked to abstain from drinking within 10 min of any post-exercise collection time point. Concentrations of cortisol, IL-1β, and uric acid were assessed via enzyme-linked immunosorbent assays , following manufacturers’ instructions. For cortisol, the assay range was 0.012-3.000 µg/dL and the sensitivity <0.007 µg/dL, for IL-1β the range was 3.13–200 pg/mL and the sensitivity <0.37 pg/mL, and for uric acid the range was 0.07–5 mg/dL with a sensitivity of 0.07 mg/dL. All samples were tested in duplicate. Intra-assay deviation was <10% for all kits.2), lean mass (kg), and fat mass (%) were considered.Body composition was evaluated by using bioelectrical impedance analysis . Weight (kg), body mass index , as between-subjects factor were applied to statistically evaluate changes in parameters defining the body composition , lean mass, and fat mass) and functional measures .Repeated measure ANOVAs with EPOCH  as within-subjects factor, and GROUP . Statistical analysis was performed in three phases. Variables’ changes were analyzed PRE and POST the first and last training sessions for both groups. Cortisol, IL-1β, and uric acid were analyzed by means of RM-ANOVA with SESSION  and TIME  as within-subjects factors.® and RT), as between-subjects factor. Significant interaction was evaluated through Bonferroni post hoc tests.Furthermore, to compare the amount of changes between PRE and POST values in the two SESSIONS between groups, we computed for each parameter the percentual changes as follows (POST-PRE)/PRE*100 in each session and group. The obtained Δ%cortisol, Δ%IL-1β, and Δ%uric acid of the two groups were compared by means of ANOVAs with SESSION , as within-subjects factor, and GROUP .The third analysis searched for possible relationships among cortisol, IL-1β, and uric acid log values in the two sessions and groups. To this aim, Pearson’s correlations were applied as follows. POST 1 cortisol values  were correlated with (1) POST_24 cortisol values—to investigate for possible relationships between values after the first and the last training sessions; (2) POST_1 IL-1β and POST 1 uric acid values—to investigate for possible relationships among salivary measures after training within the same session. The same analysis was repeated considering POST 24 cortisol, POST_1 IL-1β, POST_24 IL-1β, POST_1 uric acid, and POST_24 uric acid, as dependent variables. Moreover, Pearson’s correlations were also applied to evaluate potential relationships among Δ%Cortisol, Δ%IL-1β, and Δ%uric Acid, in SESSION_1 and SESSION_24, in each group. The level of significance was lowered according to Bonferroni’s correction for multiple comparisons (p = 0.05/3 = 0.0167).At last, the functional measures were correlated with the salivary measures. Two kinds of analyses were run. In the first analysis, Pearson’s correlation were applied to investigate possible relationship in the two groups among cortisol, IL-1β and uric acid log values at POST 1 and POST 24  with baseline measures of 1RM power clean, squat clean, and military press. Furthermore, the percentage changes of functional measures from baseline to ending epochs were computed as follows (endline–baseline)/baseline*100 in each group. The obtained parameters were Δ%power clean, Δ%squat clean, and Δ%military press. In order to evaluate the possible relationship between changes in functional parameters  and changes in Δ%cortisol, Δ%IL-1β, and Δ%uric Acid, in SESSION 1 and SESSION 24, Pearson’s correlations were applied separately for each group. The level of significance was lowered according to Bonferroni’s correction for multiple comparisons (p < 0.05 (except for multiple comparisons as previously explained). The effect size measures were presented through partial eta squared , with cut-off points of 0.10, 0.25, 0.40 representing small, medium, and high effect, respectively [The statistical analyses were performed with SPSS . Data are presented as means ± standard error. The probability level taken to indicate significance was ectively .Participants’ body weight was comparable in the two groups and did not change after training, as well as the BMI and the lean mass, as shown by the statistical analyses. Fat mass significantly decreased in both groups after the training period, as shown by the significant main effect of EPOCH.The results of ANOVAs on functional measures  showed a significant main effect of EPOCH, indicating a higher performance in both groups after the training period. No differences between group and no significant EPOCH*GROUP interactions were found in any of these parameters.® and RT groups are shown in Salivary cortisol values of CrossFit® group showed significant main effects of the factors SESSION  = 96.88, p < 0.001, η2 = 0.92) and TIME  = 164.74, p < 0.001, η2 = 0.95), and a significant interaction between them  = 45.66, p < 0.001, η2 = 0.84). Bonferroni post hoc revealed a significant increase in cortisol values from PRE to POST in both SESSIONS . No significant differences appeared between PRE_1 and PRE_24, whilst values in POST_24 were significantly lower than those in POST_1 (p = 0.0002).The result of ANOVA on data of CrossFitp < 0.01, η2 = 0.74), indicating a significant decrease from PRE to POST. No differences between sessions and no significant interactions between TIME and SESSION appeared.ANOVA on the RT group showed a significant main effect of TIME  = 25.31, p < 0.05, η2 = 0.28) and a significant interaction between TIME and GROUP  = 5.17, p < 0.01, η2 = 0.22). Bonferroni post hoc revealed significant differences between groups in both sessions . Furthermore, results showed a significant decrease in the percentage changes in CrossFit® group from SESSION 1 to SESSION 24 (p = 0.003).Comparing Δ%cortisol values between the two groups B, ANOVA Salivary IL-1β are represented in ® group showed a significant main effects of the factors SESSION  = 18.17, p < 0.01, η2 = 0.67) and TIME  = 3001.60, p < 0.0001, η2 = 0.99), and a significant interaction between them  = 7.11, p < 0.05, η2 = 0.44). Post hoc tests revealed a significant decrease from PRE to POST in both SESSIONS . No significant differences appeared between PRE_1 and PRE_24, whilst values in POST_24 were significantly lower than those in POST_1 (p < 0.0001).ANOVA on IL-1β values of CrossFitp < 0.05, η2 = 0.43) and TIME  = 1206.89, p < 0.0001, η2 = 0.99) and a significant interaction between them  = 6.74, p < 0.05, η2 = 0.43). Post hoc tests revealed a significant decrease from PRE to POST in both SESSIONS . A significant decrease was observed between PRE_1 and PRE_24 (p = 0.001).ANOVA on IL-1β values of RT group showed significant main effects of the factors SESSION  = 6.87, The results of the statistical analysis on Δ%IL-1β values between the two groups failed to find significant differences B.Salivary uric acid values are shown in ® group’s data  showed a significant main effect of the factor SESSION  = 9.81, p < 0.05, η2 = 0.52), indicating a significant increase from SESSION 1 to SESSION 24. Further, a significant increase in uric acid values appeared from PRE to POST  = 20.93, p < 0.01, η2 = 0.70).The results of ANOVA on CrossFitp < 0.01, η2 = 0.60) and a significant SESSION*TIME interaction  = 5.08, p < 0.05, η2 = 0.36). Bonferroni post hoc comparison revealed a significant increase from PRE to POST in both sessions .ANOVA on uric acid values of RT group showed a significant main effect of TIME  = 13.58, p < 0.05, η2 = 0.20) and a significant interaction between TIME and GROUP  = 5.91, p < 0.05, η2 = 0.25). Bonferroni post hoc revealed a significant decrease in the percentage changes in RT group from SESSION 1 (35.52 ± 8.50%) to SESSION 24 (22.93 ± 6.75%) (p = 0.005). No differences appeared in Δ%uric acid values in CrossFit® group .ANOVA comparing Δ%uric acid values between the two groups B showed p = 0.001; squat clean: r = −0.76, t = −3.36, p = 0.009; military press: r = −0.82, t = −4.05, p = 0.03). IL-1β values at POST_1 positively correlated with baseline military press values . Uric acid_log values at POST_24 negatively correlated with baseline squat values .In the RT group only, cortisol levels at POST_24 inversely correlated with all baseline functional measures  and changes in Δ%cortisol, Δ%IL-1β, and Δ%uric acid in all groups.p = 0.005). Furthermore, POST_1 uric acid values positively correlated with POST_24 uric acid values .In the RT group, a significant negative relationship appeared between IL-1β and uric acid values in SESSION 1; namely, the higher the POST 1 IL-1β values, the lower the POST_1 uric acid values  and Δ%Uric Acid in SESSION 1 and SESSION 24 .In the CrossFit® and RT trainings induced homogeneous responses in IL-1β levels, heterogeneous responses in cortisol and uric acid levels were documented. Both programs supported a fat mass lowering effect and an anti-inflammatory pattern. In the RT group, an inverse correlation between baseline functional measures and ending cortisol/uric acid levels suggested a favorable downregulation. Instead, the stressful reactions might have been more profound in the CrossFit® group, possibly because of its higher metabolic demand. An overall summary of the major results is offered in While CrossFit®, whereas only a few studies analyzed these extreme conditioning programs from a metabolic or hormonal standpoint [The majority of the precedent studies focused on orthopedic (injuries) or psychological aspects of CrossFitandpoint .® program and the resistance program affected cortisol, IL-1β, and uric acid levels differently.-SESSION 1: from PRE to POST-SESSION 24: from PRE to POSTAcute responses were considered as follows:Index of chronic adaptations is expressed as Δ% ((POST-PRE)/PRE*100).The optimal balance between training stimulus and recovery time has always attracted both coaches and athletes, since beneficial adaptations can be translated into enhanced athletic performance. In particular, the purpose of this study was to investigate whether the CrossFit® group.As for cortisol levels, the present results suggest a lesser stress for the RT group with respect to the CrossFit®, utilizing short-intense aerobic intervals, increased significantly from PRE to POST in both SESSIONS, therefore augmenting the availability of substrates for metabolism [® [The primary function of cortisol secretion in response to exercise is to increase the availability of substrates for metabolism . It was tabolism . In regatabolism . Presenttabolism . Acute rtabolism  to no chtabolism . The obtolism [®  or RT [4olism [® . On the olism [® . Typicalolism [® . Therefo® training. However, no significant differences were found in IL-1β levels between the two groups in both sessions. The lack of homogenous responses to these loadings might be related to an extrinsic exercise-depending conditioning mechanism, giving the complexity of multiple abilities required by CrossFit®. In this regard, previous studies reported inconclusive findings on the clinical and biochemical traits elicited by strength/power training or HIFT, possibly because of the various methods of assessment, different athletes , or simply, different regimes of training [Skeletal muscle can be considered the largest organ in the human body. Its endocrine-like function is remarkable, as skeletal muscles are able to release several cytokines . In facttraining . Finding® group. Conversely, in the RT group, being a uniform regimen in terms of training load and volume, it is possible that the total antioxidative capacity  was not® and RT. Both investigated programs exerted a fat-mass loss and an overall anti-inflammatory effect, which was more pronounced in the RT group, as demonstrated by a chronic downregulation of stress reactions. In fact, CrossFit® appeared to have a more profound catabolic impact than RT, especially as concerns the acute responses. It is likely that the higher intensity with a lesser recovery time between exercises  could be the pivotal trigger. Whether this might be physiologically deleterious remains to be ascertained. Certainly, the studied approach might help preventing overtraining circumstances by monitoring biomarkers of stress response. Ultimately, in order to define a complete picture of the dynamics of training and rule out speculations, multiple supplementary assessments should be performed as regards blood lactate and fatigue responses.In this study an ecological, three-biomarker approach related to functional measures was implemented to monitor stress responses in athletes performing strength/power trainings—CrossFitA greater understanding of adaptive conditions will depend on future, well-controlled studies, focusing on a multitude of potential stressors, even outside of training, including environmental, social aspects, or cognitive demands.® or other RT regimens. These training programs may be associated to peculiar adaptations that should be favorably harnessed, on a tailored basis, in order to maximize individual metabolic gains.As such, these preliminary indications might inspire more research alongside additional markers of catabolic status of the participants. Further studies on HIFT or differently stimulated strength/power performances should be warranted, considering the increasing numbers of fitness enthusiasts practicing CrossFit