Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases.An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Strain A9 can secrete a thermostable β-glucosidase that has high glucose tolerance• A coculture of strain A9 and C. thermocellum showed high cellulose degradation• Strain A9 achieves biological saccharification without addition of β-glucosidase• The online version contains supplementary material available at 10.1007/s00253-022-11818-0. This bottleneck occurs because cellulosic biomass is resistant to enzymatic degradation and the hydrolytic enzymes required are expensive. β-Glucosidase is a hydrolytic enzyme that can produce glucose from cellobiose and cellooligosaccharides —an anaerobic thermophilic bacterium—is the most potent cellulose-degrading bacterium known to produce “cellulosomes”  bearing heterologous β-glucosidase , which was assembled in the cellulosome through a dockerin module of CelS. The production of reducing sugars from microcrystalline cellulose, such as Avicel, was enhanced using this system, which resolved the issue of feedback inhibition by hydrolyzing the accumulated cellobiose into glucose. However, to use recombinant C. thermocellum, it is still necessary to optimize suitable β-glucosidases for the cellulosomes. Additionally, biosafety issues must be considered when using genetically modified microbes in industrial applications . BM7CO medium was degassed by boiling, then bubbled with high-purity CO2, and finally anaerobically distributed to Hungate tubes  and/or serum bottles. Clostridium thermocellum ATCC 27405T was obtained from the American Type Culture Collection (ATCC) and was grown on BM7CO medium containing 10 g/l microcrystalline cellulose Sigmacell type 20 . T. celere-type strain JW/YL-NZ35 (DSM8682T) was obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Strain JW/YL-NZ35 was grown on modified DSMZ616 (YTG) medium composed of (per liter): 0.36 g of Na2HPO4·2H2O, 0.08 g of KCl, 5.0 g of yeast extract, 10.0 g of Bacto™ tryptone , 5.3 g of Na2CO3, 0.2 g of cysteine-HCl, 0.2 g of Na2S, and 0.0005 g of resazurin (pH 9.0) containing 5.0 g of D-glucose or cellobiose. The YTG medium was also degassed, followed by bubbling with high-purity N2. Escherichia coli BL21 (DE3) and the plasmid pET19b  served as the cloning and expression host and vector, respectively.Samples of soil and wastewater sediment were collected from the layer 20 cm under the draining wastewater systems at different sampling sites at the Pilot Plant Development and Training Institute, King Mongkut’s University of Technology Thonburi, Thailand. The soil and wastewater sediment samples were stored in resealable zipper storage bags. The basal medium (BM7CO)  as the substrate (Ahamed and Vermette Approximately 1 g of each of the collected samples of soil and wastewater sediment was inoculated directly into 10 ml of BM7CO medium containing 0.5% (w/v) cellobiose. After inoculation, the cultures were incubated at 60°C for 2–3 days. The cultures were inoculated into the same fresh medium, and the enrichment culture was repeated five times. To select cultures with high β-glucosidase activity, the supernatants of the enrichment cultures were collected by centrifugation at 10,806 × Genomic DNA from strain A9 was extracted using a NucleoSpin® microbial DNA kit . The 16S rRNA gene was amplified using the bacterial domain-specific primers 27F and 1525R     by PCR. The PCR product was digested with BamHI and Bpu1102, and then ligated into pET19b. The recombinant E. coli clone was inoculated into Luria-Bertani medium supplemented with 100 μg/ml ampicillin and incubated at 37°C. Isopropyl-β-D-thiogalactopyranoside was added to induce protein expression. The recombinant enzyme was purified using the Profinia affinity chromatography system . The homogeneity of the purified protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Molecular mass standards were from Bio-Rad Laboratories.Two oligonucleotide primers for β-glucosidase (GenBank accession number: GFR35525.1) designed from the draft genome sequence data of strain A9, 5′-GGGGATCCATGCAAAAATACACTTTCCC-3′ with a pNPG. The reaction mixture contained 10 μl of enzyme and 100 μl of 1 mM pNPG in 100 mM sodium acetate buffer, pH 6.0. After incubation at 60°C for 10 min, the reaction was stopped by adding 200 μl of 0.4 M Na2CO3. The released p-nitrophenol was measured using a spectrophotometer at 405 nm  was also assayed at 60°C for 10 min. The reaction mixture contained 50 μl of enzyme and 50 μl of 50 mg/ml cellooligosaccharide in 100 mM sodium acetate buffer, pH 6.0. The concentration of glucose released from the cellooligosaccharides was determined using a glucose oxidase–peroxidase assay kit . One U of enzyme activity was defined as the amount of enzyme that liberated 1 μmol of glucose per min per ml. To determine the mode of action toward cellooligosaccharides, reaction mixtures were collected after 10, 20, 30, 40, 50 and 60 s. Each substrate (10 mg/ml) was incubated with enzyme (0.5 U) at 60°C. The hydrolysis products were analyzed using thin-layer chromatography. Protein concentrations were determined using a Coomassie protein assay kit  with bovine serum albumin as the standard.β-Glucosidase activity was determined by measuring the hydrolysis of pNPG as substrate in buffers with pH values 4.0–9.0. The buffers used were 100 mM sodium acetate for pH 4.0–6.0, 100 mM phosphate for pH 6.0–8.0, and 100 mM Tris-HCl for pH 8.0–9.0. The optimal temperature for enzyme activity was determined at pH 6.0 (in sodium acetate buffer) from 40 to 80°C. Thermostability was determined by preincubating the enzyme (1 μg protein) without substrate in sodium acetate buffer (pH 6.0) for 1 h at 50–80°C; the residual β-glucosidase activity for each condition was then assayed at 60°C to determine thermostability. Also, enzyme in sodium acetate buffer (pH 6.0) was incubated at 60°C for 7 days; a sample of the incubated enzyme was collected every 24 h to measure the residual β-glucosidase activity. Glucose inhibition of β-glucosidase activity was measured by adding glucose at different concentrations (0–1.0 M) to the standard reaction mixture with pNPG as the substrate. The glucose concentration required to inhibit 50% of the initial β-glucosidase activity  was determined. The relative activity (%) was defined as the value relative to the activity in the absence of glucose.The optimum pH for enzyme activity was determined at 60°C with Native PAGE was performed at pH 8.3 using 10% acrylamide as the resolving gel with a 5% stacking gel (pH 6.8). After electrophoresis, the gel was soaked in 100 mM sodium acetate buffer (pH 6.0) for 10 min at room temperature. β-Glucosidase activity in the gel was detected by staining with esculin mixed with ferric ammonium citrate or using 4-methylumbelliferyl-β-D-glucoside . For staining with esculin mixed with ferric ammonium citrate, the gel was incubated in 100 mM sodium acetate buffer containing 1 g/l esculin and 2.5 g/l ferric ammonium citrate for 10–30 min at 50°C . In the YTG medium, cellobiose or other carbohydrates were used as carbon sources instead of glucose. Cell growth was measured by cell turbidity determined using a spectrophotometer at 600 nm (Thermo Fisher Scientific) and 10-mm pathlength cuvettes. The total cell growth was determined by measuring the total protein concentration following a previous report  in accordance with the manufacturer’s instructions.C. thermocellum ATCC27405T stock culture was inoculated by syringe into 5 mL of BM7CO medium containing 10 g/l cellulose and was incubated at 60°C in anaerobic conditions. The subculture of C. thermocellum ATCC27405T was inoculated again by syringe into BM7CO medium containing 50 g/l Sigmacell type 20 at 60°C for 2 days. To avoid the possibility of competitive growth inhibition of C. thermocellum in the coculture, the subculture of strain A9 or T. celere-type strain JW/YL-NZ35 was inoculated after cultivation of C. thermocellum for 2 days. The cocultures of C. thermocellum and strain A9 or type strain JW/YL-NZ35 were incubated at 60°C for 10 days in anaerobic conditions. The β-glucosidase activity and the concentration of accumulated glucose in the culture supernatants were monitored by an enzymatic assay using pNPG and high-performance liquid chromatography , respectively.For subculturing, Thermobrachium celere strain A9 and T. celere type strain JW/YL-NZ35 are BMAP00000000 and CAVN000000000, respectively.The GenBank accession numbers for the genome sequences of pNPG and 4-MUG are used to screen for microbes producing β-glucosidase; however, these chemicals are unstable over long periods of incubation at >50°C, and, therefore, they are not suitable for use as substrates for the screening of thermophilic microbes producing β-glucosidase. Thus, esculin, which is stable during incubation at high temperatures, was used as the substrate. The principle of the assay is the formation of a dark brown color, which is a result of the chelation of esculetin, produced from esculin by β-glucosidase, with ferric ions in the medium . The cultures grew well on the cellobiose medium. To isolate bacteria that produced high β-glucosidase activity in the culture supernatants, enrichment culturing was performed at least five times. Single colonies were separated by the anaerobic Hungate roll tube technique using cellobiose as the sole carbon source in an agar medium supplemented with esculin and ferric ammonium citrate. Generally, T. celere type strain JW/YL-NZ35 (DSM8682T)  was compared with other sequences from the GenBank database. Homology and phylogenetic analyses of sequences showed that the 16S rRNA gene of strain A9 had 100% identity with that of 35 DSM868T , were 98.4% and 85.8%, respectively; these are above the defined thresholds for species delineation of 95–96% for ANI and 70% for GGDC  in the National Institute of Technology and Evaluation (NITE), Chiba, Japan.The whole-genome sequence of strain A9 was deposited in GenBank with accession number BMAP00000000. ANI values, dDDH values, and AAI were calculated between strain A9 and T. celere is also known as Caloramator celer  and BacDive (https://bacdive.dsmz.de/strain/2516), which are popular worldwide databases for standardized bacterial information. We have used T. celere as the species name of strain A9 in this report to avoid confusion between genera.T. celere type strain JW/YL-NZ35 was observed in cellobiose-containing medium based on YTG medium  to stationary (16 h of cultivation) phases were analyzed for β-glucosidase activity by zymography with the same stain Fig. . A largeose Fig. , and no ium Fig. . Zymograium Fig.  in the cium Fig. . These dT. celere type strain JW/YL-NZ35 have predicted β-glucosidases belonging to glycoside hydrolase (GH) families 1 (GenBank accession number: GFR35525.1) and 30 (GenBank accession number: GFR35524.1), and GH families 1 (GenBank accession number: CDF59168.1), 3 (GenBank accession number: CDF57462.1), and 30 (GenBank accession number: CDF59169.1), respectively. However, β-glucosidase activity is primarily found in GH families 1 and 3, and is less common in GH family 30  was 1,347 bp, encoding 448 amino acids with a calculated protein mass of 52 kDa. In SDS-PAGE analysis, the molecular mass of the recombinant β-glucosidase (rTcBG1)  Fig.  agreed wG1) Fig.  and 5-2.G1) Fig.  of rTcBGG1) Fig. ; after iG1) Fig. . rTcBG1 G1) Fig. , which s Fig.  agC. thermocellum cultures are supplemented with thermostable β-glucosidases   Fig. , which wC. thermocellum or coculture of C. thermocellum with T. celere type strain JW/YL-NZ35 did not efficiently hydrolyze cellulose and had low β-glucosidase activity , which is a relatively novel xylanase that can be exo-acting and liberates xylose and xylobiose from the non-reducing end of xylan , the β-glucosidases (GFR35525.1 and CDF59168.1) of strain A9 and T. celere type strain JW/YL-NZ35 have a signal peptide sequence at the N-terminus; however, at least one other β-glucosidase listed in Table To develop an efficient saccharification system using natural biomass for practical applications, it is necessary to investigate anaerobic thermophilic microbes that can secrete β-glucosidases into the culture supernatant to work in cooperation with cellulosomes and cellulases. To date, few reports have investigated the screening and isolation of aerobic microbes that can produce β-glucosidase (Ahamed and Vermette http://www.cazy.org), β-glucosidases are mainly found in GH families 1 and 3   when cocultured with lT Table . If straESM1 (PDF 129 KB)Below is the link to the electronic supplementary material.


In this research, we describe the properties of three-component composite foam scaffolds based on poly(ε-caprolactone) (PCL) as a matrix and hydroxyapatite whiskers (HAP) and L-Lysine as fillers (PCL/HAP/Lys with wt% ratio 50/48/2). The scaffolds were prepared using a thermally induced phase separation technique supported by salt leaching (TIPS-SL). All materials were precisely characterized: porosity, density, water uptake, wettability, DSC, and TGA measurements and compression tests were carried out. The microstructure of the obtained scaffolds was analyzed via SEM. It was found that the PCL/HAP/Lys scaffold has a 45% higher Young’s modulus and better wettability compared to the PCL/HAP system. At the same time, the porosity of the system was ~90%. The osteoblast hFOB 1.19 cell response was also investigated in osteogenic conditions (39 °C) and the cytokine release profile of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α was determined. Modification of PCL scaffolds with HAP and L-Lysine significantly improved the proliferation of pre-osteoblasts cultured on such materials. Tissue engineering and regenerative medicine are important fields in biomedical engineering. Progress in this area creates the need for novel scaffold materials and reproducible fabrication techniques . BiopolyIn recent years, aliphatic polyesters like polylactides, polyglycolide, polycaprolactone, and their copolymers have been widely investigated for biomedical applications . The useAmongst biopolymers, poly(ε-caprolactone) (PCL) and its composites have been considered attractive candidates to produce scaffolds for tissue engineering applications. Their mechanical properties, thermoplastic processability, biodegradability, and biocompatibility towards bone within the human body have led to them being extensively used to design scaffolds for damaged bone regeneration . PCL-bas2 . Th. Th24]. The internal pore structures of the samples were observed using FE-SEM microscopy. From this figure, it is possible to observe that all scaffolds (neat PCL and the composites) present a porous structure. SEM measurements  show scaAs listed in The mechanical properties of the foams were investigated by a compression test. The compressive stress and Young’s modulus of the PCL-based foams are listed in The static contact angle (CA) and water uptake were also measured. The results are shown in -5wt.%) was defined. TGA analysis revealed that the thermal stability of a composite that contains L-Lysine is similar to that of neat PCL foams, while the T-5wt.% value for a composite without L-Lysine is 3.4 °C lower than that of neat PCL.Based on the TGA analysis, the hydroxyapatite and L-Lysine content in composite foams was determined from the difference in mass loss at 900 °C. TGA and DTG curves are presented in c) occurring with a cooling rate of 10 °C/min had the highest value for neat PCL (55.8 J/g), while in composites the enthalpy was lower (−27.3 J/g for both samples). The temperature of melt crystallization was slightly higher for both composites than for the neat PCL foam. These results indicate an influence of the HAP content on the nucleating activity.The composite samples were first heated to 100 °C and maintained at this temperature for 5 min under nitrogen to erase the previous thermal history. The cooling and second heating DSC curves are shown in c) of neat PCL foam and composite foams was determined using Equation (3) and the results are shown in On second heating, DSC curves’ endothermic melting peaks are visible. The results show that the neat PCL melting temperature is 58.3 °C; this value is about 0.9 °C lower than for composite foams. The crystallinity degree  higher as compared to the proliferation rate induced by the reference (PCL) composite. The MNoC identified within PCL/HAP/Lys reached 2.2 × 105 ± 2.1 × 102 (on day 7), 3.3 × 105 ± 6.5 × 102 (on day 14), and 3.6 × 105 ± 3.0 × 102 (on day 21) and were significantly higher as compared to MNoC for the control (PCL) composite: 0.3 × 105 ± 3.2 × 102, p = 0.004 (on day 7), 0.3 × 105 ± 6.3 × 102, p = 0.004 (on day 14) and 0.4 × 105 ± 6.7 × 102, p = 0.004 (on day 21). In addition, osteoblasts cultured with PCL/HAP exhibited a higher proliferation rate, compared to the PCL scaffold. We have also shown that PCL/HAP/Lys-dependent proliferation was accompanied by successful colonization of the composite by osteoblasts , through day 18  and at the end of the experiment   A.p = 0.0003 (on day 4) and 724.9 ± 21.5 pg/m mL and 30.9 ± 1.1 pg/m mL, p = 0.004 (on day 21), respectively , and the production of this mediator occurred as early as on day 4 of culture and remained at a similar level throughout the whole period of the assay B. Moreovectively C.p = 0.02, 8.6 ± 0.3 U/mL, p = 0.02, 10.4 ± 0.1 U/mL, p = 0.02, respectively)  activity serves as an early marker for the osteoblast differentiation involved in bone calcification and hard tissue formation. Osteoblastic differentiation was assessed by measuring ALP activity after 7, 14, and 21 days of hFOB 1.19 osteoblasts cocultured with the PCL-based foam scaffolds. We have observed the upregulation of ALP production in cell cultures induced by PLA/HAP/Lys and PLA/HAP scaffolds, whereas no such effect was observed for cells cultured with control (PCL) scaffolds. The PCL/HAP scaffold was modified with L-Lysine-stimulated hFOB 1.19 cells to produce significantly higher ALP concentrations than those detected in cell cultures exposed to PCL composites on days 7, 14, and 21  . AlthougThe poly(ε-caprolactone) used in this research work was CAPA 6800 , purchased from Perstorp . Apatite whiskers (HAP) obtained as multiphasic calcium phosphate were prepared using a methodology described earlier ,24,36. Lw/w) was dissolved in 1,4-dioxane under stirring conditions for 24 h to obtain a homogeneous solution. After this time, the required amounts of HAP or HAP/Lys were suspended in the polymer solution and sonicated for 30 min (at 60 °C) in order to obtain a homogeneous suspension. Then, 1.5 g of NaCl and 0.75 mL of the polymer solution/suspension were added to 24-well plates. All scaffolds were produced using NaCl salt having a size of 500–600 µm. The 24-well plates were placed in a freezer for 24 h. Frozen samples were transferred to a freeze-drying vessel and submitted to a solvent removal process (at −80 °C and vacuum ∼10–20 Pa for 24 h). Dried samples were then taken out and put in demineralized water to dissolve the salt and remove it from the scaffolds. The salt leaching process was supported by slow mixing on a magnetic stirrer. Water (in a 5 L beaker) was changed three times over 24 h. After leaching, the scaffolds were dried at 60 °C for 24 h.PCL (as the reference scaffold sample) and PCL-based composite scaffolds (PCL/HAP and PCL/HAP/lys) were produced using a thermally induced phase separation technique supported by a salt leaching process (TIPS-SL). Designation of the samples are given in The microstructure of the obtained porous composites was tested by scanning electron microscopy with field emission . Before the study, the samples were covered with a conductive material (25 nm gold film) using a sputter coater . Imaging of composites was visualized in high vacuum conditions using an ETD detector (Everhart–Thornley detector combined with Nova NanoSEM 200) at 15 kV accelerating voltage and at magnifications of 100× and 250×.The compressive strength of the foams was assessed using a Universal testing machine  fitted with a 1 kN head. The compression speed was set at 2 mm/min. The barrel-shaped samples were cut into specimens 4 mm high and with a diameter of ~15 mm. The samples were tested to obtain a maximum strain of 90%. At least 10 samples of each kind were measured. The compressive stress and Young’s modulus were determined at a strain in the range between 5% and 90%.pΦ is the porosity, scρ is the scaffold density obtained from the buoyancy method using a Hildebrand Electronic Densimeter H-300S , and bρ is the bulk density of the composite scaffold . At least 10 samples of each kind were measured.The porosity of the scaffolds was calculated using the following Equation (1) :(1)Φp=, w is the weight fraction of PCL in composite foams, and ΔmH100% is the melting enthalpy of 100% crystalline PCL (139 J/g) [Samples were analyzed on the Mettler Toledo DSC1 system, coupled with a Huber TC 100 intracooler , under the following conditions: mass: ~5.5 mg; nitrogen flow: 60 mL/min; heating or cooling rate: 10 °C/min, temperature range: 0 °C to 100 °C. After the heating cycle, the samples were thermally equilibrated at 100 °C for 5 min and cooled down to 0 °C. A second heating scan was also performed. The crystallinity degree (Xc) of PCL in the materials was determined from the melting peak area of second heating according to the following Equation (3) :(3)Xc=Δ139 J/g) . Experimv/v), where A is 5 mM formic acid and 5 mM ammonium formate in an acetonitrile/water (9:1) solution. Twelve dilution points have been designated. For the obtained results, the relationship between the area under the peak in the HPLC chromatogram and the concentration was determined using Shimadzu LCsolution software . The results of three separate measurements were averaged.In order to establish a calibration curve for lysine, three identical solutions of lysine were prepared, for which chromatographic analyses (HPLC) were performed. A standard lysine solution (1 mg/mL) was prepared by dissolving the amino acid (L-Lysine hydrochloride) in a mixture of mobile phase solution A/water, 1:1 (v/v); B: 5 mM ammonium formate and 5 mM formic acid in a acetonitrile/water mixture of 7.5:2.5 (v/v), pH 7.5. The volumetric flow rate was 0.25 mL/min. Peaks were detected at a wavelength of 223 nm. The injection volume was 50 µL for each sample. The mass of lysine released was calculated on the basis of the value of the peak area of the released lysine and the previously determined calibration curve for lysine.The composite foam  was weighed and placed in Eppendorf tubes, to which 1 mL of water was added. The composite foam was incubated at 37 °C with gentle shaking (400 rpm) for 24 h. The lysine release from the composite was tested at the following time points: 0, 1, 2, 3, 4, 5, 10, 15, 30, 60, 90, 120, 180, 240, 300, 360, 420, 480, 720, 1080, and 1440 min. The analysis of the released lysine was performed using a NEXERA X2 chromatography system  consisting of two pumps (LC 30AD), a ELSD LTII detector, an SPD-M20A detector, an automatic dispenser (SIL 30AC), a column thermostat (CTO-20AC), and a degasser membrane (DGU-20A5R). HPLC analysis was performed with a linear 0–100% B gradient for 30 min on a bioZen™  2.65 µm Glycan (2.1 mm × 250 mm) column, where A: 5 mM ammonium formate and 5 mM formic acid in an acetonitrile/water mixture of 9:1  to ensure that they form confluent and homogeneous monolayers. The medium was changed every three days under aseptic conditions. Passaging of confluent cell monolayers was carried out at 80–90% confluence using a 0.5% trypsin–0.05 mM EDTA solution (Gibco).CRL-11372™, the human fetal osteoblastic cell line (hFOB 1.19) used in this study, was obtained from the American Type Culture Collection . The cells were cultured in a 1:1 mixture of phenol-free Dulbecco’s modified Eagle’s medium and Ham’s 12-F medium . Media were supplemented with 2.5 mL of L-Glutamine (100X), 10% heat-inactivated fetal bovine serum (FBS) , and 0.3 mg/mL of geneticin (G418) (Sigma-Aldrich). Cells were cultured at 34 °C, in a humidified atmosphere containing 5% CO2 for 2 h. Following the initial soaking, 20 µL of fresh cell suspension (5 × 105 cells) was added to the center of each scaffold. To induce proliferation and encourage differentiation, composites with hFOBs were cultured in an osteogenic medium , 0.3 mg/mL geneticin, 50 μg/mL ascorbate-2-phosphate , 1 μM dexamethasone (Sigma-Aldrich), and 10 mM β-glycerophosphate (Sigma-Aldrich) at 39 °C. The medium was changed every third day for 21 days. All supernatants collected on days 4, 7, 14, 18, and 21 were stored at −80 °C until further evaluation of the soluble markers  and DAPI (Thermo Fisher Scientific) to visualize cytoskeletal F-actin filaments and the nucleus, respectively. The scaffolds were observed with a confocal macroscope  with objective 5×/0.50 LWD and imaged with the following fluorescence conditions: Texas Red®-X (Ex596nm/Em615nm) and DAPI (Ex360nm/Em460nm). Leica Application Suite X software  was used for cell imaging. Confocal analysis was performed in the Laboratory of Microscopic Imaging and Specialized Biological Techniques at the Faculty of Biology and Environmental Protection at the University of Lodz, Poland.To visualize osteoblasts within PCL-based foam composites, on days 7, 14, and 21 the scaffolds were washed with PBS and fixed with a 3.7% formaldehyde (Sigma-Aldrich) solution for 20 min at room temperature. Following permeabilization  the cells were counterstained with phalloidin conjugated with Texas Red® i3x Multi-Mode Microplate Reader .The proliferation of osteoblasts was evaluated after 7, 14, and 21 days of incubation with PCL-based foam scaffolds using CyQUANT Cell Proliferation Assay . At the selected time points, cells within the foam composites were washed with PBS and frozen at −80 °C. At the time of the assay, samples were thawed at room temperature and lysed in a buffer containing CyQUANT-GR dye, prepared according to the manufacturer’s instructions. Fluorescence signals  were detected using a SpectraMax2SO4. Absorbance values were determined at 450 nm using the Multiskan EX reader (Thermo Scientific).The concentrations of IL-1β, IL-6, and TNF-α were determined using specific sandwich ELISA kits  in accordance with the manufacturer’s instructions. The minimum detectable levels were 3.91 pg/mL for IL-1β, 9.38 pg/mL for IL-6, and 15.6 pg/mL for TNF-α. In brief, wells on a 96-well half area plate  were coated overnight at room temperature with capture antibodies in PBS, then washed in PBS/0.05% Tween 20 and blocked with PBS containing 1% BSA for 1 h. After washing the wells with PBS/0.05% Tween 20, samples were added and incubated for 2 h. Standard curves were also established using serial dilutions of known concentrations of recombinant human cytokines. Afterwards, wells were washed with PBS/0.05% Tween 20 and incubated for 2 h with biotinylated monoclonal antibody diluted 1:60 in PBS/1% BSA. Following washing, a streptavidin–horseradish peroxidase (SaV-HRP) solution (diluted 1:40) was added and incubated for 20 min. After washing the wells with PBS/0.05% Tween 20, a mixture of tetramethylbenzidine (TMB) and hydrogen peroxide was added. The colorimetric reaction was stopped with 2N HThe ALP activity was measured in previously obtained cell lysates by a p-NPP (para-nitrophenylphosphate) hydrolysis assay. Then 100 µL p-NPP (4 µg/µL) were added to 100-µL samples in a 96-well plate and incubated at 37 °C for 30 min. Then, a 2 M NaOH solution was added to stop the reaction. The optical density at 405 nm was determined using the Multiskan EX reader (Thermo Scientific). The standard curve (range: 0 to 10 IU/mL) of ALP  was used to calculate the enzyme activity in the samples.p < 0.05. All analyses were performed using GraphPad Prism 7 software .Intergroup outcomes were compared for statistical significance using a one-way ANOVA . In all cases, significance was accepted at In this study, we successfully prepared a ternary foam scaffold using PCL, hydroXyapatite, and L-Lysine (50/48/2 wt% ratio). The scaffold had a ~45% higher Young’s modulus than the PCL/HAP (50/50 wt% ratio) reference sample and porosity at a level of over 90%. The incorporation of L-Lysine as a filler causes a significant improvement of the compressive stress compared to the PCL and PCL/HAP systems. At the same time, the contact angle decreased from 88° for PCL to 78° for PCL/HAP and 73° for PCL/HAP/Lys. It was found that L-Lysine was released from the PCL/HAP/Lys scaffold within 20 h. We have shown that PCL and PCL-modified composites enable cell growth and proliferation. Moreover, the bone biocompatibility of PCL scaffolds can be improved by adding hydroxyapatite and/or L-Lysine. It is worth emphasizing that, in our research, the modification of PCL/HAP scaffold with L-Lysine resulted in the significant intensification of pre-osteoblasts’ proliferation and supported sequential differentiation, as judged by IL-6, IL-1β, and alkaline phosphatase production by the cells. The modification of scaffolds with HAP and L-Lysine resulted in efficient scaffold colonization, which in turn should translate to high-density bone tissue formation and the enhanced regenerative potential of such composites. It is known that the coating of material surfaces with L-Lysine provides cationic site, which lead to increased adhesion and improved cytobiocompatibility. Overall, the biological assessment of the PCL/HAP/Lys scaffolds points to them as promising candidates for future biomedical engineering applications.


Candida albicans. Importantly, the resulting aneuploidy also conferred cross-tolerance to caspofungin, a first-line echinocandin antifungal, as well as to hydroxyurea, a common chemotherapeutic agent. Exposure to a range of tunicamycin concentrations induced similar ER stress responses. Extra copies of one Chr2 gene, MKK2, affected both tunicamycin and caspofungin tolerance, while at least 3 genes on chromosome 2  affected only tunicamycin and not caspofungin responses. Other Chr2 genes (RNR1 and RNR21) affected hydroxyurea tolerance but neither tunicamycin nor caspofungin tolerance. Deletion of components of the protein kinase C (PKC) or calcineurin pathways affected tolerance to both tunicamycin and caspofungin, supporting the idea that the ER stress response and echinocandin tolerance are regulated by overlapping stress response pathways. Thus, antifungal drug tolerance can arise rapidly via ER stress-induced aneuploidy.How cells exposed to one stress are later able to better survive other types of stress is not well understood. In eukaryotic organisms, physiological and pathological stresses can disturb endoplasmic reticulum (ER) function, resulting in “ER stress.” Here, we found that exposure to tunicamycin, an inducer of ER stress, resulted in the acquisition of a specific aneuploidy, chromosome 2 trisomy (Chr2x3), in  Candida albicans is an opportunistic human fungal pathogen. It is a commensal colonizer of the gastrointestinal tract of approximately 45% to 60% of healthy adults  and flucytosine  stress experimentally. It blocks N-linked glycosylation by inhibiting the transfer of UDP-N-acetylglucosamine to dolichol phosphate in the ER of eukaryotic cells, thus resulting in misfolded proteins and ER stress , a product of R stress . Recentle to TUN .C. albicans. Exposure of lab strain SC5314 to subinhibitory and inhibitory concentrations of TUN for short and long time periods yielded TUN-tolerant colonies/adaptors. A total of 62 tolerant adaptors were sequenced, and most of them had duplication of chromosome 2. Importantly, chromosome 2 trisomy enabled cross tolerance to TUN (ER stress inducer), CSP , and HU (chemotherapeutic drug). We identified the following chromosome 2 genes associated with tolerance to the three drugs: ALG7, RTA2, and RTA3 were associated with TUN tolerance; RNR1 and RNR21 were associated with HU tolerance; and MKK2 was associated with TUN and CSP tolerance. The calcineurin pathway and the PKC pathway also were associated with both TUN and CSP tolerance. We suggest that aneuploidy arises rapidly under many stress conditions and has the potential to confer multidrug tolerance, largely because of its concerted effect on the copy number of many genes.In this study, we asked if aneuploidy is a general mechanism for TUN tolerance in C. albicans lab strain SC5314 is susceptible to TUN, with growth slightly inhibited by 1 μg/ml (P > 0.05) and completely inhibited by 4 μg/ml of TUN (P < 0.001) in broth microdilution assays (see says see .10.1128/mBio.02272-21.1FIG S1595nm) was measured every 15 min using a plate reader . Data are represented as the mean ± SD of three biological replicates. In B, the plates were photographed after 48 h. In C, approximately 1 million cells of SC5314 were spread onto YPD agar plates supplemented with TUN. The plates were incubated at 37°C for 5 days. In D, 18 and 5 adaptors, picked up from the plates containing 4 μg/ml and 8 μg/ml of TUN, respectively, were compared with the parent for tolerance to TUN using spot assays. In E, SC5314 was grown in YPD broth containing 1 μg/ml of TUN YPD broth without drug (control) for 24 h. A total of 120 colonies were randomly selected and tested using spot assays performed on YPD agar plates containing TUN as indicated. The parent strain is indicated by a blue circle. The tolerant colony identified is indicated by a red circle. In F, SC5314 was grown in YPD broth containing 1 μg/ml of TUN YPD broth without drug (control) for 24 h as described in FIG S1, PDF file, 2.3 MB.Characterization of tunicamycin adaptors. SC5314 was grown in YPD broth (A) or streaked onto YPD agar (B), tunicamycin was added at the concentrations indicated, and growth was monitored at 37°C. In A, optical density -agar plates containing 4 μg/ml or 8 μg/ml TUN. After 5 days, ∼2,300 colonies (0.23%) appeared on plates with 4 μg/ml TUN, and only 6 colonies appeared on 8 μg/ml TUN  was grown in 1.5 ml YPD broth containing 1 μg/ml of TUN for 24 h (24-h exposure) and then diluted 1:1,000 to another tube at the same TUN concentration for an additional 24 h (48-h exposure). Each culture was washed, diluted with distilled water, and plated onto YPD agar plates (no drug selection) and 120 (24 h) and 95 (48 h) colonies were tested for TUN susceptibility using spot assays. Only one of the 24-h colonies  or YPD broth supplemented with subinhibitory concentrations of TUN . After the 10th passage, cells were plated onto YPD plates (no drug selection), and 18 colonies from each culture were randomly chosen for further analysis. Among them, 0 (0%), 2 (11.1%), 15 (83.3%), and 18 (100%) colonies, derived from passage in YPD broth containing 0 μg/ml, 0.5 μg/ml, 1 μg/ml, and 2 μg/ml, respectively, yielded isolates that had acquired TUN tolerance to TUN .Taken together, these results show that under strong selection using inhibitory concentrations of TUN (4 μg/ml and 8 μg/m), all the adaptors (100%) obtained tolerance to TUN. Subinhibitory concentrations of TUN (1 μg/ml) yielded only a small proportion of colonies (1.1% at 24 h and 3.2% at 48 h) with TUN tolerance when the exposure time was short (1 to 2 days), while a higher proportion of selected colonies were adaptors that resulted when exposure time was 5 to 10 times longer .n = 62) adaptors obtained from the 3 approaches were sequenced and analyzed by Ymap to detect ploidy and allele specificity n = 62) acificity C, D, ind10.1128/mBio.02272-21.2FIG S2FIG S2, PDF file, 2.0 MB.Ymap analysis of tunicamycin-tolerant adaptors. Karyotypes of all sequenced TUN-tolerant adaptors obtained from YPD plates supplemented with 4 μg/ml or 8 μg/ml tunicamycin (A); from 24 h or 48 h evolution in YPD broth supplemented with 1 μg/ml tunicamycin (B); or from 10-day passage in YPD broth supplemented with 0.5 μg/ml, 1 μg/ml, and 2 μg/ml tunicamycin (C) were visualized using Ymap . DownloaCopyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the C. albicans aneuploid collection, in which each strain is trisomic for a single chromosome  . Among tromosome . Importa10.1128/mBio.02272-21.3FIG S3FIG S3, PDF file, 2.8 MB.Test of aneuploid strains for tolerance to tunicamycin. TUN tolerance was tested using a collection of aneuploid strains carrying one trisomic chromosome per strain . Only thCopyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To ask why both subinhibitory and inhibitory concentrations of TUN yielded the same aneuploid adaptors, we investigated the transcriptomes of cells treated with subinhibitory (1 μg/ml) and inhibitory (4 μg/ml) concentrations of TUN using transcriptome sequencing (RNA-seq). Cells treated with 1 μg/ml had 2,152 differentially expressed genes compared with the no-drug treatment control; 1,121 genes were more abundant and 1,031 genes were less abundant than in the no-drug control cells. In the 4-μg/ml TUN-treated cells, there were 2,634 differentially expressed genes, including 1,325 genes that were more abundant and 1,309 genes that were less abundant than in the control cells see , left.10.1128/mBio.02272-21.9TABLE S3Table S3, PDF file, 0.7 MB.Differentially expressed genes in TUN treatment Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Genes involved in several biological processes reminiscent of ER stress were commonly enriched in the two treatments. Induced genes were enriched in processes such as protein glycosylation, cellular lipid biosynthesis, response to endoplasmic reticulum stress, and the endoplasmic-reticulum-associated protein degradation (ERAD) pathway. Repressed genes were enriched in ribosome biogenesis, ribosome assembly, RNA processing, and gene expression  (ress(es) . Consistress(es)  and wereress(es) . FurtherALG7  at 15-min time intervals for 48 h. Download FIG S4, PDF file, 0.9 MB.Broth microdilution assay. Growth of tunicamycin adaptor colonies and the parent strain in YPD broth supplemented with caspofungin  or tunicamycin  using drug concentrations as indicated. For each strain, approximately 2.5 × 10Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We also asked if aneuploidy generally caused cross-tolerance to azole antifungals, the most widely used antifungal drugs. Neither the Chr2x3 nor the Chr2x4 adaptors grew well; in fact, both aneuploids were more susceptible to fluconazole  similar to the parent, while both aneuploid strains had reduced resistance (larger zone of inhibition radii) relative to the parent. Download Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Aneuploid strains are generally less stable than euploid mutants, as they can rapidly revert to euploidy when grown on rich medium , 26. Ane10.1128/mBio.02272-21.6FIG S6FIG S6, PDF file, 1.5 MB.Tolerance to drugs is unstable under nonselective conditions. Approximately 100 cells from a Chr2x3 strain were spread onto YPD without drug and incubated at 37°C for 24 h. Yellow arrow, a small colony; cyan arrow, large colony (A). The small and large colonies and the parent were assayed for tolerance to tunicamycin (TUN), caspofungin (CSP), and hydroxyurea (HU) (B). The plates were photographed after 48 h at 37°C. Download Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the RTA2 is a downstream effector of the calcineurin pathway and is required for the TUN-induced ER stress response  were associated with TUN tolerance. In the C. albicans genome, CaALG7 is on Chr2, CaPRE7 is on Chr4, and no obvious homolog of YBR085C-A is evident. In C. albicans, since Chr2 duplication was the major mechanism of TUN tolerance and Chr4x3 was not associated with TUN tolerance, we focused on identifying genes on Chr2 that affect TUN tolerance. The essential gene ALG7 was haploinsufficient; deleting one ALG7 allele was sufficient to cause TUN hypersensitivity. ALG7 encodes a UDP-GlcNAc:dolichol phosphate N-acetylglucosamine-1-phosphate transferase that mediates the first step in the protein N-glycosylation pathway  stress response, gene copy number, and transcription and translation levels that are generally proportional to gene copy number variations , 39. Alt40–46–51–MKK2 is associated with tolerance to both TUN and CSP, and as shown previously , are activated by the PKC pathway  was monitored by a plate reader  at 30-min time intervals for 24 h. Data are represented as the mean ± SD of three biological replicates.Approximately 2.5 × 107 cells/ml was spread onto YPD plates supplemented with 4 μg/ml and 8 μg/ml TUN or without TUN (control) and incubated at 37°C for 5 days. Adaptors were randomly chosen, streaked onto YPD plates and incubated at 37°C for 36 h. For each adaptor, several colonies with similar sizes were selected and frozen at −80°C.A total of 100 μl of 1 × 103 cells/ml of SC5314 were inoculated into 1.5 ml of YPD broth containing 1 μg/ml TUN and incubated for 24 h at 37°C (24-h exposure), and 1.5 μl of the culture was transferred to another 1.5-ml YPD broth aliquot supplemented with 1 μg/ml TUN and incubated for another 24 h . The 24 h- and 48 h-exposure cultures were washed and diluted with distilled water, and then ∼300 cells were spread onto YPD plates and incubated at 37°C for 36 h. Randomly, 120 colonies (24-h exposure) or 95 colonies (48-h exposure) from each plate were chosen for further analysis.A total of 2.5 × 103 cells/ml of SC5314 were inoculated into 1.5 ml of YPD broth supplemented with 0 μg/ml (control), 0.5 μg/ml, 1 μg/ml, or 2 μg/ml TUN. After 24 h of incubation at 37°C, a 1.5-μl culture was transferred to another 1.5-ml YPD broth aliquot containing the same TUN concentration. After 10 passages over 10 days, the cultures were washed and diluted with sterile water. Approximately 100 cells were spread onto YPD plates and incubated at 37°C for 36 h, and 18 colonies were randomly chosen from each plate for further analysis.A total of 2.5 × 10600 of 0.2 in 50 ml of YPD broth. The culture was incubated in a shaker at 37°C until the OD600 reached 1.0. The culture was divided into three batches, namely, control (only dimethyl sulfoxide [DMSO] was added), subinhibitory treatment (1 μg/ml TUN), and inhibitory treatment (4 μg/ml TUN) and then incubated with shaking for 3 h at 37°C. Cultures were collected by centrifugation, washed, and flash frozen in liquid nitrogen.SC5314 was inoculated to a starting ODThe total RNA was extracted for 9 independent samples, corresponding to 3 conditions and 3 biological replicates. Total RNA extraction and purification, library construction, and sequencing were performed as described previously .t test in GraphPad Prism (version 5.01).The significance of differences between growth curves was determined using a two-tailed paired Strains used in this study are listed in 10.1128/mBio.02272-21.7TABLE S1Table S1, DOCX file, 0.02 MB.Strains used in this study Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02272-21.8TABLE S2Table S2, DOCX file, 0.02 MB.Primers used in this study Copyright © 2021 Yang et al.2021Yang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-10358 (DNA sequences) and E-MTAB-10391 (RNA sequences).All sequence data are available in the ArrayExpress database at EMBL-EBI (


Therefore, hematological abnormalities might occur because of these toxic particles, but few studies on this issue have been reported. According to Cochrane guidance, we performed a systematic review on the relationship between exposure to PM2.5 and the risk of hematological disorders. Ten articles were included in this review. Anemia was found among children and elderly populations with 2- to 5-year PM2.5 exposure. Young children from mothers exposed to air pollution during pregnancy had a higher incidence of leukemia similar to the elderly. Supporting these data, outdoor workers also showed abnormal epigenetic modifications after exposure to very high PM2.5 levels. Adults living in high PM2.5 areas for 2 years were more likely to develop thrombocytosis. Finally, elderly populations with 7- to 8-year PM2.5 exposure showed increased risks of venous thromboembolism. In conclusion, the associations between PM2.5 and hematological aberrations among high-risk people with long-term exposure were reported.Particulate matter 2.5 (PM Both he blood , 7–10. Fhe blood , 11. In he blood . The annpulation . Childrepulation . As PM2.host database. The keywords were separated into three categories. Searching group1 used “air pollution” or “particulate matter” or “PM2.5” and “anemia.” In group2, the terms “air pollution” or “particulate matter” or “PM2.5” and “leukemia” were searched. In group3, “air pollution” or “particulate matter” or “PM2.5” and “thrombosis” or “coagulation” were used. In group4, “air pollution” or “particulate matter” or “PM2.5” and “lymph nodes” were used. In group5, “air pollution” or “particulate matter” or “PM2.5” and “spleen” were added. The inclusion criteria were studies on PM2.5 in relation to hematological diseases, such as anemia, leukemia, and thrombosis in humans, and publications between 2014 and 2021. Duplicated articles, irrelevant titles, the non-English languages, and unavailable full papers were excluded. Afterwards, eligible articles were archived to the EndNote X7.7.1 for Windows . Interesting information from matched articles of the PM2.5 related to hematologic diseases were extracted to identify the risk factors and assessed by three independent hematologists. The search algorithms are shown in This systematic review followed the Cochrane guidelines. The search used PubMed®, Science Direct, Mendeley, Google Scholar, and EBSCO2.5 exposure to erythrocyte disorders, especially anemia. According to the WHO criteria, anemia was defined as a hemoglobin (Hb) level lower than 13.0 g/dl in men, 12.0 g/dl in women, and 11.0 g/dl in children (2.5 (range 24.97–28.84 μg/m3) during the 4-year study. Mild to moderate anemia in children was related to areas with high 24-h PM2.5 concentrations with the means between 25 (3 (2.5 of over 50 μg/m3  (2.5 levels (>11.1 ± 2.8 μg/m3). Interestingly, elderly subjects with 2- to 5-year exposure to PM2.5 pollution showed declines in Hb levels of approximately 0.81 ± 0.06 g/dl. Honda et al.  in the cells. Inflammation may inhibit differentiation and proliferation of erythroid precursor cells and enhance an erythropoietin (Epo) resistant state (2.5 and PM0.1) and the deformation of murine erythrocytes was found (2.5 (100 s for 8 days) increased erythrocyte distortion, which finally led to hemolytic anemia  = 1.00–1.29] than chronic myeloid leukemia and chronic lymphocytic leukemia. The effects of PM2.5 and other components are related not only to adult leukemia but also to childhood hematological malignancy as reported in studies by Lavigne et al.  and increasing 5mC levels in people exposed to PM2.5 (p = 0.005). This study is limited by the exposure times to PM2.5. In addition, high 5hmC and 5mC levels, which indicated epigenetic modification, were detectable among people in Beijing  and increment in PM2.5 (every 10 μg/m3 increase of PM2.5)  and PE  events after long-term exposure to PM2.5 (2.5 may be from increased inflammatory cytokine (IL-6) levels, oxidative stress, platelet activation, stimulated coagulation pathway, and reduced fibrinolysis (2.5 that can cause disseminated intravascular coagulation (DIC) through coagulation activation in rats . Finally, a shift of hemostatic balance to a pro-thrombotic/pro-coagulation state is induced by exposure to PM2.5   found a to PM2.5 . The mecinolysis . These cinolysis . In anim2.5 exposure is one of the most important avoidable hazards to human health. The effects on hematological systems have been less well-studied. There were fewer than 20 available entries in the web search according to the Cochrane recommendation as the damages are not easily analyzed and are mostly unnoticed. All available studies found harmful effects of long-term exposure to PM2.5 to the hematopoietic system. Hematological parameter changes in people exposed to medium to high PM2.5 concentrations for more than a year are summarized in 2.5 pollution may be from increased inflammatory responses affecting each hematological component. For the public, avoidance of exposure and reduction in PM2.5 pollution should be encouraged. Interventions should be focused on the high-risk groups including the elderly with cardiovascular diseases, young children, and pregnant women. Personal facemasks can minimize inhaled small particles and reduce pollution exposure time. Air purifiers also lessen indoor pollution concentrations. Improving air quality has to be addressed for the general population. Decreasing PM2.5 pollution is essential to prevent hematological adverse events. Further studies are needed to determine whether reducing PM2.5 exposure can decrease these disorders.High PMThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.KF and SC: conception of the article and updating of the article as per the suggestions. KF, SC, VD, VT, and DS: writing-first draft preparation. KF, SC, VD, VT, DS, PR, and TU: writing-review and editing. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. Methods: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. Results: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 μM vs. 17 μM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 μM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 μM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 μM, respectively. However, RA/SC does not affect the growth of Escherichia coli. Conclusions: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.Background: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of  Vibrio is a genus of ubiquitous bacteria that inhabit a wide range of aquatic and marine environments and cause seafood contamination and infectious diseases in humans. More than 30 species of Vibrio have been discovered to date and V. cholerae, V. parahaemolyticus, V. alginolyticus, V. vulnificus, V. fluvialis, V. hollisaea, V. mimicus, and V. metschnikovii are among the most frequently isolated human pathogens. These bacteria are found in environments of moderate or high salinity, including in seawater and seafood, and they are agents of human vibriosis , a, a3+-VmF2+ by RA e. A sign3+ binding site    rather th VmFbpA , indicat3+ indicated that RA interacts with Fe3+ -s-triazine (TPTZ) to Fe2+ to a colored product 2-, and [Fe(Cit)2]5-) as well as other citrate complexes  with a Kd value in the micromolar range [E. coli and V. metschnikovii were inhibited by 1000 μM RA, Fe3+ was reduced to Fe2+. Subsequently, adding citrate chelates free Fe2+ into Fe2+-citrate complexes. These Fe2+-citrate complexes can be taken up by E. coli via the Fec(CD)E/B transporter system, whereas the Fe2+-citrate complexes are not bioavailable for V. metschnikovii. This could be why E. coli is more resistant to RA and the combination of RA and SC than V. metschnikovii. Therefore, RA combined with SC could inhibit V. metschnikovii without deleterious effects on indigenous gastrointestinal bacteria.Interestingly,  citrate ,31 and h citrate , which ihnikovii . Furtherar range . When FbV. metschnikovii. SC chelates Fe3+ to Fe3+-citrate complexes, but V. metschnikovii has a siderophore-based CeuBC/A transporter that is responsible for Fe3+-enterochelin uptake (V. metschnikovii releases the siderophore enterochelin to absorb Fe3+ under iron-restricted conditions. Enterochelin is the strongest siderophore and binds to Fe3+ with extremely strong affinity (Ka = 1052 M−1) [Ka = 1025 M−1) [3+ was not reduced to Fe2+. If Fe3+ is chelated by citrate, it can be reclaimed by enterochelin. This explains why the growth of V. metschnikovii was not affected by SC alone.Moreover, the following reasons may explain why SC did not affect the growth of n uptake . V. mets052 M−1) . This va025 M−1) . TherefoVibrio species. Since we focused on non-cholera Vibrio infections in this study, V. vulnificus and V. parahaemolyticus were selected for the further antibacterial experiments. The results showed that FbpA is a highly conserved protein with a sequence identity of ≈31%  and ume , which contain RA and SC, respectively. Due to its antibacterial properties, umeboshi was traditionally used as a medicine in the middle and early modern ages and is currently used as a natural preservative to prevent the spoilage of items in lunch boxes and rice balls. In the 19th and early 20th centuries, umeboshi was found to be an effective treatment for cholera and was used as an anti-cholera medicine in Japan. Tetsumoto (1934) found that umeboshi exhibited strong bactericidal activity against V. cholerae, as compared to citric acid, which has a similar pH [V. cholerae, although it needs to be confirmed by further experiments.Additionally, the results from a pilot experiment performed in Japan several decades ago support our experimental data . The trasumoto 19 found thRA  was purchased from Combi-Blocks . All other reagents used in this study were purchased from Fujifilm Wako Pure Chemical Co., Ltd.  unless otherwise stated.Twenty spices, including Sanshō (Zanthoxylum piperitum), oregano (Origanum vulgare), thyme (Thymus vulgaris), allspice (Pimenta dioica), rosemary , basil (Ocimum basilicum), coriander (Coriandrum sativum), parsley (Petroselinum crispum), cumin (Cuminum cyminum), cumin seeds, ginger , cinnamon (Cinnamomum verum), paprika (Capsicum annuum), garam masala, garlic (Allium sativum), nutmeg (Myristica fragrans), turmeric (Curcuma longa), chili pepper (Capsicum annuum), black pepper (Piper nigrum), and white pepper (Piper nigrum) used were the products of S&B Foods .Vibrio species  were obtained from Japan Collection of Microorganisms, RIKEN  and were handled in a safety cabinet to avoid contamination. All E. coli strains (BL21(DE3) and KRX) were purchased from Novagen  and Promega , respectively, and were handled in a clean bench to avoid contamination. A total of 0.50 g (±0.01) of each spice was ground into powder, extracted in 20 mL boiling Milli-Q (Milli-Q Advantage), and incubated in a water bath at 95 °C for 3 h. The insoluble fraction was removed using a 0.45 μm polyethersulfone (PES) filter. The filtrate was evaporated to powder and stored at −30 °C.E. coli BL21(DE3). Large-scale overexpression of 6× His-tev-VmFbpA was performed at 30 °C in 2 L of LB medium with isopropyl β-D-1-thiogalactopyranoside  at an OD600 of about 0.6. After the induction, the cells were cultivated at 30 °C for 20 h and harvested by centrifugation at 4000× g and 4 °C for 20 min. The cells were suspended in lysis buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM imidazole, 0.50 mg/mL lysozyme, and 1.2 U/mL DNase I). Subsequently, the cells were disrupted by sonication using 70% amplitude, 0.5 s on/off, for 7 min, and the resulting cell lysate was centrifuged at 40,000× g and 4 °C for 30 min. The supernatant was loaded onto a Ni-nitrilotriacetic acid (Ni-NTA) column (bed volume 2 mL), washed and eluted sequentially by wash buffer 1 (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 10 mM imidazole), wash buffer 2 (wash buffer 1 supplemented with 20 mM imidazole), elution buffer 1 (wash buffer 1 with 50 mM imidazole), elution buffer 2 (wash buffer 1 with 100 mM imidazole), elution buffer 3 (wash buffer 1 with 150 mM imidazole), and elution buffer 4 (wash buffer 1 with 250 mM imidazole). Based on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, all fractions containing 6× His-tev-VmFbpA were collected and stored at 4 °C before use.The gene encoding VmFbpA (WP_004394209.1) without the signal peptide ‘MQKKLLSTVALVSSLITAPLLHA’ was cloned into a modified pET-28a vector, where the thrombin cleavage site had been replaced by a TEV cleavage site, and overexpressed in A total of 6× His-tev-VmFbpA were dialyzed overnight against lysis buffer at 4 °C to lower the imidazole concentration. The 6× His-tag was cleaved by adding 6× His-TEV protease . Then, the reaction mixture was reloaded on the Ni-NTA column and the eluent, which contained mainly 6× His-tag-free VmFbpA, and was collected and concentrated with a Vivaspin 20  to a protein concentration of approximately 1.5 mg/mL. Then, the sample was loaded onto a Superdex 200 10/300 GL column  and separated using gel filtration buffer A (20 mM Tris-HCl (pH 8.0) and 100 mM NaCl) using an ÄKTA purifier  for the final purification. Protein samples were adjusted to a final protein concentration of 0.3 mM by ultrafiltration using a Vivaspin 20  and stored at 4 °C.3+-bound VmFbpA, ferric chloride hexahydrate (FeCl3·6H2O) was added to purified VmFbpA to a final concentration of 4.2 mM. Next, EDTA at a final concentration of 10 mM was added to form apo VmFbpA. The samples were incubated with 50 mM sodium bicarbonate (NaHCO3) at 4 °C for 20 h and then concentrated to a protein concentration of approximately 0.7 mg/mL. Each sample was loaded onto a Superdex 200 10/300 GL column and separated using gel filtration buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaHCO3, and 150 mM NaCl). Fractions were collected according to the peak measured at 280 nm. The samples were adjusted to a final protein concentration of 0.1 mM by ultrafiltration using a Vivaspin 20 (MWCO 10 K) and stored at 4 °C.To form Fe3. The column was extensively washed with washing buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaHCO3, 150 mM NaCl, and 20 mM imidazole). Spice extract or RA (200 μL) was loaded onto the column and incubated at 4 °C for 60 min. The solvent for spice extracts and RA was 50 mM Tris-HCl (pH 8.0), 50 mM NaHCO3, and 150 mM NaCl. The column was subsequently washed extensively with washing buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaHCO3, 150 mM NaCl, and 20 mM imidazole) and eluted with 300 µL elution buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaHCO3, 150 mM NaCl, and 250 mM imidazole). The Fe concentration of eluent was determined by ICP-MS as reported previously [3 were used as the negative control group, and samples lacking rosemary extract or RA as the positive control group. Unless indicated, all buffers contained 0.3% dimethyl sulfoxide (DMSO) [Two-hundred microliters of 0.3 mM apo 6× His-tev-VmFbpA were loaded onto a 40 μL Ni-NTA slurry resin (bed volume: 20 μL) in a Micro Bio-Spin column (Bio-Rad) and incubated at 4 °C for 30 min with 3.0 mM FeCleviously  and adjue (DMSO)  to solub3+-VmFbpA lacking RA was used as the positive control. The Fe concentration of the eluents was determined by ICP-MS as reported previously [Rosemary extracts and RA were used to evaluate VmFbpA inhibitory activity. Two-hundred microliters of rosemary extracts (1.56 to 25.0 mg/mL) and RA (2.34 μg/mL to 2.40 mg/mL), with a two-fold dilution gradient were performed. Apo VmFbpA was used as the negative control and Feeviously . The inh50) was calculated by reading the RA concentration at the inhibitory rate is 50%.The inhibitory rate was plotted in a scatter plot as a function of RA concentration and fitted by a logarithmic function model. The 50% inhibitory concentration  and 200 µL 0.3 mM apo 6× His-tev-VmFbpA with 3 mM FeCl3 were used in the pull-down assay to assess the interaction between apo VmFbpA and RA.RA  and 200 µL 0.3 mM apo 6× His-tev-VmFbpA without FeCl3 and RA were used as the negative controls and samples lacking RA were used as the positive control. Samples without preloading of apo 6× His-tev-VmFbpA were used to determine the background.The absorbance spectra of the eluents at 300–700 nm were measured using a UV–vis spectrophotometer (JASCO V-630) at room temperature. Samples without FeCl3 were used for UV-vis spectra analysis. The absorbance at 200–700 nm was measured using a UV-vis spectrophotometer (Jasco V-630) at room temperature. Solutions of 0, 10, 20, 30, and 40 μM FeCl3 without 20 μM RA were used as the controls. The buffers did not contain DMSO.Solutions of 20 μM RA supplemented with 0, 10, 20, 30, and 40 μM FeCl3 supplemented with 0, 0.625, 1.25, 5, 10, 20, and 40 μM RA were used for analysis. First, 0.25% o-phenanthroline (2 mL) was added to each sample, and the pH was adjusted to 3.5 with hydrochloric acid (HCl). Milli-Q water was added to a final volume of 25 mL. After 1 h incubation, the absorbance at 510 nm was measured using a microplate spectrophotometer . The concentration of Fe2+ was quantified using ferrous sulfate heptahydrate (FeSO4·7H2O) as the standard. The buffers did not contain DMSO.Solutions of 40 μM FeCl3+ VmFbpA samples were dialyzed overnight at 4 °C against dialysis buffer (20 mM Tris-HCl (pH 8.0), 25 mM NaHCO3, and 50 mM NaCl, 20 mM imidazole, 0.3% DMSO). ITC was performed using MicroCal iTC200 . RA (0.5 mM) or Fe3+ (0.1 mM) diluted in dialysis buffer was injected 20 times by a motor-driven syringe into 200 µL 0.01 mM Apo VmFbpA. The first injection volume was 0.1 μL, and the subsequent injection volumes were 2.0 μL. The solution in the titration cell was stirred at 750 rpm throughout the experiment. In addition, titration of RA (0.5 mM) into Fe3+-VmFbpA (0.1 mM) was performed as described above. The reference cell of the microcalorimeter was filled with 200 µL of the corresponding dialysis buffer. The titrations were performed at room temperature. The data were analyzed by MicroCal LLC iTC 200 for Windows .The 6× His-free apo/FeInitial crystallization screening was performed in 96-well Violamo Protein Crystallization Plates (As One) using commercially available kits, namely Crystal Screen HT, Index HT (Hampton Research), and Wizard I and II  at 293 K. A crystallization drop was prepared by mixing 0.5 µL protein solution and 0.5 µL reservoir solution and was equilibrated against 40 µL reservoir solution. After two-dimensional grid optimization of the crystallization conditions (pH versus precipitant concentration), they were further optimized by the sitting-drop vapor-diffusion method in 24-well Cryschem Plates (Hampton Research). A crystallization drop was prepared by mixing 1.0 µL protein solution and 1.0 µL reservoir solution and was equilibrated against 500 µL reservoir solution.v/v) glycerol. X-ray diffraction experiments for apo VmFbpA crystals were performed on beamlines BL44XU at SPring-8  and AR-NE3A at Photon Factory . The final data set was collected from an apo VmFbpA crystal at a wavelength of 0.899995 Å, a crystal-to-detector distance of 319.90 mm, an oscillation angle of 0.1°, and an exposure time of 0.1 s per image using an EIGER 16M detector at BL44XU at SPring-8. The structure solution and refinement were described in the Crystals of apo VmFbpA were obtained using a reservoir composition of 0.25 M ammonium tartrate dibasic, 25% PEG3350, 100 mM Tris-HCl (pH 7.0). Each crystal was picked up in a mounting loop and cooled in a cold nitrogen-gas stream using a cryoprotectant consisting of 0.25 M ammonium tartrate dibasic, 25% PEG3350, 100 mM Tris-HCl (pH 7.0), and 20% (3+-binding site)  platform using AutoDock (version Vina 1.1.2) . Three ang site)  as the dng site)  was creaV. metschnikovii was prepared by culturing the bacteria in NB liquid medium  and flash freezing in liquid nitrogen after adding 50% glycerol  at the OD600 of 0.6. Next, 10 µL V. metschnikovii cell from the glycerol stock were added to 10 mL NB medium containing 0.3% DMSO and 1000, 500, 100, 50, 25, and 0 μM RA. The culture was performed at 37 °C. The optical density at 600 nm was monitored every h (0–10 h) by a microplate spectrophotometer .The glycerol stock of E. coli (BL21(DE3) or KRX) was prepared by culturing the bacteria in NB medium and flash freezing in liquid nitrogen after adding 50% glycerol  to an OD600 of 0.6. E. coli (BL21(DE3) or KRX) (10 μL) from the glycerol stock was added to 10 mL NB medium containing 0.3% DMSO and 1000, 500, 100, 50, 25, and 0 μM RA. The culture was performed at 37 °C. The optical density at 600 nm was monitored every hour (0–10 h) by a microplate spectrophotometer .A glycerol stock of V. metschnikovii (10 μL) from the glycerol stock were added to 10 mL NB medium containing 0.3% DMSO and 1000, 500, 100, 50, 25, and 0 μM RA. The culture was performed at 37 °C for 7 h followed by centrifugation at 40,000 g for 15 min. The weight of precipitate (V. metschnikovii) was measured using a precision balance. The remaining iron in supernatant was measured using ICP-MS as reported previously [Conc,BK is the original iron concentration in NB medium, FeConc,Remain is the remaining iron concentration after culture of V. metschnikovii, mvibrio is the weight of V. metschnikovii after culture, and V is the volume of NB medium.eviously . The Fe V. metschnikovii (10 μL) from the glycerol stock was added to 10 mL NB medium containing 1000 μM RA with 10 and 50 μmol FeCl3, respectively. The positive control group was V. metschnikovii without supplementation of RA and FeCl3. The negative control group was V. metschnikovii without FeCl3. Culture was performed at 37 °C. The optical density at 600 nm was monitored every hour (0–10 h) by a microplate spectrophotometer .V. metschnikovii (10 μL) from the glycerol stock was added to 10 mL of NB medium supplemented with RA and SC. Culture was performed at 37 °C for 7 h. Optical density at 600 nm was measured using a microplate spectrophotometer .E. coli (BL21(DE3) or KRX) (10 μL) from the glycerol stock was added to 10 mL NB medium supplemented with RA and SC. Culture was performed at 37 °C for 7 h. The optical density at 600 nm was measured using a microplate spectrophotometer .V. vulnificus and V. parahaemolyticus were prepared as for V. metschnikovii. Cells (10 μL) from the glycerol stock were added to 10 mL NB medium supplemented with RA and SC. Culture was performed at 37 °C for 7 h. The optical density at 600 nm was measured using a microplate spectrophotometer .Glycerol stocks of p ≤ 0.05 was considered to be statistically significant.The statistical significance was analyzed by one-way ANOVA followed by a Tukey HSD (honestly significant difference) post hoc test using R program (Version 3.6.0). Vibrio activity of RA. The growths of V. metschnikovii, V. vulnificus, and V. parahaemolyticus were inhibited significantly by RA and further inhibited by the supplementation of SC. The combination of RA and SC has high potential to be applied as a food preservative and bacteriostatic agent in the future. Moreover, such new bacteriostatic agents will reduce the excessive and inappropriate use of antibiotics in livestock, aquaculture, and plant agriculture as well as human and veterinary medicine. Further studies should evaluate the utility of RA/SC in various areas.The advent of multidrug resistance among pathogenic bacteria is imperiling the utility of antibiotics. The inhibition of FbpA and iron uptake shows promise as an antibacterial. In this study, we have revealed the molecular mechanism of anti-


Tenebrio molitor is one of the main stored product pests. This study characterized oregano essential oil (OEO) by gas chromatography (GC/FID and GC/MS) and assessed its insecticidal properties against T. molitor. Mortality, survival, respiration, and behavioral response in larva, pupa, and adult of this insect were determined. The major components of OEO were carvacrol (25.6%), p-cymene (12.3%), linalool (8.71%), thymol (7.22%), γ-terpinene (7.21%), caryophyllene oxide (4.67%), α-pinene (2.71%), and eucalyptol (2.69%). OEO caused high contact toxicity in larvae (LD50 = 3.03 µg insect–1), pupae (LD50 = 5.01 µg insect–1), and adults (LD50 = 5.12 µg insect−1) of T. molitor. Survival rates were 100% in larvae, pupae, and adults of T. molitor not treated with OEO, declining to 65–54%, 38–44%, 30–23%, and 6–2% in insects treated with LD25, LD50, LD75, and LD90, respectively. Low respiration rates of T. molitor at different developmental stages was observed after OEO exposure. Additionally, OEO exposure affects behavioral avoidance response and causes repellency in larvae and adults. These findings show that OEO exerts insecticidal and repellent effects against T. molitor, suggesting a potent alternative to synthetic insecticides for controlling the beetle. Tenebrio molitor (L.) (Coleoptera: Tenebrionidae) is one of the main stored product pests of grains, flour, bran, and pasta worldwide. Tenebrio molitor infests many stored agricultural commodities [T. molitor include temperature treatment [The yellow mealworm beetle, modities , contamimodities , and maymodities , making reatment , sun trereatment , controlreatment , and fumreatment . Howeverreatment , environreatment , and resreatment  have limPlant essential oils (EOs) are proposed for pest control in stored grains and display several insecticidal activities . EOs altCymbopogon citratus essential oil is lethal to Ulomoides dermestoides Fairmaire (Tenebrionidae) [Hazomalania voyronii essential oil to Tribolium confusum  (Tenebrionidae) [Zingiber pupureum essential oil to Lasioderma serricone Fabricius (Anobiidae) [The biological activity on insects caused by EOs varies according to plant species, and their efficacy has been demonstrated in coleopteran stored product pests ,15,23. Pionidae) , Hazomalionidae) , and Zinobiidae) , favorinobiidae) , Annonacobiidae) , Lauraceobiidae) , Meliaceobiidae) , and Poaobiidae)  are the Origanum vulgare (L.)  is an important aromatic plant rich in terpenoid components used in medical treatments [T. molitor, deleterious effects caused by synthetic insecticides have been investigated [T. molitor populations. We assume that OEO causes biocidal effects in larva, pupa, and adult of T. molitor, which could be due to its capability to affect the survival, respiration, and behavior of this pest.Oregano, eatments , the fooeatments , and agreatments . Oreganoeatments , with loeatments . In T. mstigated ; howeverT. molitor. This contributed to understanding how this bio-insecticide controls the yellow mealworm and how it can serve to manage synthetic insecticide resistance.The purpose of this research was to characterize the main components of OEO and assayed the mortality, survival, respiration, and behavioral effects on A total of 25 components were identified in OEO, accounting for 95.79% of the total composition: carvacrol (25.28 ± 0.34%), p-cymene (11.9 ± 0.33%), linalool (8.71 ± 0.66%), thymol (7.23 ± 0.22%), γ-terpinene (6.88 ± 0.38%), caryophyllene oxide (4.67 ± 0.08%), α-pinene (2.71 ± 0.02%), eucalyptol (2.66 ± 0.27%), camphene (1.96 ± 0.01%), α-phellandrene (1.87 ± 0.02%), β-pinene (1.71 ± 0.02%), thymol methyl ether (1.68 ± 0.18%), camphor (1.67 ± 0.09%), β-bisabolene (1.64 ± 0.08%), carvacrol methyl ether (1.64 ± 0.01%), terpinen-4-ol (1.56 ± 0.26%), borneol (1.46 ± 0.07%), α-thujene (1.42 ± 0.02%), terpinolene (1.37 ± 0.15%), aromandrene (1.35 ± 0.02%), cis-sabinene hydrate (1.35 ± 0.11%), α-terpineol (1.34 ± 0.19%), α-terpinene (1.23 ± 0.01%), cuminaldehyde (1.23 ± 0.01%), and β-myrcene (1.16 ± 0.02%) ; Table 1p > 0.05), demonstrating the toxicity of OEO to T. molitor and allowing estimates of toxicological endpoints (T. molitor larvae (LD50 = 3.03 µg insect−1), followed by pupae (LD50 = 5.01 µg insect−1) and adults (LD50 = 6.12 µg insect−1). Mortality was less than 1% in the control.The dose–mortality data were suitable for a model probit fit  and decreased from 99.9% (control) to 55.6% with LD25, 44.9% with LD50, 28.5% with LD75, and 6.31% with LD90. For T. molitor pupae, survival rate differed significantly  and decreased from 99.9% (control) to 65.3% with LD25, 53.9% with LD50, 30.7% with LD75, and 2.42% with LD90 of OEO. Survival rates of T. molitor adults differed significantly  and decreased from 99.9% (control) to 54.4% in insects exposed with LD25, 38.9% with LD50, 23.7% with LD50, and 3.32% with LD90 of OEO.Survival rates of al doses . SurvivaT. molitor was influenced by exposure to OEO at the LD50 and LD90. Respiration rate of T. molitor larvae differed between the control group (11.6 μL CO2 h−1), LD50 (8.29 μL CO2 h−1), and LD90 (5.52 μL CO2 h−1) 1 h after exposure, but after 3 h, the respiration rate decreased to 10.1 μL CO2 h−1 in the control group, followed by LD50 with 6.91 μL CO2 h−1, and LD90 with 4.63 μL CO2 h−1 (p < 0.001) and time (p < 0.001), but the interaction between treatments × time did not differ (p = 0.646) (T. molitor pupae differed between control group (14.2 μL CO2 h−1) and LD50 (13.1 μL CO2 h−1), and LD90 (10.2 μL CO2 h−1) 1 h after exposure. Respiration rates decreased from 12.9 μL CO2 h−1 in the control group to 10.9 μL CO2 h−1 in mealworm beetles exposed to LD50 and 7.55 μL CO2 h1− with LD90 of OEO, after 3 h (p < 0.001) and time (p < 0.001), but the interaction between treatments × time did not differ (p = 0.132) (T. molitor adult differed between the control group (20.6 μL CO2 h−1) and OEO with 20.1 μL CO2 h−1 to LD50 and 18.1 μL CO2 h−1 to LD90 1 h after exposure. After 3 h, respiration rates decreased to 19.1 μL CO2 h−1 in the control group, 17.7 μL CO2 h−1 in mealworm beetles exposed to LD50 and 14.2 μL CO2 h−1 with LD90 of OEO (p < 0.001), time (p < 0.001), but the interaction between treatments × time did not differ (p = 0.632) (The respiration rate of  CO2 h−1 A. There = 0.646) . Respirafter 3 h B. There = 0.132) . Respira0 of OEO C. There = 0.632) .2,15 = 51.05, p < 0.001) and T. molitor adults  with LD50 and LD90 of OEO      B. Restin< 0.001) F. The wa< 0.001) C. The wa< 0.001) F.T. molitor under laboratory conditions. Twenty-five components were identified. Carvacrol, p-cymene, linalool, thymol, γ-terpinene, caryophyllene oxide, α-pinene, and eucalyptol were the major components, in agreement with preliminary studies on terpenoids from OEO [Drosophila melanogaster Meigen (Diptera: Drosophilidae) in response to exposure to carvacrol [Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) exposed to p-cymene and linalool [Sitophilus granarius (L.) (Coleoptera: Curculionidae) exposed to thymol [T. molitor was due to the presence of terpenoids, resulting in rapid insect lethality, as reported for other insects exposed to EOs [This work investigated OEO chemical composition and assessed the bioactive effects caused by this EO on from OEO ,37,38. Sfrom OEO . Terpenofrom OEO  and playfrom OEO . In partarvacrol , Helicovlinalool ,44, and o thymol . Regardid to EOs ,29,46.T. molitor in a dose–dependent manner, as also studied in other insects exposed to EOs [50 = 5.17 µg insect−1), pupae (LD50 = 5.17 µg insect−1), and adults (LD50 = 5.17 µg insect−1) of T. molitor and exerted a lethal effect by topical application. The different developmental stages of T. molitor exposed to high doses of OEO displayed altered locomotor activity, lost mobility, followed by paralysis and death. In this context, the symptoms of this insect were consistent with the recognizable effect on nervous system [Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae) [Nezara viridula (L.) (Hemiptera: Pentatomidae) [Plutella xylostella (L.)  [T. molitor with potential as ecofriendly safe alternative to control this stored pest.OEO caused mortality in d to EOs ,48,49. Os system ,35. Expotomidae) , and Pluralidae) . OverallT. molitor survival is promoted by interaction of OEO attaching to the contact exposure and penetrating through respiratory system or insect cuticle, leading to the suppression nerve conduction. Short periods of exposure to OEO from 24 to 48 h, were needed to induce lethality in T. molitor and associated with the quick action of this bioinsecticide. In this research, the effects on T. molitor between lethal doses of OEO occurred at various periods. These time differences occur due to OEO components’ abilities to ingress the insect’s body during respiration [Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae) [Anagasta khueniella Zeller  [Rhyzopertha dominica Fabricius (Coleoptera: Bostrichidae) [T. molitor is another indication of the potential of OEO to protect stored products.High variability in piration  and penepiration , exertinpiration . Similarmelidae) , Anagastralidae)  and Rhyzichidae) . The rapT. molitor, indicating physiological stress. Inhaled insecticides enter in the insect’s body through spiracles and tracheae and can affect the respiratory processes [Demotispa neivai Bondar (Coleoptera: Chrysomelidae) exposed to neem essential oil [S. granarius exposed to cinnamon essential oil [T. molitor exposed to garlic essential oil [T. molitor have low respiration rate when exposed to OEO with possible energy reallocated to other physiological functions and fitness costs.OEO compromises the respiration of rocesses ,16,21. Trocesses ,47. A derocesses . A similtial oil , S. grantial oil , and T. tial oil  decreasitial oil ,21,57. TT. molitor. Changes in the locomotion of T. molitor caused by OEO may be due to the toxic effect of this EO on the nervous system. Altered behavioral responses have been observed in different insects after toxic compounds exposure [T. molitor, larvae and adults exposed to contaminated surfaces by OEO gradually increase the distance walked, velocity, and reduce the resting time, indicating repellency. Indeed, the walking pattern changes in the different concentrations of OEO may be due to its feedback effect on neuron transmission through channels modulation that exerts action potentials along nerve axons and synapsis [T. molitor are dependent on the concentrations of OEO, causing irritability.The OEO also affected the behavioral response of exposure ,29,58 wiexposure ,60,61. Isynapsis ,62. The Tenebrio molitor individuals were obtained from the Institute of Applied Biotechnology for Agriculture of the Federal University of Viçosa . Larvae and adults were kept in plastic bottles (750 mL) at 26 ± 2 °C and 55 ± 25% relative humidity under a 12:12 h light/dark cycle. The insects were fed on wheat bran , pieces of sugarcane , and chayote fruits . Newly-emerged last instar larvae, pupae, and adults (less than 24 h old) were employed in the experiments.O. vulgare (cv. hirtum) fresh plants and produced on an industrial scale by steam distillation (using a Clevenger-type apparatus) was supplied from Ferquina Industry and Commerce Ltd.a. .OEO was obtained from −1; injector temperature, 220 °C, detector temperature, 240 °C; column temperature to start at 40 °C , with a ramp of 3 °C min−1, to 240 °C, held isothermally at 240 °C for 12 min; injection, 0.1 mL (1% w/v in dichloromethane); split ratio, 1:10; and column pressure, 118 kPa. To each component identified, amount was determined as a relative percentage of total area of the chromatogram. For qualitative analysis, GC-MS analyses were performed on a Shimadzu GCMS-QP5050A chromatograph equipped with a Rtx-5MS capillary column , coated with Crossbond (35% diphenyl and 65% dimethyl polysiloxane) and the same operative conditions used for GC-FID. A 0.1 mL aliquot of OEO (in 1% w/v in dichloromethane) was injected in splitless mode (1:10 ratio). Ionization was performed at 70 eV and mass spectral data were acquired in the scan mode in m/z range 40–400 Da. The OEO components were identified by comparison of their Kovats indexes from original literature [3–C24n-alkanes) obtained from National Institute of Standards and Technology (NIST08 and NIST11) libraries.The OEO was analyzed in triplicate using gas chromatography (GC) to quantitative and qualitative chemical analyses. For quantitative analysis, GC analyses were conducted using a Shimadzu GC-17A Series instrument  equipped with a capillary column (Supelco DB-5 30 m × 0.22 mm × 0.25 μm film) and Flame Ionization Detector (FID). The operation conditions were the following: carrier gas, helium at a flow rate of 1.5 mL minterature ,37,38, r−1). Serial dilutions and a control (acetone) were used to assess the toxicity of essential oil to T. molitor, construct dose–mortality curve and calculate lethal doses . Acetone was employed as a control. For each OEO dilution, one microliter (1 µL) was applied on the body of one larva, pupa or adult of T. molitor, using a micropipette. Subsequently, the insects were individualized in Petri dishes (90 mm diameter), covered with a perforated cap for ventilation, and fed (larva or adult) on wheat bran. For each development stage, three replicates with 50 mealworm beetles were utilized for each OEO dilution and the number of dead insects was counted after 48 h of exposure.The OEO was prepared in 2.5 mL of acetone to obtain six dilutions  obtained in the dose–mortality relationship of OEO was evaluated. Tenebrio molitor were exposed to the lethal doses and individualized in glass tubes (2.5 × 120 mm). Acetone was employed as a control. Three replicates of 50 mealworm beetles were utilized for each OEO lethal dose and the number of live insects was counted every 6 h for 2 d.The time–mortality for larvae, pupae, and adults of T. molitor after exposure to OEO (LD50 and LD90) according to the procedure of dose–mortality test and those untreated with acetone used as control. Carbon dioxide (CO2) (μL of CO2 h−1/insect) was measured with a respirometer of the type CO2 TR3C . One T. molitor  was introduced into a 25 mL glass chamber in a completely closed system. CO2 production was measured by 8 h at 26 ± 3 °C after insect acclimatization. Oxygen gas was injected through the glass chamber for 2 min at a flow of 150 mL min−1 to quantify the CO2 produced in the chamber. This airflow forces the CO2 molecules to pass through an infrared reader coupled to the system, allowing continuous measurement of the CO2 produced by insects in each chamber. Fifteen mealworm beetles of each developmental stage were used for LD50 and LD90 of OEO and control.Respiration was evaluated for 3 h in individuals  of T. molitor were kept in Petri dish lined with filter paper  on the bottom, from here termed arena. To prevent the mealworm beetles escape, the inner part of the top of the Petri dish was coated with Teflon® PTFE . The behavioral avoidance response  was performed in arenas with half treated with 250 µL of OEO (LC50 and LC90) or control (acetone). The individual (larva or adult) T. molitor beetle was released in the center of an arena half-treated with OEO (in filter paper) for 10 min and recorded using the Videotrack automated system . Sixteen T. molitor (larvae or adults) were used per OEO lethal dose or control. Mealworm beetles were considered irritated when they spent 50% of the time in half or repelled when they spent <1 min in half of the treated area with OEO [Individuals (larvae and adults) of with OEO ,63.p < 0.05). The behavioral avoidance response of T. molitor was subjected to one-way ANOVA and means compared with the Tukey’s test (p < 0.05). Data on respiration rate and behavioral avoidance response were analyzed using with SAS 9.0 software. Raw data are availability in The dose–mortality relationship was determined with probit analysis using with SAS v.9.0 software  to estimT. molitor. OEO caused toxicity, low survival, reduced respiration rate, and altered behavioral responses in different developmental stages. The chemical characterization shows that terpenoids contained in OEO may act synergistically through integument or respiratory systems to exert neurotoxicity on T. molitor. Furthermore, this research suggests that OEO is not only an alternating source of synthetic insecticides, but also might be utilized as innovative tool for effectively managing T. molitor populations.Overall, the results indicate that OEO has a wide range of detrimental effects on