Eucalyptus urophylla S.T. Blake wood at 150, 170 and 190 °C, for 2 and 4 h, separately. The effects of HT on physical and mechanical properties, wood color, dimensional stability and chemical change were investigated. The results indicate that: Mass loss (ML) of wood at a moderate temperature of 150 °C was small, but increased remarkably when temperature exceeds 170 °C. A maximum ML of 5.83% was observed at 190 °C/4 h; the velocity of water vapor adsorption and equilibrium moisture content (EMC) of HT wood decreased significantly, and varied considerably with treating severity; absolute dry density of HT wood decreased, presenting a similar tendency with ML, but the reduction was greater than ML; HT reduced the tangential and radial swelling ratio and swelling coefficients of wood, and improved the dimensional stability by 71.88% at 190 °C; modulus of rupture (MOR) and modulus of elasticity (MOE) of HT wood varied significantly in severer conditions, but there were no obvious changes in a moderate conditions at 150 °C; there was a slight color change at 150 °C, but wood color became more dark and uniform with treating severity; HT decreased the relative content of hydroxyl groups in wood components, improving wood dimensional stability. Color change of wood may be caused by variations of chromophoric groups and its own structure of lignin due to HT. Moderate temperature HT at 150 °C improved dimensional stability and color uniform of wood, but without reducing mechanical stress. This is a practical HT condition for Eucalyptus urophylla S.T. Blake.Eucalyptus plantations wood have great potential application in high quality solid wood product. In order to improve the overall characteristics, heat treatments (HT) were carried out using  Eucalyptus plantations are planted widely in south China owing to their advanced growth characteristics, such as good response to fertilization , pests’ EMC) reduces by 50%, some mechanical properties decrease by 0–30%, wood color generally changes to brown or dark and presents various color stability, and biological durability increase [Suitable drying technology can reduce drying stress and improve wood quality ,11, but increase ,22,23,24Eucalyptus.globulus wood was higher than that of control specimens when wood HT at 180 °C [Eucalyptus grandis treated at temperature between 140 to 220 °C [Many heat treatments have been increasingly employed to Eucalypt plantation woods to improve their performance for high quality solid wood products ,26. The t 180 °C . Anothero 220 °C . Howevero 220 °C ,30. The o 220 °C . Color oo 220 °C , which io 220 °C . Moreoveo 220 °C ,35,36. MEucalyptus urophylla S.T. Blake wood were heat treated at 150, 170, and 190 °C, for 2 and 4 h, respectively. Our objective was to systemically determine the effect of HT on some physical and mechanical properties, wood color, dimensional stability, and chemical change in Eucalyptus urophylla S.T.Eucalyptus plantations, a fast-growing tress, are not suitable for many applications. However, as one of the least expensive woods, there is a significant interest in heat treating it to enhance its dimensional stability, reduce its color change, and increase its biological durability, but without negatively impacting the strength properties. Thus, in this study, Eucalyptus urophylla S.T. Blake in this survey were obtained from Guangxi Provence, China, which were eight-year-old plantation-grown tress having an average annual ring width of 3.5 mm and a basic density of 0.513 g·cm−3. The trees were produced into 1000 mm (L) logs and air dried in the wood LAB of Nanjing Forestry University for 1 year prior to tests. Thereafter, the logs were sawn and planed into small lumbers with dimensions of 20 (R) × 20 (T) × 1000 (L) mm3. Then, the small lumbers were produced into specimens with two dimensions of 20 (R) × 20 (T) × 2 0(L) mm3 and 20 (R) × 20 (T) × 300 (L) mm3. The large specimens were used for mechanical test and color measurement. The initial mass and dimensions were measured after specimen preparation, which were used for determination of moisture content and density, etc. Defect free specimens were selected and the initial MCs of all specimens were about 11.5%. The Prior to HT, all specimens were first dried at 60 °C for 8 h and then at 103 °C until constant mass were obtained, and then the mass, dimensions, and color of oven-dried specimens were measured. Thereafter, specimens were randomly divided into seven treatment groups in addition to one control group. Seven replicates in each treatment group were heat treated under atmospheric pressure in a lab-used chamber  at 150, 170, and 190 °C, for 2 and 4 h, respectively. The HT specimens were cooled down to room temperature in a sealed Ziploc bag. Finally, the mass, dimensions and color of HT wood were measured again.MLs of treated specimens were calculated from the mass changes according to Equation (1) using the small samples of 20 (R) × 20 (T) × 20 (L) mm3.Mo (g) and Mh (g) are the oven-dried mass of specimens prior to and after HT, respectively.The EMC), which are calculated according to Equation (2).Mo (g) is the initial mass of oven-dried specimens, Me (g) is the mass after equalization in the conditioning chamber.These properties were determined according to GB/T-1931. The oven-dried specimens of treated and control wood were kept at 20 ± 2 °C and 65 ± 3% relatively humidity (RH) in a conditioning chamber until weight constancy was achieved. During the moisture adsorption process, mass, and dimensions in tangential and radial direction of all specimens were measured by an electronic balance (JA21002)  and a digital caliper (CD-20CPX) . The capacity of treated and control wood for moisture adsorption are described by equilibrium moisture content (Do) was calculated in this study to evaluate the influence of HT according to Equation (3).Mo (g) and Vo (cm3) are the mass of volume of oven-dried specimens, respectively.Density of wood depends on water in specimens. Therefore, density of wood at oven-dried conditions  in tangential or radial directions of treated and controlled wood after equalization in the conditioning chamber. The swelling ratio  is calculated according to Equation (4).Le is the dimensions in tangential or radial directions after equalization in the conditioning chamber, Lo is the oven-dried dimension in tangential or radial directions.Wood dimensional stability is evaluated by swelling ratio . The bending strength, also termed as Modulus of rupture (MOR), and modulus of elasticity (MOE) of specimens were measured according to GB/T 1936.1-2009. The large samples of 20 (R) × 20 (T) × 300 (L) mm3 before and after HT at the same locations. Surfaces of the treated specimens were sanded down 1 mm and brushed cleanly before color measuring. The color measurements were performed using a portable sphere spectrophotometer (X-rite SP60) . The CIE L*, a* b* space coordinates were determined and the color changes, ΔE*, as a function of HT were calculated according to Equation (5).L*, Δa*, and Δb* are the changes in lightness, green-red and blue-yellow chromatic coordinate prior to and after HT, respectively. Wood color was measured at the surface of the large samples of 20 (R) × 20 (T) × 300 (L) mm−1 with 16 scans. Wood powder samples were analyzed in diffuse reflectance in 4000–400 cm−1 region.The changes in the chemical structure  of contrp < 0.05).A multiple comparison was first applied to an analysis of variance (ANOVA) using SPSS to assess the effect of HT on wood properties, and significant differences between mean values of control and treated samples were determined using Duncan’s multiple range tests (ML results are displayed in ML of wood significantly (p < 0.05), but the temperature is more effectively. Similar trend for ML was also reported in Cademartori’s study [The ’s study , which w’s study .EMC) after long-term (4 weeks) vapor sorption of control and HT wood are presented in EMC decreased considerably from 11.71% in the control to 3.27% in the treatment of 190 °C/4 h, showing a decrement of 72.1%. However, for HT wood at 150 and 170 °C, there were no significant differences (p < 0.05) in EMC, indicating both temperature and duration has little effect of EMC in these temperatures. For the treatment at 190 °C, treating duration also has no significant effect (p < 0.05) on EMC of HT wood.The MC changing in the short-term (24 h) and equilibrium moisture content  in density among the groups of 150 °C/4 h, 170 °C and 190 °C.EMC after long-term (4 weeks) vapor sorption of control and HT wood are shown in EMC, there are significant reductions in the swelling of HT wood. Compared to control wood, the tangential swelling ratios of HT wood at 150, 170, and 190 °C decreased by 46.15%, 57.52%, and 76.56%, respectively. Thus, heat treatment improves dimensional stability of wood remarkably. However, in same treating temperatures, treating duration has no significant effect (p < 0.05) on tangential swelling  and at swelling b.The radial swelling of HT wood present similar trend with that in tangential direction, i.e., swellings decrease after HT and become significant with the severity of treating temperature and duration. Comparing with control wood, the radial swelling ratios of HT wood at 150, 170, and 190 °C decreased by 12.56%, 40.70%, and 67.24%, respectively. The reduction is slightly smaller than that in tangential direction, except the 150 °C conditions, which resulted in small reduction of swelling.The relation between swelling ratio and moisture content of control and HT wood are shown in p < 0.05), however, they decrease significantly (p < 0.05) when treating conditions became severe. For the 190 °C conditions, MOR decreased by 31.7% compared with the control wood. The reduction of MOR in severe HT is in agreement with the previous report [p < 0.05) improvements of MOE between control and HT wood at 150 °C/2 h, 150 °C/4 h, and 170 °C/2 h. However, MOE of HT wood t at 170 °C/4 h, 190 °C/2 h, and 190 °C/4 h increased significantly (p < 0.05) compared with the control wood. For the 190 °C conditions, there was a 19.64% improvement of MOR compared with the control wood. Despite of the changes of MOR or MOE, wood generally become brittle after heat treatment which is a problem for its further processing.s report . The reds report ,42. In ca*, b*, L*, and ΔE* of wood before and after HT are presented in a* decreased with HT severity and presented minus values, indicating wood color become green after HT. In contrast, Δb* showed an opposite tendency, which increased with HT severity and presented plus values, this means wood color become more yellow along with treating severity. Lightness changes affect wood color remarkably. ΔL* presenting minus values decreased significantly with treating severity, indicating that wood color become darker along with the severer treating. The darkening of wood is attributed to the increase of lignin content in the cell walls, but also to the depolymerization of the hemicelluloses and the increase of low molecular weight sugars produced there from [E* of wood are small at 150 °C, become greater with the improvement of temperature, and reached the maximum value at 190 °C/4 h. For the 150 and 170 °C conditions, effects of treating duration on ΔE* are not obvious, however it became significant at the severer conditions in 190 °C. Color of eucalyptus urophylla became dark, but the color deviation also became small, indicating wood color is more uniform. The darken color benefits for its further application in indoor decoration products, but the color of HT wood is not UV-stable.The changes of color coordinate in ere from . The tot−1, 2900 cm−1, 1600 cm−1, and 1050 cm−1, which demonstrate the stretching vibration peak of –OH, C–H bond, C=O bond as well as benzene ring of lignin, and C–O bond, respectively. Additionally, the peak at 1270 cm−1 is the stretching vibration peak of the structural unit of guaiacyl and the peak at 1505 cm−1 is corresponding to stretching vibration of the lignin benzene ring [−1 of HT wood deceased, indicating heat treatment reduce the relative content of hydroxyl groups of wood components. The reduction of hydroxyl groups decreases water absorption capacity of wood, thereby improving dimensional stability of wood. There are almost no changes in the absorption peaks of C–O, C=O, and C–H, indicating that heat treatments have little effect on them. Color changes of wood are related to the chromophoric groups, such as carbonyl, carboxyl, and alkene conjugated with benzene ring of lignin. There were no significant changes in the peak at 1505 cm−1, indicating lignin benzene ring remained basically stable after heat treatment. However, the changes at 1600 cm−1 absorption peak occur, which are attributed to the conjugated carbonyl group connected to the benzene ring due to the heat treatment [−1 also has a slight decrease in peak intensity. All these changes indicate the chromophoric groups and its own structure of lignin varied due to the heat treatment, which affects color of wood.The ene ring . Comparereatment . In addiEucalyptus urophylla S.T. Blake wood were heat treated at 150, 170, and 190 °C, for 2 and 4 h, respectively. The physical and mechanical properties, wood color, dimensional stability, and chemical change of HT wood were investigated. The results are summarized as: Mass loss (ML) of wood at a moderate temperature of 150 °C was small, but increased remarkably when temperature exceeds 170 °C and the maximum reduction of ML is 5.83% for the conditions of 190 °C/4 h; both velocity of water vapor adsorption and equilibrium moisture content (EMC) of HT wood decreased significantly, and they varied considerably with treating severity. EMC of HT wood at 190 °C/4 h is 3.27%, which decreased by 72.10% in contrast to the control wood; absolute dry density of HT wood decreased, showing a similar tendency with ML, but the reduction was greater than ML; The tangential and radial swelling ratio and swelling coefficients of HT wood declined, and the dimensional stability of HT wood at 190 °C conditions was improved by 71.88%; there were no obvious changes in MOR and MOE of HT wood in a moderate condition. MOR of HT wood decreased significantly at the severer conditions, but MOE of HT wood increased performing inversely with MOR. Despite of the changes of MOR or MOE, wood generally becomes brittle after heat treatment; wood color changed slightly in the 150 °C conditions, but it became darker and uniform after severe treating. Wood’s color after HT is not UV-stable; the relative content of hydroxyl groups in wood components was reduced after HT, resulting in improvement of wood dimensional stability. Color change of wood may be caused by variations of chromophoric groups and its own structure of lignin, as well as depolymerization of the hemicelluloses owing to heat treatment. Moderate temperature HT at 150 °C improves wood dimensional stability and color uniform but without negatively reducing mechanical stress. This is a practical HT condition for Eucalyptus urophylla S.T. Blake.


The cultivated hexaploid varieties are thought to originate from a hybrid of wild chrysanthemums, among which the diploid Chrysanthemum makinoi has been mentioned. Using a combination of Oxford Nanopore long reads, Pacific Biosciences long reads, Illumina short reads, Dovetail sequences, and a genetic map, we assembled 3.1 Gb of its sequence into nine pseudochromosomes, with an N50 of 330 Mb and a BUSCO complete score of 92.1%. Our ab initio annotation pipeline predicted 95,074 genes and marked 80.0% of the genome as repetitive. This genome assembly of C. makinoi provides an important step forward in understanding the chrysanthemum genome, evolution, and history.Chrysanthemum is among the top 10 cut, potted, and perennial garden flowers in the world. Despite this, to date, only the genomes of two wild diploid chrysanthemums have been sequenced and assembled. Here, we present the most complete and contiguous chrysanthemum  Chrysanthemum morifolium Ramat. and its related varieties and species. One of the key factors of its success as an ornamental crop is the diversity available in petal colors and flower shapes , Chrysanthemum indicum (syn. D. indicum), Chrysanthemum lavandulifolium (syn. D. lavandulifolium), and Chrysanthemum zawadskii (syn. D. zawadskii), predominantly in their hexaploid form. The hexaploid Chrysanthemum vestitum and tetraploid C. indicum were later again suggested as major donors based on comparative morphology, cytology, interspecific hybridization, and molecular systematics  genebank. Cuttings were grown in greenhouses at Wageningen University and Research (WUR-Unifarm) according to standard procedures.The C. makinoi leaves using a modified  and 150-bp paired-end reads on an Illumina HiSeq 2500 . Samples were processed using the NEBNext v2.2.10  k-mer coC. makinoi was also sequenced by GenomeScan across eight SMRTcells using a PacBio “Sequel SMRT Cell 1M v2” sequencer. Sample preparation was done based on the “PacBio SMRTbell Express Kit v1” protocol. The final library was selected using the Blue Pippin protocol for fragments larger than 15 kb. Primer and polymerase were attached using the “Sequel Binding and Internal Ctrl Kit2.1” kit and purification was done using the PacBio “Procedure & Checklist—AMPure® PB Bead Purication of Polymerase Bound SMRTbell® Complexes” protocol. Sequencing was performed for 10 h on seven of the cells and 20 h for the remaining cell with the recommended amount of “DNA Internal Control Complex 2.1”. The raw data were assessed with the SMRT Link Analysis server v5.1.0.26367 by GenomeScan.High molecular weight DNA of C. makinoi cultivated in a greenhouse under long-day conditions, 20-h light/4-h dark cycle, or under short-day conditions, 11-h light/13-h dark cycle, at Dekker Chrysanten . All collected plant tissues were frozen immediately in liquid N2 and stored at −70°C until the RNA was extracted and isolated using the RNeasy mini kit  and library prepped using the PCR-cDNA sequencing kit  according to the manufacturer’s instructions. The samples were sequenced separately on an Oxford Nanopore GridION using nine flow cells in total, according to the standard protocol. Quality control was done using NanoComp v1.9.2 (Fonseca and Manning) with duplicate read IDs removed.Four tissues  used in the study were obtained from a Q ≥ 7) that had a length above 20 kb and the “fail” folder (Q < 7) with a length over 50 kb. PacBio reads over 30- kb long were also added into this dataset. This combination of long reads was assembled using SMARTdenovo v1.0.0 . Final pseudo-molecule level scaffolding was performed using ALLMAPS v0.9.14  and filtered to keep only the reads from the “pass” folder  chloroplast  and consisted of 3,924,770 reads. Illumina HiSeq yielded 113.2, 142.0, 133.7, and 120.0 Gb of raw data for the 270, 350, 400, and 500 bp insert size libraries, respectively. Between 90.5% and 94.6% of reads in each insert size had a quality “q” score of greater than or equal to 30. PacBio sequencing resulted in 70 Gb of data with an average subread length of 15.5 kb and an N50 of 24.1 kb. This meant a coverage of approximately 30.6× (assuming a haploid genome size of 3.1 GB).Nanopore sequencing resulted in 443.25 Gb of data with a read N50 of 22.6 kb. After base calling, removing adaptors and filtering for reads over 20 kb in length from the “pass” folder, which had a The nanopore cDNA sequencing resulted in datasets with 4.8–7.9 million reads, an average N50 of 1.2–1.4 kb and between 5.0 and 7.9 Gb total .K = 31) were extracted from the paired-end HiSeq Illumina reads, counted using Jellyfish v2.2.10 . Of the LTRs, 27.1% could be identified as Copia and 7.4% as Gypsy. A similar analysis in C. nankingense marked 69.6% of their assembly as repetitive and found LTRs to make up 67.7% of the identified tandem repeats, with 36.5% being Copia and 30.9% being Gypsy  amplifying Gyspy over Copia (Conyza canadensis) and globe artichoke (Cynara Cardunculus var. scolymus) showed the reverse  suggested that in chrysanthemum the abundances of Copia and Gypsy were similar, with Copia being slightly more abundant and undergoing amplification slightly earlier  in dicotyledons (approximately 122–164 MYA) and another whole-genome triplication (WGT-1), before the split between asterids I and II, approximately 53–62 MYA (C. makinoi as both the genes (red ring) and repetitive sequences (orange ring) are evenly distributed across. In fact, instead of clustering by region, the repetitive sequence density in C. makinoi has a positive Pearson correlation value of 0.60 with gene density. A possible explanation for this correlation is that chrysanthemum, like other Asteraceae, has LTRs driving a lot of diversity  that found, using optical techniques, a strong enrichment for LTRs and that the majority of repetitive sequences identified did not show a preference for centromeric or peri-centromeric regions  No. JP131333 are available for download at www.chrysanthemumgenome.wur.nl/, along with a genome browser. All the raw data as well as the assembly and annotations files can also be found at ENA under PRJEB44800. The plant accession is available through the NARO Genebank.The final assembly and annotation files for G3 online.jkab358_Supplementary_Figure1Click here for additional data file.jkab358_Supplementary_Figure2Click here for additional data file.jkab358_Supplementary_Figure3Click here for additional data file.jkab358_Supplementary_Tables-CaptionsClick here for additional data file.


Enterococcus faecalis and vancomycin-resistant Entercococcus faecium in vitro, in a dose-dependent manner, by disrupting the integrity of the bacterial cell membranes. Moreover, BT effectively reduced the bacterial load in mouse organs when combined with conventional antibiotics in a peritonitis infection model. Thus, BT has shown potential as a therapeutic agent against E. faecalis- and vancomycin-resistant E. faecium-related infections.Widespread antibiotic resistance has been reported in enterococcal pathogens that cause life-threatening infections. Enterococci species rapidly acquire resistance and the pace of new antibiotic development is slow. Drug repurposing is a promising approach in solving this problem. Bithionol (BT) is a clinically approved anthelminthic drug. In this study, we found that BT showed significant antimicrobial and antibiofilm effects against  Enterococci are Gram-positive, anaerobic bacteria that are associated with many life-threatening infections, such as bacteremia, endocarditis, and abscesses. Enterococci have developed antibiotic resistance to vancomycin (VAN), linezolid, and daptomycin (DAP) between 1980 and 2010. However, the development of antimicrobial agents is time consuming, while the emergence of antibiotic resistance in enterococci is rapid .Enterococcus faecalis and Enterococcus faecium are the two major pathogens belonging to this family. However, E. faecalis has a greater ability to form biofilms than E. faecium. The worldwide prevalence of E. faecalis biofilm associated infections is reported to range from 57.2 to 100%, with 93% reported in the United States of America  and formation of persister cells  and its persister cells in vitro and in vivo, by disrupting bacterial cell membrane lipid bilayers (Bithionol (BT), a clinically approved anthelminthic drug, was found to be active against methicillin-resistant bilayers . The toxbilayers , and it in vitro and in vivo antimicrobial effects of BT against E. faecalis and VAN-resistant E. faecium. In this study, we aimed to describe the antimicrobial and antibiofilm effects of BT against enterococci in vitro, and in a mouse peritonitis infection model in vivo. Moreover, using previously reported methods . Clinical isolates of E. faecalis and E. faecium were obtained from urine, blood, and pleural effusion samples collected from patients at the Third Xiangya Hospital of Central South University, Changsha, China and identified using colony morphology and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The antimicrobial susceptibility of the clinical isolates was analyzed using bioMerieux ATB (France). The resistant pattern and other details are described in E. faecalis and E. faecium were grown in brain heart infusion (BHI) broth medium  at 37°C. Cation-adjusted Mueller-Hinton II (MH) broth  was used for antimicrobial susceptibility testing. BT and antibiotics  were purchased from MedChemEpress . BT was dissolved in dimethyl sulfoxide (DMSO), and 0.05% DMSO was used as a vehicle control throughout the experiments. The inoculum was quantified by adjusting the bacterial suspension to 0.5 McFarland (∼1 × 108 CFU/mL) and then diluted to the final concentration required for each assay.5 CFU/mL in MH broth. Equal aliquots of bacterial suspension and two-fold diluted antimicrobial agents  were mixed in a 96-well plate to obtain a final concentration of ∼1 × 105 CFU/mL. After incubation at 37°C for 16–24 h, the minimal antimicrobial concentration necessary to inhibit the growth of the test bacteria was considered the minimum inhibitory concentration (MIC). Then, 20 μL of suspension from 1 × MIC to 64 × MIC was spread onto 5% sheep blood agar  for overnight culture, and the minimum bactericidal concentration (MBC) was identified as the lowest concentration of an antimicrobial agent that killed 99.9% of the test bacteria.Antimicrobial susceptibility tests were performed according to the recommendations of the Clinical and Laboratory Standards Institute 2020 (CLSI) guidelines . The bacterial cells were cultured overnight at 37°C with shaking at 180 rpm, followed by adjustment to 0.5 McFarland and further diluted to ∼2 × 10E. faecalis and E. faecium were sub-cultured with BHI broth to the exponential phase. The bacteria were then diluted with BHI broth containing two-fold diluted antimicrobial agents to obtain a final concentration of ∼1 × 106 CFU/mL in 50 mL centrifuge tubes. BHI containing 0.05% DMSO was used as a control. The tubes were incubated at 37°C with shaking at 180 rpm, and 10 μL of the bacterial suspension was removed from the tubes to perform bacterial live cell counting at the indicated time points over a period of 24 h  was measured.Fifty microliters of BHI containing 0–10 μg/mL of BT were added to a microplate. Enterococcal cultures in the exponential phase were diluted with BHI, and 50 μL of each bacterial suspension was diluted and added to these wells to obtain a final concentration of ∼1 × 108 CFU/mL. Fluorescent dyes SYTOX, DiSC3(5), and PI were added to the bacterial suspensions to obtain final concentrations of 5, 2, and 10 μM, respectively, and incubated at room temperature in the dark for 30 min. Fifty microliters of the bacteria/fluorescent dye mixture were added to each well containing an antimicrobial agent, and the fluorescence intensity was measured every 5 min using a spectrophotometer  for 30–60 min with excitation/emission wavelengths of 485 nm/525 nm, 622 nm/670 nm, and 535 nm/617 nm for SYTOX, DiSC3(5), and PI, respectively (Fifty microliters of 1× PBS (pH 7.4) containing two-fold dilution of BT or melittin (positive control) at the indicated concentrations were added to a black 96-well plate . Exponential-phase enterococcal cells were washed with 1× PBS, and their concentrations were adjusted to ∼1 × 10ectively .8 CFU/mL, cultured with 5 × MIC of BT with shaking at 180 rpm for 1 h, centrifuged, and washed with 1× PBS. As a control, the bacteria were exposed to BHI in 0.05% DMSO. The specimens were observed using SEM  and TEM .Enterococcal cultures in the mid-logarithmic growth phase were diluted in BHI broth to ∼1 × 106 CFU/mL. After incubation for 16–20 h, the fractional inhibitory concentration (FIC) index was calculated as follows: FIC = MICA in combination/MICA alone + MICB in combination/MICB alone. FIC ≤ 0.5 indicates synergy, 0.5 < FIC ≤ 4 indicates no interaction, and FIC > 4 indicates antagonism.The antibiotic synergy test was performed as previously described . Briefly6 CFU/mL, at different incubation temperatures (25 and 37°C), and for different incubation periods (24 and 48 h) to detect the biofilm-forming ability of E. faecalis ATCC 29212 and E. faecium U101. Biofilm biomass was quantified using crystal violet (CV) staining. Briefly, planktonic cells were removed by washing with 1× PBS, and 100 μL of 0.25% CV (w/v) was added to each well, incubated at room temperature for 10 min, and washed with 1× PBS to remove unbound CV. The wells were air-dried, and A570 was measured  (BHI-g) to a final concentration of ∼1 × 10measured .E. faecalis and E. faecium were diluted with BHI-g broth containing serially diluted BT to obtain a final concentration of ∼1 × 106 CFU/mL for biofilm inhibition determination. After incubation at 37°C  or 25°C (for E. faecium) for 24 h, the planktonic cells were removed with 1× PBS, and the biofilms were stained with CV as described above.Overnight cultures of E. faecalis and E. faecium were diluted 1:200 with BHI-g broth and incubated at 37°C for E. faecalis or 25°C for E. faecium for 24 h to monitor biofilm eradication. Planktonic cells were removed by washing with 1× PBS, and the remaining biofilms were treated with serially diluted BT. After a 24 h incubation, planktonic cells were removed, and 200 μL of 2H-tetrazolium-5-carboxanilide  containing phenazine methosulfate  was added to each well. After incubation at 37°C for 2 h, A490 was detected as the metabolic activity of live cells in the biofilms . Female, 6-week-old, outbred ICR mice  with a mean weight of 25 g were used in this study. BT was dissolved in a Kolliphor/ethanol mixture  and diluted 1:10 with saline to the indicated concentrations before use. Enterococci were cultured overnight in BHI broth and washed with saline. Each mouse was intraperitoneally (i.p.) injected with 500 μL of 1 McFarland bacterial suspension containing 5% mucin. After 30 min of injection, antibiotics or BT were administered as previously reported : vehicleE. faecalis ATCC 29212 and E. faecium U101 were determined as described above. Then, 5 μL of bacterial suspension in the wells of 0.5 × MIC was 1,000-fold diluted with MH broth for further antimicrobial susceptibility testing as described above for the next-day antimicrobial susceptibility test. The protocol was followed for a 15-day period.Resistance induction was performed as previously described  with minE. faecalis ATCC 29212 and E. faecium U101 from exponential cultures were washed in 1× PBS and inoculated onto BHI plates (n = 10 plates/group) containing 1–16 × MIC of BT, at a density of 5 × 107 CFU per plate. After incubation at 37°C for 48 h, the colonies on the plate were counted, and one-step FOR was calculated as: CFU (after incubation)/CFU (inoculated) × 100%.One-step frequency of resistance (FOR) was performed as previously described  with mint-test or one-way ANOVA. A significance level of 0.05 was used for all statistical tests.All statistical analyses were performed and graphs were constructed using GraphPad Prism (v8.0). All assays were performed at least in triplicates. The data were analyzed using Student’s E. faecalis and E. faecium ranged from 1–4 μg/mL to 0.5–2 μg/mL, respectively. Unlike VAN (a bacteriostatic antibiotic for enterococci with MBC > 32 μg/mL), BT also showed effective bactericidal effects against enterococci with MBCs of 2–8 μg/mL. Furthermore, BT showed the same activity even against VAN-resistant E. faecalis and E. faecium (VRE) and other clinical isolates with different resistance patterns , and PI are sensitive to changes in cell membrane integrity. In the present study, the antimicrobial peptide melittin was used as a positive control as it kills bacterial cells by disrupting their cell membranes and causing cell membrane depolarization . TreatmeOX Green  and PI  reduction in the BT + GEN (4 or 8 μg/mL) and BT + TOB (4 or 8 μg/mL) after 4 h treatment  could significantly reduce the live cells by Δlog10 CFU/mL of 1.38 and 1.49, respectively  values of 0.17–1.49 μg/mL  with MICs of 0.5–2 μg/mL. However, BT showed weaker antimicrobial effects against E. faecalis than against E. faecium, as evidenced by MICs of 1–4 μg/mL. By staining with SYTOX, DiSC(3)5, and PI, we further confirmed that the underlying mechanism of the anti-enterococcal effect of BT involved the disruption of the bacterial cell membrane. Interestingly, BT could also effectively kill E. faecalis and E. faecium clinical strains that are resistant to DAP (an Food and Drug Administration (FDA)-approved antibiotic that targets the bacterial cell membrane)  . Thus, wE. faecalis and E. faecium. BT can completely kill stationary-phase MRSA and its biofilm persisters within 24 h  caused by GEN cannot be ignored , BT could have a synergistic effect with a wide range of conventional antibiotics such as CRO and TEC , and YW (9th author) designed and performed the experiments and wrote the manuscript. YiL, LZ, and SL performed the experiments and data collection. XZ, YaL, and LX performed the experiments and revised the manuscript. All authors read and approved the final 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.


Malassezia pachydermatis to antifungal agents.Lifestyle factors such as hair length, the frequency of ear cleaning and bathing, age, cat rearing, and sex may contribute to opportunistic yeast infections in the external ear canal of cats. This study aimed to determine the prevalence of commensal yeast organisms in cats’ external ear canals, evaluate their predisposing lifestyle factors, and test the susceptibility of M. pachydermatis was evaluated using Fisher’s exact test. The susceptibility of M. pachydermatis to antifungal agents was also analyzed. M. pachydermatis was the most frequently recovered yeast species, with a prevalence of 50.94 % . There was an association between hair length and a positive culture for M. pachydermatis (p = 0.0001). The odds of a negative culture for M. pachydermatis among short-haired cats was 11.67  times higher than that among long-haired cats (p = 0.0002). There was also an association between the frequency of ear cleaning and the presence of M. pachydermatis (p = 0.007). The odds of a negative culture for M. pachydermatis in cats that were receiving ear cleaning at intervals of ≤ 2 weeks was 5.78  times greater than that of cats receiving ear cleaning at intervals greater than 2 weeks or never (p = 0.0055). Ranges of minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations for itraconazole, ketoconazole, miconazole, and terbinafine against M. pachydermatis were ≤ 0.063–4 and ≤ 0.063–≥32, ≤ 0.063–8 and 0.125–≥32, ≤ 0.063–≥32 and 0.5–≥32, and ≤ 0.016–1 and 0.125–8 µg/ml, respectively.A total of 53 cats  seronegative for feline leukemia virus and feline immunodeficiency virus were enrolled in this study. Their mean age (± standard deviation) was 6.04 (± 3.49) years. Fungal cultures and polymerase chain reaction tests were performed to identify the yeast species derived from the external ear canal. The association between lifestyle factors and the presence of M. pachydermatis was the most commonly identified yeast organism in the external ear canal of healthy cats. Hair length and the frequency of ear cleaning played a role in the colonization of M. pachydermatis. The M. pachydermatis isolates had various MIC levels for common fungicides. Malassezia spp. is a commensal yeast found on the mucosa and skin of mammals. Overgrowth of this organism may cause a local or systemic disease in humans and cats : 6.04 ± 3.49 years), sex , body weight (mean ± SD): 5.08 ± 1.48 kg), breed , and cat rearing , are shown in Table M. pachydermatis, Malassezia nana, Malassezia cuniculi, Malassezia furfur, Candida ciferrii, and other yeast species . The prevalence  of M. pachydermatis was 50.94 % , 1.89 % (0.05–10.07 %), and 1.89 % (0.05–10.07 %), respectively. The prevalence (95 % CI) of C. ciferrii was 9.42 % (3.13–20.66 %). The prevalence (95 % CI) of each of the remaining yeasts  was 1.89 % (0.05–10.07 %).In the present study, 10 yeast species were identified from the external ear canal of cats: M. pachydermatis among short-haired and long-haired cats was 11.32 % (4.27–23.23 %) and 39.62 % (26.45–54.0 %), respectively (Table M. pachydermatis (p = 0.0001). The odds (95 % CI) of a negative culture for M. pachydermatis among short-haired cats was 11.67 (3.22–42.24) times higher than that among long-haired cats (p = 0.0002).The prevalence (95 % CI) of M. pachydermatis in cats that had been receiving ear cleaning at intervals of ≤ 2 weeks and > 2 weeks or never was 11.32 % (4.27–23.03 %) and 49.06 % (35.06–63.16 %), respectively. There was an association between ear cleaning frequency and the presence of M. pachydermatis (p = 0.007). The odds (95 % CI) of a negative culture for M. pachydermatis in cats that were receiving ear cleaning at ≤ 2-week intervals was 5.78 (1.67–19.94) times higher than among cats receiving ear cleaning at > 2-week intervals or never (p = 0.0055).The prevalence (95 % CI) of M. pachydermatis in cats aged ≤ 6 years and > 6 years was 26.42 % (15.26–40.33 %) and 24.53 % (13.75–38.28 %), respectively. The prevalence (95 % CI) of M. pachydermatis in cats that were bathed at intervals of ≤ 1 month and > 1 month or never was 18.87 % (9.44–31.97 %) and 32.08 % (19.92–46.32 %), respectively. The prevalence (95 % CI) of M. pachydermatis in indoor-only cats and cats with outdoor access was 49.06 % (35.06–63.16 %) and 1.89 % (0.05–10.07 %), respectively. The prevalence (95 % CI) of M. pachydermatis in male cats and female cats was 30.19 % (18.34–44.34 %) and 20.75 % (10.84–34.11 %), respectively. There was no association between a positive culture of M. pachydermatis and lifestyle factors such as age (p = 0.7846), frequency of bathing (p = 0.0537), cat rearing (p = 0.1917), and sex (p = 0.7786)  of 15.26–40.3 % and 2M. pachydermatis, from 27 cats are shown in Table M. pachydermatis from these cats were analyzed for antifungal susceptibility. The ranges of the minimum inhibitory concentrations (MICs) for itraconazole (ITZ), ketoconazole (KZ), miconazole (MZ), and terbinafine (TERB) against all isolates of M. pachydermatis were ≤ 0.063–4, ≤ 0.063–8, ≤ 0.063–≥32, and ≤ 0.016–1 µg/ml, respectively. The ranges of the minimum fungicidal concentrations (MFCs) for ITZ, KZ, MZ, and TERB against all isolates of M. pachydermatis were ≤ 0.063–≥32, 0.125–≥32, 0.5–≥32, and 0.125–8 µg/ml, respectively.The results of the antifungal susceptibility testing for the most common yeast, Malassezia pachydermatis, Malassezia nana, Malassezia cuniculi, Malassezia furfur, Candida ciferrii, Aureobasidium melanogenum, Cystobasidium calyptogenae, Moesziomyces aphidis, Rhodotorula mucilaginosa, and Sympodiomycopsis sp. The most common positive cultured yeast was M. pachydermatis, with a prevalence (95 % CI) of 50.94 % (36.84–64.94 %). Because of its high abundance, M. pachydermatis was selected in this study of risk factors and susceptibility testing. Lifestyle factors such as hair length and the frequency of ear cleaning contributed to the risk of having a positive culture for M. pachydermatis from the cats’ external ear canals. This study is the first report of the MICs and MFCs of ITZ, KZ, MZ, and TERB against M. pachydermatis in cats.In this study, the identified organisms from the external ear canals of 53 cats were M. nana, M. cuniculi, and M. furfur). Molecular identification of yeast species is a quick method to confirm yeast species.All 53 enrolled cats were apparently healthy, as confirmed by taking their history and conducting a general physical examination, complete blood count testing, blood chemistry testing, and serology testing for FeLV and FIV infection. All positive cultured organisms from the external ear canal of the cats were considered commensal organisms. The cat’s cerumen was initially cultured on various cultured media to increase the variety of isolated yeast organisms followed by polymerase chain reaction (PCR) and DNA sequencing. Conventional yeast identification using culture media is often time-consuming, and it is difficult to differentiate lipid-dependent yeast species  and M. nana (16.98 %) in healthy cats from this study was higher than that in previous reports. This discrepancy may be explained by the methods of sample collection and geographic differential effects. Previous studies performed sample collection on the unilateral ear canal [M. pachydermatis in this study. Moreover, the presence of M. nana has been primarily noted to be in cat external ear canals compared to other body parts, suggesting that this organism is adapted to this anatomical site [The  animals , 9, 16. ectively . The precal site .M. cuniculi, M. furfur, C. ciferri, Aureobasidium melanogenum, Cystobasidium calyptogenae, Moesziomyces aphidis, Rhodotorula mucilaginosa, and Sympodiomycopsis sp. in the external ear canal of cats. The genus Candida is an opportunistic pathogen that causes candidiasis and candidaemia in both humans and animals [Aureobasidium melanogenum and Moesziomyces species are opportunistic pathogenic yeasts that cause fungemia [Rhodotorula spp. is an emergent opportunistic pathogen, particularly in immunocompromised humans [Cystobasidium calyptogenae and Sympodiomycopsis sp. have been reported as environmental yeast organisms in water and soil [Furthermore, this study was the first to report the prevalence of  animals . Aureobafungemia –22 and tfungemia . Rhodotod humans , 25. Howand soil .It should be noted that the yeasts isolated in this study were present on the skin of the ear canals of healthy cats. The pathogenic roles of these isolated organisms may be related to the host immune response as well as to yeast-specific virulence factors , 27. CatM. pachydermatis in the external ear canal of cats and lifestyle factors. Age, bathing frequency, cat rearing, and sex are not commonly considered to be predisposing factors for M. pachydermatis in cats; however, this study confirmed that hair length and the frequency of ear cleaning contribute to the risk of a positive culture for M. pachydermatis from cats’ external ear canals. This finding may be explained by the anatomy associated with hair length. For example, long-haired cats have hairs in the ear canal, which may increase its local humidity and the chance of yeast accumulation in the ear canal. Humidity and the presence of lipids and sebum may predispose cats to yeast overgrowth, leading to inflammation and the development of pathologic lesions [This study investigated the association between the presence of  lesions .M. pachydermatis were evaluated using results modified from a previous report [M. pachydermatis in our study was 0.063 µg/ml , which was similar to a previous report of antifungal susceptibility levels of ITZ against M. pachydermatis isolated from otitis and dermatitis dogs and cats [M. pachydermatis isolated from the skin and scales of dogs [M. pachydermatis isolates in our study were consistent with previous reports [The MICs of ITZ, KZ, MZ, and TERB against s report –31. The and cats , 31. The of dogs . The MIC reports , 31. The reports , 31.M. pachydermatis (76 %) showed the same range of MICs for MZ, with many reports at 0.063-16 µg/ml [M. pachydermatis from cats. This could be due to antifungal resistance caused by inappropriate uses of MZ, which is commonly included in ear treatment solutions [Most isolates of 16 µg/ml , 31–35. olutions .M. pachydermatis were 0.016-1 µg/ml, similar to previous reports [M. pachydermatis from the external ear canal of healthy cats was comparable to most studies in animals. Interpretative breakpoints are not available for Malassezia yeasts; nonetheless, high MIC values were recognized for the isolates. Further studies of antifungal susceptibility testing obtained from clinically related isolates and reports of treatment failure are necessary.The MICs of TERB against  reports , 36, 37.M. pachydermatis was the most commonly isolated yeast organism from cats’ external ear canals. Hair length and frequency of ear cleaning played a role in the presence of M. pachydermatis. All M. pachydermatis isolates had various MIC levels against common fungicides.The present study revealed that A prospective cross-sectional study was performed at Kasetsart University Veterinary Teaching Hospital, Faculty of Veterinary Medicine, Bangkok, Thailand, from March to December 2020. Fifty-three healthy cats were enrolled in this study. None of the cats had an illness or required any kind of medical treatment for at least 3 months prior to the study. The age (≤ 6 years and > 6 years), sex , breed, and body weight of each cat were recorded. In addition, the lifestyle factors of the individual cats were assessed, including their hair length (short-haired and long-haired), frequency of ear cleaning (≤ 2 weeks and > 2 weeks or never), frequency of bathing (≤ 1 month and > 1 month or never), and history of cat rearing (indoor only and outdoor access). A general physical examination, complete blood count, and blood chemistry testing  were performed on all cats. A commercially available FeLV-FIV rapid immunochromatography test assay  was used to determine FeLV and FIV infection.Bilateral otoscopic examination of the ear pinnae and ear canal was performed on all of the cats. The appearance of the ear pinnae and the otopedal reflex were characterized and recorded according to the criteria of the clinical findings of the external ear canal, including the history of pruritus (absent or present), amount of cerumen , the color of the cerumen , ear canal stenosis (absent or present), erythematous (absent or present), and the otopedal reflex (absence or present) , 39. SteAn external ear canal swab was inoculated on culture media consisting of Sabouraud dextrose agar (SDA) , Leeming and Notman agar (LNA) , and mycMalassezia yeasts were presumptively identified to the species level according to their lipid dependency and utilization from various lipid tests, including growth on lipid-free medium SDA, the utilization of castor oil , utilization of Tween 60-esculin agar (TE slant), and a Tween assimilation test [All isolates of ion test .DNA extraction was modified from a previous report . In brieThe internal transcribed spacer (ITS) region of the ribosomal DNA gene of all DNA samples was amplified using the primer set SR6R (5′-AAGTATAAGTCGTAACAAGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) . The PCRhttps://bioedit.software.informer.com). Sequence similarity was searched using the BLASTn tool of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The species identity of each yeast isolate was determined based on the sequence similarity of the ITS sequence. DNA alignment and phylogenetic analysis were performed using Molecular Evolution Genetics Analysis . Gene sequencing of all representative isolates was submitted to GenBank .The sequences were assembled bidirectionally and manually corrected for consensus sequences using BioEdit v.7.2.5  [M. pachydermatis to the following antifungal agents was tested: ITZ , KZ , MZ , and TERB . The final concentrations of the antifungal agents ITZ, KZ, and MZ were prepared at 10 different concentrations . TERB was prepared in concentrations of 0.016, 0.031. 0.063, 0.125, 0.25, 0.5, 1, 2, 4, and 8 µg/ml. The broth microdilution method was performed in 96-well microtiter plates . The M. pachydermatis inoculum size was measured by spectrophotometry at 530 nm and adjusted with culture medium to obtain a final cell concentration of 5 × 103–1 × 104 CFU/ml. Candida parapsilosis ATCC 22,019 was included as the quality control isolate. After incubating at 30 °C for 72 h, MIC was determined by naked-eye visualization, and MFC was determined after 72 h of incubation by transferring 200 µl of suspension from all wells showing no visible growth to newly fresh SDB with 1 % Tween 80. The tubes were incubated at 30 °C, and the MFC was defined as the lowest concentration showing complete growth inhibition as demonstrated by the clear subculture tubes.Antifungal susceptibility testing of  M27-A3) , using a M27-A3) . Sabourap < 0.05. The prevalence and 95 % CI of each yeast organism in the external aural canal in long-haired and short-haired cats were determined. A normality test was performed for all data using the Shapiro-Wilk test. Fisher’s exact test was used to analyze the association between lifestyle factors and the presence of M. pachydermatis.Significance was set at


GM of Cr during Cr plating was 1.86 mg/m3. GM of TSP and heavy metals differences were statistically significant for each process and date variation. Average ventilation volume for all hoods ranged from 1.20 to 4.98 m3/s. In the hood 30 cm from bath, ventilation was 0.1 times lower. Increasing ventilation volume of the hood was the most influential factor, followed by machine operation time and workload. The high concentration was due to low ventilation suction flow. We can improve health to reduce exposure by resolving the fundamental cause of risk occurrence.This study is to evaluate exposure to harmful substances, such as particulate and heavy metals, by considering various factors, in anodizing and electroplating surface treatment process. Exposure evaluation studies on seven heavy metals  and total suspended particulates (TSP) were conducted. Heavy metals were analyzed using inductively coupled plasma mass spectrometry (ICP-MS). This study also checked the ventilation volume of the hood with a thermal anemometer. Measurement was conducted for 8 h and 8 days. The sample number, N, of the heavy metals and TSP was 107. Geometric mean (GM) of TSP during Cr plating process was 6.15 mg/m Anodizing is applied to improve corrosion by an anode + oxidizing, thereby undergoing degreasing, neutralization as a pre-treatment process, coloring, sealing, polishing, and non-polishing as a post-treatment process. The oxide film is formed by soft anodizing and hard anodizing. As a post-treatment process, the product is completed through neutralization, water cleaning, drying, and packaging3. Al exposure was significantly correlated with various neurological disorders, and contact dermatitis in workers exposed to Al alloy and Al dust also has been reported. Al toxicity can occur as a result of the interaction between Al and the plasma membrane with target established by the body4. Al inhaled through the respiratory tract is replaced by magnesium and iron, resulting in intercellular exchange, cell growth, and secretion functions. The changes in neurons by Al are similar to the degenerative lesions observed in Alzheimer's patients, and complication of Al toxicity may have neurotoxic effects, such as nerve atrophy of the cerebellum, black matter, and striatum5. Similarly, high concentrations of Al are very toxic to aquatic animals, especially gill-respiring organisms such as fish, and can destroy plasma and bloodstream ions, and cause osmotic disorders6.The most important exposed materials in the anodizing process are heavy metals. During the process of oxide film formation, abundant Al depending on the type of Al alloy are used, and exposure to other heavy metals is possible. The artificial exposure route of Al is through the respiratory tract, Al is related to potroom asthma, chronic bronchitis, pulmonary fibroids, and granulomatous lung diseases upon exposure through inhalation7.Chromium plating was used by an electrolytic solution of chromic acid during the plating process. Workers can be exposed to Hexavalent chromium (Cr (VI)) during the mixing of acid powders and carrying products10. Cr (VI) compounds are produced from other airborne forms of Cr industries that use Cr (VI) compounds, such as steel passivation, electroplating, stainless steel welding, and paints production, Cr-based pigments, fungicides, and anti-corrosion compounds as a by-product11. Exposure to Cr during electroplating is a characteristic cause of occupational asthma. Sensitivity to Cr in electroplates may occur in situations where exposure levels are likely to be within the current exposure standards12.Hexavalent chromium (Cr (VI)) compounds are occupational carcinogens that cause lung, nasal, and sinus cancers13). For anodizing, the surface treatment principle is different; the surface of the metals is anodized by shooting oxygen from the anode14. The reactions occur simultaneously during anodizing3 .This study evaluated the exposure of each process of business sites performing Al anodizing and Cr electroplating. Exposure evaluation studies on seven heavy metals  and TSP were conducted. This study also evaluated harmful substances by checking the ventilation volume of the hoods. The sample number, N, for TSP and heavy metals is 107.Table The workplace in this study addressed all processes for anodizing and Cr plating, and the processes can be largely divided into pre-treatment, anodizing, Cr plating, and post-treatment processes. There are three types of surface treatment baths: small, medium and large. But small baths are rarely used in the process. So, to reflect realistic exposure, medium and large baths were evaluated except for small baths Fig. . Drying,Samples were collected at a height of 1.5 m to represent the breathing zone of workers. The measurement time proceeded from 8:30 am to 5:30 pm for 8 days, and the measurement was conducted for total of 8 h, excluding breaktime. As a control, the anodizing and plating control sample and drying and packaging control sample were placed in the workplace to check the blank.PVC (Polyvinyl Chloride) filter  in the three-piece 37 mm closed face plastic cassette  was used to measure fine dust generated during the operation of forming an oxide film and plating, which were the main processes during anodizing and electroplating. These were connected by two types of air sampling pump  operating at an airflow rate of 2 ℓ / min. The sample was wrapped with Parafilm  before and after the sample was collected so as not to be exposed to outside air and then stored to room temperature.To analyze the TSP, PVC filter was contained in a desiccator for more than 24 h, and then weighed using an analytical balance having 0.01 μg sensitivity . Before measuring weight of the filter, static electricity was removed and weight change according to daily temperature and humidity change was corrected using a blank filter. Weight was measured 3 times per sample, and the average value was used as the final weight concentration. High flow rate pump was calibrated before and after sampling using a flow calibrator .15. After the weighing TSP, the remaining was also used to measure heavy metals, as described for TSP measurement. After sampling, PVC filter was folded four times into a microwave vessel, and then 4 ml of 70% nitric acid solution  was added. Samples were extracted using a PVC filter and PC(Polycarbonate) filter method stored in a library in a microwave  for 2 times. The temperature was gradually raised to 200 °C for 20 min and then maintained for 20 min. The pressure was set to 800 psi, and the power to 900–1050 W. After this process, the vessel was cooled in the microwave for 20 min and at room temperature for 50 min. The extracted sample was diluted to 40 ml with 1% nitric acid solution, and quantitative analysis was performed using an inductively coupled plasma mass spectrometer . The conditions show under the Table The air sampling and analysis of heavy metals were based on the NIOSH method 73002) was more than 0.99 and it showed the linearity16. The values are Cr 0.003, Mn 0.019, Co 0.003, Zn 0.017, Ni 0.013, Ag 0.014, Pb 0.006. Cd 0.008, Fe 0.005, Al 0.004, Ba 0.002, As 0.034, Sr 0.013, Na 0.004, Mg 0.032, Cu 0.010, K 0.037 µg/sample.Quality assurance and Quality controls (QA/QC) for heavy metals was performed. LODs of heavy metals was determined by triple standard deviations from seven replicates at the lowest level of the standard solution (1 to 20 μg/L), and a determination coefficient (r3/s), and wind speed (m/s) according to distance. There are two types of thermal anemometers ; this study used a one-way type. Figure A thermal anemometer  was used to measure the hood characteristics, ventilation volume (mAir sample concentrations were expressed in terms of arithmetic mean (AM), standard deviation (SD), geometric mean (GM), geometric standard deviation (GSD), and median and range (min–max). The Shapiro–Wilk test confirmed that the TSP and heavy metals were log-normally distributed. Measured air concentrations of TSP and heavy metals in each process were compared using analysis of variance (ANOVA), and the Bonferroni method was used for post-hoc analysis. The date variations for TSP and heavy metals in each process were compared using ANOVA. Multivariate multiple linear regression was performed to find factors that affect TSP and heavy metals concentration. Response variables were TSP and heavy metals, and explanatory variables were workload, number of workers, and amount of ventilation volume. P < 0.05 was considered statistically significant. Statistical analysis was performed using R software v.3.6.3. . Graphs were plotted using Sigmaplot 14.0 .3). As a result of ANOVA test, GM concentrations of every process were statistically significant, showing that the process samples have different exposure aspects during product creation. Differences in GM concentrations and daily variation were statistically significant among the anodizing and electroplating, post-treatment, pre-treatment, and other processes. For the pre-treatment process, the average TSP concentration of degreasing was similar to that of etching and neutralization; however, the range was much higher than that of etching and neutralization. Anodizing and electroplating have similar soft and hard anodizing aspects, whereas Cr electroplating has a much higher concentration than all the processes. During the post-treatment process, the average TSP concentration of sealing has the highest average, whereas coloring and polishing have a wide concentration range. In the “other” processes, the TSP average concentration was generally lower. However, the drying and packaging and Cr cleaning processes have sufficient concentrations , 4.85(7.96), 151.40(11.09) and 85.52(4.82) µg/m3, respectively. Pb during Cr plating was 1.13(3.61) µg/m3. Al concentrations were high in most processes. GM of Al during medium-sized soft anodizing, large-sized soft anodizing, medium-sized hard anodizing, and large-sized hard anodizing were 528.97(6.80), 553.31(1.81), 545.98(2.75), and 520.79(1.34) µg/m3, respectively. Differences in GM concentrations and daily variation of the GMs of heavy metals were statistically significant. When the proportion of heavy metals was confirmed, the analysis was performed with seven basic heavy metals, excluding the 10 other heavy metals. Pre-treatment processes confirmed that Al occupied the largest proportion of heavy metals, followed by Cr, and occupied most of the anodizing and electroplating processes; for Cr plating, however, Cr occupied the largest proportion. The post-treatment process revealed that the Cr concentration occupied a high rate while polishing. In other processes, Al occupied the highest rate, and Ni occupied the highest rate in the drying and packaging processes  types and ten other types. Table 2 was 0.994, indicating a strong relationship. The ratio of seven kinds of heavy metals causing adverse health effects relative to the total measured substances in the air was not relatively high. The Cr concentration accounted for 62.65% in degreasing, drying and packaging, and Cr cleaning and for 20–40% in soft and hard anodizing  and etching & neutralization (1.77 m3/s) were ventilated. For anodizing and electroplating, medium-sized soft anodizing was 1.76 m3/s, and large soft anodizing was 2.14 m3/s. The medium-sized and large-sized hard anodizing had average ventilation volumes of 2.12 and 2.33 m3/s, respectively. Ventilation volume for Cr plating was 2.94 m3/s and was the largest than in any other processes. For the post-treatment process, the medium-sized and large-sized colorings are 1.22 and 1.31 m3/s, respectively. The ventilation of the hood 30 cm away from the bath was very low. The 30 cm away inhalation volume was 0.1 times lower than the average ventilation volume.The workplace was equipped with a hood for every process, and the total suction flow rate for all the processes was 600 mMultiple linear regression analysis was conducted by setting TSP and heavy metals (N = 107) as response variables, and environmental factors were expected to affect the concentrations of TSP and heavy metals in the workplace as explanatory variables Table . Regress3. This study conducted a two-week measurement to reduce date variation to improve the limit of one-time work environment measurements.This workplace conducts work environment measurements conducted by external work environment measurement agencies twice a year. The agencies can evaluate intensive personal exposure, but cannot determine the primary cause of the occurrence of harmful substances. Most acids  were not detected. Cr (VI) was also not detected, but Ni, sulfuric acid, and sodium hydroxide were 0.001–0.003, 0.021–0.035 and 0.349–0.453 µg/m3). Some Zn processes exceeded the OEL of Korea, whereas others had values close to those allowed in the working environment. The levels of Pb in anodizing and electroplating processes were higher than the OEL of Korea (Pb = 50 µg/m3). Cd, Al, and Ba did not exceed the OEL of Korea  = 5,000 µg/m3. Ba = 500 µg/m3). However, in the case of Al, high concentrations were medium-sized and large-sized soft anodizing, and medium-sized and large-sized hard anodizing. Concentrations were also relatively high in coloring and sealing. The principal factor affecting the concentration was ventilation volume, following machine operation time and workload. The ventilation volume of the hood in the workplace was low; thus, the suction flow rate of the hood had to be improved.The exposure values of the anodizing and electroplating processes, which handle more heavy metals were higher. The concentrations of some types of heavy metals in all the processes were below the OEL of Korea; however, the Cr processes exceeded the OEL of Korea, which far exceeded the OEL of Korea (Cr(VI) = 10 µg/m−6 ± 38.4E−6, 81.3E−6 ± 89.5E−6, 274.2E−6 ± 118.4E−6, 206.2E−6 ± 173.4E−6, 73.3E−6 ± 44.8E−6, and 69.9E−6 ± 44.5E−6 mg/m3. Heavy metals concentrations were very low, whereas TSP levels were relatively high17. When checking the distribution of sample concentrations in Cr and Cr (VI) by samples for 10 plants (n = 48), arithmetic mean (AM) range of total and Cr (VI) concentrations were 0.89–523.7 and 0.09–113.2 µg/m3, respectively. Moreover, degreasing had the highest GM 21.6 µg/m3, followed by the plating process (13.4 µg/m3) and “other” process (2.36 µg/m3). Cr (VI) concentration with plating had the highest GM (4.15 µg/m3), followed by degreasing (1.86 µg/m3) and “other” process (1.28 µg/m3). Total Cr and Cr (VI) showed a high correlation of 0.9118. In the soil around the electroplating, Cr, Cu, Pb, Zn, and Cd were detected at concentrations of 22.7–453, 9.51–120, 18–108, 24.1–143, and 0.130–0.850 mg/kg, respectively. Therefore, heavy metals in air can be detected at high concentrations in the surrounding environment for the electroplating plant19. Individual samples were measured for Ni and Cr at three factories in the electroplating industry, and the concentrations in air and urine were checked. Arithmetic mean (AM) and Standard deviation(SD) of Ni and Cr were 7.4 ± 6.4 and 3.0 ± 3.6 µg/m3 in the air. Additionally, creatinine concentrations were 30.1 ± 19.5 and 76.3 ± 54.5 µg/g in urine. Individual exposure was not very high in air, and the correlation with urine concentrations was weak20. The factors influencing individual Cr concentration in Cr plating factories were confirmed by regression analysis. The exposure coefficients for wearing/not wearing gloves, smoking, and exposure after recently had a skin disease were 1.85, 0.33, and 0.45, respectively. Washing hands before going to the bathroom reduced the Cr concentration coefficient to − 0.3221.According to Previous research, measured concentrations of TSP, Cr, Ni, Cu, Mn, Co, and Pb in the metal finishing industry were 0.32 ± 0.11, 109.9E-6 ± 17.3E-6, 65.3E-6 ± 88.9E-6, 144.5E-6 ± 143.6E-6, 46.2E-6 ± 25.8E-6, 35.9E-6 ± 22.0E-6, and 205.1E-6 ± 95.1E-6, respectively. Additionally, PM1 of Cr, Ni, Cu, Mn, Co, and Pb were 73.2 E-6 ± 11.7 E-6, 55.7 E-6 ± 78.0 E-6, 102.1 E-6 ± 103.5 E-6, 39.6 E-6 ± 20.9 E-6, 23.8 E-6 ± 22.6 E-6, and 161.6 E-6 ± 65.6 E-6 mg/m3, respectively. Although smaller particle size resulted in lower concentration, the adverse effect on health was greater at smaller particle sizes. Additionally, finer particles can move the alveoli of the lungs22. The diameter distribution of Cr (VI) less than 10 µm in the Cr plating plant was investigated, and the mass median diameters of Cr particles in the two electroplating tanks were 5.11 µm and 6.38 µm, respectively. The Cr (VI) diameter distribution in the general manufacturing industry was 1.67 µm. Therefore, the electroplating tank had a relatively high mass distribution23.At a metal finishing industry, PM2.5 of Cr, Ni, Cu, Mn, Co, and Pb were 86.7E3/s. On the other hand, the ventilation flow rate of individual hoods is relatively low. In other words, it can be inferred that the hood or duct is poorly engineered or maintained due to aging. The reason why the deviation increased in high concentration level is because we mainly evaluated TSP. Large particles are measured due to leakage from the ventilation problem, and high outliers were observed.The ventilation flow rate of the designed overall hood is 600 m24.Calculating the workload of the detailed process the precise amounts of chemical substances used in the electrolytes is difficult, because most materials are trade secrets. TSP was investigated in all the processes while making metal products. The quantitative composition of heavy metals particle size PM0.3 to PM10 using a real-time measuring instrument  found that smaller particle sizes correlate to higher quantitative composition concentration in all the processes; among these, a higher quantitative composition concentration was measured as the particle size decreased in degreasing, Cr plating, and Ni plating3 with a passivating bath process and 33,249 particles/cm3 without a passivating bath process based on 8 h TWA (Time-Weighted Average). Additionally, 5,645 and 4,947 particles/cm3 were generated when working with and without the rotary abraser bath process, respectively. Thus, further research on nanoparticles is important25.Checking the concentration of dust by particle size to provide information that can explain the extent of inhalation of smaller particles into the human body is necessary. A study for sampling nanoparticle size in electroplating revealed that the generation of nanoparticles were 64,327 particles/cmMost of the work environment measurement agency data at this workplace were undetected. Firstly, identifying the fundamental cause of the high concentration in the processes is difficult. Work environment measurement system is intensively focused on personal exposure. Obtaining a sufficient number of samples N of data is hard. Therefore, to confirm the reliability of work environment measurement system, conducting simultaneous personal and area exposure measurements would improve the results. suction flow rate was exceptionally low and difficult to collect when measured from a distance of 30 cm.3/s. In addition, ventilation plans must be taken to reduce indoor pollutants by adding an external ventilation system. Therefore, improving the suction flow rate of the hood is necessary.Few studies have measured and evaluated exposure to TSP and heavy metals related to anodizing and electroplating. In this study, we have confirmed that the exceptionally high concentrations of TSP and heavy metals are primarily caused by low ventilation suction flow rates. Concentrations were similar among pre-treatment and anodizing processes, especially high in the Cr plating process, and primarily high in the coloring and polishing processes among the post-treatment process. Concentrations in other processes remained relatively high. Although in most processes, the Al concentration was the highest, Cr concentration was high in the Cr plating and polishing processes. This study provides data on the risk of exposure for anodizing and electroplating and improves the reliability of work environment measurement data, thereby improving health through the need for fundamental exposure. Since the amount of work was also different every day for each process, this showed a large variation for each sample. The reason why the ventilation amount differs for each process is primarily due to the size of the hood. The amount of ventilation may vary depending on the size of the hood. Secondly, because of the aging of the hood, foreign substances in the duct connected to the hood and foreign substances in the hood slot have been formed over a long period of use, but they have not been cleaned or maintained. It can be a data that can raise the problem of a one-time work environment measurement agency data. Room humidity, temperature, and product characteristics were predicted the variables affecting date concentration.The ventilation volume of the hood should be increased to make the total volume similar to 600 mSupplementary Information.


Porphyrins, corrins, and tetrapyrroles constitute macrocycles in essential biomolecules such as heme, chlorophyll, cobalamin, and cofactor F430. The chemical synthesis as well as the enzymatic synthesis of these macrocycles starts from pyrrole derivatives. We here show that pyrrole and dimethyl pyrrole can be formed under the simulated volcanic, hydrothermal conditions of Early Earth, starting from acetylene, propyne, and ammonium salts in the presence of NiS or CoS as catalysts. Tetrapyrroles can be found in almost all modern domains of life with important functions, being mainly involved in respiration (heme), photosynthesis (chlorophyll), amino acid synthesis , and methane formation (cofactor F430) ,2,3.The basic structure of tetrapyrroles can be seen in Ludwig Knorr discovered in the 1880s that α-amino ketones (1) and a compound with an electron-withdrawing group next to a carbonyl group (2) yield the corresponding substituted pyrroles (Knorr pyrroles) (3) . Kle. KleLudw product .Paal–Knorr reaction, developed in 1884, is another example of the formation of a pyrrole derivative [The rivative ,8. Here,rivative .Trovimov et al.  acted as a putative transition metal catalyst for the reaction, and the molar variations of Na2S to NiSO4 resulted in free sulfide ions in the solution. For the formation of dimethylpyrrole, propyne was used instead of acetylene.All chemicals were purchased from Sigma-Aldrich GmbH  in the highest purity available. Acetylene 2.6 (acetone free) was purchased from Linde AG , and CO 2.5 and argon 4.6 were purchased from Westfalen AG . In a typical run run 1, , a 125 m × 10−22 ,24,25 in2SO4 solution (1 M), NaOH solution (1 M), and solid Ca(OH)2. For safety reasons (danger of explosion) and for technical reasons, the reactions were carried out at low gas pressure. Bottles were filled with ~1 bar at room temperature, leading to a total absolute pressure of ~2.5 bar at 105 °C. After the defined reaction time, the reaction mixture was allowed to cool down and transferred into a test tube. After centrifuging at 3000 rpm for 10 min, the supernatant was extracted with 3 × 1 mL ethyl acetate. The organic phases were dried over Na2SO4 and directly analyzed with gas chromatography–mass spectrometry (GC–MS). GC–MS analysis was performed with a GC-2010, coupled with MS-QP2010 Ultra  with a 30 m × 0.25 mm × 0.25 µm fused silica capillary column  and an AOC-20i auto injector. Additionally, 1 mL of supernatant was freeze dried and treated with 250 µL ACN and N-(tert-butyldimethylsilyl)-N-methyltrifluoracetamide each, and heated at 70 °C for about an hour.Reactions were carried out at 105 °C. pH variations were achieved through the addition of HTemperature program and settings for ethyl acetate phase: 0–6 min at 40 °C; 6–25 min at 40–280 °C, 10 °C/min; injector temperature, 260 °C; interface temperature, 260 °C; column flow rate, 1 mL/min; scan interval, 0.5 s; and injection volume, 1.0 µL.Temperature program and settings for silylated products: 0–6 min at 90 °C; 6–25 min at 90–310 °C, 10 °C/min; injector temperature, 260 °C; interface temperature, 260 °C; column flow rate, 1 mL/min; scan interval, 0.5 s; and injection volume, 0.5 µL.Peak assignment was achieved by comparison with the retention times and mass spectra of purchased reference compounds, as well as with data from the National Institute of Standards and Technology (NIST) spectral library. Pyrrole showed a retention time of 5.4 min; dimethylpyrrole a retention time of 8.8 min. Quantification was performed by external calibration using known concentrations of pyrrole and dimethylpyrrole, respectively. Runs without a transition metal compound or with argon instead of acetylene were performed for comparison.3 forms pyrrole , as shown by comparison with the reference material. The highest mass peak was observed at 67.0 u. A second characteristic peak was detected at 41.0 u, which belongs to the C-C=Nanalysis .4, 1 mmol Na2S, 1 mmol NH4Cl, 1 mL 1 M NaOH, 60 mL C2H2, 60 mL CO, 105 °C)  see  was set 4 and CoSO4 were used instead of NiSO4. The pyrrole formation in experiments using cobalt was about 50% compared to experiments with nickel. In the case of iron, no formation of pyrrole could be observed . When the amount of acetylene was increased to 120 mL (5.14 mmol), the yield of pyrrole decreased to 50% as compared to run 1, indicating that pyrrole formation was not limited by C2H2 in our setting.In the above-described setup, acetylene was present with a small molar excess compared to NH− and therefore a blockage of catalytic nickel sites. This is supported by runs, with Ni(OH)2 instead of NiSO4, under otherwise identical parameters, which show no formation of pyrrole . Notably, pyrrole formation could already be observed after the short reaction time of one hour, and showed its maximum after one day (24 h) . A longe4, NiOH, and NiS, only NiS supported pyrrole formation  instead of acetylene under experimental conditions, which led to the formation of pyrrole. Indeed, one of the products in this reaction was dimethylpyrrole. Dimethylpyrrole can be found at a retention time of 8.8 min. The typical masses were 95 u, which is the molecular peak, 94 u as M-1, and 80.0 u, which results from the loss of one methyl group . AccordiIn addition to pyrrole and dimethylpyrrole, further nitrogen-containing molecules were detected in trace amounts in single runs; for example, methylthiazole and pyridinol .2S and NiSO4, and after one day of reaction time. The conditions of our experiments were chosen to fit a scenario where life possibly emerged near volcanic exhalations [We here showed that pyrrole and dimethyl pyrrole can be abiotically formed from acetylene or methylacetylene, ammonium chloride, and metal sulfides under aqueous conditions at 105 °C. The best yields for pyrrole were achieved at a pH of 9.1, an equimolar concentration of Naalations  or hydroalations . Nowadayalations ,34. In o2 and water vapor [2 and CO. Ammonia, which does not fit in this highly oxidized atmosphere, could be formed out of NO3− due to reduction driven by FeS/H2S [2 and CO2 by electric discharges under oxygen-free conditions [The most common belief regarding the atmosphere on early Earth is that it mostly consisted of COer vapor ,36, with FeS/H2S . Nitratenditions  and subsThe yields of pyrrole and dimethylpyrrole in our experiments are comparatively low. For safety reasons, due to the danger of explosion, experiments were performed at low pressure. At sub-seafloor level (>1000 bar), yields would be increased ,40.The synthesis of pyrrole can be seen as a first step in the formation of larger porphyrin systems. A first achievement in this direction could represent the synthesis of dimethylpyrrole in our experiments when starting from propyne instead of acetylene. Different side chains could then be achieved by using the corresponding starting materials, either to enhance the reactivity of the molecule for polymerization, or to match the side chains of biological tetrapyrrole structures. As transition metal sulfides first served as catalysts in this reaction system, a later substitution of the sulfide through the newly formed pyrrole structures could be imagined for the evolution of the potent catalyst factor F430, cobalamin, and also at a later stage, when oxygen was available, hemoglobin and chlorophyll.


Both of the formulations prepared by solvent diffusion method exhibited a spherical shape under transmission electron microscopy (TEM) and similar zeta potential value of –11 mV. The particles size, encapsulation efficiency (EE), drug loading (DL) of REP-NLCs-Small and REP-NLCs-Large size preparations were about 79 nm and 325 nm, 96.83% and 98.60%, 4.41% and 3.05%, respectively. Besides, both REP-NLCs showed good colloidal stability and had no burst release phenomenon compared with REP-Sol. SPIP demonstrated the improved membrane permeability for NLCs compared with REP-Sol, especially NLCs-Small size preparation. The bioavailability increased sequentially in REP-Sol, REP-NLCs-Large, and REP-NLCs-Small, and the difference between each other was statistical significant. Our investigations demonstrate that NLCs with small particles size of 50–100 nm, such as 79 nm, are able to enhance absorption performance of a poorly soluble repaglinide compared with large particles size, such as 325 nm, by significantly improving the absorption in jejunum, and colon of rats and thus well improving oral bioavailability.Repaglinide-loaded nanostructured lipid carriers (REP-NLCs) with different particle sizes were successfully designed and prepared to investigate the permeation and absorption ability by  The former requires relatively electro-neutral, hydrophilic surface of particles as well as size less than 200 nm , is an oral medicine used to treat type 2 diabetes with very low aqueous solubility, poor oral bioavailability, and short terminal elimination half-life  technique was carried out utilizing gravimetric method to correct drug concentration for the first time. The prepared NLCs were characterized by particle size, polydispersity index, zeta potential, morphology, encapsulation efficiency (EE), and drug loading (DL), and besides, the colloidal stability of prepared NLCs in differently simulated fluids was also investigated. Finally, in situ SPIP was used to study the intestinal permeability of prepared NLCs and pharmacokinetics was carried out to evaluate their oral absorption performances in vivo in rats. This study demonstrates the possibility to overcome mucus and epithelium barriers meantime for orally administered NPs.In this study, different particles size repaglinide-loaded NLCs as a model of oral administration were prepared to investigate the effect of particles size on bioavailability and intestinal absorption. Besides, we prepare repaglinide-loaded NLCs using glyceryl monostearate and medium-chain triglyceride (MCT) as carriers for the first time and intestinal absorption investigation of repaglinide by 2.2.1.Repaglinide raw material was obtained from Tianjin Jinkang Pharmaceutical Ltd. . The commercial tablet of repaglinide was purchased from Novo Nordisk Co. . MCT was obtained from Lipoid GMBH . Glycerin monostearate and Tween 80 were purchased from Tianjin Bodi Chemical Company . Poloxamer 188 (F68) was provided by BASF SE . Sodium dodecyl sulfate (SDS) was obtained from Anhui Shanhe Pharmaceutical Excipients Co., Ltd. . Pepsin was purchased from Shanghai Macklin Biochemical Co., Ltd. . Trypsin was obtained from Dalian Meilun Biological Technology Co., Ltd. . All other reagents used in this study were high-performance liquid chromatography (HPLC) grade.2.2.Repaglinide-loaded nanostructured lipid carriers (REP-NLCs) were prepared by solvent diffusion method. Optimal amounts of MCT, glycerin monostearate, and repaglinide were dissolved in 3 ml (5 ml in formulation with larger size) ethanol at 75 °C under stirring. In addition, 10 ml aqueous solution containing 0.5% F68 (w/v) (1% Tween 80 in formulation with larger size) was heated to the same temperature with the same stirring rate. After some time, the organic phase was dispersed quickly in aqueous phase. The mixed liquid was stirred for 20 min (or 5 min in formulation with larger size) to get emulsion or pre-emulsion which was sonicated at 237.5 W (or 285 W with larger size) for 8 min (or 6 min in formulation with larger size) with 2 s/4s . Then, the solution was rapidly transferred into a brine bath with –20 °C to solidify the lipid droplets (stirring 25 min before this step in formulation with larger size).2.3.2.3.1.The particle size, zeta potential, and polydispersity index (PDI) were measured by a Zetasizer , which was based on a technique of dynamic light scattering.2.3.2.W1, W2, and W3 represent the number of REP encapsulated in the NLCs, the total amount of REP in NLCs suspensions, and the weighing of excipients, respectively.EE and DL of NLCs were evaluated by determining the total amount of REP in NLC suspensions and encapsulated REP in NLCs. 0.8 μm membrane was used to remove free REP from NLC suspensions. The content of total REP in NLC suspensions and the encapsulated REP in NLCs was determined after ethanol disruption and deionized water with equal volume dilution by HPLC. The EE and DL of the NLCs were calculated using 2.3.3.To investigate the colloidal stability of NLCs in different media, 200 μl of NLCs was added to 2 ml of different media including pH 1.2 hydrochloric acid, pH 6.8 phosphate buffer, pH 7.4 phosphate buffer, fetal bovine serum (FBS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF). The mixtures were incubated in a water bath at 37 °C with constant shaking. At 0, 2, 4, 6, 8, 12, 24 h, and the average particle size was determined by Zetasizer . In addition, the stability of REP-NLCs in seven days following storage at 4 °C was evaluated by measuring the particle size by Zetasizer . The morphology of REP-NLCs was observed by JEM-2100 transmission electron microscope .2.3.4.In vitro release of REP-NLCs and REP-Sol was investigated by dialysis bag diffusion technique. pH 7.4 phosphate buffer was utilized as release media, with 0.01% SDS (w/v) to achieve the sink condition. One milliliter REP-NLCs of 0.6 mg REP or REP-Sol with equal amount of REP dissolved by ethanol was dialyzed against 50 ml media using a dialysis membrane (MW cutoff 12–14 kDa) in water bath at 37 °C with constant shaking. At 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, and 24 h, dialyzed solution of 1 ml was withdrawn and the fresh media of same volume was added to maintain the volume. The concentration of REP was determined by HPLC.2.4.Sprague-Dawley (SD) rats were obtained from the Laboratory Animal Center of Shenyang Pharmaceutical University . All the animal experiments in this study were carried out according to the guidelines for the Care and Use of Laboratory Animals approved by the Institutional Animal Ethical Care Committee (IAEC) in Shenyang Pharmaceutical University. Before experiments, the rats were fasted for 12 h with free access to water.2.5.2.5.1.Twelve rats weighing 210–240 g were randomly divided into three groups. A single dose of 1.32 mg/kg commercial tablets of REP and REP-NLCs  was orally administered by an oral gavage by a stainless steel with a blunt end. At 0.083, 0.167, 0.250, 0.333, 0.500, 0.750, 1, 2, 4, 6, 8, and 12 h, blood of about 300 μl was collected from orbit and centrifuged at 13,000 rpm for 10 min to obtained plasma samples. The samples were stored at –20 °C until analysis.2.5.2.3PO4 solution and 100 µl deionized water were added. The mixture was vortexed for 1 min and extracted with 3 ml of n-hexane–ether absolute  by vortexing for 3 min. After centrifugation at 3500 rpm for 10 min, the organic phase was transferred to another tube and evaporated to dryness at 37 °C under a stream of nitrogen. The residue was reconstituted using 100 µl of the initial mobile phase and then vortexed for 10 min. A 5 µl supernate after centrifuging at 13,000 rpm for 10 min was injected into the UPLC–MS–MS system.One hundred microliters internal standard (IS)  and 100 µl methanol were added to 100 µl rat plasma sample. Then, 100 µl of 0.05 M H2.5.3.TM system  with a Phenomenex Kinetex C18 . A binary gradient elution was performed at a flow rate of 0.2 ml/min. The solvent A was methanol and solvent B was aqueous solution with 0.05% formic acid and 0.1% acetic acid. The mobile phase started at 60% of solvent A and kept 0.6 min and increased to 85% solvent A using 0.02 min, holding this state until 2.2 min. It was then decreased to 60% solvent A at 2.22 min and allowed to equilibrate at 60% solvent A to 3.4 min. The total run time was 3.4 min. The injection volume was 5 μl and the column temperature was 35 °C. Mass spectrometric detection was operated with a triple quadrupole detector with electrospray ionization in positive ion mode (ESI+) and quantitative analysis with multiple reaction monitoring (MRM). The ion pairs used for monitoring were 453.4 → 230.3 for REG and 324.3 → 127.2 for gliclazide (IS). The mass spectrometric parameters were optimized and set as follows: cone voltage: 40 V (REG) and 35 V (IS); collision voltage: 25 V (REG) and 19 V (IS); capillary voltage: 3.5 kV; source temperature: 120 °C; desolvation temperature: 350 °C; desolvation gas flow: 550 L/h; cone gas flow: 50 L/h.The concentration of REP in rat plasma was determined using a validated UPLC–MS/MS method after liquid–liquid extraction by ether absolute and n-hexane  with gliclazide as IS. The chromatographic separations were carried out on an ACQUITY UPLC2.5.4.The main pharmacokinetic parameters were calculated by DAS 2.1.1 software. The plasma concentration–time curves were obtained by origin 8.0 software.2.6.Ka) and effective permeability (Peff) of repaglinide were calculated according to the following equations:The previous SPIP studies about repaglinide are relatively infrequent except for Yin et al. investigating recirculating perfusion by phenol red to correct  and r is the radius of intestinal segment (cm).2.7.p < .05 was thought to be statistically significant difference, and **p < .01 was thought to be very significant difference.All quantitative data were shown as means ± standard deviation (SD). One-way ANOVA was used to evaluate differences among multiple groups in the experiments by variance analysis in the SPSS software . *3.3.1.We successfully prepared NLCs with different particles size by solvent diffusion method. Ethanol is nontoxic and is easily soluble and diffused in water with minimal interfacial tension. In our investigations, type of stabilizer, volume ratio of organic phase and aqueous phase, stirring time before sonication and sonication time could affect the particle size. Besides, the heated temperature with properly upper than melting point of solid lipid and low cooling temperature contributed to get small and stable particles. We had to note that the length of ultrasonic probe in solution and diameter of ultrasonic tube were important for particle size and homogeneity of particles. The NLCs with oleic acid as liquid lipid were instable and would precipitate after one day, which may be due to the incompatibility between oleic acid and repaglinide. We could not get stable colloidal solution no matter that the liquid lipid was oleic acid or MCT, and the stabilizers was Tween 80, F68, Span 80, lecithin, or their mixture with proper HLB value and different ratios.in vivo performances  due to the shrinkage of hydration layer probably, and besides, the distribution of particles size was homogeneous  of REP-NLC-Small size and REP-NLC-Large size were increased to 3.02-fold (p < .01) and 1.93-fold (p < .01). The Cmax of REP-NLC-Small size and REP-NLC-Large size was also significantly increased than marketed repaglinide tablets (p < .01 for both NLCs). Besides, compared with REP-NLC-Large size, REP-NLC-Small size showed significantly better bioavailability and Cmax improvement (p < .01). The three groups had no significant difference in Tmax and T1/2 (p> .05).The pharmacokinetic study of marketed tablet of repaglinide and REP-NLCs after single-dose oral administration was investigated in SD rats. The plasma concentration of repaglinide from high to low was REP-NLC-Small size, REP-NLC-Large, marketed repaglinide observing from 3.4.in situ intestinal perfusion is thought the most similar to in vivo conditions, such as innervation, intact blood supply, active enzymes and transporters , ileum (p < .01) and colon (p < .01) compared with REP-Sol. Ka and Peff values of REP-NLC-Large size showed significant difference and improvement in the ileum (p < .05) and colon (p < .05) compared with REP-Sol. There were no significant differences for two kinds of REP-NLCs and REP-Sol in duodenum. It was interesting that there was high absorption performance for REP-NLC-Small size among duodenum, jejunum, ileum, and colon, and besides, compared with REP-NLC-Large size, REP-NLC-Small size showed significantly high absorption performances in jejunum (p< .05) and colon (p < .01).The absorption performances of REP-NLCs and REP-Sol in duodenum, jejunum, ileum, and colon were studied by an SPIP study. The obtained n et al. . The Ka The results showed that NLCs could significantly improve intestinal absorption of REP, and REP-NLC-Small size demonstrated better intestinal absorption potential than REP-NLC-Large size, which might be due to passing through epithelium easily for small particle size except for good permeability of mucus due to lower negative-charge surface as well as hydrophilicity of two formulations. Considering that the better intestinal absorption may cause higher drug concentration in plasma, we can understand the corresponding pharmacokinetic results.2. The surface is covered with columnar epithelial cells, including enterocytes, M cells contained in Peyer’s patch, and goblet cells secreting substance composing mucus, except for buccal and rectal with stratified squamous epithelium  and large intestine (colon and rectum) has approximately 6 m long with different diameters in different segments (Rabanel et al., um Deli, . The majum Deli, . Tight jum Deli, . NPs thrum Deli, . It is kum Deli, . For endum Deli, . All of Ka and Peff value for REP-Sol, REP-NLC-Large, and REP-NLC-Small were significantly increased sequentially whether they were in jejunum or ileum or colon while had no significant difference in duodenum. Besides, the tightness of intercellular junction in jejunum and ileum is lower while in colon it is intermediate tightness and villous of small intestine increases surface area compared with colon without villous (Deli, Besides, considering the factor-particle size, the maximum uptake of enterocytes for particles size is 50–100 nm, while M cells can up to micrometer and ∼100 nm is more efficiently than larger size. Although M cell has only about 1% mucosal surface of intestine, its uptake ability predominates over enterocytes (Shakweh et al., us Deli, , which mThe result of better permeation in colon for NLC-Small size than NLC-Large size may give us an inspiration to prepare formulation with small particle size to cure colonic diseases to solve the problem of many drugs absorbed from upper gastrointestinal tracts.4.in vivo pharmacokinetics indicated that the bioavailability increased sequentially in REP-Sol, REP-NLC-Large, and REP-NLCs-Small, and the differences between each other were statistical significant. This study demonstrates the possibility to overcome mucus and epithelium barriers meantime for orally administered NPs and get possible candidate drugs formulation to cure colonic diseases by preparing low negative-charge NPs with 50–100 nm size.In this study, repaglinide-loaded NLCs with different particles size of 79 nm and 325 nm were successfully prepared. The two formulations were same at zeta potential, shape, colloidal stability, and had no burst release phenomenon. SPIP study using gravimetric method developed successfully to correct drug concentration demonstrated the improved significantly membrane permeability for REP-NLC-Large in Ileum and colon and REP-NLC-Small in jejunum, ileum, and colon compared with REP-Sol. Besides, intestinal permeability of NLC-Large and NLC-Small also had significant differences in jejunum and colon, and NLC-Small was significantly better. The