R-(+)-mephedrone and S-(−)-mephedrone. There are some preliminary data suggesting that the enantiomers of mephedrone may display enantioselective pharmacokinetics and exhibit different neurological effects. In this study, enantiomers of mephedrone were resolved via chromatographic chiral recognition and the absolute configuration was unambiguously determined by a combination of elution order and chiroptical analysis . A chiral liquid chromatography tandem mass spectrometry method was fully validated and was applied to the analysis of whole blood samples collected from a controlled intranasal administration of racemic mephedrone hydrochloride to healthy male volunteers. Both enantiomers showed similar kinetics, however, R-(+)-mephedrone had a greater mean Cmax of 48.5 ± 11.9 ng/mL and a longer mean half-life of 1.92 ± 0.27 h compared with 44.6 ± 11.8 ng/mL and 1.63 ± 0.23 h for S-(−)-mephedrone, respectively. Moreover, R-(+)-mephedrone had a lower mean clearance and roughly 1.3 times greater mean area under the curve than S-(−)-mephedrone. Significant changes in the enantiomeric ratio over time were observed, which suggest that the analytes exhibit enantioselective pharmacokinetics. Even though the clinical significance of this finding is not yet fully understood, the study confirms that the chiral nature, and consequently the enantiomeric purity of mephedrone, can be a crucial consideration when interpreting toxicological results.Mephedrone, which is one of the most popular synthetic cathinones, has one chiral centre and thus exists as two enantiomers:  R-(+)-mephedrone (R-MEPH) and S-(−)-mephedrone (S-MEPH) [Mephedrone (4-methylmethcathinone) is a popular synthetic cathinone which is structurally and chemically similar to other stimulant drugs, such as amphetamine . With a (S-MEPH) , as showEven though recreational use of mephedrone in Europe began in 2009, its synthesis was first described in 1929 . The maiEnantiomers usually differ in their biochemical activity which may result in one enantiomer being biologically active while the other may be inactive or may even produce adverse effects . To our In a recently published study describing detection of racemic mephedrone in human whole blood and plasma, mephedrone was detected for 6 h post administration and showed rapid absorption and elimination . Herein,R2 > 0.999 was achieved for both enantiomers in all three validation runs -methcathinone and R-(+)-methcathinone (S-(−)-methcathinone. Therefore, (S)-absolute configuration can be confidently assigned to the second eluting peak collected from (±)-mephedrone samples. Indirectly, this result also confirms that the first eluting peak of (±)-mephedrone is the R-(+)-mephedrone, a conclusion based on the structural homology between mephedrone and methcathinone.Alongside collected fractions, 1 mg/mL solutions of pure methcathinone enantiomers were analysed by circular dichroism (CD) spectroscopy. athinone B, fully In order to highlight individual changes in analyte concentration, ER, used to show the proportion of enantiomers in a mixture , was detEquation (1). Equation for calculating enantiomeric ratio.t-test, statistically significant changes from the expected value of 1.0 start to emerge at 45 min  and continue until 360 min . Calibration standards and quality control samples had mean ER ± SD of 0.992 ± 0.056 across all runs.ER is expected to be 1.0 if both enantiomers are present in a matrix at 1:1 ratio . ER calculated for each participant at each timepoint between 5 and 360 min is presented in Whole blood drug concentrations were fitted with a single-dose first-order elimination phase model and calculated pharmacokinetic parameters are summarised in 1/2 of 1.63 ± 0.23 h compared to 45 ± 11 min and 1.92 ± 0.27 h for R-MEPH, respectively. Differences in the area under the curve (AUC) and clearance (CL) were also observed. R-MEPH resulted in roughly 1.3 times greater mean AUC compared to S-MEPH and had lower mean clearance of 126 ± 22 mL min−1 kg−1 compared with 164 ± 29 mL min−1 kg−1 for the other enantiomer. Mean Vd was similar for R-MEPH (21.0 ± 4.5 L kg−1) and S-MEPH (23.1 ± 4.6 L kg−1).Both enantiomers showed similar kinetics but S-MEPH peaked earlier at 39 ± 8 min and had shorter tt-test was performed to check if the pharmacokinetic parameters obtained for R-MEPH and S-MEPH were significantly different. At 95% confidence level, Cmax, kel, t1/2, AUC, CL and Vd were all statistically different, with the two-tailed p-value of 0.001, 0.004, 0.011, 0.002, 0.005, 0.002, respectively. The only parameter which did not show statistically significant difference was Tmax .Paired max, had a greater AUC and a longer t1/2 than S-MEPH in each of the participants. These results are in agreement with previous research that found R-MEPH to be a predominant analyte in pooled human urine samples and in in vitro experiments performed in human liver microsomes [The whole blood concentrations of R-MEPH reached a higher Ccrosomes . In the crosomes . R-MEPH crosomes . “Streetcrosomes . Should kel of 0.006 ± 0.001 min−1 and nearly identical t1/2 of 2.12 ± 0.33 h for the racemic mephedrone and 1.92 ± 0.27 h for R-MEPH. Cmax of 101 ± 45.4 ng/mL was observed for racemic mephedrone, which was 2.26 times greater than the Cmax of S-MEPH and 2.08 times greater than the Cmax of R-MEPH.Recently published  mean racChanges in the ER over time showed whole blood samples collected between 45 and 360 min to have a statistically different ER from the value of 1.0. Differences in concentrations of S-MEPH and R-MEPH may be a result of pharmacokinetic processes occurring at different rates during drug absorption, distribution, metabolism or excretion . Some drEven though chiral analysis is considered important and sometimes mandatory for accurate interpretation of analytical findings , it is y3 hydrochloride (MEPH-d3), S-(−)-methcathinone and R-(+)-methcathinone were purchased as certified reference materials from Sigma-Aldrich . Racemic mephedrone hydrochloride in powder form used for the human administration was purchased from Chiron . The chemical structure, purity and racemic nature of the powder was confirmed by mass spectrometry, circular dichroism (CD) spectroscopy and nuclear magnetic resonance.Racemic mephedrone hydrochloride, racemic mephedrone-d® DAU High Flow  cartridges were purchased from Chromatography Direct .All solvents were HPLC grade unless stated otherwise. Methanol , acetonitrile (ACN), isopropyl alcohol (IPA), dichloromethane (DCM), sodium phosphate monobase, sodium phosphate diabase, formic acid, acetic acid and ammonium hydroxide  were purchased from Fisher Scientific . Diethylamine (DEA) and ethanol  were purchased from Sigma-Aldrich . Ultrapure water (18 MΩcm) was prepared on an ELGA Purelab Maxima HPLC water purification system . Xtrackt®  containing 12.5 mg of sodium fluoride and 10 mg of potassium oxalate (NaF/KOx).Drug-free whole blood was collected by trained phlebotomists into 5 mL VacutainersEthical approval for the collection of drug-free matrix was granted by the Research Ethics Committee at King’s College London (HR 16/17 4237).® containing NaF/KOx preservative at 0 min (before administration on Day 1), 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 2 h, 2.5 h, 3 h, 5 h, 6 h, Day 2 and Day 3. Samples were stored at +4 °C until analysis.All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Riverside National Research Ethics Service (16/LO/1342). Details of the administration study have been recently published . BrieflyWhole blood samples were collected from six participants but for the purpose of this study, samples from five participants (referred to here as M2–M6) were analysed. Whole blood samples from participant M1 were discarded due to stability concerns  as they v/v) at 160, 200, 400, 700, 1000, 2000, 4000 ng/mL. Working solution used for the preparation of the quality control (QC) samples at low (Low), medium (Med) and high (High) levels were also made in the same solvent at 200, 800, 3000 ng/mL, respectively. Internal standard (IS) solution, used for quantification of analytes in the samples, contained racemic MEPH-d3 at 50 ng/mL in MeOH:water (50:50 v/v).Working solutions, used for the preparation of the calibration curve, contained mephedrone only and were made in MeOH:water , half of their concentration was used for plotting calibration curves and assessing the accuracy of the QCs. Calibration curves were constructed by plotting concentration against peak area ratio .Calibration standards and QCs were prepared fresh on the day of sample analysis.v/v/v) and dried under nitrogen at 50 °C. Samples were then reconstituted with 100 µL of 0.1% DEA in EtOH:MeOH (20:80 v/v).Sample extraction has been described before . BrieflySample analysis was performed by liquid chromatography–tandem mass spectrometry using a Xevo TQ-S triple quadrupole mass spectrometer  coupled to an Acquity ultra performance liquid chromatograph system . Optimised MS conditions have been published elsewhere .3 were monitored using selected reaction monitoring (SRM) as detailed in ® AD-3 column  held at room temperature. The flow rate was 0.1 mL/min with 0.1% DEA in EtOH:MeOH (20:80 v/v) as mobile phase used in an isocratic mode. The total run time was 10 min.Mephedrone and MEPH-dR-(+)-methcathinone and S-(−)-methcathinone, were analysed by CD alongside fractions of mephedrone. Methcathinone differs from mephedrone by the absence of the 4-methyl group on the benzene ring  semi-preparative column . The separation was performed on Agilent HPLC 1050 system  equipped with a manual injector coupled to a diode array detector. The column was held at room temperature and 0.1% DEA in EtOH:MeOH (20:80 v/v) was used as mobile phase in an isocratic mode. An injection of a mephedrone solution prepared at 5 mg/mL was performed. To check the purity of collected fractions, fractions were diluted 1 in 10 with mobile phase and injected on the analytical CHIRALPAK® AD-3 column . Since one enantiomer is sufficient to establish the configuration of both, only one peak  was isolated and characterised. Pure fractions were then dried overnight under vacuum at room temperature, dissolved in MeOH and combined.To isolate pure enantiomers in milligram quantities, racemic mephedrone was injected onto a 250 × 10 mm, 5 µm LuxCD spectroscopy  was used to analyse the combined fraction. One-millimetre Quartz Suprasil rectangular cells  were used in the region of 450–180 nm. The instrument was flushed continuously with pure evaporated nitrogen throughout the experiment. The following parameters were employed: 2 nm spectral bandwidth, 1 nm stepsize and 1.0 s accumulation time per point. The CD spectra were solvent baseline corrected and measured at +23 °C. The CD spectra were smoothed using the Savitzky–Golay method for better presentation. Data processing was done using APL Prodata Viewer (version 4.2.15) and spectra were modified in Origin (version 6.0). The intensities are presented as ellipticity values (mdeg).Pharmacokinetic data were determined by using a non-compartmental pharmacokinetics data analysis software ).Cmax) and the time after dosing when it occurred (Tmax) were observed directly from the data. The elimination rate constant (kel) was calculated using a log-linear regression of the elimination phase, which was achieved by plotting analyte concentration versus time and using data points which produced r2 of at least 0.97. Elimination half-life (t1/2) was calculated as ln(2)/kel. Clearance (CL) was calculated as dose/AUC. Volume of distribution (Vd) was calculated as CL/kel. Both CL and Vd were adjusted for the individual’s weight and are reported here as apparent CL and Vd because absolute bioavailability of mephedrone is unknown.Peak concentration . The data set (n = 5) at each time point for the concentrations of mephedrone were normally distributed according to the D’Agostino–Pearson normality test .One sample t-test and paired Validation experiments determined selectivity, linearity, inter- and intra-day precision and accuracy, limit of detection (LOD), lowest limit of quantification (LLOQ), recovery, matrix effect and carryover. The method was validated for mephedrone according to the Food and Drug Administration guidelines  and recomax, had a greater AUC and longer t1/2.In this study, enantiomers of (±)-mephedrone were quantified in human whole blood samples from a controlled drug administration. Mephedrone exhibited enantioselective pharmacokinetics whereas R-MEPH reached a higher C


Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600 MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance.High pressure processing  is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including 10 was observed for all strains after 1 min 600 MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400 MPa for 1 min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains.None of the tested strains were tolerant to 600 MPa. A reduction of more than 5 logL. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes. Listeria monocytogenes is a well-known foodborne pathogen that may cause listeriosis, a severe infection with high hospitalization rate which can be fatal in humans and some animal species [L. monocytogenes is generally found in agricultural, aquacultural, and food processing environments [L. monocytogenes (absence in 25 g product). Food processing aimed at L. monocytogenes inactivation is challenging due to its pronounced ability to adapt to different environments, and to survive under various stress conditions [L. monocytogenes, and maintain a high quality of the product. Only very small or no quality changes, such as loss of color, nutritional value, flavor, and texture, have been reported for HPP products [ species . Althoug species . L. monoronments . Listerironments , 4. The ronments ), which nditions . In addiproducts  . Severaproducts . Dependiproducts . HPP hasproducts , meat [1products , cheese products , fish [1products  and vegeproducts .Cronobacter sakazakii (previously Enterobacter sakazakii) [Salmonella enterica [L. monocytogenes [L. monocytogenes strains, pressure tolerance varies between 300 and 500 MPa [L. monocytogenes strains. In this study, we selected 10 L. monocytogenes strains, isolated from either foods, food processing environments or clinical sources, to test their tolerance towards HPP at 400 and 600 MPa, compare their genomes, and investigate whether genetic traits may be associated with pressure tolerance.It has been shown that HPP has varying effects on different target organisms. For example, a pressure of 300 MPa is sufficient to inactivate most Gram-negative bacteria, while more than 400 MPa is needed for an inactivation of Gram-positive bacteria , 16. It kazakii) , 24 diffenterica , and L. ytogenes , 20. In  500 MPa . To our 10 colony forming units per milliliter (cfu/ml) of the treated samples was significantly larger  to 2.07 log10 cfu/ml for the most pressure sensitive strain (EGD-e). Similar results were obtained at 600 MPa. Here, the 10 strains exhibited an average log10 cell number reduction of 7.06 cfu/ml, with a range of 5.42 to 8.27 log10 cfu/ml  after pressure treatment at 400 and 600 MPa for 1 min showed that pressure tolerance differs between strains. The variance in log10 cfu/ml for treated samples was significantly lower than the control for all strains (p < 0.02), except for strain RO15 (p = 0.15) , comparing the pressure (400 MPa) treated samples of each strain to the control samples, showed that the mean logL. monocytogenes strains for which no genome sequences were available in public databases. Genomes of strains RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 were sequenced using Illumina MiSeq equipment and strain RO15 was sequenced using Pacbio RSII. Sequences of these seven strains were assembled and three additional strains  were used for comparative genome analysis. Assembly of PacBio long reads of strain RO15 resulted in one continuous 3,042,507 bp sized chromosome , and clIn addition to genome sequencing, transcriptome analysis using RNA-seq for strains RO15 and ScottA provided a basic view of transcriptional activity of the genomes. Differential expression analysis indicated that virulence genes and heat shock genes were upregulated after high pressure treatment in strain ScottA Table S.High PacBio sequencing coverage allowed us to analyze DNA methylation modification motifs in strain RO15 in addition to assembling the genome. Using the long read modification data, 11 methylated sequence motifs were detected in the genome Table . None ofWe predicted one type II cytosine-5 DNA methyltransferase gene (OCPFDLNE_00657), and three type II N4-cytosine or N6-adenine DNA methyltransferase genes  in the strain RO15 genome. In addition, genes for a type IV methyl-directed restriction enzyme (OCPFDLNE_00324) and type II restriction enzymes  were predicted. These predicted methyltransferases and restriction enzymes had significant  BLASTP hits in the REBASE protein sequences database [In the genome assembly we also identified a circular phage as an independent contig in strain RO15. While looking after phage-originating genome parts, prophage prediction revealed five prophage regions (10.7 kb to 47.9 kb sized) in the RO15 genome Fig. . ProphagL. monocytogenes, all 200 complete L. monocytogenes genomes in RefSeq database [L. monocytogenes strains, while prophages 1 and 5 were seen in the majority of the L. monocytogenes strains. The comparison indicated that these prophage regions were common locations of variance between L. monocytogenes strains.Genome alignment of the selected nine strains against the strain RO15 Fig.  showed ldatabase  were dow10 cfu/ml with the 400 MPa pressure treatment (Table addB gene (OCPFDLNE_02427) located in the chromosome encoding an ATP-dependent helicase.Annotation of CRISPR systems revealed that the RliB-CRISPR system was seen in all the strains, which is in line with previous RliB-CRISPR system studies , 31. IntListeria anti-CRISPR genes, i.e. acrIIA1, acrIIA2, acrIIA3, and acrIIA4 [acrIIA4 was not seen in strains MB5, RO4, and EGD-e, but the rest of the anti-CRISPR genes were present. RO15 contains two copies of acrIIA1  and acrIIA2 (OCPFDLNE_02582)  (OCPFDLNE_03068) was slightly different in barotolerant strain RO15 compared to the other strains  (OCPFDLNE_00980) amino acid difference was seen for strains 2HF33 and RO15  are closely related to each other and cluster separately from the serovar 1/2a strains in the phylogenetic tree. In addition, there was also a clear difference in accessory genome for serovar 4b strains compared to serovar 1/2a strains  against strain RO15 using Student’s t-test (Table SL. monocytogenes strains and found in some strains (Table STo test any significant association between pressure tolerance and (clusters of) genes in the accessory genome, we performed a pan-genome wide association analysis using Scoary v1.6.16 . Based oL. monocytogenes strains, compared their genomes and predicted genome features related to pressure tolerance.HPP is commonly used in the food industry to inactivate foodborne pathogens and spoilage organisms. Depending on the type of food product, up to ~ 600 MPa pressure is applied . It has 10 reduction in all selected strains, which is generally considered sufficient for food safety regulations. According to the Food Safety and Inspection Service (United States Department of Agriculture), 5 log10 reduction is considered a full lethality treatment for L. monocytogenes [L. monocytogenes strains tested [L. monocytogenes [Pressure treatment using 600 MPa for 1 min caused more than 5 logytogenes . This sus tested . Howevers tested , 40. Howytogenes . In thisORF2110 and ORF2819) and lack lmo0737 marker gene sequence as described by Doumith et al. [lmo0737 homologue was present suggesting that the serotype of strain RO15 is indeed PCR-serogroup 1/2a. The phylogenetic tree that we created based on the core genome showed that there is considerable genetic variation between serovars. Serovar 4b strains (ScottA and F2365) clustered separately from the other strains  were only seen in strains that contain inactivated CRISPR/Cas system . Since the existence of a self-targeting spacer indicates that the activity of the CRISPR/Cas systems is inhibited , it can s Table S. CRISPR/RNA-seq revealed the general transcriptome profile of strains RO15 and ScottA. Significantly upregulated heat-shock genes after pressure treatment (chaperone and clp protease genes) in ScottA Table S might inL. monocytogenes strains are more tolerant to pressure at 400 MPa [A previous study concluded that antibiotic resistant  400 MPa . Here, aIn this study we noticed that barotolerance is manifested at pressures lower than 600 MPa. Strain RO15 was identified as the most barotolerant strain for 400 MPa 1 min pressure treatment. Genome sequence of seven new strains and genome comparison of 10 strains revealed that the three most barotolerant strains have CRISPR-Cas genes and anti-CRISPR genes in their genomes. In addition, the average number of prophages was slightly higher in barotolerant strains compared to barosensitive strains. Furthermore, we have predicted 10 phage genes that might be related to pressure tolerance based on the pan-genome association test. Therefore, we conclude that prophages and inhibited prophage defense systems may be linked to pressure tolerance. The observation described above may argue for the use of phage cocktails as a pretreatment before HPP and will allow to reduce pressure and holding time.9 cfu/mL. Aliquots (10 mL) of the cultures were packaged in sous-vide plastic pouches and sealed without using vacuum, and 10 mL was transferred to 15 mL falcon tubes to serve as untreated controls.Strains Scott A (CIP103575) and EGD-e (CIP107776) were obtained from Centre de Ressources Biologiques de l’Institut Pasteur, Paris, France, and strain F2365 (LMG23356) from Laboratorium voor Microbiologie, UGent, Gent, Belgium. Strains RO4, RO15, AB120 and AB199 are from Dunarea de Jos University of Galati, Romania, and have been isolated in the Promise FP7 project either from food illegally introduced to Romania or from meat processing environments. All other strains were isolated from the environment as indicated in Table HPP was carried out using the QFP 2 L-700 . The cylindrical pressure vessel had 10 × 25.4 cm dimensions, 2 L capacity and 690 MPa upper pressure limit. Temperature of the pressure medium (water) was tracked with a K-type thermocouple located on the external surface of the samples. A holding time of 1 min was used, pressures of 400 and 600 MPa, and ambient vessel water temperature (20–22 °C). Due to adiabatic heating, water temperatures in the middle of the vessel at the end of pressure treatment had risen to 31–33 °C after the 400 MPa treatment, and 36–38 °C after the 600 MPa treatment. Straight after pressure treatment, 400 MPa pressurized and untreated samples were serially ten-fold diluted in TSBYE and plated in triplicate on tryptic soy agar with 0.6% yeast extract  by using a spiral plater . Pressurized samples at 600 MPa were additionally plated manually (100 μL) without being diluted. TSAYE plates were incubated at 37 °C for 48 h prior to counting the colonies and estimating bacterial inactivation. Two consecutive trials of the methodology were performed (Exp. 1 and 2).DNA of each strain was extracted from 5 ml of a culture grown overnight in BHI broth at 37 °C with aeration on a rotary shaker. Wizard Genomic DNA purification kit  was used according to the manufacturer’s instructions.L. monocytogenes strain RO15 was sequenced using PacBio RSII  and Illumina Miseq . Genomic DNA PacBio library was prepared according to the manufacturer’s Template Preparation and Sequencing Guide using DNA Template Prep Kit 2.0 and DNA/Polymerase Binding Kit P6. The Nextera XT protocol was used for the enzymatic genomic DNA fragmentation and Illumina sequencing library preparation. Pacbio reads were assembled using HGAP3 protocol [dnaA gene. Cutadapt v1.8.1 [protocol  in SMRTPprotocol  and finat v1.8.1  was usedt v1.8.1 . Short it v1.8.1 . AverageL. monocytogenes strain RO4, AB199, and AB120, Nextera DNA Library  preparation and for strain 2HF33, MB5, and C7, Nextera XT DNA Library preparation was performed. Paired-end sequencing was performed using Illumina Miseq. RTA v1.18.54 and bcl2fastq v2.17.1.14 were used for base calling, demultiplexing and converting data to fastq format. The Cutadapt v1.8.1 [For t v1.8.1  was usedt v1.8.1 . Spades t v1.8.1  with defFor both strain RO15 and ScottA, 10 ml of cells were grown in TSBYE until early stationary phase (each triplicated with new cultures). Samples were packed in sterile sous-vide pouches and packaged without applying vacuum. They were put in the refrigerator 30 min before pressure treatment. After treatment at 200 and 400 MPa for 2, 8 and 60 min at 20–22 °C, samples were transferred to sterile 15 ml Falcon tubes. From each sample, 0.5 ml were transferred to a 2 ml Eppendorf tube prefilled with 1 ml RNA Protect . They were vortexed, incubated for 5 min at room temperature, and frozen at − 80 °C. Cells previously stored in RNA Protect were pelleted by centrifugation for 10 min at 5000 g. RNA extraction was performed with NucleoSpin RNA kit  according to manufacturer’s instructions with some modifications in the cell disruption phase. The cell pellets were suspended with 700 μl RA1 buffer and 7 μl β-mercaptoethanol . Cells were then mechanically disrupted using Lysing Matrix B tubes  and FastPrep 24 tissue homogenizer  at 6 m/s for 3 × 30 s. Cells were rested on ice for 5 min between cycles. After spinning the cells briefly and transferring the supernatant to NucleoSpin filter column, manufacturer’s protocol was followed. Quantity and quality of RNA extractions were analyzed using the Agilent 2100 Bioanalyzer and RNA 6000 Nano kit .Ribosomal RNAs were removed from the total RNAs  with Ribo-Zero rRNA Removal kit for bacteria  according to manufacturer’s instructions with 1/3 (ScottA) or 1/2 (RO15) volumes of kit solutions. rRNA-depleted RNA was purified with RNeasy MinElute Cleanup Kit  according to modified protocol of the Ribo-Zero kit manual and eluted in 12 μl of RNA-free water. Eight μl of rRNA-depleted RNA was used to prepare RNA sequencing libraries with SENSE Total RNA-seq Library Prep Kit for Illumina  according to manufacturer’s instructions. In reverse transcription and ligation phase, incubation time was extended to 2 h. After second-strand synthesis, purification and size-selection of the libraries were performed with 13 μl of Bead Diluent and 27 μl of Purification Solution. PCR amplification program was slightly modified by increasing the denaturation times: from 30 to 60 s in the beginning, and from 10 to 30 s during the cycles; cycle number was increased to 40. Concentration of amplified libraries was measured with Qubit fluorometer and dsDNA HS assay kit , and size distribution visualized with Fragment Analyzer and High Sensitivity NGS Fragment Analysis kit . Aliquots of amplified libraries were pooled twice for both strains. After pooling, libraries were concentrated using Amicon Ultra 100 K columns . To remove fragments under 200 bp, size selection of ScottA pools was performed using BluePippin and 2% agarose gel cassette . In size selection of RO15 library pools, bead purification with 0.9 x AMPure XP beads  and PEG/NaCl precipitation on MyOne™ carboxylic acid beads  were additionally used. Concentration of the pooled libraries was measured with Qubit fluorometer, and libraries were sequenced with NextSeq 500 .Quality filtering and adapter trimming for RNA-seq reads was done using Trimmomatic v0.36  using thhttps://bigsdb.pasteur.fr/listeria/listeria.html). Antibiotic resistance genes were detected using Resistance Gene Identifier (RGI) tool with CARD database [Assembled genomes were annotated using Prokka v1.13  with defdatabase . Heuristdatabase . Whole gdatabase . The ANIdatabase .The pan-genome analysis was done using Roary Pan-genome Pipeline  with defAdditional File 1 : Supplementary Text 1.L. monocytogenes strain selection, including Table S1, Figure S1.Additional File 2 : Table S2. Gene count data and differentially expressed gene list for both strains RO15 and ScottA based on RNA-Seq.Additional File 3 : Table S3. Table shows Average Nucleotide Identity (ANI) (based on BLAST) calculation results of each strain. For strains ScottA and F2365 the ANI score with other strains was lower than 95. ANI score between ScottA and F2365 was higher than 95. For the other strains ANI scores between each other were more than 98.Additional File 4 : Figure S2. Genome comparison of 200 L. monocytogenes strains mapped against strain RO15. The genomes were aligned using BLAST and visualized as ring figures. a) Each ring represents the whole genome alignment to strain RO15 (100 most similar genomes (based on BLAST) were displayed in (a) for simplification), b) ring figure zoomed in the prophage 1 region, c) ring figure zoomed in the prophage 2 region, d) ring figure zoomed in the prophage 3 region, e) ring figure zoomed in the prophage 4 region, f) ring figure zoomed in the prophage 5 region. The Refseq identifiers of 200 L. monocytogenes strains can be seen in figure. Genome features can be seen in Table SAdditional File 5 : Table S4 and Figure S3. Table S4 shows identified antibiotic resistance genes and percentage identity of matching region in all strains using CARD database. Figure S3 shows average distance trees from alignments of protein sequences of identified antibiotic resistance genes.Additional File 6 : Table S5. T-test against RO15 log10 reduction.Additional File 7 : Table S6. Barotolerant strains specific genes based on pan-genome wide association analysis. Table shows genes that have p < 0.01 based on pan-genome wide association analysis for barotolerant strains. Gene IDs of strain RO15 was used for this table. Orthologs of these genes with 95% identity were also seen in strains MB5 and 2HF33. In addition, the list of other publicly available L. monocytogenes genomes, where these genes were seen, are also provided.


Ovarian cancer is difficult to diagnose early and has high rates of relapse and mortality. Therefore, the treatment of ovarian cancer needs to be improved. Recently, several studies have been conducted in an attempt to develop anticancer drugs from naturally derived ingredients. Compared to traditional chemotherapy, natural compounds can overcome drug resistance with lower side effects. Fucosterol, a phytosterol present in brown algae, reportedly possesses many bioactive effects, including anticancer properties. However, the anticancer effects of fucosterol in ovarian cancer remain unexplored. Therefore, we investigated the effects of fucosterol on progression in human ovarian cancer cells. Fucosterol inhibited cell proliferation and cell-cycle progression in ovarian cancer cells. Additionally, fucosterol regulated the proliferation-related signaling pathways, the production of reactive oxygen species, mitochondrial function, endoplasmic reticulum stress, angiogenesis, and calcium homeostasis. Moreover, it decreased tumor formation in a zebrafish xenograft model. These results indicate that fucosterol could be used as a potential therapeutic agent in ovarian cancer.  Ovarian cancer, a gynecological malignancy, has a high mortality rate . Most ovSeaweed is readily available worldwide and belongs to the marine plant group. Recent studies have focused on the biological and pharmacological activities of seaweeds, as well as their secondary metabolites, for the development of new drugs. Various bioactive compounds, such as phlorotannins, diterpenes, and polysaccharides, produced in seaweed have already been well examined; however, fucosterol, a plant sterol, needs further investigation . FucosteTherefore, we investigated the suppressive effect of fucosterol on the progression of ovarian cancer in vitro and in vivo. This study aimed to (1) identify the effects of fucosterol on cell viability and apoptosis in ovarian cancer cells (ES2 and OV90 cells); (2) investigate the regulatory effects of fucosterol on cancer cell progression and the production of reactive oxygen species (ROS) in ES2 and OV90 cells; (3) identify the fucosterol-mediated mitochondrial and endoplasmic reticulum (ER) function in ovarian cancer cells; (4) investigate the synergistic effects of fucosterol in combination with cisplatin or paclitaxel on cell apoptosis and angiogenesis; and (5) evaluate the effects of fucosterol on zebrafish embryos in vivo. Collectively, the present study presents the first evidence of the anticancer effects of fucosterol in human ovarian cancer cells, indicating the possibility of developing this compound as a novel therapeutic agent to inhibit progression in ovarian cancer. p < 0.001; ES2 cells) and 24.2%  in a dose-dependent manner, compared to non-treated cells  dose-dependently decreased the phosphorylation of cyclin D1 (CCND1), which is associated with cell-cycle regulation  significantly decreased the viability to 31.3%  and OV90  cells compared with vehicle-treated ovarian cancer cells, as assessed using flow cytometric analysis . The G0/G1 stage was decreased from 66.6% to 40.1% by fucosterol in OV90 cells, but a significant change was not observed in OV90 cells. In addition, the G2/M stage was decreased from 37.6% to 19.6% in ES2 cells and from 21.7% to 15.6% in OV90 cells A,B. Furtanalysis C,D.p < 0.001) and 328.1% (p < 0.01), respectively, in comparison with vehicle-treated ovarian cancer cells (p < 0.01) and OV90  compared with the control cells (p < 0.001) and 1770.0% (p < 0.001) in ES2 and OV90 cells, respectively, compared with vehicle-treated control cells. Next, we identified any alterations in the apoptotic protein expression induced by fucosterol , cleaved caspase 9 , and cytochrome c  in comparison with the DMSO-treated cells. To investigate the changes in intracellular and mitochondrial calcium levels, human ovarian cancer cells (ES2 and OV90 cells) were stained with Fluo-4 and Rhod-2, respectively, after the treatment with fucosterol, using both for flow cytometry analysis A,B. In ber cells A. Consisol cells B. We theol cells C. Fucostcosterol D–F. Westp < 0.01; OV90: up to 0.5-fold, p < 0.01), P70S6K , and S6  decreased gradually following treatment with various doses of fucosterol  , p-JNK , and p-P38  in ES2 and OV90 cells compared with vehicle-treated ovarian cancer cells  A–C. In aer cells D–F. Theser cells . In bother cells A. p-CCNDer cells B. Furtheer cells C. p-P70Ser cells D. P-S6 ser cells E. Fucoster cells F. These er cells G. Fucoster cells H. Overalp < 0.05; OV90: up to 4.2-fold, p < 0.01), ATF6α , p-PERK , GADD153 , p-eIF2α , and GRP78  compared with DMSO-treated control ovarian cancer cells . In ER-stress-induced cells, the unfolded protein response (UPR) proteins were activated in the ER transmembrane, well-known as ER stress sensors, including inositol-requiring enzyme 1α (IRE1α), activating transcription factor 6α (ATF6α) and PKR-like ER-resident kinase (PERK), growth-arrest- and DNA-damage-inducible gene 153 (GADD153), phosphorylated eukaryotic translation-initiation factor 2α (p-eIF2α), and glucose-regulated protein 78 (GRP78). In ES2 and OV90 cells, Western blot analysis demonstrated that fucosterol increased UPR signaling proteins, including IRE1α  mRNA expression was reduced by fucosterol. Additionally, co-treatment of fucosterol with cisplatin markedly decreased VEGFA expression compared to each treatment in ES2 and OV90 cells  and combined effects of fucosterol with the chemotherapeutic drugs, we performed Western blot analysis using the ovarian cancer cells A–C. In b90 cells D. In added cells E. Relatireatment F. Furtheol cells G. FLT-1 eatments H,I. Thesp < 0.001) and 39.4% (p < 0.001) in 60 and 100 μM fucosterol-treated ES2 cells, respectively. In OV90 cells, the ratio of tumor formation decreased to 60.4% (p < 0.001) and 38.5% (p < 0.001) with the 60 and 100 μM fucosterol treatments, respectively. Furthermore, we examined vascular formation in transgenic zebrafish (fli1:EGFP) treated with fucosterol to examine the anti-angiogenic effects in vivo  for 24 h. Notably, embryo viability and development were not affected at all fucosterol concentrations A. Moreov in vivo C. The vaResearch on the development of novel cancer drugs has significantly increased with the goal to enhance the survival rate in cancer patients. However, owing to the side effects of synthetic drugs and the recurrence of the disease, the development of anticancer drugs from new natural sources such as plants and marine algae is crucial . This isFucosterol has various biological activities, including anticancer, antidiabetic, antioxidant, hepatoprotective, anti-hyperlipidemic, anti-fungal, and blood-cholesterol-reducing activities ,8. The c2+ overload can affect the release of pro-apoptotic factors by damaging the mitochondria. In cancer cells, mitochondrial malfunction results in increased cytosolic Ca2+ levels by inducing apoptosis [2+ concentration in both the cytoplasm and mitochondria. In general, low levels of pro-apoptotic signals are detected in poor prognosis cancers [Conventional anticancer agents inhibit the cell proliferation of cancer cells in order to suppress the development of cancer ,23. In mpoptosis . The res cancers . Cleaved cancers . In our  cancers . As prev cancers . Activat cancers , and sup cancers . In our  cancers .ER and angiogenesis are important for the management of cancer progression. First, the ER plays a pivotal role in the synthesis, folding, modification, and transport of proteins . MitochoVEGFA, VEGFB, VEGFC, VEGFD, FLT-1, and FLT-4, were synergistically decreased by fucosterol in combination with cisplatin or paclitaxel. These results indicate that fucosteol suppresses angiogenesis and metastasis of ovarian cancer. In agreement with the results from angiogenesis-associated genes, vascular development was interrupted in response to fucosterol in the fli1 transgenic zebrafish embryo. Furthermore, fucosterol has no cytotoxicity in normal cell lines including normal colon fibroblast cell [According to the reports for clinical outcomes in ovarian cancer, most chemotherapeutic failures are based on angiogenesis-derived metastasized tumors and ovarian cancer as metastatic disease . Althougast cell  and normast cell , which cIn conclusion, the present study reported the first evidence of the anticancer effects of fucosterol in human ovarian cancer cells. To provide comprehensive mechanistic insight into the anticancer function of fucosterol in ovarian cancer cells, we provide a graphical illustration in Fucosterol  was purchased from the Cayman Chemical Company  and dissolved in dimethyl sulfoxide (DMSO) before use. The antibodies used in this study are listed in ES2 and OV90 cell lines were purchased from the American Type Culture Collection  and maintained as previously described .Cell Proliferation ELISA, BrdU Kit , was used for to evaluate cell growth. Briefly, the ES2 and OV90 cells were seeded in 96 well plates and then incubated for 24 h in serum-free 5A McCoy’s medium. Cells were then treated with various concentrations of fucosterol to a final volume of 100 µL/well. After 48 h of incubation, 10 mM BrdU was added to the cell culture and the cells were incubated for an additional 2 h at 37 °C.2 incubator and probed with mouse anti-human monoclonal PCNA at a final dilution of 1:100 (2 µg/mL). Next, the cells were incubated with goat anti-mouse IgG Alexa 488 at a dilution of 1:200 for 1 h at room temperature. The cells were then washed using 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and overlaid with 4′,6-diamidino-2-phenylindole (DAPI). For the primary antibody, the images were captured using an LSM710  confocal microscope.The effects of fucosterol on the expression of proliferating-cell nuclear antigen (PCNA) were determined using immunofluorescence microscopy. Briefly, ES2 and OV90 cells were incubated with or without fucosterol (100 µM) at 37 °C for 48 h in a CO5 cells) were seeded on 6 well plates and treated with fucosterol at different doses of 0, 20, 40, 60, 80, and 100 μM for 48 h at 37 °C in a CO2 incubator. The supernatants were removed from the culture dishes and the adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation, washed with PBS, and resuspended using 1× binding buffer at a dilution of 1 × 106 cells/mL. Next, 100 μL of the cell suspension (1 × 106 cells) was transferred to a 5 mL culture tube and incubated with 5 μL FITC Annexin V and 5 μL PI for 15 min at room temperature in the dark. After this, 400 μL of 1× binding buffer was added to a 5 mL culture tube. The fluorescence intensity was determined using the FACS Calibur (BD Biosciences).Fucosterol-induced ovarian cancer cell (ES2 and OV90) apoptosis was analyzed using the FITC Annexin V apoptosis detection kit I . Briefly, cells (5 × 105 cells) were seeded in 6 well plates and treated with fucosterol in a dose-dependent manner for 48 h at 37 °C in a CO2 incubator. The supernatants were removed from the culture dishes and the adherent cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA). The cells were collected by centrifugation, washed with PBS, and resuspended using 1 × binding buffer at a dilution of 1 × 106 cells/mL. Next, 100 μL of the cell suspension (1 × 106 cells) was transferred to a 5 mL culture tube and incubated with 5 μL of RNase A and 5 μL of PI for 30 min at room temperature in the dark. Subsequently, 300 μL of 1× binding buffer was added to a 5 mL FACS tube. The fluorescence intensity was determined using FACSCalibur (BD Biosciences).In ES2 and OV90 cells, fucosterol’s effects on the cell-cycle stages were evaluated using PI. The cells .Intracellular ROS production was estimated using 2′,7′-dichlorofluorescin diacetate , which is converted to fluorescent 2′,7′-dichlorofluorescin (DCF) in the presence of peroxides. ES2 and OV90 cells were detached with trypsin-EDTA, collected by centrifugation, and washed with PBS. The cells were treated with 10 µM DCFH-DA for 30 min at 37 °C. The cells were then washed twice with PBS and treated with different doses of fucosterol for 1 h at 37 °C in a CO2 incubator. Supernatants were removed from the culture dishes and adherent cells were detached with trypsin-EDTA. The cells were collected by centrifugation. Collected cells were resuspended using 3 μM Fluo-4 and incubated at 37 °C in a CO2 incubator for 20 min. The stained cells were washed with PBS and fluorescent intensity was analyzed using a flow cytometer (BD Bioscience).ES2 and OV90 cells were seeded on 6 well plates and incubated for 24 h in serum-free medium when the cells attained 70%-80% confluency. The cells were then treated with different concentrations of fucosterol for 48 h at 37 °C in a CO2 incubator. Supernatants were removed from the culture dishes and adherent cells were detached using trypsin-EDTA. The cells were collected by centrifugation, with the collected cells resuspended using 3 μM Rhod-2 and incubated at 37 °C in a CO2 incubator for 20 min. The stained cells were washed with PBS. The fluorescent intensity was analyzed using a flow cytometer (BD Bioscience).ES2 and OV90 cells were seeded on 6 well plates and incubated for 24 h in the serum-free medium when the cells attained 70%–80% confluency. Next, the cells were treated with different concentrations of fucosterol for 48 h at 37 °C in a CO2 incubator. Supernatants were removed from the culture dishes, and adherent cells were detached using trypsin-EDTA. The cells were collected by centrifugation. Collected cells were resuspended in a staining solution, which included 200 × JC-1 and 1 × staining buffer, and incubated at 37 °C in a CO2 incubator for 20 min. The stained cells were collected by centrifugation and washed once with 1 × JC-1 staining buffer. After washing, the cells were centrifuged once more and resuspended in 1 mL staining buffer. The fluorescence intensity was analyzed using FACSCalibur (BD Biosciences).Changes in the JC-1 MMP were determined using a mitochondrial staining kit (Sigma). ES2 and OV90 cells were seeded on 6 well plates and incubated for 24 h in the serum-free medium until 70% confluency was attained. Cells were then treated with fucosterol at various doses for 48 h at 37 °C in a COThe protein concentrations in whole-cell extracts were determined using the Bradford protein assay  with BSA as the standard. Proteins were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to nitrocellulose membranes. Blots were developed using enhanced chemiluminescence detection  and quantified by measuring the intensity of light emitted from correctly sized bands under ultraviolet light using a ChemiDoc EQ system and Quantity One software (Bio-Rad). Immunoreactive proteins were detected using goat anti-rabbit polyclonal antibodies against phosphorylated and total proteins at a 1:1000 dilution and separated using 10% SDS-PAGE. As a loading control, total protein and α-tubulin (TUBA) were used to normalize results for the detection of target proteins. Multiple exposures of each Western blot were used to ensure the linearity of chemiluminescent signals.Total cellular RNA was isolated from ovarian cancer cells (ES2 and OV90) using the Trizol reagent  and purified using an RNeasy Mini Kit  according to the manufacturer’s guidelines. The quantity and quality of total RNA were determined using spectrometry and denaturing agarose gel electrophoresis, respectively.Specific primers for human VEGFA, VEGFB, VEGFC, VEGFD, FLT-1, and FLT-4 were designed based on sequences in the GenBank database using Primer 3 (ver. 4.0.0). All primers were synthesized by Bioneer . Gene expression levels were measured using SYBR Green (Sigma) and a StepOnePlus Real-Time PCR System . The PCR conditions were 95 °C for 3 min, followed by 40 cycles at 95 °C for 20 sec, 64 °C for 40 sec, and 72 °C for 1 min using a melting curve program (increasing the temperature from 55 to 95 °C at 0.5 °C per 10 sec) and continuous fluorescence measurements. Sequence-specific products were identified by generating a melting curve in which the CT value represented the cycle number at which a fluorescent signal was significantly greater than the background, and the relative gene expression was quantified using the 2-ΔΔCT method. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the endogenous control to standardize the amount of RNA in each reaction.To confirm the toxicity of fucosterol, zebrafish embryos were treated with fucosterol at various doses. In brief, embryos were exposed to 0.003% phenylthiourea (PTU) for 14 h before fucosterol treatment to suppress pigmentation. The embryo shell was removed and then treated with various doses of fucosterol in 24 well plates for 24 h. After 24 h of treatment, viability and development were observed under light microscopy (Carl Zeiss). Pictures were obtained by fixing zebrafish embryos onto a glass slide with 3% methylcellulose (Sigma). For the angiogenesis assay, transgenic Fli1:GFP larvae were treated with fucosterol for 48 h, and then GFP expression patterns of 10 zebrafish larvae per condition were evaluated. Pictures were obtained using a fluorescence microscope .Xenograft studies were performed as previously described with modifications . Fertilip-value less than or equal to 0.05 was considered significant. Data are presented as least-square means (LSMs) with standard errors [All quantitative data were subjected to least-squares analysis of variance (ANOVA) using the general linear model procedures of the Statistical Analysis System . Western blot data were corrected for differences in the sample loading using total protein or TUBA data as a covariate. All tests of significance were performed using the appropriate error terms corresponding to the expected value to the mean squares for error. A d errors .


The aim of the study was to evaluate glucose metabolism, as measured by glycated hemoglobin (HbA1c) levels and the need for antidiabetic medical treatment, in patients with acromegaly resistant to first-generation somatostatin receptors ligands (SRLs) treated with pasireotide long-acting release (LAR) in real-world clinical practice. Biochemical control of acromegaly, as measured by growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels, was also assessed.Two-center retrospective cohort of consecutive patients with acromegaly treated with first-generation SRLs at maximum doses, who had not achieved biochemical disease control. After SRLs were discontinued, patients were given pasireotide LAR 40 mg i.m. every 28 days. The dose was increased to 60 mg i.m. in patients for whom adequate control was not achieved after 3 months. Patients were given dietary and lifestyle advice, and antihyperglycemic treatment was modified as needed.Biochemical disease control parameters (GH and IGF-1 concentration), as well as HbA1c level at baseline and after 6 months.me =-1.56 µg/L, range -21.38–3.62, p <0.001) during 6 months of pasireotide LAR treatment. A worsening of metabolic status was observed, with an increase of median HbA1c , and antihyperglycemic treatment intensification in 23 (59.0%) patients. The median decline in IGF-1 concentration was: -283.0 µg/L, range -682.7–171.6, p <0.001. IGF-1 reached the age- and gender-specific upper level of normal in 23 (59%) patients.In total, 39 patients with acromegaly were enrolled. GH concentration decreased (ΔPasireotide LAR is an effective therapeutic option in patients with acromegaly refractory to first-generation SRLs. However, this therapy may result in pasireotide LAR-associated hyperglycemia, which requires early and aggressive antidiabetic medical therapy to prevent glucose homeostasis alterations. Acromegaly is usually caused by pituitary adenoma secreting growth hormone (GH). Excess GH accompanied by increased insulin-like growth factor 1 (IGF-1) levels results in cardiovascular and metabolic complications. As the morbidity and mortality rate in acromegaly is significantly associated with IGF-1 and GH level, the aim of medical therapy is to ensure target IGF-1 level within age- and gender-specific normal ranges and to lower and normalize GH levels . BiochemThe treatment of choice is selective transsphenoidal adenomectomy performed by an experienced neurosurgeon. The average cure rate is 65%, but adenoma size, extrasellar extension, and cavernous sinus invasion are the most reliable factors predicting a complete tumor resection in individual patients , 4.First-generation somatostatin receptor ligands (SRLs) (octreotide or lanreotide) are used either as preoperative or adjuvant therapy. In patients for whom this treatment failed , pasireotide long-acting release (LAR) is a reliable second-line therapeutic option. Pasireotide LAR is a multireceptor-targeted SRL, binding to four of five somatostatin receptor subtypes SSTR 1,2,3,5), with the highest affinity for SSTR-5. Somatostatin receptors are also expressed in pancreatic islet cells; therefore, pasireotide LAR adversely affects glucose metabolism . This si,3,5, witThe aim of the study was to evaluate glucose metabolism, as measured by glycated hemoglobin A1c (HbA1c) levels and the need for antidiabetic medical treatment, in patients with acromegaly resistant to first-generation SRLs treated with pasireotide LAR in real-world clinical practice. Biochemical control of acromegaly, as measured by GH and IGF-1 levels, was also assessed.the National Health Fund. After discontinuation of treatment with SRLs, patients were given pasireotide LAR 40 mg i.m. every 28 days. The dose was increased to 60 mg i.m. in 24 patients (61.5%) for whom adequate control was not achieved after 3 months. Patients were given dietary and lifestyle advice, and antihyperglycemic treatment was modified when needed.Between January 2019 and June 2020, we enrolled all 39 patients with acromegaly treated with first-generation SRLs at maximum doses, for whom biochemical disease control had not been achieved. Patients were from two tertiary referral centers. A history of type 1 diabetes mellitus was the only exclusion criterion from the study. During the recruitment of our patients switching to pasireotide LAR was the only option of second line medical therapy in Poland as the pegvisomant or cabergoline were not reimbursed by The study protocol was reviewed and approved by the local Bioethics Committees.HbA1c level and biochemical disease control parameters (GH and IGF-1 levels) were analyzed at baseline (V0) and after 6 months (V6). Using the diabetes diagnostic criteria published by the World Health Organization , patientNormal glucose tolerance (NGT): fasting plasma glucose (FPG) ≤5.5 mmol/L and 120 min glucose <7.8 mmol/LPrediabetes (IFG or IGT): FPG ≥5.6 mmol/L or 120 min glucose ≥7.8 mmol/L but <11.1 mmol/LDiabetes: FPG ≥7.0 mmol/L or 120 min glucose ≥11.1 mmol/L.OGTT was not performed at V0 if the diagnosis of diabetes was known earlier . Additionally, at V6 OGTT was performed in all patients except for those with diabetes already diagnosed at V0 (n=4).Additionally, patients were categorized by their acromegaly biochemical control status, defined as:IGF-1 below upper level of normal (ULN) vs. IGF-1 ≥ULNGH concentration below 2.5 µg/L vs. ≥2.5 µg/L IGF-1 <ULN and GH concentration <2.5 µg/L vs. IGF-1 ≥ULN and GH concentration ≥2.5 µg/L.® . The linear range was 18–179 mmol/mol (3.8%–18.5%) and coefficient of variation (CV) <2.5%.HbA1c was measured using the ion-exchange high performance liquid chromatography (HPLC) method, certified by the National Glycohemoglobin Standardization Program (NGSP) and the International Federation of Clinical Chemistry (IFCC), and the Bio-Rad D-10 Hemoglobin Testing System® analyzer . GH was calibrated against the pure 22kDa recombinant growth hormone (WHO IRP 98/574). Functional sensitivity was 0.05 µg/L and limit of detection was 0.03 µg/L, respectively. Serum IGF-1 was measured by enzyme-linked immunosorbent assay . Analytical sensitivity was 9.75 µg/L. IGF-1 values were compared with age- and gender-standardized normal values.Random serum GH was determined using the electrochemiluminescence (ECLIA) immunoassay on the ElecsysDescriptive statistics were used to present the study population at baseline. For categorical variables, the number and percentage of occurrences were reported; for continuous variables, the following were provided: mean and standard deviation (SD), median and the range as well as the number of observations.S) and the Mann–Whitney exact test were applied to examine the relationships between changes in HbA1c and age, baseline GH or IGF-1 levels and gender.The dynamics of evaluated parameters were analyzed using the Wilcoxon exact test and McNemar–Bowker test. Spearman’s Rho correlation coefficient  patients were female Table 1.All but six patients had a history of one or two transsphenoidal procedures. Median GH level was 3.8 µg/L (range 0.6–27.3) and in its concentration was below 2.5 µg/L in 14 patients (35.9%). Median IGF-1 was 491 µg/L (range 216–1199), and in all patients IGF-1 exceeded the ULN (range 1.0–4.9).trend =0.053). Median HbA1c percentage at baseline was 5.60% (range 5.0%–7.5%). HbA1c correlated with age .Normal glucose tolerance was observed in 15 (38.4%) patients. In total, 20 (51.3%) patients were diagnosed as prediabetic and were given dietary advice or metformin or both . Diabetes was present in four (10.3%) patients Figure 1During the study period, the glucose metabolism status worsened  . The mean HbA1c percentage did not differ at 6 months between controlled and uncontrolled patients regardless of the criteria for control of acromegaly .After 6 months of treatment with pasireotide LAR median HbA1c increased by Δp<0.001) Figure 2During 6 months of pasireotide LAR treatment, antihyperglycemic therapy was intensified in 23 (59%) patients (p<0.001) Figure 3me =-1.56 µg/L, range -21.4–3.62, p=0.001) during 6 months of pasireotide LAR treatment  were receiving the 60 mg of pasireotide LAR, whereas remaining 15 patients (38.5%) the dose of 40 mg. The IGF-1 level within ULN and GH < 2.5 µg/L were confirmed in 23 (59.0%) and 21 (53.8%) patients, respectively, whereas both criteria were fulfilled in 16 (41%) patients Table 3.Pasireotide LAR is an effective treatment option for patients with acromegaly resistant to first-generation SRLs , 8. Our Our study confirms the increased risk of pasireotide LAR-associated impairment of glucose metabolism, which is well known and an expected adverse event of such a treatment, and has been observed in previous clinical trials , 7, 15. As hyperglycemia and diabetes are long-term risk factors for cardiovascular disease, early and effective management of this complication is important. Furthermore, proper management of hyperglycemia increases the chance of maintaining long-term compliance with pasireotide LAR .According to Samson , it is pIn the Colao et al. study, 44.4% of patients in the pasireotide LAR group received antidiabetic medications, with metformin and sulfonylureas the most commonly used . In our The mechanism of pasireotide LAR-related hyperglycemia needs to be understood to introduce optimal treatment . InsulinConsidering the above, it is essential to closely monitor blood glucose levels in patients treated with pasireotide LAR. Hyperglycemia should be managed with proper antidiabetic drugs. No specific recommendation has been published for acromegaly patients with hyperglycemia . HoweverWe have also found that the age of the patients positively correlated with HbA1c at baseline and that HbA1c was not affected by the acromegaly control status. This observation reflects the situation in the general population and is also consistent with the findings of the French Acromegaly Registry study .We analyzed the impact of 6 months’ pasireotide LAR treatment on acromegaly parameters and glucose metabolism. Some studies show that FPG and HbA1c levels rise rapidly for the first 3 months and then remain stable without further antidiabetic therapy , 23, 25.Our study was conducted in clinical practice in Poland where, for years, only the first-generation SRLs (octreotide and lanreotide) were reimbursed for treatment of acromegaly , 33. GH-We confirm that treatment with pasireotide LAR may result in carbohydrate metabolism disorders, which in most patients requires intensification of antidiabetic therapy. Therefore, all patients treated with pasireotide LAR should be given dietary and lifestyle advice and have glucose-related parameters closely monitored. These findings do not undermine our other observation that pasireotide LAR is an effective therapeutic option in patients with acromegaly refractory to first-generation SRLs. Additionally, if properly managed, it is also a safe therapeutic option.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies were conducted according to Good Clinical Practice guidelines and the Declaration of Helsinki. The studies involving human participants were reviewed and approved by Bioethics Committee at the Medical University of Warsaw, Warsaw, Poland. The patients/participants provided their written informed consent to participate in this study.PW and MB conceived the concept of the study. PW, MK, AW-L, KS, AJ-P, and MB contributed to the study design. PW, MK, and KS organized the database. KS performed the statistical analysis. PW, MK, KS, and MB wrote the manuscript. All authors contributed to the article and approved the submitted version.Recordati Rare Diseases. The funder provided medical editorial assistance and provided article processing fee. The funder had no role in the study design, data collection and analysis, preparation of the manuscript, or decision to publish.The National Health Fund covered the costs of treatment as well as laboratory and imaging tests for all study patients. The authors declare that this study received funding from 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.


Random skin flaps are often used in plastic surgery, but the complications of marginal flap ischemia and necrosis often limit their wider clinical application. Apigenin (Api) is a flavonoid found in various fruits and vegetables. Api has been shown to promote angiogenesis, as well as reduce oxidative stress, membrane damage, and inflammation. In this study, we assessed the effects of Api treatment on random skin flap survival. Dorsal McFarlane skin flaps were transplanted into rats, which were randomly divided into three groups: control , low-dose Api (20 mg/kg), and high-dose Api (50 mg/kg). Seven days after the surgery, the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) were measured. Histological analyses were performed to determine flap survival and tissue edema. H&E staining was performed to assess the histopathological changes in skin flaps, and the levels of microvascular density (MVD) were determined. Laser doppler flowmetry was used to assess microcirculation blood flow. Flap angiography was performed by injection of lead oxide/gelatin. The expression levels of vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interlukin-1β (IL-lβ) were evaluated by immunohistochemistry. Rats in the high-dose Api group exhibited higher average flap survival area, microcirculatory flow, increased SOD activity, and higher VEGF expression levels compared with the other two groups. Furthermore, the levels of MDA and pro-inflammatory cytokines were significantly decreased in rats treated with high-dose Api. Our findings suggest the potential usefulness of Api in preventing skin flap tissue necrosis. In contrast to axial pattern flaps, random skin flaps lack axial vascularization, due to anatomical differences in blood supply. Flap design and direction are not affected by the distribution of axial blood vessels; therefore, flaps are designed in a way to minimize tissue or organ dislocation during plastic surgery and cosmetic defect repairment. Since random skin flaps lack specific blood vessels, flap survival relies on blood supply from the pedicle muscle skin. Hence, low perfusion pressure in these tissues impacts flap survival, and marginal flap ischemia and necrosis, observed in 10–15% of cases , limit tThe development of interventions to prevent or treat skin flap necrosis remains an unmet clinical need. The length-to-width ratio of the flap should not exceed 2:1 because the lack of flap vascularization increases the risk of distal ischemia and necrosis. In the face and neck, the length-to-width ratio can be increased to 2.5:1, due to the good vascularization of these tissues. In random skin flaps, the scarfskin of the terminal necrotic area is characterized by marked inflammatory cell infiltration and coagulation necrosis . FurtherApigenin (Api) or 4′,5,7-trihydroxyflavone is a naturally occurring flavonoid, abundant in various vegetables, fruits, beans, and tea leaves, among which celery has the highest content. Api contains 4′ hydroxyl groups at positions 5 and 7, as well as a C2C3 double bond largely responsible for its unique physicochemical properties . AlthougDue to its anti-inflammatory, antioxidant, and pro-angiogenic effects, Api treatment has been considered as a method to improve random skin flap survival . In thisEthical approval for this study was provided by the Laboratory Animal Ethics Committee of Wenzhou Medical University, Wenzhou, China  on February 28, 2018 (no. WYDW2017-0509). Pathogen-free Sprague–Dawley male rats  were obtained from the Wenzhou Medical University Laboratory Animal Center. Api (purity ≥98%) was purchased from Solarbio life sciences . Api was dissolved in 4% (v/v) DMSO, and corn oil was added to achieve a concentration of 4 mg/ml.Rats (n = 60) were divided into three groups: high-dose (50 mg/kg) Api, low-dose (20 mg/kg) Api, and control . Rats were not fed the night before the experiment. The next day, rats were anesthetized by intraperitoneal injection of pentobarbital sodium saline solution (40 mg/kg). After back hair removal, the modified McFarlane (3 × 9 cm) flap was applied to the middle back of each rat . The pedFlap survival was macroscopically examined at days 1, 3, 5, and 7; changes in color, texture, tissue elasticity, hair growth, and tissue necrosis were recorded. Skin flap necrosis was defined as blackened color, stiff texture, shrunken tissue, reduced elasticity, and absence of bleeding after tissue puncture. The flap survival area was assessed 7 days after the operation by measuring the surviving area and total area of each flap using weighing paper and the weights of the flap using an electronic scale. Flap survival quantification was achieved using the following formula: flap survival area (%) = paperweight of the flap survival area/paperweight of the total flap surface area × 100.Rats were sacrificed 7 days after the operation by anesthetic overdose. Tissue samples (1 × 1 cm) were obtained from the proximal, middle, and terminal skin flaps , fixed in 4% paraformaldehyde for 24 h, and paraffin-embedded. Tissue sections were prepared, followed by dewaxing, dehydration, staining, and microscopic examination. The degrees of tissue edema, necrosis, and neutrophil infiltration were evaluated under a light microscope . Microvascular density (MVD) was used to assess angiogenic aggregation. Five optical fields were randomly selected for each tissue section, and the microvessel number in the cross-section was counted; the average number of microvessels per square millimeter area was calculated and the microvessel density per unit area was used to evaluate MVD.Seven days after the operation, rats were anesthetized and restrained. The blood flow in each area of the flap  was measured using a laser Doppler imaging system. Microcirculatory blood flow in the local tissues was expressed as perfusion units (PU).Rats were sacrificed 7 days after the operation by anesthetic overdose. Pre-warmed (37°C) isotonic saline was injected into the carotid artery to clean blood vessels, and the blood was drained through the jugular vein; body temperature was monitored throughout this process. Subsequently, gelatin/lead oxide solution (100 ml/kg) was injected into the carotid artery; perfusion was continued until the solution reached the limbs, ears, and corneas. The dorsal flap and cutis were dissected and subjected to X-ray angiography.Paraffin-embedded sections were stained using the Elivison two-step method. Sections were incubated with normal goat serum blocking solution and left to stand at room temperature (20–25°C) for 25 min. Tissue sections were then incubated with a mouse anti-rat VEGF primary antibody (1:100 in 50 μl) overnight at 4°C. Subsequently, sections were incubated at 37°C for 45 min, washed with phosphate-buffered saline (PBS), and incubated with a goat anti-mouse secondary antibody (1:50 in 50 μl) for 1 h at 37°C. After washing with PBS, tissue sections were incubated with diaminobenzidine for 10 min. Sections were observed under low-power and high-power light microscopes. Five optical fields per section were imaged and used for quantification of VEGF expression. The basic principle of field selection in the immunohistochemical analysis was to randomly select five fields that did not overlap under a 400× light microscope. Similar methods were used to detect the expression level of TNF-α,IL-6 and IL-1β.Blood was centrifuged at 5,000 × g for 15 min, serum was collected, and serum TNF-α and IL-6 levels were measured using an ELISA kit according to the manufacturer’s instructions. Optical absorbance was measured at 450 nm.Ischemia reperfusion injury typically occurs within 24–48 h of flap transplantation. The rats were sacrificed by anesthetic overdose 7 days after the surgery. Tissue samples (0.5 × 0.5 cm) were collected from the middle area (zone 2), where partial flap necrosis was observed. Samples were homogenized, weighed, and diluted in an ice bath to 10% of their initial concentration. The MDA level and SOD activity were measured according to the manufacturer’s instructions.t-test. p-values < 0.05 were considered statistically significant.Statistical analyses were performed using SPSS version 19 . Data were expressed as mean ± standard error of the mean (SEM). The mean values of different groups were compared using the Student’s p < 0.01).On postoperative day 1, all animals presented some degree of swelling near the incision edges of zone 3, accompanied by dark brown patches. However, there was no profound tissue swelling in zones 1 and 2. Seven days after the operation, we observed a demarcation line between the healthy area of the flap and the necrotic tissue. The necrotic tissue, especially in the distal flap, was dark and hard, with reduced tissue elasticity and granulation. Macroscopic examination revealed that the extent of necrosis was greater in the control group compared to that in Api-treated rats . The fla2) and low-dose Api group (22.81 ± 3.75/mm2) were significantly higher than that in the control group  and high-dose Api group (17.37 ± 5.83/mm2) was significantly lower than that in the control group  and high-dose Api group (345.65 ± 71.87 PU) compared with that in the control group . The levels of IL-6 in the high-dose Api, low-dose Api, and control groups were 819.83 ± 115.31 IA, 1,996.33 ± 230.22 IA, and 3,710.17 ± 225.38 IA, respectively (p < 0.01). Similarly, IL-1β levels in the high-dose Api, low-dose Api, and control groups were 721.67 ± 105.91 IA, 1,293.83 ± 239.67 IA, and 2,253.33 ± 373.67 IA, respectively (p < 0.01). The levels of TNF-α in the high-dose Api, low-dose Api, and control groups were 1,196.33 ± 159.29 IA, 2,402.5 ± 271.01 IA, and 4,223.00 ± 206.92 IA, respectively  and low-dose Api group (125.83 ± 18.65 pg/ml) compared with those in the control group  and high-dose Api group (68.52 ± 3.42 units·mg-1·protein-1) were significantly higher compared with that in the control group (24.81 ± 4.23 units·mg-1·protein-1; Api is a flavonoid found at higher levels in celery. Several lines of evidence have shown that it promotes tissue healing and angiogenesis, and prevents venous thrombosis. Additionally, Api can reduce oxidative stress and inflammation and has an excellent safety profile in humans . A previIn this study, we showed that intragastrical administration of Api in rats induced the expression of VEGF, promoting angiogenesis. Additionally, it reduced oxidative stress and tissue inflammation, as well as inhibited ischemia reperfusion injury in skin flaps, reducing tissue edema, and improving flap survival. Our team has been exploring methods to intervene in the necrosis of ischemic flaps and has found that azadirachtin A , dexmedeThe excision of a large tissue area from the donor is vital for sufficient blood supply in the skin flap and subcutaneous adipose tissue , which, Inflammatory responses also play decisive roles in the survival of random skin flaps. Necrotic areas at the distal flap are characterized by profound coagulative necrosis and inflammatory cell infiltration. Additionally, the extent of tissue necrosis has been associated with the degree of inflammatory responses . Hence, Mounting evidence suggests that Api has remarkable anti-inflammatory effects. Du H et al. showed that the Api-rich plant celery strongly suppressed inflammation in rat models, mitigating acute myocardial infarction . MoreoveIschemia reperfusion injury is another factor promoting ROS production and oxidative stress and is considered to be the leading cause of skin flap necrosis . As an aWe also found that Api upregulated VEGF expression. VEGF levels have been associated with vascular regeneration in skin flaps. Early during angiogenesis, VEGF promotes vascular endothelial cell proliferation and differentiation . PreviouCompared with other flavonoids, such as quercetin  and kaemWe conclude that Api improves flap survival, but the underlying mechanism remains to be elucidated fully. Based on existing research, we believe that this mechanism may be related to the TLR4 or caveolin-1/VEGF pathway, which we will consider in future research.Given its potent antioxidative, angiogenic, and anti-inflammatory capabilities, Api has potential applications for the treatment of ischemic flap tissue in rats. Further studies are necessary to determine the optimal dose and evaluate the safety of Api as a drug, as well as to yield a more detailed understanding of the drug’s mechanisms of action. This study was an experimental investigation performed exclusively in rats; whether Api can be applied to the recovery and reconstruction of human skin flap transplantation requires further investigation.


S. aureus and E. coli in both suspension and adhesion and its cytotoxicity over time (1 to 24 days). Results show that the AuNRs nano-functionalization improves the antibacterial activity with 100% bacterial reduction after 24 h. The toxicity study, however, indicates a 4.38-fold cell number decrease at 24 days. Although further optimization on nano-functionalization process are needed for cytotoxicity, these data indicated that Au-NRs nano-functionalization is a very promising method for improving the antibacterial properties of HA.Hydroxyapatite (HA) is the main inorganic mineral that constitutes bone matrix and represents the most used biomaterial for bone regeneration. Over the years, it has been demonstrated that HA exhibits good biocompatibility, osteoconductivity, and osteoinductivity both in vitro and in vivo, and can be prepared by synthetic and natural sources via easy fabrication strategies. However, its low antibacterial property and its fragile nature restricts its usage for bone graft applications. In this study we functionalized a MgHA scaffold with gold nanorods (AuNRs) and evaluated its antibacterial effect against  Bone tissue engineering (BTE) requires the use of biomaterials with adequate mechanical properties and mostly with specific features able to mimic the original tissue structure.Furthermore, the biomaterials must also provide, beyond biocompatibility, osteoconductivity, and osteoinductivity, a number of other factors such as good degradation rate, porosity, and surface characteristics, to improve cell adhesion, migration, proliferation, and differentiation ,2. HA isTherefore, some efforts are still necessary to obtain HA-based materials that are mechanically strong, chemically inert, adequately biodegradable, and especially capable of offering a good antibacterial activity.2O), zinc (Zn), zinc oxide (ZnO), titanium (Ti), titanium dioxide (TiO2), copper (Cu), copper oxide (CuO), and gold (Au) [Recently, several evidences focused on HA scaffold matrix functionalization with nanoparticles (NPs), including metal NPs, to improve physical–chemical, mechanical, and biological properties . Nanotecold (Au) . The addold (Au) ,15,16, aold (Au) , probes old (Au)  etc. Allold (Au) ,20. Moreold (Au) ,22,23. HThus, the combination of Au nanostructures with HA materials stimulated the design of a new scaffold with multiple properties, optimizing antimicrobial and chemical properties without any degradation of the HA porosity.S. aureus and E. coli in both suspension and adhesion, and its cytotoxicity.In this study we developed a new gold nanorod (AuNR)_MgHA scaffold, obtained by surface functionalization of MgHA with AuNRs, and evaluated its antibacterial effect against The AuNRs were synthetized according to the process described by Calabrese et al.  and are Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), both in suspension and in adhesion (9 at 24 h and (4.0 ± 0.4) × 1010 at 48 h for S. aureus, (1.2 ± 0.4) × 109 at 24 h and (2.6 ± 0.6) × 1010 at 48 h for E. coli, in suspension; whereas, in AuNR_MgHA scaffolds for both bacteria no colonies were found after 24 h. The number of suspended bacterial cells alone was similar to that of bacteria grown on MgHA scaffolds, (3.4 ± 1.0) × 109 and (4.2 ± 0.5) × 1010 CFU/mL at 24 h and 48 h, respectively, for S. aureus, and (1.8 ± 0.7) × 109 at 24 h and (2.4 ± 0.6) × 1010 at 48 h for E. coli.The antibacterial properties of AuNR_MgHA scaffolds were tested against adhesion A,B. Data4 at 24 h and (5.9 ± 0.7) × 105 at 48 h for S. aureus, and (8.7 ± 1.1) × 105 at 24 h and (3.4 ± 0.8) × 106 at 48 h for E. coli. On the other hand, in AuNR_MgHA scaffolds no colonies were found at both time points for both bacteria  × 10bacteria A,B.These results were in agreement with the live/dead staining B which sIn term of bactericidal mechanism, many works have reported on the significant antibacterial (against both Gram+ and Gram–) and antifungal activity of AuNPs . On the st to the 24th day, in a statistically significant manner (p < 0.01). In further detail, the MTS results of the AuNR_MgHA scaffolds show that cell viability decreased about 1.02-fold at day 1, 1.8-fold at day 7, 4.0-fold at day 14, and 5.2-fold at day 24, compared to the MgHA scaffolds  by MTS assay and DAPI staining. Data are reported in caffolds A. These caffolds B, displaThese results suggest that the AuNR_MgHA scaffolds are not able to stimulate hADSCs proliferation, indeed they reduce their growth probably due to their cytotoxicity. These data are consistent with a lot of in vitro studies on AuNP cytotoxicity, even though the mechanism of this is not yet clear . In factThe morphology of AuNR_MgHA with cells was analyzed by SEM. In Since the surface chemical properties of implanted materials are key features in their biological response, high-sensitive surface techniques are required for their characterization. X-ray photoelectron spectroscopy (XPS) is one of these techniques, which is widely employed for qualitative and quantitative analysis of biomaterials, probing the outermost atomic monolayers of their surface ,36. The 4 phase [XPS representative high-resolution spectra are reported in 4 phase .http://www.xpsfitting.com/search/label/Phosphorus, accessed on 17 February 2021). Furthermore, the C1s peak is characterized by specific features: the band at about 285 eV could be attributed to aliphatic C–C carbons of the amino acid side chains, the band located at about 285.5 eV could be due to C–N carbons of the main peptide/lysine side chains, while the peak located at 287.4 eV could arise from the N–C=O peptide bond and to O–C=O carbons of the glutamate side chains [These XPS profiles are relatively broad, because of the presence of a further oxide phase for P2p peaks and the presence of carbonates for the Ca2p peaks  and produced according to the process described in [The AuNR_MgHA scaffold was obtained by a synthetic process reported in . AuNRs wribed in . To prep2 window energy-dispersive X-ray (EDX) spectrometer, was used. UV–Vis optical absorption spectra were recorded with a Jasco V-560 homemade photoreactor equipped with UV light .Transmission electron microscopy (TEM) analysis was performed using the bright field in conventional TEM parallel beam mode. An ATEM JEOL JEM-2010 , equipped with a 30 mmMorphological analyses were acquired both by a Philips XL-30-FEI SEM  and a Zeiss EVO MA10  equipped with an LaB6 electron gun, at the operating voltage of 20 kV. Phases and structures present in the synthesized samples were investigated by X-ray diffraction (XRD) analysis. XRD patterns were recorded, in the 2θ range from 20° to 80°, using a Bruker D8 advance X-ray diffractometer  with Cu Kα radiation (1.5406 Angstrom).The chemical composition and the bonding configurations of the scaffolds were investigated by means of X-ray photoelectron spectroscopy (XPS). The spectra were acquired at room temperature using a Thermo Scientific K-Alpha system, equipped with a monochromatic Al Kαsource (1486.6 eV) and operating with a hemispherical analyzer operating in constant-pass energy (CAE) mode. The pass-energy was set at 200 eV for survey scans and at 50 eV for the XPS core-level spectra. A spot size diameter of about 400 μm was adopted while a charge neutralizer (low energy e-beam 0.2 V at 100 µA) was used for charge compensation. Binding energy shifts were calibrated keeping the C1s position fixed at 284.5 eV.S. aureus (ATCC29213) and E. coli (ATCC19138) were purchased from the American Type Culture Collection , cultured in Tryptone Soya Broth  and Luria-Bertani Broth , respectively, and maintained in 20% glycerol at −80 °C.All scaffolds were decontaminated before being used, through the use of a UV lamp for 12 h.S. aureus and E. coli overnight cultures were inoculated in fresh medium and incubated for 6–8 h at 37 °C under shaking at 150 rpm. Then bacteria were centrifugated and resuspended in phosphate-buffered saline (PBS) three times. Starting from this bacterial solution having an equivalent turbidity (McFarland standard) of 0.5 (approximately 2 × 108 CFU/mL), serial dilutions were made to finally obtain 2 × 105 bacteria/mL in TSB (Tryptic Soy Broth) medium.Bacterial semi-exponential cultures were used for all bactericidal assays. Specifically, Bacteria suspension was added to the pre-decontaminated scaffolds in 24-well plates (1 mL/well), and incubated at 37 °C under gentle shaking . MgHA scaffolds were used as positive control of normal bacterial growth.S. aureus and E. coli (after 24 h and 48 h) were placed in a new well, washed three times in PBS to remove non-adherent bacteria, placed in a glass tube containing 1 mL of PBS, and sonicated for 60 s in an ultrasonic bath (frequency 35 KHz). After, bacteria suspensions were diluted 1:10 in PBS and 100 μL of each dilution were spread on agar medium and incubated overnight at 37 °C. Plates in the range of 30–300 colonies were evaluated for CFU count by the following Equation (1):The antibacterial effect of the scaffolds was evaluated both in suspension and in adhesion, after 24 and 48 h of incubation by Colony Forming Units (CFU) assay. For bacteria in adhesion, the scaffolds incubated with Live/dead BacLight bacterial viability assay was also performed as follows: 6 μL of dye, SYTO 9 and propidium iodide (1:1), were added to 200 µl of bacterial suspension and incubated for 15 min at 37 °C . The sam2 until about 70–80% confluence was achieved. The hADSCs at the third passage (P3) were used for the following experiments.The cell line hADSCs  used in this study has been derived from human lipoaspirate tissue and cryopreserved from primary cultures. hADSCs were chosen because they give rise to bone cells under appropriate conditions, as we previously demonstrated, both in vitro and in vivo ,42. The 6 cells diluted in 50 μL of culture medium were slowly seeded onto the scaffold surfaces, placed in 24-well plates, and incubated at 37 °C for 4 h. After 4 h, the culture medium was added until the scaffolds were completely covered and maintained in culture for 24 days. The medium was wholly substituted twice a week, and the scaffolds analyzed at specific time points  through DAPI  staining.1 × 10For MTS assay we added the MTS solution to the cell culture media and incubated for 2 h in standard culture conditions. After briefly shaking the plate, the absorbance at 490 nm was read using a synergy HT plate reader .DAPI staining was performed as follows. hADSCs-AuNR_MgHA scaffolds, at several time points, were fixed in 4% PFA, dehydrated, embedded in paraffin, and cut into 5 μm-thick sections. Sections were permeabilized in 0.3% Triton X-100 for 10 min, washed in phosphate-buffered saline (PBS), and the nuclei stained with DAPI  in PBS for 5 min. Successively, the slides were mounted, with fluorescent mounting medium , and digital images were acquired using a Leica DMI4000B fluorescence microscope. Four sections from each scaffold were analyzed and the nuclei counted using Fiji image J recognition software.p < 0.05 was considered to be significant.Data were analyzed either as raw data or as mean ± standard error (SE), as appropriate. Differences between several time points of the hADSCs-AuNR_MgHA scaffolds were evaluated by using one-way ANOVA with a post-hoc Tukey HSD test, where appropriate. S. aureus and E. coli and its cytotoxicity over time (1 to 24 days). Our findings show that AuNR_MgHA scaffolds are already able to reduce the bacteria both in suspension and adhesion by 100% after 24 h, are showing good antibacterial properties against both bacterial strains. On the contrary, the cytotoxicity results demonstrated a noticeable cell number reduction over time, with a 4.38-fold decrease demonstrated from day 1 to day 24, suggesting that AuNR functionalization induces a cytotoxic effect on hADSCs. The cytotoxicity findings are in accordance with some previous literature evidences which report on how the toxicity of AuNRs on different cell models may be due to several factors, such as size, shape, surface area/porosity concentration, surface charges, aggregation, surface modifications, and host–cell interactions. In the light of this evidence and concerning the relevant antibacterial activity of AuNR_MgHA scaffolds, our future studies will be directed towards better understanding the AuNR cytotoxicity mechanism. Therefore, AuNRs of different sizes, shapes, concentrations, and surface modifications will be tested on several cell lines in order to improve their biocompatibility. Additionally, it will be important to understand the long term behavior of AuNPs in vivo before their application in BTE.In this work we developed an AuNR_MgHA scaffold, through the surface functionalization of MgHA with AuNRs, and tested it for both its antibacterial effect against