Hydrogels, such as fibrin, offer a promising delivery vehicle to introduce cells into the intervertebral disc (IVD) to regenerate damaged disc tissue as a potential treatment for low back pain. However, fibrin lacks key extracellular matrix (ECM) components, such as collagen (Col) and hyaluronan (HA), normally found in native nucleus pulposus (NP) tissue. The overall aim of this work was to create a fibrin-based hydrogel, by incorporating Col and HA into the matrix to enhance NP-like matrix accumulation using articular chondrocytes (CC). Firstly, we assessed the effect of fibrin concentrations on hydrogel stability, and the viability and proliferation kinetics of articular chondrocytes. Secondly, we investigated the effect of incorporating Col and HA to enhance NP-like matrix accumulation, and finally, examined the influence of various HA concentrations. Results showed that increasing fibrin concentration enhanced cell viability and proliferation. Interestingly, incorporation of HA promoted sGAG accumulation and tended to suppress collagen formation at higher concentrations. Taken together, these results suggest that incorporation of ECM components can enhance the bioactivity of fibrin-based hydrogels, which may help advance the clinical potential of commercial cell and biomaterial ventures in the treatment of IVD regeneration. Low back pain (LBP) is one of the most prevalent pathologies worldwide , placing® chondrospheres). However, it has been shown that when cells are directly injected into the target site, cell leakage and increased cell death due to shear forces may occur, inhibiting treatment success [These challenges have motivated the exploration of various cell types, including chondrocytes ,7 and st success ,12,13,14 success ,18,19,20 success ,23,24,25Fibrin is perhaps one of the most clinically used hydrogels, and has received particular attention as a sealant and adhesive in surgery ,27,28. FIn addition, incorporation of extracellular matrix (ECM) constituents into fibrin gels, such as collagen and hyaluronic acid (HA), have been shown to enhance matrix deposition by chondrocytes ,34. CollIn the present study, we investigated fibrin hydrogels of different compositions in an attempt to identify a suitable composition to promote NP-like extracellular matrix accumulation that may be compatible with commercial ventures. The first objective explored the effects of fibrin concentration  on hydrogel stability and the viability and proliferation kinetics of articular chondrocytes. We next sought to enhance the bioactivity of fibrin-based hydrogels by incorporating key matrix components (Col and HA), and further investigated the effect on matrix formation by articular chondrocytes. Finally, we examined the influence of incorporating different HA concentrations (2.5 and 5 mg/mL) into fibrin-based hydrogels.At the initiation of culture, all constructs possessed an average diameter of 5.46 mm (±0.19) a,b. AfteFibrinogen concentration was observed to significantly influence cell proliferation c,d. For To assess whether incorporation of ECM components was retained after encapsulation, acellular hydrogels were evaluated over 21 days. All hydrogel formulations maintained their shape a and iniCell viability and proliferation was investigated using a combination of semi-quantitative confocal image analysis and biochemical quantification of DNA content. From confocal images of live/dead staining a, cell vCell morphology was investigated using fluorescence staining of actin with phalloidin and nuclei staining with DAPI. Results revealed that incorporating hyaluronic acid promoted cell spreading immediately after cell encapsulation into the material a—top. HoMatrix deposition was analysed through biochemical analysis and histological staining for sGAG and collagen. Total sGAG accumulation was higher in both FH (250.2 µg ± 23.5) and FCH (233.8 µg ± 45.6) hydrogels compared to materials without HA incorporation (169.9 µg ± 12.7 in F group and 182.4 µg ± 19.4 in FC group), a. On a pThe highest amount of collagen was accumulated by cells encapsulated in an FC double matrix, and found to be higher compared to the FCH triple matrix  a. On a pLive/dead images were captured at day 0 and day 21, showing good viability at the onset of culture, with increasing density of green fluorescent cells in FH-5 group after three weeks of culture This group also exhibited the highest degree of contraction after 21 days (27.8% ± 3.7%) compared to both FH-2.5 (3.5% ± 3.1%) and F (3.3% ± 3.7%) groups a. Semi qp < 0.001) (p < 0.05). However, no significant differences were detected between groups on a per cell basis when normalising the total collagen to DNA content (p < 0.001) , compared to all other groups (< 0.001) a,b. In a content c,d. The < 0.001) e.Histology revealed higher sGAG content in the periphery of constructs containing hyaluronic acid f—left. OVarious cell types have been successfully encapsulated into fibrin hydrogels, such as chondrocytes, stem cells, and fibroblasts ,39,40,41Lower fibrin concentrations (12.5 mg/mL) resulted in significant contraction, which was minimised for concentrations at or above 25 mg/mL. Similar observations have been reported by Eyrich et al., who found that a final fibrin concentration of 25 mg/mL or higher, a calcium concentration of 20 mM and pH between 6.8 and 9 produced a stable, transparent gel for up to three weeks . The conCell proliferation was found to be enhanced for increasing fibrinogen concentrations. Previous work has reported reduced permeability with increasing concentration, which would be indicative of a diminished nutrient supply for encapsulated cells , and mayFor lower fibrin concentrations, increased cell death was observed, which could be a result of the increased contraction due to lower fibre density . Being sThe enhanced proliferation of chondrocytes in fibrin gels may also be due to increased cell–matrix interaction of fibrin with CD44 surface receptors ,49. CellIncorporation of collagen into fibrin-based hydrogels enhanced total collagen accumulation after three weeks in culture, compared to all other groups. Specifically, we observed enhanced col1 deposition at the periphery of hydrogels and enhanced levels of collagen type II within the pericellular matrix (PCM) of chondrocytes. It has been shown that fibrin can interact with CD44 and the integrin αvβ3. αvβ3 is an integrin which has no intracellular effect on chondrocytes when present or knocked out, indicating no major role when attachment of chondrocytes occurs . The affIncorporation of hyaluronic acid into fibrin–collagen matrices appeared to suppress levels of total collagen and increased levels of sGAG, possibly due to the activation of CD44 receptors. Collagen–cell interactions mainly activate pathways involved with cell adhesion, cell migration, and stress fibre assembly in AF and NP cells, which are not primarily involved in matrix deposition . MahapatIn the final stage of this study, the effect of different amounts of hyaluronic incorporated into the fibrin matrix was investigated. Previous work has investigated the effect of different concentrations of HA (0.1–3.0 mg/mL) on chondrocytes in alginate hydrogels . The autAnother possible explanation for the observed enhancement of sGAG with higher concentrations of HA is due to a physical change in hydrogel network, rather than a cellular activation alone. HA is inherently difficult to retain in hydrogel systems, and easily diffuses into the surrounding media , which wThis study consisted of three different stages . In all Streptococcus equi , and collagen type I, rat tail high concentration .The following components were used for the fabrication of different hydrogel compositions: fibrinogen type I-S , thrombin from bovine plasma , hyaluronic acid sodium salt from 2, pH 7) and allowed to gel  in 10,000 KIU/mL aprotinin solution  containing 19 mg/mL sodium chloride at 37 °C, and crosslinked using a pre-warmed thrombin solution . Thrombin (5 U/mL) was added and crosslinked using pre-warmed fibrinogen at a specified concentration to obtain a final concentration of 50 mg/mL fibrinogen. For fibrin–collagen–hyaluronic hydrogels, a thrombin–hyaluronic acid mix  was combined with neutralised collagen in a similar fashion as described for fibrin–collagen hydrogels, and crosslinked using pre-warmed fibrinogen.Specific fibrin-based hydrogels  were fabricated at 37 °C, and allowed to crosslink for 60 min in a humidified atmosphere inside a 3% agarose mould see . To creaFibrin–hyaluronic acid hydrogels were created as described in the previous 3 cells/cm2 in media consisting of low-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum (FBS), penicillin (100 U/mL)–streptomycin (100 µg/mL) , and amphotericin B  at 37 °C and 5% CO2, and cultured until passage 1 or 2.Juvenile porcine chondrocytes (pCC) were isolated from articular cartilage of 4-month-old female porcine donors (~50 kg). Tissue was finely minced and digested in collagenase for 12 h at 37 °C. Digested tissue/cell suspensions were passed through a 40 µm cell strainer to remove tissue debris, and washed three times by repeated centrifugation at 650 g for 5 min. Cell viability was determined with a haemocytometer and trypan blue exclusion. Cells were seeded at an initial density of 5 × 106 cells/mL. This was combined with thrombin solution (1:1 ratio) and allowed to gel in an 3% agarose mould, pre-soaked in cell culture media to ensure nutrient supply for cells for 30 min at 37 °C to produce cylindrical constructs (Ø5 mm × 3 mm thickness), and a final cell seeding density of 4 × 106 cells/mL.For fibrin encapsulation, pCCs were suspended in fibrinogen solutions  at a cel6 cells/mL at matrix concentrations, described in For encapsulation, pCCs were suspended in pre-warmed fibrinogen solution and combined with collagen–thrombin or collagen–HA–thrombin solution, respectively, to obtain a final cell seeding density of 4 × 106 cells/mL, and allowed to gel in a 3% agarose mould pre-soaked in media for 60 min at 37 °C to produce cylindrical constructs (Ø5 mm × 3 mm thickness). All samples were allowed to equilibrate overnight before initiation of experiments.For encapsulation of pCCs into different concentrations of fibrin–hyaluronic acid hydrogels, cells were suspended in fibrinogen solution (100 mg/mL) with or without incorporation of 5 mg/mL HA. This was combined at a ratio of 1:1 with 5 U/mL thrombin–HA solution, to obtain a final cell seeding density of 4 × 10All constructs were maintained at 37 °C and 5% oxygen conditions in 2 mL of chemically defined medium (CDM) consisting of lgDMEM supplemented with penicillin (100 U/mL)–streptomycin (100 µg/mL) , 40 µg/mL L-proline, 50 µg/mL L-ascorbic acid-2-phosphate, 1.5 mg/mL BSA, 1× insulin–transferrin–selenium, 100 nM dexamethasone , and 5% FBS. Medium was exchanged twice weekly and sampled for biochemical analysis for a total duration of 21 days.Hydrogel contraction kinetics were determined via image analysis using a digital camera (Canon Powershot SX240HS) at each feeding period (twice weekly). The average diameter of three constructs was determined using image analysis software .® Viability/Cytotoxicity Assay Kit (Invitrogen). Briefly, hydrogels were cut in half and washed in phosphate buffered saline , followed by incubation in PBS containing 2 μM calcein AM  and 4 μM ethidium homodimer-1 . Hydrogels were again washed in PBS, imaged with a Leica SP8 scanning confocal microscope at 515 and 615 nm and assessed using Leica Application Suit X (LAS X) Software.Cell viability was assessed using a LIVE/DEADCell shape was analysed on fixed samples . After fixation, samples were thoroughly washed in PBS and permeabilised using 0.5% Triton-X. Fluorescent stains were diluted in 1.5% BSA solution  at 1:40, and for DAPI (ex/em = 364/454 nm), a 1:1000 dilution was used). Stains were incubated for 1 h  and 10 min (DAPI) at room temperature, avoiding artificial light, imaged with a Leica SP8 scanning confocal at 358/524 and 540/565 nm channels, and assessed using Leica Application Suit X (LAS X) Software.n = 3 for each group). DNA content of each sample was quantified using the Hoechst bisbenzimide 33258 dye assay, with a calf thymus DNA standard. sGAG content was quantified using the dimethylmethylene blue dye-binding assay , with a chondroitin sulphate standard. Total collagen content was determined by measuring the hydroxyproline content. Samples were hydrolysed at 110 °C for 18 h in 38% HCl, and assayed using a chloramine-T assay [To quantify the accumulation of biochemical constituents, samples were digested with 125 μg/mL papain in 0.1 M sodium acetate, 5 mM L-cysteine-HCl, 0.05 M EDTA, pH 6.0  at 60 °C under constant rotation for 18 h , embedded in paraffin wax, sectioned at 7 μm, and affixed to microscope slides. Sections were stained with 1% alcian blue 8GX in 0.1 M HCL to assess sGAG content and picrosirius red to assess collagen distribution . Collagen types 1 and 2 were evaluated using a standard immunohistochemical technique. Briefly, sections were treated with peroxidase, followed by treatment with chondroitinase ABC (Sigma-Aldrich) in a humidified environment at 37 °C to enhance permeability of the extracellular matrix. Sections were incubated with goat serum to block non-specific sites and collagen type I , collagen type II  primary antibodies  were applied overnight at 4 °C. Next, the secondary antibody  (Sigma-Aldrich B7151) was added for 1 h, followed by incubation with ABC reagent  for 45 min. Finally, sections were developed with DAB peroxidase (Vector Labs) for 5 min. Positive and negative controls were included in the immunohistochemistry staining protocol for each batch.p < 0.05.Statistical analyses were performed using GraphPad Prism  software with 3–4 samples analysed for each experimental group. The sGAG/collagen ratio was determined by dividing sGAG (µg) by collagen (µg). One-way ANOVA was used for analysis of variance with Tukey’s multiple comparisons test to compare between groups. Results are displayed as mean ± standard deviation, with significance accepted at a level of In this work, we explored the incorporation of ECM molecules into fibrin-based hydrogels in order to develop a suitable injectable system to facilitate cellular delivery. Significant hydrogel contraction was observed using low (12.5 mg/mL) fibrin concentrations, with limited contraction for concentrations at or above 25 mg/mL. With the incorporation of ECM components, cell viability was seen to be maintained when HA was incorporated. HA also enhanced sGAG accumulation and tended to suppress collagen formation at higher concentrations. There did not appear to be any significant benefit of incorporating collagen in the fibrin matrix. Lastly, we showed that there is an overall increase in the sGAG to collagen ratio when increasing HA concentration in the fibrin matrix to 5 mg/mL. Taken together, these results suggest that incorporation HA can enhance the bioactivity of fibrin-based hydrogels, which may help advance the clinical potential of commercial cell and biomaterial ventures in the treatment of IVD regeneration.


Dinophysis spp. are a major source of diarrheic toxins to marine food webs, especially during blooms. This study documented the occurrence, in late May 2016, of a massive toxic bloom of the Dinophysis acuminata complex along the southern coast of Brazil, associated with an episode of marked salinity stratification. The study tracked the daily vertical distribution of Dinophysis spp. cells and their ciliate prey, Mesodinium cf. rubrum, and quantified the amount of lipophilic toxins present in seston and accumulated by various marine organisms in the food web. The abundance of the D. acuminata complex reached 43 × 104 cells·L−1 at 1.0 m depth at the peak of the bloom. Maximum cell densities of cryptophyceans and M. cf. rubrum  were recorded on the first day of sampling, one week before the peak in abundance of the D. acuminata complex. The diarrheic toxin okadaic acid (OA) was the only toxin detected during the bloom, attaining unprecedented, high concentrations of up to 829 µg·L−1 in seston, and 143 ± 93 pg·cell−1 in individually picked cells of the D. acuminata complex. Suspension-feeders such as the mussel, Perna perna, and barnacle, Megabalanus tintinnabulum, accumulated maximum OA levels  during early bloom stages, whereas predators and detritivores such as Caprellidae amphipods (154.6 µg·Kg−1), Stramonita haemastoma gastropods (111.6 µg·Kg−1), Pilumnus spinosissimus crabs (33.4 µg·Kg−1) and a commercially important species of shrimp, Xiphopenaeus kroyeri (7.2 µg·Kg−1), only incorporated OA from mid- to late bloom stages. Conjugated forms of OA were dominant (>70%) in most organisms, except in blenny fish, Hypleurochilus fissicornis, and polychaetes, Pseudonereis palpata , which contained mostly free-OA throughout the bloom. Although algal toxins are only regulated in bivalves during toxic blooms in most countries, including Brazil, this study indicates that human seafood consumers might be exposed to moderate toxin levels from a variety of other vectors during intense toxic outbreaks. Dinophysis blooms have increased worldwide over the past two decades  inlet in Santa Catarina State, southern Brazil . Surrounded by hills (up to 250 m high), its geographic SE-NE orientation provides natural shelter from prevailing waves and winds, especially the stronger ones coming from the south. Due to these favorable attributes, Armação do Itapocoroy  harbors Dinophysis bloom. Seawater sampling was carried out from a floating platform, anchored at a depth of 4.5 ± 0.5 m and deployed 200 m from the low tide level. Samples (~1.5 L) were taken every meter along a vertical profile, from the surface to the bottom, using an EN-470 manual diaphragm pump  equipped with anti-reflux valves and coupled to a 20 mm-diameter hose. Daily samples were taken over 12 days, followed by two sampling operations after 3- and 10-day intervals of the 12th sampling day. Additionally, single depth-integrated water samples (2.0 L) were taken with a 5 m long hose, to compare the efficiency of both sampling strategies for monitoring of extreme bloom events. Plankton net (20-µm mesh size) samples were also collected, and water temperature and salinity were measured along a vertical profile, at 0.5 m intervals from surface to bottom, using a multiparameter YSI Professional Plus probe. Secchi depth was used to estimate water column light penetration or transparency.An intensive sampling effort was conducted between late May and mid-June 2016, when the Santa Catarina coastal zone was affected by a dense Perna perna; n > 5), gastropods , barnacles , amphipods , crabs , shrimp , polychaetes  and fish  were manually collected from raft mooring cables at 0–1 m depth, for quantification of the toxin levels incorporated in their tissues. The species collected and sampling frequency depended on their availability in the environment during the study period. Whenever possible, at least two individuals of each species were collected, packed in 50 mL plastic tubes and immediately immersed in an ice bath until return to the laboratory. Before freezing, soft tissues of mussels, barnacles and gastropods were removed from their shells. Fish muscles (flesh) and viscera were dissected and individually stored at −18 °C. The remaining organisms were frozen and analyzed whole.In parallel, bivalve mollusks (Water samples: Aliquots (300 mL) of both depth-discrete and integrated samples were fixed with 1% Lugol’s iodine solution, and used for quantitative phytoplankton analysis. Plankton net samples, fixed with a 4% formalin solution , were used for analysis of cell morphology and phytoplankton identification. In the laboratory, additional 300 mL aliquots of each sample were gently vacuum-filtered in duplicate and immediately frozen. One fiberglass filter  was allocated for the analysis of photosynthetic pigments, and the second one to determine the amount of lipophilic toxins contained in SPM. In an ice bath, each filter sample was soaked in HPLC grade methanol (99.5%) and exposed to an ultra-sonic probe  for 30 s. The extract was then passed through a 13 mm × 0.22 µm PVDF syringe filter  to remove any cell debris, and the filtrate collected into plastic microtubes (1.5 mL), which were maintained frozen. Aliquots (400 mL) of the filtrate from depth-integrated samples collected from 1 May on were stored frozen in plastic bottles for future spectrophotometric determination of dissolved inorganic nutrient (DIN) concentrations.Samples of marine fauna: The protocol for toxin extraction was adapted from the official analytical method harmonized by the European Union [v:v) to 1.0 ± 0.5 g of homogenate from selected tissues or whole body, exposed to an ultra-sonic probe  until complete tissue disruption, and centrifuged at 2000× g for 10 min. The supernatant was filtered through a 0.22-µm syringe filter directly into a 2.0 mL glass vial, and kept frozen for the analysis of diarrheic toxins in their free form. Subsequently, 1 mL aliquots of the extract were subjected to alkaline hydrolysis by the addition of 2.5 M sodium hydroxide in a 76 °C thermal bath for 40 min, followed by the addition of 2.5 M hydrochloric acid to neutralize the solution and convert the conjugated (metabolized) toxins into their free toxin forms. The amount of conjugated toxins was obtained by subtracting the concentration of free toxins initially measured in the non-hydrolyzed extract from the total concentration of toxins obtained in the hydrolyzed extract.an Union . MethanoDinophysis spp. and M. cf. rubrum cells was performed using a 20 mL aliquot of the Lugol-fixed sample, after settling the particles for 24 h in a Utermöhl chamber [−1). Other phytoplankton groups  were quantified in volumes ranging from 10 to 20 mL (depending on sample turbidity) by counting all cells contained in 5–10 random microscope fields of view (LOD: 2100–8400 cell·L−1) after 24-h settlement. Additionally, a minimum of 100 cells of Dinophysis spp. were picked with a micropipette from in natura plankton net samples at the bloom apex , and placed into 1.5 mL plastic microtubes containing methanol 99.5% (HPLC grade) for determination of the toxin cellular quota.Counting of  chamber . Cell co43−), nitrite (N-NO2−), nitrate (N-NO3−), ammonium (N-NH4+), and silicate (SiO2−) using colorimetric methods [r2 > 0.90).Aliquots (25 mL) of the filtrate samples were used to determine the concentrations of phosphate , composed of a quaternary gradient pump, an automatic thermostat injector, a column oven (set at 40 °C) and a photodiode detector (DAD). Samples were eluted in a mixture of (A) methanol:acetone:pyridine (50:25:25) and (B) acetonitrile:acetone (80:20), at 1.0 mL·min−1. The proportion of B increased from 0 to 40% in 18 min, and then to 100% within the following 4 min of analysis, remaining at 100% for an extra 16 min before returning to the initial conditions (0% B) for an additional 2 min period . Pigment identification was performed by evaluating retention times after the chromatographic separation in a Waters Symmetry® C8 column , as well as the absorbance spectrum (350–750 nm scan), following methods of Zapata et al. [a (chl-a) concentration was calculated using a linear regression obtained from successive dilutions of the analytical standard   with r2 > 0.98.The methanolic extracts were injected (100 μL) into a Chromaster liquid chromatography (LC) system  coupled to a triple quadrupole mass spectrometer, MS  equipped with a turbo ion spray ionization source, following the EURLMB protocol [−1 flow rate, the initial proportion of 80:20% (A:B) increased to 100% B during the first 8 min of analysis, thus remaining for 3.5 min, and returning to the initial condition by the end of the analysis (13 min). Compounds were separated on a C18 column , maintained at 20 °C. Identification of individual toxins was achieved from their retention time and the mass spectra of the transition ions present in the samples in relation to the same parameters obtained for the analytical standards. High-purity nitrogen, heated up to 500 °C, was used as the nebulizing gas. The electron spray (ESI) ion source operated in negative mode, and toxins were scanned for transition ions (Q1 → Q3) of characteristic mass/charge (m/z) ratios. Optimized MS parameters were selected for each toxin of interest (Toxins were measured using a 1260 LC system (Agilent Technologiesprotocol . Brieflyinterest .r2 > 0.95) to the following concentrations: 3.49, 13.96, 55.86, and 223.44 ng·mL−1.Toxin quantification was carried out using an external standard from a calibration curve generated with certified reference material  dissolved in methanol for OA, and in mussel tissue matrix (CRM-DSP-Mus-b) for DTX-1. Quantification of OA was based on the equation obtained by fitting a linear regression , using the statistical package for preliminary analysis. Data were statistically analyzed with R studio software [Graphs were constructed in SigmaPlotsoftware , using tsoftware ) for comsoftware ) allowed


Enterococcus faecium strains (MZF1 to MZF5), which were isolated from homemade Tunisian meat (dried ossban). The estimated median genome sizes were about 2,582,641 ± 109,039 bp, with a median G+C content of 40% ± 0.4%.Here, we report the first draft genome sequences of five bacteriocinogenic and potentially probiotic  Enterococcus faecium strains (MZF1 to MZF5), which were isolated from homemade Tunisian meat (dried ossban). The estimated median genome sizes were about 2,582,641 ± 109,039 bp, with a median G+C content of 40% ± 0.4%.Here, we report the first draft genome sequences of five bacteriocinogenic and potentially probiotic  Enterococcus spp. are Gram-positive lactic acid bacteria that are ubiquitous in nature  but are mostly considered to be of fecal origin, as they are common members of the intestinal microbiota of humans, mammals, reptiles, birds, and insects   . Recentld ossban , 6. Meatus MZF16 , confirmGenomic DNAs were extracted from overnight cultures in MRS broth using a GeneJet genomic DNA purification kit , as recommended by the manufacturer, and assessed using the double-stranded DNA (dsDNA) high-sensitivity kit on a Qubit fluorometer  and 1% agarose gel electrophoresis. Sequencing libraries were prepared using the Nextera XT DNA sample preparation kit . Genomic sequencing was performed by the Laboratory of Microbiology Signals and Microenvironment (LMSM) genomics platform  using a MiSeq instrument (Illumina) with a 2 × 250-bp paired-end read protocol.De novo assembly was performed with Unicycler v.0.4.7 , 3 rRNAs, 52 ± 2 tRNAs, and 1 transfer-messenger RNA (tmRNA). BAGEL4 and antiSMASH5 predicted six bacteriocin biosynthetic gene clusters associated with antimicrobial activity  along with regulatory genes, immunity proteins, and transporters that were differentially detected in the five genomes . No virulence factors  that correlate with human pathogenicity were identified. We found two intrinsic antibiotic resistance genes, namely, aac6′ (aminoglycosides) and msrC (macrolides and streptogramin B antibiotics), and one acquired resistance determinant, eatA . Altogether, the genetic features of these five strains are of interest, serving as candidates for investigation into bacterial antagonism associated with the production of multiple bacteriocins.The average (± standard deviation) total size of the draft genomes was 2,582,641 ± 109,039 bp arranged into 90 ± 30 contigs . The gen genomes . MLST idThe draft sequences of the genomes have been deposited at DDBJ/ENA/GenBank under the accession numbers cited in


Enterobacter aerogene (n = 1), 8.3% for Serratia fonticola (n = 1), 16.7% for Serratia marcesens (n = 2), 16.7% for Klebsiella pneumoniae (n = 2), and 50% (n = 6) for Enterobacter cloacae. The endpoint PCR results showed 11 colonies positive for blaBIL (91.7%), 11 for blaSHV (91.7%), 11 for blaCTX (97.7%), 12 for blaDHA (100%), four for blaVIM (33.3%), two for blaOXA (16.7%), two for blaIMP (16.7%), one for blaKPC (8.3%), and one for blaTEM (8.3%) gen; all samples were negative for blaROB, blaCMY, blaP, blaCFX and blaLAP gene. The sequencing analysis revealed a specific genotype for Enterobacter cloacae ; Serratia marcescens ; Klebsiella pneumoniae ; Serratia fonticola ; and, Enterobacter aerogene . Our results indicate that beta-lactam-resistant bacteria have acquired integrons with a different number of genes that provide pan-resistance to beta-lactam antibiotics, including penicillins, oxacillins, cefalosporins, monobactams, carbapenems, and imipenems.Beta-lactam resistant bacteria, which are commonly resident in tertiary hospitals, have emerged as a worldwide health problem because of ready-to-eat vegetable intake. We aimed to characterize the genes that provide resistance to beta-lactam antibiotics in Enterobacteriaceae, isolated from five commercial salad brands for human consumption in Mexico City. In total, twenty-five samples were collected, grown in blood agar plates, and the bacteria were biochemistry identified and antimicrobial susceptibility testing was done. The carried family genes were identified by endpoint PCR and the specific genes were confirmed with whole genome sequencing (WGS) by Next Generation Sequencing (NGS). Twelve positive cultures were identified and their microbiological distribution was as follows: 8.3% for  Enterobactrias [The intake of ready-to-eat salad greens from commercial sources is a worldwide health practice that might be a potential cause of gastrointestinal (GI) disease. Because of traditional agricultural techniques, fresh foods, such as fruits and vegetables, are usually contaminated with a great deal of diverse bacteria . Frequenbactrias . Beta-labactrias .bla) are classified depending on their structure or resemblance in the amino acid sequence (Classes A-D) [blaTEM, blaSHV, blaCTX, blaCFX, blaLAP, blaROB, and blaKPC [blaIMP (Imipenem-resistant) and blaVIM  [blaCMY has activity against cephamycin [blaFOX against cefoxitin, blaMOX against moxalactam, and blaLAT against latamoxef. Other C group beta-lactamases were named according to the place of isolation, such as blaMIR-1  or blaDHA . The C blaBIL beta-lactamase owes its name to the patient from which it was isolated  [blaOXA gene [blaOXA with weak resistance to carbapenems has been reported [The enzymatic degradation of beta-lactam rings by beta-lactamases is the most effective resistance mechanism that bacteria have for fighting back against the antibacterial effect of beta-lactam antibiotics . The genses A-D)  or accorses A-D) . There ases A-D) . Classesses A-D) . The memses A-D) ,12. The d blaKPC ; howeverd blaKPC . The memtamases) . Class Ctamases)  was the phamycin , blaFOX   . Class DOXA gene . A worldreported ,20. The reported ,22. Recently, the Mexican health system has been promoting the intake of vegetables, fruits, and herbs to prevent and reduce obesity, which is a health problem in Mexico City . This loWe obtained the beta-lactamase integrons and DNA sequences from the National Center for Biotechnology Information. We used all the sequences of beta-lactamase families reported in order to determine the conserved sequence in each gene family encoding beta-lactamases. The integrons and DNA alignments were made with ClustalW software   accordin2 were collected from the vegetables of five different salad brands, labelled as suitable for human consumption and sold in marketplaces in Mexico City. The samples were cultured in BHI  incubated at 37 °C for 24 h.Under sterile conditions, samples of 2 cmAfter its incubation, 10 μL from the BHI were taken and subcultured in MacConkey agar , and then incubated at 37 °C for 24 h. The colonies obtained were subcultured in blood agar and incubated at 37 °C for 24 h.8). To adjust the scale, a CrystalSpec Nephelometer  was used. The inoculum was deposited in BBL Crystal Enteric/Nonfermenter ID Kit plates, incubated at 37 °C for 18 h, without CO2 and 40 to 60% humidity. Finally, the plates were read using a BBL Crystal AutoReader  and the results were analyzed with the BBL Crystal MIND Software .The bacterial identification was made from the pure colonies that were obtained from blood agar using Gram staining and oxidase, Indol and biochemistry tests. The BD BBL Gram Stain Kit  was used. Oxidase and Indol tests were carried out by using BBL DrySlide Oxidase kit  and BBL DrySlide Indole kit  according to the manufacturer’s specifications. The bacterial identification was performed using the BBL Crystal Enteric/Nonfermenter ID system . Briefly, bacterial suspensions were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth . That inoculum was adjusted to a Mac Farland 1.0 scale (Expected CFU/mL 3.0 × 108), and cultured in Müeller Hinton 150 × 15 mm2 media BD BBL . The antibiotic discs were applied with the Sensi-Disc Designer Dispenser System. The antibiotics panel was conformed by ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), mezlocillin (75 µg), carbenicillin (100 µg), piperacillin/tazobactam (100/10 µg), cefazolin (30 µg), cefaclor (30 µg), cefepime (30 µg), cefoperazone (75 µg), and cefotetan (30 µg) from Becton Dickinson Antimicrobial susceptibility testing was carried out by the Kirby–Bauer method under the Clinical and Laboratory Standards Institute protocol (NCCLS standards). The pure colonies obtained from blood agar were resuspended in bacterial suspensions were made depositing two or three medium-sized colonies (2 to 3 mm) in BBL Crystal Inoculum Broth . The obtained inoculum was adjusted while using a Mac Farland 0.5 reading (Expected CFU/mL 1.5 × 10Aliquots of 5 mL of Luria–Bertani broth (LB) were inoculated with the isolated bacteria and incubated overnight at 37 °C with continuous shaking (200 rpm). The bacterial culture was centrifuged at 4000 rpm for five minutes at room temperature (RT). The bacterial pellet was resuspended in 1 mL of RNase and DNase-free deionized water and then heated at 94 °C for 10 min, followed by thermal shock in ice. Finally, the sample of lysed bacterial was kept at −80 °C until use.g for one minute, then the column was replaced into a new collector tube, loaded with 500 µL of wash R1 buffer, and centrifuged at 9300× g for a min. Again, the column was replaced into a new collector tube, loaded with 700 µL of wash R2 buffer and centrifuged at 9300× g for one minute; then, the column was replaced into a new collector tube and centrifuged at 12,000× g for 4 min. Finally, the column was replaced into a new 1.5 mL Eppendorf tube and 60 µL of elution buffer prewarmed to 80 °C was added, incubated for 3 min at RT, and centrifuged at 9300× g for 2 min. The eluted DNA solution was quantified by absorbance and its integrity was verified by 2%-agarose gel electrophoresis. The sample was kept at −20 °C until use.Samples of 500 µL of overnight bacterial culture was subjected to genomic DNA isolation by using an RTP pathogen kit  following the manufacturer’s instructions. Briefly, a bacterial pellet was collected by centrifugation at 13,000 rpm for 5 min, suspended with 400 µL of resuspension buffer, and placed into an extraction L tube to be incubated at 37 °C for 10 min and switched to 95 °C for 10 min. Then, 400 µL of binding buffer were added and loaded onto the RTA spin filter set, incubated for one minute, and centrifuged at 9300× g for 10 min, then 10 mL of resuspension buffer (R3) and 10 mL of lysis buffer (L7) were added to the bacterial pellet, and incubated at RT for 5 min. Then, 10 mL of precipitation buffer (N3) was added and centrifuged at 12,000× g for 10 min at RT. The supernatant was placed onto a pre-equilibrated maxi-column using 30 mL equilibrium buffer (EQ1) and eluted by gravity. Then, 60 mL of wash buffer (W8) was added, eluted by gravidity, and the eluent was discarded. Finally, 15 mL of elution buffer (E4) was added, eluted by gravity, and the eluent was added with 10.5 mL of isopropanol and centrifuged at 12,000× g for 30 min at 4 °C. The DNA pellet was washed with 5 mL of 70% ethanol and centrifuged at 12,000× g for 5 min at 4 °C. The DNA pellet was dried at RT for 10 min and finally resuspended in 250 µL of sterile RNase- and DNase-free water. The Plasmid DNA concentration was quantified by digital spectrophotometry  and its integrity was verified by electrophoresis in 2% agarose gel, and the plasmid DNA sample was kept at −20 °C until use.Samples of overnight bacterial culture (100 mL) were subjected to a plasmid DNA isolation by using PureLink HiPure Plasmid Midiprep Kit  following the manufacturer’s instructions. Briefly, the bacterial culture was harvested by centrifugation at 4000× 2, and 500 mM KCl, pH 8.3), 200 nM each dNTP, 10 μM each primer, 1 U Taq DNA polymerase , and 2 μL of crude extract or 10 ng of DNA (genomic or plasmid). The PCR conditions were 94 °C for 5 min, then 35 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and finally 72 °C for 10 min. The PCR products were analyzed in 2% agarose gel electrophoresis and the image was digitized in a Gel Logistic 2200 Digital Imaging System .The endpoint PCR to identify the β-Lactamase gene family was made using the primers sense and antisense described in The WGS was performed from indexed libraries that were prepared using a standard Illumina Nextera XT DNA Sample Preparation Kit FC-131-1096) for small genomes and was sequenced on the MiSeq platform . Adapters and barcodes were trimmed by the default setting in the Illumina experiment manager, generating 300 bp paired-end reads. The quality of the unprocessed reads was assessed using FastQC High Throughput Sequence QC Report v:0.11.5  -1096 for.The metagenomic analysis for the taxonomic classification of bacteria was done by using the software Kraken taxonomic sequence classification system Version 0.10.5-beta  . The betThe bioinformatics analysis included 1007 complete sequences of different genes encoding beta-lactamases reported in the Genbank, of which 936 sequences were used for primer design after discarding 71 repeated sequences .From the aligned sequences, we constructed fourteen phylogenetic , which aEnterobacter aerogenes (n = 1), 8.3% of Serratia fonticola (n = 1), 16.7% of Serratia marcescens (n = 2), 16.7% Klebsiella pneumoniae (n = 2), and 50% (n = 6) of the samples were identified as Enterobacter cloacae  showed resistance to mezlocillin. The antibiotic resistance towards cephalosporins was as follows: 75% (n = 9) were resistant to cefazolin, 67% (n = 8) to cefaclor, 8.3% (n = 1) to cefotetan and 8.3% (n = 1) to cefoperazone showing medium resistance, and 100% were sensitive to cefepime. As to the beta-lactam and beta-lactamase inhibitor combination (sulbactam or tazobactam), 33% (n = 4) showed resistance and 33% (n = 4) medium resistance to ampicillin + sulbactam, and 100% were sensitive to piperacillin + tazobactam. In the antimicrobial susceptibility testing, 100% of the isolated Enterobacteriaceae strains showed resistance to ampicillin and carbenicillin, and 8.3% (n = 11) of characterized Endobacteriaceae strains were positive to blaBIL, 91.7% (n = 11) to blaSHV, 83.3% (n = 10) to blaCTX, 75% (n = 9) for blaDHA, 33.3% (n = 4) to blaVIM, 16.7% (n = 2) to blaOXA, 16.7% (n = 2) to blaIMP, 8.3% (n = 1) to blaKPC and 8.3% (n = 1) to blaTEM ; Serratia marcescens ; Klebsiella pneumoniae ; Serratia fonticola  and Enterobacter aerogene . However, in the NGS analysis, all of the bacterial strains tested were negative to blaBIL  to six (blaOXA + blaVIM + blaSHV + blaCTX + blaDHA + blaBIL) gene families of beta-lactamases (The strains that were isolated from ready-to-eat vegetable salads showed a broad pattern of resistance to beta-lactam antibiotic. The analysis of the different families of gene responsible for this resistance pattern showed a mixed genotype of multiple gene families encoding beta-lactamase and were specific for each strain, conformed from three (ctamases .The implementation of food safety measures has been shown to have a great cost–benefit impact in the developing world, decreasing healthcare costs by $25.5 USD for each $1.0 USD invested in hygiene measures . To contOne molecular mechanism by which bacteria acquire resistance is the production of beta-lactamase enzymes that annihilate the bactericidal effect of beta-lactam drugs. Some Enterobacteriaceae, which are multi-resistant to a wide range of beta-lactam drugs that produce extended-spectrum beta-lactamases, classified as ESBL-producing Enterobacteriaceae, represent a major threat to human health. Usually, ESBL-producing Enterobacteriaceae are resistant to a wide variety of penicillins and cephalosporins, including broad-spectrum cephalosporins. The USA Health Agency reported 140,000 annual infections caused by Enterobacteriaceae during 2013, and 18% of these cases were resistant to beta-lactams that are associated to ESBL enzyme production, 12% of Enterobacteriaceae infections were caused by Klebsiella, and 6% by Escherichia coli, both of which are ESBL-producing. It has been estimated that multi-resistant ESBL-producing Enterobacteriaceae cause 1700 annual deaths in the USA. Patients with bacteremia caused by ESBL-producing Enterobacteriaceae have a rate of mortality 57% higher than that of patients with bacteremia caused by non-resistant Enterobacteriaceae ,39. Due Enterobacter cloacae, Enterobacter aerogenes, Serratia fonticola, Serratia marcescens, and Klebsiella pneumoniae. All of the organisms isolated showed multi-resistance to beta-lactam antibiotics, tested by the Kirby–Bauer method. The antibiogram results showed resistance to penicillin and cephalosporin. Ampicillin is considered to be a broad-spectrum penicillin due to its use in gastrointestinal infections for some Enterobacteriaceae [Pseudmonas spp. [Pseudomonas spp. and Klebsiella spp. [In this study, we isolated Enterobacteriaceae with high pathogenic potential from ready-to-eat vegetables that are sold in supermarkets located in Mexico City. The isolated Enterobacteriaceae were eriaceae . Howevernas spp. . Our stulla spp.  and, witlla spp. . The fooblaDHA gene family, which is responsible for cephalosporin resistance [blaSHV gene family associated with penicillin and cephalosporin resistance, also related to two grand phenomena of resistance: first, to ESBL-producing Enterobacteriaceae and resistance to beta-lactamase inhibitors [blaCTX gene family member, causing resistance to penicillin and broad spectrum cephalosporins, and this also was associated to ESBL-producing Enterobacteriaceae. We found that 8.3% of all strains carry the blaTEM gene family; in previous works, associates to ESBL-producing Enterobacteriaceae have been found [blaVIM gene family was present in 33.3% of Enterobacteriaceae isolated, blaIMP were in 16.7%, blaOXA in 16.7%, and blaKPC were positives in 8.3%, all of which have been associated with resistance to carbapenem [Our results with bacterial lysates or plasmid coincided with the identification of the gene families encoding beta-lactamases that are involved in the multi-resistance pattern to beta-lactam antibiotics. We demonstrated that 92% of all Enterobacteriaceae isolated carry at least one member of the beta-lactamase gene family that is associated with cephalosporin resistance, and that 100% were positive to the sistance . In addihibitors ,45,46. Trbapenem ,47,48. TAll of these results together demonstrate that ready-to-eat vegetables for human intake in Mexico City are contaminated with potentially pathogenic multi-resistant Enterobacteriaceae; their intake might lead to GI diseases with a high difficulty of treatment. Therefore, food processing under strict safety measures and quality standards must be followed by food industries and supervised by the Health National Agency in order to avoid GI disease outbreaks. In addition, our results provide insight into the beta-lactam pan-resistance mechanism of Enterobacteriaceae. Our methodological approach can be implemented by food enterprises as a routine safety control of food processing.


Background We conducted a first-in-Japanese, phase I study of ontuxizumab, a humanized, anti-endosialin monoclonal antibody, to confirm its tolerability, safety, and pharmacokinetics, and identify exploratory efficacy. Methods This was a multicenter, multiple-dose, open-label study in Japanese patients aged ≥20 years with solid tumors, including gastric cancer (GC) or advanced hepatocellular carcinoma (HCC), who had failed standard chemotherapy. The study comprised two parts: part 1  and part 2 . Results Fifteen patients were treated in part 1, and 31 in part 2 (16 patients with GC and 15 with HCC). In part 1, the most common treatment-related, treatment-emergent adverse event (TEAE) was fatigue (20%); no patients had grade ≥ 3 treatment-related TEAEs. In part 2, the most common treatment-related TEAEs were constipation, malaise, hiccups, and increased bilirubin; treatment-related grade 3 TEAEs occurred in two patients with HCC. In part 1, no patients achieved a partial response, and 6/15 (40%) had stable disease (SD). In part 2, 2/15 patients (13.3%) with GC and 8/15 (53.3%) with HCC had SD. Tumor shrinkage was observed in 5/15 HCC patients (33.3%). Conclusions Ontuxizumab, up to a dosage of 12 mg/kg weekly, was generally safe and well tolerated in this population, with no dose-limiting toxicities. The maximum tolerated dose was not reached; 8 mg/kg weekly or 12 mg/kg biweekly were the recommended dosages. We observed long-term disease stabilization in GC and extraskeletal chondrosarcoma, and tumor shrinkage in gastrointestinal stromal tumor and HCC. Trial registration: NCT01773434 .The online version of this article (10.1007/s10637-018-0713-7) contains supplementary material, which is available to authorized users. Endosialin  is an 80.9 kDa sialic acid-rich transmembrane glycoprotein of the C-type, lectin-like receptor family . It is a8) is an Endosialin mRNA and protein expression have been associated with multiple human cancers, including colorectal, gastric, renal, breast, pancreatic, lung, endometrial, ovarian, and neuroectodermal tumors, hepatocellular carcinoma (HCC), and metastatic malignant melanoma , 5–10. DEndosialin is implicated in tumor-cell vascular adhesion and migration, neoangiogenesis, local invasion, and metastasis , 12–16. Tem1 knockout mice showed no obvious phenotype and demonstrated normal wound healing, transplanted tumors grew more slowly, were less invasive, and fewer metastases developed than in wild-type mice [Although ype mice . Thus, bype mice , endosiaOntuxizumab is a humanized, anti-endosialin, IgG1κ monoclonal antibody with a structure comprising two heavy chains and two light chains with disulfide bonds. In vivo, ontuxizumab significantly affected syngeneic tumor growth and tumor metastasis in human CD248 knock-in mice. Compared with untreated tumors, the blood vessels of ontuxizumab-treated tumors were shortened and distorted. Additionally, CD248 levels on the cell surfaces of neovascular pericytes were significantly reduced due to CD248 internalization. This was accompanied by reduced smooth muscle α-actin expression, depolarization of pericytes and endothelium, and ultimately, dysfunctional microvessels .The first-in-human study of ontuxizumab (MORAb-004-001) was conducted in the US, and was an open-label, phase I trial in patients with solid tumors  who had failed standard chemotherapy . This stThe present study (MORAb-004-J081–103) is a first-in-Japanese study of ontuxizumab. After confirmation of tolerability, the study expanded cohorts to further characterize the tolerability, safety, and PK of ontuxizumab and to identify the exploratory efficacy and PK of ontuxizumab.This was a multicenter, multiple-dose, open-label, phase I study of ontuxizumab in Japanese patients with solid tumors  who had failed standard chemotherapy. The study was conducted in two parts: a dose-escalation portion to assess the tolerability and safety of ontuxizumab monotherapy in patients with solid tumors (study part 1); and a cohort-expansion portion (study part 2), which was designed, in part, to assess the PK relationships of ontuxizumab in patients with gastric cancer or HCC.Ontuxizumab was administered as an intravenous infusion. The stock solution was 5 mg/mL, which was diluted with normal saline, as required, before administration. In study part 1, patients received weekly administrations of ontuxizumab on days 1, 8, 15, and 22 of a 4-week cycle. The dose started at 2 mg/kg and escalated up to 12 mg/kg. Treatment at the next dose started if no DLTs were observed. In study part 2, each patient at 4 or 8 mg/kg received ontuxizumab infusions on days 1, 8, 15, and 22 of a 4-week cycle. Each patient at 12 mg/kg received biweekly ontuxizumab infusions on days 1 and 15 of a 4-week cycle. Patients repeated this cycle at the same dosage until disease progression, unless they met the discontinuation criteria. In study part 2, three dose levels were examined, based on the safety profile obtained in study part 1: 4 or 8 mg/kg (administered on a weekly basis), and 12 mg/kg (administered once every 2 weeks). Study part 2 consisted of cohort A, which comprised patients with a histologic diagnosis of gastric cancer, and cohort B, which comprised patients with a histologic diagnosis of HCC.No patients had intracranial involvement or metastases. All patients had failed or were resistant to standard chemotherapy and had no appropriate therapies available. All patients gave written informed consent to participate.The protocol, informed consent form, and related documents were approved by the relevant Institutional Review Boards. The study was conducted in accordance with Good Clinical Practice guidelines, as outlined in the Principles of the World Medical Association Declaration of Helsinki, and the Pharmaceutical Affairs Law of Japan.Included patients were Japanese males or females, aged ≥20 years, with solid tumors, gastric cancer, or a clinically confirmed diagnosis of advanced HCC (Child-Pugh class A). Exclusion criteria are shown in supplementary methods (Online Resource 1). All patients had Eastern Cooperative Oncology Group (ECOG) performance status 0–1 and adequate organ function. Patients with a preserved tumor biopsy sample taken before entry into the study had to provide written informed consent for the sample to be used.The primary study objective was to investigate the tolerability and safety of multiple intravenous infusions of ontuxizumab in Japanese patients with solid tumors (gastric cancer or HCC). Secondary study objectives were to determine the maximum tolerated dose (MTD) of ontuxizumab as defined by DLTs; establish the serum PK of ontuxizumab; detect any anti-drug antibody (ADA) response to multiple intravenous infusions of ontuxizumab; and describe changes in objective measurements of tumor size after treatment with ontuxizumab.Safety assessments included monitoring and recording all adverse events (AEs) and serious adverse events (SAEs), various laboratory parameter investigations, and physical examinations.For DLT evaluation, the severity (grade) of AEs was classified according to the latest version of Common Terminology Criteria for Adverse Events (CTCAE) version 4.0–Japan Clinical Oncology Group (JCOG). A DLT was defined as any grade ≥ 3 hematologic or non-hematologic toxicity (CTCAE version 4.0–JCOG definition) related to ontuxizumab administration, with the following exceptions: grade ≥ 3 anaphylactic or anaphylactoid reactions; infusion-related toxicities that could be treated or controlled to grade ≤ 2 by maximal medical management within 48 h ; abnormal laboratory parameters not requiring medical treatment; and pretreatment grade 2 liver function test abnormalities that progressed to grade 3 during the study, if the reason for progression was considered by the investigators to be the underlying disease and not ontuxizumab. The MTD was the highest dose at which no more than one of six patients experienced a DLT in cycle 1.Blood samples were collected and serum ontuxizumab and ADA concentrations were measured by electrochemiluminescent immunoassay.Tumor assessment based on Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1 was performed for evaluable tumor lesions. The same modality was used to characterize each identified and reported lesion at baseline and through follow-up, by suitable computed tomography (CT), magnetic resonance imaging (MRI), or alternatives.The following items were assessed for tumor response based on RECIST: best overall response (BOR) — i.e., the best response recorded from the start of treatment until study end; objective response rate (ORR) — defined as the proportion of patients with a BOR of complete response (CR) or partial response (PR), and disease control rate (DCR) — defined as the proportion of patients with a BOR of CR, PR, or stable disease (SD). To achieve a BOR of SD, measurements at ≥7 weeks after the administration had to meet the SD criteria.For tumor assessment, CT and MRI with contrast agents were recommended, except in patients with a history of allergy to oral or intravenous contrast agents. If patients were allergic to intravenous iodinated contrast agents, CT with oral contrast agents or, for the abdomen and pelvis, MRI with intravenous, gadolinium-based contrast agents were considered. Low-dose CT images from positron emission tomography CT, CT images used for absorption correction, or ultrasonography were not used for tumor assessment.A tumor with a longest diameter of ≥10 mm was regarded as a target lesion (or when CT scans had a slice width > 5 mm). In the case of lymph node involvement, a tumor with a shortest diameter of ≥15 mm was regarded as a target lesion.All statistical analyses were performed using SAS software version 9.3 . The safety analysis set comprised patients who received at least one administration of the study drug and had ≥1 post-dose safety assessment. Safety analyses other than DLTs were based on the safety analysis set. The number (percent) of patients with treatment-emergent AEs  and treatment-emergent SAEs was summarized by Medical Dictionary for Regulatory Activities system organ class and preferred term.The DLT analysis set comprised patients who registered in study part 1, received at least three administrations of the study drug in cycle 1, and had a DLT assessment. Patients with DLTs were included in the DLT analysis set, regardless of the number of administrations. DLT analyses were based on the DLT analysis set. The number (percent) of patients who experienced DLTs was summarized, and DLTs were also summarized by type.The PK analysis set comprised patients who received at least one administration of study drug and had sufficient PK data to derive at least one PK parameter. The safety analysis set was used for individual ontuxizumab concentration listings. The PK analysis set was used for summaries of ontuxizumab serum concentrations and summaries and analyses of PK parameters.max), time to Cmax, area under the plasma concentration-time curve (AUC), clearance, and volume of distribution after single and multiple doses.Summary statistics were tabulated for serum ontuxizumab concentrations according to study part , dose, and time point. Linear and semi-log plots of serum ontuxizumab concentration-time profiles for individual patients were displayed by study part and dose group. Using non-compartmental methods , serum ontuxizumab concentrations were analyzed to determine PK parameters, including peak plasma concentration   discontinued ontuxizumab due to AEs, and 14 discontinued due to disease progression. All 31 patients in study part 2 received at least one dose of ontuxizumab. One patient with HCC (4 mg/kg weekly) discontinued ontuxizumab due to AEs, and the remaining 30 patients discontinued due to disease progression.n = 15; study part 2, n = 31) received ontuxizumab. In study part 1, the median number of cycles was 2.0 (range: 1 to 8), and the median exposure duration was 51.0 days. In study part 2, the median number of cycles was 2.0 (range: 1 to 4) in patients with gastric cancer, and 2.0 (range: 2 to 36) in patients with HCC; median exposure duration was 49.0 days and 51.0 days, respectively. The median percentage of received versus planned doses was 100.0% (range: 75.0%–100.0%).A total of 46 patients , followed by constipation, decreased appetite, hyperkalemia, infusion-related reactions, and rash  , followed by nausea, vomiting, pyrexia, hypoalbuminemia, increased aspartate aminotransferase (AST), and increased alanine aminotransferase  (Table max had been attained (Table ½) values increased from 62.6 to 168 h with increasing ontuxizumab dosage from 2 to 12 mg/kg on cycle 1, day 1 (study part 1). In study parts 1 and 2, serum ontuxizumab concentrations reached steady-state approximately 1008 h after the start of repeated-dose administration (at cycle 2).In study parts 1 and 2, intravenously administered ontuxizumab on cycle 1, day 1, and on cycle 1, day 22 , or cycle 2, day 1 (12 mg/kg biweekly) was eliminated slowly from serum with a long half-life after Ced Table . Mean elmax and AUC(0–168h) at both cycle 1, day 1 and cycle 1, day 22 increased in an almost dose-proportional manner  after weekly administration of ontuxizumab 4 mg/kg and 8 mg/kg at cycle 1, day 22 were 8710 and 24,900 μg•h/mL in patients with gastric cancer, and 10,200 and 20,100 μg•h/mL in patients with HCC. Mean values of AUC(0–336h) after biweekly administration of ontuxizumab 12 mg/kg on cycle 2, day 1 were 39,700 and 42,700 μg•h/mL in patients with gastric cancer and HCC, respectively.In study part 1, Cmin on cycle 2, day 15 for ontuxizumab 12 mg/kg biweekly administration was between the trough concentrations of 4 mg/kg and 8 mg/kg weekly administration in patients with gastric cancer or HCC.In study part 2, the mean value of CIn study part 1, no patients achieved a BOR of a PR. Six of 15 patients 40%) had a BOR of SD, while the remaining nine had progressive disease. Tumor stabilization or shrinkage was observed in patients with gastric cancers and extraskeletal chondrosarcoma, while tumor shrinkage was observed in one patient with gastrointestinal stromal tumor (GIST) (Online Resource 2). The DCR was 40%  with gastric cancer and 8 of 15 (53.3%) with HCC had a BOR of SD. The DCR was 13.3% in patients with gastric cancer, and 53.3% in those with HCC Table . Tumor s.3% with In patients with HCC in study part 2, non-vascular stromal cells (11/15), perivascular cells (8/15), capillary endothelial cells (7/15), and lymphatic endothelial cells (5/15) showed endosialin expression. Conversely, gastric cancers in study parts 1 and 2 expressed endosialin in perivascular cells (12/17), capillary endothelial cells (10/17), non-vascular stromal cells (9/17), lymphatic endothelial cells (8/17), and endothelial cells (1/17).This multiple dose, open-label, phase I study was the first to examine the safety and tolerability of ontuxizumab in Japanese patients with solid tumors who had failed standard chemotherapy; an associated aim was to determine the recommended ontuxizumab dosage for future clinical trials. The study was conducted in two parts: a dose-escalation phase (study part 1), and an expansion phase (study part 2).Ontuxizumab was generally well tolerated in Japanese patients with solid tumors, up to a dosage of 12 mg/kg weekly, and in Japanese patients with gastric cancer and HCC, across the dosages studied in study part 1. No DLTs were observed up to 12 mg/kg weekly; therefore, the MTD was not reached. A dosage of 12 mg/kg weekly, the MTD of the previous phase I study , was alsIn study part 1, the most common drug-related TEAE was fatigue, while in study part 2, the most frequent TEAEs were vomiting, constipation, and pyrexia in patients with gastric cancer, and hiccups in patients with HCC. These results are consistent with previous findings from phase I and II studies in non-Japanese patients , 24. No Although one patient had a positive ADA response during the study, this was transient and not associated with significant safety concerns. Moreover, no treatment-related SAEs were reported.max and AUC(0–168h), increased in an approximately dose-proportional manner on cycle 1, day 1 and cycle 1, day 22 across the dose range of 2–12 mg/kg. After multiple-dose administration, t½ values increased with increasing dosage, thus indicating slow clearance of ontuxizumab, especially at higher dosages.Systemic exposure to ontuxizumab, as assessed by Cmax and AUC(0–168h) values in the present study were similar to values in the US study of ontuxizumab in non-Japanese patients [Although data are limited, saturable clearance may play a role in ontuxizumab PK. Indeed, after administration of ontuxizumab 2, 4, 8, and 12 mg/kg weekly on cycle 1, day 22, Cpatients . Thus, nmax, AUC(0–168h), or AUC(0–336h) were observed between patients with gastric cancer and those with HCC. This is in contrast to previous studies of trastuzumab and bevacizumab, in which median values for AUC, Cmax, and Cmin at steady state were approximately 30–40% lower in patients with gastric rather than other cancers [In study part 2 of the present study, no clinically significant differences in C cancers , 26. Themin on cycle 2, day 15 for 12 mg/kg biweekly dosing was within the range of the 4 mg/kg and 8 mg/kg weekly dosing schedules in patients with gastric cancer or HCC. The ontuxizumab serum trough concentration with 12 mg/kg biweekly administration was between the trough concentrations for the 4 mg/kg and 8 mg/kg weekly schedules (data not shown). Additionally, the clearance values at dosages of 8 mg/kg weekly and 12 mg/kg biweekly in patients with gastric cancer were 0.0197 and 0.0198 L/h, respectively. Similarly, the clearance values were 0.0266 and 0.0210 L/h, respectively, in patients with HCC. These results demonstrate that clearance was saturated at dosages above 8 mg/kg weekly  had a BOR of SD. Tumor stabilization was observed, especially in patients with gastric cancer, extraskeletal chondrosarcoma, or GIST, from the percent change in the sum of longest tumor diameters. In part 2, although complete or partial responses were not observed, tumor shrinkage occurred in five of 15 patients with HCC.A previous examination of 50 human tumor cell lines and 250 clinical specimens of human cancer, including 20 cancer subtypes, revealed that endosialin was expressed in tumor cells, perivascular cells, and stromal cells in sarcoma; further investigation with 11 types of carcinoma showed that endosialin expression originated from perivascular and stromal cells, and not from carcinoma cells [In a previous examination of gastric cancer tissue samples, CAF-endosialin positivity and CAF-endosialin intensity were significantly correlated with several clinicopathologic factors; moreover, both a higher positive rate and a stronger intensity of endosialin expression in CAFs were associated with poorer recurrence-free survival, cancer-related survival, and overall survival . RecentlEndosialin expressed on hepatic stromal cells, such as hepatic stellate cells (HSCs), was previously implicated in the initiation and progression of liver metastatic cancers and/or HCC tumors. However, a recent examination with HSCs and an HCC tumor cell co-culture system reported that endosialin-expressing and fully activated HSCs impaired HCC tumor growth, suggesting an inverse causal relationship between HSC-expressed endosialin and HCC growth . ConversOntuxizumab, up to a dosage of 12 mg/kg weekly, was generally safe and well tolerated in Japanese patients with solid tumors. There were no DLTs, and the MTD was not reached. No clinically significant differences in PK parameters were evident between patients with gastric cancer and those with HCC. According to the safety and PK results of the present study, 8 mg/kg weekly or 12 mg/kg biweekly are the recommended dosages for future studies. Long-term disease stabilization was observed in gastric cancer and extraskeletal chondrosarcoma, and tumor shrinkage was noted in GIST and HCC. All these conditions were resistant and progressive after standard chemotherapies.ESM 1(DOCX 18 kb)ESM 2(DOCX 67 kb)


O-glucoside (M3G), delphinidin-3-O-glucoside (D3G), petunidin-3-O-glucoside (Pt3G), peonidin-3-O-glucoside (Pn3G), and cyanidin-3-O-glucoside (C3G) were identified. Juice from “Merlot” had significantly higher (p < 0.05) content of all individual anthocyanins as compared to “Ruby.” During the long-term storage, total, and individual anthocyanins from both cultivars degraded following first-order reaction kinetics at the rate strongly dependent on temperature. At the end of the storage, noticeably higher loss of anthocyanins (95–99.9%) was observed at 25 and 35°C as compared to storage at 5°C . Considerably lower rate of decay was observed at 5°C (k = 0.01–0.04) as compared to 25 (k = 0.04–0.14) and 35°C (k = 0.05–0.14) storage temperatures. The most temperature sensitive anthocyanin compounds were C3G (Ea = 66.5 kJ/mol) and D3G (Ea = 63.3 kJ/mol). At higher storage temperatures, significant (p < 0.05) and strong negative correlations were observed between anthocyanin concentrations and the levels of haze, polymeric and brown color development during storage. Storing grape juice, at lower temperature conditions could reduce the continuous loss of biologically active anthocyanins as well as the development of haze and brown color.This study evaluated the degradation kinetics of selected anthocyanins and the change in polymeric color, browning index, and haze development of grape juices from “Merlot” and “Ruby” grape cultivars stored at 5, 25, and 35°C for up to 360 days. Five major anthocyanins namely malvidin-3- Grape juice is very important in the international juice trade because it is used not only as a single beverage but also as an ingredient for blending or making other products such as juice beverages, jams, jellies, pie fillings, and some confectionaries . WorldwiColor is one of the most crucial quality parameters of grape juice and its derivatives . A typicIn previous studies, anthocyanin degradation in different juice products was commonly evaluated as total monomeric anthocyanin, and little information is available on the fate of individual anthocyanin compounds in grape juice after long-term storage at different temperatures. Moreover, there is very limited information, to allow evaluation of the relation between anthocyanin degradation and the change in haze accumulation, polymeric color development, and browning of grape juice with time. Hence, the objectives of this study were: (1) to determine the degradation kinetics of individual and total anthocyanins contained in grape juice stored at different temperatures for up to 360 days, and (2) to evaluate the relation between anthocyanin content and the change in polymeric color, juice browning and haze development during storage.The grape juice of both grape cultivars (“Ruby” and “Merlot”) was obtained from a local juice processor and stored at −35°C until use. Standards were purchased as follows: M3G from Chromatographic Specialities Inc. , Pt3G, Pn3G, and D3G from Polyphenols Laboratories  and C3G from Extrasynthese . Potassium chloride, hydrochloric acid, sodium acetate, methanol, acetonitrile, and phosphoric acid were purchased from Sigma Aldrich .18 column by solid phase extraction protocol  according to the manufacturer's recommendation .An aliquot of 100 mL juice was filled into amber bottles and pasteurized in a water bath at 70°C for 15 min. The headspace was flushed with nitrogen gas closed tightly using screw caps equipped with rubber septa, first sealed with Parafilm M  film and stored at 5, 25, 35°C. Two bottles of juice samples were removed at 0, 5, 10, 20, 30, 60, 120, 210, 280 and 360 days and kept at −80°C until analyses. Extraction and purification of anthocyanin pigments were performed using 100% methanol and CThe total monomeric anthocyanin content and polymeric color were determined by pH differential and bisulfite bleaching methods, respectively , 22. A U18 Phenomenex  Luna column  maintained at 25°C. The flow rate was 0.35 mL/min with a total run time of 42 min and an injection volume of 20 mL. The eluents were 5% (vol/vol) formic acid in water (A) and 5% (vol/vol) formic acid in methanol (B). A linear gradient profile was used with the following proportion of solvent A applied at time t (min): : , , , , , , , . The standards were prepared in methanol, and their concentration was as follows 0.25-25 mg/L C3G, M3G, D3G, Pt3G, and Pn3G. Each analysis was carried out in duplicate.Analyses of selected individual anthocyanins were performed according to a previously described method  using LCBrowning index (BI) which shows the ratio of the loss of total anthocyanin pigment to the development of brown color was calculated using Equation 1. Absorbance was measured after diluting the juice with distilled water (1:1).Turbidity measurements were carried out using a turbidimeter . Undiluted juice samples were poured in a 10 mL cell holder, which was cleaned and air-dried, and haze sediments were re-suspended by gently rocking the sample holder just prior to measurement. All samples were allowed to equilibrate to room temperature before measuring their turbidity. Results were expressed in nephelos turbidity units (NTU).Ae (Equation 3) first devised by (Instead of the commonly used first-order model (Equation 2), which assumes the complete degradation of anthocyanins after long-term storage, a first-order model which assumes an equilibrium value, vised by  was applvised by  and stravised by , 27 to eAo is the initial anthocyanin content, Ae is the equilibrium value, At is the anthocyanin content after treatment time t (min) and k is the rate constant (day−1).where t1/2) (the time needed for 50% degradation of anthocyanins) and activation energy (Ea), a measure of temperature dependence, were calculated using the Equations , respectively.The half-life (k is rate constant (day−1), ko is the frequency factor (day−1), R is gas constant (8.314 J/mol.K) and T1 and T2 are absolute temperatures in Kelvin (K).where p < 0.05) differences between means was evaluated using Tukey's test.The experimental design consisted of a three-factor  storage trial with two replications. Nonlinear regression analysis was performed using SYSTAT 11  and Minitab 16  statistical software applications. Analysis of variance (ANOVA) was applied to compare the initial concentration of individual anthocyanins between the two cultivars. The possibility of significant (p < 0.05) content of all individual anthocyanins as compared to “Ruby.” The differences in anthocyanin concentrations are presumably due to genetic variation between the two cultivars, is in accordance with a study conducted by Liang et al.  were indicated in Table p < 0.05)  were > 0.96 especially at 25 and 35°C, demonstrating a direct correlation between anthocyanin concentration decrease and storage time at each temperature  up to 280 days of storage Figures , 2. The e Figure . While ne Figure , strawbee Figure , sour che Figure , and pome Figure  reportedk = 0.01–0.04) as compared to higher storage temperatures 25°C (k = 0.04–0.14) and 35°C (k = 0.05–0.14)  and D3G (t1/2 = 69 days) at 5°C  and D3G (Ea = 63.3 kJ/mol) Table . Similares Table  where hies Table . On the es Table . Aglycones Table . GeneralThe development of polymeric color, haze formation, and browning of juice samples was also monitored up to 360 days of storage and presented in Figure 2 bleaching (p < 0.001) negative correlation  between polymeric color and total anthocyanin content at 35°C  negative correlation between BI and total anthocyanin content at 5 (r2 = 0.72), 25 (r2 = 0.84) and 35°C (r2 = 0.96)  Table  and appl6) Table  for 180 C r2 = 0.6 (Table 6) Table , 43.p < 0.05) correlation between haze development and anthocyanins concentration particularly in “Merlot” juices. Moreover, in agreement with (Haze formation can limit the shelf life of products that the consumer expects to be clear including fruit juices. Haze also fouls process equipment surfaces with deposits that are difficult to remove by in-place cleaning . Irrespeent with , haze shent with –48. Anotent with . During ent with . The incent with .As presented in Figure The results of this research provided evidence on the degradation kinetics of individual as well as total anthocyanin pigments contained in grape juice when stored at different temperatures for long periods of time. The five major individual anthocyanins identified from “Merlot” and “Ruby” grape juices were M3G, D3G, Pt3G, Pn3G, and C3G. During the long-term storage, total, and individual anthocyanins degraded following first-order reaction kinetics at the rate strongly dependent on temperature. The individual anthocyanins exhibited different degradation rates and kinetic parameters, which might be related to their chemical structure. At higher storage temperatures D3G and C3G were found the most unstable and temperature sensitive anthocyanins. Higher temperatures and longer storage periods not only reduced anthocyanin concentrations but also increased polymeric color accumulation, brown color development, and haze formation. Therefore, the findings could be adapted to industrial applications for preserving the biologically active anthocyanins and authentic color of grape juice by storing them at low temperatures for limited storage periods.BM conducted all the experiments, data analyzing and writing the first draft of the manuscript. HPVR and RAS contributed by advising methods and experimental design, supervising data analysis, and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Thin magnetic wires can present excellent soft magnetic properties (with coercivities up to 4 A/m), Giant Magneto-impedance effect, GMI, or rectangular hysteresis loops combined with quite fast domain wall, DW, propagation. In this paper we overview the magnetic properties of thin magnetic wires and post-processing allowing optimization of their magnetic properties for magnetic sensor applications. We concluded that the GMI effect, magnetic softness or DW dynamics of microwires can be tailored by controlling the magnetoelastic anisotropy of as-prepared microwires or controlling their internal stresses and domain structure by appropriate thermal treatment.  Magnetic sensors allowing control and monitoring of various processes and functions are essentially relevant for a great number of industries, such as data storage, home entertainment, energy harvesting and conversion, informatics, telecommunications, aircrafts, aerospace, automobiles, electronic surveillance, medicine, biology, etc. over many decades ,2,3. The essential part of magnetic sensors is appropriate magnetic material. Particularly, soft magnetic materials are used for a wide number of magnetic sensors . One of the most prospective families of soft magnetic materials presenting a number of advantages such as excellent magnetic softness, fast and inexpensive fabrication process, dimensionality suitable for various sensors applications and good mechanical properties is the family of amorphous and nanocrystalline materials prepared using rapid melt quenching ,5,6. Best magnetic softness as well as physical properties linked to soft magnetic properties  are reported for amorphous and nanocrystalline wires or ribbons ,7,8,9,10It is worth noting that the observation of both single domain wall propagation and above mentioned GMI are not restricted to amorphous and nanocrystalline wires and were reported in crystalline magnetic wires ,18. HoweThe main interest in the GMI effect is related to one of the largest among the non-cryogenic effects, impedance sensitivity to an external magnetic field (up to 10%/A/m) reported for magnetic microwires ,19,20. SThe main features of the GMI effect are satisfactorily explained in terms of classical electrodynamics considering the skin effect of a magnetically soft conductor ,8,9,10. However, like magnetic permeability, GMI effect presents tensor origin ,27,28,29The miniaturization of the sensors and devices requires employment of thinner soft magnetic wires and hence an extension of the frequency range towards the GHz band ,23. In tOn the other hand, different families of magnetic wires can present fast and controllable electric current or magnetic field driven domain wall (DW) propagation ,16,32,33As recently reported elsewhere ,39,40, tConsequently, from the actual state of art on DW dynamics in magnetic microwires, we can assume that the DW velocity can be tailored by the chemical composition of metallic nucleus as well as by the thermal treatment ,38,39,40Given the attractive magnetic properties of amorphous wires, the development of magnetic sensors using magnetic wires has aroused great interest in recent years.Thus, various magnetic sensors employing stability either GMI effect or magnetic bistability of amorphous wires have been developed ,41,42,43Consequently, preparation and processing of thin magnetic wires with improved magnetic properties is essentially relevant for development of magnetic sensors. Therefore, in this paper we will provide the routes allowing optimization of either GMI effect or domain wall dynamics in magnetic microwires.d, ranging from 10 up to 25 μm, prepared using the Taylor-Ulitovsky technique described elsewhere [We studied Fe-, Ni- and Co- based microwires with metallic nucleus diameters, lsewhere ,15,16,20Z of a magnetic conductor is expressed by [dcR is the DC electrical resistance, k = (1 + j)/δ, where 0J and 1J are the Bessel functions, r is the wire’s radius and δ the penetration depth given by:σ is the electrical conductivity, μϕ is the circular magnetic permeability and f is the frequency of the current along the sample. The electrical impedance essed by ,8:(1)Z=RZ evaluation we used a micro-strip sample holder previously described elsewhere [-values have been measured using a vector network analyzer from the reflection coefficient, S,11 using expression [Z0 = 50 ohm is the characteristic impedance of the coaxial line. For the impedance lsewhere ,45. The pression :Z =Z0 that corresponds to viscous DW propagation regime. The origin of deviations from linear v(H) dependencies at high-field region has been discussed elsewhere [We considered only the linear region of lsewhere ,16. Proplsewhere .aσ, up to 500 MPa. All the annealing processes have been performed at temperatures below the crystallization, observed at above 450–500 °C.Prepared microwires have been studied in as-prepared state and after annealing. Conventional and stress annealing processes have been performed in a conventional furnace. During the stress-annealing, a mechanical load has been attached to one end of the microwire and axially placed via the furnace nozzle. Such mechanical load allowed to apply tensile stress during the annealing, As discussed elsewhere ,9,10,11,sλ, either by selection of the appropriate chemical composition of the metallic alloys  [The primary parameter that allows to optimize the GMI effect is the minimization of the magnetostriction coefficient, ≤ x ≤ 1) ,47,48 or≤ x ≤ 1) .xFe1 − x (0 ≤ x ≤ 1) binary system, the sλ values can be changed from high and positive sλ values (sλ ≈ 35 − 40 × 10−6) for the Fe-rich compositions  to negative λs values  for Co-rich alloy (x = 1) [sλ values can be tuned in NixFe1 − x alloys: λs values decrease with increasing of Ni-content is reported elsewhere [sλ values correspond to almost paramagnetic ordering for ambient temperature [In the alloys based on Co (x = 1) ,48. Similsewhere ,48,49. Hsλ values and sign on hysteresis loops of magnetic microwires is shown in One of the examples illustrating the influence of the The other important parameter that affects the magnetic softness and hence the GMI effect is the value and distribution of the internal stresses within the metallic nucleus. Such stresses are originated by rapid solidification of metallic alloy itself as well as by the different thermal expansion coefficients of the metallic alloy and glass coating ,52,53,54iσ increase with increasing of the glass-coating thickness [ρ between the metallic nucleus diameter d and the total microwire diameter, D (ρ =d/D) [sλ values can present different magnetic properties. As an example, influence of the internal stresses controlled through the ρ–ratio for both families of microwires (Co-rich and Fe-rich) is shown in The common viewpoint on internal stresses distribution ,53,54 cohickness ,52, i.e.ρ =d/D) ,54. ConsIt is worth mentioning that the glass-coating thickness  also affects the thermal exchange conditions between the metallic nucleus and the ambient  and henccH ≈ 5 A/m. As can be seen from Consequently, both Co-rich microwires present high GMI ratio see a. On thef is one of the conditions for GMI effect observation: f must be high enough in order to ensure that the skin depth is below the sample radius. One more important parameter for GMI ratio optimization is the frequency. Sufficiently enough frequency ΔZ/Z(H) dependencies measured at different frequencies in as-prepared Fe3.6Co71.3Ni0.2B13.4Si10.4C0.9Mo0.2 (d = 9.6 μm and 11 μm) microwires are shown in mΔZ/Z, defined as a maximum ΔZ/Z obtained at a given frequency. Both samples have high GMI ratio, however, for the frequency range below 600 MHz the microwire with d = 11 μm presents superior mΔZ/Z values (f > 600 MHz the thinner sample (d = 9.6 μm) presents higher mΔZ/Z values.m values c. HowevemΔZ/Z(f) dependencies for the microwires of the same composition must be related to the relationship between the wire diameter and the optimal frequency for the GMI performance: for achievement of the significant GMI effect, the penetration skin depth must be below the wire radius [Observed difference in e radius . Conseque radius . mΔZ/Z(f) dependencies can be affected by the interfacial layer between the metallic nucleus and the glass coating observed in glass-coated microwires [It is worth mentioning that this approach has some limitations because the crowires . Indeed,Consequently, a number of various factors must be considered for tuning the magnetic properties in order to meet the requirements of any industrial applications. Thus, thermal treatment in some case can considerably affect magnetic properties of magnetic microwires prepared by Taylor-Ulitovsky method ,40. The other factor that must be addressed from the viewpoint of application is the cost of magnetic microwires. In this regard, Co belongs to critical raw materials . ConsequIn this case the nanocrystallization of Fe-rich microwires can be a useful route for improvement of magnetic softness ,62,63. HTherefore, search of routes allowing optimization of functional magnetic properties of amorphous microwires becomes essentially important for applications.Consequently, below we will present our last results on optimization of magnetic softness, GMI effect and domain wall dynamics of magnetic microwires.3.6Co69.2Ni1B12.5Si11Mo1.5C1.2, d = 22.8 μm, D = 23.2 μm). Below we present a systematic study of the effect of annealing conditions on hysteresis loops and GMI effect of the given microwire.One of the most common methods allowing stress relaxation is annealing. Recently, a few attempts to tailor magnetic properties and GMI effect of Co and Fe-rich microwires have been reported ,68,69,70sλ values Fe3.6Co69.2Ni1B12.5Si11Mo1.5C1.2 microwire presents drastic magnetic hardening: an order of magnitude coercivity rising from 5 A/m up to 90 A/m  the GMI ratio is higher than that of as-prepared sample  dependencies measured at low f-values (10–100 MHz) is considerably affected by the annealing: a double-maximum dependence typical for wires with circumferential magnetic anisotropy is observed for as-prepared sample, while for annealed sample ΔZ/Z(H) dependence has a shape of decay from H = 0 (see ΔZ/Z(H) dependencies can be observed even in annealed samples  dependence for each sample) and mΔZ/Z (Tann) dependencies are plotted. From these dependencies it can be deduced that from the viewpoint of the GMI ratio optimization the conventional furnace annealing can be suitable at certain annealing conditions. Additionally, observation of double-maximum ΔZ/Z(H) dependencies at higher frequencies (above 200 MHz) can be attributed to existence of thin outer shell with transverse magnetic anisotropy. In this case bulk hysteresis loop reflects only the overall magnetic anisotropy modification induced by annealing. However, for GMI effect the magnetic anisotropy in thin surface layer (with thickness comparable to the skin depth at a given frequency) is more relevant.These experimental results are resumed in As recently reported ,68,69,70annT, ann,taσ) on hysteresis loop of Fe3.6Co69.2Ni1B12.5Si11Mo1.5C1.2 microwire subjected to stress-annealing is illustrated by cH, reduction and remanent magnetization, r/M0M, increase  present lower r/M0M-values [The influence of the annealing parameters  dependencies shown in mΔZ/Z improvement is observed for all frequency range (up to 1 GHz). It is interesting that the optimum frequency for as-prepared sample is about 100 MHz, however, for stress-annealed samples the optimal frequency shifts to about 150 MHz. The highest mΔZ/Z ratio (of about 230%) is observed for the sample annealed at aσ =236 MPa. However, the sample annealed under aσ = 472 MPa presents the highest mΔZ/Z in the frequency range f ≥ 200 MHz  dependencies.Consequently, we can resume that the stress-induced anisotropy allows tuning of both the GMI ratio value and the 3.6Co69.2Ni1B12.5Si11Mo1.5C1.2 microwires present rectangular shape. Therefore, it is expected that the remagnetization process runs by single domain wall propagation as described elsewhere [Most of hysteresis loops of annealed and stress-annealed Felsewhere ,14.annT = 350 °C the DW velocity v up to 3000 m/s can be observed (annT (aσ (3.6Co69.2Ni1B12.5Si11Mo1.5C1.2 microwires is that the magnetic field range at which the single DW propagation can be observed is shifted to low –field region (see aσ = 0 MPa) samples. Experimental results on DW dynamics of studied sample are presented in observed a. The DWed (Tann b and σa Tann (σa c. One ofgion see b as compv(H) dependencies previously attributed to a viscous regime [S is the DW mobility and 0H is the critical propagation field, below which the DW propagation cannot be observed.Furthermore, the DW dynamics in all annealed samples can be described by linear s regime ,36,37,38v and S-values increase rising the ann  with high and positive magnetostriction coefficient (sλ ≈ 38 × 10−6) [Below we will present some last experimental results on tuning of GMI effect and DW dynamics of an amorphous Fe-rich microwire with quite typical composition (Fe × 10−6) ,73 by po75B9Si12C4 samples present rectangular hysteresis loops, as reported elsewhere for microwires with positive sλ values [cH decreasing after annealing is observed (see annT = 350 °C).As can be appreciated from s values ,36,37,38annT) Fe75B9Si12C4 sample  dependencies of both samples (see ΔZ/Z(H) dependence with ΔZ/Z maximum at H = 0 is observed for all frequencies. Such kind of ΔZ/Z(H) dependence with decay from H = 0 is predicted for axial magnetic anisotropy  [Rather different magnetic anisotropy of as-prepared and stress-annealed Feples see a,b. As os loops) . annT = 350 °C for σa = 190 MPa) Fe75B9Si12C4 microwire presents rather unusual and irregular ΔZ/Z(H) dependence for intermediate frequency range (100–400 MHz) (see ΔZ/Z(H) dependence of stress-annealed Fe75B9Si12C4 microwire is observed (see f ≤ 50 MHz) the single peak ΔZ/Z(H) dependence of stress-annealed Fe75B9Si12C4 microwire is observed  dependence for intermediate f –values  see a,b. Howerved see b. On therved see a. Similalues see .ΔZ/Z(H) dependence recently has been attributed to as the superposition of the double-peak ΔZ/Z(H) dependence typical for transverse magnetic anisotropy and single-peak dependence reported for axial magnetic anisotropy [ΔZ/Z(H) dependencies rising the frequency can be explained considering frequency dependence of the penetration depth, δ, given by Equation 2. Indeed, as followed from Equation 2 and reported previously elsewhere [δ with f increasing is expected. On the other hand, it is commonly considered that the domain structure of magnetic wires consists of inner axially magnetized core and outer domain shell [r/MoM with the radius of the inner axially magnetized core, cR, given as [R is the metallic nucleus radius. Observed irregular isotropy . Evolutilsewhere , a decrein shell ,77,78. Tgiven as : Rc = R. Consequently, from cR(σa) dependence we can assume an increase of the outer domain shell volume with transverse magnetization orientation in expense of a decreasing in the inner axially magnetized core volume. From the hysteresis loops presented in 75B9Si12C4 microwires is elevated mΔZ/Z values observed in a wide frequency range evidenced from the mΔZ/Z(f) dependencies shown in mΔZ/Z ≥ 100% are observed for the frequency between 200 MHz and 1 GHz  dependencies of Fe75B9Si12C4 microwires is resumed in v, and DW mobility, S, is observed  and even up to S ≈ 27 m2/A∙s . Even more remarkable effect of stress-annealing on S- values (S up to 40 m2/A∙s) is reported recently for annT = 325 °C [Previously it was shown that the DW dynamics can be considerably improved by minimization of the magnetoelastic anisotropy ,36,37,38rved see a. It is rved see  by stresTann see b. Howevelity see . The DW = 325 °C .S, given by [β is the viscous damping coefficient, 0μ is magnetic permeability of vacuum. The interpretation of the effect of conventional annealing on DW dynamics involves the magnetoeslastic anisotropy contribution to the domain wall mobility, given by ,80,81:S βe, is associated to the micro-eddy currents circulating nearby moving domain wall;The eddy current contribution, βr, related to the Gilbert damping parameter, α;The magnetic relaxation damping, The structural relaxation contribution originated from the interaction of mobile defects with the local magnetization .Although generally three contributions have been discussed elsewhere ,81, i.e.βr, which is related to the anisotropy constant, K through the DW width δw [A is the exchange interaction constant. As mentioned above, in amorphous materials the main origin of magnetic anisotropy is the magnetoelastic anisotropy, meK, given by:sλ is the magnetostriction coefficient, and iσ - the internal stress.The most evident interpretation is related to the magnetic relaxation damping, width δw βr ≈ 2απIn the latter case the annealing influence must be related to the internal stresses relaxation ,79,81.Indeed, elevated internal stresses are intrinsically related to the preparation method involving rapid melt quenching of metallic nucleus surrounded by the glass coating ,52,53,54The interpretation of the stress-annealing influence of DW dynamics in magnetic microwires is recently given considering the similarity of the stress-annealing induced magnetic anisotropy and transverse magnetic field on DW dynamics . Indeed,Aforementioned examples provide the routes for optimization of GMI effect and DW dynamics in Co-and Fe-rich microwires. We concluded that the GMI effect, magnetic softness or DW dynamics of microwires can be tailored by controlling the magnetoelastic anisotropy of as-prepared microwires either through tuning of their internal stresses, magnetostriction coefficient and hence domain structure or by appropriate thermal treatment. The impact of stress-annealing on magnetic properties and domain wall (DW) propagation and giant magneto-impedance (GMI) effect in Co and Fe- rich microwires is experimentally studied. Observed stress-induced anisotropy is considerably affected by annealing conditions .cH ≈ 4 A/m) into rectangular with cH ≈ 90 A/m upon annealing without stress is observed in Co-rich microwires. However, considerable MI effect improvement at certain annealing conditions is observed. Even more remarkable improvement of the GMI effect is observed in Co-rich glass-coated microwires subjected to stress annealing.Considerable magnetic hardening and transformation of linear hysteresis loop with low coercivity (ΔZ/Z(H) dependencies for as-prepared and stress-annealed samples present considerable difference. A remarkable effect of stress-annealing on hysteresis loops is attributed to the domain structure modification: Rising the volume of the outer domain shell with transverse magnetic anisotropy. We assumed that this outer domain shell with transverse magnetic anisotropy affects the travelling DW in a similar way as application of transversal bias magnetic field allowing enhancement the DW velocity. Accordingly, stress annealing of Fe-rich microwires allowed us to achieve the magnetic anisotropy distribution beneficial for optimization of either the GMI effect or the DW dynamics.Remarkable improvement of DW mobility and GMI ratio are achieved by stress-annealing in Fe-rich microwires. Furthermore, the shape of Consequently, versatile properties of magnetic microwires  can be optimized by appropriate postprocessing.