Glycine max (L.) Merr.) into farming systems raises concerns that transgenes might escape from the soybeans via pollen into their endemic wild relatives, the wild soybean (Glycine soja Sieb. et Zucc.). The fitness of F1 hybrids obtained from 10 wild soybean populations collected from China and transgenic glyphosate-resistant soybean was measured without weed competition, as well as one JLBC-1 F1 hybrid under weed competition. All crossed seeds emerged at a lower rate from 13.33–63.33%. Compared with those of their wild progenitors, most F1 hybrids were shorter, smaller, and with decreased aboveground dry biomass, pod number, and 100-seed weight. All F1 hybrids had lower pollen viability and filled seeds per plant. Finally, the composite fitness of nine F1 hybrids was significantly lower. One exceptional F1 hybrid was IMBT F1, in which the composite fitness was 1.28, which was similar to that of its wild progenitor due to the similarities in pod number, increased aboveground dry biomass, and 100-seed weight. Under weed competition, plant height, aboveground dry biomass, pod number per plant, filled seed number per plant, and 100-seed weight of JLBC-1 F1 were lower than those of the wild progenitor JLBC-1. JLBC-1 F1 hybrids produced 60 filled seeds per plant. Therefore, F1 hybrids could emerge and produce offspring. Thus, effective measures should be taken to prevent gene flow from transgenic soybean to wild soybean to avoid the production F1 hybrids when releasing transgenic soybean in fields in the future.The releasing of transgenic soybeans (The online version of this article (10.1007/s11248-020-00230-x) contains supplementary material, which is available to authorized users. Since 2012, 80% of consumed soybeans have been imported, reaching 88.03 million tons in 2018 (http://data.stats.gov.cn), most of which were herbicide-resistant soybeans. Meanwhile, domestic transgenic soybeans have also been developed under governmental support. On January 21, 2020, China’s Ministry of Agriculture issued a biosafety certificate for production and field planting in the south of China of the glyphosate-resistant transgenic soybean SHZD32-01 developed by Shanghai Jiaotong University, representing a first step toward commercialized production of transgenic soybeans in China.The cultivation area of transgenic soybeans have considerably increased since their commercial release in 1996. In 2018, transgenic soybeans were planted on 95.9 million hectare, reaching their highest level of adoption worldwide, covering 50% of the global biotech crop area, and 78% of the global soybean area James . HerbiciGlycine soja Sieb. et Zucc.)  Merr.), is the progenitor of soybean and is extensively distributed in the Far East of Russia, the Korean peninsula, China, and Japan .Ten wild soybean populations were collected from six provinces, namely Heilongjiang, Jilin, Liaoning, Hebei, Henan, and Jiangsu, and the Inner Mongolia Autonomous Region during 2015–2016 (Table The glyphosate-resistant (GR) soybeans (T14R 1251–70) were provided by the National Soybean Improvement Center of Nanjing Agricultural University. The GR soybean, contains one single-copy 16 Table . The croAt the beginning of May of the next year (the second year for IMBT F1 and HNSQ F1) after crossed seeds were obtained by artificial hybridization, 20 filled seeds of each wild soybean population and transgenic soybean were selected from the 10 mother plants (two seeds from each plant). Then single filled seed of each wild soybean population and transgenic soybean were sown at 1 cm depth in individual pots  previously filled with a mixture of wasteland soil and organic cultivated soil  at a 1:1 (v/v) ratio. The filled crossed seeds were also selected and sown using the same method. Owing to limited filled crossed seeds obtained after artificial hybridization, six filled crossed seeds of JSCZ, HNSQ, and IMBT, 10 filled crossed seeds of LNTL, 15 filled crossed seeds of HLJHRB-2, JLBC-2, LNSY, and HBHD, 20 filled crossed seeds of HLJHRB-1, and 24 filled crossed seeds of JLBC-1 were randomly selected and each seed sown directly into individual plastic pots. The experiments were replicated five times. Number of wild soybeans and crossed seeds sown in the experiments are disclosed in detail in supplementary Table Pots were laid out in a completely randomized design in the same replicate in the greenhouse. Wild soybean seed coats were sturdy and durable under natural state, so the embryo-dorsal seed coats (on the opposite of hilum) of wild soybeans and crossed seeds were carefully nicked with a razor blade prior to sowing , to break the limit of imperviousness of the seed coat. Experimental pots were exposed to natural light and photoperiods, approximately 13 light/11 dark, and the temperature fluctuated from 22 to 28 °C during the experiment. The pots were watered as needed and hand weeded.Emergence number of transgenic soybeans, wild soybeans, and crossed seeds was recorded when the cotyledon emerged from the soil and completely turned green approximately 2 weeks after sowing. Emergence rate (%) = (emergence number / number of seeds sown) × 100. The length and width of cotyledons and true leaves were measured using vernier calipers  when the first trifoliolate leaf was not completely spread. The size of cotyledons and true leaves was calculated as the length Χ width of cotyledons + the lenght Χ width of true leaves.cp4-epsps gene. The DNA of each F1 seedling was extracted with a Plant Genomic DNA Kit , according to the manufacturer's instructions. A set of primer pairs for PCR analysis was designed using the sequence of the cp4-epsps gene included in the transgenic soybeans with Primer 6.0 Software .All emerging F1 seedlings were tested by PCR to confirm whether they contained the Each PCR reaction involved a 20-µL reaction solution containing 10 µL of Premix Taq Version 2.0 plus dye , 6 µL of double distilled water, 1 µL of forward primer (10 µM), 1 µL of reverse primer (10 µM), and 2 µL of 20–30 ng/µL genomic DNA. PCR amplification was performed on a Whatman Biometra TGRADIENT Thermocycler at 95 °C for 5 min for the initial denaturation, 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 50 s, elongation at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Amplified DNA products were separated on 1% agarose gels at 120 V for 30 min, stained with 10,000 × SolarRed, and visualized under UV light. Wild and transgenic soybeans were used as negative and positive controls, respectively.cp4-epsps gene were transplanted individually into pots with holes at the bottom , containing the same growth media as described previously when the second trifoliolate leaf spread completely. On the third day after transplanting, a bamboo pole  was inserted into the pots with wild soybeans and F1 seedlings for the plants to climb. Pots were watered and hand-weeded as needed. No chemicals were applied during the experiment. Seedlings were grown under natural conditions exposed to natural light (approximately 14–11 light) and temperature (approximately 15–35 °C) from the date of transplanting to harvesting (from the end of June to the end of November). Adjacent pots were separated by 60 cm. Pots were laid out in a completely randomized design in the net house, and no sexually compatible Leguminosae species were present for a 100-m radius around the experiment. The plant height was measured from the top of the plant to the cotyledonary node when the third trifoliolate leaf spread completely.At least 15 plants uniform in size of wild soybeans, transgenic soybeans (the transplanting number were the same with wild soybean in the same year), and 10–29 F1 plants , each individual plant was separately harvested (cut from cotyledonary node). Each plant was sun dried to a constant weight and the aboveground dry biomass was weighed. The number of pods of each harvested plant was counted. All seeds were hand-peeled from the pods. Then, the number of filled seeds was counted for each plant. After being sun dried for 10 days in a greenhouse, 100 filled seeds were randomly counted from 10 plants and weighed for each wild soybean, transgenic soybean, and F1 hybrid.Setaria viridis (L.) Beauv., Digitaria sanguinalis (L.) Scop., and Echinochloa colona (L.) Link., 0.25 g of seeds from Eleusine indica (L.) Gaertn., and 0.2 g of seeds from Amaranthus retroflexus L. were well mixed and then sown evenly on the surface of pots with holes in the bottom . The pots contained the same media as those in the experiment without competition. These pots were watered and cultured in the net house.On the same day of sowing wild soybeans and JLBC-1 F1 hybrids in the experiment without weed competition in 2017, 0.5 g of seeds each from cp4-epsps gene were individually transplanted into the pots with weeds. The methods for measuring performance variables, including plant height, aboveground dry biomass, pod number per plant, and filled seed number per plant, were the same as those used in the experiment without weed competition.We imposed interspecific competition by transplanting JLBC-1 F1 hybrids and its progenitors, JLBC-1 and transgenic soybeans, into recently established stands of weeds in 2017. Fifteen uniformly sized wild soybean of JLBC-1, transgenic soybean, and JLBC-1 F1 plants with the Separation of the means of each measured variable was performed following the method described by Liu et al.  using SPThe means of each variables and composite fitness for the F1 hybrids and the respective wild soybeans without weed competition were separated using a t-test for independent samples.With weed competition, the performance of all variables among F1 hybrids, wild soybeans, and transgenic soybean were separated using Duncan’s multiple range test. The composite fitness of JLBC-1, its F1 hybrid and transgenic soybean was the mean of the relative variable estimates for plant height, aboveground dry biomass, pod number per plant, filled seeds per plant, and 100-seed weight without weed competition compared with those under weed competition. The difference in composite fitness of JLBC-1, its hybrids and transgenic soybean without and with weed competition were separated using a t-test for independent samples.P < 0.01 for nine F1 hybrids except for IMBT, it’s was P < 0.05)  fragments from the transgenic soybeans were highly conserved in the F1 hybrids . However, LNTL F1 hybrids exhibited cotyledons and true leaves of similar sizes. HNSQ F1 hybrids had similar cotyledons size and true leaves length. HLJHRB-2 F1 hybrids had cotyledons of similar length. Nine of the 10 F1 hybrids were significant shorter at 71.55–15.39% (P < 0.01) the height of their wild soybeans, except for HNSQ F1 hybrids, which were 17.88 cm tall, similar to HNSQ. Seven of the 10 F1 hybrids had significantly lower aboveground dry biomass (61.34–18.44%) than their respective wild parents (P < 0.01). HNSQ F1 and HLJHRB-1 F1 hybrids were 12.69 and 11.43% (P < 0.05) lower. IMBT F1 hybrids were an exception at 90.39% higher than its wild parent . F1 hybrids of HLJHRB-1, JLBC-1, LNTL, HLJHRB-2, and HNSQ produced 15.88–77.56% fewer pods per plant than their wild progenitors (P < 0.01). JSCZ F1, JLBC-2 F1, LNSY F1, HBHD F1, and IMBT F1 produced 188.82–397.55 pods per plant, which was similar to the number produced by their wild parents.The pollen germination rates of wild soybeans ranged from 71.13% to 95.93% in the in vitro experiments at 100 min, while the pollen germination rates of F1 hybrids were 49.57–82.17%, which were significantly lower than those of their wild parents (P < 0.01) at 169.80–757.80. HNSQ F1, IMBT F1, JLBC-2 F1, and JSCZ F1 hybrids exhibited better performance, which difference with wild parents were 22.92, 36.23, 39.90, and 46.98%, respectively. The filled seed number of HLJHRB-1 F1, HLJHRB-2 F1, and LNSY F1 hybrids was approximately 50% of that of their wild parents. JLBC-1 F1 and LNTL F1 hybrids produced 77.08 and 91.17% fewer seeds than their wild parents.All 10 F1 hybrids produced significantly fewer filled seeds per plant than their wild parents (P < 0.01 or 0.05). However, IMBT F1 hybrids had a large 100-seed weight (5.52 g), which was 256.13% greater than that of its wild parent. Besides larger seeds, the seed coat color of IMBT F1 hybrids turned to the color of transgenic soybean. Pictures of the seeds are shown in Supplementary Fig. Compared with their respective wild parents, LNTL F1, HLJHRB-2 and JLBC-2 F1 hybrids had no significant differences in 100-seed weight. In the other six F1 hybrids, 100-seed weight was significantly lower (7.52–13.87%) than that of their wild progenitors (P = 0.4590)  Fig. . It had Weeds grew vigorously and similarly in each pot in the whole experiment. After being harvested, weeds occurred in JLBC-1 F1 hybrid and its progenitor wild soybean and transgenic soybean at 95.62, 98.16, and 103.27 g, but there was no significant difference in aboveground dry biomass. This implied that the interference of weeds on JLBC-1 F1 hybrid and its progenitors was similar.P < 0.05). The plant height, aboveground dry biomass, and 100-seed weight were lower by 59.00, 83.55, and 90.97%, respectively, than its paternal parent transgenic soybean (P < 0.05). Meanwhile, JLBC-1 F1 produced 65.10% more pods per plant (P < 0.05) and 24.79% more filled seeds per plant than its paternal parent transgenic soybean  than wild soybeans by 15.39–71.55%, except for HNSQ F1, which was similar to its wild progenitor. The results implied that most F1 hybrids had a disadvantage in plant height compared with maternal wild soybeans at the seedling stage. The results on plant height of F1 hybrids were similar to that of the previous study . This result implied that these nine F1 hybrids were weaker in resource capture and biomass production rate compared with their wild progenitors. However, IMBT F1 hybrids exhibited 90.39% higher aboveground dry biomass than its wild parent. In the current research, the difference in aboveground dry biomass between F1 hybrids, and their wild soybean relatives could be determined by the genotypes of wild soybeans rather than other variation and interactions in environmental or experimental condition. Wild soybeans harbor a high level of genetic variation, and most of the variation was found within the populations and groups, but significant genetic differentiation was also detected among different eco-geographical groups  has three clear genetic categories: small-seeded (100-seed weight under 2.0 g), medium-seeded (100-seed weight of 2.01–2.5 g), and large-seeded (100-seed weight of 2.51–3.0 g). The semi-wild soybean (Glycine gracilis) usually had more than 3.0 g 100-seed weight , which originated from reciprocal hybridization between wild and cultivated soybeans  were found between the maternal wild soybeans  and paternal cultivated soybeans. These novel plants produced more seeds with a mean of 4.6 g of 100-seed weight (4.6 times that of wild soybeans). Beside the 100-seed weight, these hybrid plants distinctly differed from their maternal wild soybean populations by having thicker mean diameter for basal stem, higher yield per plant, and aboveground dry mass weight of 18.3 g . Similarly, IMBT F1 hybrids had 1.90 times the aboveground dry biomass weight of maternal wild soybeans. The results indicate that gene flow from transgenic soybeans to certain wild soybeans may generate herbicide resistant G. gracilis. Therefore, the risk of gene flow from transgenic soybeans to different wild soybean populations must be evaluated before commercial release.Wang and Li  providedHelianthus annuus from different places and cultivated H. annuus demonstrated the variable responses to stressful environments. The initial phases of introgression could vary radically in different populations growing under diverse conditions , and 100-seed weight of F1 hybrids showed greater variability among wild soybeans and transgenic soybeans under benign conditions. The composite fitness ranged from 0.52 to 0.84, with one exception reaching 1.28 across genetic backgrounds. Mercer et al.  found thP < 0.05). Nonetheless, JLBC-1 F1 hybrids produced 60 filled seeds per plant. From a mechanistic standpoint, the extent of gene introgression depends on the interactions between recombination and selection Below is the link to the electronic supplementary material.


The feedstock materials with the desired compositions were high-energy ball-milled under argon atmosphere for 50 h. The end products of the powders obtained after milling revealed a short-range order structure with a broad amorphization range (2 at% ≤ B ≤ 25 at%). The behaviors of these glassy systems, characterized by the supercooled liquid region, and reduced glass transition temperature, were improved upon increasing B molar fraction. The results had shown that when B content increased, the saturation magnetization was increased, where coercivity was decreased.Metallic glasses, with their short-range order structure, exhibit unique characteristics that do not exist in the corresponding crystalline alloys with the same compositions. These unusual properties are attributed to the absence of translational periodicity, grain boundaries, and compositional homogeneity. Cobalt (Co)-based metallic glassy alloys have been receiving great attention due to their superior mechanical and magnetic properties. Unluckily, Co-Ti alloys and its based alloys are difficult to be prepared in glassy form, due to their rather poor glass-forming ability. In the present work, the mechanical alloying approach was employed to investigate the possibility of preparing homogeneous (Co Life in the 21st century cannot depend on limited groups of materials; instead, it is dependent on unlimited families of advanced materials . Metalli6 K/s [for). Moreover, RS cannot be used with those systems that have a drastic gap difference between their melting points, such as Al (661 °C) and Ta (3020 °C) [There are different approaches used for preparing amorphous and metallic glassy alloys, such as the atomic disordering of crystalline lattices, solid-state amorphization reaction between pure elements, and solid-state transformations from metastable phase ; however6 K/s . Unlucki3020 °C) .60Nb40 powders by milling a mixture of pure elements [Apart from RS approach, a wide range of amorphous and metallic glassy alloys have been prepared by mechanical alloying (MA) technique that was established by Koch et al. in 1983, when they prepared amorphous Nielements . In contelements . Since tWithin the last decade, Co-based metallic glassy alloys have received great attention due to their very useful soft magnetic properties. Besides, they have stimulated much interesting research based on their ultrahigh strength, particularly bulk metallic glass (BMG). This is attributed to their excellent soft magnetic behaviors ,15,16,1775Ti25)100−xBx (x: 0–25 at%) nanopowders via MA technique, using high-energy ball milling (BM). In this work, the GFA of this new ternary system has been investigated as a function of boron (B) additives, in the range between 2 at% and 30 at%. Furthermore, the effect of B-concentration on the thermal stability and magnetic properties of the Co-Ti base is reported. The detailed chemical analysis, conducted by inductively coupled plasma (ICP) spectrometry of the starting composition is given in at% in The present study was carried out in part to fabricate novel metallic glassy-(Co75Ti25)100−xBx, exemplified by (Co75Ti25)75B25 powders, obtained after 6 h of MA time, is presented in The typical morphological characteristics of (CoWith further MA time (50 h), the powders were disintegrated into finer particles a,b, wher75Ti25)100−xBx  obtained after 50 h of BM time are displayed collectively in The X-ray diffraction (XRD) patterns of MA-(Co75Ti25)90B10, and (Co75Ti25)85B15 are displayed together with their NBDPs in The FE-HRTEM micrographs taken for the end-products of (Co75Ti25)75B25 powders, which implies the formation of amorphous structure , intensive FE-HRTEM/EDS investigations were conducted for all the samples. The FE-HRTEM micrograph of 70B30 powders obtained after 50 h of MA time is presented in The FE-SEM-EDS elemental maps for the alloying elements of Co d, Ti Fie, and B 75Ti25)100−xBx amorphous alloys, indexed by their glass transition temperature (Tg), crystallization temperature (Tx), and supercooled liquid region (∆Tx = Tx − Tg). However, differential thermal analysis (DTA) technique was employed to investigate their corresponding melting behaviors, characterized by the melting temperature (Tm), liquids temperature (Tl), and reduced glass transition temperature (Trg = Tg/Tl). 75Ti25)100−xBx powders, while their corresponding DTA traces are displayed in Figure 10a–e. The heating rates used for conducting DSC experiments were 40 °C/min, where it was 10 °C/min for all of DTA experiments. In the DSC measurements, all the samples were isothermally heated up to the desired temperatures before cooling down to room temperature. Then, second heating runs were carried out with the same heating rates to establish baselines.Differential scanning calorimetry (DSC) technique was used to characterize the crystallization behavior of as-MA  without any structural or compositional changes. The second events, however, were characterized by sharp pronounced exothermic peaks, taken place at a higher temperature (x). At these Tx, the metallic glassy phase is crystallized into the long-range order phase. The thermodynamics parameters of Tg and Tx are used to describe the thermal stability of metallic glassy materials, where ∆Tx is used to characterize their GFA. Wide ∆Tx indicates that the system has a good GFA.The DSC thermograms presented in perature , known a75Ti25)100−xBx powders revealed different GFA and crystallization behaviors, as presented in 75Ti25)95B5 glassy powders possessed low values of ∆Tx (63 °C) and Tg (412 °C), as presented in 75Ti25)90B10, revealed wider Tx (102 °C), and higher Tx value (511 °C), as shown in x (x of (Co75Ti25)70B20 has a lower value of (82 °C) when compared with that measured value of (Co75Ti25)75B25 (121 °C), as indexed in Depending on B concentrations, metallic glassy 75B25 metallic glassy system revealed the best GFA and possessed high thermal stability. In contrast to these full-metallic glassy systems, nanocomposite (Co75Ti25)70B30 powders, which consisted of a glassy phase coexisted with nano-spheres of unprocessed crystalline B particles (x (79 °C) and Tx (513 °C) values, as indexed in 75Ti25)70B30, as detected by HRTEM/EDS  and liquids (Tl) temperature, and calculate the Trg = Tg/Tl. All the DTA curves of (Co75Ti25)100−xBx systems presented in m and Tl temperature, respectively 95B5 system were 1119 °C and 1184 °C  and Tl (1224 °C), but corresponding Trg of this composition has not been improved and remained at the level of (0.35), as presented in m and Tl tended to increase with increasing the B content to be 1181 °C and 1268 °C ((Co75Ti25)80B20), and 1271 °C and 1324 °C ((Co75Ti25)75B25), as presented in rg value could be detected, as shown in 75Ti25)70B30 system, which consisted of a glassy phase coexisted with nano-spheres of unprocessed crystalline B particles showed the highest value of Tm (1319 °C) and Tl (1398 °C), as indexed in rg (0.31), as presented in In parallel to ∆Tectively . The ons 1184 °C a, respec be 0.35 c. Howeve be 0.35 b led to rg should be far above 0.5. The current (Co75Ti25)100−xBx system in all range of B content showed low Trg values, laid in the range between 0.31 to 0.41, as displayed in For excellent GFA systems prepared by rapid solidification technique, Ts) and coercive force (H) of (Co75Ti25)100−xBx metallic glassy systems were obtained according to the measured hysteresis loops, as depicted in 75Ti25)100−xBx metallic glassy system revealed a rather modest value of Bs (0.48 T), with a low coercivity value of about 15 kA m−1, as presented in −1), indicating an improvement in the soft magnetic characteristics. Further improvement in the increase in Bs was attained upon increasing the concentration B alloying element to 10 at% (0.73 T), 15 at% (0.82 T), and 20 at% (0.94 T), as depicted in The saturation magnetization (B−1) for (Co75Ti25)80B20 (75Ti25)75B25 system possessed the best soft magnetic characteristics, as indexed by its high Bs value (1.1 T) and a very low H (9.3 kA m−1), as presented in 75Ti25)70B30 system showed a lower Bs value (0.87 T) and a relatively high H value (13.8 kA m−1), as indexed in In parallel, the coercivity force, indexed by H was monotonically decreased with increasing the B concentration to be (11.5 kA m25)80B20 . Metalli80−xTaxSi5C15  glassy/nanocrystalline system possesses promising soft magnetic behavior, i.e., a minimum coercivity (Hc) of 1.2 kA m−1, which is notably lower than a minimum value obtained for Co80Si5C15 (3.3 kA m−1) [In addition to the current system of this study, there are a considerable number of recent reports which show the possibility of preparing metallic glassy soft magnetic materials through mechanical alloying and other approaches. For example, Co kA m−1) . Recentl kA m−1) . They po kA m−1) .70Co30 has been reported by N. Loudjani et al. [0.25Co0.25Ni0.25Cr0.125Mn0.125)100–xBx system with B concentration ranged from 9 at% to 13 at%, using melt spinning approach [c = 2.5–7.0 A m−1, μi = 4910–15,830, and Bs = 0.40–0.48 T [s and Hc were varied from 159.6 to 166.8 emu/g, and 87 to 109.2 Oe, respectively [Furthermore, the effect of MA on the magnetic properties of mechanically alloyed Nii et al. . Their ri et al. . More reapproach . They po0–0.48 T . An inte0–0.48 T . This alectively .75Ti25)100−xBx (x: 2–30 at%).High purity elemental powders of Co , Ti , and B  were used as the starting feedstock materials. The powders were handled, balanced, and then mixed inside the He-atmosphere (99.99%) glove box  to get an amount of 25 g with the desired nominal composition of  and well-sealed together and with 75 tool steel balls (11 mm in diameter) in the glove box under He-atmosphere (99.99%), using a ball-to-powder weight ratio was 20:1. The vials were then fixed on a high-energy ball mill , where the ball milling (BM) process was carried out for 15, 30, 45, and 60 h at ambient temperature. After each milling run, a small amount (<500 mg) was discharged from the vial for different analyses. Then, the BM process was resumed under the same operating conditions.The crystal structures of all samples were investigated by X-ray diffraction (XRD) with CuKα radiation, using 9kW Intelligent X-ray diffraction system, provided by SmartLab-Rigaku . The local structure of the synthesized materials was studied by 200 kV-field emission high-resolution transmission electron microscopy/scanning transmission electron microscopy (HRTEM/STEM) supplied by JEOL-2100F , and equipped with Energy-dispersive X-ray spectroscopy (EDS) supplied by Oxford Instruments .The morphological characteristics of the milled and consolidated samples were investigated through field-emission scanning electron microscope (FE-SEM), using 15 kV- JSM-7800F, JEOL . The local elemental analysis was investigated by energy-dispersive X-ray spectroscopy  system interfaced with the FE-SEM.Differential scanning calorimeter (DSC) and differential thermal analysis (DTA), provided by Setaram-France, using a heating rate of 40 °C/min and 10 °C/min, respectively, employed to investigate the glass transition temperature, glass-forming ability, and thermal stability indexed by the supercooled liquid region and crystallization temperature of the metallic glassy samples.s) of the as-consolidated samples was measured at room temperature, using a vibrating sample magnetometer (VSM) with a maximum applied magnetic field of 670 kA m−1. The coercive force was measured with a B-H loop tracer.The magnetization (B75Ti25)100−xBx systems in a very wide range of (2 ≤ x ≤ 30 at%), using high-energy ball milling technique. Based on the results of the present work, we can conclude that:(1)75Ti25)100−xBx was improved upon increasing the B molar fraction in the range between 2 at% to 25 at%.The GFA of (Co(2)75Ti25)100−xBx systems.The effect of elemental B of enhancing the GFA is attributed to the chemical bonding between the alloying elements  in the alloy. This can be realized upon considering that electronegativity, which is a very important factor for glass formation, is directly related to the chemical bonding. Electrons of metalloid B element, which transferred to metallic Co-Ti may lead to the formation of very strong covalent bonding and hence led to improve the GFA of (Co(3)75Ti25)100−xBx, (Co75Ti25)75B25 system revealed excellent thermodynamics properties, as indexed by its high GFA as characterized by the widest ∆Tx (121 °C), and high thermal stability, indicated by its highest Tx value (633 °C). Moreover, this glassy system revealed the highest Trg value (0.41), which indicates its high GFA when compared with other compositions in the (Co75Ti25)100−xBx systems.Among the as-prepared metallic glassy alloys of (Co(4)75Ti25)75B25 system, where coercivity was decreased to a very low level of 9.3 kA m−1.The results have shown that when B content increased, the saturation magnetization was increased to reach to 1.1 T for (CoDue to the absence of deep eutectic compositions in the Co-Ti binary phase diagram, it is very difficult to obtain a metallic glassy phase for this system and its based alloys, using the melting and casting and melt spinning techniques. This is in contrast to the Co-Zr binary system, which possesses several deep eutectic compositions, allowing a wide glass-formation range. The present work has been addressed in part to employ the MA approach for preparing metallic glasses of ternary (Co


In vitro models able to represent osteoarthritis (OA) synovial tissue (ST) inflammation are lacking. Therefore, we aimed to characterize OA ST and to compare mechanical and enzymatic digestion procedures to find the one that better preserve the heterogeneity of the main OA synovial cell populations: fibroblasts and macrophages. We demonstrated that typical macrophage phenotypical markers, like CD68, CD80 and CD163, were higher expressed on cells isolated with mechanical than enzymatic procedure. Moreover, we found that typical cytokines of inflammatory macrophages  and anti-inflammatory macrophages  were also higher on mechanically isolated cells. Synovial fibroblasts were well conserved in both procedures. The definition of an OA ST model in vitro that better preserve the heterogeneity of specific cell populations represents a prerequisite for defining the real effects of new cell therapies or drugs for OA treatment, and could contribute to the reduction or avoidance of animal models.There is a lack ofin vitromodels able to properly represent osteoarthritis (OA) synovial tissue (ST). We aimed to characterize OA ST and to investigate whether a mechanical or enzymatic digestion procedures influence synovial cell functional heterogeneity in vitro. Procedures using mechanical nondigested fragments (NDF), synovial digested fragments (SDF), and filtrated synovial digested cells (SDC) were compared. An immunophenotypic profile was performed to distinguish synovial fibroblasts , macrophages , M1-like , and M2-like  synovial macrophages. Pro-inflammatory (interleukin 6 IL6), tumor necrosis factor alpha (TNFα), chemokine C-C motif ligand 3 (CCL3/MIP1α), C-X- motif chemokine ligand 10 (CXCL10/IP10) and anti-inflammatory (interleukin 10 (IL10)), transforming growth factor beta 1 (TGFβ1), C-C motif chemokine ligand 18 (CCL18) cytokines were evaluated. CD68 and CD163 markers were higher in NDF and SDF compared to the SDC procedure, while CD80, CD86, and CD206 were higher only in NDF compared to the SDC procedure. Synovial fibroblast markers showed similar percentages. TNFα, CCL3/MIP1α, CXCL10/IP10, and CCL18 were higher in NDF compared to SDC, but not compared to SDF. IL10 and TGFβ1 were higher in NDF than SDC at the molecular level, while IL6 did not show differences among procedures. We demonstrated that NDF isolation procedures better preserved the heterogeneity of specific OA synovial populations , fostering their use for testing new cell therapies or drugs for OA, reducing or avoiding the use of animal models. Synovial inflammation is a key feature of osteoarthritis (OA), as well as of other arthritis conditions  ,2. It isMacrophages are mainly located in the synovial lining layer and their number significantly increases from low to high grade synovial inflammation ,7,10. ThTherefore, understanding the dual role of macrophages in driving and resolving joint inflammation is a mandatory step in advancing OA management ,21 and dDifferent studies focused onin vitro synovial fibroblast cell monolayers or on cell lines , but both these models do not take into consideration the role of synovial macrophages or extracellular matrix (ECM) ,31. OtheTo overcome this challenge, in this study we investigated whether different isolation procedures  influence cell functional heterogeneity among synovial isolated cells. We proposed a protocol that permits the study of fresh human synovial cell populations and demonstrated that this method is useful in obtaining synovial fibroblasts and macrophages that adequately represent the pathological ST in vitro.2; disease duration: 8 ± 3 years; Kellgren/Lawrence grade 3/4 [ST was obtained from 15 OA patients undergoing total knee replacement surgery, selected through clinical and radiological examinations . The stST specimens were collected from at least two different areas, out of approximately 15 selected areas that showed macroscopically inflamed characteristics (like hypervascularity and hyperplasia) and were fixed in 4% formalin at room temperature for 16 h and then embedded in paraffin. Serial tissue specimen sections (4 µm thick) of each sample were prepared and routinely stained with hematoxylin-eosin. The histopathological features of each ST specimen were evaluated according to the synovitis scoring system described by Krenn . This scSerial sections were deparaffinized, baked at 95 °C in epitope retrieval solution for 5–20 min  and incubated for 10 min at room temperature with Protein Block Serum-Free (Dako) to inhibit nonspecific staining. Then, slides were incubated for 1.5 h at room temperature with monoclonal anti-human-CD55 , -CD68 , -CD86 , and -CD206  diluted in Tris-buffered saline (TBS) containing 0.1% bovine serum albumin (BSA). CD55 and CD68 single staining signals were developed with a biotin/streptavidin amplified, alkaline phosphatase-based, detection system , using Fast Red as a substrate .CD86/CD206 double staining was evaluated by a commercial kit , containing a mixture of anti-mouse, anti-rabbit labelled with either Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP), and using as substrates diaminobenzidine (DAB)  and Fast Red .After nuclear counterstaining with hematoxylin, sections were mounted with glycerol gel and analyzed by brightfield microscope . Negative controls were performed using isotype controls (Dako). Semiquantitative analysis of immunohistochemically stained slides were performed on 20 microscopic fields (×200 magnification) for each section. The analysis was performed using Red/Green/Blue (RGB) with Software NIS-Elements 5.21.00 and Eclipse 90i microscope (Nikon Instruments Europe BV). Briefly, we acquired the total number of blue-stained nuclei and the total number of positive-stained red/brown cells. The data were expressed as percentage of positive cells for CD55, CD68, CD86, and CD206, respectively.3/each) with surgical forceps or scissors and equally divided into two parts for: mechanical nondigested fragments (NDF) cell isolation, a standardized procedure previously described [Clostridium histoliticum (400 U/mL)  for 1 h at 37°C in slow rotation. At the end of the enzymatic digestion, synovial digested cells (SDC) (at the bottom of the tube) were separated from synovial digested fragments (SDF) using a 70 µm cell strainer . NDF, SDC, and SDF were cultured in petri dishes and grown in OPTIMEM  supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 15% FBS  in a humidified atmosphere at 37°C with 5% CO2. After 7 days, fragments were removed from NDF and SDF cultures and medium changed from all isolated cells. We performed (passage 0) all the evaluations indicated in To select synovial homogeneous tissue containing the most abundant cell populations (macrophages and fibroblasts) in sufficient amounts, we isolated synovial cells following both mechanical and enzymatic procedures, as summarized in escribed , and enzCD14-positive and-negative synoviocytes were also isolated from SDF isolated cells in 4 cases out of 8, using anti-CD14-coated magnetic beads , as previously described ,20.6 ± 1.5 × 105; SDF: 1.1 × 106 ± 1.2 × 105; SDC: 1.2 × 106 ± 1.1 × 105, cells were 99% live), and characterized by flow cytometry using markers expressed by synovial fibroblasts (SF): monoclonal anti-human-CD55 , -CD73, -CD90 , -CD106 ; by synovial macrophages (SM): monoclonal anti-human-CD14 , -CD68 , -CD80 , -CD163 , -CD86 , -CD206 , as well as by endothelial  and mononuclear cells  . Briefly, cells were harvested, washed twice with phosphate- buffer saline (PBS), centrifuged, washed in PBS supplemented with 2% BSA and 0.1% sodium azide (flow cytometry buffer), and aliquots of 1 × 105 cells were then incubated with specific primary antibodies at 4°C for 30 min. Cells were washed twice with a flow cytometry buffer and incubated with polyclonal rabbit anti-mouse immunoglobulins/FITC conjugate (DakoCytomation) at 4 °C for 30 min. After two final washes, cells were analyzed using a FACS CantoII Cytometer (BD). For isotype control, a nonspecific mouse IgG was substituted to the primary antibody.Freshly isolated synovial cells from NDF, SDF, and SDC (passages 0) were detached at day 10 by trypsin (Life Technologies),counted with eosin  using multiplex bead-based sandwich immunoassay kits  following the manufacturer’s instructions. Briefly, we added 50μL to each well of the diluted standards (fourfold dilution series), controls and samples in triplicate and added 50μL of coupled beads and the plate was incubated at room temperature for 30 min. The plate was then washed three times with 100μLwash buffer and incubated with 25 μL of detection antibodies for 30 min. Finally, the plate was washed three times and incubated with 50 μL of streptavidin-PE for 30 min and measured in a reader .The concentrations of IL6, CXCL10/IP10, IL10, CCL3/MIP1α, TNFα, CCL18, and TGFβ1 were simultaneously evaluated in the supernatants of 10Total RNA was extracted from NDF, SDF, and SDC isolated human synovial cells (passages 0), using RNA PURE reagent  according to the manufacturer’s instructions, and then treated with DNase I . Reverse transcription was performed using SuperScript VILO (Life Technologies) reverse transcriptase and random hexamers, following the manufacturer’s protocol. −∆Ct and expressed as a percentage of the reference gene since this was expressed in the same amount in all conditions tested. Forward and reverse oligonucleotides for PCR amplification of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), CD80, CD86, CD163, CD206, IL6,TNFα, CCL3/MIP1α, CXCL10/IP10, IL10, TGFβ1, and CCL18 are described in p < 0.05 were considered statistically significant. Values were expressed as median, minimum and maximum.Statistical analysis was performed using nonparametric tests since the data did not have a normal distribution. Mann–Whitney U test was used to compare CD55 versus CD68 and CD14-positive versus CD14-negative cells. Friedman and Dunn’s post-hoc test was used to compare CD68, CD86, and CD206 or NDF, SDF, and SDC. GraphPad Prism 8  was used for the analysis, and values of ST from 15 patients with OA were first scored on hematoxylin-eosin-stained slides, as reported by Krenn , to selep < 0.0001) higher percentage of CD55 (mean ± SD64.4 ± 6.8) compared to CD68 positive cells (mean ± SD 24.7 ± 7.4) and we found a lower percentage of CD86 (mean ± SD 16.5 ± 6.2) and CD206 (mean ± SD 13.6 ± 5.7) compared to CD68-positive cells.To identify the distribution of different macrophage subsets in OA ST with moderate-grade synovitis, we evaluated the expression of CD86 and CD206 in 6 out of 8 different cases. As we previously demonstrated , we confp = 0.0424 and p = 0.0183, respectively). In all three procedures CD3, CD31, and CD45RA typical markers of T lymphocytes, endothelial and hematopoietic cells, showed a very low percentage (not more than 2%), while CD34 was absent.Isolated synovial cells from NDF, SDF, and SDC were evaluated by flow cytometry at passage 0. As shown in p = 0.0071, p = 0.0370, p = 0.014, p = 0.014, respectively) in NDF than in SDC isolated cells. Only CD163 was significantly higher in SDF compared to SDC (p = 0.04).We then focused on typical markers of M1- and M2-like synovial macrophages CD80, CD86, CD163, and CD206 that were analyzed both at molecular and protein levels. As shown in p = 0.0397, p = 0.0483, p = 0.0436, p = 0.0035, respectively) and as mean fluorescent intensity  in NDF compared to SDC isolated cells  and protein levels , while IL6 did not show any difference.We then evaluated typical factors released by synovial M1- and M2-like macrophages. As shown in p = 0.011, p = 0.0486, p = 0.0140, respectively) and only CCL18 at protein levels (p = 0.0044) (p = 0.006) at the molecular level.Interestingly, also typically released factors of M2-like macrophages, such as IL10, TGFβ1, and CCL18, were significantly higher in NDF compared to SDC at molecular ( 0.0044) . TGFβ1 wp = 0.0043, p = 0.0043, p = 0.0022, p = 0.0043) compared to CD14-negative cells. As shown in p = 0.0079, p = 0.0006, p = 0.0159, p = 0.0286) compared to CD14-negativesynoviocytes, but lower than NDF or SDF  and we confirmed their expression only on CD14-positive synoviocytes . As showF or SDF . MoreoveF or SDF  and protF or SDF  levels, F or SDF .ST from different diseases is widely studied by histological or immunohistochemical evaluations or by focusing on isolated cells . SynovitFirst, we defined the grade of synovitis of the OA samples analyzed, and then selected only samples with Krenn score moderate-grade synovitis that had a higher percentage of both synovial fibroblasts and macrophages. These scored synovial samples were used to isolate cells using both mechanical and enzymatic procedures. Our data demonstrated that the two synovial isolation procedures did not differently affect (at passage 0) the percentage of positive isolated cells on typical markers of synovial fibroblasts , suggesting that both mechanical or enzymatic treatments had no specific impact on surface markers, confirming a high percentage of isolated synovial fibroblasts compared to macrophages . HoweverIt has been shown on fresh PBMC ,26,27 thWhen we focused on typical markers of M1- or M2-like macrophages , we observed a significant decrease of these cell populations in SDC compared to NDF. A similar trend was also observed with SDF, even if it did not reach the statistical significance at both molecular and protein levels, except for CD163. M1- and M2-like synovial macrophages seemed entrapped in the fragments and the enzymatic digestion partially contributed to facilitate their release in culture. In fact, the mechanically and enzymatically treated fragments showed no difference in the percentage of M1- and M2-like synovial macrophages, showing a similar recovery of these cells. However, the enzymatic procedure (SDF) could have the advantage of shortening the time necessary for the release of cells in culture.The analysis of released factors typical of M1- and M2-like synovial macrophages from the same number of isolated cells evidenced, both at molecular and protein levels, that TNFα, CCL3/MIP1α, CXCL10/IP10 (M1 released factors), and CCL18 (M2 released factor) were significantly higher in NDF compared to SDC, but not different from SDF. All these factors were significantly higher also in pure populations of CD14-positive OA synovial cells compared to CD14-negative ones, confirming that they are mainly released by M1-like synovial macrophages. The same trend was also confirmed using NDF and SDF procedures, that better preserved M1- and M2-like synovial macrophages in culture, and showed a high ratio of M1/M2 as found in OA synovial fluid and peripheral blood ,47,48 onBy contrast, we found that IL6, a typical cytokine produced by synovial M1-like macrophages , showed IL10 and TGFβ1 were significantly higher in NDF than SDC at molecular level but not at the protein level, even if the percentage of M2-like synovial macrophages (directly involved in the production of IL10 and TGFβ1) was significantly higher in NDF, but not different from SDF. Moreover, we also found that, both at molecular and protein levels, IL10 and TGFβ1 showed the same expression both in CD14-positive and negative OA synovial cells, and that IL10 was released in low amount as previously reported ,51,52. TTGFβ1 has been shown to be localized in the lining layer of OA ST  and prodCCL18 was the only typical M2 macrophage cytokine that we found significantly higher in the NDF procedure compared to SDC, but not different than the SDF procedure, demonstrating that this cell population was well preserved in culture. It has been shown that CCL18 is associated with OA disease severity and directly correlated with radiographic grading , suggestThe limit of this study is the number of collected synovial samples from each patient that influenced the total amount of cells obtained for performing the experiments. By contrast, the strength of this study is based on the use of a mixed population of synovial fibroblasts and macrophages. This avoids the use of a restricted number of synovial explants, which do not adequately represent the overall composition of the OA ST (characterized by patched inflamed areas) and limits the evaluation at different experimental points. Moreover, the mixed cell populations model could also contribute to defining the interactions and signaling among the cells. We have previously demonstrated  that lowThis study underlines the importance of defining the correct synovial cell isolation procedure to obtain an in vitro cell culture model that adequately preserves the cell populations and retains the features of synovial pathological native tissue. The NDF isolation procedure can retain synovial fibroblasts and a high ratio of M1/M2-like macrophages, as we found in ST, suggesting that an OA-like environment was maintained in the in vitro culture, and providing new indications and future directions for synovial OA research in vitro models. The definition of an in vitro model approximate to the pathological condition represents a prerequisite for defining the real effects of new cell therapies or drugs for OA treatment, and could contribute to the reduction or avoidance of animal models.


Adenia kirkii (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of  Ribosome-inactivating proteins (RIPs) are toxic enzymes widely distributed in the plant kingdom, but also present in some fungal and bacterial species ,2,3. RIPFurther studies showed that RIPs can deadenylate a range of polynucleotides, such as DNA, tRNA, mRNA, and viral RNA and the term polynucleotide:adenosine glycosidases was proposed ,9; afterRIPs are structurally divided into two main groups: type 1 RIPs, characterized by a single polypeptide chain of about 30 kDa with enzymatic activity, and type 2 RIPs with molecular weight of about 60 kDa, consisting of an enzymatically active A chain, similar to type 1 RIPs, linked through a disulfide bond to a B chain with lectin properties. The B chain has strong affinity for sugar moieties on cell surface and can facilitate the entry of the toxin into the cell, thus conferring to many type 2 RIPs high cytotoxicity ,12,13.Several studies indicate that RIPs have a role in protecting plants from viral and fungal infection or in plant senescence . HoweverAdenia genus are among the most lethal plant toxins. In addition to modeccin and volkensin, extracted from the roots of Adenia (Modecca) digitata (Harv.) Engl and Adenia volkensii Harms, respectively  leucine was purchased by GE Healthcare . Flasks and plates were from Falcon . The pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone  was purchased from Vinci-Biochem .Human neuroblastoma-derived NB100 cell line was from long term culture in our department  and was Rabbit sera against ricin, volkensin, type 1 RIPs , and ste® Aqueous One Solution Cell Proliferation Assay (Promega), which contains the tetrazolium compound  and an electron coupling reagent . The mitochondrial potential changes were detected using the Mitochondria Staining Kit (Sigma-Aldrich).Caspase activity was evaluated using the luminescent kit Caspase-Glo™3/7 Assay . Morphological membrane changes were detected using Annexin V-EGFP/PI detection kit . Viability was measured using the colorimetric CellTiter 96The liquid scintillation cocktail was the Ready-Gel .Pre-casted gels, molecular weight standards, and buffer strips used for electrophoretic analysis were obtained from GE Healthcare. DAPI-Antifade was from Resnova SRL, Genzano di Roma, Italy. Yeast RNA was purchased from Roche Diagnostics S.L. . Single-stranded salmon sperm DNA was purchased from Sigma-Aldrich. The water used was prepared with a Milli-Q apparatus . Other reagents used were from Merck , Carlo Erba , and Sigma. All reagents were of analytical grade, and when possible RNase-free.A. kirkii caudex (446 g) was decorticated and homogenized with an Ultra-Turrax  with 5 mL/g of phosphate-buffered saline . After overnight stirring at 4 °C, the extract was strained through cheesecloth and centrifuged at 18,000× g at 4 °C for 30 min. The supernatant  was subjected to affinity chromatography on Sepharose CL-6B matrix , pre-treated with 0.2 M HCl for 150 min at 50 °C (acid-treated Sepharose CL-6B), and equilibrated with PBS. The sample was loaded onto the acid-treated Sepharose CL-6B column (7cm h × 5cm Ø) and, after wash with PBS to eliminate the unbound material, the retained protein was eluted stepwise with 0.2 M galactose in PBS . The deribed in .Lectins from acid-treated Sepharose CL-6B were analyzed by 8–25% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the PhastSystem  both under reducing and non-reducing conditions.The volume eluted from acid-treated Sepharose CL-6B (38 mL) was concentrated to 2 mL on YM10 membrane  under nitrogen pressure and loaded into a Sephacryl S-100 column (94 cm h × 1.5 cm Ø)  in PBS. Peak fractions of the S-100 protein peaks were analyzed on 8–25% PhastGel gradient, following the supplier’s protocol. The protein fractions corresponding to the purified lectins were collected and analyzed on a 4–15% PhastGel gradient.v/v) 2-mercaptoethanol (reducing conditions), or 1 mg/mL iodoacetamide (non-reducing conditions) for 20 min at 37 °C. The gel was stained with 0.1% (w/v) Coomassie Brilliant Blue G250 in 50% methanol and 10% acetic acid, following the protocol recommended by the manufacturer . Densitometric analysis of gels was carried out using ImageJ software, version 1.53a . For electrophoretic analysis, proteins were incubated in sample buffer  containing 0.5% (3H]leucine incorporated into new synthesized proteins was measured by β-counter (Beckman Instruments). The experiments were conducted in duplicate, and IC50 values were calculated by linear regression. Specific activity is expressed as units (U) per mg of protein, where one U is the amount of proteins (in μg) that inhibits 50% protein synthesis in 1 mL of reaction mixture. Total activity was calculated as the specific activity per whole basic-fraction proteins (mg) normalized to the total proteins of the crude extract.The effect of lectins on protein synthesis was determined through a cell-free system, based on a rabbit reticulocyte lysate. Experiments were carried out both under non-reducing and reducing conditions with the addition of 1% 2-mercaptoethanol for 30 min at 37 °C. Samples were diluted and added to the reaction mixture, as previously described . The radHemagglutinating activity was determined in 96 wells microtiter plates. Each well contained 50 μL of a 2% suspension of human erythrocytes  and 2-fold serial dilutions of the lectins, in a final volume of 100 μL. The plates were gently shaken and after about 1 h at 25 °C, the presence/absence of agglutination was visually examined.w/v) were added, and the samples were vigorously vortexed for 30 s. RNA was extracted by phenolization, treated with 2 M aniline acetate (pH 4.5) on ice for 10 min in the dark, and precipitated with ethanol. The pellet was resuspended in 20 μL of sterile water and the concentration was determined by spectrophotometer at 260 nm. Ribosomal RNA was analyzed using 5% (w/v) polyacrylamide in denaturating conditions with 7 M urea. RNA samples were incubated in loading buffer containing 150 mg/mL sucrose, 7 M urea, 0.4 μg/mL bromophenol blue, and 1XTBE buffer . After boiling the samples for 30 s, the run was performed at 15 mA for 1 h 50 min, approximately, using TBE buffer. The gel was stained with ethidium bromide (20 mg/mL) in TBE buffer for 20–30 min and RNA bands were analyzed by UV-transilluminator (254–312 nm) including in the imaging instrument GelDoc (Biorad).N-glycosylase activity of kirkiin was conducted as previously described . BrieflyAdenine polynucleotide glycosylase activity was determined by measuring the adenine release from salmon sperm DNA (ssDNA) according to the method reported in  with a fw/v) polyacrylamide-7 M urea gel at 15 mA for 75 min and stained with ethidium bromide.On TMV, the adenine polynucleotide glycosylase activity of kirkiin was assayed as described in . BrieflyE. coli plasmid pCR 2.1 (Invitrogen). 200 ng of the plasmid were incubated with 3 μg of kirkiin at 37 °C for 1 h in a final volume of 10 μL of 10 mM Tris-HCl (pH 7.8), 50 mM NaCl, and 50 mM KCl in presence/absence of 5 mM MgCl2. The samples were analyzed on 0.8% agarose gel electrophoresis in TAE buffer  and visualized by gel red staining.The endonuclease activity of the RIP was assayed on the 2 × 6H2O, and 3 mM NaN3 were added. The absorption was measured at 405 nm with the Multiskan EX microtiter plate reader .ELISA assay was performed as described previously , using 2The cytotoxicity of kirkiin was assessed by evaluating both protein synthesis inhibition and viability reduction.3H]leucine incorporation in neosynthesized proteins. NB100 cells (2 × 104/well) were seeded onto 24-well plates in 250 µL of complete medium in the absence (control cultures) or presence of scalar dilutions (from 1 × 10−15 to 1 × 10−11 M) of kirkiin. After 72 h, cell protein synthesis was evaluated as previously described [−11 M), in a range between 4 and 48 h. The IC50 and IT50 (kirkiin concentration and time required to inhibit cell protein synthesis by 50%) were calculated using linear regression analysis.Protein synthesis inhibition was evaluated through L-[escribed . In addi® Aqueous One Solution Cell Proliferation), based on the cellular conversion of a tetrazolium salt into a colored formazan. NB100 cells (2 × 103/100 μL complete medium) were seeded in 96-well microtiter plates. After 24 h, cells were incubated with scalar dilutions of kirkiin (from 1 × 10−15 to 1 × 10−11 M) and left for 72 h. In addition, time course experiments were conducted on cells exposed to kirkiin (1 × 10−11 M), in a range between 4 and 48 h. After the indicated times, the medium was removed and CellTiter 96 Aqueous One Solution Reagent (1: 6 in complete medium). After 1 h of incubation at 37 °C, the absorbance at 492 nm was measured. The EC50 and ET50 (kirkiin effective concentration and time required to reduce cell viability by 50%) were calculated using linear regression analysis. The results are the means of at least three experiments performed in triplicate.Cell viability was evaluated through the colorimetric cell cytotoxicity assay (CellTiter 963/100 μL complete medium) was conducted through phase contrast microscopy, directly in 24-well plate, using an inverted microscope Nikon Eclipse TS100 . For the nuclear analysis, NB100 cells (2 × 104/500 μL complete medium) were seeded directly on a coverslip in 24-well plates 48 h prior to the experiment. After treatment with kirkiin for 48 h, cells were fixed with methanol/acetic acid 1:3 for 20 min. The analysis was conducted under Nikon Eclipse E600W fluorescence microscope with pretreatment of cells with 7 μL DAPI/antifade .The morphological analysis of treated cells (2 × 104/500 μL complete medium) were seeded directly on a coverslip in 24-well plates 48 h prior the experiments. After treatment with kirkiin for 48 h, cells were stained with 500 μL of JC-1 dye (1:100 in complete medium) and incubated at room temperature for 10 min in the dark. The cells were then washed three times with staining buffer purchased from the kit. The coverslips were inverted on glass slide and the cells were observed under Nikon Eclipse E600W fluorescence microscope.The mitochondrial membrane potential (Δѱm) was measured using the cationic, lipophilic dye JC-1  contained in Mitochondria Staining Kit (Sigma). JC-1 selectively enters the mitochondria and reversibly change color from green to red as the membrane potential increases. Cells (2 × 106/3 mL complete medium) were seeded in 25 cm2 flasks, and after incubation with kirkiin for 48 h, the cells were pelleted at 400× g for 5 min, washed twice in cold PBS, pelleted, and resuspended in 294 µL of binding buffer provided by the kit. Annexin V-EGFP (3 µL) and PI (3 µL) were added. After 10 min incubation in the dark at room temperature, cells were analyzed by flow cytometry FACSAria (BD) using the FACSDiva software.Apoptotic cell death was assessed using a flow cytometry Annexin V-EGFP/PI detection kit and by a luminescent reagent detecting caspase activity. Before flow cytometry, cells (2 × 103/100 µL complete medium) were seeded in 96-well microtiter plates. After incubation with kirkiin for the indicated amounts of time, 50 µL/well of caspase kit reagent (1:2 in complete medium) was added. The plates were shaken at 420 rpm for 1 min and then incubated for 20 min at room temperature in the dark. The luminescence was acquired (integration time 10 s) by a Fluoroskan Ascent FL (Thermo Labsystems) and the values were normalized for cell viability.The caspase-3/7 activity was assessed by the luminescent Caspase-Glo™3/7 Assay as described in . Brieflyt-distribution. The Dunnett’s test was used in addiction to ANOVA, when necessary.Statistical analyses were conducted using XLSTAT-Pro software, version 6.1.9, 2003 . The results are presented as the means ± S.D. of three different experiments. The data were analyzed using ANOVA/Bonferroni test or Student’s


Enterococcus faecium (8 strains), Enterococcus faecalis (3) and Enterococcus hirae (4). They were hemolysis-negative (γ-hemolysis) with a biofilm-forming ability, which was evaluated as low-grade biofilm formation, susceptible to conventional antibiotics and mainly susceptible to lantibiotic bacteriocins, namely, gallidermin and nisin; they even showed a higher susceptibility to gallidermin than to nisin. They were also susceptible to enterocin–durancin, but most strains showed resistance to enterocin A/P. This study indicated that bacteriocins can play a key role in preventing and/or protecting from undesirable bacterial multiplication or contamination in the food industry and that they have great potential for further experimental applications.This study investigated eight types of Slovak dry fermented meat products  that are available on the market and were produced by three different producers in different regions of Slovakia. The total counts of enterococci in these products ranged from 2.0 up to 6.0 cfu/g (log10). Three species were identified among the 15 selected enterococcal strains;  Enterococcus belong in the family Enterococcacae, order Lactobacillales, class Bacilli and phylum Firmicutes. They are also involved in the lactic acid bacteria group because of their lactic acid production [in vitro studies, as well as in vivo/in situ studies, have been conducted on enterococci, which confirmed their anti-contaminant bacteria effectivity [Sausages and salami are dry fermented meat products that are very popular and widely consumed among the populations of many countries. In this field, the production of especially traditional fermented meat products is mostly associated with the Mediterranean countries . Howeveroduction . On the ectivity ,11,12,132/air). The total count of enterococci was expressed in colony-forming units per millilitre (cfu/mL). Representative colonies were picked up, inoculated on Brain-Heart-Blood Agar  to check their purity. Pure colonies were submitted for identification using a matrix–assisted laser desorption ionisation time-of-flight spectrometry identification system  based on protein “fingerprints” [Eight different types of Slovak dry fermented meat products made by three different producers that were available on the market were used in this study, namely, the salami types known as Bratislavska, Nitran, Kalinka, Plesnivec, Scipak, Tokaj and Lovecka, as well as Kľusovska sausage. Their general/brief description is as follows: Bratislavska salami is made with a 96% mixture of meats (pork and beef), salt, antioxidants and a starter culture. Plesnivec salami is also produced with a starter culture, prepared from a mixture of meats (mostly pork), additives, dextrose and spices. Similarly, Kalinka salami contains 45% pork, 23% beef, 22% bacon/fat, garlic, spice, salt and a starter culture. The next products are produced without a starter culture: Scipak belongs among the products containing 51% pork, 27% beef, 17% bacon/fat, salt and pepper; Nitran salami is also made from a pork and beef mixture (96%), salt, spice mixture, garlic, a stabiliser and an antioxidant (ascorbic acid). Tokaj salami contains pork and spices. Lovecka salami with a smoked flavor contains pork and spices and smoked Kľusovska sausage is made from pork, bacon/fat, spices and dried vegetables. The products were bought on the market and taken to our laboratory. They were sampled and treated using the standard microbiological dilution method following the International Organization for Standardization (ISO) protocol; samples (10 g) were mixed in 90 mL of peptone water , treated using a Stomacher  and diluted (1:9). Dilutions were spread onto M-Enterococcus agar plates  and cultivated at 37 °C for 48 h in a 5% gaseous atmosphere  were used; for E. faecalis, 5′-ATCAAGTACAGTTAGTCTT-3′ and 5′-ACGATTCAAAGCTAACTG-3′ were used, following the protocol according to Woodford et al. [E. hirae DCH5 [E. faecium EK13, as well as E. faecalis CCM 4224 . The identified strains were maintained on M-Enterococcus agar . Strains were stored using the Microbank system  and used for subsequent analyses.Besides the MALDI-TOF identification, the polymerase chain reaction (PCR) genotyping method  was applied, followed by agarose electrophoresis in 0.8% agarose gel  buffered with 1×TAE  containing 1 µg/mL ethidium bromide (Sigma-Aldrich). The molecular mass standard  was used according to the manufacturer’s instructions. A DNA template from each strain was isolated by means of rapid alkaline lysis . The seqGGAAT-3′ ; for E. d et al. . The posrae DCH5  and our N-acetyl-β-glucosamonidase, α-manosidase and α-fucosidase. Inocula (65 µL) of McFarland Standard 1 suspensions were pipetted into each well of the kit. Enzyme activities were evaluated after 4 h of incubation at 37 °C and after the addition of Zym A and Zym B reagents. Color intensity values from 0 to 5 and their relevant value in nanomoles were assigned for each reaction according to the color chart supplied with the kit.The commercial API-ZYM system  was used to test the enzyme production. According to the manufacturer‘s recommendation, the following enzymes were tested: alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naftol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, Hemolysis was detected by streaking the cultures onto BH agar  and supplemented with 5% defibrinated sheep blood. Plates were incubated at 37 °C for 24 h in an incubator. The presence or absence of clear zones around the colonies was interpreted as α- and β-hemolysis respectively, while γ-hemolysis indicated negative strains .w/v) to obtain a suspension corresponding to 1 × 108 cfu/mL. A 100 µL aliquot from that dilution was transferred into 10 mL of Brain Heart Infusion/Broth . A 200 µL volume of the dilution was inoculated into polystyrene microtiter plate wells  and incubated for 24 h at 37 °C. The biofilm that formed in the microtiter plate wells was washed twice with 200 µL of deionized water and dried at 25 °C for 40 min. The remaining attached bacteria were stained for 30 min at 25 °C with 200 µL 0.1% (m/v) crystal violet in deionized water. The dye solution was aspirated away, and the wells were washed twice with 200 µL of deionized water. After the water removal, the plate was dried for 30 min at 25 °C and the dye bound to the adherent biofilm was extracted with 200 µL 95% ethanol. A 150 µL aliquot was transferred from each well into a new microplate well for absorbance (A) at 570 nm using an Apollo 11 Absorbance Microplate reader LB 913 . Each strain and condition was tested in two independent tests with 12 replicates. Sterile BHI was included in each analysis as a negative control. Streptococcus equi subsp. zooepidemicus CCM 7316 was used as a positive control . Biofilm formation was classified as highly positive (A570 ≥ 1.0), low-grade positive (0.1 ≤ A570 < 1.0) or negative (A570 < 0.1) according to Chaieb et al. [A quantitative plate assay ,21 was uE. faecium CCM 7419 [E. faecalis CCM 4224  and E. hirae DCH5 [Following the European Food Safety Authority (EFSA) rules for determining antibiotic profiles, the Clinical Laboratory Standard Institute (CLSI)  system wCCM 7419 , E. faecrae DCH5 .E. faecium EK13 (CCM 7419). Durancin ED26E/7 was prepared as previously described by Lauková et al. [Enterococcus durans [600 absorbance up to 0.800 nm). A dilution of bacteriocins in phosphate buffer  was dropped on the soft agar surface with each tested enterococcal indicator strain. The plates were incubated at 37 °C for 18 h. Clear inhibition zones around the doses of bacteriocins were checked and the inhibition activity was expressed in arbitrary units per milliliter (AU/mL); this means the reciprocal of the highest two-fold dilution of bacteriocins demonstrating complete growth inhibition of the indicator strain. Tests were performed twice. The positive control was the principal indicator strain Enterococcus avium EA5 (our isolate from piglets); its growth was inhibited by an activity of 25,600 AU/mL.Ent A/P and durancin ED26E/7 are bacteriocins classified in group II . It is pá et al. . It is ps durans . Gallides durans . BecauseTotal enterococcal count in Kalinka salami reached 2.0 cfu/g (log10), while in Scipak and Tokaj salami, it was 3.3 and 3.0 cfu/g, respectively; in Kľusovska sausage, it was determined to be 3.48 cfu/g. In Bratislavska and Nitran salami, we counted enterococci in volumes of 4.8 and 4.4 cfu/g (log10), respectively. Lovecka and Plesnivec revealed high enterococcal amounts ; however, this could have been due to the starter cultures used.E. faecium (8 strains), E. faecalis (3 strains) and E. hirae , Nitran (EF1NS), Scipak (EF2SC) and Kalinka , as well as Kľusovska sausage (EFKL5) and Plesnivec salami . However, in Plesnivec salami, we also identified E. faecalis (EEPL1S) and E. hirae (EHPL2) strains. E. faecalis was also found in Kľusovska sausage (EEKL2) and Scipak salami (EE1Sc). E. hirae strain species were isolated from Tokaj salami  and Plesnivec salami  and nine strains were assessed and allotted on the basis of secure genus identification/probable species identification (2.000–2.299). They were allotted to three species: i EHPL2, . E. hiraE. hirae EHPL2, which was resistant to tetracycline. While some strains were resistant to clindamycine (Da), namely three E. hirae strains , the other strains were susceptible to Da, and except for two E. faecalis strains  that were resistant to gentamicin, the rest were susceptible to Gn. One E. hirae (EHTOK 2), one E. faecalis EEKL2) and two E. faecium  were resistant to Amp. EHPL2 showed resistance to three antibiotics, namely, Azm, T and Da, while the remaining strains were resistant to at least one antibiotic (The strains were hemolysis negative (γ-hemolysis). They were susceptible to vancomycin, novobiocin, chloramphenicol, erythromycin and penicillin. They were resistant to kanamycin, rifampicin and streptomycin. Thirteen out of 15 strains were also resistant to Azm, except EHLov1 and EHTOK2, which were susceptible to Azm  did not form a biofilm (570 < 1.0) biofilm-forming strains ; the lowest value was measured in E. faecium strain EF1NS isolated from Nitran salami (0.113 ± 0.30).Four strains out of the 14 tested (one strain not tested) representing three species  up to 25,600 AU/mL  and EF4Kal (biofilm not formed)). The range of nisin activity against enterococci varied from 800 AU/mL  up to 6400 AU/mL , the growth of 14 strains was inhibited with activity ranging from 400 up to 6400 AU/mL , with α-chymotrypsin (EEKL 2–5 nmoL) and especially no endo-β-N-acetylglucosaminidase. The latter enzyme is one that needs to proliferate in vivo. This enzyme cleaves mannose-type glycans in glycoproteins between the N-acetylglucosamine residues of the pentasaccharide core [With regard to enzymes as disease markers, enterococci from dry fermented meat products appear not to cause spoilage because they produced no trypsin  were susceptible to treatment with lantibiotic bacteriocins, namely, gallidermin and nisin, where they showed a higher susceptibility to gallidermin than to nisin. They were also susceptible to enterocin–durancin. The results achieved represent a very promising concept in the fight against contaminant, biofilm-forming bacterial strains in the processing of meat products or the products themselves. Moreover, this study also contributes to knowledge of bacteriocin spectra and their potential range of practical applications.


The results showed that NA significantly reduced the level of IL-8 produced in both LPS and IL-1β stimulated cells, confirming the anti-inflammatory effect of NA also on intestinal inflammation. Moreover, it was demonstrated that NA treatment had a restoring effect on several metabolites whose levels were modified by treatments with IL-1β or LPS. This study points out a possible use of NA as anti-inflammatory compound and might be considered as a promising starting point in understanding the beneficial effect of NA in IBD.Inflammatory bowel diseases (IBD) are the most common gastrointestinal inflammatory pathologies. Previous work evidenced a lower content of nicotinic acid (NA) in feces of IBD patients compared to healthy subjects. In the present study, we aimed to understand the effects of NA on intestinal inflammation, as several studies reported its possible beneficial effect, and investigate its influence on inflammation-driven metabolism. NA was tested on a Caco-2 in-vitro model in which inflammation was induced with interleukin-1β (IL-1β) and lipopolysaccharide (LPS), two mayor proinflammatory compounds produced in IBD, that stimulate the production of cytokines, such as interleukin 8. A metabolomics approach, with gas chromatography–mass spectrometry (GC-MS) and nuclear proton magnetic resonance ( Inflammatory bowel disease (IBD) is a chronic inflammatory disease responsible for profound changes in the gastrointestinal tract. These changes are due, at least in part, to the recruitment of inflammatory cells, particularly myeloid cells such as neutrophils and monocytes, and include exhaustion of nutrients, increased oxygen consumption, and generation of large quantities of reactive nitrogen and oxygen intermediate . The dysTo investigate the effect of LPS, IL-1β, and NA on differentiated Caco-2 cell monolayers, experiments were initially carried out to assess cell viability after treatment and to choose a non-toxic concentration for each compound. As reported in To estimate the effect of LPS, IL-1β, and NA on inflammation, an ELISA assay was carried out to measure the levels of IL-8 released after treatments with each compound. In the first experiment, cells were treated with LPS (50 µg/mL) and LPS in combination with NA (100 µg/mL) and with IL-1β (25 ng/mL) and IL-1β plus NA , IL-1β (25 ng/mL), and NA (200 µg/mL) alone or in combination for 48 h. Cells and cell culture media metabolites extracts were analyzed with both GC-MS and he GC-MS . Metabolhe GC-MS , Table 1Results show that alanine, glutamine, lactic, and uric acid were increased in the cell culture medium after IL-1β treatment, showing an opposite trend compared to the intracellular levels. Conversely, glycine, ornithine, and glucose levels were decreased. As IL-1β, LPS increased serine, glutamine, and cholesterol levels in the cell culture medium while glucose levels were decreased. , Table 1The effect of NA treatment alone on Caco-2 model was also investigated. NA treatment alone caused the increase of ATP, glycerolphosphate, lactic acid, and glycerophosphocholine and a decrease of fumaric acid, glycine, pyruvic acid, and serine at the intracellular level , Table 1Regarding the effects of NA on rescuing metabolism after inflammation, levels of glutamine, isoleucine, ornithine, and glycerophosphocholine were increased and glutamic acid levels were reduced after IL-1β+NA treatment compared to the IL-1β treatment alone , Table 1Moreover, at the intracellular level, the IL-1β+NA treatment was able to reduce levels of alanine, 3-hydroxybutyric acid and to increase ATP and proline levels when compared to the IL-1β treatment alone. The treatment with LPS+NA caused an increase in the levels of glycerophosphocholine, ATP, glycerolphosphate, glycine, and proline and a decrease of 3-hydroxybutyric acid and alanine levels, compared to LPS treatment alone at the intracellular level , Table 1+, NADPH, and NADP+, play essential roles in energy metabolism in the living cell [The metabolic profile is altered in conditions of active inflammation, such as those associated with IBD and has become an area of significant interest for research . Our preing cell . The desing cell . Among aing cell ,16. Moreing cell  and it hing cell . In the ing cell ,6,19. Ining cell . As knowing cell , but in ing cell ,23. In ting cell ,25. Stiming cell . Similaring cell . Furthering cell  as showning cell . In the ing cell . As ment2, replacing the medium twice a week. Caco-2 cells  were cultured in Dulbecco’s modified Eagle’s medium low glucose with L-glutamine and sodium pyruvate  supplemented with 10% heat-inactivated bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were cultured in monolayers, at 37 °C, in a humidified atmosphere of 5% CO4 cells/mL in 100 μL) and exposed to different concentrations of the compounds alone: 10, 25, 50 and 100 µg/mL for LPS from Escherichia coli O111:B4 , 10 and 25 ng/mL for IL-1β  and 100, 150, 200 and 300 µg/mL for NA . NA was dissolved in the cell medium. After 48 h incubation, the medium was removed and 100 μL of MTT solution (2.5 mg/mL in fresh medium) were added and left for 2 h at 37 °C. Formazan crystals were then solubilized by adding 100 μL of dimethyl sulfoxide (DMSO). The absorbance was read at 570 nm using a microplate reader .The MTT assay was assessed on Caco-2 cells to evaluate the viability of the cells in the presence of the tested compounds. Cells were seeded in 96-well plates  and following the manufacturer’s instructions. Sample absorbance values were read at 450 nm in a microplate reader .g for 30 min at 4 °C. For GC-MS analysis, 400 μL of supernatant was aliquoted and dried in an EppendorfTM Concentrator Plus overnight. Dried pellets were derivatized with 50 μL of a solution of methoxamine in pyridine . After 1 h at 70 °C, 50 μL of MSTFA  were added and left at room temperature for one hour. Successively, 50 μL of hexane were added and samples were transferred in a vial for the GC-MS analysis. A pool of all samples was created and used as quality control (QC.) For 1H-NMR analysis, 700 μL of supernatant was aliquoted and dried in an EppendorfTM Concentrator Plus overnight. Dried hydrophilic cells and medium extracts were redissolved in 690 μL of potassium phosphate buffer in D2O  and 10 μL of TSP  as a chemical shift reference . An aliquot of 650 μL was analyzed by 1H-NMR. To perform metabolomics analysis, the growth medium was removed from the Petri dishes and aliquoted (500 μL) in eppendorf tubes to be treated similarly to the cells. Cells were washed with 3 mL of physiological solution, and intracellular metabolites were extracted with 1.2 mL of cold methanol/water (80:20) and shacked for 15 min at low temperature (4 °C). Then cells were harvested by scraping and transferred in eppendorf tubes. To ensure the complete lysis of the cells, the extraction was combined with 10 min of ultrasonic treatment at a controlled temperature (4 °C). The growth medium was extracted as described above. Briefly, 500 μL of medium were aliquoted in eppendorf tubes and centrifuged at 5500× g, 10 min, 4 °C, to remove cell debris and dead cells. Then 1.2 mL of cold methanol/water with the internal standard (80:20) was added to the supernatant of the medium and the extraction procedure followed as described for the cells. Cell suspensions and growth medium were centrifuged at 5500× v/v). Cells were scraped on ice, incubated for 15 min on ice, centrifuged at 12,500× g at 4 °C for 7 min and then stored at −20 °C prior to quantification. The calibration curve was generated using bovine serum albumin (BSA) in Cell Lytic M reagent, at different standard concentrations (0.1–5 mg/mL). 500 μL of Bradford Reagent solution  were added and incubated with 10 μL of standard and protein sample at room temperature for 5 min. The absorbance was measured at 595 nm using a microplate reader  at a controlled temperature of 37 °C. Total proteins were extracted with the Bradford assay, with some modifications. Cell pellets were added with 500 μL Cell Lytic M lysis buffer  for protein extraction, supplemented with mammalian protease and phosphatase inhibitor cocktail  equipped with a 30 m × 0.25 mm ID, fused silica capillary column, with a 0.25 μM TG-5MS stationary phase . The injector and transfer line temperatures were at 250 °C and 280 °C, respectively. The gas flow rate through the column was 1 mL/min. The column initial temperature was kept at 60 °C for 3 min, then increased to 140 °C at 7 °C/min, held at 140 °C for 4 min, increased to 300 °C at 5 °C/min, and kept for 1 min. Identification of metabolites was performed using the standard NIST 08 and GMD mass spectra libraries and, when available, by comparison with authentic standards. Peak detection and deconvolution, filtering, and normalization were performed using a pipeline on Knime . The tot1H-NMR analysis was performed using a Varian UNITY INOVA 500 spectrometer operating at 499.839 MHz for proton and equipped with a 5 mm double resonance probe . 1H-NMR spectra were acquired at 300 K with a spectral width of 6000 Hz, a 90° pulse. The acquisition time was of 1.5 s, the relaxation delay was of 2 s, and for each sample, 512 FID were collected into 64 K data points. The residual water signal was suppressed by applying a presaturation technique with low power radiofrequency irradiation for 2 s. After Fourier transformation with 0.3 Hz line broadening and a zero-filling to 64 K, 1H-NMR spectra were manually phased and baseline corrected using ACD Lab Processor Academic Edition . Spectral chemical shift referencing on the TSP CH3 signal at 0.00 ppm was performed on all spectra. Metabolites were identified and quantify of each NMR spectra of samples using the Chenomx NMR Suite 7.1  [ Canada) . The tott-test was performed to test statistical significance. All experimental data are the mean of three experiments.GraphPad Prism software  was used to perform the univariate statistical analysis on the data. Specifically, a Student’s In conclusion, this in vitro study demonstrated that NA could exert a counteracting effect in intestinal inflammation, by reducing IL-8 production after inflammatory stimuli. Furthermore, data presented here also showed how NA plays a role in metabolic rewiring, by restoring metabolites levels, altered in the inflammation process. Taken together, our data pinpointed a potential strategy to counteract intestinal inflammation, although, future studies are needed to clarify the mechanism behind the NA effects in IBD. Surely, to test the real potentiality of NA as an anti-inflammatory compound in IBD it should be tested as well in in-vivo and human studies.


P > 0.10). Serum triglycerides tended to be greater in horses consuming CSFAs compared to SB (P = 0.10); however, serum cholesterol was not different (P = 0.45). In horses consuming SB, cecal pH decreased below baseline (hour 0) at hours 2, 4, and 6 (P < 0.01), whereas cecal pH in horses consuming CSFAs was below baseline at hours 4 and 6 (P < 0.01). There were no treatment or time effects on cecal pH (P > 0.10). Cecal concentrations of total VFAs were greater in horses consuming SB compared to CSFAs at hour 2 (P = 0.01). Cecal concentrations of acetate, propionate, acetate:propionate (A:P), and butyrate were affected by time (P < 0.01). Propionate was less at hour 2 in horses fed CSFAs compared to horses fed SB (P = 0.04). A treatment × time interaction was detected for total cecal LCFAs concentration (P < 0.01); LCFAs concentration was greater at hour 2 for horses consuming CSFAs compared to horses fed SB (P = 0.02). SB oil and CSFAs have similar effects on the digestion of DM, NDF, ADF, GE, CF, and CP.Calcium salts of fatty acids (CSFAs) frequently are fed to ruminants, but their fate in the equine digestive system is unknown. The purpose of this study was to compare Enertia s/f, a proprietary CSFAs, and soybean (SB) oil with respect to impact on apparent total tract nutrient digestion and cecal fermentation parameters in horses. Eight cecally cannulated Quarter Horses were used in a crossover design in which horses consumed a diet for 32 d consisting of 1.5% body weight (BW) (as-fed) smooth bromegrass hay and 0.5% BW (as-fed) pelleted concentrate containing 4.9% CSFAs or 4.1% SB oil. Fecal samples were collected every 4 h from day 30 to 32 of each period and analyzed for apparent total tract digestibilities of dry matter (DM), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), crude fat (CF), and gross energy (GE) using acid detergent insoluble ash as an internal marker. Cecal digesta was obtained at 0, 2, 4, 6, 8, 10, and 12 h following the morning meal on day 29 of each period and analyzed for pH and concentrations of volatile fatty acid (VFA) and long-chain fatty acids (LCFAs). Serum was collected on day 33 of each period following a 16-h fast and analyzed for triglycerides and cholesterol. Apparent total tract digestibilities of DM, NDF, ADF, CP, CF, and GE were unaffected by lipid source ( The gastrointestinal tract (GIT) of horses evolved to accommodate continuous grazing. Many performance horses have greater energy requirements than what can be met by forages alone; therefore, concentrates are often added to diets to increase energy density. Unfortunately, large amounts of dietary concentrates have the potential to cause digestive upset . Fat canThe amount of liquid fat added to animal diets often is limited by pellet durability and palatability . In concDigestibility of diets containing CSFAs in horses is unknown, so the purpose of this study was to compare the influence of a proprietary CSFAs to soybean (SB) oil, a common source of supplemental fat in equine diets, on digestibility of dry matter (DM), organic matter (OM), neutral detergent fiber (NDF), acid detergent fiber (ADF), crude protein (CP), crude fat (CF), and gross energy (GE) in horses.All animal procedures were approved by the Institutional Animal Care and Use Committee at Kansas State University. Eight Quarter Horses  with a mean BW of 535 ± 26.2 kg were used. All horses were fitted with a permanent cecal cannula  in the form of Enertia s/f and were provided in a pelleted concentrate. The CSFAs, which had less CF (84%) than SB (100%), was added at a level calculated to bring CF in both concentrates to 4.1%. Diets consisted of 1.5% BW smooth bromegrass hay and 0.5% BW pelleted concentrate (as-fed basis), divided equally between two meals fed at 0700 and 1900 hours , and 3. Cecal samples from all horses were collected 0, 2, 4, 6, 8, 10, and 12 h following the morning feeding on day 29 of each period. Approximately 50 mL cecal contents were obtained through cannulae via gravity flow, strained through four layers of cheesecloth, and collected into a container . Immediately after collection, pH of strained cecal fluid was measured using a portable pH meter . One milliliter aliquots of strained cecal fluid from each horse for each time point were transferred in duplicate into microcentrifuge tubes. Deproteinization was achieved with 250 µL of 25% (wt/vol) meta-phosphoric acid solution and samples were stored at −18°C until volatile fatty acid (VFA) analysis. Twenty-five milliliters of remaining strained cecal fluid were transferred to a 25 mm × 150 mm glass screw-top tube with a Teflon lined cap  and stored at −18°C until analysis of long-chain fatty acids (LCFAs). Following final cecal collection in each period, there was a 12-h buffer period where no collections were obtained. On day 30 of each period, bedding was removed from stalls. From day 30 to 32, feces were collected from stall floors every 6 h. Feces contaminated with cecal fluid or urine were discarded. Each day, feces from each horse were homogenized and a sample was transferred to a 3.8-liter Ziploc bag . Samples from each day were combined and homogenized into 1 sample for each horse (approximately 2.3 kg) and stored at −18°C for further analysis of DM, OM, NDF, ADF, CP, CF, and GE.Following fecal collections, horses were fasted for 16 h on day 33 and approximately 15 to 20 mL whole blood was obtained via jugular venipuncture using a 38.1-mm, 20-gauge needle. Blood was collected into 10-mL non-heparinized red top Vacutainer serum tubes , left at room temperature until a clot was identified, and centrifuged at 1,400 × g at room temperature for 20 min. Serum was removed using a transfer pipette and approximately 1 mL was deposited into 1.5-mL microcentrifuge tubes in quadruplicate. Tubes were stored at −18°C for future analysis of serum triglycerides (TGs) and cholesterol.Fecal samples were dried at 55°C in a forced-air oven for 48 h and ground using a Wiley mill . Samples from hay and concentrates were ground through a 1-mm screen using a Wiley mill. Subsamples (0.5 g) of feedstuffs and feces were dried at 105°C in a forced-air oven to determine DM. Separate 3-g subsamples of hay, concentrate, and feces were combusted in a muffle oven at 450°C for 8 h for quantification of ash and OM . Dried fAdiabatic bomb calorimetry was used to determine GE in dried feed and feces . The CF Total dietary compositions were determined using this equation:3PO4; Supelco #1-1965; Agilent Technologies, Santa Clara, CA) with a flame ionization detector (compressed air set at 200 mL/min and H2 set at 20 mL/min). Nitrogen was used as the carrier gas with a flow rate of 60 mL/min. The detector and injector were set at 250°C and the column was at a constant temperature of 130°C. VFAs were quantified by comparing to known standards  containing acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate.Following deproteinization, strained cecal samples were frozen for at least 24 h, later thawed, homogenized, and centrifuged at 17,000 × g for 15 min. The aqueous supernatant was transferred into 12 mm × 32 mm gas chromatography (GC) vials, vortexed , and analyzed using a packed column  with a flame ionization detector was used. Hydrogen was used as the carrier gas with a flow rate of 1 mL/min and a split ratio of 1:100. The injection and detector temperatures were set at 250°C. Initial oven temperature was 140°C. Temperature increased at 2°C/min until 200°C was reached, and this was followed by an increase of 4°C/min until a final oven temperature of 245°C was achieved. Fatty acids were quantified by comparing to known standards  containing C6:0, C8:0, C10:0, C11:0, C12:0, C14:0, C14:1, C15:0, C15:1, C16:0, C16:1, C17:0, C17:1, C18:0, C18:1n9t, C18:1n10t, C18:1n11t, C18:1n9c, C18:1n11c, C18:2n6t, C18:2n6c, C20:0, C18:3n6, C20:1, C18:3n3, CLA 9c11t, C21:0, CLA 10t12c, CLA 9c11c, CLA 9t11t, C20:2, C22:0, C20:3n6, C22:1n9, C20:3n3, C20:4n6, C23:0, C22:2, C24:0, C20:5n3, C24:1, C22:5n3, and C22:6n3.For fatty acid analysis (C8 to C24), cecal samples were removed from a −18°C freezer and transferred directly to a precooled drying chamber of a lyophilizer  programmed with vacuum of 100 mTorr, shelf temperature of −10°C, and condenser chamber temperature of −70°C. After 48 h, samples were removed and interesterified to create methyl-esters . InteresSerum TGs and cholesterol were measured at the Animal Health and Diagnostic Center at Cornell University using the triglyceride GPO-PAP method and cholesterol CHOD-PAP method, respectively .P < 0.05, and a tendency was considered to be present when 0.05 < P < 0.10. Differences among LSMEANS were determined using the PDiff option of SAS. A Bonferroni adjustment was used to correct for multiplicity.Data were analyzed using the MIXED procedure of SAS (Version 9.4). For digestibility variables  and serum concentrations of TGs and cholesterol, the model included fixed effect of treatment and random effects of replicate (horse) and period to determine the least-squares means (LSMEANS), and experimental unit was horse. For pH, VFAs, and LCFAs, a repeated measures statement was utilized and the model included fixed effect of treatment and treatment by hour interaction and random effect of replicate (horse) and period to determine LSMEANS. Linear and quadratic contrasts for seven equally spaced time points were included to determine hour trends. Repeated statement included horse as the subject, time, and autoregressive(1) as the covariance structure. The 101 Kenward–Rogers correction was applied for degrees of freedom estimation for all analyses. Significance was declared at P > 0.10). DM intake was not different between treatments (P > 0.10), and both concentrates were consumed in their entirety within 30 min of feeding throughout the duration of the experiment. Apparent total tract digestibilities of DM, NDF, ADF, CP, CF, and GE were unaffected by lipid source . In contrast, concentrations of TGs in the serum tended to be greater in horses fed CSFAs compared to those consuming SB  and was not different from baseline at hours 8, 10, and 12 (P > 0.10). Cecal pH of horses consuming CSFAs was below baseline at hours 4 and 6 (P < 0.01), but was not different from baseline at hours 8, 10, and 12 (P > 0.10). There were no treatment effects on cecal pH (P > 0.10).There was an effect of time on cecal pH  and were not different from baseline at hour 12 (P > 0.10) in horses consuming SB. In horses consuming CSFAs, total cecal VFA concentrations were above baseline at hours 4, 6, and 8 (P < 0.001) and were not different from baseline at hours 2, 10, and 12 (P > 0.10). Total cecal VFA concentrations tended to be greater in horses consuming SB compared to horses consuming CSFAs at hour 2 (P = 0.07). By hour 4, total cecal VFA concentrations were not different in horses consuming the two diets, which continued through hour 12 (P > 0.10).There was a quadratic effect of time on total cecal VFAs concentration  in horses consuming SB. Elevation of cecal acetate concentration above baseline in horses consuming CSFAs occurred at hours 4, 6, and 8 (P < 0.01) and returned to baseline at hours 10 and 12 (P > 0.10). Cecal propionate concentrations were elevated above baseline at hours 4, 6, 8, and 10 (P < 0.03) in horses consuming SB and were elevated above baseline at hours 4, 6, and 8 in horses consuming CSFAs (P < 0.01). Horses consuming SB tended to have greater cecal acetate concentrations (P = 0.08) and greater propionate concentrations at hour 2 compared to those consuming CSFAs (P = 0.04). In horses consuming CSFAs, cecal A:P increased above baseline at hour 2 (P < 0.001); however, cecal A:P was not different from baseline in horses consuming SB at any time point (P > 0.10). Lipid source did not have an effect on A:P (P > 0.10). Cecal butyrate concentration was elevated above baseline at hours 4 and 6 in horses consuming CSFA and at hour 8 in horses consuming SB (P < 0.05); however, differences were not noted between horses consuming the two diets (P > 0.10). Cecal concentrations of valerate and isobutyrate were affected by time (P < 0.05). In horses consuming SB, cecal concentrations of valerate were increased at hour 2 (P < 0.01) compared to baseline, but not different at any other time point.Cecal concentrations of acetate, propionate, and butyrate, as well as acetate:proprionate ratio (A:P), changed over time . Tendencies for increased cecal concentrations of saturated LCFAs were detected at hours 2 and 8 (P = 0.07) in horses consuming CSFAs compared to horses consuming SB. There was also a treatment × time interaction in cecal concentrations of palmitic acid (C16:0), oleic acid (C18:1n9c), and linoleic acid (C18:2n6c), with greater concentrations in horses consuming CSFAs compared to those consuming SB at hour 2 (P < 0.05). There was no observed effect of treatment  on cecal concentrations of conjugated linoleic acid (C18:2n9c11t), heneicosylic acid (C21:0), conjugated linoleic acid (C12:2n9c11c), behenic acid (C22:0), dihomogamma linolenic acid , erucic acid (C22:1n9), alpha-linolenic acid (C20:3n3), arachidonic acid (C20:4n6), tricosylic acid (C23:0), docosadienoic acid (C22:2), lignoceric acid (C24:0), and nervonic acid (C23:1).Treatment × time interactions were observed in cecal concentrations of total LCFAs, saturated LCFAs, and unsaturated LCFAs . Mineral oil and electrolytes were mixed with 18.9 liters of water and administered via nasogastric tube. Flunixin (1.03 mg/kg), xylazine (0.29 mg/kg), and butorphanol (0.01 mg/kg) were administered intravenously and feed was withheld for 12 h. Hay was slowly reintroduced starting 12 h after symptoms subsided and grain was reintroduced after 24 h. Because hindgut disturbances may have occurred, the initial feeding period was extended by 1 wk for all horses to allow normal motility and fermentation to be restored in the colic patient. The acclimation period of period 2 was also extended 1 wk; thus, both acclimation periods were 28 d rather than the originally planned 21 d. No other signs of colic were noted throughout the experiment; however, this horse was found deceased the morning following the conclusion of this experiment. Technicians at the Veterinary Health Center were unable to identify the cause of death through standard necropsy procedures. Data obtained from this horse were not outliers compared to other horses on study; therefore, data collected from this horse were included in the statistical analyses.Although treatments were formulated to be iso-fat, proximate analysis revealed that SB concentrate was slightly greater in CF. This discrepancy could be attributed to analytical variation, improper mixing at the feed mill, or incomplete dissociation of Ca from the fatty acids.This experiment was the first of its kind to evaluate the effect of CSFAs on nutrient digestibility in the horse. In dairy cows supplemented with 4.5% added soy fatty acids or soy calcium soaps, no differences in DM, NDF, ADF, CP, nor CF digestibilities were detected . FurtherThough digestibilities of nutrients were not different between horses on the two diets in this study, the coefficients differ from what has been reported by others. Digestibility of DM may be slightly decreased in a cecally cannulated horse compared to an intact horse , but thiIn contrast to what was noted for DM, NDF, and ADF, digestibility of CP in the present study was greater than what has been reported in previous work . DelobelSerum was collected for the measurement of TGs and cholesterol after a 16-h fast in order to obtain basal values and to decrease variability due to differences in nutrient absorption rates between animals . ConcentIn ruminants, unsaturated LCFAs depress fiber fermentation more than saturated LCFAs . BecauseThere is interest in increasing energy density in diets of performance horses. Increasing starch has the potential to elicit hindgut disturbances and increase excitability, both of which are undesirable. Recently, interest has shifted to increasing dietary fat as it does not cause cecal acidosis and has been shown to decrease reactivity; however, the amount of added fat in a concentrate is limited by palatability and pellet quality. CSFAs can provide a means to add more fat prior to pelleting, but the effects of CSFAs in horses have not been reported. CSFAs may not be absorbed in the foregut with the same efficiency as SB oil; however, there are no deleterious effects on total tract digestibility of fiber in the diet. Further research is warranted to determine if greater dietary inclusion rates of CSFAs would yield the same results. Based on the results of the current study, it appears that CSFAs can be included at 4% added fat in the concentrate of horses with similar effects on nutrient digestibility to a similar inclusion of SB oil.