Gracilaria corticata and G. edulis. Proximate composition of the dried seaweeds revealed a higher content in carbohydrates (8.30 g/100 g), total crude protein (22.84 g/100 g) and lipid content (7.07 g/100 g) in G. corticata than in G. edulis. Fatty acids profile showed that G. corticata samples contain higher concentrations of saturated fatty acids, such as palmitic and stearic acids, and polyunsaturated ones such as α-linolenic and docosahexaenoic acids. Contrariwise, G. edulis contained higher amounts of monounsaturated oleic acid. Total amino acid content was 76.60 mg/g in G. corticata and 65.42 mg/g in G. edulis, being the essential amino acid content higher in G. edulis (35.55 mg/g) than in G. corticata (22.76 mg/g). Chlorophyll a was found in significantly higher amounts in G. edulis (17.14 μg/g) than G. corticata, whereas carotenoid content was significantly higher in G. corticata (12.98 μg/g) than in G. edulis. With respect to physical properties, both water- and oil-holding capacities were similar in both seaweeds, whereas swelling capacity was higher in G. edulis. In view of the results, the present study suggests that G. corticata and G. edulis contains important nutrients for human health and are possible natural functional foods.The present study sought to evaluate the nutritional composition and physicochemical properties of two dried commercially interesting edible red seaweeds,  Seaweeds are very important natural resources from the oceans that are employed as human foods and animal feeds in their whole form, and as sources of polysaccharides , carotenoids, lipids, vitamins, minerals, dietary fiber, proline and amino acids for use in food and pharmaceutical industry . SeaweedGracilaria contains a broad diversity of valuable contents for human nutrition and are one of the world’s most cultivated and valuable marine seaweed [n-3 polyunsaturated fatty acid (PUFA) to reduce the risk of cardiovascular diseases [n-3 PUFAs act as excellent antioxidants, strengthening the cell membrane, repairing damaged cells and tissues, improving heart function and fighting against cancer [n-3 PUFAs were also found to prevent the growth of atherosclerotic plaque that affects blood clotting and blood pressure and improve the immune function, while n-6 PUFAs decrease low-density lipoprotein cholesterol and may also decrease high-density lipoprotein, cholesterol which reduces heart disease risk [Seaweeds are considered healthy foods because, despite their low caloric content, they are rich in important nutrients such as protein, essential amino acids, vitamins, minerals and some bioactive compounds . Seaweed seaweed . Its lip seaweed , but it diseases ,5. In pat cancer . n-3 PUFase risk .Gracilaria species are aspartic acid, alanine, glutamic acid and glutamine. These amino acids provide the typical flavor of algae and accumulate in response to stress conditions [Gracilaria is also a good source of both soluble and insoluble dietary fiber, so it can be employed as a potential alternative to cereal-based fiber in Western countries [With respect to their protein content, the most abundant amino acids in nditions . Gracilaountries . Solubleountries . Insolubountries . Polysacountries .Gracilaria can concentrate minerals from seawater and reach a mineral content 10–20 times higher than that of terrestrial plants [Seaweeds such as l plants ,12. Chlol plants . Chloropl plants . Moreovel plants ,16,17.Gracilaria is influenced by different factors such as seaweed species, habitat, maturity stage, season, water temperature and the sampling conditions and method employed in the determinations [Gracilaria edulis and G. corticata is abundantly available in almost all seasons in Palk Bay, on the southeast coast of India, rather than other Gracilaria sp. Both G. edulis and G. corticata are commercially important and commonly edible seaweeds in India. These two algae exhibited in a previous work high biological activities  of G. corticata and G. edulis was 8.40 g/100 g and 10.40 g/100 g, respectively. With respect to proximate composition, important differences were obtained for the two seaweeds investigated. Carbohydrates and fat content were significantly higher in G. corticata, whereas protein content was significantly higher in the case of G. edulis.The crude polysaccharide content found for G. corticata and G. edulis was 5.49 ± 0.30 g/100 g and 3.92 ± 0.13 g/100 g, respectively , margaric acid (C17:0) and stearic acid (C18:0). With respect to PUFAs, linoleic acid (C18:2n-6), α-linolenic acid (C18:3n-3), stearidonic acid (C18:4n-3) and DHA (C22:6n-3) were found in both seaweeds. In the case of monounsaturated fatty acids (MUFAs), only oleic acid (C18:1) was detected in relevant amounts in both G. corticata and G. edulis. Margaric, linoleic and stearidonic acids were found in similar amounts in G. corticata and G. edulis. Palmitic, stearic, α-linolenic acid were found in higher amounts in G. corticata than in G. edulis. Overall, in G. corticata, SFAs accounted 49.4% of total fatty acids, MUFAs accounted a 3.3% and PUFAs accounted a 47.3%, whereas in the case of G. edulis, SFAs accounted 43.9% of total fatty acids, MUFAs accounted a 27% of total fatty acids, and PUFAs accounted a 29%. With respect to fatty acids profile, total fatty acid content, expressed as g fatty acids methyl esters (FAME)/100 g total fat, of ectively . The maiG. corticata and G. edulis is shown in G. corticata (76.60 ± 5.14 mg/g), than in G. edulis (65.42 ± 3.58 mg/g). These values are comparable to their corresponding crude protein content of 22.84 ± 0.87 and 25.29 ± 0.67 g/100 g, respectively, indicating that the amount of non-protein nitrogenous materials in these red seaweeds is low. The protein of G. corticata and G. edulis. Total EAAs where significantly higher in G. edulis (35.55 ± 1.75 mg/g) than in G. corticata (22.76 ± 1.81 mg/g), whereas total NEAAs where higher in G. corticata (36.14 ± 3.33 mg/g) than in G. edulis (29.86 ± 1.83 mg/g). The EAAs/total amino acid ratio suggests that more than 50% of the amino acids found in G. edulis are EAAs. The results also indicate a good ratio of essential amino acids to non-essential amino acids in G. corticata (0.62 ± 0.54 mg/g) and G. edulis (1.19 ± 0.95 mg/g). It was noted that a much higher concentration of the essential amino acid threonine (20.57 ± 0.62 mg/g) was found in G. edulis than in G. corticata. Contrariwise, alanine content was much higher in G. corticata (21.11 ± 0.54 mg/g) than in G. edulis (1.46 ± 0.18 mg/g). Aspartic acid content was similar in both seaweeds.Nine essential amino acids (EAAs), and 11 non-essential amino acids (NEAAs), were found in both G. corticata and G. edulis is shown in G. corticata showed a higher content of Mg (463.23 ± 8.87 mg/kg) and Fe (1072.48 ± 20.97 mg/kg) than G. edulis. Moreover, G. edulis was found to possess more of trace elements like Zn (42.73 ± 2.12 mg/kg) and Cu (14.61 ± 0.46 mg/kg) than G. corticata. In view of the present results, both G. corticata and G. edulis contain an adequate amount of minerals, which suggests that these seaweeds could act as important sources of mineral supplements which are essential for human nutrition.The mineral content of G. corticata and G. edulis, the presence of water-soluble vitamins  and fat-soluble vitamins (vitamin A and vitamin E) was found, as shown in G. corticata had a higher vitamin A (2.67 ± 0.31 mg/g vs. 2.14 ± 0.17 mg/g) and vitamin B9 contents (1.00 ± 0.07 mg/g vs. 0.45 ± 0.06 mg/g) than G. edulis, whereas G. edulis showed a significantly higher content of vitamin B2 (1.54 ± 0.39 mg/g vs. 0.05 ± 0.01 mg/g) and vitamin B6 (4.77 ± 0.23 mg/g vs. 3.79 ± 0.30 mg/g) than G. corticata. For both G. corticata and G. edulis at 1 mg/mL concentration indicate the presence of three major compounds, chlorophyll a, chlorophyll b and carotenoids, in G. corticata (Retention factor (Rf) value = 0.97, 0.92 and 0.95, respectively) and G. edulis . G. corticata and G. edulis contained 8.96 ± 0.39 µg/g and 17.14 ± 0.55 µg/g of chlorophyll a and 7.74 ± 0.33 µg/g and 8.44 ± 0.63 µg/g of chlorophyll b, respectively. With respect to the carotenoid content, it was higher for G. corticata (12.82 ± 0.50 µg/g) than for G. edulis (2.99 ± 0.56 µg/g). The methanolic extracts of G. corticata and G. edulis. In general, as temperature varied, the SWC and WHC of G. corticata and G. edulis powder varied, due to an increase in the solubility of the dietary fiber and the presence of protein in G. corticata and G. edulis. However, it also reaches significant differences for the case of SWC, whereas no statistical differences were obtained for WHC or OHC. The SWC of G. edulis were higher than G. corticata at both 25 °C and 37 °C . G. corticata and G. edulis, values of 4.03 ± 0.39 and 4.09 ± 0.28 g/g, respectively, were obtained at 25 °C, reduced to 3.96 ± 0.58 g/g in G. corticata and 3.64 ± 0.18 g/g in G. edulis at 37 °C. In this study, both G. corticata and G. edulis exhibited similar OHC values (about 2 g/g) at both 25 °C and 37 °C. With respect to the WHC of G. corticata and G. edulis was lower than most results obtained for Gracilaria sp. in general, such as the 12.15 g/100 g obtained for G. acerosa [G. edulis [G. edulis [G. changii [G. edulis showed a higher ash content in a DW basis than G. corticata. Similarly, it was reported an ash content of 8.70 g/100 g in G. edulis [G. changii [G. changii [G. corticata and G. edulis may have the potential to be used as a source of dietary fiber in the nutraceutical industries.With respect to proximate content, the moisture of  acerosa  the 19.2. edulis , and the changii ,21. TherGracilaria spp. than those found in the present work. Thus, it was reported a crude protein content of 6.68 g/100 g for G. edulis [G. acerosa [G. changii [G. cervicornis [G. corticata and G. edulis indicates that these seaweeds may be considered as potential marine plant sources of protein [Other authors found much lower crude protein contents in  changii  or 19.70vicornis . Moreove protein . Protein protein .G. corticata and G. edulis was markedly lower than that reported [G. edulis (10.2 g/100 g) or the 29.44 g/100 g reported for G. changii [Gracilaria species, such as G. acerosa, for which was reported a carbohydrate content of 1.05 g/100 g [The total carbohydrate content of both reported  for G. e changii . However g/100 g . The widdulis 10. g/100 g  g/100 g .G. corticata) and 4.71 g/100 g DW seaweed (G. edulis). These results are lower than those obtained by other authors [G. edulis of 8.30 g/100 g but significantly that the 0.3% reported for G. changi [G. fisheri and G. tenuistipitata [Gracilaria content in fat can widely vary depending on the species and source. In general, seaweeds have a low fat content ; that ma authors , whose r. changi , or the tipitata . Thus, GGracilaria species in previous works, such as 29.08 g/100 g [G. debilis [Gracilaria species varies due to seasonal variations, physiochemical factors, environmental conditions and extraction methods. Additionally, the variations in the polysaccharide content of Gracilaria can vary depending on atmospheric temperature at the time of extraction [G. corticata and G. edulis may exert varied biological activity [Polysaccharides are polymers composed of at least 10 monosaccharides linked by glycosidic bonds . Recentl g/100 g , 27.20 g g/100 g , 21.40 g g/100 g , and 32. debilis , in the traction . Hence tactivity .G. edulis. According to this work [G. changii [G. changii for DHA content, in which DHA were found as the most abundant fatty acids, with a 48.36% of total fatty acids. The results of the present study revealed that both seaweeds are rich in SFAs and especially in PUFAs, which provide important health benefits. With respect to most commonly found n-3 PUFA, eicosapentaenoic acid (EPA) and DHA, it is common that their contents vary dramatically from Gracilaria spp. and even into the same species [Gracilaria spp. is inconstant, because it was found in G. gracilis [G. changii [G. edulis [Gracilaria sp. [Gracilaria changii [With respect to the fatty acids composition, those of seaweeds often differ from those of terrestrial plants whereby seaweeds have a higher proportion of PUFAs than terrestrial vegetables. Red seaweeds are particularly rich in SFAs and PUFAs which have nutritional applications that lead to their extensive use in food, feed, cosmetic, biotechnological and pharmaceutical applications ,31. Varihis work , the mos changii . However species . No EPA gracilis , but it  changii  or G. ed changii .G. corticata and G. edulis was lower than those found in a previous work [G. changii. The EAAs/total amino acid ratio was higher than those previously reported [The protein composition found in this work for ous work , which rreported ,19,26. Areported , was in G. corticata showed a higher content of Mg (463.23 mg/kg) and Fe (1072.48 mg/kg) than G. edulis. Moreover, G. edulis was found to possess more of trace elements like Zn (42.73 mg/kg) and Cu (14.61 mg/kg) than G. corticata. Both seaweeds had a higher or similar content of minerals like Zn, Cu, Mg and Fe when compared with the content of G. acerosa [G. edulis [G. fisheri and G. tenuistipidatata [G. changii [G. edulis which were lower than those found for other previous works as G. changii [G. corticata and G. edulis contain an adequate amount of minerals, which suggests that these seaweeds could act as important sources of mineral supplements which are essential for human nutrition.Seaweeds are one of the richest sources of minerals and trace elements, because the cell-wall polysaccharides and proteins of seaweed contain sulfate, anionic carboxyl and phosphate groups which act as binding sites for metal retention . With reFe 1072.4 mg/kg thpidatata  or G. ch changii , with th changii . The abi changii . In viewG. corticata and G. edulis, the presence of water-soluble vitamins  and fat-soluble vitamins (vitamin A and vitamin E) was found. G. corticata had a higher vitamin A (2.67 mg/g vs. 2.14 mg/g) and vitamin B9 contents (1.00 mg/g vs. 0.45 mg/g) than G. edulis, whereas G. edulis showed a significantly higher content of vitamin B2 (1.54 mg/g vs. 0.05 mg/g) and vitamin B6 (4.77 mg/g vs. 3.79 mg/g) than G. corticata. With respect to previously published works, the vitamin content reported for Gracilaria species is widely different between the different authors [G. corticata and G. edulis showed a significantly higher vitamin A content  than those previously reported for G. acerosa [G. edulis [For both  authors . PerhapsG. edulis or G. acerosa, respectively. The variation in vitamin content may be due to some environmental factors such as salinity, atmospheric temperature, seasonality and methods of preservation and processing [Another important difference was found for the case of vitamin C that showed a higher content than those previously reported ,8 for G.ocessing . G. corticata and G. edulis contained 8.96 µg/g and 17.14 µg/g of chlorophyll a and 7.74 µg/g and 8.44 µg/g of chlorophyll b, respectively. In a previous work describing the composition of several seaweeds [a and b in red seaweeds ranged from 68 to 162 µg/g and from 25 to 46 µg/g, respectively. Specifically, for Gracilaria spp., it was reported high chlorophyll a of 577.89 µg/g and low chlorophyll b of 1.11 µg/g in G. changii [seaweeds  it was r changii . G. changii than in the present study (74.22 µg/g) [β-carotene, lutein, zeaxanthin and antheraxanthin have been identified in red seaweed, including Gracilaria species. Further, seaweed carotenoids, especially β-carotene, are preferred by the market of natural products, because they are a mixture of cis and trans isomers, which may possess anticancer activity [With respect to carotenoid content, a higher total carotenoid content was reported for 22 µg/g) . Carotenactivity .G. edulis, higher than those found in the present study. Similarly, a SWC at 37 °C of 7.68 mL/g in G. changii was reported [G. acerosa [The SWC, WHC and OHC properties of seaweeds are generally related to their content and type of polysaccharides as well as protein which links to the cell wall of polysaccharide . Previoureported , whereasG. corticata and G. edulis, a similar observation was also made in a previous work [G. edulis of 3.08 g/g. Other authors found better WHC than in the present work for other Gracilaria spp., such as G. fisheri, for which a WHC of 5.53 g/g was reported [G. changii [With respect to the WHC of ous work , that re changii , for whi changii .Gracilaria spp., it was reported [G. edulis showed an OHC of 1.64 g/g, which is very similar to the OHC values of the present study. Moreover, for G. changii [G. corticata and G. edulis could be considered as a source of food ingredients including proteins, dietary and soluble fiber [OHC is another functional property of food ingredients used in formulated foods for consumption. Ingredients with high OHC values allow the stabilization of food emulsions and high-fat food products . For othreported  that G.  changii  OHCs of  changii . The resle fiber .G. edulis and G. corticata were collected by hand from the Thondi Coast (Latitude: 9° 44′ N and Longitude: 79° 00′ E), Palk Bay, on the southeast coast of India. Freshly collected seaweeds were washed thoroughly in seawater and transported to the laboratory immediately. Epiphytes, sediment particles and other debris were removed by washing thoroughly using potable water and thoroughly washed with distilled water immediately after washing with potable water. Seaweeds were identified using a standard manual [Samples of the commercially important and commonly edible red seaweeds d manual . The vouGracilaria species was determined in all cases following Association of Official Analytical Chemists (AOAC) methods [G. corticata and G. edulis the ash content by heating at 550 °C for 24 h in a muffle furnace , moisture content by heating at 105 °C for 24 h , total fat by Soxhlet extraction with petroleum ether (AOAC 991.36) and protein by the Kjeldahl method (N × 6.25) (AOAC 981.10) [G. corticata and G. edulis was determined by the phenol–sulfuric acid method [The proximate composition of each  methods . AOAC me 981.10) . The totd method . All meaG. corticata and G. edulis using 2 g samples of each seaweed according as previously described [G. corticata and G. edulis were dissolved and homogenized with distilled water under constant stirring for 2 h at 100 °C. The residues obtained were then removed by centrifuging the sample at 6300 g for 10 min using a CPR 30 plus centrifuge . The obtained supernatant was then precipitated by addition of an ethanol-in-water solution (1:3 v/v), followed by subsequent washing with 30 mL of acetone. The precipitated polysaccharides were then collected and subsequently air-dried, re-dissolved in distilled water and washed with acetone. Afterwards, the collected polysaccharides were stored at −20 °C in a deep freezer  for further use. Isolation were performed in triplicate for each seaweed and results are expressed as g/100 g seaweeds in a dry weight basis.Polysaccharides were separated from escribed . PowdereG. corticata and G. edulis were mixed with 5 mL of a chloroform:methanol solution (2:1 v/v), tightly covered with aluminum foil and kept at room temperature for 24 h. After this period, solutions were filtered through 11 µm Whatman No. 1 filter paper, and the filtered extract was placed in a pre-weighed and oven-dried beaker. The beaker was weighed with lipids, and the difference in weight was taken as total lipid content and expressed as a percentage [Portions of 500 mg each of powdered rcentage . Afterwarcentage . For thiG. corticata and G. edulis was determined based on a reversed-phase high performance liquid chromatography (HPLC) analysis method [g (Remi Lab World) for 20 min at 4 °C. The supernatant was collected, and protein content was precipitated by adding 10% v/v trichloroacetic acid (TCA). The protein pellet was resuspended in 1 N NaOH and hydrolyzed by heating the solution with 6 N HCl in a boiling water bath for 24 h. After incubation, the supernatant was collected by centrifuging the sample at 3500 g for 15 min. The supernatant was then filtered and neutralized by the addition of 1 N NaOH. The filtered supernatant was diluted to 1:100 (v/v) with deionized water. The sample was subjected to reversed-phase HPLC analysis  with UV and fluorescence detectors. One µl of sample was injected into a Denali C18 5-mm column (4.6 mm × 150 mm) at 23 °C with detection at 254 nm. The mobile phase used was 20 mM sodium acetate/triethylamine (0.018% v/v) in phase A. The pH was adjusted to 7.2 using 1–2% acetic acid. In phase B, 20% of 100 mM sodium acetate (pH 7.2) with 1–2% acetic acid was used; 40% acetonitrile was used as the mobile phase. The protein composition was determined in triplicate for each seaweed and expressed as mg of amino acid/g of seaweed in a DW basis.Protein composition of s method . Two graG. corticata and G. edulis was performed according to the European Standards, with minor modifications according to the method previously described [Zn, Cu, Mg and Fe analysis of escribed . One graescribed . Zn, Cu,escribed  for metaG. corticata and G. edulis was determined by the method previously described [The vitamin content of escribed ; vitamina and b and carotenoid content in the G. corticata and G. edulis extracts [a and b and carotenoid content.Thin-layer chromatography (TLC) was used to screen chlorophyll extracts . The moba and b determination, 1 g each of G. corticata and G. edulis powder was extracted with 96% CH3OH, and the supernatant was collected by centrifugation at 1000 g for 1 min. After filtration, the supernatant was again filtered with Whatman No. 1 filter paper, centrifuged at 2300 g (Remi Lab World) for 10 min, and the absorbance of the collected supernatant was measured using a UV–Vis spectrophotometer [a and b contents were calculated using the following formulas in triplicate for each seaweed and expressed as µg/g of DW:666 = absorbance at 666 nm; A653 = absorbance at 653 nm. Carotenoid content was determined according to [G. corticata and G. edulis powder was extracted with 5 mL of acetone and incubated in the dark for 45 min, and the supernatant was collected by centrifugation at 10,000 g for 5 min. The supernatant was then stored in a refrigerator, and the extraction repeated with acetone until it became colourless. The supernatant was pooled and made up to 10 mL with acetone, and the absorbance of the collected supernatant was measured at 450 nm using a UV–Vis spectrophotometer . Carotenoid content was calculated using the following formula in triplicate for each seaweed and expressed as µg/g of DW:Afterwards, for chlorophyll otometer . The chlrding to . One graG. corticata and G. edulis powder, the SWC, WHC and OHC were determined for each powder. The SWC of G. corticata and G. edulis was assessed based on a method previously described [G. corticata and G. edulis were taken and mixed with 20 mL of distilled water and stirred vigorously. The influence of temperature on SWC was determined by maintaining the tubes at 25 °C and 37 °C overnight. The SWC of G. corticata and G. edulis was calculated using the following formula and expressed as ml of swollen sample per g of DW:In order to determine the physicochemical properties of the escribed . BrieflyG. corticata and G. edulis was assessed by a modified method [G. corticata and G. edulis was put into two sets of centrifuge tubes, and 20 mL of deionized water was added. The tubes were kept separately in an incubator shaker for 24 h at 25 and 37 °C. The supernatant was discarded after centrifuging the tubes at 12,000 g for 30 min (Remi Lab World). The wet weight of G. corticata and G. edulis was noted. The samples were dehydrated by keeping them in an oven at 160 °C for 2 h, and the dry weight of the sample was noted. The WHC was determined in triplicate for each seaweed and calculated using the following formula and expressed as the weight in g of water held by 1 g of DW sample:The WHC of d method . BrieflyG. corticata and G. edulis was assessed according to previous methods [G. corticata and G. edulis was taken in a tube and mixed with 10.5 g of corn oil. The tubes were placed in a shaker at room temperature for 30 min. The oil supernatant was collected by centrifugation at 3000 g for 30 min (Remi Lab World). The OHC was determined in triplicate for each seaweed and was determined using the following formula and expressed as the number of g of oil held by 1 g of DW sample:The OHC of  methods ,38. About-test was used to compare composition values between G. edulis and G. corticata. One-way analysis of variance (ANOVA) and Duncan’s test were used to compare the effects of temperature on the physicochemical properties. All determinations were performed using SPPS version 14 . A positive significant variation was defined at the significance level of p < 0.05.All results were expressed as mean ± SD. Paired sample G. corticata and G. edulis) were found to be rich sources of polysaccharides. Because of the potential as prebiotics, antioxidant and anti-inflammatory compounds, carbohydrates from seaweeds has are compounds in high demand by consumers today. The physicochemical properties and proximate composition revealed that both G. corticata and G. edulis have appreciable levels of ash, protein, carbohydrate, fatty acid, essential and non-essential amino acid and vitamin content. It is suggested that both the seaweeds tested have great potential as potential food supplements and may be used in the food industry as a source of ingredients with an appreciable amount of nutritional value. Since both red seaweeds were found to be a good source of essential nutrients, their commercial value can be enhanced by marketing them as value-added products. However, depending on their composition, G. edulis and G. corticata have important differences that make it more adequate for certain cases. G. edulis showed higher concentration of essential amino acids, chlorophyll, vitamin B2 and Zn. Thus, it could be a good nutrient for low-protein diets or people whose need to reduce their oxidative status, because of its content in chlorophyll and Zn. Contrariwise, G. corticata showed higher PUFA content, carotenoids and minerals as Fe and Mg. Thus G. corticata is more adequate than G. edulis for people who need to reinforce their intake of such nutrients.The polysaccharide content of the investigated dried red seaweeds (


Chronic hyperglycaemia is a major risk factor for diabetes-induced cardiovascular dysfunction. In a hyperglycaemic state, excess production of reactive oxygen species (ROS), coupled with decreased levels of glutathione, contribute to increased lipid peroxidation and subsequent myocardial apoptosis. N-acetylcysteine (NAC) is a thiol-containing antioxidant known to protect against hyperglycaemic-induced oxidative stress by promoting the production of glutathione. While the role of NAC against oxidative stress-related cardiac dysfunction has been documented, to date data is lacking on its beneficial effect when used with glucose lowering therapies, such as metformin (MET). Thus, the aim of the study was to better understand the cardioprotective effect of NAC plus MET against hyperglycaemia-induced cardiac damage in an H9c2 cardiomyoblast model. H9c2 cardiomyoblasts were exposed to chronic high glucose concentrations for 24 h. Thereafter, cells were treated with MET, NAC or a combination of MET and NAC for an additional 24 h. The combination treatment mitigated high glucose-induced oxidative stress by improving metabolic activity i.e. ATP activity, glucose uptake (GU) and reducing lipid accumulation. The combination treatment was as effective as MET in diminishing oxidative stress, lipid peroxidation and apoptosis. We observed that the combination treatment prevented hyperglycaemic-induced cardiac damage by increasing GLUT4 expression and mitigating lipid accumulation via phosphorylation of both AMPK and AKT, while decreasing nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), as well as protein kinase C (PKC), a known activator of insulin receptor substrate-1 (IRS-1), via phosphorylation at Ser307. On this basis, the current results support the notion that the combination of NAC and MET can shield the diabetic heart against impaired glucose utilization and therefore its long-term protective effect warrants further investigation. According to the latest IDF report 2017), the global prevalence of diabetes is rising all over the world 7, the gl,4,5,6. FMetformin is an insulin sensitizing agent known to partially protect the heart from high glucose induced oxidative stress . Howeverv/v foetal bovine serum (FBS)) and containing 33 mM glucose  for subsequent experimental analysis.A 10 mM stock solution of both MET and NAC  was made in tissue culture (TC) grade water  and thereafter, 2 mM working stock solutions were prepared and filter-sterilised. Final concentrations of 1 µM MET and/or 1 mM NAC were prepared in Dulbecco’s Modified Eagle Medium (DMEM) (supplemented with 1% v/v FBS) under standard tissue culture conditions (37 °C in humidified air and 5% CO2) in a 75 cm2 flask and media was refreshed every two days. Upon 80% confluency, cells were split and seeded in DMEM for 48 h in either a 6-well plate (2 × 105 cells/well) for protein, 96-well plate (0.8 × 105 cells/well) for ATP or 24-well plate (1 × 105 cells/well) for all other analyses. Subsequently, H9c2 cells were cultured in glucose-free DMEM without phenol red , supplemented with 1% bovine serum albumin (BSA) for 30 min, before hyperglycaemia was induced by exposing H9c2 cardiomyoblasts to 33 mM glucose  for 24 h, as per the method described by Jadaun et al. -D-glucose (DOG) uptake was measured in H9c2 cells to assess myocardial glucose uptake. The principle behind the assay entails the addition of radioactively labelled DOG to H9c2 cardiomyoblasts, followed by quantifying DOG by means of a scintillation counter. To assess NAC and MET ability to improve GU in H9c2 cardiomyoblasts after high glucose exposure, DOG uptake was performed as per the previously published protocol . Brieflyv/v) paraformaldehyde for 15 min and washed three times in PBS. Thereafter, cells were incubated in freshly prepared 0.7% (v/v) ORO staining solution for 30 min at room temperature. Following a 5 min rinse in distilled water, cells were immersed in 100% isopropanol for 20–30 s to extract the dye. Supernatants were then transferred to a 96-well plate for measurement of optical density for lipid accumulation at 490 nm with a BioTek EL×800 plate reader using Gen 5 software. For normalisation, cells were treated with 70% ethanol, then the solution was aspirated before the addition of 400 µL crystal violet (CV) for 5 min at room temperature. The CV was removed, and cells washed with PBS. Thereafter, 70% ethanol was added to extract the CV and 100 µL transferred to a clean 96-well plate for the measurement of optical density at 570 nm.Cardiac lipid accumulation is associated with decreased cardiac function. To quantify lipid accumulation, Oil red O (ORO) staining , a widely used lysochrome (fat-soluble) diazo dye for staining and quantifying neutral intracellular lipids, was adapted as previously described . Briefly® software.Intracellular production of ROS was detected using 2’,7’-dichlorflourescein diacetate (DCFH-DA) fluorescent dye , as per the previously described protocol [g for 5 min at 4 °C. Twenty microliter of the cell lysates were then transferred to a fresh 96-well plate with the addition of 200 µL WST and 20 µL Enzyme Working Solution (supplied in the kit), where after the cells were incubated at 37 °C for 20 min prior to measuring of SOD activity. Absorbance was read at an excitation and emission wavelength of 485 ± 20 nm/528 ± 20 nm with a Biotek ELX 800 plate reader using Gen 5 software.Superoxide dismutase (SOD) is an endogenous antioxidant which scavenges the superoxide anion into hydrogen peroxide and oxygen. SOD activity was measured by means of a Biovision SOD activity kit , according to manufacturer’s instructions. Briefly, H9c2 cardiomyoblasts were lysed by the addition of 100 µL of 0.1 M trizma/hydrochloride (Tris/HCl) and 0.1 mg/mL phenylmethylsulfonyl fluoride (PMSF). Lysed cells were then centrifuged at 15,000× The Glutathione Luminescent-based assay is an assay design for the quantification and detection of glutathione (GSH) levels, a known antioxidant. Total glutathione content was assessed using a 7-amino-4-chloromethylcoumarin CellTrackerTM Blue dye  assay kit following the manufacturer’s guidelines. Briefly, cells cultured in white 96-well TC plates, were lysed in 50 µL GSH lysis reagent. Cells were mixed using a orbital shaker for 5 min, where after 50 µL of a luciferin generation reagent was added and cells incubated for an additional 15 min at room temperature. GSH fluorescent intensity was determined using the BioTek FL×800 plate reader with an excitation at 485 ± 20 nm and emission at 528 ± 20 nm.™ TBARS assay kit, as previously described [g. Next, supernatants of samples and standards were inserted into a new 96-well plate to measure the absorbance at 532 nm using a BioTek ELX800 plate reader (Gen 5 software).The thiobarbituric acid reactive substances (TBARS) assay is an established method that quantifies lipid peroxides based on the production of a reactive aldehyde, malondialdehyde (MDA). Lipid peroxidation was assessed by using an OxiSelectescribed ,23. BrieJC-1 stain was used to assess mitochondrial membrane depolarization, as previously described ,23. Brie™) protein assay .Caspase 3/7 activity was determined as per previously described protocol [Protein expression was determined by Western blot analysis as previously described . Brieflyp < 0.001). Treatment with either NAC , MET  or MET+NAC  was able to significantly improve the metabolic activity of cardiomyoblasts exposed to HG. The combination of MET+NAC was as effective as the MET treatment and more effective than NAC alone   on H9c2 cardiomyoblasts over a period of 24 h. Cells cultured in HG displayed a significant decrease in ATP activity compared to the normal glucose  control  .p < 0.01). No significant difference was observed in cells exposed to NAC in comparison to the HG control. Interestingly, enhanced GU was displayed by cells treated with the combination of MET+NAC when compared to the HG control  as well as the cells treated with MET alone   . The comp < 0.001). Treatment with either NAC, MET or the combination of MET+NAC was able to decrease lipid accumulation when compared to the HG control . MET+NAC treatment was as effective at reducing lipid content as treatment with MET only (Increase in cellular lipid accumulation can lead to cardiac dysfunction. H9c2 cardiomyoblasts exposed to HG displayed a significant increase in lipid content in comparison to the NG control (91% increase MET only .p < 0.001). Treatment with either NAC  or MET  or the combination MET+NAC  drastically decreased the production of intracellular ROS when compared to the HG control . The combination of MET+NAC  was able to decrease the production of MDA to a similar effect as either NAC  or MET  when compared the HG control  in the diabetic heart. High glucose-induced lipid peroxidation was evident in H9c2 cardiomyoblasts exposed to HG when compared to the NG control  and GSH content  when compared to the normal control. This effect was abolished when cells were treated with NAC (p < 0.001), MET (p < 0.001) and the combination of MET+NAC (p < 0.001); (p < 0.05) or MET (p < 0.01) alone.Increased oxidative stress is the main initiator of cardiac dysfunction in the diabetic heart. This study showed that H9c2 cardiomyoblasts cultured in HG displayed a significant reduction in SOD activity (31% compared to 100%  0.001); A,B. Treap < 0.001). Treatment with MET  significantly prevented mitochondrial depolarisation, whereas NAC as well as the combination of MET+NAC only showed a trend towards decreased depolarisation . Metformin treatment significantly  decreased this effect to a similar degree in cells exposed to the combination treatment of MET+NAC , whereas NAC treatment showed a trend towards reduced activity when compared to the HG control (10.63 ± 1.96 versus 102.63 ± 1.39). MET+NAC was able to reduce DNA fragmentation to a similar degree as the MET treatment . Treatment with MET+NAC (p < 0.01), MET (p < 0.01) as well as NAC (p < 0.01), was able to decrease caspase activity, however the treatments were unable to completely reduce caspace 3/7 activity to the level of the NG control (Caspase 3/7 activity assay is used to measure cellular apoptotic death. High glucose treatment significantly increased caspase-3/7 activity in H9c2 cardiomyoblasts when compared to the NG control ( control .p < 0.001). H9c2 cardiomyoblasts exposed to MET (p < 0.001) and the combination of MET+NAC (p < 0.001) were able to significantly upregulate the expression of p-AMPK(Thr172). The combination treatment was similar to MET treatment only (p < 0.05). HG exposed H9c2 cells presented with a significant increase in p-NF-kB(Ser536) expression when compared to cells cultured in NG (p < 0.001). NAC (p < 0.001), MET (p < 0.001), as well as the combination of MET+NAC (p < 0.001) were able to significantly attenuate the phosphorylation of NF-kB(Ser536). Interestingly, NAC treatment only was better at attenuating p-NF-kB protein expression compared to either MET or the combination of MET+NAC treatment , whilst NF-kB, a transcription factor, regulates the expression of genes involved in inflammation in response to increased glucose levels. The expression of p-AMPK(Thr172) in H9c2 cardiomyoblasts was significantly diminished following HG exposure when compared to the NG control (ent only A but indp < 0.001). Treatment with NAC (p < 0.01), MET (p < 0.001) as well as MET+NAC (p < 0.001) significantly mitigated the phosphorylation of PKC when compared to the HG control (p < 0.001). Treatment with NAC (p < 0.01), MET (p < 0.001) and the combination of MET+NAC (p < 0.05) were able to alleviate this effect (Increased serine phosphorylation of protein kinase (p-PKC(Ser660)) has been linked with insulin resistance. H9c2 cardiomyoblasts cultured in HG significantly increased the expression of p-PKC(Ser660) when compared to the NG control ( control A. Furthes effect B.p < 0.001). Interestingly, MET, NAC showed an increasing trend, whereas the combination (MET+NAC) treatment significantly (p < 0.05) increased AKT expression (p < 0.001). However, this reduction was significantly reversed following treatment with NAC (p < 0.001), MET (p < 0.001) and that of MET+NAC (p < 0.001) when compared to the HG control (Phosphorylated protein kinase B (pAKT) signalling plays a central role in insulin-stimulated GLUT4 expression. In the present study, H9c2 cardiomyoblasts cultured in HG presented with a significant decrease in the expression of AKT when compared to the NG control , the authors reported that MET and NAC therapy are beneficial within a disease state . The aut16, the aApart from abnormal redox cycling induced by chronic glucose exposure, excessive amounts of ROS will interfere with cellular processes and oxidatively degrade membrane lipids in a process called lipid peroxidation. As such, lipid peroxidation can be seen as one of the major mechanisms associated with accelerated myocardial damage in the diabetic state. In a study published in 2013 by Basha and Priscilla, the authors showed that NAC pre-treatment at a dose of 10 mg/kg daily for 14 days in rats displayed robust antioxidant properties against isoproterenol induced myocardial lipid peroxidation and mitochondrial dysfunction similarly, while modulating calcium handling during ischemic reperfusion injury . In anotApart from increased oxidative stress and lipid peroxidation, intracellular lipid accumulation plays an important role in the development of cardiac dysfunction . The heaIt is further acknowledged that glucose homeostasis is maintained by insulin which facilitates the uptake of glucose into peripheral tissue via the GLUT4 transporter. Improved glucose disposal accounts for the redistribution of GLUT4 to the plasma membrane and subsequent regulation and maintenance of glucose homeostasis. Mechanistically, it is known that the NF-kB(Ser536)/p-PKC(Ser660)/p-IRS(Ser307) signalling pathway plays a major role in regulating glucose homeostasis in cardiac cells, while p-AMPK/p-AKT signalling ensures cellular homeostasis through increased GLUT4 uptake 41). As such, increased p-PKC(Ser660)/p-IRS(Ser307) signalling has been linked to augmented lipid accumulation and decreased GU in the diabetic heart [. As suchThe findings from this study offer plausible validation that NAC was able to attenuate high glucose-induced oxidative stress, lipid peroxidation and subsequent myocardial apoptosis in H9c2 cardiomyoblasts. However, though effective, the combination of MET and NAC did not diminish the protective effects of the MET treatment alone, which makes the combination of MET and NAC a promising candidate to further investigate their potential to protect the diabetic heart from developing cardiomyopathy. In addition, based on the results obtained we speculate that the combinatory effect of NAC and MET might be more beneficial in improving glucose uptake and increasing the endogenous antioxidant activity. If this holds true then such combination therapy would be worth pursuing, however, this requires further investigation. The authors acknowledge the following limitations to the study; (1) that the efficacy of the combined treatment was only tested in a single H9c2 cardiomyoblast model and (2) lipid toxicity is a phenomenon that occurs over a period of time and that the present study only investigated a single episode of hyperglycaemia. As such, future studies would be needed to confirm such findings on an in vivo rat model.


Mw (+61%) and polydispersity  as well as lower gT value (−16 °C) in relation to the A-POSS-free copolymer (EA-0). Differential scanning calorimetry (DSC) measurements (one glass transition process) confirmed statistic chain structure of the EA-POSS materials. Replacement of EA-0 by the EA-POSS copolymers in a SATs recipe caused simultaneous improvement of their self-adhesive features, i.e., adhesion (+70%), tack (+21%) and cohesion (+1590%). Moreover, the POSS-based copolymers improved the shear strength of thermally cured Al/SAT/Al overlap joints; the best mechanical resistance (before and after accelerated ageing tests) was observed for the sample containing 0.5 mol % of A-POSS (an increment range of 50–294% in relation to the A-POSS-free joints). Thermogravimetric analysis (TGA) revealed markedly improved thermal stability of the A-POSS-based SATs as well.New organic-inorganic hybrid copolymers (EA-POSSs) based on butyl acrylate, glycidyl methacylate, hydroxybutyl acrylate, acryloiloxybenzophenone and acryloxypropyl-heptaisobutyl-POSS (A-POSS) were prepared via free-radical solution polymerization (FRP) and applied as a component of thermally curable structural self-adhesive tapes (SATs). The EA-POSS with 0.25, 0.5 or 1 mol % of A-POSS exhibited significantly higher dynamic viscosity (ca. +104%),  Depending on the functionality  and structure  of POSSs, they can be used as fillers, comonomers, cross-linking agents or initiators [gT and thermal stability, reduced thermal conductivity), barrier (better corrosion resistance) and other properties of polymeric materials [Polyhedral oligomeric silsesquioxanes (POSSs) create a class of organosilicon materials with an empirical formula RSiOitiators . Incorpoitiators ,9,10,11 itiators ,13,14,15itiators . Applicaitiators . Moreoveitiators ,19,20 anitiators ,25,26,27aterials . It is naterials ,31,32,33aterials , while raterials . Biomediaterials , structuaterials  and cyanaterials  are knowaterials . A relataterials ,41,42. Uaterials ), it see34H72O14Si8, Mw: 929.61 g/mol, Hybrid Plastics, USA, The following components were used for preparation of hybrid epoxyacrylate copolymers (EA-POSSs): n-butyl acrylate (BA), glycidyl methacrylate (GMA), 4-hydroxybutyl acrylate (HBA) , 4-acryloyloxy benzophenone , acryloxypropyl-heptaisobutyl-POSS , BA (7.5–8 moles), GMA (1 mol), HBA (1 mol) and ABP (0.001 mol) in ethyl acetate using AIBN as an initiator (0.1 wt part/100 wt parts of the monomers). The copolymerization process was realized at 78 °C for 5 h in a glass reactor equipped with a mechanical stirrer. The prepared products  contained 50 wt % of solids. Composition of the EA-0 and EA-POSS is tabulated in Mw, Mn) and polydispersity (PDI) of the copolymers; the GPC apparatus contained the differential refractive index detector (Dn-2010 RI WGE Dr. Bures), the multiangle laser light scattering detector  and the columns: PSS 100 Å, PSS 500 Å, PSS 1000 Å and PSS 100000 Å . The GPC tests were performed using THF and polystyrene standards.Dynamic viscosity of the EA-0 and EA-POSS solutions was measured at 23 °C by means of the DV-II Pro Extra viscometer . Gel permeation chromatography (GPC-MALLS) was used for determination of molecular masses (2) using the medium pressure mercury lamp . The UV-exposition was controlled with the radiometer . Base weight and thickness of the UV-photocrosslinked SAT layers were 150 g/m2 and 100 µm, respectively. Self-adhesive properties of the thermally uncured SATs were tested according to AFERA 4001 (adhesion to a steel substrate), AFERA 4015 (tack) and AFERA 4012 (cohesion). These parameters were evaluated using three samples of each adhesive tape. Differential scanning calorimetry  was used for determination of the glass transition temperature (gT) of the SATs, enthalpy of SAT curing processes (ΔH), onset temperature of the curing reactions (iT), and maximum temperature of the curing reactions (pT). Samples (ca. 10 mg) were analyzed using standard aluminum pans at the temperature range of −80–300 °C (heating rate of 10 °C/min). Two DSC measurements for each composition were carried out. The SATs were compounded using the EA-0 or EA-POSS copolymers (50 wt parts), the epoxy resin (50 wt parts), the latent curing agent (1.5 wt parts), and the polymethylalkylsiloxane (0.75 wt part). The compositions were applied onto polyester foils (samples for self-adhesive tests) or siliconized paper (other tests), dried at 110 °C for 10 min and UV-irradiated for 40 s  were prepared using the SATs and degreased 2024 aluminum panels (100 × 25 × 1.6 mm); the joints were thermally cured at 170 °C for 60 min. Shear strength of the Al/SAT/Al systems was measured at room temperature according to the ASTM D1002-10 standard (ten samples of each system) using the Z010 machine . A long-lasting thermal ageing test was conducted acc. to MMM-A-132B (shear strength of the joints was measured after their storage at 82 °C for 192 h). Humidity resistance of the joints was determined (MMM-A-132B) after their 30-day exposure  in the climatic chamber . A fluid immersion test was realized acc. to MMM-A-132B as well; the overlap joints were immersed at 23 °C for 7 days in the aviation turbine fuel .−1 (oxirane groups) were monitored and EGC values were calculated according to Equation (1) [A(0)—the initial intensity of the peak at 915 cm−1; A(t)—the intensity of the peak at 915 cm−1 after thermal curing of the sample. The mentioned peaks were normalized in relation to the reference peaks at 1182 cm−1 (C-O-Ar bonds of Bisphenol A-based diglycidyl ether [α) of the thermally cured SATs was calculated using DSC data according to Equation (2) [TH—total enthalpy of a SAT curing process (J/g); ΔresH—enthalpy of a post-curing process of the thermally cured SAT (in a Al/SAT/Al joint). Epoxy group conversion (EGC) in thermally cured SATs was analyzed using the FT-IR technique; variations of the absorbance value at 915 cmtion (1) :(1)EGC=(yl ether ). Additi2 atmosphere) was measured using the TG Libra analyzer . Samples (ca. 10 mg) were heated in aluminum pans at the temperature range of 25–800 °C (10 °C/min).Thermal stability of the thermally cured SATs , Mw reaches its highest value (ca. 703 kg/mol) in relation to the EA-0 and the other samples. Moreover, the PDI values (Mw/Mn) for EA-POSS-0.25 (6.63 a.u.), as well as for EA-POSS-0.5 (5.54 a.u.) and EA-POSS-1 (5.72 a.u.), were markedly higher than for the reference sample (3.22 a.u.) and samples with higher A-POSS content . Generally, the GPC measurements indicate that the former group of the modified copolymers (with the lower A-POSS doses) does not have a unimodal molecular weight distribution (the high PDI). Nevertheless, all the EA-POSS samples exhibited only one gT value , EA-POSS-0.5 (−36 °C) and EA-POSS-1 (−35 °C). This was probably a result of the higher Mw and PDI values of these copolymers in relation to the reference and other samples with A-POSS; a mixture of copolymer chains with markedly different length should generally be characterized by lower crystallinity and higher molecular mobility.Selected physico-chemical features  of the epoxyacrylate copolymers (EA) chemically modified with monoacryloxypropyl-heptaisobutyl-POSS (A-POSS) are presented in gT value revealed by the DSC technique [gT value (−18 °C), and this result was quite similar to gT of the reference SAT sample with the unmodified EA-0 copolymer . Thus, it can be claimed that ER is thermodynamically miscible with EA-0 and EA-POSS-0.25. In contrast, the other samples exhibited two gT values, indicating partial immiscibility of the epoxy resin and the epoxyacrylate copolymers prepared using 0.5–5 mol % of A-POSS. It should be noted that the first/lower gT decreased (while the second/higher gT increased) with increasing content of A-POSS; generally, the differences between the two recorded gT values were largest for SAT-POSS-2.5 (30 °C) and SAT-POSS-5 (28 °C). The observed phenomenon of the main reduction in gT values  and the epoxyacrylate copolymer (EA-0) are miscible components of SAT compositions due to the polarity of the two comonomers, i.e., glycidyl methacrylate and 4-hydroxybutyl acrylate. The ER/EA-0 mixture was characterized by one echnique . In thisecrement . Additio. −10 °C , the secTg value  probablypolymers . The secSS cages  embeddedH) (185 J/g and 193 J/g), as well as higher onset temperature of the process (iT)  in relation to the A-POSS-free SAT sample  and reduction of the iT value (107 °C for SAT-POSS-5). Interestingly, the mentioned relations are typical for SAT systems modified with fillers [pT) for A-POSS-modified SAT samples confirm this thesis.Generally, SATs based on EA copolymers with the lowest doses of A-POSS (SAT-POSS-0.25 and SAT-POSS-0.5) exhibited markedly lower enthalpy of cationic curing process of epoxy groups (Δ fillers . Arguablαuv) of the adhesive films. It was previously reported that a decrement of αuv could improve adhesion of pressure-sensitive tapes [αuv values). In the case of tack, this parameter value increased with increasing A-POSS concentration up to 2.5 mol % ; at higher modifier contents, it was reduced to 17.3 N (SAT-POSS-5). In relation to the tack measurement methodology, this parameter probably decreased as a result of the relatively high stiffness of the SAT-POSS-5 film as well. The influence of POSS structures on the adhesion of epoxy adhesives has not been widely described in the literature; moreover, there are no scientific papers concerning POSS-modified pressure-sensitive tapes and pressure-sensitive structural adhesives. Thus, the present research on SATs containing EA-POSS copolymers should be considered pioneering in this field. The basic self-adhesive features of the thermally uncured SATs are presented in ve tapes . Arguabl−1 was not observed for SAT-0) to 0.5 a.u. for SAT-POSS-5. Arguably, a large size of A-POSS cages disturbed the curing process of epoxy groups via the creation of effective structural hindrances. Nevertheless, due to chemical incorporation of the cages into EA copolymer chains, this modifier did not cause such a dramatic decrement in the EGC parameter in comparison to the SATs modified with micro- and nanofillers [α; calculated using DSC data) for the thermally cured SATs  were quite similar, i.e., 0.95 a.u. for SAT-0 and ca. 0.98 a.u. for the samples with A-POSS  show that the thermally initiated curing process of epoxy groups was effectively blocked by the A-POSS presence in the EA copolymers. As a result, EGC should be taken into consideration as a more reliable parameter describing thermal crosslinking efficiency of SATs in the Al/SAT/Al joints. The SATs containing A-POSS-based epoxyacrylate copolymers were applied onto 2024 T3 aluminum panels and the prepared Al/SAT/Al overlap joints were thermally cured at 170 °C for 60 min. As can be seen in ofillers . On the h A-POSS . Post-cugT increment due to limitation of polymer chain segments mobility [gT values  were probably related to the epoxyacrylate copolymer (without or with A-POSS), while the latter gT values represent the Bisphenol A-based epoxy component or phase. As can be seen, the cured SAT-POSS-0.25 exhibited higher gT1 (96 °C) and lower gT2 . The above-mentioned initial limitation of gT2 was probably affected by the dramatic EGC reduction observed for the samples with the lowest A-POSS content. On the other hand, it is known that POSS molecules increase molecular rigidity of copolymers [gT2 value increases at higher A-POSS concentrations. It has been presented in the literature that increasing the crosslinking density of thermosetting polymers (DSC data) results in a mobility . The rea 148 °C;  than thepolymers ; thus, tτ) values recorded for the thermally cured Al/SAT/Al joints are presented in τ to 13.4 MPa , 11.5 MPa , and 10.4 MPa . Generally, it is noteworthy that the highest τ values were registered for the samples containing the copolymers with the highest Mw  probably improved the shear strength of the tested joints.Shear strength  in comparison to Al/SAT-0/Al (4.4 MPa) and other A-POSS-modified samples. Nevertheless, even the lowest dose of the cage-type modifier drastically improved the analyzed parameter of the thermally aged joints . The high thermal stability of POSS/epoxy systems  has been widely described in the literature ; in this case, T50 was also relatively higher in comparison to the reference samples (+32 °C) and the other A-POSS-based materials. It should be noted that SAT-POSS-0.25 was based on the copolymer exhibited the highest Mw  probably resulted in the relatively highest thermal stability of the SAT-POSS-0.25 polymeric joint. On the other hand, a Mw value and A-POSS concentration arguably influence T5 and T50 for the materials with a higher content of the modifier . It seems that increments of these parameters should improve thermal stability of the cured SATs. Therefore, the highest T5 and T50 values were simultaneously observed for the samples with the higher Mw  and with the higher A-POSS dose . In this case, the lowest thermal stability (except for the reference sample) was observed for the materials containing copolymers with the medium values of Mw and A-POSS content (SAT-POSS-1 and SAT-POSS-2.5).Considering the e . The reτ values than the A-POSS-free system ; moreover, τ increased with increasing A-POSS concentration and the best mechanical resistance was noted for Al/SAT-POSS-2.5/Al  and Al/SAT-POSS-5/Al . The A-POSS cages possibly limited migration of water vapor throughout the polymeric layer during storage of the joints at high humidity; the hydrophobic features of POSS were described in [τ and A-POSS-content in the applied EA copolymers) was revealed for the non-aged Al/SAT/Al joints and the joints immersed for 7 days in fuel. In both cases, the highest shear strength values were observed for the samples based on SAT-POSS-0.5 (14.0 MPa and 12.5 MPa) and the percentage increment values (vs. non-aged and aged Al/SAT-0/Al samples) reached ca. 51% and 76%, respectively. The applied immersion medium probably did not act as a destruction agent of the Al/SAT/Al joints , but as a plasticizer of the polymeric films. Thus, the shear strength of the reference and A-POSS-based overlap joints was uniformly reduced after their immersion test in the aviation fuel.Shear strength values for Al/SAT/Al joints (after their exposition in the climatic chamber) are presented in ribed in . InteresMw and polydispersity, as well as the gT value of the copolymers. Considering the fact that DSC measurements revealed only one gT value for all of the copolymers, it can be claimed that EA-POSS have statistic chain structures [Mw and gT of the applied EA-POSS copolymers, as well as on epoxy group conversion in thermally cured SATs (containing the mixture of EA-POSS and Bisphenol A-type epoxy resin). In the case of the shear strength of Al/SAT/Al joints, the best mechanical resistance was generally observed for samples containing 0.5 mol % of A-POSS . Specifically, these joints exhibited higher or similar shear strength  in comparison to the reference sample and the other A-POSS-based systems. Higher resistance of the joints to water vapor (than Al/SAT-POSS-0.5/Al) was only recorded for the samples containing the highest concentration of the A-POSS modifier (2.5 or 5 mol %).In this paper, the influence of acryloxypropyl-heptaisobutyl-POSS on selected features of epoxyacrylate copolymers (EA-POSS) was studied. Moreover, several physico-chemical, thermal and mechanical properties of thermally curable structural self-adhesive tapes with the copolymers (SATs), as well as Al/SAT/Al overlap joints were investigated. It was revealed that even a relatively small content of A-POSS  significantly affected dynamic viscosity, ructures . Replace


Stigma is commonly experienced by people living with HIV/AIDS and by those providing care to HIV/AIDS patients. Few intervention studies have explored the impact of workplace policies and/or quality improvement on stigma. We examine the contribution of health care workplace policies, procedures and quality assurance initiatives, and self- and peer-assessed individual nurse practices, to nurse-reported HIV/AIDS-stigma practices toward patients living with HIV/AIDS and nurses in health care settings. Our sample of survey respondents (n = 1157) included managers (n = 392) and registered/enrolled nurses (n = 765) from 29 facilities in 4 countries . This is one of the first studies in LMIC countries to use hierarchical linear modeling to examine the contributions of organizational and individual factors to HIV/AIDS stigma. Based on our results, we argue that organizational interventions explicitly targeting HIV/AIDS stigma are required to reduce the incidence, prevalence and morbidity of HIV/AIDS. Despite the fact that 30 years have passed since the HIV virus was first identified, those diagnosed with HIV/AIDS continue to face significant discrimination and stigma . Stigma Effectively reducing stigma is considered a critical intervention to halt the HIV/AIDS epidemic. However, most interventions tested in lower- or middle-income countries (LMICs) have focused on community approaches oriented toward individuals, such as educational, behavioural and cognitive strategies. In a recent systematic review  of interStigma-inducing policies may be engrained in the workplace. Based on this, the Global Business Coalition on HIV/AIDS formally encouraged companies without formal HIV/AIDS programmes to establish them . NotableOnly ten of 48 studies included in Stangl et al.’s  review rTo date, few intervention studies have explored the impact of workplace policies and/or mandates for total quality improvement on stigma toward people living with HIV/AIDS and/or their health care providers. Feyissa et al.  conducteA number of researchers have explored the role of quality assurance initiatives (distinct from workplace policies) on HIV/AIDS-related outcomes, such as mother-to-child transmission of HIV/AIDS , 15 and In summary, few studies have explicitly tested the impact of organizational policies and procedures on HIV/AIDS stigma . None that we could find studied the impact of quality assurance initiatives on HIV/AIDS stigma. The majority of interventions targeted individuals rather than organizations ; Stangl Does the presence of organizational-level HIV/AIDS-related workplace policies and procedures predict nurse-reported HIV/AIDS stigma toward nurses and patients?Does the presence of quality assurance initiatives predict nurse-reported HIV/AIDS stigma toward nurses and patients?Does the quality of individual level nursing practices predict nurse-reported HIV/AIDS stigma toward nurses and patients?The original study, a prospective quasi-experimental study evaluating the impact of newly-established leadership hubs on nurses’ care of adults living with HIV/AIDS in Jamaica, Kenya, South Africa and Uganda was conducted between 2008 and 2012; HIV/AIDS is highly prevalent in all of these countries. Data were collected pre- and post- establishment of leadership hubs. We direct readers to Edwards et al. [Our hypotheses were that, in the study countries: (1) the presence of organization-level HIV/AIDS-related workplace policies and procedures would predict fewer incidents of nurse-reported HIV/AIDS stigma toward nurses and patients; (2) the presence of quality assurance initiatives would predict fewer incidents of nurse-reported HIV/AIDS stigma toward nurses and patients, and; (3) a higher quality of individual level nursing practices (whether self-assessed or peer-assessed) would predict fewer incidents of nurse-reported HIV/AIDS stigma toward nurses and patients.For a comprehensive description of the leadership hub intervention, refer to Edwards et al. . This inA random, stratified sample of health institutions was selected in each district. Each national, provincial and parish/district hospital was provided with the protocol to randomly sample staff for inclusion in the study. All eligible staff employed in health centres were asked to participate; criteria for eligibility included fluency in English, employment in the health centre for a minimum of 3 months, profession of registered or enrolled nurse, and employment as a staff nurse or manager. Although institutions sampled were the same both pre- and post- intervention, individuals sampled pre-intervention were not necessarily the same as those sampled post-intervention. Only one wave of data was required to answer our secondary question; we elected to use post-intervention data because they were collected more recently. As noted by Edwards et al. , there wThe baseline and follow-up questionnaires were identical. A complete description of the questionnaire is included in Edwards et al. . For ourIn my clinical care practice: I assess my patients’ comfort in disclosing his/her HIV/AIDS status to family members”), and; (2) the clinical conduct of their coworkers (e.g. “On my work unit: Nurses and midwives assess their patients’ comfort in disclosing his/her HIV/AIDS status to family members”). Respondents were instructed to circle the response category indicating the frequency with which they  or other nurses and midwives on their unit  provided the identified type of clinical care. The two clinical conduct scales each contained 12 parallel items measured via five-point Likert scale ranging from 1 (rarely) to 5  [Each nurse participant reported on: (1) their own clinical conduct  .At your workplace is there a quality assurance or quality improvement initiative in place to monitor the occurrence of occupational exposure to HIV/AIDS?” One item in the workplace policy scale was: “At your workplace, are there policies or procedures outlining the standard treatment of staff following exposure to HIV/AIDS in the workplace?” [The scales used to evaluate workplace quality assurance initiatives and workplace policies each contained 6-items with response options of yes, no and unsure. For example, one item in the quality assurance scale was “kplace?” . Like Edkplace?”  we electobserving that a nurse kept her distance when talking to an HIV/AIDS patient”), and; the second evaluated stigmatization of nurses providing care to people with HIV/AIDS by community members and nursing co-workers (e.g. “observing someone say that nurses who care for HIV/AIDS patients spread the disease”). A four-point scale used to capture responses ranged from 1 = never to 4 = most of the time; however, the post-intervention data revealed little variation in either of the sub-scale scores. This potential limitation of low variance has been addressed by previous authors [or nurses providing care) was observed; 2 = two or more stigmatizing events were observed, with at least one each toward patients and nurses providing care.We evaluated two distinct dimensions of HIV/AIDS-stigma that could be experienced in the health care system. The 10-item and 9-item scales respectively, evaluated stigmatizing experience in the past three months: the first evaluated stigmatization of people with HIV/AIDS by nurses  often had fewer than 10 nursing staff. This exclusion still provided the minimum sample of 25 facilities required at level 2 . It is nWe built the model step-wise by adding variables at level 1 first, then level 2, using model deviance statistic as a selection criterion. Predictor variables at the individual-level, utilizing responses from staff nurses, included self-assessment of clinical conduct  and perceptions of coworkers’ clinical conduct . At the facility level we included the measures of quality assurance initiatives  and workplace policies (Policies and Procedures) to determine whether having organizational workplace policies and procedures and quality assurance initiatives predicts HIV/AIDS-stigma behaviours toward nurses and patients. Manager responses within each organization were aggregated to the institutional level to represent the institution. Stigmatization—the outcome variable—was at the individual level, using data from staff nurses. Per this selection criteria, the variables described above were introduced into the model in the order of Clinical Conduct, Co-Worker Clinical Conduct, Policies and Procedures and Quality Assurance. The fitted models described, including a full univariate model that ignores the individual-facility hierarchical structure for comparison, are presented in Table p value in the regression output was less than 0.05.Variables included in model estimations were considered as significantly contributing to prediction of the outcome variable when the The analytic sample n = 1157) included managers n = 392) and registered/enrolled nurses (n = 765) from 29 facilities—5 in South Africa, 10 in Uganda, 7 in Jamaica and 7 in Kenya. Ten of the facilities were health centers (Level 3 WHO Institutions), 8 (Level 4) were at the district/parish level and 11 were national/provincial (Level 5). Across the 29 facilities, a range of 1-88 and a median of 10 managers responded per institution. The managers in the sample were 89% female and 77% reported interacting with HIV/AIDS patients daily. Seventy percent of managers in the sample worked in national/provincial (Level 5) institutions, 18% in district/parish (Level 4) institutions and 12% in health centers (Level 3). The nurses in the sample were 93% female and 67% reported interacting with HIV/AIDS patients daily. See Table 157 inclu and regiWe calculated an ICC2 of 0.60 for our ordinal measure of stigma, supporting its use as a level 1 variable. See Table The model testing results are presented in Table To our knowledge, this is one of the first studies in LMIC countries to use HLM to analyze nested data, and to examine the contributions of organizational and individual factors to perceived stigma toward nurses caring for and patients affected by HIV/AIDS. Results reinforce the importance of using institutional policies to support destigmatizing approaches by nurses. Although other authors –34 have While this analysis focused on policies directed toward nursing care, human resource management (HRM) policies are also important as they influence health workers’ perceptions of how their rights and employment experiences will be safeguarded. While measures of HRM were included in the original surveys, these data did not meet the statistical requirements for an HRM analysis. Additional HRM findings from the study have been published in a separate manuscript .Future research questions using HLM techniques could be significantly advanced by planning this approach from the outset of the study—prior to data collection. This could allow for partitioning of variance between the actions, characteristics and/or other factors at each individual level (nurse data), unit level (manager data and unit characteristics) and facility level . However, this requires considerably larger samples sizes  than may be required for more conventional analyses. More broadly, inclusion of measures of intercessions introduced by external bodies, such as governmental ministries responsible for health outcomes (e.g. media campaigns) or professional nursing associations , may further add to our understanding of factors implicated in the reduction of HIV/AIDS stigma. In our view, the additional costs for studies designed to account for multiple avenues of influence on stigma are offset by the benefits of an advanced understanding of effective stigma reduction.HLM also requires an adequate sample size within organizations and this presents another challenge. For a study similar to ours meeting this sample size requirement would exclude many of the smaller but most predominant health care institutions in LMICs. This is because staffing numbers in these peripheral health units are often minimal; and yet, the vast majority of health care services are delivered by health unit staff. Thus, to better understand facets of stigmatization at this important, peripheral level, it may be necessary to include all allied health staff, not just one particular professional group . In some of our earlier analysis, we found that stigmatization patterns reported by nurses in these peripheral health facilities differed from those of district and provincial hospitals. Surprisingly, reports of stigmatizing behaviours by nurses and their experience of stigmatization were lower in health units than in either district or provincial hospitals. This may reflect closer ties to the community of service and perhaps better knowledge of what policies exist. However, it seems unlikely to reflect stronger quality assurance approaches given the resource constraints in these settings. Had there been sufficient numbers of respondents at the health unit level, we may have found a significant interaction effect between stigmatization scores and the presence of organizational policies and procedures.In our study, our analysis may have yielded more specific results if the tool used to evaluate HIV/AIDS stigma and discrimination had elicited more varied responses from study participants. Issues with measures of stigma have been reported by both Stangl et al. , who notAs noted above, a limitation of HLM was the requirement for a minimum of 10 individual-level responses per institution; for this reason many institutions  had to be removed from the analytic sample. Additionally, we were unable to directly connect an employee to their manager, which prevented us from conducting a more complex analysis that could have accounted for variance at three levels . The role of manager likely varied across facilities—managers in WHO level three facilities would be more likely to be involved in direct clinical care while those in WHO level 5 facilities may have been directly involved in crafting policies and procedures and in designing and implementing quality assurance initiatives. In future studies, where feasible, information on reporting structure could be recorded. This would allow us to conduct analyses that could parse out the role of managers in reducing HIV/AIDS stigma.Stigma remains a barrier to optimal management of HIV/AIDS. The potential for organization-level interventions to reduce stigma, whether against patients living with HIV/AIDS or their health care providers (or both), has been under-explored. We argue that workplace policy and quality assurance initiatives explicitly targeted at HIV/AIDS stigma are required to reduce the incidence, prevalence and morbidity of HIV/AIDS and to realize the potential of innovation in HIV/AIDS diagnosis and treatment.


Escherichia coli is one of the main responsible pathogens for moderate and serious infections in hospital and community environments, being involved in the rapid evolution of fluoroquinolones and third-generation cephalosporin resistance. The aim of this study was to evaluate the effect of combined treatments of phages and antibiotics in the inactivation of E. coli. For this, ciprofloxacin at lethal and sublethal concentrations was added at different times  and was tested in combination with the phage ELY-1 to inactivate E. coli. The efficacy of the combined treatment varied with the antibiotic concentration and with the time of antibiotic addition. The combined treatment prevented bacterial regrowth when the antibiotic was used at minimum inhibitory concentration (MIC) and added after 6 h of phage addition, causing less bacterial resistance than phage and antibiotic applied alone . Combined treatment with phage and antibiotic can be effective in reducing the bacterial density and it can also prevent the emergence of resistant variants. However, the antibiotic concentration and the time of antibiotic application are essential factors that need to be considered in the combined treatment.The emergence of antibiotic resistance in bacteria is a global concern. The use of bacteriophages (or phages) alone or combined with antibiotics is consolidating itself as an alternative approach to inactivate antibiotic-resistant bacteria. However, phage-resistant mutants have been considered as a major threat when phage treatment is employed.  Escherichia coli is a non-pathogenic commensal bacterium characterized by its diversity and versatility since it is able to colonize human and other animal gastrointestinal systems. However, this species developed some variants that colonize outside the gastrointestinal system. These strains harbor many virulence factors, causing severe diseases such as diarrhea, urinal tract infections, septicemia, pneumonia and meningitis [E. coli strains involved in enteric disease are divided into enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), Shiga-toxin-producing enteroaggregative E. coli (STEAEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), adherent invasive E. coli (AIEC) and diffusely adhering E. coli (DAEC) [E. coli is divided into neonatal meningitis-associated E. coli, uropathogenic E. coli (UPEC) and sepsis-causing E. coli [E. coli strains is currently increasing, especially against beta-lactam antibiotics and quinolones [E. coli, which is the main representative species of this family [E. coli strains, specifically antibiotic resistant ones, are among the main bacteria responsible for moderate and serious infections in the hospital and community environments [ningitis . E. colii (DAEC) ,3,4,5. T E. coli . Additioinolones ,8. Accors family ,10. Acturonments .Infections with resistant bacteria, namely those caused by Gram-negative bacteria, are difficult to treat, causing severe illness and requiring costly and sometimes toxic alternatives such as last resort antibiotics such as colistin, which is used against Gram-negative bacteria. Nevertheless, bacterial strains resistant to last resort antibiotics have been isolated worldwide ,12. The E. coli, even antibiotic resistant strains [E. coli mutants [The use of phages as antibacterial agents can be a very promising alternative for the treatment of infections, to be used alone or in combination with antibiotics. Phage treatment is based on the use of lytic phages to combat bacterial infections, mainly those caused by multidrug-resistant bacteria. There are several potential advantages of the application of phage therapy over antibiotics. Phages are usually highly specific to a single species or even strain of bacteria and therefore cause much less damage to the normal flora. Phages have limited impact in natural bacterial communities because they are self-replicating as well as self-limiting, they replicate exponentially as bacteria and decline when bacteria number decreases . Until t strains ,28,29 an mutants . Pseudomonas aeruginosa infection in endocarditis and highly synergistic with ciprofloxacin [Pseudomonas fluorescens SBW25 by the combined therapy [P. aeruginosa PAO1 population density and in limiting its recovery rate [Staphylococcus aureus phage-resistant isolates were extinct [E. coli inactivation efficacy and on the development of resistance, testing different antibiotic application times and phage/antibiotic doses.The emergence of phage-resistant mutants is nowadays a major concern regarding the use of phages to control bacterial infections. It has been shown that the use of phage cocktails can reduce the development of phage-resistant mutants ,32,33,34floxacin . In this therapy . Accordi therapy . Torres-ery rate . In anot extinct . In a pr extinct . However extinct ,53. The  extinct . The dyn extinct . If phag extinct . Thus, iE. coli Top10 [E. coli through the measurement of bioluminescence, thus avoiding the laborious and time-consuming conventional method of counting colony-forming units (CFU).The genetically transformed bacterium li Top10  was usedSalmonella enterica serovar Typhimurium (ATCC 13311 and ATCC 14028), E. coli (ATCC 25922 and ATCC 13706), Aeromonas hydrophila (ATCC 7966), Vibrio fischeri (ATCC 49387), Vibrio parahaemolyticus (DSM 27657), Vibrio anguillarum (DSM 21597), Photobacterium damselae damselae (DSM 7482), Shigella flexneri (DSM 4782), Listeria innocua (NCTC 11288), Listeria monocytogenes (NCTC 1194), Aeromonas salmonicida (CECT 894) and five strains of Salmonella enterica serovar Enteriditis. Salmonella Enteriditis was isolated from food (kindly provided by Controlvet Laboratory). E. coli , Enterobacter cloacae, Citrobacter freundii, Proteus mirabilis, Providencia sp. and P. aeruginosa were isolated in previous works from water samples collected in Ria de Aveiro [E. coli, used as phage host in the phage treatment experiments, was stored at −80 °C in 10% glycerol. Before each assay, a stock culture of E. coli was aseptically inoculated in 30 mL of Tryptic Soy Broth  and was grown overnight at 25 °C and 120 rpm stirring. Then, an aliquot (300 µL) of this culture was transferred to 30 mL of fresh TSB and grown overnight at 25 °C under stirring (120 rpm), to reach 108 relative light units (RLU), corresponding approximately to 8 log colony forming units (CFU)/mL.Other bacterial strains were used to determine the phage host range: s DSM 276, Vibrio 8–9 cells per mL.The other bacterial strains used in this study were maintained in solid Tryptic Soy Agar  at 4 °C. Before each assay, one isolated colony was aseptically transferred to 30 mL of TSB and incubated as described above. Then, an aliquot of each culture was subcultured in 30 mL of fresh TSB to reach an optical density (O.D.) of 600 nm = 0.8, corresponding to about 10E. coli (108 CFU/mL) was serially diluted (10−1–10−8) in phosphate buffered saline , 2.7 mM KCl (Sigma), 8.1 mM Na2HPO4·2H2O, and 1.76 mM KH2PO4 (Sigma), pH 7.4). The non-diluted (100) and diluted aliquots were read on a luminometer (1 mL)  to determine the bioluminescence signal. Simultaneously, 1 mL of each dilution was pour-plated in TSA and incubated at 25 °C for 24 h. Three independent experiments were performed and the results were averaged. An overnight culture of the bioluminescent E. coli strain [The phage ELY-1 was isolated in a previous work from water samples collected from the Corte das Freiras Aquaculture . The phai strain .4 (Sigma), 20 mM Tris-HCl (Sigma), 2% (w/v) gelatin, pH 7.5). Three hundred microliters of the phage stock were added to thirty milliliters of TSB with double concentration and one milliliter of the E. coli in exponential growth phase. The suspension was grown overnight and incubated at 25 °C at 50 rpm. The lysate was centrifuged at 13,000 rpm for 30 min at 4 °C to remove intact bacteria or bacterial debris. Phage suspension was stored at 4 °C and the titer was determined by the double-layer agar method [The phage suspension was prepared from the phage stock previously prepared in SM buffer (0.1 M NaCl (Sigma), 8 mM MgSOr method . Successr method  and platPhage host range was determined by spot test according to Vieira et al. (2012) . The plaE. coli is resistant to ampicillin, chloramphenicol, kanamycin, piperacillin and susceptible to ciprofloxacin. As ciprofloxacin showed a synergistic effect in our previous study [The bioluminescent us study , contrarA stock solution of ciprofloxacin  was prepared following the manufacture instructions and stored in the freezer at −80 °C.E. coli. A control of bacteria without antibiotic was also included. The MIC was defined as the lowest concentration that showed no growth in the Mueller Hinton broth. The results were interpreted according to EUCAST [The minimum inhibitory concentration (MIC) for ciprofloxacin was determined in triplicate by the microdilution method in Mueller Hinton broth according to European Committee on Antimicrobial (EUCAST) . Differeo EUCAST . Three iE. coli inactivation was determined using the phage and ciprofloxacin at 1 × MIC, 2 × MIC, 1/5MIC and 1/10MIC, at a multiplicity of infection (MOI) of 1 and 100 in TSB. To obtain a MOI of 1, 6.5 µL of 108 URLs of the overnight E. coli culture  and 12 µL of 108 PFU/mL of the phage suspension  were inoculated in 30 mL of TSB (B + P). To obtain a MOI of 100, a fresh culture of bacteria was inoculated as described above and 300 µL of 108 PFU/mL of the phage suspension  were inoculated in sterilized glass Erlenmeyer flasks with 30 mL of TSB. When the assays were performed with ciprofloxacin (B + P + Cip), the same phage and bacterium concentrations were used. For these assays, three controls were included: bacterial control (BC), antibiotic control (B + CipC) and the phage control (PC). The bacterial control was inoculated with bioluminescent E. coli but not with phage, the phage control was inoculated with phage and without bacteria, the antibiotic control was inoculated with the bioluminescent E. coli and antibiotic. The controls and test samples were incubated at 25 °C and 50 rpm stirring. Aliquots of test sample and bacterial and phage controls were collected at time zero and after 6, 12, 18, 24 and 36 h of incubation for bioluminescence signal measurement  and phage quantification . The bioluminescence signal was measured in the luminometer  in triplicate. The phage titer was determined, in duplicate, through the double agar layer method and plates were incubated for 12 h at 25 °C and expressed in PFU/mL. Three independent experiments were performed.Bioluminescent E. coli inactivation was determined using phage and ciprofloxacin at 1 × MIC and at MOI of 100 in TSB. The bacterial and phage suspension was inoculated as described , the concentrations of phage and bacterium described above were used  and Filippov et al. (2011) [The development of resistant mutants of . (2011) ,66  aliquots of 100 µL from the 10−1 to 10−5 dilutions of the bacterial culture were plated on TSA plates and incubated at 25 °C for 48 h. To determinate the frequency of E. coli mutants resistant to the antibiotic (with phage), aliquots of 100 µL from the 100 to 10−2 dilutions of the bacterial culture were plated on TSA plates by double agar layer method and incubated at 25 °C for 48 h. The calculation of the frequency of mutants was done by dividing the number of resistant bacteria (obtained from the ten isolated colonies) by the total number of sensitive bacteria. This formula was used to calculate the frequency of antibiotic-resistant mutants, phage-resistant mutants and the frequency of mutants for the mixture of phage and antibiotic.To determinate the frequency of phage-resistant bacteria, ten isolated colonies from a plate with sensitive bacteria were selected and inoculated into ten tubes with 5 mL of TSB, grown at 25 °C for 18 h. The ten previously prepared TSB cultures of bacteria were also used to determine the development of phage-resistant bacteria in the presence of ciprofloxacin. To determine the phage-resistant mutants, aliquots of 100 µL from the 10p < 0.05 was considered statistically significant and Tukey’s multiple comparison test was used for a pairwise comparison of the means. The existence of significant differences on bacterial concentration in killing curves assays was analyzed using a two-way analysis of variance (ANOVA) with repeated measures. The significance of the differences was evaluated by comparing the results obtained in the test samples and control samples for the different times between treatments of each of the three independent assays, along the incubation time. One-way ANOVA was used to examine differences between the concentration of resistant bacteria in the presence of the antibiotics, in the presence of the phage alone and in the presence of both simultaneously.Statistical analysis was performed using GraphPad Prism 7.04. Normal distribution was achieved by Kolmogorov–Smirnov test and homogeneity of variance was assessed by Levene’s test. A value of E. coli to ciprofloxacin was 0.25 mg/mL .E. coli bioluminescent, E. coli BC30, E. coli ATCC 25922, S. Typhimurium ATCC 13311 and S. Enteriditis CVD) of the 32 strains (E. coli ATCC 25922 and S. Typhimurium ATCC 13311) of the 32 strains tested. Phage infected E. coli ATCC 25922 and S. Typhimurium ATCC 13311 with an efficacy of 2.27 × 103 and 3.45 × 10−3, respectively  after 12 h of phage treatment (B + P), relative to the bacterial control (BC)  to those obtained in the bacterial control after 36 h of incubation  AI–DI. Thcubation AI–DI.E. coli inactivation was similar in both MOIs   to those obtained in bacterial control after 36 h of incubation (p < 0.05) relative to the treatment with antibiotic alone at a sublethal concentration (B + CipC1/10MIC and B + CipC1/5MIC) were observed during 36 h of incubation. The bacterial inactivation in the treatment with antibiotic at a sublethal concentration (B + CipC1/10MIC and B + CipC1/5MIC) was significantly lower than that observed in the treatment with phage, with or without the antibiotic . The bacterial inactivation in the treatment with phage and antibiotic at a sublethal concentration (B + P + Cip1/10MIC and B + P + Cip1/5MIC) was significantly higher  than that observed when the phage was used alone (B + P) after 24 and 36 h of incubation. No decrease in bacterial inactivation was observed for the treatment with ciprofloxacin at these two sublethal concentrations (B + P + Cip1/5MIC and B + P + Cip1/10MIC) when compared with the bacterial control (BC). When the phage ELY-1 was combined with antibiotic at sublethal concentrations (1/10 and 1/5 of MIC), the bioluminescent  > 0.05) AI,BI. Th > 0.05) AI,BI. Hocubation AI,BI. FoE. coli bioluminescence after 12, 18 and 24 h of incubation (p < 0.05), but the pattern of variation between treatments was similar  for both MOIs. When the phage was combined with ciprofloxacin at lethal concentrations (B + P + Cip1 × MIC and B + P + Cip2 × MIC), the increase in the MOI from 1 to 100 promoted a decrease in p < 0.05).The maximum rate of bacterial inactivation for the mix of the phage and ciprofloxacin at 1 × MIC (B + P + Cip1 × MIC) was 2.5 log RLU, achieved after 18 h of treatment at MOI of 100. In this treatment, as well as in the treatment with the antibiotic alone (B + CipC1 × MIC), no regrowth of bacteria was observed until the end of the treatment CI. When p < 0.05) was obtained after 18 h of incubation and was similar to that obtained in the antibiotic control (B + Cip2 × MIC) (p > 0.05) were observed for both MOIs between treatment with antibiotic alone (B + CipC2 × MIC) and treatment with antibiotic and phage (B + P + Cip2 × MIC) (p < 0.05) than that observed when the phage was used alone (B + P). When the phage was combined with antibiotic at 2 × MIC (B + P + Cip2 × MIC) or the antibiotic was used alone (B + CipC2 × MIC), no regrowth of bacteria was observed until the end of the treatment.When the phage was combined with ciprofloxacin at 2 × MIC (B + P + Cip2 × MIC), the maximum rate of bacterial inactivation  DI. Durin2 × MIC) DI. Howevp > 0.05), but, when the phage was incubated in the presence of the host without antibiotic, a significant increase  was observed  remained constant throughout the experiment  than that observed in the phage control (PC) (p < 0.05) was observed in the phage concentration (increase 2.0 log PFU/mL for MOI of 1 and 0.5–1.0 log PFU/mL for MOI of 100). However, for both MOIs, when the phage was incubated in the presence of the host and ciprofloxacin at lethal concentration (B + P + Cip1 × MIC and B + P + Cip2 × MIC), the phage concentration remained constant  throughout the experiment (p > 0.05) between these treatments (B + P + Cip1 × MIC and B + P + Cip2 × MIC) and phage control (PC) were observed. When the phage was incubated in the presence of the host (B + P) and antibiotic at sublethal concentrations (B + P + Cip1/10MIC and B + P + Cip1/5MIC), the phage concentration was significantly higher  AII,BII. periment CII,DII. E. coli in the BC decreased 2 log RLU, remaining constant until the end of treatment  than that obtained in the other tested conditions   AI–CI. Whp > 0.05) to that obtained when the antibiotic was added at the same time of the phage (decrease 2.1 log RLU). However, at the end of the treatment, the bacterial inactivation in this treatment was significantly higher  than that obtained in treatment with phage without antibiotic   and was similar  AI–CI.p > 0.05) to that obtained in bacteria control (BC) and to that obtained in the treatment with phage without antibiotic (B + P) (p > 0.05)  CI. In th > 0.05) CI. p > 0.05) (p < 0.05) was observed in the phage concentration (The phage control (PC) remained constant throughout the experiment  AII–CII. ntration AII–CII. p < 0.05). The number of produced phages in the three conditions (B + P + Cip6h B + P + Cip12h and B + P + Cip18h) was similar  to that obtained without the antibiotic addition (B + P) and significantly higher than that observed when the antibiotic was added at the same time of the phage  , a significant increase was observed in the phage concentration  AII–CII. E. coli showed different rates of emergence of resistant mutants when subjected to ciprofloxacin alone, phage alone and to the mix of the phage plus ciprofloxacin  was significantly higher  than that obtained when phage was used alone (3.43 × 10−5) and when the mixture of phage and ciprofloxacin (4.00 × 10−5) was used. However, when bioluminescent E. coli was treated with ciprofloxacin at 1 × MIC (3.95 × 10−6), the rates of emergence of resistant mutants were significantly different  from those obtained when phage was used alone (3.43 × 10−5) and when the mixture of phage and ciprofloxacin at 1 × MIC (4.04 × 10−7) was used. The frequency of E. coli resistant mutants in the presence of the phage alone (3.43 × 10−5) was similar  to that obtained when the phage was used in combination with ciprofloxacin at 1/5MIC (4.00 × 10−5). However, the frequency of E. coli resistant mutants in the presence of the phage alone was significantly higher  than that obtained when the phage was used in combination with ciprofloxacin at 1 × MIC (4.04 × 10−7).The bioluminescent floxacin . The devE. coli, even in commensal strains, there is a need to develop alternative treatments. Several studies have demonstrated that phages can be used to control pathogenic bacteria [E. coli; nevertheless, bacterial regrowth after treatment was observed. Thus, in the present study, we try to understand if the development of bacterial regrowth can be circumvented by the use of different antibiotic and phage concentration combinations and also by the application of antibiotic after different times of phage addition. We showed that: (1) phage and antibiotic combinations could result in positive effects in the inactivation of bacteria, preventing bacterial regrowth; (2) the efficacy of the combination depends greatly on the concentration of antibiotic and on the time of antibiotic addition; (3) the efficacy of the combination is not greatly influenced by the initial phage concentration; and (4) the combination effectively decreases the development of bacterial resistance, not only against the antibiotic but also against the phage.With the emergence of antibiotic resistance in common bacteria, such as bacteria ,72,73,74bacteria ,33,75,76bacteria , we showPhage specificity is one of the major advantages of phage treatment, since the non-target bacterial populations should remain undisturbed. The phage ELY-1 forms completely cleared zones on 5 of the 34 tested strains. However, EOP results indicate that the phage only forms lysis plaques on two of the tested bacterial strains. Other authors such as Mirzaei and Nilsson (2015) obtained similar results, stating that the spot test cannot be used for identification and selection of phages and should be replaced by the EOP assays . PositivThe kinetic theory indicates that the MOI could be critical to control pathogenic bacteria . SeveralIn this study, the combination of phages with antibiotics at sublethal and lethal concentrations, do not increase the efficacy of bacterial inactivation relative to the treatment with phages alone. However, the results demonstrated that the combination of antibiotics with phages is an effective alternative to prevent bacterial regrowth when used at 1/5MIC, 1 × MIC or 2 × MIC. The combination with the antibiotic at 1/5MIC delayed the development of resistant bacteria, bacterial regrowth was observed only after 24 h, whereas in the treatment with the phage alone it was observed after 12 h of incubation. When phage ELY-1 was combined with ciprofloxacin at lethal concentrations (1 × MIC and 2 × MIC), after 12 h of treatment, the bacterial reduction by the combined treatment (B + P + Cip1 × MIC and B + P + Cip2 × MIC) was higher than that obtained with the antibiotic alone (B + CipC) but was lower than that caused by the phage alone (B + P). However, when phage was combined with ciprofloxacin at 2 × MIC, the difference was not as high as that observed when the antibiotic was used at MIC. The results indicate that the efficiency of the combined therapy with antibiotics and phages depend on the ability of the host bacteria to replicate the phages, which is affected by the antibiotic concentration. In fact, the combined treatment affects the phage production according to the antibiotic concentration. When the ciprofloxacin was added at sublethal concentrations (B + P + Cip1/10MIC and B + P + Cip1/5MIC), the number of phages in the presence of the host increased significantly . However, in the treatment with ciprofloxacin at 1 × MIC and 2 × MIC, the concentration of phages remained constant (phage concentration similar to that of phage control). Although the antibiotic addition avoids phage replication by the bacteria when used at high concentrations, the initial quantity of phages was enough to allow the bacterial inactivation. However, the bacterial inactivation in these conditions is delayed relative to the treatment with the phage alone or with the combined treatment with sublethal antibiotic concentrations. E. coli was effectively inactivated when the antibiotic was added after the phage for the three tested times but the prevention of bacterial regrowth was more effective when the antibiotic was added after 6 h of phage addition. When the antibiotic was added 12 h after the phage, the bacterial regrowth was prevented, but not as effectively as observed after 6 h and, when the antibiotic was added 18 h after, bacterial regrowth was not prevented. This sequential approach to add the phage and antibiotic in combined treatment allows the bacteria to replicate the phages efficiently. The number of produced phages in the three conditions was similar to that obtained without the antibiotic addition (increase of around 2 log in both conditions) and significantly higher than that observed when the antibiotic was added at the same time as the phage (maximum increase of around 0.5 log). Torres-Barceló et al. (2014) obtained similar results using the combination of streptomycin and the phage LUZ7 against P. aeruginosa when the antibiotic streptomycin was added after 12 h of phage addition [Considering the results obtained in these experiments, the next step was to evaluate the efficacy of bacterial inactivation using the combined treatment but adding the antibiotic after phage addition . The addition .The results of this study demonstrated that the combination of antibiotics with phages is an effective alternative to prevent bacterial regrowth, controlling the emergence of resistance to the phages but also to the antibiotic. Similar results have been obtained by other authors ,46,49,87−7 and 4.0 × 10−5, respectively) than that observed when only the antibiotic was used , for both conditions. Contrarily, other studies demonstrated that phage–antibiotic combinations cause the same resistance as phage and antibiotic introduced individually [The results of this study, as indicated by some other authors ,42,45, svidually ,42,45. H−7) than that observed when the antibiotic was not added (3.4 × 10−5). However, this reduction was not detected for ciprofloxacin at a sub-inhibitory concentration (1/5MIC). In this case, the concentration of phage-resistant mutants in the presence of ciprofloxacin (4.00 × 10−5) was similar to that when phages were tested alone (3.43 × 10−5). Nevertheless, in our previous study [E. coli, the addition of ciprofloxacin at a sub-inhibitory concentration (1/10MIC) during phage treatment reduced the emergence of phage-resistant mutants. Further studies, using different antibiotic concentrations and different bacteria and phages, are necessary to clarify the reason for this different behavior.Relative to the emergence of phage-resistant mutants, when ciprofloxacin was added at a MIC together with the phage, the frequency of phage-mutants was lower (4.0 × 10us study , using aE. coli strain used in this study, when using the combined therapy with the phage ELY-1 and the antibiotic ciprofloxacin, the efficiency of inactivation and the prevention of resistant mutants is higher when the antibiotic is used at 1 × MIC and added 6 h after phage addition. The combined treatment can be used to inactivate E. coli, including antibiotic resistant strains, which are among the main ones responsible for moderate and serious infections in hospital and community environments.We can state that the combined treatment with phages and antibiotics is effective in reducing the bacterial density but also in preventing the emergence of phage-resistant variants. However, the antibiotic concentration and the time of antibiotic application are essential factors to be considered in the combined treatment. In the case of the