Pourouma cecropiifolia Martius) was investigated by high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). For this purpose, suitable extraction and liquid chromatographic methods were developed. Anthocyanins, flavonols and chlorogenic acids were found mainly in the peel. Besides the main anthocyanins, i.e. delphinidin 3-glucoside, cyanidin 3-glucoside and cyanidin 3-glucoside, several minor anthocyanins were identified in the peel. Among these, cyanidin 3,5-diglucoside, delphinidin 3-galactoside, cyanidin 3-rutinoside, cyanidin 3-glucoside, malvidin 3-glucoside, pelargonidin 3-glucoside, peonidin 3-glucoside and petunidin 3-glucoside were characterized on the basis of their fragmentation patterns in MS/MS experiments. The total anthocyanin content in the peel was 420.26 ± 3.07 mg kg-1 fresh weight. The pulp contained mainly 5-O-caffeoylquinic acid (210.39 ± 3.43 mg kg-1 fresh weight). Rutin was the predominant flavonol found in Amazon grape (peel 155.45 ± 2.06 mg kg-1 fresh weight and pulp 2.64 ± 1.21 mg kg-1 fresh weight). Total polyphenols content was higher in the peel than in the pulp. The phenolic profile of Amazon grape fruit ( Fruits are excellent sources of bioactive components, mainly secondary plant metabolites such as carotenoids and polyphenols. Recently, anthocyanins, along with other phenolics, have attracted much interest since they are major antioxidants in our diet and may impart health benefits linked to their antimicrobial, anti-inflammatory, and anticarcinogenic activities, insulin secretion ability, and neuroprotective effects . InteresPourouma cecropiifolia Martius), which belongs to the Moraceae family, is a tropical fruit that has a sweet and juicy flesh with a characteristic flavor which resembles Muscat grape with a mild wintergreen mint aroma. It is the only species of Pourouma that is cultivated for the consumption of the fruits in several countries located in the western Amazon Basin since pre-Hispanic times and is also referred to as mapati. The dark purple spherical drupes (2-4 cm) are eaten fresh or processed into jams, jellies, marmalades and wine. It is a dioecious tree that grows to 12 m and starts producing fruits in the third year + ion at m/z 611 resulted in fragments at 449 and 287, corresponding to the aglycone cyanidin and the loss of two hexose moieties. Compounds 8 and 11 had [M]+ ions at 535 Da. CID of the molecular ion of compound 11 led to the formation of a predominant fragment at m/z 287 and another fragment of lower intensity at m/z 449 formed by the loss of 86 a.m.u., which can be ascribed to release of the malonyl moiety. This fragmentation behavior is in accordance with literature data for cyanidin 3-glucoside; the more stable cyanidin 3-glucoside (compound 8) yielded only a product ion at m/z 287, showing that the acyl linkage to the 6”-position of the sugar is more labile than the corresponding linkage to the 3”-position [The identification of major anthocyanins  was baseO-caffeoylquinic acid) was the predominant hydroxycinnamate, with contents of 685.44 ± 5.31 and 210.39 ± 3.43 mg kg-1 fresh weight in the peel and pulp, respectively. The LC-MS/MS data showed the presence of neochlorogenic (3-O-caffeoylquinic acid), chlorogenic, 4,5-O-dicaffeoyl quinic and 5-O-feruloyl quinic acids in Amazon grape extracts  was obtained from Waters . Sodium hydroxide, water (HPLC grade), methanol (HPLC grade), acetonitrile (HLPC grade), formic acid and acetic acid were purchased from Fisher Scientific . Folin-Ciocalteu reagent (2N), sodium carbonate, gallic acid and polyamide for column chromatography were supplied by Sigma–Aldrich ; cyanidin 3-glucoside chloride, cyanidin 3-rutinoside chloride and cyanidin 3,5-diglucoside chloride were obtained from Extrasynthèse . Chlorogenic acid, (+)-catechin, (-)-epicatechin, rutin, quercetin-3-glucoside, and quercetin-3-galactoside were purchased from ChromaDex . A CRipe fruits (3 kg) were harvested in December 2009 from trees cultivated in Manaus, Amazon, Brazil. The most uniform fruits were separated and frozen at -70 °C until analysis. The average weight of the fruits was 10.4 g .g, 4 °C). The samples were extracted another three times using the same procedure. For the spectrophotometric determination of total polyphenols, the supernatants were combined, adjusted to a defined volume, and filtered through 0.45 µm filter. For high-performance liquid chromatography analysis, the combined supernatants were evaporated to dryness at 40 °C under vacuum. The residue was dissolved in water containing 0.1% formic acid (pH adjusted to 1.5 with formic acid) and transferred to a polyamide column. The resin (10 g) was eluted with aqueous 0.1% formic acid solution (60 mL) to remove sugars and then with 0.1% formic acid in methanol (200 mL) to recover the polyphenols. The eluate was evaporated at 40 °C under vacuum to remove the organic solvent and the residue dissolved in water containing 0.1% formic acid. A 10 µL aliquot was injected for HPLC analysis of anthocyanins [Frozen fruits were peeled manually using a stainless steel knife. Polyphenols were extracted by homogenizing frozen peel (12 g) and frozen pulp (22 g) in 80% aqueous methanol containing 0.1% formic acid 70 mL) for 30 s. The mixture was sonicated for 20 min and centrifuged for 30 min . After washing with water containing 0.1% formic acid (10 mL), the polyphenol fraction 1 was collected. Polyphenol fraction 2 was recovered after eluting the C18 Sep-Pak cartridge with ethyl acetate. Both fractions were evaporated to dryness using a rotary evaporator. The residue was dissolved in 80% aqueous methanol (0.1% formic acid) [For the analysis of non-anthocyanin polyphenols, the extraction was performed as described above and the residue obtained after evaporation of methanol was dissolved in water containing 0.1% formic acid (pH adjusted to 1.5 with formic acid). The aqueous solution was extracted four times with 50 mL of ethyl acetate and the organic phase was evaporated to dryness. The residue was dissolved in water and the pH adjusted to 7 with 1 N NaOH solution. It was applied to a Cet al. [2CO3, 10 mL) was added, and the flask was made up to volume with distilled water. The absorbance at 765 nm was measured after 1 h, and the measurement was compared to a standard curve of gallic acid. Concentrations were expressed as milligrams of gallic acid equivalents per 100 g of fresh weight ± SD for triplicate fruit extracts.The total phenols content of the extracts was measured using a modified colorimetric Folin-Ciocalteu assay as described by Singleton et al. . One mL -1 (peak width 0.2 min). Quantification was conducted by LC-DAD with external calibration using a set of seven standard dilutions of cyanidin-3-glucoside at 520 nm. A stock solution of 1 mg/mL in 80% methanol in aqueous 0.1% formic acid was prepared and stored at -20 °C. The calibration curves were linear over the range of 1 to 200 µg/mL with a correlation coefficient of ≥ 0.99. For quantification, peak areas were correlated with concentrations in accordance with the calibration curves. Data are reported as means ± standard deviations of triplicate independent analyses.Anthocyanin analysis was conducted using a 1200 Series Agilent Technologies HPLC  which was equipped with a model G1315D diode array detector (DAD), a model G1379B degasser, a model G1312A binary gradient pump, a model G1329A thermoautosampler and a model G1316A column oven. The reversed-phase separation was performed on a 250 mm × 4.6 mm i.d. Symmetry C18 column, particle size 5 µm  with a Nova-Pak 4 µm C18 guard column 3.9 × 20 mm operating at 35 °C and at a flow rate of 1.0 mL/min. The compounds were separated with gradient elution of (A) 4.5% aqueous formic acid and (B) 80% acetonitrile in solution A. The gradient program was: 0-15% B (9 min), 15-45% B (22 min), 45-100% B (6 min), 100-0% B (1 min), 2 min 0% B. The injection volume was 10 uL. Monitoring was performed at 520 nm and the diode array detector was set at an acquisition range from 200 nm to 700 nm at a spectral acquisition rate of 1.25 scans s18 precolumn (Phenomenex) operating at 20 °C and at a flow rate of 0.5 mL/min. The compounds were separated with gradient elution of (A) 2% aqueous acetic acid solution and (B) 0.5% aqueous acetic acid solution and acetonitrile . Samples were eluted with the following gradient: 0% B (5 min), 0-40% B (10 min), 40-60% B (40 min), 60-80% B (10 min), 80-100% B (10 min), 100% B (30 min), and 100-0% B (2 min). The injection volume was 20 uL. Monitoring was performed at 280 and 320 nm and the diode array detector was set at an acquisition range from 200 nm to 700 nm at a spectral acquisition rate of 1.25 scans s-1 (peak width 0.2 min). Quantification was performed by LC-DAD using external calibration with a set of seven standard dilutions of rutin at 320 nm and chlorogenic acid at 280 nm. Data acquisition, peak integration, and calibrations were performed with Agilent Chemstation software. A stock solution of 1 mg/mL in 100% methanol was prepared and stored at -20 °C. The calibration curves were linear over the range of 1 to 500 µg/mL with a correlation coefficient of ≥ 0.99. For quantification, peak areas were correlated with concentrations in accordance with the calibration curves. Data are reported as means ± standard deviations of triplicate independent analyses.The analysis of phenolic acids and flavonols was conducted with a 1200 Series Agilent Technologies HPLC  equipped with a model G1315D diode array detector (DAD), a model G1379B degasser, a model G1312A binary gradient pump, a model G1329A thermoautosampler and a model G1316A column oven. The reversed-phase separation was performed on a 250 mm × 4.6 mm i.d. Luna C18(2) column, particle size 5 µm  with an AQ 4 × 20 mm CThe chromatographic system consisted of an Agilent 1200 Series HPLC unit comprising a degasser, binary pump, autosampler, thermostated column compartment, and diode array detector  connected to a linear ion trap mass spectrometer 4000 QTRAP system  which was equipped with an ESI Turbo V™ source. The reversed-phase separation of anthocyanins was performed on a 250 mm × 4.6 mm i.d. Symmetry C18 column, particle size 5 µm  with a Nova-Pak C18 guard column 4 µm 3.9 × 20 mm using the same conditions as described above. For the HPLC-MS analysis of anthocyanins, the effluent from the column after the DAD detector was directly introduced into the electrospray ion source (ESI). The tuning of MS instrument parameters was performed by compound optimization using a 100 ng/mL solution of cyanidin 3-glucoside dissolved in 80% methanol/20% formic acid (0.1%) solution. The optimization of ion source parameters was conducted using a flow rate of 1.0 mL/min. High-purity nitrogen gas (99.995 %) was used as the nebulizing (GS1) and heating gas (GS2). The values for optimum spray voltage, source temperature, GS1, GS2 and curtain gases were +3.0 kV, 600 °C, 40, 40 and 20 psi, respectively.m/z 50 to 1300 in 1 s. LIT fill time was set at 20 ms. The IDA threshold was set at 100 cps, above which enhanced product ion (EPI) spectra were collected from the eight most intense peaks. The EPI scan rate was 4000 amu s-1. Collision-induced dissociation (CID) spectra were acquired using nitrogen as the collision gas under a collision energy of 20 eV (CES15). The other MS parameters used were as follows: declustering potential (DP), 96 V; entrance potential (EP), 10V; and collision exit potential (CXP) 3V. An information-dependent acquisition (IDA) method, EMS → 4 EPI, was used to profile the anthocyanins in positive mode. The spectra were obtained over a range from m/z 50 to 1300 in 1 s. LIT fill time was set at 20 ms. The IDA threshold was set at 100 cps, above which enhanced product ion (EPI) spectra were collected from the eight most intense peaks. The EPI scan rate was 4000 amu s-1. Collision-induced dissociation (CID) spectra were acquired using nitrogen as collision gas under a collision energy of -20 eV. The other MS parameters were as follows: declustering potential (DP), -70 V; entrance potential (EP), -10V; and collision exit potential (CXP), -7V. Data acquisition was interfaced to a computer workstation Analyst 1.5 .The separation of the non-anthocyanin phenolic compounds was performed on a 250 mm × 4.6 mm i.d. Luna C18(2) column, particle size 5 µm , using the same conditions described above. The mass spectra were recorded in negative mode; the flow rate was maintained at 0.5 mL/min with the pneumatically assisted electrospray probe using high-purity nitrogen gas (99.995%) as the nebulizing (GS1) and heating gas (GS2). The values for optimum spray voltage, source temperature, GS1, GS2, and curtain gases were -4 kV, 600 °C, and 50, 30, and 25 psi, respectively. An information-dependent acquisition (IDA) method, EMS → 4 EPI, was used to identify phenolic compounds. Both Q1 and Q3 were operated at low and unit mass resolution. The spectra were obtained over a range from This study clearly shows that LC-ESI-MS/MS is a powerful technique enabling fast separation and characterization of polyphenols reported herein for the first time in Amazon grape. The high sensitivity of this hyphenated technique allowed expanding previous characterization of Amazon grape to additional eight minor anthocyanins, four quercetin glycosides, catechin, epicatechin, procyanidin B and four hydroxycinnamic acid derivatives. In this study, only peel and pulp samples were investigated for their profile of phenolic compounds. It would be desirable to characterize also the polyphenols present in the seeds, which constitute approximately 20% of the total fruit weight and are by-products of processing, to assess their potential as a source of natural bioactive components.


Lactobacillus mucosae AN1 and Lactobacillus fermentum SNR1 were assessed for anti-inflammatory properties in carrageenan (acute) and complete Freund’s adjuvant-induced inflammation (chronic) models in the present study. The two probiotic strains were administered orally along with feed to the Wistar albino male rats as whole cell as well as microencapsulated form. The following experiments were performed to evaluate the anti-inflammatory properties of probiotic strains and the results were observed that the encapsulated and unencapsulated probiotic strains have exhibited statistically significant decrease in paw thickness. Percentage of inhibition in paw thickness of microencapsulated probiotic bacteria (Group VIII), unencapsulated strains (Group IX) were revealed 85 ± 13% and 77 ± 25%, respectively. In Hematoxylin and Eosin staining, results were revealed that the probiotic strains were exhibited anti-inflammatory effects on inflammation-induced paw tissues. qRT-PCR studies revealed upregulation of anti-inflammatory cytokine genes and down-regulation pro-inflammatory cytokine genes in probiotic-treated rat paw tissues. Further, the expression of anti-inflammatory and pro-inflammatory cytokines were examined using immunohistochemistry and ELISA methods. The probiotic administered rat paw tissue in different groups have exhibited the low level of lipid peroxides formation and higher anti-oxidant activities when compared to the control and inflammation control tissues.The potent antioxidant probiotic strains  Lactic bacteria are the major probiotic bacteria that included in functional foods since they are considered as generally regarded as safe status and various benefits to the host organisms. However, probiotics should be able to survive in the host’s gastrointestinal tract . FAO defLactobacillus mucosae is a Gram-positive, rod-shaped species, an obligate anaerobe, catalase-negative, non-motile and it has mucus-binding activity. Lactobacillus mucosae is a probiotic bacteria was first identified using a gene probe designed for the mucus-binding gene and it was significantly enhanced the adherent ability of L. reuteri strains to mucin  at 25°C temperature, 50% relative humidity standard conditions were maintained. After 2 weeks of acclimatization period, rats divided into nine groups of six animals in each group and experimental duration for 40 days. During experimental period body weights and daily feed intake were recorded. The present study was carried out according to the principles and guidelines of animal care (Reg. No. 1159/c/07/CPCSEA). All the protocols used in this study was approved by the Animal Ethics Committee, Pondicherry University (Ref. No. PU/SLS/AH/IAEC/2016/08).Lactobacillus mucosae , Lactobacillus fermentum  were isolated from sheep milk and fermented rice gruel, respectively (Probiotic bacteria ectively . These pL. mucosae), Group V (Encapsulated L. mucosae), Group VI (Unencapsulated L. fermentum), Group VII (Encapsulated L. fermentum), Group VIII (Encapsulated L. mucosae and L. fermentum), and Group IX (Unencapsulated L. mucosae and L. fermentum). Animals were administered with the specified probiotic strain combinations consisting, 1 mL of culture 108 CFU/ mL/day/oral and microencapsulated probiotic strains were administered for the period of 40 days. Then animal organs such as paw tissues and blood were collected for the biochemical estimation.During the acclimatization period, all the rats were fed with normal rat feed and nine groups were divided. Each group composed of six rats such as Group I (Control); Group II (Inflammation control) Group III (Diclofenac sodium treated). The probiotic bacteria was administered to the rats in the following groups, Group IV (Unencapsulated L. mucosae AN1 and L. fermentum SNR1 cultures were suspended with 3% (w/v) sodium alginate and gently mixed for 20 min to obtain the suspension. It was injected through the fine gauge syringes into sterile calcium chloride with sodium chloride. After formation of the beads, recovered by filtration, washed with sterile water, air dried and stored at 4°C in aseptic conditions for administration to the specified rat groups  and stained with hematoxylin for 3–5 min, then washed in water for 5 min. After washing, tissue sections were differentiated in 1% acid alcohol (1% HCl in 70% ethanol) for 5 min and washed again in water until the sections were blue in color by ammonia solution. The tissue sections were rinsed with water and stained using 1% Eosin for 15 min and followed by water wash for 5 min then dehydrated with increasing concentration of ethanol. Tissue sections were cleared by xylene, mounted with DPX then observed under the light microscope (20×) .Blood samples were collected from all the rat groups then WBC, RBC cell counts, hemoglobin content and erythrocyte sedimentation rate (ESR) were estimated, C-reactive protein (CRP) levels were analyzed using ELISA method .α (proinflammatory cytokines) in pg/mL by ELISA reader (SpectraMax M2e). IL-6, TNF-α, and IL-10 ELISA kits were procured from Ray Biotech, United States and experiments were performed according to the manufacturer’s instructions and recommendations  and IL-6, TNF-ndations .Determination of the protective effects of microencapsulated probiotic strains and inflammation in the paw tissues were assessed using immunohistochemistry. Rat paw tissue sections were coated with 3-Aminopropyl triethoxysilane (HiMedia) then fixed in 20% acetone and embedded in paraffin. After that tissue sections were treated with IL-6 , TNF-α , and IL-10  for immunohistochemistry analysis was performed according to the manufacturer’s recommendations. Tissue sections were deparaffinized in xylene and gradual rehydration with ethanol (100–60%), then incubated in 1% sodium citrate buffer (pH 6.5) in a microwave oven at the optimum temperature for 25 min. Tissue sections were incubated in 3% Hydrogen peroxide for 15 min and washed in PBS for 10 min. Then incubated in humidity conditions by adding a secondary anti-rabbit antibody  for 8 h at 4°C and section slides were rinsed 3–4 times with PBS for 5 min. After washing with PBS, the tissue sections were stained with 3, 3′-Diaminobenzidine and counterstained using hematoxylin. The tissue sections were dehydrated in ascending grades of ethanol (60–100%) then cleared with xylene, mounted using DPX and observed using light microscopy (20×). Control and inflammation control tissue sections were processed along with IL-6, TNF-α, and IL-10 antibodies in independent experiments. Negative control tissue sections were stained with secondary antibody alone in this experiment .Rat paw tissues (50 mg) were homogenized using liquid nitrogen and 1 mL of TRIzol reagent  was added. After TRIzoL treatment, RNA was suspended in 50 μL Diethylpyrocarbonate. Purity of RNA was determined based on the 260/280 nm ratio. The residual DNA was removed by treating RNA with DNase I (Promega). 1μg of DNase treated RNA was transcribed to cDNA by reverse transcriptase .−1, fluorescence and final step to 35°C. The sequence of the primers used in this study was described in Table The qRT-PCR reaction was performed in Light Cycler instrumentation by relative quantification software with fluorescence detector. In PCR reaction, each cycle as follows; 10 μL reaction composed of 5 μL of 2× SYBR Green PCR master mix, 2.50 μL of cDNA (30 ng/μL for genes), 0.5 μL of primer at 10 μM and 1.50 μL of PCR water. PCR conditions briefly, initial denaturation temperature at 95°C for 5 min, amplification and quantification of PCR cycles as follows; 10 s at 955°C, 15 s annealing temperature and 15 s at 72°C with fluorescence, melt curve program at 60–955°C with a temperature rate of 0.15°C sQuantitative data analysis by qRT-PCR is based on the number of cycles required for amplification generated fluorescence to reach the specific Ct values. RT- PCR amplification efficiencies were determined for each set of primers with the slope of a linear regression . StandarLipid peroxidation was assessed by Thiobarbituric acid reactive substances (TBARS) in inflammation induced rat paw tissues. Stock solution contained the equal volume of 15% Trichloroacetic acid in Hydrochloride, 1:2 ratio of test sample and stock reagent were mixed in a centrifuge tube and boiled for15 min. After that precipitates were removed by the centrifugation and absorbance was read at 532 nm against blank. Experimental values were denoted as μM of malondialdehyde formed per min per mg of protein .Superoxide dismutase: 100 μL of tissue homogenate was incubated with ethanol and chloroform in an incubator shaker for 15 min then centrifuged for 5 min. Collected supernatant was mixed with 0.1 M Tris-hydrochloride, sterile water and 1 mM pyrogallol in 0.05 M Tris-hydrochloride, optical density (OD) at 420 nm was measured at 0, 1, and 2 min. Control samples containing 0.5 mL of distilled water treated against the blank and enzyme activity was represented as unit per mg protein .Catalase: 100 μL of tissue homogenate was taken and 0.5 mL of 0.01 M PBS and 0.2 M Hydrogen peroxide were added and incubated for 10 min at room temperature. After incubation, the reaction was stopped by 1.0 mL of 5% dichromate acetic acid (1:3) and hydrogen peroxide in the range of 0–20 μM was treated. The sample were heated 10 min and the established green color measured at 570 nm. Catalase enzyme activity represented as μM of Hydrogen peroxide consumed per min per mg of protein .Glutathione S-transferase: Assay mixture was composed of 0.1 M phosphate buffer, 1 mM of 1-Chloro- 2, 4-dinitrobenzene, 1 mM EDTA and 1 mM of glutathione. Reaction was initiated by addition of test samples and absorbance was read at 345 nm. Glutathione S-transferase enzyme activity was expressed as μM of 1-Chloro- 2, 4-dinitrobenzene utilized per min per mg of protein .L. mucosae AN1 and L. fermentum SNR1 on DPPH radical scavenging were performed. Reaction mixture consists 2 mL of 2, 2-diphenyl-1-picrylhydrazyl  (0.2 mM in ethanol) and 2 mL of AN1 and SNR1 strains independently (108 CFU/mL). The reaction mixture was incubated at 37°C for 30 min and scavenged DPPH was observed by measuring the decrease in OD at 517 nm. Ascorbic acid used as a standard drug and scavenging activity of bacterial strains (AN1 and SNR1) were determined using the following formula.The antioxidant properties of + solution was added with 500 μL of L. mucosae AN1, L. fermentum SNR1 strains independently (108 CFU/mL), incubated for 5 min and the measured absorbance at 734 nm. Three independent experiments were carried out and the percentage inhibition of absorbance was calculated using ascorbic acid standard curve  exhibited moderate destruction and less degenerative changes in the morphology of rat paw tissue sections , revealed significantly increased in inflammatory leukocyte infiltration than the control rats (Group I). Necrotic and degenerative changes were found in control group (Group I) and inflammation control (Group II) rats, while in histopathological analysis of probiotic strains s Figure .In inflammation control (Group II), there was found increased levels of ESR and C-reactive protein levels were observed than probiotic administered groups (Groups IV–IX). RBC, hemoglobin, WBC and platelet counts were found to be normal in all the groups Table .α were high in inflammation control (Group II) was found to be 53.01 ± 0.105 and 525.00 ± 0.220 pg/mL, respectively, and 26.016 ± 0.025 pg/mL of IL-10 levels were determined. In diclofenac sodium treated (Group III) rats interleukin IL6, TNFα, and IL-10 levels were determined to be 66.10 ± 0.120, 475.02 ± 0.162, and 32.265 ± 0.030 pg/mL, respectively. On the contrary, L. mucosae AN1 and L. fermentum SNR1 administered groups were exhibited anti-inflammatory properties, particularly in group VIII which was treated with the combination of encapsulated AN1 and SNR1 strains. Interleukin (cytokines) levels such as IL6, TNFα, and IL10 in group VIII rats serum was determined to be 47.15 ± 0.120, 431.01 ± 0.720, 42.005 ± 0.580 pg/mL, respectively  rats paw tissue sections were revealed high expression of IL-6 than IL-10 in staining. Microencapsulated probiotic strains administered group (VIII) was revealed high expressions of IL-10 (an anti-inflammatory cytokine) and low expressions of IL-6 (a proinflammatory cytokine) were observed in the immunohistochemical staining. In Group XI, interleukins IL-10 and IL-6 expression levels were observed to be low when compared to the Group VIII paw tissue sections Figures .α, COX2, iNOS, and IL1β) and anti-inflammatory (IL10) cytokines gene expressions in the paw tissues of probiotic strains administered groups were analyzed using reverse transcription quantitative PCR (qRT-PCR). Results were observed that in the normal control group I proinflammatory cytokine genes were upregulated and anti-inflammatory (IL10) genes were downregulated. In inflammation control (Group II) cytokine genes such as IL-6, TNF- α, COX-2, iNOS, and IL-1β were upregulated and relative mRNA expression was found to be 75, 92, 40, 35, and 53%, respectively, whereas anti-inflammatory cytokine (IL-10) was down-regulated and expression of IL-10levels found to be 135%. The diclofenac sodium reference drug-treated Group III rats were exhibited up-regulation of anti-inflammatory cytokine  and down-regulation of proinflammatory cytokines such as IL-6, TNF- α, COX-2, iNOS, and IL-1β; gene expression levels 78, 90, 40, 38, 60% were observed, respectively. In group VIII, revealed 240% of relative mRNA expression of IL-10 was recorded and down-regulation of proinflammatory cytokines such as IL-6, TNF- α, COX-2, iNOS, and IL-1β and their relative mRNA expressions were observed78, 140, 55, 44, and 56% respectively. Among all the probiotic administered animal groups, VIII and IX groups have exhibited better anti-inflammatory properties than the remaining Wistar rat groups   Table .−1 mg of protein−1. Probiotic bacteria administered groups such as group IV, V, VI, VII, and IX were exhibited least TBARS formation of 1.50 ± 0.72, 0.092 ± 0.54, 0.62 ± 0.18, 0.65 ± 0.12, and 0.74 ± 0.22 min−1 mg of protein−1, respectively, in the inflammation induced paw tissue homogenates  such as 2.00 ± 0.127, 2.17 ± 0.080, and 0.104 ± 0.02 per min per mg of protein were observed, respectively. In probiotic administered groups, particularly in group VIII, lipid peroxides formation was found to be 0.40 ± 0.02 min2O2 utilized per min per mg of protein, respectively. In probiotic bacteria administered groups which are exhibited high catalase activity in group V (Encapsulated L. mucosae AN1) and VIII (Encapsulated L. mucosae AN1 and L. fermentum SNR1) were determined to be 2.15 ± 0.52, 2.24 ± 0.07 μM H2O2 utilized per min per mg of protein, respectively.Superoxide dismutase, Glutathione S-transferase and Catalase in rat paw tissues were assessed for antioxidant properties. Superoxide dismutase activity was determined as 1.890 ± 0.07 units per mg of protein and in the probiotic administered groups paw tissue homogenates such as IV, V, VII, and VIII were revealed the better activity of 3.11 ± 0.15, 3.15 ± 0.617, 3.15 ± 0.65, 3.02 ± 0.35 units/mg of protein, respectively. Glutathione S-transferase activity of tissues in rat groups such as group I, group II, and group III were determined to be 0.42 ± 0.052, 0.68 ± 0.029, 2.12 ± 0.04 μM of 1-Chloro- 2, 4-dinitrobenzene utilized per min per mg of protein, whereas in probiotic bacteria treated tissue homogenates of group V and VIII were found to be high activity of 2.25 ± 0.08, 2.22 ± 0.63 μM of 1-Chloro- 2, 4-dinitrobenzene utilized per min per mg of protein, respectively. Catalase activity of group I, II and III tissues was determined as 0.625 ± 0.011, 0.832 ± 0.64, 1.5 ± 0.02 μM HL. mucosae AN1 and L. fermentum SNR1 were exhibited maximum DPPH free radical scavenging ability was determined to be 66.53 ± 1.15, 67.25 ± 1.64%, respectively. Scavenging ability of the strains AN1 and SNR1 against ABTS radicals were analyzed and determined to be 75.00 ± 1.73 and 80.83 ± 1.81%, respectively. The probiotic strains have exhibited similar antioxidant activities when compared with ascorbic acid control in this study.Antioxidant properties of in vivo approach to investigate antioxidant, anti-inflammatory characteristics of Lactobacillus mucosae AN1, Lactobacillus fermentum SNR1 in Wistar albino rats. Probiotic bacteria modulates immunity by interacting with the indigenous gut microflora for immunomodulation  and unencapsulated strains (Group IX) administered rats have exhibited better paw edema inhibition. Previous studies were also reported that sodium alginate microencapsulated L. sporogenes and B. bifidum strains inhibited carrageenan-induced paw edema  administered groups (IV–IX) were revealed normal levels in all the parameters such as hemoglobin content, WBC, RBC, platelet counts, ESR and CRP levels. In hematoxylin and eosin staining results were revealed that the morphology of epidermis of inflamed tissues (Group II), paw edema was clearly observed. The probiotic administered groups have exhibited no inflammation in the epidermis of tissue sections (Group IV to IX).In blood analysis, ESR and CRP levels were elevated in group I, II, and III animals, whereas in probiotic  rats rat paw tissues of lipid peroxides formation was very low levels whereas in inflammation control (Group II) was reported high in lipid peroxide formation. Antioxidant enzyme activities of probiotic administered rat paw tissues were observed high when compared to control tissue homogenates. The probiotic strains administered groups were observed that there were significant in reduced levels of lipid peroxides formation and increased antioxidant enzyme activities when compared to control and diclofenac sodium drug-treated rats. In operties . L. gassse model .L. mucosae AN1 and L. fermentum SNR1) were significantly decreased the paw edema inflammation and in the hematoxylin-eosin staining, the morphology of the probiotic administered groups were exhibited effective anti-inflammatory properties. Immunohistochemistry staining results revealed that anti-inflammatory cytokine IL-10 expressions were high when compared to proinflammatory cytokine IL-6 in the rat paw tissue sections. qRT-PCR and ELISA results were revealed that there is upregulation of anti-inflammatory cytokine and down-regulation of proinflammatory cytokines. Therefore the probable mechanism in antioxidant probiotic bacteria administered Wistar albino rats could be inhibition of prostaglandins synthesis due to high expression of anti-inflammatory cytokines. Hence, L. mucosae AN1 and L. fermentum SNR1 revealed antioxidant and anti-inflammatory properties they may be used in the animal model for their beneficial health.In summary, the probiotic strains (RA designed this research work, executed the experiments, interpreted the results and wrote the manuscript. DA provided technical assistance during the animal study. All the authors have contributed equally and approved this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 °C, with an optimum pH of pH 7–7.5. The activity was enhanced by divalent cations, such as Mg2+, Mn2+, or Co2+, and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 Å, which possesses a β-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the pKa of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and β-helix polysaccharide lyases.A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from  These degrading enzymes have been isolated from microbial populations based on their enzymatic activities. However, a more powerful tool for the detection of novel enzymes is whole-genome sequencing3.Polysaccharides, such as cellulose, starch, chitin, and alginate, are widely distributed in nature and constitute renewable biopolymer resources with a potential for bioconversion into many useful biological and chemical products. Identifying new enzymes for the synthesis, degradation, and modification of these polysaccharides is an important task for bioconversion. Microbes possess many polysaccharide-degrading enzymes with metabolic pathways that depend on their habitats. Polysaccharide-degrading enzymes, such as glycoside hydrolases and polysaccharide lyases, are classified into families in the CAZy databasePaenibacillus sp. str. FPU-7 (P. str. FPU-7), a chitinolytic bacterium, and characterised the secreted chitinases of the bacterium4. The genus Paenibacillus is the largest genus of aerobic endospore-forming bacteria (after Bacillus), is widely distributed in the environment, including in the air, rhizosphere, soil, and marine environments, and has diverse physiological characteristics, including several polysaccharide-degrading enzymes5. The draft genome sequence of P. str. FPU-7 suggested that the bacterium potentially contains enzymes with novel functions7. In this study, we experimentally determined the function of a putative protein, as a novel alginate lyase (PsAly), whose gene is found beside two characterised chitinase genes (chiC and chiD) in the genome.As reported previously, we have isolated a Gram-positive bacterium, Pseudomonas aeruginosa and Azotobacter vinelandii. The polysaccharide is a heteropolymer consisting of two uronic acids, α-L-guluronic acid (G) and β-D-mannuronic acid (M). These residues are covalently linked with 1,4-glycosidic bonds in three blocks: a homopolymeric G block (PG), a homopolymeric M block (PM), and a heteropolymeric block (MG)8. In brown algae and alginate-producing bacteria, the M residue can be epimerised to its C5 epimer, the G residue. In polymer form, alginate is used as a biomaterial for hydrogels in medical applications, including tissue engineering and drug delivery, thanks to its biocompatibility and gelation abilities. Alginate oligosaccharides are composed of 3 to 25 monomers and have several biological properties, including antioxidant, neuroprotective, antibacterial, and antitumor capacities9, as well as promoting growth and root elongation in plants10. Bacterial alginate lyases can degrade alginate into oligosaccharides or monosaccharides. Lyases catalyse a β-elimination reaction of glycoside bonds to yield a 4,5-unsaturated sugar, 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid, at the non-reducing end11. Due to their industrial potential, a number of alginate-degrading bacteria and alginate lyases have been isolated and identified. These alginate lyases are classified into 10 polysaccharide-degrading enzyme families (PLs) in the CAZy database: PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL32, PL34, and PL3611. These families have the same lyase activities but diverse structures; PL5, PL15, and PL17 share an (α/α)6-barrel fold as a catalytic domain; PL6 has a β-helix fold; PL7, PL14, and PL18 alginate lyases share a β-jelly roll fold. Alginate lyases generally degrade alginates into oligosaccharides in an endolytic manner13, while some exotype lyases degrade alginates in an exolytic manner16. Each alginate lyase has a different substrate specificity; some alginate lyases prefer PM, whereas others prefer PG or MG11.Alginate, a linear anionic polysaccharide, is widely distributed in the cell walls of brown algae and is synthesised by several bacteria, including In this paper, we describe the functions and structure of a novel alginate lyase, PsAly. The crystal structure of this alginate lyase was determined at a resolution of 0.89 Å. The basic structural fold is a single right-handed β-helix fold. Right-handed β-helix folds are commonly found in carbohydrate-active enzymes. The number of coils (10 coils for PsAly) is different among these β-helix folds, and the active site architecture is different from that of other polysaccharide lyases with β-helix folds. Amino acid sequence similarity and site-directed mutagenesis studies indicated the putative residues involved in substrate binding and catalysis. Our results provide an insight into the structural basis for the catalysis of alginate lyase and β-helix fold polysaccharide lyases.P. str. FPU-7 is a known chitin-degrading bacterium4. In this bacterium, seven chitinase genes (chiA-F and W) have been cloned and their enzymatic properties have been characterised17. In this study, we re-examined the draft genome of P. str. FPU-7 and detected a gene (accession number: LC490364) with 1,014 bp and a GC content of 55.7% for a hypothetical protein of unknown function composed of 337 amino acid (aa) residues next to the chitinase genes chiC and chiD  showed no significant sequence identity (<30%) with those of any functionally characterised proteins classified using the Pfam19 database or BLAST20 search program. To identify the function of this protein, the recombinant protein without the signal peptide was prepared using an E. coli expression system.hiD Fig. . A signachiC and chiD. As such, the degradation activity of several polysaccharides  were examined by detecting the increase of the reducing ends of the reaction products using 3,5-dinitrosalicylic acid (DNS). The recombinant protein exhibited significant degradation activity for alginate , including a C-terminal His6 tag, was purified by immobilised metal affinity chromatography and anion-exchange chromatography Fig. . Analysiate Fig. . This prsay Fig.  and exhisay Fig.  indicateTm) of PsAly was 52.9 ± 0.4 °C. After preincubation at temperatures from 4 to 50 °C for 1 h, significant decreases in activities were observed over 37 °C, although the enzyme was relatively stable at temperatures from 4 to 37 °C  and Km (0.14 ± 0.01% [w/v]), were determined using the Michaelis–Menten equation  of alginate lyases using a single species of substrate. However, the specific activity of PsAly is similar to those of other well-studied alginate lyases27 (Table S2). Most alginate lyases exhibit maximal activities at pH 7 to 9 (Table S2). The optimal pH (7.0 to 7.5) of PsAly was also within this range. The Km value of PsAly (0.14% or 7.9 mM of monomer uronic acid) is similar to those of other alginate lyases. For example, the Km values of FsAlyPL6 of Flammeovirga sp. NJ-0424, Aly-SJ02 of Pseudomonas sp. SM052427, AlgMsp of Microbulbifer sp. 6532A28, and AlyA1PL7 of Zobellia galactanivorans29 are 0.050% to 0.16% (varied with substrates), 0.047% to 0.28%, 3.4 mM, and 1.7 to 6.2 mM, respectively. The catalytic rate constant (kcat = 14 1/s) of PsAly is also similar to those of other alginate lyases, such as FsAlyPL624 (5.0 to 63 1/s), AlgMsp28 (42 1/s), and AlyA1PL729 (13 to 20 1/s).The apparent highest activity of PsAly was initially obtained at 67 °C from the activity-temperature profile by measuring the initial velocities at various temperatures Fig. , whereas °C Fig. . The kin °C Fig. . The act °C Fig. . The hal °C Fig.  and the  °C Fig. . The enzn2+ Fig. . The speion Fig. . In geneion Fig. . However nm Fig. , and by  nm Fig. . The vis nm Fig. . TLC ind nm Fig.  and PM . PsAly folds into a right-handed β-helix structure comprising 10 β-stranded coils and one α-helix cap at the N-terminus, with overall dimensions of 26 × 28 × 52 Å ; and mannuronan C-5 epimerase11. The number of coils, for example, 10 coils for PsAly, vary among these β-helix folds; pectate lyase Pe11A and Pel3A have 8 coils; LnbX has 15 coils; PL6 alginate lyase consists of double right-handed β-helix fold domains, each with 12 and 8 coils , P. chitinolyticus KCCM 41400 , P. durus DSM 1735 (Acc. No. AIQ11428), and P. lautus E7593-69 (Acc. No. AYB44622). These AlyGC and AlyF mainly hydrolyse PG in an exolytic manner, while PsAly prefers to hydrolyse PM in an endolytic manner. The active cleft structures are also different. The cleft of PsAly is an open-ended form, while the cleft structures of AlyGC and AlyF were semi-closed forms  with 10 coils, although Pel9A is a PL9 pectate lyase, not an alginate lyase. The root mean square (rms) deviations were 2.22 Å for the superimpositioning of 216 Cα atoms of PsAly onto those of Pel9A. In the PsAly structure, 32 β-strands formed a triple β-helix structure with three distorted parallel β-sheets, named PB1, PB2, and PB3, according to the naming of those of Pel9A  Fig. . The cry2 Å Fig. . A singlrms Fig. . The pril9A Fig. . The β-sl9A Fig. . The T1 l9A Fig. . At the l9A Fig. .Figure 434  analysis, sodium or calcium ions were appropriate to occupy these binding sites. After refinements with replacement of these ions on the sites, the Fo–Fc electron density map showed strong negative peaks  were those of bacterial uncharacterised proteins , mainly from Gram-positive bacteria such as Paenibacillus sp., Streptomyces sp., and Ruminococcus sp. The conserved residues are concentrated near the cation binding sites on the cleft , with a low amino acid sequence identity between PsAly and SjAly (24%)  Fig. . Althoug4%) Fig. . On the 4%) Fig. . Their f4%) Fig. , suggest37. In previously characterised polysaccharide lyases, various side-chains of amino acid residues, such as histidine, tyrosine, lysine, and arginine, have been proposed to form the Brønsted base, whereas arginine, tyrosine, and lysine residues and a water molecule have been proposed to be the acid catalyst37. Previous reports have also proposed that polysaccharide lyases with β-helix folds utilise a divalent cation, for example Ca2+, to neutralise the negative charge of the carboxyl group of the substrate and reduce the pKa of the C5 proton37. Indeed, the activity of PsAly was enhanced by several divalent cations and inhibited by EDTA  lyases, such as PsAly, which cleave β-1,4-glycosidic bonds, the proton of C5 and the lone pair of the scissile glycosidic oxygen O4 are arranged on the same face, such that the proton transfer reactions should occur on the same side (syn elimination). To proceed with the reaction, the saccharide binding is limited as follows: the carboxy group of the mannuronate residue should interact with the bound divalent cation; the proton of C5 and the lone pair of O4 of the glyosidic bond are positioned to face the conserved amino acid residues on the active site. Considering the position of the ligand and the mutagenic results, the sidechains of Tyr184 and/or Lys221 are the most likely candidates for the base and/or acid catalysts  by the adjacent amino group.In general, polysaccharide lyases cleave a glyosidic bond in two steps: β-elimination with the general-base-catalysed abstraction of the proton from C5 of the substrate sugar ring, and the protonation of O4 of the glycosidic bond to release the products (E1cB-elimination reaction)DTA Fig. . On the sts Fig. . The adjsts Fig.  most lik39 program to obtain the oligosaccharide binding structure. In the simulation results, hexamannuronate (6M) was bound on the bottom surface of the cleft with the lowest binding free energy   Fig. , althougol) Fig. . Howeverol) Fig.  and the ol) Fig. . Althougol) Fig. ; the hydnuronate M was bouP. str. FPU-7 was investigated. Alginate was found to be degraded by the supernatant of the cells at stationary phase. The activity of the supernatant was 0.031 ± 0.009 U/mg, determined using the DNS method. The Luria–Bertani (LB) medium used did not contain alginate. This suggests that the secretion of alginate-degrading enzymes is constitutive. So far, alginate lyases have been classified into 10 PL families: PL5, PL6, PL7, PL14, PL15, PL17, PL18, PL32, PL34, and PL36. The genes of these family enzymes could not be found in the draft genome of P. str. FPU-7. The N-terminal amino acid sequence of PsAly indicates that PsAly is a secretory enzyme. The degradation activity of P. str. FPU-7 may result from the constitutive secretion of PsAly.The alginate degradation activity of the cell culture supernatant of 40. All other chemicals and reagents were of analytical grade and purchased from FUJIFILM Wako Pure Chemical Corporation or Sigma , unless stated otherwise.Low viscosity sodium alginate  was purchased from FUJIFILM Wako Pure Chemical Corporation . Medium and high viscosity sodium alginate  were purchased from Nacalai Tesque Inc. . PM, PG, and MG (DP = 20–30) were prepared from sodium alginate  using Gacesa and Wusteman’s method19, BLAST20, and ClustalW41 programs. The N-terminal signal peptide of PsAly was predicted by using the SignalP 5.0 server at the Technical University of Denmark 18.Primary sequence analysis was performed using the Pfam databasePsAly gene and the purification of the enzymes are described in the Supplementary Information. The protein concentration was determined by UV spectrophotometry using the theoretical molar extinction coefficient ε280 = 37,485 (1/M cm) according to the ExPASy ProtParam tool server (http://web.expasy.org/protparam/)42.The procedures for the subcloning of the 2-terminal amino acid sequence analysis and gel permeation chromatography of PsAly are described in the Supplementary Information.The procedures for the NHg for 10 min and the clear fractions were transferred to clean tubes for assays. The degradation of the polysaccharides was determined using the DNS assay43. The DNS reagent consisted of 1% (w/v) DNS, 30% (w/v) potassium sodium tartrate, and 0.4M NaOH. The aliquots (100 μl) were mixed with equal volumes (100 μl) of DNS reagent, boiled for 5 min, cooled to 20 °C, and then observed for a red colour or measured at an absorbance of 525 nm43.The reaction mixtures (500 μl each) were prepared with 0.01 mg/ml purified protein, 1% (w/v) soluble or insoluble polysaccharides, such as chitin , chitosan , curdlan, cellulose , carboxymethyl cellulose sodium salt , xylan , laminarin , sodium alginate (500 cps), pectin , heparin sodium, xanthan gum , or gellan gum , and 50 mM Tris buffer pH 7.5. These reaction mixtures were incubated at 37 °C for 12 h. Then, the reaction mixtures were centrifuged at 15,000 g for 10 min and the supernatant was transferred to a tube. The concentration of the unsaturated alginate oligosaccharides was determined using the extinction coefficient of 6150 1/M cm44. The conditions of the samples and mass analyser  are described in the Supplementary Information.The reactions were conducted at 37 °C for 24 h with a 100 μl solution of 50 mM Tris buffer pH 7.5, 0.4% (w/v) alginate (500 cps), and 0.01 mg/ml enzyme. After incubation, two volumes of ethanol were added to the solution. Then, the mixture was centrifuged at 15,000 The differential scanning fluorimetry of PsAly was performed with a SYPRO Orange Protein Gel Stain dye . The procedure and analysis for the differential scanning fluorimetry are described in the Supplementary Information.45. Standard assays were performed at 37 °C in a total volume of 500 μl of 50 mM Tris pH 7.5, 0.2 μM of PsAly, and 0.4% (w/v) alginate (500 cps). The thermal stability, kinetics of thermal inactivation, activity-temperature profile, activity-pH profile, and pH stability were determined by carrying out assays under various conditions. To determine the pH-activity profile, the following 50 mM buffer systems were used: sodium acetate, pH 5.5 and 6.0; imidazole, pH 6.0–7.5; and Tris, pH 7.0–9.0. To determine the pH stability, the following 50 mM buffer systems were used: glycine, pH 2.0, 3.0, 10.0, 11.0, and 11.5; sodium acetate, pH 4.0 and 5.0; sodium phosphate, pH 6.0 and 7.0; Tris, pH 8.0; N,N-bis(2-hydroxyethyl)glycine, pH 9.0. To determine the activity-temperature profile, reactions were performed at different temperatures, ranging from 4 °C to 75 °C. To determine the pH and thermal stabilities, the enzyme was preincubated for 1 h at a given pH or temperature and subjected to the standard assay. The thermal inactivation of PsAly was obtained at 37, 47, 57, and 67 °C. Aliquots were removed after incubation for 0, 5, 15, 30, and 60 min; 2, 4, 8, and 24 h ; or 0, 0.5, 1, and 3 min (at 67 °C) and then subjected to the standard assay. The effect of cations and metal chelation on enzyme activity was determined by adding 5 mM ethylenediaminetetraacetic acid (EDTA), NaCl, KCl, MgCl2, MnCl2, CoCl2, NiSO4, CuSO4, Zn(O2CCH3)2, or 1 mM CaCl2 to the reactions. The kinetic parameters (kcat and Km) were determined by nonlinear fitting to the Michaelis–Menten equation with alginate (500 cps) in the range of 0.025%–0.4% (w/v), and the activity was measured as described above. The substrate specificity of PsAly was evaluated using 0.4% (w/v) alginate , PM, PG, or MG as the substrate.The alginate lyase activity of PsAly was determined by measuring the increase in absorbance at 235 nm with the formation of a carbon-carbon double bond of the productThe reaction mixture of PsAly and 0.4% (w/v) alginate (500 cps) was monitored by an Ostwald viscometer . The reaction products of PsAly with 0.4% (w/v) alginate (500 cps), PM, or PG were also analysed by thin layer chromatography (TLC) . These procedures are described in the Supplementary Information.46  consisted of 2 μl of protein solution 1 mg/ml) and 2 μl of reservoir solution (0.5 ml) containing 30% (w/v) PEG8000 and 0.1M imidazole pH 6.5. For cryoprotection, a protein crystal was soaked in the reservoir solution containing 30% (v/v) PEG400. X-ray diffraction images were acquired for the native and Se-Met derivative crystals at −173 °C under a nitrogen gas stream with a MAR MX 225HS or 225HE detector  and synchrotron radiation  at the BL-26B1, BL-26B2, or BL-38B1 stations of SPring-8 (Japan), integrated and scaled using HKL-2000 mg/ml an47 programs implemented in the HKL2MAP program package. The partial model obtained was manually rebuilt using the Coot ver. 0.8 program. Then, the model was refined using the Phenix.refine program in the PHENIX package48 against the native dataset 49 and the DALI program32. Structural alignments were conducted by superimposition using a fitting program in Coot. The conservation of the amino acid sequence on the surface of the crystal structure was estimated using the ConSurf server35. Multiple sequence alignment (150 sequences) was automatically built using the UNIREF90 database by the ConSurf server. The bound metal ions were analysed using CheckMyMetal34 . Structural figures were prepared by PyMol .Phase determination and initial model building were performed using the processed Se-Met anomalous dataset Table  and the et Table . Several39. The crystal structure of PsAly was used as the target molecule and oligosaccharides, such as dimannuronate to hexamannuronate (2M to 6M), were used as the ligand. The structure of 6M was built by linking two trimannuronate molecules from the crystal structure of alginate ABC transporter complexed with unsaturated tetramannuronate50 (PDB: 4XIG), followed by energy minimisation with AutoDock version 4.251 software. The other oligosaccharide models (2M to 5M) were prepared from the model of 6M.Docking simulations were performed using the SwissDock serverThe residues Lys128, Arg156, Tyr184, Asp188, Lys191, Tyr195, Lys221, Tyr244, Asp250, Lys252, and His278 in PsAly were replaced with an alanine, an asparagine, or a phenyl alanine residue, such as K128A, R156A, Y184F, D188N, K191A, Y195F, K221A, Y244F, D250N, K252A, and H278A, using site-directed mutagenesis with the expression plasmid for PsAly and the appropriate primers (Table S3). The mutagenesis, the evaluation of the purity of the enzymes, and the structural conformations of the enzymes by far-UV CD spectroscopy are described in the Supplementary Information.P. str. FPU-7 is described in the Supplementary Information.The measurement of the alginate degradation activity of the culture supernatant of aly gene in P. str. FPU-7 has been deposited in DDBJ/EMBL/GenBank database under accession number LC490364.The nucleotide sequence of the The coordinates and structure of PsAly are available in the Protein Data Bank (PDB) under accession number 6KFN.Supporting information


Ruminococcaceae UCG-014 (P < 0.001) and Lactobacillus (P = 0.043) were increased and Ruminococcaceae UCG-005 (P = 0.012) was increased first and then decreased when the alfalfa particle size decreased, while Lachnospiraceae NK4A136 group (P = 0.016), Ruminococcaceae NK4A214 (P = 0.044), Christensenellaceae R-7 group (P = 0.019), Lachnospiraceae other (Family) (P = 0.011) and Ruminococcaceae UCG-013 (P = 0.021) were decreased. The relative abundance of Methanobrevibacter was increased from 62.48% to 90.40% (P < 0.001), whereas the relative abundance of Methanosphaera was reduced from 35.47% to 8.62% (P < 0.001). In conclusion, as the particle size of alfalfa meal decreased, both the bacterial and archaeal population in the caecum of rabbit experienced alterations, however archaea response earlier than bacteria to the decrease of alfalfa meal particle size.This work was aimed to investigate the effects of the different particle size of ground alfalfa hay on caecal microbial and archeal communities of rabbits. One hundred-twenty New Zealand rabbits (950.3 ± 8.82 g) were allocated into four treatments, with five replicates in each treatment and six rabbits in each replicate. The particle sizes of the alfalfa meal in the four treatment diets were 2,500, 1,000, 100 and 10 µm respectively, while the other ingredients were ground through a 2.5 mm sieve. High-throughput sequencing technology was applied to examine the differences in bacteria and methanogenic archaea diversity in the caecum of the four treatment groups of rabbits. A total of 745,946 bacterial sequences  and 539,227 archaeal sequences  were recovered from twenty-four caecal samples, and were clustered into 9,953 and 2,246 OTUs respectively. A total of 26 bacterial phyla with 465 genera and three archaeal phyla with 10 genera were identified after taxonomic summarization. Bioinformatic analyses illustrated that Firmicutes (58.69% ∼ 68.50%) and Bacteroidetes (23.96% ∼ 36.05%) were the two most predominant bacterial phyla and Euryarchaeota (over 99.9%) was the most predominant archaeal phyla in the caecum of all rabbits. At genus level, as the particle size of alfalfa decreased from 2,500 to 10 µm, the relative abundances of  In recent years, the rabbit breeding industry has also prospered, and more and more attention has been paid to rabbit research. Rabbit is a monogastric herbivore animal that has a certain ability to digest fiber. Although the digestibility is not as high as other herbivores  in the caecum of rabbits. Physical structure, especially particle size, is another important characteristic of fiber that influences caecal microorganism in rabbits. The intestinal microorganism is always in the dynamic change, and there are many factors affect the composition of the gut microbiota, especially fiber plays an important role in the balance of intestinal microflora structure. Previous work  showed tIn addition, most of the existing reports about particle size of diet fiber in rabbits are descriptions on the retention time of chyme in the caecum, the digestion of nutrients and the growth performance . Yet limThe experimental protocol used in the present study was approved by the Animal Policy and Welfare Committee of the Agricultural Research Organization of Sichuan Province, China, and was in accordance with the guidelines of the Animal Care and Ethical Committee of the Sichuan Agricultural University (Permission code SYXK (chuan) 2014-187).ad libitum access to water and diets during the whole experimental period. Neither feed nor drinking water was medicated with antibiotics, but a coccidiostatic (robenidine) was provided in the feed. In this study, the diets  with average body weight (950.3  ± 8.82 g), weaned at 35 days of age were selected and allocated into 4 treatments, with five replicates in each treatment and six rabbits in each replicate, and the rabbits were kept in three pairs with two rabbits per cage. Rabbits were housed in the same building  in flat-deck cages (600 × 250 × 330 mm) for the 49 d experiment. The temperature inside the house was maintained at 15–25 °C and rabbits had he diets  were forCaecal samples were thawed at room temperature and kept on ice during the extraction process. According to the manufacturer’s instructions, total genomic DNA was extracted from caecal samples using a DNeasy PowerSoil Kit  that included a step of the microbiological cells to be mechanically broken. The quality and concentration of the extracted DNA was detected, respectively, using 0.8% agarose gel electrophoresis and a NanoDrop ND-1,000 spectrophotometer . All extracted DNA samples were diluted to 10 ng/µL using sterile ultrapure water and stored at −20 °C until used for real-time PCR and Illumina sequence analyses.2, each deoxynucleoside triphosphate at 0.4 µM, each primer at 1.0 µM, 0.5 U of KOD-Plus-Neo  and 10 ng template DNA. The PCR amplification program consists of initial denaturation at 94 °C for 1 min, followed by 30 cycles of denaturation at 94 °C for 20 s, annealing at 54 °C for 30 s, elongation at 72 °C for 30 s, and a final extension at 72 °C for 5 min. Three replicates of PCR reactions for each sample were combined together, PCR products mixed with 1/6 volume of 6× loading buffer were loaded on 2% agarose gel for detection. We used a 2 step process for library preparation. First, 16S amplicons were generated using PCR to generate 200–400 bp amplicons. Second, we added unique barcodes to samples using emulsion PCR (EmPCR) to prevent chimera formation  and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) as the primer pair for the bacteria , while sormation , and theormation . This meormation . The elehttp://drive5.com/uparse/) and QIIME  and lead to false positive results for overestimation of diversity. Sequences were clustered into operational taxonomic units (OTUs) at 97% identity threshold using UPARSE algorithms (https://www.arb-silva.de) database  function hclust. To identify if there were significant differences among different groups, permutational multivariate analysis of variance was performed based on the dissimilarity matrix.Then the filtered OTU is removed from the OTU table in the process of evolutionary tree reconstruction, and the OTU table is resampled so that each sample has the same sequence number. Phylogenetic diversity (Faith’s PD18) was calculated using Picante . WeighteYij is an observation on the dependent variable ij, μ is the overall population mean, Ti isthe fixed effect of alfalfa meal particle size, and eij is the random error associated with the observation ij. Tukey-Kramer multiple comparison tests were performed after differences were detected. Differences between means with P < 0.05 were accepted as statistically significant differences.The effects of alfalfa meal particle size on cecal microbial communities were analyzed using the MIXED procedure of SAS v9.4 . The model used for the analysis was P > 0.05). As for the archaea, the Shannon-Wiener index of group 1,000, 100 and 10 µm were significantly decreased compared with group 2,500 µm (P = 0.044). However, no significant difference was obtained among 1,000, 100 and 10 µm groups. The above results suggested that rabbit caecum alpha diversity of bacteria and archaea experienced different alterations when the alfalfa particle size decreased.A rarefaction test was performed at the OTU level and the results are presented . As can n = 24) per sample, and the mean length of tag N50 is 304  ± 6 base pairs (bp). Based on the principle that the similarity is greater than 97%, the obtained effective sequences were clustered into a total of 9,953 OTUs, the average OTU was 1,896  ± 251  per sample. A total of 2107 OTUs were shared in all of four treatments, while 1,390, 587, 632 and 1,791 OTUs were exclusively found in group 2,500, 1,000, 100 and 10 µm, respectively  using weighted unifrac clustering in P < 0.01). However, the bacterial community among 2,500, 1,000 and 100 µm groups could not be obvious isolated (P > 0.05), and PCo1 accounted for 32.2% (P = 0.995) but could be separated thoroughly from group 2,500 and 1,000 µm (P = 0.025), and the PCo1 accounted for 98.7% , Ruminococcaceae UCG-014 (13.25 ± 0.101%), Lachnospiraceae NK4A136 group (10.73 ± 0.065%) and Rikenellaceae RC9 gut group (5.00 ± 0.066%) in the 2,500 µm group. Unclassified Bacteroidales S24-7 group (14.48 ± 0.087% and 14.56 ± 0.088%), Ruminococcaceae UCG-014 (9.34 ± 0.040% and 11.55 ± 0.097%), Lachnospiraceae NK4A136 group (8.80 ± 0.046% and 9.61 ± 0.035%), unclassified Clostridiales vadinBB60 group (5.72 ± 0.060% and 4.86 ± 0.015%) and Ruminococcaceae NK4A214 group (4.71 ± 0.024% and 4.39 ± 0.027%) were the most two abundant genera in 1,000 and 100 µm groups. In the 10 µm group, Ruminococcaceae UCG-014 (33.82 ± 9.752%) and Unclassified Bacteroidales S24-7 (11.81 ± 2.876%) group were the two most abundant genera. The relative abundance of the 10 unclassified genera accounted for 29.45%, 20.55%, 30.34% and 24.86% in 2,500, 1,000, 100 and 10 µm groups, respectively , 23 , 22 (100 µm) and 20 (10 µm), respectively. The most abundant genera (relative abundance more than 4%) were Unclassified ectively .Ruminococcaceae UCG-014 (P < 0.001) and Lactobacillus (P = 0.043) were increased while the alfalfa particle size decreased, while the relative abundance of Lachnospiraceae NK4A136 group (P = 0.016), Ruminococcaceae NK4A214 (P = 0.044), Ruminococcaceae UCG-005 (P = 0.012), Christensenellaceae R-7 group (P = 0.019), Lachnospiraceae other (Family) (P = 0.011) and Ruminococcaceae UCG-013 (P = 0.021) were decreased .Methanobrevibacter, and its relative abundance was significantly increased from 62.48% to 90.40% as the particle size of alfalfa decreased from 2,500 to 10 µm (P < 0.001). The Methanosphaera was the second largest genus in the caecum of rabbits, and its relative abundance decreased from 35.47% to 8.26% with the decrease of alfalfa particle size (P < 0.001)  ; Table 2In the present work, we attempted to study the impact of different fiber particle size on the caecal microflora of rabbits. Variations in the microbial community structure of caecum in rabbits were observed according to the decrease of particle size of alfalfa hay, and the archaea responsed earlier than bacteria when the particle size of alfalfa decreased.Effects of fiber particle size on growth performance and digestibility of nutrients in rabbit has been assessed in previous studies ; howeverWe also discovered that microbial abundance varied with the different particle size of alfalfa, this is consistent with previous studies on poultry . FurtherSuccinivibrionaceae UCG-002 was significant increased with the reduction of particle size of alfalfa. Therefore, we assumed that Succinivibrionaceae UCG-002 might be a microorganism involved in protein digestion. But further work is needed to confirm this speculation. In addition, it was more interesting that the relative abundance of Tenericutes (2.11%) in the 10 µm group was significantly higher than the other three groups and became one of the major microbe of the caecum. Nevertheless, Proteobacteria are commonly found in the gastrointestinal tract of animals and exist as the dominant bacteria of some animals . In the Bacteroidales S24-7 and Ruminococcaceae UCG-014 were the two most abundant bacteria in rabbit caecum. However, Ruminococcus and Alistipes. This might have resulted from the different rabbit species and diets used in two studies. It was reported that the specific role of Unclassified Bacteroidales S24-7 in the intestine was to breakdown starch, complex polysaccharides and fiber  had the same changes trend among the core genera. Furthermore, with the exception that Ruminococcaceae UCG-005 was increased first and then decreased as the particle size of alfalfa decreased, the abundance of other genera (abundance >1%) of Firmicutes (mainly Lachnospiraceae NK4A136 group and Ruminococcaceae NK4A214 group) were significantly lowered when particle size decreased. This led to no statistical difference in the abundance of Firmicutes among treatments. of host . It was bacillus . Our prebacillus  also fouMethanogens belong exclusively to the domain of archaea. They play a key role in the final stage of microbial organic matter decay in the digestive system . Thus, tMethanobrevibacter and Methanosphaera genus usually were the two most abundantly distributed genera  of the NCBI nucleotide database under accession number Ruminococcaceae UCG-014 and Lactotobacillus increased whereas the Lachnospiraceae NK4A136 group, Ruminococcaceae NK4A214 group decreased when the fiber particle size decreased from 2,500 to 10 µm. As for the archaeal populations, the Methanobrevibacter increased at the cost of redution of Methanosphaera. The gastrointestinal microbial populations could be manipulated by feeds processing technology in the aim of promoting animal production performance.In summary, as the particle size of alfalfa meal decreased, both the bacterial and archaeal population in the caecum of rabbit experienced variations. However, the changes in the archaea are produced earlier in time. The bacterial populations changed when the alfalfa fiber particle size decrease from 100 to 10 µm, whereas the archaeal populations changed while the fiber particle size decrease from 1,000 to 100 µm. In terms of bacteria, 10.7717/peerj.7910/supp-1Table S1Click here for additional data file.10.7717/peerj.7910/supp-2Table S2Click here for additional data file.10.7717/peerj.7910/supp-3Figure S1Click here for additional data file.10.7717/peerj.7910/supp-4Figure S2Click here for additional data file.10.7717/peerj.7910/supp-5Figure S3Principal co-ordinate analysis (PCoA) scores plot generated from rabbits caecum sample by a unweighted UniFrac analysis at the 97% similarity level. A and B represent bacteria and archaea, respectively.Click here for additional data file.


Streptomyces roseosporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, auroramycin has antifungal activities, which is unique among structurally similar polyene macrolactams, such as incednine and silvalactam. In this work, we employed different engineering strategies to target glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. Auroramycin analogs with variations in C-, N- methylation, hydroxylation and extender units incorporation were produced and characterized. By comparing the bioactivity profiles of five of these analogs, we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among structurally similar polyene macrolactams, is key to its antifungal activity.Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in  Natural products (NPs) are an important source for pharmacological applications with a significant proportion of current drugs being natural product or natural product-derived . Advance1) is a polyene macrolactam that is doubly glycosylated. Sugars are attached in the order of xylosamine and 3, 5-epi-lemonose to the polyketide core. Compared to structurally similar natural products  in addition to a P450 (AurS12) as its potential auxiliary partner, DesVIII . The fou13 genes . To assiaurS4 and aurS5 as deletion of either gene yielded aglycon 2  to func6 and 7 respectively  yielded the expected products ly Figs. c, 1b. Hoo 7 Fig. c. The la8, Fig. 9. Analogs 8 and 9 were produced at an approximately 1:2 ratio with yields less than 5% of auroramycin. Substrate preference of Dszs AT for mCoA to mmCoA was observed previously to be approximately more than 46,000-fold . See latter for assignment of analog 5.To the crude ethyl acetate extracts from 200 agar fermentation plates (~ 6 L), cold ethyl acetate (20 mL × 2) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution and yellow solid. Cold methanol (10 mL) was then added to the yellow solid, sonicated and centrifuged to separate the yellow solution. The obtained white/pale yellow solid  was found to be mainly analog 4 was obtained as a white/pale yellow solid . [α]D25: − 301.6° ; 1H and 13C NMR: see Additional file ν = 3353, 2953, 2925, 2871, 1630, 1577, 1525, 1459, 1384, 1284, 1234, 1167, 1129, 1079, 1058, 985, 960 cm−1; UV/VIS (1:1 CH2Cl2:MeOH): λmax (log ε) = 317.5 (1.25) nm; HRMS m/z: 780.5161 .To the crude ethyl acetate extracts from 125 agar fermentation plates (~ 4 L), cold ethyl acetate (20 mL × 2) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution and yellow solid. Cold methanol (15 mL) was then added to the yellow solid, sonicated and centrifuged to separate the yellow solution and analog 5 was obtained as a white/pale yellow solid . [α]D25: − 233.5° ; 1H and 13C NMR: see Additional file ν = 3350, 2925, 2854, 1712, 1628, 1530, 1463, 1386, 1285, 1147, 1077, 1057, 985, 961 cm−1; UV/VIS (MeOH): λmax (log ε) = 317.5 (1.74) nm; HRMS m/z: 623.4056 .To the crude ethyl acetate extracts from 184 agar fermentation plates (~ 6 L), cold ethyl acetate (30 mL) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution and yellow solid. Cold methanol (5 mL) was then added to the yellow solid, sonicated and centrifuged to separate the yellow solution and analog 6 was obtained in an approximate 17:1 ratio together with a hydroxylated product as a brown solid . See Additional file m/z: 780.5156 .To the crude ethyl acetate extracts from 200 agar fermentation plates (~ 4 L), cold diethyl ether 10 mL × 2) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution. Analog 0 mL × 2 7 was obtained in an approximate 5:1 ratio together with a hydroxylated product as a brown solid . See Additional file m/z: 780.5161 . Due to constraints of the sensitivity of the compounds to light and acid, further purification was not performed.To the crude ethyl acetate extracts from 191 agar fermentation plates (~ 4 L), cold diethyl ether (10 mL × 2) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution. Analog 2O (10–15 mL × 2) was added, sonicated for 1–2 min and centrifuged to separate the yellow solution and brown solid. The brown solid was washed with cold 5:1 Et2O:acetone (2 mL × 3) and analog 10 was obtained as a brown solid . [α]D25: –261.9° ; 1H and 13C NMR: see Additional file ν = 3333, 2926, 2867, 1661, 1515, 1456, 1382, 1274, 1238, 1156, 1097, 1058, 988, 962 cm−1; UV/VIS (MeOH): λmax (log ε) = 317.5 (1.90) nm; HRMS m/z: 778.5361 .To the crude ethyl acetate extracts from 180 agar fermentation plates (~ 6 L), cold Etwww.eurofins.com/epds), according to methodology described by the Clinical and Laboratory Standards Institute (CLSI) .Measurements against indicated fungal strains were conducted at Eurofins Panlabs Taiwan  values were determined by the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute with slight modifications. Briefly, purified auroramycin analogs were dissolved in DMSO, then diluted in Mueller–Hinton broth containing 0.2% DMSO. The organisms were tested at 5 × 10Additional file 1. Table S1. Putative functions of genes in the auroramycin biosynthetic gene cluster. Table S2. List of strains and plasmids used in this study. Table S4.1H and 13C NMR data of auroramycin analog 4 and analog 5. Tabe S5.1H and 13C NMR data of auroramycin analog 10. Figure S1. Alignment of sanger sequencing of aurS9 deletion mutant (edited) against wild type genome. Figure S2. Alignment of sanger sequencing of aurS11 deletion mutant (edited) against wild type genome. Figure S3. Alignment of sanger sequencing of aurS4 deletion mutant (edited) against wild type genome. Figure S4. Alignment of sanger sequencing of aurS10 deletion mutant (edited) against wild type genome. Figure S5. Alignment of sanger sequencing of aurS12 deletion mutant (edited) against wild type genome. Figure S6. Alignment of sanger sequencing of aurS13 deletion mutant (edited) against wild type genome. Figure S7. Alignment of sanger sequencing of aur01 deletion mutant (edited) against wild type genome. Figure S8. Alignment of sanger sequencing of AT mutant (edited) against wild type genome. Figure S9 Alignment of glycosyltransferases. Figure S10 Purification and characterization of His-tagged AurS13 expressed alone or His-tagged AurS13 co-expressed with AurS12 (no His-tag). Figure S11. Analytical data for the structural assignment of auroramycin analog 3. Figure S12. Analytical data for the structural assignment of auroramycin analog 6 and minor hydroxylated product. Figure S13. Analytical data for the structural assignment of auroramycin analog 7 and minor hydroxylated product. Figure S14. Representative microdilution assay plates for compound 1, 3-7, 10 with (A) Staphylococcus aureus N216, (B) S. aureus Z172, (C) Enterococcus faecalis ATCC 51299, (D) Pseudomonas aeruginosa ATCC 700603 and (E) E. coli ATCC 25922.Additional file 2: Table S3. Oligonucleotides used in this study.


With the rising tide of antibiotic resistant bacteria, extending the longevity of the current antibiotic arsenal is becoming a necessity. Developing local, controlled release antibiotic strategies, particularly for difficult to penetrate tissues such as bone, may prove to be a better alternative. Previous efforts to develop an osteoconductive local antibiotic release device for bone were created as solid molded composites; however, intimate contact with host bone was found to be critical to support host bone regrowth; thus, an osteocondconductive antibiotic releasing bone void filling putty was developed. Furthermore, a controlled releasing polymer matrix was refined using pendant-functionalized diols to provide tailorable pharmacokinetics. In vitro pharmacokinetic and bioactivity profiles were compared for a putty formulation with an analogous composition as its molded counterpart as well as four new pendant-functionalized polymers. A best-fit analysis of polymer composition in either small cylindrical disks or larger spheres revealed that the new pendant-functionalized polymers appear to release vancomycin via both diffusion and erosion regardless of the geometry of the putty. In silico simulations, a valuable technique for diffusion mediated controlled release models, will be used to confirm and optimize this property. Listed by the World Health Organization (WHO) as one of the top three threats to global public health , more thPeriprosthetic Joint Infection (PJI): Despite the clinical success of Total Joint Replacements (TJR) in relieving pain and improving quality of life, up to 10% of implants will fail early (<10 years), requiring a revision procedure to remove the original device and infected bone and replace components . A steadLocal controlled-release antibiotic delivery to bone: Although osteomyelitis—a serious bone infection accompanied by inflammation, cytokine up-regulation, and bone resorption—is increasingly linked to antibiotic resistant bacteria, current clinical tools to address it are tedious, costly, and often ineffective, compromising the patient’s overall bone health, healing, and recovery. Systemic antibiotic prophylaxis is considered the current clinical standard of care; however, studies are lacking to support this approach . While cControlled drug delivery directly to bone presents an unsolved pharmacokinetic challenge. A strictly biomaterials-based approach has proven inadequate to address infection ; improveThe synthesis of monomers and polymers is based on previously described procedures ,30.tert-butyl protected succinic acid , 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl  and anhydrous Dimethyl formamide  were added into a 250 mL flask and the mixture was stirred for 10 min in an ice bath. To this activated carboxylic acid, compound 1  dissolved in anhydrous DMF (5 mL) was added and the reaction was stirred overnight. After reacting, DMF was removed under vacuum and the product was extracted with Ethyl acetate . The organic layer was washed with water , saturated NaHCO3 solution  and brine  and dried over anhydrous Na2SO4. The product was purified by column chromatography (15% EtOAc and 85% Hexane).Synthesis of 3a : Mono-te1H NMR  ppm 0.04–0.06  0.88–0.90  1.45  2.56  2.67  3.48  3.56  3.74 .Yield: 70%: t-butyl dimethyl silane) was performed by reaction with iodine (20% weight of product) in MeOH (15 mL) overnight. Na2S2O3 was added dropwise to quench the reaction until the solution turned from brown to colorless. The solvent was removed under vacuum and the product was extracted with CH2Cl2  and dried over anhydrous Na2SO4. The product was purified via column chromatography .Deprotection of TBDMS  ppm 0.04–0.06  0.88–0.90  1.45  2.56  2.67  3.48  3.56  3.74 .Diol 3a: Yield: 61%, 2Cl2) and completely dried in high vacuum line.To synthesize diols from ester derivatives of functionalized carboxylic acids , diethan1H NMR : ppm 1.16 , 2.39–2.50 , 3.50–3.59 , 3.78–3.90 .Diol 3b: Yield = 76%, 1H NMR  ppm 2.65–2.74 , 2.95–3.00 , 3.42 , 3.54–3.57 , 3.70 , 3.85 , 7.21–7.32 .Diol 3c: Yield = 63%, 1H NMR  ppm 2.2  2.4  2.65  3.53–3.62  3.81  3.91  4.12 , 6.14  6.82–6.89  7.50 .Diol 3d: 3  was added and stirred at 110 °C until completely dissolved. Soybean oil (43.6 g) was added dropwise via funnel over 30 min. After addition, the reaction was stirred for another one hour at 110 °C under vacuum. EtOAc was added to dilute the reaction mixture, which was then washed with 15% NaCl solution (3×). The product was purified via column chromatography (5% MeOH and 95% CH2Cl2).To synthesize the soybean oil monomer, diethanolamine  was added into a 500 mL round bottom flask. Subsequently, NaOCH1H NMR  ppm 5.30–5.41  3.76–3.85  3.49–3.85  2.76–2.81  2.37–2.40  2.00–2.07  1.63  1.26–1.1.39  0.87–0.99 .Diol 3e: 2 (3×). Anhydrous CH2Cl2 (2 mL for 1 mmol of diacid) was syringed into the reaction flask. The mixture was homogenized by warming to 40 °C for 1–2 min. Subsequently, the mixture was cooled on ice and diisopropylcarbodiimide (DIC)  was added dropwise by syringe. The mixture was stirred for 48 h at room temperature. The polymer was purified by precipitation from cold iso-propanol/methanol or dialysis against MeOH and dried under vacuum. Total equivalents of diols were equivalent to that of diacid if more than one functionalized diol was used in the reaction.Polyesterification of functionalized diols and diacids  was accop(A-C4):1H NMR  ppm 1.15  2.39  2.62  3.60–3.70  4.22–4.26 . Mn = 60 KDa, polydispersity index (PDI) = 1.1, Td: 238 °C, Tg: 5.3 °C.p(A-C10):1H NMR  ppm 1.14  1.29  1.56–1.64 (m. 4 H) 2.22–2.35  2.39  2.66–2.77  3.58–3.64  4.18–4.26  Mn: 78 KDa, PDI: 1.19, Td: 283 °C, Tg: −32 °C.1H NMR  ppm 0.87  1.27  1.59  2.04  2.16  2.39–2.30  2.59  2.76  3.61  4.11–4.08  4.19  5.34 , 6.11  6.87–6.80  7.49  Mn: 11.9KDa, PDI: 2.13, Td: 320 °C, Tg: −20 °C.p(SC-C6): 2Cl2 (6 mL), trifluoroacetic acid (TFA) (3 mL) and triisopropylsilane (50 μL) and stirred for 2 h. After the stirring incubation, TFA and CH2Cl2 were removed under vacuum and the polymer was precipitated into diethyl ether twice to obtain pure polymer.Finally, the tBu-protected p(FD-C10) was deprotected by dissolving the polymer (1 g) in regular CH1H NMR  ppm 1.25–1.36  1.53–1.65  2.22–2.35  2.64–2.76  2.94–3.01  3.52  3.58–3.69  4.12  4.18–4.26  7.16–7.24  7.24–7.31  Mn: 40KDa, PDI: 1.34, Td: 273 °C, Tg: −16 °C.p(FD-C10): Structures of small molecules and polymers were confirmed by 300 and 500 MHz Nuclear magnetic resonance (NMR) proton spectra via a Varian NMRS instrument . Deuterated chloroform was used as a solvent. Chemical shifts, δ (ppm), were referenced to the residual proton signal.The molecular weight and polydispersity index for each custom polymer was measured via gel permeation chromatography (GPC). GPC analysis in tetrahydrofuran (THF) was performed on a Waters 150-C Plus instrument  equipped with refractive index and light scattering detector. Polystyrene was used as a standard. GPC analysis in DMF was performed on an HLC-8320 instrument  equipped with refractive index and UV detectors using polystyrene as a standard. The flow rate of the eluent was 1 mL/min for both GPC instruments.2 from room temperature to 600 °C with a heating rate of 10 °C /min. Alternatively, the glass transition temperature was tested by differential scanning calorimeter (DSC) using TA Q2000 differential scanning calorimeter  with heating and cooling rates set at 10 °C/min.The polymer’s decomposition temperature was measured by thermal gravimetric analysis (TGA) with a TA Q500 thermal gravimetric analysis instrument . Each polymer was heated in Nd,l-lactide-co-glycolide)  dissolved in N-Methyl-2-pyrrolidone , Poly ethyleneglycol ; and polycaprolactone (PCL-10kD); calcium chloride as a poragen, and vancomycin. Note that vancomycin from two different manufacturers was used in different formulations: (1) Vancomycin HCl Sterile Lyophilized Powder for Injection ; and (2) Vancomycin HCl; Research Products International (RPI), Mount Prospect, IL, USA. PLGA was dissolved in NMP (200 μL) and added to a homogenous mixture of molten PCL and PEG at ~75 °C. BVF, calcium chloride and vancomycin were mixed into the polymer composition and 20 μL of 1× phosphate buffered saline (PBS) was added to produce a putty-like composite. Additional, ABVF putty formulations were created using custom-made polymers (as described above). Briefly, polymers were heated to ~120 °C and ProOsteon 500R™ , calcium chloride, and vancomycin  were added. ProOsteon 500R™  is a cancellous bone like, osteoconductive matrix consisting of calcium carbonate and hydroxyapatite. The addition of NMP and PBS were not necessary to create a putty-like composite from the custom polymers. With the exception of formulation 2, which was shaped into a single large sphere, all formulations were made as single batches and partitioned into replicates (as indicated in Antibiotic-loaded bone void filling (ABVF) putties were fabricated similarly to our previous molded formulations ,24,25 wiBoth the small cylindrical disk as well as the larger sphere were imaged using scanning electron microscopy imaging. Additionally, the cylindrical disk was imaged using Micro computed tomography (micro CT) at the NDSU (North Dakota State University) Electron Microscopy Center core facility .The sample was hot glued to a glass rod and placed into a GE Phoenix v|tome| xs X-ray computed tomography system with a 180 kV high power nanofocus X-ray tube xs|180 nf, high contrast GE DXR250RT flat panel detector, and molybdenum target . One thousand projections were acquired at a voltage of 80 kV and a current of 300 µA. Voxel size was 6.4 µm. Acquired images were reconstructed into a volume data set using GE datos|x 3D computer tomography software Version 2.2 . The reconstructed volume was then viewed and manipulated using VGStudio Max .Samples for scanning electron microscopy were cut with a razor blade to expose the interior surfaces, attached to cylindrical aluminum mounts with colloidal silver paint  and coated with gold  or with carbon (Cressington Inc.). Images were obtained with a JEOL JSM-6490LV scanning electron microscope or JEOL JSM-7600F field-emission scanning electron microscope .Three molded ABVF cylindrical disks from both putty formulations 1 and 3 were released after curing 24 h at 4 °C, whereas the three remaining ABVF disks were released after one week at 4 °C. The ABVF putty cylindrical disk released after 24 h weighed an average of 45.6 mg (STD (standard deviation): 4.925). Cylindrical disks were placed in individual 2 mL microcentrifuge tubes containing 2 mL of PBS for six weeks at a constant temperature of 37 °C. PBS was replaced at 24, 48, and 72 h and every week thereafter for a total of six week as previously described ,31. At tThe amount of vancomycin released into PBS or the amount of residual vancomycin left in the device after release was determined using a custom-built detector as previously described . BrieflyStaphylococcus aureus strain 49230 (12 × 107 CFU). Standard vancomycin concentrations  and PBS blanks were prepared in same fashion as release solution. Plates were inverted and incubated at 37 °C for up to 20 h before diameter of zone of inhibition was recorded via electronic calipers.A standard Kirby Bauer ZOI assay was performed ,34. In an = 3) and without (n = 3) vancomycin and from formulation 4 (n = 2) were assessed for their putty-like characteristics using a Bose 3200 load frame  with supplied compression surfaces. A 225N load cell with Wintest software  was used for data collection. Supplied Bose compression surfaces were used in the compressive tests. However, a custom designed and built fixture was used to test the tackiness of the samples in a tensile lap test. This fixture consisted of two 16 gauge 304 stainless blanks, which were placed in grips. The blank held in the bottom grip had a toggle clamp bolted to it, which provided consistent sample thicknesses. The toggle clamp had a foot, which used a roller bearing to provide clamping force but provide minimal drag to the test. Each sample (26–41 mm3) was compressed to create a uniform cylinder of 2.45 mm in diameter and 4.8–8.9 mm in height. Samples were tested on the same day they were fabricated.ABVF constructs from formulation 1 with  contains two non-natural pendant groups. The amounts of pendant-functionalized polymers were limiting, and thus all other components of the BVF putty composition were altered to maintain the ratios previously reported [p(FD-C10) had a honey-like consistency and was light sensitive; thus, it was protected from light until it was used in ABVF fabrication. These samples were released or tested the same day and were protected from light during release. Duplicate formulations without vancomycin were made as a negative control (data not shown). There was no bioactivity from any of the control formulations without drugs. Incorporating polymers with different pendant groups and therefore various physical properties as the bulk matrix of an antibiotic-loaded BVF device showed (i) that there was no intrinsic antimicrobial activity for this set of polymers; (ii) p(FD-C10), p(SC-C6), and p(A-C4) were all able to release vancomycin for over one month, with p(SC-C6) (formulation 5) exhibiting the lowest burst release  (formulation 4), were mechanically evaluated as model formulations to characterize the new composition as a putty. Each sample was compressed into a cylinder prior to testing as described in Materials and Methods. A combination of compression  and custom polymer pression  and tenspression  were usepression B. Formulpression C. Tensilpression .Osteomyelitis is a complex condition with multiple etiologies that necessitate new, tailorable strategies . PreviouTg) than the previous polyesters and hence allow fabrication of drug-encapsulated putties at lower temperatures. This is critical for future efforts, which may encapsulate heat sensitive drugs. The polyester, p(SC-C6), with pendant groups having long aliphatic chains lead to polymers with very low Tg. Additionally, the nature of the diacid also influences the Tg of the polymers. Polymerization of the functionalized diols with long chain diacids such as sebacic acid lowers the Tg of the polyesters such as p(A-C10). However, unlike polypeptides, the ester backbone ensures hydrolytic degradation and bioresorption, the rate of which can be tuned by the identity of the pendant group. In addition, the polyesters were designed to avoid any hydrogen bonding, which makes the polymers easier to process. The modular synthesis of the polyester platform has enabled the design of such polymers for various applications such as thermoresponsive polymers [Previously, a platform of peptidomimetic polyesters whose physical and chemical properties can be modulated over a wide range of properties was developed . These ppolymers ; polyest4) will be more hydrophilic and have a higher modulus than the corresponding polyesters made from sebacic acid (C10). Therefore, these high molecular weight polymers can be mixed with the bone filler without the use of any organic solvent. The release profiles of a putty-encapsulated therapeutic can be gated utilizing polyester pendant groups   to a firS. aureus enters the osteoblast to avoid antibiotics and the host immune system. Currently, in silico efforts are underway using Schrodinger software  to create relevant polymer models of vancomycin release.Using a combination of confirmed and in silico derived models is a powerful approach in controlled-drug delivery formulation development, providing a mechanism to consider each element of the composition  to attain a particular pharmacokinetic profile by theoretical means . UnfortuTg custom polymers, a promising putty-like composition was created to locally deliver bioactive vancomycin over 42 days, ultimately facilitating effective treatment for orthopedic S.aureus infection. Using the strategies presented here, additional polymers can be developed to tailor the pharmacokinetic profile of vancomycin release and begin to tackle the most challenging chronic and invasive infections.The work presented here represents a logical extension of our previous moldable antibiotic-releasing, polymer controlled BVF device . By incl