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During collagen biosynthesis, proline is post-translationally converted to hydroxyproline by specific enzymes. This amino acid, unique to collagen, plays a crucial role in stabilizing the collagen triple helix structure and could serve as an important biomarker for collagen content and quality analysis. Hydroxyproline has four isomers, depending on whether proline is hydroxylated at position 4 or 3 and on whether the cis- or trans- conformation is formed. Moreover, as extensive hydrolysis of collagen is required for its amino acid analysis, epimerization may also occur, although to a lesser extent, giving a total of eight possible isomers. The aim of the present study was to develop a reversed-phase high-performance liquid chromatography-UV-mass spectrometry (RPLC-UV-MS) method for the separation and quantification of all eight hydroxyproline isomers. After the chiral derivatization of the hydroxyproline isomers with Nα-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA), to enable their UV detection, the derivatized diastereoisomers were separated by testing different C18 column technologies and morphologies and optimizing operative conditions such as the mobile phase composition (solvent, additives), elution mode, flow rate and temperature. Baseline resolution of all eight isomers was achieved on a HALO® ES-C18 reversed-phase column (150×1.5 mm, 2.7 μm, 160 Å) using isocratic elution and MS-compatible mobile phase. The optimized method was validated for the quantification of hydroxyproline isomers and then applied to different collagen hydrolysates to gain insight and a deeper understanding of hydroxyproline abundances in different species (human, chicken) and sources (native, recombinant)., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Caterina Temporini reports financial support was provided by Italian Ministry of University and Research. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

This work describes the development of a flow injection method to determine hydroxyproline (HYP), one of collagen's most abundant amino acids. Collagen is a protein with several applications and high nutritional value. Evaluating the feasibility of using collagen from fish skin over its mammalian source is essential. The determination of HYP requires the pre-treatment and hydrolysis of the fish skin to break down collagen into its amino acids, and the HYP value quantified relates to the collagen content. The determination was based on the HYP oxidation with permanganate in an alkaline medium and the consequent decrease of colour intensity registered. Under optimal conditions, the developed method enables the determination of the HYP within the dynamic range of 23.8 to 500 mg L -1 , with a limit of detection (LOD) of 2.6 mg L -1 and a limit of quantification (LOQ) of 23.8 mg L -1 . Different samples were processed, and the digests were analysed by the proposed method and with the conventional procedure with good correlation (relative error < 7%). Moreover, the analyte quantification is performed faster, simpler, and more accurately, with less toxic solutions. The reproducibility of the developed method was also evaluated by calculating the relative standard deviation of the calibration curve slope (RSD < 1%).

The detection of non-protein nitrogen adulterants is a major challenge in dairy testing. As a marker molecule of animal hydrolyzed protein, the presence of non-edible L-hydroxyproline (L-Hyp) molecules can be used to identify low-quality milk containing components of animal hydrolyzed protein. However, it is still difficult to detect L-Hyp directly in milk. The Ag@COF-COOH substrate in this paper can be used to realize label-free L-Hyp sensitive detection based on the hydrogen bond transition mechanism. To explore the mechanism, the binding sites of hydrogen bond interaction have been verified experimentally and computationally, and the charge transfer process was also explained in terms of HOMO/LOMO energy level. In conclusion, the quantitative models for L-Hyp in an aqueous environment and in milk were developed. The limit of detection (LOD) of L-Hyp in an aqueous environment could reach 8.18 ng/mL, with R 2 of 0.982. The linear range of quantitative detection in milk was 0.5-1000 μg/mL and the LOD was as low as 0.13 μg/mL. In this work, a hydrogen bond interaction based Surface-enhanced Raman spectroscopy (SERS) method for the label-free detection of L-Hyp was proposed, which complemented the application of SERS technology in the detection of dairy products., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The red dragon fruit (Hylocereus polyrhizus) extract (RDFE) is frequently used for a variety of therapeutic purposes (e.g., boosting the immune system, promoting a healthy gastrointestinal system, improving wound healing). We investigated the effects of a topical cream containing 7.5% RDFE on hydroxyproline and fibroblast growth factor 2 (FGF-2) levels and wound healing. On Day 0, we divided a total of 36 albino male Wistar rats into two equal groups. Using an 8-mm punch biopsy, we created a circular excision to fascial depth on the back of each rat. On Day 1, we treated the control group (n = 18) with 20 mg of base cream and the RDFE group (n = 18) with 20 mg of 7.5% RDFE cream. We measured hydroxyproline and FGF-2 levels in the wound tissue using an ELISA method on Days 3, 7, and 14. We found that on Day 3, hydroxyproline levels were significantly lower in the treatment group than in the control group (p = .031). We also found a significant correlation between FGF-2 levels in the treatment group and wound diameter (p = .02). On the basis of the results of this study, we concluded that using a topical cream containing 7.5% RDFE has the potential to accelerate wound healing by increasing levels of hydroxyproline and FGF-2 in the wound., Competing Interests: None of the authors have any conflicts of interest to disclose., (Copyright © 2023 International Society of Plastic and Aesthetic Nurses. All rights reserved.)

Objective: JWH133, a cannabinoid type 2 receptor agonist, was tested for its ability to protect mice from bleomycin-induced pulmonary fibrosis. Methods: By using a random number generator, 24 C57BL/6J male mice were randomly divided into the control group, model group, JWH133 intervention group, and JWH133+a cannabinoid type-2 receptor antagonist (AM630) inhibitor group, with 6 mice in each group. A mouse pulmonary fibrosis model was established by tracheal instillation of bleomycin (5 mg/kg). Starting from the first day after modeling, the control group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution, and the model group mice were intraperitoneally injected with 0.1 ml of 0.9% sodium chloride solution. The JWH133 intervention group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg, dissolved in physiological saline), and the JWH133+AM630 antagonistic group mice were intraperitoneally injected with 0.1 ml of JWH133 (2.5 mg/kg) and AM630 (2.5 mg/kg). After 28 days, all mice were killed; the lung tissue was obtained, pathological changes were observed, and alveolar inflammation scores and Ashcroft scores were calculated. The content of type Ⅰ collagen in the lung tissue of the four groups of mice was measured using immunohistochemistry. The levels of interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in the serum of the four groups of mice were measured using enzyme-linked immunosorbent assay (ELISA), and the content of hydroxyproline (HYP) in the lung tissue of the four groups of mice was measured. Western blotting was used to measure the protein expression levels of type Ⅲ collagen, α-smooth muscle actin (α-SMA), extracellular signal regulated kinase (ERK1/2), phosphorylated P-ERK1/2 (P-ERK1/2), and phosphorylated ribosome S6 kinase type 1 (P-p90RSK) in the lung tissue of mice in the four groups. Real-time quantitative polymerase chain reaction was used to measure the expression levels of collagen Ⅰ, collagen Ⅲ, and α-SMA mRNA in the lung tissue of the four groups of mice. Results: Compared with the control group, the pathological changes in the lung tissue of the model group mice worsened, with an increase in alveolar inflammation score (3.833±0.408 vs. 0.833±0.408, P <0.05), an increase in Ashcroft score (7.333±0.516 vs. 2.000±0.633, P <0.05), an increase in type Ⅰ collagen absorbance value (0.065±0.008 vs. 0.018±0.006, P <0.05), an increase in inflammatory cell infiltration, and an increase in hydroxyproline levels [(1.551±0.051) μg/mg vs. (0.974±0.060) μg/mg, P <0.05]. Compared with the model group, the JWH133 intervention group showed reduced pathological changes in lung tissue, decreased alveolar inflammation score (1.833±0.408, P <0.05), decreased Ashcroft score (4.167±0.753, P <0.05), decreased type Ⅰ collagen absorbance value (0.032±0.004, P <0.05), reduced inflammatory cell infiltration, and decreased hydroxyproline levels [(1.148±0.055) μg/mg, P <0.05]. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group showed more severe pathological changes in the lung tissue of mice, increased alveolar inflammation score and Ashcroft score, increased type Ⅰ collagen absorbance value, increased inflammatory cell infiltration, and increased hydroxyproline levels. Compared with the control group, the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK proteins in the lung tissue of the model group mice increased, while the expression of type Ⅰ collagen, type Ⅲ collagen, and α-SMA mRNA increased. Compared with the model group, the protein expression of α-SMA (relative expression 0.60±0.17 vs. 1.34±0.19, P <0.05), type Ⅲ collagen (relative expression 0.52±0.09 vs. 1.35±0.14, P <0.05), P-ERK1/2 (relative expression 0.32±0.11 vs. 1.14±0.14, P <0.05), and P-p90RSK (relative expression 0.43±0.14 vs. 1.15±0.07, P <0.05) decreased in the JWH133 intervention group. The type Ⅰ collagen mRNA (2.190±0.362 vs. 5.078±0.792, P <0.05), type Ⅲ collagen mRNA (1.750±0.290 vs. 4.935±0.456, P <0.05), and α-SMA mRNA (1.588±0.060 vs. 5.192±0.506, P <0.05) decreased. Compared with the JWH133 intervention group, the JWH133+AM630 antagonistic group increased the expression of α-SMA, type Ⅲ collagen, P-ERK1/2, and P-p90RSK protein in the lung tissue of mice, and increased the expression of type Ⅲ collagen and α-SMA mRNA. Conclusion: In mice with bleomycin-induced pulmonary fibrosis, the cannabinoid type-2 receptor agonist JWH133 inhibited inflammation and improved extracellular matrix deposition, which alleviated lung fibrosis. The underlying mechanism of action may be related to the activation of the ERK1/2-RSK1 signaling pathway.

Context: Oxidative stress leads to deleterious processes in the liver that resulted in liver diseases. Objective: To evaluate antioxidant activity and hepatoprotective potential of ethanolic leaves extract of Citrus reticulate against hepatic dysfunction induced by thioacetamide (TAA). Materials and Methods: Flavonoid constituents were isolated from the ethanol extract by chromatographic techniques and identified by the spectroscopic analyses. Antioxidant activity was determined using DPPH assay. Hepatotoxicity was induced in rats via intraperitoneal injection of TAA and the ethanol extract was orally administrated at a dose of 100 mg/kg/day for four weeks. Serum biomarkers, hepatic antioxidant enzymes, tumour necrosis factor-alpha (TNF-α), hepatic hydroxyproline levels, and histopathology were examined. Results: Ten known flavonoids were identified, among of them, 6,3`-dimethoxyluteolin and 8,3`-dimethoxyluteolin possessed the highest antioxidant activity. The substantially elevated serum enzymatic levels of ALT, ALP, and bilirubin were found to be restored towards normalisation significantly by the plant extract. Furthermore, the markers including MDA, GSH, SOD, NO, and protein carbonyl which were close to oxidative damage, were restored. Meanwhile, the extract treatment decreased TNF-α level and also was able to reverse the induced fibrosis by significantly reducing the hydroxyproline content. Moreover, histopathological studies further substantiate the protective effect of the extract. Conclusion: C. reticulate leaves extract is a rich source of phytochemicals with in vitro and in vivo protective effects.

Introduction: The root bark of Berberis aristata has been utilized by indigenous peoples for wound treatment for centuries. The mature root barks are crushed into a paste and applied to the wound's surface., Objective: The focus of this research is to analyse the wound healing activities of an ethanolic extract of Berberis aristata, as well as to use molecular docking to establish the likely mechanism of the potent phytochemical. There is no scientific evidence to support the usage of root bark extract of Berberis aristata., Methods: The Herbal ointment, which comprises (1%, 2%, and 4% w/w) ethanolic extract of root bark, was developed to test the wound healing ability of incision and excision wounds, and the molecular mechanism was established using Auto-Dock software., Results: Epithelization stage, wound index, % wound contraction area, hydroxyproline content, DNA estimate, and histopathological assessments were performed on the incision wound model. Tensile strength was assessed in an excision wound model. TLC was used to identify the samples after successive extractions with different solvents based on polarity., Conclusion: Berberine and tetrahydropalmatine were major active phytoconstituent found in root barks of Berberis aristata as secondary metabolites. Animals treated with 4% w/w formulation demonstrated considerable wound contraction, epithelization time, and wound index in the excision model. In contrast, to control and standardize the concentrations of hydroxyproline, total amino acids, and DNA in recovering tissue were higher. At 4% w/w extract formulation, the parameters studied indicated a substantial result. Berberine and tetrahydropalmatine, active metabolites which are present in the ethanolic extract of Berberis aristata, were found to be responsible for wound healing. Based on ligand interactions, the findings verified Berberis aristata ethnomedicinal claim in a wound healing capacity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objectives: To evaluate the interactions of two phosphate ester monomers [10-methacryloyloxydecyl dihydrogen phosphate (10-MDP) and dipentaerythritol penta-acrylate phosphate (PENTA)] with hydroxyapatite and collagen and understand their influence on dentine bonding., Methods: Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, nuclear magnetic resonance, ultraviolet-visible, and molecular docking were applied for separately evaluating the interactions of two monomers with hydroxyapatite and collagen. Hydrophilicity tests and morphological observation were employed to characterize pretreated dentine. Microtensile bond strength (μTBS) and nanoleakage were investigated to evaluate the bonding performance. Hydroxyproline assay, in situ zymography, and matrix metalloproteinase-9 (MMP-9) activity assay were used to confirm the MMP inhibition., Results: Chemoanalytic characterization confirmed the interactions of 10-MDP and PENTA with hydroxyapatite and collagen. The interactions of PENTA were weaker than 10-MDP. PENTA possessed better dentine tubule sealing after etching than 10-MDP. Dentine treated with PENTA was more hydrophilic than 10-MDP. 10-MDP and PENTA treating significantly increased the initial μTBS than the control group without primer conditioning. μTBS decreased significantly during aging, and the decrease was more severe in the PENTA group than 10-MDP. The 10-MDP and PENTA groups exhibited relatively less fluorescence than the control. The relative inhibition percentages of MMP-9 decreased in the order of 10-MDP-Ca salt, 10-MDP and PENTA. The 10-MDP, PENTA, and 10-MDP-Ca salt groups showed significantly lower hydroxyproline contents than the control., Conclusions: Although PENTA adsorbed on hydroxyapatite, it did not form a stable calcium salt. The interactions of 10-MDP with hydroxyapatite and collagen are different than those of PENTA., Clinical Significance: The sealing of dentinal tubules by PENTA and the inhibition of MMP by 10-MDP and its calcium salts contribute to improving the dentine bonding durability., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objective: To assess dentin collagen denaturation from phosphoric acid and enzyme treatments using collagen hybridizing peptide (CHP) and to investigate the effect of collagen denaturation on bio-stabilization promoted by proanthocyanidins (PA)., Methods: Human molars were sectioned into 7-µm-thick dentin films, demineralized, and assigned to six groups: control with/without PA modification, H 3 PO 4 -treated collagen with/without PA modification, enzyme-treated collagen with/without PA modification. PA modification involved immersing collagen films in 0.65% PA for 30 s. H 3 PO 4 and enzyme treatments were used to experimentally induce collagen denaturation, which was quantitated by fluorescence intensity (FI) from the fluorescently-conjugated-CHP (F-CHP) staining (n = 4). FTIR was used to characterize collagen structures. All groups were subject to collagenase digestion to test the bio-stabilization effect of PA on denatured collagen using weight loss analysis and hydroxyproline assay (n = 6). Data were analyzed using two-factor ANOVA and Games-Howell post hoc tests (α = 0.05)., Results: FTIR showed collagen secondary structural changes after denaturation treatments and confirmed the incorporation and cross-linking of PA in control and treated collagen. F-CHP staining indicated high-degree, medium-degree, and low-degree collagen denaturation from H 3 PO 4 -treatment (FI = 83.22), enzyme-treatment (FI = 36.54), and control (FI = 6.01) respectively. PA modification significantly reduced the weight loss and hydroxyproline release of all groups after digestion (p < 0.0001), with the results correlated with FI values at r = 0.96-0.98., Significance: A molecular method CHP is introduced as a sensitive technique to quantitate dentin collagen denaturation for the first time. PA modification is shown to effectively stabilize denatured collagen against collagenase digestion, with the stabilization effect negatively associated with the collagen denaturation degree., (Copyright © 2022 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) impart structural heterogeneities that can alter plasma proteins' functions in various pathophysiological processes. However, the identification and mapping of PTMs in untargeted plasma proteomics is still a challenge due to the presence of diverse components in blood. Here, we report a label-free method for identifying and mapping hydroxylated proteins using tandem mass spectrometry (MS/MS) in the human plasma sample. Our untargeted proteomics approach led us to identify 676 de novo sequenced peptides in human plasma that correspond to 201 proteins, out of which 11 plasma proteins were found to be hydroxylated. Among these hydroxylated proteins, Immunoglobulin A1 (IgA1) heavy chain was found to be modified at residue 285 (Pro 285 to Hyp 285 ), which was further validated by MS/MS study. Molecular dynamics (MD) simulation analysis demonstrated that this proline hydroxylation in IgA1 caused both local and global structural changes. Overall, this study provides a comprehensive understanding of the protein profile containing Hyp PTMs in human plasma and shows the future perspective of identifying and discriminating Hyp PTM in the normal and the diseased proteomes., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Immunosuppressed patients sometimes require colorectal surgery. We investigated whether adipose tissue-derived stem cells contributed to anastomosis healing in rats immunosuppressed with the mTOR inhibitor everolimus., Materials and Methods: Sixty male Sprague-Dawley rats were randomly divided into 4 groups of 14 each, with all groups undergoing descending colon anastomosis; the 4 remaining rats were used for stem cell retrieval. Group 1 (control) underwent surgery only, group 2 received stem cell injection, group 3 received everolimus only, and group 4 received everolimus plus stem cell injection. After treatment, each group was randomly divided into 2 equal subgroups according to the day of euthanasia (posttreatment day 4 or day 7). We measured anastomosis bursting pressure and tissue hydroxyproline level and performed histopathological evaluation., Results: At both posttreatment days 4 and 7, median weight loss in group 3 was higher than in group 1, group 3 had higher severity of intraabdominal adhesion than group 4, and group 2 had mean hydroxyproline level higher than the other groups. At posttreatment day 4, mean bursting pressure was significantly different in group 1 versus groups 2 and 4 (P = .002) and group 2 versus groups 3 and 4 (P < .001). No significant differences were shown in pathological analysis except for vascular proliferation on day 7 (P = .003)., Conclusions: Injection of adipose tissue-derived stem cells in the anastomosis site prevented anastomosis leakage by contributing to healing. Injection of adipose tissue-derived stem cells in the anastomosis region, especially in the early period after solid-organ transplant in recipients and after gastrointestinal surgery in immunosuppressed patients, may help reduce mortality and morbidity.

Radiocarbon dating of bone and charcoal from sites dating to the Middle and Upper Paleolithic is challenging due to low residual levels of radiocarbon. This means that small amounts of contaminating carbon can wield a great influence over accuracy unless they are fully removed. The site of Kabazi II in the Crimea is important because radiocarbon dates previously obtained from bones in archaeological horizons that date to the Western Crimean Mousterian (WCM) are surprisingly young. We redated the same samples using a single compound dating method that focuses on extracting and dating the amino acid hydroxyproline. We show that single amino acid dates produce significantly older determinations than those that use bulk collagen pretreatment procedures. Our results suggest that instead of dating to 35,000-40,000 cal BP, the bones actually date to >50,000 cal BP. This implies that the WCM at this site is much older than previously thought. In light of these current findings, we considered the dates of other key Crimean sites and concluded that in the absence of reliable pretreatment methods, it would be wise to consider many of them minimum ages. We conclude that there is little robust evidence to suggest Neanderthals were present in the Crimea after 40,000 cal BP., Competing Interests: Declaration of competing interest The authors declare there is no conflict of interest., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

Purpose: Type II pneumocyte (alveolar epithelial cells type II [AECII]) senescence has been implicated in the progression of lung fibrosis. The capacity of senescent cells to modulate pulmonary macrophages to drive fibrosis is unexplored. Insulin-like growth factor-1 receptor (IGF-1R) signaling has been implicated as a regulator of senescence and aging., Methods and Materials: Mice with an AECII-specific deletion of IGF-1R received thoracic irradiation (n ≥ 5 per condition), and the effect of IGF-1R deficiency on radiation-induced AECII senescence and macrophage polarization to an alternatively activated phenotype (M2) was investigated. IGF-1R signaling, macrophage polarization, and senescence were evaluated in surgically resected human lung (n = 63)., Results: IGF-1R deficient mice demonstrated reduced AECII senescence (senescent AECII/field; intact: 7.25% ± 3.5% [mean ± SD], deficient: 2.75% ± 2.8%, P = .0001), reduced accumulation of M2 macrophages (intact: 24.7 ± 2.2 cells/field, deficient: 15.5 ± 1.2 cells/field, P = .0086), and fibrosis (hydroxyproline content; intact: 71.9 ± 21.7 μg/lung, deficient: 31.7 ± 7.9, P = .0485) after irradiation. Senescent AECII enhanced M2 polarization in a paracrine fashion (relative Arg1 mRNA, 0 Gy: 1.0 ± 0.4, 17.5 Gy: 7.34 ± 0.5, P < .0001). Evaluation of surgical samples from patients treated with chemoradiation demonstrated increased expression of IGF-1 (unirradiated: 10.2% ± 4.9% area, irradiated: 15.1% ± 11.5%, P = .0377), p21 (unirradiated: 0.013 ± 0.02 histoscore, irradiated: 0.084 ± 0.09 histoscore, P = .0002), IL-13 (unirradiated: 13.7% ± 2.8% area, irradiated: 21.7% ± 3.8%, P < .0001), and M2 macrophages in fibrotic regions relative to nonfibrotic regions (unirradiated: 11.4 ± 12.2 CD163 + cells/core, irradiated: 43.1 ± 40.9 cells/core, P = .0011), consistent with findings from animal models of lung fibrosis., Conclusions: This study demonstrates that senescent AECII are necessary for the progression of pulmonary fibrosis and serve as a targetable, chronic stimuli for macrophage activation in fibrotic lung., (Published by Elsevier Inc.)

Previously we developed and characterized a novel hydrogel film wound dressing containing Sodium Alginate and Pectin loaded with Simvastatin with multi-functional properties. This study investigated the in-vivo efficacy of the developed wound dressing on type I diabetic wound model. Experiments were performed on male Wistar rats for the period of 21-days. Animals developed diabetes after intraperitoneal injection (50 mg/kg) of Streptozotocin then randomly divided into different groups. On days 7, 14, and 21 of post-wounding, animals were euthanized and the wounds tissue were harvested for analysis. The wound healing rate, hematology and histological analysis, hydroxyproline assay, and Vascular Endothelial Growth Factor A measurements were noted. The results revealed that the wound dressing healed the wounded area significantly (p < 0.05) higher than the control after 21-day treatment and wound closure was ~99% without any adverse systemic reactions. Histological analysis qualitatively revealed an enhanced re-epithelialization and collagen deposition. Moreover, results also showed an improved rate of collagen synthesis and angiogenesis in the group treated with the hydrogel film loaded with Simvastatin. Thus, the present study demonstrated that developed film holds great potential for the acceleration of diabetic wound healing by its pro-angiogenic effect, faster re-epithelialization and increased collagen deposition., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Gelatin and collagen are considered halal-critical ingredients as they are typically derived from either bovine or porcine animals. Current analytical methods for determining the sources of gelatin and collagen suffer from limitations in terms of robustness and false positives in peptide matching. Thus, the aim of this study was to investigate the utility of monitoring hydroxyproline, a signature amino acid for gelatin and collagen, for identifying potentially haram foodstuffs. To determine the hydroxyproline profiles among animal- and plant-based samples, one-way univariate analysis of variance followed by pair-wise comparison was used to establish statistical significance. Multivariate chemometric analysis through principal component analysis revealed a discrete distribution pattern among 59 samples due to hydroxyproline variability. Finally, inter- and intra-laboratory comparisons demonstrated the validity and robustness of hydroxyproline determination according to ISO 17025. Thus, this preliminary identification technique will aid the identification of potentially haram foodstuffs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Objective: To observe the alteration of clinical features of intrahepatic lymphocyte subsets in C57BL/6N-TG (1.28HBV)/Vst hepatitis B virus (HBV) transgenic mice composite carbon tetrachloride (CCl(4)) with intraperitoneal injection under the background of hepatitis B to induce liver fibrosis mice model, and analyze their correlation with serum HBV DNA and liver tissue hydroxyproline (Hyp) content. Methods: HBV-Tg mice were intraperitoneally injected with 10% CCl(4) to induce the rapid formation of hepatic fibrosis. Serum HBV DNA, HBsAg, HBeAg levels and liver tissue HBsAg expressional conditions were used to evaluate the virological characteristics of mice model. The degree of hepatic inflammation and fibrosis in mice were observed by HE, Sirius Red staining and liver tissue hydroxyproline (Hyp) content. Intrahepatic T lymphocyte, B lymphocyte, CD4+T lymphocyte, CD8+T lymphocyte, natural killer (NK) cell and natural killer T (NKT) cells distribution were observed by flow cytometry. One-way analysis of variance was used for intergroup data comparison, and LSD was used for pairwise comparison. Pearson's correlation analysis was used to analyze the correlation between the above lymphocyte subsets and serum HBV DNA and liver tissue Hyp content. Results: Serum HBsAg, HBeAg and liver tissue HBsAg had equal positive expression in the HBV-Tg composite CCl(4) mice model group, and the serum HBV DNA load was > 1 × 10(6) IU / ml. Compared with the wild-type control group, liver tissue Hyp content of the composite model group was significantly higher [(196.39 ± 38.14) μg /g and (347.67 ± 59.53) μ g/g, P < 0.01). The degree of inflammation and fibrosis in liver tissues was aggravated, and the proportion of all intrahepatic CD4+T, NK and NKT cells was significantly reduced ( P < 0.01), while the proportion of CD8+T lymphocytes (30.58% ± 2.89% vs. 46.50% ± 2.24%, P < 0.01) and B lymphocytes (28.82% ± 2.24% vs. 37.10% ± 8.59%, P < 0.05) was significantly increased. Serum HBV DNA level was positively correlated with the proportion of intrahepatic T lymphocytes ( r = 0.413, P < 0.05), and negatively correlated with the proportion of NK cells ( r = -0.419, P < 0.05). Liver tissue Hyp content was negatively correlated with the proportion of all CD4+T lymphocytes ( r = -0.871), NK cells ( r = -0.716), and NKT cells ( r = -0.876) (all P < 0.01), and positively correlated with the proportion of all CD8 + T lymphocytes ( r = 0.852), and B lymphocytes ( r = 0.593) (all P < 0.01). Conclusion: HBV-Tg composite CCl4 mice model can induce positive HBV virological indicators, liver inflammation and fibrosis in mice model of hepatitis B coexisting with fibrosis. This model has the features of immune disorder of liver lymphocyte similar to human disease, and the immune disorder of intrahepatic lymphocytes is correlated with HBV viral load and liver fibrosis degree.

Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC-MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl- N -hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.

Hydroxyproline is a common variation of proline, with diverse biological roles. The hydroxylation of proline gives rise to several (natural and/or synthetic) isomeric forms, including both positional isomers and stereoisomers. While mass spectrometry is widely touted as a very selective analytical technique, the identification of closely related isomers often poses a challenge. In these cases, allied technologies become helpful in providing full characterization. Here, infrared multiple photon dissociation (IRMPD) spectroscopy is used to differentiate between three isomers, namely cis -3-hydroxyproline, cis -4-hydroxyproline, and trans -4-hydroxyproline. In contrast to the protonated species which show only minor variations in their IRMPD spectra, lithiated species were found to display significant spectral differences, making their differentiation more straightforward. The conformational origin of these spectral differences was investigated by complementary quantum-chemical calculations.

Examination of collagen turnover in the liver, lungs, and spleen of BALB/c mice revealed the age-related differences in the levels of hydroxyproline and its fractions. The highest level of total hydroxyproline was observed in the lungs and spleen of young (2-month-old) mice. In the liver, this level attained maximum at the age of 6 months at the expense of elevation of protein-bound hydroxyproline relatively its level in 2-month-old mice. At the age of 12 months, the levels of total hydroxyproline in the liver and spleen were lower than in 6-month-old mice. The decrease in the collagen turnover rate in the liver of 12-month-old mice reflected lower levels of hydroxyproline fractions in comparison with the corresponding values in 6-month-old mice. The rates of collagen turnover in organs differed in mice of different ages: it was maximum in the lungs and spleen of young animals and in the liver of middle-aged mice.

Objective: To explore the expression and significance of miR-223 in mice with pulmonary fibrosis., Materials and Methods: The rats were separated into a control group (n=15), a sham operation group (n=15), and a model group (n=45) (which was then divided into a 3-day group, a 7-day group, and a 14-day group, with 15 rats in each group). The rat model of pulmonary fibrosis was established. The rats in the model group were injected with bleomycin solution, while those in the control group and sham operation group were given the same operation and injected with the same amount of normal saline. After observing the pulmonary function indexes of the rats on the 3rd, 7th and 14th days after modeling, the rats were sacrificed by cervical dislocation, the pulmonary inflammation and fibrosis of the rats were observed, and the HYP (hydroxyproline) content and miR-223 expression level were determined. Pearson correlation analysis was employed to analyze the correlation between miR-223 and HYP., Results: The pulmonary inflammation score of the model group was significantly higher than that of the sham group and the control group, and the pulmonary inflammation of the model group significantly increased with the increase of time (p<0.05). The pulmonary fibrosis score in the model group was markedly higher than that in the rest two groups, and the pulmonary fibrosis in the model group elevated significantly with the passage of time (p<0.05). The relevant pulmonary function indexes of the model group rats were significantly lower than those of the other two groups, and the pulmonary function of the model group rats gradually decreased with time (p<0.05). As to the HYP, it presented notably higher content in the model group than in the remaining two groups, and its content in the model group rats increased significantly with time (p<0.05). The expression of miR-223 decreased with the increase of fibrosis (p<0.05), and the expression level of miR-223 was negatively correlated with the HYP content (p<0.05)., Conclusions: MiR-572 targeted CDH1 to promote cell metastasis in WT by suppressing EMT.

Build-up of extracellular matrix in liver fibrosis results in changes on endogenous molecules expression that may be studied through the fluorescence characterization of ex vivo liver samples. To the best of our knowledge, no investigations have provided in-depth evidence and discussion on the changes of the endogenous fluorescence in ex vivo tissue due to the effects of the preservation media. In this work, we contrast and analyze the endogenous fluorescence from tryptophan, vitamin A, hydroxyproline and elastin cross-links potential biomarkers of the liver fibrosis, in in vivo measurements and liver samples preserved on formaldehyde, and two standard preservation media. As it is known, chemical changes in tissue, caused by formaldehyde fixation, alter the endogenous fluorescence spectra. We propose the use of phosphate-buffered saline (PBS), and Iscove's Modified Dulbecco's Medium (IMDM) to elude the fluorescence changes. PBS and IMDM showed to maintain the endogenous fluorescence characteristics similar to in vivo conditions. The results of this work point the way for a more reliable assessment of endogenous fluorescence in ex vivo hepatic studies.

Background: Liquiritigenin (LQ), an aglycone of liquiritin in licorice, has demonstrated antioxidant, anti-inflammatory and anti-tumor activities. Previously, LQ was found to inhibit liver fibrosis progression., Purpose: Phosphatase and tensin homolog (PTEN) has been reported to act as a negative regulator of hepatic stellate cell (HSC) activation. However, the roles of PTEN in the effects of LQ on liver fibrosis have not been identified to date., Methods: The effects of LQ on liver fibrosis in carbon tetrachloride (CCl 4 ) mice as well as primary HSCs were examined. Moreover, the roles of PTEN and microRNA-181b (miR-181b) in the effects of LQ on liver fibrosis were examined., Results: LQ markedly ameliorated CCl 4 -induced liver fibrosis, with a reduction in collagen deposition as well as α-SMA level. Moreover, LQ induced an increase in PTEN and effectively inhibited HSC activation including cell proliferation, α-SMA and collagen expression, which was similar with curcumin (a positive control). Notably, loss of PTEN blocked down the effects of LQ on HSC activation. PTEN was confirmed as a target of miR-181b and miR-181b-mediated PTEN was involved in the effects of LQ on liver fibrosis. LQ led to a significant reduction in miR-181b expression. LQ-inhibited HSC activation could be restored by over-expression of miR-181b. Further studies demonstrated that LQ down-regulated miR-181b level via Sp1. Collectively, we demonstrate that LQ inhibits liver fibrosis, at least in part, via regulation of miR-181b and PTEN., Conclusion: LQ down-regulates miR-181b level, leading to the restoration of PTEN expression, which contributes to the suppression of HSC activation. LQ may be a potential candidate drug against liver fibrosis., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Autologous chondrocytes are generally evaluated by mechanical, histological, and biochemical methods. However, the analyzed data are from discontinuous measurements at an end point, during which living chondrocytes are destroyed and stained. To solve the problem, we developed a label-free and noninvasive method of estimating extracellular matrix (ECM) production that results from chondrocyte metabolic activities using a cultured-chondrocyte-based gate ion-sensitive field-effect transistor (ISFET) in this study. Using the cultured-chondrocyte-based gate ISFET sensor, we found that the change in electrical signal (Δ V out ), which indicated the change in interfacial pH at the chondrocyte/gate nanogap (ΔpH int ), gradually increased over 3 weeks upon adding a biologically active substance, l-ascorbic acid phosphate magnesium salt n-hydrate (APM), which was attributable to chondrocyte metabolic activities. The increase in Δ V out (ΔpH int ) caused by the chondrocyte metabolic activities was also confirmed by the observation of the suppression of chondrocyte metabolism upon adding 2-deoxy-d-glucose, which was clearly monitored using the cultured-chondrocyte-based gate ISFET sensor. In particular, Δ V out increased monotonically with increasing APM concentrations, indicating that the addition of APM increased Δ V out (ΔpH int ), the amount of which depended on the APM concentration. Moreover, the amounts of hydroxyproline and sulfated glycosaminoglycan (sGAG) as the main components of ECM, which were quantified by colorimetry using various APM concentrations, were found to have a linear relationship with Δ V out (ΔpH int ) of the cultured-chondrocyte-based gate ISFET sensor. Therefore, a platform based on the cultured-chondrocyte-based gate ISFET is suitable for a label-free, noninvasive, and real-time measurement system to analyze ECM production by autologous chondrocytes before transplantation.

Remodeling of the extracellular matrix of the liver in mice with BCG-induced granulomatosis after 2-month course of intraperitoneal injections of isonicotinic acid hydrazide, oxidized dextran conjugated with isonicotinic acid hydrazide, dextrazide, and its liposome-encapsulated form manifested in modification of the proteoglycan composition and hydroxyproline fractions associated with changes in hyaluronidase and MMP activities. The antifibrotic effect of dextrazide and its liposome-encapsulated form manifested in reduction of hydroxyproline fractions that reflected the process of collagen synthesis. Administration of isonicotinic acid hydrazide was followed by a decrease in hydroxyproline fractions reflecting collagen synthesis (antifibrotics effect) and degradation (profibrotic effect), which can be explained by its hepatotoxic activity.

Purpose: Liver fibrosis is the hallmark of chronic nonalcoholic steatohepatitis (NASH) and is characterised by the excessive deposition of extracellular matrix proteins. Early detection and accurate staging of liver fibrosis is critically important for patient management. One of the earliest pathological markers in NASH is the activation of hepatic stellate cells (HSCs) which may be exploited as a marker of fibrogenesis. Activated HSCs secreting factors such as integrin α v β 3 propagate fibrosis. The purpose of the current study was to assess the utility of the integrin α v β 3 imaging agent [ 18 F]FtRGD for the early detection of fibrosis in a diet-induced model of NASH longitudinally using PET imaging., Procedures: Mice were fed with either standard chow diet (SD), high-fat diet (HFD), or a choline-deficient, L-amino acid-defined high-fat fibrogenic diet (CDAHFD) to mimic the clinical pathology of liver disease and followed longitudinally for 10 weeks to assess the development of liver fibrosis using [ 18 F]FtRGD positron emission tomography (PET) imaging. Standard blood biochemistry, histological measures, and qPCR were used to quantify integrin α v β 3 , smooth muscle actin, and collagen types 1 and 6 to assess the extent of NASH pathology and accurately stage liver fibrosis., Results: The CDAHFD fibrogenic diet predictably developed hepatic inflammation and steatosis over the 10 weeks studied with little NASH pathology detected in high fat diet-treated animals. Stage 1 fibrosis was detected early by histology at day 21 and progressed to stage 2 by day 35 and stage 3 by day 56 in mice fed with CDAHFD diet only. Noninvasive imaging with [ 18 F]FtRGD correlated well with integrin α v β 3 and was able to distinguish early mild stage 2 fibrosis in CDAHFD animals compared with standard chow diet-fed animals at day 35. When compared with high fat diet-fed animals, [ 18 F]FtRGD was only able to distinguish later moderate stage 2 fibrosis in CDAHFD animals at day 49., Conclusions: The diet-induced progression of liver fibrosis was confirmed using histology and correlated well with the mRNA of integrin α v β 3 and extracellular matrix protein expression. [ 18 F]FtRGD showed very good correlation between liver uptake and integrin α v β 3 expression and similar detection sensitivity to the current clinical gold standard modalities for staging of liver fibrosis., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 S. V. Hartimath et al.)

Cholestatic liver disease is recognized by extreme collagen formation and deposition, which is mediated by free radicals. The aim of the current study was to investigate the probable hepatoprotective effects of hydroalcoholic extract of watercress (WC) against oxidative stress and liver injury in bile duct ligation (BDL)- induced cholestatic rats. A total of 32 male Wistar rats were divided into four groups; sham control (SC), BDL, SC + hydroalcoholic extract of WC and BDL + hydroalcoholic extract of WC. WC-treated rats received daily WC 500 mg/kg/day for 10 days. Biochemical tests, hepatic oxidative stress markers, and antioxidant enzymes activity were estimated. Further, liver hydroxyproline content was assayed and histological analysis was made. The BDL model markedly elevated the protein carbonyl (PCO) and hydroxyproline contents and decreased the glutathione peroxidase (GPx) activity. Hydroalcoholic extract of WC significantly decreased the surge in liver PCO and hydroxyproline levels and increased the reduced GPx enzyme activity contents in the hepatic tissue. As determined by hematoxylin and eosin staining, BDL considerably induced hepatocyte necrosis. Moreover, these changes were significantly attenuated by the hydroalcoholic extract of WC treatment. Our data indicate that the hydroalcoholic extract of WC extract attenuated liver damage in BDL rats by decreasing the hydroxyproline content and histopathological indexes. Also, it reduced oxidative stress by preventing the hepatic protein oxidation and enhancing the activity of the GPx enzyme via antioxidative effect and free-radical scavenging. Our findings suggest that hydroalcoholic extract of WC could be a beneficial new curative agent for cholestatic liver damage., (© 2019 Wiley Periodicals, Inc.)

The effects of topically administered snake (Deinagkistrodon acutus) oil and its main fatty acid components on skin photodamage were explored. Epidermal thickness, mice body weight, antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase), inflammatory cytokines (tumor necrosis factor alpha and interleukin-6), skin histology, collagen content, and metalloproteinase-1 indicators were analyzed. The results show that topical application of both snake oil and its main fatty acids recovered ultraviolet B (UVB) irradiation induced antioxidant enzymes depletion, prevented metalloproteinase-1. Snake oil and its main fatty acids suppressed dermal infiltration of inflammatory cells and reduced inflammatory cytokines levels. Notably, there was no significant difference in the antioxidant activity but a significant difference in the anti-inflammatory activity between fatty acids and snake oil under the same dose. Finally, snake oil and its main fatty acids inhibited UVB-induced histological damage such as epidermal thickening, collagen fiber and elastic fiber destruction. Our study demonstrated for the first time in KM mice that snake oil exhibited prominent photoprotection activity by protecting the activity of antioxidant enzymes and inhibiting inflammatory factors, as well as reducing the generation of MMP-1. What's more, the main fatty acids in snake oil play an important role in preventing photo-damage especially in protecting antioxidant enzyme activity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Growing demand for gelatin has increased interest in using alternative raw materials. In this study, different animal skins; namely frog, tuna and chicken skins; were utilized in gelatin extraction by previously optimized extraction procedures. Quality characteristics and functional properties of the resultant gelatins were comparatively investigated. Frog skin gelatin had the highest protein content with 77.8% while the highest hydroxyproline content was found in chicken skin gelatin with 6.4%. Frog skin gelatin showed a significantly higher melting point (42.7 °C) compared to tuna and chicken gelatins. Bloom value was also significantly higher in frog skin gelatin compared to that of chicken and tuna skin gelatins. Results showed that processing waste like skins of different animals may present opportunities in gelatin production as high quality alternatives. This study may help the industry by providing one hand comparable data over potentially significant sources., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Objective: To study the preventive and therapeutic effects of safflower water extract on systemic scleroderma (SSc) in mice and its mechanism., Methods: Sixty BALB/C mice were randomly divided into the control group, model group, prednisone group and safflower low, middle, high dose groups, 10 mice in each group.The control group was injected with normal saline, and the other five groups were subcutaneously injected with bleomycin hydrochloride with 100 μl at the concentration of 200 μg /ml on the back, once a day for 28 days to establish the SSc models.At the same time, the control group and model group were treated with normal saline (10 ml/kg), the prednisone group was treated with prednisone 4.5 mg/kg (10 ml/kg), and the low, middle, and high dose safflower groups were treated with safflower at the doses of 1.5, 3, 6 g/kg (10 ml/kg), and all groups were treated for 28 days.After 28 days, all mice were decapitated. The blood samples and back skin of the BLM injection part were collected.After that, all the tissue slices were taken to measure the dermal thickness, and the content of hydroxyproline (HYP) in the skin tissues was detected by hydrolysis method.The contents of tissue growth factor (CTGF) and transforming growth factor-β (TGF-β ) in the skin tissues and the levels of interleukin-6 (IL-6) and interleukin-17 (IL-17) in serum were determined by ELISA., Results: Compared with the control group, the dermal thickness of the model group was increased(P＜0.05), the contents of CTGF, TGF-β and HYP in the skin tissues and the levels of IL-6 and IL-17 in the serum of the model group were increased(P＜0.05); compared with the model group, the dermal thickness in the prednisone group and safflower groups was decreased (P＜0.05), the levels of CTGF, TGF-β and HYP in the skin tissues and the serum levels of IL-6 and IL-17 in the prednisone group and safflower groups were decreased (P＜0.05)., Conclusion: Safflower water extract can improve skin condition (or dermal thickness) in SSc mice, and its mechanism may be related to reducing immune inflammatory response.

Background: In developing countries, esophageal burns are quite common. They are caused by the ingestion of corrosive substances that may lead to esophageal perforation in the short-term and stricture formation in the long-term. Prevention of stricture progression in the esophagus is the main aim of the treatment for corrosive esophageal burns. We aimed to investigate the protective and anti-inflammatory effects of methylene blue (MB) treatment on corrosive esophageal burns., Methods: Twenty-eight rats were used in the study and randomly divided into four equal groups; group 1 (Sham), group 2 (control), group 3 (topical treatment), and group 4 (topical plus systemic treatment). Except for group 1 (Sham group), all three groups received sodium hydroxide (NaOH) in order to generate esophageal burns. In addition, group 2 was given normal saline, group 3 topical MB, and group 4 topical and systemic MB., Results: Hydroxyproline levels were found to be lower in each of the treatment groups as compared to the control group (p=0.005 for group 3 and p=0.009 for group 4). There were no differences in the tumor necrosis factor-α (TNF-α) levels between the groups. The stenosis index (SI) in the treatment groups was also lower than the control group (p=0.016 for group 3 and p=0.015 group 4). The histopathologic damage score (HDS) was prominently lower in group 4 as compared to the control group (p=0.05)., Conclusion: MB is effective in treating tissue damage caused by corrosive esophageal burns and in preventing esophageal stenosis. Complication rates of corrosive esophageal burns may be decreased by using MB in the initial treatment stage.

Background: Oils from marine organisms have a different fatty acid composition. Fish oil (FO) has a high content of eicosapentaenoic and docosahexaenoic acids esterified to triacylglycerols; while in krill oil (KO), fatty acids are primarily esterified to phospholipids. This study aimed to compare the efficacy of two different, marine-derived omega-3 fatty acid sources in the wound healing of colon anastomoses rat model., Methods: For the study, we used 42 male Wistar albino rats. The rats were divided into six groups with seven rats in each group-CO3: left colonic anastomosis (control group), sacrificed on the third day; KO3: left colonic anastomosis + oral KO, sacrificed on the third day; FO3: left colonic anastomosis + oral FO, sacrificed on the third day; CO7: left colonic anastomosis (control group), sacrificed on the seventh day; KO7: left colonic anastomosis + oral KO, sacrificed on the seventh day; FO7: left colonic anastomosis + oral FO, sacrificed on the seventh day. Peritoneal adhesions, anastomotic bursting pressures, hydroxyproline levels, and histological examination of the anastomotic tissue were evaluated., Results: On day 7, bursting pressure and hydroxyproline measurements of the KO group was significantly higher than the FO group (p=0.012; p=0.002, respectively). Also, on day 7, a statistically significant difference was observed between the groups according to inflammatory cell infiltration, fibroblast activity, neoangiogenesis, and collagen deposition in favor of the KO group (p=0.023; p=0.028; p=0.016; p=0.012, respectively)., Conclusion: Both KO and FO supplementation in patients before colorectal surgery may reduce some risk of anastomotic leakage; and KO might be a better alternative and excellent omega-3 source.

Collagen II (COLII), the most abundant protein in vertebrates, helps maintain the structural and functional integrity of cartilage. Delivery of COLII from animal sources could improve cartilage regeneration therapies. Here we show that COLII can be purified from the Capra ear cartilage, a commonly available bio-waste product, with a high yield. MALDI-MS/MS analysis evidenced post-translational modifications of the signature triplet, Glycine-Proline-Hydroxyproline (G-P-Hyp), in alpha chain of isolated COLII (COLIIA1). Additionally, thirty-two peptides containing 59 Hyp residues and a few G-X-Y triplets with positional alterations of Hyp in COLIIA1 are also identified. Furthermore, we show that an injectable hydrogel formulation containing the isolated COLII facilitates chondrogenic differentiation towards cartilage regeneration. These findings show that COLII can be isolated from Capra ear cartilage and that positional alteration of Hyp in its structural motif, as detected by newly developed mass spectrometric method, might be an early marker of cartilage disorder., Competing Interests: The authors declare no competing interests.

The objective of this study was to evaluate the effects of the hot-water extract of defatted Camellia oleifera seeds (CSE) on body and liver fat accumulation in rats. Forty rats were divided into 5 groups and each group was fed either an isocaloric control diet or a high-fat liquid diet with 0% (H), 0.12% (H1), 0.24% (H2), or 0.48% CSE (H3) for 8 weeks. Ingestion of the high-fat liquid diet increased abdominal and liver fat accumulation, although no difference was found in body weights compared with rats fed the control diet. We found that rats fed the H2 and H3 diets had lower plasma alanine aminotransferase activities than the H group in the fourth and eighth weeks. At the end of the study, the H2 and H3 groups also had lower epididymal and retroperitoneal fat masses, and all CSE groups had lower circulatory leptin levels than the H group. CSE consumption decreased hepatic fat accumulation in terms of liver triglycerides and a histopathology analysis, and ameliorated high-fat diet-induced elevation of hepatic tumor necrosis factor-α levels. We also found that CSE groups had lower malondialdehyde and hydroxyproline levels in the liver. Our results suggested that CSE may exert beneficial effects through decreasing body fat accumulation and hepatic steatosis and regulating adipokine levels in diet-induced nonalcoholic fatty liver disease.

Hydroxyproline (Hyp) is an imino acid posttranslationally formed by sequence-specific hydroxylases in the repeating collagen Gly-Xaa-Yaa triad present in all collagen types of all species. In both Xaa- and Yaa-positions, Pro is the most common residue, often oxidized to 4-Hyp in the Yaa- and rarely to 3-Hyp in the Xaa-positions. Here we describe the qualitative and quantitative analysis of 3- and 4-Hyp-isomers by separating the free imino acids either with hydrophilic interaction chromatography (HILIC) or after derivatization with reversed-phase chromatography (RPC). In both cases the compounds were detected by electrospray-ionization mass spectrometry.

Multiple reaction monitoring (MRM) mass spectrometry may be regarded as the gold standard methodology for quantitative mass spectrometry and has been adopted for the analysis of small molecules especially within the pharmaceutical industry. It can also be applied to the analysis of peptides and proteins and to measurement of the basic building blocks of proteins, amino acids. Here we describe the application of MRM mass spectrometry to the measurement of hydroxyproline after acid hydrolysis of various animal tissues. We show that measurement of hydroxyproline provides an accurate and reliable estimate of the collagen content of such tissues and may be a useful indicator of meat tenderness.

Influence of different drying method on yield, physicochemical and functional properties of gelatin from rohu (Labeo rohita) swim bladder were investigated. Freeze-drying presented the highest gelatin yield (54.51 g/100 g, dry weight basis), followed by vacuum-drying (48.95 g/100 g) and spray-drying (41.76 g/100 g), respectively (P < 0.05). All gelatin samples showed glycine as the major amino acid followed by proline, glutamic acid, alanine, arginine and hydroxyproline, respectively. Freeze-dried gelatin (FDG) and spray-dried gelatin (SDG) showed α (α 1 and α 2 ), β-chains and γ-chain as the predominant components, whereas an absence of β- and γ-chains found in vacuum-dried gelatin (VDG). FTIR spectra revealed that the loss of the triple-helix was found in all gelatins which indicated the conversion of collagen to soluble gelatin. FDG and SDG had the highest surface hydrophobicity, protein solubility, emulsifying, foaming and gelation property than VDG (P < 0.05). Moreover, SDG showed slightly higher surface hydrophobicity, protein solubility, emulsifying and foaming properties as compared to FDG (P < 0.05). However, no significant difference was found in gelation properties of FDG and SDG (P > 0.05). Therefore, freeze-drying or spray-drying could be an appropriate drying method for preparation of gelatin from rohu swim bladder with better functionalities., (Copyright © 2018. Published by Elsevier B.V.)

Background: Protein concentration and quality in cat food can vary considerably, and the impact on feline urine composition and nutrient supply is of high practical relevance. In the present study, 6 canned diets with varying protein concentrations and qualities were fed to 10 healthy adult cats. Protein quality in the diet differed depending on the amount of collagen-rich ingredients. Hydroxyproline concentrations were 2.56-4.45 g/kg dry matter in the high quality and 3.76-9.44 g/kg dry matter in the low quality diets. Protein levels were 36.2, 43.3 and 54.9% in the high quality and 36.7, 45.0 and 56.1% in the low quality groups. Each diet was fed for 6 weeks, using a randomized cross-over design. In the last 2 weeks of each feeding period, urine and faeces of the cats were collected., Results: Renal calcium (Ca), oxalate (Ox) and citrate excretion were unaffected by the dietary protein concentration, possibly mediated by a high urine volume (24.2-34.2 ml/kg bodyweight (BW)/day) in all groups. However, renal Ox excretion was lower when the high quality diets were fed (P = 0.013). Urinary relative supersaturation (RSS) with calcium oxalate (CaOx) was low in general, but reduced in the high quality groups (P = 0.031). Urinary RSS values for magnesium ammonium phosphate (MAP) were high (2.64-5.00) among all groups. Apparent digestibility of crude protein and most minerals was unaffected by the different diets. Feed intake was higher in the low quality groups (P = 0.026), but BW of the cats did not differ depending on dietary protein quality. BW of the cats increased with increasing dietary protein concentrations (P = 0.003)., Conclusion: In conclusion, a high protein canned diet might not be a specific risk factor for CaOx urolith formation in cats. In contrast, all diets resulted in high RSS MAP values, which might be critical concerning MAP crystallization. Protein quality had a minor, but significant impact on urine composition, necessitating further research on this subject. A lower energy supply when feeding a low protein quality can be assumed. Changes in BW were only small and require a careful interpretation.

Purpose: To investigate the possible effects of argan oil on the healing of colorectal anastomoses., Methods: n Group 1 (sham), laparotomy was performed and the colon was mobilized. In the control (Group 2) and argan oil (Group 3) groups, colonic resection and anastomosis were applied. To the control and sham groups, 2 mL of 0.9% NaCl was administred rectally, and in the argan oil group, 2 mL/day argan oil was applied rectally for 7 days., Results: The mean bursting pressures of the argan oil and sham groups were significantly higher than the values in the control group. A significant difference was determined between the tissue hydroxyproline and prolidase levels of control group and other groups. Histopathologically, argan oil showed significant beneficial effects on colonic wound healing. In the argan oil and sham groups, the tissue malondialdehyde and fluorescent oxidation product levels were found to be lower and total sulfhydryl levels were higher than the control group., Conclusions: The rectally administered argan oil was observed to have significantly ameliorated wound healing parameters and exerted a significant antioxidant effect. This is the first study in the literature about the beneficial effects of argan oil on colorectal anastomoses.

Objective: Wound healing consists of several continuous phases in which various cells and chemical intermediates are involved. Milk as a rich source of nutritional elements has proved to have potential benefits for treatment of various diseases. The present study was designed to investigate the healing effect of low-fat cow's milk on an open skin wound model in the rabbit., Methods: The 2%, 5%, and 10% (w/w) ointments of lyophilized powder of low-fat milk were prepared in the eucerin base and were applied twice daily in the treatment groups. Phenytoin 1% ointment was used as a standard control. The healing effect of the milk ointment (MO) was evaluated through the measurement of wound surface area, the extent of tissue tension, and the content of hydroxyproline. Histological evaluation of skin tissue specimens was also performed using hematoxylin and eosin staining., Results: The results showed that the healing rate in the treatment group was significantly higher than that of untreated group and eucerin group (p < 0.01). The best healing effect was seen in 5% milk ointment with the shortest healing time (15 days) and the highest tissue tension in comparison to other groups. Although the tissue hydroxyproline content in this group was slightly lower than that of the phenytoin group, this difference was not significant. Histologic, findings indicated increased collagen fibers, increased fibroblasts and an evident decrease in inflammatory cells in that group., Conclusion: It can, therefore, be concluded that low-fat cow's milk has significant beneficial effects on skin wound healing. Therefore, it may be used as a healing agent in different types of the wound in humans after certain clinical trials., (Copyright © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to assess the effects three electrospun fiber membranes on postoperative adhesion formation in rat surgical models., Materials and Methods: The PEG-PHBV (polyethylene glycol and poly-β-hydroxybutyrate valerate), PLA(Polylactic acid) and CS-PEG(chitosan and polyethylene glycol) were used for submicrometric membranes fabrication by electrospinning. A total of 64 SD male rats were randomly divided into 4 groups, one control group and 3 treatment groups, each consisting of 16 rats. The rats were underwent median laparotomy and standardized abrasion of the cecum. The indicated membranes were applied intraperitoneally at the end of the surgical procedure in the treatment groups, while the controls left without treatment. Two and four weeks after surgery respectively, a relaparotomy was made for adhesion grading and histopathological examination, Hydroxyproline (Hyp) levels at the adhesion sites in each group were measured., Results: The electrospun submicrometric membranes significantly reduced not only the incidence of adhesion but also the adhesion grading scales (p < 0.05 compared to the control group) at both two weeks and four weeks after the initial operation. Furthermore, the fibrosis grades were also higher in control groups as compared with the submicrometric membrane groups. Hyp levels in the control group was significant higher than the three experiment groups at both 2 and 4 weeks time points (p < 0.05). There were no statistical differences between the different treatment groups at either time points., Conclusions: Three electrospun submicrometric membranes were soft, flexible, easy to handle, and effectively reduced adhesion formation in rat modes.

3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than 5-fold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP) n domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser, and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines.

Purpose: To compare platelet rich plasma (PRP) and fibrin glue about the effect of anastomotic healing., Methods: Thirty six Wistar-Albino male rats diveded into 3 groups according to control(Group1), PRP (Group 2) and fibrin glue(Tisseel VH) (Group 3). The colon was transected with scissor and subsequently an end to end anastomosis was performed using continuous one layer 6/0 vicryl sutures. Postoperative 7th day effect of anastomotic healing measuring with tissue hydroxyproline(TH) level and anastomotic bursting pressure(ABP); moreover comparison of cytokine (IL-6 and IL-10) and procalcitonin levels on 1st,3rd and 7th days., Results: There was no statistically significant difference of the ABP and hydroxyproline levels between PRP and fibrin glue on the 7th day. There was no statistically significant difference between levels of proinflammatory cytokine (IL-6) (P=0.41), anti-inflammatory cytokine (IL-10) (P=0.35), and procalcitonin levels (P=0.63) on 1, 3 and 7 days., Conclusion: Fibrin glue and platelet rich plasma are shown to be effective in healing intestinal anastomoses without superior to each other.

Liver fibrosis is a major health concern worldwide. Inhibitors of Signal Transducer and Activator of Transcription 3 (STAT3) have been reported to attenuate experimental liver fibrosis. Therefore, the aim of this study was to investigate the potential ameliorative effect of cucurbitacin-B (Cucu-B) against CCl 4 -induced liver fibrosis in mice. Treatment with Cucu-B (5 mg/kg) preserved hepatocellular membrane integrity and amended the metabolic function as indicated by preventing the rise of serum liver function markers. This was confirmed histologically. CCl 4 -induced oxidative stress was improved by Cucu-B treatment (1 and 5 mg/kg). Furthermore, Cucu-B treatment ameliorated the fibrotic state as evidenced by inhibiting the rise of hydroxyproline liver content and mitigating the overexpressions of collagen-1α, α-smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) as well as the downexpression of matrix metalloproteinase-2 (MMP-2) mRNA. Importantly, STAT3 activity was inhibited by Cucu-B as confirmed by decreased phosphorylation of STAT3 without changing total STAT3 expression. This was substantiated by the reduced Bcl-2 together with increased Bax mRNA expressions with subsequent elevation of Bax/Bcl-2 ratio. In conclusion, Cucu-B hampers CCl 4 -induced liver fibrosis in mice. This can be attributed-at least partly-to inhibition of oxidative stress, inflammation and STAT3 signalling., (© 2017 John Wiley & Sons A/S.)

In this work, a novel core-shell magnetic molecularly imprinted polymers (MMIPs) for the measurement of L-Hydroxyproline (Hyp) in dairy products was prepared. The derivative of Hyp using N-hydroxysuccinimidyl rhodamine B ester (RBS) as derivatization reagent was employed as template to prepare RBS-Hyp-MMIPs (Fe 3 O 4 @MIPs for RBS-Hyp). A new analytical procedure of in situ derivatization with MMIPs (ISD-MMIPs) has been developed for the specific extraction and determination of Hyp in dairy products by ultra high performance liquid chromatography tandem mass spectrometry. The RBS-Hyp-MMIPs was characterized by fourier transform infrared spectrometer and transmission electron microscopy, and evaluated by adsorption experiments. The adsorption process followed Langumuir adsorption isotherm with maximum adsorption capacity of RBS-Hyp on RBS-Hyp-MMIPs at 96 mg/g. In addition, RBS-Hyp-MMIPs showed a short equilibrium time (15.0 min), rapid magnetic separation (5 s) and high stability (retained 95.3% after six cycles). Under the optimized conditions, good linearity was observed with the limits of detection (S/N > 3) and limits of quantification (S/N > 10) at 0.1 and 0.5 ng/mL, respectively. On account of the specific extraction performance of RBS-Hyp-MMIPs, not any interference peak from real sample matrix was observed in the chromatograms of milk powder, liquid milk and milk drink. The proposed procedure was successfully applied for selective determination of Hyp from dairy products with satisfactory validation results, which is of great significance to food safety., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This study investigates the effects of replacing dietary casein by soya protein on the underlying mechanisms involved in the impaired metabolic fate of glucose and lipid metabolisms in the heart of dyslipidaemic rats chronically fed (8 months) a sucrose-rich (62·5 %) diet (SRD). To test this hypothesis, Wistar rats were fed an SRD for 4 months. From months 4 to 8, half the animals continued with the SRD and the other half were fed an SRD in which casein was substituted by soya. The control group received a diet with maize starch as the carbohydrate source. Compared with the SRD-fed group, the following results were obtained. First, soya protein significantly (P<0·001) reduced the plasma NEFA levels and normalised dyslipidaemia and glucose homoeostasis, improving insulin resistance. The protein levels of fatty acid translocase at basal state and under insulin stimulation and the protein levels and activity of muscle-type carnitine palmitoyltransferase 1 were normalised. Second, a significant (P<0·001) reduction of TAG, long-chain acyl CoA and diacylglycerol levels was observed in the heart muscle. Third, soya protein significantly increased (P<0·01) GLUT4 protein level under insulin stimulation and normalised glucose phosphorylation and oxidation. A reduction of phosphorylated AMP protein kinase protein level was recorded without changes in uncoupling protein 2 and PPARα. Fourth, hydroxyproline concentration decreased in the left ventricle and hypertension was normalised. The new information provided shows the beneficial effects of soya protein upon the altered pathways of glucose and lipid metabolism in the heart muscle of this rat model.

Amino pyridine-based rhodamine conjugate (APR) has been developed as a first chemosensor for selective detection and quantification of L-4-Hydroxyproline (Hyp). The "turn-on" fluorescence property of the chemosensor makes it unique for easy estimation of Hyp in collagen and biological samples.

A method for the determination of hydroxyproline (Hyp) in liver tissue of mice by hydrophilic interaction chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry (HILIC-HRMS) was developed. The liver tissue samples of normal mice and liver fibrosis mice induced by carbon tetrachloride were hydrolyzed by concentrated hydrochloric acid. After filtrated and diluted by solution, the diluent was separated on an Hypersil GOLD HILIC column (100 mm×2.1 mm, 3 μm). Water-acetonitrile (28:72, v/v)were used as the mobile phases with isocratic elution. Finally, the target analytes were detected in positive model by HRMS equipped with an electrospray ionization source. The linear range of hydroxyproline was from 0.78 to 100.00 μg/L with the correlation coefficient ( R 2 ) of 0.9983. The limit of quantification was 0.78 μg/L. By detecting the spiked samples, the recoveries were in the range of 97.4%-100.9% with the relative standard deviations (RSDs) between 1.4% and 2.0%. In addition, comparison of the measurement results by this method and the chloramine T method was proceeded. It was found that the linear correlation between the two methods was very good, and the Pearson correlation coefficient was 0.927. And this method had simpler operation procedure and higher accuracy than chloramine T method. This method can be used for the quick determination of hydroxyproline in liver tissue samples.

Objective: To investigate the effects of carvedilol on inflammation, apoptosis, and hepatic fibrosis caused by biliary cirrhosis and its mechanisms in mice., Materials and Methods: 60 male C57/BL6 mice were randomly divided into sham-operation group (Sham group, n=20), biliary cirrhosis group (BDL group, n=20) and carvedilol group (CAR group, n=20). The CAR group was treated with gavage using 12.5 mg/kg carvedilol, once a day for 14 consecutive days, while the Sham group and BDL group were treated with gavage using the equivalent normal saline. After that, the mice in Sham group received the laparotomy under chloral hydrate anesthesia, followed by direct abdominal closure. The mice in BDL group and CAR group received the common bile duct ligation after anesthesia for modeling. After modeling, the survival rate of mice in each group was detected, and the blood and liver tissues were taken for detection. The morphological changes in liver tissues and apoptosis in mice in each group were detected and compared. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), superoxide dismutase (SOD), malondialdehyde (MDA), hydroxyproline, and α-smooth muscle actin (α-SMA) were also detected. The mRNA expression levels of pro-inflammatory factors, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), transforming growth factor β-1 (TGF-β1), α-SMA and collagen-1 were detected via reverse transcription-polymerase chain reaction (RT-PCR). The protein expression levels of CHOP (CCAAT-enhancer-binding protein homologous protein), activating transcription factor 4 (ATF4), ATF6, inositol-requiring enzyme 1 (IRE1), phosphorylated Jun N-terminal kinase (pJNK), α-SMA, and collagen-1, were detected via Western-blotting., Results: Our study showed that carvedilol could significantly alleviate the biliary cirrhosis in mice, and improve the survival rate of mice. The ALT, AST and TBIL levels, severity of cirrhosis, and number of apoptotic cells in CAR group were significantly lower than those in BDL group. The levels of α-SMA and hydroxyproline in CAR group were also significantly lower than those in BDL group. The activity of SOD in CAR group was significantly higher than that in BDL group; the above differences were statistically significant (p<0.05). In addition, it was also found that carvedilol could down-regulate the mRNA expression levels of iNOS, COX-2 and TGF-β1, down-regulate the mRNA and protein expression levels of α-SMA and collagen-1, and negatively regulate the ATF4-CHOP, ATF6-CHOP and IRE1-pJNK signaling pathways., Conclusions: Carvedilol has a significant effect on alleviating the biliary cirrhosis in mice, and its relevant mechanism may be that carvedilol inhibits the endoplasmic reticulum stress through the negative regulation of ATF4-CHOP, ATF6-CHOP and IRE1-pJNK signaling pathways, which needs to be confirmed by further in vitro experiments.

Background: Perioperative adjuncts are utilized across surgical specialities with the goal of improving patient outcomes. High-dose oxygen and extended warming are shown to increase wound collagen deposition during abdominal surgery. Prostacyclin is shown to improve limb salvage and patency rate in infrainguinal bypass (IIB) surgery. This study evaluated the impact of these adjuncts on healing and perfusion post IIB surgery., Methods: This randomized controlled study allocated patients undergoing IIB surgery into three treatment arms (perioperative high-dose oxygen, extended warming, and a synthetic prostacyclin) or a control group. The primary outcome was accumulation of hydroxyproline (OHP, collagen surrogate marker) as collected in polytetrafluoroethylene implants on day 5. Secondary outcomes included levels of growth factors and cytokines, and tissue oxygenation of the wound and foot as measured by hyperspectral technology and ankle-brachial pressure index. Clinical outcomes were observed to day 30, with long-term follow-up of 12 mo., Results: Seventy-one patients completed the study. Comparing treatment groups with the control at day 5, there were no differences in OHP, growth factors or cytokines levels, or improvement in tissue oxygenation at the surgical incision. However, there was more flow to the foot (HT-SUM (%) change) in the Ilomedin group compared to control (0% versus -14.6%, P = 0.045). HT-deoxy was higher at the peripheries in the oxygen and temperature groups, suggesting decreased tissue oxygenation., Conclusions: The perioperative treatments did not dramatically improve oxygenation or healing of the surgical wound in IIB surgery; however, Ilomedin may result in greater flow to the peripheries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Disc nucleus pulposus (NP) matrix homeostasis is important for normal disc function. Mechanical overloading seriously decreases matrix synthesis and increases matrix degradation. The present study aims to investigate the effects of resveratrol on disc NP matrix homeostasis under a relatively high-magnitude mechanical compression and the potential mechanism underlying this process. Porcine discs were perfusion-cultured and subjected to a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days in a mechanically active bioreactor. The non-compressed discs were used as controls. Resveratrol was added along with culture medium to observe the effects of resveratrol on NP matrix synthesis under mechanical load respectively. NP matrix synthesis was evaluated by histology, biochemical content (glycosaminoglycan (GAG) and hydroxyproline (HYP)), and expression of matrix macromolecules (aggrecan and collagen II). Results showed that this high-magnitude mechanical compression significantly decreased NP matrix content, indicated by the decreased staining intensity of Alcian Blue and biochemical content (GAG and HYP), and the down-regulated expression of NP matrix macromolecules (aggrecan and collagen II). Further analysis indicated that resveratrol partly stimulated NP matrix synthesis and increased activity of the PI3K/Akt pathway in a dose-dependent manner under mechanical compression. Together, resveratrol is beneficial for disc NP matrix synthesis under mechanical overloading, and the activation of the PI3K/Akt pathway may participate in this regulatory process. Resveratrol may be promising to regenerate mechanical overloading-induced disc degeneration., (© 2017 The Author(s).)