Hydroxyproline Analysis Research Articles

Hydroxyproline as a Marker of the Efficiency of Skin Wound Therapy in Ischemic Conditions

The aim of the study was to analyze the level of hydroxysoplin in tissues as a marker of healing efficiency of experimental skin wound under ischemia conditions. A wound was reproduced according to our developed method on 90 rats Vistar. There were 3 groups of rats of 30 individuals. Therapy was not carried out in the group «Control», local treatment with multi-component means was performed in the «First experimental» group, magnetotherapy was performed with the above-mentioned agent in the «Second experimental» group. The hydroxylol content was estimated based on Newman’s, Logan’s technique modified by Cissel. The results were compared with planimetric, visual and morphological data. The formation of scar was observed in each series on the 10th day, there was recorded the predominance of mature connective tissue over granulation in the experimental groups in contrast to the «Control» group. There was a strong correlation between the hydroxyprolin content and the wound area in all groups. The article marked the reliability of the method of quantitative analysis of hydroxyproline, which made it possible to increase the objectivity of the representation of the course of the wound process in conjunction with other methods of research.

407 The OGT/O-GlcNAc Axis Regulates Fibrosis Resolution in Idiopathic Pulmonary Fibrosis

OBJECTIVES/GOALS: Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by dysregulated collagen accumulation in the lung parenchyma. Our goal is to investigate the role of O-linked N-Acetylglucosamine (O-GlcNAc) transferase (OGT) in pulmonary fibrosis to ultimately discover novel therapies for fibrosis resolution. METHODS/STUDY POPULATION: Lung tissue from IPF and non-IPF donors was subjected to immunohistochemistry (IHC) to assess O-GlcNAc levels. Primary human lung fibroblasts were treated with OGT or O-GlcNAcase (OGA) inhibitors followed by transforming growth factor-beta 1 (TGF-β1) stimulation to assess O-GlcNAc regulation of fibroblast-to-myofibroblast transition (FMT) markers [alpha smooth muscle actin (α-SMA) and type 1 and type 3 collagen (COL1α1, COL3α1)] In Drosophila melanogaster, OGT knockdown (KD)/overexpression (OE) was conditionally induced to assess pericardin, a type IV collagen-like protein, regulation by immunofluorescence. Lastly, a mouse model of bleomycin-induced pulmonary fibrosis was examined following OGT KD and assessed for fibrosis resolution via histology, hydroxyproline assay, and western blotting. RESULTS/ANTICIPATED RESULTS: O-GlcNAc staining was increased in IPF lung tissue compared to non-IPF control lungs. In primary human lung fibroblasts, TGF-α1 administration resulted in increased FMT markers (α-SMA, COL1α1, and COL3α1), which were reduced or increased by OGT or OGA inhibition, respectively. Genetic manipulation in the Drosophila models showed decreased pericardin expression with OGT KD compared to the wild-type, whereas OGT OE increased pericardin compared to control. Additionally, OGT KD in bleomycin treated aged mice resulted in reduced collagen levels at the transcript and protein level and concurrent fibrosis resolution as assessed by Masson’s trichrome staining and total hydroxyproline analysis. Collectively, showing OGT/O-GlcNAc regulating collagen in fibrosis resolution. DISCUSSION/SIGNIFICANCE: These data suggest that the OGT/O-GlcNAc axis regulates collagen deposition in pulmonary fibrosis, and we show that O-GlcNAc is implicated in the pathogenesis of IPF. We identified OGT as a therapeutic target to overcome current drug limitations, opening new horizons for biomedical treatment.

Collagen-EGCG Combination Synergistically Prevents UVB-Induced Skin Photoaging in Nude Mice.

Ultraviolet (UV)radiation is a major cause of skin photoaging through generating excessive oxidative stress and inflammation. One of the strategies is to use photo-chemoprotectors, such as natural products with antioxidant and anti-inflammatory properties, to protect the skin from photo damage. The present study investigates the photoprotective potentials of topical administration of unhydrolyzed collagen, epigallocatechin gallate (EGCG), and their combination against ultraviolet B (UVB)-induced photoaging in nude mice. It is found that both the solo and combined pretreatments could recover UVB-induced depletion of antioxidative enzymes, including superoxide dismutase and glutathione peroxidase (GSH-Px), as well as an increase of lipid peroxide malondialdehyde and inflammatory tumor necrosis factor-α. Meanwhile, the UVB-stimulated skin collagen degradation is attenuated significantly with drug treatments, which is evidenced by expression analysis of matrix metalloproteinase-1 and hydroxyproline. Additionally, the mouse skin histology shows that the drug-pretreated groups possess decreased epidermis thickness and normal collagen fiber structure of the dermis layer. These results demonstrate that both EGCG and collagen can protect the skin against UVB-induced skin photoaging. Synergistically, the combination of them shows the maximum prevention to skin damage, showing its potential in the application of anti-photoaging formulation products.

Baicalin alleviates bleomycin-induced early pulmonary fibrosis in mice via the mitoKATP signaling pathway

Bleomycin (BLM), a frequently employed chemotherapeutic agent, exhibits restricted clinical utility owing to its pulmonary toxicity. Meanwhile, baicalin (BA)—an active ingredient extracted from the roots of Scutellaria baicalensis Georgi —has been shown to alleviate BLM-induced pulmonary fibrosis (PF). Hence, the objective of this study was to examine the protective effects of BA in the context of BLM-induced early PF in mice and elucidate the underlying mechanism(s). We established an in vivo BLM (3.5 mg/kg)-induced PF murine model and in vitro BLM (35 μM)-damaged MLE-12 cell model. On Day 14 of treatment, the levels of fibrosis and apoptosis were evaluated in mouse lungs via hydroxyproline analysis, western blotting (COL1A1, TGF-β, Bax, Bcl-2, cleaved caspase-3), and Masson, immunohistochemical (α-SMA, AIF, Cyto C), and TUNEL staining. Additionally, in vitro, apoptosis was assessed in MLE-12 cells exposed to BLM for 24 h using the Annexin V/PI assay and western blotting (Bax, Bcl-2, cleaved caspase-3, AIF, Cyto C). To elucidate the role of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the protective effect of BA, we utilised diazoxide (DZX)—a mitoKATP agonist—and 5-hydroxydecanoate sodium (5-HD)—a mitoKATP inhibitor. Results revealed the involvement of mitoKATP in the protective effect of BA in BLM-induced PF. More specifically, mitoKATP activation can attenuate BLM-induced PF progression and mitigate alveolar epithelial type II cell death by reducing mitochondrial ROS, maintaining the mitochondrial membrane potential, and impeding the mitochondrial apoptotic pathway. Collectively, the findings offer pharmacological support to use BA for the treatment or prevention of BLM-induced PF and suggest that mitoKATP might serve as an effective therapeutic target for this condition.

Effect of X-ray irradiation combined with PD-1 inhibitor treatment on lung tissue injury in mice

PurposeTo determine the effect of X-ray irradiation combined with PD-1 immune checkpoint inhibitor administration on lung tissue injury in a mouse model and its potential mechanism. MethodsIn all, 20 C57BL/6J mice were randomly divided into four groups with five mice in each group: control group, PD-1 inhibitor group, irradiation group, and irradiation combined with PD-1 inhibitor group. Hematoxylin-eosin staining of the lung tissue was performed 30 days after the end of irradiation to evaluate the morphological and pathological changes in the tissue. Masson staining and analysis of hydroxyproline were used to evaluate the degree of pulmonary fibrosis. The levels of transforming growth factor-β1 (TGF-β1) and tumor necrosis factor α(TNF-α) were evaluated by Enzyme-Linked immunosorbent assay (ELISA). CD3+, CD4+, and CD8+ T lymphocytes in the lung tissue were detected by immunohistochemistry. The expression levels of TGF-β1, Smad3, cGAS, and STING in the lung tissue were evaluated by Western blotting. ResultsThe lung injury scores and pulmonary fibrosis indices in the irradiation group were higher than those in the control group. Meanwhile, lung pneumonia score, pulmonary fibrosis index, percentage of CD4 cells and expression of TGF-β1, p-Smad3, and STING in the lung tissue of mice in irradiation combined with PD-1 inhibitor group were higher than those in the other three groups. ConclusionLung injury and pulmonary fibrosis were induced by whole chest X-ray irradiation in mice, and PD-1 inhibitor could aggravate lung injury and pulmonary fibrosis in mice. Thus, radiotherapy combined with PD-1 inhibitors may affect the immune inflammatory microenvironment in the lung tissues of mice by activating TGF-β1/Samd3 and cGAS/STING signaling pathways, thus aggravating lung tissue damage induced by radiation.

The longitudinal and regional analysis of bleomycin-induced pulmonary fibrosis in mice by microcomputed tomography

IntroductionMicrocomputed tomography (micro-CT) is powerful for assessment of the progression of lung fibrosis in animal model, but current whole lung analysis (WLA) methods are time-consuming. Here, a longitudinal and regional analysis (LRA) method was developed to assess fibrosis easily and quickly by micro-CT. MethodFirstly, we investigated the distribution pattern of lesions in BLM-induced pulmonary fibrosis mice. Then, the VOIs for LRA were selected based on the anatomical locations and we compared the robustness, accuracy, repeatability, analysis time of LRA to WLA. Additionally, LRA was applied to assess different stages of pulmonary fibrosis, and was validated with conventional endpoint measurements (such as lung hydroxyproline and histopathology). ResultsThe lesions of fibrosis in 66 bleomycin (BLM)-induced pulmonary fibrosis mice were mostly in the middle and upper parts of lungs. By applying LRA, the percentages of high-density voxels in selected volumes of interest (VOIs) were well correlated with that in WLA both at Day 7 and Day 21 after bleomycin induction (R2 = 0.8784 and 0.8464, respectively). The relative standard deviation (RSD) of the percentage of high-density voxels in the VOIs was lower than that of WLA (P < 0.05). The cost time of LRA was shorter than that of WLA (P < 0.05) and the accuracy of LRA was further confirmed by the histological analysis and biochemical quantification of hydroxyproline. ConclusionLRA is probably an easier and more time-saving method to assess fibrosis formation and evaluate treatment efficacy.

Irradiation Combined with PD-1 Inhibitor Aggravates Immune-Related Pneumonitis of the Non-Irradiated Lung in a Preclinical Model

<h3>Purpose/Objective(s)</h3> The combination of radiation with PD-1 inhibitors for lung cancer is a trend. However, the combination is limited by immune-related adverse effects, particularly checkpoint inhibitor pneumonitis (CIP). The incidence and the mechanism of CIP of the non-irradiated lung caused by radiation plus PD-1 inhibitors remain unknown. This study was designed to evaluate the incidence of CIP and lung tissue morphology in the preclinical model induced by radiation combined with a PD-1 inhibitor (αPD-1). <h3>Materials/Methods</h3> Fifty-four C57BL/6 mice were divided randomly into the normal group, the irradiation combined with the αPD-1 group (RT + αPD-1 group), and the single irradiation group (RT group). Mice in the RT + αPD-1 group were treated with right lung irradiation (8 Gy once) on day1 and anti-mouse PD-1 monoclonal antibody three times a week. Mice in the RT group received right lung irradiation alone. The mice were euthanized on day10, day15, and day20 after the end RT. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining. Lung fibrosis was evaluated by Masson staining and analysis of hydroxyproline. <h3>Results</h3> Histopathological examination revealed that the inflammation in the irradiated lung is more severe than the unirradiated site. With the extension of time, the lung inflammation of mice in the RT group and RT + αPD-1 group was significantly aggravated, and the whole lung fibrosis appeared on day20. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions, and hydroxyproline contents in lung tissues were all significantly higher in mice administered αPD-1 plus irradiation than in the other groups. In particular, the inflammation is significantly worse in the unirradiated site of the mice administered PD-1 inhibitor plus irradiation than in the RT group on day15 and day20. <h3>Conclusion</h3> Radiotherapy combined with PD-1 inhibitors could aggravate CIP on both sides. Further studies on the mechanism are under investigation. Our findings highlight the potential risk of CIP in non-irradiated lung in patients treated with radiotherapy and PD-1 inhibitors.

Human Occupation of the North American Colorado Plateau ∼37,000 Years Ago

Calibrating human population dispersals across Earth’s surface is fundamental to assessing rates and timing of anthropogenic impacts and distinguishing ecological phenomena influenced by humans from those that were not. Here, we describe the Hartley mammoth locality, which dates to 38,900–36,250 cal BP by AMS 14C analysis of hydroxyproline from bone collagen. We accept the standard view that elaborate stone technology of the Eurasian Upper Paleolithic was introduced into the Americas by arrival of the Native American clade ∼16,000 cal BP. It follows that if older cultural sites exist in the Americas, they might only be diagnosed using nuanced taphonomic approaches. We employed computed tomography (CT and μCT) and other state-of-the-art methods that had not previously been applied to investigating ancient American sites. This revealed multiple lines of taphonomic evidence suggesting that two mammoths were butchered using expedient lithic and bone technology, along with evidence diagnostic of controlled (domestic) fire. That this may be an ancient cultural site is corroborated by independent genetic evidence of two founding populations for humans in the Americas, which has already raised the possibility of a dispersal into the Americas by people of East Asian ancestry that preceded the Native American clade by millennia. The Hartley mammoth locality thus provides a new deep point of chronologic reference for occupation of the Americas and the attainment by humans of a near-global distribution.

Irradiation combined with PD-1 inhibitor aggravates immune-related pneumonitis of the non-irradiated lung in a preclinical model.

e14569 Background: The combination of radiation with PD-1 inhibitors for lung cancer is a trend. However, the combination is limited by immune-related adverse effects, particularly checkpoint inhibitor pneumonitis (CIP). The incidence and the mechanism of CIP of the non-irradiated lung caused by radiation plus PD-1 inhibitors remain unknown. This study was designed to evaluate the incidence of CIP and lung tissue morphology in the preclinical model induced by radiation combined with a PD-1 inhibitor (αPD-1). Methods: Fifty-four C57BL/6 mice were divided randomly into the normal group, the irradiation combined with the αPD-1 group (RT + αPD-1 group), and the single irradiation group (RT group). Mice in the RT + αPD-1 group were treated with right lung irradiation (8 Gy once) on day1 and anti-mouse PD-1 monoclonal antibody three times a week. Mice in the RT group received right lung irradiation alone. The mice were euthanized on day10, day15, and day20 after the end RT. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining. Lung fibrosis was evaluated by Masson staining and analysis of hydroxyproline. Results: Histopathological examination revealed that the inflammation in the irradiated lung is more severe than the unirradiated site. With the extension of time, the lung inflammation of mice in the RT group and RT + αPD-1 group was significantly aggravated, and the whole lung fibrosis appeared on day20. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions, and hydroxyproline contents in lung tissues were all significantly higher in mice administered αPD-1 plus irradiation than in the other groups. In particular, the inflammation is significantly worse in the unirradiated site of the mice administered PD-1 inhibitor plus irradiation than in the RT group on day15 and day20. Conclusions: Radiotherapy combined with PD-1 inhibitors could aggravate CIP on both sides. Further studies on the mechanism are under investigation. Our findings highlight the potential risk of CIP in non-irradiated lung in patients treated with radiotherapy and PD-1 inhibitors. Research Sponsor: This work was supported by the National Natural Science Foundation of China [No. 81972853, No. 81572279] and Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2019ZD009).

Coomassie brilliant blue G-250 dye attenuates bleomycin-induced lung fibrosis by regulating the NF-κB and NLRP3 crosstalk: A novel approach for filling an unmet medical need

Pulmonary fibrosis (PF) is a life-threatening disorder with a very poor prognosis. Because of the complexity of PF pathological mechanisms, filling such an unmet medical need is challenging. A number of pulmonary diseases have been linked to the activation of NF-κB and the NLRP3 inflammasome. Coomassie brilliant blue G-250 (CBBG) is proved to be a safe highly selective P2×7R antagonist with promising consequent inactivation of NLRP3 inflammasome. This is the first report to investigate the effect of CBBG on the bleomycin-induced lung fibrosis in rats. Our findings revealed that CBBG resulted in a significant improvement in histological features and oxidative status biomarkers of bleomycin-exposed lung tissue. Additionally, CBBG repressed collagen deposition as indicated after the analysis of hydroxyproline, TGF-β, PDGF-BB, TIMP-1, MMP-9, Col1a1, SMA and ICAM-1. It also exhibited anti-inflammatory potential as revealed by the determination of TNF-α, IL-1β, IL-18, MCP-1 in the lung tissue. In the bronchoalveolar lavage, the total protein and the LDH activity were substantially reduced. The lung protective effects of CBBG might be attributed on the one hand to the inhibition of NLRP3 inflammasome and on the other hand to the inactivation of NF-κB. Decreased levels of phospho-p65 and its DNA-binding activity as well as the analysis of TLR4 confirmed NF-κB inactivation. Caspase-1 activity is suppressed as a consequence of inhibiting NLRP3 inflammasome assembly. To conclude, CBBG may act as a primary or adjuvant therapy for the management of PF and therefore it may pose an opportunity for a novel approach to an unmet medical need.

Captopril inhibits matrix metalloproteinase activity and improves dentin bonding durability.

We investigated the inhibitory effects of captopril on matrix metalloproteinases (MMPs) and its effect as a primer on dentin bonding durability. One hundred fifty human third molars were selected. Flat surfaces of the middle dentin were exposed, etched 15s, and followed by pretreatment with a primer for 60s, including distilled water (control, the negative control primer), 2% chlorhexidine digluconate (CHD,the positive control primer), and captopril solution. Inhibitory effects of primers on MMPs were evaluated by hydroxyproline and gelatinase activity tests. All primers were applied on dentin followed by bonding. Some of the samples were sliced into slabs, placed in a fluorescent solution containing gelatin, and incubated for in situ zymography. Some were cut into sticks, and after aging for 1day, 12months, or 24months, microtensile bonding strength was tested. Some were cut into slabs, aged for 1day, 12months, or 24months, and taken out for nanoleakage tests to reveal interface defects. Hydroxyproline and gelatinase activity analyses showed that captopril exerted better inhibitory effects on MMPs, relative to 2% CHD (p < 0.05). A 0.2% captopril aqueous solution (0.2% CapW) was chosen to apply to the dentin. In situ zymography showed that inhibitory effects of captopril on gelatinase were significantly higher compared to 2% CHD (p < 0.01). Microtensile strength revealed that the bonding effects of the 0.2% CapW group lasted longer, compared to the control and 2% CHD groups (p < 0.05). Interface defects, detected by nanoleakage, were significantly reduced in the 0.2% CapW group, compared to the control and 2% CHD groups (p < 0.05). Captopril inhibits dentin MMP activities and effectively improves dentin bonding durability. Captopril is a promising dentin bonding primer for improving bonding durability.

Effect of PD-1 Inhibitor Combined with X-Ray Irradiation on the Inflammatory Microenvironment and Lung Tissue Injury in Mice.

PurposeThis study was designed to evaluate the effects of PD-1 inhibitor on lung tissue morphology and the immune system in a mouse model of radiation-induced lung injury (RILI) and to assess interactions between radiation therapy and PD-1 inhibition.MethodsTwenty C57BL/6 mice were divided randomly into four groups of five mice each. Mice were treated with an anti-mouse PD-1 monoclonal antibody, whole thorax irradiation, both or neither. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining; lung fibrosis was assessed by Masson staining and analysis of hydroxyproline; CD3+, CD4+, and CD8+ T lymphocytes in lung tissues were detected immunohistochemically; and the concentrations of transforming growth factor-β1 (TGF-β1) and interleukin-6 (IL-6) in lung tissue were evaluated by cytokine multiplex analysis.ResultsLung injury scores and indicators of pulmonary fibrosis were higher in mice administration whole thorax irradiation than in control mice. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions and hydroxyproline contents in lung tissues were all significantly higher in mice administered PD-1 inhibitor plus irradiation than in the other three groups. Similarly, the percentages of CD3+ and CD8+T cells and the concentrations of IL-6 and TGF-β1 in lung tissue were significantly higher in mice treated with radiation and PD-1 inhibitor than in the other groups. However, PD-1 inhibitor and irradiation interacted significantly only in the elevation of TGF-β1 level.ConclusionWhole thorax X-ray irradiation in mice can cause pulmonary injury and fibrosis, which could be exacerbated by PD-1 inhibitors. Radiotherapy combined with PD-1 inhibitors may aggravate RILI by synergistically upregulating TGF-β1 expression, thereby affecting the immune-inflammatory microenvironment in the lungs.

Analysis of a Urinary Biomarker Hydroxyproline for Clinical Assessment in Osteoporosis Patients

The various degradation products derived from bone collagen are pyridinoline, deoxypyridinoline, N- telopeptides, and hydroxyproline are excreted in the urine. Among that hydroxyproline is an important biomarker used to correlate the osteoporosis condition. A simple method of reversed phased ultra-fast liquid chromatography (RP UFLC) with a PDA detector. Hydroxyproline a UV inactive molecule is derivatized with Fluorenyl methyl chloroformate (FMOC) to form a UV active adduct. FMOC- Hydroxyproline adduct and Chlorthalidone as an internal standard were spiked to healthy adult urine and processed through strata Phenomenex C18 cartridges. Extracts from cartridges were collected and analysed on Phenomenex C18 column (250mm ×4.6mm i.d., 5µm particle size) as a stationary phase. The mobile phase was composed of acetonitrile and diethylamine (DEA) buffer pH 9.0 in the ratio 50:50v/v at a flow rate of 0.8 ml/min. Elutes were analyzed using a PDA detector at a detection wavelength of 265nm. The proposed method was validated as per US-FDA Guidance. In this study, the chromatographic peaks of hydroxyproline and chlorthalidone show better resolution with a retention time of 2.5 and 6.4 min respectively. Hydroxyproline shows excellent linearity with 0.9994 of the correlation coefficient (R2). The newly developed and validated bioanalytical method established is conveniently used for the clinical assessment of hydroxyproline in biological fluids & is used to quantify the biomarker concentration (hydroxyproline) in a urine sample and correlate to disease condition (osteoporosis).

Dopamine Synthesis and D1 Receptor Agonism are Essential for Resolution of Experimental Lung Fibrosis

Rationale Idiopathic Pulmonary fibrosis (IPF) is characterized by progressive and ultimately fatal end-stage lung scarring associated with uncontrolled deposition, stiffening and remodeling of fibrous connective tissue proteins. We have previously shown that agonism of the dopamine receptor D1 attenuates experimental lung fibrosis in mice, and that the lungs of IPF patients display reduced transcript and protein levels of DOPA decarboxylase (DDC), the enzyme that catalyzes the biosynthesis of dopamine. Here we investigate the role of endogenous dopamine production and signaling in the resolution of experimental lung fibrosis. Methods Using a self-resolving model of experimental lung fibrosis, we administered bleomycin (1.1U/kg) to 10-week old mice intratracheally at day 0, and collected lung tissue from sham and bleo groups at day 21 to establish the level of baseline fibrosis. We then administered the DDC inhibitor carbidopa (20mg/kg, IP), the D1 receptor antagonist SCH 39166 (0.5mg/kg, SC), or vehicle control daily from day 21 to 42. Lung tissue was collected for hydroxyproline and histological analysis. In order to investigate dopamine synthesis and signaling ex vivo, we generated precision-cut lung slices (PCLS) from healthy mice and stimulated them +/- TGFβ for 72 hours and collected total RNA from the lung slices and conditioned media to measure dopamine production by ELISA. Results As previously observed, lung tissue collected at day 21 post injury displayed enhanced markers of fibrosis measured by hydroxyproline (478% increase over sham) and H&E stained histology. By day 42 the vehicle control mice resolved this fibrosis. In contrast, the mice treated with carbidopa or SCH 39166 maintained the fibrosis observed at day 21 (514% and 592% increase compared to sham, respectively). Consistent with our prior observation in IPF patient lung, PCLS cultured ex vivo with TGFβ expressed significantly less Ddc (75% reduction compared to control) and total dopamine abundance in the conditioned media dropped from ~120ng/mL to ~32ng/mL. Conclusions Dopamine synthesis through the activity of DDC, and agonism of the dopamine receptor D1 are essential to pulmonary fibrosis resolution after injury. PCLS represents a useful model to further investigate the molecular mechanisms leading to reduced expression of DDC, and test pharmacological approaches to stimulate dopamine signaling as a potential therapy for IPF.

Alveolar epithelial TET2 is not involved in the development of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology with minimal treatment options. Repetitive alveolar epithelial injury has been suggested as one of the causative mechanisms of this disease. Type 2 alveolar epithelial cells (AEC2) play a crucial role during fibrosis by functioning as stem cells able to repair epithelial damage. The DNA demethylase Tet methylcytosine dioxygenase 2 (TET2) regulates the stemness of multiple types of stem cells, but whether it also affects the stemness of AEC2 during fibrosis remains elusive. To study the role of TET2 in AEC2 during fibrosis, we first determined TET2 protein levels in the lungs of IPF patients and compared TET2 expression in AEC2 of IPF patients and controls using publicly available data sets. Subsequently, pulmonary fibrosis was induced by the intranasal administration of bleomycin to wild-type and AEC2-specific TET2 knockout mice to determine the role of TET2 in vivo. Fibrosis was assessed by hydroxyproline analysis and fibrotic gene expression. Additionally, macrophage recruitment and activation, and epithelial injury were analyzed. TET2 protein levels and gene expression were downregulated in IPF lungs and AEC2, respectively. Bleomycin inoculation induced a robust fibrotic response as indicated by increased hydroxyproline levels and increased expression of pro-fibrotic genes. Additionally, increased macrophage recruitment and both M1 and M2 activation were observed. None of these parameters were, however, affected by AEC2-specific TET2 deficiency. TET2 expression is reduced in IPF, but the absence of TET2 in AEC2 cells does not affect the development of bleomycin-induced pulmonary fibrosis.

Evaluation of urinary hydroxyproline and creatinine level in patients with benign mandibular odontogenic tumor.

BackgroundOdontogenic tumors are relatively common oro‐facial tumors seen in our environment with challenges encountered with management in terms of inadequate infrastructure and high cost of treatment. They are often associated with bone resorption with concomitant collagen degradation and excretion of their by‐products in serum or urine. The aim of this present study was to evaluate urinary hydroxyproline level in patients with benign mandibular odontogenic tumors.Materials and methodsTwenty‐two consecutive patients with histologically diagnosed mandibular odontogenic tumors were recruited. Twenty‐two controls who matched the study group for sex and age were also recruited. The study group had CT‐Scan of their lesions done. All participants were required to fast 12 hours overnight and their early morning second void urine collected between 7 a.m and 8 a.m. The collected urine samples were stored frozen at −20°C until analysis. Colorimetric method of analysis of urinary hydroxyproline and creatinine were done using Biovision hydroxyproline kit and Randox creatinine kit, respectively. The results were recorded as urinary hydroxyproline alone (μg/μl) and as urinary hydroxyproline/creatinine ratio.ResultsThe mean age of the participants was 28.45 ± 6.8 years. The mean duration of the tumors in the study group was 5.9 ± 4.4 years. A mean urinary hydroxyproline/ creatinine ratio of 0.081 ± 0.129 was noted in the study group as compared to 0.016 ± 0.006 that was noted among healthy Nigerian who served as controls in the study.ConclusionThere was a significant increase in urinary hydroxyproline level in patients with odontogenic tumors when compared with healthy Nigerians.

Sensitive Electrochemical Analysis of Hydroxyproline in Achilles Tendon Collagen and Human Urine

L-hydroxyproline (Hyp) is one of the significant amino acids present in connective tissue proteins such as collagen, elastin, and gelatin. The quantitative analysis of Hyp levels in bodily fluids is critical to assist with diagnosing diseases and early treatments. In the present study, for the first time, we report on a facile electrochemical method for the detection of Hyp using gold nanoparticles (AuNPs), which were electrochemically deposited on a glassy carbon electrode (GCE). The electrochemical behavior of the AuNPs/GCE for the oxidation of Hyp was examined using cyclic voltammetry, demonstrating higher electrocatalytic activity in contrast to GCE and bulk Au electrodes. Additionally, the mechanism for the electrochemical oxidation of Hyp was investigated using in situ Fourier transform infrared (FT-IR) spectroscopy. Moreover, the electrochemical sensing performance of the AuNPs was investigated using differential pulse voltammetry (DPV), exhibiting a low limit of detection (0.026 mM) and high sensitivity (8.5 μA (mM cm2)−1). The interference of other amino acids present in collagen and urine has been further tested, demonstrating high selectivity and good reproducibility. The novel electrochemical sensing approach described in the present study may lead to a facile non-enzymatic technique for the sensitive detection of Hyp, a significant biomarker.

A Preliminary estimation of baseline urinary hydroxyproline/creatinine levels in a study population of healthy Nigerians

Background: Hydroxyproline is one of the biochemical markers that can be measured objectively as an indicator of normal biological processes or pathological processes. It is usually raised in disease conditions that are associated with bone resorption. Aim: To determine the urinary hydroxyproline/creatinine levels in a study population of healthy Nigerians. Methods: This study recruited 22 consenting participants who served as control for another study at the University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla, Enugu. All participants were required to fast for at least 12 hours overnight and their early morning second void urine collected between 7am and 8am. The collected urine samples were stored frozen at -20oC until analysis. Colorimetric method of analysis of urinary hydroxyproline and creatinine were done using Biovisionhydroxyproline kit and Randoxcreatinine kit respectively. Bivariate analysis was conducted on the collated data using statistical package of social science (SPSS) version 19. The results were recorded as urinary hydroxyproline alone (μg/μL) and as urinary hydroxyproline/creatinine ratio. Results: The mean urinary hydroxyproline level of 0.020±0.013μg/μL and urinary hydroxyproline/ creatinine ratio of 0.016±0.006 were noted for healthy Nigerians. Conclusions: The urinary hydroxyproline levels in the study population of healthy Nigerians are within normal values reported in other healthy populations.

The importance of collagen composition and biomechanics for the porcine aortic root

A thorough understanding of the aortic root structure and biomechanics is necessary when performing aortic valve-sparing procedures in patients with aortic root aneurysms. This study aimed to evaluate the amount of collagen and biomechanics at different levels and segments of the aortic root. Ten aortic roots from healthy pigs were excised including the aortic annulus, the sinuses of Valsalva, and the sinotubular junction (STJ). Specimens were further divided into three circumferential segments; left coronary (LC)-, right coronary (RC)-, and non-coronary (NC) sinus. Collagen was determined using hydroxyproline analysis and specimens were tested biomechanically for stress–strain relations. The annulus showed significantly larger average maximum stiffness (9.6 ± 4.5 N/mm) compared with the sinus (4.5 ± 2.0 N/mm) and STJ (4.8 ± 1.8 N/mm). The average collagen content was likewise higher in the annulus (4.0 ± 1.0 mg/ml) compared with the sinus (2.4 ± 0.6 mg/ml) and STJ (2.2 ± 0.5 mg/ml) for all three segments. The NC sinus segment exhibited a significantly larger maximum stiffness and stress under static conditions compared with the RC. These results suggest that the aortic root is heterogeneous in both structure and biomechanical properties and that it varies both in levels and segments of the aortic root. Future surgical approaches should consider enhanced strength parameters for specific areas of the aortic root to achieve the best results when performing aortic valve-sparing techniques. From this study, we conclude that the aortic annulus needs special attention to imitate normal physiologic properties during aortic valve-sparing surgery due to its higher maximum stiffness, stress, and load. Modified future surgical procedures could potentially prevent recurrent aneurysmal formation.

Long-Term Effects With Potential Clinical Importance of Botulinum Toxin Type-A on Mechanics of Muscles Exposed.

Botulinum toxin type-A (BTX-A) is widely used for spasticity management and mechanically aims at reducing passive resistance at the joint and widening joint range of movement. However, recent experiments on acute BTX-A effects showed that the injected rat tibialis anterior (TA) muscle’s passive forces increased, and the length range of active force exertion (lrange) did not change. Additionally, BTX-A was shown to spread into non-injected muscles in the compartment and affect their mechanics. Whether those effects persist in the long term is highly important, but unknown. The aim was to test the following hypotheses with experiments conducted in the anterior crural compartment of the rat: In the long term, BTX-A (1) maintains lrange, (2) increases passive forces of the injected TA muscle, and (3) spreads into non-injected extensor digitorum longus (EDL) and the extensor hallucis longus (EHL) muscles, also affecting their active and passive forces. Male Wistar rats were divided into two groups: BTX-A and Control (0.1 units of BTX-A or only saline was injected into the TA). Isometric forces of the muscles were measured simultaneously 1-month post-injection. The targeted TA was lengthened, whereas the non-targeted EDL and EHL were kept at constant length. Hydroxyproline analysis was done to quantify changes in the collagen content of studied muscles. Two-way ANOVA test (for muscle forces, factors: TA length and animal group) and unpaired t or Mann-Whitney U test (for lrange and collagen content, where appropriate) were used for statistical analyses (P < 0.05). BTX-A caused significant effects. TA: active forces decreased (maximally by 75.2% at short and minimally by 48.3%, at long muscle lengths), lrange decreased (by 22.9%), passive forces increased (by 12.3%), and collagen content increased (approximately threefold). EDL and EHL: active forces decreased (up to 66.8%), passive force increased (minimally by 62.5%), and collagen content increased (approximately twofold). Therefore, hypothesis 1 was rejected and 2 and 3 were confirmed indicating that previously reported acute BTX-A effects persist and advance in the long term. A narrower lrange and an elevated passive resistance of the targeted muscle are unintended mechanical effects, whereas spread of BTX-A into other compartmental muscles indicates the presence of uncontrolled mechanical effects.