Injectable antibacterial conductive hydrogels with dual response to an electric field and pH for localized “smart” drug release

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Jin Qu, Xin Zhao, Peter X. Ma, Baolin Guo
Injectable hydrogels with multistimuli responsiveness to electrical field and pH as a drug delivery system have been rarely reported. Herein, we developed a series of injectable conductive hydrogels as "smart" drug carrier with the properties of electro-responsiveness, pH-sensitivity, and inherent antibacterial activity. The hydrogels were prepared by mixing chitosan-graft-polyaniline (CP) copolymer and oxidized dextran (OD) as a cross-linker. The chemical structures, morphologies, electrochemical property, swelling ratio, conductivity, rheological property, in vitro and in vivo biodegradation, and gelation time of hydrogels were characterized. The pH-responsive behavior was verified by drug release from hydrogels in PBS solutions with different pH values (pH = 7.4 or 5.5) in an in vitro model. As drug carriers with electric-driven release, the release rate of the model drugs amoxicillin and ibuprofen loaded within CP/OD hydrogels dramatically increased when an increase in voltage was applied. Both chitosan and polyaniline with inherent antibacterial properties endowed the hydrogels with excellent antibacterial properties. Furthermore, cytotoxicity tests of the hydrogels using L929 cells confirmed their good cytocompatibility. The in vivo biocompatibility of the hydrogels was verified by H&E staining. Together, all these results suggest that these injectable pH-sensitive conductive hydrogels with antibacterial activity could be ideal candidates as smart drug delivery vehicles for precise doses of medicine to meet practical demand.Statement of SignificanceStimuli-responsive or "smart"hydrogels have caused great attention in the field of biotechnology and biomedicine, especially on designing novel drug delivery systems. Compared with traditional implantable electronic delivery devices, the injectable hydrogels with electrical stimuli not only are easy to generate and control electrical field, but also they could avoid frequent invasive surgeries which offer a new avenue for chronic diseases. In addition, designing a drug carrier with pH-sensitive property could release drug efficiently in targeted acid environment and it could reinforce the precise doses of medicine. Furthermore, caused by opportunistic microorganisms and rapid spread of antibiotic-resistant microbes, infection is still a serious threat for many clinical utilities. To overcome these barriers, we designed a series of injectable antibacterial conductive hydrogels based on chitosan-graft-polyaniline (CP) copolymer and oxidized dextran (OD), and demonstrated their potential as "smart" delivery vehicles with electric field and pH-responsive properties for triggered and localized release of drugs.

Graphical abstract

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Crimped elastomer scaffolds prepared through MEW with non-linear extension behaviour mimicking that of ligaments and tendons

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Gernot Hochleitner, Fei Chen, Carina Blum, Paul D. Dalton, Brian Amsden, Jürgen Groll
Ligaments and tendons are comprised of aligned, crimped collagen fibrils that provide tissue-specific mechanical properties with non-linear extension behaviour, exhibiting low stress at initial strain (toe region behaviour). To approximate this behaviour, we report fibrous scaffolds with sinusoidal patterns by melt electrowriting (MEW) below the critical translation speed (CTS) by exploitation of the natural flow behaviour of the polymer melt. More specifically, we synthesised photopolymerizable poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (p(LLA-co-ε-CL-co-AC)) and poly(ε-caprolactone-co-acryloyl carbonate) (p(ε-CL-co-AC)) by ring-opening polymerization (ROP). Single fibre (fØ=26.8±1.9µm) tensile testing revealed a customisable toe region with Young's Moduli ranging from E=29±17MPa for the most crimped structures to E=314±157MPa for straight fibres. This toe region extended to scaffolds containing multiple fibres, while the sinusoidal pattern could be influenced by printing speed. The synthesized polymers were cytocompatible and exhibited a tensile strength of σ=26±7MPa after 10⁴ cycles of preloading at 10% strain while retaining the distinct toe region commonly observed in native ligaments and tendon tissue.Statement of SignificanceDamaged tendons and ligaments are serious and frequently occurring injuries worldwide. Recent therapies, including autologous grafts, still have severe disadvantages leading to a demand for synthetic alternatives. Materials envisioned to induce tendon and ligament regeneration should be degradable, cytocompatible and mimic the ultrastructural and mechanical properties of the native tissue. Specifically, we utilised photo-cross-linkable polymers for additive manufacturing (AM) with Melt Electrowriting (MEW). In this way, we were able to direct-write cytocompatible fibres of a few micrometres thickness into crimp-structured elastomer scaffolds that mimic the non-linear biomechanical behaviour of tendon and ligament tissue.

Graphical abstract

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Long-term in vivo evolution of high-purity Mg screw degradation — Local and systemic effects of Mg degradation products

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Yiqiang Yu, Hua Lu, Jiao Sun
Magnesium (Mg) based materials are the focus of research for use as degradable materials in orthopedics and cranio-maxillofacial surgery. However, corrosion rate control and biosecurity are still the key issues that need to be solved prior to their clinical applications. In the present study, as-rolled high-purity magnesium (HP Mg, 99.99 wt%) screws were implanted in rabbit tibiae for up to 52 weeks in order to investigate their long-term in vivo degradation and the local and systemic effects of their degradation products. A series of long-term monitoring were performed at various time points (4w, 12w, 26w and 52w) after implantation using numerous investigations such as micro-CT assay, histomorphometric analysis, local micro-environment testing and biochemical analysis of serum and urine. It was revealed that HP Mg screws had a uniform degradation morphology and a slow degradation rate in vivo during the period of 52 weeks. Their degradation products not only increased the local pH values but also changed the local Mg²⁺ ions concentration and gas cavity area in the peri-implant tissues in a dynamic manner. More importantly, both the new bone formation and bone-implant contact rate were increased at bone-implant interfaces at 26 weeks and 52 weeks post-implantation. Furthermore, neither abnormal elevation of serum magnesium and urine magnesium level, nor liver and kidney dysfunction were detected during the monitoring period of 26 weeks. All these results of long-term investigation suggest that HP Mg screws possess a slow degradation rate, desirable bone repair capacity and long-term local/systemic biosafety, and consequently may have good potential for application as bone fixation devices.Statement of significanceThe corrosion resistance control and biosecurity issues of Mg alloys limited their clinical applications in some extent. Mg purification is another effective way to improve corrosion resistance of Mg-based materials. However, the long-term in vivo degradation of high-purity magnesium (HP Mg) and the local and systemic effects of its degradation products have not been fully investigated yet, which are the key factors to determine the clinical application prospect of HP Mg. Especially the changes in peri-implant microenvironment may greatly influence the local physiological response and bone repair. In this study, the long-term evolution tendency of in vivo degradation behavior of HP Mg screws was discovered from the view of space-time. Furthermore, not only the dynamic changes of local microenvironment and the long-term evolution process of bone repair, but also the dynamic systemic responses were systematically revealed. Conclusions of this study may help us to further understand the long-term in vivo evolution of HP Mg degradation and the local/systemic effects of its degradation products and help to guide the design of biodegradable bone fixation material.

Graphical abstract

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Development of tannin-inspired antimicrobial bioadhesives

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Jinshan Guo, Wei Sun, Jimin Peter Kim, Xili Lu, Qiyao Li, Min Lin, Oliver Mrowczynski, Elias B. Rizk, Juange Cheng, Guoying Qian, Jian Yang
Tissue adhesives play an important role in surgery to close wounds, seal tissues, and stop bleeding, but existing adhesives are costly, cytotoxic, or bond weakly to tissue. Inspired by the water-resistant adhesion of plant-derived tannins, we herein report a new family of bioadhesives derived from a facile, one-step Michael addition of tannic acid and gelatin under oxidizing conditions and crosslinked by silver nitrate. The oxidized polyphenol groups of tannic acid enable wet tissue adhesion through catecholamine-like chemistry, while both tannic acid and silver nanoparticles reduced from silver nitrate provide antimicrobial sources inherent within the polymeric network. These tannin-inspired gelatin bioadhesives are low-cost and readily scalable and eliminate the concerns of potential neurological effect brought by mussel-inspired strategy due to the inclusion of dopamine; variations in gelatin source (fish, bovine, or porcine) and monomer feeding ratios resulted in tunable gelation times (36 s to 8 min), controllable degradation (up to 100% degradation within a month), considerable wet tissue adhesion strengths (up to 3.7 times to that of fibrin glue), excellent cytocompatibility, as well as antibacterial and antifungal properties. The innate properties of tannic acid as a natural phenolic crosslinker, molecular glue, and antimicrobial agent warrant a unique and significant approach to bioadhesive design.Statement of SignificanceThis manuscript describes the development of a new family of tannin-inspired antimicrobial bioadhesives derived from a facile, one-step Michael addition of tannic acid and gelatin under oxidizing conditions and crosslinked by silver nitrate. Our strategy is new and can be easily extended to other polymer systems, low-cost and readily scalable, and eliminate the concerns of potential neurological effect brought by mussel-inspired strategy due to the inclusion of dopamine.The tannin-inspired gelatin bioadhesives hold great promise for a number of applications in wound closure, tissue sealant, hemostasis, antimicrobial and cell/drug delivery, and would be interested to the readers from biomaterials, tissue engineering, and drug delivery area.

Graphical abstract

http://ift.tt/2FLqyD6

Enhancing anti-tumor efficiency in hepatocellular carcinoma through the autophagy inhibition by miR-375/sorafenib in lipid-coated calcium carbonate nanoparticles

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Pengxuan Zhao, Minsi Li, Yao Wang, Yan Chen, Chuanchuan He, Xiaojuan Zhang, Tan Yang, Yao Lu, Jia You, Robert J. Lee, Guangya Xiang
Sorafenib is a first-line drug for hepatocellular carcinoma (HCC). Autophagy has been shown to facilitate sorafenib resistance. miR-375 has been shown to be an inhibitor of autophagy. In this study, miR-375 and sorafenib were co-loaded into calcium carbonate nanoparticles with lipid coating (miR-375/Sf-LCC NPs). The nanoparticles had high loading efficiency and were ∼ 50 nm in diameter. Besides, the NPs could increase the stability and residence time of both drugs. Moreover, we demonstrated that autophagy was activated in HCC cells by sorafenib but not by miR-375/Sf-LCC NPs. In vitro, miR-375/Sf-LCC NPs exhibited pH-dependent drug release and potent cytotoxicity. In vivo, miR-375/Sf-LCC NPs increased miR-375 and sorafenib uptake in tumor (2 folds compared with Lipofectamine 2000-miR-375 and 2-5 folds compared with free sorafenib). Furthermore, miR-375/Sf-LCC NPs showed greatly enhanced therapeutic efficacy in an HCC xenograft model. These findings suggest that miR-375/Sf-LCC NPs may be a promising agent for the HCC therapy.Statement of SignificanceHepatocellular carcinoma (HCC) is the most common primary liver tumor and the third leading cause of cancer mortality globally. In this manuscript, miR-375 and sorafenib were co-loaded into calcium carbonate nanoparticles with lipid coating (miR-375/Sf-LCC NPs) to treat HCC. We demonstrated that miR-375/Sf-LCC NPs can deliver sorafenib and miR-375 into HCC cells and tumor tissues, increase drug retention time in tumor, significantly inhibit autophagy and produce enhanced anti-tumor effect.

Graphical abstract

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Photooxidatively crosslinked acellular tumor extracellular matrices as potential tumor engineering scaffolds

Publication date: Available online 17 March 2018
Source:Acta Biomaterialia
Author(s): Wei-Dong Lü, Rui-Fang Sun, Ye-Rong Hu, Jan-Rong Lu, Lu Gu, Zhi-Gang Liu, Guang-Yan Lei, Zhun Qiang, Lin Cai
Acellular tumor extracellular matrices (ECMs) have limitations when employed as three-dimensional scaffolds for tumor engineering. In this work, methylene blue-mediated photooxidation was used to crosslink acellular tumor ECMs. Photooxidative crosslinking greatly increased the stiffness of acellular tumor ECM scaffolds but barely altered the Amide III band of the secondary structure of polypeptides and proteins. MCF-7, HepG2 and A549 cells cultured on photooxidatively crosslinked acellular tumor ECM scaffolds exhibited greater cell number per scaffold, more IL-8 and VEGF secretion, and increase migration and invasion abilities than cells cultured on uncrosslinked acellular tumor ECM scaffolds. The three tumor cell lines cultured on the stiffer photooxidatively crosslinked acellular matrices acquire mesenchymal properties (mesenchymal shift) and dedifferentiated phenotypes. Furthermore, the malignant phenotypes induced in vitro when cultured on the crosslinked scaffold promoted the in vivo tumor growth of BALB/c nude mice. Finally, the dedifferentiated cancer cells, including MCF-7, HepG2 and A549 cells, were less sensitive to chemotherapeutics. Thus, photooxidatively crosslinked acellular tumor ECMs have potentials as three-dimensional (3D) tumor engineering scaffolds for cancer research.Statement of SignificanceNatural material scaffolds have been successfully used as 3D matrices to study the in vitro tumor cell growth and mimic the in vivo tumor microenvironment. Acellular tumor extracellular matrices (ECMs) are developed as 3D scaffolds for tumor engineering but have limitations in terms of elastic modulus and cell spheroid formation. Here we use methylene blue-mediated photooxidation to crosslink acellular tumor ECMs and investigate the influence of photooxidative crosslinking on structural, mechanical and biological characteristics of acellular tumor ECM scaffolds. It is the first study to evaluate the feasibility of photooxidatively crosslinked acellular tumor ECMs as 3D scaffolds for cancer research and the results are encouraging. Moreover, this study provides new research areas in regard to photodynamic therapy (PDT) for Cancer.

Graphical abstract

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Effects of Tunable, 3D-Bioprinted Hydrogels on Human Brown Adipocyte Behavior and Metabolic Function

Publication date: Available online 16 March 2018
Source:Acta Biomaterialia
Author(s): Mitchell Kuss, Jiyoung Kim, Dianjun Qi, Shaohua Wu, Yuguo Lei, Soonkyu Chung, Bin Duan
Obesity and its related health complications cause billions of dollars in healthcare costs annually in the United States, and there are yet to be safe and long-lasting anti-obesity approaches. Using brown adipose tissue (BAT) is a promising approach, as it uses fats for energy expenditure. However, the effect of the microenvironment on human thermogenic brown adipogenesis and how to generate clinically relevant sized and functioning BAT are still unknown. In our current study, we evaluated the effects of endothelial growth medium exposure on brown adipogenesis of human brown adipose progenitors (BAP). We found that pre-exposing BAP to angiogenic factors promoted brown adipogenic differentiation and metabolic activity. We further 3D bioprinted brown and white adipose progenitors within hydrogel-based bioink with controllable physicochemical properties and evaluated the cell responses in 3D bioprinted environments. We used soft, stiff, and stiff-porous constructs to encapsulate the cells. All three types had high cell viability and allowed for varying levels of function for both white and brown adipocytes. We found that the soft hydrogel constructs promoted white adipogenesis, while the stiff-porous hydrogel constructs improved both white and brown adipogenesis and were the optimal condition for promoting brown adipogenesis. Consistently, stiff-porous hydrogel constructs showed higher metabolic activities than stiff hydrogel constructs, as assessed by 2-deoxy glucose uptake (2-DOG) and oxygen consumption rate (OCR). These findings show that the physicochemical environments affect the brown adipogenesis and metabolic function, and further tuning will be able to optimize their functions. Our results also demonstrate that 3D bioprinting of brown adipose tissues with clinically relevant size and metabolic activity has the potential to be a viable option in the treatment of obesity and type 2 diabetes.Statement of SignificanceOne promising strategy for the treatment or prevention of obesity-mediated health complications is augmenting brown adipose tissues (BAT), which is a specialized fat that actively dissipate energy in the form of heat and maintain energy balance. In this study, we determined how pre-exposing human brown adipose progenitors (BAP) to angiogenic factors in 2D and how bioprinted microenvironments in 3D affected brown adipogenic differentiation and metabolic activity. We demonstrated that white and brown adipogenesis, and thermogenesis were regulated by tuning the bioprintable matrix stiffness and construct structure. This study not only unveils the interaction between BAP and 3D physiological microenvironments, but also presents a novel tissue engineered strategy to manage obesity and other related metabolic disorders.

Graphical abstract

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Genipin cross-linked type II collagen/chondroitin sulfate composite hydrogel-like cell delivery system induces differentiation of adipose-derived stem cells and regenerates degenerated nucleus pulposus

Publication date: Available online 16 March 2018
Source:Acta Biomaterialia
Author(s): Xiaopeng Zhou, Jingkai Wang, Weijing Fang, Yiqing Tao, Tengfei Zhao, Kaishun Xia, Chengzhen Liang, Jianming Hua, Fangcai Li, Qixin Chen
Nucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent lower back pain. Although adipose-derived stem cell (ADSC)-based therapy is regarded to be promising for the treatment of degenerated NP, there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSC (CCSA) delivery system with genipin as the cross-linking agent. The induction effect of the scaffold on ADSC differentiation was studied in vitro, and a rat coccygeal vertebrae degeneration model was used to investigate the regenerative effect of the CCSA system on the degenerated NP in vivo. The results showed that the CCSA delivery system cross-linked with 0.02% genipin was biocompatible and promoted the expressions of NP-specific genes. After the injection of the CCSA system, the disc height, water content, extracellular matrix synthesis, and structure of the degenerated NP were partly restored. Our CCSA delivery system uses minimally invasive approaches to promote the regeneration of degenerated NP and provides an exciting new avenue for the treatment of degenerative disc disease.Statement of significanceNucleus pulposus (NP) degeneration is usually the origin of intervertebral disc degeneration and consequent low back pain. Stem cell-based tissue engineering is a promising method in NP regeneration, but there is a lack of viable cell carriers to transplant ADSCs into the NP while maintaining cell function. In this study, we developed a type II collagen/chondroitin sulfate (CS) composite hydrogel-like ADSCs (CCSA) delivery system with genipin cross-linking. Although several research groups have studied the fabrication of injectable hydrogel with biological matrix, our study differs other works. We chose type II collagen and CS, the two primary native components in the NP, as the main materials and combined them according to the natural ratio of collagen and sGAG in the NP. The delivery system is pre-loaded with ADSCs and can be injected into the NP with needle, followed by in situ gelation. Genipin is used as a cross-linker to improve the bio-stability of scaffold, with low cytotoxicity. We investigated the stimulatory effects of our scaffold on ADSCs differentiation in vitro and the regenerative effect of the CCSA delivery system on degenerated NP in vivo.

Graphical abstract

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Measuring zinc in biological nanovesicles by multiple analytical approaches

Publication date: July 2018
Source:Journal of Trace Elements in Medicine and Biology, Volume 48
Author(s): Francesco Piacenza, Antje Biesemeier, Marco Farina, Francesco Piva, Xin Jin, Eleonora Pavoni, Lorenzo Nisi, Maurizio Cardelli, Laura Costarelli, Robertina Giacconi, Andrea Basso, Elisa Pierpaoli, Mauro Provinciali, James C.M. Hwang, Antonio Morini, Andrea di Donato, Marco Malavolta
Exosomes are nanovesicles known to mediate intercellular communication. Although it is established that zinc ions can act as intracellular signaling factors, the measurement of zinc in circulating nanovesicles has not yet been attempted. Providing evidence of the existence of this zinc fraction and methods for its measurement might be important to advance our knowledge of zinc status and its relevance in diseases.Exosomes from 0.5 ml of either fresh or frozen human plasma were isolated by differential centrifugation. A morphological and dimensional evaluation at the nanoscale level was performed by atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM).Energy Dispersive X-Ray Microanalysis (EDX) revealed the elemental composition of exosomes and their respective total Zinc content on a quantitative basis. The zinc mole fraction (in at%) was correlated to the phosphorous mole fraction, which is indicative for exosomal membrane material. Both fresh (Zn/P 0.09 ± 0.01) and frozen exosomes (Zn/P 0.08 ± 0.02) had a significant zinc content, which increased up to 1.09 ± 0.12 for frozen exosomes when treated with increasing amounts of zinc (100–500 μM; each p < 0.05). Interestingly, after zinc addition, the Calcium mole fractions decreased accordingly suggesting a possible exchange by zinc.In order to estimate the intra-exosomal labile zinc content, an Imaging Flow Cytometry approach was developed by using the specific membrane permeable zinc-probe Fluozin-3AM. A labile zinc content of 0.59 ± 0.27 nM was calculated but it is likely that the measurement may be affected by purification and isolation conditions. This study suggests that circulating nano-vesicular-zinc can represent a newly discovered zinc fraction in the blood plasma whose functional and biological properties will have to be further investigated in future studies.



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Acute effects of PM 2.5 on lung function parameters in schoolchildren in Nanjing, China: a panel study

Abstract

The association between exposure to ambient particulate matter (PM) and reduced lung function parameters has been reported in many works. However, few studies have been conducted in developing countries with high levels of air pollution like China, and little attention has been paid to the acute effects of short-term exposure to air pollution on lung function. The study design consisted of a panel comprising 86 children from the same school in Nanjing, China. Four measurements of lung function were performed. A mixed-effects regression model with study participant as a random effect was used to investigate the relationship between PM_2.5 and lung function. An increase in the current day, 1-day and 2-day moving average PM_2.5 concentration was associated with decreases in lung function indicators. The greatest effect of PM_2.5 on lung function was detected at 1-day moving average PM_2.5 exposure. An increase of 10 μg/m³ in the 1-day moving average PM_2.5 concentration was associated with a 23.22 mL decrease (95% CI: 13.19, 33.25) in Forced Vital Capacity (FVC), a 18.93 mL decrease (95% CI: 9.34, 28.52) in 1-s Forced Expiratory Volume (FEV₁), a 29.38 mL/s decrease (95% CI: -0.40, 59.15) in Peak Expiratory Flow (PEF), and a 27.21 mL/s decrease (95% CI: 8.38, 46.04) in forced expiratory flow 25–75% (FEF_25–75%). The effects of PM_2.5 on lung function had significant lag effects. After an air pollution event, the health effects last for several days and we still need to pay attention to health protection.

http://ift.tt/2G2C8bX

Heavy metal accumulation by Corchorus olitorius L. irrigated with wastewater

Abstract

Many agricultural soils in Egypt irrigated with untreated wastewater. Herein, we investigated the effect of untreated industrial wastewater irrigation on the soil and fodder plant Corchorus olittorius (Jew mallow). It also aimed to assess its effect on the growth measurements as well as analyses of soils, irrigation waters, and plants for heavy metal and nutrient concentrations. Significant differences between irrigation waters and soil irrigated with fresh and wastewater were recognized. Wastewater irrigation leads to remarkable reduction in the growth parameters and reduced its vegetative biomass. The concentration of Pb, Cd, Cr, Cu, Fe, and Zn were high significant and above phytotoxic concentrations in leaves (edible part) and roots of wastewater-irrigated plant. The present study indicated that Jew mallow plant tends to phytostabilize (Cd, Ni, and Mn) in its root and had the ability to translocate (Pb, Cu, Cr, Fe, and Zn) to its leaves. Higher concentrations of Cd, Cu, Cr, Pb, Fe, Mn, Ni, and Zn in the roots than leaves indicate that the roots are hyper-accumulators for Pb, Cr, Cu, Fe, and Zn more than the leaves. The research study recommended that there is a need to protect the soil from contamination through regular monitoring and not to cultivate Jew mallow in wastewater-irrigated soil and that it had a high capacity to accumulate heavy metals in its edible part and causes several harmful health effects for consumers.

http://ift.tt/2tW0lMv

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells

Publication date: Available online 17 March 2018
Source:Free Radical Biology and Medicine
Author(s): Annie R. Knight, Emma Taylor, Roman Lukaszewski, Karina Tveen Jensen, Helen E. Jones, Jane E. Carré, Michail N. Isupov, Jennifer A. Littlechild, Stephen J. Bailey, Emily Brewer, Timothy J. McDonald, Andrew R. Pitt, Corinne M. Spickett, Paul G. Winyard
The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.

Graphical abstract

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Stress responsive mitochondrial proteins in Arabidopsis thaliana

Publication date: Available online 17 March 2018
Source:Free Radical Biology and Medicine
Author(s): Yan Wang, Oliver Berkowitz, Jennifer Selinski, Yue Xu, Andreas Hartmann, James Whelan
In the last decade plant mitochondria have emerged as a target, sensor and initiator of signalling cascades to a variety of stress and adverse growth conditions. A combination of various 'omic profiling approaches combined with forward and reverse genetic studies have defined how mitochondria respond to stress and the signalling pathways and regulators of these responses. Reactive oxygen species (ROS)-dependent and -independent pathways, specific metabolites, complex I dysfunction, and the mitochondrial unfolded protein response (UPR) pathway have been proposed to date. These pathways are regulated by kinases (sucrose non-fermenting response like kinase; cyclin dependent protein kinase E 1) and transcription factors from the abscisic acid-related, WRKY and NAC families. A number of independent studies have revealed that these mitochondrial signalling pathways interact with a variety of phytohormone signalling pathways. While this represents significant progress in the last decade there are more pathways to be uncovered. Post-transcriptional/translational regulation is also a likely determinant of the mitochondrial stress response. Unbiased analyses of the expression of genes encoding mitochondrial proteins in a variety of stress conditions reveal a modular network exerting a high degree of anterograde control. As abiotic and biotic stresses have significant impact on the yield of important crops such as rice, wheat and barley we will give an outlook of how knowledge gained in Arabidopsis may help to increase crop production and how emerging technologies may contribute.

Graphical abstract

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Rosmarinic Acid Attenuates β-amyloid-Induced Oxidative Stress via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in PC12 Cells

Publication date: Available online 16 March 2018
Source:Free Radical Biology and Medicine
Author(s): Hua Rong, Yini Liang, Yingcai Niu
Oxidative stress is an important pathogenic factor in Alzheimer's disease (AD). Recently, nuclear factor E2-related factor 2 (Nrf2) has emerged as a master regulator for the endogenous antioxidant response, and thus represents an attractive therapeutic target against AD. The aim of this study is to test the hypothesis that rosmarinic acid (RosA) attenuates amyloid-β (Aβ)-evoked oxidative stress through activating Nrf2-inducible cellular antioxidant defense system. Here, we reported that RosA attenuated Aβ-induced cellular reactive oxygen species (ROS) generation and lipid hydroperoxides (LPO). Interestingly, knockdown of Nrf2 by plasmid-based short hairpin RNA (shRNA) abrogated, at least in part, RosA-mediated neuroprotection in Aβ-challenged PC12 cells. Mechanistically, RosA enhanced the nuclear translocation of Nrf2 and binding to antioxidant response element (ARE) core element but did not induced Nrf2 transcription. Simultaneously, RosA induced a set of Nrf2 downstream target genes encoding phase-II antioxidant enzymes. Furthermore, RosA enhanced protein kinase B (Akt) phosphorylation, glycogen synthase kinase-3β (GSK-3β) phosphorylation at Ser9, and Fyn phosphorylation. Noteworthy, pharmacological inhibition or gene knockdown studies demonstrated that Akt locate upstream of GSK-3β and regulate Nrf2 through Fyn in the context of PC12 cells pre-incubated with RosA following exposed to Aβ. Conversely, the antioxidant effects of RosA could be blocked by Akt inhibitors LY294002, GSK-3β inhibitor LiCl, Nrf2 shRNA, or Fyn shRNA in Aβ-challenged PC12 cells. Consequently, the antioxidant effects of RosA are mediated predominantly by Akt/GSK-3β/Fyn pathway through increased activity of Nrf2. These results suggest, although do not prove, that RosA can be a promising candidate for neuroprotective treatment of AD.

Graphical abstract

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Keto-enol-based modification on piperlongumine to generate a potent Cu(II) ionophore that triggers redox imbalance and death of HepG2 cells

Publication date: Available online 16 March 2018
Source:Free Radical Biology and Medicine
Author(s): Fang Dai, Cui-Hong Yuan, Yuan Ji, Yu-Ting Du, Xia-Zhen Bao, Ling-Xi Wu, Xiao-Ling Jin, Bo Zhou
Altered redox status including higher levels of copper in cancer cells than in normal cells inspired many researchers to develop copper ionophores targeting this status. We have recently found that flavon-3-ol (3-HF) works as a potent Cu(II) ionophore by virtue of its keto-enol moiety. To further emphasize the significance of this moiety for developing Cu(II) ionophores, we herein designed a β-diketo analog of piperlongumine, PL-I, characterized by the presence of high proportion of the keto-enol form in dimethylsulfoxide and chloroform, and identified its keto-enol structure by NMR and theoretical calculations. Benefiting from deprotonation of its enolic hydroxyl group, this molecule is capable of facilitating the transport of Cu(II) through cellular membranes to disrupt redox homeostasis of human hepatoma HepG2 cells and trigger their death.

Graphical abstract

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Characterization of centriole duplication in human epidermis, Bowen’s disease, and squamous cell carcinoma

Publication date: Available online 16 March 2018
Source:Journal of Dermatological Science
Author(s): Saori Watanuki, Harumi Fujita, Keisuke Kouyama, Masayuki Amagai, Akiharu Kubo
BackgroundCentrosomes contain two centrioles: a pre-existing mature centriole and a newly formed immature centriole. Each centriole is duplicated once within a cell cycle, which is crucial for proper centrosome duplication and cell division.ObjectiveTo describe the centrosome duplication cycle in human epidermis, Bowen's disease (BD), and squamous cell carcinoma (SCC).MethodsImmunofluorescent staining of centriolar proteins and Ki-67 was used to evaluate cell cycles and the number of centrioles. Centrobin and Outer dense fiber of sperm tails 2 (ODF2) were used as markers for immature and mature centrioles, respectively.ResultsNormal human primary epidermal keratinocytes in a monolayered culture have one centrobin⁺ centriole (CTRB¹⁺ cells) supposed in G0/G1 phases or have two centrobin⁺ centrioles (CTRB²⁺ cells) supposed in S−G2 phase. In a three-dimensional culture and in vivo human epidermis, the majority of suprabasal cells were CTRB²⁺ cells, in spite of their non-proliferative Ki-67⁻ nature. The tumor mass of BD and SCC contained CTRB¹⁺ cells and Ki-67⁺ proliferating and Ki-67⁻ non-proliferative CTRB²⁺ cells. Clumping cells in BD had increased numbers of centrioles, with an approximate 1:1 to 2:1 ratio of centrobin⁺ to ODF2⁺ centrioles.ConclusionsThe cell cycle arrest of suprabasal cells is distinct from the G0 arrest of monolayered epithelial cells. Tumor mass of BD and SCC contained non-proliferative cells with the characteristics of the suprabasal cells of normal epidermis. A constant ratio of the number of centrobin⁺ to ODF2⁺ centrioles indicates that multiple centrioles were induced by cell division failure rather than centriole overduplication in clumping cells.



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ESCMID Postgraduate Education Course Regional Capacity Building for Integration of Next-Generation Sequencing in the Clinical Microlab

Publication date: Available online 17 March 2018
Source:Microbes and Infection
Author(s): Yair Motro, João André Carriço, Alexander W. Friedrich, John WA. Rossen, Jacob Moran-Gilad




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Rapid response to an emerging infectious disease - Lessons learned from development of a synthetic DNA vaccine targeting Zika virus

Publication date: Available online 17 March 2018
Source:Microbes and Infection
Author(s): Sagar B. Kudchodkar, Hyeree Choi, Emma L. Reuschel, Rianne Esquivel, Jackie Jin-Ah Kwon, Moonsup Jeong, Joel N. Maslow, Charles C. Reed, Scott White, J. Joseph Kim, Gary P. Kobinger, Pablo Tebas, David B. Weiner, Kar Muthumani
Vaccines are considered one of the greatest advances in modern medicine. The global burden of numerous infectious diseases has been significantly reduced, and in some cases, effectively eradicated through the deployment of specific vaccines. However, efforts to develop effective vaccines against infectious pathogens such as influenza, HIV, dengue virus (DENV), chikungunya virus (CHIKV), Ebola virus, and Zika virus (ZIKV) have proven challenging. Zika virus is a mosquito-vectored flavivirus responsible for periodic outbreaks of disease in Africa, Southeast Asia, and the Pacific Islands dating back over 50 years. Over this period, ZIKV infections were subclinical in most infected individuals and resulted in mild cases of fever, arthralgia, and rash in others. Concerns about ZIKV changed over the past two years, however, as outbreaks in Brazil, Central American countries, and Caribbean islands revealed novel aspects of infection including vertical and sexual transmission modes. Cases have been reported showing dramatic neurological pathologies including microcephaly and other neurodevelopmental problems in babies born to ZIKV infected mothers, as well as an increased risk of Guillain-Barre syndrome in adults. These findings prompted the World Health Organization to declare ZIKV a public health emergency in 2016, which resulted in expanded efforts to develop ZIKV vaccines and immunotherapeutics. Several ZIKV vaccine candidates that are immunogenic and effective at blocking ZIKV infection in animal models have since been developed, with some of these now being evaluated in the clinic. Additional therapeutics under investigation include anti-ZIKV monoclonal antibodies (mAbs) that have been shown to neutralize infection in vitro as well as protect against morbidity in mouse models of ZIKV infection. In this review, we summarize the current understanding of ZIKV biology and pathogenesis and describe our efforts to rapidly develop a vaccine against ZIKV.



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Boy with Hand Blisters

Publication date: Available online 17 March 2018
Source:The Journal of Emergency Medicine
Author(s): William Sveen, Shane McWhirter, Kari Schneider




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Blend of organic acids and medium chain fatty acids prevents the inflammatory response and intestinal barrier dysfunction in mice challenged with enterohemorrhagic Escherichia coli O157:H7

Publication date: May 2018
Source:International Immunopharmacology, Volume 58
Author(s): Jun Wang, JinXin Lu, XiaoWei Xie, Jia Xiong, Ningning Huang, Hongkui Wei, Siwen Jiang, Jian Peng
Impaired epithelial barrier function disrupts immune homeostasis and increases inflammation in intestines, leading to many intestinal diseases. The blend of organic acids (OAs) and medium chain fatty acids (MCFAs) has been shown to have synergistic bactericidal effect. In this study, we demonstrated that two blends of OAs and MCFAs (OM1 and OM2) could prevent the inflammatory response and intestinal barrier dysfunction in enterohemorrhagic Escherichia coli (EHEC)-infected mice. Treatments of OM1 and OM2 significantly reduced the body weight loss and production of IL-6 and TNF-α induced by EHEC. Mice treated with OM1 and OM2 showed decrease in serum D-lactic concentration, DAO activity and bacterial transfer to liver and spleen. Furthermore, OM1 and OM2 increased the expression of tight junction proteins occludin and ZO-1, mucus protein MUC-2, and host defense peptides mBD1, mBD2 and mBD3. Finally, OM1 and OM2 increased the population of Lactobacillus spp. and Bifidobacterium spp., but decreased that of E. coli in the cecum. These findings indicate that OM1 and OM2 may be used to develop a prophylactic agent for intestinal inflammation and injury in enteric pathogen infection.



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