Global Medical Discovery features paper: DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis

Significance Statement

This study analyzed the expression of the Ku protein and its DNA-binding activity during early apoptosis. In the study, Ku was neither cleaved nor degraded during early apoptosis induced by etoposide treatment. In addition, Ku was found to bind cleaved chromosomal DNA and/or nucleosomes in apoptotic cells.

The DNA-binding properties of Ku were assessed by two methods, each of which is based on electrophoresis mobility shift assay (EMSA). One commonly applied method, EMSA with 15-bp ³²P-labeled DNA probe (RI-EMSA), employs radiolabeled DNA probes. The other method, EMSA combined with western blot (WB-EMSA), employs unlabeled DNA probes followed by western blot and detection using anti-Ku antiserum. This study showed that both WB-EMSA and RI-EMSA are useful for evaluating Ku-DNA binding activity.

The significance of this work lies in its contribution to the understanding of the mechanism of interaction of Ku with DNA during apoptosis.

Figure legends:

1. RI-EMSA: Protein extract obtained from cells treated with etoposide for the duration indicated was incubated with ³²P-labeled, 15-bp dsDNA probes in the presence of closed circular DNA.
2. Western blot: Protein extract obtained from either control or etoposide-treated cells was run on a 10% SDS polyacrylamide gel under reducing conditions. Ku70 and Ku80 were detected using human polyclonal antiserum.
3. WB-EMSA: Extracts from etoposide-treated HL-60 cells were separated by 6% native PAGE, and then analyzed by western blot with polyclonal antiserum raised against Ku (lane 1). Extracts from untreated HL-60 cells were incubated with 160-bp DNA, followed by western blot (lane 2). Extracts from untreated or etoposide treated HL-60 cells were incubated with 15-bp DNA, followed by western blot (lanes 3–6).
4. Comparison of WB-EMSA to conventional RI-EMSA. The diagram of RI-EMSA is a part of Figure A, and the figure of WB-EMSA is a part of Figure C.

About The Author

Katsuya Iuchi is currently an assistant professor at the Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School. His research focuses on the mechanism of cell death and lipid peroxidation of biomembranes. Prior to joining Nippon Medical School, he was a postdoctoral fellow in the Sodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technology Agency. He received his Ph.D. in chemistry from Kwansei Gakuin University, Japan in 2009.  

Journal Reference

Exp Cell Res. 2016;342(2):135-44.

Iuchi K¹, Yagura T².

Show Affiliations

1. Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan. Electronic address: iuchi@nms.ac.jp.
2. Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan.

Abstract

Ku-protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku-protein during apoptosis remains unclear. Here, we show characterization of Ku-protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the non-treated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion,Ku is intact and retains DNA binding activity in early apoptotic cells.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Global Medical Discovery features paper: Identification of Palmitoylated Transitional Endoplasmic Reticulum ATPase by Proteomic Technique and Pan Antipalmitoylation Antibody

J Proteome Res. 2016 Mar 4;15(3):956-62. doi: 10.1021/acs.jproteome.5b00979.

Caiyun Fang,^† Xiaoqin Zhang,^† Lei Zhang, Xing Gao, Pengyuan Yang, and Haojie Lu^*

Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China

 

Abstract

Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis, and carcinogenesis. For high-throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labeling of azide/alkynyl-palmitate analogs are commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via a TS-6B-based method. In total, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins, ATP1A1, ZDHHC5, and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4, exportin-2, etc. More importantly, the pan antipalmitoylation antibody was developed and verified for the first time. Our homemade pan antipalmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique for analyzing protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by Western blotting with anti-VCP antibody and the homemade pan antipalmitoylation antibody.

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Global Medical Discovery features paper: Synthesis, antimicrobial activity of Schiff base compounds of cinnamaldehyde and amino acids

Significance Statement

Microorganism infection is a continue threat to human health. About the control the infection, using antimicrobial agent is one of the effective options. Now, natural antimicrobial agents are focused extensively because they are low-toxic and safe. Cinnamaldehyde is a natural antimicrobial substance, which is extracted from bark of  Cinnamomum genus trees. Cinnamaldehyde possesses good bioactivity and could inhibit the growing of many kinds of fungi and bacteria. Cinnamaldehyde is categorized as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration. However, Cinnamaldehyde has several drawbacks, such as its strong odor, high volatility and water insolubility, which limited its application.

In our research, cinnamaldehyde was modified by amino acids to synthesize a new class of Schiff base compounds. Bioactivity results indicated that the Schiff base compounds remarkably inhibited the growth of the tested bacteria and fungi, and they have broad-spectrum antimicrobial activity. Compared with control compounds, i.e. Ciprofloxacin and Fluconazole, most of the Schiff base compounds exhibited better bioactivity than the two control compounds.

The key finding of this study is that, the new class of cinnamaldehyde Schiff base compounds, which could be easily synthesized, have great bioactivity, low-toxicity, low odor and good water solubility. Therefore, cinnamaldehyde Schiff base compounds were potential to be antibacterial agents or food preservatives.

Moreover, the further analysis on antimicrobial activity and chemical structure were conducted in our research. Results from the structure-activity relationship suggest that both p-Cl of benzene ring from cinnamaldehyde and the number of –COOK from amino acid salts significantly contributed to their antimicrobial activity.

Our research results provided an alternative option for exploring of new antimicrobial agents. 

About The Author

Prof. Shujun Li received her Bachelor Degree in Forest Products Chemical Processing from Northeast Forestry University, China in 1995. She received her Master Degree in 1998, also in the same major, and received her Ph. D in 2001, majored in wood science and technology.

Currently, she serves as a full Professor of Forest Products Chemical Processing Department at Northeast Forestry University, Harbin, China. Her research interests include biomass conversion and application in a high-value way. 

 

About The Author

Miss Hui Wang received her bachelor degree in 2013 from Lanzhou University of Technology, Lanzhou, China. She is now a doctoral candidate at Northeast Forestry University, and her current research is synthesis of cinnamaldehyde derivatives and its bioactivity. 

Journal Reference

Bioorg Med Chem Lett. 2016 Feb 1;26(3):809-13.

Wang H¹, Yuan H¹, Li S², Li Z¹, Jiang M¹. 

Show Affiliations

1. Key Laboratory of Bio-Based Material Science and Technology of the Ministry of Education, Northeast Forestry University, Harbin 150040, China.
2. Key Laboratory of Bio-Based Material Science and Technology of the Ministry of Education, Northeast Forestry University, Harbin 150040, China. Electronic address: lishujun@nefu.edu.cn.

Abstract

The purpose of this study was to synthesize hydrophilic cinnamaldehyde Schiff base compounds and investigate those bioactivity. A total of 24Schiff base compounds were synthesized using a simple approach with 3 cinnamaldehyde derivates and 8 amino acids as raw materials. The structures of synthesized compounds were confirmed using FTIR, (1)HNMR, HRMS purity and melting point. The antimicrobial activities of newcompounds were evaluated with fluconazole and ciprofloxacin as the control against Aspergillus niger, Penicillium citrinum, Escherichia coli and Staphylococcus aureus.

Findings show that major compounds exhibited significant bioactivity. Results from the structure-activity relationship suggest that both -p-Cl on benzene ring of cinnamaldehyde and the number of -COOK of amino acid salts significantly contributed to antimicrobialactivity.

Copyright © 2015 Elsevier Ltd. All rights reserved.

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Global Medical Discovery features paper: Inkjet-Print Micromagnet Array on Glass Slides for Immunomagnetic Enrichment of Circulating Tumor Cells

Significance Statement

To advance cancer studies and enable oncologists to make more accurate diagnosis, prognosis, and develop personalized therapies, Prof. John X.J. Zhang and Dr. Peng Chen led a team of bioengineers from the Thayer School of Engineering at Dartmouth and the University of Texas at Austin in developing an innovative system that combines inkjet-printing, microfluidic technology, and immunoassay for capturing and manipulating circulating tumor cells (CTCs). This miniaturized bio-analytical system can be further integrated with fluorescent microscope for cancer cell imaging, with fluorescent in-situ hybridization (FISH) and PCR for molecular level cancer studies. It opens up a great many possibilities to optimize cancer diagnostic and tumor management.

“This work provides a new angle, other than traditional tissue biopsy and medical imaging, to approach cancer. A fast, simple and less painful blood test may eventually provide equivalently accurate disease information about individual cancer patient in a timely manner, ” said Zhang. circulating tumor cells detach from primary tumor site, shed into the blood circulation system, and may initiate the deadly cancer metastasis process. Through efficient detection of these cells, especially at an early stage, physicians will be able to design the best diagnosis and treatment strategy for each individual cancer patient.

Immunomagnetic assay has been successfully used to separate rare circulating tumor cells from blood. However, traditional immunomagnetic assay is often limited in the low magnetic field gradient and low density of effective magnetic traps. In this work, Dr. Chen developed a novel method using inkjet-printing technology to fabricate microscale magnetic structures that can be easily deposited on an arbitrary substrate.

This inkjet-printing technology is a versatile but cost-effective approach for rapid prototyping with high accuracy and flexibility. Such microscale magnets, when placed in an external magnetic field, largely enhance the magnetic field and the attractive force applied on the target circulating tumor cells, and hence facilitates the detection. In our experiments with COLO205 (a human colorectal cancer cell line) as the separation target, the inkjet-printed micro-magnets integrated assay increased the system sensitivity by 26% compared with using normal glass slide as the substrate.

Dr. Chen’s research is focused on developing miniaturized high-performance bio-analytical systems for point-of-care and globally relevant medical diagnostic applications. “This project is to implement advanced engineering fabrication, sensing, and manipulating techniques with multiplexing immunoassay to separate these rare circulating tumor cells from whole blood.” Zhang said. “The ultimate goal is to deliver a highly sensitive, reliable, affordable, and portable cancer screening platform with high throughput. The next step – bringing the technology from lab bench to clinics is a big challenging step, but is also the most rewarding step.

This work has the potential to revolutionize cancer risk assessment, cancer management, and increase the cure rate for cancers such as breast cancer, lung cancer, and prostate cancer.” This paper is published on Annals of Biomedical Engineering (DOI: 10.1007/s10439-015-1427-z).

      

About The Author

Dr. Peng Chen received his PhD degree in Biomedical Engineering from the University of Texas at Austin. He is now working as a postdoc research associate at the Center for Applied NanoBioscience and Medicine at the University of Arizona. His research interests include (1) miniaturized microfluidic system for detection and analysis of rare circulating tumor cells (CTCs), and (2) vertical flow paper based microfluidic device as point-of-need multiplex diagnostic and surveillance tool for bio-threat detection. He has published 10 peer-reviewed journal papers and 10 conference proceedings. He now serves the editorial board of Scientific Reports (Nature publishing group). Email: pengchen@email.arizona.edu

About The Author

Prof. John Zhang, Ph.D., is a Professor at Thayer School of Engineering at Dartmouth, and a Fellow of American Institute for Medical and Biological Engineering (AIMBE). He received his Ph.D. from Stanford University, and was a Research Scientist at MIT. His key contribution is in developing miniature medical systems to improve global health, through innovations in bio-inspired nanomaterials, lab-on-chip design, and advanced nanofabrication technologies for probing complex biological networks critical to human development and diseases such as cancer. He received the Wallace Coulter Foundation Early Career Award for developing handheld microphotonic imaging scanners and microsystems for early oral cancer detection; NSF CAREER award for the invention of plasmonic scanning probes design for controlled perturbation and imaging at sub-cellular level; and DARPA Young Faculty Award for patterning plasmonic surface on MEMS for biomarker sensing applications. He has published over 120 peer reviewed papers and proceedings, presented over 45 invited seminars worldwide, and filed over 60 US and international patents. He is an alumnus of NAE Frontiers of Engineering programs, an Associate Editor for Biomedical Microdevices, IEEE/ASME Journal of Microelectromechanical Systems, and has published a textbook for undergraduates titled “Molecular Sensors and Nanodevices: Principles, Designs and Applications in Biomedical Engineering”. Email: john.zhang@dartmouth.edu

Reference

Ann Biomed Eng. 2016;44(5):1710-20.

Peng Chen¹, Yu-Yen Huang², Gauri Bhave¹, Kazunori Hoshino³, Xiaojing Zhang^4,5

Show Affiliations

¹Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, 78712, USA.

²Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755, USA.

³Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA.

⁴Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, 78712, USA.

⁵Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755, USA. john.zhang@dartmouth.edu.

We report an inkjet-printed microscale magnetic structure that can be integrated on regular glass slides for the immunomagnetic screening of rare circulating tumor cells . circulating tumor cells detach from the primary tumor site, circulate with the bloodstream, and initiate the cancer metastasis process. Therefore, a liquid biopsy in the form of capturing and analyzing circulating tumor cells may provide key information for cancer prognosis and diagnosis. Inkjet printing technology provides a non-contact, layer-by-layer and mask-less approach to deposit defined magnetic patterns on an arbitrary substrate. Such thin film patterns, when placed in an external magnetic field, significantly enhance the attractive force in the near-field close to the circulating tumor cells to facilitate the separation. We demonstrated the efficacy of the inkjet-print micromagnet array integrated immunomagnetic assay in separating COLO205 (human colorectal cancer cell line) from whole blood samples. The micromagnets increased the capture efficiency by 26% compared with using plain glass slide as the substrate.

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Global Medical Discovery features paper: Impact of patient characteristics on the clinical efficacy of mongersen (GED-0301), an oral Smad7 antisenseoligonucleotide, in active Crohn’s disease

Significance Statement

Crohn’s disease (CD) is a chronic condition characterized by segmental, transmural inflammation, which, although most common in the terminal ileum and right colon, can affect any part of the alimentary tract. In addition to the commonly observed signs and symptoms of Crohn’s disease, local complications as well as manifestations outside the digestive tract can also occur as a result of Crohn’s disease -associated inflammation, underscoring the importance of effective treatment options.

Subgroup analyses from prospective, randomised, controlled trials in patients with active Crohn’s disease have shown that patient demographics and disease characteristics such as disease duration and treatment history can impact clinical outcomes. Our retrospective study further evaluated the impact of patient baseline clinical and disease characteristics on the efficacy of mongersen, an oral, locally active Smad7 antisense oligonucleotide that targets Smad7 in the ileum and colon. In addition to the impact of human serum C-reactive protein (hsCRP) values, disease duration, and disease activity at baseline on mongersen over 10 weeks, our analysis also looked at how sex, body mass index, smoking status, history of Crohn’s disease-related intestinal resection, steroid status, and immunosuppressant use at baseline impacted treatment efficacy.

Our results showed that patients with Crohn’s Disease Activity Index (CDAI) scores ≤260 at baseline had significantly higher clinical remission rates (defined as a CDAI <150) with mongersen 40 mg/day and 160 mg/day treatment, whereas patients with CDAI scores >260 at baseline achieved clinical remission most frequently with the highest mongersen dose (160 mg/day), suggesting that greater disease activity can impact clinical benefit. Baseline disease characteristics such as hsCRP (<3 mg/L or ≥3 mg/L) and disease duration (<5 or ≥5 years) did not appear to significantly impact efficacy of mongersen treatment in our study.

About The Author

Giovanni Monteleone received his medical degree from the University Magna Graecia of Catanzaro (Italy) and completed his internship, residency, and a fellowship in gastroenterology at the same university. He joined the faculty at the University of Rome Tor Vergata in 2003, becoming an Assistant Professor of Gastroenterology, then Professor of Gastroenterology. He is the Head of Gastroenterology Unit at the Policlinico Tor Vergata in Rome. Dr. Monteleone’s basic research is mostly focused on mechanisms involved in the control of mucosal immune homeostasis and inflammatory signals that sustain and amplify pathological processes in the gastrointestinal tract. In particular, he is interested in understanding how immune cells and non-immune cells cross-talk in the gastrointestinal mucosa and which molecules mediate such an interplay. So far, these studies have contributed to delineate novel pathways of intestinal mucosal damage and paved the way for the development of anti-inflammatory compounds, which are now ready to move into the clinic. Dr. Monteleone is also interested in the immune-inflammatory networks involved in the colitis-associated colon carcinogenesis. He is author of >300 peer-reviewed articles, books, and book chapters, and serves as an editorial board member or a reviewer for several journals.

 

Journal Reference

Aliment Pharmacol Ther. 2016 ;43(6):717-24.

Monteleone G¹, Di Sabatino A², Ardizzone S³, Pallone F¹, Usiskin K⁴, Zhan X⁴, Rossiter G⁴, Neurath MF⁵. 

Show Affiliations

1. Department of Systems Medicine, University of Tor Vergata, Rome, Italy.
2. First Department of Internal Medicine, St. Matteo Hospital Foundation, University of Pavia, Pavia, Italy.
3. Department of Surgery, “L. Sacco” University Hospital, Milan, Italy.
4. Celgene Corporation, Warren, NJ, USA.
5. Department of Medicine, University of Erlangen-Nürnberg, Erlangen, Germany.

Abstract

BACKGROUND:

In a phase 2 study, mongersen, an oral antisense oligonucleotide targeting Smad7, was effective in inducing clinical remission in approximately 60% of patients with active Crohn’s disease (CD).

AIM:

In a post hoc analysis to evaluate those patient disease characteristics that may have influenced the efficacy and safety of mongersen therapy.

METHODS:

Patients with steroid-dependent/resistant, active Crohn’s disease were randomised to mongersen 10, 40 or 160 mg/day or placebo for 2 weeks; patients were followed for 10 weeks. Clinical remission [Crohn’s Disease Activity Index (CDAI) score <150] and clinical response (CDAI score reduction ≥100 points) were assessed at weeks 2, 4 and 12 for these subgroups: disease duration <5/≥5 years, human serum C-reactive protein (hsCRP) <3/≥3 mg/L, and CDAI at baseline ≤260/>260. Additional patient baseline and disease characteristics were explored.

RESULTS:

Clinical remission and response rates were significantly higher in patients receiving mongersen 40 and 160 mg/day but not 10 mg/day vs. placebo and independent of disease duration and hsCRP. Patients with baseline CDAI ≤260 had significantly higher remission rates with 40 and 160 mg/day. In patients with baseline CDAI >260, remission rates were statistically greater with 160 mg/day and numerically better with 40 mg/day vs. placebo. Adverse event rates were similar across treatment groups. Mongersen was safe and well tolerated.

CONCLUSIONS:

Patients with higher CDAI scores achieved clinical remission most frequently with the highest mongersen dose. Disease duration and baseline human serum C-reactive protein did not appear to significantly impact efficacy of mongersen in this study (EudraCT Number: 2011-002640-27.).

© 2016 The Authors. Alimentary Pharmacology & Therapeutics published by John Wiley & Sons Ltd.

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Global Medical Discovery features paper: Nanostructured material formulated acrylic bone cements with enhanced drug release

Significance Statement

Current commercial polymethylmethacrylate (PMMA) based antibiotics-loaded bone cements exhibit very low drug release as most of the antibiotics are embedded inside the polymerized bone cement.  This results in a rapid decrease in antimicrobial activity below the effective therapeutic level within one to several days. To increase the therapeutic window to several weeks for reducing the risk of post-operative joint infection, mesoporous silica nanoparticles (MSN) functional bone cement was developed to enable a sustained release of antibiotics.

Our results show that the novel formulation can be designed to tailor a desired release profile, e.g. up to 70% drug release for several weeks in comparison with commercial bone cement showing 10% or lower drug release and peak concentration only on day one.  MSN with uniform nano-porous channels built up effective diffusion nano-networks which enabled the drug molecules to be released sustainably. Moreover, the mechanical properties of bone cements were well preserved in the presence of MSN in formulation even after drug release.

This formulation is expected to deliver the next generation bone cement with tailor-designed antibiotics release profiles and prolonged antibacterial activity.  The key commercial advantages to potential bone cement manufacturers and end users are sustained antibiotics release, reduction in standard antibiotics dosage, minimised use of intravenous doses and creating new opportunities to use other benefit ingredient for local targeted delivery.  Once commercialized, the socio-economic impact and savings in medical resources and costs are expected to be significant as the risk of post-operative infections can be reduced.       

About The Author

Reginald Tan received PhD degree from University of Cambridge in 1989. He is Director (Research) of the Institute of Chemical and Engineering Sciences (ICES, Singapore) and concurrently a Professor in the Department of Chemical and Bimolecular Engineering, National University of Singapore. His current research is in crystallisation science and modelling, and formulation sciences.

About The Author

Shoucang SHEN obtained PhD degree from National University of Singapore at 2001. Currently, he is a senior scientist in Institute of Chemical and Engineering Sciences (ICES, Singapore). His research interest includes formulation of poorly soluble drugs and controlled release of active ingredients, as well as the exploration of nanotechnology.

About The Author

Ng Wai Kiong obtained PhD degree from National University of Singapore and Diplom Ingenieur from Technical University of Clausthal, Germany. He is Team Leader (Formulation Sciences) in the Crystallisation and Particle Sciences Division, Institute of Chemical and Engineering Sciences (ICES, Singapore).

Reference

Mater Sci Eng C Mater Biol Appl. 2016 ;58:233-41.

Shen SC¹, Ng WK², Dong YC², Ng J², Tan RB³.

Show Affiliations

¹Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore. Electronic address: shen_shoucang@ices.a-star.edu.sg.

²Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore.

³Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore; Department of Chemical and Biomolecular Engineering, The National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore. Electronic address: reginald_tan@ices.a-star.edu.sg.

Abstract

To improve antibiotic properties, poly(methyl methacrylate) (PMMA)-based bone cements are formulated with antibiotic and nanostructured materials, such as hydroxyapatite (HAP) nanorods, carbon nanotubes (CNT) and mesoporous silica nanoparticles (MSN) as drug carriers. For nonporous HAP nanorods, the release of gentamicin (GTMC) is not obviously improved when the content of HAP is below 10%; while the high content of HAP shows detrimental to mechanical properties although the release of GTMC can be substantially increased. As a comparison, low content of hollow nanostructured CNT and MSN can enhance drug delivery efficiency. The presence of 5.3% of CNT in formulation can facilitate the release of more than 75% of GTMC in 80 days, however, its mechanical strength is seriously impaired. Among nanostructured drug carriers, antibiotic/MSN formulation can effectively improve drug delivery and exhibit well preserved mechanical properties. The hollow nanostructured materials are believed to build up nano-networks for antibiotic to diffuse from the bone cement matrix to surface and achieve sustained drug release. Based on MSN drug carrier in formulated bone cement, a binary delivery system is also investigated to release GTMC together with other antibiotics.

Copyright © 2015 Elsevier B.V. All rights reserved.

Go To Materials Science and Engineering: C

Global Medical Discovery features paper: Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Significance Statement

Arteries have the remarkable ability to adjust the amount and force of blood that flows through them. Such adjustments are made possible by contraction or relaxation of arterial smooth muscle cells (ASMCs) within the walls of the arteries. Many hormonal and neuronal actions serve to adjust the contraction of ASMCs, to modulate blood flow and pressure. At the cellular level, contraction of ASMCs depends on the flow of calcium ions into the cells through specialized protein pores or “channels” on the cell’s plasma membrane. Opening of voltage-sensitive calcium channels (VSCCs) involves positive changes to the voltage across the plasma membrane. Relaxation of ASMCs occurs when membrane voltage is maintained around -60 millivolts (negative inside compared to outside) by a flux of potassium ions out of the cells through channels that selectively conduct potassium; this negative voltage inhibits the opening of VSCCs. Among the many types of potassium channels on the ASMC plasma membrane, Kv7 channels, are particularly well suited as targets for hormonal and neuronal regulation of ASMC contraction to adjust blood pressure and blood flow.

Membrane voltage, and hence the flux of calcium ions via VSCCs, is very sensitive to the opening (activation) or closing (e.g. blocking) of Kv7 channels. These channels are tetrameric assemblies constituted by four Kv7 channel alpha subunits. There are five different types of Kv7 channel alpha subunits, named Kv7.1 through 7.5. In ASMCs, Kv7 channels are composed of four Kv7.4 subunits, four Kv7.5 subunits, or by some combination of Kv7.4/7.5 subunits (i.e. heterotetrameric channels). Altering the activity of the Kv7 channels with chemicals that bind directly to the channels has been shown to influence arterial contractility and diameter. However, physiological regulation of Kv7 channel activity is still poorly understood. In particular, it is unclear how the subunit composition of the channels influences the regulation of their activity.

In the article by Mani et al. (PMID: 26700561), the activity of ASMC Kv7 channels was monitored in response to activation of cell surface receptors and intracellular signaling regulators to better understand the physiological regulation of these channels. Furthermore, to study how different channel subunits respond to these stimuli, cultured rat aorta ASMCs (A7r5 cells) that naturally express only Kv7.5 subunits were used, and compared with the responses of ASMCs freshly isolated from rat arteries, which predominantly express heterotetrameric Kv7.4/Kv7.5 channels. In addition, human Kv7 channel alpha subunits were artificially introduced into A7r5 cells to compare regulation of human Kv7.4 or human Kv7.5 channels expressed individually or expressed together.

The results indicated that activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), a well-known vasodilatory stimulus, robustly increased the activity of Kv7.5 channels. Activation of cAMP/PKA using chemicals, or by engagement of beta adrenergic receptors that activate this signaling pathway, similarly increased the activity of naturally occurring Kv7.5 channels or of artificially introduced human Kv7.5 channels in A7r5 cells. In contrast, activity of freshly isolated rat artery Kv7.4/Kv7.5 channels or artificially introduced human Kv7.4/7.5 channels were only modestly enhanced, and human Kv7.4 channels were insensitive to activation of this signaling pathway. It was further demonstrated that the changes in activity of the channels by the signaling pathway are dependent on temporary addition of a phosphate group to the Kv7.5 channel subunits. No phosphate additions were detectable by activation of the signaling pathway in cells with artificially introduced Kv7.4 channels.

In summary, these results suggest that the responsiveness of arterial smooth muscle Kv7 channel subunits to intracellular cAMP/PKA signal activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. The differences in Kv7 channel subunit response may have important implications in terms of arterial function, as the Kv7 channel subunit expression patterns may differ among vascular beds and may change during development or with disease.

      

Figure Legend: Phosphorylation of Kv7.5 alpha subunits enhances efflux of potassium ions from arterial smooth muscle cells. Schematic diagram illustrating the signal transduction pathway whereby activation of β-adrenergic receptors (β-Adr) leads to elevation of cytosolic concentrations of cyclic adenosine monophosphate (cAMP) and hence to activation of Protein Kinase A. Protein kinase A can catalyze the transfer of phosphate (P) to Kv7.5 alpha subunits, which increases the opening of channels containing these subunits (Kv7.5 homotetramers or Kv7.4/Kv7.5 heterotetramers; solid arrows). Opening of the channels increases efflux of potassium ions (K+), which promotes smooth muscle relaxation. Kv7.4 alpha subunits are not phosphorylated by Protein Kinase A, and the activity of Kv7.4 homotetramers is not altered by this mechanism (dashed arrow).

 

About The Author

Bharath Mani received his DVM degree in 2001 and MS degree in 2003 from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, and Ph.D (Pharmacology) degree in 2012 from Loyola University Chicago, USA. His doctoral research examined the role of arterial smooth muscle Kv7 channel function in influencing arterial contraction status.

He is now a postdoctoral fellow at the University of Texas Southwestern Medical Center at Dallas, Texas, USA. His current research investigates neuroendocrine regulation of metabolic and cardiovascular function.

Journal Reference

Mol Pharmacol. 2016; 89(3):323-34.

Mani BK, Robakowski C, Brueggemann LI, Cribbs LL, Tripathi A, Majetschak M, and Byron KL.

Loyola University Chicago, Dept. of Molecular Pharmacology & Therapeutics, Maywood, IL 60153, USA.

Abstract

Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth musclecell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth musclecells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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Global Medical Discovery features paper: Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions

Journal Reference

Biochem Pharmacol. 2016;103:129-39.

Saenz-de-Viteri M¹, Fernández-Robredo P², Hernández M³, Bezunartea J⁴, Reiter N⁴, Recalde S², García-Layana A⁵.

Show Affiliations

1. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain.
2. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
3. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain. Electronic address: mahersan@unav.es.
4. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain.
5. Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.

 

Abstract

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under  normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under  normal and  oxidative  stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5days after treatment. Finally, none of the drugs affected migration.

In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Global Medical Discovery features paper: Low-density lipoprotein transport through an arterial wall under hyperthermia and hypertension conditions–An analytical solution

About The Author

Professor Kambiz Vafai received his B.S. in Mechanical Engineering from the University of Minnesota at Minneapolis in 1975. He received his M.S. degree in Mechanical Engineering from the University of California at Berkeley in 1977 and his Ph.D. degree in Mechanical Engineering also from the University of California, Berkeley in 1980. He is currently serves as Distinguished Professor of Mechanical Engineering at the University of California, Riverside, USA.

Professor Vafai holds 12 US patents associated with electronic cooling and medical applications. His research interests include transport through porous media, multiphase transport, aircraft brakes, biomedical applications, microcantilever-based biosensors, biofilms, electronic cooling, macromolecule transport through arteries, cooling enhancement investigations, modeling of tissue and organs, natural convection in complex configurations, analysis of porous insulations, heat flux applications, free surface flows, flat-shaped heat pipes, thermal design and modeling, and feasibility, optimization, and parametric studies for various engineering applications and power electronics. He is one of the highest cited in his fields of research.

He is currently Editor in Chief- Journal of Porous Media and Editor in Chief- Special Topics & Reviews in Porous Media

Journal Reference

J Biomech. 2016;49(2):193-204.

Iasiello M¹, Vafai K², Andreozzi A³, Bianco N³.

Show Affiliations

1. Department of Mechanical Engineering, University of California, Riverside, CA 92521, USA; Dipartimento di Ingegneria Industriale, Università degli Studi di Napoli Federico II, P.le Tecchio, 80, Napoli 80125, Italy.
2. Department of Mechanical Engineering, University of California, Riverside, CA 92521, USA. Electronic address: vafai@engr.ucr.edu.
3. Dipartimento di Ingegneria Industriale, Università degli Studi di Napoli Federico II, P.le Tecchio, 80, Napoli 80125, Italy. 

Abstract

An analytical solution for Low-Density Lipoprotein transport through an  arterial wall  under hyperthermia conditions is established in this work. A four-layer model is used to characterize the arterial wall. Transport governing equations are obtained as a combination between Staverman-Kedem-Katchalsky membrane equations and volume-averaged porous media equations. Temperature and solute transport fields are coupled by means of Ludwig-Soret effect.

Results are in excellent agreement with numerical and analytical literature data  under  isothermal conditions, and with numerical literature data for the hyperthermia case. Effects of hypertension  combined with hyperthermia, are also analyzed in this work.

Copyright © 2015 Elsevier Ltd. All rights reserved.

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Global Medical Discovery features paper: Effect of collagen-glycosaminoglycan scaffold pore size on matrix mineralization and cellular behavior in different cell types

Significance Statement

Bone tissue engineering has emerged as one of the leading fields in tissue engineering and regenerative medicine. Its success relies on understanding the interplay between progenitor cells, regulatory signals, and the biomaterials/scaffolds used to deliver them. Subtle changes in scaffold architecture can have significant effects on cellular activity. Optimising the design of bioactive scaffolds is guided by an understanding of the behaviour and responses of cells to their surrounding environment. Pore size is an essential architectural consideration in construct development; therefore, it is crucial to identify the optimal pore size for augmented tissue formation.

Using a series of collagen-glycosaminoglycan (CG) scaffolds with a homogenous mean pore size ranging from 85 µm up to 325 µm, we identified key differences in osteoblast and mesenchymal stem cell (MSC) behaviour in response to pore size. Scaffolds with the largest pore size (325 µm) facilitated superior osteoblast attachment, migration, scaffold infiltration and matrix deposition. MSC response was similar to osteoblasts but cell motility, proliferation, and scaffold infiltration was reduced. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for collagen-glycosaminoglycan scaffolds compared to MSCs.

This study, for the first time within the literature, compares two very different cell types head to head to investigate individual cell behaviour in response to a single parameter. The findings elucidate fundamental mechanisms underlying the differences between the two cell types and highlight the importance of tailoring scaffold micro-architecture and cell type for cell-specific applications.

About The Author

Dr. Ciara Murphy received her PhD in area of bone tissue engineering from the Royal College of Surgeons in Ireland (RCSI) in 2010. Subsequently, she joined the Orthopaedic & Biotechnology Research (ORB) Group in the Children’s Hospital at Westmead, Sydney, Australia, where she focused her post-doctoral research on developing biologic delivery systems that utilised tissue engineering technologies, including collagen-based scaffolds, as novel therapies for bone healing.

In 2014, she was awarded the New Investigator Recognition Award (NIRA) at the International Orthopaedic Research Society (ORS) for her postdoctoral work. She returned to Ireland in 2015 joining University College Dublin (UCD) as an Assistant Professor in the School of Medicine and a Principal Investigator in the UCD Centre for Biomedical Engineering. Her research focuses on developing advanced biomaterials as innovative platforms for targeted therapeutic delivery, disease model systems and 3-D studies of cell-matrix interactions. 

 

About The Author

A/Prof Garry Duffy leads a multidisciplinary team of biomaterials, stem cell and drug delivery scientists within the Tissue Engineering Research Group (TERG), based in the Royal College of Surgeons in Ireland, with a large focus on chronic diseases. The long-term goal of his lab is to develop advanced biomaterials to facilitate targeted delivery and future clinical translation of cell based therapeutics.  As well as the DRIVE project, Garry also leads the Advanced Materials for Cardiac Regeneration (AMCARE) project, an €8.6 million FP7-funded research programme with the goal of using smart biomaterials and minimally-invasive surgical devices for targeted delivery of stem cells to treat the infarcted myocardium.

About The Author

A/Prof Aaron Schindeler is a Senior Research Scientist at The Children’s Hospital at Westmead and the Director of Basic Research in the Centre for Children’s Bone & Musculoskeletal Health (CCBMH). He joined the orthopaedic research department in 2003 and since then has tackled a range of research questions looking at traumatic bone injuries and genetic diseases affecting. Aaron leads a multidisciplinary team of scientists, engineers, and medical and allied health professionals. Key research areas for him include reducing the risk and impact of fracture and implant infection, cell and genetic therapies for brittle bone disease, studying the metabolic muscle weakness associated with neurofibromatosis type 1, and bone tissue engineering using novel biomaterials and 3D printing. 

About The Author

Prof Fergal O’Brien is a leading innovator in the development of advanced biomaterials for drug delivery and tissue repair. He is Professor of Bioengineering & Regenerative Medicine, Deputy Director for Research and heads the Tissue Engineering Research Group based in the Royal College of Surgeons in Ireland. He is also a PI and Deputy Director of the €58 million SFI-funded Advanced Materials and Bioengineering Research (AMBER) Centre. He is currently a member of the World Council of Biomechanics, Biomaterials Topic Chair for the Orthopaedic Research Society and President of the Section of Bioengineering of the Royal Academy of Medicine in Ireland.

Since his faculty appointment in 2003, he has published over 150 journal articles in leading peer-reviewed international journals and supervised 30 doctoral candidates to completion. He has a current h-index of 47.  Accolades include a Fulbright Scholarship (2001), New Investigator Recognition Award by the Orthopaedic Research Society (2002), Science Foundation Ireland, President of Ireland Young Researcher Award (€1.1. million, 2004), Engineers Ireland Chartered Engineer of the Year (2005), European Research Council (ERC) Investigator Award (€2 million, 2009),  Anatomical Society New Fellow of the Year (2014) and Fellowship of Engineers Ireland (2013) and the European Alliance for Medical & Biological Engineering Science (2016).

 

Journal Reference

J Biomed Mater Res A. 2016;104(1):291-304. 

Murphy CM^1,2,3, Duffy GP^2,3,4, Schindeler A^5,6, O’brien FJ^2,3,4.

Show Affiliations

1. School of Medicine & Medical Science, University College Dublin, Dublin, Ireland.
2. Tissue Engineering Research Group, Department of Anatomy, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland.
3. Trinity Centre for Bioengineering, Trinity College Dublin (TCD), Dublin, Ireland.
4. Advanced Materials and Bioengineering Research Centre (AMBER) RCSI & TCD, Dublin, Ireland.
5. Orthopaedic Research & Biotechnology Unit the Children’s Hospital at Westmead.
6. Discipline of Paediatrics and Child Health, University of Sydney, Sydney, Australia.

Abstract

We have previously examined osteoblast behavior on porous collagen-glycosaminoglycan (CG) scaffolds with a range of mean pore sizes demonstrating superior cell attachment and migration in scaffolds with the largest pores (325 μm). Scaffolds provide a framework for construct development; therefore, it is crucial to identify the optimal pore size for augmented tissue formation. Utilizing the same range of scaffolds (85 μm – 325 μm), this study aimed to examine the effects of mean pore size on subsequent osteoblast differentiation and matrix mineralization, and to understand the mechanism by which pore size influences behavior of different cell types. Consequently, primary mesenchymal stem cells (MSCs) were assessed and their behavior compared to osteoblasts.

Results demonstrated that scaffolds with the largest pore size (325 μm) facilitated improved osteoblast infiltration, earlier expression of mature bone markers osteopontin (OPN) and osteocalcin (OCN), and increased mineralization. MSCs responded similarly to osteoblasts whereby cell attachment and scaffold infiltration improved with increasing pore size. However, MSCs showed reduced cell motility, proliferation, and scaffold infiltration compared to osteoblasts. This was associated with differences in the profile of integrin subunits (α2) and collagen receptors (CD44), indicating that osteoblasts have a stronger affinity for collagen-glycosaminoglycan scaffolds compared to MSCs.

In summary, these results reveal how larger pores promote improved cell infiltration, essential for construct development, however the optimal scaffold pore size can be cell type specific. As such, this study highlights a necessity to tailor both scaffold micro-architecture and cell-type when designing constructs for successful bone tissue engineering applications.

© 2015 Wiley Periodicals, Inc.

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Global Medical Discovery features paper: Highly specific quantification of microRNA by coupling probe-rolling circle amplification and Förster resonance energy transfer

Significance Statement

The first microRNA (miRNA) was discovered in C. elegans in 1993. Subsequently, miRNAs have been identified as a class of small non-coding RNAs containing around 21 nucleotides and virtually exist in all mammalian cells. Currently, miRNAs have been regarded as a class of potential diagnostic markers or therapeutic targets for cancer, cardiovascular and others diseases.

The short length, the similarity of nucleotide sequence and extremely low level in living cells have seriously limited the detection of miRNAs with ultimate sensitivity, high specificity and superior accuracy. Up to date, such a limitation has not been adequately overcome by reported methods. In this paper, the authors presented a novel approach (pRCA-FRET) for quantitative detection of miRNA based on a sequential combination of padlock probe-rolling circle amplification (p-RCA) and förster resonance energy transfer (FRET). First, the padlock probe is specifically ligated and circularized with the target miRNA to produce a long ssDNA by p-RCA in the presence of a DNA polymerase. In this step, p-RCA can superiorly distinguish the mismatch between miRNA and padlock probe and increase the specificity towards target miRNAs. Then, FRET probes labeled with florescent groups Cy3 and Cy5 are used to hybridize the ssDNA from p-RCA. After the excitation of the donor fluorophore Cy3, detectable readouts from the fluorescence emission of Cy5 are generated by FRET. Consequently, the target miRNA can be quantified by analyzing the intensity of fluorescent emission. Compared to previous methods, the sensitivity and operational simplicity have obviously been improved in the step of FRET without complex PCR amplification, accompanying a further enhancement of specificity for miRNA detection.

Using pRCA-FRET, the detection limit of miRNA quantification is markedly reduced from fM level to 103 aM, and remarkable specificity is exemplified to differentiate single-base mismatch between target miRNA and similar miRNAs. Accordingly, this pRCA-FRET has the potential to quantify low amount of miRNA with excellent sensitivity and specificity for the exploration of the biological functions of miRNAs and their clinical applications.  

About The Author

Prof. Dr. Yijun Chen received a B.S. degree in pharmaceutical science at China Pharmaceutical University, Nanjing, China, in 1982 and completed his Ph.D. degree in medicinal and natural products chemistry at University of Iowa, Iowa, USA, in 1996 under the guidance of Professor John P. N. Rosazza. After his postdoctoral research with Professor Edward A. Dennis at University of California, San Diego, he worked as a Senior Scientist at MicroGenomics, Inc., California, USA from 1998 to 1999. From 1999 to 2006, he served as a Senior Research Investigator at Lead Discovery and Enzyme Technology Departments of Bristol-Myers Squibb Pharmaceutical Research Institute in New Jersey, USA. Since 2007, he has been appointed as an Endowed Professor and Director of Laboratory of Chemical Biology at China Pharmaceutical University, and he presently is also an Adjunct Professor of Department of Chemical Biology, Rutgers University, USA. Currently, he has published over 80 peer-reviewed papers and invented more than 40 patents. The research of Dr. Chen’s group focuses on discovery and validation of novel antitumor targets using chemical proteomics approach, elucidation and manipulation of biosynthetic pathways for microbial secondary metabolites and development of biocatalytic routes for chiral drug intermediates. 

Journal Reference

Anal Biochem. 2016 Jun 1;502:16-23.

Wu X¹, Zhu S¹, Huang P¹, Chen Y².

Show Affiliations

1. State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China.
2. State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing, Jiangsu Province, 210009, People’s Republic of China. Electronic address: yjchen@cpu.edu.cn. 

Abstract

MicroRNA (miRNA) plays vital roles in various biological processes. In general, sensitivity and specificity are the major parameters for the quantification of miRNA. In this study, padlock probe-rolling circle amplification and Förster resonance energy transfer (pRCA-FRET) were coupled for specific and quantitative detection of miRNA. pRCA-FRET showed superior specificity to differentiate single-base mismatch and excellent sensitivity with a detection limit of 103 aM. The current method has the potential to quantify low amounts of miRNA in the same family for studies on their biological functions.

Copyright © 2016 Elsevier Inc. All rights reserved.

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Global Medical Discovery features paper: Diagnostic Utility of Flow Cytometry Analysis of Reactive T Cells in Nodular Lymphocyte-Predominant Hodgkin Lymphoma

About The Author

James Huang, M.D.

Oakland University William Beaumont School of Medicine, Rochester, MI, USA

James Huang undertook his early medical studies at Hunan Medical University, Changsha, China, completing a research fellowship at Harvard Medical School, a pathology residency at Dartmouth-Hitchcock Medical Center, a hematopathology fellowship in University of Nebraska Medical Center. He is currently an Associate Professor of Pathology, Co-Director of Diagnostic Medicine Clerkship, and Director of Clinical Pathology Elective at Oakland University William Beaumont School of Medicine. He is also a senior staff hematopathologist at Beaumont Health.

Dr. Huang is on the Editorial Board of a number of journals including International Journal of Pathology and Clinical Research, the Clinics in Oncology, and Pathology- Remedy Open Access.  

About The Author

James David, M.D.

Oakland University William Beaumont School of Medicine, Rochester, MI, USA

James David graduated from Oakland University William Beaumont School of Medicine. This research was his capstone project for which he received a competitive scholarship award form Oakland University William Beaumont School of Medicine. He is currently in residency program in ophthalmology at Louisiana State University-Ochsner, Louisiana.  

Journal Reference

Am J Clin Pathol. 2016 Jan;145(1):107-15.

David JA¹, Huang JZ².

Show Affiliations

1. From the Department of Pathology, Oakland University William Beaumont School of Medicine, Rochester, MI;
2. From the Department of Pathology, Oakland University William Beaumont School of Medicine, Rochester, MI; Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, MI. james.huang@beaumont.edu.

Abstract

OBJECTIVES:

This study aims to define the diagnostic utility of flow cytometric features of T cells in  nodular  lymphocyte- predominant Hodgkin lymphoma (NLPHL).

METHODS:

Cases were retrospectively identified based on diagnosis with NLPHL (n = 30 samples), classic  Hodgkin lymphoma (CHL; n = 33), and reactive lymphoid hyperplasia (RLH; n = 43). Pathology slides were reviewed. Flow cytometry list mode data were reanalyzed.

RESULTS:

The mean proportion of CD4 + CD8 + T cells (8.4%) in cases of NLPHL was significantly higher than seen in CHL (1.0%) or RLH (0.6%). Of the T cells, 28.4% were CD57 + in NLPHL, significantly higher (P < .05) than in CHL (3.2%) or RLH (3.2%). Based on receiver operating characteristic curve analysis, when using a cutoff of 3.0% of CD4 + CD8 + T cells, the diagnostic sensitivity for NLPHL is 83.3% with a specificity of 97.4%. The diagnostic sensitivity was 96.7% with a specificity of 98.7% when using a cutoff of 12% for CD57 + T cells.

CONCLUSIONS:

Increased portions of CD57 + T cells and CD4 + CD8 + T cells are highly suggestive of the possibility of NLPHL. In addition, NLPHL diagnosis appears unlikely if neither CD57 + T cells nor CD4 + CD8 + T cells are increased. Future prospective studies including cases of progressive transformation of germinal center and T-cell/histiocyte-rich large B-cell lymphoma will further define the  utility  of  flow cytometry of T cells in NLPHL.

© American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

 

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Global Medical Discovery features paper: Kir3 channels undergo arrestin-dependant internalization following delta opioid receptor activation

Significance Statement

Protein-protein interactions are of great importance for virtually all biological processes and whether transient or stable, they support the formation of multimeric complexes. Monitoring such interactions may help us characterize signaling and trafficking behaviours of these complexes and sometimes allows us to elucidate new signaling pathways for a known protein. A better and detailed knowledge of these different aspects of complex function is essential not only to understand the majority of physiological processes but also for the development of new therapeutic ligands.

In this article, we characterized protein-protein interactions within a complex involved in opioid analgesia which is formed by delta opioid receptors (DORs), heterotrimeric G protein (Gαoβ1γ2) and their effector, the G protein-gated inwardly rectifying potassium channel (GIRK/Kir3). It is well established that sustained DOR stimulation by an agonist triggers a series of adaptive changes that reduce receptor ability to signal and this desensitization may contribute to analgesic tolerance. Although there is considerable information of how desensitization reduces receptor ability to interact and activate the G protein, much less is known on how desensitization modifies the channel standing in the complex. We therefore focused on how interactions between the channel and other complex components were modified by sustained receptor activation.

Our results show that DORs, G protein and Kir3 channels form a constitutive complex at the plasma membrane. This complex undergoes rapid conformational rearrangements upon acute DOR stimulation and maintains its integrity over more prolonged periods of receptor activation. During this time, the DOR/G protein/Kir3 complex undergoes additional conformational changes imposed by βarrestin 2 (βarr2) recruitment and association with receptors and channels. This interaction not only induces DOR removal from the membrane but also that of the channel. Both signaling partners are concomitantly internalized via a clathrin and dynamin-dependent mechanism.

Conclusion: Taken together, these data show that DORs and Kir3 channels form a constitutive complex which is recognized and internalized as a signaling unit by βarr2.

Contribution to the advancement of knowledge: Kir3 channels removal from the membrane represents an additional level of regulation of opioid receptor signaling that had not been previously described. Moreover, given active Kir3 channels participation in opioid analgesia, their removal from the membrane may constitute an additional and powerful mechanism of tolerance. Thus, it is reasonable to expect that development of DOR ligands that activate the channel but could prevent complex interaction with βarr2 could lead to the production of opioid analgesics that preserve their therapeutic efficacy.

Highlights

– Kir3.1/3.2 channels, G proteins and DORs form a complex.

– The complex maintains its integrity over prolonged periods of receptor stimulation.

– βarr2 is recruited to DORs and channels mediating their internalization as a unit.

– DOR-Kir3 channel internalization is clathrin/dynamin dependent.

Figure Legend. Mechanism of analgesia induced by Kir3 channels at the synaptic cleft.

When released into the synaptic cleft, neurotransmitters such as endogenous opioids (pink) activate the DOR receptor (red) of the postsynaptic neuron, which in turn activate the Kir3 channel (green). Activation of Kir3 channels produces hyperpolarization at the postsynaptic membrane thereby reducing the transmission of nociceptive impulses.

About The Author

Dr. Karim Nagi received his Bachelor’s degree in Biology from the Lebanese University, Tripoli, Lebanon (2005-2008). He then moved to Canada where he completed one year in basic research in Molecular Cardiology and Genetics at Sacré-Cœur Hospital’s Research Center, Montreal, Canada. In 2010, he entered the MSc program at the Department of Pharmacology, University of Montreal under the supervision of Prof. Pineyro at Sainte-Justine Hospital Research Center. By the end of his first year, he was offered accelerated switch to the PhD program which he completed in 2015. During this time Dr Nagi applied a variety of approaches in biochemistry and neuroscience to investigate the network properties of GPCR signaling with particular focus in the analgesic actions of opioid receptor ligands and their potential to induce tolerance.

Dr. Nagi has authored 7 publications in peer-reviewed journals and 50+ presentations. In addition, he received eight awards for the best oral and poster presentations in scientific conferences, two travel awards to international congresses and his studies were supported by a number of fellowship awards including CHU Sainte-Justine and Foundation of Stars fellowship, fellowship from the Department of Pharmacology and a fellowship from the Faculty of Graduate and Postdoctoral Studies, University of Montreal. He was also granted a Recognition Award for the Best Scientific Contribution of the year (2014-2015) among students in the Department of Pharmacology.

After completing graduate studies, Dr. Nagi continued his training as a postdoctoral fellow in the Department of Cellular Biology, Duke University, Durham, USA under the supervision of Prof. Marc G. Caron. His current research focuses on characterizing different biased receptors signaling and regulation.

Working at these different institutions with a world-renowned reputation in pharmacological research, Dr. Nagi has developed expertise in GPCRs pharmacology, BRET-based biosensor development for drug screening, molecular biology and biased signaling.

About The Author

Graciela Pineyro, MD, Ph.D. 

Prof. Graciela Pineyro is a Full Professor at the Department of Psychiatry, University of Montreal, Montreal, Canada. In 1991, she received her medical degree with specialty in Pharmacology from the Faculty of Medicine, National University, Uruguay. She then moved to Canada where she obtained a Ph.D. in Neurosciences from McGill University, Montreal, Canada (1997) followed by postdoctoral training in molecular pharmacology in the Department of Biochemistry, University of Montréal (1997-2001). During her career she was supported by different fellowships and awards including McGill Major Fellowships (Canada), Fogarty-NIH International Fellowship (USA) and Postdoctoral Fellowships from Medical Research Council of Canada and Heart and Stroke Foundation of Canada.

Today, she is head of a pharmacology laboratory with research focus on molecular determinants of analgesic efficacy of opioids, as well as cellular and molecular bases of analgesic tolerance. She has substantially contributed to the notion of biased signaling showing that delta opioid receptors adopt ligand-specific conformations with distinct signaling and trafficking properties. Insights from her research have provided the basis for the rational development of novel opioid analgesics with a reduced side effects profile.

Prof. Pineyro has authored 37 peer-reviewed publications, 7 book chapters and 100+ presentations, and holds 2 licensed patents.

As an independent investigator, she has received the New Investigator Award from Fond de Recherche en Santé du Québec and her research has been continuously funded by Canadian Institutes of Health Research and Natural Sciences and Engineering Research Council of Canada.

About The Author

Iness Charfi, MSc 

Iness Charfi received her Bachelor in Pharmacy in 2008 at Monastir University (Tunisia). In 2009, she joined Prof. Graciela Pineyro’s Lab as an MSc student in Neuropsychopharmacology at the Sainte-Justine Hospital Research Center, through the Department of Pharmacology, University of Montreal (Canada). After graduating in 2012, she started a PhD in the same laboratory. Throughout her training, she focused on the mechanistic understanding of the molecular basis of delta opioid receptor post-endocytic trafficking, in order to better understand the development of analgesic tolerance to opioids. Iness has been awarded a number of prizes and fellowships, including two presentation awards for best oral and poster presentations at scientific meetings. Her fellowship awards include a CHU Sainte-Justine and Fondation of Stars fellowship, a recruitment fellowship from the Department of Pharmacology, University of Montreal and a fellowship award from the FRSQ.

 

Journal Reference

Cell Mol Life Sci. 2015 Sep;72(18):3543-57.

Karim Nagi^1,2, Iness Charfi^1,2 and Graciela Pineyro^1,2,3

Show Affiliations

1. Sainte-Justine Hospital Research Center, Montreal, Quebec, H3T 1C5, Canada.
2. Department of Pharmacology, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.
3. Department of Psychiatry, Faculty of Medicine, University of Montreal, Montreal, Quebec, H3T 1J4, Canada.

Abstract 

Kir3 channels control excitability in the nervous system and the heart. Their surface expression is strictly regulated but mechanisms responsible for channel removal from the membrane remain incompletely understood. Using transfected cells, we show that Kir3.1/3.2 channels and delta opioid receptors (DORs) associate in a complex which persists during receptor activation, behaving as a scaffold that allows beta-arrestin (βarr) to interact with both signaling partners. This organization favored co-internalization of DORs and Kir3 channels in a βarr-dependent manner via a clathrin/dynamin-mediated endocytic path. Taken together, these findings identify a new way of modulating Kir3 channel availability at the membrane and assign a putatively novel role for βarrs in regulating canonical effectors for G protein-coupled receptors.

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