Biosciences Institute Research Articles

Inquérito sorológico para paramixovirus ofídico em serpentes cativas

O manejo de serpentes em cativeiro é um desafio permanente para os criadores, principalmente no que tange ao controle de doenças. Parasitoses, problemas gastrointestinais e viroses respiratórias têm sido as principais enfermidades registradas em serpentários e coleções particulares. A maior preocupação é a paramixovirose ofídica, causada pelo Ophidian paramyxovirus (OPMV), que acomete primariamente o sistema respiratório podendo envolver também o nervoso, sendo muitas vezes fatal e que já foi descrita em colubrídeos, elapídeos, viperídeos, boídeos e pitonídeos. O OPMV é transmitido por aerossóis e pelo contato direto com secreções respiratórias, fômites, ectoparasitas tais como os carrapatos e as excretas digestivas. Não há tratamento específico nem vacinas disponíveis para esta virose. Os procedimentos laboratoriais utilizados para o diagnóstico da infecção incluem: isolamento viral em cultivo celular; microscopia eletrônica; imunoistoquímica; reação em cadeia da polimerase (PCR); ou pode-se ainda trabalhar com a quantificação de anticorpos pelo teste de inibição de hemaglutinação (HI) e pelo teste de ELISA (Enzyme Linked Immuno Sorbent Assay) com bloqueio na fase líquida. O presente estudo empregou a técnica de ELISA com bloqueio de fase líquida para investigar a presença de anticorpos anti-OPMV em 42 serpentes, das quais 36 peçonhentas e seis não peçonhentas alojadas no Centro de Estudos de Venenos e Animais Peçonhentos da UNESP - CEVAP. O sangue foi coletado da veia caudal ventral e o soro foi enviado ao Laboratório de Virologia do Instituto de Biociências de Botucatu - UNESP. A prevalência de soropositivos foi de 9,5% (quatro animais), todos pertencentes à espécie Crotalus durissus terrificus. Destes, três apresentavam os maiores títulos de anticorpos (forte positivo) e estavam alojados no serpentário semiextensivo e um, com o menor título (fraco positivo), no intensivo. É sabido que no ambiente semiextensivo, onde os animais estão em contato direto e com a mesma fonte de água, a possibilidade de contágio aumenta. O resultado dos animais fortemente positivos sugere a existência de contato recente com o vírus, porém, sem que os animais expressem sinais de doença. A serpente considerada fraca positiva, alojada no cativeiro intensivo, foi sobrevivente de um surto ocorrido há 12 anos no CEVAP, o que demonstra que os anticorpos podem persistir por anos. Ainda não se sabe se os anticorpos são protetores ou apenas sinalizadores do contágio e se estes animais podem ser considerados como reservatórios da doença. A paramixovirose ofídica é uma doença silenciosa, altamente contagiosa e com muitos casos de morte súbita. Por fim, estudos do tipo caso-controle deverão ser conduzidos para se saber se a presença destes anticorpos indica resistência viral ou reservatório. Neste último caso, a adequada profilaxia da doença incluirá o isolamento dos animais sorologicamente positivos.

Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria.

As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P < 0.001). Over 1800 proteins were identified, of which 152 were different (P < 0.05) by at least 1.3 fold and ≤ 15 fold between the high and low FE phenotypes. Data were analyzed for a modified differential expression (DE) metric (Phenotypic Impact Factors or PIF) and interpretation of protein expression data facilitated using the Ingenuity Pathway Analysis (IPA) program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P < 0.00001). Within the top up and down 5% PIF molecules in the dataset, there were 15 mitoproteome proteins up-regulated and only 5 down-regulated in the high FE phenotype. Pathway enrichment analysis also identified mitochondrial dysfunction and oxidative phosphorylation as the number 1 and 5 differentially expressed canonical pathways (up-regulated in high FE) in the proteomic dataset. Upstream analysis (based on DE of downstream molecules) predicted that insulin receptor, insulin like growth receptor 1, nuclear factor, erythroid 2-like 2, AMP activated protein kinase (α subunit), progesterone and triiodothyronine would be activated in the high FE phenotype whereas rapamycin independent companion of target of rapamycin, mitogen activated protein kinase 4, and serum response factor would be inhibited in the high FE phenotype. The results provide additional insight into the fundamental molecular landscape of feed efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE.Funding provided by USDA-NIFA (#2013–01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville).

Sarcopenic Obesity Alters Ecm-related Gene Expression In Mouse Skeletal Muscle

Sarcopenic obesity is a metabolic syndrome where excess adipose tissue and a decline of muscle due to age results in an increased risk of disability. This is a concern in the United States because older adults are the fastest growing obese population. The extracellular matrix (ECM) in skeletal muscle is essential because it assists in stability, force transmission, and muscle remodeling. Both sarcopenia and obesity are associated with increased fibrosis, however it is not known how these co-morbidities interact during sarcopenic obesity to affect the ECM. PURPOSE: To determine if skeletal muscle ECM-related gene expression is impaired in sarcopenic obese mice. METHODS: Twelve young (3-4 months old) male C57/BL6J mice and twelve aged (22-24 months old) male mice were randomly assigned a normal chow or a high-fat diet (HFD, 60% fat) at 4 weeks of age. The gastrocnemius was excised for further analysis. ECM-related markers were determined by qPCR. Data were analyzed by two-way ANOVA and post hoc Fisher’s LSD. RESULTS: There were significant interactions in collagen I, collagen III, fibronectin, and matrix metalloproteinase 2 (MMP-2) (p<0.05). There was a 9-fold increase in collagen I gene expression in young HFD mice compared to young lean mice (p<0.05). However, there was no differences in collagen I in aged HFD and aged lean mice. There was a 3-fold increase in collagen III in young HFD mice compared to young lean mice (p<0.05). However, there was a 73% reduction in collagen III in aged HFD mice compared to aged lean mice (p<0.05). There was a 2-fold increase in fibronectin in young HFD mice compared to young lean mice (p<0.05). However, there was no difference in fibronectin in aged HFD and aged lean mice. There was a 2-fold increase in MMP-2 in young HFD mice compared to young lean mice (p<0.05). However, there was a 45% reduction in MMP-2 in aged HFD mice compared to aged lean mice (p<0.05). There was a main effect of diet to decrease MMP-9 and myoD in obese mice (p<0.05). There was a main effect of age to increase myoD in aged mice (p<0.05). There were no differences in TGF-β, myogenin, and TIMP-1. CONCLUSIONS: Aging and obesity altered ECM-related gene expression that may be partially responsible for the reduction in muscle function observed in sarcopenic obese individuals. The study was funded by the Arkansas Bioscience Institute.

Differential Effects of Sarcopenic Obesity on Markers of Protein Synthesis in Hearts of C57BL/6J Mice

In 1990, 23.6% of the elderly population (60+ years old) was obese (≥30 BMI). By 2010, it increased to 39.5%. Pathological, cardiac hypertrophy is highly associated with both age and obesity. As the prevalence of obesity in the elderly population continues to rise, a firmer understanding of how age and obesity interact to affect cardiac function is needed. PURPOSE: To examine how sarcopenic obesity affects markers of protein synthesis. METHODS: Twenty-four C57BL/6J mice were evenly distributed into either a normal chow (17% kcals from fat) or high-fat (60% kcals from fat) diet after weaning. Six mice from each diet were euthanized at 3-4 months of age (young) or 22-24 months of age (aged). The classification of the 4 groups is as follows: Young Lean (YL), Young Obese (YO), Aged Lean (AL), Aged Obese (AO). Hearts were excised, snap-frozen, and processed for protein expression via western blot. All protein targets are reported as a ratio of phosphorylated to total protein. RESULTS: There was an interaction of age and obesity on heart mass:tibia length. Heart mass:tibia length was 11 % higher in YO compared to YL and 37% greater in AO compared to AL (p < 0.05). Also, AO heart mass:tibia length was 40% greater compared to YO (p < 0.05). There was a main effect of age to increase both pAkt/Akt (p < 0.05) and pmTOR/mTOR (p < 0.05). There was an interaction of age and obesity on p4EBP-1/4EBP-1 expression. p4EBP-1/4EBP-1 was lower in YO compared to YL, AL, and AO (p < 0.05). We detected an interaction of age and obesity on pp70S6K/p70S6K expression. pp70S6K/p70S6K was 47% higher in YO compared to YL (p < 0.05); however, pp70S6K/p70S6k expression was not different between AL and AO mice. We observed an interaction of age and obesity on pAMPK/AMPK expression. pAMPK/AMPK expression was 329% higher (p < 0.05) in YL compared to YO but 46% lower (p < 0.05) in AO compared to AL. CONCLUSION: Sarcopenic obesity induced differential expression of protein synthesis markers. Obesity mediates cardiac hypertrophy through different pathways in the aging and young groups. Cardio protective processes may be downregulated in the sarcopenic obese group as represented by the loss of AMPK in that group. This work was supported by a grant from the American Biosciences Institute and the animals were a kind gift from Rigel Pharmaceuticals.

Litter Size Reduction Alters the Lipid Metabolism of Adult Male Rat Offspring of Dams Fed a Diet with Caloric Restriction

Recent studies have showed that nutritional changes during critical periods of development can lead to permanent alterations in the metabolism of the offspring which can become more susceptible to chronic diseases in adult age. The aim of this study was to evaluate the effect of litter size on the body weight and the lipid metabolism of adult male offspring rats born to caloric restricted dams. Dams were fed with either ad libitum standard diet (C) or a 30% caloric restricted diet (R) throughout gestation. After birth, litters were reduced to eight pups or four pups in both C and R dams. All dams were fed ad libitum during lactation. After weaning (3 wk), the male rat offspring born to control dams (C8 and C4) and male rat offspring born to restricted dams (R8 and R4) were maintained in individual cages until they were 20 weeks old. An interaction between the diet of control or restricted dams and the litter size was observed for the hepatic lipid % (P=0.015). Reduction of litter size decreased the hepatic lipid % in control rats but increased it in rats to restricted dams. The caloric restriction in the dams decreased the hepatic lipids only in the large litter offspring, but no effect on the small litter was observed. There was no interaction between litter size reduction and the diet of the dams (P=0.689). The reduction of litter size induced an increase (P=0.035) of carcass weight, regardless the diet of the dams. No effects of caloric restriction during gestation and of the litter size on body weight (P=0.187) and on blood cholesterol (P=0.171) were found in C or R adult male rat offspring. It is concluded that the effect of reduction of litter size on hepatic lipid metabolism depends on the diet of the dams during gestation. The increase of carcass weight resulting from litter size reduction is not affected by the diet of the dams.Support or Funding InformationResearch supported by Institute of Biosciences

Aqueous Co-Electropolymerization of Thieno[3,4-b]-1,4-Dioxin-2-Methanol and a Synthesized Derivative

Thiophene-containing conducting polymers have been used in a range of applications, including supercapacitors, amperometric biosensors, and microfluidic pumping systems.1 This versatility is based on the substituents of the thiophene and desired chemical properties that can be tailored through various organic syntheses.2 One particular thiophene polymer of significant interest is poly(3,4-ethylenedioxythiophene) (PEDOT) due to its high conductivity, low oxidation potential, reversible electrochemical doping, and stability in aqueous environments.3 Hydrophobic in nature, electropolymerization of PEDOT in non-aqueous media does not require additional reagents or processing. However if the application requires aqueous media, as is the case for co-electropolymerization with enzymes, then solubilizing reagents, such as beta cyclodextrin (β-CD), and solubilization processes, such as lengthy sonication, are required. PEDOT also lacks additional functional groups that could be used to easily derivatize its structure and tailor its chemical properties. A commercially available thiophene derivative that is similar to PEDOT in structure but soluble in aqueous media is thieno[3,4-b]-1,4-dioxin-2-methanol (EDOT-CH2-OH). Electrochemically deposited PEDOT-CH2-OH in aqueous conditions exhibits characteristics such as increased surface area and charge capacity similar to that of PEDOT films. Additionally the hydroxyl group allows for further modification by established synthetic protocols. One such reaction is esterification of the hydroxyl group with succinic anhydride yielding 4-((2,3-dihydrothieno[3,4-b]-1,4-dioxin-2-yl)-methoxy)-4-oxobutanoic acid (EDOT-CH2-OCO-(CH2)2-COOH).4 The extended carboxylic acid moiety from the polymer backbone provides a location for covalent attachment of enzymes and other molecules of interest. Despite the functional advantages of the monomer EDOT-CH2-OCO-(CH2)2-COOH its dissolution in aqueous conditions is pH dependent and a pristine film results in lower charge capacity than PEDOT-CH2-OH. Recent developments on the electropolymerization of PEDOT-CH2-OH and EDOT-CH2-OCO-(CH2)2-COOH in aqueous conditions on gold electrodes will be presented. In this work variations of pH and molar ratios of the monomers were made to optimize electropolymerization and electrochemical behavior. It was observed that although an alkaline pH allows for rapid and complete dissolution of EDOT-CH2-OCO-(CH2)2-COOH in aqueous conditions, the resulting solution did not produce electropolymerized films. It is believed that charge repulsion between the monomers prevents film deposition. In acidic conditions the dissolution of EDOT-CH2-OCO-(CH2)2-COOH took more time but resulted in stable films. Therefore it was of interest to determine the optimal pH and molar ratio between both monomers that resulted in facile co-electropolymerization and films with the electrochemical behavior of PEDOT-CH2-OH and chemical functionality of EDOT-CH2-OCO-(CH2)2-COOH. Films were deposited by cyclic voltammetry using a three electrode electrochemical cell with gold as the working electrodes, a Ag/AgCl in saturated KCl as the reference, and Pt foil as the counter electrode. ACKNOWLEDGMENTS Research was partially supported through the National Science Foundation (CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. REFERENCES 1. (a) Roncali, J., Conjugated poly(thiophenes): synthesis, functionalization, and applications. Chem. Rev. 1992, 92 (4), 711-38; (b) Roncali, J.; Blanchard, P.; Frère, P., 3,4-Ethylenedioxythiophene (EDOT) as a versatile building block for advanced functional π-conjugated systems. Journal of Materials Chemistry 2005, 15 (16), 1589; (c) Hiller, M.; Kranz, C.; Huber, J.; Baeuerle, P.; Schuhmann, W., Amperometric biosensors produced by immobilization of redox enzymes at polythiophene-modified electrode surfaces. Adv. Mater. (Weinheim, Ger.) 1996, 8 (3), 219-22; (d) Nash, C. K.; Fritsch, I., Poly(3,4-ethylenedioxythiophene)-Modified Electrodes for Microfluidics Pumping with Redox-Magnetohydrodynamics (MHD): Improving Compatibility for Broader Applications by Eliminating Addition of Redox Species to Solution. Anal. Chem. (Washington, DC, U. S.) 2015, Ahead of Print. 2. (a) Perepichka, I. F.; Besbes, M.; Levillain, E.; Sallé, M.; Roncali, J., Hydrophilic Oligo(oxyethylene)-Derivatized Poly(3,4-ethylenedioxythiophenes): Cation-Responsive Optoelectroelectrochemical Properties and Solid-State Chromism. Chemistry of Materials 2002, 14 (1), 449-457; (b) Brisset, H.; Navarro, A.-E.; Moustrou, C.; Perepichka, I. F.; Roncali, J., Electrogenerated conjugated polymers incorporating a ferrocene-derivatized-(3,4-ethylenedioxythiophene). Electrochemistry Communications 2004, 6 (3), 249-253. 3. Groenendaal, L. B.; Jonas, F.; Freitag, D.; Pielartzik, H.; Reynolds, J. R., Poly(3,4-ethylenedioxythiophene) and its derivatives: past, present, and future. Adv. Mater. (Weinheim, Ger.) 2000, 12 (7), 481-494. 4. Luo, S.-C.; Ali, E. M.; Tansil, N. C.; Yu, H.-h.; Gao, S.; Kantchev, E. A. B.; Ying, J. Y., Poly(3,4-ethylenedioxythiophene) (PEDOT) nanobiointerfaces: thin, ultrasmooth, and functionalized PEDOT films with in vitro and in vivo biocompatibility. Langmuir : the ACS journal of surfaces and colloids 2008, 24 (15), 8071-8077.

Whey and Pea Protein Influence Energy Metabolism and Appetite Response to a Greater Extent Than Beef Protein

More than 30% of young adults in the US are obese. A primary contributor to obesity is an unhealthy diet related to an imbalance of macronutrients. Diets higher in protein (PRO) are associated with health benefits such as increased energy expenditure (EE) and reduced food intake. The objective of this pilot study was to determine if protein source at breakfast influences EE in young men (n=5; ages 18–35). Participants consumed four isocaloric protein‐based beverages (whey (WP), pea (PP), beef (BP); 275 kcal‐62%PRO:23%CHO:15% Fat) for breakfast in randomized, crossover design with a one‐week washout period between beverages. Each test day EE, appetite and food preference were assessed at 0, 15, 30, 60, 120, 180, and 240 min postprandial. Data was analyzed using a 2‐way ANOVA for the effect of protein source over time. One‐way ANOVA was used to analyze area under the curve (AUC). Resting EE AUC was 8% lower in BP vs PP and 5% lower vs WP. Thermic effect of feeding AUC was 77% lower (P = 0.08) in BP compared to WP; PP was 43% lower than WP. Postprandial carbohydrate oxidation was higher with PP (31%) compared to WP and there was no difference between BP and WP. Postprandial fat oxidation was 23% higher in WP vs BP and PP. WP was more satiating compared to BP and PP, however participants had a higher craving for sweet foods following PP and a higher desire for a snack following BP. Taken together, these data suggest that protein source (animal versus plant) is not a predictor of postprandial EE and appetite response.Support or Funding InformationArkansas Biosciences Institute

Protective effects of sea cucumber surface-associated bacteria against Vibrio harveyi in brown-marbled grouper fingerlings

AEI Aquaculture Environment Interactions Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections AEI 8:147-155 (2016) - DOI: https://doi.org/10.3354/aei00169 Protective effects of sea cucumber surface-associated bacteria against Vibrio harveyi in brown-marbled grouper fingerlings Norfarrah Mohamed Alipiah1,*, Nur Hasli Syahidda Ramli1, Chen-Fei Low1, Mariana Nor Shamsudin1,2, Fatimah Md. Yusoff1,3 1Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia *Corresponding author: norfarrah_80@yahoo.com ABSTRACT: Brown-marbled grouper Epinephelus fuscoguttatus (Forsskål) fingerlings are more vulnerable to diseases than the adult grouper because the fingerlings’ adaptive immune system is still in the development stage. The mortality rates are approximately 20 to 70% during outbreaks of vibriosis in aquacultures of grouper fingerlings. Studies were conducted to identify alternative treatments with low impacts on humans, animals and the environment as well as treatments that minimize the use of antibiotics in aquaculture. In this study, we report the first use of surface-associated bacteria isolated from marine invertebrates to increase fingerlings’ resistance against infections. Twenty-two surface-associated bacteria were isolated from the sea cucumber Stichopus badionotus and were identified with 16S rRNA gene sequencing. Three of the surface-associated bacteria had inhibitory activities against pathogenic Vibrio harveyi and V. parahaemolyticus. The fingerlings treated with the surface-associated bacteria Exiguobacterium acetylicum for 12 d prior to the challenge experiment with pathogenic V. harveyi showed significantly higher survival rates and increases in antibody titres compared to the control group. This study illustrats a symbiotic interaction between E. acetylicum and grouper fingerlings. E. acetylicum colonized the scales of grouper fingerlings and enhanced the fish’s immune response against the pathogen V. harveyi. KEY WORDS: Epinephelus fuscoguttatus · Surface-associated bacteria · Surface colonizer · Symbiotic relation · Bacterial attachment · Bio-control · Marine bacteria · Immune response Full text in pdf format Supplementary material PreviousNextCite this article as: Mohamed Alipiah N, Ramli NHS, Low CF, Shamsudin MN, Yusoff FM (2016) Protective effects of sea cucumber surface-associated bacteria against Vibrio harveyi in brown-marbled grouper fingerlings. Aquacult Environ Interact 8:147-155. https://doi.org/10.3354/aei00169 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AEI Vol. 8. Online publication date: February 25, 2016 Print ISSN: 1869-215X; Online ISSN: 1869-7534 Copyright © 2016 Inter-Research.

Development of bioactive hydrogel foams to accelerate healing of chronic wounds

Development of bioactive hydrogel foams to accelerate healing of chronic wounds

Generation of hemophilia A mouse induced pluripotent stem cells using polycistronic lentiviral vector in serum- and feeder-free culture conditions

Event Abstract Back to Event Generation of hemophilia A mouse induced pluripotent stem cells using polycistronic lentiviral vector in serum- and feeder-free culture conditions Rozita M. Sakri1, Syahril Abdullah2, 3 and Mohamed Saifulaman M. Said1* 1 Universiti Teknologi MARA, Faculty of Applied Sciences, Malaysia 2 Universiti Putra Malaysia, Genetics & Regenerative Medicine Research Centre, Faculty of Medicine & Health Sciences, Malaysia 3 Universiti Putra Malaysia, Institute of Bioscience, Malaysia Induced pluripotent stem cells (iPSCs) research has opened up exciting possibilities in many research areas especially in the field of regenerative medicine. The ability to reprogram back somatic cells into embryonic stem (ES)-like state has made patient-specific cells therapy possible unhindered ethical and practical dilemmas associated with the use of embryonic stem cells [1]. There have been several reports on the generation of iPS cells from murine somatic cells in vitro, however, there are still many challenges to overcome; (i) the induction efficiency is extremely low, (ii) multiple transgene integrations which increase the risk of tumorigenicity of iPS cells produced, (iii) partially differentiated iPS colonies due to incomplete reprogramming, (iv) the use of feeder cells and medium containing serum add complexity and variability in nutrients and factors that contribute to cell growth and the maintenance of pluripotency [2,3,4,5]. To overcome these issues, we utilized polycistronic lentiviral vector carrying the Yamanaka’s factors (Oct4, Sox2, Klf4 and c-Myc) for reprogramming of murine fibroblasts in serum- and feeder- free defined culture conditions. In this study, primary fibroblasts from hemophilic A B6;129S4-F8tm1Kaz/J and wild-type C57BL/6 mouse were transduced with an optimal multiplicity of infection of the virus produced. Suspected iPS cell colonies were picked within 20 days post-transduction. The results showed that iPS cells derived from the wild-type mouse fibroblast were successfully generated in serum- and feeder-free defined culture conditions. The generated wild-type mouse iPS cells are similar to embryonic stem cells in many aspects including morphology, their properties of self-renewal and pluripotency, and in vitro differentiation into the three primary germ layers. However, we were not able to generate iPS cells from hemophilia A mouse fibroblast with the similar lentiviral vector using the same reprogramming manner. We obtained neuronal-like morphology instead of the expected pluripotent cell colonies. The emerging of neuronal-like cells post-transduction is speculated as an incomplete reprogramming process [6]. These partially reprogrammed cells could not maintain the stem cells-like characteristic and eventually undergo differentiation. Hence, these data could provide an insight of reprogramming process. Acknowledgements Special thanks to Faculty of Applied Sciences, UiTM and Genetics & Regenerative Medicine Research Centre, UPM, for their assistance. This study was supported by ERGS Grant, MOHE. Keywords: induced pluripotent stem cell, cellular reprogramming, polycistronic lentivirus Conference: 6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting, Seberang Jaya, Penang, Malaysia, 17 Nov - 18 Nov, 2016. Presentation Type: Poster Topic: Gene Therapy, Reprogramming and Pluripotency Citation: Sakri RM, Abdullah S and Said MM (2016). Generation of hemophilia A mouse induced pluripotent stem cells using polycistronic lentiviral vector in serum- and feeder-free culture conditions. Front. Bioeng. Biotechnol. Conference Abstract: 6th Malaysian Tissue Engineering and Regenerative Medicine Scientific Meeting (6th MTERMS) 2016 and 2nd Malaysian Stem Cell Meeting. doi: 10.3389/conf.FBIOE.2016.02.00028 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Dec 2016; Published Online: 19 Dec 2016. * Correspondence: Dr. Mohamed Saifulaman M Said, Universiti Teknologi MARA, Faculty of Applied Sciences, Shah Alam, Selangor, 40450, Malaysia, dsaifulaman@salam.uitm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Rozita M Sakri Syahril Abdullah Mohamed Saifulaman M Said Google Rozita M Sakri Syahril Abdullah Mohamed Saifulaman M Said Google Scholar Rozita M Sakri Syahril Abdullah Mohamed Saifulaman M Said PubMed Rozita M Sakri Syahril Abdullah Mohamed Saifulaman M Said Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Integrin-specific hydrogels for increased vascularization and bone regeneration of critically-sized bone defects

Event Abstract Back to Event Integrin-specific hydrogels for increased vascularization and bone regeneration of critically-sized bone defects Jose Garcia1, 2 and Andres J. Garcia1, 2 1 Georgia Institute of Technology, Woodruff Scool of Mechanical Engineering, United States 2 Georgia Institute of Technology, Institute of Bioengineering and Biosciences, United States Introduction: Non-healing bone defects and fractures represent a serious clinical problem with over 600,000 bone replacement procedures performed each year. The lack of functional vasculature following trauma or resection of bone has emerged as a major limitation that prevents full regeneration of the defect[1]. While numerous scaffolds have been engineered to increase regeneration of the vascular network, the majority use growth factors at supraphysiological levels which increase the cost of potential therapies. Engineering scaffolds that inherently promote revascularization based on the material’s interaction with surrounding tissue can be an effective strategy for healing the defect. In this study, we investigated how integrin specificity in the biomaterial influences vascularization of defects with the hypothesis that integrin specificity will enhance vascularization. Methods: Hydrogel Synthesis: Integrin-specific hydrogels were engineered by functionalizing 4-arm PEG-maleimide macromers with either the α2β1-specific ‘GFOGER’ peptide[2] or the αvβ3-targeting ‘RGD’ peptide followed by functionalization with vascular endothelial growth factor (VEGF) and cross-linked with a protease-degradable peptide. Vasculogenic protein analysis: Human mesenchymal stem cells (hMSCs) were encapsulated within integrin-specific hydrogels and cultured in osteogenic differentiation media for 7 days. Conditioned media was then assayed for VEGF by ELISA. Critically-sized murine radial defect: For in vivo studies, hydrogels were implanted within a 2.5 mm long critically-sized murine radial bone defect. At 8 weeks, animals were perfused with a radiopaque polymer (Microfil) and µCT analysis performed for vasculature. Bone formation was also analyzed by µCT at 4 and 8 weeks. Results and Discussion: hMSCs cultured within GFOGER-functionalized hydrogels in osteogenic differentiation media exhibited increased secretion of VEGF compared to hMSCs within RGD hydrogels (Fig. 1). In vivo, GFOGER-functionalized VEGF-free hydrogels exhibited significantly increased vascular volume (Fig. 2A), density (Fig. 2B) and resulted in a larger number (counts) of thicker blood vessels compared to RGD-functionalized VEGF-free hydrogels (Fig. 2C-D). The levels of vasculogenesis for VEGF-free GFOGER hydrogels were similar to those of RGD hydrogels delivering 50 ng of VEGF. VEGF-free GFOGER hydrogels also exhibited significantly increased bone volume at 4 and 8 weeks compared to VEGF-free RGD hydrogels (0.028 ± 0.008 mm3 vs 0.004 ± 0.001 mm3 and 0.06 ± 0.02 mm3 vs 0.01 ± 0.006 mm3 for 4 and 8 weeks respectively, mean ±SEM, p<0.05) Conclusion: Whereas growth factors are routinely used to increase vascular regeneration in vivo, their high cost as well as their detrimental effects at supraphysiological levels demands exploration of more viable strategies. Integrin-specificity is a crucial aspect of vascularization and represents a useful tool in engineering constructs for this purpose. Cellular interaction with α2β1-specific hydrogels caused increased secretion of VEGF which correlated with increased vascularization and bone regeneration of a bone defect in vivo. Future biomaterial should consider integrin specificity to reduce or even avoid the use of growth factors. Dr. Nick Willet; Amy Clark; Funding provided by NIH grants R01 AR062368 and R01 AR062920.

Promoting the utilization of plant, animal and microbial genetic resources for research and development in biotechnology: evidence on researchers' preferences for specific attributes from Korean genebanks

With regard to the survey data of Korean researchers using genetic resources from three genebanks administered by the Korea Research Institute of Bioscience and Biotechnology, we conducted conjoint and cluster analyses to estimate the relative importance of genetic resource (microbial, plant and animal) attributes among the researchers. Our results indicate that Korean researchers view price (cost of acquiring an accession), high new functionality (functional properties of germplasms for specific applications and uses) and completely uncovered genomic information (about mutation, genetic transformation, genomic function and pathways) as far more important in decision-making about R&amp;D use of microbial, plant and animal genetic resources, respectively, than other attributes. Furthermore, this study shows that researchers conducting R&amp;D in the microbial and plant genetic resource sectors especially prefer resources from specific domestic environments and Korean indigenous species, respectively. The study also sheds light on different patterns of researcher segments in terms of utilities of attributes and subgroups of researchers who have common needs in the three genetic resource sectors. We proposed some policy and strategic implications based on the results of this study.

Abstract 3456: MUC16/CA125, TNFα and IFNγ are co-expressed in malignant gynecologic neoplasms

Abstract MUC16 is a very large, heavily glycosylated cell surface mucin that carries the CA125 epitope, a well-known molecular marker for human epithelial ovarian cancer. MUC16 is primarily found at the apical surface of normal simple epithelia serving various functions including lubrication and protection against pathogenic infections. MUC16 is aberrantly over-expressed in many diseases including cancers of the ovary, uterus, breast, and pancreas. MUC16 also may support disease progression and ectopic cell colonization in cancer and endometriosis via binding to cell surface proteins including mesothelin and Siglec-9. We demonstrate that MUC16 expression is highly stimulated by the proinflammatory cytokines, tumor necrosis factor α (TNFα) and interferon γ (IFNγ), in multiple cell lines of various origins, an effect mediated by NFκB. MUC16 mRNA and protein expression is mildly stimulated by low concentrations of TNFα (2.5ng/ml) or IFNγ (20 IU/ml) alone. Interestingly, combined treatment with both cytokines at these concentrations resulted in a moderate to large stimulation of MUC16 mRNA and protein expression in a variety of cell contexts indicating that this may be a general response. To further confirm our in vitro results, the multi-cancer tissue-microarrays were used to perform immunohistochemistry testing with anti-Ca125, anti-TNFα and anti-IFNγ antibodies, respectively. Our results demonstrated that MUC16 expression is positively correlated with the TNFα and IFNγ expressions in multiple human cancers, however the most significant positive correlations between MUC16 expression with the TNFα and IFNγ expressions were observed in ovarian and breast cancer. Of the 94 tumors stained, 64 (70.3%) were positive for TNFα, IFNγ, and MUC16 expression. Only 8 cases (8.8%) were negative for all three protein markers, while 18 cases (19.8%) were MUC16 negative but positive for the proinflammatory cytokines, and 1 case (1.1%) was MUC16 positive but negative for both TNFα and IFNγ cytokines. These studies provide in vivo documentation of the synergistic effect tumor necrosis factor α and interferon γ have on MUC16 expression across several disease sites demonstrating that MUC16 is a target of proinflammatory cytokines, which may contribute to mucosal defense, and progression of MUC16-expressing tumors. Altogether, our data for first time provides an explanation for elevated CA 125 expression in various cancers. (Supported by Rice Institute of Biosciences and Bioengineering Medical Innovation Award to DDC, RCB, and PEC, in part by the M.D. Anderson SPORE in Ovarian Cancer NCI P50 CA83639, National Cancer Institute R01 CA135354). Citation Format: Micaela Morgado, Margie N. Sutton, Mary Simmons, Jinsong Liu, Zhen Lu, Pamela E. Papadopoulos, Robert C. Bast, Daniel D. Carson. MUC16/CA125, TNFα and IFNγ are co-expressed in malignant gynecologic neoplasms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3456. doi:10.1158/1538-7445.AM2015-3456

Michael Jackson, Brian Ford-Lloyd and Martin Parry (eds.): Plant genetic resources and climate change

‘‘The climate change that has been predicted is an enormous challenge for society worldwide’’. This sentence taken from the preface to the book ‘‘Plant genetic resources and climate change’’ by its editors discloses the rationale for this book. Thirty-four contributors in sixteen chapters report on solid experience and recent scientific advancement that might help devise strategies or develop sustainable systems able to mitigate the effects of the oncoming climate change on crop plants growth, and hence on food production. This book comes almost 25 years after a volume by the same authors with a similar title: Climatic change and plant genetic resources, edited by CABI in 1990, and which gathered contributions presented at the Second International Workshop on Plant Genetic Resources, held in Birmingham, UK, in April 1989. The 1990 book mostly dealt with the effects of the green house effect on domesticated plants. In the latest 25 years, though, knowledge on the oncoming climate change has dramatically increased, and the effects of the global climate change have become evident. A new picture of the present-day knowledge and perspectives was, therefore, necessary. Three chapters of the 2014 book deal with the impact of climate change on agriculture sensu lato. Chapter 1 describes the strong interplay between food security and plant genetic resources (PGRs) by exploring the different potential uses of PGRs in contributing to crop adaptation and to crop resilience to external inputs. This chapter also outlines possible scenarios for future rice breeding in relation to climate change. Chapter 3 deals with the complex picture of the drivers of climate change, and analyses the different models that make it possible to explain it. Which future will us experience is not well understood yet, so different possible scenarios are reported and reviewed. Chapter 4, after analysing several concurring impacts of climate change on different areas, including social ones, urges for a substantial reduction of greenhouse gases. It is now clear that, if not moderated, these emissions are projected to considerably increase the risk of hunger for large segments of the world population. Six chapters concern different aspects of plant genetic resources conservation and use. Chapter 2 reviews the present day knowledge on PGRs conservation and use. Many important developments have come forth in this science since the pioneering efforts of the ‘‘genetic resources movement’’, thanks to the introduction of molecular tools for the description and use of PGRs and as a consequence of devising novel approaches to their rational use. Important achievements facilitating access to PGRs derived directly D. Pignone (&) Institute of Bioscience and Bioresources, National Research Council, Bari, Italy e-mail: domenico.pignone@igv.cnr.it

Studies Toward Lab-on-a-Chip Separations and Detection Using Redox Magnetohydrodynamic Microfluidics

Low cost, miniaturized, simple, and rapid isolation and detection of target chemicals and biological analytes from a complex mixture is of great interest to the point-of-care (POC) diagnostics community. On-chip redox- magnetohydrodynamics (MHD) microfluidics is being studied for complex chemical and biological mixture separations as a promising lab-on-a-chip application. Redox MHD is a particularly attractive pumping technique for this purpose because it does not rely on an external pump and, more importantly for separations, produces a flat flow profile within a small fluid volume.1 In Redox-MHD, a force (FMHD ) acts on ions with ionic current density of j, perpendicular to the magnetic field (B). The resultant force acts on the surrounding fluid causing the fluid to flow according to right hand rule FMHD = j x B. Fluid flow may also be programmed by activating different electrodes of a microfabricated chip resting on a magnet and both permanent magnets and electromagnets can be used, allowing for wide flexibility and tunability in application design.2,3 Previously, studies used a mixture of redox species, K3Fe(CN)6 and K4Fe (CN)6, producing j without any bubble formation from solvent electrolysis and achieving very flat flow profiles.1 More recently, redox species in solution were replaced with the conducting polymer poly (3, 4 ethylenedioxythiophene) (PEDOT) directly deposited on the electrodes. These, too, produced a flat flow profile and, in addition, produced higher fluid velocities since they generate about 10 to 1000 times more current.4 To apply these chips for separation purposes, we have modified a glass lid and chip surface between the pumping and detecting electrode with acrylic polymer (silicone acrylate and butyl acrylate) to serve as the stationary phase.5 Long wavelength (366 nm) UV polymerization of the acrylate monomer solution directed the polymerization between these electrodes. Here, we will report on our results addressing the effect of stationary and the mobile phase on fluid control, speed, and flow profile, as well as considerations for optimizing pumping parameters for efficient separation. We will also report on detection methods to quantify the separation of model analytes. One such method involves the use of fluorescence microscopy and single photon counting detection to directly follow the flow and separation of fluorescent analytes inside these microfluidic devices.6 Acknowledgments Research was supported partially through the National Science Foundation (CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.

Redox Cycling Behavior of Catecholamines and Their Mixtures at Different Diffusion Distances: Steps Toward Quantitative Speciation

Catecholamines (dopamine, DA; norepinephrine, NE and epinephrine, EP) are neurotransmitters of great importance in the mammalian brain. The electrochemical oxidation of these catecholamines has been studied before and can undergo a disproportionation reaction.1,2 During electrochemical oxidation, a catecholamine undergoes a reversible 2-electron transfer process and is oxidized to its o-quinone form. At physiological pH, the o-quinone deprotonates and undergoes homogeneous intramolecular cyclization to form the leucoaminochrome. The leucoaminochrome can oxidize to form the aminochrome which is not electroactive by reacting with the o-quinone form and re-generating the starting catecholamine species. The degree of cyclization reaction will change the reversibility of the total reaction. Different catecholamines have different apparent rate constants of the cyclization reaction (kDA = 0.13 ± 0.05 s-1, kNE = 0.98 ± 0.52 s-1, kEP = 87 ± 10 s-1, in phosphate buffer at pH 7.43), which provide the possibility for distinguishing the three catecholamines from each other in an electrochemical analysis. The electrochemical method of redox cycling has the ability of recycling electrochemically reversible species, thus providing amplified signals and the selectivity of reversible species over irreversible ones.4 In the redox cycling process of catecholamines, the oxidation and subsequent reduction reactions take place at different electrodes, namely the generator and collector electrodes, respectively. The time between the two reactions is controlled by the time the species diffuse across the gap between those electrodes. The extent of reversibility of the heterogeneous electron transfer reaction during redox cycling is then determined by the relationship of diffusion time and cyclization reaction time.5 We will report the electrochemical redox cycling behaviors of individual and binary mixtures of DA, NE, and EP using microfabricated gold microelectrode arrays with various gap widths between the generators and collectors. The catecholamines showed decreasing collector signals as gap width increased with increasing cyclization rates, which is a reflection of how much o-quinone survives diffusion time from the generator. With increasing gap width, the collector signals of the three catecholamines all decreased because of decreasing collection efficiencies, as expected. However, the signals decreased at different extents, depending on the cyclization rates. The collector signal for EP became undetectable at a smaller gap width than that for NE, while the collector signal for DA was still measurable. In the binary mixture of catecholamines, o-quinone and leucoaminochrome from different catecholamines can also react, thus make the collector signal decrease further, compared to solutions containing a single kind of catecholamine. Acknowledgments Research was supported partially through the National Science Foundation (CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000.

Microfluidics with Alternating Current-Redox Magnetohydrodynamics at Modified Electrodes for Cell Identification

A decrease in white blood cell count below the normal range is known as leucopenia. Neutropenia is an absolute neutrophil count of less than 1500 cells per microliter of blood. This is the most common and clinically significant form of leucopenia. In cases where patients have a count of less than 500 cells per microliter of blood, the body’s normal flora in the digestive tract can cause fatal infections and sepsis. We are developing a cost-effective method to improve monitoring of the patients’ leukocyte counts and composition. Leukocyte cell types (granulocytes, lymphocytes, and monocytes ) are identified via image-based classification. Microfluidics is an ideal platform for evaluation of neutrophil count, and large numbers of fluorescently-stained cells (using proflavine 0.01% w/v) may be rapidly imaged using a high-speed microscopy setup. Each cell image is quantified using image texture-based analysis. A linear discriminant analysis-based predictive classification algorithm is trained and used to group each cell into one of these three classes based solely on image texture features. This approach has been used in various other biomedical imaging applications to objectively classify cells and tissues.1 We will report on interfacing this image-based classification method with a self-contained microfluidic pumping approach that offers parallel, uniform fluid flow, redox-magnetohydrodynamics (RMHD). RMHD has shown the ability to move fluid in a flat flow profile across macroscopic dimensions,2 so that fluid elements travel downstream at the same rate and without distortion. This feature facilitates the counting and monitoring of the composition of cells in biological samples. The MHD phenomenon produces a body force, FB , whose direction and magnitude determines fluid flow, and is governed by the cross product FB = j x B, where j is theionic current density in solution and B is the magnetic flux density. By activating different individually-addressable microelectrodes on a chip exposed to an electrolyte and placed on a permanent magnet, and by setting the sign and magnitude of the electronic current, the ionic current density can be manipulated, and therefore the flow can be programmed.3 Early work on MHD microfluidics, reported limitations because of electrode corrosion and solvent electrolysis, resulting in bubble formation.4,5 Subsequently, redox species were added to the solution to prevent these problems, and was coined redox-MHD (RMHD).6,7 However the redox species can react with the sample and interfere with detection. Replacing the solution redox species with electrode-tethered ones, such as poly(3,4-ethylenedioxythiophene) (PEDOT), a conducting polymer, resolves the complications and provides higher current and fluid speed. Synchronizing a sinusoidal current at the modified electrode with that of the magnetic field sustains pumping at high velocities, and is called AC-RMHD.8 The frequencies are much lower than AC-MHD at bare electrodes where inductive heating is a problem.9 The performance of AC-RMHD at PEDOT-modified electrodes will be presented as it pertains to integration with image-based cell classification. Characterization of PEDOT-modified electrodes and how biological material is affected as it flows using AC-RMHD will be described. Linear fluid flow is established between oppositely polarized, gold microband electrodes with lengths of 1.5 cm and 2.5 cm and separated by gaps as large as 5.6 mm. The solution consists of a phosphate buffer mixed with blood pipetted onto the electrode chip. The solution is contained by a rectangular cutout of a poly(dimethylsiloxane) film and sealed on top with a glass cover slip. Acknowledgments Research was supported through the National Science Foundation (CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. References (1) Muldoon, T. J.; Roblyer, D.; Williams, M. D.; Stepanek, V. M. T.; Richards–Kortum, R.; Gillenwater, A. M. Head & Neck 2012, 34, 305-312. (2) Sahore, V.; Fritsch, I. Anal. Chem. 2013, 85, 11809-11816. (3) Weston, M. C.; Fritsch, I. Sens. Actuat. B 2012, 173, 935-944. (4) Jang, J.; Lee, S. S. Sens. Actuat. A 2000, 80, 84-89. (5) Lemoff, A. V.; Lee, A. P. Sens. Actuat. B 2000, 63, 178-185. (6) Anderson, E. C.; Weston, M. C.; Fritsch, I. Anal. Chem. 2010, 82, 2643–2651. (7) Arumugam, P. U.; Fakunle, E. S.; Anderson, E. C.; Evans, S. R.; King, K. G.; Aguilar, Z. P.; Carter, C. S.; Fritsch, I. J. Electrochem. Soc. 2006, E185-E194. (8) Nash, C. K. Modified-Electrodes for Redox-Magnetohydrodynamic (MHD) Pumping for Microfluidic Applications. Ph.D. Dissertation, University of Arkansas, Fayetteville, AR, 2014. (9) Eijkel, J. C. T.; Dalton, C.; Hayden, C. J.; Burt, J. P. H.; Manz, A. Sens. Actuat. B 2003, 92, 215-221.

New Advances and Opportunities of Magnetohydrodynamic Microfluidics

An important need in miniaturizing technology for applications in point-of-care medicine and environmental monitoring is the ability to manipulate small volumes of fluids to perform sample preparation and multiple steps for chemical analysis in an automated fashion. We are pioneering the use of magnetohydrodynamics (MHD) for this purpose, which offers combined advantages of other microfluidic methods: it pumps both aqueous and non-aqueous solutions; easily starts, stops, and reverses direction; finely tunes fluid speeds up to mm/s; offers flat flow profiles and stirring capabilities; and can pump in a loop. Probably most unique to MHD microfluidics is its programmability: magnitude, direction, and pattern of fluid flow can be altered without constructing new devices. In our presentation, we will report the most recent advances in development of MHD microfluidics technology. MHD propels fluids when ions moving perpendicular to a magnetic field experience a force in the direction that is perpendicular to both, and impart momentum on the surrounding fluid. The relationship of the MHD force, FB , on the ionic current density, j, and the magnetic flux density, B, vectors is expressed by the right-hand rule, j x B = FB . We make use of individually-addressable microelectrodes patterned on a chip that is placed onto a magnet, to control the magnitude and direction of the ionic current in a solution through electrochemistry. Thus, the programmability of MHD microfluidics is realized. Several advances have made MHD microfluidics more viable for lab-on-a-chip applications. Early studies in salt solutions reported complications from electrode corrosion and bubble formation (from solvent electrolysis).1,2 These problems resulted from inefficient conversion of electronic current in the external circuit to ionic current in the solution. Adding chemical species that are more easily reduced and oxidized (redox species) than the solvent and supporting electrolyte eliminate these problems,3,4 introducing the concept of redox-MHD (RMHD). Thus, the ability to tune fluid velocity by MHD could be investigated unhindered, leading to discovery of flat flow profiles and spiral stirring.5,6 The concentration of the redox species, electron transfer kinetics, and mass transport limit the maximum current, and therefore the maximum achievable fluid velocity. However, adding high concentrations of redox species to the solution is not always compatible with biological samples and detection schemes. Two new developments maintain the advantages of RMHD microfluidics and yet avoid the interferences that occur by adding redox species to a sample. The first modifies electrodes with redox polymers (e.g. a conducting polymer of poly(3,4-ethylenedioxythiophene), PEDOT) so that adding redox species to solution is no longer necessary. Immediate access to that charge at the electrode surface allows for high currents and therefore high velocities. The second sustains fluid flow even when the charge at the modified electrodes depletes, by a recharging process by manipulating both B and j sinusoidally and in synchrony (AC-RMHD).7 The frequencies are much lower than in prior AC-MHD studies in supporting electrolyte at bare electrodes where inductive heating is a problem.8 Our presentation will describe advances in RMHD and in AC-RMHD with the emphasis on applications involving analysis of biological samples. New approaches and materials for modifying the electrodes will be discussed. Enhancements in manipulating the fluid flow through electrochemical cell, electrode, and magnet design will be addressed. Also, recent developments will be described toward interfacing the unique properties of RMHD microfluidics (i.e. flat flow profile and pumping in a loop) with technology for cell identification of leukocytes in blood,9which is of interest for point-of-care monitoring of patients undergoing chemotherapy. ACKNOWLEDGMENTS Research was supported through the National Science Foundation (CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. REFERENCES (1) Jang, J.; Lee, S.S. Sens. Actuat. A 2000, 80, 84-89. (2) Lemoff, A.V.; Lee, A.P. Sens. Actuat. B 2000, 63, 178-185. (3) Anderson, E.C.; Weston, M.C.; Fritsch, I. Anal. Chem. 2010, 82, 2643–2651. (4) Arumugam, P.U.; Fakunle, E.S.; Anderson, E. C.; Evans, S. R.; King, K. G.; Aguilar, Z. P.; Carter, C.S.; Fritsch, I. J. Electrochem. Soc. 2006, E185-E194. (5) Sahore, V.; Fritsch, I. Anal. Chem. 2013, 85, 11809-11816. (6) Sahore, V.; Fritsch, I. Microfluid. Nanofluid. 2014, onlne (DOI 10.1007/s10404-014-1427-6). (7) Nash, C.K. Modified-Electrodes for Redox-Magnetohydrodynamic (MHD) Pumping for Microfluidic Applications. Ph.D. Dissertation, University of Arkansas, Fayetteville, AR, 2014. (8) Eijkel, J.C.T.; Dalton, C.; Hayden, C. J.; Burt, J. P. H.; Manz, A. Sens. Actuat. B 2003, 92, 215-221. (9) Muldoon, T.J.; Roblyer, D.; Williams, M.D.; Stepanek, V.M.T.; Richards–Kortum, R.; Gillenwater, A.M. Head & Neck 2012, 34, 305-312.

Conducting Polymers Covalently Linked to Enzymes and Mediators and Electropolymerized on Microelectrode Arrays

Enzymes have proven to be effective electrode surface catalysts for the oxidation and reduction of fuels and oxidants, respectively, in biofuel cell applications.1 Due to low power densities enzymatic biofuel cells (EBFCs) are best suited for low power miniature systems.2 Microfabricated arrays of individually addressable electrodes can serve as an ideal platform for this application. As the size of the cell and electrode arrays decrease it is necessary to simplify the overall cell design using as few components as possible. One way to minimize the number of components is to remove the separator that is usually required to keep the anodic and cathodic compartments separate. This can be accomplished by immobilizing the enzymes and electron mediators to the electrode surface.3 There are three main ways of accomplishing immobilization: adsorption, entrapment, and covalent linkage. Adsorption of the mediator and enzyme can include weak interacting forces that may be disturbed upon shear flow and suffers from poor spatial control. Entrapment is an immobilization strategy in which the species of interest is held inside a 3D polymer matrix on the electrode surface. This can be accomplished in several different ways but the two most common methods are drop casting and electropolymerization. Drop casting is suitable for large area electrodes but is impractical for interdigitated microelectrodes. Electropolymerization can affect a much higher spatial resolution and can allow for faster processing. However, entrapment methods may result in the species diffusing away and an overall inefficient cell. Covalent linkage of mediators and enzymes provides strong bonds to the polymer backbone while maintaining high spatial resolution, electroactivity, and catalytic activity. Therefore it is of interest to use electropolymerizable monomers with functional groups for covalent coupling with enzymes and mediators. The electropolymerizable monomers studied in this present work are 3,4-ethylenedioxythiophene (EDOT) and functionalized derivatives such as hydroxymethyl 3,4-ethylenedioxythiophene (HM-EDOT), ferroceneacetic acid derivatized 3,4-ethylenedioxythiophene (FcAA-EDOT)4 , and carboxylic acid functionalized 3,4-ethylenedioxythiophene (COOH-EDOT)5 . The resulting electropolymerized conducting polymers (PEDOT, HM-PEDOT, FcAA-PEDOT, COOH-PEDOT) were then used to either immobilize enzymes of interest such as horseradish peroxidase (HRP) and glucose oxidase (GLOX) or serve as electron mediators to shuttle electrons from the enzymes’ catalytic center to the electrode surface. Pre and post polymerization covalent linkage was studied as well as combinations of the different monomers polymerized to a single electrode. Electropolymerizations were carried under aqueous conditions. Electrochemical and spectroscopic characterization of the modified surfaces will be described, with an emphasis on the facilitation of electron transfer between the electrodes and enzymes and cross communication with neighboring electrodes. Acknowledgments Research was partially supported through the National Science Foundation (CHE-0719097 and CBET-1336853) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000. REFERENCES 1. Barton, S. C.; Gallaway, J.; Atanassov, P., Enzymatic biofuel cells for implantable and microscale devices. Chem. Rev. (Washington, DC, U. S.) 2004, 104(10), 4867-4886. 2. Minteer, S. D.; Liaw, B. Y.; Cooney, M. J., Enzyme-based biofuel cells. Current Opinion in Biotechnology 2007, 18(3), 228-234. 3. Heller, A., Miniature biofuel cells. Physical Chemistry Chemical Physics 2004, 6(2), 209. 4. Brisset, H.; Navarro, A.-E.; Moustrou, C.; Perepichka, I. F.; Roncali, J., Electrogenerated conjugated polymers incorporating a ferrocene-derivatized-(3,4-ethylenedioxythiophene). Electrochemistry Communications 2004, 6(3), 249-253. 5. Luo, S.-C.; Ali, E. M.; Tansil, N. C.; Yu, H.-h.; Gao, S.; Kantchev, E. A. B.; Ying, J. Y., Poly(3,4-ethylenedioxythiophene) (PEDOT) nanobiointerfaces: thin, ultrasmooth, and functionalized PEDOT films with in vitro and in vivo biocompatibility. Langmuir : the ACS journal of surfaces and colloids 2008, 24 (15), 8071-8077.

A Diet High in Protein Reduces Weight Gain, Food Intake and Liver Weight in Obese Zucker Rats

It has become a challenge to find effective treatments and preventive strategies to reduce obesity and diabetes (T2DM). There are several approaches to treating these diseases, including nutrition therapy. Diets high in protein are linked with weight reduction and improved insulin resistance. Therefore, the objective of this study was to examine the effects of a long‐term, high protein (pro) diet (40%) vs a moderate pro diet (20%) on body composition and metabolism using a genetic model of obesity/T2DM. Male, lean (L) and obese (O) Zucker rats (n = 8‐10 per group) were housed 2/cage and placed in one of four groups: L20, L40, O20 or O40. Animals were provided ad libitum access to diet and water for 12 weeks. At 12 weeks, animals were fasted for 8 hours, then trunk blood and tissues were collected. L had lower food intake (P &lt; 0.05) than O, and O40 had significantly lower food intake (P &lt; 0.05) than O20. Animals fed 40% pro had significantly lower weight gain (P &lt; 0.05) than those fed 20% pro. L had more muscle mass and lower fat mass than O (P &lt; 0.05). However, L40 had significantly lower fat mass than L20. O40 had lower liver weights (P &lt; 0.05) than O20. There was no difference in fasting glucose and O had higher fasting insulin. Fasting free fatty acids were higher in animals fed the 20% pro diet. There was no difference in basal phosphorylation of Akt, AMPK, mTOR or 4E‐BP1. There was no difference in basal BCAA between L20 and O20. L40 and O40 had significantly higher basal levels of BCAA (P &lt; 0.05). We conclude that higher protein diets have the potential to reduce weight gain and alter metabolism in obese rats. Funded by Arkansas Biosciences Institute.