NanoDrop Spectrophotometer Research Articles

Selective Recognition of H3.1K36 Dimethylation/H4K16 Acetylation Facilitates the Regulation of All-trans-retinoic Acid (ATRA)-responsive Genes by Putative Chromatin Reader ZMYND8

ZMYND8 (zinc finger MYND (Myeloid, Nervy and DEAF-1)-type containing 8), a newly identified component of the transcriptional coregulator network, was found to interact with the Nucleosome Remodeling and Deacetylase (NuRD) complex. Previous reports have shown that ZMYND8 is instrumental in recruiting the NuRD complex to damaged chromatin for repressing transcription and promoting double strand break repair by homologous recombination. However, the mode of transcription regulation by ZMYND8 has remained elusive. Here, we report that through its specific key residues present in its conserved chromatin-binding modules, ZMYND8 interacts with the selective epigenetic marks H3.1K36Me2/H4K16Ac. Furthermore, ZMYND8 shows a clear preference for canonical histone H3.1 over variant H3.3. Interestingly, ZMYND8 was found to be recruited to several developmental genes, including the all-trans-retinoic acid (ATRA)-responsive ones, through its modified histone-binding ability. Being itself inducible by ATRA, this zinc finger transcription factor is involved in modulating other ATRA-inducible genes. We found that ZMYND8 interacts with transcription initiation-competent RNA polymerase II phosphorylated at Ser-5 in a DNA template-dependent manner and can alter the global gene transcription. Overall, our study identifies that ZMYND8 has CHD4-independent functions in regulating gene expression through its modified histone-binding ability.

Boiling, Freezing - Thawing and Simple Chemical Extraction Methods Are Comparable to Costly Kit - Based Bacterial DNA Extraction Methods as Evidenced by Conventional and Real-Time PCR

Background: Bacterial DNA extraction is a preliminary step for PCR studies carried out on bacteria for diagnosis and detection of antimicrobial resistance genes. Objectives: Testing efficiency of two physical methods (boiling and freezing-thawing) and two chemical methods (SDS and tween based methods) for extraction of bacterial DNA suitable for conventional as well as real time PCRs in comparison to a costly ready to use kit method. Methodology: Escherichia coli ATCC 25922 was submitted for five DNA extraction methods including QIAamp ready kit, boiling, freezing-thawing in TE, SDS method, and tween method. DNA purification by ethanol precipitation and wash was carried out after tested physical and chemical methods. The extracted DNAs were assessed by Nanodrop spectrophotometer, conventional PCR followed by Uviband quantification, and semiquantitative SYBR green based RT- PCR. Results: Nanodrop assessment showed that QIAamp kit and tween method produced the highest DNA yields; 78.1 ng/µl and 49.72 ng/µl, respectively, while SDS method gave the lowest. Both QIAamp kit and tween methods produced comparable DNA in concentration and purity. All tested methods extracted DNA amplifiable by PCR. In conventional PCR, the concentrations ranged from 287.31 to330.46 ng/5µl for boiling and QIAamp kit, respectively. In RT-PCR, the highest CT values were obtained by OIAmp kit, followed by tween, freezing-thawing in TE, SDS, and boiling methods with narrow range of variation (from 30.80 to 31.85). No significant statistical differences were detected between tested methods in the results of PCRs either conventional or real time. Conclusion: Boiling in water, freezing-thawing in TE, tween and SDS based methods followed by DNA precipitation provide rapid, effective, less laborious and truly cost-effective methods for extraction of bacterial DNA suitable for PCR studies.

Surfactant-Assisted Nanodrop Spectrophotometer Determination of Iron(III) in a Single Drop of Food, Biological, and Environmental Samples

A surfactant-assisted nanodrop spectrophotometric (NDS) method has been developed for the determination of the iron(III) content in single drops (1 μL) of food, biological, and or environmental sample using disodium 1-nitroso-2-naphthol-3,6-sulfonate (Nitroso-R salt) as a complexing agent and Tween-80 as non-ionic surfactant at pH 4.0. This method is based on the formation of a complex between the Fe(III) present in a sample and the Nitroso-R-salt in the presence of a surfactant to form a green-colored Fe(III)–Nitroso-R salt complex, which can be measured using a NDS method at a λ max = 710 nm. This system was found to obey Beer’s law at concentrations in the range of 50–5000 μg/L with slope, intercept and correlation coefficient values of 0.683, 0.102, and 0.986, respectively. The molar absorptivity of the complex in terms of the Fe(III) content was determined to be 4.86 × 10 5 L·mol –1 ·cm –1 . The detection limit and %RSD values of the method were found to be 17 × 10–3 mg/L and ±1.3706%, respectively. This newly developed method was successfully applied to the determination of the Fe(III) content in single drops of food, biological, and environmental samples, and the results were compared with those obtained by atomic absorption spectrometry.

Sap Extract Papaya for Autolitic Debridement Chronic Wound

Introduction: The high cost of drugs or specific wound dressings for chronic wounds can be healed at least in two weeks depending on the stage of the wound and blood sugar levels. Hence the need for an innovative processing of natural resources (NR) to produce a chronic wound care products that are accessible to all people. One of the current trends is to use protease enzymes (sap extract papaya) that serves as autolitic debridement in the treatment of chronic wounds. The aims of this research was to indentify characteristics of protease enzyme present in sap of papaya. Method: This study was a descriptive exploratory that doing pure protease enzyme extract of papaya sap. Results: Molecular weight measurement using a kit Pre-stained Protein Markers (Broad Range) for SDS PAGE, Nacalai tesque No. 02525 showed a homogeneous molecular weight that slightly above 28 kD. Measurements of the enzyme levels using NanoDrop Spectrophotometer ND kit-1000 (with a distilled water marker) showed a relatively homogeneous enzyme level with a range between 43% - 61% and 53, 88% in average. The addition of papaya sap extract into each tube containing 7 ml gelatin obtained results: the control group there were clots perfectly on gelatin; groups with the addition of 2 drops (± 0.5 ml) obtained 50% hydrolyzed gelatin; and groups with the addition of 4 drops (± 0.5 ml) obtained 75% hydrolyzed gelatin. Discussion: The results showed that papaya sap extraction synonymous with protease enzyme. Papaya sap extraction activity (protease enzymes) on hydrolyzing gelatin shows a qualitative picture. Therefore, it can be used as autholitic debridement. Further, the following study is essential to identify structure and activity of the enzyme with a more determinant (temperature, time, and dose).Keywords: sap papaya extraction, protease enzymes

Sap Extract Papaya for Autolitic Debridement Chronic Wound

Introduction: The high cost of drugs or specific wound dressings for chronic wounds can be healed at least in two weeks depending on the stage of the wound and blood sugar levels. Hence the need for an innovative processing of natural resources (NR) to produce a chronic wound care products that are accessible to all people. One of the current trends is to use protease enzymes (sap extract papaya) that serves as autolitic debridement in the treatment of chronic wounds. The aims of this research was to indentify characteristics of protease enzyme present in sap of papaya.Method: This study was a descriptive exploratory that doing pure protease enzyme extract of papaya sap. Results: Molecular weight measurement using a kit Pre-stained Protein Markers (Broad Range) for SDS PAGE, Nacalai tesque No. 02525 showed a homogeneous molecular weight that slightly above 28 kD. Measurements of the enzyme levels using NanoDrop Spectrophotometer ND kit-1000 (with a distilled water marker) showed a relatively homogeneous enzyme level with a range between 43% - 61% and 53, 88% in average. The addition of papaya sap extract into each tube containing 7 ml gelatin obtained.Results: the control group there were clots perfectly on gelatin; groups with the addition of 2 drops (± 0.5 ml) obtained 50% hydrolyzed gelatin; and groups with the addition of 4 drops (± 0.5 ml) obtained 75% hydrolyzed gelatin.Discussion: The results showed that papaya sap extraction synonymous with protease enzyme. Papaya sap extraction activity (protease enzymes) on hydrolyzing gelatin shows a qualitative picture. Therefore, it can be used as autholitic debridement. Further, the following study is essential to identify structure and activity of the enzyme with a more determinant (temperature, time, and dose).

Bacteria-killing ability of fresh blood plasma compared to frozen blood plasma

In recent years, the bacteria-killing assay (BKA) has become a popular technique among ecoimmunologists. New variations of that assay allow researchers to use smaller volumes of blood, an important consideration for those working on small-bodied animals. However, this version of the assay requires access to a lab with a nanodrop spectrophotometer, something that may not be available in the field. One possible solution is to freeze plasma for transport; however, this assumes that frozen plasma samples will give comparable results to fresh ones. We tested this assumption using plasma samples from three species of birds: chickens (Gallus gallus), ash-throated flycatchers (Myiarchus cinerascens), and western bluebirds (Sialia mexicana). Chicken plasma samples lost most or all of their bacterial killing ability after freezing. This did not happen in flycatchers and bluebirds; however, frozen plasma did not produce results comparable to those obtained using fresh plasma. We caution researchers using the BKA to use fresh samples whenever possible, and to validate the use of frozen samples on a species-by-species basis.

Abstract LB-288: Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis

Abstract Background: Formalin fixed paraffin embedded (FFPE) tissues remains the standard method for fixation and storage of tissues for clinical pathology use. Protein extraction from these tissues remains challenging due to the reduced quality and amount of extracted proteins. Despite multiple successful attempts, isolation of proteins from FFPE tissue sections necessitates routine use of xylene, a highly toxic organic solvent. We previously showed that proteins can be efficiently extracted with a novel technique that utilizes hot distilled water as a substitute for xylene with a quality adequate for western blot analysis. However, its major drawback is the need to use an entire or major part of the paraffin-embedded tissue block. To address these issues we developed a new xylene-free method for protein extraction from FFPE tissue sections of around 8 μm thickness. Methods: A total of 44 different types of FFPE tissues sections of 8 μm thickness were obtained from various archived FFPE specimens which include: 17 colorectal cancer (5-6 years), 7 breast cancer (3 years), 3 thyroid cancer (2 years), 4 ovarian cancer (2 years), 8 uterine cancer (1 year), and 5 prostate cancer (1 year). Deparaffinization was conducted by gentle treatment of each section with hot distilled water (≈90°C) for less than 10 seconds. Deparaffinized tissues were then placed in a cell lysis buffer and Laemli buffer and incubated at 100°C for 5-10 minutes. The extracted proteins were quantified using NanoDrop Spectrophotometer and evaluated using western blot analysis for the presence of AKT and beta-actin. Results: Using this method, a significant amount of proteins was successfully isolated with an average amount of 2.670 μg/μl ± 0.623 or 1068 μg/8 μm tissue section. Compared to the standard protein extraction method using xylene the amount of proteins extracted in this experiment is at least four times greater. Protein extracts were of good quality and efficiently analyzed by western blot experiment. Moreover, the isolated proteins showed a similar migration pattern compared to the positive control protein on SDS-PAGE but with better bands’ intensity and clarity. Protein kinase B (PKB/AKT) was successfully identified in all specimens, and beta-actin protein was resolved with an efficiency higher than 80%. Hence, this technique has enabled us to efficiently extract and detect selected proteins from archived samples for up to 6 years. Conclusion: We developed an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissues sections and adequate for western blot analysis. Hence, utilizing this technique can further aid in the identification of tissue protein biomarkers of various diseases, monitor cancer progression, and improve the diagnosis. It remains to be determined if this method of protein extraction form FFPE is adequate for use in proteomic analyses. Citation Format: Anthony Mansour, Noha Bejjani, Carole Dagher, Rajaa Chatila, Wissam H. Faour. Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis. [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 LB-288. doi:10.1158/1538-7445.AM2015-LB-288

Different protein expression systems can influence the direction of the immune responses against HCV core protein in animal model

Introduction: Hepatitis C virus (HCV) infection is a major public health problem which influences about 170 million people worldwide. Different types of vaccines have been designed using HCV structural proteins to control the viral infection. The core nucleocapsid protein is one of the most conserved proteins and a desirable target for HCV vaccines, especially the protein-based vaccines. In current study, we generated the core protein recombinantly in prokaryotic (Escherichia coli) and eukaryotic (Leishmania tarentolae) expression systems and compared the humoral immune responses stimulated by each protein in a BALB/c mice model. Methods: The expression of HCV core protein was performed using the prokaryotic pET-28a/ BL21 expression system and also the eukaryotic LEXSY expression system. The recombinant core proteins expressed in E. coli and Leishmania were purified using reverse staining method and affinity chromatography under native conditions, respectively. The purified core proteins were detected by SDS-PAGE and Western blotting using anti-His antibody and were assessed by NanoDrop spectrophotometer. Finally, the abilities of both recombinant core proteins to induce the effective humoral immune responses were evaluated using indirect ELISA. Results: Our data indicated a clear band of ~ 21 kDa for the purified HCV core proteins in both expression systems, confirmed by SDS-PAGE and Western blotting. The mice immunization with both recombinant core proteins was able to produce high levels of antibody isotypes (IgG1 and IgG2a) in comparison with the controls (p < 0.05). In addition, the level of IgG2a response was significantly higher in the group immunized with the core protein purified from leishmania compared to the protein generated from E. coli (p < 0.05). Conclusion: The recombinant core protein generated by the leishmania expression system could induce a Th1-biased immune response with respect to the increase of IgG2a to IgG1 ratio.

Association between tissue miR-141, miR-200c and miR-30b and bladder cancer: a matched case-control study.

To evaluate the expression of microRNAs in tissue samples from patients with bladder cancer and to compare it with healthy adjacent tissue samples as controls. Thirty five tissue samples from patients with newly diagnosed untreated bladder transitional cell carcinoma and 35 adjacent normal urothelium were collected during 2013 to 2014. TRIzol reagent was used to isolate total RNA including microRNAs. RNA concentration and purity were determined using a nanodrop spectrophotometer. Also 1% agarose gel electrophoresis was used to assess integrity of RNA. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) method was performed using the PARSGENOME microRNA RT-PCR system. Data was analyzed by STATA 11. A couple of patients were female the remainder were male. Mean age of patients were 71.06 ± 11.43 years. The expression level of miR-30b, miR-141 and miR-200c in case group were significantly higher than that of control normal tissue samples. miR-141 had higher expression rate in malignant tissue than two other miRNAs (P < .001). There was a more expression rate of miR-200c, miR-141 and miR-30b in bladder cancer tissues than healthy adjacent control tissues. Further studies are needed to draw final conclusion.

Changes in miRNA expression profile between stress-vulnerable and stress-resilient rats in Chronic Mild Stress (CMS) - an animal model of depression.

Event Abstract Back to Event Changes in miRNA expression profile between stress-vulnerable and stress-resilient rats in Chronic Mild Stress (CMS) - an animal model of depression. Dariusz Zurawek1*, Agata Faron-Gorecka1, Maciej Kusmider1, Magdalena Kolasa1, Paulina Pabian1, Joanna Solich1, Kinga Szafran1, Piotr Gruca2, Mariusz Papp3 and Marta Dziedzicka-Wasylewska1 1 Institute of Pharmacology Polish Academy of Sciences, Department of Pharmacology Laboratory of Biochemical Pharmacology, Poland 2 Institute of Pharmacology Polish Academy of Sciences , Department of Pharmacology Laboratory of Behavioural Pharmacology, Poland 3 Institute of Pharmacology Polish Academy of Sciences, Department of Pharmacology Laboratory of Behavioural Pharmacology, Poland Prolonged, chronic stressful events are considered to be one of the major causes of depression, however many people and animals exposed to stress seem to be resilient. Only a few studies have focused on stress resilience phenomenon. Depression may result from impairment in neuronal plasticity which may be associated with altered miRNA expression level - a class of newly discovered posttranscriptional regulators of gene expression. We used Chronic Mild Stress (CMS) paradigm which is well characterised animal model of depression. We observed that in this model about 30% of rats exposed to chronic, stressful conditions demonstrate resilience to stress what is manifested as not altered consumption of a palatable 1% sucrose solution by this group of animals. We investigated a global miRNA expression profile in serum of rats subjected to chronic mild stress to identify the potential, non-invasive molecular markers differentiating animals that demonstrated resilience to stress from stress-reactive ones. After two weeks of CMS procedure we identified three groups of rats based on their sucrose consumption: control (12,1±0,6g), anhedonic (5,3±0,4g, p<0,0001) and stress resilient (11,6±0,4g,). Animals were sacrificed and the serum was collected, from which miRNA was purified. The RNA concentration and quality was measured with NanoDrop spectrophotometer. RT-PCR based TaqMan Array Rodent MicroRNA Cards were used to screening the changes in miRNA expression induced by stress conditions and response to stress. Using Expression Suite Software analysis we found that stress significantly altered the expression of one miRNA in anhedonic rats as compared to control group, while in stress-resilient animals the expression of 16 miRNAs was changed. This data indicate that resilient animals can actively cope with stressful conditions leading to "žstress-resistant" phenotype. miRNAs may play important role in stress resilience by pleiotropic modulation of expression of many genes. The study was supported by National Science Center Grant UMO-2012/07/B/NZ7/01164, Poland. Keywords: Depression, miRNA, Animal Models, Chronic mild stress, stress-resilience Conference: XII International Conference on Cognitive Neuroscience (ICON-XII), Brisbane, Queensland, Australia, 27 Jul - 31 Jul, 2014. Presentation Type: Poster Topic: Emotional and Social Processes Citation: Zurawek D, Faron-Gorecka A, Kusmider M, Kolasa M, Pabian P, Solich J, Szafran K, Gruca P, Papp M and Dziedzicka-Wasylewska M (2015). Changes in miRNA expression profile between stress-vulnerable and stress-resilient rats in Chronic Mild Stress (CMS) - an animal model of depression.. Conference Abstract: XII International Conference on Cognitive Neuroscience (ICON-XII). doi: 10.3389/conf.fnhum.2015.217.00376 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: 19 Feb 2015; Published Online: 24 Apr 2015. * Correspondence: Mr. Dariusz Zurawek, Institute of Pharmacology Polish Academy of Sciences, Department of Pharmacology Laboratory of Biochemical Pharmacology, Krakow, Poland, zurawek@if-pan.krakow.pl 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 Dariusz Zurawek Agata Faron-Gorecka Maciej Kusmider Magdalena Kolasa Paulina Pabian Joanna Solich Kinga Szafran Piotr Gruca Mariusz Papp Marta Dziedzicka-Wasylewska Google Dariusz Zurawek Agata Faron-Gorecka Maciej Kusmider Magdalena Kolasa Paulina Pabian Joanna Solich Kinga Szafran Piotr Gruca Mariusz Papp Marta Dziedzicka-Wasylewska Google Scholar Dariusz Zurawek Agata Faron-Gorecka Maciej Kusmider Magdalena Kolasa Paulina Pabian Joanna Solich Kinga Szafran Piotr Gruca Mariusz Papp Marta Dziedzicka-Wasylewska PubMed Dariusz Zurawek Agata Faron-Gorecka Maciej Kusmider Magdalena Kolasa Paulina Pabian Joanna Solich Kinga Szafran Piotr Gruca Mariusz Papp Marta Dziedzicka-Wasylewska 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.

Microparticles engineered to highly express peroxisome proliferator-activated receptor-γ decreased inflammatory mediator production and increased adhesion of recipient monocytes.

Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-γ (PPARγ). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPARγ reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPARγ microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPARγ microparticles also changed monocyte mRNA levels of several genes including those under PPARγ control. Overall, the delivery of PPARγ from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.

Abstract B51: Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection

Abstract Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of infants. We constructed a tissue microarray (TMA) including 47 diagnostic NB cases and performed immunohistochemical (IHC) staining for a number of biomarkers. Differential IHC staining was identified between duplicate tissue cores for a proportion of NB cases. Differential IHC staining occurs frequently in cancer specimens, and can be due to technical factors, tissue artefacts, or underlying biological differences between tumor sub-regions. As laser capture microdissection (LCM) allows isolation of cells from formalin-fixed paraffin-embedded (FFPE) tissue slides, we applied LCM to FFPE NB samples, to investigate the basis of differential IHC staining. Material and Methods: NSE, NB84, ALK, TPD52 and MGMT proteins were detected within diagnostic NB TMA cores and whole NB sections using IHC. Cells were isolated from FFPE sections using LCM, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Extracted genomic DNA was quantified using a Nanodrop spectrophotometer, and DNA quality was assessed by a Bioanalyzer 2100. All DNA samples were tested using PCR with TPD52 and ALK primers. Results including clinical and sample variables were analysed using SPSS version 19. Results: Of 47 NB cases within the TMA, 35 cases were represented by duplicate tissue cores, whereas the remaining 12 cases were represented by single cores. IHC analysis of 5 biomarkers showed visually different IHC staining in tumour tissue between duplicate tissue cores in 15/35 (43%) cases. Whole tissue sections from 6 of these 15 cases were then examined, which reproduced differential IHC staining. Isolation of NB cells by LCM was successfully performed after substantial optimisation. A total of 14 regions from 8 NB cases were microdissected, producing DNA yields ranging from 0.12 - 4.05 pg/ µm2 tissue. There were no significant associations between total yields of extracted DNA, and microdissected tissue areas, or tissue sample ages. All DNA samples were successfully amplified by TPD52 but not by ALK PCR primers. Conclusions: Genomic DNA can be reliably extracted from microdissected FFPE NB tissues, although only one of 2 genes could be successfully PCR amplified. Downstream assays of DNA extracted from FFPE samples may therefore have particular design requirements. Further application of this approach may help improve our understanding of the level of tumour heterogeneity present in NB samples at diagnosis. Citation Format: Le M. Thwe, Laurence C. Cantrill, Daniel R. Catchpoole, Loretta Lau, Jennifer A. Byrne. Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B51.

SPION-mediated soil DNA extraction and comparative analysis with conventional and commercial kit-based protocol

Direct isolation of soil DNA comes as an emerging technology to understand the microbial diversity of a particular environment circumventing the dependency on culturable methods. Soil DNA isolation is tough due to the presence of various organic components present in soil which interfere in extraction procedure. Here, we report a novel direct soil DNA extraction protocol utilizing bare superparamagnetic iron oxide nanoparticles and its comparison with conventional and commercial kit-based soil DNA extraction methods. The quality, quantity and feasibility of the recovered DNA from all the three methods towards various molecular techniques were checked. Our magnetic nanoparticle-based soil DNA extraction successfully yields pure DNA without any RNA or protein contamination as revealed by the nanodrop spectrophotometer and agarose gel electrophoretic study. Different methods of soil DNA extraction were evaluated on the basis of PCR, denaturing gradient gel electrophoresis and real-time PCR. Soil DNA extracted using conventional method fails to carry out critical molecular biology techniques where as magnetic nanoparticle-based soil DNA extraction gave good results which is comparable to commercial kit. This comparative study suggests that protocol described in this report is novel, less time consuming, cost effective with fewer handling steps and yields high quantity, good quality DNA from soil.

Immune Activation Is Associated With Increased Gut Microbial Translocation in Treatment-Naive, HIV-Infected Children in a Resource-Limited Setting

Gut damage resulting in microbial translocation (MT) is considered a major cause of immune activation (IA) in HIV infection, but data in children are limited, particularly in the absence of antiretroviral therapy. Sixty perinatally HIV-infected, antiretroviral therapy-naive children, aged 2-12 years, were evaluated for plasma levels of lipopolysaccharide, DNA sequences encoding bacterial 16 second ribosomal DNA (16S rDNA) and soluble CD14 concurrently with markers of CD4 and CD8 T-cell IA and immune exhaustion (IE), CD4 counts, and plasma viral load. At study entry, participants were classified into immune categories (ICs): IC1 (CD4% > 25), IC2 (CD4% 15-25), and IC3 (CD4% < 15). Age-matched HIV-uninfected children served as controls. Data were evaluated at study entry and at 12 months. Levels of MT, IA, and IE were increased in patients as compared with controls, were highest in patients in IC3 group, and did not change over 12 months. MT products lipopolysaccharide and 16S rDNA correlated with each other and each correlated with plasma viral load, soluble CD14, and T-cell IA and IE. There was a correlation of IA with IE. CD4 counts and percentage were inversely correlated with MT products and underlying CD4 activation. In a natural history cohort of HIV-infected children not on therapy, MT was more pronounced in the most severely immunocompromised patients and was associated with IA. Strategies to reduce MT may help to reduce IA and prevent CD4 depletion.

Gene expression study of Saccharomyces cerivisae GPH1 gene in response to chemical modulators

Article history: The aim of the present study was to evaluate the gene expression of GPH1 gene of Saccharomyces cerivisae after it was induced to different stress conditions. The S. cerivisae was procured from MTCC and grown in YPD medium with different modulators like NaCl, Glucose, Calcium chloride, Sodium chloride, Potassium Nitrate, Arabinose and Dipotassium hydrogen orthophosphate. The RNA was isolated and analyzed on formaldehyde gel electrophoresis and determined by Nanodrop spectrophotometer. The cDNA was synthesized and used for Real Time PCR. Quantification was based on a standard curve, prepared from samples of known template concentration. The concentration of unknown sample is then determined by Ct value. There is a varied response to glycogen metabolism in S. cerivisae to environmental stress conditions such as nutrient limitation and heat shock. This response is mediated, in part, by the regulation of the glycogen metabolic genes. The results indicated that gene expression was high in modulator 3 and lowest in 2. Environmental stress induces a number of glycogen metabolic genes, including GPH1, which encodes glycogen phosphorylase. This study confirmed induction patterns of gene encoding glycogen phosphorylase GPH1 after treatment with modulators. This study gives an insight to the transcriptional control of GPH1 after modulator treatment.

Association of single nucleotide polymorphisms of CACNA1A gene in migraine.

INTRODUCTION:Migraine is a chronic, neurovascular polygenic disease where genetic and environmental factors are involved in its etiology. Dysfunction of neuronal ion transportation can provide a model for predisposition for common forms of migraine. Mutations in genes encoding ion channels disturb the rhythmic function of exposed tissue that may also explain the episodic nature of migraine. Our aim was to study the single nucleotide polymorphisms of CACNA1A gene in migraine patients.MATERIALS AND METHODS:The subjects were the patients of migraine, in the age range of 18-80 years, diagnosed by a Neurologist, as per the diagnostic criteria of International Headache Society (IHS) Classification 2004 after excluding other causes of headache by clinical examination and relevant investigations.The controls were the age and sex matched healthy persons from the same population excluding the relatives of patients. Only those patients and the controls, who voluntarily participated in the study, were taken and their blood samples were taken for the study. Deoxyribonucleic acid (DNA) extraction was performed according to the manufacturer's protocol for Qiagen DNA extraction kits (Qiagen, Hilden, NRW, Germany). DNA content was quantified by spectrophotometric absorption (Nanodrop Spectrophotometer, BioLab, Scoresby, VIC, Australia). Polymerase chain reaction was performed using an iCycler Thermal Cycler (Bio.Rad, Hercules, CA, USA). The polymorphic analysis of CACNA1A gene was carried out by two methods: Restriction fragment length polymorphism and sequencing.RESULTS:The study included a total of 25 patients of migraine, diagnosed on out-patient department basis as per IHS Classification 2004 and compared with age and sex matched 25 healthy controls. Most of the patients 23 (92%) were below the age of 50 years. 20 of the patients (80%) were females and 5 (20%) were males. The polymorphic analysis of CACNA1A gene revealed the presence of only the wild form of the gene for the codon E993V in both case and control groups.CONCLUSION:In our study, we could not find any polymorphism of CACNA1A gene in the selected patients. Instead the wild type of genotype was found in both patients and controls. This negative result presented here, implies that if the CACNA1A gene is involved in typical migraine (with and without aura), its contribution is very modest and therefore difficult to discern. Nevertheless, there are other genes that could be considered potential candidates for typical migraine susceptibility for which further research is needed.

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

Preconcentration and Determination of Lead by Solidification of Floated Organic Drop Coupled with Nanodrop Spectrophotometry

The liquid-phase microextraction based on solidification of floating organic microdrop coupled with UV-visible spectrophotometer for preconcentration and determination of lead in the aqueous samples has been developed. In this technique, 20μL of 1-undecanol containing dithizone as the chelating agent (10-3mol L-1) was transferred to the water samples containing lead ions, and the solution was stirred for 25 min. The sample vial was cooled in an ice bath for 5 min. The solidified extract was transferred into a conical vial where it melted immediately, and then the organic phase was analyzed by Nanodrop spectrophotometer. Factors that influence the extraction and complex formation, such as pH, concentration of dithizone, extraction time, effect of ionic strenght, temperature and stirring rate were optimized. Under the optimized conditions, a preconcentration factor of 356, detection limit of 0.006 μgL-1, limit of quantification 0.018 μgL-1and a good relative standard deviation of 2.1% were obtained. The procedure was applied to sea water, mineral water and well water.

Identification of Biochemically Distinct Properties of the Small Ubiquitin-related Modifier (SUMO) Conjugation Pathway in Plasmodium falciparum

Small ubiquitin-related modifiers (SUMOs) are post-translationally conjugated to other proteins and are thereby essential regulators of a wide range of cellular processes. Sumoylation, and enzymes of the sumoylation pathway, are conserved in the malaria causing parasite, Plasmodium falciparum. However, the specific functions of sumoylation in P. falciparum, and the degree of functional conservation between enzymes of the human and P. falciparum sumoylation pathways, have not been characterized. Here, we demonstrate that sumoylation levels peak during midstages of the intra-erythrocyte developmental cycle, concomitant with hemoglobin consumption and elevated oxidative stress. In vitro studies revealed that P. falciparum E1- and E2-conjugating enzymes interact effectively to recognize and modify RanGAP1, a model mammalian SUMO substrate. However, in heterologous reactions, P. falciparum E1 and E2 enzymes failed to interact with cognate human E2 and E1 partners, respectively, to modify RanGAP1. Structural analysis, binding studies, and functional assays revealed divergent amino acid residues within the E1-E2 binding interface that define organism-specific enzyme interactions. Our studies identify sumoylation as a potentially important regulator of oxidative stress response during the P. falciparum intra-erythrocyte developmental cycle, and define E1 and E2 interactions as a promising target for development of parasite-specific inhibitors of sumoylation and parasite replication.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays