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https://www.readbyqxmd.com/read/28449002/purification-and-biochemical-and-kinetic-properties-of-an-endo-polygalacturonase-from-the-industrial-fungus-aspergillus-sojae
#1
Dante Fratebianchi, Ivana Alejandra Cavello, Sebastián Fernando Cavalitto
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3...
April 28, 2017: Journal of Molecular Microbiology and Biotechnology
https://www.readbyqxmd.com/read/28448897/a-performance-evaluation-of-nextera-xt-and-kapa-hyperplus-for-rapid-illumina-library-preparation-of-long-range-mitogenome-amplicons
#2
Joseph D Ring, Kimberly Sturk-Andreaggi, Michelle A Peck, Charla Marshall
Next-generation sequencing (NGS) facilitates the rapid and high-throughput generation of human mitochondrial genome (mitogenome) data to build population and reference databases for forensic comparisons. To this end, long-range amplification provides an effective method of target enrichment that is amenable to library preparation assays employing DNA fragmentation. This study compared the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and the KAPA HyperPlus Library Preparation Kit (Kapa Biosystems, Wilmington, MA) for enzymatic fragmentation and indexing of ∼8500bp mitogenome amplicons for Illumina sequencing...
April 5, 2017: Forensic Science International. Genetics
https://www.readbyqxmd.com/read/28448765/digital-assays-part-i-partitioning-statistics-and-digital-pcr
#3
Amar S Basu
A digital assay is one in which the sample is partitioned into many small containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, …). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotypes and phenotypes. Part I of this review begins with the benefits and Poisson statistics of partitioning, including sources of error...
April 1, 2017: SLAS Technology
https://www.readbyqxmd.com/read/28448738/site-specific-recruitment-of-epigenetic-factors-with-a-modular-crispr-cas-system
#4
Tobias Anton, Sebastian Bultmann
Dissecting the complex network of epigenetic modifications requires tools that combine precise recognition of DNA sequences with the capability to modify epigenetic marks. The CRISPR/Cas system has been proven to be a valuable addition to existing methodologies that fulfill these tasks. So far, sequence-specific editing of epigenetic modifications such as DNA methylation and histone posttranslational modifications relied on direct fusions of enzymatically inactivated Cas9 (dCas9) with epigenetic effectors. Here, we report a novel, modular system that facilitates the recruitment of any GFP-tagged protein to desired genomic loci...
February 23, 2017: Nucleus
https://www.readbyqxmd.com/read/28448622/goniodysgenesis-variability-and-activity-of-cyp1b1-genotypes-in-primary-congenital-glaucoma
#5
María T García-Antón, Juan J Salazar, Rosa de Hoz, Blanca Rojas, Ana I Ramírez, Alberto Triviño, José-Daniel Aroca-Aguilar, Julián García-Feijoo, Julio Escribano, José M Ramírez
Mutations in the CYP1B1 gene are currently the main known genetic cause of primary congenital glaucoma (PCG), a leading cause of blindness in children. Here, we analyze for the first time the CYP1B1 genotype activity and the microscopic and clinical phenotypes in human PCG. Surgical pieces from trabeculectomy from patients with PCG (n = 5) and sclerocorneal rims (n = 3) from cadaver donors were processed for transmission electron microscopy. Patients were classified into three groups depending on goniodysgenesis severity, which was influenced by CYP1B1 enzymatic activity...
2017: PloS One
https://www.readbyqxmd.com/read/28448610/characterization-and-expression-analysis-of-galnts-in-developing-strongylocentrotus-purpuratus-embryos
#6
Amber L Famiglietti, Zheng Wei, Thomas M Beres, Adina L Milac, Duy T Tran, Divya Patel, Robert C Angerer, Lynne M Angerer, Lawrence A Tabak
Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates...
2017: PloS One
https://www.readbyqxmd.com/read/28448564/insights-into-the-biochemical-defence-and-methylation-of-the-solitary-bee-osmia-rufa-l-a-foundation-for-examining-eusociality-development
#7
Aneta Strachecka, Jacek Chobotow, Jerzy Paleolog, Aleksandra Łoś, Michał Schulz, Dariusz Teper, Halina Kucharczyk, Maciej Grzybek
We examined age-related biochemical and histological changes in the fat bodies and hemolymph of Osmia rufa males and females. We analysed solitary bees during diapause, in October and in April; as well as the flying insects following diapause, in May and June. The trophocyte sizes, as well as the numbers of lipid droplets were the greatest at the beginning of diapause. Subsequently, they decreased along with age. Triglyceride and glucose concentrations systematically decreased in fat body cells but increased in the hemolymph from October to June...
2017: PloS One
https://www.readbyqxmd.com/read/28448143/effect-directed-analysis-of-toxicants-in-sediment-with-combined-passive-dosing-and-in-vivo-toxicity-testing
#8
Hongxue Qi, Huizhen Li, Yanli Wei, W Tyler Mehler, Eddy Y Zeng, Jing You
Identifying key toxicants in sediment is a great challenge, particularly if non-target toxicants are involved. To identify the contaminants responsible for sediment toxicity to Chironomus dilutus in Guangzhou reach of the Pearl River in South China, passive dosing and in vivo toxicity testing were incorporated into effect-directed analysis (EDA) to account for bioavailability. Fractionation of sediment extracts was performed with gel permeation chromatography and reverse phase liquid chromatography sequentially...
April 27, 2017: Environmental Science & Technology
https://www.readbyqxmd.com/read/28448139/suppression-of-nitrite-oxidizing-bacteria-in-intermittently-membrane-aerated-biofilms-a-model-based-explanation
#9
Yunjie Ma, Carlos Domingo-Félez, Benedek Gy Plosz, Barth F Smets
Autotrophic ammonium oxidation in membrane-aerated biofilm reactors (MABRs) can make treatment of ammonium-rich wastewaters more energy-efficient, especially within the context of short-cut ammonium removal. The challenge is to exclusively enrich ammonium-oxidizing bacteria (AOB). To achieve nitritation, strategies to suppress nitrite-oxidizing bacteria (NOB) are needed, which are ideally grounded on an understanding of underlying mechanisms. In this study, a counter-diffusion nitrifying biofilm reactor was operated under intermittent aeration...
April 27, 2017: Environmental Science & Technology
https://www.readbyqxmd.com/read/28448124/bioinspired-engineering-of-poly-ethylene-glycol-hydrogels-and-natural-protein-fibers-for-layered-heart-valve-constructs
#10
Qian Li, Yun Bai, Tao Jin, Shuo Wang, Wei Cui, Ilinca Stanciulescu, Rui Yang, Hemin Nie, Linshan Wang, Xing Zhang
Layered constructs from poly(ethylene glycol) (PEG) hydrogels and chicken eggshell membranes (ESMs) are fabricated, which can be further crosslinked by glutaraldehyde (GA) to form GA-PEG-ESM composites. Our results indicate that ESMs composed of protein fibrous networks show elastic moduli ~3.3-5.0 MPa and elongation percentages ~47-56%, close to human heart valve leaflets. Finite element simulations reveal obvious stress concentration on a partial number of fibers in the GA-crosslinked ESM (GA-ESM) samples, which can be alleviated by efficient stress distribution among multiple layers of ESMs embedded in PEG hydrogels...
April 27, 2017: ACS Applied Materials & Interfaces
https://www.readbyqxmd.com/read/28448026/lerlic-ms-ms-for-in-depth-characterization-and-quantification-of-glutamine-and-asparagine-deamidation-in-shotgun-proteomics
#11
Xavier Gallart-Palau, Aida Serra, Siu Kwan Sze
Characterization of protein deamidation is imperative to decipher the role(s) and potentialities of this protein posttranslational modification (PTM) in human pathology and other biochemical contexts. In order to perform characterization of protein deamidation, we have recently developed a novel long-length electrostatic repulsion-hydrophilic interaction chromatography-tandem mass spectrometry (LERLIC-MS/MS) method which can separate the glutamine (Gln) and asparagine (Asn) isoform products of deamidation from model compounds to highly complex biological samples...
April 9, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28448016/zebrafish-in-situ-spinal-cord-preparation-for-electrophysiological-recordings-from-spinal-sensory-and-motor-neurons
#12
Rosa L Moreno, Megan Josey, Angeles B Ribera
Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied...
April 18, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447989/fabrication-of-extracellular-matrix-derived-foams-and-microcarriers-as-tissue-specific-cell-culture-and-delivery-platforms
#13
Anna Kornmuller, Cody F C Brown, Claire Yu, Lauren E Flynn
Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries...
April 11, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447988/increased-recovery-time-and-decreased-lps-administration-to-study-the-vagus-nerve-stimulation-mechanisms-in-limited-inflammatory-responses
#14
Erwan Le Maître, Priya Revathikumar, Johanna Estelius, Jon Lampa
Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner...
March 29, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447981/isolation-characterization-and-purification-of-macrophages-from-tissues-affected-by-obesity-related-inflammation
#15
Joselyn N Allen, Adwitia Dey, Ruth Nissly, James Fraser, Shan Yu, Gayathri Balandaram, Jeffrey M Peters, Pamela A Hankey-Giblin
Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention...
April 3, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28447783/real-time-detection-reveals-responsive-co-transcriptional-formation-of-persistent-intramolecular-dna-and-intermolecular-dna-rna-hybrid-g-quadruplexes-stabilized-by-r-loop
#16
Yang Zhao, Jia-Yu Zhang, Zong-Yu Zhang, Tan-Jun Tong, Yu-Hua Hao, Zheng Tan
G-quadruplex (GQ) structures are implicated in important physiological and pathological processes. Millions of GQ-forming motifs are enriched near transcription start sites (TSSs) of animal genes. Transcription can induce formation of GQs which in turn regulates transcription. The kinetics of the formation and persistence of GQs in transcription is crucial for the role they play, but has not yet been explored. We established a method based on the fluorescence resonance energy transfer (FRET) technique to monitor in real-time the co-transcriptional formation and post-transcriptional persistence of GQs in DNA...
April 27, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28447456/numerical-modeling-of-electroosmotic-push-pull-perfusion-and-assessment-of-its-application-to-quantitative-determination-of-enzymatic-activity-in-the-extracellular-space-of-mammalian-tissue
#17
Yangguang Ou, Stephen G Weber
Many sampling methods have been developed to measure the extracellular concentrations of solutes in the extracellular space of mammalian tissue. However, few have been used to quantitatively study the various processes, such as enzymatic degradation, that determines the fate of these solutes. For a method to be useful in this pursuit, it must be able to 1) perfuse tissue and collect the perfusate for quantitative analysis of the solutes introduced and reaction products produced, 2) control the average residence time of the active solutes, and 3) have the appropriate spatial resolution for the process of interest...
April 27, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28447385/ultrasound-enhances-calcium-absorption-of-jujube-fruit-by-regulating-the-cellular-calcium-distribution-and-metabolism-of-cell-wall-polysaccharides
#18
Huanhuan Zhi, Qiqi Liu, Juan Xu, Yu Dong, Mengpei Liu, Wei Zong
BACKGROUND: Ultrasound has been applied in fruit pre-washing processes. However, it is not sufficient to protect fruit from pathogenic infection throughout the entire storage period, and sometimes ultrasound causes tissue damage. The goal of this study was to investigate the effects of calcium chloride (10 g L(-1) ) and ultrasound (350 W at 40 kHz), separately and in combination, on fruit quality, antioxidant status, tissue Ca(2+) content and distribution along with cell wall metabolism at 20 °C for six days...
April 26, 2017: Journal of the Science of Food and Agriculture
https://www.readbyqxmd.com/read/28446800/oxidative-stress-evoked-damages-on-rat-sperm-and-attenuated-antioxidant-status-on-consumption-of-aspartame
#19
I Ashok, P S Poornima, D Wankhar, R Ravindran, R Sheeladevi
Although several studies on toxic effect of aspartame metabolite have been studied, controversial reports over the use of aspartame owing to the fact that it releases methanol as one of its metabolite during metabolism exist. This present study is proposed to investigate whether aspartame (40 mg kg(-1) b.wt) administration for 90 days could induce oxidative stress and alter antioxidant status of epididymal sperm in Wistar strain male albino rats. To mimic the human methanol metabolism, methotrexate (MTX)-treated rats were included to study the effects of aspartame...
April 27, 2017: International Journal of Impotence Research
https://www.readbyqxmd.com/read/28446681/minute-virus-of-mice-mvm-inhibits-transcription-of-the-cyclin-b1-gene-during-infection
#20
Matthew S Fuller, Kinjal Majumder, David J Pintel
Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent pre-mitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others previously reported, depends upon the significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function thus preventing mitotic entry...
April 26, 2017: Journal of Virology
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