Purification and Characterization of Recombinant Expressed Apple Allergen Mal d 1

Mal d 1 is the primary apple allergen in northern Europe. To explain the differences in the allergenicity of apple varieties, it is essential to study its properties and interaction with other phytochemicals, which might modulate the allergenic potential. Therefore, an optimized production route followed by an unsophisticated purification step for Mal d 1 and respective mutants is desired to produce sufficient amounts. We describe a procedure for the transformation of the plasmid in competent E. coli cells, protein expression and rapid one-step purification. r-Mal d 1 with and without a polyhistidine-tag are purified by immobilized metal ion affinity chromatography (IMAC) and fast-protein liquid chromatography (FPLC) using a high-resolution anion-exchange column, respectively. Purity is estimated by SDS-PAGE using an image-processing program (Fiji). For both mutants an appropriate yield of r-Mal d 1 with purity higher than 85% is achieved. The allergen is characterized after tryptic in gel digestion by peptide analyses using HPLC-MS/MS. Secondary structure elements are calculated based on CD-spectroscopy and the negligible impact of the polyhistidine-tag on the folding is confirmed. The formation of dimers is proved by mass spectrometry and reduction by DTT prior to SDS-PAGE. Furthermore, the impact of the freeze and thawing process, freeze drying and storage on dimer formation is investigated.

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Development and Validation of an LC-MS/MS Method for Simultaneous Determination of Canagliflozin and Metformin HCl in Rat Plasma and its Application

Current Pharmaceutical Analysis ◽

10.2174/1573412915666190312161823 ◽

2020 ◽

Vol 16 (6) ◽

pp. 752-762

Author(s):

Vivek Nalawade ◽

Vaibhav A. Dixit ◽

Amisha Vora ◽

Himashu Zade

Keyword(s):

Internal Standard ◽

Rat Plasma ◽

Precipitation Method ◽

Herbal Extracts ◽

One Step ◽

Precision And Accuracy ◽

Development And Validation ◽

The Impact ◽

Metformin Hcl

Background: Food and herbal extracts rich in Quercetin (QRT) are often self-medicated by diabetics and can potentially alter the pharmacokinetics (PK) of Metformin HCl (MET) and Canagliflozin (CNG) leading to food or herb-drug interactions and reduced therapeutic efficacy. However, the impact of these flavonoids on the pharmacokinetic behaviour of MET and CNG is mostly unknown. Methods: A simple one-step protein precipitation method was developed for the determination of MET and CNG from rat plasma. The mobile phase chosen was MeOH 65% and 35% water containing 0.1% formic acid at a flow rate of 1mL/min. Results: The retention time of MET, internal standard (Valsartan) and CNG was 1.83, 6.2 and 8.2 min, respectively. The method was found to be linear in the range of 200 - 8000 ng/mL for CNG and 100 = 4000 ng/ml for MET. Precision and accuracy of the method were below 20% at LLOQ and below 15% for LQC, MQC, and HQC. Conclusion: The method was successfully applied for the determination of PK of MET and CNG by using 100 μL of rat plasma. QRT co-administration affects the PK parameters of MET and CNG. This alteration in PK parameters might be of significant use for clinicians and patients.

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One-step synthesis of PCL/Mg Janus micromotor for precious metal ion sensing, removal and recycling

Journal of Materials Science ◽

10.1007/s10853-019-03390-2 ◽

2019 ◽

Vol 54 (9) ◽

pp. 7322-7332 ◽

Cited By ~ 8

Author(s):

Dongmei Zhang ◽

Dan Wang ◽

Jieai Li ◽

Xiaoyi Xu ◽

Hui Zhang ◽

...

Keyword(s):

Precious Metal ◽

Metal Ion ◽

Ion Sensing ◽

One Step ◽

Metal Ion Sensing

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The Science of Ageism: Two Steps Forward, One Step Back

Innovation in Aging ◽

10.1093/geroni/igaa057.2222 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

pp. 646-646

Author(s):

Jerin Lee ◽

Natalie Shook

Keyword(s):

Psychological Health ◽

Well Being ◽

Census Bureau ◽

Complex Nature ◽

The Past ◽

Growing Pains ◽

Research Findings ◽

One Step ◽

Prejudicial Attitudes ◽

The Impact

Abstract The past two decades have been marked by a rapidly aging population in the U.S. (U.S. Census Bureau, 2018), making prejudicial attitudes toward older adults (i.e., ageism) and the impact of such attitudes more relevant. As such, ageism researchers have worked tirelessly to not only understand this normalized and insidious form of bias, but also develop efforts to combat it. This symposium will feature four ageism researchers who will showcase both the growing pains and novel contributions of ageism research, ranging from the impact of ageism on psychological health to ageism interventions to issues related to the measurement of ageism. Specifically, Dr. Ayalon will present findings regarding difficulties with the assessment of exposure to ageism and the consequences of ageism for psychological well-being. Dr. Horhota will share research demonstrating challenges associated with confronting ageism. Dr. Levy will present a model showcasing factors associated with the reduction of ageism. Ms. Lee will discuss research findings examining the construct validity of several ageism measures. These talks highlight theoretical and real-world implications associated with the complex nature of ageism, providing important directions for enriching ageism research going forward.

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Relationship between Phenolic Compounds, Antioxidant Properties, and the Allergenic Protein Mal d 1 in Different Selenium-Biofortified Apple Cultivars (Malus domestica)

Molecules ◽

10.3390/molecules26092647 ◽

2021 ◽

Vol 26 (9) ◽

pp. 2647

Author(s):

Sabrina Groth ◽

Christoph Budke ◽

Timo Weber ◽

Susanne Neugart ◽

Sven Brockmann ◽

...

Keyword(s):

Phenolic Compounds ◽

Antioxidant Properties ◽

Total Phenolic ◽

Allergenic Protein ◽

Antibody Recognition ◽

Allergenic Potential ◽

Allergenic Proteins ◽

Other Substances ◽

Mal D 1 ◽

Apple Cultivars

Notable parts of the population in Europe suffer from allergies towards apples. To address this health problem, the analysis of the interactions of relevant allergens with other substances such as phenolic compounds is of particular importance. The aim of this study was to evaluate the correlations between the total phenolic content (TPC), polyphenol oxidase (PPO) activity, antioxidant activity (AOA), and the phenolic compound profile and the content of the allergenic protein Mal d 1 in six apple cultivars. It was found that the PPO activity and the content of individual phenolic compounds had an influence on the Mal d 1 content. With regard to the important constituents, flavan-3-ols and phenolic acids, it was found that apples with a higher content of chlorogenic acid and a low content of procyanidin trimers and/or epicatechin had a lower allergenic potential. This is probably based on the reaction of phenolic compounds (when oxidized by the endogenous PPO) with proteins, thus being able to change the conformation of the (allergenic) proteins, which further corresponds to a loss of antibody recognition. When apples were additionally biofortified with selenium, the composition of the apples, with regard to TPC, phenolic profile, AOA, and PPO, was significantly affected. Consequently, this innovative agronomic practice seems to be promising for reducing the allergenic potential of apples.

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Solvent-Induced Hysteresis Loop in Anionic Spin Crossover (SCO) Isomorph Complexes

Magnetochemistry ◽

10.3390/magnetochemistry7060075 ◽

2021 ◽

Vol 7 (6) ◽

pp. 75

Author(s):

Emmelyne Cuza ◽

Samia Benmansour ◽

Nathalie Cosquer ◽

Françoise Conan ◽

Carlos J. Gómez-García ◽

...

Keyword(s):

Metal Ion ◽

Spin Crossover ◽

Magnetic Behaviour ◽

Magnetic Studies ◽

Tridentate Ligands ◽

Tripodal Ligand ◽

Single Crystal Structures ◽

One Step ◽

Lower Temperature ◽

Step Transition

Reaction of Fe(II) with the tris-(pyridin-2-yl)ethoxymethane (py3C-OEt) tripodal ligand in the presence of the pseudohalide ancillary NCSe- (E = S, Se, BH3) ligand leads to the mononuclear complex [Fe(py3C-OEt)2][Fe(py3C-OEt)(NCSe)3]2·2CH3CN (3), which has been characterised as an isomorph of the two previously reported complexes, Fe(py3C-OEt)2][Fe(py3C-OEt)(NCE)3]2·2CH3CN, with E = S (1), BH3 (2). X-ray powder diffraction of the three complexes (1–3), associated with the previously reported single crystal structures of 1–2, revealed a monomeric isomorph structure for 3, formed by the spin crossover (SCO) anionic [Fe(py3C-OEt)(NCSe)3]− complex, associated with the low spin (LS) [Fe(py3C-OEt)2]2+ cationic complex and two solvent acetonitrile molecules. In the [Fe(py3C-OEt)2]2+ complex, the metal ion environment involves two py3C-OEt tridentate ligands, while the [Fe(py3C-OEt)(NCSe)3]− anion displays a hexacoordinated environment involving three N-donor atoms of one py3C-OEt ligand and three nitrogen atoms arising from the three (NCSe)− coligands. The magnetic studies for 3 performed in the temperature range 300-5-400 K, indicated the presence of a two-step SCO transition centred around 170 and 298 K, while when the sample was heated at 400 K until its complete desolvation, the magnetic behaviour of the high temperature transition (T1/2 = 298 K) shifted to a lower temperature until the two-step behaviour merged with a gradual one-step transition at ca. 216 K.

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Zinc limitation in Klebsiella pneumoniae profiled by quantitative proteomics influences transcriptional regulation and cation transporter-associated capsule production

BMC Microbiology ◽

10.1186/s12866-021-02091-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

A. Sukumaran ◽

S. Pladwig ◽

J. Geddes-McAlister

Keyword(s):

Klebsiella Pneumoniae ◽

Metal Ion ◽

Ion Homeostasis ◽

Cellular Protein ◽

Pcr Analysis ◽

Phenotypic Analysis ◽

Metal Ion Homeostasis ◽

Capsule Production ◽

The Impact

Abstract Background Microbial organisms encounter a variety of environmental conditions, including changes to metal ion availability. Metal ions play an important role in many biological processes for growth and survival. As such, microbes alter their cellular protein levels and secretion patterns in adaptation to a changing environment. This study focuses on Klebsiella pneumoniae, an opportunistic bacterium responsible for nosocomial infections. By using K. pneumoniae, we aim to determine how a nutrient-limited environment (e.g., zinc depletion) modulates the cellular proteome and secretome of the bacterium. By testing virulence in vitro, we provide novel insight into bacterial responses to limited environments in the presence of the host. Results Analysis of intra- and extracellular changes identified 2380 proteins from the total cellular proteome (cell pellet) and 246 secreted proteins (supernatant). Specifically, HutC, a repressor of the histidine utilization operon, showed significantly increased abundance under zinc-replete conditions, which coincided with an expected reduction in expression of genes within the hut operon from our validating qRT-PCR analysis. Additionally, we characterized a putative cation transport regulator, ChaB that showed significantly higher abundance under zinc-replete vs. -limited conditions, suggesting a role in metal ion homeostasis. Phenotypic analysis of a chaB deletion strain demonstrated a reduction in capsule production, zinc-dependent growth and ion utilization, and reduced virulence when compared to the wild-type strain. Conclusions This is first study to comprehensively profile the impact of zinc availability on the proteome and secretome of K. pneumoniae and uncover a novel connection between zinc transport and capsule production in the bacterial system.

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Isolation and structural elucidation of dimeric epigallocatechin-3-gallate autoxidation products and their antioxidant capacity

European Food Research and Technology ◽

10.1007/s00217-021-03846-3 ◽

2021 ◽

Author(s):

Julian Alfke ◽

Uta Kampermann ◽

Svetlana Kalinina ◽

Melanie Esselen

Keyword(s):

Antioxidant Capacity ◽

Antioxidant Properties ◽

Cd Spectroscopy ◽

Trolox Equivalent Antioxidant Capacity ◽

Published Data ◽

Dietary Polyphenols ◽

Cellular Effects ◽

The Impact

AbstractDietary polyphenols like epigallocatechin-3-gallate (EGCG)—which represents the most abundant flavan-3-ol in green tea—are subject of several studies regarding their bioactivity and health-related properties. On many occasions, cell culture or in vitro experiments form the basis of published data. Although the stability of these compounds is observed to be low, many reported effects are directly related to the parent compounds whereas the impact of EGCG degradation and autoxidation products is not yet understood and merely studied. EGCG autoxidation products like its dimers theasinensin A and D, “P2” and oolongtheanin are yet to be characterized in the same extent as their parental polyphenol. However, to investigate the bioactivity of autoxidation products—which would minimize the discrepancy between in vitro and in vivo data—isolation and structure elucidation techniques are urgently needed. In this study, a new protocol to acquire the dimers theasinensin A and D as well as oolongtheanin is depicted, including a variety of spectroscopic and quadrupole time-of-flight high-resolution mass spectrometric (qTOF-HRMS) data to characterize and assign these isolates. Through nuclear magnetic resonance (NMR) spectroscopy, polarimetry, and especially circular dichroism (CD) spectroscopy after enzymatic hydrolysis the complementary atropisomeric stereochemistry of the isolated theasinensins is illuminated and elucidated. Lastly, a direct comparison between the isolated EGCG autoxidation products and the monomer itself is carried out regarding their antioxidant properties featuring Trolox equivalent antioxidant capacity (TEAC) values. These findings help to characterize these products regarding their cellular effects and—which is of special interest in the flavonoid group—their redox properties.

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Open problems in traceability: from raw materials to finished food products

Journal of Agricultural Engineering ◽

10.4081/jae.2013.272 ◽

2013 ◽

Vol 44 (2s) ◽

Cited By ~ 1

Author(s):

Lorenzo Comba ◽

Fabrizio Dabbene ◽

Paolo Gay ◽

Cristina Tortia

Keyword(s):

Supply Chain ◽

Raw Materials ◽

Product Recalls ◽

Open Problems ◽

Food Traceability ◽

Clear Sign ◽

One Step ◽

Risk Notification ◽

Production Period ◽

The Impact

Even though the main EU regulations concerning food traceability have already entered to force since many years, we still remark very wide and impacting product recalls, which often involve simultaneously large territories and many countries. This is a clear sign that current traceability procedures and systems, when implemented with the only aim of respecting mandatory policies, are not effective, and that there are some aspects that are at present underestimated, and therefore should be attentively reconsidered. In particular, the sole adoption of the so-called “one step back-one step forward traceability” to comply the EC Regulation 178/2002, where every actor in the chain handles merely the data coming from his supplier and those sent to his client, is in fact not sufficient to control and to limit the impact of a recall action after a risk notification. Recent studies on lots dispersion and routing demonstrate that each stakeholder has to plan his activities (production, transformation or distribution) according to specific criteria that allow pre-emptively estimating and limiting the range action of a possible recall. Moreover, these new and very recently proposed techniques still present some limits; first of all the problem of traceability of bulk products (e.g. liquids, powders, grains, crystals) during production phases that involve mixing operations of several lots of different/same materials. In fact, current traceability practices are in most cases unable to deal efficiently with this kind of products, and, in order to compensate the lack of knowledge about lot composition, typically resort to the adoption of very large lots, based for instance on a considered production period. Aim of this paper is to present recent advances in the design of supply chain traceability systems, discussing problems that are still open and are nowadays subject of research.

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Does Learners’ Proficiency Level Affect Oral Corrective Feedback Preferences?

International Journal of Linguistics ◽

10.5296/ijl.v8i4.9766 ◽

2016 ◽

Vol 8 (4) ◽

pp. 150 ◽

Cited By ~ 1

Author(s):

Nesa Bandarchian Rashti ◽

Maryam Danaye Tous

Keyword(s):

Error Correction ◽

Corrective Feedback ◽

Good Deal ◽

Potential Effect ◽

Factors Affecting ◽

Proficiency Level ◽

Oral Corrective Feedback ◽

One Step ◽

Feedback Preferences ◽

The Impact

<p>For decades now, there has been a good deal of research on factors affecting students’ oral corrective feedback preferences. Although it has been proven that learners’ characteristics such as their verbal intelligence and attitude toward error correction are highly effective in students’ preferred type of oral corrective feedback, the claims regarding the impact of learners’ proficiency level on their choice of oral corrective feedback have yet to be fully substantiated. In order to take this line of research one step forward, it is important to examine the potential effect of students’ level of proficiency in error correction literature. To this end, this paper aims to shed light on lower and higher level learners’ opinion about corrective feedback issues in an EFL context.</p>

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