",isbn:"978-1-80356-552-1",printIsbn:"978-1-80356-551-4",pdfIsbn:"978-1-80356-553-8",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!0,isSalesforceBook:!1,hash:"4c2e03f295fbc697350f0bf3bf89a14f",bookSignature:"Associate Prof. Murat Eyvaz, Dr. Ahmed Albahnasawi, M.Sc. Ercan Gürbulak and MSc. Mesut Tekbaş",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/11840.jpg",keywords:"Aridity and Drought, Precipitation and Evapotranspiration, Land Use, Human Activity, Desertification, Desert, Soil Structure, Water Treatment, Water Scarcity, Irrigated Agriculture, Remote Sensing, Climate Change",numberOfDownloads:47,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 10th 2022",dateEndSecondStepPublish:"May 11th 2022",dateEndThirdStepPublish:"July 10th 2022",dateEndFourthStepPublish:"September 28th 2022",dateEndFifthStepPublish:"November 27th 2022",remainingDaysToSecondStep:"11 days",secondStepPassed:!0,currentStepOfPublishingProcess:3,editedByType:null,kuFlag:!1,biosketch:"Dr. Murat Eyvaz has co-authored many journal articles and conference papers and has taken part in many national projects. He serves as an editor in 51 journals and a reviewer in 125 journals indexed in SCI, SCI-E, and other indexes. He has four patents on wastewater treatment systems. Dr. Eyvaz's research interests include applications in water and wastewater treatment facilities, electrochemical treatment processes, and filtration systems at the lab.",coeditorOneBiosketch:"Dr. Albahnasawi is a pioneering researcher in environmental sciences and engineering, he has co-authored numerous journal articles and conference papers on water and wastewater treatment, and waste remediation. Recently, his research interests are the application and designing of Microbial Fuel Cell integrated with Fenton oxidation for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",coeditorTwoBiosketch:"Dr. Gurbulak is a pioneering researcher in environmental sciences and engineering. He has co-authored numerous journal articles and conference papers on water and wastewater treatment, and advanced waste remediation technologies. His research interests are the application and designing of hydrothermal processes for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",coeditorThreeBiosketch:"Dr. Tekbaş is a pioneering researcher in environmental sciences and engineering, he has co-authored numerous journal articles and conference papers on water and wastewater treatment, and advanced waste remediation technologies. His research interests are the application and designing of supercritical water oxidation processes for wastewater treatment/solid waste management and electrochemical analyses.",coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"170083",title:"Associate Prof.",name:"Murat",middleName:null,surname:"Eyvaz",slug:"murat-eyvaz",fullName:"Murat Eyvaz",profilePictureURL:"https://mts.intechopen.com/storage/users/170083/images/system/170083.png",biography:"Dr. Murat Eyvaz is an associate professor in the Environmental Engineering Department, Gebze Technical University, Turkey. His research interests include applications in water and wastewater treatment facilities, electrochemical treatment processes, filtration systems at the lab and pilot-scale, membrane processes (forward osmosis, reverse osmosis, membrane bioreactors), membrane manufacturing methods (polymeric membranes, nanofiber membranes, electrospinning), spectrophotometric analyses (UV, atomic absorption spectrophotometry), chromatographic analyses (gas chromatography, high-pressure liquid chromatography). He has co-authored many journal articles and conference papers and has taken part in many national projects. He serves as an editor and reviewer for many indexed journals. Dr. Eyvaz has four patents on wastewater treatment systems.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"5",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}}],coeditorOne:{id:"323629",title:"Dr.",name:"Ahmed",middleName:null,surname:"Albahnasawi",slug:"ahmed-albahnasawi",fullName:"Ahmed Albahnasawi",profilePictureURL:"https://mts.intechopen.com/storage/users/323629/images/system/323629.png",biography:"Dr. Ahmed Albahnasawi is a post-doctorate fellow in the Environmental Engineering Department, Gebze Technical University, Turkey. His graduate work focused on the investigation of the treatability of the sequential anoxic-aerobic batch reactors followed by ceramic membrane for textile wastewater treatment. Based on his Ph.D. research, Dr. Albahnasawi published three journal articles and participated in three international conferences. His research interests include the application and design of a microbial fuel cell integrated with Fenton oxidation for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}},coeditorTwo:{id:"176699",title:"M.Sc.",name:"Ercan",middleName:null,surname:"Gürbulak",slug:"ercan-gurbulak",fullName:"Ercan Gürbulak",profilePictureURL:"https://mts.intechopen.com/storage/users/176699/images/system/176699.png",biography:"Dr. Ercan Gürbulak is a research associate in the Environmental Engineering Department, Gebze Technical University, Turkey. He received his bachelor’s degree in Environmental Engineering from Marmara University, Turkey, in 2005. He completed his MSc and Ph.D. at Gebze Technical University in 2008 and 2019, respectively. His research interests include the application and design of hydrothermal processes for industrial wastewater treatment/solid waste management and monitoring of organic micropollutants by both chromatographic and spectrophotometric analyses.",institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical University",institutionURL:null,country:{name:"Turkey"}}},coeditorThree:{id:"189677",title:"MSc.",name:"Mesut",middleName:null,surname:"Tekbaş",slug:"mesut-tekbas",fullName:"Mesut Tekbaş",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRIbNQAW/Profile_Picture_1644828776099",biography:null,institutionString:"Gebze Technical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Gebze Technical 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1. Introduction
\n
Shared research core facilities can provide support to campus-wide investigators by providing research infrastructure for the production and purification of recombinant proteins for a variety of research applications. We have designed a research support structure for investigators pursuing research in structural and functional studies that require high yields of pure proteins, particularly suited for structural studies including biomolecular nuclear magnetic resonance (NMR) and small angle X-ray scattering.
\n
The Escherichia coli (E. coli) expression platform is commonly used for recombinant expression of proteins. The E. coli system has several advantages over yeast, insect cells, or mammalian cell expression systems: E. coli are relatively easy to handle, the doubling time is short, media are low-cost and there are abundantly established methods for protein expression [1, 2, 3, 4]. The E. coli expression platform is also well-suited for stable isotope labeling of proteins for biological NMR studies [5, 6, 7, 8, 9]. Structural studies of proteins demand large quantities of high purity protein. Meeting these requirements can be challenging, however, advancements in high-throughput technologies for recombinant expression of proteins have greatly advanced in the last decade or more, in large part due to efforts from large structural genomics and structural proteomics centers [1, 4, 10, 11, 12]. The lessons learned and technologies developed from these centers can allow for rapid assessment of different expression strategies, which can be transferred and scaled down to smaller-scale centers and academic labs [3, 4, 13].
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In addition to a demand for large quantities of highly pure protein, structural studies also often demand high solubility and stability of the protein in solution. To address this need, a high-throughput fluorescence-based thermal-shift assay, also known as differential scanning fluorimetry (DSF), has been implemented at the large structural genomics and structural proteomics centers [14]. DSF was originally developed as a high-throughput drug discovery assay to screen for small molecules that bind to and stabilize target proteins [15, 16, 17]. The DSF screen has been further adapted to optimize buffer conditions by varying the pH, buffer components, detergents, reducing agents and small molecules to screen for conditions that increase the stability and conformational homogeneity of a protein [14, 17, 18, 19, 20], which is key in obtaining high-quality structural data.
\n
We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology and have used these in our own research [21, 22, 23, 24, 25, 26, 27, 28, 29]. The Integrative Structural Biology effort within the Biomolecular Research Center, a shared core facility, allows researchers at Boise State University and collaborating institutions to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics can impact their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers and research laboratories. Here we outline a general workflow of essential steps in protein expression and purification that includes plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer conditions (Figure 1).
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Figure 1.
A protein expression and purification workflow from plasmid to stable purified protein.
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\n
2. Materials
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All reagents listed in this chapter are commonly available from commercial vendors. A chemical hygiene plan including storage, shelf life, and safety of all chemicals should be in place at the institution.
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2.1 Preparation of chemically competent cells
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Ice.
Lysogeny broth (LB) medium: 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl. Sterilize by autoclaving and store at room temperature.
Super optimal broth (SOB, a.k.a. Hanahan’s Broth) medium for DH5α cells: 20 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl, 0.186 g/L KCl. Adjust the pH to 7.0 with NaOH. Sterilize by autoclaving and store at room temperature.
Culture tubes and flasks.
Incubator/shaker.
Centrifuge tubes.
Serological pipettes.
Repeating pipettor.
Dimethyl sulfoxide (DMSO).
Competent Cell (CC) buffer: 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 15 mM CaCl2, 55 mM MnCl2, 250 mM KCl, pH 6.7. Dissolve all components except MnCl2 and adjust the pH to 6.7 with KOH. Then add the MnCl2 and filter sterilize the solution over a 0.22 μm filter.
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2.2 Transformation of cells for expression of desired plasmid
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Ice.
LB or super optimal broth with catabolite repression (SOC).
Culture tubes and flasks.
Incubator/shaker.
Centrifuge tubes.
Serological pipettes.
Competent cells.
LB-agar plates containing the appropriate antibiotic.
Plasmid DNA.
Heat block set.
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2.3 Calculating efficiency of competent cells
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Transformed colonies on LB-agar plate (see Section 3.3).
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2.4 Inoculating overnight cultures
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LB.
15 mL conical tube.
Sterile inoculating loop.
Appropriate antibiotics.
Shaker/incubator.
Sterile aluminum foil or culture tube cap.
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2.5 Glycerol stocks
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50% glycerol solution (autoclaved).
Make the 50% glycerol solution by diluting 100% glycerol into water.
Screwtop cryogenic vials.
Liquid nitrogen.
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2.6 DNA plasmid purification
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Resuspension buffer: 50 mM Tris-HCl, pH 8.0; 10 mM ethylenediaminetetraacetic acid (EDTA), 20 μg/mL RNase A.
Precipitation buffer: 3 M potassium acetate, 2 M glacial acetic acid, 4°C.
Wash buffer: 70% ethanol.
95% (or 100%) ethanol.
TE buffer: 10 mM Tris-HCl, pH 8.0; 0.1 mM EDTA.
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\n
2.7 Testing for protein expression and solubility in E. coli
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\n
LB.
Appropriate antibiotics.
Incubator/shaker.
Microcentrifuge tubes.
Centrifuge.
Isopropyl β-D-1-thiogalactopyranoside (IPTG).
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2.8 Lysing cells
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Induced cells suspended in lysis buffer with a protease inhibitor cocktail, 0.1 mg/mL DNase I, 1 mg/mL lysozyme and 0.1 mg/mL 4-(2-Aminoethyl) benzenesulfonyl fluoride (AEBSF).
Sonication buffer.
Ice-saltwater bath.
Probe sonicator equipped with ½ inch tip.
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2.9 Gel electrophoresis, protein quantification
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Electrophoresis system.
4–12% Bis-Tris mini gel.
Sample loading buffer: 10% glycerol, 0.14 M Tris Base, 0.1 M Tris-HCl, 2% lithium dodecyl sulfate (LDS), 0.5 mM EDTA, 0.02% Blue G250; 0.006% phenol red, 1.25% 2-mercaptoethanol, pH 8.5.
Running buffer: 50 mM 2-(N-morpholino)ethanesulfonic acid (MES), 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.2.
Coomassie Blue stain.
Protein molecular weight marker.
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2.10 Testing lysis conditions for solubility
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\n
Buffer A: 50 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1 mg/mL lysozyme.
Buffer B: 50 mM Tris pH 7.5, 2 M NaCl, 5 mM EDTA, 1 mg/mL lysozyme.
Buffer C: 50 mM Tris pH 7.5, 100 mM NaCl, 50% detergent, 1 mg/mL lysozyme.
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2.11 Large-scale expression of recombinant proteins
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\n
LB.
IPTG.
Culture tubes and flasks.
Incubator/shaker.
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2.12 Uniform 15N/13C labeling of recombinant proteins
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\n
LB.
IPTG.
Culture tubes and flasks.
Incubator/shaker.
10X M9 medium: 340 mM Na2HPO4, 220 mM KH2PO4, 85.5 mM NaCl, pH 7.4.
2.13 Protein purification using immobilized metal affinity chromotography (IMAC)
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Immobilized metal affinity chromatography (IMAC) is a common method for affinity purification. A genetically encoded 6-histidine repeat affinity tag can be introduced to the carboxy or amino terminal end of the protein during cloning, which has high affinity for metal ions. The protocol given here is for affinity purification by immobilization of nickel ions with a chelator molecule, nitrilotriacetic acid (NTA) that is covalently bound to agarose; commonly known as Ni-NTA agarose. The following buffers are meant to represent a general starting point. Depending on the pI of your recombinant protein and the propensity to nonspecifically interact with the column material or resident E. coli proteins, modifications may need to be made. Additional purification may be necessary, especially when purifying proteins that bind to nucleic acids. A lithium wash may be added to the Ni-NTA purification to remove nucleic acids. Ion exchange, heparin affinity, size exclusion chromatography are often added in addition to a nickel affinity purification step.
Lysis buffer: 0.1 M Tris-HCl, 0.1 M NaCl, pH 8.1.
Wash buffer: Lysis buffer plus 5–20 mM imidazole.
Elution buffer: Lysis buffer plus 100–300 mM imidazole.
Probe sonicator.
1 mg/mL lysozyme.
Protease inhibitor cocktail.
AEBSF.
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2.14 Differential scanning fluorimetry to assess protein stability
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Low ionic strength buffer (e.g., 10 mM Tris-HCl).
qPCR machine with filter set that matches fluorescent dye and equipped with a ramp rate of minimum 1°C/min.
Inoculate 5 mL of LB (or SOB if preparing DH5α cells) with 10 μL of appropriate E. coli cells2 and grow overnight at 37°C and 250 rpm in a shaking incubator.
Use the overnight culture to inoculate 250 mL of LB (or SOB if preparing DH5α cells) and incubate at 30°C until the optical density at 600 nm (OD600) is between 0.4–0.6.
Chill the culture for at least 10 min on ice. For steps 4–10, keep the cell suspension on ice.
Spin the cell suspension for 10 min at 6000× g.
Gently resuspend the pellet in 50 mL ice-cold CC buffer into 50-mL conical tubes. Resuspend with a 10-mL serological pipette and avoid introducing bubbles.
Incubate the cell suspension on ice for at least 10 min.
Spin for 10 min at 6000× g at 4°C.
Gently resuspend the pellet in 9.4 mL ice-cold CC buffer and add 0.7 mL DMSO.
Incubate the cell suspension on ice for at least 10 min.
Distribute the cell suspension in 50–200 μL aliquots in 1.5-mL microcentrifuge tubes.3
Flash freeze the cell suspension in liquid nitrogen and store the tubes at −80°C.
At −80°C the cells will be competent for at least 6 months.
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3.2 Transformation of E. coli cells with plasmid DNA
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\n
Take competent cells out of −80°C and thaw on ice (approximately 20–30 min).
For each transformation, remove two LB-agar plates (containing the appropriate antibiotic) from storage at 4°C and warm to room temperature; optionally warm to 37°C in an incubator.
Mix 10–100 pg. DNA into 20–50 μL of competent cells in a 1.5 mL microcentrifuge tube.
Gently mix by flicking the bottom of the tube with your finger a few times.
Incubate the competent cell/DNA mixture on ice for 20–30 min.
Heat shock each tube at 42°C for 45–60 s.
Put the tubes back on ice for 2 min.
Add 1 mL of LB medium (without antibiotic) to the bacteria and grow at 37°C and 250 rpm in a shaking incubator for 45 min.
Plate 50 μL of the transformed cells onto one of the 10 cm LB-agar plate containing the appropriate antibiotic and the remaining 950 μL onto the second 10 cm LB-agar plate.
Incubate plates at 37°C overnight.
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3.3 Calculating transformation efficiency of competent cells
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\n
Count the number of colony forming units (CFUs) on the LB-agar plate after transformation (see Section 3.2).
Calculate the transformation efficiency (TrEff) in CFUs/μg of DNA using Eq. (1).
Add 5–10 mL of liquid LB to a culture tube and add the appropriate antibiotic to at correct concentration. A good negative control is LB media plus antibiotic without any bacteria inoculated. You should see no growth in this culture after overnight incubation.
Using a sterile inoculating loop, select a single colony from your LB-agar plate for plasmid purifications and a swipe from 10 to 20 colonies for protein expression (Section 3.2).
Add the inoculating loop to the liquid LB with antibiotics and swirl.
Loosely cover the culture with sterile aluminum foil or a culture tube cap.
After incubation, check for growth, which is characterized by a cloudy haze in the media.
For overnight cultures, incubate bacterial culture at 30°C for 12–16 h in a shaking incubator.4
For long-term storage of the bacteria, you can proceed with Section 3.5.
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3.5 Preparation of a glycerol stock
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\n
Follow Section 3.2 for transforming and plating E. coli cells.
Follow Section 3.4 for inoculating an overnight culture.
Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top cryogenic vial5 and gently mix.
Submerse the glycerol stock tube into liquid nitrogen and store at −80°C. The stock is now stable for years, as long as it is kept at −80°C. Subsequent freeze and thaw cycles reduce shelf life.
To recover bacteria from your glycerol stock: open the tube and use a sterile loop, toothpick, or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock thaw. Streak the bacteria onto an LB-agar plate.
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3.6 Plasmid DNA purification
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\n
Preheat the TE Buffer in the incubator at 37°C.
Spin 5 mL of the overnight LB culture at 6000× g for 10 min at 4°C. Discard supernatant.
Add 250 μL Resuspension Buffer containing RNase A to the cell pellet and resuspend the pellet by pipetting until homogeneous.
Add 250 μL Lysis Buffer. Mix gently by inverting the capped tube until homogeneous. Do not vortex. Incubate the tube at room temperature for 5 min.
Add 350 μL Precipitation Buffer. Mix immediately by inverting the tube until homogeneous. Do not vortex. Centrifuge the lysate at 16,000× g for 10 min at 4°C.
Add 2–2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8) to the supernatant. Invert the microcentrifuge tube to mix. Let stand for 2 min at room temperature.
Centrifuge solution at high speed (at least 16,000× g) for 15–30 min at 4°C. Discard supernatant.
Open and invert the tube on a paper towel to drain it out.
Wash pellet by adding 500 μL cold 70% ethanol.
Centrifuge solution at high speed (at least 16,000× g) for 5 min at room temp. Discard supernatant by pipetting it out of the tube.
Dry the pellet by inverting over paper towel for 5–20 min.
Resuspend dry DNA with TE.
Store plasmid DNA at 4°C (short term) or store the DNA in aliquots at −20°C (long term.)
\n
\n
3.7 Testing for soluble protein expression in E. coli
\n
The following protocol is written for proteins expressed under the control of the lac, tac, or T7 promoters. The method as described is a generic protocol that can be expanded to test expression in different strains of E. coli, induction temperatures, concentrations of IPTG, or even in the presence of ligands or cofactors.
\n
3.7.1 Protein expression
\n
\n
Transform plasmid into an E. coli expression strain following Section 3.2.
Inoculate a liquid LB culture following Section 3.4.
Grow cells for a few hours at 37°C, shaking at 250 rpm. Make sure the tubes are tilted.
Monitor the turbidity. Once the culture reaches an OD600 of 0.4–0.6 (takes ~2–4 h, depending on the sample), take out 2 mL of the culture. Measure the actual OD600.
Transfer the equivalent of 1 mL of cells at OD600 = 0.8 in a 1.5-mL microcentrifuge tube.6
Collect cells by centrifugation at 16,000× g on a tabletop centrifuge for at least 1 min. Carefully remove all of the supernatant. This is the uninduced sample. Store the cells at −20°C.
Add IPTG to a final concentration of 1.0 mM to the remaining culture. Continue shaking at 250 rpm for 12–16 h at 18°C.
Measure the OD600. Collect cells by centrifugation in two tubes containing the equivalent of 1 mL of cells at OD600 = 0.8 and remove the supernatant. These are the induced samples; one tube will be used to test for expression and the second for solubility. Store the cells at −20°C until ready to test for expression.
\n
\n
3.7.2 Testing for expression
\n
\n
Take the tube of uninduced and one tube of induced cells and resuspend each in 100 μL of 1X SDS polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer.
Boil the samples for 10 min, then cool down to room temperature.
Centrifuge for 5 min at 16,000× g on a tabletop centrifuge at room temperature.
Take 10 μL of each sample from the top of tube taking care not to disturb the pellet.
Analyze the results using SDS-PAGE following Section 3.9 (Figure 2), with western blotting if necessary.
\n
Figure 2.
SDS-PAGE gel of pre- and post-induction of an RNA binding protein (RBP) in both rich (LB) and minimal (M9) media. The arrow indicates the recombinant RBP.
\n
\n
3.7.3 Testing for solubility
\n
\n
Resuspend the remaining induced cell pellet in 50 μL of lysis buffer containing protease inhibitors and 1 mg/mL of lysozyme.
Follow Section 3.8.1 for freeze-thawing to lyse the cells.
Spin down in a microcentrifuge at maximum speed for 10 min at 4°C.
Carefully transfer all of the supernatant into a new microcentrifuge tube. Add 50 μL of 2X SDS-PAGE buffer. This is the soluble fraction.
Resuspend the pellet in 100 μL of 1X SDS-PAGE buffer. This is the insoluble fraction.
Boil the samples for 10 min, then cool down to room temperature.
Centrifuge for 5 min at 16,000× g at room temperature.
Analyze 15 μL of each sample using SDS-PAGE following Section 3.9.
\n
\n
\n
3.8 Lysing cells
\n
Traditionally cell lysis can be done with physical disruption or reagent-based methods. Freeze-thaw protocol works best for small volumes (less than 1 mL) in 1.5 mL microcentrifuge tubes. Sonication can be done with smaller volumes using a microtip.
\n
3.8.1 Freeze-thaw
\n
\n
Freeze the samples to be lysed (typically 0.1–1.0 mL in a 1.5 mL microcentrifuge tube) in a − 80°C freezer, leave for 15 min.
Thaw immediately in a 42°C water bath. Vortex vigorously to mix well.
Repeat the two previous steps three more times (four freeze-thaw-vortex cycles in all).
Spin the tubes for 5 min at maximum speed in a microcentrifuge.
Separate the supernatant (contains soluble protein) from the pellet (contains insoluble protein) by pipetting out the supernatant to a clean tube.
\n
\n
3.8.2 Sonication
\n
\n
Prepare ice-saltwater bath by sprinkling salt over packed ice in a container.
Place a 50-mL conical tube containing the cell pellet suspended in lysis buffer securely in the ice-saltwater bath.
Insert clean tip of a sonicator in the sample without contacting sides or bottom of the tube.
Set the output power, cycle, and timer to the optimal settings (e.g., five short bursts of 15 s followed by intervals of 30 s for cooling).
Keep the suspension at all times on ice.
\n
\n
\n
3.9 Gel electrophoresis
\n
\n
Add ~100 μg of protein to SDS sample buffer.
Heat the sample at 70°C for 10 min.
Load the entire volume of sample onto a 4–12% Bis-Tris mini gel.
Run the gel at 200 V for 35 min.
At the end of the electrophoresis, wash the gel in deionized water three times.
Stain the gel with Coomassie Blue stain for 1 h.
Wash the gel with deionized water extensively until the water is clear.
\n
\n
3.10 Testing lysis conditions for solubility
\n
The solubility of a protein depends strongly on the composition of the lysis buffer. Using the procedure described below, the solubility of a specific protein can be tested under neutral (Buffer A), high salt (Buffer B), and with detergent included (Buffer C).
Follow Section 3.7.1 for the best expressing condition and collect four induced samples.
Spin down the cells for 5 min at 12,000× g in a microcentrifuge.
To each cell pellet, add 100 μL of the appropriate buffer (see Section 2.10).
Vortex to resuspend the cells.
Lyse cells using the freeze-thaw method (Section 3.8.1).
To the supernatant, add 25 μL of 4X SDS-PAGE loading buffer.
To the cell pellet, add 125 μL of 1X SDS-PAGE loading buffer.
Heat all samples to 95°C for 5 min.
Vortex briefly and then centrifuge for 5 min at maximum speed.
Load 20 μL on an SDS-PAGE gel; avoid disturbing the pellet.
\n
\n
3.11 Large-scale expression of recombinant proteins
\n
\n
Transform plasmid into an E. coli expression strain following Section 3.2.
Inoculate a liquid LB culture for an overnight growth following Section 3.4.
The next day, use the overnight culture to inoculate 1 L of LB with the appropriate antibiotic.
Grow cultures at 37°C and 250 rpm shaking until the OD600 is ~0.6–0.8.
Induce expression of protein by adding IPTG to a final concentration of 0.1 mM.
Lower the temperature to 18°C and continue 250 rpm shaking for 12–16 h.
Follow Sections 3.7.1 and 3.7.2 to test for protein expression.
Harvest the cells by centrifugation at 6000× g.
Suspend cells in lysis buffer and store at −20°C.
\n
\n
3.12 Uniform 15N/13C labeling of recombinant proteins
\n
This protocol is for proteins expressed under the control of the lac, tac, or T7 promoters.
\n
3.12.1 Day 1
\n
\n
Transform 10 μL of competent BL21(DE3) cells (or derivatives) with 10 ng of plasmid DNA and plate cells on LB-agar containing the appropriate antibiotics (See Section 3.2).
\n
\n
3.12.2 Day 2
\n
\n
Prepare 50 mL of unlabeled defined medium for overnight culture as follows, in a 200 mL culture flask:\n
5 mL 10X M9 medium.
5 mL 10X ammonium chloride.
0.75 mL 20% glucose.
50 μL of each CaCl2, MgSO4, thiamine and biotin.
antibiotic at working concentration.
autoclaved water to 50 mL.
Inoculate a 5 mL culture (LB with appropriate antibiotic) with several freshly grown colonies (ca. 10–20).
Incubate cells in tilted tubes for a few hours at 37°C and 250 rpm in a shaking incubator, until the culture is visibly turbid.
Prewarm 50 mL of unlabeled defined medium to 30°C. While warming, centrifuge the LB culture (5 min, 4000× g, 30°C) and discard supernatant.
Resuspend cell pellet in 50 mL unlabeled defined media, for a starting OD600 of ~0.03–0.08. Grow the culture overnight at 30°C in a shaking incubator.
\n
\n
3.12.3 Day 3
\n
\n
Prepare 500 mL of 13C, 15N labeled defined medium as follows, in a 2 L baffled flask:\n
50 mL 10X M9 medium.
0.5 g 15NH4Cl dissolved in 5 mL water and sterile filtered.
1.5 g 13C glucose dissolved in 10 mL water and sterile filtered.
500 μL of each CaCl2, MgSO4, thiamine and biotin.
antibiotic at working concentration.
autoclaved water to 500 mL.
Prewarm the 500 mL of 13C, 15N labeled defined medium.
Centrifuge the overnight 50-mL unlabeled defined medium culture (5 min, 4000× g, 30°C) and discard supernatant.
Resuspend the cell pellet in 500 mL of 13C, 15N labeled defined medium, for a starting OD600 of 0.03–0.08.
Grow culture at 37°C and 250 rpm in a shaking incubator until cells reach mid-log growth (OD600 ~ 0.5–0.8).
Once cells reach mid-log growth (OD600 ~ 0.5–0.8), measure the OD600. Calculate the corrected volume (in mL) to take for the sample aliquot equivalent of 1 mL of cells at OD600 = 0.8 (See Section 3.7.1 for details).
Transfer aliquot to a microcentrifuge tube, and spin it down at maximum speed for at least 1 min at room temperature. Remove the supernatant. This is an uninduced sample. Store the uninduced cells at −20°C.
Induce protein expression by adding IPTG based on the optimal values of IPTG concentration, incubation time and incubation temperature (See Section 3.7).
After the induced cells have grown for the proper length of time, dilute 200 μL of the culture 10-fold with 1X PBS and measure the OD600. To prepare an induced sample, take an aliquot containing the equivalent of 1 mL of cells at OD600 = 0.8 and immediately process it as described in Section 3.7.2.
Harvest the cells by centrifugation at 6000× g for 20–30 min at 4°C. Discard supernatant. Store the pellet at −20°C until ready for cell lysis.
\n
\n
\n
3.13 Protein purification using IMAC
\n
\n
Resuspend cell pellet in ~35 mL of lysis buffer containing AEBSF, a protease inhibitor cocktail, and 1 mg/mL lysozyme.
Lyse cells (See Section 3.8).
Remove 75 μL of lysate and pipette into a 1.5 mL microcentrifuge tube. Centrifuge the 75 μL aliquot for 10 min at 12,000× g at room temperature.
Separate the supernatant into a new vial and treat with 25 μL of 4X SDS PAGE sample buffer. To the remaining pellet, add 100 μL of 1X SDS PAGE.
Boil separated and SDS buffer-treated samples for 10 min and store at room temperature for further SDS-PAGE analysis.
Centrifuge the remaining lysate (ca. 35 mL) at 16,000× g at 4°C for 20–30 min.
Filter the supernatant over a 0.4-micron syringe filter.
Pre-equilibrate the appropriate amount of Ni-NTA agarose for the amount of protein expressed in desired equilibration buffer; typically, 1–2 mL of settled agarose washed with two column volumes (CVs) of sterile, deionized water followed by two CVs of the buffer.
Bind the filtered lysate to the Ni-NTA agarose either batch or column loading. For batch loading, combine the filtered lysate and Ni-NTA agarose and gently rock for 30–60 min prior to pouring into the column. For column loading, pack Ni-NTA agarose into the column and pass the filtered lysate through the column. Collect the flow through eluent for SDS-PAGE analysis.
Wash the column with 15 CVs of cold lysis buffer. Collect wash eluent for SDS-PAGE analysis.
A step gradient consisting of 15 CVs each of 5, 10, and 20 mM imidazole may be used to determine best washing conditions. Collect wash eluents for SDS-PAGE analysis.
Wash the column with 20 CVs elution buffer, collecting 1 mL fractions.
Evaluate all collected samples by SDS-PAGE (see Section 3.9)7 (Figure 3).
Pool fractions containing pure recombinant protein and dialyze into an appropriate buffer.
Clean Ni-NTA agarose by washing with 0.5 M NaOH for 30 min. Wash with five CVs sterile deionized water and store in 30% ethanol for long-term storage. The Ni-NTA agarose may be re-used for the same protein multiple times.
\n
Figure 3.
SDS-PAGE gel of a typical Ni-NTA purification of an RBP (arrow indicates the recombinant RBP). (A) Samples appear in the following order: MW markers, Lysate, Supernatant, Pellet, Flowthrough, Wash #1: 50 mM Tris-Cl, 100 mM NaCl, pH 7.7; washes #2–4: 10 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. (B) MW markers, Elutions #1–9: 200 mM Imidazole, 50 mM Tris-Cl, 100 mM NaCl, pH 7.7. Some protein elutes from the column in the wash steps. All fractions are kept and can be pooled after SDS-PAGE analysis.
\n
\n
3.14 Differential scanning fluorimetry to assess protein stability
\n
\n
Prepare 1500 μL of 18 μM protein in dilution buffer.
Add 1.5 μL of 5000X dye.
Pipette up and down gently to mix.
Divide the protein plus dye solution among 10 vials: 140 μL per vial (some stock solution will remain).
Add 80 μL additive to be screened to one of nine vials.
Add 80 μL of dilution buffer in the tenth vial as a control.
Pipette up and down gently to mix.
Transfer 50 μL of protein plus dye plus additive solution (or control solution) to the 96-well PCR plate in triplicate.8
Cover PCR plate with a sheet of optically clear adhesive and seal each well.
Spin 96-well PCR plate in a centrifuge equipped with a plate holder at 800× g for 2 min at room temperature.
Place 96-well PCR plate into qPCR machine and run the following program:\n
select total volume per well 50 μL
select experiment type melting curve
set the following temperatures: an initial 2 min hold at 25°C, increase in increments of 0.5–1.0°C and hold each for 30 s,9 to a final temperature of 95°C (with a 2 min hold).
Export data for further analysis.
Data can be plotted as the fluorescence vs. temperature (Figure 4).
After buffer optimization, structural data can be collected such as a 1H, 15N 2D NMR spectrum (see Figure 5 for example of spectrum).
\n
Figure 4.
DSF analysis of an RBP in buffer (10 mM Tris-Cl) with different additives. (A) A graph of the fluorescence at 602 nm at increasing temperatures for the surveyed additive screen. The inflection point preceding the peak is the melting temperature. (B) A first derivative plot with a four-point smoothing applied helps to visualize the melting temperature, where the peak is the melting temperature. The legend provides a key for both A and B.
\n
Figure 5.
1H, 15N 2D NMR spectrum of an RBP prepared using the methods described here.
\n
\n
\n
4. Conclusion
\n
We have described the workflow for protein expression and purification used in our shared core laboratory. These methods for growing and handling bacterial cultures work well for plasmid amplification, mini-expression screening, optimized larger-scale protein production, protein isolation and purification, and characterization of optimized experimental solution buffer conditions. Future methods can be added as needed by the users of the core and the university research community.
\n
Acknowledgments
\n
This publication was made possible by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants P20GM109095 and P20GM103408. The authors wish to acknowledge Jackson Wall for careful reading and suggestions.
\n
Conflict of interest
Authors have no conflict of interest.
\n',keywords:"protein expression, recombinant protein, isotype enrichment, core facility, protocols",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/64334.pdf",chapterXML:"https://mts.intechopen.com/source/xml/64334.xml",downloadPdfUrl:"/chapter/pdf-download/64334",previewPdfUrl:"/chapter/pdf-preview/64334",totalDownloads:1078,totalViews:0,totalCrossrefCites:1,totalDimensionsCites:2,totalAltmetricsMentions:0,impactScore:1,impactScorePercentile:71,impactScoreQuartile:3,hasAltmetrics:0,dateSubmitted:"February 19th 2018",dateReviewed:"October 10th 2018",datePrePublished:"November 8th 2018",datePublished:"December 4th 2019",dateFinished:"November 7th 2018",readingETA:"0",abstract:"We have established and optimized standard operating procedures for growing and handling bacterial cultures in a shared core laboratory to support Integrative Structural Biology. The Integrative Structural Biology effort within the Biomolecular Research Center allows researchers to generate new knowledge about protein and RNA structure and function. We aim to understand how biomolecules assemble into stable structures and how structural dynamics impacts their function. Here we describe specific procedures for growing and handling bacterial cultures for overexpression and isolation of recombinant proteins, 15N/13C uniform labeling of recombinant proteins, protein isolation and purification, and analysis of protein solubility that are ideal for implementation in a shared research core laboratory that serves a multitude of diverse customers and research laboratories.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/64334",risUrl:"/chapter/ris/64334",book:{id:"7240",slug:"growing-and-handling-of-bacterial-cultures"},signatures:"Lisa R. Warner, Olga Mass, Nancy Donnelly Lenn, Briana R. Grantham and Julia Thom Oxford",authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Materials",level:"1"},{id:"sec_2_2",title:"2.1 Preparation of chemically competent cells",level:"2"},{id:"sec_3_2",title:"2.2 Transformation of cells for expression of desired plasmid",level:"2"},{id:"sec_4_2",title:"2.3 Calculating efficiency of competent cells",level:"2"},{id:"sec_5_2",title:"2.4 Inoculating overnight cultures",level:"2"},{id:"sec_6_2",title:"2.5 Glycerol stocks",level:"2"},{id:"sec_7_2",title:"2.6 DNA plasmid purification",level:"2"},{id:"sec_8_2",title:"2.7 Testing for protein expression and solubility in E. coli",level:"2"},{id:"sec_9_2",title:"2.8 Lysing cells",level:"2"},{id:"sec_10_2",title:"2.9 Gel electrophoresis, protein quantification",level:"2"},{id:"sec_11_2",title:"2.10 Testing lysis conditions for solubility",level:"2"},{id:"sec_12_2",title:"2.11 Large-scale expression of recombinant proteins",level:"2"},{id:"sec_13_2",title:"2.12 Uniform 15N/13C labeling of recombinant proteins",level:"2"},{id:"sec_14_2",title:"2.13 Protein purification using immobilized metal affinity chromotography (IMAC)",level:"2"},{id:"sec_15_2",title:"2.14 Differential scanning fluorimetry to assess protein stability",level:"2"},{id:"sec_17",title:"3. Methods",level:"1"},{id:"sec_17_2",title:"3.1 Preparation of chemically competent cells",level:"2"},{id:"sec_18_2",title:"3.2 Transformation of E. coli cells with plasmid DNA",level:"2"},{id:"sec_19_2",title:"3.3 Calculating transformation efficiency of competent cells",level:"2"},{id:"sec_20_2",title:"3.4 Inoculating cultures",level:"2"},{id:"sec_21_2",title:"3.5 Preparation of a glycerol stock",level:"2"},{id:"sec_22_2",title:"3.6 Plasmid DNA purification",level:"2"},{id:"sec_23_2",title:"3.7 Testing for soluble protein expression in E. coli",level:"2"},{id:"sec_23_3",title:"3.7.1 Protein expression",level:"3"},{id:"sec_24_3",title:"3.7.2 Testing for expression",level:"3"},{id:"sec_25_3",title:"3.7.3 Testing for solubility",level:"3"},{id:"sec_27_2",title:"3.8 Lysing cells",level:"2"},{id:"sec_27_3",title:"3.8.1 Freeze-thaw",level:"3"},{id:"sec_28_3",title:"3.8.2 Sonication",level:"3"},{id:"sec_30_2",title:"3.9 Gel electrophoresis",level:"2"},{id:"sec_31_2",title:"3.10 Testing lysis conditions for solubility",level:"2"},{id:"sec_32_2",title:"3.11 Large-scale expression of recombinant proteins",level:"2"},{id:"sec_33_2",title:"3.12 Uniform 15N/13C labeling of recombinant proteins",level:"2"},{id:"sec_33_3",title:"3.12.1 Day 1",level:"3"},{id:"sec_34_3",title:"3.12.2 Day 2",level:"3"},{id:"sec_35_3",title:"3.12.3 Day 3",level:"3"},{id:"sec_37_2",title:"3.13 Protein purification using IMAC",level:"2"},{id:"sec_38_2",title:"3.14 Differential scanning fluorimetry to assess protein stability",level:"2"},{id:"sec_40",title:"4. Conclusion",level:"1"},{id:"sec_41",title:"Acknowledgments",level:"1"},{id:"sec_44",title:"Conflict of interest",level:"1"}],chapterReferences:[{id:"B1",body:'Vincentelli R, Romier C. Expression in Escherichia coli: Becoming faster and more complex. Current Opinion in Structural Biology. 2013;23(3):326-334'},{id:"B2",body:'Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: Advances and challenges. Frontiers in Microbiology. 2014;5:172'},{id:"B3",body:'Vincentelli R, Bignon C, Gruez A, Canaan S, Sulzenbacher G, Tegoni M, et al. Medium-Scale Structural Genomics: Strategies for Protein Expression and Crystallization.Accounts of Chemical Research. 2003;36(3):165-172'},{id:"B4",body:'Berrow NS, Büssow K, Coutard B, Diprose J, Ekberg M, Folkers GE, et al. Recombinant protein expression and solubility screening in Escherichia coli: A comparative study. Acta Crystallogr Sect D Biol Crystallogr. 2006;62(10):1218-1226'},{id:"B5",body:'Muchmore DC, McIntosh LP, Russell CB, Anderson DE, Dahlquist FW. Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods in Enzymology. 1989;177:44-73'},{id:"B6",body:'Fiaux J, Bertelsen EB, Horwich AL, Wüthrich K. Uniform and residue-specific 15N-labeling of proteins on a highly deuterated background. Journal of Biomolecular NMR. 2004;29(3):289-297'},{id:"B7",body:'Skrisovska L, Schubert M, Allain FH-T. Recent advances in segmental isotope labeling of proteins: NMR applications to large proteins and glycoproteins. Journal of Biomolecular NMR. 2010;46(1):51-65'},{id:"B8",body:'Tugarinov V, Kay LE. Ile, Leu, and Val methyl assignments of the 723-residue malate synthase G using a new labeling strategy and novel NMR methods. Journal of the American Chemical Society. 2003;125(45):13868-13878'},{id:"B9",body:'Freiburger L, Sonntag M, Hennig J, Li J, Zou P, Sattler M. Efficient segmental isotope labeling of multi-domain proteins using Sortase A. Journal of Biomolecular NMR. 2015;63(1):1-8'},{id:"B10",body:'Edwards AM, Arrowsmith CH, Christendat D, Dharamsi A, Friesen JD, Greenblatt JF, et al. Protein production: Feeding the crystallographers and NMR spectroscopists. Nature Structural Biology. 2000;7:970-972'},{id:"B11",body:'Yee A, Gutmanas A, Arrowsmith CH. Solution NMR in structural genomics. Current Opinion in Structural Biology. 2006;16(5):611-617'},{id:"B12",body:'Busso D, Thierry JC, Moras D. The Structural Biology and Genomics Platform in Strasbourg: An Overview. Methods in Molecular Biology. New York, NY: Humana Press; 2008. pp. 523-536'},{id:"B13",body:'Gräslund S, Nordlund P, Weigelt J, Hallberg BM, Bray J, Gileadi O, et al. Protein production and purification. Nature Methods. 2008;5(2):135-146'},{id:"B14",body:'Reinhard L, Mayerhofer H, Geerlof A, Mueller-Dieckmann J, Weiss MS. Optimization of protein buffer cocktails using Thermofluor. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications. 2013;69(Pt 2):209-214'},{id:"B15",body:'Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature Protocols. 2007;2(9):2212-2221'},{id:"B16",body:'Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. Journal of Biomolecular Screening. 2001;6(6):429-440'},{id:"B17",body:'Senisterra GA, Markin E, Yamazaki K, Hui R, Vedadi M, Awrey DE. Screening for ligands using a generic and high-throughput light-scattering-based assay. Journal of Biomolecular Screening. 2006;11(8):940-948'},{id:"B18",body:'Nettleship JE, Brown J, Groves MR, Geerlof A. Methods for Protein Characterization by Mass Spectrometry, Thermal Shift (ThermoFluor) Assay, and Multiangle or Static Light Scattering. New York, NY: Humana Press; 2008. pp. 299-318'},{id:"B19",body:'Ericsson UB, Hallberg BM, DeTitta GT, Dekker N, Nordlund P. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Analytical Biochemistry. 2006;357(2):289-298'},{id:"B20",body:'Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(43):15835-15840'},{id:"B21",body:'Fallahi A, Kroll B, Warner LR, Oxford RJ, Irwin KM, Mercer LM, et al. Structural model of the amino propeptide of collagen XI alpha1 chain with similarity to the LNS domains. Protein Science. 2005;14(6):1526-1537'},{id:"B22",body:'Warner LR, Fallahi A, Kroll B, Irwin KM, Yingst S, Mercer LM, et al. Modeling and characterization of the amino propeptide of collagen α1(XI), a regulatory domain in collagen fibrillar architecture. Materials Research Society Symposium Proceedings, Structure and Mechanical Behavior of Biological Materials. 2005;874:41-46'},{id:"B23",body:'Oxford JT, DeScala J, Morris N, Gregory K, Medeck R, Irwin K, et al. Interaction between amino propeptides of type XI procollagen alpha1 chains. The Journal of Biological Chemistry. 2004;279(12):10939-10945'},{id:"B24",body:'Medeck RJ, Sosa S, Morris N, Oxford JT. BMP-1-mediated proteolytic processing of alternatively spliced isoforms of collagen type XI. The Biochemical Journal. 2003;376(pt 2):361-368'},{id:"B25",body:'Warner LR, Brown RJ, Yingst SM, Oxford JT. Isoform-specific heparan sulfate binding within the amino-terminal noncollagenous domain of collagen α1(XI). The Journal of Biological Chemistry. 2006;281(51):39507-39516'},{id:"B26",body:'Ryan RE, Martin B, Mellor L, Jacob RB, Tawara K, McDougal OM, et al. Oncostatin M binds to extracellular matrix in a bioactive conformation: Implications for inflammation and metastasis. Cytokine. 2015;72(1):71-85'},{id:"B27",body:'Kahler RA, Yingst SMC, Hoeppner LH, Jensen ED, Krawczak D, Oxford JT, et al. Collagen 11a1 is indirectly activated by lymphocyte enhancer-binding factor 1 (Lef1) and negatively regulates osteoblast maturation. Matrix Biology. 2008;27(4):330-338'},{id:"B28",body:'Oxford JT, DeScala J, Morris N, Gregory K, Medeck R, Irwin K, et al. Interaction between amino propeptides of type XI procollagen α1 chains. The Journal of Biological Chemistry. 2004;279(12):10939-10945'},{id:"B29",body:'Gregory KE, Oxford JT, Chen Y, Gambee JE, Gygi SP, Aebersold R, et al. Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI. The Journal of Biological Chemistry. 2000;275(15):11498-11506'}],footnotes:[{id:"fn1",explanation:"The stock solution of 10 mg/mL is above the solubility limit of biotin, do not sterile filter this solution. Simply make the solution with previously sterilized water."},{id:"fn2",explanation:"Some cell lines have a resident plasmid, such as BL21(DE3) pLysS or pLysE cells and require addition of antibiotics for selection of cells containing those plasmids."},{id:"fn3",explanation:"A repeating pipettor or a multichannel pipettor speeds up the aliquoting process greatly. This will minimize the time that the competent cells are manipulated, thus increasing their competency. Expect competency of ca. 107–108 CFU/μg of plasmid DNA."},{id:"fn4",explanation:"For some applications (especially culturing cells in minimal defined media) cultures should never be overgrown; growing overnight cultures at a reduced temperature, 25–30°C, is suggested."},{id:"fn5",explanation:"Snap top tubes are not recommended."},{id:"fn6",explanation:"In order to have equal loading on an SDS-PAGE gel, the same amount of cells need to be harvested for gel analysis. To harvest the same number of cells each time, calculate the volume in mL needed of your culture that would be the equivalent of 1 mL of OD600 = 0.8. E.g. X mL = 0.8/OD600 of your culture."},{id:"fn7",explanation:"Store lysate cell pellet at −20°C until SDS-PAGE has confirmed that full extraction of desired protein from the pellet is accomplished. Keep all buffers and protein samples at 4°C during purification. Batch vs. column choice will depend on binding properties of the individual protein. The SDS-PAGE of the step gradient imidazole washes will illustrate what is the highest imidazole concentration that can be used as an initial wash to clean off non-binding contaminants. If large amounts of protein remain in the cell pellet, alternative growing methods, such as IPTG concentration adjustment, or alternative purification methods including purification under denaturing conditions, should be considered. Additional purification may be necessary, such as ion exchange, or heparin binding column chromatography."},{id:"fn8",explanation:"Excess solutions are suggested to account for loss due to transfers and sticking to the sidewall of the tubes."},{id:"fn9",explanation:"Slower ramp rates will provide better melting temperature resolution, however not all instruments are equipped with fine temperature resolution."}],contributors:[{corresp:null,contributorFullName:"Lisa R. Warner",address:null,affiliation:'
Biomolecular Research Center, Boise State University, Boise, Idaho, USA
Biomolecular Research Center, Boise State University, Boise, Idaho, USA
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1. Introduction
Wood is a gift from nature. Wood is a sustainable and renewable bio-composite material, which has a long history of serving human beings in the form of fuel, construction materials, furniture, paper, sports equipment, musical instruments, and transportation components. Wood is “manufactured” in a living tree with aims to grow it by transporting water and minerals and providing strength and rigidity to anchor it to the ground. A tree is optimally “designed” to resist the loads created by gravity, wind, snow, and others, rather than produce lumber and boards. The anatomical structure of wood is adapted to generate maximum strength in the stressed directions; yet in other directions, the strength is quite low [1]. This results in the anisotropic nature of wood, i.e., the properties of wood in a given direction are different from those in another. In addition, wood, as a biomaterial, maintains its fairly high variability of anatomical structures and physical properties; therefore, it requires a very large sampling size in research practice.
The dimensions of solid wood are entirely dependent on the dimensions of trees. The largest tree in the world is, in terms of the overall volume of its trunk, reported to be the Giant Sequoia (Sequoiadendron giganteum), which takes about 2300–2700 years to form its current dimensions, roughly 84 m in height and 11 m in diameter at the base [2]. On the other hand, the free span of composite glue laminated timber arch beams used in the Richmond Olympic Oval, Vancouver, Canada reaches 100 m with a depth of 1.6 m [3]. Undoubtedly, natural wood fails to meet the requirements for constructing modern timber structures, suggesting a need for “man-made” wood products. Furthermore, to address climate change and protect the earth’s environment, logging in old-growth forests has been, in the last several decades, restricted or almost banned in most of the countries in the world. Consequently, available trees are largely from faster-growth plantations, which usually produce small-diameter logs of low-density wood and large-percentage juvenile wood. Traditional large-diameter solid timber, which was often used for long-span wood buildings in the past, has been phasing out. However, there is an increasing demand for using wood, as a green building material, to construct large and tall residential, commercial, and industrial buildings. In order to address the foregoing challenges of sourcing raw wood materials and catering to the market demands, an ever-growing number of value-added wood-based commodities and building materials have been created through the advances of technology. Contemporarily, modern engineered wood products (EWPs) have been widely used in construction [4].
An EWP is a product fabricated with wood materials and adhesives and/or fasteners (such as nails) targeted mainly for structural applications. An EWP has gone through an engineering design, which is often inspired by nature, and innovative technology. With the great efforts made by scientists and engineers in the last century, EWPs have grown into an extended family. Figure 1 illustrates commonly used EWPs and their respective abbreviations.
Figure 1.
Major types of engineered wood products and their abbreviations (source: images obtained from archiproducts.com, canac.ca, diy.com, globalsources.com, leben.co.in, nrcan.gc.ca, and structurecraft.com).
EWPs offer many advantages over traditional solid timber products [5, 6]: (1) EWPs can reach a size that is not confined by the tree dimension. Theoretically, EWPs are only limited in width and length under transportation considerations; (2) EWPs accommodate a wide spectrum of species and sizes of trees, allowing more efficient utilization of raw wood materials in the form of fibers, strands, veneers, and lumber; (3) EWPs have more uniform and reliable properties than solid wood, since the strength-reducing defects present in solid wood can be removed to a large degree or placed in a less critical zone(s) in the products; (4) EWPs exhibit greater dimensional stability and tolerances than sawn timber, due to the use of adhesives, dry wood elements, heat and pressure during their manufacturing processes; and (5) EWPs can make themselves much easier to adapt to market requirements than solid wood due to their designability. Figure 2 illustrates the yields of raw wood material usage from logs to various EWPs. It can be found that laminated strand lumber (LSL) and oriented strand board (OSB) have a higher yield (larger than 75%) than plywood, laminated veneer lumber (LVL), and parallel strand lumber (PSL) (less than 65%). It can also be reasonably estimated that finger-jointed lumber, glue laminated timber (GLT), cross laminated timber (CLT), nail laminated timber (NLT), or dowel laminated timber (DLT) has a yield being less than that of sawn lumber due to the loss of wood materials during their manufacturing. A higher raw wood material yield means less waste and lower production cost, suggesting that EWPs are a great solution to the utilization of wood resources.
Figure 2.
Raw wood material yields of sawn lumber and EWPs from logs (data from [7, 8]).
However, there are some disadvantages associated with EWPs [5, 6], one of which is that the process of manufacturing of an EWP requires more variables to manipulate than that of sawn lumber. Thus, highly automated equipment and technologically intense processes are essential in the production of an EWP, which significantly increases the capital cost of establishing an EWP mill. Therefore, the production of an EWP with the existing technologies is very costly compared to that of sawn lumber. Another shortcoming of most EWPs is that the use of adhesives in those glue-bonded EWPs causes a negative impact on the ecological image of wood as a natural biomaterial [5].
From Figure 1, it can be easily distinguished that there exist two groups, i.e., beam-like and panel-like EWPs. The beam-like EWPs is a group of relatively large length and depth compared to width, which is commonly used for beams and columns. The beam-like EWPs include finger-jointed lumber, GLT, LVL, PSL, LSL, and oriented strand lumber (OSL). The panel-like EWPs are of relatively small thickness and large width and length, which are usually used for floors, walls, and roofs. The panel-like EWPs can be further classified into two sub-groups, in terms of thickness: (1) thick-panel-like EWPs, containing CLT, NLT, and DLT, and (2) thin-panel-like EWPs, consisting of plywood and OSB. However, there is not a widely accepted criterion for sorting EWPs according to their dimensions and shape. For example, GLT can be used flat as panels for decking like NLT. For another example, LSL can be manufactured in the form of panels at the beginning and then ripped along the panel length direction to make beam-like or lumber-like products. In addition to those EWPs listed in Figure 1, there are many other types of EWPs such as I-joist (or I-beam), timber concrete composite (TCC), and fiberglass-reinforced GLT. It is noteworthy that Figure 1 also lists two types of fiber-based EWPs, which are made of wood fibers as “fibers” and plastic or cement as “matrix”. These two fiber-based EWPs are not commonly used for structural components in wood buildings nowadays, but they can be used for ceilings and decking. Wood plastic composite (WPC) has found its applications in the automotive, marine, and construction industries. Wood cement fiberboard (WCF) has also been steadily invading the housing and construction market.
With advancing wood-based nanotechnology, nanomaterials, such as nanocrystalline cellulose (NCC), have been derived from woody biomass and other cellulose sources such as straws. NCC is the celluloses in their crystalline form, which can be extracted by removing the amorphous sections from the celluloses and processed into solid flake, liquid, and gel forms. NCC can be employed, for instance, to reinforce the adhesive bond in EWPs. There is another innovative EWP called scrimber that is made of “scrims”, a kind of interconnected loose webs. The steps of manufacturing scrims include crushing small diameter logs into webs, drying the webs, applying an adhesive into the webs, cutting the webs to the required length and width, laying up the webs into a mat, and pressing the mat into a billet using a radio frequency heating press [5]. However, scrimber has not been well commercialized because of the application of large quantity of adhesive (causing the high cost of production), and damaged wood generated while preparing scrims (reducing the strength of wood).
EWPs (usually excluding fiber-based ones) can be also classified into parallel and cross-laminated groups. The parallel-laminated EWPs include LSL, OSL, LVL, PSL, GLT, NLT, and DLT; meanwhile, cross-laminated EWPs contain OSB, plywood, and CLT. The parallel-laminated EWPs are usually used for load-carrying members such as beams and headers; while the cross-laminated EWPs are used for floor plates and sheathing sheets. The way of lamination inspires a philosophy of designing EWPs, which will be briefly outlined in Section 2 “Stress” of this chapter.
Another way of grouping EWPs is rooted in the wood elements that make them, which include fiber, strand, veneer, and lumber-based EWPs, shown in Figure 3. It should be noted that PSL is made of long veneer strands (also called veneer strips). Thus, PSL is classified into the group of veneer-based EWPs rather than the group of strand-based EWPs albeit it has “strand” in its name. The wood elements largely govern, in terms of their dimensions, shape, moisture content (MC), species, the physical and mechanical properties of the product made from them. For instance, the density of an LVL made of yellow poplar is just slightly greater than that of yellow poplar veneer, which is attributed to the use of an adhesive(s) and slightly densified veneer during manufacturing. For another example, the equilibrium moisture content (EMC) of plywood is usually lower than the wood from which it is made due to the use of adhesive, dried veneer, and heat and pressure in the course of manufacturing. However, the EMC of GLT is very similar to that of the lumber used for making it, since only a relatively small amount of adhesive is applied in comparison to the volume of GLT itself, and the room temperature is applied during its manufacturing process.
Figure 3.
Classification of EWPs in terms of the wood elements used for making them.
In the course of discussing EWPs, it is worth introducing another two terms: structural composite lumber (SCL) and mass timber products (MTPs). SCL refers to those products that combine dried strands, veneer, or other small wood elements bonded with an exterior structural adhesive(s) to form thick-panel-like or beam-like EWPs [6, 9]. SCL basically includes LVL, LSL, OSL, and PSL. One outstanding characteristic of SCL is that the grain of the wood elements used is essentially aligned parallel to the length direction of its products with an aim to maximize its structural properties in this direction. Thus, SCL products are broadly used for beams and columns in wood buildings. MTPs connote a family of EWPs of a large section size, which can be employed to make strong but light load-bearing components for structural applications such as floors and walls [9]. MTPs basically include lumber-based EWPs, i.e., CLT, GLT, NLT, and DLT. However, MTPs also include SCL, TCC, and other large-size EWPs such as fiberglass-reinforced GLT, see Figure 4. Among EWPs, the lumber-based MTPs also possess other unique features, such as wood-look appearance, environmental friendliness, and low carbon emission. MTPs have been, since the mid-1990s, attracting architects, engineers, and builders to employ them in their design and construction of tall and large buildings with an aim to compete with or even substitute steel and concrete.
Figure 4.
Classification of commonly used EWPs.
The philosophy of designing a timber structure/component/connection is largely rooted in two aspects: safety, limiting the maximum load-carrying capacity (i.e., strength) and serviceability, restricting excessive deflection (i.e., deformation) [10, 11]. The reaction of a material (such as wood) or a product (such as EWP) to the action of external forces is indicated by its mechanical properties, or otherwise known as engineering properties, including tensile strength, compressive strength, shear strength, bending capacity, ductility, and creep. When an external load is applied on a wood component/connection/structure, it causes the stresses inside the wood component/connection/structure, generating deformations and eventually leading to failure. Failure can be, from an engineering point of view, defined as a fracture, when stress exceeds the strength of the wood component/connection/structure or failure as deformation exceeds the design value.
Under certain assumptions, such as ignorance of natural growth characteristics (e.g., knots and eccentricity) and growth ring curvature, a three-dimensional Cartesian coordinate system can be introduced in Figure 5, representing the wood/lumber longitudinal or EWP major direction (X), wood/lumber tangential or EWP minor direction (Y), and wood/lumber radial or EWP thickness direction (Z). On this basis, an orthotropic model can be built to simulate the mechanical behavior of wood, lumber, and EWPs in scientific research and engineering design. As mentioned above, the mechanical properties of natural wood vary much more than those of EWPs, which is illustrated in Figure 6. The lumber product has a relatively wide distribution of strength due to the nature of its biomaterial, and the EWP has a fairly narrow range since it goes through an engineering design during its manufacturing. Overall, the EWP has greater design stress, 5th percentile, and mean values than sawn lumber. The following two sections will discuss these two basic mechanical terms (stress and deformation) from the standpoint of tree growth and wood uses.
Figure 5.
Cartesian coordinate system for wood/lumber (left), LVL (middle) or PSL (right).
Figure 6.
Probability density functions for strength of lumber and EWP.
2. Stress
The strength of wood is a measure of its resistance to failure. If the stress applied to the wood exceeds the strength of the wood, will break. Stress accompanies wood during its formation in a living tree and its services over its life span. As mentioned, a tree is subjected to various types of stresses during its growth. Mattheck and Kubler depicted external loading and internal stresses distributed in a tree [1], Figure 7. By neglecting the weights of the stem and branches, and loads generated by wind and snow, they simplified their model by only considering the weights of the upper and lateral crowns (F1 and F2). The compressive stresses (σ1) produced by F1 act on the area (A1) of the stem above the lateral branch, equaling to F1/A1. The bending moment generated by F2 is applied on the lateral branches, which increases linearly towards the stem. Thus, the stem below the branch joint bears both bending moment (tensile and compressive stresses) and axial compressive stress (σ1).
Figure 7.
Combined axial stresses and bending stresses in a tree generated by its crown weights [1].
These stresses refer to the mechanical stresses permanently supported by wood in a living tree during its growth, which are called tree growth stresses. The tree growth stresses result from the combined effects of the increase of dead weight and maturation of cell walls [12]. There is an interesting phenomenon that can be viewed in a leaning stem when it is subjected to a bending moment. In this situation, the stem tries to resume its original, usually upright, position, thus, it needs to counteract the bending moment. In such a stem, the growth stresses often differ on its two opposite sides, resulting in abnormally wide growth rings appearing in the upper or lower side of a leaning stem, Figure 8. The wood of such abnormally wide growth rings is called reaction wood. Reaction wood in hardwoods or softwoods is named tension wood or compression wood, respectively. Understanding compression wood is of great importance since softwood lumber is commonly used in construction of wood buildings. Compression wood has a relatively large longitudinal shrinkage compared to normal wood, which can reach 1–2% [13], around 10 times as large as that of normal wood. As a result, warping and even cracks, often emerge in softwood lumber. In comparison to normal wood, compression wood has a relatively high density but similar strength, resulting in a low strength-to-weight ratio [13].
Figure 8.
Reaction wood in a leaning stem: (a) tension wood in hardwood, and (b) compression wood in softwood (modified from [12]).
Wood is a complicated hollow structure consisting of substances and voids. The substance is the basic building materials constructing cell walls made of an ordered association of cellulose, hemicellulose, and lignin, with an average density of 1.5 g/cm3 under oven-dry conditions [13]. The voids in wood appear in the form of cell lumens, pit openings, pit cavities, and intercellular spaces. The density of wood is largely governed by these voids, i.e., if a wood species has a larger volume of voids, its wood has a lower value of density. Figure 9 illustrates the tensile strength values of wood at various levels. At the molecular level, the strength of wood is extremely high in the longitudinal direction, with estimates exceeding 7000 MPa [14]. Wood strength is about 15 times larger than that of structural steel, which has a strength of 400–550 MPa. Bundles of cellulose molecules form so-called microfibrils, the basic cell wall elements constituting cell walls in association with hemicellulose and lignin. From the composite theory point of view, wood is a natural composite, i.e., nature’s fiberglass, in which celluloses are the “fibers” and hemicellulose and lignin are the “matrix”. Microfibrils have a strength of about 480 MPa and individual cells are estimated to have a strength of about 140 MPa [14]. The tensile strength of clear softwoods in the longitudinal direction has values ranging from 40 to 200 MPa [5]. However, lumber has, in the same longitudinal direction, a tensile strength of only 15–40 MPa [5] due to the existence of many strength-reducing characteristics such as knots, the slope of grain, checks, and splits. In the derivation of the design value of lumber, its characteristic strength properties, such as characteristic tensile strength in the longitudinal direction, are determined using the lower 5th percentile value of the Weibull distribution, which is much lower than the mean. From here, an allowable property value can be calculated by dividing the 5th percentile with a property reduction factor (n), or a so-called adjustment factor. For example, allowable tensile strength is equal to the 5th percentile divided by n, where n = 2.1 [15]. Finally, design value can be derived by multiplying the allowable property value with modification factors such as load duration, service condition, size, treatment, system, and other factors. For instance, the tensile strength of wood in the longitudinal direction can be as low as 2.5 MPa in the design of a structural component, which is fully attributed to the biomaterial nature of wood and its service conditions.
Figure 9.
Tensile strength of wood at various levels.
The above discussion suggests that human beings have not fully utilized the strength of wood. Contributed by the recent technological progress, the optimized use of wood has been improved to a certain degree in terms of strength. Song et al. selected three hardwood species (basswood, oak, and poplar) and two softwood species (western red cedar and eastern white pine) as test materials, and took two steps to transform bulk natural wood directly into super strong and tough densified wood [16], Figure 10. Step 1 used a chemical treatment to partially remove lignin/hemicelluloses and Step 2 mechanically hot-pressed the chemically treated wood at 100°C to reduce its thickness by about 80%. They discovered that the tensile strength of densified wood reached about 550 MPa, which was 12 times as large as that of natural wood. This value is higher than that of microfibrils (about 480 MPa). They indicated that most of the densified wood consisted of well-aligned cellulose nanofibers, greatly enhanced hydrogen bond formation among neighboring nanofibers. Their research provides a promising method of maximizing the use of wood strength by removing most voids and some lignin/hemicellulose. As mentioned in Section 1, NCC derived from wood attracts increasing attention due to the non-renewability of petroleum and the global promotion of green materials and products. NCC made from bleached softwood kraft pulp exhibits a diameter of 10 nm and a length of 150 nm [17]. The Young’s modulus of NCC can reach about 137 GPa [18]; while that of Douglas fir clear wood, one of the strongest softwoods, reaches only about 14.5 GPa [19]. This is one of the key reasons for applying NCC as high-performance “fibers” to “matrix” to produce composite materials of increased stiffness and tensile strength.
Figure 10.
Two-step processing approach for making densified wood (modified from [16]).
EWPs are fabricated through an engineering process, the basic principle of which is to place stronger materials at the most stressed zones [5, 7, 9]. Each lamination, such as lumber and veneer, must be graded visually or mechanically. Figure 11 illustrates two lumber-based EWPs, GLT, and CLT. GLT here is made of sawn lumber of various visual grades, notably select structural (SS, the highest grade), No. 1, No. 2, and No.3 (the lowest grade here). The GLT in Figure 11 (left) is intended to be used as a beam subject to the bending of a simple span. Therefore, an unbalanced layup is designed with an aim to optimally and efficiently use lumber by locating SS-grade lumber on the bottom face and No. 3 lumber in the core layers. Figure 11 (right) depicts the basic idea of designing CLT, in which lumber grades in its major strength axis are required to be at least 1200f-1.2E MSR or visually graded No. 2, where No. 3 is the minimum lumber grade required in the minor strength axis [20]. This design gives CLT two-way action capacities, suitable for floor uses.
Figure 11.
Design of GLT (left) and CLT (right) [9].
Figure 12 further justifies the principles of engineering design in wood in terms of two basic veneer-based EWPs, namely plywood and LVL. Veneer can be, according to the size and number of defects (such as knots), sorted into three grades (high, mid, and low). In the construction of plywood and LVL, the best quality veneer is used as top and bottom laminations; however, the laminating approach differs. The cross-lamination of veneer enables plywood to be relatively strong in both the length (X1) and width (Y1) directions when loaded on its face, enabling it be widely used as sheathing materials. In LVL, the grain of each layer of veneer runs in the same direction, i.e., parallel-lamination. As a result, the strength in the length direction (X2) of LVL is much stronger than that in the width direction (Y2), making it a more suitable material for beam and column uses.
Figure 12.
Design of veneer-based products.
Assuming the same wood species, veneer quality, adhesive type, and other manufacturing parameters (except the laminating approach) are used in the fabrication of plywood and LVL, it can be reasonably predicted from Figure 12 that the modulus of elasticity (MOE) and modulus of rupture (MOR) are the largest in the direction X2, the second largest in X1, the second smallest in Y1, and the smallest in Y2. This is verified by a group of undergraduate students at the University of New Brunswick, Canada, who made 5-layer poplar plywood and LVL panels in their laboratory using a phenol-formaldehyde adhesive at two levels of pressure. Each panel they made had a width of about 150 mm and a length of about 300 mm, from which small specimens were cut for bending tests. Figure 13 summarizes their results indicating that there is a notable difference in MOE and MOR between plywood and LVL in either the major (X1 or X2) or minor (Y1 or Y2) direction. Plywood has fewer degrees of difference in MOR and MOE than LVL between the major and minor directions, suggesting a more uniform structure in plywood. However, LVL has a much higher MOR and MOE in the major direction (X1) than that in the minor one (X2), indicating its one-way strength capacities.
Figure 13.
The means and standard deviations of MOR and MOE of plywood and LVL.
As discussed above, one of the advantages of EWPs over sawn lumber is that they can be engineering-designed. For instance, a cylindrical LVL was inspired by the hierarchical structure of a wood cell wall. A cell wall consists of three major layers in its secondary wall, and each layer has many lamellae containing microfibrils at different angles [21], Figure 14 (left). The middle layer in the secondary wall has 30–50 lamellae, occupying about 75% of the thickness of the cell wall. The microfibrillar angle in the middle layer ranges from 10 to 30 degrees relative to that of the longitudinal axis of the cell (almost parallel to the longitudinal direction of wood). Therefore, the middle layer governs the properties of the cell wall and furthermore the properties of wood, and provides considerably more strength and stiffness parallel to its axis than perpendicular. Yamauchi et al. used 2.5-mm-thick Japanese cedar veneer and a resorcinol resin to make cylindrical LVLs using the spiral-winding method, i.e., by laying neighboring veneer sheets at ±10 degrees to form an interlocked grain structure [22], Figure 14 (right). The cylindrical LVL specimens they made were about 300 mm in the outer diameter, 25 mm in the wall thickness, and 3600 mm in the specimen length. They discovered that (1) the MOE of cylindrical LVLs was the same as that of solid lumber of the same species; and (2) as the number of veneer plies used in cylindrical LVLs increased from 6 to 10, its MOR increased; yet MOE remained almost unchanged. Yamauchi et al. concluded that the interlocked grain structure they applied could effectively prevent a decrease in MOE and indicated that cylindrical LVL was suitable for structural uses, especially for posts in construction [22].
Figure 14.
The hierarchical structure of a mature cell wall (left) (adapted from [21]) and a cylindrical LVL (right) (adapted from [22]).
3. Deformation
Wood and EWPs may undergo dimensional changes due to variation in ambient temperature and relative humidity, and stresses caused by external loads. The interaction of the surrounding atmosphere and loading conditions can create an enhanced level of deformation in wood. This section briefly explains how the change in moisture content of wood affects the shrinkage or swelling of wood and aims to increase the awareness of how the moisture-induced deformation impacts the structural performance of a timber building.
As a hygroscopic material, wood and its products can absorb and release moisture, resulting in dimensional changes. Figure 15 illustrates the possible changes in moisture content (MC) of wood during the construction and use of wood buildings. The initial MC is the MC at the time of the manufacturing of a wood product, which is usually less than 19% for lumber and 4–15% for EWPs [10, 23]. The initial MC of strand-based or veneer-based EWPs, such as OSB and LVL, ranges from 4–6% [10]. However, the initial MC of lumber-based EWPs, such as GLT and CLT, varies from 11–15% [10]. The difference in initial MC between strand/veneer-based and lumber-based EWPs can be attributed to the dimension and shape of wood elements, amount and type of adhesive, and heat and pressure applied during manufacturing. The MC can significantly increase during construction if the wood components are not well protected from moisture/water, which can cause a large change in dimension and shape as well. Proper cautions and measures must be used to minimize such a large dimensional change. After a wood building is completed and occupied, its in-service MC may vary from 7–15% [23], depending on the surrounding temperature and relative humidity, before eventually reaching equilibrium moisture content (EMC). The EMC fluctuates between the low and high in-service MC, resulting in some dimensional changes in wood from time to time.
Figure 15.
Variation of moisture content of wood during the construction and use of wood buildings [23].
As discussed above, the wood components of a low MC at the time of delivery may get wet on a construction site, generating a swelling value that cannot be ignored. Figure 16 illustrates a floor joist that is supported by a wood frame wall on the right end and by a masonry block on the left [10]. During the service of the floor joist, differential wood movements may occur between the two ends, Figure 16 (upper). To ensure that the floor is at a horizontal level, the movements at the two ends must be the same. To address this, a wooden sill beam, just like the interior beam, can be added to the concrete wall, as shown in Figure 16 (lower), which provides an equal amount of wood movement in the vertical direction. This example provides a hint to designers, i.e., it is important to identify, in the course of designing a wood building, the locations where potential differential wood movements could affect structural integrity and serviceability.
Figure 16.
Detailing to account for vertical movement due to the change in moisture content [10].
Wood is indeed an anisotropic material. Thus, its dimensional change varies from one direction to another. The dimensional change in the longitudinal direction is as low as 0.1–0.2% for mature wood [13]; thus, it can be ignored. However, the dimensional change in the transverse (i.e., radial, tangential or in-between) direction can reach as high as 12% or so [13]; therefore, it must be taken into account during the design of wood buildings. However, it is not practical and sometimes impossible to estimate the dimensional change in the transverse direction because the grain orientation in the cross-section of lumber cannot be predicted in the construction of a wood building. To resolve this problem, the Canadian standard CSA O86 “Engineering design in wood” specifies Eq. (1) to estimate the dimensional change of a member made of sawn lumber or lumber-based EWPs such as GLT and CLT [11]:
S=D×Mi−Mf×cE1
where, S is the dimensional change (mm) due to moisture; D is the actual dressed dimension (mm) (i.e., thickness, width, or length); Mi is the lesser of the initial moisture content of the fiber saturation point (28%); Mf is the final moisture content; and c is a coefficient. As for lumber, c = 0.002 or 0.00005 for the dimensional change perpendicular to the grain (i.e., the transverse direction) or parallel to grain (i.e., the longitudinal direction), respectively.
Scientists at FPInnovations, Canada, conducted a study by monitoring the vertical movement in a 4-storey wood-frame building over 22 months [24]. The floors consist of 38 mm by 240 mm “S-Dry” dimension lumber joists with a concrete topping. The walls consist of 38 mm by 140 mm “S-Dry” solid sawn plates and studs. Double top plates and double bottom plates are used in all storeys. The stud length of all storeys is 2.44 m. The joist spacing is 400 mm; joist spans are 3.75 m; and stud spacing is 400 mm. The scientists calculated the vertical wood movement including shrinkage from an initial MC of 19% to the final MC of 8%, using the above equation and deformation by assuming the specified roof and floor dead loads to be 0.5 kPa and 1.3 kPa, respectively. Deformation generated by stress includes instantaneous deformation and creep deformation. The equations for calculating these deformations are provided in the report by Doudak et al. [24]. Figure 17 summarizes the estimated vertical movement values at each storey, indicating that the accumulated shrinkage over 4 stories accounts for about 90% of the total vertical movement. This suggests the vertical deformation generated due to the change in the moisture content of wood is critical and must be considered in the design of a wood building. The actual vertical movement measured in this 4-storey building was about 40 mm, which is in good agreement with the estimated value (about 38 mm). They concluded that it was possible to make a good estimation of vertical movement to avoid the potential problems of structural integrity, serviceability, and building envelope over the lifespan of this wood building. The scientists also found that the use of EWPs could reduce the accumulated shrinkage to about 80% of the total vertical movement, based on their monitoring of another 5-storey building with floor joists made of LVL flanges and OSB webs. The total vertical movement of this 5-storey building was about 30 mm, which was 75% of that of the 4-storey building they studied.
Figure 17.
Estimated vertical movement due to changes in MC and axial compressive load (source: data from [24]).
There are two basic wood framing construction methods, notably platform framing and balloon framing [25], Figure 18. In the platform framing, all vertical structural elements of the exterior bearing walls and partitions consist of single studs extending the full height of the frame. Meanwhile, in the balloon framing, the studs of the exterior walls and some of the interior walls are continuous from the foundation sill plate to the top plate below the roof framing. In comparison to balloon framing, platform framing is more commonly used for modern structures due to its simplicity and ease of erection; but its vertical movement due to MC changes is much larger. Balloon framing is rarely used nowadays since the length of sawn lumber available is not sufficient due to changes in forest resources with the production of smaller trees compared to older times. However, with advancing technologies, many modern EWPs have been invented and manufactured, providing sufficiently sized materials with less moisture-induced wood movements. This gives an opportunity for people to rethink the use of the balloon framing method in building construction.
Figure 18.
Light frame construction methods: platform framing and balloon framing (modified from [25]).
CLT panels can, attributed by their inherent two-way spanning capabilities, eliminate the necessity of placing beams underneath the panels, as with other MTPs. This facilitates the emergence and application of a post-and-panel mass timber construction system [9]. Therefore, this system significantly reduces the building height and construction time, as well as overall costs [26]. In this type of modern construction system, the CLT floor plates are point-supported by GLT and PSL columns. This may cause two potential issues, i.e., excessive vertical wood movement because of MC change and crushing of CLT panels due to axial loads, if there are no proper connectors joining CLT plates and columns. These issues occur because the vertical direction of the building is the thickness direction of CLT (i.e., the transverse direction of lumber), along which larger accumulated wood movements and lower compressive strengths exist in CLT. To address these issues, a so-called HSS steel connector was developed and employed, Figure 19 (lower), which can directly transfer load from upper columns to lower columns and provide some tolerance for dimensional change. The development of various types of metal connectors is of great importance in the design and construction of wood buildings with EWPs.
Figure 19.
Column-to-column connection used in the Brock Commons Tallwood House in the University of British Columbia, Vancouver, Canada (upper: on-site installation of columns connected to CLT panels (source: www.fastepp.com); lower: HSS steel connectors [26]).
4. Endnotes
With climate change being an inevitable and urgent global challenge, it is essential to address such issues with real-life content, for instance, the global warming impacts caused by buildings and constructions. The World Green Building Council reports that building construction and operation account for 39% of greenhouse gas (GHG) emissions annually [27]. Among this 39%, 11% is from embodied carbon and 28% from operational carbon. The embodied carbon of a building is defined as the amount of carbon emitted during its construction. Whereas, operational carbon is defined as the amount of carbon emitted during the operation of a building. As innovative building technologies continue to develop, operational carbon will be significantly reduced, and embodied carbon can be responsible for almost 50% of total new construction emissions from now to 2050 [28]. In the next 40 years, with a doubled urban population, a building area of 2.48 trillion square feet is required to fit the needs of urban population growth [29]. The combination of considerable global CO2 emissions from the building sector and the increasing demand on buildings reveals that actions should be taken immediately to mitigate emissions from the embodied carbon. One of the answers to this global challenge is to increase the use of wood and wood-based products in the construction sector. As a biomaterial, wood possesses its natural ability to mitigate carbon dioxide (CO2). During the growth of trees, wood is produced, sequestering carbon. After trees are harvested, wood can be processed into various products. These products can be therefore used in construction of buildings, which store carbon over their lifespan. The recently released report “The state of mass timber in Canada 2021” from the Government of Canada [30] indicates “As high-value wood products, mass timber can play an instrumental role in the circular economy by providing a renewable source of building materials and contributing to a lower carbon footprint for the construction sector.” In the last two decades, the development of mass timber was rapid in Canada, which can be viewed through the number of completed mass timber projects, Figure 20, with 10 projects in 2007 and upwards of 60 projects in 2018. It should be noted that each project listed in this figure must meet two criteria, i.e., a minimum floor area of 300 m2 and structural use of MTPs.
Figure 20.
The number of completed mass timber projects per year in Canada [30].
Figure 21 illustrates the percentage share of each EWP in major markets over a time horizon [31]. The data in this figure are outdated, only depicting the status of EWPs in 2006, but it still provides some insight into the market shares of each EWP. The sheathing and industry plywood markets are in the stages of “decline” and “maturity”, respectively. This is mainly due to the decreasing volume of veneer quality logs and the rapid development of OSB. Sheathing OSB is in “rapid growth”, which has taken a big market share from plywood because of its high yield of using raw wood materials. Framing lumber takes 90% or more market shares since SCL is still much more expansive than sawn lumber, resulting in its failure to completely replace lumber [6]. Glulam, a commonly used name for GLT, has passed its plateau of market demand, moving into the “decline” stage. The main reason for this decline could be the emergence of other EWPs such as LVL, LSL, and PSL. Albeit LVL stays at the end of the “expansion”, it could have reached its maximum market share. This implies that the cost of producing LVL will, just like manufacturing plywood, definitely increase, since premium logs for producing veneer are becoming scarce [6]. LSL and PSL are in “expansion” due to their high raw material yield from logs to the final products. Figure 21 does not show the market shares of CLT, NLT, and DLT, but it can be reasonably speculated that these lumber-based EWPs are in the “development” and “expansion phase”, and will enter “rapid growth” quickly, particularly for CLT. As technologies advance, the cost of manufacturing EWPs will be further reduced. For example, adoption of the artificial intelligence in production can lead to an increased yield of raw material usage from logs and reduced labor costs. Therefore, EWPs will be more competitive to sawn lumber and make inroads into more market shares.
Figure 21.
Percentage share in major markets of EWPs over time horizon (source: photos obtained from [31]).
It can be well foreseen that EWPs will have a bright future in construction because (1) EWPs are designable, producing an optimal structural performance for construction; (2) EWPs have more uniform strength properties and fewer changes in dimensions and shape, making them more suitable building materials; (3) EWPs provide a wide selection of dimensions, allowing designers and builders to design and build tall timber, wood-concrete, and wood-steel hybrid structures; and (4) EWPs fall into the category of environmentally friendly and recyclable products, contributing to a lower carbon footprint for the construction sector.
Acknowledgments
This piece of work was financially supported by the New Brunswick Innovation Foundation (Canada) under its New Brunswick Innovation Research Chair Initiative Program and the Natural Sciences and Engineering Research Council of Canada under its Discovery Grant Program (RGPIN 2017-03994).
\n',keywords:"wood, engineered wood products, classification, growth stresses, strength evolution, design principle, moisture-induced deformation, market shares",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/79485.pdf",chapterXML:"https://mts.intechopen.com/source/xml/79485.xml",downloadPdfUrl:"/chapter/pdf-download/79485",previewPdfUrl:"/chapter/pdf-preview/79485",totalDownloads:183,totalViews:0,totalCrossrefCites:0,dateSubmitted:"July 13th 2021",dateReviewed:"October 12th 2021",datePrePublished:"November 27th 2021",datePublished:"April 28th 2022",dateFinished:"November 27th 2021",readingETA:"0",abstract:"Wood, as a natural, sustainable, and renewable bio-composite material, has a long history of serving humanity as construction materials. With the advance in technologies, many modern engineered wood products (EWPs) have been invented, produced, and used in construction, such as laminated veneer lumber, oriented strand board, and cross laminated timber. This chapter first introduces the classification, rationales, and pros and cons of EWPs. Secondly, it discusses the stress-related topics, including growth stresses in living trees, the evolution of wood strength from the molecular level to the actual design implementation. Thirdly, this chapter discusses moisture-induced deformation with examples. Finally, it mentions the benefits of using EWPs and their market shares.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/79485",risUrl:"/chapter/ris/79485",signatures:"Meng Gong",book:{id:"10584",type:"book",title:"Engineered Wood Products for Construction",subtitle:null,fullTitle:"Engineered Wood Products for Construction",slug:"engineered-wood-products-for-construction",publishedDate:"April 28th 2022",bookSignature:"Meng Gong",coverURL:"https://cdn.intechopen.com/books/images_new/10584.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",isbn:"978-1-83962-772-9",printIsbn:"978-1-83962-771-2",pdfIsbn:"978-1-83962-790-3",isAvailableForWebshopOrdering:!0,editors:[{id:"274242",title:"Dr.",name:"Meng",middleName:null,surname:"Gong",slug:"meng-gong",fullName:"Meng Gong"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:[{id:"274242",title:"Dr.",name:"Meng",middleName:null,surname:"Gong",fullName:"Meng Gong",slug:"meng-gong",email:"meng.gong@unb.ca",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/274242/images/system/274242.jpg",institution:{name:"University of New Brunswick",institutionURL:null,country:{name:"Canada"}}}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Stress",level:"1"},{id:"sec_3",title:"3. Deformation",level:"1"},{id:"sec_4",title:"4. Endnotes",level:"1"},{id:"sec_5",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Mattheck C, Kubler H. Wood—The Internal Optimization of Trees. Berlin, Germany: Springer-Verlag; 1997'},{id:"B2",body:'National Parks Conservation Association. Introduction to Giant Sequoia, Sequoia National Park, California, USA; 2019.'},{id:"B3",body:'Canadian Wood Council. The Richmond Olympic Oval. Canadian Wood Council, Ottawa, ON, Canada; 2019. Available from: https://cwc.ca/case-studies [Accessed: June 30, 2021]'},{id:"B4",body:'McKeever DR. Engineered wood products: A response to the changing timber resource. In: Pacific Rim Wood Market Report. Madison, Wisconsin, USA: USDA Forest Products Laboratory; 1997'},{id:"B5",body:'Thelandersson S, Larsen HJ. Timber Engineering. Chichester, West Sussex, UK: John Wiley & Sons Ltd; 2003'},{id:"B6",body:'Hsu WE. Structural Composite Lumber Manufacturing. CreateSpace Independent Publishing Platform, Scotts Valley, California, USA; 2016'},{id:"B7",body:'Smulski S. Engineered Wood Products: A Guide for Specifies, Designers and Users. Madison, Wisconsin, USA: PFS Research Foundation, Inc.; 1997'},{id:"B8",body:'Schuler A. Innovative use of wood promotes market development and supports forest sustainability: A win-win situation for society, forest products industry, and forest owners. UNECE Timber Committee Sixtieth session, Item 4 “Innovative uses of wood”, Economic Commission for Europe, Economic and Social Council, United Nations, September 24-27, Geneva, Switzerland. 2002'},{id:"B9",body:'Gong M. Lumber-based mass timber products in construction. In: Timber Buildings and Sustainability. London, UK: IntechOpen; 2019'},{id:"B10",body:'Canadian Wood Council. Introduction to Wood Design. Canadian Wood Council, Ottawa, ON, Canada; 2011'},{id:"B11",body:'CSA Group. Engineering Design in Wood. CSA O86-14. Canadian Standards Association, Mississauga, ON, Canada; 2014'},{id:"B12",body:'Gril J, Jullien D, Bardet S, Yamamoto H. Tree growth stress and related problems. Journal of Wood Science. 2017;63:411-432'},{id:"B13",body:'Shmulsky R, Jones PD. Forest Products and Wood Science: An Introduction. 6th ed. West Sussex, UK: John Wiley & Sons Ltd.; 2011'},{id:"B14",body:'Canadian Wood Council. Structure and Properties of Wood. Slide-Cassette Presentation. Ottawa, Canada: Canadian Wood Council and Forintek Canada Corp; n.d.'},{id:"B15",body:'ASTM International. Designation D1990-19. Standard Practice for Establishing Allowable Properties for Visually Graded Dimension Lumber from In-Grade Tests of Full-Size Specimens. West Conshohocken, PA, USA: ASTM International; 2019'},{id:"B16",body:'Song JW, Chen CJ, Zhu SZ, et al. Processing bulk natural wood into a high-performance structural material. Nature. 2018;554:224-228. DOI: 10.1038/nature25476'},{id:"B17",body:'Landry V, Alemdar A, Blanchet P. Nanocrystalline cellulose: morphological, physical, and mechanical properties. Forest Products Journal. 2011;61(2):104-112'},{id:"B18",body:'Julkapli NM, Bagheri S. Progress on nanocrystalline cellulose biocomposites. Reactive and Functional Polymers. 2017;112:9-12'},{id:"B19",body:'Bodig J, Jayne BA. Mechanics of Wood and Wood Composites. New York, NY, USA: Van Nostrand Reinhold Company Inc.; 1982'},{id:"B20",body:'Karacabeyli E, Gagnon S. Canadian CLT Handbook, 2019 Edition, Special Publication SP-532E. Pointe-Claire, QC, Canada: FPInnovations; 2019'},{id:"B21",body:'Côté WA Jr. Wood Ultrastructure—An Atlas of Electron Micrographs. Seattle, WA, USA: University of Washington Press; 1967'},{id:"B22",body:'Yamauchi H, Miura I, Sasaki T, Koizumi A, Kawai S, Sasaki H. Manufacturing condition and mechanical properties of cylindrical LVL manufactured by the spiral-winding method. Journal of the Society of Materials Science. 1998;47(4):350-355'},{id:"B23",body:'McLain R, Steimle D. WW-WSP-10. Accommodating Shrinkage in Multi-Story Wood-Frame Structures. Washington DC, USA: WoodWorksTM, Wood Products Council; 2019'},{id:"B24",body:'Doudak G, Lepper P, Ni C, Wang J. Vertical Movement in Wood Platform Frame Structures: Movement Prediction. Vancouver, BC, Canada: FPInnovations; 2013'},{id:"B25",body:'Allen E, Iano J. Fundamentals of Building Construction: Materials and Methods. 6th ed. Hoboken, New Jersey, USA: John Wiley & Sons, Inc.; 2014'},{id:"B26",body:'Canadian Wood Council. Brock Commons Tallwood House: A Case Study. Canadian Wood Council, Ottawa, ON, Canada; 2019. Available from: https://cwc.ca/case-studies [Accessed: July 8, 2021]'},{id:"B27",body:'World Green Building Council. Global Status Report for Buildings and Construction. 2019. Available from: https://www.worldgbc.org/news-media/2019-global-status-report-buildings-and-construction [Accessed: February 16, 2021]'},{id:"B28",body:'Architecture 2030. New buildings: Embodied carbon. 2020. https://architecture2030.org/new-buildings-embodied [Accessed: November 18, 2020]'},{id:"B29",body:'Green M. How timber will define the skylines of our future. “Online Education Series “The future of Sustainable Buildings”, October 27” Soprema, Drummodville, QC, Canada; 2020.'},{id:"B30",body:'Natural Resources Canada. The State of Mass Timber in Canada 2021. Ottawa, ON, Canada; 2021'},{id:"B31",body:'UNECE/FAO. Forest Products Annual Market Review 2019-2020. Geneva, Switzerland: United Nations Economic Commission for Europe (UNECE) and the Food and Agriculture Organization (FAO); 2020'}],footnotes:[],contributors:[{corresp:"yes",contributorFullName:"Meng Gong",address:"meng.gong@unb.ca",affiliation:'
University of New Brunswick, Canada
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Given the neurotoxic substances found in the human body, certain people have been regarded as having a propensity to epileptic seizures. In many situations, the neurotransmission processes of these toxins are similar to the physiopathology of epilepsy. Epileptic models have been developed to induce seizures in animals, allowing researchers to study convulsive seizure mechanisms. Pentylenetetrazol, kainic acid, pilocarpine, penicillin, aluminum, bicuculline, picrotoxine, 4-aminopyridine, strictine, domoic acid, and other compounds fall under this category. However, there are some drugs used in clinical practice that can cause neurotoxicity as well. In this chapter, the predominant substances and drugs involved in epileptogenesis through neurotoxicity effects are reviewed. Throughout this chapter, we attempt to describe the mechanisms documented in the literature, in which epileptic seizures cause neurotoxicity in the brain by themselves, as shown with excitotoxicity mediated by glutamate and ions involved.",signatures:"Carmen Rubio, Artemio Rosiles-Abonce, Elisa Taddei and Moisés Rubio-Osornio",authors:[{id:"419945",title:"Ph.D.",name:"Moisés",surname:"Rubio-Osornio",fullName:"Moisés Rubio-Osornio",slug:"moises-rubio-osornio",email:"ruomon@gmail.com"},{id:"419955",title:"Dr.",name:"Carmen",surname:"Rubio",fullName:"Carmen Rubio",slug:"carmen-rubio",email:"macaru4@yahoo.com.mx"},{id:"420551",title:"Dr.",name:"Artemio",surname:"Rosiles-Abonce",fullName:"Artemio Rosiles-Abonce",slug:"artemio-rosiles-abonce",email:"artemioabonce@outlook.com"},{id:"420552",title:"Dr.",name:"Elisa",surname:"Taddei",fullName:"Elisa Taddei",slug:"elisa-taddei",email:"elisa.taddei@uabc.edu.mx"}],book:{id:"10736",title:"Neurotoxicity",slug:"neurotoxicity-new-advances",productType:{id:"1",title:"Edited Volume"}}},{id:"79254",title:"Neurotoxic Effects of Insecticides Chlorpyrifos, Carbaryl, Imidacloprid, in Different Animal Species",slug:"neurotoxic-effects-of-insecticides-chlorpyrifos-carbaryl-imidacloprid-in-different-animal-species",abstract:"Insecticides are pesticides used to control insects in agriculture, ornamental gardens, homes, and veterinary medicine. Although the toxic effects on the environment and the health of living beings are not fully understood, these pesticides have become the first options for crop protection in agriculture. After herbicides, insecticides are the most extensively used pesticides in agriculture, with large quantities consumed on every continent, primarily in America. Chlorpyrifos, carbaryl, and imidacloprid are among the top ten most used insecticides. Amidst organophosphates, chlorpyrifos has been reported to be used in over fifty food crops. Carbaryl is a carbamate employed as an insecticide, fungicide, herbicide, and nematicide. Similarly, neonicotinoids are the most used insecticide on a global scale. Neonicotinoids include imidacloprid, the second most frequently used pesticide, surpassed only by glyphosate. It is used because it is less toxic to humans. However, insects appear to be less resistant to its compounds. Evidence suggests that these insecticides persist in soils for a long time and have neurotoxic effects in animal species not intended to receive its consequences. Thus, this chapter’s aim is to describe these three pesticides effects and contrast them with the most recent findings regarding their neurotoxic effects in various animal species.",signatures:"Alejandra Mora-Gutiérrez, Carmen Rubio, Ángel Alonso Romero-López and Moisés Rubio-Osornio",authors:[{id:"419945",title:"Ph.D.",name:"Moisés",surname:"Rubio-Osornio",fullName:"Moisés Rubio-Osornio",slug:"moises-rubio-osornio",email:"ruomon@gmail.com"},{id:"419955",title:"Dr.",name:"Carmen",surname:"Rubio",fullName:"Carmen Rubio",slug:"carmen-rubio",email:"macaru4@yahoo.com.mx"},{id:"419956",title:"M.Sc.",name:"Alejandra",surname:"Mora-Gutiérrez",fullName:"Alejandra Mora-Gutiérrez",slug:"alejandra-mora-gutierrez",email:"mvzalejandramora@gmail.com"},{id:"426668",title:"Dr.",name:"Angel",surname:"Romero-López",fullName:"Angel Romero-López",slug:"angel-romero-lopez",email:"alonso.romerolopez@correo.buap.mx"}],book:{id:"10736",title:"Neurotoxicity",slug:"neurotoxicity-new-advances",productType:{id:"1",title:"Edited Volume"}}}],collaborators:[{id:"97601",title:"Dr.",name:"Janneth",surname:"Gonzalez",slug:"janneth-gonzalez",fullName:"Janneth Gonzalez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Pontifical Xavierian University",institutionURL:null,country:{name:"Colombia"}}},{id:"419945",title:"Ph.D.",name:"Moisés",surname:"Rubio-Osornio",slug:"moises-rubio-osornio",fullName:"Moisés Rubio-Osornio",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435480",title:"Dr.",name:"Felipe",surname:"Rojas-Rodríguez",slug:"felipe-rojas-rodriguez",fullName:"Felipe Rojas-Rodríguez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435481",title:"Dr.",name:"Andrés",surname:"Pinzón",slug:"andres-pinzon",fullName:"Andrés Pinzón",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435482",title:"Dr.",name:"Daniel",surname:"Fuenmayor",slug:"daniel-fuenmayor",fullName:"Daniel Fuenmayor",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435483",title:"Dr.",name:"Tábata",surname:"Barbosa",slug:"tabata-barbosa",fullName:"Tábata Barbosa",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435484",title:"Dr.",name:"Diego Vesga",surname:"Jimenez",slug:"diego-vesga-jimenez",fullName:"Diego Vesga Jimenez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435485",title:"Dr.",name:"Cynthia",surname:"Martin",slug:"cynthia-martin",fullName:"Cynthia Martin",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435486",title:"Dr.",name:"George E.",surname:"Barreto",slug:"george-e.-barreto",fullName:"George E. Barreto",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"435487",title:"Dr.",name:"Andrés",surname:"Aristizabal-Pachón",slug:"andres-aristizabal-pachon",fullName:"Andrés Aristizabal-Pachón",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null}]},generic:{page:{slug:"editorial-policies",title:"Editorial Policies",intro:'
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\r\n\tTransforming our World: the 2030 Agenda for Sustainable Development endorsed by United Nations and 193 Member States, came into effect on Jan 1, 2016, to guide decision making and actions to the year 2030 and beyond. Central to this Agenda are 17 Goals, 169 associated targets and over 230 indicators that are reviewed annually. The vision envisaged in the implementation of the SDGs is centered on the five Ps: People, Planet, Prosperity, Peace and Partnership. This call for renewed focused efforts ensure we have a safe and healthy planet for current and future generations.
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\r\n\tThis Series focuses on covering research and applied research involving the five Ps through the following topics:
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\r\n\t2. Health and Wellbeing focusing on SDG 3 on Good Health and Wellbeing and SDG 6 on Clean Water and Sanitation
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\r\n\t3. Inclusivity and Social Equality involving SDG 4 on Quality Education, SDG 5 on Gender Equality, and SDG 16 on Peace, Justice and Strong Institutions
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\r\n\t
\r\n
\r\n\t4. Climate Change and Environmental Sustainability comprising SDG 13 on Climate Action, SDG 14 on Life Below Water, and SDG 15 on Life on Land
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\r\n\t
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\r\n\t5. Urban Planning and Environmental Management embracing SDG 7 on Affordable Clean Energy, SDG 9 on Industry, Innovation and Infrastructure, and SDG 11 on Sustainable Cities and Communities.
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\r\n\tThe series also seeks to support the use of cross cutting SDGs, as many of the goals listed above, targets and indicators are all interconnected to impact our lives and the decisions we make on a daily basis, making them impossible to tie to a single topic.
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Chatterjee Memorial Research Prize-2019” and he is also the recipient of 'Dr.Raja Ramanna State Scientist Award 2015” by Government of Karnataka. He is a Fellow of the Royal Society of Biology (FRSB), London and Honorary Fellow of Karnataka Science and Technology Academy, Department of Science and Technology, Government of Karnataka.",institutionString:"BLDE (Deemed to be University), India",institution:null},{id:"243660",title:"Dr.",name:"Mallanagouda Shivanagouda",middleName:null,surname:"Biradar",slug:"mallanagouda-shivanagouda-biradar",fullName:"Mallanagouda Shivanagouda Biradar",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/243660/images/system/243660.jpeg",biography:"M. S. Biradar is Vice Chancellor and Professor of Medicine of\nBLDE (Deemed to be University), Vijayapura, Karnataka, India.\nHe obtained his MD with a gold medal in General Medicine and\nhas devoted himself to medical teaching, research, and administrations. He has also immensely contributed to medical research\non vascular medicine, which is reflected by his numerous publications including books and book chapters. Professor Biradar was\nalso Visiting Professor at Tulane University School of Medicine, New Orleans, USA.",institutionString:"BLDE (Deemed to be University)",institution:{name:"BLDE University",country:{name:"India"}}},{id:"289796",title:"Dr.",name:"Swastika",middleName:null,surname:"Das",slug:"swastika-das",fullName:"Swastika Das",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/289796/images/system/289796.jpeg",biography:"Swastika N. Das is Professor of Chemistry at the V. P. Dr. P. G.\nHalakatti College of Engineering and Technology, BLDE (Deemed\nto be University), Vijayapura, Karnataka, India. She obtained an\nMSc, MPhil, and PhD in Chemistry from Sambalpur University,\nOdisha, India. Her areas of research interest are medicinal chemistry, chemical kinetics, and free radical chemistry. She is a member\nof the investigators who invented a new modified method of estimation of serum vitamin E. She has authored numerous publications including book\nchapters and is a mentor of doctoral curriculum at her university.",institutionString:"BLDEA’s V.P.Dr.P.G.Halakatti College of Engineering & Technology",institution:{name:"BLDE University",country:{name:"India"}}},{id:"248459",title:"Dr.",name:"Akikazu",middleName:null,surname:"Takada",slug:"akikazu-takada",fullName:"Akikazu Takada",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/248459/images/system/248459.png",biography:"Akikazu Takada was born in Japan, 1935. After graduation from\nKeio University School of Medicine and finishing his post-graduate studies, he worked at Roswell Park Memorial Institute NY,\nUSA. He then took a professorship at Hamamatsu University\nSchool of Medicine. In thrombosis studies, he found the SK\npotentiator that enhances plasminogen activation by streptokinase. He is very much interested in simultaneous measurements\nof fatty acids, amino acids, and tryptophan degradation products. By using fatty\nacid analyses, he indicated that plasma levels of trans-fatty acids of old men were\nfar higher in the US than Japanese men. . He also showed that eicosapentaenoic acid\n(EPA) and docosahexaenoic acid (DHA) levels are higher, and arachidonic acid\nlevels are lower in Japanese than US people. By using simultaneous LC/MS analyses\nof plasma levels of tryptophan metabolites, he recently found that plasma levels of\nserotonin, kynurenine, or 5-HIAA were higher in patients of mono- and bipolar\ndepression, which are significantly different from observations reported before. In\nview of recent reports that plasma tryptophan metabolites are mainly produced by\nmicrobiota. He is now working on the relationships between microbiota and depression or autism.",institutionString:"Hamamatsu University School of Medicine",institution:{name:"Hamamatsu University School of Medicine",country:{name:"Japan"}}},{id:"137240",title:"Prof.",name:"Mohammed",middleName:null,surname:"Khalid",slug:"mohammed-khalid",fullName:"Mohammed Khalid",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/137240/images/system/137240.png",biography:"Mohammed Khalid received his B.S. degree in chemistry in 2000 and Ph.D. degree in physical chemistry in 2007 from the University of Khartoum, Sudan. He moved to School of Chemistry, Faculty of Science, University of Sydney, Australia in 2009 and joined Dr. Ron Clarke as a postdoctoral fellow where he worked on the interaction of ATP with the phosphoenzyme of the Na+/K+-ATPase and dual mechanisms of allosteric acceleration of the Na+/K+-ATPase by ATP; then he went back to Department of Chemistry, University of Khartoum as an assistant professor, and in 2014 he was promoted as an associate professor. In 2011, he joined the staff of Department of Chemistry at Taif University, Saudi Arabia, where he is currently an assistant professor. His research interests include the following: P-Type ATPase enzyme kinetics and mechanisms, kinetics and mechanisms of redox reactions, autocatalytic reactions, computational enzyme kinetics, allosteric acceleration of P-type ATPases by ATP, exploring of allosteric sites of ATPases, and interaction of ATP with ATPases located in cell membranes.",institutionString:"Taif University",institution:{name:"Taif University",country:{name:"Saudi Arabia"}}},{id:"63810",title:"Prof.",name:"Jorge",middleName:null,surname:"Morales-Montor",slug:"jorge-morales-montor",fullName:"Jorge Morales-Montor",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/63810/images/system/63810.png",biography:"Dr. Jorge Morales-Montor was recognized with the Lola and Igo Flisser PUIS Award for best graduate thesis at the national level in the field of parasitology. He received a fellowship from the Fogarty Foundation to perform postdoctoral research stay at the University of Georgia. He has 153 journal articles to his credit. He has also edited several books and published more than fifty-five book chapters. He is a member of the Mexican Academy of Sciences, Latin American Academy of Sciences, and the National Academy of Medicine. He has received more than thirty-five awards and has supervised numerous bachelor’s, master’s, and Ph.D. students. Dr. Morales-Montor is the past president of the Mexican Society of Parasitology.",institutionString:"National Autonomous University of Mexico",institution:{name:"National Autonomous University of Mexico",country:{name:"Mexico"}}},{id:"217215",title:"Dr.",name:"Palash",middleName:null,surname:"Mandal",slug:"palash-mandal",fullName:"Palash Mandal",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/217215/images/system/217215.jpeg",biography:null,institutionString:"Charusat University",institution:null},{id:"49739",title:"Dr.",name:"Leszek",middleName:null,surname:"Szablewski",slug:"leszek-szablewski",fullName:"Leszek Szablewski",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/49739/images/system/49739.jpg",biography:"Leszek Szablewski is a professor of medical sciences. He received his M.S. in the Faculty of Biology from the University of Warsaw and his PhD degree from the Institute of Experimental Biology Polish Academy of Sciences. He habilitated in the Medical University of Warsaw, and he obtained his degree of Professor from the President of Poland. Professor Szablewski is the Head of Chair and Department of General Biology and Parasitology, Medical University of Warsaw. Professor Szablewski has published over 80 peer-reviewed papers in journals such as Journal of Alzheimer’s Disease, Biochim. Biophys. Acta Reviews of Cancer, Biol. Chem., J. Biomed. Sci., and Diabetes/Metabol. Res. Rev, Endocrine. He is the author of two books and four book chapters. He has edited four books, written 15 scripts for students, is the ad hoc reviewer of over 30 peer-reviewed journals, and editorial member of peer-reviewed journals. Prof. Szablewski’s research focuses on cell physiology, genetics, and pathophysiology. He works on the damage caused by lack of glucose homeostasis and changes in the expression and/or function of glucose transporters due to various diseases. He has given lectures, seminars, and exercises for students at the Medical University.",institutionString:"Medical University of Warsaw",institution:{name:"Medical University of Warsaw",country:{name:"Poland"}}},{id:"173123",title:"Dr.",name:"Maitham",middleName:null,surname:"Khajah",slug:"maitham-khajah",fullName:"Maitham Khajah",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/173123/images/system/173123.jpeg",biography:"Dr. Maitham A. Khajah received his degree in Pharmacy from Faculty of Pharmacy, Kuwait University, in 2003 and obtained his PhD degree in December 2009 from the University of Calgary, Canada (Gastrointestinal Science and Immunology). Since January 2010 he has been assistant professor in Kuwait University, Faculty of Pharmacy, Department of Pharmacology and Therapeutics. His research interest are molecular targets for the treatment of inflammatory bowel disease (IBD) and the mechanisms responsible for immune cell chemotaxis. He cosupervised many students for the MSc Molecular Biology Program, College of Graduate Studies, Kuwait University. Ever since joining Kuwait University in 2010, he got various grants as PI and Co-I. He was awarded the Best Young Researcher Award by Kuwait University, Research Sector, for the Year 2013–2014. He was a member in the organizing committee for three conferences organized by Kuwait University, Faculty of Pharmacy, as cochair and a member in the scientific committee (the 3rd, 4th, and 5th Kuwait International Pharmacy Conference).",institutionString:"Kuwait University",institution:{name:"Kuwait University",country:{name:"Kuwait"}}},{id:"195136",title:"Dr.",name:"Aya",middleName:null,surname:"Adel",slug:"aya-adel",fullName:"Aya Adel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/195136/images/system/195136.jpg",biography:"Dr. Adel works as an Assistant Lecturer in the unit of Phoniatrics, Department of Otolaryngology, Ain Shams University in Cairo, Egypt. Dr. Adel is especially interested in joint attention and its impairment in autism spectrum disorder",institutionString:"Ain Shams University",institution:{name:"Ain Shams University",country:{name:"Egypt"}}},{id:"94911",title:"Dr.",name:"Boulenouar",middleName:null,surname:"Mesraoua",slug:"boulenouar-mesraoua",fullName:"Boulenouar Mesraoua",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94911/images/system/94911.png",biography:"Dr Boulenouar Mesraoua is the Associate Professor of Clinical Neurology at Weill Cornell Medical College-Qatar and a Consultant Neurologist at Hamad Medical Corporation at the Neuroscience Department; He graduated as a Medical Doctor from the University of Oran, Algeria; he then moved to Belgium, the City of Liege, for a Residency in Internal Medicine and Neurology at Liege University; after getting the Belgian Board of Neurology (with high marks), he went to the National Hospital for Nervous Diseases, Queen Square, London, United Kingdom for a fellowship in Clinical Neurophysiology, under Pr Willison ; Dr Mesraoua had also further training in Epilepsy and Continuous EEG Monitoring for two years (from 2001-2003) in the Neurophysiology department of Zurich University, Switzerland, under late Pr Hans Gregor Wieser ,an internationally known epileptologist expert. \n\nDr B. Mesraoua is the Director of the Neurology Fellowship Program at the Neurology Section and an active member of the newly created Comprehensive Epilepsy Program at Hamad General Hospital, Doha, Qatar; he is also Assistant Director of the Residency Program at the Qatar Medical School. \nDr B. Mesraoua's main interests are Epilepsy, Multiple Sclerosis, and Clinical Neurology; He is the Chairman and the Organizer of the well known Qatar Epilepsy Symposium, he is running yearly for the past 14 years and which is considered a landmark in the Gulf region; He has also started last year , together with other epileptologists from Qatar, the region and elsewhere, a yearly International Epilepsy School Course, which was attended by many neurologists from the Area.\n\nInternationally, Dr Mesraoua is an active and elected member of the Commission on Eastern Mediterranean Region (EMR ) , a regional branch of the International League Against Epilepsy (ILAE), where he represents the Middle East and North Africa(MENA ) and where he holds the position of chief of the Epilepsy Epidemiology Section; Dr Mesraoua is a member of the American Academy of Neurology, the Europeen Academy of Neurology and the American Epilepsy Society.\n\nDr Mesraoua's main objectives are to encourage frequent gathering of the epileptologists/neurologists from the MENA region and the rest of the world, promote Epilepsy Teaching in the MENA Region, and encourage multicenter studies involving neurologists and epileptologists in the MENA region, particularly epilepsy epidemiological studies. \n\nDr. Mesraoua is the recipient of two research Grants, as the Lead Principal Investigator (750.000 USD and 250.000 USD) from the Qatar National Research Fund (QNRF) and the Hamad Hospital Internal Research Grant (IRGC), on the following topics : “Continuous EEG Monitoring in the ICU “ and on “Alpha-lactoalbumin , proof of concept in the treatment of epilepsy” .Dr Mesraoua is a reviewer for the journal \"seizures\" (Europeen Epilepsy Journal ) as well as dove journals ; Dr Mesraoua is the author and co-author of many peer reviewed publications and four book chapters in the field of Epilepsy and Clinical Neurology",institutionString:"Weill Cornell Medical College in Qatar",institution:{name:"Weill Cornell Medical College in Qatar",country:{name:"Qatar"}}},{id:"282429",title:"Prof.",name:"Covanis",middleName:null,surname:"Athanasios",slug:"covanis-athanasios",fullName:"Covanis Athanasios",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/282429/images/system/282429.jpg",biography:null,institutionString:"Neurology-Neurophysiology Department of the Children Hospital Agia Sophia",institution:null},{id:"190980",title:"Prof.",name:"Marwa",middleName:null,surname:"Mahmoud Saleh",slug:"marwa-mahmoud-saleh",fullName:"Marwa Mahmoud Saleh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/190980/images/system/190980.jpg",biography:"Professor Marwa Mahmoud Saleh is a doctor of medicine and currently works in the unit of Phoniatrics, Department of Otolaryngology, Ain Shams University in Cairo, Egypt. She got her doctoral degree in 1991 and her doctoral thesis was accomplished in the University of Iowa, United States. Her publications covered a multitude of topics as videokymography, cochlear implants, stuttering, and dysphagia. She has lectured Egyptian phonology for many years. Her recent research interest is joint attention in autism.",institutionString:"Ain Shams University",institution:{name:"Ain Shams University",country:{name:"Egypt"}}},{id:"259190",title:"Dr.",name:"Syed Ali Raza",middleName:null,surname:"Naqvi",slug:"syed-ali-raza-naqvi",fullName:"Syed Ali Raza Naqvi",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259190/images/system/259190.png",biography:"Dr. Naqvi is a radioanalytical chemist and is working as an associate professor of analytical chemistry in the Department of Chemistry, Government College University, Faisalabad, Pakistan. Advance separation techniques, nuclear analytical techniques and radiopharmaceutical analysis are the main courses that he is teaching to graduate and post-graduate students. In the research area, he is focusing on the development of organic- and biomolecule-based radiopharmaceuticals for diagnosis and therapy of infectious and cancerous diseases. Under the supervision of Dr. Naqvi, three students have completed their Ph.D. degrees and 41 students have completed their MS degrees. He has completed three research projects and is currently working on 2 projects entitled “Radiolabeling of fluoroquinolone derivatives for the diagnosis of deep-seated bacterial infections” and “Radiolabeled minigastrin peptides for diagnosis and therapy of NETs”. He has published about 100 research articles in international reputed journals and 7 book chapters. Pakistan Institute of Nuclear Science & Technology (PINSTECH) Islamabad, Punjab Institute of Nuclear Medicine (PINM), Faisalabad and Institute of Nuclear Medicine and Radiology (INOR) Abbottabad are the main collaborating institutes.",institutionString:"Government College University",institution:{name:"Government College University, Faisalabad",country:{name:"Pakistan"}}},{id:"58390",title:"Dr.",name:"Gyula",middleName:null,surname:"Mozsik",slug:"gyula-mozsik",fullName:"Gyula Mozsik",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/58390/images/system/58390.png",biography:"Gyula Mózsik MD, Ph.D., ScD (med), is an emeritus professor of Medicine at the First Department of Medicine, Univesity of Pécs, Hungary. He was head of this department from 1993 to 2003. His specializations are medicine, gastroenterology, clinical pharmacology, clinical nutrition, and dietetics. His research fields are biochemical pharmacological examinations in the human gastrointestinal (GI) mucosa, mechanisms of retinoids, drugs, capsaicin-sensitive afferent nerves, and innovative pharmacological, pharmaceutical, and nutritional (dietary) research in humans. He has published about 360 peer-reviewed papers, 197 book chapters, 692 abstracts, 19 monographs, and has edited 37 books. He has given about 1120 regular and review lectures. He has organized thirty-eight national and international congresses and symposia. He is the founder of the International Conference on Ulcer Research (ICUR); International Union of Pharmacology, Gastrointestinal Section (IUPHAR-GI); Brain-Gut Society symposiums, and gastrointestinal cytoprotective symposiums. He received the Andre Robert Award from IUPHAR-GI in 2014. Fifteen of his students have been appointed as full professors in Egypt, Cuba, and Hungary.",institutionString:"University of Pécs",institution:{name:"University of Pecs",country:{name:"Hungary"}}},{id:"277367",title:"M.Sc.",name:"Daniel",middleName:"Martin",surname:"Márquez López",slug:"daniel-marquez-lopez",fullName:"Daniel Márquez López",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/277367/images/7909_n.jpg",biography:"Msc Daniel Martin Márquez López has a bachelor degree in Industrial Chemical Engineering, a Master of science degree in the same área and he is a PhD candidate for the Instituto Politécnico Nacional. His Works are realted to the Green chemistry field, biolubricants, biodiesel, transesterification reactions for biodiesel production and the manipulation of oils for therapeutic purposes.",institutionString:null,institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"196544",title:"Prof.",name:"Angel",middleName:null,surname:"Catala",slug:"angel-catala",fullName:"Angel Catala",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/196544/images/system/196544.jpg",biography:"Angel Catalá studied chemistry at Universidad Nacional de La Plata, Argentina, where he received a Ph.D. in Chemistry (Biological Branch) in 1965. From 1964 to 1974, he worked as an Assistant in Biochemistry at the School of Medicine at the same university. From 1974 to 1976, he was a fellow of the National Institutes of Health (NIH) at the University of Connecticut, Health Center, USA. From 1985 to 2004, he served as a Full Professor of Biochemistry at the Universidad Nacional de La Plata. He is a member of the National Research Council (CONICET), Argentina, and the Argentine Society for Biochemistry and Molecular Biology (SAIB). His laboratory has been interested for many years in the lipid peroxidation of biological membranes from various tissues and different species. Dr. Catalá has directed twelve doctoral theses, published more than 100 papers in peer-reviewed journals, several chapters in books, and edited twelve books. He received awards at the 40th International Conference Biochemistry of Lipids 1999 in Dijon, France. He is the winner of the Bimbo Pan-American Nutrition, Food Science and Technology Award 2006 and 2012, South America, Human Nutrition, Professional Category. In 2006, he won the Bernardo Houssay award in pharmacology, in recognition of his meritorious works of research. Dr. Catalá belongs to the editorial board of several journals including Journal of Lipids; International Review of Biophysical Chemistry; Frontiers in Membrane Physiology and Biophysics; World Journal of Experimental Medicine and Biochemistry Research International; World Journal of Biological Chemistry, Diabetes, and the Pancreas; International Journal of Chronic Diseases & Therapy; and International Journal of Nutrition. He is the co-editor of The Open Biology Journal and associate editor for Oxidative Medicine and Cellular Longevity.",institutionString:"Universidad Nacional de La Plata",institution:{name:"National University of La Plata",country:{name:"Argentina"}}},{id:"186585",title:"Dr.",name:"Francisco Javier",middleName:null,surname:"Martin-Romero",slug:"francisco-javier-martin-romero",fullName:"Francisco Javier Martin-Romero",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bSB3HQAW/Profile_Picture_1631258137641",biography:"Francisco Javier Martín-Romero (Javier) is a Professor of Biochemistry and Molecular Biology at the University of Extremadura, Spain. He is also a group leader at the Biomarkers Institute of Molecular Pathology. Javier received his Ph.D. in 1998 in Biochemistry and Biophysics. At the National Cancer Institute (National Institute of Health, Bethesda, MD) he worked as a research associate on the molecular biology of selenium and its role in health and disease. After postdoctoral collaborations with Carlos Gutierrez-Merino (University of Extremadura, Spain) and Dario Alessi (University of Dundee, UK), he established his own laboratory in 2008. The interest of Javier's lab is the study of cell signaling with a special focus on Ca2+ signaling, and how Ca2+ transport modulates the cytoskeleton, migration, differentiation, cell death, etc. He is especially interested in the study of Ca2+ channels, and the role of STIM1 in the initiation of pathological events.",institutionString:null,institution:{name:"University of Extremadura",country:{name:"Spain"}}},{id:"217323",title:"Prof.",name:"Guang-Jer",middleName:null,surname:"Wu",slug:"guang-jer-wu",fullName:"Guang-Jer Wu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/217323/images/8027_n.jpg",biography:null,institutionString:null,institution:null},{id:"148546",title:"Dr.",name:"Norma Francenia",middleName:null,surname:"Santos-Sánchez",slug:"norma-francenia-santos-sanchez",fullName:"Norma Francenia Santos-Sánchez",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/148546/images/4640_n.jpg",biography:null,institutionString:null,institution:null},{id:"272889",title:"Dr.",name:"Narendra",middleName:null,surname:"Maddu",slug:"narendra-maddu",fullName:"Narendra Maddu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/272889/images/10758_n.jpg",biography:null,institutionString:null,institution:null},{id:"242491",title:"Prof.",name:"Angelica",middleName:null,surname:"Rueda",slug:"angelica-rueda",fullName:"Angelica Rueda",position:"Investigador Cinvestav 3B",profilePictureURL:"https://mts.intechopen.com/storage/users/242491/images/6765_n.jpg",biography:null,institutionString:null,institution:null},{id:"88631",title:"Dr.",name:"Ivan",middleName:null,surname:"Petyaev",slug:"ivan-petyaev",fullName:"Ivan Petyaev",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Lycotec (United Kingdom)",country:{name:"United Kingdom"}}},{id:"423869",title:"Ms.",name:"Smita",middleName:null,surname:"Rai",slug:"smita-rai",fullName:"Smita Rai",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Integral University",country:{name:"India"}}},{id:"424024",title:"Prof.",name:"Swati",middleName:null,surname:"Sharma",slug:"swati-sharma",fullName:"Swati Sharma",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Integral University",country:{name:"India"}}},{id:"439112",title:"MSc.",name:"Touseef",middleName:null,surname:"Fatima",slug:"touseef-fatima",fullName:"Touseef Fatima",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Integral University",country:{name:"India"}}},{id:"424836",title:"Dr.",name:"Orsolya",middleName:null,surname:"Borsai",slug:"orsolya-borsai",fullName:"Orsolya Borsai",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca",country:{name:"Romania"}}},{id:"422262",title:"Ph.D.",name:"Paola Andrea",middleName:null,surname:"Palmeros-Suárez",slug:"paola-andrea-palmeros-suarez",fullName:"Paola Andrea Palmeros-Suárez",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Guadalajara",country:{name:"Mexico"}}}]}},subseries:{item:{id:"17",type:"subseries",title:"Metabolism",keywords:"Biomolecules Metabolism, Energy Metabolism, Metabolic Pathways, Key Metabolic Enzymes, Metabolic Adaptation",scope:"Metabolism is frequently defined in biochemistry textbooks as the overall process that allows living systems to acquire and use the free energy they need for their vital functions or the chemical processes that occur within a living organism to maintain life. Behind these definitions are hidden all the aspects of normal and pathological functioning of all processes that the topic ‘Metabolism’ will cover within the Biochemistry Series. Thus all studies on metabolism will be considered for publication.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/17.jpg",hasOnlineFirst:!0,hasPublishedBooks:!0,annualVolume:11413,editor:{id:"138626",title:"Dr.",name:"Yannis",middleName:null,surname:"Karamanos",slug:"yannis-karamanos",fullName:"Yannis Karamanos",profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002g6Jv2QAE/Profile_Picture_1629356660984",biography:"Yannis Karamanos, born in Greece in 1953, completed his pre-graduate studies at the Université Pierre et Marie Curie, Paris, then his Masters and Doctoral degree at the Université de Lille (1983). He was associate professor at the University of Limoges (1987) before becoming full professor of biochemistry at the Université d’Artois (1996). He worked on the structure-function relationships of glycoconjugates and his main project was the investigations on the biological roles of the de-N-glycosylation enzymes (Endo-N-acetyl-β-D-glucosaminidase and peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase). From 2002 he contributes to the understanding of the Blood-brain barrier functioning using proteomics approaches. He has published more than 70 papers. His teaching areas are energy metabolism and regulation, integration and organ specialization and metabolic adaptation.",institutionString:null,institution:{name:"Artois University",institutionURL:null,country:{name:"France"}}},editorTwo:null,editorThree:null,series:{id:"11",title:"Biochemistry",doi:"10.5772/intechopen.72877",issn:"2632-0983"},editorialBoard:[{id:"243049",title:"Dr.",name:"Anca",middleName:null,surname:"Pantea Stoian",slug:"anca-pantea-stoian",fullName:"Anca Pantea Stoian",profilePictureURL:"https://mts.intechopen.com/storage/users/243049/images/system/243049.jpg",institutionString:null,institution:{name:"Carol Davila University of Medicine and Pharmacy",institutionURL:null,country:{name:"Romania"}}},{id:"203824",title:"Dr.",name:"Attilio",middleName:null,surname:"Rigotti",slug:"attilio-rigotti",fullName:"Attilio Rigotti",profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institutionString:null,institution:{name:"Pontifical Catholic University of Chile",institutionURL:null,country:{name:"Chile"}}},{id:"300470",title:"Dr.",name:"Yanfei (Jacob)",middleName:null,surname:"Qi",slug:"yanfei-(jacob)-qi",fullName:"Yanfei (Jacob) Qi",profilePictureURL:"https://mts.intechopen.com/storage/users/300470/images/system/300470.jpg",institutionString:null,institution:{name:"Centenary Institute of Cancer Medicine and Cell Biology",institutionURL:null,country:{name:"Australia"}}}]},onlineFirstChapters:{paginationCount:17,paginationItems:[{id:"81647",title:"Diabetes and Epigenetics",doi:"10.5772/intechopen.104653",signatures:"Rasha A. 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