**4.3 Anastomotic stenosis**

It can sometimes be treated endoscopically, when surgery is contraindicated, by performing, as was found in a case report by Deng and team [13] with minimally invasive endoscopic approach was adopted to repair the obstruction. A needle knife was used to puncture the linear white scar, and contrast agent was injected under endoscopy and fluoroscopic guidance. Fluoroscopically, the proximal bowel was identified and a dual knife-mediated membrane puncture was performed. A guidewire was then passed through the incision into the proximal bowel and progressive pneumatic dilatation was performed successively with a controlled radial expansion balloon dilator until a 1.8-cm-diameter dilation was achieved. After conventional balloon dilatation, the endoscope easily passed through the anastomosis without any patient discomfort. There were no postoperative signs of immediate or delayed complications (**Figure 13**).

**Figure 12.** *Illustration of a simple abdominal X-ray exam in a patient with bowel obstruction.*

**Figure 13.** *Colonoscopic aspect of a anastomotic stenosis.*


**5.1 Efficient and effective use of water**

When water demand increases and water availability decreases one of the most widely used solution towards decreasing water more consumption is using the available water effectively and more efficiently. Water demand management considers measures to improve efficiency of water use.

Among sectors, agriculture is the leading sector in terms of water consumption. Climate change is expected to directly and indirectly increase demand for agricultural irrigation. Adaptation measures to climate change in the agricultural sector include changes of agrotechnical practices (e.g., use of crop rotation, advancing sowing dates) and introduction of new cultivars (heat-wave- and drought-tolerant crops). Soil moisture should also be conserved (e.g. through mulching). Besides, timing and frequency of irrigation need to be optimized considering the crop requirements. This is important for reducing irrigation return flows which in turn deteriorates the quality of the receiving water.

Managing the Effects of the Climate Change on Water Resources and Watershed Ecology 271

hydrological conditions and ecological conditions of the basins. Pre-screening in terms of engineering works focus on costs of the work and on the length of water transmission lines. Besides, head loss/energy consumption of the pumps, natural and artificial barriers along

Inter-basin water transfer depends on the availability of excess water from where the water is withdrawn. Especially the climatic conditions of both basins gain importance. If both basins face drought conditions in the same years, water transfer among them should not be considered as a feasible option. Both basins must be surveyed prior to realization of water transfer regarding their hydrological characteristics. During these surveys, long-term hydrological data must be analyzed. Watershed ecology is equally important. Socio-cultural conditions and economical characteristics should also be taken into consideration and sustainability should be kept in mind during water withdrawals. There are still contradicting opinions on inter-basin water transfer. They argue that inter-basin water transfer may no longer be viable in a future with climate change, as climate change stresses almost every source of freshwater. Also taking more water from the natural system has biological, ethical, and increasingly legal limitations (Karakaya and Gonenc, 2005; Hall et al., 2008). Consequently, it is advised to consider inter-basin water transfer to be considered as

Natural aquatic ecosystems are among the important water resources supporting life. It is very important to maintain the ecological flows of these systems. Ecological flows are usually determined by some practical statistical approaches, assumptions and methods supported by scientific research conducted at site. During these studies it must be considered that aquatic ecosystems are in interaction with terrestrial ecosystems. Thus, any change in aquatic or terrestrial ecosystem will have an effect on the other one. For example, the decrease in surface water levels will affect the groundwater levels and dependent ecosystems. Evapotranspitation increase due to climate change has also effect on the decrease of groundwater levels. As this condition may lead to change in the vegetation cover which in turn lead to habitat change regulation of groundwater use becomes more important. As renewal of groundwater lasts long, planning must be done prior to facing

Changes in water quality in water resources will necessitate revision of existing waterrelated infrastructure. New components of the infrastructure should be designed according to possible extremes that would occur. Resilience of the infrastructure should also be

Water treatment systems must be designed and operated according to drinking water standards under raw water inflow with varying water quality. On the other hand, different wastewater treatment options that seem not feasible today may be available in a world with higher annual average temperature. One example is the upflow anaerobic sludge blanket (UASB) process that is used to treat municipal wastewater in warmer countries such as India currently. Such technologies that are more cost-efficient could be applied in higher

latitudes once further meteorological conditions change due to climate change.

the pipeline and its vicinity are also important factors to be considered.

**5.4 Maintaining the sustainability of watershed ecosystems**

the last solution to water scarcity.

water scarcity.

enhanced.

**5.5 Revision of infrastructure**

Industrial water consumption may also be reduced by developing less water using technologies as well as in-plant control measures. Clean technologies should be preferred due to their optimized water consumption.

Domestic uses may be decreased by encouraging public to use water-saving home appliances, through water pricing, legal sanctions and raising public awareness. In the big cities in developing countries, water loss through leakages in the water distribution lines constitutes a significant amount. Thus, it must be aimed to decrease water losses below 10% by renewing the old pipelines.


Bismuth - Advanced Applications and Defects Characterization

*Edited by Ying Zhou, Fan Dong and Shengming Jin*

Bismuth (Bi) is a post-transition metal element with the atomic number of 83, which belongs to the pnictogen group elements in Period 6 in the elemental periodic table. As a heavy metal, the hazard of Bi is unusually low in contrast to its neighbors Pb and Sb. This property, along with other typical characteristics like strong diamagnetism and low thermal conductivity, makes Bi attractive in industrial applications. There are more than 100 commercial bismuth products, from pharmaceutical to industrial catalysts. Based on the wide applications of Bi materials, this book goes further and mainly focuses on the potential uses of Bi-based materials, which consist of nine chapters. In addition, a special chapter concerning the defect in bismuth is also presented.

ISBN 978-1-78923-262-2

Published in London, UK © 2018 IntechOpen © miriam-doerr / iStock


**7.3. Observation**

Observation is the main tool to gather ongoing information of students' performance. It can take place in a variety of settings, across many activities, and employ a number of different tools to record information including checklists, anecdotal records, frequency count tables, and rubrics [28]. Observational assessments empower measurement of students' behavior, skills, and abilities in ways not possible via traditional assessment (quiz, test, or exam). When planning to observe students, instructors should consider whom they want to observe, what to observe, and how to evaluate and document what they see.


Photoperiod sensitivity is an important trait responsible for regional and seasonal adaptability of rice varieties. In this study, we detected two novel alleles at the *E1*/*Ghd7* locus. It is known that many other loci are involved in the photoperiod sensitivity pathway of flowering in rice. Recent studies showed that photoperiod sensitivity loci, *Se1*/*Hd1*, *OsPRR37* and *Ghd8*, each have a large allelic variation [49,54–57]. Therefore, analyzing the functional and inter-locus interactions should be advanced, which will lead to practice the fine-tuning of flowering time in rice breeding programs. In addition, we elucidated that the *E1* and *Ghd7* are the same locus. Until now, numerous genetic information has been accumulated for each locus (*E1*, *Ghd7*). For further genetic analyses of the *E1*/*Ghd7* locus, however, all information about *E1* and *Ghd7* should be available.

#### **4. Materials and Methods**

#### *4.1. Photoperiod Sensitivities of T65, T65m, and T65*

The Taiwanese *japonica* rice variety "Taichung 65 (T65)", and its isogenic line T65m and its near-isogenic line T65w were used. T65 harbors a photoperiod sensitivity allele *E1* at the *E1* locus, while T65m harbors a photoperiod-insensitivity allele *e1*, which is derived from the cross with Bozu5 [16,19,34,35]. T65w is a chromosome substitution line which was developed by introducing the *E1* region of *O. rufipogon* (W107) into the genetic background of T65 [36]. Five seeds were sown on field soil in a 3.6 L pot and covered with granulated soils. Seedlings were thinned to one plant per pot 14 days after sowing, and were grown under two photoperiod conditions, SD (12-h light/12-h dark) and LD (14-h light/10-h dark). Photoperiod treatments were conducted using two growth cabinets without temperature control. In addition to natural daylight (8:00–18:00), supplementary artificial light was used for the 12-h and 14-h light conditions. The degree of photoperiod sensitivity of each line was expressed as a difference of DH between SD and LD. The experiment was conducted by using five plants per line with three replications from 1 May to October 16. Heading date was recorded for each plant when the first panicle emerged from the sheath of the flag leaf.

#### *4.2. Chromosomal Location of the E1 Locus*

Two F2 populations from crosses of T65 × T65w, comprising 205 plants, and T65w × T65m, comprising 125 plants, were subjected to genetic analysis of heading date. They were grown in a paddy field of Kyoto University, Kyoto, Japan (35◦01- N). Seeds were sown on April 25 in 2007, and seedlings were transplanted on May 16 in 2007. The progeny test was conducted with 40 F3 lines (sowing on April 30 in 2008 and transplanting on May 21 in 2008). Each F3 line with approximately 25 plants was the progeny of an F2 plant randomly selected from the F2 population. To narrow down the candidate region of the *E1* locus, the F4 progenies derived from several F3 recombinants between RM1253 and RM3635 were cultivated (sowing on May 1 in 2009 and transplanting on May 22 in 2009).

#### *4.3. Expression Analysis of E1*/*Ghd7*

Plants of T65, T65m, and T65w were grown in a growth cabinet with a temperature controller under a LD condition (14.5-h light, 30 ◦C/9.5-h dark, 25 ◦C) at 70% relative humidity. Seedlings were grown on sand in 3.6 L pots (two plants/pot) with additional liquid fertilizer (Kimura's B Culture Solution, Nippon Medical and Chemical Instruments Co., Ltd., Osaka, Japan). Thirty days before flowering, leaves were collected at 4-h intervals during that day. Total RNAs were extracted with Trizol reagent (Life Technologies Inc., Gaithersburg, Maryland, USA) according to the manufacturer's protocols. Total RNA was subjected to DNA digestion by treatment with RNase-free DNase I (Takara Bio Inc.). The transcriptor first-strand cDNA synthesis kit (Roche Applied Science, Indianapolis, Indiana, USA) was used to reverse-transcribe cDNA from 1 μg of RNA using anchored-oligo (dT)18 primers. Real-time PCR analysis was performed by TaqMan PCR using a LightCycler 1.5 (Roche Applied Science) according to the manufacturer's instructions. The primer sets of *Ghd7* and *UBQ* genes and Universal Probe Library probes of each gene were designed with ProbeFinder version 2.45 (Roche; https://www.roche-applied-science.com/). Primer and probe sequences are shown in Table S5. Expression analysis using the standard curve method was performed to determine the expression level

of each gene. The relative expression level of each gene was calculated using *UBQ* gene. The RNA gene standards for the seven genes were applied to their plasmids prepared by the pGEM-T Easy Vector System (Promega Corp., Madison, Wisconsin, USA) using PCR amplicons from the total RNA of T65.

#### *4.4. Identification of the Genotype at the E1*/*Ghd7 Locus*

To identify the *E1*/*Ghd7* genotype, we developed two DNA markers based on an SNP and a TE insertion. CAPS marker analysis was based on a nucleotide substitution from GAG (Glu) to TAG (stop codon) in exon 1, producing an additional restriction enzyme (*Spe* I) site. A pair of PCR primers (Ghd7\_CAPS\_F: 5- -CCAACTTGCCCTGTCTTCTT-3- , Ghd7\_CAPS\_R: 5- -AGCTGCTGCAAGCCAGTAAT-3- ) was designed to amplify the 950 bp. PCR was performed with a 20 μL reaction mixture containing 2 μL template DNA, 10× PCR buffer, 25 mM MgCl2, 2 mM of each deoxyriboside-triphosphate (dNTP), 0.2 μL *Taq* DNA polymerase (5 U/μL), 4 μL of a 2.5 mM solution of each primer, and 3.2 μL H2O. PCR conditions were as follows: 94 ◦C for 5 min, followed by 35 cycles (1 min at 94 ◦C, 1 min at 60 ◦C, and 2 min at 72 ◦C) with a final extension of 7 min at 72 ◦C. The amplified products were digested with *Spe* I at 37 ◦C for 6 h. After digestion, the nonfunctional allele produced three fragments (81, 287, and 582 bp), whereas the functional allele produced two fragments (81 and 869 bp). Amplicons and digested amplicons were separated on 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide, and the DNA fragments were visualized under UV light. Insertion and deletion (INDEL) marker analysis was based on an 1897-bp copia-like TE insertion. A pair of PCR primers (Ghd7\_INDEL\_F: 5- -CGTTTCAGCAATAGCATTATGG-3- , Ghd7\_INDEL\_R: 5- -GCGGGTAGTCATCGAACAG-3- ) was designed to amplify the 824 bp in the wild type and the 2721 bp in the insertion type. PCR was performed with a 20 μL reaction mixture containing 2 μL template DNA, 10× PCR buffer, 25 mM MgCl2, 2 mM of each dNTP, 0.2 μL *Taq* DNA polymerase (5 U/μL), 4 μL of a 2.5 mM solution of each primer, and 3.2 μL H2O. PCR conditions were as follows: 94 ◦C for 5 min, followed by 35 cycles (1 min at 94 ◦C, 1 min at 60 ◦C, and 2 min at 72 ◦C) with a final extension of 7 min at 72 ◦C. Amplicons were separated on 1% agarose gel. After electrophoresis, the gel was stained with ethidium bromide, and the DNA fragments were visualized under UV light.

#### *4.5. Haplotype Patterns of the Chromosomal Region around the E1*/*Ghd7 Locus*

Three DNA polymorphisms (two single-nucleotide substitutions and a 20 bp deletion) within the 100-kb chromosomal region surrounding *Ghd7* existed among 44 Hokkaido, 50 Japanese-core-collection [58] and 71 Aikoku-related varieties, which are considered to the derivatives of "Aikoku" variety based on their names. The Japanese rice core collection is a limited set of accessions representing, with a minimum set repetitiveness, the genetic diversity among Japanese rice varieties [58]. The two substitutions (Hap\_SNP1 and Hap\_SNP2) were detected by CAPS marker analyses. Hap\_SNP1 harbors a G-to-T substitution, resulting in changing the recognition site of the restriction enzyme *Hpy188* I. Hap\_SNP2 harbors a C-to-T substitution, resulting in changing the recognition site of *Hpy188* I. Primer pairs for Hap\_SNP1 and Hap\_SNP2 were designed to amplify 623 and 295 bp, respectively (Table S6). In addition, two polymorphism surveys of the *copia*-like TE insertion and the SNP in the first exon of *Ghd7* were performed. The CAPS and INDEL marker analyses to detect the insertion and SNP were described above.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/8/12/550/s1, Figure S1: Pedigree of the varieties, which harbor *e1-ret*/*ghd7-0ret* allele. Bold with underline indicates that the varieties harbor *e1-ret*/*ghd7-0ret* allele. Gray indicates varieties whose allele is unknown. Other indicates that the varieties harbor *e1*/*ghd7-0a* allele., Table S1: Frequency distributions of days to heading in F3 lines (T65 × T65w), Table S2: Frequency distributions of days to heading in F3 lines (T65m × T65w), Table S3: Lists of Hokkaido and Japanese core collection varieties used in this study, Table S4: Lists of Aikoku-related varieties used in this study, Table S5: Primer and probe sequences for expression analysis, Table S6 Primer sequences for haplotype analysis. **Author Contributions:** Conceptualization, H.S., Y.O. and T.T. (Takatoshi Tanisaka); Methodology, H.S., Y.O. and T.T. (Takatoshi Tanisaka); Formal analysis, H.S., T.T. (Takuji Tsukiyama) and M.T.; Investigation, H.S. and C.X.; Writing—original draft preparation, H.S.; Writing—review and editing, H.S., Y.O. and T.T. (Takatoshi Tanisaka); Project administration, H.S. and T.T. (Takatoshi Tanisaka).

**Acknowledgments:** We thank the genetic resources center, NARO (National Agriculture and Food Research Organization) for providing the seeds of Hokkaido, Japanese-core-collection and Aikoku-related varieties. T65m was kindly provided from Shigetoshi Sato in Ryukyu University and Kuo Hai Tsai in National Chung-Hsing University. T65w was kindly provided from Yoshio Sano in Hokkaido University. We also would like to thank Syo Asano and Shinsuke Hamada for their supports of our experiments.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**Acknowledgement**

Financial support for this study was provided by Italian Ministry of Agriculture, Food and Forestry through the project GERMOLI "Salvaguardia e valorizzazione del GERMoplasma OLIvicolo delle collezioni del CRA-OLI"


**Performance and Heat Index of West African Dwarf (WAD) Rams Fed with** *Adansonia digitata* **Bark (Baobab) as Supplement**

Idayat Odunola Agboola *Federal University of Agriculture, Abeokuta, Ogun State, Nigeria*


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List of Illustrations

291

# List of Illustrations

If not indicated otherwise, the photographs were made by the respective author.

Cover: Daniele Barbaro and Andrea Palladio, representation of the Orthographia and Sciographia in Barbaro's commentary of Vitruvius from 1567. In: Barbaro 1567, I 32 (© CISA A. Palladio).

Content: Chapter header: Basilica (Palazzo della Ragione), Vicenza. Photograph by Thorsten Bürklin.

Foreword: Chapter header: See Armando Dal Fabbro, fig. 11, detail.

# Armando Dal Fabbro

The Empty Space, the Enclosure and the *boîte à miracles:*  Let's Go Back to Palladio

Chapter header: See fig. 3, detail.

# Thorsten Bürklin

# Radical Pragmatism

 Chapter header: Basilica (Palazzo della Ragione), Vicenza, in *I Quattro Libri dell'Architettura*. In: Palladio 1570, III 42 (Cap. XX) (© CISA A. Palladio), detail.

# Damiana Lucia Paternò

Firmitas, Utilitas, Venustas, Economy. The Four Principles of Palladio's Way of Building

Chapter header: See Fig. 10, detail.

# Francesco Marcorin

Palladio's Drawings as a Means of Knowledge: Looking at the Past Through Modern Eyes

Chapter header: See Fig. 11, detail.

Fig. 19 Andrea Palladio, *I Quattro Libri dell'Architettura*, details and section of the Pantheon in Rome. In: Palladio 1570, IV 82–83 [84, 81] (Cap. XX) (© CISA A. Palladio).

# Patrizio M. Martinelli

Palladian Façades: Inhabited Thresholds and Theatrical Urban Micro-Cosms

Chapter header: See Fig. 1.

# Sören Fischer

Architecture within Architecture. Strategies of Spatial Design in Andrea Palladio's Villas

Chapter header: See fig. 1, detail.

# Renata Samperi

Corners and Design Process in Palladio's Architecture

Chapter header: See Fig. 11, detail.

# Martin Ebert

# Fact and Fiction

Chapter header: See Fig. 1.

# Dikkie Scipio

Let's Talk About Palladio. A Brave Attempt to Understand a 16th-Century Architect

Chapter header: See Fig. 7, detail.

About the Authors

307

# About the Authors

# Armando Dal Fabbro

is an architect and Professor of Architectural and Urban Composition at IUAV University of Venice, where he is the coordinator of the PhD program in Architectural Composition. He carries out research on the themes of composition and figuration in architectural design. He has received several awards, including the Grand Prix of the VI International Architecture Biennale in Krakow, the Mention of Honor and the Bene Merentibus Medal of the Polytechnic University of Krakow. In 2021, he was appointed a member of the National Academy of San Luca in Rome.

# Damiana Lucia Paternò

obtained a PhD in Preservation of Architectural Heritage in 2013 at Politecnico di Milano. She was a post-doctoral research fellow at IUAV University of Venice, and since 2010, she has been collaborating with CISA Centro Internazionale di Studi di Architettura Andrea Palladio in Vicenza. Currently she works as an architect for the Ministero della Cultura. She published *Palladio nel tempo. Trasformazioni, autenticità, mito tra Ottocento e Novecento*. Other publications include reflections on history and theory of restoration in Italy.

## Dikkie Scipio

is an architect and Professor of Architectural Design at MSA Münster School of Architecture, FH Münster University of Applied Sciences. She is the co-founder of KAAN Architecten, and an expert in complex projects and processes, focusing on museums and architectural heritage. Scipio is also a writer, columnist, international lecturer, and a member of several boards and juries. As a spokeswoman for integrated art and craftsmanship in architecture her views have been featured in many publications and documentaries.

## Francesco Marcorin

is the curator of the Palladio Museum in Vicenza. He obtained a PhD in Architectural History from IUAV University of Venice. In 2017, he continued his studies in New York, as an Ayesha Bulchandani Intern at the Frick Collection, and as a Weinberg Fellow at Columbia University. In 2018 and 2019, he was Adjunct Professor with the Union College International Program (New York-Florence). His publications and conference papers have dealt with the architects Michele Sanmicheli and Andrea Palladio, the reception of late-antiquity architecture during the Renaissance, and the antiquities of Verona.

## Martin Ebert

is Professor of Construction at MSA Münster School of Architecture, FH Münster University of Applied Sciences. He also runs an architecture and design practice in London. His teaching and work explore the role of structure and order in the creation of architectural space and form. This is evidenced in his interior and housing designs and chairs and tables.

## Patrizio M. Martinelli

is Assistant Professor of Interior Architecture and Architecture at Northumbria University, Newcastle upon Tyne (UK). His research and creative work, published in monographs and international journals, and showcased in exhibitions in UK and USA, focuses on modernism, domestic and urban interiors, adaptive reuse, and the concept of theatricality of interior space.

# Renata Samperi

is Professor of History of Architecture at Università degli Studi di Ferrara, Dipartimento di Architettura. Her research covers the fifteenth, sixteenth, and seventeenth centuries, centering on selected topics of Roman architecture and the work of architects such as Alberti, Giuliano da Sangallo, Peruzzi, Palladio, and Borromini. Its central focus is the reconstruction of the complex architectural processes based on the cross-analysis of data from archival documents, surveys, and direct observation. In 2019, she published a book on corners in Palladio's architecture.

# Sören Fischer

is the head of the Prints and Drawings Collection at Museum Pfalzgalerie Kaiserslautern. He curates and researches early modern art as well as art from the 19th to the 21st century. In 2011, he completed his doctorate on illusionist wall paintings in Venetian villas of the 16th century. He has published a book and written several essays on illusory architecture and its capacity of designing and manipulating interior space, particularly in the buildings of Andrea Palladio.

# Thorsten Bürklin

is Professor of History and Theory of Architecture at MSA Münster School of Architecture, FH Münster University of Applied Sciences. His research focuses on the Renaissance, the perception of architectural space, and contemporary architectural and urban theory. He published a book on Palladio's architecture (*Palladio, der Bildermacher*).

Impressum

Funding: FH Münster University of Applied Sciences, MSA Münster School of Architecture, and the Palladio Museum – Centro Internazionale di Studi di Architettura Andrea Palladio

Bibliographic information published by the Deutsche Nationalbibliothek The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.d-nb.de

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# First published in 2024 by transcript Verlag, Bielefeld © Thorsten Bürklin, Martin Ebert (eds.)

Cover layout: Vera Lohmann, Münster Cover illustration: Daniele Barbaro/Andrea Palladio Book design: Vera Lohmann, Münster Copy-editing: Transcript Verlag, Bielefeld Typeset: Melior LT Pro, Camber Pro Printed by Majuskel Medienproduktion GmbH, Wetzlar Print-ISBN 978-3-8376-6672-4 PDF-ISBN 978-3-8394-6672-8 https://doi.org/10.14361/9783839466728 ISSN of series: 2702-8070 eISSN of series: 2702-8089

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**The Maximum Non-Linear Feature Selection of Kernel Based on Object Appearance**

Mauridhi Hery Purnomo1, Diah P. Wulandari1, I. Ketut Eddy Purnama1 and Arif Muntasa2 *1Electrical Engineering Department – Industrial Engineering Faculty, Institut Teknologi Sepuluh Nopember, Surabaya 2Informatics Engineering Department – Engineering Faculty, Universitas Trunojoyo Madura Indonesia*


**Contents**

The Impact of Different Pretreatment Processes (Freezing, Ultrasound and High Pressure) on the Sensory and Functional Properties of Black Garlic (*Allium sativum* L.) Reprinted from: *Molecules* **2022**, *27*, 6992, doi:10.3390/molecules27206992 .............. **131**


**User, Gesture and Robot Behaviour Adaptation for Human-Robot Interaction**

Md. Hasanuzzaman1 and Haruki Ueno2

*1Department of Computer Science & Engineering, University of Dhaka,Dhaka 2National Institute of Informatics (NII), Tokyo 1Bangladesh 2Japan*


*2.5. Detection of Oxidized Proteins (OXs) by Resin-Assisted Capture RAC (OX–RAC)*

The detection of OXs by OX–RAC was performed on three biological replicates using a previously described protocol [6]. Captured proteins were eluted with 30-μL elution buffer containing 10 mM HEPES, 0.1 mM EDTA, 0.01 mM neocuproine, 0.1% sodium dodecyl sulfate (SDS), and 100 mM 2-mercaptoethanol for 20 min at room temperature. Proteins in a 10-μL aliquot of each eluent were resolved on a 12.5% SDS—polyacrylamide gel electrophoresis (PAGE) gel. Each gel was then stained with silver (Pierce Silver Stain), and each gel slice was independently analyzed by MS. A protein was considered to be oxidized when its relative amount in the DTT-treated lysates was significantly more than that in the DTT-untreated lysates (*p <* 0.05 according to the results of an unpaired *t*-test).

## *2.6. In-Gel Proteolysis and MS Analysis*

The proteins in the gel were reduced with 2.8mM DTT (60 ◦C for 30 min), modified with 8.8 mM iodoacetamide in 100 mM ammonium bicarbonate (in the dark and at room temperature for 30 min) and digested in 10% acetonitrile and 10 mM ammonium bicarbonate with modified trypsin (Promega, Beit Haemek, Israel) overnight at 37 ◦C. A second trypsin digestion was carried out for another 4 h at 37 ◦C.

The tryptic peptides were desalted using C18 tips (Home-made, 3M) dried and resuspended in 0.1% formic acid.

The peptides were resolved by reverse-phase chromatography on 0.075 × 180-mm fused silica capillaries (JW) packed with Reprosil reversed phase material (Dr Maisch GmbH, Ammerbuch, Germany). The peptides were eluted with linear 60 min gradient of 5 to 28% 15 min gradient of 28 to 95% and 25 min at 95% acetonitrile with 0.1% formic acid in water at flow rates of 0.15 μL/min. MS was performed by Q Exactive HF mass spectrometer (Thermo Fisher Scientific represented by BARGAL analytical instruments, Shoham, Israel) in a positive mode using a repetitively full MS scan followed by collision induces dissociation (HCD) of the 18 most dominant ions selected from the first MS scan. The mass spectrometry data were analyzed using the MaxQuant software <IP_ADDRESS>, The Max Plank Institute of Biochemistry, Munich, Germany [24] vs. *Entamoeba histolytica* and *Lactobacillus acidophilus* proteomes from the Uniprot database with 1% FDR (false discovery rate). The data were quantified by label free analysis using the same software. Statistical analysis of the identification and quantization results was done using Perseus <IP_ADDRESS> software, The Max Plank Institute of Biochemistry, Munich, Germany [25]. A *t*-test between the groups with or without DTT was carried out, with the Benjamini–Hochberg correction for multiple testing. Proteins were considered as significantly changed if their *p*-value < 0.05, *q*-value < 0.05, and the fold change between the groups ≥ 1.