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## Meet the editors

Assist. Prof. Dr. Eng. Oana-Maria Boldura is licensed in Genetic Engineering with specialization in Molecular Biology (2005). He has an MSc in Genetic Manipulation (2007) and Pharmacy (2014) and a PhD in Biotechnology (2010). Since 2015 he has been Assistant Professor at Banat University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania" Timisoara, Faculty of Veterinary Medicine, Department

of Chemistry, Biochemistry and Molecular Biology. He is also a researcher in the "Horia Cernescu" Research Unit. His main scientific competences are in the fields of molecular biology, biotechnology, genomics, and biosecurity with technical aptitudes in manipulation and experimentation of nucleic acids and proteins (purification, sequencing, gene expression studies, SDS-PAGE analysis of protein fractions, "Lab-on-chip" electrophoresis, and immunological testing). His areas of interest include molecular forensic methods and apoptotic process pathways.

Dr. Cornel Baltă, MD, DVM, is a licensed veterinarian with both a PhD and MD in Veterinary Medicine. He is a researcher at the Institute of Life Sciences, at "Vasile Goldis" Western University Arad, Romania. Dr. Baltă's main scientific competences are in the fields of laboratory animals, molecular biology, physiology, histology, and clinical Laboratory. He has technical aptitudes in nucleic acid examination by electrophoresis techniques, PCR and

qPCR, and qualitative and quantitative determination of proteins by Western Blot and microelectrophoresis. His areas of interest include molecular mechanisms involved in the evolution of liver and gastrointestinal diseases, and bone regeneration and remodeling using different types of polymers.

Dr. Nasser Sayed Awwad obtained a PhD in Inorganic and Radiochemistry from Ain Shams University in 2000 and a postdoctorate degree from Sandia National Labs, Albuquerque, New Mexico, in 2004. He was Associate Professor of Radiochemistry in 2006 and Professor of Inorganic and Radiochemistry in 2011. Currently he is a professor at King Khalid University (KKU), Abha, Kingdom of Saudi Arabia. He has published two book

chapters, edited and coedited several books, and published seventy-five papers in indexed journals. He has supervised MSc and PhD students in Radioactive and Wastewater Treatment. He participated in twenty-six international conferences in South Korea, the United States, Lebanon, Kingdom of Saudi Arabia, and Egypt. He participated in six big projects with King Abdulaziz City for Science and Technology (KACST), Kingdom of Saudi Arabia, and Sandia National Labs, Albuquerque, New Mexico, about the conditioning of radioactive sealed sources for wastewater treatment. Dr. Awwad is a leader of two research groups about utilization of nanomaterials for treatment of inorganic and organic pollutants and the influence of dand f- block elements interactions with drugs on its bioactivities. He is a member of the Arab Society of Forensic Sciences and Forensic Medicine, the American Chemical Society, and the Permanent Committee for the Development of Scientific Research Strategies and Policies and Committee for the Development of International Cooperation Management, both at KKU. He is also Rapporteur of the Permanent Committee for Nuclear and Radiological Protection and Rapporteur of the Permanent Committee of the Center of Talent and Innovation and Entrepreneurship, both at KKU. He is also head of the Scientific Research and International Cooperation Unit, Faculty of Science, KKU.

Contents

**Section 1**

and Chronic Myeloid Leukemia

*by Alice Abdel Aleem*

**Preface XI**

DNA Based Molecular Tools **1**

**Chapter 1 3**

**Chapter 2 17**

**Chapter 3 43**

**Chapter 4 61**

**Chapter 5 85**

**Chapter 6 97**

**Chapter 7 111**

Detection of the Species Composition of Food Using Mitochondrial DNA:

Molecular Markers and Their Optimization: Addressing the Problems

Ambient Biobanking Solutions for Whole Blood Sampling,

*by Armaity Nasarabadi Fouts, Alejandro Romero, James Nelson,*

Latest Implications of Next-Gen Sequencing in Diagnosis of Acute

*by Oana Maria Boldura, Cristina Petrine, Alin Mihu and Cornel Balta*

DNA Sequencing Resolves Misdiagnosed and Rare Genetic Disorders

Biological Evidence Analysis in Cases of Sexual Assault *by Benito Ramos González, Miranda Córdova Mercado, Orlando Salas Salas, Juan Carlos Hernández Reyes, Martín Guardiola Ramos, Elton Solis Esquivel, Gerardo Castellanos Aguilar and Porfirio Diaz Torres*

Molecular Tools for Gene Analysis in Fission Yeast

Challenges and Possibilities of a Modern Laboratory *by Małgorzata Natonek-Wiśniewska and Piotr Krzyścin*

of Nonhomology Using Decapod COI Gene *by Deepak Jose and Mahadevan Harikrishnan*

Transportation, and Extraction

*Mike Hogan and Shanavaz Nasarabadi*

*Francisca Sosa-Jurado, Nancy Martínez-Montiel,*

*by Irma Pilar Herrera-Camacho, Lourdes Millán-Pérez-Peña,*

*Rebeca Débora Martínez-Contreras and Nora Hilda Rosas Murrieta*

## Contents

Preface

The analysis of nucleic acids has applications in several fields. In agriculture, it has been used to develop organisms (plant or animal) with improved characteristics. In medicine, it has been used to identify changes in the structure of DNA, hereditary or acquired, which predict or lead to the appearance of numerous pathological conditions. Across the world, techniques based on nucleic acid analysis are used in medical diagnostic laboratories because once standardized, they can provide accurate results in a very short time. Some of these functional studies can even be performed with samples commonly found in clinical pathology laboratories. Also, DNA-based methods are used in microbiology, ecology, food science, and forensics. Continuous improvements in technology coupled with creative minds seeking to understand the inner workings of the cell have begun to unravel the importance of genome-wide coding variation.

The first section of this book is a collection of chapters covering experimental

methods used in molecular biology, the techniques adjacent to these methods, and the steps of analysis before and after obtaining raw DNA data. This section also includes examples of practical applications from different fields, including bioinformatics.

In the second section of this book, the chapters cover the parameters of analytical methods and chromatography techniques for the identification, separation, purification, and quantitative estimation of complex mixtures of organic compounds. In addition, the analytical method is suitable for the quantitative estimation of targeted analytes present in pharmaceutical drugs thus playing a vital role in the development and manufacture of pharmaceuticals. Also, this section discusses how the sample preparation is one of the most labor-intensive and time-consuming operations in sample analysis. The section also presents efficient micro sample preparation techniques for high-performance liquid chromatography, especially online automated in-tube solid phase microextraction (IT-SPME). Moreover, this section covers one of the industrial applications for ion chromatography via preparation of an organic–inorganic composite using the sol–

gel process to improve the cation exchanger's properties and efficiency.

book project possible.

The editors of this book wish to thank the authors for their valuable contributions. We would also like to thank Author Service Manager Mrs. Dolores Kuzelj and Commissioning Editor Mrs. Iva Simcic. Their help and professionalism made this

**Dr. Eng. Oana-Maria Boldura**

Faculty of Veterinary Medicine,

"King Michael I of Romania",

Department of Chemistry, Biochemistry and Molecular Biology, Banat University of Agricultural Sciences and Veterinary Medicine

Assistant Professor,

Timisoara, Romania,


*3.1.2 The effect on the cumulative days (*�*1,0)*

Now we look at the effect of the changes in SKEW on the cumulative abnormal returns (i.e., CARs) over days �1 and 0. **Table 4** reports the results for both the NYSE (Panel A) and the NASDAQ (Panel B). Cumulative ΔSKEW represents the contemporaneous cumulative changes in SKEW over days �1 and 0. We present further evidence that SKEW has a statistical and economic effect on stock returns around analyst recommendations. That is, we find similar return patterns for Cumulative ΔSKEW to those on separated single days (i.e., day �1 and day 0). Taking the NYSE for example, for upgrades, the significantly *positive* difference (i.e., Diff = 0.83% with *t*-statistic = 8.93) indicates that the CAR (with a value of 2.53%) is stronger when the cumulative change in SKEW is *positive* (i.e., Cumulative ΔSKEW>0) than that (with a value of 1.70%) when the cumulative change in SKEW is *negative* (i.e., Cumulative ΔSKEW<0). For downgrades, the significantly *positive* difference (i.e., Diff = 0.57% with *t*-statistic = 4.89) also indicates that the CAR (with a value of �1.87%) is stronger when the cumulative change in SKEW is *positive* (i.e., Cumulative ΔSKEW> 0) than that (with a value of �2.45%) when the cumulative change in SKEW is *negative* (i.e., Cumulative ΔSKEW<0).

Next, we find that the corresponding magnitudes of the CARs are larger for the NASDAQ than the NYSE. In summary, these results provide further evidence supporting the hypothesis that abnormal returns around analyst recommendation revisions are closely correlated with contemporaneous changes in SKEW.

### *3.1.3 Additional tests*

To further validate the event results obtained in Section 3.1, we apply a simple regression model (i.e., univariate model) to test whether SKEW could act as one measure of investors' greed or fear. We write the regression model as [7]:

$$AR\_{i,t} = a + \beta \Delta \text{SKEW}\_t + \varepsilon\_{i,t},\tag{3}$$

In the regression analysis, we take day 0 for example and present the results in **Table 5**. From Panel A, we observe that the regression coefficient on the changes in SKEW (i.e., ΔSKEW) is positive and significant, with a value of 0.11 (*t*-statistic = 9.81) for upgrades and 0.09 (*t*-statistic = 5.85) for downgrades. These results suggest that an

#### **Table 4**.

*The effect of changes in SKEW on the cumulative abnormal returns (CARs) for the NYSE (Panel A) and the NASDAQ (Panel B).*

#### **Table 5.**

*Regression results for the abnormal returns (ARs) and the changes in SKEW (ΔSKEW) on recommendation revision days.*

increase in SKEW is associated with an increase in abnormal returns regardless of recommendation upgrades or downgrades. Consistent with the event results presented in Sections 3.1.1 and 3.1.2, we argue that SKEW should be a proper measure of investors' greed.

We also examine the effect of positive and negative changes in SKEW on stock abnormal returns by separatingΔSKEW into the positive part (i.e., ΔSKEWþ) and the negative part (i.e., ΔSKEW�). We estimate the following regression:

$$AR\_{i,t} = a + \beta\_1 \Delta \text{SKEW}\_t^+ + \beta\_2 \Delta \text{SKEW}\_t^- + \varepsilon\_t,\tag{4}$$

where ΔSKEWþ and ΔSKEWþ are defined as:

ΔSKEWþ *t* = ΔSKEW*t*, if ΔSKEW*t* > 0; otherwise ΔSKEWþ *t* = 0, and

ΔSKEW� *t* = ΔSKEW*t*, if ΔSKEW*t* < 0; otherwise ΔSKEW� *t* = 0.

Panel B in **Table 5** reports the results. Again, regardless of upgrades or downgrades, we observe positive and significant estimated coefficients on both ΔSKEWþ and ΔSKEW�. We also use Eqs. (3) and (4) to test the effect of changes in SKEW on the day prior to recommendation revisions (i.e., day �1), and obtain qualitatively similar results.10 In summary, these results provide further evidence that SKEW could be considered as an investors' greed indicator.


**7. Therapeutic potential for targeting ERR**α **in NAFLD treatment**

Fatty liver and associated diseases including ASH/NASH are comorbid diseases with diabetes, particularly type 2 diabetes (T2D) which accounts for 95% of all diabetes cases [104]. Fatty liver disease occurs in 80% of T2D patients [87, 90]. While simple fatty liver is readily reversible, NASH can progress to more morbid forms of liver pathologies including fibrosis/cirrhosis and even liver cancer. Currently, there is no therapy besides caloric restriction for the treatment of fatty liver disease [105]. A potential therapy is under development in clinical trials that activates FXR, a bile acid receptor. In a completed clinical trial (NCT01265498), FXR ligand obeticholic acid treatment led to NAFLD resolution in 21% of the subjects vs. 13% in placebo treated subjects after 72 weeks of treatment. While these clinical trials are underway, studies exploring molecules that play roles in liver lipid dysfunction have the potential to discover novel therapies. In mouse models, caloric restriction is capable of curing fatty liver disease [105].

The function of ERRs as master regulators for metabolism made them interesting targets for the treatment of T2D, as activating ERRs has the potential to improve overall mitochondrial respiratory function and suppress hepatic glucose output. Earlier studies, tethered with the elucidation of LBD structures, have focused on developing agonists or antagonists. These studies show that ERRs are constitutively active and identified several estrogen-related hydrophilic molecules that can bind and block the establishment of active conformations. These molecules such as the diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT) have been described as antagonists for ERRs, where DES binds to all three ERRs, and 4-OHT does not bind to ERRα [106, 107].

Due to the lack of endogenous ligand, XCT-790 was identified as a potent inverse agonist for ERRα and is used in many studies as inhibitors of ERR functions [108]. Using the NIH compound library in a couple high throughput screening studies, it was found that several pesticides contain ERRβ genes promoting activities, and these compounds may act as potential "ligands" for ERRs [109, 110]. The activity of ERRα has been reported to be antagonized by the organochlorine pesticides chlordane and toxaphene [111, 112]. Screening approaches also identified small molecules with the ability to alter ERR transcriptional activity. An example is the discovery of compound 11 that potently inhibits ERRα's transcriptional activity by preventing binding of ERRα to PGC-1α and suppressing the proliferation of different cancer cell lines [113].

Troglitazone also interferes with binding of ERRα and γ to PGC-1α. Troglitazone was recently discovered to be an inverse agonist for ERRα and γ [114]. Troglitazone is an FDA approved therapy for T2D. In hepatocytes, troglitazone inhibits oleic acid induced liquid buildup. Thus, activity of troglitazone towards ERRα likely plays a role in this effect. In addition, pharmacological inhibition of either ERRα or ERRγ has led to improved insulin response and better tolerance to diet induced metabolic changes [79, 80].

Beyond identification of ligand-like molecules that can serve as either antagonist or agonist for ERR, effort was put into blocking its binding to DNA. Pyrrole-imidazole (Py-Im) polyamides are a class of synthetic ligands for the sequence-specific recognition in double-helical DNA minor groove [84]. Polyamides targeted at the ERRE (ERR-PA) were designed to block binding of ERR onto the promoters of genes regulated by it. An *in vitro* study showed that there was over a 50% reduction in basal and maximal respiration with 0.2 μM ERR-PA treatment and around 70% reduction with 1 μM, emphasizing the dose dependency of the polyamide molecule [84]. ERR-PA was highly effective at reducing liver steatosis in multiple NAFLD models, including one with deletion of *Pten*, the negative regulator of


**3.3.5 Biodegradation of fibres**

The degradable properties of both coated and uncoated OPEFB fibre were monitored through aging in two different soils and in contact with moisture and fungus for about 3 months. Figure 15 shows the effect of fungus in the degradation of the fibre, the black part of the fibre was affected by the fungus. Most part of the uncoated fibres was influenced by fungus and the fungus spread over the surface of the fibres. In comparison the coated fibre was less affected by fungus and it only decayed at the end parts of fibres. Otherwise, the colour of the fibres was shown as the water sorption in the uncoated fibres. Water is the important factor for the growth of the fungus that increased the biodegradation of the fibres.

Fig. 15. Fungus biodegradation of the fibres after 3 month a) Sheet of OPEFB fibre, b) Sheet of coated OPEFB fibre

The degradable properties of both coated and uncoated OPEFB fibre were monitored through aging in two different soils and in contact with moisture and fungus for about 3 months. Figure 15 shows the effect of fungus in the degradation of the fibre, the black part of the fibre was affected by the fungus. Most part of the uncoated fibres was influenced by fungus and the fungus spread over the surface of the fibres. In comparison the coated fibre was less affected by fungus and it only decayed at the end parts of fibres. Otherwise, the colour of the fibres was shown as the water sorption in the uncoated fibres. Water is the important factor for the growth of the fungus that increased the biodegradation of the

Fig. 15. Fungus biodegradation of the fibres after 3 month a) Sheet of OPEFB fibre, b) Sheet

Fig. 14. Tensile test result of MTS fabrics and coated OPEFB sheets

Uncoated fibre Coated fibre

a b

**3.3.5 Biodegradation of fibres**

fibres.

of coated OPEFB fibre

The fibres were also placed in to the silty sand and peat soil to estimate the weight loss of the fibres in soil. The discrete fibre and fibre sheet were weighed before test. The fibres also were coated with ABS solution for 24 hours and all the specimens were placed on soils for about 3 months.

Figure 16 shows the coated and uncoated OPEFB fibre after aging in the silty sand soil. The fibre sheets decayed after 3 months and it is shown that the uncoated fibre had the separate structure due to the biodegradation of the fibre.The coating was protected the fibres from biodegradation; the shape of the coating fibres was kept.

Fig. 16. a) before decay b) decayed ABS coated c) decayed uncoated fibre sheet in the silty sand soil after 3 months

The same results are shown in Figure 17 for the fibres in organic clay soil. The uncoated sheet fibre in organic clay separated from each other and did not have the textile structure. The uncoated fibre lost its weight due to biodegradation in the soil.

Fig. 17. a) before decay b) decayed ABS coated c) decayed uncoated fibre sheet in the organic clay soil after 3 months

The results of these losses are graphically plotted in Figure 18. Loss of weight of the discrete fibres was higher than the fibre sheet due to their larger contact surface with the soils and environment factors.

The result shows that in all condition coating decreases the biodegradation of the fibres both in discrete fibre and fibre sheet. The decay of the fibres in three conditions had

Application of Thermoplastics in Protection of Natural Fibres 345

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**5. Acknowledgment**

closely in this project.

www.astm.org.

**6. References**

approximately the same result. The weight loss result indicate the influence of coating on protecting OPEFB fibres from biodegradation, around 50% decrease on weight loss were estimated from tests after three months.

Fig. 18. Biodegradation of the fibres inside Silty sand, Organic soil and be in contact with fungus after 3 months