**2.2 Oil productivity**

Oleaginous microalgae (**Table 1**) are a promising source for the production of renewable biofuels because of their efficient photosynthetic capabilities. Moreover, microalgal growth requires less area in comparison to the terrestrial plants, and they are capable to channel the majority of the acquired energy into cell division, which increases the biomass yield [23]. Microalgae can be subdivided into four different groups depending on the carbon source (inorganic and organic), namely, autotrophic, mixotrophic, heterotrophic, and photoheterotrophic [24].

The synthesis of triacylglycerol in microalgae takes place mostly in the chloroplast and endoplasmic reticulum through multiple enzymatic reactions [25]. Fatty acid synthesis in the chloroplast, assembly of glycerolipids in endoplasmic reticulum, and accumulation of TAGs into the oil bodies are the three major steps involved in the accumulation of lipids in the microalgae [26]. It has been proven that facilitate the synthesis of high amounts of lipids is influenced by different stress conditions

#### **Table 1.**

*Some prominent oleaginous microalgae.*

such as physical, chemical, or environmental, individually or in combination [27]. Under the aforementioned stress conditions, microalgae can switch their metabolism towards the synthesis of neutral lipids in the form of TAGs, which serves as a form of carbon and energy storage [28–30]. Microalgae employ the de novo pathway to synthesize lipids. It starts in the chloroplast by CO2 fixation into sugars, which are further metabolized to acetyl-CoA, which acts as a precursor of fatty acid synthesis [31].

Marine microalgae have a higher content of PUFA in comparison to the freshwater species because they need to produce more unsaturated fatty acids to survive in the salty marine environment [32]. Thus, cultivation of marine microalgae can render higher economic interest to the cultivars. According to reported literature, the marine oleaginous diatom Fistulifera solaris when cultivated in photoautotrophic conditions can produce 135.7 mg/(L·day) EPA. On the otherhand, the heterotrophic growth of the marine diatom Nitzschia laevis, when supplemented with glucose, resulted in EPA production of 174.6 g/(L·day) [33].

## **2.3 Extraction of oil**

During lipid extraction, the microalgal biomass is exposed to an organic eluting solvent which extracts the lipids out of the cell cytoplasm. A lipid extraction technology for microalgal oil production needs to be highly specific towards the lipids in order to avoid the co-extraction of non-lipid contaminants, viz. protein and carbohydrates. The lipid extraction technology should be more selective towards acylglycerols than other lipid fractions as they are not readily convertible to biodiesel, such as polar lipids and non-acylglycerol neutral lipids (free fatty acids, hydrocarbons, sterols, ketones, carotenes, and chlorophylls) [34]. Moreover, the technology should be efficient (both time and energy saving), non-reactive with the lipids, relatively cheap (capital cost and operating cost), and safe (environmentally and mechanically) [35]. Dewatering of the microalgal biomass beyond a paste consistency (200 g dried microalgal biomass/L culture) is energy consuming, so, it will be economically friendly if the selected lipid extraction technology is effective for the wet feedstock, i.e. concentrate or disrupted concentrate with concentrations between 10 and 200 g dried microalgal biomass/L culture [36].

### *2.3.1 Solvent extraction*

The principles underlying solvent extraction of microalgal lipids are based on the concept of chemistry 'like dissolving like'. The long hydrophobic fatty acid chains interact with neutral lipids through weak van der Waals forces, thus forms globules in the cytoplasm [37]. The mechanism for organic solvent extraction is depicted in **Figure 1**. When a microalgal cell is exposed to a non-polar organic solvent, such as hexane or chloroform, the organic solvent penetrates through the cell membrane into the cytoplasm and interacts with the neutral lipids trough van der Waals forces to form an organic solvent-lipids complex. This organic solvent–lipids complex, driven by a concentration gradient, diffuses across the cell membrane. The neutral lipids are thus extracted out of the cells and remain dissolved in the non-polar organic solvent. However, some neutral lipids remain as a complex with polar lipids in the cytoplasm. The complex is strongly linked via hydrogen bonds to the proteins in the cell membrane. The van der Waals interactions formed between non-polar organic solvent and

#### **Figure 1.**

*Schematic representation depicting the solvent extraction of oil from oleaginous microalgae. 1. Penetration of the solvent inside the cell cytoplasm. 2.Solvent interaction with the lipids. 3. Formation of lipid-solvent complex. 4. Diffusion of the complex through the cell membrane. 5. Diffusion of the complex in the surrounding bulk organic solvent.*

neutral lipids are inadequate to disrupt these membrane-based lipid–protein associations. On the other hand, polar organic solvent (viz. methanol or isopropanol) is able to disrupt the lipid–protein associations by forming hydrogen bonds with the polar lipids in the complex [38].

**Figure 2** depicts the extraction steps generally, undertaken for laboratory-scale production of microalgal oil and finally trans-esterified to biodiesel using an organic solvent mixture.


**3. Forms of inflammation or damage**

**Neutrophilic inflammation** can be induced by cigarette smoke, fine dust, bacteria, and viruses, resulting in the release of neutrophilic mediators, including IL-8, which signal through its receptor on neutrophils. Macrophages are activated as well, and attract Th17 cells to release IL-17, which stimulates the release of IL-6 and IL-8 from epithelial cells. Neutrophils are maintained in the airway by TNF-α and GM-CSF (granulocyte-macrophage colony-stimulating factor) and release neutrophil elastase. Neutrophils also generate oxidative stress, which further activates inflammation and induces corticosteroid resistance. The neutrophilic inflammation in COPD is unresponsive to corticosteroids, even in high doses. Other therapies directed toward neutrophilic inflammation, including antibodies against TNF-α, have been largely clinically ineffective as well. A CXCR2 (chemokine receptor-2) antagonist, which blocks the chemotactic effect of IL-8 and related chemokines, is at least able to reduce the neutrophils in the sputum of COPD patients but has no clinical benefit on lung function, symptoms, or exacerbations. Neutrophil elastase is a major proteinase in primary granules in neutrophils that participates in the microbicidal activity. It induces airway remodeling with increased mucin secretion and impaired ciliary motility, it interrupts epithelial repair by promoting cellular apoptosis, and it activates inflammation by increasing cytokine expression [1, 4–8, 14].

**Eosinophilic inflammation** is well-known in patients with bronchial asthma. Some people have clinical features from both diseases, bronchial asthma and COPD. In case of such overlap, airway epithelial cells can release the upstream cytokines TSLP and IL-33 in response to cigarette smoke and virus infection, which recruit Th2 and type 2 innate lymphoid cells, which secrete IL-5, resulting in eosinophilic inflammation. Eosinophils may be attracted into the lungs by CCL5 (chemokine (C-C motif) ligand) and maintained in the lungs by IL-5 and GM-CSF. Patients with so-called eosinophilic COPD may have a better response to corticosteroid therapy and more reversibility to bronchodilators, and these patients show an increase in sputum eosinophils and an increased FeNO, which are characteristic features of asthma [1, 5–7, 14].

#### *COPD and Inflammation DOI: http://dx.doi.org/10.5772/intechopen.107863*

**Protease-antiprotease imbalance** means that inhaled pollutants, such as cigarette smoke and fine dust, activate immune cells like macrophages and T-helper cells, which release inflammatory messengers. These signals cause neutrophilic granulocytes to migrate into the bronchial mucosa. Together with the macrophages, they release cell-damaging proteases. And at the same time, protective antiprotease is disabled. According to current knowledge, this imbalance of proteases and antiproteases favors the formation of pulmonary emphysema. Various proteases produced by inflammatory cells and epithelial cells are elevated in many people with COPD [3, 19].

**Oxidant-antioxidant imbalance** leads to inflammatory reactions due to oxidative stress. As a result, pulmonary cells are damaged, and inflammation is further promoted. This accelerates the development of COPD or emphysema. Oxidative stress also increases mucus production, the formation of proteases, and the migration of neutrophilic granulocytes into the bronchial mucosa. An oxidant-antioxidant imbalance also occurs in other lung diseases, such as pulmonary fibrosis or bronchial asthma [3, 5, 15, 19].

**Apoptosis** is critical for the maintenance of normal tissue homeostasis and is in equilibrium with proliferation and differentiation. There is increasing evidence that disturbance of the balance between apoptosis and proliferation in lung tissue contributes to the pathogenesis of COPD. Several experimental studies in animal models of COPD provide more insight into the association between cigarette smoking, apoptosis, and the development of emphysema. Epithelial cells in the small airways express TGF-β, which then induces local fibrosis. VEGF (vascular endothelial growth factor) appears to be necessary to maintain alveolar cell integrity, and blockade of VEGF receptors in rats induces apoptosis of alveolar cells and an emphysema-like pathology [19].


**8. Acknowledgements**

The research was funded by the Austrian Science Fund (FWF): TRP140-N23-2010. The authors would also like to thank Dr. Mark Blackburn†(Fitzwilliam Museum, UK) for providing images for 2D image analysis and Mario Schlapke (Thüringisches Landesamt für Denkmalpflege und Archäologie, Weimar, Germany) for providing the coins for 3D scanning.

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**8**

*Tunisia*

**Non-Rigid Objects Recognition: Automatic**

Mehrez Abdellaoui1, Ali Douik1 and Kamel Besbes2

*1National Engineering School of Monastir,*

*2Faculty of Sciences of Monastir,*

**Human Action Recognition in Video Sequences**

Non-rigid objects recognition is an important problem in video analysis and understanding. It is nevertheless a challenging task to achieve due to the properties carried out by the nonrigid objects, and is more complicated by camera motion as well as background variation. Human body recognition in video sequences is the best application of the non-rigid objects recognition due to the large capacities of the human body in doing actions and poses. These difficulties prohibit practical attempts toward conceiving a robust global model for each action class. Human body recognition is highly interesting for a variety of applications: detecting relevant activities in surveillance video, summarizing and indexing video sequences. It relies, however, on the interpretation of the body movements and classifies

A considerable amount of previous work has addressed the question of human action categorization and motion analysis. One line of work is based on the computation of correlation between volumes of video data (Efros et al., 2003). Another popular approach is to track body parts at first and then uses the obtained motion trajectories to perform action recognition (Ramanan & Forsyth, 2004). The robustness of the approach is highly dependent on the tracking system. Alternatively, researchers have considered the analysis of human actions by looking at video sequences as space-time intensity volumes (Bobick & Davis, 2001). Some researchers have also explored unsupervised methods for motion analysis such as hierarchical dynamic Bayesian network model (Hoey, 2001; Zhong et al., 2004). Another approach uses a video representation based on spatiotemporal interest points (STIPs). In spite of the existence of a fairly large variety of methods to extract interest points (IPs) from static images Harris corner detector (Harris & Stephens, 1988), Scale invariant feature transform (Lowe, 1999), Salient regions (Kadir & Brady, 2003) …, less work has been done on STIPs detection in videos. In 2005, Laptev (Laptev, 2005) present a STIPs detector based on the idea of the Harris IPs operators. They detect local structures in space-time where the image values have significant local variations in space and time dimension. IPs extracted with such methods had been used as features for human action classification. These points are particularly interesting because they focus the initial information contained in any image in a few specific points. The integration of the time component can perform filtering on the

IP and keep only those who also have a temporal discontinuity.

**1. Introduction**

them in different events.


**3.4 Phenotypes and response to corticosteroids with bronchodilators**

COPD with peripheral blood eosinophilia may have a particularly favorable response to inhaled corticosteroid (ICS)/long-acting β2-adrenergic agonist (LABA) therapy, perhaps because of the inflammation profiling that responds well to corticosteroids. However, prospective studies are required to evaluate the role of blood

*New Perspectives in Pharmacological Therapy for COPD: Phenotype Classification and… DOI: http://dx.doi.org/10.5772/intechopen.106949*

#### **Table 1.**

*Effects of airway eosinophilia and airway hyperresponsiveness as treatable traits on the management of COPD. Illustrated based on refs. [12, 55].*

eosinophils as a biomarker of inhaled therapy response in COPD [110]. Eosinophil counts in the peripheral blood are not always associated with those in the airways. For this reason, patients with COPD who have sputum eosinophilia were enrolled to examine involvement of eosinophilic inflammation in response to corticosteroids and LABA [12, 55, 105, 106]. Inhaled indacaterol (a LABA) caused a greater increase in FEV1 [107] and IC [108] in these patients than those shown in other previous reports. Addition of inhaled ciclesonide (a corticosteroid) to indacaterol caused much higher increases in FEV1 and IC, and values of CAT score and frequency of on demand use of procaterol (a short-acting β2-adrenergic agonist) were markedly reduced (**Table 1**) [12]. These results indicate that not only ICS but also LABA is effective in improving lung function, symptoms, and quality of life in COPD with airway eosinophilia [109]. Since airway inflammation induced by neutrophils and oxidative stress may be the main pathogenesis of COPD, ICS is generally considered to be not so beneficial to this disease. However, ICS/LABA is beneficial to airway eosinophilic inflammation in COPD, similar to that in asthma. Indacaterol, a strong partial β2-adrenergic agonist, is probably effective for COPD because of higher values of its intrinsic efficacy close to a full agonist [111–115]. In these patients with COPD who have sputum eosinophilia, there was no deference in response to indacaterol for FEV1 and IC between these subjects with and without AHR; in contrast, addition to ciclesonide caused greater increases in FEV1 and IC in these subjects without AHR than in these subjects with AHR [12]. However, mechanisms underlining this reduced responsiveness to corticosteroids in COPD with AHR have not been investigated in detail. Therefore, airway eosinophilia and AHR affect symptom suitability (exacerbations) and responsiveness to corticosteroids and β2-adrenergic agonists in COPD (**Table 1**, **Figure 2**).


Preface

The development of thermal appliances and equipment that make use of non-conventional energy sources is currently receiving a significant amount of focus within the realm of thermal energy research. The ability to store thermal energy is a fundamental prerequisite for such applications. When it comes to thermal energy storage (TES), the use of phase change material (PCM) for latent heat storage is more desirable than sensible heat storage. This is due to its potential to store a large amount of energy for a given volume at a reduced temperature swing. Other reasons include its compactness, uniform energy storage, and delivery, and temperature swings.

This book provides readers with a comprehensive overview of PCMs and their current applications. It begins with a discussion of the basic concept of phase-change energy storage and the categorization of phase-change energy materials. Next, the book explores the essential features that influence the use of PCMs, followed by an in-depth discussion of the techniques for the thermal analysis of PCM. Other topics addressed include recent uses of PCMs in electronic cooling, battery thermal management, and textile and solar energy conversion.

Section 1 provides a brief introduction to PCMs and the different techniques used to enhance their thermal performance. Section 2 discusses the design of a heat exchanger that is based on latent heat as well as methods for conducting thermal analysis of PCMs. Section 3 discusses the application of PCMs in battery thermal management. Section 4 explores the use of PCMs to cool electronic devices and Section 5 discusses the applications of PCMs in the textile industry. Section 6 focuses on other recent applications of PCMs.

I would like to express my thanks to IntechOpen for the care and attention it has shown in publishing this book on schedule.

> **Manish Rathod** Department of Mechanical Engineering, Sardar Vallabhbhai National Institute of Technology, Surat, India

**1**

Section 1

Introduction to Phase Change

Material

Section 1


**Monoclonal Antibodies and Their Functional Fragments in Research, Diagnosis and Therapy 2.0**

Editors

**Menotti Ruvo Annamaria Sandomenico**

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**2.3.** *T. brucei* **choline kinase activity assay**

High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 µl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 µl from each well of the working plates was added to 110 µl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2. 3 µg of purified *Tb*CK was added to each well in 30 µl of the same buffer and the plates were mixed and incubated for 5 min at room temperature. A fur‐ ther 30 µl of buffer containing PEP (1 mM final), ATP (0.5 mM final), NADH (0.5 mM final) and pyruvate kinase and lactate dehydrogenase(PK/LDH) (5 units/ml final) was added, and the reaction was started by addition of 30 µl choline (0.5 mM final) to rows 1-11, 30 µl buffer alone was added to row 12 (negative control) and this was used as an intra-plate control (background rate) in conjunction with row 1 (maximal rate). Following mixing the change in absorbance at 341 nm was monitored for 10 min at room temperature. For testing inhibition of the coupling enzymes (PK/LDH), standard buffer conditions were used but the assay con‐ tained 1 mM PEP, 0.1 mM ADP and 0.5 mM NADH. The PK/LDH was titrated to give a change in absorbance of approximately 0.05 absorbance units/min in the absence of inhibi‐ tor.

### **2.4. Differential scanning fluorimetry with** *Tb***CK**

of a 96-well plate, at 10 mM in 5% DMSO, allowing the two outside columns for positive

Large-scale recombinant expression and purification of *Tb*CK was conducted using the con‐ struct pET-15bTEV-*Tb*CK in BL21 Rosetta (DE3) cells as described previously [23], except the cells were grown in tryptone phosphate broth [37], harvested by centrifugation at 3500 g for 20 min at 4°C and affinity purified with either a HisTrap™ FF crude column (enzyme

Briefly, pelleted cells were suspended in buffer A (50 mMTris/HCl, pH 8.0, 300 mMNaCl and 10 mM imidazole) and lysed in the presence of DNase I by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTrap™ FF crude column column (GE Healthcare) pre-loaded with Ni2+. Unbound proteins were re‐ moved by washing the column with 15 column volumes of buffer A containing 32.5 mM imidazole and *Tb*CK was eluted with 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mMTris/HCl, pH 8.0, 300 mMNaCl, glycerol

Alternatively, pelleted cells were suspended in 50 mMTris/HCl, pH 8.0, 300 mMNaCl and 5 mM imidazole and lysed by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTALON Cartridge (Clontech).Unbound proteins were removed by washing the column with 10 column volumes of loading buffer, *Tb*CK was eluted with 15 mM imidazole and a final clearing wash of 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mM HEPES pH 8.0, 300

Typical yields were > 10 mg per litre of bacterial culture,*Tb*CK was stable and freeze thaw‐

High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 µl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 µl from each well of the working plates was added to 110 µl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2. 3 µg of purified *Tb*CK was added to each well in 30 µl of the same buffer and the plates were mixed and incubated for 5 min at room temperature. A fur‐ ther 30 µl of buffer containing PEP (1 mM final), ATP (0.5 mM final), NADH (0.5 mM final) and pyruvate kinase and lactate dehydrogenase(PK/LDH) (5 units/ml final) was added, and the reaction was started by addition of 30 µl choline (0.5 mM final) to rows 1-11, 30 µl buffer alone was added to row 12 (negative control) and this was used as an intra-plate control (background rate) in conjunction with row 1 (maximal rate). Following mixing the change in

and negative controls.

416 Drug Discovery

(15% w/v) and stored at -80°C.

**2.2. Recombinant expression and purification of** *Tb***CK**

mMNaCl and 15% glycerol prior to storage at -80°C.

ing did not lead to any significant loss of activity.

**2.3.** *T. brucei* **choline kinase activity assay**

activity assay) or a HisTALON Cartridge (thermal shift analysis).

Differential scanning fluorimetry was set up in 96 well PCR plates using a reaction volume of 100 µL. Samples contained 2.1 µM *Tb*CK, 6 mM MgCl2, 50 mM HEPES pH 8.0, 80 mMNaCl, 5.25% glycerol (v/v) and 1.4 x Sypro Orange (Invitrogen), Maybridge Ro3 com‐ pounds were screened at 1 mM concentration with a final DMSO concentration of 0.5% (v/ v).Two controls with eight repetitions per plate were used for the thermal shift experiments: 0.5% DMSO; 0.5 mM ATP, 0.5% DMSO.

Differential fluorimetric scans were performed in a realtime PCR machine (Stratagene Mx3005P with software MxPro v 4.01) using a temperature scan from 25°C to 95°C at 0.5°C min-1.Data were then exported to Excel for analysis using "DSF analysis" modified from the template provided by Niesen et al. [38].Tm values were calculated by non-linear regression, fitting the Boltzmann equation to the denaturation curves using GraFit. *Tb*CK Tm, in the presence of 6 mM MgCl2 and 0.5% DMSO, 41.21 ± 0.03°C (n > 60), Tm for *Tb*CK and 0.5 mM ATP = 44.46 ± 0.05°C (n > 60).


**1. Introduction**

Nowadays, with the advent of smart energy environments, the demand for cyber-physical systems [1] and the growth in switch-mode converter applications [2–6], electromagnetic compatibility (EMC) issues are becoming more and more significant [7]. Among many methods of improving EMC of converters, we may use improved control techniques. In addition to the primary function of controlling energy conversion, such a control technique can reduce the level of conducted electromagnetic interference (EMI) by spreading the harmonics on a broader frequency range [6,8]. A Field-Programmable Gate Array (FPGA) may be used for the cyber-physical implementation of such controls. It should be added that FPGA may be much more flexible and may cover more applications than other commonly used control devices such as microcontrollers (*μC*) or digital signal processors (DSP). In particular, FPGAs allow for the building of hardware circuits of the modified Pulse Width Modulators (PWM), unlike *μC* or DSP's, where the built-in PWM circuit cannot be changed.

In a typical PWM circuit, the user may set the fundamental switching frequency (*f sw*) and the duty cycle (*D*). The parameter *D* controls the output voltage of the converters and thus affects the energy conversion process. The *fsw* of the PWM signal is practically irrelevant to the voltage transfer function of the converter, and of the primary energy conversion. However, it affects the losses in the switch-mode converter and the parameters of the reactance elements. Conducted EMI generated by the converter are grouped around the *fsw* and its harmonics [8,9]. Any modification of *fsw* leads to changes in EMI emission.

Traditionally, pseudo-random modulators (RanM) can be used as a switch control strategy, which can reduce the level of EMI [8,10]. In such type of modulation, the frequency of the PWM signal is randomly changed in the selected range. As a consequence, disturbance energy is distributed more evenly across a wider spectrum. The development of a random modulation requires a combination of changes in the frequency *f sw* (or the period) of the PWM signal with a pseudo-random number generator. Therefore, the range of pseudo-random numbers must be adapted to the specific hardware platform. In addition, the probability density function (PDF) of such a pseudo-random stream should be analysed in terms of the emission of conducted disturbances.

The design of RanM in FPGA may be challenging. The modern graphical programming tools such as LabVIEW may facilitate the design process. Nevertheless, in LabVIEW, there is still some inconvenience associated with fixed-point operations, and with the lack of some arithmetic operations, e.g., divide. Therefore, this manuscript demonstrates how to provide an FPGA-based control system for a DC/DC converter that limits the level of conducted EMI. The presented algorithms, based on LabVIEW engineering software, do not change the essential functions or parameters of the converter. During the design of algorithms, we take into account the PDF of frequency changes, and we propose a method of shaping the PDF in FPGA without arithmetic division and using only fixed-point operations. For the presented algorithms, we perform the EMI evaluation in an experimental system. The CISPR-A frequency band was considered as the primary frequency range for tests.


*3.1. Assessment of SUVmax, MTV, and TLG on 18F-FDG Uptake*

In all 85 patients, the median values of SUVmax, MTV, and TLG on 18F-FDG and TB were 6.0 (range, 3.1–21.0), 17.8 cm3 (range, 1.1–379 cm3), 75.4 gcm3/mL (range, 3.9–2550 g·cm3/mL), and 65 cm (range, 6.6–230.6 cm), respectively. According to histological types, the median values of SUVmax, MTV, and TLG on 18F-FDG and TB in adenocarcinoma were 6.7 (range, 3.1–21.3), 11.3 cm3 (range, 1.1–276 cm3), 58.3 g·cm3/mL (range, 3.9–1398 g·cm3/mL), and 53 cm (range, 6.6–230.6 cm), respectively, and those in non-adenocarcinoma displayed 8.4 (range, 3.2–20.4) 24.8 cm3 (range, 1.1–379 cm3), 105.6 g·cm3/mL (range, 4.0–2550 g·cm3/mL), and 75 cm (range, 18.2–228.3 cm), respectively. The difference of each parameter for SUVmax, MTV, and TLG on 18F-FDG and TB was not significant between adenocarcinoma and non- adenocarcinoma. The SUVmax correlated significantly with MTV (*r* = 0.49, *p* < 0.01), TLG (*r* = 0.56, *p* < 0.01), and TB (*r* = 0.33, *p* < 0.01). Figure 1 is a representative PET image showing the assessment of SUVmax, MTV, and TLG on 18F-FDG.

The discriminative value of various SUVmax, MTV, and TLG cutoffs for 18F-FDG uptake were explored in the context of OS and PFS (Figure 2) [13]. For prognosis in OS and PFS analyses, the most discriminative cutoffs based on log-rank test for SUVmax, MTV, and TLG were 6.0, 5.0, and 20, respectively. The TLG cutoff of 20 was significant in the OS analysis, but not in the PFS analysis, although it was the most favorable TLG cutoff. The SUVmax cutoff of 6 was not significant in either OS or PFS analysis, but still seemed better considering the results of the log-rank test. The 12 cm cutoff for TB was based on a previous study [13].

**Figure 1.** Imaging from a positron emission tomography (PET) scan of non-small cell lung cancer (NSCLC), indicating the measurement of SUVmax, metabolic tumor volume (MTV) and total lesion glycolysis (TLG) on 2-deoxy-2-[18F]fluoro-d-glucose (18F-FDG).


**1. Introduction**

RNA interference (RNAi) is a powerful gene-silencing process that holds great promise in the field of cancer therapy. Small double-stranded RNAs, i.e., synthetic small interfering RNAs (siRNAs) suppress the expression of a target gene by triggering specific degradation of the complementary mRNA sequence [1], and therefore siRNA therapeutics have become an increasingly important strategy for anticancer therapy [2]. For siRNA therapy against tumors, siRNAs are designed for targeting mRNAs transcribed from a tumor-causing gene, and siRNAs are introduced into the cytoplasm of tumor cells to cause cleavage of the target mRNA. For example, polo-like kinase 1 (PLK1) is a key regulator for cell mitosis, and its expression is elevated in many types of human tumors [3,4]. The inhibition of PLK1 expression by PLK1 siRNA can lead to death of tumor cells with multiple stages of mitosis [5], and therefore PLK1 is expected to be one of potential targets for siRNA therapy against tumors. However, the success of siRNA therapy relies on the development of safe and efficacious delivery systems that can introduce siRNAs into target tumor cells [2,6,7].

For effective transfection of siRNAs into tumor cells, siRNA carriers such as cationic liposomes are currently the most widely validated means [8]. Many different cationic lipids have been synthesized for lipid-based gene delivery [9] and shown activity in delivering siRNA into cells [10]. For siRNA delivery by cationic liposomes, cationic cholesterol derivatives have often been used [11]. Recently, we reported that cationic liposomes composed of cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol), *N*-(2-(2-hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol), or cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) could efficiently suppress the expression of target genes by siRNA in cells [12,13]. Regarding the liposomal formulation, we previously reported that cationic liposomes composed of OH-Chol/DOPE and OH-C-Chol/DOPE exhibited high gene silencing efficacies compared with cationic nanoparticles composed of OH-Chol/Tween80 and OH-C-Chol/Tween80, respectively [14]. DOPE is thought to improve transfection efficiency by destabilizing the endosomal membrane [15,16], thereby facilitating the release of siRNAs into the cytoplasm. Therefore, a combination of cationic cholesterol derivative with DOPE in cationic liposomes might be a suitable formulation in siRNA delivery. However, selective delivery of siRNAs into tumor cells by cationic liposomes must be achieved for clinical applications.

Folate receptors (FRs) have been found to be overexpressed in a wide range of tumors, including ovary, uterus, lung, kidney, breast, colon, prostate, and brain cancers [17–19]. The following four isoforms of FR have been identified: folate receptor alpha (FR-α), beta (FR-β), delta (FR-Δ), and gamma (FR-γ); and FR-α and FR-β are attached to the cell by a GPI-anchor [18]. Among these FR isoforms, FR-α is the most widely studied as a biomarker for tumors, because a few normal tissues have been found to express FR-α, although most express the protein at much lower levels than are detected in FR-α-positive carcinoma. Therefore, FR-mediated tumor targeting has emerged as an attractive method of active targeting of siRNAs into tumor cells by cationic liposomes. When folic acid (FA) or its conjugates bind to FRs, they are taken up into the cells via receptor-mediated endocytosis. Therefore, FA-polyethylene glycol (PEG)-modification has been employed in cationic liposomes to facilitate the uptake of siRNA lipoplexes into tumor cells. Generally, PEGylated cationic liposomes can significantly reduce nonspecific gene transfer. However, conjugation of folate to the PEG chain can restore the cellular association with FR-positive tumors. To the best of our knowledge, there have been no reports about the effect of cationic lipids in FA-PEG-modified cationic liposomes on FR-targeting, although several studies have investigated the application of FA-PEG-modified cationic liposomes for siRNA delivery into FR-expressing cells [20–23]. In this study, to examine the effect of cationic lipid type in FA-PEG-modified cationic liposomes on gene-silencing effects, we selected three kinds of cationic cholesterol derivatives, HAPC-Chol, OH-Chol, and OH-C-Chol, and prepared three types of FA-PEG-modified cationic liposomes composed of cationic cholesterol derivatives and DOPE for the evaluation of gene-silencing effects. Here, we found that in FR-selective siRNA delivery, the cationic lipid type in FA-PEG-modified cationic liposomes affected an optimal amount of FA-PEG2000-DSPE in liposomal formulation, and the optimized formulation of FA-PEG-modified cationic liposomes by in vitro transfection was not necessarily correlated with formulation by in vivo transfection.

#### **2. Materials and Methods**

#### *2.1. Materials*

*N*-(2-(2-Hydroxyethylamino)ethyl)cholesteryl-3-carboxamide (OH-Chol) and cholesteryl (2-((2 hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) were synthesized as described previously [14,24,25]. Cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) was synthesized as described previously [26]. Methoxy-poly(ethyleneglycol)-distearylphosphatidylethanolamine (PEG2000- DSPE/SUNBRIGHT DSPE-020CN, and PEG5000-DSPE/SUNBRIGHT DSPE-050CN, and PEG mean molecular weight, 2000 and 5000, respectively) and 1,2-dioleoyl-*sn*-glycero-3-phosphoethanolamine

(DOPE, COATSOME ME-8181) were obtained from NOF Co. Ltd. (Tokyo, Japan). All other chemicals were of the highest grade available.

#### *2.2. Small Interfering RNAs*

The siRNAs targeting nucleotides of firefly pGL4 luciferase (Luc), enhanced green fluorescent protein (EGFP), human polo-like kinase 1 (PLK1), and non-silencing siRNA control (Cont) as a negative control were synthesized by Sigma Genosys (Tokyo, Japan). Alexa Fluor®488-labeled AllStars Negative Control siRNA (AF-siRNA) was obtained from Qiagen (Valencia, CA, USA). The siRNA sequences for Luc siRNA: sense strand: 5 -GGACGAGGACGAGCACUUCUU-3 and antisense strand: 5 -GAAGUGCUCGUCCUCGUCCUU-3. The siRNA sequences for EGFP siRNA: sense strand: 5 -GGCUACGUCCAGGAGCGCACCTT-3 and antisense strand: 5 -GGUGCGCUCC UGGACGUAGCCTT-3. The siRNA sequences of the PLK1 siRNA were as reported previously [27]. The siRNA sequences of the Cont siRNA were as reported previously [28].

## *2.3. Preparation of Cationic Liposomes and siRNA Lipoplexes*

FA-PEG-distearoylphosphatidylethanolamine (FA-PEG-DSPE) (mean MW of PEG: 2000 and 5000) was synthesized as described previously [29]. Cationic liposomes were prepared from HAPC-Chol/ DOPE (composition designated as LP-HAPC), OH-Chol/DOPE (composition designated as LP-OH), and OH-C-Chol/DOPE (composition designated as LP-OH-C), at a molar ratio of 3:2. PEG2000 and FA-PEG2000-modified cationic liposomes were incorporated at 1, 2, or 3 mol% PEG2000-DSPE and FA-PEG2000-DSPE, respectively, into the cationic liposomal formulation, as shown in Table 1. PEG5000- and FA-PEG5000-modified LP-HAPC (LP-HAPC-PEG5000 and LP-HAPC-FA-PEG5000) were incorporated with 1 mol % PEG5000-DSPE and FA-PEG5000-DSPE, respectively, into the formulation of LP-HAPC.

For the preparation of cationic liposomes by a thin-film hydration method, cationic cholesterol derivative, DOPE, and PEG-DSPE or FA-PEG-DSPE were dissolved in chloroform, and then the chloroform was evaporated under vacuum on a rotary evaporator at 60 ◦C to obtain a thin film. The thin film was hydrated with water at 60 ◦C by vortex mixing. The liposomes were sonicated in a bath-type sonicator (Bransonic® 2510J-MTH, 100W, Branson UL Trasonics Co., Danbury, CT, USA) to produce liposomes with a final size of approximately 100 nm.

In in vitro transfection, to prepare cationic liposome/siRNA complexes (siRNA lipoplexes), each cationic liposome was added to 50 pmol siRNA at a charge ratio (+:−) of 7:1 with vortex mixing for 10 s and left at room temperature for 15 min. The charge ratio (+:−) of cationic liposome:siRNA is expressed as the molar ratio of cationic lipid to siRNA phosphate.

#### *2.4. Size and* ζ*-Potential of Cationic Liposomes and siRNA Lipoplexes*

The siRNA lipoplexes were formed by addition of cationic liposomes to 5 μg Cont siRNA at a charge ratio (+:−) of 7:1 with vortex mixing for 10 s. The particle size distributions of cationic liposomes and siRNA lipoplexes were measured by the cumulant method using a light-scattering photometer (ELS-Z2, Otsuka Electronics Co., Ltd., Osaka, Japan) at 25 ◦C after diluting the dispersion to an appropriate volume (~1.5 mL) with water. The ζ-potentials of cationic liposomes and siRNA lipoplexes were measured by electrophoresis light-scattering methods using ELS-Z2 at 25 ◦C after diluting the dispersion with an appropriate volume (~1.5 mL) with water.

#### *2.5. Cell Culture*

Human nasopharyngeal tumor KB cells (also known as a subline of human cervix adenocarcinoma HeLa) were obtained from the Cell Resource Center for Biomedical Research, Tohoku University (Miyagi, Japan). KB cells were cultured in RPMI-1640 medium with 10% heat-inactivated fetal bovine serum (FBS) and 100 μg/mL kanamycin in a humidified atmosphere containing 5% CO2 at 37 ◦C.

For the preparation of KB cells stably expressing firefly pGL4 luciferase, KB cells were plated on 35-mm culture dishes. Twenty-four hours later, the cells were transfected with 2 μg of plasmid DNA encoding firefly pGL4 luciferase under the control of a cytomegalovirus (CMV) promoter (pGL4.51[luc2/CMV/Neo] Vector, Promega, Madison, WI, USA) using Lipofectamine 2000 transfection reagent (Invitrogen, Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). The transfected cells were selected in medium with 1200 μg/mL G418 sulfate. G418-resistant colonies were subcultured and established as a permanent cell line expressing firefly pGL4 luciferase (KB-Luc).

For the preparation of KB cells stably expressing EGFP, KB cells were transfected with 2 μg of plasmid DNA encoding EGFP under the control of a CMV promoter (pEGFP-C1, Clontech, Palo Alto, CA, USA) using Lipofectamine 2000 transfection reagent. The transfected cells were selected in medium with 1 mg/mL G418 sulfate. G418-resistant colonies were subcultured and established as a permanent cell line expressing EGFP (KB-EGFP).

#### *2.6. Gene Silencing E*ff*ect by FA-PEG-Modified siRNA Lipoplexes in KB-Luc Cells*

The KB-Luc cells were seeded in 6-well culture plate at a density of 3 × 105 cells per well, 24 h prior to transfection. The siRNA lipoplexes were formed by the addition of cationic liposomes into 50 pmol Cont siRNA or Luc siRNA with vortex mixing for 10 s and left at room temperature for 15 min. For siRNA transfection, each siRNA lipoplex was diluted in 1 mL of folate-deficient RPMI-1640 medium (Invitrogen) supplemented with 10% FBS and then the mixture was added to the cells (final 50 nM siRNA concentration). Lipofectamine RNAiMAX lipoplexes (Invitrogen) were prepared in accordance with the manufacturer's protocol and then added to the cells. Forty-eight hours after the transfection, luciferase activity was measured as counts per sec (cps)/μg protein using the luciferase assay system (PicaGene MelioraStar-LT Luminescence Reagent, Toyo Ink Mfg. Co. Ltd., Tokyo, Japan) and BCA reagent (Pierce™ BCA Protein Assay Kit, Pierce, Rockford, IL, USA). Luciferase activity (%) was calculated as relative to the luciferase activity (cps/μg protein) of untransfected cells.

#### *2.7. Gene Silencing E*ff*ect by FA-PEG-Modified siRNA Lipoplexes in KB-EGFP Cells*

The KB-EGFP cells were seeded in 6-well culture plate at a density of 3 × 105 cells per well, 24 h prior to transfection. The siRNA lipoplexes were formed by addition of cationic liposomes into 50 pmol Cont siRNA or EGFP siRNA with vortex mixing for 10 s and left at room temperature for 15 min. For siRNA transfection, each siRNA lipoplex was diluted in 1 mL of folate-deficient RPMI-1640 medium supplemented with 10% FBS and then the mixture was added to the cells (final 50 nM siRNA concentration). Lipofectamine RNAiMAX lipoplexes were prepared in accordance with the manufacturer's protocol and then added to the cells. Forty-eight hours after transfection, EGFP expression levels in the cells were determined by examining fluorescence intensity using a

FACSVerseTM flow cytometer (Becton Dickinson, San Jose, CA, USA) equipped with a 488-nm argon ion laser. Data for 10,000 fluorescence events were obtained by recording forward scatter (FSC), side scatter (SSC), and green (530/30 nm) fluorescence. EGFP expression level (%) was calculated as relative to the fluorescence intensity of untransfected cells.


*2.7. Statistical Analysis*

All data were presented as mean ± SEM. Normality of the data and homogeneity of variances were determined by the Shapiro–Wilk and Bartlett tests, respectively. Because we euthanized di fferent rats at each time point, statistical di fferences between groups were determined using Two-Way ANOVA and Bonferroni test (to assess treatment and time-specific changes) using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). The Mann–Whitney test was used to determine statistical di fferences in sperm motility and DNA oxidation among groups. Di fferences with a *p*-value of ≤0.05 were considered significant.


**Henri¨ette E. van Heemstra, Willem F. Scholte, Angela Nickerson and Paul A. Boelen**

Can Circumstances Be Softened? Self-Efficacy, Post-Migratory Stressors, and Mental Health among Refugees

Reprinted from: *Int. J. Environ. Res. Public Health* **2021**, *18*, 1440, doi:10.3390/ijerph18041440 . . . **273**


**Cell Signaling in Model Plants**

Editors

**Jen-Tsung Chen Parviz Heidari**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Jen-Tsung Chen National University of Kaohsiung Taiwan

Parviz Heidari Shahrood University of Technology Iran

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: https://www.mdpi.com/ journal/ijms/special issues/CSMP).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

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# **11th EASN International Conference on Innovation in Aviation & Space to the Satisfaction of the European Citizens**

Edited by

Spiros Pantelakis, Andreas Strohmayer and Liberata Guadagno Printed Edition of the Special Issue Published in *Aerospace*

www.mdpi.com/journal/aerospace


**2. Methods and materials**

#### **2.1 Patients**

A retrospective clinical chart review was performed on all of the author's patients who had nasal surgery from January 2016 through December 2018 at the ENT unit of Pantai Hospital Kuala Lumpur (PHKL). All patients had severe nasal obstruction with chronic rhinosinusitis and were followed up for a minimum of 6 months post-surgery. The data revealed that 53 patients out of the 116 patients (45.6%) underwent concurrent open rhinoplasty and ESS by the same surgeon at PHKL. Patients who underwent rhinoplasty and ESS at different sittings (54.4%) were excluded from the study because the SNOT 22 subjective scoring system which was used only for the evaluation of patient symptoms in the concurrent group before and after surgery was sufficient, and therefore the need to compare with patients who underwent rhinoplasty and ESS at different sittings was not necessary. A history of nasal trauma and snoring was documented.

Patients with primary nasal dysfunction and sinus complaints were seen by the same surgeon. All the patients underwent ENT workup which included history, head and neck examination, nasal endoscopy, and CT scans of paranasal sinuses

and were treated with oral antibiotics and topical nasal steroids prior to the CT scan and a full facial analysis including standardized photography.

### **2.2 Evaluation**

The main complaints of the patients prior to surgery were chronic nasal obstruction, postnasal drip, headaches with occasional voice changes, and snoring. External nasal examination was performed to detect a twisted/crooked/saddled nose.

Nasal endoscopy revealed that all these patients had significant anterior septal deviation involving the internal nasal valve, in addition to posterior septal deviation. Nasal endoscopic examination was performed to detect the grading of septal deviations, namely, I, II, III, IV, and V (**Figure 4**), and diseased mucosal or polypoidal tissue (grade 1, 2, 3) involving the paranasal sinuses. If there was evidence of mucopurulent discharge from the paranasal sinuses on nasal endoscopy on admission, the patients were commenced on systemic antibiotics prior to surgery.

#### **2.3 Surgical technique**

All the cases were performed as an inpatient procedure by a one surgeon and two procedure approach under general anesthesia at Pantai Hospital Kuala Lumpur. At the time of induction, all patients received IV antibiotics (ceftriaxone 1 gm) and steroids (dexamethasone 8 mg). The CT scans of the paranasal sinuses were reviewed again in OR prior to performing the surgery. A throat pack was inserted, and the nasal cavity was packed with soaked spacers for vasoconstriction. Infiltration was performed at the nasal dorsum, alar rim, septum, and greater palatine fossa transorally with levobupivacaine (20 cc), adrenaline (0.2 mg), and aqua (1.8 cc). Surgery was initiated with ESS procedure followed by open rhinoplasty approach, but in gross septal deviations, the septoplasty was performed prior to the ESS.

For the open rhinoplasty approach, an inverted transcolumellar V-shaped incision was made, and the SMAS elevated all the way to the dorsum of the nose (**Figure 5**). The domes are divided in the midline, and the upper lateral cartilages released laterally, creating excellent exposure of the septum. Bilateral submucoperichondrial flaps are elevated, exposing the entire cartilaginous and anterior bony septum. The cartilaginous and bony septum is then resected by paramedian

#### **Figure 4.**

*The five areas of the internal nose most commonly involved in nasal septal deviations. Open approach rhinoplasty is indicated in anterior deviations of nasal septum involving areas I, II, and III along with significant internal nasal valve involvement, whereas closed approach is indicated for posterior septal deviations restricted to areas IV and V only.*

**157**

were used for the skin.

**Figure 5.**

*nasal dorsum.*

*Concurrent Rhinoplasty and Endoscopic Sinus Surgery DOI: http://dx.doi.org/10.5772/intechopen.89415*

osteotomy, separating cartilaginous septum from maxillary crest and fracturing bony septum as posterior as possible leaving behind the cribriform plate and sphenoid rostrum. Extracorporeal approach was performed on all patients with gross high septal deviation, which requires complete removal of the entire cartilaginous septum, which is then straightened and returned to the nose. In revision rhinoplasty cases where adequate quadrangular cartilage and septal bone grafts were not available, conchal cartilage graft was harvested. Bilateral spreader grafts are then placed on the dorsal part of the septum. K-wire drill was used to drill multiple holes on the septal bone graft for use as spreader/ columella strut/columella extension graft. Straight 4/0 Monosyn mattress sutures were used to secure the spreader graft. Then lateral osteotomies are performed by external subcutaneous method if required. Nasal spine if deviated more than 30° is gauged out or drilled. Neo-septum with spreader graft is inserted in the nose. Areas of fixation are the caudal end of the nasal bones, upper lateral cartilage, and maxillary crest. A hole is drilled through the nasal bones and the nasal spine and suturing the neo-septum with Monosyn 4/0 sutures. Other required steps like columellar strut, rim grafts, and tip grafts are performed. Soft silicon splints are placed along either side of septum and sutured in place with through-and through 3/0 Monosyn sutures. Curve Monosyn 4/0 and 5/0 were used for tip plasty (transcrural, intercrural, shield graft), dorsal augmentation, caudal augmentation, septum augmentation, and alar rim suturing. Ethicon 6/0 sutures

*Close-up view of open rhinoplasty via a transcolumellar incision and elevation of SMAS all the way to the*

For the ESS, the mucosa on the lateral wall of the nose and the anterior face of the sphenoid was infiltrated and the diseased sinuses addressed by performing ethmoidectomy, middle meatal antrostomy, sphenoidotomy or frontal sinusotomy. Prior to performing middle meatal antrostomy, an uncinectomy was performed

The nasal and sinus cavities were packed with Nasapore. Steri-Strip was applied

externally on the nasal dorsum along with Denver splints which were removed between 7 and 10 days postoperatively. Nasal cavity suction was performed on the third postoperative day along with the removal of the nasal septal splints and patient sent home the same day. The ESS was performed using a technique adapted

using thru-cut instruments along with a microdebrider.

*Concurrent Rhinoplasty and Endoscopic Sinus Surgery DOI: http://dx.doi.org/10.5772/intechopen.89415*

#### **Figure 5.**

*Rhinosinusitis*

**2.2 Evaluation**

**2.3 Surgical technique**

and were treated with oral antibiotics and topical nasal steroids prior to the CT scan

The main complaints of the patients prior to surgery were chronic nasal obstruction, postnasal drip, headaches with occasional voice changes, and snoring. External

Nasal endoscopy revealed that all these patients had significant anterior septal deviation involving the internal nasal valve, in addition to posterior septal deviation. Nasal endoscopic examination was performed to detect the grading of septal deviations, namely, I, II, III, IV, and V (**Figure 4**), and diseased mucosal or polypoidal tissue (grade 1, 2, 3) involving the paranasal sinuses. If there was evidence of mucopurulent discharge from the paranasal sinuses on nasal endoscopy on admission, the patients were commenced on systemic antibiotics prior to surgery.

All the cases were performed as an inpatient procedure by a one surgeon and two procedure approach under general anesthesia at Pantai Hospital Kuala Lumpur. At the time of induction, all patients received IV antibiotics (ceftriaxone 1 gm) and steroids (dexamethasone 8 mg). The CT scans of the paranasal sinuses were reviewed again in OR prior to performing the surgery. A throat pack was inserted, and the nasal cavity was packed with soaked spacers for vasoconstriction. Infiltration was performed at the nasal dorsum, alar rim, septum, and greater palatine fossa transorally with levobupivacaine (20 cc), adrenaline (0.2 mg), and aqua (1.8 cc). Surgery was initiated with ESS procedure followed by open rhinoplasty approach, but in

gross septal deviations, the septoplasty was performed prior to the ESS.

For the open rhinoplasty approach, an inverted transcolumellar V-shaped incision was made, and the SMAS elevated all the way to the dorsum of the nose (**Figure 5**). The domes are divided in the midline, and the upper lateral cartilages released laterally, creating excellent exposure of the septum. Bilateral submucoperichondrial flaps are elevated, exposing the entire cartilaginous and anterior bony septum. The cartilaginous and bony septum is then resected by paramedian

*The five areas of the internal nose most commonly involved in nasal septal deviations. Open approach rhinoplasty is indicated in anterior deviations of nasal septum involving areas I, II, and III along with significant internal nasal valve involvement, whereas closed approach is indicated for posterior septal*

nasal examination was performed to detect a twisted/crooked/saddled nose.

and a full facial analysis including standardized photography.

**156**

**Figure 4.**

*deviations restricted to areas IV and V only.*

*Close-up view of open rhinoplasty via a transcolumellar incision and elevation of SMAS all the way to the nasal dorsum.*

osteotomy, separating cartilaginous septum from maxillary crest and fracturing bony septum as posterior as possible leaving behind the cribriform plate and sphenoid rostrum. Extracorporeal approach was performed on all patients with gross high septal deviation, which requires complete removal of the entire cartilaginous septum, which is then straightened and returned to the nose. In revision rhinoplasty cases where adequate quadrangular cartilage and septal bone grafts were not available, conchal cartilage graft was harvested. Bilateral spreader grafts are then placed on the dorsal part of the septum. K-wire drill was used to drill multiple holes on the septal bone graft for use as spreader/ columella strut/columella extension graft. Straight 4/0 Monosyn mattress sutures were used to secure the spreader graft. Then lateral osteotomies are performed by external subcutaneous method if required. Nasal spine if deviated more than 30° is gauged out or drilled. Neo-septum with spreader graft is inserted in the nose. Areas of fixation are the caudal end of the nasal bones, upper lateral cartilage, and maxillary crest. A hole is drilled through the nasal bones and the nasal spine and suturing the neo-septum with Monosyn 4/0 sutures. Other required steps like columellar strut, rim grafts, and tip grafts are performed. Soft silicon splints are placed along either side of septum and sutured in place with through-and through 3/0 Monosyn sutures. Curve Monosyn 4/0 and 5/0 were used for tip plasty (transcrural, intercrural, shield graft), dorsal augmentation, caudal augmentation, septum augmentation, and alar rim suturing. Ethicon 6/0 sutures were used for the skin.

For the ESS, the mucosa on the lateral wall of the nose and the anterior face of the sphenoid was infiltrated and the diseased sinuses addressed by performing ethmoidectomy, middle meatal antrostomy, sphenoidotomy or frontal sinusotomy. Prior to performing middle meatal antrostomy, an uncinectomy was performed using thru-cut instruments along with a microdebrider.

The nasal and sinus cavities were packed with Nasapore. Steri-Strip was applied externally on the nasal dorsum along with Denver splints which were removed between 7 and 10 days postoperatively. Nasal cavity suction was performed on the third postoperative day along with the removal of the nasal septal splints and patient sent home the same day. The ESS was performed using a technique adapted

from Stammberger [12] and Kennedy [13]. The ESS instruments included high-definition Spice monitor, 4 mm endoscopes (0, 30 and 70°), and powered instruments (debrider by Medtronic). IGS was used in revision sinus surgery cases. All patients received postoperative antibiotics and nasal rinse. The one surgeon team performed the postoperative endoscopic debridement and nasal function and documented aesthetic alterations with standardized postoperative photography.


*3.3.5. Stop codon depletion in antisense strand and stop codon translation: ND4l and ND5*

Annotations in GenBank for *Aleurodicus dispersus*' ND4l and ND5 do not match proteins homologous to the corresponding NADH dehydrogenase subunits. For ND4l, the peptide translated from frame 0 of the antisense of that gene (this frame includes five stops) yields a short alignment with the regular protein in *Aleurodicus dugesii* (24 residues, 87% similarity, e value 8.5, not shown). Hence, the annotated ORF in *Aleurodicus dispersus* is probably a stop-codon-depleted antisense sequence (the corresponding frame in *Aleurodicus dugesii* has four stops), which does not code for the regular ND4l gene. This stop codon depletion in *Aleurodicus dispersus* introduced five stops in the sense strand frame that apparently codes for ND4l according to the above-described alignment.

For ND5, the peptide translated from the +1 frame of the antisense of the GenBank-annotated sequence is homologous over its complete length to NADH dehydrogenase subunit 5 of *Aleurodicus dugesii* (89% similarity, e value 0, not shown). This frame has a single stop codon that aligns with serine in *Aleurodicus dugesii* (see discussion of insertion of serine at stops in previous section).

> codon percentages decrease in 11 among 13 protein coding genes of *Aleurodicus dispersus*, as compared to *Aleurodicus dugesii*. This is a significant majority of cases according to a one tailed sign test using a binomial distribution and assuming equal probability of getting more or less stop codons in any of these mitogenes (P = 0.00562). This overall stop codon depletion occurs in all seven "recoded" genes. Stop codon depletion occurs qualitatively in four among the six genes with regular, unchanged coding structure. This tendency is not statistically significant for this subgroup of genes when using the robust, but blunt nonparametric sign test. A paired t test between mean percentages of stop codons averaged across frames indicates also for these six genes a statistically significant decrease in stops in *Aleurodicus dispersus*, as compared to *Aleurodicus dugesii*. This result suggests that stop codon depletion occurred across all or at least most of this genome, and for most frames, not only for genes whose coding structure was altered, and not only for frames

> **Table 1.** TAR stop codons in the six frames of mitogenome-encoded genes of *Aleurodicus dugesii* and *Aleurodicus*

Directed Mutations Recode Mitochondrial Genes: From Regular to Stopless Genetic Codes

http://dx.doi.org/10.5772/intechopen.80871

65

Presumably, unknown mechanisms associated with replication depleted stop codons in this species' mitogenome, perhaps cumulatively over several replication or DNA edition cycles. Total stop codon depletion in some frames produced new ORFs. Natural selection against stop codons presumably enhanced unknown enzymatic phenomena, eliminating stop codons in these frames. It seems plausible that these frames in usual mitogenomes code for proteins translated by stop suppressor tRNAs. Specific unknown conditions in *Aleurodicus dispersus* may favor enhanced expression of peptides coded by frames that usually include stops in other mitogenomes, such as *Aleurodicus dugesii*. These constraints would have ultimately caused genomic stop codon depletion in *Aleurodicus dispersus*. In regular mitogenomes, stop codon translation downregulates expression of these unusual peptides in favor of proteins coded by regular ORFs, but in *Aleurodicus dispersus*, this hierarchy may be inexistent (when two stopless ORFs occur in a gene) or reversed (as in several mitogenes of *Lepidochelys olivacea* [9]), with translation of the unusual peptide not necessitating stop codon suppression, and translation of regular mitochondrial proteins requiring tRNAs with anticodons matching stop codons.

who became ORFs.

*dispersus*.

#### *3.3.6. Stop codon depletion in antisense strand, frameshift, and stop translation: ND4*

A further mitochondrial gene for which the GenBank annotation does not produce the expected protein for *Aleurodicus dispersus* is ND4. Alignment analyses detect peptides homologous with regular NADH dehydrogenase subunit 4 when translating frames +1 and +2 of the antisense of the GenBank-annotated ND4 gene. Blastp alignment analyses detect homology with the regular ND4-encoded protein of *Aleurodicus dugesii*; the regular protein is encoded in antisense frame +1 until residue 297 (89% similarity, e value 3 × 10−126, not shown). This alignment includes a single stop, matching tyrosine in *Aleurodicus dugesii*. Part of the remaining protein is coded by a stopless stretch of antisense strand frame +2, where residues 341–427 align with the regular protein from *Aleurodicus dugesii* (73% similarity, e value 2 × 10−21). Hence, the annotated ORF in *Aleurodicus dispersus* is a stop-codon-depleted antisense frame that codes for an apparently different protein, the actual NADH dehydrogenase subunit 4 is encoded by two frames, one containing a stop, on the opposite strand.


**4. Management of mycotoxin prevention**

Food safety is a key component in public health issue, and a mycotoxin is a huge food safety risk in developing countries. Prevention is the most important and effective way in reducing fungal growth and mycotoxin production to ensure food safety. The following steps that explain prevention and control of mycotoxin occurrence include good agricultural practices (GAP) in field, control practices of harvesting and storage, physical methods (cleaning, milling, etc.), implementation of biotechnological application, biological control through the use of controlled atmosphere during storage, detoxification/degradation, and fermentation techniques.

Pre-harvesting is considered first and one of the most important stages to prevent mold growth and mycotoxin synthesis. Several strategies are available for the produce of healthy products and reduce the mold formation at pre-harvesting, including selection of plants according to the soil structure and production capacity, use of plant which is resistant to fungi and insects, irrigation time, make fertilization, use of insecticides to prevent insect damage [87].

Harvesting at the appropriate time periods (low moisture and full maturity) is essential for reducing the risk of a mycotoxin contamination since overmaturity creates sensitivity to mold growth. Additionally, suitable harvesting equipment and procedures should be used, and crops should be dried after maturity to both reduce grain moisture to safe levels [88].

The latest technological advances provided new paths in mycotoxin control strategies that include the use of a controlled atmosphere with inhibitory or a protective effect and use of naturally occurring compounds under different conditions and essential oils with antioxidant properties to decrease fungal growth and mycotoxin production in grains during storage [89]. Moreover, these strategies also include using regularly cleaned transport vehicles to prevent cross contamination of products; monitoring of temperature, humidity, aeration and pest infestation periodic during storage [90]; using mold inhibitors (propionic acid) to contaminated food and feed; and application of disinfectant such as sodium hypochlorite to storage area [91].

Some studies have shown that using physical methods (dehulling, washing, sorting, and cleaning of visible moldy seed) reduces different mycotoxin species in foods regardless of grain genre [70]. Scudamore and Pascale et al. [92] and Patel [93] observed a reduction of T-2 (62%) and HT-2 (53%) and DON (50%) in wheat seeds after cleaning. Scudamore and Patel also reported a 32% reduction in fumonisin levels in corn in an industrial enterprise [94]. Moreover, milling is an important effect in the reduction of *Fusarium* mycotoxins in grains especially wet milling of maize which has shown to result in the degradation of mycotoxins [95].

One of the best applicable strategies for the prevention of mycotoxin formation is the cultivation of fungal infestation-resistant plants and improvement of the genetic composition to suppress mycotoxin production [96]. The benefits of biotechnological applications were observed with Aflasafe. Aflasafe is a biocontrol product that includes a blend of four fungal species covered over grains which reduce aflatoxigenic fungi that produce AFs in maize and groundnuts (https:// aflasafe.com/).

Mycotoxins are resistant to heat and cannot be completely destroyed under normal cooking process. On the other hand, mycotoxin reduction has been determined after heating, and this may be the result of reactions changing the chemical structure [70]. Ryu et al. reported heat treatment (at temperature 120–160°C) causes a reduction between 66 and 83% of ZEN [97]. Scott and Lawrence also reported a reduction of 60–100% of fumonisins with a heat treatment at 190°C (60 min) and 220°C (25 min).

Biological control of mycotoxins via detoxification/degradation offers a promising alternative method [98]. Recently the effectiveness of fermentation for the reduction and elimination of mycotoxins has also been proven. Studies documented in the literature generally show that mycotoxins are reduced by conversion, detoxification, binding, degradation, and decontamination after food fermentation [99]. Modification of the chemical structure of the mycotoxin molecule, removal or detoxification/inactivation, and adhesion to bacterial cell walls provide a reduced toxicity during fermentation [99]. Implementation of these preventive methods cannot solve the problem alone; also it must be an integral part of an integrated food safety management system based on the hazard analysis and critical control point (HACCP).


*6.2. Results and Discussion*

forwards (players 9–11).

Table 2 depicts the overall values of the distance covered, average values, standard deviation and entropy of the fractional coefficient, and entropy of the heat maps.

**Table 2.** Descriptive statistics of the overall results for each player.

Players 7 and 8 covered the largest overall distance (!8CW8 and !8CWWY km, respectively). On the other hand, the goalkeeper (player 1: VCW\<4) and central defenders (player 3: XCZW km; player 4: XCVWW km) covered smaller overall distances. Both cases are in line with the literature [30,42,43]. Generally, the largest distances are covered by midfielders since they act as links between defence and attack [36,42]. Bangsbo [44] reported that elite defenders and forwards cover approximately the same average distance, which is significantly less than the distance covered by midfield players. This study shows that central defenders, excluding goalkeepers since they are more constrained than other players, cover (with a large difference) a smaller distance than any other tactical position.

In terms of heat maps entropy, the results are in line with the overall distance. All the midfielders have a larger entropy than the remaining teammates (player 7: E  8CPZ; player 8: E  8CPPX; player 6: E  8CVZ8). On the other hand, the goalkeeper (player 1) presents the lower entropy value (E  CW!W), followed by the right forward (player 10) with E  8C8P, and the central defender (player 3) with E  8C\V. These results can be easily explained by the tactical roles of each position. In football, midfielders act as a link between the defenders and the forwards [42]. Therefore, they present a higher level of participation in the periods of time with or without ball possession. Also, as the goalkeeper and the central defenders have different roles in specific confined *TR*, they present a smaller spatial distribution (lower heat maps entropy). Conversely, lateral positions (defenders and forwards) have a larger *TR*. In some cases, the lateral defenders participate in offensive attempts. The inverse is observed in the lateral forwards players, because they regularly help in the defensive moments. Hence, the low values of entropy from player 10 can be explained by his reduced participation in the defensive phase.

The fractional coefficients show that the midfielders are the most unpredictable players. Players 6 and 7 are characterised by values close to H :) ,  CY\. These values are in line with the combination linear-random trajectories. This tendency makes sense since midfielders cover more distances. On the other hand the goalkeeper's trajectory is defined by larger fractional coefficient values (H :) ,  C\Y). This result is in line with the combination constant-chaotic. In point of fact, this also makes sense since the goalkeeper stays most of the time around the same tactical region. The remaining players are somewhere between the combination constant-chaotic and linearrandom, with more tendency for the linear-random.

Going further on this analysis, the attraction domain previously defined was considered so as to study the number of times that each player remained within their stability region. In a quantitative point-of-view, if a player's trajectory is classified as stable (based on : ") , *and* H:) ,), then the stability is defined as 1. Otherwise, the stability is defined as -1. Putting differently, a player that is as often within the stable region and the unstable one, will have an overall stability level of 0. From this analysis it is possible to obtain the stability values *per* player on the 5-axis and I-axis coordinates (see Figure 13).

From the results shown, it is possible to observe that the goalkeeper (player 1) is the more unstable elements in both axes. On the other hand, defenders (players 2–5) are the elements with higher stability values. These values can be supported by the specific tactical missions of each player. Defenders should keep a large defensive stability by remaining in their tactical position, giving some equilibrium to the team. As a point of interest, a considerable number of goals suffered results from the defensive instability. Therefore, defenders should maintain their trajectories within their specific regions so as to ensure the possibility of recovering the ball in the offensive attempts by the opponent team. In contrast, the goalkeeper's *TR* is evidently smaller than all his teammates. As such, at many moments of the match (mainly in the offensive situations) the goalkeeper moves outside his *TR*, towards his remaining teammates. Such movements decrease the goalkeeper's stability.

**Figure 13.** Players' stability levels at 5-axis and I-axis coordinates.

Using both concepts (predictability and stability) it is possible to observe that one player can be highly predictable (in terms of trajectory) while unstable (going outside his *TR*). On the other hand, a highly unpredictable player can be very stable if he stays most of the time inside his *TR*. Therefore, those two concepts are different and they provide an interesting set of information for coaches and their staff. The predictability level can be used to classify the oscillations during the football match while the stability level can be used to identify player's responsiveness to his *TR*. To illustrate the relationship between predictability and stability, let us present Figure 14. The 3D chart on Figure 14 depicts how the level of stability on the *x*-axis, : , is related to the level of stability on the *y*-axis, : 7, and the level of predictability represented by the fractional coefficient H:.

As one may observe, the relationship between these measures is represented by a plane. Moreover, as already concluded from Figure 13, although players are more stable in the *x*-axis, there is a clear dependency between the stability on both axes. On the other hand, the level of predictability seems to vary in a significant manner depending on the positional main role of players. For instance, it is possible to divide the points into four clusters, wherein the goalkeeper

(red circle) represents the first cluster, the defenders represent the second cluster (blue triangles), the central players represent the third cluster (green lozenges) and the forwards represent the forth cluster (purple squares).

The information retrieved from the fractional coefficient cannot be compared neither with the outcome provided by the total distance covered nor with the heat maps entropy. The distance covered can be the same for all players, without providing a specific characteristic about the behaviour of the player. The heat maps entropy only provides information about the spatial distribution of players on the field. This distribution may also be similar for two players without describing their trajectory over time. Only understanding the specific properties of each football player's trajectory can improve the performance analysis and, likewise, improve the quality of the football training.

#### *6.3. Practical Remarks*

The information retrieved from the fractional coefficient cannot be compared with either the outcome provided by the total distance covered, or with the heat maps entropy. The distance covered can be the same for all players, without providing any specific characteristic about the behaviour of the player. The heat maps entropy only provides information about the spatial distribution of players on the field. This distribution may also be similar for two players without describing their trajectory over time. Only understanding the specific properties of each football player's trajectory can improve the performance analysis and, likewise, to improve the quality of the football training.

We should note that we are not redefining the concept of variability. Instead, this work proposes to analyse such variability by studying the regularity of players in returning to their own *TR* (stability), and by studying how predictable their trajectory may be (predictability). Stability, in science, is defined as its resistance to perturbations. In fact, this is a typical property shared by many dynamical systems, in which we could state that the stability is not only the resistance to a perturbation, but also the ability to return to the equilibrium point (or initial position) [45]. The existence of a stable equilibrium point implies the existence of a "restoring force" which is directed towards the equilibrium point. For instance, in the simple pendulum case study, this is a combination of the tension in the string and the force of gravity. Nevertheless, as opposed to the simple pendulum, the results presented here classify football players as non-linear dynamical systems, thus presenting chaotic or even stochastic trajectories. Although we assume that there is a steady-state point from which players converge, the truth is that they converge to an equilibrium point which is defined by their *TR*. In other words, a player's orbit spirals in towards the equilibrium. The same can be said about the predictability of players. By definition, predictability is the degree to which a correct estimation of a system's state can be made. This is in line with the concept of predictability in mathematics, wherein a process is classified as predictable if it is possible to know the "next" state at the present time.

In many situations, the choice regarding the players from the first team is based on each player's specific properties so as to adjust the team against their opponents. As such, one may choose between more stable players to focus on the defence, or more unpredictable ones to focus on the attack. The fractional coefficient can also be a useful method to improve the understanding about

decision-making in sports. The main techniques used so far for this specific issue have been the approximate entropy and the Lyapunov exponent. Nevertheless, the applicability of such methods depends on the variable that better explains the level of predictability. For a more specific tactical analysis, one should go further into understanding the fractional coefficient variability by resorting to stability theory. This is very important in understanding the player's regularity on returning to his own *TR*. The stability confined to on attraction region has a great potential for use by coaches to classify the tactical oscillations of players, thus adjusting or readjusting the desired tactical behaviours. Also, the opponent coach can use this information to identify some unstable points and exploit them during the match. Nevertheless, it should be highlighted that neither the fractional coefficient, nor the stability analysis *per se*, are the ultimate answer to one's needs in the context of football. Such team sport, as a complex and dynamic game, should be analysed using collective nonlinear methods. The classical perspective of the performance analysis has been overtaken using new technologies to improve the understanding of the individual and tactical parameters, mainly trying to explain the process variables. For the collective analysis, some metrics have been proposed based on the position of players over time [3,46,47]. Nevertheless, for the individual performance, the researchers have been emphasizing on the notational information (*i.e.*, product variables) and kinematical information [43]. This paper provides a new take-home message on the individual performance of a football match, with the main purpose being the understanding of the specific properties of each player and their dynamical behaviour during the match.

It is noteworthy that the herein proposed methodology proposed here was evaluated using one match. Its usefulness for coaches and sports analysts needs to be further assessed over multiple matches, with and without professional players. Note, however, that this requires the use of automatic tracking systems, such as *AMISCO Pro* and *ProZone* [48]. These systems provide online information to coaches and their staff about players' movements (e.g., energy spent by a player). Nevertheless, despite of their efficiency and autonomous properties, player-to-player occlusion, similar player appearance, number of players changing over time, variability of players' motion and noises or video blur present themselves as open problems [49]. Therefore, although generally autonomous, these tracking systems still require some human input as well as continual online verification by an operator to make sure that players are correctly tracked by the computer program [48]. Hence, beyond their expensive devices (e.g., many high-definition video cameras), those systems may benefit from the outcome provided by the fractional calculus methodology provided in this paper and previously presented in Couceiro *et al.* [17], to accurately and autonomously estimate a given player's position over time.

#### **7. Conclusions**

New technological devices and mathematical methods have been used recently to analyse the performance of football players. Despite these developments, a gap still remains on understanding a player's dynamical behaviour during the match. Some of the most important variables one may look at are inherent in a player's variability, which one may classify based on the predictability and stability of his trajectory. This study proposed an approach to measure the predictability and stability levels of player's trajectories based on the concepts inherent in Fractional Calculus. Furthermore, the variability of each player was measured using the well-known Shannon's entropy and the approximate entropy. The fractional coefficient, explaining a given player's trajectory, was used in order to estimate his predictability. The addition of a new parameter, herein denoted here as the stability vector, gave rise to an attraction domain defining the player's stability. The results showed that the goalkeeper was the most predictable and unstable player. The most unpredictable players were the midfielders while the most stable players were the defenders. All this information can be used by coaches to adjust and readjust the team's strategy, as well as the tactical behaviour of players.

## **Acknowledgments**

This work was supported by a PhD scholarship (SFRH/BD /73382/2010) by the Portuguese Foundation for Science and Technology (FCT). Also, this paper reports research work carried out within the project "Towards a technological approach of the match analysis: Using tactical metrics to evaluate football teams" from the Instituto de Telecomunicações, granted by the Portuguese Foundation for Science and Technology (FCT) with the ref. PEst-OE/EEI/LA0008/2011.

## **Conflicts of Interest**

The authors declare no conflict of interest.

### **References**

Reprinted from *Entropy*. Cite as: Wei, Z.; Wang, Y.; Wei, G.; Wang, T.; Bourquin, S. The Entropy of Co-Compact Open Covers. *Entropy* 2013, *15*, 2464–2479.

#### *Article*


*3.4. Winery Wastewater*

Winery wastewater with different compositions was tested using MFCs made up of two single-chamber air cathodes [80]. A wire made up of titanium along with carbon fiber was put in place of generic anodes, while on the other hand, cathodes were made up of platinum-coated carbon cloth of 0.4 mg Pt/cm2 . The white wine wastewater resulted in less promising results with an energy generation capacity of 263 mW/m2 ; albeit a significant quantity of COD (90%) and BOD (95%) removal was achieved with a CE of 15%. Comparatively, red wine wastewater had 111 W/m2 , with a recorded 27% COD removal, whereas a maximum BOD removal of 83% was achieved; hence, a lower CE of 9% was observed. These experiments indicated that different wastewater, even from the same industry, can have different usable substrates with differentiated compositional characteristics, which determines the MFC's power output. This was facilitated by diverse and different microbial populations found in the anode; besides, the negative influence on MFC performance was due to a high concentration of polyphenolic compounds in some winery wastewater.


**2.3 Transport layer and above**

It's typical for the transport layer to ensure end-to-end communication. In networks based on the IP for the network layer, the Transmission Control Protocol (TCP) is often used for the transport layer [28]. Networks which do not make use of IP for the network layer, or networks which use IP but do not use TCP for the transport layer, occasionally have the own error-correction mechanism of theirs in the transport layer [29, 30].

The authors do not go over the session layer (or higher layers), as they are not the topic of the research.