**Preface to "Probabilistic and Fuzzy Approaches for Estimating the Life Cycle Costs of Buildings"**

The construction sector is a major consumer of natural resources and incurs high costs. Life cycle cost (LCC) makes it possible for the whole life performance of buildings and other structures to be optimized. The introduction of the idea of thinking in terms of a building life cycle resulted in the need to use appropriate tools and techniques to assess and analyze costs throughout the life cycle of a building. Traditionally, estimates of LCC have been calculated based on the historical analysis of data and have used deterministic models. The concepts of probability theory can also be applied to life cycle costing, treating the costs and timings as a stochastic process. If any subjectivity is introduced to the estimates, then the uncertainty cannot be handled using probability theory alone. The theory of fuzzy sets is a valuable tool for handling such uncertainties.

In this Special Issue, a collection of 11 contributions provide an updated overview of the approaches for estimating the life cycle cost of buildings. In the first paper the importance of information and communication technology use in life cycle cost management are considered. The research assumes that the most critical implementation factor is the investment cost. The second paper uses multiple criteria analysis to define the factors influencing the sustainable construction industries in the EU member states, the UK and Norway. Construction development of Commercial and Recreational Complex Building Projects (CRCBPs) is one of the community needs, but the implementation of these projects is usually very costly. The results and findings of the third article can be considered by CRCBPs in both the private and public sectors for properly effective risk identification, evaluation, and mitigation. The next four papers (4–7) consider the broadly understood maintenance phase of the building, with particular emphasis on the costs incurred at this stage. The eighth article deals with the partial outputs of large-scale infrastructure project risk assessment, specifically in the field of road and motorway construction. A partial section of the research was focused on the analysis of the probability distribution of the input variables, especially "the investment costs". The research topic of the ninth article addresses a part of the evaluation of railway infrastructure project efficiency within its life cycle by using the cost–benefit analysis method. The contractor selection problem, with emphasis on the life cycle costing method as the criterion of choosing the most appropriate company, is discussed in the tenth paper. In the last article, the parameters of tuned mass dampers are optimized to improve the performance level of steel structures during earthquakes.

> **Edyta Plebankiewicz** *Editor*


**Meet the editor**

Carol and Harris Bernstein received their PhDs in Genetics from the University of California (Davis) and from the California Institute of Technology, respectively. They subsequently met and got married while at Yale in 1962. They were faculty members at the University of Arizona College of Medicine for 43 years, retiring in 2011. Their research interests focus on DNA

damage and its underlying role in sex, aging and in the early steps of initiation and progression to cancer. In their research, they have used organisms including bacteriophage T4, Neurospora crassa, Schizosaccharomyces pombe and mice, as well as human cells and tissues. They have authored (jointly and singly) more than 140 scientific publications, including articles in major peer reviewed journals, book chapters and invited reviews.

Contents

**Preface VII**

**Section 1 Molecular Basis of Meiosis 1**

**Chromatin Fiber 3**

Elvira Hörandl

Philip Bell

**Section 3 Teaching Meiosis 99**

**Section 2 Origin and Function of Meiosis 15**

Chapter 2 **Meiosis and the Paradox of Sex in Nature 17**

Harris Bernstein and Carol Bernstein

Chapter 4 **Meiosis: Its Origin According to the Viral Eukaryogenesis Theory 77**

**Reproduction Project 101**

Chapter 1 **Intrinsic Homology-Sensing and Assembling Property of**

Chapter 3 **Evolutionary Origin and Adaptive Function of Meiosis 41**

Chapter 5 **Teaching of Meiosis and Mitosis in Schools of Developing Countries: How to Improve Education with a Plant**

Erica Duarte-Silva, Adriano Silvério and Angela M. H. D. Silva

Jun-ichi Nishikawa, Yasutoshi Shimooka and Takashi Ohyama

## Contents

#### **Preface XI**

**Countries: How to Improve Education with a Plant Reproduction Project 101** Erica Duarte-Silva, Adriano Silvério and Angela M. H. D. Silva

Preface

meiosis be taught?" .

somes is a central molecular step in meiosis.

Meiosis is the key process underlying sexual reproduction in eukaryotes, occurring in sin‐ gle-celled eukaryotes and in most multicellular eukaryotes including animals and most plants. Thus meiosis is of considerable interest, both at the scientific level and at the level of natural human curiosity about sexual reproduction. Improved understanding of important aspects of meiosis has emerged in recent years and major questions are starting to be an‐ swered, such as "How does meiosis occur at the molecular level?", "How did meiosis and sex arise during evolution?", "What is the major adaptive function of meiosis and sex?". In addition, changing perspectives on meiosis and sex have led to the question: "How should

The first chapter in this book reviews a number of recent studies indicating that doublestranded DNA molecules can distinguish "self" from "non-self" and consequently can un‐ dergo self-assembly in a pair-wise manner even in a solution of heterogeneous doublestranded DNA species. These studies used experimental approaches involving electrophoretic analysis and atomic force microscopy, analysis of liquid-crystalline aggre‐ gates, and a technique involving parallel single molecule magnetic tweezers applied to iso‐ lated double-stranded DNA. In addition, this chapter reviewed evidence that this type of selective association between homologous DNA molecules also applies to nucleosomes. Self-assembly of homologous double-stranded DNA molecules and homologous nucleo‐

The next three chapters are concerned with the origin and function of meiosis. The first two of these chapters emphasize that central enzymatic features of meiosis were already present in the prokaryotic ancestors of eukaryotes. One of these chapters went further, indicating that the prokaryotic ancestor was likely able to undergo transformation, a process of DNA transfer between individual bacteria. In transformation, the central proteins catalyzing this process are homologous to proteins with similar functions in meiosis. The other of the first two chapters emphasizes the triggering role of DNA damage in initiating meiosis as well as the purging role of deleterious mutations in diploid-haploid cycles. The major function of meiosis, dealt with extensively in these two chapters, is DNA repair of unavoidable damag‐ es in germ line DNA. Especially discussed is the need for meiosis-provided homologous re‐ combinational repair of double-strand breaks and cross-links in the germ line. Both chapters discuss difficulties with earlier proposals that the main selective advantage of meiosis is the long term benefit of producing genetic variation. The third chapter in this section points out characteristics of the eukaryotic cell (such as coding for mRNA capping enzymes and phage-type RNA polymerases in mitochondria) that could have been derived from symbio‐ genic complex viruses. These characteristics led the author to propose a viral eukaryogene‐

## Preface

Meiosis is the key process underlying sexual reproduction in eukaryotes, occurring in sin‐ gle-celled eukaryotes and in most multicellular eukaryotes including animals and most plants. Thus meiosis is of considerable interest, both at the scientific level and at the level of natural human curiosity about sexual reproduction. Improved understanding of important aspects of meiosis has emerged in recent years and major questions are starting to be an‐ swered, such as "How does meiosis occur at the molecular level?", "How did meiosis and sex arise during evolution?", "What is the major adaptive function of meiosis and sex?". In addition, changing perspectives on meiosis and sex have led to the question: "How should meiosis be taught?" .

The first chapter in this book reviews a number of recent studies indicating that doublestranded DNA molecules can distinguish "self" from "non-self" and consequently can un‐ dergo self-assembly in a pair-wise manner even in a solution of heterogeneous doublestranded DNA species. These studies used experimental approaches involving electrophoretic analysis and atomic force microscopy, analysis of liquid-crystalline aggre‐ gates, and a technique involving parallel single molecule magnetic tweezers applied to iso‐ lated double-stranded DNA. In addition, this chapter reviewed evidence that this type of selective association between homologous DNA molecules also applies to nucleosomes. Self-assembly of homologous double-stranded DNA molecules and homologous nucleo‐ somes is a central molecular step in meiosis.

The next three chapters are concerned with the origin and function of meiosis. The first two of these chapters emphasize that central enzymatic features of meiosis were already present in the prokaryotic ancestors of eukaryotes. One of these chapters went further, indicating that the prokaryotic ancestor was likely able to undergo transformation, a process of DNA transfer between individual bacteria. In transformation, the central proteins catalyzing this process are homologous to proteins with similar functions in meiosis. The other of the first two chapters emphasizes the triggering role of DNA damage in initiating meiosis as well as the purging role of deleterious mutations in diploid-haploid cycles. The major function of meiosis, dealt with extensively in these two chapters, is DNA repair of unavoidable damag‐ es in germ line DNA. Especially discussed is the need for meiosis-provided homologous re‐ combinational repair of double-strand breaks and cross-links in the germ line. Both chapters discuss difficulties with earlier proposals that the main selective advantage of meiosis is the long term benefit of producing genetic variation. The third chapter in this section points out characteristics of the eukaryotic cell (such as coding for mRNA capping enzymes and phage-type RNA polymerases in mitochondria) that could have been derived from symbio‐ genic complex viruses. These characteristics led the author to propose a viral eukaryogene‐

sis theory. By this theory, meiosis is derived from the processes by which a postulated viral ancestor of the nucleus infected new hosts.

The last chapter of the book presents a practical approach to teaching meiosis and gives ex‐ amples of how a low cost program can be set up. This program will allow students to under‐ stand the general processes employed by different types of plants that undergo meiosis. It emphasizes gross morphology and aspects of morphology that can be observed with a hand lens or under a microscope.

**Carol Bernstein and Harris Bernstein**

**Section 1**

**Molecular Basis of Meiosis**

Department of Cellular and Molecular Medicine University of Arizona, USA


**1. Introduction**

To date, vaccines have been able to overcome the evolution of antibiotic-resistant strains, which makes vaccination one of the most cost-effective measures for fighting infectious diseases [1]. *Mycobacterium tuberculosis* (*Mtb*) is included as 'critical' in the WHO priority list of research and development for new antibiotics effective against current resistant strains of tuberculosis (TB) [2]. Concurrently, new efficacious vaccines will be a very important tool to fight antimicrobial resistant TB (AMR TB) and are called to play an important role against this serious health issue [1].

The potential of using TB vaccines to combat AMR TB has generally been undervalued. This could be partly due to the lack of integral efficacy of the present vaccine Bacillus Calmette-Guérin (BCG) failing to reduce the numbers of TB cases, which makes new efficacious vaccines against respiratory forms of TB a critical necessity to help combat AMR TB. Currently, one of the biggest threats in TB is the emergence of multidrug-resistant (MDR) *Mtb* strains, resistant to isoniazide and rifampicin, and extensively drug-resistant (XDR) strains, resistant to at least four of the core anti-TB drugs. In 2018, half a million people were diagnosed with MDR-TB and is estimated that fifty million people were infected with MDR *Mtb* strains, creating a reservoir for future cases of active TB making treatment extremely difficult [2]. AMR poses a threat in TB; both the World Health Organization (WHO) and International Centres for Disease Control and Prevention (CDC) have expressed concern about antibiotic treatments for TB. New TB vaccines are necessary to complement existing and in-development pipeline TB treatment and diagnostic strategies. Considering there has been no evidence to suggest that molecular mechanisms of drug resistance in *Mtb* could affect immune control susceptibility, it is likely that vaccine efficacy against MDR-TB and drug-sensitive TB will be equivalent [3].

In the present work we review the current use of BCG and the non-specific effect against other pathogens and we summarized the diversity of new TB vaccine candidates in clinical trials and their indications. We discuss the need to keep the pipeline of new TB vaccine candidates and the current clinical trial designs employed in the field for efficacy determination of new TB vaccines which include designs for prevention of infection (POI), prevention of disease (POD), and prevention of recurrence of TB (POR), or trials for evaluating the therapeutic effect of TB vaccine candidates when applied in combination with current TB drug regimens with the aim to shorten duration of treatment. The pipeline includes new prime TB vaccines for administration at birth, which are expected to protect better than BCG, and for use in adolescents and adults, as revaccination strategies in individuals previously vaccinated with BCG at birth, as per WHO Preferred Product Characteristics (PPC) for new TB vaccines [3,4]. Novel TB vaccine strategies, which are safe and effective, are imperative in the global efforts to halt dissemination of drug-sensitive and AMR TB [3].


**Conflict of interest**

**3.2. Gliptin on vascular inflammation**

84 Ultimate Guide to Insulin

increasing endothelial production of NO [34].

activation of the inflammatory process.

**4. Conclusion**

Renna et al. suggest that incretin system dysfunction, as happens in patients with diabetes mellitus or metabolic syndrome, allows activation of inflammatory response in different levels. The consequence is the creation of a vascular microenvironment that is conducive to the creation, perpetuation, progression, and destabilization of vascular injury, with either a simple eutrophic mechanism of vascular remodeling or the generation of an atherosclerotic lesion [32]. Several mechanisms may underlie these results: (1) increase the circulating levels of GLP-1 [33]. The cardiovascular actions of GLP-1 may occur either directly through the GLP-1 receptor or through a GLP-1 receptor-independent effect of the degradation product of GLP-1 [38]; (2) DPP-IV also degrades GIP and potentially cytokines and certain chemokines (including stromal-derived factor 1-α). Thus, other substrates of DPP-IV may be responsible for the improvement in endothelial function. Alternatively, DDP-IV inhibition might improve endothelial function by influencing insulin and glucose levels. Insulin causes vasodilation by

The improvement in endothelial function and oxidative stress could result in a decrease in

Other authors have suggested that the DDP-IV inhibitors may have anti-inflammatory effects,

There is sufficient evidence to show that insulin resistance and hyperinsulinism produce significant changes at the vascular level [7, 25, 32, 36, 37]. The proposed mechanisms are (1) the IGF-1 receptor, (2) through the coactivation between IG-1 and AT1R, (3) by activating nuclear transcription factors such as NF-KB or AP-1, (4) by dimerization of IL-6R, and (5) from the activation of oxidative cascades such as NADP (H) oxidase, peroxynitrites, or superoxide dismutase (SOD). However, the effects of hyperglycemia are more erratic: moderate hyperglycemia is sufficient to induce adverse structural changes in the mesenteric vasculature, but

It is more interesting that the blocking of these pathways has significant effects on the activation/deactivation of vascular remodeling, independent of the correction or not, of hyperinsulinism or insulin resistance. This shows that intracellular cascades, in most of these

On the other hand, it is likely that the vascular remodeling associated with insulin resistance, due to the stimulation of growth factors, from the pathways, is due to changes in vascular hemodynamics or to the increase in peripheral resistances, as in the case of arterial hypertension.

The new drugs, which modify the inflammatory modulating response, such as tocilizumab (anti-IL-6R alpha) and canakinumab (anti-IL-1b), will be drugs that could further modify the cardiovascular risk of these patients in the future, since it could modify the vascular inflammatory

such as reduced activation of TNF-alpha during macrophage activation [33, 35].

more severe hyperglycemia is essential to cause endothelial dysfunction.

mechanisms, have no feedback from insulin or glucose receptors.

The authors have no "conflict of interest" to declare.


Preface

Globally, the attainment of the United Nations Sustainable Development Goals (SDGs) established in 2015, especially poverty alleviation, zero hunger, clean water and sanitation, decent work and economic growth, climate action, and life on land, are both directly and indirectly associated with grasslands. The underlying reason is that grasslands, also referred to as prairies, savannas, steppes, and meadows, play a strategic role in sustaining diverse ecosystems and provide a wide array of crucial benefits to the environment, wildlife, livestock, and human communities. For instance, grasslands play an important role in carbon sequestration wherein the grasses store bulk quantities of carbon in the soil through their deep root systems. Grasslands are also significant in sustaining biodiversity (plants, birds, and animal species), controlling floods, providing water through effective regulation of rainwater, and preventing erosion and improving infiltration and hotspots of recreational activity such as hiking, birdwatching, and so on. Moreover, grasslands are precious sources of feed for livestock, herbs for traditional medicines, and rearing spots for honeybees. They also have cultural value in terms of spiritual and religious sites. However, grasslands have remained quite vulnerable to invasive weed species, anthropogenic disturbances (e.g., land conversion for carrying out modern input-intensive farming, uncontrolled grazing by livestock, etc.), and numerous types of climate change. This book, *Grasslands – Conservation and Development*, has been tailored to address these issues. It suggests practices for conserving grasslands that ensure the protection and sustainable management of grassland ecosystems by maintaining the biodiversity and ecological integrity for persistent provision of ecosystem services, as well as grassland development initiatives intended for improving the productivity and sustainability of grassland areas for agricultural purposes.

The book is organized into two sections. Section 1, "Novel Grasslands Conservation Approaches", synthesizes fundamental concepts, recent advancements, and knowledge on sustainable management, biologically viable protection, economic utilization, effective restoration, and viable preservation of grasslands. Section 2, "Grasslands Development Initiatives", includes chapters that discuss recently developed initiatives that ensure the conversion of grasslands into more profitable, selfsustaining, and ecologically balanced natural or improved entities.

Chapter 1 discusses the fundamentals and advancements of grassland conservation and development using artificial intelligence (AI) tools. It suggests that AI-based tools might be developed and optimized as viable, reliable, and cost-effective methods to monitor and assess grassland production potential under changing pedo-climatic scenarios. Moreover, it elaborates that digitalized monitoring using automated tools might assist in recognizing native grass species as well as invasive species and analyzing soil fertility status for formulating effective management and conservation options. For instance, unmanned aircraft systems (UAS) integrating numerous types of cameras and sensors hold the potential to precisely collect scattered spatial and temporal information with remarkably high resolution in the visible and infrared

spectrum. Likewise, this chapter forwards the idea that AI, deep learning (DL), and machine learning (ML) could aid in the construction of 2D and 3D models for establishing grass species composition, total vegetation cover, barren patches, and nutritional quality of grass species. Furthermore, ML and AI algorithms may be employed for localizing grass species from data supplied by images and sensors. Using these approaches, grass species can be localized down to the centimeter scale and maps can be constructed to illustrate grass cover distribution on large swathes of grasslands.

Chapter 2 examines grasslands in terms of carbon sink, ecological barriers and watershed for low riparian regions, feedstock, mineral extraction sites for drilling and mining, and numerous associated benefits like wool, herbs for traditional medicines, tourism and leisure, and more in China and Pakistan. Additionally, it highlights that grassland ecosystems have been persistently degraded by anthropogenic disturbances (land use changes, tourism, intensive grazing, fire, vegetation clearance, invasive weeds such as knapweed, parthenium, Johnsongrass, etc.) and climate change drivers (heat, drought, chilling, salinity, and shifting of rainfall patterns) in both countries. Moreover, it suggests that for conserving grasslands, nitrogen (N) and carbon (C) in these countries' soils are integral for maintaining the primary productivity of grass species; hence estimating the extent of numerous interventions on N and C cycling along with grass–microbe interactions become imperative from socioeconomic and environmental perspectives. The chapter also compiles recent knowledge on the productivity status and persistent degradation of grasslands in China and Pakistan. Finally, the chapter analyzes the prevalence of invasive weeds in grasslands, grass– microbe interactions and their effect on the growth of plant species, microclimate, and availability of nutrients as well as synthesizes recent advances in C and N dynamics in grasslands ecosystems.

Chapter 3 proposes that grasslands in lowlands and mountains either in natural form or developed landscape can provide an added value in terms of ecotourism opportunities, owing to having huge esthetic and recreational potential compared to uniform agricultural areas. It has been objectively highlighted that grasslands characterized by high species and habitat diversity-based ecotourism are a form of nature-based tourism whereby people visit natural or developed areas for recreation, sightseeing, permitted and controlled hunting, on-site purchase of organic products, and more, and are usually managed by adopting sustainable practices. Moreover, it highlights that ecotourism can generate multifaceted economic advantages for local communities such as the direct sale of products to tourists. This chapter highlights a variety of negative impacts such as the high number of tourists, which leads to overuse of resources. Finally, the authors describe pronounced challenges to the development of grasslands for ecotourism, such as lack of community cooperation, careless herders, need of hefty investment, and absence of trained human capital along with climate change and loss of biodiversity.

Chapter 4 reports that prescribed grassland burning in Japan (commonly referred to as Noyaki in Japanese) for the purpose of grassland conservation has been conducted for over a thousand years by local residents. It highlights that biomass burning has emerged as one of the most significant sources of airborne substances including mercury. Additionally, during Noyaki, the chapter authors sampled and measured gaseous mercury from the Noyaki plumes to better understand mercury emission and cycle in the local environment. The chapter reports on the remarkable elevation of

atmospheric mercury concentrations, demonstrating the emission of gaseous mercury from the Noyaki. Lastly, the chapter discusses possible origins, novel information on the results inferred, and future research directions.

Chapter 5 highlights that many conventional farming approaches in developing nations segregate livestock and crop production, ignoring the synergistic advantages and sustainable land management possibilities that may be gained by combining the two. It suggests that in order to increase agricultural output and foster grassland conservation, the idea of merging livestock and crop systems must be considered. By highlighting the advantages and challenges of the approach, this chapter draws attention to the potential benefits of integration, including enhanced soil fertility, efficient resource use, increased productivity, and better protection of grassland ecosystems. Moreover, the chapter explores the importance and viability of a variety of integrated agricultural methods, including agro-pastoral, mixed, and silvopastoral systems, in a variety of geographical settings. Finally, this chapter culminates with solid suggestions to educate policymakers, academics, and practitioners about the need to integrate livestock and crop production to achieve long-term agricultural sustainability in low-income nations.

Chapter 6 focuses on determining the quality of grasses using spectroscopy techniques, specifically, spectrophotometry. It points out that chemical methods used to determine the nutritional quality of grasses produce chemical residues, are timeconsuming, and are costly to use when analyzing large crop extensions. Thus, the chapter authors suggest that spectroscopy, which is a non-destructive technique, can establish the nutritional quality of grass easily and accurately. This chapter describes the techniques focused on the use of spectroscopy and machine learning models to predict and determine the quality of grasses. Moreover, it compiles different methods and results focusing on the quality of the grasses. In general, it shows that the most used spectroscopic method is the near-infrared analysis. In addition, the statistical model most used by the authors to predict the quality of grasses is the partial least squares regression. Moreover, this chapter interestingly portrays spectroscopy as an effective tool that opens the way to new types of technologies for determining the quality of grasses.

Chapter 7 provides an overview of the fundamental aspects of cation exchange capacity (CEC) and its significance as a soil fertility indicator, particularly in evaluating pasture quality. It highlights the limitations of conventional methods and emphasizes the need to explore alternative approaches for rapid pasture quality assessment. In addition, this contribution focuses on the growing importance of hyperspectral information (HSI) as a valuable tool for efficient and accurate CEC estimation. It also discusses the advantages and challenges associated with using HSI data for CEC estimation and its potential to enhance decision-making in pasture and forage production. This chapter highlights that HSI technology allows for the capture and analysis of a wide spectrum of light reflectance information at various wavelengths, providing valuable insights into the physical and chemical characteristics of the soil.

Chapter 8 delves into the intricate relationship between agro-pastoralism and grassland ecosystems in Tanzania's Western Serengeti and Ugalla ecosystems. This chapter reveals that despite the acknowledged contribution of agro-pastoralism to rural well-being and economic development, its impact on the delicate balance of grassland ecosystems remains unclear in these crucial Tanzanian landscapes. Moreover, it illuminates agro-pastoralism's environmental, social, and economic dimensions in these regions. Guided by research questions exploring current conditions, potential solutions, and the path toward sustainable grassland resource utilization, the study employs a systematic approach to develop grasslands for promoting agro-pastoralism. Furthermore, it highlights challenges pertaining to the progressive expansion of agropastoral activities, leading to trade-offs between ecosystem services and productivity. Finally, this chapter concludes with the identification of agro-pastoral clusters across the area, revealing variations in economic activities and their impact on grassland utilization.

Key Features of the book:

I would like to acknowledge the strategically vital and pertinent intellectual support furnished by my mentor Dr. Asif Iqbal (Associate Professor, Department of Agronomy, University of Agriculture Faisalabad, Pakistan) and Dr. Chunjia Li (Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, China).

#### **Muhammad Aamir Iqbal**

Faculty of Agriculture, Department of Agronomy, University of Poonch Rawalakot, Azad Jammu & Kashmir, Pakistan

Section 1


**6. Surgical treatment of endometriosis**

*Molecular Bases of Endometriosis - The Integration Between Research and Clinical Practice*

concentrations do not exclude the disease, cases with elevated preoperative levels may aid in patient follow-up and clinical suspicion of recurrence of endometriosis. More recently, some cytokines have been studied as new nonsurgical endometriosis markers. Interleukin-6 (IL-6) appears to perform better than other cytokines in discriminating patients with endometriosis [4]. The first imaging test to be applied to the patient with a history and physical examination suggestive of endometriosis is transvaginal pelvic ultrasound, preferably with intestinal preparation. A study by Abrão et al. [5], evaluating the accuracy of this test, demonstrated a sensitivity of 94% and a specificity of 98% in the identification of foci of deep endometriosis. If the test is normal, the patient may not have endometriosis or have noninfiltrative initial disease. On the other hand, if the test is conclusive for ovarian endometriosis, rectovaginal septum or rectosigmoid, or urinary tract, treatment may be indicated without additional imaging tests. For evaluation of endometriomas larger than 2 cm, transvaginal ultrasonography is an efficient method, according to Moore et al. [6]. The presence of ovarian masses with a doubtful diagnostic hypothesis can be better evaluated with magnetic resonance imaging (MRI). Changes suggestive of rectovaginal septum disease, uterosacral, or rectosigmoid ligaments may be confirmed by rectal echoendoscopy or MRI. Rectal echoendoscopy allows the identification of the distance between the lesion and the rectal lumen as well as extrinsic compressions and submucosal lesions of the rectum [7]. MRI also allows the identification of deep disease with invasion of the intestinal tract, but it does not make it possible to specify the intestinal layer affected by the lesion [8]. Transvaginal ultrasonography for the diagnosis of bladder endometriosis has been reported as an effective method, with sensitivity of 71.4% and specificity of 100% [9]. Ultrasonography suggestive of bladder or ureteral endometriosis can be complemented with excretory urography, which may show ureteral narrowing. Uro-resonance can be used as an alternative method to excretory urography for evaluation of renal collecting system dilatations. Although the available imaging exams presented good accuracy in the diagnosis of endometriosis, laparoscopy with lesion biopsy for anatomopathological analysis is still the gold standard in the

After videolaparoscopy, endometriosis can be classified according to the histological type of the implants, with the anatomical location of the disease peritoneum, ovary or rectum—or by the extension of the disease to the pelvic organs. The most commonly used classification is that of the American Society of Reproductive Medicine—revised in 1996 [10]. This classification rates minimal, mild, moderate, or severe endometriosis due to the extent of disease in the peritoneum and ovaries, as well as the presence of tube-ovarian and Douglas sack bottom block. This classification, although with some limitations, is quite useful in the orientation of postsurgical treatment, especially when the patient's complaint is

The therapeutic approach to endometriosis varies, depending on the patient's complaint, pelvic pain or infertility, although such complaints are often associated. Gonadotropin-releasing hormone (GnRH) analogues, GnRHa, may be indicated for three months and then continue with oral contraceptives. If the patient has

**5. Critical analysis of treatments for endometriosis**

**2**

infertility.

diagnosis of endometriosis.

**4. Classification of endometriosis**

Surgical treatment of endometriosis involves procedures of low complexity, such as the treatment of superficial foci and the release of tendon adhesions, to complex interventions in the ovaries, Douglas sacs, intestines, bladder, and ureters, requiring, in some cases, a multidisciplinary team. For several years, the surgical treatment of endometriosis was based on the oncological principles of radical removal of the lesions. This principle is still used when it comes to cases of intestinal or ureteral stenosis or ovarian masses of doubtful characteristics. However, we currently know that there is no correlation between the disease with the severity of the symptoms, as well as the reproductive prognosis and long-term recurrence of pain [11]. In addition, many patients present infertility associated with pain, requiring that the surgical procedure be conservative. Based on these considerations, some authors recommend surgical treatment only for patients who do not respond to drug treatment, as well as for those who wish to become pregnant spontaneously [12]. There are few published randomized clinical trials evaluating the outcome of surgical treatment of symptomatic endometriosis. A review by Vercellini et al. [12] describes symptomatic improvement after conservative treatment of around 60–80%, with recurrence of symptoms and reoperation rate ranging from 12 to 58% between studies.

For the patient with infertility, follicle ablation and adhesiolysis appear to improve fertility in the minimal and mild degrees of disease [1]. In cases of moderate or severe degrees, there are no randomized clinical trials or meta-analyses available to answer if resection of foci would increase gestation rates.

#### **7. Molecular genetics and endometriosis**

Endometriosis exhibits similarity with cancer since endometrial cell implants require neovascularization to establish, grow, and invade tissues. In addition, the etiopathogenic theories of endometriosis involve growth factors and cytokines associated with regulation of cell multiplication and neoangiogenesis that may act on carcinogenesis. It is estimated that 1% of cases of endometriosis is related to cancer, and for some types of endometriosis, its benign nature has been questionable [13, 14].

Although the definitive diagnosis of endometriosis necessitates a surgical intervention, called video-laparoscopy, several findings in the physical, imaging and laboratory examinations can already predict, with a high degree of reliability, that the patient has this disease. During this surgical procedure, it is possible to visualize lesions suggestive of the disease and to obtain a tissue specimen for anatomopathological analysis and confirmation of the diagnosis of endometriosis [15]. The classification used for endometriosis is that of the American Society of Reproductive Medicine (ASRM), revised in 1996, which rates this disease in minimal (stage I), mild (stage II), moderate (stage III), or severe (stage IV) [16]. Currently, the most common treatments are surgery, ovarian suppression therapy, or the association of both [13, 15].

The cause of endometriosis remains unknown. However, there is evidence of immunological [17, 18], environmental [19], and genetic [18, 20] factors involved in its pathogenesis.

Regarding the immune response, the role of cytokines in the development of endometriosis [21–23] is highlighted, and elevated levels of several of them have been found in patients with endometriosis [23, 25]. The same group of investigators [24, 26] evaluated the levels of cytokines involved in the Th1 immune response patterns (interleukin (IL)-2, tumor necrosis factor (TNF)-alpha and interferon (IFN) and Th2 (IL-4 and IL-10) in patients with endometriosis (n = 65) and in those without the disease (n = 33). Podgaec et al. [24] observed elevation in IFNgamma and IL-10 levels in patients with endometriosis, evidencing the coexistence of both responses. However, when considering the ratio of cytokine levels to these responses, IL-4 and IL-10 predominated, thus reflecting a possible shift to the Th2 immune response component. In the subsequent study, 18 cytokine levels were associated with the clinical symptoms of endometriosis. Patients with endometriosis who had depth dyspareunia and infertility exhibited elevated levels of TNF-alpha and IL-2, respectively. These cytokines are related to the Th1 immune response, and almost 70% of the patients who presented these results exhibited deep endometriosis. The authors conclude that when specific clinical data are associated with elevated production of certain cytokines, there is a Th1 response pattern that may be associated with deep endometriosis. Induction of Th1 immune response was also reported by Fairbanks et al. [25], who showed elevated levels of IL-12 in patients with severe endometriosis.

The contribution of environmental factors to the development of endometriosis was reviewed by Bellelis et al. [19] who related their influence and diet to the genetics of this disease. They concluded that the mechanism by which dioxin and its similes (2,3,7,8-tetrachlorodibenzo-p-dioxin/TCDD and polychlorinated biphenyls/ PCBs) act to alter endometrial physiology is uncertain and speculative. They also state that there is insufficient evidence regarding the use of diets as preventive factors or even adjuvants in the treatment of endometriosis.

The genetic and hereditary basis of endometriosis was evidenced in the study by Bellelis et al. [19] in which approximately 5.3% of the patients reported a firstdegree family history with a history of endometriosis. Familial aggregation, a high concordance rate in monozygotic twins, and a 4–7% risk for first-degree relatives support a contribution of genetic factors to the pathogenesis of this disease [14]. In this context, the identification of genetic variants or single nucleotide polymorphisms (SNPs), responsible for susceptibility to endometriosis, has been the subject of investigation in recent years [26–28]. Different classifications were proposed for endometriosis candidate genes.

### **8. Conclusions**

What are the objectives of the genetic study of individuals? There is a great interest of the medical community and also much concern of the lay press about the potential benefits and harms of genetic screening, gene therapy, and even the possibility of cloning individuals. The current use of genetic tests for the detection and treatment of endometriosis is still at an early stage, but very important. The determination of susceptibility markers will be increasingly explored in clinical studies, and their uses will be much more defined.

Still, it seems increasingly likely that major changes will occur over the next decade in how we evaluate and treat our patients. In particular, surgeons and clinicians will have the opportunity to use a number of new tests to predict the future appearance of endometriosis in patients still free of the disease. They may have the power to explore the best therapeutic modality for a particular patient according to their genetic makeup. And they will be able to more specifically target prevention

**5**

**Author details**

Giovana Aparecida Gonçalves

provided the original work is properly cited.

1 Federal University of São Paulo (UNIFESP), Brazil

2 Father Albino University Center (UNIFIPA), Brazil

\*Address all correspondence to<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Introductory Chapter: Changes in the Approach of the Patient with Endometriosis…*

measures for family members of people already affected by the disease. It should be understood that molecular diagnosis, especially in asymptomatic individuals, does not mean disease, but an increased risk of developing this disease. Ethical implications exist and should not be underestimated. Patients should be advised about the likely implications of such tests not only after, but especially before the achievement

A major step has already been overcome and we currently have basic tools for a new leap in understanding human pathologies responsible for much of the world's mortality. Bridging the great barrier that still separates this basic knowledge from

*DOI: http://dx.doi.org/10.5772/intechopen.85608*

clinical practice is still a much greater challenge.

of these.

*Introductory Chapter: Changes in the Approach of the Patient with Endometriosis… DOI: http://dx.doi.org/10.5772/intechopen.85608*

measures for family members of people already affected by the disease. It should be understood that molecular diagnosis, especially in asymptomatic individuals, does not mean disease, but an increased risk of developing this disease. Ethical implications exist and should not be underestimated. Patients should be advised about the likely implications of such tests not only after, but especially before the achievement of these.

A major step has already been overcome and we currently have basic tools for a new leap in understanding human pathologies responsible for much of the world's mortality. Bridging the great barrier that still separates this basic knowledge from clinical practice is still a much greater challenge.


**Mary Frohlich**

Catholic Theological Union, Chicago, IL 60615, USA<EMAIL_ADDRESS>
Received: 10 March 2020; Accepted: 14 April 2020; Published: 16 April 2020

**Abstract:** In the period now being called the Anthropocene, the fatal vulnerabilities of the modern way of constructing selfhood are becoming ever more evident. Joanna Macy, who writes from a Buddhist perspective, has argued for the need to "green" the self by rediscovering its participation in ecological and cosmic networks. From a Christian perspective, I would articulate this in terms of an imperative to rediscover our spiritual personhood as radical communion in both God and cosmos. In this paper, "self" refers to an ever-restless process of construction of identity based in self-awareness and aimed at maintaining one's integrity, coherence, and social esteem. I use the term "person," on the other hand, to refer to a relational center that exists to be in communion with other persons. How—within the conditions of the dawning Anthropocene—can the tension between these two essential aspects of human existence be opened up in a way that can more effectively protect human and other life on Earth? This would require, it seems, harnessing both the self-protective and the self-giving potentials of human beings. The proposed path is to give ourselves over into the rhythms of the Spirit, being breathed in to selfless personal communion and out to co-creation of our refreshed selfhood.

**Keywords:** Anthropocene; selfhood; personhood; Spirit; Christology; breathing; self-loss; transformed self


*4.3. Enzyme-Responsive Gatekeepers*

MSN drug release can also be modulated by the enzymatic cleavages of ester, peptide, urea, and oxamide bonds decorated on theMSN surface. Several enzymes such as esterase, protease, galactosidase, amylase, lipase, etc. have been exploited for enzyme responsive controlled drug release [166]. In this regard, Patel et al. have introduced ester bonds between MSN and the adamantine capping moiety, to employ the enzymatic role of porcine liver esterase for the controlled release of cargos [167]. Mondragón et al. have exploited protease cleavable ε-poly-l-lysine moiety to seal the camptothecin encapsulated MSN and reported the reduced viability of human cervix epitheloid carcinoma cells upon treatment of that nanosystem [168]. They have also reported some enzyme-responsive hydrolyzed starch products as saccharides to be used for controlled drug release [169]. There are various other protease-responsive moieties that have been used to cap the MSN pores and improve the drug release, e.g., protease-responsive biotin-avidin [170], arginine-rich protamine proteins [171], matrix metalloproteinase (MMP) degradable gelatin [172], avidin with MMP9-sensitive peptide linker (RSWMGLP) [173], poly (ethylene glycol) diacrylate moiety with protease-sensitive peptide linker (CGPQGIWGQGCR) [174]. Furthermore, cyclodextrin gatekeepers and HRP-polymer nanocapsules have also been employed on the MSN surface for enzyme-responsive drug release [175,176].

## *4.4. Magnetic Responsive Delivery System*

One of the e ffective ways to exploit external stimuli is to exert the magnetic field on MSN, either to have magnetic guidance by applying the permanent magnetic field, or to increase the temperature by applying an alternating magnetic (AM) field [177,178]. In this regards, iron oxide has been widely exploited as the required magnetic component. There are mainly two ways that have been used to conjugate iron oxide with MSN, either using iron oxide core coated with mesoporous silica or MSN capped with iron oxide nanoparticles [179,180]. The most employed strategy consists on encapsulating superparamagnetic iron oxide nanoparticles (SPIONs) of ca. 5–10 nm within the MSN network during their synthesis [181,182]. These SPIONs are able to convert the magnetic energy into heat and can increase the local temperature of the system upon application of the AM field. If the surface of MSN has already been coated with temperature responsive moieties acting as gatekeepers, e.g., poly (N-isopropylacrylamide), pore opening and drug release from MSN can be triggered by applying an AM field [183]. Taken together, upon application of an AM field, SPIONs encapsulated in MSN can increase the local temperature up to a certain point, to change the conformation of the temperature responsive gatekeepers and open the pore entrances to release the anti-cancer drugs e fficiently without having any premature leakage. There are several reports showing the controlled release of anti-cancer therapeutics by applying a magnetic stimulus [180,184,185]. Moreover, there are a few FDA-approved SPIONs for using as imaging agents and EU-approved iron oxide nanoparticles to use in glioblastoma therapy; these can be further exploited in magnetic responsive drug delivery [20].

Another strategy for the design of the magnetic responsive delivery system consists of the functionalization of drug-loaded MSN with a single DNA strand and then mixing this with SPIONs functionalized with the complementary DNA strand, to allow DNA hybridization that can act as a capping agent [186]. The reason behind selecting the DNA sequence is its melting temperature of 47 ◦C. Thus, upon application of an AM field, SPIONs encapsulated into the MSN network can increase the local temperature that subsequently trigger the double-stranded DNA melting and open the pores of MSN to release the drug. Interestingly, when the magnetic field is switched o ff, the DNA hybridization occurs again, thus closing the pores and stopping the drug release. This mechanism smartly provides the chance of exploiting the on-o ff drug release mechanism.


**4. Parasite culture system**

340 Current Topics in Tropical Medicine

al., 1997). In addition to the wide geographical distribution and their use in folk medicine, the interest in these compounds and plant extracts is based on the fact that it is easy to isolate secondary metabolites and to propagate the plant, which has a short reproductive cycle. Thus, considering the *in vitro* schistosomicidal activity of the amide piplartine, the importance of more research on the biological activity of the natural compounds isolated

Schistosome species are dioecious (having male and female reproductive organs in separate individuals) platyhelminthes and have complex life cycles comprising multiple morphologically distinct phenotypes in definitive mammalian and intermediate snail hosts. *S. mansoni* is one of the most common etiological agents of human schistosomiasis and is the most widely used schistosome model for chemotherapeutic studies.Schistosome infection of humans (or another definitive host) occurs by direct contact with freshwater containing freeswimming larval forms of the parasite, known as cercariae. Cercariae penetrate the intact human skin and transform into schistosomula, which reside in the skin for up to 72 hours before entering a blood vessel. Within the vascular system, schistosomula migrate via complex routes to their nal venous destination, where they mature into male and female adults. The mature flukes dwell in the human portal vasculature, depositing eggs in the intestinal wall that either pass to the gut lumen and are expelled in the faeces or travel to the liver and trigger immune-mediated granuloma formation and peri-portal fibrosis. Egg production commences 5 to 6 weeks after infection and continues for the life of the worm. The life cycle is completed when the eggs passed in the faeces hatch in the water, releasing the larval form miracidia, which then infect freshwater snails of the *Biomphalaria* spp. The infected snails, bearing schistosomal sporocysts, release cercariae into the water, which in

To complete a life cycle in the laboratory, *S. mansoni* is commonly maintained using rodents, ranging from hamsters to mice, as the definitive hosts and *Biomphalaria glabrata* as the intermediate host snail species (Figure 1). Infections of rodents of the same gender, 3 to 4 weeks of age and weighing 18 to 25 g, with *S. mansoni* are commonly initiated by subcutaneous injection of 100 to 150 cercariae (infective larvae). At 42, 49 or 56 days postinfection, animals are sacrificed with CO2, dissected, and miracidia are hatched from *S. mansoni* eggs taken from animal livers. Each intermediate host snail is exposed to approximately 10 miracidia. All animals should be handled in strict accordance with good animal practice adhering to the institutional guidelines for animal husbandry. Thus, all studies should have a statement from their ethics committee or institutional review board

The use of *in vivo* animal models in drug discovery and the techniques used for these studies in the laboratory have recently been described in detail elsewhere by Keiser (2010) and Ramirez et al. (2007). This chapter will focus on the techniques used for *in vitro* studies. Many methods have been described that aim to determine the antischistosomal activity of drugs *in vitro*. The assessment of the viability of different stages of schistosome, tegumental changes, oviposition, toxicity in mammalian cells and other parameters are important in the search for antischistosomal substances. The techniques detailed here will be the key to better

**3.** *Schistosoma mansoni* **life cycle and maintenance in the laboratory**

from the family Piperaceae and other plants is apparent.

turn penetrate the skin of their denitive host (Gryseels et al., 2006).

indicating the approval of the research.

assess the methodology employed during screening tests.

*In vitro* studies with schistosomula, juvenile and adult worms of *S. mansoni* are frequently used in screening strategies for the discovery of new antischistosomal drugs (Abdulla et al., 2009; Keiser, 2010; Mølgaard et al., 2001; Peak et al., 2010; Ramirez et al., 2007; Smout et al., 2010; Yousif et al., 2007). Parasites at different stages might show differences with regard to drug sensitivity. The *in vitro* methods currently utilised have recently been reviewed, and following the establishment of the *S. mansoni* life cycle in the laboratory, *in vitro* parasite culture techniques were developed (Keiser, 2010; Ramirez et al., 2007). For *in vitro* trials, parasites of different ages are used, such as 3-h-old and 1-, 3-, 5- and 7-day-old schistosomula, 21 day-old juveniles, and 42- to 56-day-old adults. Figure 1 shows the life cycle of *S. mansoni* in the laboratory, illustrating the collection points for *in vitro* chemotherapeutic studies.

Fig. 1. Life cycle of *S. mansoni*, illustrating the collection points for *in vitro* chemotherapeutic studies. Black arrow: maturation of parasite within final host. Blue arrow: aquatic phase

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 343

Obtaining sufficient quantities of schistosomula directly from skin or lung tissue for most research purposes is time consuming and involves working with mammalian hosts. These difficulties led to the development of techniques for transforming cercariae and maintaining schistosomula. In fact, schistosomula can be obtained by transforming cercariae using simple techniques, such as centrifugation, vortexing, repeated aspiration through a syringe needle, or chemical stimulation, and these schistosomula are easily maintained *in vitro* for several days (Basch, 1981). These *in vitro* strategies are advantageous because they confer uniformity in parasite maturation, which cannot be achieved *in vivo* due to the variation in the time required for individual parasites to penetrate the host skin and enter the vasculature. In addition, the use of mechanically obtained schistosomula is an alternative method that reduces, refines and replaces the use of animals in laboratory research in accordance with animal protection principles

Among the techniques available for the production of schistosomula, the current methods most commonly used for the removal of tails from cercariae are the repeated aspiration through a syringe needle, based on Colley and Wikel (1974), and the use of a Vortex mixer, based on Ramalho-Pinto et al. (1974). In our laboratory, for example, schistosomula are mechanically transformed according to Ramalho-Pinto et al. (1974) and cultured *in vitro* in 169 medium, as described by Bash (1981). This method is recommended over the syringepassage technique, which severely stresses and damages the parasites. In addition, the mechanical transformation procedure in a Vortex mixer is simpler than the passage of the parasites under pressure many times through a needle. Briey, to obtain cercariae, *B. glabrata* snails infected with miracidia are exposed to incandescent light for 2 h, and then cercariae are collected, concentrated in glass conical centrifuge tubes and cooled in a ice bath for 10 minutes to reduce the motility of the worms. The ice-cold cercarial suspension is centrifuged, resuspended in RPMI 1640 medium with 25 mM HEPES, 200 UI/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, and vortexed for 2 minutes to trigger tail loss. The resulting cercarial bodies are isolated from free tails by centrifugation through a 60% Percoll gradient (Lazdins et el., 1982) or decantation (Ramalho-Pinto et al., 1974). Microscope examination is used to assess the quantity and quality of purified schistosomula. Finally, schistosomula are cultivated in 169 medium containing antibiotics and supplemented with 10% bovine foetal serum at 37 ºC in a 5% CO2 atmosphere. Schistosomula are cultured until day 7, which corresponds to the lung-stage worm. For *in vitro* drug screening assays, schistosomula are transferred into 96-well culture microplates, with approximately 50 parasites per well, and maintained in 200 µl 169 medium under the

After penetrating the definitive host, significant morphological, physiological, and biochemical changes occur in the developing schistosomula (Gobert et al., 2007; Skelly & Alan Wilson, 2006). Although mechanically transformed schistosomula are different from the schistosomula that have penetrated the skin, these larval cultures have been maintained in *vitro* for different amounts of time to produce all the mammalian host stages, including skin- and lung-stage schistosomula and paired, mature adult males and females (Basch, 1981). Mechanically transformed schistosomula are now commonly used in studies of behaviour, development, metabolic activity, biochemistry, molecular biology, immunology,

**4.2 Schistosomula**

(Broadhead & Bottrill, 1997).

conditions described above.

#### **4.1 Juvenile and adult schistosomes**

Adult worms have been more commonly used for antischistosome drug discovery. Today, *in vitro* chemotherapeutic studies using juvenile worms are also highly recommended. For these assays, each rodent is commonly infected with either 100-150 or 400-500 cercariae. Rodents exposed to 400-500 cercariae are sacriced 21 days after infection for juvenile recovery, while mice exposed to 100-150 cercariae are sacriced 42 to 56 days after infection for adult recovery. Juveniles or adults are collected using a perfusion technique with Hanks' balanced salt solution (HBSS), Dulbecco's Modified Eagle's Medium (DMEM), or Roswell Memorial Park Institute (RPMI) 1640 medium, containing an anticoagulant such as heparin at a concentration range of 5-20 U/ml (Smithers & Terry, 1965). The worms are washed in RPMI 1640 medium, kept at pH 7.5 with 25 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES) containing 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. After washing, ten to fifteen juveniles or one pair of adult worms (male and female coupled) are transferred to each well of a 24-well culture plate containing 2 ml of the same medium supplemented with 10% bovine foetal serum and incubated at 37 ºC in a humid atmosphere containing 5% CO2. Only viable, contractile worms showing total tegument integrity as assessed by light microscopy should be included in the different investigations (Figure 2).

Fig. 2. *In vitro* assay models with *Schistosoma mansoni* adult worms.

#### **4.2 Schistosomula**

342 Current Topics in Tropical Medicine

Adult worms have been more commonly used for antischistosome drug discovery. Today, *in vitro* chemotherapeutic studies using juvenile worms are also highly recommended. For these assays, each rodent is commonly infected with either 100-150 or 400-500 cercariae. Rodents exposed to 400-500 cercariae are sacriced 21 days after infection for juvenile recovery, while mice exposed to 100-150 cercariae are sacriced 42 to 56 days after infection for adult recovery. Juveniles or adults are collected using a perfusion technique with Hanks' balanced salt solution (HBSS), Dulbecco's Modified Eagle's Medium (DMEM), or Roswell Memorial Park Institute (RPMI) 1640 medium, containing an anticoagulant such as heparin at a concentration range of 5-20 U/ml (Smithers & Terry, 1965). The worms are washed in RPMI 1640 medium, kept at pH 7.5 with 25 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid (HEPES) containing 200 U/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. After washing, ten to fifteen juveniles or one pair of adult worms (male and female coupled) are transferred to each well of a 24-well culture plate containing 2 ml of the same medium supplemented with 10% bovine foetal serum and incubated at 37 ºC in a humid atmosphere containing 5% CO2. Only viable, contractile worms showing total tegument integrity as assessed by light microscopy should be included

**4.1 Juvenile and adult schistosomes**

in the different investigations (Figure 2).

Fig. 2. *In vitro* assay models with *Schistosoma mansoni* adult worms.

Obtaining sufficient quantities of schistosomula directly from skin or lung tissue for most research purposes is time consuming and involves working with mammalian hosts. These difficulties led to the development of techniques for transforming cercariae and maintaining schistosomula. In fact, schistosomula can be obtained by transforming cercariae using simple techniques, such as centrifugation, vortexing, repeated aspiration through a syringe needle, or chemical stimulation, and these schistosomula are easily maintained *in vitro* for several days (Basch, 1981). These *in vitro* strategies are advantageous because they confer uniformity in parasite maturation, which cannot be achieved *in vivo* due to the variation in the time required for individual parasites to penetrate the host skin and enter the vasculature. In addition, the use of mechanically obtained schistosomula is an alternative method that reduces, refines and replaces the use of animals in laboratory research in accordance with animal protection principles (Broadhead & Bottrill, 1997).

Among the techniques available for the production of schistosomula, the current methods most commonly used for the removal of tails from cercariae are the repeated aspiration through a syringe needle, based on Colley and Wikel (1974), and the use of a Vortex mixer, based on Ramalho-Pinto et al. (1974). In our laboratory, for example, schistosomula are mechanically transformed according to Ramalho-Pinto et al. (1974) and cultured *in vitro* in 169 medium, as described by Bash (1981). This method is recommended over the syringepassage technique, which severely stresses and damages the parasites. In addition, the mechanical transformation procedure in a Vortex mixer is simpler than the passage of the parasites under pressure many times through a needle. Briey, to obtain cercariae, *B. glabrata* snails infected with miracidia are exposed to incandescent light for 2 h, and then cercariae are collected, concentrated in glass conical centrifuge tubes and cooled in a ice bath for 10 minutes to reduce the motility of the worms. The ice-cold cercarial suspension is centrifuged, resuspended in RPMI 1640 medium with 25 mM HEPES, 200 UI/ml penicillin, 200 µg/ml streptomycin, and 0.25 µg/ml amphotericin B, and vortexed for 2 minutes to trigger tail loss. The resulting cercarial bodies are isolated from free tails by centrifugation through a 60% Percoll gradient (Lazdins et el., 1982) or decantation (Ramalho-Pinto et al., 1974). Microscope examination is used to assess the quantity and quality of purified schistosomula. Finally, schistosomula are cultivated in 169 medium containing antibiotics and supplemented with 10% bovine foetal serum at 37 ºC in a 5% CO2 atmosphere. Schistosomula are cultured until day 7, which corresponds to the lung-stage worm. For *in vitro* drug screening assays, schistosomula are transferred into 96-well culture microplates, with approximately 50 parasites per well, and maintained in 200 µl 169 medium under the conditions described above.

After penetrating the definitive host, significant morphological, physiological, and biochemical changes occur in the developing schistosomula (Gobert et al., 2007; Skelly & Alan Wilson, 2006). Although mechanically transformed schistosomula are different from the schistosomula that have penetrated the skin, these larval cultures have been maintained in *vitro* for different amounts of time to produce all the mammalian host stages, including skin- and lung-stage schistosomula and paired, mature adult males and females (Basch, 1981). Mechanically transformed schistosomula are now commonly used in studies of behaviour, development, metabolic activity, biochemistry, molecular biology, immunology,

Antischistosomal Natural Compounds: Present Challenges for New Drug Screens 345

Current methods utilised to assess schistosomal viability have recently been reviewed, and most of these methods involve microscopic techniques (Keiser, 2010; Ramirez et al., 2007). The phenotypic changes are scored by using a viability scale. For example, a scale of 0 – 4, where 4= normally active, 3= slowed activity, 2= minimal activity, 1= absence of motility apart from gut movements, and 0= total absence of mobility, is based on standard procedures for compound screening at the Special Programme for Research and Training in Tropical Diseases, World Health Organization, WHO-TDR (Ramirez et al., 2007). Alternatively, as described by Manneck et al. (2010, 2011), drug activity is defined as 3= totally vital, normally active, and no morphological changes; 2= slowed activity, primary morphological changes and visible granularity; 1= minimal activity, severe morphological changes and granularity; 0= all worms dead, severe morphological changes and granularity; the granularity is characterised only for schistosomula. The regular movement of both larval and adult schistosomes has proven to be a valuable trait in assessing schistosome viability *in vitro* because lack of movement is a good indicator of death. Worm death is usually dened as no movement observed for at least 2 min of examination (Manneck et al., 2010). In this context, the viability of worm during the culture period is also assessed by motor activity reduction, and it is defined as "slight " or "significant". This subjective criterion is commonly used by several research groups (Braguine et al., 2009; de Melo et al., 2011; de Moraes et al., 2011; Magalhães et al., 2009, 2010; Moraes et al., 2011, Parreira et al., 2010; Pereira et al., 2011; Xiao et al., 2007). Size measurements of parasites are also employed to study phenotypic

In addition to the phenotypic approaches, another *in vitro* drug-screening assay method is based on microcalorimetry. Manneck et al. (2011) analysed the effects of drugs on the metabolic activity of schistosomula and adult *S. mansoni* by comparing their heat ow. In this study, a multi-channel isothermal microcalorimeter equipped with 48 measuring channels was used to monitor the heat production by schistosomes as a result of their metabolism over time. The results show that microcalorimetry can be a valuable tool to study antischistosomal drugs, and the microcalorimetric measurements conrmed, in part, the results of the phenotypic evaluation. However, the level of agreement between microscopy and microcalorimetry data requires further investigation (Manneck et al., 2011). In the following section, other methods are described that are used to determine the effect of

Phenotypic changes are determined as mentioned above. However, because of the lack of standardisation between laboratories, the replication of results obtained by microscopic means is not always possible. In an effort to avoid the subjective nature of quantifying schistosome viability from the microscopic examination of phenotype alone, further adaptations have been developed and are based on the differentiating potential of some colorimetric vital dyes. Diamidinophenylindole (DAPI) has been used as a differential stain of dead schistosomula during microscopy; in addition, the low DAPI concentration (1 µg/ml) in the medium proved not to be toxic to the schistosomula, nor did it cause any background fluorescence (Van Der Linden & Deelder, 1984). Trypan blue has also been shown to be a reliable dye for differentially staining dead schistosomula (Harrop & Wilson, 1993) and by means of a methylene blue dye exclusion test (Gold, 1997). The tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is also a vital dye that has been successfully used to assess the viability of worms. The use of this assay on helminths was pioneered by Comley et al. (1989), and several nematode

changes.

drugs on schistosomula and adult *S. mansoni.*

and in vaccine development and drug screening protocols (Abdulla et al., 2009; Gobert et al., 2007; Harrop & Wilson, 1993; Peak et al., 2010). There is evidence that mechanically transformed schistosomula are structurally similar to their lung schistosomula counterparts (Chai et al., 2006), and after 7 days in culture, the larvae have the morphological features of lung worms and are capable of maturation when introduced into the portal vein of mice (Harrop & Wilson, 1993). Furthermore, mechanically transformed schistosomula are able to develop steadily until adult worm pairing (Basch, 1981). Because of these reasons, drugscreening assays in our laboratory are based on mechanically transformed schistosomula of different ages *in vitro* (3-h-, 1-, 3-, 5- and 7-day-olds). It takes roughly 3 h for the cercariae to secrete the contents of their acetabular glands; the 1- to 7-day-olds correspond to the skinand lung-stage schistosomula.