**2.4. 22q11 deletion syndrome (DiGeorge syndrome, velocardiofacial syndrome, conotruncal anomaly face syndrome)**

Molecular studies demonstrate that the vast majority of patients with clinical diagnosis of DiGeorge, velocardiofacial, conotruncal anomaly face syndromes share a common genetic cause, namely a 22q11.2 deletion [43–45]. The prevalence is 1 in 4000 to 6000 live births, but the severity of the phenotype is variable and in some the features may go unrecognized [46]. CHD are estimated to occur in 75–80% of patients with a 22q11.2 deletion. The most common CHDs associated with 22q11.2 deletion include tetralogy of Fallot (TOF) and aortic arch anomalies. Other conotruncal lesions include pulmonary atresia with ventricular septal defect (VSD), and truncus arteriosus [47, 48]. In cases of truncus arteriosus, the truncal valve tends to be more dysplastic when the 22q11 deletion is present [49].

Identifying the cardiac patient with a 22q11.2 deletion early in life can provide substantial benefits to the child and family. Currently, it is recommended that infant with interrupted aortic arch type B, truncus arteriosus, TOF and isolated aortic arch anomalies undergo testing for a 22q11.2 deletion [22]. Prenatal diagnosis can be performed using FISH technology on samples obtained from chorionic villus sampling or amniocentesis if cardiac defects are present. Non-cardiac lesions are not easily diagnosed but, absent fetal thymus has been reported [50]. Many children initially present without cardiac symptoms in the form of recurrent infections. This may point to an underlying immunodeficiency associated with 22q11.2 deletion [51]. The risk of recurrence is in 50% as this is transmitted in an autosomal dominant inheritance if either parent carries the deletion. If neither parent carries the deletion, there may still be germline mosaicism, but the risk of recurrence is small (1%) [22, 48].


**4. Role of continuous miner in Indian coal mechanization**

The new era of mechanized underground coal mining in India started with the implementation of continuous miner in 2002 [44]. Since then, there has been a steady

#### **Table 2.**

*Evolution of continuous miner.*

increase in continuous miners implemented in Indian coal mines with increasing mechanization and safety in underground mines. But most of the 213 underground coal mines are still highly dependent on conventional mining. This is high time to adopt complete mechanized mining in all underground coal mines with continuous miner technology.

Even though continuous miner is a major change in mechanization and safety in Indian coal mines, but still are not able to increase underground coal production due to adverse strata conditions, lack of implementing the suitable mining method prior to existing geo-mining conditions, lack of understanding of the caving nature of overlying strata before starting depillaring, which eventually results in a loss in production by leaving the number of pillars untouched in safety point of view. Undoubtedly, the future of Indian underground coal mining lies in successful pillar extraction with continuous miners. This is high time to study the stability parameters influencing the continuous miner panel workings in underground coal mines. Evolution of continuous Miner [45–48] is shown in **Table 2**.


Contents

Preface

Geochemistry has always been a multidisciplinary scientific field, characterized by bringing together and synthesizing knowledge and tools from numerous related subjects. It is also a dynamic field of knowledge which, from its formal origins in the 19th century to the present day, has encountered and confronted new challenges. Without abandoning research in mature fields, such as mineral exploration or the geochemical characterization of tectonic processes, the interest of geochemistry has been directed, in recent decades, to some of the most imminent challenges for our society, such as the effects and possible mitigation of climate change or the impact of our society's habits on the health of people and ecosystems. As has happened since the beginning of this scientific discipline, the development of these new fields of geochemistry is linked to advances in the available instrumental techniques for an increasingly precise characterization of the distribution of elements, substances, mineral species, and isotopes in the different geological and environmental matrices. All the aspects mentioned above about geochemistry are reflected in this book, in which researchers from five continents make use of advanced analytical techniques (i.e. Mössbauer spectroscopy, Itrax X-Ray Fluorescence core scanning), and tools and concepts of nearby or auxiliary disciplines (geophysics, remote sensing,

biology) to advance the knowledge of complex geochemical problems.

producing this book.

We would like to thank all contributors greatly for their collaboration and effort in

**Luis Felipe Mazadiego, Eduardo De Miguel Garcia, Miguel Izquierdo-Díaz and Fernando Barrio-Parra**

Escuela Técnica Superior de Ingenieros de Minas y Energía,

Universidad Politécnica de Madrid,

Madrid, Spain


Another issue is psychometric test validity when assessing learners in classrooms. As Phakiti & Isaacs (2021) argued, "classroom assessment scores cannot be correlated with other external test scores because classroom assessment is used to help the students to improve their skills and overcome any learning difficulties through instructional support" (2021: 9). Assessment quality should be the approach. In their model, the authors proposed the following components for what they called *assessment quality* rather than *validity.* This model includes the following components: (1) validity, (2) reliability, (3) practicality, (4) authenticity, (5) ethics, (6) fairness, and (7) effect. It also proposes to start with the intended learning outcomes, and that classroom activities and classroom assessment need to be aligned with the outcomes.

Yet, the challenges classroom instructors continue to face are related to institutional policies as well as the rise in standard tests assessment. This generates in some cases the need for teachers to focus on the tests rather than on the learning process and improvement of their students. Another issue is that alternative forms of assessment require more time spent in the planning stages of the activities. The development of some assessment instruments such as rubrics and group activities to assess different language skills may also require cooperation between teachers. Depending on the class composition, there will be a variety of

### 8 Pedagogical implications of assessment in multilingual contexts

challenges related to group work and task completion. However, these activities are more aligned with providing learners with opportunities to engage with their context. Awareness and understanding of assessment practices, institutional policies, and students' academic needs as well as their sociocultural context should be considered in the design of assessment that fosters students' learning through assessment quality criteria.

### **3.2 Computer-mediated communication and assessment in multilingual contexts**

The role of technology in assessment has evolved over the last two decades. The language learner of the 21st Century is constantly learning through different computer-mediated communication outlets. Social media technology and our ability to text and communicate in different languages has revolutionized and expanded the way we communicate and use languages (Thorne et al. 2021; Crystal 2009; Thorne 2008) and how we assess language learners through the use of videogames (Thorne et al. 2012; Gee 2014). As global citizens, we have moved quickly from texting with limited characters and communicating a message through short texts, to smart phones and social media in which we can basically generate entire documents to share with a wide audience through multimodal approaches to literacy. This has also facilitated processing information in different ways and through multiple languages including the use of technology and *netspeak* (Crystal 2009). These technologies have challenged our reality and altered how we communicate in real time with others around the world. Learners are now more interested in learning foreign languages due to their particular interests. Watching *anime* cartoons, reading comic books such as *Manga* for Japanese learning, and playing videogames, are examples of the motivations for younger generations to use language. Assessment practices are also challenged by these new ways of learning. As such, in multilingual contexts learners are tested in two or more languages simultaneously and their performance is assessed on how well they complete a given task instead of how well they validate and use one of their languages. In this regard, Larsen-Freeman (2018) fittingly points out that "while this type of assessment may not be widely adopted any time soon, computer adaptive testing may well lend itself to more developmentally sensitive, self-referenced assessment, instead of approaches that resemble traditional standardized exams" (2018: 63).

Recent events such as the pandemic and natural disasters are forcing language educators to rethink assessment during these challenging times. Alternative assessments become the way in which the world continues to collaborate through

synchronous and asynchronous instruction. This has also generated new opportunities to engage with learners in different ways that were not previously considered by more traditional assessment methods.

The present volume aimed to provide a snapshot of some alternative assessments that address different realities and needs and initiate a dialogue on future research and additional modalities of alternative assessments in language learning in different communities of practice with other learner profiles. The limited scope of the research findings covered in this volume was due to the contexts the authors were operating mostly in Higher Education in the United States, and the Amazon of Peru and the languages involved (e.g. Heritage language learners of Spanish, Portuguese). Further research in language assessment should include additional language profiles in multiple communities of practice such as sign language learners, heritage language learners of other languages different from Spanish in the U.S. for instance, and study of language assessment in indigenous communities around the globe, to identify a few. Another area of much needed research would be assessment of language learning in hybrid and fully online educational settings. Additionally, pedagogical and assessment challenges (and solutions) remain to be fully explored when addressing disparities between formal and informal assessment practices, validity issues and teaching training. An interesting line of inquiry worth pursuing when advancing our knowledge on alternative assessments for language development would be to determine learners' (and also instructors') dispositions by assessing how receptive they are to the proposed assessments, how willing they are to continue to learn about them, apply them, and be influenced by them. The research findings presented in this volume, though yielding more questions than answers, provides a promising research agenda and dialogue for scholars interested in assessment of language learning.

 It seeks to initiate a conversation among scholars about inclusive practices in language assessments. Whether the student is a second language learner, a heritage language learner, a multilingual language speaker, a community member, the authors in the present volume provide examples of assessment that do not follow a single universal or standardized design but an applicable one based on the needs and context of a given community. The contributors in this volume are scholars from different disciplines and contexts in Higher Education. They have created and proposed multiple lower-stakes assignments and accommodated learning by being flexible and open without assuming that learners know how to do specific tasks. Each chapter provides different examples on Justice, Equity, Diversity, and Inclusion (JEDI) assessment practices based on observation, examination, and integrative notions of diverse language scenarios. It may be of interest to researchers and practitioners in the fields of curriculum and instruction, language learning, and applied linguistics as well as those in the field of language teaching in general. Thus this volume broadens the scope of research in the area of multilingual assessment.


**Author details**

*The Human Auditory System - Basic Features and Updates on Audiological Diagnosis and Therapy*

about sound field changes when the IACCE3 value changed. ASW changes between (ASW (0.03) and ASW (0.45)), which were more evident in the right hemisphere, affected both right and left hemispheres. They are coherent while the N2 latency of SVR significantly prolonged in both left and right hemispheres under changes of subjective diffuseness in IACCE3 found by Ando et al. [20]. Different sites are activated

Cerebral specialization has been reported to be determined by focused conscious decisions. For instance, Floel et al. [23] conducted a spatial—visual focus experiment and used a Doppler ultrasound system and magnetic resonance imaging (MRI) equipment to observe the brain reactions of right-handed participants; the researchers found that both spatial recognition and speech functions were activated in the right hemisphere, which corresponded to clinical

Nevertheless, for CBW researches, we conclude that α-waves (8–12 Hz) mainly responds to the emotional reactions; β-waves (13–30 Hz) reacts to the auditory matter drift (**Figures 6** and **7**). But the left hemisphere leads focus or attention on the varying of situational conditions (**Figures 8** and **9**), and the right one blends with imaginable feeling and experience. Hemispheric specialization has to pay attention to the conditioned response, conscientious and careful detail to setup each

I would like to express my thanks to the graduated students, Yong-Shang Chen and Qi-Wen Lin who have, in brainwaves' experiments, helped me in the course of preparing this study. In particular, I wish to thank Professor Em. Yoichi Ando, who kindly gave me directions on my brain research and data analysis methodology. I am also indebted to the Ministry of Science and Technology Taiwan, for their 2 years period (2007 and 2012) of financial support to complete this research. Meanwhile, the application conformed to academic ethics for the conduct of

research. Moreover, during the brainwave experiments, the subjects were assured of their safety and told the procedure was non-invasive. Special thanks are due to my many colleagues for their participation in the experiments involving the subjective

ASW apparent source width, a sound perception of the subjective dif-

CBW continuous brainwave, a term to distinguish from an evoked poten-

SVR slow vertex response, an evoked potential is a direct result after a specific sensory stimulus in the period of 10–500 ms LEV listener envelopment, a sound perception of the subjective diffuse-

SD semantic differences, a method of questionnaire employed the scale of responses caused by a psychological affection

τe effective delay of autocorrelation function (ACF)

tial (EP) or evoked response within EEG

ness occurred after 80 ms of stimulus

fuseness occurred from beginning to 80 ms of stimulus Δt1 delay gap between direct and first reflection in a defuse sound field IACCE3 binaural initial (<80 ms) interaural cross-correlation function

by brainwaves during focused and ambient use of the brain.

experiment results.

brainwaves' experiment.

**Acknowledgements**

**Glossary of symbols**

judgments and the brainwaves' recordings.

ΑCF autocorrelation function

**46**

Chiung Yao Chen Department of Architecture, Chaoyang University of Technology, Taiwan

\*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, provided the original work is properly cited.


**Hypothesis 3 (H3).** *Environmental influences have a positive effect on sustainable consumption behavioural intention.*

In the framework of the European Union's sustainable policies, the promotion of sustainable development thinking has been a key issue. The promotion of the sustainability idea supports alternative consumption patterns; it increases consumers' knowledge and awareness connected with environmental and social problems, changes consumption and purchase behaviours, and enhances the acceptance level of sustainable consumption (Radziszewska 2019). In previous research, attention was paid to different contextual variables that have an impact on individuals' sustainable consumption behaviour, such as the price of green products, the availability of infrastructure, policy incentives (Wang et al. 2014), situation (Pimdee 2020), economic factors (Wang et al. 2019), etc. However, as far as we know, there are very few studies presenting empirical evidence of the effect of the SC construct on SCB. For instance, Sousa et al. (2022) confirm the existence of a positive relationship between companies' green communication and green purchase intentions. The findings of Piligrimiene et al. ˙ (2020) showed that the respondents had a positive attitude to green product promotion and to the application of other "engaging mechanisms" for sustainable consumption promotion. Therefore, it is hypothesised that:

**Hypothesis 4 (H4).** *SC promotion has a positive effect on sustainable consumption behavioural intention.*

## *2.3. Influence of Environmental Knowledge, Materialism, Environmental Influences, Promotion and Sustainable Consumption Behavioural Intention on Sustainable Consumption Behaviour*

Based on the theoretical background, we also decided to study the direct effect of the influencing factors selected by us on SCB. The same approach was adopted by other researchers as well (Saari et al. 2021; Wang et al. 2014; Brandão and Cupertino de Miranda 2022; Le 2020; Joshi and Rahman 2019; Nekmahmud et al. 2022). Most empirical results supported the conventional view that knowledge and behaviour are positively related (Wu et al. 2016). Figueroa-García et al. (2018) revealed that sustainable consumption behaviour was determined by environmental influences (in particular, the influence of family and friends, as well as that generated by cultural factors such as traditions). In conformity with other research, Matharu et al. (2021) concluded that sustainable consumption intention had a significant positive effect on sustainable consumption behaviour. In addressing this issue, the present study has presented the following five hypotheses:

**Hypothesis 5a (H5a).** *Environmental knowledge has a positive effect on sustainable consumption behaviour.*

**Hypothesis 5b (H5b).** *Materialism has a negative effect on sustainable consumption behaviour.*

**Hypothesis 5c (H5c).** *Environmental influences have a positive effect on sustainable consumption behaviour.*

**Hypothesis 5d (H5d).** *SC promotion has a positive effect on sustainable consumption behaviour.*

**Hypothesis 5e (H5e).** *Sustainable consumption behavioural intention has a positive effect on sustainable consumption behaviour.*

#### *2.4. The Mediating Effect of Sustainable Consumption Behavioural Intention*

As emphasised by Figueroa-García et al. (2018), it is important to not only evaluate the direct effect of one construct on another, but also the indirect effects produced by mediating constructs. This evaluation is performed by measuring the total effect, which is the sum of all direct and indirect effects. Because of the Theory of Planned Behaviour (TPB), which is one of the most influential theories among the studies on consumer behaviour, we considered examining the mediating role that behaviour intention has in the relationships between antecedents and SCB. In this study, we have referred sustainable consumption behavioural intention to people's willingness to act and consume more sustainably by making sacrifices or by paying more (Saari et al. 2021). Since the behavioural intention construct is often considered a mediator that facilitates some expected outcomes (e.g., attitude, subjective norms, perceived behavioural control), we have derived four mediation hypotheses:

**Hypothesis 6a (H6a).** *Sustainable consumption behavioural intention mediates the relationship between environmental knowledge and sustainable consumption behaviour.*

**Hypothesis 6b (H6b).** *Sustainable consumption behavioural intention mediates the relationship between materialism and sustainable consumption behaviour.*

**Hypothesis 6c (H6c).** *Sustainable consumption behavioural intention mediates the relationship between environmental influences and sustainable consumption behaviour.*

**Hypothesis 6d (H6d).** *Sustainable consumption behavioural intention mediates the relationship between SC promotion and sustainable consumption behaviour.*

#### *2.5. The Moderating Role of Age*

Scholars have argued that demographic variables, such as age, gender, income, etc., have a significant moderating influence on consumers' behaviour (Wu et al. 2016). As regards the effect of age on the occurrence of different SCB forms in particular, the results are contradictory. On the one hand, some studies found that, compared to younger customers, older customers tended to be more ecologically conscious (Han et al. 2009; Yahya et al. 2015). Accordingly, young consumers had a poor environmental attitude and low environmental concern and environmental knowledge (Dhir et al. 2021). Similarly, Witek and Ku ´zniar (2021) found that young people cared the least about being perceived as environmentally friendly. On the other hand, other studies explained that younger people were more environmentally concerned and made their decisions having considered the effect of their choice on the environment (Akehurst et al. 2012; Sheoran and Kumar 2022). As emphasised by Tripathi and Singh (2016), age could be an important variable while examining sustainable consumption, but inconsistencies of this kind in the previous literature need further research. Based on the above discussions, it is assumed that age can moderate all direct relationships developed in this study. Therefore, the following nine hypotheses have been postulated:

**Hypothesis 7a (H7a).** *Age moderates the relationship between environmental knowledge and sustainable consumption behavioural intention.*

**Hypothesis 7b (H7b).** *Age moderates the relationship between materialism and sustainable consumption behavioural intention.*

**Hypothesis 7c (H7c).** *Age moderates the relationship between environmental influences and sustainable consumption behavioural intention.*

**Hypothesis 7d (H7d).** *Age moderates the relationship between SC promotion and sustainable consumption behavioural intention.*

**Hypothesis 8a (H8a).** *Age moderates the relationship between environmental knowledge and sustainable consumption behaviour.*

**Hypothesis 8b (H8b).** *Age moderates the relationship between materialism and sustainable consumption behaviour.*

**Hypothesis 8c (H8c).** *Age moderates the relationship between environmental influences and sustainable consumption behaviour.*

**Hypothesis 8d (H8d).** *Age moderates the relationship between SC promotion and sustainable consumption behaviour.*

**Hypothesis 8e (H8e).** *Age moderates the relationship between sustainable consumption behavioural intention and sustainable consumption behaviour.*


**2. DNA repair machineries in pathogenic eukaryotic cells**

#### **2.1 Identification of DNA repair machineries**

In order to identify amino acids sequences of *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis* proteins related to DNA repair factors, we performed similarity searches in the Eupath database (http://eupathdb.org/eupathdb/) using the *Saccharomyces cerevisiae* DNA repair proteins from HRR, NHEJ, BER, NER and MMR machineries as probes [reviewed in Lopez-Camarillo et al., 2009]. Putative gene products were selected from BLAST analysis against each parasite database using the Blosum 62 scoring matrix and the following criteria: (i) at least 20% identity and 35% homology to the query sequence and (ii) e-value lower than 0.002, unless a portion of the protein showed a very strong similarity. All sequences, as well as the *E. histolytica* sequences obtained from previous work [López-Camarillo et al., 2009], were then verified by BLAST against *S. cerevisiae* and *Homo sapiens* databases to confirm their identity. Additionally, we also retrieved data from published reports about *G. lamblia*,

DNA repair mechanisms, and genome evolution. *Entamoeba histolytica* and *Giardia lamblia* (syn. *G. intestinalis, G. duodenalis*) are intestinal parasites that cause diarrheal diseases. *E. histolytica* is responsible for fulminating dysentery, bloody diarrhea, weight loss, fatigue, abdominal pain, which affect 50 million people and provoke 100,000 deaths in developing countries each year. In some cases, *E. histolytica* trophozoites can cross the intestinal wall and use the blood stream to reach different vital organs of the human body, usually the liver (but also the lungs, brain or spleen) to provoke liver abscesses, which can be fatal if untreated [Guo et al., 2007]. *G. lamblia*  is another contributor to the enormous burden of diarrheal diseases with over 250 million symptomatic human infections per year worldwide. This anaerobic flagellated protozoa colonises and reproduces in the small intestine of several vertebrates, including human, causing giardiasis, commonly known as Beaver fever, which is characterized by diarrhea, excess gas, stomach or abdominal cramps, upset stomach, and nausea.  Individuals become infected by *E. histolytica* and *G. lamblia* through ingesting or coming into contact with food, soil, or water that have been contaminated by the feces of an infected human or animal. *Plasmodium falciparum* is the protozoan parasite responsible for human malaria, which is one of the most severe infectious diseases with 240 million cases in 2009 and more than 1 million deaths in children each year in Africa alone. The presence of the parasite in red blood cells lead them to stick to blood vessels through a process called cytoadherence, which produce the obstruction of the microcirculation and dysfunction of multiple organs, typically the brain in cerebral malaria. Symptoms usually include fever and headache, in severe cases progressing to coma, and death (Kokwaro, 2009). Trichomoniasis caused by *Trichomonas vaginalis* is the most common nonviral sexually transmitted disease (STD) in the world [WHO, 1995]. It has long been recognized as a frequent cause of vaginitis in women and urethritis in men, but data now link it to cervical cancer and bad pregnancy outcomes [Cotch et al., 1997], as well as to an enhanced risk for human immunodeficiency virus transmission [Sorvillo & Kerndt , 1998]. Here, we combined the use of genomic approaches based on bioinformatic analysis of parasite genome sequence with the review of published reports to perform a comparative description of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis*, which

cause high morbidity and mortality in many developed and developing countries.

In order to identify amino acids sequences of *E. histolytica*, *G. lamblia*, *P. falciparum* and *T. vaginalis* proteins related to DNA repair factors, we performed similarity searches in the Eupath database (http://eupathdb.org/eupathdb/) using the *Saccharomyces cerevisiae* DNA repair proteins from HRR, NHEJ, BER, NER and MMR machineries as probes [reviewed in Lopez-Camarillo et al., 2009]. Putative gene products were selected from BLAST analysis against each parasite database using the Blosum 62 scoring matrix and the following criteria: (i) at least 20% identity and 35% homology to the query sequence and (ii) e-value lower than 0.002, unless a portion of the protein showed a very strong similarity. All sequences, as well as the *E. histolytica* sequences obtained from previous work [López-Camarillo et al., 2009], were then verified by BLAST against *S. cerevisiae* and *Homo sapiens* databases to confirm their identity. Additionally, we also retrieved data from published reports about *G. lamblia*,

**2. DNA repair machineries in pathogenic eukaryotic cells**

**2.1 Identification of DNA repair machineries**

*P. falciparum* and *T. vaginalis* (**Table 1**). The absence of a given sequence in the table indicates that the corresponding gene was not identified in the parasite genome or that the sequence was too divergent to be detected by our *in silico* strategy.

None of the protozoan parasites studied here has the complete DNA repair pathways reported in yeast. HRR is the most conserved pathway suggesting that it is the mayor DSB repair pathway in these protozoan parasites. *E. histolytica*, *G*. *lamblia*, *P. falciparum* and *T. vaginalis* genomes contain most of the RAD52 epistasis group genes, although their functional relevance remains to be determined. Homologs for RAD50, RAD51, MRE11, RAD54 and RPA (lacking the RAD52 interacting domain) have been previously reported in *P. falciparum* [Voss et al., 2002; Malik et al., 2008]. In agreement with its participation in DNA repair, the *PfRad51* gene is overexpressed in the mitotically active schizont stage and in response to methyl methane sulfonate [Bhattacharyya & Kumar, 2003]. In. *T. vaginalis*, RAD50 y MRE11 were previously published as components of the meiotic recombination machinery, although meiosis has not been observed in this organism [Malik et al., 2008]. Ramesh et al. [2005] and Malik et al. [2008] identified the *Rad50/Mre11*, *Rad52* and *Dmc1 genes* involved in meiotic recombination machinery by HRR in *Giardia*. Intriguingly, *G. lamblia* and *P. falciparum* lack the *nsb1* homologue (*xrs2* in Yeast) that is a component of the MRN complex involved in DSB detection and 3´ ssDNA tails conversion. Recently, we published the *E. histolytica* RAD52 epistasis group involved in HRR [Lopez-Casamichana et al., 2007, 2008]. Interestingly, RT-PCR assays evidenced that some genes were down-regulated, whereas others were up-regulated when DSB were induced by UV-C irradiation, which revealed an intricate transcriptional modulation of *E. histolytica* RAD52 epistasis group related genes in response to DNA damage. Particularly, *Ehrad51* mRNA expression was 16-, 11- and 4-fold increased at 30 min, 3 h and 12 h, respectively. DNA microarrays assays confirmed the activation of *EhMre11, EhRad50*, and *EhRad54* genes at 5 min after DSB induction, suggesting that they represent early sensors of damage in HRR pathway [Weber et al., 2009]. Additionally, the molecular characterization of EhRAD51 showed that the presence of all the functional domains reported in yeast and human homologues. EhRAD51 was upregulated and redistributed from cytoplasm to the nucleus of trophozoites at 3 h after DNA damage and it was able to catalyze specific single-strand DNA (ssDNA) transfer to homologous double strand DNA (dsDNA) forming the three-stranded pairing molecule called D-loop structure, confirming that it is a *bonafide* recombinase in *E. histolytica* [Lopez-Casamichana et al., 2008].

*G. lamblia* and P*. falciparum* only have three of the eight factors of the NHEJ pathway (including the MNR complex also involved in HRR), which strongly suggest that they preferably use HRR to repair DSB. In contrast, almost all NEHJ pathway factors have been identified in *E. histolytica* and *T. vaginalis*, including the LIF1 ligase, RAD27 nuclease and MRE11/RAD50/NSB1 proteins. However, *E. histolytica* genome does not contain a homologous gene for KU80 subunit [López-Camarillo et al., 2009] and *T. vaginalis* lacks both *ku70* and *ku80* genes [Carlton et al., 2007]. As these proteins form a single KU complex that recognizes DSB sites and recruits other DNA repair factors, our findings could appear contradictory. The absence of conserved KU proteins has also been reported in *Encephalitozoon cunili* [Gill & Fast, 2007] and yeast [Hefferin & Tomkinson, 2005], thus it is possible that these organisms use highly divergent KU proteins to perform the NHEJ pathway.

The other key DNA repair mechanisms represented by BER, NER and MMR pathways operate to repair aberrant bases or nucleotides from a ssDNA using the complementary strand as template for DNA synthesis. As in *E. histolytica* [Lopez-Camarillo et al., 2009], the *G. lamblia* BER pathway appears to be largely incomplete, lacking *apn1*, *mag1, ogg1*, *rad10*, *mus81* and *mms4* genes.  Then the absence of OGG1 could indicate that they do not suffer oxidative damage to mitochondrial DNA. In contrast, *Plasmodium* Flap endonuclease-1 (PfFEN-1) and Pf DNA Ligase I (PfLigI) have enzymatic activities similar to other species [Gardner et al., 2002; Casta et al., 2008], indicating that BER pathway should be functional in this parasite although several components are lacking.

Most genes involved in NER pathway are represented in *E. histolytica* [Lopez-Camarillo et al., 2009]*, G. lamblia, P. falciparum and T. vaginalis* genomes suggesting that this mechanism could be potentially active in these eukaryotic parasites. PfXPB/RAD25, PfXPG/RAD2 and PfXPD/RAD3 have been previously reported in *P. falciparum* [Gardner et al., 2002; Bethke et al., 2007; Casta et al., 2008]. Additionally, the overexpression of *EhDdb1, EhRad23* and *EhRad54* genes after UV-induced DNA damage in *E. histolytica* [Weber et al., 2009] suggested that these genes could be involved in chromatin remodeling complexes as their homologues in human and yeast. *E. histolytica*, *G. lamblia* and *T. vaginalis* have various *rad3* genes to form the NEF3 complex (RAD2, RAD3, RAD25) of the BER pathway. Particularly, we identified six *rad3* genes and an additional truncated gene in *T. vaginalis*. On the other hand, all the parasites studied here lack almost one of the components of the TFIIH complex subunits (TFB1, TFB2 or TFB3).

As in bacteria, *Drosophila melanogaster*, *H. sapiens* and many other organisms [Lisby & Rothstein, 2005], *E. histolytica*, *G. lamblia* [Ramesh et al., 2005], *P. falciparum* [Bethke et al., 2007] and *T. vaginalis* [Malik et al., 2008] have almost all *S. cerevisiae* MMR genes, including the components of the MUTS (MSH2/MSH6) heterodimer, which strongly suggest that MMR could be an active DNA repair pathway in these parasites. Notably, *E. histolytica* and *P. falciparum* have two *msh2* genes. However, neither *E. histolytica* nor *P. falciparum* present the *msh3* gene that is required for the formation of the MUTS (MSH2/MSH3) heterodimer. PfMSH2-1, PfMSH2-2, PfMSH6, PfMLH1 and PfPMS1 proteins potentially participating in MMR have been previously reported in *P. falciparum*. Inhibition of *PfMSH2-2* gene increased mutation rate and microsatellite polymorphism, indirectly demonstrating its relevance in MMR and microsatellite slippage prevention. Moreover, antimalarial drug resistance has been recently related to a defective DNA mismatch repair, mainly in PfMutLα content [Castellini et al., 2011], which demonstrated the relevance of this mechanism for the parasite biology.

highly reduced form of mitocondria called mitosomes [Tovar et al., 1999, 2003]. Then the absence of OGG1 could indicate that they do not suffer oxidative damage to mitochondrial DNA. In contrast, *Plasmodium* Flap endonuclease-1 (PfFEN-1) and Pf DNA Ligase I (PfLigI) have enzymatic activities similar to other species [Gardner et al., 2002; Casta et al., 2008], indicating that BER pathway should be functional in this parasite although several

Most genes involved in NER pathway are represented in *E. histolytica* [Lopez-Camarillo et al., 2009]*, G. lamblia, P. falciparum and T. vaginalis* genomes suggesting that this mechanism could be potentially active in these eukaryotic parasites. PfXPB/RAD25, PfXPG/RAD2 and PfXPD/RAD3 have been previously reported in *P. falciparum* [Gardner et al., 2002; Bethke et al., 2007; Casta et al., 2008]. Additionally, the overexpression of *EhDdb1, EhRad23* and *EhRad54* genes after UV-induced DNA damage in *E. histolytica* [Weber et al., 2009] suggested that these genes could be involved in chromatin remodeling complexes as their homologues in human and yeast. *E. histolytica*, *G. lamblia* and *T. vaginalis* have various *rad3* genes to form the NEF3 complex (RAD2, RAD3, RAD25) of the BER pathway. Particularly, we identified six *rad3* genes and an additional truncated gene in *T. vaginalis*. On the other hand, all the parasites studied here lack almost one of the components of the TFIIH complex subunits

As in bacteria, *Drosophila melanogaster*, *H. sapiens* and many other organisms [Lisby & Rothstein, 2005], *E. histolytica*, *G. lamblia* [Ramesh et al., 2005], *P. falciparum* [Bethke et al., 2007] and *T. vaginalis* [Malik et al., 2008] have almost all *S. cerevisiae* MMR genes, including the components of the MUTS (MSH2/MSH6) heterodimer, which strongly suggest that MMR could be an active DNA repair pathway in these parasites. Notably, *E. histolytica* and *P. falciparum* have two *msh2* genes. However, neither *E. histolytica* nor *P. falciparum* present the *msh3* gene that is required for the formation of the MUTS (MSH2/MSH3) heterodimer. PfMSH2-1, PfMSH2-2, PfMSH6, PfMLH1 and PfPMS1 proteins potentially participating in MMR have been previously reported in *P. falciparum*. Inhibition of *PfMSH2-2* gene increased mutation rate and microsatellite polymorphism, indirectly demonstrating its relevance in MMR and microsatellite slippage prevention. Moreover, antimalarial drug resistance has been recently related to a defective DNA mismatch repair, mainly in PfMutLα content [Castellini et al., 2011], which demonstrated the relevance of this mechanism for the parasite biology.

**Gene name** *E. histolytica G. lamblia P. falciparum T. vaginalis S. cerevisiae*

*rad50* C4M2L7 Q6WD96 C6KSQ6 A2FAD3 P12753

*nbs1* C4M874 - - A2DHF7 P33301

*rad52* C4M197 Q6WD95 - - P06778

Pf11\_0087\*\*

C4M5L7 - - A2GIB8 P38953

PFA0390w\*\* A2ECB0 P32829

A2FXT7 P25454

P25301

Q86CI9 A8BR27\*

**Homologous recombination repair (HRR) pathway**

C4LVX7 C4M8N7\*

C4LVM6 C4M7S7

*rad51* C4M4K4 Q86C21 Q8IIS8

*mre11* Q86C23

*rad54 rad54b*

*rad51c rad57*

components are lacking.

(TFB1, TFB2 or TFB3).

DNA Repair in Pathogenic Eukaryotic Cells:

each graph.

**proteins**

Insights from Comparative Genomics of Parasitic Protozoan 375

Fig. 1. **Conservation of DNA repair pathways between** *E. histolytica* **and** *G. lamblia* **(A),** *P. falciparium* **(B) and** *T. vaginalis* **(C).** Amino acids sequences from orthologous proteins were compared by Blast and the percentage of identity was determined through pair wise

alignment of the most conserved region. Average identity of all pathways is indicated above

**2.3 DNA repair activity in cell free lysates evidences the functionality of DNA repair**

Although insights about the activity of DNA repair proteins in protozoa have been mainly obtained from experimental evidence based in heterologous expression and characterization of recombinant proteins, some reports showed that DNA repair activity could be detected in whole cell extracts, supporting the notion that DNA repair pathways already operates *in vivo*. For instance, Haltiwanger et al., (2000) reported the characterization of an AP

Table 1. Comparison of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum*, *T. vaginalis* and *S. cerevisiae*. \* fragment, \*\* PlasmoDB database.

#### **2.2 Conservation of DNA repair pathways**

To investigate the degree of conservation of DNA repair pathways in protozoan parasites, we next determined the values of Smith-Waterman identity scores between *E. histolytica* proteins and their corresponding orthologues in *G. lamblia*, *P. falciparum* and *T. vaginalis* by BLAST analysis based in pairwaise sequence alignments and calculated the mean value for each DNA repair machinery (**Fig. 1**). Data of the MNR complex which participate in HRR and NHEJ pathways were included in both mechanisms. DSB repair pathways were generally more conserved than Excision Repair mechanisms. Considering amino acids identity, mean values for HRR and NHEJ pathways were higher in *E. histolytica*/*P. falciparum* comparison, suggesting that *E. histolytica* machinery was closer to *P. falciparum* than to *G. lamblia* and *T. vaginalis* machineries. The comparison *E. histolytica*/*G. lamblia* evidenced that HRR is highly conserved between both parasites, whereas components of the other pathways were more divergent. In the case of *E. histolytica*/*P. falciparum* comparison, NHEJ appeared to be more conserved that HRR, while the identity of HRR and NHEJ factors was very similar in *E. histolytica*/*T. vaginalis*. In all the parasites, the RAD51 recombinase is the most conserved protein (51%, 58% and 64% when *E. histolytica* protein sequence was compared with *G. lamblia*, *P. faciparum* and *T. vaginalis* orthologues, respectively), which is consistent with its relevant role in HRR mechanism.

 Comparison of DNA repair machineries from *E. histolytica*, *G. lamblia*, *P. falciparum*,

Q6WD97 Q8ILI9

To investigate the degree of conservation of DNA repair pathways in protozoan parasites, we next determined the values of Smith-Waterman identity scores between *E. histolytica* proteins and their corresponding orthologues in *G. lamblia*, *P. falciparum* and *T. vaginalis* by BLAST analysis based in pairwaise sequence alignments and calculated the mean value for each DNA repair machinery (**Fig. 1**). Data of the MNR complex which participate in HRR and NHEJ pathways were included in both mechanisms. DSB repair pathways were generally more conserved than Excision Repair mechanisms. Considering amino acids identity, mean values for HRR and NHEJ pathways were higher in *E. histolytica*/*P. falciparum* comparison, suggesting that *E. histolytica* machinery was closer to *P. falciparum* than to *G. lamblia* and *T. vaginalis* machineries. The comparison *E. histolytica*/*G. lamblia* evidenced that HRR is highly conserved between both parasites, whereas components of the other pathways were more divergent. In the case of *E. histolytica*/*P. falciparum* comparison, NHEJ appeared to be more conserved that HRR, while the identity of HRR and NHEJ factors was very similar in *E. histolytica*/*T. vaginalis*. In all the parasites, the RAD51 recombinase is the most conserved protein (51%, 58% and 64% when *E. histolytica* protein sequence was compared with *G. lamblia*, *P. faciparum* and *T. vaginalis* orthologues,

C0H4L8

A2EP54 P25847

**Mismatch repair (MMR) pathway**

B1N4L6

**2.2 Conservation of DNA repair pathways**

*T. vaginalis* and *S. cerevisiae*. \* fragment, \*\* PlasmoDB database.

respectively), which is consistent with its relevant role in HRR mechanism.

*msh2* C4M9J9

Fig. 1. **Conservation of DNA repair pathways between** *E. histolytica* **and** *G. lamblia* **(A),** *P. falciparium* **(B) and** *T. vaginalis* **(C).** Amino acids sequences from orthologous proteins were compared by Blast and the percentage of identity was determined through pair wise alignment of the most conserved region. Average identity of all pathways is indicated above each graph.

#### **2.3 DNA repair activity in cell free lysates evidences the functionality of DNA repair proteins**

Although insights about the activity of DNA repair proteins in protozoa have been mainly obtained from experimental evidence based in heterologous expression and characterization of recombinant proteins, some reports showed that DNA repair activity could be detected in whole cell extracts, supporting the notion that DNA repair pathways already operates *in vivo*. For instance, Haltiwanger et al., (2000) reported the characterization of an AP

DNA Repair in Pathogenic Eukaryotic Cells:

1993; Gilbert et al., 1997].

**4. Duplicated genes: The case of** *rad3*

Insights from Comparative Genomics of Parasitic Protozoan 377

Gene duplicates represent for 8-20% of the genes in eukaryotic cells, and the rates of gene duplication are estimated at between 0.2% and 2% per gene per million years. Gene duplications are one of the major motors in the evolution of genetic systems and may occur in homologous recombination, retrotransposition event, or duplication of an entire chromosome [Zhang, 2003]. Duplicated genes are believed to be a main system for the establishment of new gene functions generating evolutionary novelty [Long & Langley,

A detailed examination of **Table 1** revealed that several DNA repair genes are duplicated in protozoan parasites, while there is only one gene in yeast. For example, the HRR machinery includes two *rad51* genes in *P. falciparum*, two *rad54* and *mre11* genes in *E. histolytica* [Lopez-Casamichana et al., 2008], two *rpa1* genes in *T. vaginalis*, and two *sgs1* genes in *G. lamblia* and *P. falciparum*. We also identified two *rad27* genes in *P. falciparum* and *G. lamblia* NHEJ pathway, two *E. histolylica ntg1* and *P. falciparum pcna* genes in the BER pathway, as well as two *msh2* genes for the MMR pathway in *E. histolytica* and *P. falciparum*. But the most duplicated gene was the *rad3* gene from the NER mechanism, since there are three genes in *E. histolytica*, two in *G. lamblia* and six in *T. vaginalis*, whereas *P. falciparum* has only one *rad3* gene, alike yeast. Remarkably, gene duplication is evident for many other genes in *T. vaginalis* and reflexes the massive gene expansion inside the large genome of this pathogen [Hartl & Wirth, 2006]. In yeast, the RAD3 protein is involved in mitotic recombination and spontaneous mutagenesis, becoming essential for cell viability in the absence of DNA injury. Furthermore, this protein participates in the repair of UV-irradiated DNA via NER, and constitutes a subunit of RNA polII initiation factor TFIIH [Moriel-Carretero & Aguilera, 2010]. *S. cerevisiae* RAD3 is related to the *H. sapiens* XPD, also known as ERCC2. Defects in human XPD result in a wide range of diseases, including Xeroderma pigmentosum (XP), Cockayne's syndrome, and Trichothiodystrophy characterized by a wide spectrum of symptoms ranging from cancer

In order to describe the inferred evolutionary relationships among the most abundant duplicated gene found through the analysis of DNA repair machineries from the human pathogens studied here, we have undertaken a phylogenetic analysis of RAD3 helicase orthologues in *S. cerevisiae*, *E. histolytica*, *T. vaginalis*, *G. lamblia* and *P. falciparum*. We evaluated the minimum evolution of RAD3 proteins through the construction of Neighbor-Joining phylogenetic tree using the *MEGA* version 5.05 [Tamura et al., 2011]. The robustness was established by bootstrapping test, involving 500 replications of the data based on the criteria of 50% majority-rule consensus (**Fig. 3**). Two main branches that came from a common ancestor can be observed. On one branch, *T. vaginalis* RAD3 parologues are clustered into two sister proteins pairs (A2E1B9 and A2ELX1, A2E4I6 and A2DDD4), that have each evolved from the same ancestor. Besides, *E. histolytica* C4M6T8 is closer to *T. vaginalis* A2E4I6 and A2DDD4, than to its own paralogues. The other branch supports *T. vaginalis* A2G2G8 that is closely related to yeast and *P. falciparum* RAD3 proteins that came off the same node. Interestingly, these two organisms only have one *rad3* gene. This branch also includes *E. histolytica* C4M8K7 and C4M8Q4 sister proteins pair. Intriguingly, the two *Giardia* RAD3 proteins have emerged from different nodes and appeared to be more related to orthologues from other species than to each other; particularly, the branch supporting *Giardia* A8B495 also includes *Trichomonas* A2E1B9 and A2ELX1, while *Giardia* A8BYS3 is on the other branch, isolated from the other proteins, such as *Trichomonas* A2F1W2, which

susceptibility to neurological and developmental defects [Liu et al., 2008].

suggested that these proteins have evolved early.

endonuclease activity in a *P. falciparum* cell free lysate. Authors provide evidence for the presence of class II, Mg2+–dependent and independent AP endonucleases in the extracts. Moreover, they detected that *Plasmodium* AP endonuclease(s) possessed a 3´ phosphodiesterase activity similar to those described in other class II AP endonucleases Demple et al., 1986. In a related study, it was reported that a *P. falciparum* lysate contained uracil DNA glycosylase, AP endonuclease, DNA polymerase, flap endonuclease, and DNA ligase activities Haltiwanger et al., 2000. In contrast, DNA repair activities in cell lysates have not been detected in *Entamoeba, Giardia* and *Trichomonas* parasites. These data remark the utility of cell free lysates to understand DNA repair pathways, and pointed out to the urgency to investigate endogenous DNA repair activities using whole cell extracts in parasites where no data is available.


**15. Impact of obesity on COVID 19**

People with obesity are at high risk for the contraction of COVID – 19 infection. Adipose tissue is increased in ACE2 receptors, which facilitates the SARS-CoV-2 to enter the human cells quickly [35]. The viral load is significantly increased along with the prolonged viremia due to the massive number of adipocytes in obese people. Visceral adiposity causes cytokine proliferation, which causes the low-grade inflammation to advance the cytokines storm in COVID- 19 [36]. Obesity is responsible for reducing individuals' immunity and susceptibility to infection due to various pathogenic organisms. Because increased cytokines in the circulatory system reduce adiponectin levels, thus the immune response to infection is reduced. This mechanism causes damage to the lymphoid tissue and decreasing the B and T cells in the immune system, susceptible to viral infection [37]. Obesity and procoagulant factors are interlinked together that play a vital role in causing thromboembolic complications in patients with COVID-19. Obesity also reduces pulmonary function, reducing reserve volume and respiratory system compliance, at high risk for COVID 19 complications. Obesity with dysfunctional adipose tissue is associated with type 2 diabetes, hypertension, and CVD, which impair individuals' health during COVID 19 infection [38].


d.Association

The statistical technique of association is a data mining technique. It denotes that some data (or data-driven events) are linked to other data. It's similar to the machine learning concept of co-occurrence, where the existence of one data-driven event indicates the probability of another. Correlation and association are two statistical concepts that are very similar. This means that data analysis reveals a connection between two data occurrences, such as the fact that hamburger purchases are often followed by French fries purchases.


**<IP_ADDRESS> Low molecular weight compounds with antiviral activity**

Several triterpenes from *G. lucidum* (ganoderiol F, ganodermanontriol and ganoderic acid and B) are active antiviral agents against HIV-1 virus. *In vitro* antiviral activity of influenza viruses type A and B was noticed in extracts of mycelium of mushroom *Kuehneromyces mutabilis* (Schaeff.: Fr.) (Singer & AH Sm.), while the extract and two isolated phenolic components from the mushroom *Inonotus hispidus* (Bull.; Fr.) P. Karst, as well as ergosterol peroxide, are present in many different fungal species.

#### **<IP_ADDRESS> High molecular weight compounds with antiviral activity**

Water-soluble lignins isolated from *Inonotus obliquus* (Pers.: Fr.) Pilate, inhibit HIV protease with IC 50 value of 2.5 mg/ml. Anti-HIV activity is recorded for the submerged culture media of *L. edodes* and water-soluble lignin isolated from the same fungus. Proteinpolysaccharide complex PSK and PSP from *Coriolus versicolor*, also shows antiviral activity on HIV and cytomegalovirus *in vitro*. Inhibition of HIV-1 reverse transcriptase is caused by velutin, protein from *Flammulina velutipes*, which inactivates ribosomes. MD fractions of mushroom *Grifola frondosa* showed general improvement of condition of the patients (85%) who had various symptoms of HIV and other secondary diseases (Zjawioni, 2004).

#### **1.4.2 Antifungal substances**

Compounds with antibacterial and antifungal activity of mushrooms assists in their survival in their environment. These substances can be very useful in the treatment of human infections, but the official antibiotic therapeutics in the world market can be only found originating from microfungi so far. Opportunistic fungal infections are always a big problem, especially in immunocompromised patients receiving chemotherapy or in cases of transplantation of organs or bone marrow, as well as in HIV infection. During the last ten years, the interest in compounds that show antifungal activity has been increased. Among them the sordarin (tricyclic diterpene glycoside) was for the first time isolated in 1971 (Hauer and Sigg as cited in Inouye et al., 2004), and slightly more potent zofimarin was isolated for the first time in 1987 (Ogita et al. 1987 as cited in Inouye et al., 2004). In addition, suggestive is xylarin (compound SCH57404) isolated from the lignicolous fungus *Xylaria s*p. (Schneider, 1995). Many derivatisations of sordarin antibiotics have been performed in research groups of the GlaxoSmith Kline company by biotransformation with *Streptomyces avermitilis*, what resulted in the synthesis of GM237354 (Herreros et al.*,* 1998), with the MIC of 90% that was 0.015 mg/ml for isolates of *C. albicans* and 0.12 for *C. tropicalis*. Further development of these compounds has led to the azasordarin group in which the sugar component is replaced by Nsubstituted morpholine (Herreros et al., 2001 as cited in Inouye et al., 2004).

terpenoids as well as phenolic compounds as the main active components responsible for the

Presented antiviral activity of fungi is related to their whole, complex extracts, but also to the isolated compounds. Agents isolated from fungi can directly cause the inhibition of viral enzymes, the synthesis of viral nucleic acid, or adsorption and absorption of virus in mammalian cells. The most often small molecules are active in the direct antiviral effect, while the indirect effects are mediated by antiviral activity immunostimulative

Several triterpenes from *G. lucidum* (ganoderiol F, ganodermanontriol and ganoderic acid and B) are active antiviral agents against HIV-1 virus. *In vitro* antiviral activity of influenza viruses type A and B was noticed in extracts of mycelium of mushroom *Kuehneromyces mutabilis* (Schaeff.: Fr.) (Singer & AH Sm.), while the extract and two isolated phenolic components from the mushroom *Inonotus hispidus* (Bull.; Fr.) P. Karst, as well as ergosterol

Water-soluble lignins isolated from *Inonotus obliquus* (Pers.: Fr.) Pilate, inhibit HIV protease with IC 50 value of 2.5 mg/ml. Anti-HIV activity is recorded for the submerged culture media of *L. edodes* and water-soluble lignin isolated from the same fungus. Proteinpolysaccharide complex PSK and PSP from *Coriolus versicolor*, also shows antiviral activity on HIV and cytomegalovirus *in vitro*. Inhibition of HIV-1 reverse transcriptase is caused by velutin, protein from *Flammulina velutipes*, which inactivates ribosomes. MD fractions of mushroom *Grifola frondosa* showed general improvement of condition of the patients (85%)

Compounds with antibacterial and antifungal activity of mushrooms assists in their survival in their environment. These substances can be very useful in the treatment of human infections, but the official antibiotic therapeutics in the world market can be only found originating from microfungi so far. Opportunistic fungal infections are always a big problem, especially in immunocompromised patients receiving chemotherapy or in cases of transplantation of organs or bone marrow, as well as in HIV infection. During the last ten years, the interest in compounds that show antifungal activity has been increased. Among them the sordarin (tricyclic diterpene glycoside) was for the first time isolated in 1971 (Hauer and Sigg as cited in Inouye et al., 2004), and slightly more potent zofimarin was isolated for the first time in 1987 (Ogita et al. 1987 as cited in Inouye et al., 2004). In addition, suggestive is xylarin (compound SCH57404) isolated from the lignicolous fungus *Xylaria s*p. (Schneider, 1995). Many derivatisations of sordarin antibiotics have been performed in research groups of the GlaxoSmith Kline company by biotransformation with *Streptomyces avermitilis*, what resulted in the synthesis of GM237354 (Herreros et al.*,* 1998), with the MIC of 90% that was 0.015 mg/ml for isolates of *C. albicans* and 0.12 for *C. tropicalis*. Further development of these compounds has led to the azasordarin group in which the sugar component is replaced by N-

who had various symptoms of HIV and other secondary diseases (Zjawioni, 2004).

substituted morpholine (Herreros et al., 2001 as cited in Inouye et al., 2004).

obtained activity (Turkoglu et al., 2007; Barros et al., 2007; Elmastas et al., 2007).

polysaccharides and other complex molecules (Zjawioni, 2004). **<IP_ADDRESS> Low molecular weight compounds with antiviral activity**

peroxide, are present in many different fungal species.

**<IP_ADDRESS> High molecular weight compounds with antiviral activity**

**1.4.1 Antiviral substances**

**1.4.2 Antifungal substances**

Several antifungal metabolites with steroid structure have been also isolated from fungi A25822 A and B from *Geotrichum* (Gordee and Butler, 1975 as cited in Inouye et al., 2004) and from *Wallemia sebi*; Mer-NF8054 A and X from the genus *Aspergillus*. The most famous triterpene, favonol isolated from basidiomycetous *Favolashia* sp. (Anke et al., 1995 as cited in Inouye et al., 2004) is a metabolite that exhibited antifungal activity against Ascomycetes, Basidiomycetes, Zygomycetes and Oomycetes, but did not show antibacterial activity. Researchers of Merck Group have discovered four acidic terpenoids from filamentous fungi: ergokonin A (from *Trichoderma koningii*), ascosteroid (from *Ascotricha amphitricha*) arundifungin (steroid from *Arthrinium arundinis*) and enfumafungin (pentacyclic terpenoid from from mould *Trichoderma koningii*), ascosteroid (from ascomycetous *Ascotricha amphitricha*), arundifungin (steroid from mould *Arthrinium arundinis*) and enfumafungin (pentacyclic terpenoid from *Aureobasidium*), which were found to affect the biosynthesis of β-D-glucan but not the biosynthesis of steroids. Among them the best antifungal activity on *Candida* species and species of *Aspergillus* genera showed enfumafungin.


**4. Conclusions**

In this study, cement and fly ash were used as cementitious materials and waste gypsum was used as the fine aggregates, which were suitable for CLSM. The compressive strengths at 28 and 56 days were 4.71 and 6.08 MPa, respectively. The air-curing method should be applied to avoid abnormalities such as volume expansion after curing the specimen in water. With the hydrated blending reaction, the waste gypsum in the CLSM specimen continued to react with cement and fly ash. The reaction produced more ettringite, C-S-H colloids and other hydrates with age, increasing the compressive strength. In waste gypsum, some components react with CO2 in the air, such as CaO and Ca(OH)2, but they have become stable, allowing for the specimen to maintain a reasonable volume. In addition, the volume and weight of the specimen did not change significantly after being immersed in seawater and tap water, and the appearance of the specimen was intact. Additionally, it was determined that waste gypsum can entirely replace fine aggregates in construction and can significantly increase the amount of reuse, thus achieving the effect of the green circular economy.

**Author Contributions:** Conceptualization, W.-T.L., A.C. and K.K.; methodology, K.K.; validation, W.-T.L., D.M. and M.Ł.; investigation, A.C.; resources, K.-L.L.; data curation, W.-T.L. and D.M.; writing—original draft preparation, W.-T.L. and K.K.; writing—review and editing, W.-T.L. and M.Ł.; visualization, A.C. and K.-L.L.; supervision, W.-T.L. and K.-L.L.; project administration, W.-T.L. and D.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The National Science and Technology Council (NSTC) in Taiwan supported this research with grant NSTC 111-2923-E-197-003-MY3 and the Polish National Centre for Research and Development in Poland, under the M-ERA.NET 3 program, grant number M-ERA.NET3/2021/144/3D-FOAM/2022.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Publication cost of this paper was covered with founds of the Polish National Agency for Academic Exchange (NAWA): "MATBUD'2023—Developing international scientific cooperation in the field of building materials engineering" BPI/WTP/2021/1/00002, MATBUD'2023.

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


**1. Introduction**

Diabetes mellitus (DM) is a group of metabolic disorders that are characterized by hyperglycemia [1]. Hyperglycemia is caused by the combined action of insulin deficiency and/or insulin resistance in diabetic patients [2]. Long-term hyperglycemia is associated with microvascular complications and leads to cardiovascular disease, diabetic nephropathy, and other severe complications, which are the major causes of death in diabetic patients [3]. In the early stages of DM, the effective control and strict management of postprandial blood glucose levels are crucial for alleviating DM. The current effective ways to reduce

postprandial blood glucose level are as follows: (I) inhibit the activities of digestive enzymes, such as glucosidases and α-amylase, which can produce glucose from starch and other carbohydrates [4]; (II) promote the secretion of insulin, such as by inhibiting dipeptidyl peptidase 4 (DPP-4) and elevating glucagon-like peptide 1 (GLP-1) activities [5,6]; (III) reduce glucose reabsorption in the kidney by inhibiting Na+/glucose cotransporter 2 (SGLT2) activity [7]; (IV) suppress the glucose absorption into the bloodstream via the small intestinal by inhibiting the function of Na+/glucose cotransporter 1 (SGLT1) [8].

Although there are several oral hypoglycemic agents that can reduce the blood glucose level in DM, these agents are mainly chemically synthetic, costly, and can cause severe adverse side-e ffects (e.g., serious hypoglycemia and kidney damage) [9,10]. Thus, the discovery of e ffective, safer, and a ffordable drugs for DM patients has attracted significant research attention. Research suggests that certain natural herbal products can exert hypoglycemic e ffects and are safer and easier to obtain than other synthetic chemicals [11]. Convincing evidence suggests that bioactive compounds (e.g., proteins, polyphenols, tannins, and phenolic acids) can directly a ffect intestinal glucose uptake by competitive inhibition of the glucose transporter SGLT1 [12–14]. In the small intestine, SGLT1 is expressed in the apical cell membrane constituting the brush border [15], which is of primary importance for glucose absorption from the lumen to the epithelial cells of the intestine [16,17]. Ina et al. demonstrate that rice albumin could alleviate postprandial hyperglycemia by inhibiting SGLT1 function via in vivo and in vitro assays [18]. Müller et al. confirmed that the extracts of guava leaves and fruits, which contain polysaccharides, polyphenols, and other bioactive substances, can e ffectively reduce intestinal glucose transport by inhibiting the function of SGLT1 in vivo [19].

An increasing number of studies have demonstrated that non-toxic biological macromolecules, especially polysaccharides, possess prominent e fficacies for treating DM and other metabolic diseases [20–24]. In addition, Tang et al. reported that water-soluble polysaccharides from *Lycium barbarum* have a conspicuously inhibitory e ffect on glucose uptake in vitro [25], which may have been due to the inhibitory e ffect on the intestinal glucose transporter SGLT1. Fucoidan is a family of sulfated fucan predominantly existing in the cell walls of brown algae and several marine invertebrates (e.g., sea cucumbers and sea urchins) [26]. These water-soluble heteropolysaccharides are composed of various percentages of L-fucose and sulfate ester groups. Fucoidans from natural sources are usually composed of two types of chain structures, type I, with α (1 →3)-linked fucose, and type II, made up of alternating α (1 →3)- and α (1 →4)-linked fucose molecules [27]. In recent years, fucoidans isolated from di fferent sources have been extensively studied due to their diverse biological activities, including anticoagulant, anti-inflammatory, antivirus, antitumor, lipid-lowering, antidiabetic nephropathy, antimetabolic syndrome, and prebiotic e ffects [26,28]. Due to their promising therapeutic e ffects and availability from various kinds of cheap brown algae, an increasing number of studies have been devoted to the development and utilization of fucoidans in the fields of drugs and functional foods. Although the application prospects of fucoidans are promising, it is worth noting that the bioactivities of fucoidans are probably highly dependent on their structural properties (such as type of glycosidic linkages, molecular weight (MW), and branches) [26,29] and little attention has been devoted to determining the e ffects of fucoidans with various structure on attenuating postprandial hyperglycemia and its underlying mechanism.

We reported that fucoidan from *Fucus vesiculosus* (FvF) with a type II structure can significantly inhibit α-glucosidase and the glucose transport activities in the small intestine, and regulate glucose consumption and lipid metabolism via reactive oxygen species (ROS)-mediated c-Jun N-terminal kinase (JNK) and protein kinase B (Akt) signaling pathways, thus improving postprandial hyperglycemia in diabetic mice [30,31]. To explore the relationship between the inhibitory e ffect of glucose absorption and fucoidan, it is necessary to rule out the other factors related to antihyperglycemia. Therefore, we prepared three fucoidans as follows: fucoidan from *Ascophyllum nodosum* (AnF) with a type II structure [32] and mild inhibition of α-glucosidase [31], and fucoidans from *Laminaria japonica* (LjF) and *Kjellmaniella crassifolia* (KcF) with type I structures [33,34]. Additionally, we investigated the pharmacological e ffects of the above fucoidans on alleviating postprandial hyperglycemia using in vitro (Caco-2 monolayer model), semi-in vivo (everted gut sac model), and in vivo (oral glucose tolerance test (OGTT) in Kunming and leptin receptor-deficient (db/db) mice) assays. Based on previous studies [25,30], we also focused on evaluating the binding affinity of various fucoidans to SGLT1 via surface plasmon resonance (SPR). Taken together, as we have previously reported, FvF with a type II structure could alleviate the postprandial hyperglycemia. Thus, this study aimed to evaluate the effects of fucoidans with different types of fucosidic linkages on alleviating postprandial hyperglycemia and preliminarily elucidated the underlying mechanism.


**A Review of Hydrogen-Natural Gas Blend Fuels in Internal Combustion Engines**

Antonio Mariani, Biagio Morrone and Andrea Unich

*Dept. of Aerospace and Mechanical Engineering - Seconda Universitá degli Studi di Napoli Italy*

#### **1. Introduction**

16 Fossil Fuel and the Environment

This work was a cooperative research project with the Japan Petroleum Energy Center. We thank Prof. M. Arai and Prof. K. Amagai of Gunma University for helpful suggestions.

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**6. Acknowledgement**

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**7. References**

In the last ten years, the number of natural gas (NG) vehicles worldwide has rapidly grown with the biggest contribution coming from the Asia-Pacific and Latin America regions (IANGV, 2011). As natural gas is the cleanest fossil fuel, the exhaust emissions from natural gas spark ignition vehicles are lower than those of gasoline-powered vehicles. Moreover, natural gas is less affected by price fluctuations and its reserves are more evenly widespread over the globe than oil. In order to increase the efficiency of natural gas engines and to stimulate hydrogen technology and market, hydrogen can be added to natural gas, obtaining Hydrogen - Natural Gas blends, usually named as HCNG.

This chapter gives an overview of the use of HCNG fuels in internal combustion engines. The chemical and physical properties of hydrogen and natural gas relevant for use in internal combustion engines are described. Then a survey on the impact of hydrogen on natural gas engine performance and emissions is presented with reference to research activities performed on this field.

#### **2. Data reduction**

In this section the main physical quantities used in this chapter are presented and discussed.

The stoichiometric air-fuel ratio on mass basis (AFR*stoich*), defined in equation 1, is the mass of air needed to fully oxidize 1 kg of fuel, while AFR is the ratio between air and fuel mass flow rates, equation 2. The ratio between the actual AFR and the AFR*stoich*, is the relative air-fuel ratio, equation 3. If *λ >* 1 the mixture is *lean* and the oxidation takes place with excess of air respect to the stoichiometric amount; for *λ* values lower than 1 the mixture is *rich*, and the fuel oxidation is not complete. The ratio 1/*λ* is defined as the equivalence ratio *φ*, equation 4.

$$AFR\_{stoich} = \left(\frac{m\_d}{m\_f}\right)\_{stoich} \tag{1}$$

$$AFR = \frac{m\_a}{m\_f} \tag{2}$$

$$
\lambda = \frac{AFR}{AFR\_{stoich}}\tag{3}
$$

**3. Natural gas**

The main natural gas constituent is methane and the composition is strictly dependent on the origin gas field. Table 1 shows the composition of a natural gas sample obtained by the Italian

A Review of Hydrogen-Natural Gas Blend Fuels in Internal Combustion Engines 19

Natural gas has been widely investigated as fuel for road vehicles because of its lower impact

**Constituent Composition [% vol.]**

Ristovski et al. (2004) performed an experimental activity on a passenger car converted to operate either on gasoline or on compressed natural gas (CNG). Fuelling the engine by CNG, both regulated (CO, NOx and HC) and unregulated emissions (PAHs and formaldehyde) were

Prati, Mariani, Torbati, Unich, Costagliola & Morrone (2011) tested a bifuel passenger car fuelled alternatively by gasoline and natural gas on a chassis dynamometer over different driving cycles, in order to evaluate the effects of fuel properties on combustion, exhaust emissions and engine efficiency. The results showed that gasoline produced CO emissions higher than NG over the real world Artemis driving cycles, as a consequence of mixture enrichment during load transients. A detailed description of the driving cycles is reported in Barlow et al. (2009). Over the type approval New European Driving Cycle (NEDC), NG involved higher HC emissions compared to gasoline as a consequence of the higher light-off temperature for the catalytic oxidation of CH4, which is the major constituent of HC when the vehicle is fuelled by NG, while there were no differences over the Artemis driving cycles which were performed after a warming up conditioning of the vehicle. NOx emissions were higher for gasoline over all the test cycles. CO2 emissions for CNG showed a reduction between 21% and 29% over the tested driving cycles as a consequence of the reduced carbon content of the fuel and the lower fuel consumption on mass basis. A 5% fuel consumption reduction, expressed in MJ/km, is observed over the NEDC for the CNG respect to gasoline, while for the Artemis the reduction ranges between 10% and 22%. The higher gasoline consumption is the consequence of the mixture enrichment during transients. Particulate emissions referred to gasoline were higher than NG ones over the NEDC and comparable over the Artemis. Particle number observed was also higher for gasoline, with the exception

Methane 88.98 Ethane 6.85 Propane 1.27 Butane 0.24 Pentane 0.04 Hexane 0.003 Nitrogen 0.96 Carbon dioxide 1.61

distribution network, determined by means of gas chromatographic analysis.

on the environment than gasoline and more widespread resources.

Table 1. Example of natural gas composition.

lower than gasoline.

of the Artemis Motorway.

$$
\phi = \frac{1}{\lambda} \tag{4}
$$

Equation 5 defines the indicated mean effective pressure (imep), an engine parameter which evaluates the work obtained by an engine cycle, *p dV*, divided by the engine displacement. The Coefficient of Variation of imep, COV*imep*, is the ratio of the standard deviation of the indicated mean effective pressure and the average imep over a representative number of cycles, equation 6.

$$imp = \frac{1}{V\_d} \oint p\,dV\tag{5}$$

$$COV\_{imp} = \frac{\sigma\_{imp}}{\dot{image}\_{avg}}\tag{6}$$

In case the effect of mechanical efficiency has to be taken into account, the brake mean effective pressure (bmep) is considered. In 4-stroke engines, the bmep is calculated from the torque measured at the engine shaft, according to equation 7:

$$bmep = \frac{T \cdot 4\pi}{V\_d} \tag{7}$$

The stoichiometric reaction equation of a methane-hydrogen blend reads as:

$$\left(\left(\text{aCH}\_4 + \beta \, H\_2\right) + \left(2\,\text{a} + \frac{\beta}{2}\right)\left(\text{O}\_2 + 3.76 \, N\_2\right) \rightarrow \text{aCO}\_2 + \left(2\,\text{a} + \beta\right)H\_2O + \left(2\,\text{a} + \frac{\beta}{2}\right) 3.76 \, N\_2\right) \tag{8}$$

where *α* + *β* = 1. The quantities *α* and *β* represent the mole per each species in the blend, and it is immediate to observe that the reduction of the C/H ratio, compared to pure methane, brings about a theoretical reduction of the CO2.

The burning velocity represents a main property for the combustion characteristics of the fuels and is defined as the velocity at which unburned gases move through the combustion wave in the direction normal to the wave surface (Glassman & Yetter, 2008). The laminar burning velocities can be obtained using the following equation 9 (Mandilas et al., 2007) being S*s* the unstretched flame speed, *ρb* and *ρu* the burned and unburned gas densities. Equation 10 relates the unstretched flame speed, the stretched flame speed S*n*, the stretch rate *κ* and the Markstein length L*b*.

$$
\mu\_l = \mathcal{S}\_s \frac{\rho\_b}{\rho\_u} \tag{9}
$$

$$
\mathcal{S}\_s - \mathcal{S}\_\hbar = \kappa L\_\hbar \tag{10}
$$

The stretch rate *κ* is calculated from the position of the flame front, *R* = *R*(*t*), with the following equation 11 (Chen, 2009):

$$\kappa = \frac{1}{R} \frac{d\mathcal{R}}{dt} \tag{11}$$

The Markstein length characterizes the variation in the local flame speed due to the influence of external stretching and determines the flame instability with respect to preferential diffusion (Markstein, 1964).


**Conflict of interest**

The authors declare no conflict of interest.

*Exploring Standard and Alternative Sites for Left Ventricular Assist Device Inflow Cannula… DOI: http://dx.doi.org/10.5772/intechopen.114053*