Author details

4. Conclusion

Figure 12.

Acknowledgements

ITS/2017.

54

It has been demonstrated that a simple dissolve method using HCl or NaOH which

The authors express gratitude to Kemenristekdikti of the Republic of Indonesia and LPPM ITS who support the research funding through PBK-2017 No. 528/PKS/

was reacted with a mono- or bi-cationic metals or with a natural mineral can be accomplished to produce high-purity, nanocrystalline metal oxide ceramics. Pure MgO, TiO2, MgTiO3, and ZrSiO4 nano-powders were produced by the method. The main step for the metal case was on the formation of the precursor and appropriate calcination temperature. For the mineral case, purification of the phase under consideration by reacting the contaminating phase with acid or base, followed by filter-

ing, washing, and calcination, was the key of the process.

TEM micrographs of unmilled (left) and 10 h milled zircon (right) powders.

Ceramic Materials ‐ Synthesis, Characterization, Applications and Recycling

Suminar Pratapa1 \*, Ella A.D. Kiswanti1 , Dien R. Diana1 , Yufi Hariyani1 , Lisma D.K. Sari2 , Musyarofah Musyarofah1 , Triwikantoro Triwikantoro1 and Malik A. Baqiya1

1 Department of Physics, Faculty of Science, Institute of Technology Sepuluh Nopember (ITS), Surabaya, Indonesia

2 Study Program of Mathematics, STKIP PGRI, Situbondo, Indonesia

\*Address all correspondence to<EMAIL_ADDRESS>
© 2018 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.


**1. Introduction**

#### **1.1. Viruses: Special class of organisms**

Viruses form a major class of biological entities encompassing diverse environments ranging from algae in marine ecosystems to soil, plant, human and animal systems. Several metage‐

© 2015 The Author(s). Licensee InTech. 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.

nomic studies have revealed the possibility of viruses being the dominant species of our biosphere [1]. Deep sequencing efforts have shown that viruses form 106 –109 particles per millilitre of seawater [2]. It is also interesting to note that ~90% of the reads obtained from such experiments did not encode proteins, which are reported in other organisms, including viruses, that have been characterised so far. This clearly demonstrates that the actual viral diversity has not been sampled in an adequate manner so far. A crucial aspect of viral studies is the disease burden associated with them, which is known to be enormous with serious economic implications. World Health Organization documents that the global burden of communicable diseases (of which viral diseases form a major chunk) is ~15 million annually [3].

Beyond abundance aspects, study of viral evolution and genetic variations enabled the proposal of the virocentric standpoint of the evolution. Viruses gained centre stage for reasons such as being smallest replicating entities, having short generation time, large population sizes and high replication and mutation rates. Attributes such as variation in genome sizes, gene pool, shape and assembly of particles are responsible for viruses to attain pivotal role in the study of evolution [4]. It has been observed that all plausible replication and expression strategies have been employed by viruses to dynamically adapt to the ever-changing envi‐ ronments. Processes like complementation, recombination, reassortment, high mutation rate and existence as quasispecies enable the viruses to outgrow and outcompete the host immune system. The molecular forces driving these processes can be delineated by sequencing and the subsequent analyses.


**About the Editors**

**Fred´ eric ´ Dierick** holds a Ph.D. in Physiotherapy and Rehabilitation (Universite´ Catholique de Louvain (UCLouvain), Belgium, 2006). From 2003 to 2019, he worked at the Haute Ecole Louvain in Hainaut (HELHa) as a teacher-researcher and developed the Forme et Fonctionnement Humain laboratory (Physiotherapy Department, Charleroi, Belgium). In 2020, he joined the CNRFR—Rehazenter (Luxembourg, Luxembourg) as a senior researcher and manager of national and international research projects. Currently he is also project manager at CeREF (HELHa's research center). His research is devoted to human motion analysis in healthy and pathological states, especially the development of multidisciplinary methods involving low-cost sensors, medical imaging, and nonlinear time series analysis.

**Fabien Buisseret** has a degree in Physics and a Ph.D. in Science (University of Mons (UMONS), Belgium). After pursuing research in hadronic physics as a post-doctoral fellow at UMONS, he became a teacher at Haute Ecole Louvain in Hainaut, a researcher at CeREF (HELHa's research center) and a scientific collaborator at UMONS. His research interests include human motion analysis through mechanical, nonlinear, and sensor-based approaches, as well as theoretical and hadronic physics. He also participates in the creation and management of interdisciplinary projects for CeREF.

**St´ephanie Eggermont** is a civil engineer (Facult´e Polytechnique de Mons). She holds a Ph.D. in engineering sciences (Universite´ catholique de Louvain (UCLouvain), Belgium, 2013) in the field of antennas involving metamaterials. Since September 2011, she has been professor at the Haute Ecole Louvain in Hainaut and head of research of the electricity/electronics department of CeREF (HELHa research center). She is involved in various research projects aiming, in particular, at the development of intelligent and connected electronic boards and platforms in medical or cultural applications.


**Abstract**

Dengue is an acute febrile disease caused by a virus of the genus *Flavivirus*, family Flaviviridae, endemic in tropical regions of the globe. The agent is a virus with single-stranded RNA, classified into four distinct dengue virus (DENV) serotypes: DENV-1, DENV-2, DENV-3, and DENV-4. The host's innate and adaptive immune responses play an essential role in determining the natural history of viral infections, especially in dengue. In this context, it has observed in recent years that the presence of RNA interference (RNAi) in viral infection processes is increasing, as well as immune defense. The context microRNAs (miRNAs) go for stood out, as their presence during viral infection, both in the replication of the virus and in the defense against these infections, becomes increasingly noticeable, therefore, making it increasingly necessary to better understand the role of these small RNAs within viral infection by DENV and what their consequences are in aggravating the consequences of patients affected by this disease.

**Keywords:** dengue, miRNA, genetics, immunology, *Flavivirus*

### **1. Introduction**

RNA interference (RNAi) is understood as the mechanism of gene silencing through transcription or post-transcription. Post-transcription gene silencing (PTGS) operates through translational repression induced by microRNAs (miRNAs), from precursors transcribed in the nucleus [1].

The silencing machinery by microRNAs directs mRNA to the P bodies present in the cytoplasm. They are deprived of the translation machinery and conserve proteins involved in the degradation of the target mRNA [2].

The natural functions of RNAi and their related processes appear to be the protection of the genome against invasion by mobile genetic elements, such as viruses and transposons, as well as the functioning of eukaryotic organism development programs [3, 4].

These analyses led to the identification of proteins encoded by the host involved in gene silencing. Also, they revealed that several enzymes or essential factors are common to these processes. Some components have identified to serve as initiators. In contrast, others serve as effectors, amplifiers, and transmitters for the gene silencing process [5].

The RNAi is widely used to fight viruses, due to the exposure of their genetic material in the intracellular environment at various stages of their replication cycle [6, 7]. Due to the distinction between viral and cellular genomes, the

chances of cross-silencing are low. In contrast, mutational changes in the viral genome allow mechanisms to escape interference pathways [8, 9].

### **2. miRNA**

The microRNAs are defined as small single-stranded RNA molecules with approximately 19–25 nucleotides, not protein encoders, that act as mediators for the regulation of the posttranscriptional gene expression [10].

The first miRNA was described in 1993 and related to the regulation of larval development in *Caenorhabditis elegans*. However, understand that the miRNA class is the largest class of gene regulators, with around 1000 miRNAs. Promoting regulation is necessary to bind the 3'untranslated region of the target mRNA [11].

There are more than 2500 miRNAs identified in the human genome. Although transcriptional targets are predicted, most of these have not been validated, which makes this area for investigation rich [6, 12]. Studies revealed that viruses encode miRNA, for example, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and human immunodeficiency virus type 1 (HIV-1) [13].

miRNAs play essential roles in the cell, such as proliferation, differentiation, apoptosis, stress response, and transcriptional regulation. Studies have shown that miRNAs change their expression in several pathologies, increasing their importance and the need for a better understanding of this process [14].

#### **2.1 miRNA biogenesis and mechanism of action**

The miRNA biogenesis process consists of transcription, export, processing, and maturation, at the cytoplasmic and cellular levels. As a fundamental element of this miRNA transcription, RNA polymerases II and III are found, with the function of transcribing gene encoding proteins [12].

Currently, most of the identified miRNAs are intragenic and processed from introns. Simultaneously, the rest are intergenic and transcribed independently of a host gene and regulated by their promoters [15]. Sometimes, miRNAs are transcribed as a long transcript called "clusters," which can have similar regions, and, in this case, are considered a family. miRNA biogenesis can be classified into two pathways: canonical and noncanonical (**Figure 1**) [5].

What defines the choice of these mechanisms is the complementarity between the bases of miRNA and mRNA. When there is perfect parity between the bases, degradation of the mRNAs will occur [16]. On the other hand, incomplete pairings generate inhibition of the translation of the target mRNA. Since miRNAs are small molecules, there is no need for complete pairing for binding. Thus, a miRNA can act in the regulation of several target mRNAs, or several miRNAs regulate a single mRNA [17].

#### *2.1.1 Canonical pathway*

The canonical pathway of biogenesis is the dominant pathway by which miRNAs are processed. In this way, pri-miRNAs are transcribed from their genes and processed into pre-miRNAs by the microprocessor complex, consisting of a protein that binds RNA to the critical region of DiGeorge syndrome 8 (DGCR8) and a ribonuclease III enzyme, Drosha [18]. DGCR8 recognizes an N6-methyladenylated GGAC and other motifs within the pri-miRNA [19]. At the same time, Drosha cleaves the duplex pri-miRNA based on the characteristic structure of the pri-miRNA, resulting in the formation of an excess of 2 nt 3′ in the pre-miRNA [20]. Once pre-miRNAs

**135**

**Figure 1.**

sively unwound and degraded [23].

*Dengue Virus and the Relationship with MicroRNAs DOI: http://dx.doi.org/10.5772/intechopen.92453*

are generated, they are exported to the cytoplasm by an exportin 5 (XPO5)/RanGTP

*MicroRNA biogenesis and mechanism of action. The canonical miRNA biogenesis begins with the generation of the pri-miRNA transcript passing through the microprocessor complex, composed of the critical region 8 of Drosha and DiGeorge syndrome (DGCR8), and cleaves the pri-miRNA to produce the precursor miRNA (pre-miRNA). The mature miRNA is associated with the Argonaute (AGO) protein family forming a miRNAinduced silencing complex (miRISC). In noncanonical pathways, small hairpin RNA (shRNA) is initially cleaved by the microprocessor complex and exported to the cytoplasm via exportin 5/RanGT, cleaved by AGO2,*

This processing involves removing the loop from the terminal, resulting in a mature miRNA duplex. The directionality of the miRNA chain determines the name of the mature miRNA form. The 5p chain emerges from the 5′ end of the premiRNA hairpin, while the 3p chain originates from the 3′ end. Both chains derived from the mature miRNA duplex can carry in the Argonaute family of proteins. miRNA chains that do not contain incompatibilities are cleaved by AGO2 and degraded by cellular machinery that can produce a strong chain bias. Otherwise, miRNA duplexes with central mismatches or miRNA not loaded with AGO2 are pas-

complex and processed by the RNase III Dicer endonuclease [5, 22].

*but this action is independent of Dicer (modified by Tanzer et al.) [21].*

*Dengue Fever in a One Health Perspective*

**2. miRNA**

chances of cross-silencing are low. In contrast, mutational changes in the viral

The microRNAs are defined as small single-stranded RNA molecules with approximately 19–25 nucleotides, not protein encoders, that act as mediators for the

The first miRNA was described in 1993 and related to the regulation of larval development in *Caenorhabditis elegans*. However, understand that the miRNA class is the largest class of gene regulators, with around 1000 miRNAs. Promoting regulation is necessary to bind the 3'untranslated region of the target mRNA [11]. There are more than 2500 miRNAs identified in the human genome. Although transcriptional targets are predicted, most of these have not been validated, which makes this area for investigation rich [6, 12]. Studies revealed that viruses encode miRNA, for example, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV),

miRNAs play essential roles in the cell, such as proliferation, differentiation, apoptosis, stress response, and transcriptional regulation. Studies have shown that miRNAs change their expression in several pathologies, increasing their importance

The miRNA biogenesis process consists of transcription, export, processing, and maturation, at the cytoplasmic and cellular levels. As a fundamental element of this miRNA transcription, RNA polymerases II and III are found, with the function of

Currently, most of the identified miRNAs are intragenic and processed from introns. Simultaneously, the rest are intergenic and transcribed independently of a host gene and regulated by their promoters [15]. Sometimes, miRNAs are transcribed as a long transcript called "clusters," which can have similar regions, and, in this case, are considered a family. miRNA biogenesis can be classified into two

What defines the choice of these mechanisms is the complementarity between the bases of miRNA and mRNA. When there is perfect parity between the bases, degradation of the mRNAs will occur [16]. On the other hand, incomplete pairings generate inhibition of the translation of the target mRNA. Since miRNAs are small molecules, there is no need for complete pairing for binding. Thus, a miRNA can act in the regulation of several target mRNAs, or several miRNAs regulate a single

The canonical pathway of biogenesis is the dominant pathway by which miRNAs are processed. In this way, pri-miRNAs are transcribed from their genes and processed into pre-miRNAs by the microprocessor complex, consisting of a protein that binds RNA to the critical region of DiGeorge syndrome 8 (DGCR8) and a ribonuclease III enzyme, Drosha [18]. DGCR8 recognizes an N6-methyladenylated GGAC and other motifs within the pri-miRNA [19]. At the same time, Drosha cleaves the duplex pri-miRNA based on the characteristic structure of the pri-miRNA, resulting in the formation of an excess of 2 nt 3′ in the pre-miRNA [20]. Once pre-miRNAs

genome allow mechanisms to escape interference pathways [8, 9].

regulation of the posttranscriptional gene expression [10].

and human immunodeficiency virus type 1 (HIV-1) [13].

and the need for a better understanding of this process [14].

**2.1 miRNA biogenesis and mechanism of action**

pathways: canonical and noncanonical (**Figure 1**) [5].

transcribing gene encoding proteins [12].

**134**

mRNA [17].

*2.1.1 Canonical pathway*

#### **Figure 1.**

*MicroRNA biogenesis and mechanism of action. The canonical miRNA biogenesis begins with the generation of the pri-miRNA transcript passing through the microprocessor complex, composed of the critical region 8 of Drosha and DiGeorge syndrome (DGCR8), and cleaves the pri-miRNA to produce the precursor miRNA (pre-miRNA). The mature miRNA is associated with the Argonaute (AGO) protein family forming a miRNAinduced silencing complex (miRISC). In noncanonical pathways, small hairpin RNA (shRNA) is initially cleaved by the microprocessor complex and exported to the cytoplasm via exportin 5/RanGT, cleaved by AGO2, but this action is independent of Dicer (modified by Tanzer et al.) [21].*

are generated, they are exported to the cytoplasm by an exportin 5 (XPO5)/RanGTP complex and processed by the RNase III Dicer endonuclease [5, 22].

This processing involves removing the loop from the terminal, resulting in a mature miRNA duplex. The directionality of the miRNA chain determines the name of the mature miRNA form. The 5p chain emerges from the 5′ end of the premiRNA hairpin, while the 3p chain originates from the 3′ end. Both chains derived from the mature miRNA duplex can carry in the Argonaute family of proteins. miRNA chains that do not contain incompatibilities are cleaved by AGO2 and degraded by cellular machinery that can produce a strong chain bias. Otherwise, miRNA duplexes with central mismatches or miRNA not loaded with AGO2 are passively unwound and degraded [23].

#### *2.1.2 Noncanonical pathway*

Several noncanonical biogenesis pathways in miRNA are elucidated (**Figure 1**). These pathways make use of combinations of proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2. The noncanonical miRNA can be grouped into Drosha-/DGCR8-independent and Dicer-independent pathways [24, 25].

The pre-miRNAs produced by the Drosha-/DGCR8-independent pathway resemble Dicer products. On the other hand, Dicer-independent miRNAs are processed by Drosha from endogenous RNA transcripts of hairpins. These premiRNAs require AGO2 to complete their maturation in the cytoplasm. They are of insufficient length to be the substrates for Dicer. That, in turn, promotes the loading of the entire pre-miRNA in the AGO2 slicing [26, 27].

#### *2.1.3 Argonaute and TNRC6 proteins*

The proteins of the Argonaute family are related to the RISC complex, as a member of the machinery of the RNAi pathways [28]. The highly conserved between species and several organisms encode several members of the family. Usually found in the cytoplasm are concentrated close to the P bodies [29].

Such proteins, therefore, act with the transcriptional and posttranscriptional silencing pathways. The main stage of the interference mechanism is the cleavage of mRNAs; the Argonaute protein in the RISC complex catalyzes this process [28].

Argonautes are applied in transcriptional and posttranscriptional gene silencing, acting through the modulation of the degradation or inhibition of the translation of specific mRNAs, when associated with miRNAs [18].

The miRNAs associated with Argonaute proteins constitute a more massive complex called the miRNA-induced silencing complex, which will suppress the expression of mRNAs. In addition to interference at the translational level, it shows that miRNAs can induce poly(A) tail deadening [18, 19]. Studies suggest that proteins of the TNRC6 family are essential components when associated with miRISCs, for the location of cytoplasmic P bodies and the gene silencing of mRNAs [19, 20].

#### **3. miRNA and dengue virus**

The dengue virus is a virus composed of a single-stranded positive RNA belonging to the family Flaviviridae. DENV serotypes have been identified (DENV-1 to DENV-4). All serotypes are causing similar diseases and similar symptoms, without significant severity and serious diseases, such as dengue hemorrhagic fever and dengue shock syndrome. The DENV genome is approximately 11 kb in length that encodes a single polyprotein. This polyprotein is cleaved posttranslationally by the host and viral proteases into three structural proteins (capsid C; pre-membrane/ membrane, prM/M; envelope, E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [30, 31].

The DENVs enter their target cells via receptor-mediated endocytosis in a clathrin-dependent manner. An acidified endosomal vesicle, virion, undergoes conformational changes that allow fusion and endosomal membrane and release RNA from the genome into the cytosol. After initial translation and cleavage of polyprotein, DENV triggers the formation of a replication complex in the perinuclear endoplasmic reticulum, and RNA replication and protein translation occur. Protein C then packages the newly synthesized positive RNA assembled into a virion, which

**137**

*Dengue Virus and the Relationship with MicroRNAs DOI: http://dx.doi.org/10.5772/intechopen.92453*

**3.1 Mosquitoes, miRNAs, and dengue**

resistance to insecticides [34].

blood feeding [35–37].

*Ae. albopictus* [36, 37].

*3.1.2 Mosquitoes and dengue infection*

*3.1.1 Development and metabolism*

secreted [32, 33].

is covered with prM/E heterodimers. When the vesicles containing the immature virions move through the Golgi apparatus, the prM is cleaved by a furin protease. Finally, immature virions become mature or partially mature virions, which are

The functions that miRNA is involved in mosquitoes are related to posttranscriptional regulation of gene expression in physiological and immunological pathways and affect development, metabolism, host-pathogen interactions, and

The specific expression of the miRNA stage in the four stages of development (eggs, larvae, pupae, and adults) was confirmed using sequencing. Understand the role of regulated miRNAs in the development of the mosquito and the action of knocking down the expressed miRNAs in a specific way carried out in *Ae. albopictus*. The knockdown of aal-miR-286b and aal-miR-2942 decreased the hatching of embryos and the hatching rate of larvae, respectively, compared to the knock-in groups. Reduced longevity and fertility (aal-miR-1891) were observed in the knockdown groups for miR-1891 compared to the knock-in and control groups in adults. Female mosquitoes require sugar for energy metabolism and a blood meal for egg development. Recent studies have indicated that blood supply leads to the differential expression of many genes, proteins, and miRNAs. The abundance of miRNA differs under sugar-fed and blood-fed conditions; ast-miR-2796-5p was observed exclusively in sugar-fed. The depletion of aae-miR-275 in *Ae. aegypti* females led to severe defects in blood digestion, fluid excretion, and egg development. aaemiR-1890 is induced after blood feeding and reaches a peak of 24 PMB. The systemic depletion of aae-miR-1890 resulted in less egg development and deposition, suggesting that miR-1890 may be the key to mosquito blood digestion. In contrast to the upregulated miRNAs after blood feeding, some miRNAs were downregulated. For example, reduced ast-miR-989 was observed 72 h after a blood meal. aga-let7 decreased in the midgut and other parts/leftovers, but most miRNAs increased after

The viruses of the *Flavivirus* genus are transmitted by mosquitoes and cause diseases, including dengue. It is observed that *Cx. quinquefasciatus* mosquitoes with West Nile virus (WNV) showed altered miR-92 and miR-989 expressions. In *Aedes albopictus*, the aae-miR-2940 miRNA is downregulated in response to WNV infection to restrict viral replication. In studies, the expressions of 35 miRNAs of mosquitoes modulating DENV infection in *Aedes aegypti* and more than 66 miRNAs were reported. *Ae. albopictus* is differentially expressed after DENV-2 infection. Therefore, aal-miR-34-5p and aal-miR-87 contribute to antipathogenic and immunological responses during DENV-2 infection. ae-miR-375 is the key to DENV replication, which can improve DENV-2 infection in an *Ae. aegypti* cell line. aae-miR-252 is three times more expressed after DENV-2 infection in *Ae. albopictus* cell line (C6/36); this inhibited DENV replication by suppressing the expression of the envelope protein of DENV. Regarding aal-miR-281, an abundant miRNA specific to the midgut, it was found that it facilitates the replication of DENV-2 in

*Dengue Fever in a One Health Perspective*

*2.1.3 Argonaute and TNRC6 proteins*

Several noncanonical biogenesis pathways in miRNA are elucidated (**Figure 1**). These pathways make use of combinations of proteins involved in the canonical pathway, mainly Drosha, Dicer, exportin 5, and AGO2. The noncanonical miRNA can be grouped into Drosha-/DGCR8-independent and Dicer-independent path-

The pre-miRNAs produced by the Drosha-/DGCR8-independent pathway resemble Dicer products. On the other hand, Dicer-independent miRNAs are processed by Drosha from endogenous RNA transcripts of hairpins. These premiRNAs require AGO2 to complete their maturation in the cytoplasm. They are of insufficient length to be the substrates for Dicer. That, in turn, promotes the loading

The proteins of the Argonaute family are related to the RISC complex, as a member of the machinery of the RNAi pathways [28]. The highly conserved between species and several organisms encode several members of the family. Usually found

Such proteins, therefore, act with the transcriptional and posttranscriptional silencing pathways. The main stage of the interference mechanism is the cleavage of mRNAs; the Argonaute protein in the RISC complex catalyzes this process [28].

The miRNAs associated with Argonaute proteins constitute a more massive complex called the miRNA-induced silencing complex, which will suppress the expression of mRNAs. In addition to interference at the translational level, it shows that miRNAs can induce poly(A) tail deadening [18, 19]. Studies suggest that proteins of the TNRC6 family are essential components when associated with miRISCs, for the location of cytoplasmic P bodies and the gene silencing of

The dengue virus is a virus composed of a single-stranded positive RNA belonging to the family Flaviviridae. DENV serotypes have been identified (DENV-1 to DENV-4). All serotypes are causing similar diseases and similar symptoms, without significant severity and serious diseases, such as dengue hemorrhagic fever and dengue shock syndrome. The DENV genome is approximately 11 kb in length that encodes a single polyprotein. This polyprotein is cleaved posttranslationally by the host and viral proteases into three structural proteins (capsid C; pre-membrane/ membrane, prM/M; envelope, E) and seven nonstructural proteins (NS1, NS2A,

The DENVs enter their target cells via receptor-mediated endocytosis in a clathrin-dependent manner. An acidified endosomal vesicle, virion, undergoes conformational changes that allow fusion and endosomal membrane and release RNA from the genome into the cytosol. After initial translation and cleavage of polyprotein, DENV triggers the formation of a replication complex in the perinuclear endoplasmic reticulum, and RNA replication and protein translation occur. Protein C then packages the newly synthesized positive RNA assembled into a virion, which

Argonautes are applied in transcriptional and posttranscriptional gene silencing, acting through the modulation of the degradation or inhibition of the translation of

of the entire pre-miRNA in the AGO2 slicing [26, 27].

in the cytoplasm are concentrated close to the P bodies [29].

specific mRNAs, when associated with miRNAs [18].

*2.1.2 Noncanonical pathway*

ways [24, 25].

mRNAs [19, 20].

**3. miRNA and dengue virus**

NS2B, NS3, NS4A, NS4B, and NS5) [30, 31].

**136**

is covered with prM/E heterodimers. When the vesicles containing the immature virions move through the Golgi apparatus, the prM is cleaved by a furin protease.


*2.4. Physico-Chemical Characterization*

**Infrared spectroscopy.** Infrared (IR) spectra were obtained using a Nicolet iN10 MX Fourier-transform (FT)-IR microscope (Thermo Fischer Scientific, Waltham, MA, USA) with a liquid nitrogen-cooled mercury cadmium telluride (MCT) detector with a measurement range of 4000–700 cm−1. Spectra collection was performed in reflection mode at a resolution of 4 cm−1. For each spectrum, 32 scans were co-added and converted to absorbance using the OmincPicta software (Version 1, Thermo Fischer Scientific, Waltham, MA, USA).

**X-ray di**ff**raction.** Grazing incidence X-ray diffraction (GIXRD) was performed with a Panalytical Empyrean diffractometer (PANalytical, Almelo, The Netherlands), using CuK radiation (1.541874 A) equipped with a 2× Ge (2 2 0) hybrid monochromator for Cu and a parallel plate collimator on the PIXcel3D. Scanning was performed on the 2θ axis in the range of 5–80◦, with an incidence angle of 0.5◦, a step size of 0.04◦, and a time step of 3 s.

**Scanning electron microscopy.** In order to investigate the morphology and size of the nanostructured membranes, images produced by recording the resultant secondary electron beam with an energy of 30 keV of the samples were taken with a scanning electron microscope purchased from FEI (Hillsboro, OR, USA).

**Transmission electron microscopy (TEM). Electron di**ff**raction on selected area.** In order to obtain transmission electron microscopy images, the samples were placed on a carbon-coated copper grid at room temperature. The TEM images were acquired using a high-resolution TecnaiTM G2 F30 S-TWIN transmission microscope equipped with selected area electron diffraction (SAED), purchased from FEI (Hillsboro, OR, USA). This microscope operates in transmission mode at a voltage of 300 kV, while the point and line resolution are guaranteed with values of 2 Å and 1 Å, respectively. The SAED analysis for silver nanoparticles was performed in a light field using the TecnaiTM G2 F30 S-TWIN high-resolution electronic microscope equipped with SAED, purchased from FEI (Hillsboro, OR, USA).

#### *2.5. In Vitro Biocompatibility*

## 2.5.1. Cell Line

Human amniotic fluid stem cells (AFSC) were used to evaluate the biocompatibility of the prepared samples. The AFSC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin/neomycin). All cells were maintained at 37 ◦C in a humidified incubator with 5% CO2.

#### 2.5.2. MTT (3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) Assay

The evaluation of cell viability was performed by measuring the degree of reduction of a tetrazolium salt solution, MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide), to insoluble purple formazan crystals by viable cells. In this purpose, the AFSC cells were seeded in 96-well plates, with a density of 3000 cells/well under different experimental conditions. Subsequently, a volume of 10 μL of 12 mM MTT was added and the cells were incubated at 37 ◦C for 4 h. A volume of 100 μL of SDS–HCl solution was further added, vigorously pipetted to solubilize the formazan crystals, incubated for 1 h, and read at 570 nm (TECAN spectrophotometer, Männedorf, Switzerland).

#### *2.6. In Vitro Antibacterial Tests*

#### 2.6.1. Growth of Planktonic (Floating) Microorganisms in the Presence of Material

To test the effect of the obtained material on the growth of microorganisms in liquid medium (planktonic cultures), the obtained material was cut into 1-cm2 samples and sterilized by exposure to ultraviolet (UV) radiation for 30 min on each side. One fragment of the sterile material was individually deposited in a well of a six-well sterile plate. Over the deposited materials, 2 mL of liquid medium and then 20 μL of 0.5 McFarland microbial suspension (bacteria—*Staphylococcus aureus* American Type Culture Collection (ATCC) 25923 and *Pseudomonas aeruginosa* ATCC 27853) or 1McFarland (yeast—*Candida albicans* ATCC 10231) prepared in sterile physiological water, 0.9% NaCl salt, was added to the wells. The as-prepared six-well plates were incubated at 37 ◦C for 24 h. At the end of the incubation time, 200 μL of the obtained microbial suspension was transferred to 96 sterile plates, and the turbidity of the microbial cultures (absorbance) was spectrophotometrically measured at 600 nm.

#### 2.6.2. Evaluation of Adhesion and Biofilm Formation

To test the e ffect of fibrillated materials on microbial adhesion and biofilm production, the sterile material samples treated as described above were washed with sterile saline water (SSW), and the medium was changed to allow the microbial cells—adhered onto the surface of the material samples in the first 24 h of incubation—to continue biofilm development and maturation for another 24, 48, and 72 h. After the end of each incubation period, the colonized sample was washed with AFS to remove non-adherent microorganisms and deposited in a sterile tube in 1 mL of SSW. The tube was vigorously vortexed for 30 s and sonicated for 10 s to harvest the cells from the biofilm. The obtained cell suspension was diluted, and various ten-fold serial dilutions were seeded on solid culture media plates in triplicate, to obtain and quantify the number of viable cells, expressed in colony-forming units (CFU)/mL.

#### *2.7. In Vivo Biocompatibility Evaluation*

#### 2.7.1. Animals and Ethics

CD1 mice were housed in controlled-airflow cabinets with 12-h light cycles and constant temperature and humidity conditions. Animal experiments were performed in accordance with the guidelines of the Vasile Goldis Western University of Arad and approved by the Ethical Committee.

#### 2.7.2. Experimental Design and Surgical Procedures

The mice were randomly assigned to 18 groups (*n*=5): control, PET\_2.5\_ctrl, PET\_5\_ctrl, PET\_7.5\_ctrl, PET\_10\_ctrl, PET\_2.5\_NanoAg, PET\_5\_NanoAg, PET\_7.5\_NanoAg, and PET\_10\_NanoAg, for one day and seven days.

Before the experiment, the material samples (1 cm2) were sterilized under UV light for 30 min (both faces) and implanted into a subcutaneous pocket in the dorsum of the animals. For the surgical procedure, the animals were anesthetized by intraperitoneal injection of xylazine/ketamine.

Animals were allowed to recover from anesthesia, housed in individual cages, and observed daily for evidence of wound complications, such as redness, infection, edema, abscess, hematoma, encapsulation, or skin dehiscence.

On days two and seven post-surgery, the animals were euthanatized by anesthetic overdose and the implanted materials, together with the surrounding tissues, were explanted and collected for analysis. Blood was sampled by cardiac puncture to assess acute inflammatory markers, in order to be able to exclude systemic inflammation.


**1. Introduction**

A well-designed, efficiently-performing WDS is a prerequisite for both urban and rural civilizations. Various software packages are available for modelling a WDS. The public domain software that is available free of charge include EPANET, Pipes and Loops, Branch, etc., and the paid ones available includes WaterCAD, Watergems, Synergy Water, etc. [1]. The paid versions are found to be more efficient in reference to functionality and compatibility with different computational systems, a graphical user interface, and searching and optimizing algorithms [2]. However, EPANET is freely available software that offers a fair degree of accuracy and ease of use when cost is a constraint [3]. EPANET 2.0 was used by various researchers to design the WDS for different areas [4,5]. The design includes the layout of the WDS, modelling the pressure head at nodes, and the velocity of flow in the links. However, no consideration was given to maximisation of the efficiency of performance or minimisation of the capital cost of the network.

The design of a WDS by EPANET must be such that it serves as an "optimal" solution, which can be obtained by setting out objective functions of maximum efficiency of performance and minimum capital cost of the network [6,7]. The decision variables and the

**Citation:** Ajaz, M.; Ahmad, D. Application of EPANET 2.2 Software and Jal-Tantra Web System for Optimal Hydraulic Design of Water Distribution System for University of Kashmir. *Environ. Sci. Proc.* **2023**, *25*, 83. https://doi.org/10.3390/ ECWS-7-14234

Academic Editor: Lampros Vasiliades

Published: 16 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

constraints for the optimality of the network design have to be decided after conducting a study of the factors affecting the optimality of the design and consulting various codes and manuals laying down the standard range of values for those parameters [8,9]. The decision variables for optimal hydraulic design include the pressure head at nodes, the velocity of flow in the links, and the staging height of the storage tank. The check for cost optimality of the network formulated by EPANET can be conducted using the Jal-Tantra web system [10,11]. The optimality of the network can be further checked by carrying out a performance analysis using various indices [12–14].

The objective of this current study is to provide an optimally designed WDS for the University of Kashmir using EPANET 2.0 software and the Jal-Tantra web system. The existing distribution system has already served for more than 60 years and is unable to meet the requirements of the growing population of the campus. Moreover, the worn-out links and other components of the network lead to increased pipe and joint losses, reducing the efficiency of the existing distribution system. Therefore, there is an immediate need to redesign the water distribution network such that the design is an optimal solution meeting the objective functions of maximum efficiency of performance and minimum capital cost of the network, rather than just a simulation of the pressure head and velocity of flow.


*3.3. Biological Activity*

Compounds **1**–**3** were evaluated for their antimicrobial activities in 96-well plates according to the Antimicrobial Susceptibility Testing Standards outlined by the Clinical and Laboratory Standards Institute Document M07-A7 (CLSI) and our previous report [27–29]. The MIC was defined as the minimum concentration of the compound that prevented visible growth of the microbes.


**Aerogel Hybrids and Nanocomposites**

Editors

**Istv´an L´az´ar Melita Menelaou**

*Editors* Istvan L ´ az´ ar´ Department of Inorganic and Analytical Chemistry University of Debrecen Debrecen Hungary

Melita Menelaou Department of Chemical Engineering Cyprus University of Technology Limassol Cyprus

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

This is a reprint of articles from the Special Issue published online in the open access journal *Gels* (ISSN 2310-2861) (available at: www.mdpi.com/journal/gels/special issues/Aerogel Hybrids).

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